A Single Immunization with Spike-Functionalized Ferritin Vaccines Elicits Neutralizing Antibody Responses against SARS-CoV-2 in Mice
- Abigail E. PowellAbigail E. PowellDepartment of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United StatesMore by Abigail E. Powell,
- Kaiming ZhangKaiming ZhangDepartment of Bioengineering & James H. Clark Center, Stanford University, Stanford, California 94305, United StatesMore by Kaiming Zhang,
- Mrinmoy SanyalMrinmoy SanyalDepartment of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United StatesMore by Mrinmoy Sanyal,
- Shaogeng TangShaogeng TangDepartment of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United StatesMore by Shaogeng Tang,
- Payton A. WeidenbacherPayton A. WeidenbacherDepartment of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United StatesDepartment of Chemistry, Stanford University, Stanford, California 94305, United StatesMore by Payton A. Weidenbacher,
- Shanshan LiShanshan LiDepartment of Bioengineering & James H. Clark Center, Stanford University, Stanford, California 94305, United StatesMore by Shanshan Li,
- Tho D. PhamTho D. PhamDepartment of Pathology, Stanford University, Stanford, California 94305, United StatesStanford Blood Center, Palo Alto, California 94304, United StatesMore by Tho D. Pham,
- John E. PakJohn E. PakChan Zuckerberg Biohub, San Francisco, California 94158, United StatesMore by John E. Pak,
- Wah ChiuWah ChiuDepartment of Bioengineering & James H. Clark Center, Stanford University, Stanford, California 94305, United StatesChan Zuckerberg Biohub, San Francisco, California 94158, United StatesDivision of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United StatesMore by Wah Chiu, and
- Peter S. Kim*Peter S. Kim*E-mail: [email protected]Department of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United StatesChan Zuckerberg Biohub, San Francisco, California 94158, United StatesMore by Peter S. Kim
Copyright © 2021 The Authors. Published by American Chemical Society
Abstract
The development of a safe and effective SARS-CoV-2 vaccine is a public health priority. We designed subunit vaccine candidates using self-assembling ferritin nanoparticles displaying one of two multimerized SARS-CoV-2 spikes: full-length ectodomain (S-Fer) or a C-terminal 70 amino-acid deletion (SΔC-Fer). Ferritin is an attractive nanoparticle platform for production of vaccines, and ferritin-based vaccines have been investigated in humans in two separate clinical trials. We confirmed proper folding and antigenicity of spike on the surface of ferritin by cryo-EM and binding to conformation-specific monoclonal antibodies. After a single immunization of mice with either of the two spike ferritin particles, a lentiviral SARS-CoV-2 pseudovirus assay revealed mean neutralizing antibody titers at least 2-fold greater than those in convalescent plasma from COVID-19 patients. Additionally, a single dose of SΔC-Fer elicited significantly higher neutralizing responses as compared to immunization with the spike receptor binding domain (RBD) monomer or spike ectodomain trimer alone. After a second dose, mice immunized with SΔC-Fer exhibited higher neutralizing titers than all other groups. Taken together, these results demonstrate that multivalent presentation of SARS-CoV-2 spike on ferritin can notably enhance elicitation of neutralizing antibodies, thus constituting a viable strategy for single-dose vaccination against COVID-19.
Synopsis
Multivalent SARS-CoV-2 spike displayed on a self-assembling ferritin nanoparticle elicits a more robust neutralizing antibody response in mice following single immunization as compared to spike alone.
Introduction
ARTICLE SECTIONS
The emergence of SARS-CoV-2 in the human population in 2019 has caused a rapidly growing pandemic that has disrupted nearly all global infrastructures. To date, there have been over 63 million confirmed cases of COVID-19 and nearly 1.5 million deaths worldwide.(1) While some nations have controlled viral spread through social distancing, widespread testing, and contact tracing, many nations struggle to contain the growing number of cases and are still experiencing extensive community spread. Additionally, the introduction of SARS-CoV-2 into low-resource settings will lead to severe and lasting impacts on economic and healthcare systems. Long-term control of the pandemic will require one or more effective vaccines that can be made widely available across the globe.
The primary viral target for protective antibody-based vaccines against COVID-19 is the SARS-CoV-2 spike, a trimeric surface glycoprotein responsible for viral entry.(2,3) Importantly, COVID-19 patients have been shown to elicit robust neutralizing antibody responses directed at the SARS-CoV-2 spike, which suggests that this antigen could be promising in the context of a protective vaccine.(4,5) The spike protein is produced as a single polypeptide and cleaved to form the S1 and S2 subunits, which are responsible for receptor binding (S1) and fusion with the host cell membrane (S2).(3,6,7) The receptor binding domain (RBD) is a 25 kDa domain of S1 that recognizes the SARS-CoV-2 human receptor, angiotensin converting enzyme 2 (ACE2), and can form a functionally folded domain when expressed separately from the rest of S1.(8−10)
A vast array of vaccination platforms are being employed for the development of a safe and effective SARS-CoV-2 vaccine.(11−19) Several vaccine candidates are currently being investigated in Phase 3 clinical trials including two mRNA-based vaccines and two virally vectored vaccines.(20−22) Importantly, the mRNA vaccine candidates rely on lipid–nanoparticle encapsulation and require long-term cold-chain storage (−20 to −80 °C),(20,21) leading to extensive logistical challenges for distribution and administration. Virus-based vaccines including inactivated, live-attenuated, and recombinant viral vaccines can produce robust immune responses, but are known to induce off-target vector-directed immune responses(23,24) and can be associated with more frequent side effects and adverse events.(25,26)
Subunit vaccines, in which a protein antigen from the pathogen is used to elicit a protective antibody response, are an attractive option for an accessible SARS-CoV-2 vaccine for reasons including safety, manufacturing scalability, and ease of distribution to low- and middle-income nations.(26) Though typically less immunogenic than virus-based vaccines, the immunogenicity of subunit vaccinations can be significantly increased by formulation with adjuvants.(25) It has also been demonstrated that multivalent presentation of antigens markedly enhances the immune response,(27,28) and several nanoparticle-based platforms have been utilized to multimerize antigens of interest to improve the antibody response to subunit vaccine candidates.(27−30) Furthermore, two recent studies have shown that the multivalent presentation of the SARS-CoV-2 RBD(31) as well as the spike ectodomain(32) using various multimerization platforms elicits better neutralizing antibody responses than nonmultimerized forms of the same antigens.
One such multimerization platform, Helicobacter pylori ferritin, has been used to display antigens from influenza,(33,34) HIV-1,(35,36) and Epstein–Barr virus,(30) among others.(37,38)H. pylori ferritin self-assembles into 24-subunit particles with eight 3-fold axes of symmetry.(39) Fusion of a single protomer of a viral glycoprotein to the N-terminal region of an H. pylori ferritin subunit facilitates assembly of a protein nanoparticle that displays eight copies of a trimeric antigen on the surface at the 3-fold axes.(33,39) Display of antigens on ferritin generally elicits a more robust neutralizing antibody response against the target pathogen as compared to immunization with the antigen alone.(30,33,35) Importantly, two influenza-functionalized ferritin vaccines have been shown to be safe and immunogenic in clinical trials (NCT03186781 and NCT03814720),(33,34) and robust pipelines have been established for large-scale manufacturing of ferritin-based vaccines.(40)
Here, we fused the full-length spike ectodomain (residues 1–1213) to H. pylori ferritin (denoted S-Fer; Figure 1) to determine the effect of antigen multimerization on elicitation of antibodies against SARS-CoV-2. Additionally, we designed a second nanoparticle construct in which we deleted the C-terminal 70 residues of the ectodomain and expressed this truncated spike (residues 1–1143) on ferritin (SΔC-Fer). These C-terminal residues are unresolved in the cryo-EM structures of the spike trimer,(3,7) and it has been suggested that they have extensive conformational flexibility based on electron microscopy of soluble trimers and viral particles.(41−43) Additionally, this region of the spike contains an immunodominant linear epitope, as determined via analysis of convalescent sera from COVID-19 patients.(44,45) We therefore hypothesized that deleting these residues would more readily facilitate formation of spike ferritin particles and could influence immunogenicity. As points of comparison, we also expressed and purified three additional antigens: spike trimer containing a GCN4-based trimerization domain either in full-length or SΔC form (denoted S-GCN4 and SΔC-GCN4)(46) and monomeric receptor binding domain (RBD) (Figure 1).
Figure 1
Figure 1. Construct design for SARS-CoV-2 spike-functionalized ferritin nanoparticles. All constructs are based on the Wuhan-Hu-1 amino acid sequence (GenBank MN9089473) of SARS-CoV-2 spike. Spike-functionalized ferritin constructs were made by fusing spike ectodomain (residues 1–1213) or spikeΔC (residues 1–1143) to the H. pylori ferritin subunit separated by an SGG linker. A structural representation based on the spike trimer cryo-EM structure (PDB 6VXX) and the H. pylori ferritin crystal structure (PDB 3BVE) depicts the 24-subunit particle displaying spike or spikeΔC on the surface. The estimated size of the spike-functionalized ferritin particles based on structural data is ∼300 Å. The S-GCN4 and SΔC-GCN4 trimer constructs were made by fusing either the full-length spike residues (1–1213) or spikeΔC (1–1137) to a modified GCN4 trimerization domain followed by a hexahistidine tag. A structural representation of the spike trimers based on the cryo-EM structure (PDB 6VXX) is shown with an estimate length of ∼100 Å. The RBD spans residues 319–541 of the spike protein and is preceded by the native signal peptide (not shown) and followed by a hexahistidine tag.
After expressing and purifying the S-Fer and SΔC-Fer nanoparticles, we confirmed that they were stable, homogeneous, and properly folded using biophysical, structural, and binding analyses including size-exclusion chromatography multiangle light scattering (SEC-MALS), cryoelectron microscopy (cryo-EM), and biolayer interferometry (BLI). To assess the immune response in vivo, we immunized mice and characterized the antibody responses to the spike ferritin particles versus S-GCN4, SΔC-GCN4, and RBD. After a single dose, mice immunized with SΔC-Fer exhibited a significantly higher neutralizing antibody response than all nonferritin groups as determined using a spike-pseudotyped lentiviral assay.(47,48) Importantly, immunization with a single dose of S-Fer or SΔC-Fer elicited at least 2-fold higher neutralizing titers as those observed in plasma from convalescent COVID-19 patients. Taken together, these results provide insights into the development of SARS-CoV-2 subunit vaccines and demonstrate that spike-functionalized ferritin nanoparticles elicit an enhanced antibody response compared to the spike trimers or RBD alone.
Results
ARTICLE SECTIONS
Design of Spike-Functionalized Ferritin Nanoparticles
To design functionalized nanoparticles displaying the spike protein, we generated a fusion protein containing the spike ectodomain followed by H. pylori ferritin, a 19 kDa protein that self-assembles into a 24-subunit protein-based nanoparticle (Figure 1). Given the 3-fold symmetrical axes on the ferritin particle, fusion of a protomer to the N-terminus of this domain creates a functionalized particle that displays eight trimers on the surface.
We designed two versions of the spike-functionalized nanoparticle: one containing the full-length ectodomain (residues 1–1213; S-Fer) and one in which the C-terminus of the ectodomain was truncated (residues 1–1143; SΔC-Fer) (Figure 1). We sought to investigate whether deletion of this region would influence expression levels, protein stability, and/or the immune response to spike. All spike antigens contained a mutated furin cleavage site (RRAR mutated to a single alanine) plus two proline mutations at residues 986 and 987, which stabilize the spike trimer in the prefusion conformation.(3,49) Previous work has shown that stabilization of the prefusion conformation of other coronavirus spikes enhances protein expression and leads to greater neutralizing titers in the context of immunization;(50,51) thus, both vaccine and serology efforts involving soluble SARS-CoV-2 spike have utilized a stabilized form of the trimer.
For comparison, we also produced two spike ectodomain proteins fused to a trimeric coiled-coil, GCN4-pIQI(46) (denoted S-GCN4 and SΔC-GCN4; Figure 1). SΔC-GCN4 contained a deletion of residues 1138–1143 (which were present in SΔC-Fer) to eliminate a short helical segment prior to the start of the GCN4-pIQI coiled-coil. A similar truncated version of the spike trimer was recently utilized for antibody discovery.(5) We included the SARS-CoV-2 spike receptor binding domain (RBD; Figure 1) in our antigen panel because there is interest in employing the RBD as a potential vaccine candidate.(49,52,53)
Spike Ferritin Nanoparticles Can Be Expressed in Mammalian Cells and Purified to Homogeneity
The SARS-CoV-2 spike is a heavily glycosylated trimeric protein that can be challenging to produce recombinantly.(3,49,54) Production of recombinant spike ectodomain is commonly done in mammalian cell culture systems in which the protein is glycosylated during synthesis.(54) We expressed the spike ferritin nanoparticles using human Expi293F cells. Since the ferritin subunit facilitates self-assembly of the nanoparticles, production of purified spike-functionalized particles was achieved by transfecting a single plasmid followed by a two-step chromatographic purification (Figure 2A). After Expi293F transfection, we monitored expression levels of the four spike-based antigens (ferritins and trimers) with Western blots of cell culture supernatants probed with SARS-CoV-2 reactive monoclonal antibodies (mAbs) CR3022,(55−57) CB6,(58) and COVA2-15(5) (Figure S1A). Although fusing spike to ferritin increases the size of the antigen from ∼450 kDa (trimer) to ∼4 MDa (ferritin), the expression levels for all spike proteins were comparable (Figure S1A). Nanoparticles can sometimes remain intact during SDS-PAGE analysis, even under denaturing conditions, which can lead to retention of some sample within the well, causing the intensity of the monomer band to appear weaker. Therefore, for a more quantitative estimate of the protein levels in Expi cell culture supernatant, we quantified the expression levels of each spike antigen using a dot blot of unpurified cell culture supernatant blotted with SARS-CoV-2 mAb CR3022. We made standard curves of purified antigen and compared these curves to expression levels from cell culture supernatants from a set (n = 5) of small-scale protein expressions (Figure S1B).(59) This analysis indicated similar expression trends to those observed via Western blot and further confirmed that fusion of spike to H. pylori ferritin did not impact expression levels. Interestingly, it also suggests that fusion of SΔC to ferritin enhances the levels of protein expression under these conditions.
Figure 2
Figure 2. Spike ferritin nanoparticles can be expressed in mammalian cell culture and purified to homogeneity. (A) Scheme for expressing and purifying spike ferritin nanoparticle antigens in mammalian cells. Spike ferritin particle subunits are encoded in a single plasmid that is transfected into the Expi293F suspension human cell line. Expi293F cells are harvested, and culture supernatant is buffer exchanged and purified via anion exchange chromatography. Protein-containing fractions are identified via Western blot, pooled, and purified by size-exclusion chromatography (SRT SEC-1000). Purified nanoparticles are assessed using biophysical characterization methods including SDS-PAGE, analytical size-exclusion chromatography, and BLI follow by in vivo characterization of the immune responses elicited in mice. (B) SEC-MALS UV A280 (left) and light scattering signals (right) from analysis of spike-based ferritin antigens using an SRT SEC-1000 size-exclusion column. A single prominent peak in both the UV and light-scattering traces confirms that spike ferritin nanoparticle preparations are homogeneous and do not aggregate.
Since the spike ferritin particles lack an affinity purification tag, we purified them to homogeneity using anion exchange followed by size-exclusion chromatography (Figure 2A). We used SEC-MALS to assess sample quality and homogeneity following purification.(60) No evidence of aggregation was detected from the S-Fer or the SΔC-Fer samples via ultraviolet absorbance (A280) or light scattering signals (Figure 2B). Additionally, we determined that a freeze–thaw cycle does not perturb particle formation or cause sample aggregation (Figure S2A). We used the light scattering and refractive index data from spike nanoparticles to determine (Figure S2B) molecular weights of 4.2 MDa (S-Fer) and 3.1 MDa (SΔC-Fer). These values are close to those expected from the amino acid sequences and the additional mass(61) expected from ∼20 predicted N-linked glycosylation sites (Figure S2B). Taken together, these experiments confirm that functionalization of H. pylori ferritin with the SARS-CoV-2 spike did not perturb assembly of the nanoparticles.
We also produced the S-GCN4, SΔC-GCN4, and RBD in human Expi293F cells. We purified these samples using NiNTA purification followed by size-exclusion chromatography and confirmed sample purity and homogeneity using SEC-MALS (Figure S2C). We observed a main peak and a secondary peak for the S-GCN4 sample, which has also been noted in other recently reported work describing expression and purification conditions of spike trimers.(62) These populations may correspond to conformational populations of the trimer and have been suggested to potentially be related to RBDs on different protomers in “up” and “down” states.(3,7,62)
Structural and Functional Analysis Demonstrates That Spike-Functionalized Nanoparticles Are Stably Folded and Properly Display Epitopes of Interest
To confirm that spike is displayed on the surface of ferritin, we performed cryo-EM on both S-Fer and SΔC-Fer. Cryo-EM raw images showed observable densities around ferritin particles (Figure 3A and Figure S3A), indicating proper formation of the nanoparticles and display of spike on the surface. The two-dimensional class averages further showed the densities of spike surrounding ferritin (Figure 3B and Figure S3B). We chose to perform additional data collection and image processing of the SΔC-Fer particles since they had more defined spike density than the S-Fer particles. Using single-particle analysis, we determined the three-dimensional (3D) structure of the SΔC-Fer complex from ∼60 000 particles and achieved overall resolutions of 13.5 and 23.6 Å, with and without octahedral symmetry applied, respectively (Figure S3C–E). The overall shapes of the two cryo-EM maps were very similar, with a cross-correlation coefficient of 0.99 (Figure S3D,E). The map obtained with octahedral symmetry can be seen in two different views in Figure 3C. One representative trimer on the surface is highlighted, with each protomer indicated in a separate color to demonstrate the display of trimeric spike on the surface of ferritin.
Figure 3
Figure 3. Cryo-EM and BLI confirm that spike proteins are presented on the particle surface with mAb epitopes intact. (A) Representative motion-corrected cryo-EM micrograph of the SΔC-Fer nanoparticles. Circles indicate representative particles that were picked for further analysis. Micrographs demonstrate that particles are approximately 300 Å. (B) Reference-free 2D class averages of SΔC-Fer. 2D class averages confirm the presence of both ferritin particles and the display of spike on the surface seen as density surrounding the particles. (C) Reconstructed cryo-EM map of the SΔC-Fer nanoparticle in two views. A single spike trimer on the surface is highlighted with each protomer of the trimer shown in a different color. (D) BLI binding of SARS-CoV-2 mAbs to purified spike antigens. Antigens were diluted to 100 nM monomer concentration (100 nM RBD, 33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Fer and SΔC-Fer 24-mer ferritin particle). Binding of all antigens to three SARS-CoV-2 reactive mAbs indicates that spike ferritin nanoparticles display epitopes similarly to the RBD and spike trimers. Curves were fitted with an association/dissociation nonlinear regression, and fits are represented with dashed black lines; kon values for each binding reaction are shown in Figure S4A. Both S-Fer and SΔC-Fer exhibited a slight increase in signal during the dissociation step, perhaps due to rearrangements of the particles on the BLI sensor tip due to the extensive avidity present on the multimerized particles. Lack of binding to an off-target Ebola-specific antibody (ADI-15731) is presented in Figure S4B. Binding experiments were performed in at least duplicate; a representative trace and fit are shown from one replicate.
The smearing of the spike protein densities (Figure 3B and Figure S3B) is likely due to the flexibility of spike on the ferritin surface resulting from the Ser-Gly-Gly linker (Figure 1). This conclusion predicts that density for the ferritin core of the nanoparticles would not be smeared. Indeed, when single-particle analysis of the SΔC-Fer cryo-EM data set focused on only the ferritin core, excluding spike-specific density, we were able to obtain a 3.7 Å resolution structure of the ferritin core of SΔC-Fer (Figure S3F).
Since we aimed to use the S-Fer and SΔC-Fer nanoparticles as antigens for eliciting SARS-CoV-2-directed antibodies, we confirmed that they displayed properly folded spike that could recognize mAbs and ACE2. We used BLI (Figure 3D) to assess binding of three SARS-CoV-2 mAbs (CR3022, COVA2-15, and CB6) in IgG form to S-Fer and SΔC-Fer. We performed the BLI experiments at a set concentration of 100 nM monomer for each antigen which corresponds to 100 nM RBD monomer, 33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Fer and SΔC-Fer 24-mer ferritin nanoparticle. Thus, observed differences in the magnitude of binding are largely due to differences in molar concentration of antigen. We fit the BLI data using an association/dissociation nonlinear regression and extracted kon values for each mAb binding each antigen (Figure S4A). We could not obtain koff values for a subset of the binding interactions due to the undetectable dissociation of IgGs from the highly avid spike ferritin nanoparticle samples (Figure 3D). Thus, we did not determine KD values for mAbs binding to each antigen but used the kon values as a metric to compare binding (Figure S4A). The mAbs bound the functionalized nanoparticles similarly to both the spike trimers and the RBD (Figure 3D and Figure S4A), confirming that display of the spike on ferritin nanoparticles does not perturb or occlude critical conformation-specific epitopes. We observed minimal binding when a nontarget mAb (anti-Ebola glycoprotein mAb, ADI-15731(63)) was loaded on the tip (Figure S4B). We also compared binding of the three mAbs as well as the ectodomain of human ACE2, the SARS-CoV-2 receptor, to all antigens using enzyme-linked immunosorbent assays (ELISAs; Figure S5). These ELISA results were consistent with those from BLI, indicating that SARS-CoV-2 and ACE2 bind the spike-functionalized particles as well as they bind spike trimers and RBD.
Immunization with Spike-Functionalized Nanoparticles Elicits SARS-CoV-2 Neutralizing Antibodies with a Single Dose
To assess the immune response to S-Fer and SΔC-Fer nanoparticles versus the other antigens, we immunized 10 mice (n = 5 in two replicate immunizations) with 10 μg of antigen adjuvanted with 10 μg of Quil-A and 10 μg of monophosphoryl lipid A (MPLA).(64−66) We collected serum at day 21 to assess the response to a single dose of antigen. We administered a second dose of antigen at day 21 and subsequently collected serum at day 28 to determine how the initial response to each antigen was boosted (Figure 4A). Mean ELISA titers and spike-pseudotyped lentivirus neutralization titers for all antigen groups at day 21 and day 28 can be found in Table S1.
Figure 4
Figure 4. Immunization with SΔC-Fer nanoparticles elicits a stronger neutralizing response than immunization with nonferritin groups in mice. (A) Immunization schedule including a priming dose with 10 μg of antigen at day 0 and a boost with 10 μg of antigen at day 21. Serum was collected on days 0, 21, and 28. Both doses were adjuvanted with 10 μg of Quil-A and 10 μg of MPLA in a total volume of 100 μL per mouse administered via subcutaneous injection. (B) ELISA binding titers to both the RBD and full-length spike ectodomain after a single dose of antigen demonstrate that all groups elicited a SARS-CoV-2-directed antibody response following immunization. Each point represents the EC50 titer from a single animal; each bar represents the mean EC50 titer from the group (n = 10 mice per group). Error bars represent standard deviation. Points with signal less than EC50 1:100 dilution are placed at the limit of quantitation for the assay. (C) S-Fer and SΔC-Fer antigens elicit stronger neutralizing antibody responses than spike trimers alone or RBD, as indicated by spike-pseudotyped lentivirus neutralizing titers after a single dose of antigen. Immunization with a single dose of S-Fer or SΔC-Fer elicits neutralizing responses that are at least 2-fold greater on average than those found in plasma from 20 convalescent COVID-19 patients (CCP). Each point represents the IC50 titer from a single animal or patient; each bar represents the mean IC50 titer from each group (n = 10 per group, with the exception of CCP which is n = 20). Error bars represent standard deviation. Samples with neutralizing activity that was undetectable at 1:50 dilution or with an IC50 less than 1:100 dilution are placed at the limit of quantitation. (D) ELISA binding titers to the RBD and spike after two doses of antigen show that the SARS-CoV-2-specific response against both antigens was boosted in all groups. Groups and error are as defined in part B. (E) Spike-pseudotyped lentivirus neutralization following two doses of antigen indicates that although all groups had a neutralizing response following two doses, animals immunized with SΔC-Fer have the highest neutralizing titers, and these are significantly greater than S-GCN4 and SΔC-GCN4. Groups and error are as defined in part C. Statistical comparisons for panels B–E were performed using Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons. All p values are represented as follows: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Mean titers with standard deviation and values from pairwise comparisons between groups can be found in Tables S1–S3.
After a single dose (“Prime only”), all groups exhibited a detectable antibody response against both RBD and spike, as revealed by ELISA (Figure 4B). To analyze neutralizing titers, we used lentivirus pseudotyped with SARS-CoV-2 spike(47) and assessed inhibition of viral entry into HeLa cells overexpressing ACE2.(48) Neutralization with pseudotyped viruses is a common way to assess viral inhibition in a BSL2 setting since the SARS-CoV-2 replicating pathogen requires a BSL3 facility.(47,67) We validated the neutralization assay using published neutralizing mAbs against SARS-CoV-2 (Figure S6).
Analysis of antisera from immunized mice revealed that the only groups with notable neutralizing activity after a single dose of antigen were those immunized with S-Fer or SΔC-Fer (Figure 4C), underscoring that multivalent presentation greatly enhances the neutralizing antibody response in a single-dose regimen of an adjuvanted SARS-CoV-2 subunit vaccine. As illustrated in Figure 4C, the SΔC-Fer group had significantly higher neutralizing titers than all three nonferritin groups at the day 21 time point, whereas the S-Fer group elicited significantly greater neutralizing responses than SΔC-GCN4 and RBD. Importantly, antisera from mice immunized with a single dose of either S-Fer or SΔC-Fer had ∼2-fold higher mean neutralizing titers than those observed in a cohort of 20 convalescent COVID-19 plasma donors (Figure 4C, far right).
The lack of neutralizing titers after a single-dose immunization in both the RBD and SΔC-GCN4 groups and minimal neutralizing titers in the S-GCN4 group (Figure 4C) are consistent with recent reports that also found that a single dose of spike trimer or RBD was insufficient to elicit robust neutralizing antibodies in mice.(31,32,68) Consistent with the lentiviral neutralization results (Figure 4C), the only two groups with substantial ACE2-competing antibodies (as determined by a decrease in ACE2 binding to RBD on ELISA) were those immunized with spike ferritin nanoparticles (Figure S7, left). ACE2-blocking activity in the antisera of mice after a single dose of antigen (day 21) appeared to be weak, as even the nanoparticle groups with the strongest neutralizing response exhibited minimal blocking at the highest serum concentration tested (1:50 dilution), and no groups exhibited any blocking activity at a 1:500 dilution (Figure S7).
To evaluate the effect of boosting on the immune responses to these antigens, we administered a second dose of antigen on day 21 and collected serum at day 28 (“Prime + Boost”). We analyzed the post-boost serum for RBD and spike titers, ACE2 blocking activity, and neutralization potency. The immune response to all antigens was increased, and antigen-specific titers for both RBD and spike were similar among groups after the second dose (Figure 4D). ACE2 blocking activity was notably enhanced after the boost, and all five antigen groups had nearly complete ACE2 blocking at 1:50 serum dilution at that time point (Figure S7, right). While all antigen groups also had partial ACE2 blocking activity at 1:500 serum dilution, the levels of ACE2 blocking at this dilution did not relate to overall trends in neutralizing titers (compare Figure 4E and Figure S7). This discordance indicates that ACE2-blocking activity may not correlate with neutralizing titers for some antigens and suggests that neutralizing epitopes other than the ACE2 binding site exist on the SARS-CoV-2 spike (see refs (5and69)).
To assess the polarization of the immune response following two doses of antigen, we quantified the levels IgG1, IgG2a, and IgG2b titers for RBD-specific antibodies (Figure S8). IgG subclasses can provide information regarding whether a given response is biased toward a Th1- or Th2-like response.(70−73) Adjuvants are known to play a major role in influencing the polarization of vaccine immune responses, and prior work has shown that Quil-A and MPLA adjuvants can lead to a Th1-biased response.(70,71) Some vaccine candidates against SARS-CoV were found to cause pulmonary immunopathology potentially due to Th2-type responses in small animal models,(74−77) and thus one focus for SARS-CoV-2 vaccine development has been to favor a Th1-type response.(72,73) A comparison of the ratio of IgG2a/IgG1 antibody titers among antigen groups demonstrated that, except for the RBD, the antigens exhibit a balanced IgG2a and IgG1 response, which was slightly skewed toward IgG2a for both the SΔC-Fer and S-GCN4 groups (Figure S8A,B). Notably, the RBD elicited a substantially higher IgG1 response as compared to both IgG2a and IgG2b, suggesting that this antigen in combination with Quil-A/MPLA caused a Th2-biased immune response (Figure S8B,C). IgM responses did not substantially differ among antigen groups at the day 28 time point and were lower than IgG isotypes (Figure S8D), and at the same time point serum IgA levels were undetectable (data not shown).
Given that each antigen contains a non-SARS-CoV-2 fragment, we wanted to assess the off-target antibody responses directed at either the H. pylori ferritin, the GCN4 trimerization domain, or the His-tag. We observed anti-H. pylori ferritin responses in the S-Fer and SΔC-Fer groups that were similar and were equivalent to or less than anti-RBD and anti-spike titers from the same groups as determined by ELISA (Figure S9, left). The S-GCN4 and SΔC-GCN4 groups had similar anti-GCN4 responses (Figure S9, middle), suggesting that the H. pylori ferritin and GCN4 domains are equivalently immunogenic. Interestingly, when we probed the anti-His-tag response specifically, we observed that the SΔC-GCN4 group had a notably higher His-tag response as compared to either the S-GCN4 or the RBD group, even though all three antigens contained His-tags (Figure S9, right). This could suggest that this antigen either was less structurally stable or had other antigenic features that altered the overall immunogenicity to cause a higher tag-directed response. Additionally, this indicates that, for the RBD- and spike-specific titers from the S-GCN4, SΔC-GCN4, and RBD groups, the His-tag on the plated antigen likely did not impact the overall titers for the S-GCN4 and RBD groups but may have slightly increased the titers for the SΔC-GCN4 group.
Though antisera from all groups exhibited neutralizing activity after a boost, mice immunized with SΔC-Fer exhibited the highest neutralizing titers overall and had significantly higher titers than both the S-GCN4 and SΔC-GCN4 groups (Figure 4E). Importantly, the variation in response to the nonferritin groups was also considerably larger than the variability detected in mice immunized with S-Fer or SΔC-Fer nanoparticles (Figure 4E), suggesting that the multivalent presentation of spike facilitated a more consistent immune response. Furthermore, the neutralizing antibody response elicited by SΔC-Fer shows dose-dependent trends (Figure S10A). Though a decrease in dose from 10 to 1 μg led to an increased variability in the elicitation of neutralizing titers, the presence of neutralizing antibodies in a subset of mice following only 1 μg of SΔC-Fer could have important dose-sparing implications. Additionally, neutralizing titers remain stable even to 20 weeks following a single immunization with 20 μg (Figure S10B), demonstrating that this formulation may successfully achieve neutralizing antibody levels that persist in the months following vaccination. These data demonstrate that the spike ferritin nanoparticles presented here, particularly SΔC-Fer, are superior vaccine candidates to spike trimer or RBD alone.
Discussion
ARTICLE SECTIONS
Global management of the COVID-19 pandemic depends not only upon the development of safe and protective vaccine candidates but also on the rapid production and deployment of billions of doses. Given these extensive manufacturing challenges, vaccination strategies requiring only a single dose could be critical to achieving worldwide immunization against SARS-CoV-2. Toward this end, we designed and characterized two vaccine candidates based on ferritin nanoparticles (S-Fer and SΔC-Fer) that display multiple copies of the SARS-CoV-2 spike and can be readily expressed in mammalian cells. These proteins can be purified using routine methods and a production scheme similar to that for soluble spike trimers.
Immunization with the spike nanoparticles elicited neutralizing antibodies in mice after a single dose, whereas immunization with the RBD or spike trimers elicited little to no neutralizing titers with the same dose (Figure 4C). Interestingly, deletion of the C-terminal portion of the spike ectodomain enhanced the neutralizing antibody response specifically in the context of the nanoparticle (Figure 4C). After a second dose, all antigen groups elicited neutralizing antibodies; however, the SΔC-Fer immunized mice had the highest overall neutralizing titers and, importantly, had significantly higher titers than both the S-GCN4 and SΔC-GCN4 trimer groups (Figure 4E). These results provide important insight into the use of spike ferritin nanoparticles as an effective single-dose vaccine against SARS-CoV-2.
Ferritin nanoparticles are of significant interest for developing new vaccines because they self-assemble into stable structures that display trimeric proteins on 3-fold symmetry axes.(39) Unlike nanoparticle platforms that require postpurification conjugation of an antigen to a carrier or scaffold,(78) here, spike-functionalized nanoparticles were produced by transfecting a single plasmid encoding the spike fused to the ferritin subunit into mammalian cells. We built on existing work using ferritin nanoparticles to display other viral antigens for vaccine development(30,33−38) and designed nanoparticles displaying the SARS-Cov-2 spike ectodomain. Importantly, the two ferritin-based antigens that we designed (S-Fer and SΔC-Fer) expressed comparably to spike trimers (S-GCN4 and SΔC-GCN4) in mammalian cells (Figure S1), indicating that fusing the spike to ferritin does not negatively impact protein production. As glycosylation can influence proper folding of viral antigens, it is also noteworthy that expression was achieved in mammalian cells, which allows spike proteins to be produced with native-like glycosylation.(54)
These constructs could also be important for the development of nucleic acid vaccines given the self-assembly of spike ferritin nanoparticles following production in mammalian cells, expression levels comparable to those of spike trimers, and the enhanced immune response after a single dose of antigen. We administered equivalent doses of spike ferritin nanoparticle and trimer and observed an enhanced response after single-dose and two-dose vaccine regimens with the SΔC-Fer (Figure 4C,E). Thus, administration of a nucleic acid vaccine encoding a multimerized ferritin-based spike protein could potentially elicit a heightened response versus constructs encoding spike alone, although we did not test that hypothesis here.
Following purification of the spike-functionalized nanoparticles from Expi293F culture supernatant, we extensively characterized their biophysical properties to ensure that the spikes were properly folded and stable and could display important epitopes of interest. We confirmed the structure, homogeneity, and epitope conformations of spike-functionalized nanoparticles using cryo-EM (Figure 3A–C and Figure S3), SEC-MALS (Figure 2B and Figure S2), and BLI-measured binding to conformation-specific SARS-CoV-2 mAbs (Figure 3D).
Remarkably, mice immunized with a single dose of either S-Fer or SΔC-Fer elicited mean neutralizing antibody titers that were approximately 2-fold higher than mean titers observed in plasma from convalescent COVID-19 patients (Figure 4C). Additionally, mice immunized with one dose of SΔC-Fer had significantly higher neutralizing titers versus all nonferritin groups, whereas mice immunized with one dose of S-Fer had significantly higher titers than the RBD and SΔC-GCN4 group (Figure 4C). These data clearly demonstrate that equivalent doses of spike ferritin nanoparticles elicit a better neutralizing response versus RBD or trimer alone in a single-dose setting which is consistent with observations from other recent studies investigating other multimerized SARS-CoV-2 vaccine platforms.(31,32) A comparison of neutralizing antibody titers observed in these other studies reveals that, despite the use of different adjuvants, the spike ferritin nanoparticles we present here elicit similar levels of neutralizing antibodies following one or two doses.(31,32)
Mice immunized with the RBD exhibited no observable serum neutralizing activity after a single dose (Figure 4C) despite having RBD-specific antibody titers that were similar to those in other groups (Figure 4B). This discordance suggests that RBD binding titers may not be a strong correlate of neutralizing antibody responses in the context of vaccination, particularly for candidates based on the RBD alone.(52,79,80) Notably, the full-length spike ELISA binding titers from animals immunized with the RBD were significantly lower than titers from the other groups (Figure 4B), suggesting that immunization with monomeric RBD elicits antibodies to epitopes that are occluded in full-length spike, rendering them unable to neutralize virus. This supports the notion that the response to RBD may have largely focused on the non-neutralizing regions.
After a second dose of antigen, all antigens elicited detectable neutralizing antibody titers (Figure 4E). Despite the lack of detectable neutralizing signal in the RBD or SΔC-GCN4 groups after a single dose (Figure 4C), these groups exhibited titers roughly equivalent to those of the S-GCN4 group after a second dose (Figure 4E). Interestingly, mice immunized with either S-Fer or SΔC-Fer had less variability in response than the other three groups (Figure 4E), suggesting that multivalent presentation facilitates more consistent elicitation of neutralizing antibodies. Although all groups elicited a neutralizing response after the second dose, the mice immunized with SΔC-Fer had the highest overall neutralizing titers and elicited significantly higher neutralizing titers versus both spike trimers tested (Figure 4E). Taken together, these data suggest that SΔC-Fer is the best-performing antigen out of those we tested here and could be a favorable candidate for use in a subunit or nucleic acid vaccine against SARS-CoV-2.
Effective global deployment of a SARS-CoV-2 vaccine will depend on several logistical factors. The number of doses in an immunization regimen required to achieve efficacy will be of critical importance due to the number of doses required worldwide and to the ability to access patients multiple times. Therefore, assessing candidates that can achieve protection after a single dose is highly important for implementing a global vaccination strategy. Here, we have demonstrated that a dose of either S-Fer or SΔC-Fer nanoparticles adjuvanted with Quil-A and MPLA was sufficient to achieve neutralizing titers greater than those in plasma from convalescent COVID-19 patients. Additionally, both S-Fer and SΔC-Fer form nanoparticles spontaneously following expression in mammalian cells without the need for a conjugation step, showcasing the potential application of spike ferritin constructs to nucleic acid-based vaccine strategies. Taken together, our data indicate that the multivalent presentation of the SARS-CoV-2 spike is an effective way to enhance the antibody response after a single dose, providing key insight for the development of an effective and deployable vaccine to combat the COVID-19 pandemic.
Materials and Methods
ARTICLE SECTIONS
DNA Plasmid Construction and Propagation
All spike constructs were cloned from a full-length spike expression plasmid received from Dr. Florian Krammer.(49) This construct contains residues 1–1213 from the Wuhan-Hu-1 genome sequence (GenBank MN9089473), followed by a thrombin cleavage site, a T4 fibritin trimerization domain, and a hexa-histidine tag for purification. This construct was cloned out of the parent vector and into an in-house pADD2 mammalian expression vector using HiFi PCR (Takara) followed by In-Fusion (Takara) cloning with EcoRI/XhoI cut sites. This construct was used to clone subsequent spike ferritin and spike GCN4-pIQI trimer constructs. Full-length spike ferritin (S-Fer) and spikeΔCferritin (SΔC-Fer) were cloned by polymerase chain reaction (PCR) amplification of full-length spike (residues 1–1213) or spikeΔC (residues 1–1143) off the parent expression vector. This was followed by a stitching PCR in which constructs were annealed to an amplicon containing H. pylori ferritin (residues 5–168) originally generated as a gene-block fragment from Integrated DNA Technologies (IDT). The spike and ferritin subunits were separated by a SGG linker.(33) Spike ferritin amplicons were inserted into the pADD2 mammalian expression vector via In-Fusion using EcoRI/XhoI cut sites. The spike trimer constructs were cloned similarly and fused to the GCN4-pIQI(46) domain instead of ferritin. The spikeΔC trimer included residues 1–1137 followed by a glycine residue and then the GCN4-pIQI domain. The full-length spike trimer included residues 1–1213 followed by a GGGGS linker and then the GCN4-pIQI domain. Both trimer constructs contained a hexa-histidine tag for NiNTA purification.
The SARS-CoV-2 RBD construct was kindly provided by Dr. Florian Krammer.(49) This construct contains the native signal peptide (residues 1–14) followed by residues 319–541 from the SARS-CoV-2 Wuhan-Hu-1 genome sequence (GenBank MN908947.3) and a hexa-histidine tag at the C-terminus for purification. This expression plasmid (pCAGGS) contains a CMV promoter for protein expression in mammalian cells.
The variable heavy chain (HC) and variable light chain (LC) sequences for SARS-CoV-2 reactive mAbs, CR3022 (HC GenBank DQ168569, LC Genbank DQ168570), CB6 (HC GenBank MT470197, LC GenBank MT470196), and COVA-2-15 (HC GenBank MT599861, LC GenBank MT599945) were codon optimized for human expression using the IDT Codon Optimization Tool and ordered as gene-block fragments from IDT. Fragments were PCR amplified and inserted into linearized CMV/R expression vectors containing the heavy chain or light chain Fc sequence from VRC01 using In-Fusion.
Soluble human ACE2 with an Fc tag was constructed by PCR amplifying ACE2 (residues 1–615) from Addgene plasmid 1786 (a kind gift from Dr. Jesse Bloom) and fusing it to a human Fc domain from VRC01, separated by a TEV-GSGG linker using a stitching PCR step. ACE2-Fc was inserted into the pADD2 mammalian expression vector via In-Fusion using EcoRI/XhoI cut sites.
After all cloned plasmids were sequence confirmed using Sanger sequencing, plasmids were transformed into Stellar Cells (Takara) and grown overnight in 2xYT/carbenicillin cultures, with the exception of the CMV/R mAb plasmids which were grown in 2xYT/kanamycin cultures. Plasmids were prepared for mammalian cell transfection using Machery Nagel Maxi Prep columns. Eluted DNA was filtered in a biosafety hood using a 0.22 μm filter prior to transfection.
Expression and Purification of SARS-CoV-2 Antigens, mAbs, and Soluble ACE2
All antigens for immunization, mAbs, and soluble human ACE2-Fc were expressed and purified from Expi293F cells. Expi293F cells were cultured using 66% FreeStyle 293 Expression/33% Expi293 Expression medium (ThermoFisher) and grown in polycarbonate baffled shaking flasks at 37 °C and 8% CO2 while shaking at 120 rpm. Cells were transfected at a density of approximately (3–4) × 106 cells/mL. Transfection mixtures were made by adding 568 μg of maxi-prepped DNA to 113 mL of culture medium (per liter of transfected cells) followed by addition of 1.48 mL of FectoPro (Polyplus). For mAbs, cells were transfected with a 1:1 ratio of HC:LC plasmid DNA. Mixtures were incubated at room temperature for 10 min and then added to cells. Cells were immediately boosted with d-glucose (4 g/L final concentration) and 2-propylpentanoic (valproic) acid (3 mM final concentration). Cells were harvested 3–5 days post-transfection via centrifugation at 7000g for 15 min. Culture supernatants were filtered with a 0.22 μm filter.
Superfolder GFP (sfGFP) was tagged with either GCN4-Avi-His (GCN4 tag domain amino acid sequence taken from(81)) or His-tag only to quantify off-target tag antibody responses. sfGFP-His and sf-GFP-GCN4-His encoded in pET28a bacterial T7-expression vectors were transformed into BL21 cells and induced at OD 0.6 with 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 3 h at 37 °C. Cells were harvested by spinning at 7000g for 10 min, resuspended in 10 mM imidazole/1× PBS [pH 7.4], lysed by sonicating for 20 s three times, and spun at 15 000g for 20 min following lysis. sfGFP-His and sf-GFP-GCN4-His were then purified from supernatants using NiNTA resin as described below.
S-Fer, SΔC-Fer, and WT H. pylori ferritin nanoparticles were isolated using anion-exchange chromatography followed by size-exclusion chromatography using an SRT SEC-1000 (Sepax) column. Briefly, Expi293F culture supernatants were concentrated using tangential flow filtration with a GE AKTA Flux S with a 10 kDa molecular weight cutoff (MWCO) hollow fiber cartridge (UFP-10-E-4MA) and then buffer-exchanged into 20 mM Tris [pH 8.0] via overnight dialysis at 4 °C using 100 kDa MWCO dialysis tubing. For some preps, culture supernatant was dialyzed directly and not concentrated using tangential flow filtration. Dialyzed culture supernatants were filtered through a 0.22 μm filter and loaded onto a 5 mL HiTrapQ anion-exchange column (GE) equilibrated in 20 mM Tris [pH 8.0] on a GE AKTA Pure system. Ferritin nanoparticles were eluted with a sodium chloride (NaCl) gradient, and the particle-containing fractions were identified via Western blot with CR3022 for spike ferritins or SDS-PAGE followed by staining with GelCode blue stain reagent (Thermo) for WT H. pylori ferritin. Fractions were pooled and concentrated using a 100 kDa MWCO Amicon spin filter and subsequently purified on a GE AKTA Pure system using an SRT SEC-1000 SEC column equilibrated in 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco).
RBD, S-GCN4, SΔC-GCN4, sf-GFPHis, and sfGFP-GCN4-His were purified with HisPur NiNTA resin (ThermoFisher). Prior to purification, resin was washed 3× with ∼10 column volumes of wash buffer (10 mM imidazole/1× PBS [pH 7.4]). Expi293F cell culture supernatants were diluted 1:1 with 10 mM imidazole/1× PBS [pH 7.4]; resin was added to diluted cell supernatant and incubated at 4 °C. Resin/supernatant mixtures were agitated during binding using a stir-bar and a magnetic stir-plate. Resin/supernatant mixtures were added to glass chromatography columns for gravity flow purification. Resin was washed with 10 mM imidazole/1× PBS [pH 7.4], and proteins were eluted with 250 mM imidazole/1× PBS. NiNTA elutions were concentrated using Amicon spin concentrators (10 kDa MWCO for RBD, sfGFP-His, and sfGFP-GCN4-His and 100 kDa MWCO for spike trimers) followed by size-exclusion chromatography. The RBD, sfGFP-His, and sfGFP-GCN4-His were purified using a GE Superdex 200 Increase 10/300 GL column, and the S-GCN4 and SΔC-GCN4 proteins were purified using a GE Superose 6 Increase 10/300 GL column. Columns were pre-equilibrated in 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) or 1× PBS [pH 7.4].
Fractions were pooled based on A280 signals and/or SDS-PAGE on 4–20% Mini-PROTEAN TGX protein gels stained with GelCode blue stain reagent (ThermoFisher). Samples for immunizations were supplemented with 10% glycerol, filtered through a 0.22 μm filter, snap frozen, and stored at −20 °C until use.
mAbs and ACE2-hFc were purified using Protein A agarose resin (Pierce). Filtered cell culture supernatant was diluted 1:1 with 1× PBS [pH 7.4] and added to Protein A resin which was prewashed with ∼10 column volumes of 1× PBS [pH 7.4]. Resin/supernatant mixtures were batch bound at 4 °C. Proteins were eluted with 100 mM glycine [pH 2.8], and elutions were neutralized via addition of 1/10th volume 1 M Tris [pH 8.0].
Western Blot Analysis of Expi293F Culture Supernatants
Expi293F culture supernatants were collected 3 days after transfection, harvested via spinning at 7000g for 15 min, and filtered through a 0.22 μm filter. Samples were diluted in SDS-PAGE Laemmli loading buffer (Bio-Rad), boiled at 95 °C, and run on a 4–20% Mini-PROTEAN TGX protein gel (Bio-Rad). Proteins were transferred to nitrocellulose membranes using a Trans-Blot Turbo transfer system. Blots were blocked in 5% milk/PBST (1× PBS [pH 7.4], 0.1% Tween 20) and then washed with PBST. In-house-made primary antibodies CR3022, COVA2-15, and CB6 (approximate concentrations 0.8–1.3 mg/mL) were added at a 1:10 000 dilution in PBST. Blots were washed with PBST, and secondary rabbit antihuman IgG H&L HRP (abcam ab6759) was added at 1:10 000 in PBST. Blots were developed using Pierce ECL substrate and imaged using a GE Amersham imager 600.
Dot Blot Analysis of Expi293F Culture Supernatants
Purified proteins were diluted into conditioned expression medium (medium harvested 3 days post-mock-transfection) to a final concentration of 0.1 mg/mL and serially diluted 3-fold using conditioned medium as diluent. Dilution series were made in two independent replicates. Each concentration (2 μL) was dotted onto a nitrocellulose membrane to produce a standard curve. Expi293F culture supernatants from spike antigen expressions were harvested 3 days post-transfection via centrifugation at 7000g for 15 min and filtered through a 0.22 μm filter. Supernatants were spotted on the same blot as the standard curve. Blots were left to dry for 20 min in a fume hood and then blocked in 5% milk/PBST for 10 min at room temperature. CR3022 (4 μg) was added to blocking solution (0.4 μg/mL final concentration) and incubated for 1 h at room temperature. Blots were washed 16 times with 9 mL of PBST. Secondary antibody was added at 1:10 000 (abcam ab6759, rabbit antihuman IgG H&L HRP) in 5% milk/PBST and incubated for 1 h at room temperature. Blots were washed 16 times with 9 mL of PBST, developed using Pierce ECL Western blotting substrate, and imaged using a GE Amersham imager 600. Replicate protein expressions (n = 5) were performed and included in the analysis. Dots were quantified using the gel analysis protocol in Fiji (ImageJ), and curves were fitted using a linear regression in GraphPad Prism 8.4.1. Statistical analysis on expression data presented in Figure S1B was assessed using an ordinary one-way ANOVA with Tukey’s multiple comparisons tests using GraphPad Prism 8.4.1. * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.
SEC-MALS of SARS-CoV-2 Antigens
SEC-MALS was performed on an Agilent 1260 Infinity II HPLC with Wyatt detectors for light scattering (miniDAWN) and refractive index (Optilab). Purified antigen (1–10 μg) was loaded onto a Superdex 200 Increase 3.2/200 (RBD) or onto an SRT SEC-1000 4.6 × 300 mm (spike proteins) column equilibrated in 1× PBS [pH 7.4] or 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco). Columns were flowed at a rate of 0.15 mL/min (S200) or 0.35 mL/min (SRT SEC-1000). Molecular weights were determined using ASTRA 7.3.2 (Wyatt Technologies).
Cryo-EM Data Acquisition
Samples were diluted to a final concentration of ∼0.4 mg/mL for both the S-Fer and SΔC-Fer particles after purification. The samples (3 μL) were applied onto glow-discharged 200-mesh R2/1 Quantifoil grids coated with continuous carbon. The grids were blotted for 2 s and rapidly cryocooled in liquid ethane using a Vitrobot Mark IV instrument (Thermo Fisher Scientific) at 4 °C and 100% humidity. Samples were screened using a Talos Arctica cryoelectron microscope (Thermo Fisher Scientific) operated at 200 kV. The spikeΔC ferritin sample was imaged in a Titan Krios cryoelectron microscope (Thermo Fisher Scientific) operated at 300 kV with GIF energy filter (Gatan) at a magnification of 130 000× (corresponding to a calibrated sampling of 1.06 Å per pixel). Micrographs were recorded with EPU 2.6 (Thermo Fisher Scientific) with a Gatan K2 Summit direct electron detector; each image was composed of 30 individual frames with an exposure time of 6 s and an exposure rate of 7.8 electrons per second per Å2. A total of 3684 movie stacks was collected.
Single-Particle Image Processing and 3D Reconstruction
All movie stacks were first imported into Relion 3.0.6(82) for image processing. Motion-correction was performed with MotionCor2 1.3.2,(83) and the contrast transfer function was determined with CTFFIND4 4.1.13.(84) All particles were autopicked using the NeuralNet option in EMAN2 2.31,(85) yielding 152 734 particles from 3540 selected micrographs. Then, particle coordinates were imported into Relion 3.0.6, where poor 2D class averages were removed through several rounds of 2D classification. The initial model was built in cryoSPARC(86) using the ab initio reconstruction option with octahedral symmetry applied. The final 3D refinement was performed using 62 837 particles with or without octahedral symmetry applied; a 13.5 Å map and a 23.6 Å map were obtained, respectively. Resolution for the final maps was estimated with the 0.143 criterion of the Fourier shell correlation curve. A Gaussian low-pass filter was applied to the final 3D maps displayed in the UCSF Chimera 1.13.1 software package.(87)
BLI of mAbs Binding to SARS-CoV-2 Purified Antigens
BLI was performed on an OctetRed 96 system (ForteBio). Antigens and mAbs were diluted in Octet buffer (0.5% bovine serum albumin, 0.02% Tween, 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco)) and plated in 96-well flat-bottom black plates (Greiner). Tips were pre-equilibrated in Octet buffer and regenerated in 100 mM glycine [pH 1.5] prior to binding antigens. Anti-Human Fc sensor tips (Forte) were dipped into 200 nM mAb and then submerged into wells containing 100 nM (protomer or monomer concentration) of each antigen. The final concentration of each antigen was as follows: 100 nM RBD monomer, 33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Fer and SΔC-Fer nanoparticle. Background subtraction was performed using an mAb-loaded sensor tip submerged into a well containing buffer only.
ELISA with Purified mAbs and Mouse Serum
ELISA of SARS-CoV-2 antigens was performed by coating antigens on MaxiSorp 96-well plates (ThermoFisher) at 2 μg/mL in 1× PBS [pH 7.4] overnight at 4 °C. Mouse serum ELISAs were performed using RBD or full-length spike ectodomain with a T4 fibritin (foldon) trimerization domain, described in detail in ref (49). ELISAs to quantify off-target antibody responses were performed by coating either WT H. pylori ferritin particles, sfGFP-GCN4-Avi-His (GCN4-Avi-His sequence taken from ref (81)), or sfGFP-His. After coating, plates were washed 3× with PBST and blocked overnight at 4 °C with ChonBlock Blocking/Dilution ELISA buffer (Chondrex). ChonBlock was removed manually, and plates were washed 3× with PBST. Mouse serum samples, purified mAbs, and ACE2-Fc were serially diluted in diluent buffer (1× PBS, 0.5% bovine serum albumin, 2% filtered fetal bovine serum, 0.2% bovine gamma globulins (Sigma), 5 mM EDTA, 0.1% Tween) starting at 1:50 serum dilution or 10 μg/mL and then added to coated plates for 1 h at room temperature. Plates were washed 3× with PBST. For mouse serum ELISAs, HRP goat antimouse (BioLegend 405306) was added at a 1:10 000 dilution in diluent buffer for 1 h at room temperature. Isotyping experiments to quantify isotypes of RBD-specific antibodies were performed by adding a 1:10 000 dilution of either HRP antimouse IgG1 (abcam ab97240), HRP antimouse IgG2a heavy chain (abcam ab97245), HRP antimouse IgG2b heavy chain (abcam ab97250), HRP antimouse IgA alpha chain (abcam ab97235), or HRP antimouse IgM mu chain (abcam ab97230). For purified mAbs and ACE2-Fc, Direct-Blot HRP antihuman IgG1 Fc antibody (Biolegend 410604) was added at a 1:10 000 dilution in diluent buffer for 1 h at room temperature.
ACE2 blocking assays were performed by coating RBD as described for other antigens, incubating serially diluted mouse serum for 1 h at room temperature, and then adding ACE2-Fc at a final concentration of 1 μg/mL to wells for 1 h at room temperature. ACE2 binding was measured with Direct-Blot HRP antihuman IgG1 Fc at a 1:10 000 dilution, and blocking was quantified as the decrease in observed ACE2 binding as a function of serum dilution.
After incubation with secondary antibody, ELISA plates were washed 6× with PBST. Plates were developed for 6 min using One-Step Turbo TMB substrate (Pierce) and were quenched with 2 M sulfuric acid. Absorbance at 450 nm was determined using a BioTek plate reader. Background was determined using an average of wells containing no serum, mAb, or ACE2. This value was then subtracted from all wells on the plate. Background subtracted values were then imported into GraphPad Prism 8.4.1 and fitted with a three-parameter nonlinear regression to obtain EC50 or apparent KD values.
For ACE2 blocking assays, A450 values from ACE2 only wells and background wells (no serum, no ACE2) were averaged and set at either 0% ACE2 blocking (ACE2 only) or 100% ACE2 blocking (background). A450 values for each serum concentration were then normalized in GraphPad Prism 8.4.1 using these values. Percent ACE2 blocking at either 1:50 or 1:500 serum dilution can be found in Figure S6 for both day 21 and day 28 immunization time points.
Mouse Immunizations
Balb/c female mice (6–8 weeks old) were procured from The Jackson Laboratory. All mice were maintained at Stanford University according to the Public Health Service Policy for “Humane Care and Use of Laboratory Animals” following a protocol approved by Stanford University Administrative Panel on Laboratory Animal Care (APLAC-33709). Mice were immunized via subcutaneous injection of 10 μg of antigen with the exception of SΔC-Fer dosing and longevity studies in which animals were immunized with either 0.1, 1, 10, or 20 μg as specified in Figure S10. All antigen doses were formulated with 10 μg of Quil-A (InVivogen) and 10 μg of monophosphoryl lipid A (InVivogen) diluted in 1× Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) in a total volume of 100 μL per injection. Mice were immunized at day 0 and day 21. Serum was collected at days 0, 21, and 28 and processed using Sarstedt serum collection tubes. Day 0 serum was analyzed for both ELISA binding and lentiviral neutralization and showed no evidence of binding or neutralizing activity (data not shown).
Collection of Plasma from Convalescent COVID-19 Patients
Convalescent COVID-19 plasma (CCP) donor samples were obtained from residual ethylenediaminetetraacetic acid (EDTA) specimens at the time of collection, aliquoted, and stored at −80 °C. CCP samples were obtained from donors who had a clinical diagnosis of COVID-19 and either a positive RT-PCR or SARS-CoV-2 antibody test. On the day of donation, donors were required to be healthy and asymptomatic for at least 4 weeks. The date of collection ranged from 4 to 10 weeks after symptom resolution.
SARS-CoV-2 Pseudotyped Lentivirus Production and Viral Neutralization Assays
SARS-CoV-2 spike-pseudotyped lentivirus was produced in HEK293T cells via calcium phosphate transfection. Six million cells were seeded in D10 medium (DMEM + additives: 10% fetal bovine serum, l-glutamate, penicillin, streptomycin, and 10 mM HEPES) in 10 cm plates 1 day prior to transfection. A five-plasmid system(47) was used for viral production: the lentiviral packaging vector (pHAGE_Luc2_IRES_ZsGreen), the SARS-CoV-2 spike, and lentiviral helper plasmids (HDM-Hgpm2, HDM-Tat1b, and pRC-CMV_Rev1b). The spike vector contained the full-length wild-type spike sequence from the Wuhan-Hu-1 strain of SARS-CoV-2 (GenBank NC_045512). Plasmids were added to filter-sterilized water as follows: 10 μg of pHAGE_Luc2_IRS_ZsGreen, 3.4 μg of SARS-CoV-2 spike, 2.2 μg of HDM-Hgpm2, 2.2 μg of HDM-Tat1b, and 2.2 μg of pRC-CMV_Rev1b in a final volume of 500 μL. HEPES-buffered Saline (2×, pH 7.0) was added dropwise to this mixture to a final volume of 1 mL. To form transfection complexes, 100 μL of 2.5 M CaCl2 was added dropwise while the solution was gently agitated. Transfection reactions were incubated for 20 min at room temperature and then added dropwise to plated cells. Medium was removed ∼24 h post-transfection and replaced with fresh D10 medium. Virus-containing culture supernatants were harvested ∼72 h post-transfection via centrifugation at 300g for 5 min and filtered through a 0.45 μm filter. Viral stocks were aliquoted and stored at −80 °C until use.
For viral neutralization assays, ACE2/HeLa(48) cells were plated in white-walled clear-bottom 96-well plates at 5000 cells/well 1 day prior to infection or at 2500 cells/well 2 days prior to infection. Mouse serum was centrifuged at 2000g for 15 min, heat inactivated for 30 min at 56 °C, and diluted in D10 medium. Virus was diluted in D10 medium, supplemented with Polybrene, and then added to inhibitor dilutions. Polybrene was present at a final concentration of 5 μg/mL in diluted samples. Purified mAbs were filtered through a 0.22 μm filter prior to neutralization assays. Virus/inhibitor dilutions were incubated at 37 °C for 1 h. After incubation, medium was removed from cells, replaced with an equivalent volume of inhibitor/virus dilutions, and incubated at 37 °C for ∼48 h. Cells were lysed by adding BriteLite assay readout solution (PerkinElmer), and luminescence values were measured with a BioTek plate reader. Each plate was normalized by averaging RLUs from wells with cells only (0% infectivity) and virus only (100% infectivity). Normalized values were fitted with a three-parameter nonlinear regression inhibitor curve in GraphPad Prism 8.4.1 to obtain IC50 values. Fits for all serum neutralization assays were constrained to have a value of 0% at the bottom of the fit. The limit of quantitation for this assay is approximately 1:100 serum dilution. Serum samples that failed to neutralize or that neutralized at levels higher than 1:100 were set at the limit of quantitation for statistical analyses. Plots in Figure 4B–E are shown starting at the reciprocal serum dilution of the limit of quantitation (102).
Statistical Analyses
All normalization, cure-fitting, and statistical analysis described below were performed using GraphPad Prism 8.4.1.
For ELISA, binding titers of RBD and spike were determined for each mouse by performing 8-point serial serum dilutions started at 1:50 with 10-fold dilutions in duplicate, with replicates performed on separate days. Background subtracted replicates for each mouse were compiled and fitted with a three-parameter nonlinear regression activation curve to obtain an EC50 value for each serum binding from each animal to each antigen. Data were then compiled per group for each time point. Statistical comparisons between groups were performed using a Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test. Significance for comparisons between groups is indicated in Figure 4B,D: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Compiled data from Dunn’s multiple comparisons tests between groups for ELISA titers can be found in Table S2.
For lentiviral neutralization assays, serum from each animal at each time point was assessed in duplicate with a 6-point serial dilution (starting at 1:50 with 5-fold dilutions). Each assay was performed again in a separate experimental replicate. Each dilution series was normalized to 0% and 100% infectivity using the average RLU values of wells with cells only and with virus only, respectively, from each plate. Four normalized curves for each animal at each time point were compiled to obtain an IC50 value. Statistical comparisons between groups were performed using a Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test. Significance for comparisons between groups is indicated in Figure 4C,E: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Compiled data from Dunn’s multiple comparisons tests between groups for neutralization titers can be found in Table S3.
Safety Statement
No unexpected or unusually high safety hazards were encountered.
Data Availability
The cryo-EM map of the SΔC-Fer nanoparticle has been deposited in the wwPDB OneDep System under EMD accession code EMD-23220.
ARTICLE SECTIONS
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c01405.
Additional data and figures including expression levels, size-exclusion chromatography, cryo-EM, ELISA results, assay validation, and off-target antibody responses (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Author Information
ARTICLE SECTIONS
- Peter S. Kim - Department of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United States; Chan Zuckerberg Biohub, San Francisco, California 94158, United States;
http://orcid.org/0000-0001-6503-4541;
- Abigail E. Powell - Department of Biochemistry & Stanford ChEM-H, Stanford University, Stanford, California 94305, United States;http://orcid.org/0000-0001-6408-9495
- Kaiming Zhang - Department of Bioengineering & James H. Clark Center, Stanford University, Stanford, California 94305, United States;http://orcid.org/0000-0003-0414-4776
- Wah Chiu - Department of Bioengineering & James H. Clark Center, Stanford University, Stanford, California 94305, United States; Chan Zuckerberg Biohub, San Francisco, California 94158, United States; Division of CryoEM and Bioimaging, SSRL, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United States;http://orcid.org/0000-0002-8910-3078
A.E.P., P.A.W., S.T., M.S., and P.S.K. designed experiments. A.E.P. and S.T. purified antigens for immunization. M.S. performed mouse work. P.A.W. performed protein quantitation experiments and analysis. A.E.P. performed antigen characterization, serum ELISAs and neutralizations, and data analysis. K.Z. performed cryo-EM sample preparation, data collection, and image processing. S.L. assisted with image processing. K.Z., S.L., and W.C. analyzed the cryo-EM data. T.D.P. collected convalescent plasma samples. J.E.P. assisted with protein characterization methods and optimization. A.E.P. and P.S.K. wrote the manuscript. All authors assisted with editing and approved the final version of the manuscript.
The authors declare the following competing financial interest(s): A.E.P., P.A.W., and P.S.K. are named as inventors on a provisional patent application applied for by Stanford University and the Chan Zuckerberg Biohub on immunogenic coronavirus fusion proteins and related methods.
Acknowledgments
ARTICLE SECTIONS
We thank Dr. Jesse Bloom, Kate Crawford, Dr. Dennis Burton, and Dr. Deli Huang for sharing the plasmids, cells, and invaluable advice for implementation of the spike-pseudotyped lentiviral neutralization assay. We thank Dr. Florian Krammer and Fatima Amanat for providing the SARS-CoV-2 RBD and FL 2P spike plasmids for protein production. We thank Nielson Weng and Dr. Nicholas Tierney for advice on statistical analyses. We thank Dr. Duo Xu for designing and providing the structural representations of spike and ferritin in Figure 1. We thank Dr. Corey Liu and the Stanford ChEM-H Macromolecular Knowledge Center for kindly relocating the GE Amersham 600 imager to our lab space during the pandemic shutdown for us to rapidly begin this work. We thank Drs. Corey Hecksel and Patrick Mitchell for expert maintenance of Stanford-SLAC Cryo-EM Center and the SLAC National Accelerator Laboratory for supporting these studies during the pandemic shutdown. We thank members of the Kim Lab for fruitful discussions and insight on project design as well as helpful comments on the manuscript. We acknowledge BioRender for the images used in Figure 2A. The CMV/R expression vectors were received from the NIH AIDS Reagent Program. This work was supported by the Stanford Maternal and Child Health Research Institute postdoctoral fellowship (to A.E.P.), the Damon Runyon Cancer Research Foundation Merck Fellowship (DRG-2301-17 to S.T.), National Institutes of Health grants (P41GM103832, R01AI148382, P01AI120943, S10OD02160 to W.C.), Chan Zuckerberg Biohub (to W.C. and P.S.K.), the Virginia and D. K. Ludwig Fund for Cancer Research (to P.S.K.), and the Frank Quattrone and Denise Foderaro Family Research Fund (to P.S.K.).
References
ARTICLE SECTIONS
This article references 87 other publications.
- 1World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard. https://covid19.who.int/ (accessed 02 December 2020).Google ScholarThere is no corresponding record for this reference.
- 2Xia, S.; Liu, M.; Wang, C.; Xu, W.; Lan, Q.; Feng, S.; Qi, F.; Bao, L.; Du, L.; Liu, S.; Qin, C.; Sun, F.; Shi, Z.; Zhu, Y.; Jiang, S.; Lu, L. Inhibition of SARS-CoV-2 (Previously 2019-NCoV) Infection by a Highly Potent Pan-Coronavirus Fusion Inhibitor Targeting Its Spike Protein That Harbors a High Capacity to Mediate Membrane Fusion. Cell Res. 2020, 30 (4), 343– 355, DOI: 10.1038/s41422-020-0305-x[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFKhtL0%253D&md5=00a4a7fb87b1053fb1033973f3734af3Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusionXia, Shuai; Liu, Meiqin; Wang, Chao; Xu, Wei; Lan, Qiaoshuai; Feng, Siliang; Qi, Feifei; Bao, Linlin; Du, Lanying; Liu, Shuwen; Qin, Chuan; Sun, Fei; Shi, Zhengli; Zhu, Yun; Jiang, Shibo; Lu, LuCell Research (2020), 30 (4), 343-355CODEN: CREEB6; ISSN:1001-0602. (Nature Research)The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the mol. mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be assocd. with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, resp. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examd., including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
- 3Wrapp, D.; Wang, N.; Corbett, K. S.; Goldsmith, J. A.; Hsieh, C. L.; Abiona, O.; Graham, B. S.; McLellan, J. S. Cryo-EM Structure of the 2019-NCoV Spike in the Prefusion Conformation. Science 2020, 367 (6483), 1260– 1263, DOI: 10.1126/science.abb2507[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFemt70%253D&md5=27d08cbb9a43d1da051a8a92a9f68aa5Cryo-EM structure of the 2019-nCoV spike in the prefusion conformationWrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
- 4Robbiani, D. F.; Gaebler, C.; Muecksch, F.; Lorenzi, J. C. C.; Wang, Z.; Cho, A.; Agudelo, M.; Barnes, C. O.; Gazumyan, A.; Finkin, S.; Hägglöf, T.; Oliveira, T. Y.; Viant, C.; Hurley, A.; Hoffmann, H.-H.; Millard, K. G.; Kost, R. G.; Cipolla, M.; Gordon, K.; Bianchini, F.; Chen, S. T.; Ramos, V.; Patel, R.; Dizon, J.; Shimeliovich, I.; Mendoza, P.; Hartweger, H.; Nogueira, L.; Pack, M.; Horowitz, J.; Schmidt, F.; Weisblum, Y.; Michailidis, E.; Ashbrook, A. W.; Waltari, E.; Pak, J. E.; Huey-Tubman, K. E.; Koranda, N.; Hoffman, P. R.; West, A. P.; Rice, C. M.; Hatziioannou, T.; Bjorkman, P. J.; Bieniasz, P. D.; Caskey, M.; Nussenzweig, M. C. Convergent Antibody Responses to SARS-CoV-2 in Convalescent Individuals. Nature 2020, 584 (7821), 437– 442, DOI: 10.1038/s41586-020-2456-9[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFegtb3P&md5=952577f69ffe8282d0b2451b17a5ecadConvergent antibody responses to SARS-CoV-2 in convalescent individualsRobbiani, Davide F.; Gaebler, Christian; Muecksch, Frauke; Lorenzi, Julio C. C.; Wang, Zijun; Cho, Alice; Agudelo, Marianna; Barnes, Christopher O.; Gazumyan, Anna; Finkin, Shlomo; Hagglof, Thomas; Oliveira, Thiago Y.; Viant, Charlotte; Hurley, Arlene; Hoffmann, Hans-Heinrich; Millard, Katrina G.; Kost, Rhonda G.; Cipolla, Melissa; Gordon, Kristie; Bianchini, Filippo; Chen, Spencer T.; Ramos, Victor; Patel, Roshni; Dizon, Juan; Shimeliovich, Irina; Mendoza, Pilar; Hartweger, Harald; Nogueira, Lilian; Pack, Maggi; Horowitz, Jill; Schmidt, Fabian; Weisblum, Yiska; Michailidis, Eleftherios; Ashbrook, Alison W.; Waltari, Eric; Pak, John E.; Huey-Tubman, Kathryn E.; Koranda, Nicholas; Hoffman, Pauline R.; West Jr, Anthony P.; Rice, Charles M.; Hatziioannou, Theodora; Bjorkman, Pamela J.; Bieniasz, Paul D.; Caskey, Marina; Nussenzweig, Michel C.Nature (London, United Kingdom) (2020), 584 (7821), 437-442CODEN: NATUAS; ISSN:0028-0836. (Nature Research)During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. We report on 149 COVID-19-convalescent individuals. Plasma samples collected an av. of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titers; titers were <50 in 33% of samples, <1000 in 79% of samples, and only 1% of samples had titers >5000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titers, antibodies to 3 distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concns. (IC50 values) as low as 2 ng/mL. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
- 5Brouwer, P. J. M.; Caniels, T. G.; van der Straten, K.; Snitselaar, J. L.; Aldon, Y.; Bangaru, S.; Torres, J. L.; Okba, N. M. A.; Claireaux, M.; Kerster, G.; Bentlage, A. E. H.; van Haaren, M. M.; Guerra, D.; Burger, J. A.; Schermer, E. E.; Verheul, K. D.; van der Velde, N.; van der Kooi, A.; van Schooten, J.; van Breemen, M. J.; Bijl, T. P. L.; Sliepen, K.; Aartse, A.; Derking, R.; Bontjer, I.; Kootstra, N. A.; Wiersinga, W. J.; Vidarsson, G.; Haagmans, B. L.; Ward, A. B.; de Bree, G. J.; Sanders, R. W.; van Gils, M. J. Potent Neutralizing Antibodies from COVID-19 Patients Define Multiple Targets of Vulnerability. Science 2020, 369 (6504), 643– 650, DOI: 10.1126/science.abc5902[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGms7vJ&md5=59e9b7db3e2d86923e5ecbb880aa24ccPotent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerabilityBrouwer, Philip J. M.; Caniels, Tom G.; van der Straten, Karlijn; Snitselaar, Jonne L.; Aldon, Yoann; Bangaru, Sandhya; Torres, Jonathan L.; Okba, Nisreen M. A.; Claireaux, Mathieu; Kerster, Gius; Bentlage, Arthur E. H.; van Haaren, Marlies M.; Guerra, Denise; Burger, Judith A.; Schermer, Edith E.; Verheul, Kirsten D.; van der Velde, Niels; van der Kooi, Alex; van Schooten, Jelle; van Breemen, Marielle J.; Bijl, Tom P. L.; Sliepen, Kwinten; Aartse, Aafke; Derking, Ronald; Bontjer, Ilja; Kootstra, Neeltje A.; Wiersinga, W. Joost; Vidarsson, Gestur; Haagmans, Bart L.; Ward, Andrew B.; de Bree, Godelieve J.; Sanders, Rogier W.; van Gils, Marit J.Science (Washington, DC, United States) (2020), 369 (6504), 643-650CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. We isolated monoclonal antibodies from 3 convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concn. as low as 0.007μg/mL. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addn. to providing guidance for vaccine design, the antibodies described are promising candidates for COVID-19 treatment and prevention.
- 6Hoffmann, M.; Kleine-Weber, H.; Pöhlmann, S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol. Cell 2020, 78 (4), 779– 784, DOI: 10.1016/j.molcel.2020.04.022[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovVCgs7o%253D&md5=18c626bd22d3d9c280d8b5e19c116db8A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung CellsHoffmann, Markus; Kleine-Weber, Hannah; Poehlmann, StefanMolecular Cell (2020), 78 (4), 779-784.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site contg. multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
- 7Walls, A. C.; Park, Y.-J.; Tortorici, M. A.; Wall, A.; McGuire, A. T.; Veesler, D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 2020, 181 (2), 281– 292, DOI: 10.1016/j.cell.2020.02.058[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvVejsLk%253D&md5=ac8a8a208d9c26f88f702fb7634ab1abStructure, Function, and Antigenicity of the SARS-CoV-2 Spike GlycoproteinWalls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, DavidCell (Cambridge, MA, United States) (2020), 181 (2), 281-292.e6CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We detd. cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
- 8Yan, R.; Zhang, Y.; Li, Y.; Xia, L.; Guo, Y.; Zhou, Q. Structural Basis for the Recognition of SARS-CoV-2 by Full-Length Human ACE2. Science 2020, 367 (6485), 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 9Shang, J.; Ye, G.; Shi, K.; Wan, Y.; Luo, C.; Aihara, H.; Geng, Q.; Auerbach, A.; Li, F. Structural Basis of Receptor Recognition by SARS-CoV-2. Nature 2020, 581 (7807), 221– 224, DOI: 10.1038/s41586-020-2179-y[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtbs%253D&md5=33bc9151641b2adcfb0dbf446621a1dcStructural basis of receptor recognition by SARS-CoV-2Shang, Jian; Ye, Gang; Shi, Ke; Wan, Yushun; Luo, Chuming; Aihara, Hideki; Geng, Qibin; Auerbach, Ashley; Li, FangNature (London, United Kingdom) (2020), 581 (7807), 221-224CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystn.) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Addnl., we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
- 10Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; Wang, X. Structure of the SARS-CoV-2 Spike Receptor-Binding Domain Bound to the ACE2 Receptor. Nature 2020, 581 (7807), 215– 220, DOI: 10.1038/s41586-020-2180-5[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtL8%253D&md5=279c60143e8e5eb505457e0778baa8efStructure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptorLan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, XinquanNature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
- 11Corbett, K. S.; Flynn, B.; Foulds, K. E.; Francica, J. R.; Boyoglu-Barnum, S.; Werner, A. P.; Flach, B.; O’Connell, S.; Bock, K. W.; Minai, M.; Nagata, B. M.; Andersen, H.; Martinez, D. R.; Noe, A. T.; Douek, N.; Donaldson, M. M.; Nji, N. N.; Alvarado, G. S.; Edwards, D. K.; Flebbe, D. R.; Lamb, E.; Doria-Rose, N. A.; Lin, B. C.; Louder, M. K.; O’Dell, S.; Schmidt, S. D.; Phung, E.; Chang, L. A.; Yap, C.; Todd, J.-P. M.; Pessaint, L.; Van Ry, A.; Browne, S.; Greenhouse, J.; Putman-Taylor, T.; Strasbaugh, A.; Campbell, T.-A.; Cook, A.; Dodson, A.; Steingrebe, K.; Shi, W.; Zhang, Y.; Abiona, O. M.; Wang, L.; Pegu, A.; Yang, E. S.; Leung, K.; Zhou, T.; Teng, I.-T.; Widge, A.; Gordon, I.; Novik, L.; Gillespie, R. A.; Loomis, R. J.; Moliva, J. I.; Stewart-Jones, G.; Himansu, S.; Kong, W.-P.; Nason, M. C.; Morabito, K. M.; Ruckwardt, T. J.; Ledgerwood, J. E.; Gaudinski, M. R.; Kwong, P. D.; Mascola, J. R.; Carfi, A.; Lewis, M. G.; Baric, R. S.; McDermott, A.; Moore, I. N.; Sullivan, N. J.; Roederer, M.; Seder, R. A.; Graham, B. S. Evaluation of the MRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N. Engl. J. Med. 2020, 383, 1544, DOI: 10.1056/NEJMoa2024671[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFWmsr7I&md5=8ecba6b18bc615d886ca5016b7c85496Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primatesCorbett, K. S.; Flynn, B.; Foulds, K. E.; Francica, J. R.; Boyoglu-Barnum, S.; Werner, A. P.; Flach, B.; O'Connell, S.; Bock, K. W.; Minai, M.; Nagata, B. M.; Andersen, H.; Martinez, D. R.; Noe, A. T.; Douek, N.; Donaldson, M. M.; Nji, N. N.; Alvarado, G. S.; Edwards, D. K.; Flebbe, D. R.; Lamb, E.; Doria-Rose, N. A.; Lin, B. C.; Louder, M. K.; O'Dell, S.; Schmidt, S. D.; Phung, E.; Chang, L. A.; Yap, C.; Todd, J.-P. M.; Pessaint, L.; Van Ry, A.; Browne, S.; Greenhouse, J.; Putman-Taylor, T.; Strasbaugh, A.; Campbell, T.-A.; Cook, A.; Dodson, A.; Steingrebe, K.; Shi, W.; Zhang, Y.; Abiona, O. M.; Wang, L.; Pegu, A.; Yang, E. S.; Leung, K.; Zhou, T.; Teng, I-T.; Widge, A.; Gordon, I.; Novik, L.; Gillespie, R. A.; Loomis, R. J.; Moliva, J. I.; Stewart-Jones, G.; Himansu, S.; Kong, W.-P.; Nason, M. C.; Morabito, K. M.; Ruckwardt, T. J.; Ledgerwood, J. E.; Gaudinski, M. R.; Kwong, P. D.; Mascola, J. R.; Carfi, A.; Lewis, M. G.; Baric, R. S.; McDermott, A.; Moore, I. N.; Sullivan, N. J.; Roederer, M.; Seder, R. A.; Graham, B. S.New England Journal of Medicine (2020), 383 (16), 1544-1555CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates. Methods: Nonhuman primates received 10 or 100μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathol. anal. and viral quantification were performed on lung-tissue specimens. Results The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory diln. (ID50) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. conclusions Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathol. changes in the lung.
- 12Corbett, K. S.; Edwards, D.; Leist, S. R.; Abiona, O. M.; Boyoglu-Barnum, S.; Gillespie, R. A.; Himansu, S.; Schäfer, A.; Ziwawo, C. T.; DiPiazza, A. T.; Dinnon, K. H.; Elbashir, S. M.; Shaw, C. A.; Woods, A.; Fritch, E. J.; Martinez, D. R.; Bock, K. W.; Minai, M.; Nagata, B. M.; Hutchinson, G. B.; Bahl, K.; Garcia-Dominguez, D.; Ma, L.; Renzi, I.; Kong, W.-P.; Schmidt, S. D.; Wang, L.; Zhang, Y.; Stevens, L. J.; Phung, E.; Chang, L. A.; Loomis, R. J.; Altaras, N. E.; Narayanan, E.; Metkar, M.; Presnyak, V.; Liu, C.; Louder, M. K.; Shi, W.; Leung, K.; Yang, E. S.; West, A.; Gully, K. L.; Wang, N.; Wrapp, D.; Doria-Rose, N. A.; Stewart-Jones, G.; Bennett, H.; Nason, M. C.; Ruckwardt, T. J.; McLellan, J. S.; Denison, M. R.; Chappell, J. D.; Moore, I. N.; Morabito, K. M.; Mascola, J. R.; Baric, R. S.; Carfi, A.; Graham, B. S. SARS-CoV-2 MRNA Vaccine Development Enabled by Prototype Pathogen Preparedness. Nature 2020, 586, 567, DOI: 10.1038/s41586-020-2622-0[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1ejtLbO&md5=fa0483e26f66c6a899486917083d257aSARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparednessCorbett, Kizzmekia S.; Edwards, Darin K.; Leist, Sarah R.; Abiona, Olubukola M.; Boyoglu-Barnum, Seyhan; Gillespie, Rebecca A.; Himansu, Sunny; Schafer, Alexandra; Ziwawo, Cynthia T.; DiPiazza, Anthony T.; Dinnon, Kenneth H.; Elbashir, Sayda M.; Shaw, Christine A.; Woods, Angela; Fritch, Ethan J.; Martinez, David R.; Bock, Kevin W.; Minai, Mahnaz; Nagata, Bianca M.; Hutchinson, Geoffrey B.; Wu, Kai; Henry, Carole; Bahl, Kapil; Garcia-Dominguez, Dario; Ma, LingZhi; Renzi, Isabella; Kong, Wing-Pui; Schmidt, Stephen D.; Wang, Lingshu; Zhang, Yi; Phung, Emily; Chang, Lauren A.; Loomis, Rebecca J.; Altaras, Nedim Emil; Narayanan, Elisabeth; Metkar, Mihir; Presnyak, Vlad; Liu, Cuiping; Louder, Mark K.; Shi, Wei; Leung, Kwanyee; Yang, Eun Sung; West, Ande; Gully, Kendra L.; Stevens, Laura J.; Wang, Nianshuang; Wrapp, Daniel; Doria-Rose, Nicole A.; Stewart-Jones, Guillaume; Bennett, Hamilton; Alvarado, Gabriela S.; Nason, Martha C.; Ruckwardt, Tracy J.; McLellan, Jason S.; Denison, Mark R.; Chappell, James D.; Moore, Ian N.; Morabito, Kaitlyn M.; Mascola, John R.; Baric, Ralph S.; Carfi, Andrea; Graham, Barney S.Nature (London, United Kingdom) (2020), 586 (7830), 567-571CODEN: NATUAS; ISSN:0028-0836. (Nature Research)A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathol. MRNA-1273 is currently in a phase III trial to evaluate its efficacy.
- 13Smith, T. R. F.; Patel, A.; Ramos, S.; Elwood, D.; Zhu, X.; Yan, J.; Gary, E. N.; Walker, S. N.; Schultheis, K.; Purwar, M.; Xu, Z.; Walters, J.; Bhojnagarwala, P.; Yang, M.; Chokkalingam, N.; Pezzoli, P.; Parzych, E.; Reuschel, E. L.; Doan, A.; Tursi, N.; Vasquez, M.; Choi, J.; Tello-Ruiz, E.; Maricic, I.; Bah, M. A.; Wu, Y.; Amante, D.; Park, D. H.; Dia, Y.; Ali, A. R.; Zaidi, F. I.; Generotti, A.; Kim, K. Y.; Herring, T. A.; Reeder, S.; Andrade, V. M.; Buttigieg, K.; Zhao, G.; Wu, J. M.; Li, D.; Bao, L.; Liu, J.; Deng, W.; Qin, C.; Brown, A. S.; Khoshnejad, M.; Wang, N.; Chu, J.; Wrapp, D.; McLellan, J. S.; Muthumani, K.; Wang, B.; Carroll, M. W.; Kim, J. J.; Boyer, J.; Kulp, D. W.; Humeau, L. M. P. F.; Weiner, D. B.; Broderick, K. E. Immunogenicity of a DNA Vaccine Candidate for COVID-19. Nat. Commun. 2020, 11 (1), 1– 13, DOI: 10.1038/s41467-020-16505-0
- 14Wang, H.; Zhang, Y.; Huang, B.; Deng, W.; Quan, Y.; Wang, W.; Xu, W.; Zhao, Y.; Li, N.; Zhang, J.; Liang, H.; Bao, L.; Xu, Y.; Ding, L.; Zhou, W.; Gao, H.; Liu, J.; Niu, P.; Zhao, L.; Zhen, W.; Fu, H.; Yu, S.; Zhang, Z.; Xu, G.; Li, C.; Lou, Z.; Xu, M.; Qin, C.; Wu, G.; Gao, G. F.; Tan, W.; Yang, X. Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2. Cell 2020, 182 (3), 713– 721, DOI: 10.1016/j.cell.2020.06.008[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFylur7P&md5=057a1910bb9c65887d8647a1b1d8c310Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2Wang, Hui; Zhang, Yuntao; Huang, Baoying; Deng, Wei; Quan, Yaru; Wang, Wenling; Xu, Wenbo; Zhao, Yuxiu; Li, Na; Zhang, Jin; Liang, Hongyang; Bao, Linlin; Xu, Yanfeng; Ding, Ling; Zhou, Weimin; Gao, Hong; Liu, Jiangning; Niu, Peihua; Zhao, Li; Zhen, Wei; Fu, Hui; Yu, Shouzhi; Zhang, Zhengli; Xu, Guangxue; Li, Changgui; Lou, Zhiyong; Xu, Miao; Qin, Chuan; Wu, Guizhen; Gao, George Fu; Tan, Wenjie; Yang, XiaomingCell (Cambridge, MA, United States) (2020), 182 (3), 713-721.e9CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. We report the pilot-scale prodn. of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2μg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addn., BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manuf. These results support the further evaluation of BBIBP-CorV in a clin. trial.
- 15van Doremalen, N.; Lambe, T.; Spencer, A.; Belij-Rammerstorfer, S.; Purushotham, J. N.; Port, J. R.; Avanzato, V. A.; Bushmaker, T.; Flaxman, A.; Ulaszewska, M.; Feldmann, F.; Allen, E. R.; Sharpe, H.; Schulz, J.; Holbrook, M.; Okumura, A.; Meade-White, K.; Pérez-Pérez, L.; Edwards, N. J.; Wright, D.; Bissett, C.; Gilbride, C.; Williamson, B. N.; Rosenke, R.; Long, D.; Ishwarbhai, A.; Kailath, R.; Rose, L.; Morris, S.; Powers, C.; Lovaglio, J.; Hanley, P. W.; Scott, D.; Saturday, G.; de Wit, E.; Gilbert, S. C.; Munster, V. J. ChAdOx1 NCoV-19 Vaccine Prevents SARS-CoV-2 Pneumonia in Rhesus Macaques. Nature 2020, 586, 578, DOI: 10.1038/s41586-020-2608-y[Crossref], [PubMed], [CAS], Google ScholarThere is no corresponding record for this reference.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=&md5=9874b665cc7a056b8e2f928dd3112440
- 16Folegatti, P. M.; Ewer, K. J.; Aley, P. K.; Angus, B.; Becker, S.; Belij-Rammerstorfer, S.; Bellamy, D.; Bibi, S.; Bittaye, M.; Clutterbuck, E. A.; Dold, C.; Faust, S. N.; Finn, A.; Flaxman, A. L.; Hallis, B.; Heath, P.; Jenkin, D.; Lazarus, R.; Makinson, R.; Minassian, A. M.; Pollock, K. M.; Ramasamy, M.; Robinson, H.; Snape, M.; Tarrant, R.; Voysey, M.; Green, C.; Douglas, A. D.; Hill, A. V. S.; Lambe, T.; Gilbert, S. C.; Pollard, A. J.; Aboagye, J.; Adams, K.; Ali, A.; Allen, E.; Allison, J. L.; Anslow, R.; Arbe-Barnes, E. H.; Babbage, G.; Baillie, K.; Baker, M.; Baker, N.; Baker, P.; Baleanu, I.; Ballaminut, J.; Barnes, E.; Barrett, J.; Bates, L.; Batten, A.; Beadon, K.; Beckley, R.; Berrie, E.; Berry, L.; Beveridge, A.; Bewley, K. R.; Bijker, E. M.; Bingham, T.; Blackwell, L.; Blundell, C. L.; Bolam, E.; Boland, E.; Borthwick, N.; Bower, T.; Boyd, A.; Brenner, T.; Bright, P. D.; Brown-O’Sullivan, C.; Brunt, E.; Burbage, J.; Burge, S.; Buttigieg, K. R.; Byard, N.; Cabera Puig, I.; Calvert, A.; Camara, S.; Cao, M.; Cappuccini, F.; Carr, M.; Carroll, M. W.; Carter, V.; Cathie, K.; Challis, R. J.; Charlton, S.; Chelysheva, I.; Cho, J.-S.; Cicconi, P.; Cifuentes, L.; Clark, H.; Clark, E.; Cole, T.; Colin-Jones, R.; Conlon, C. P.; Cook, A.; Coombes, N. S.; Cooper, R.; Cosgrove, C. A.; Coy, K.; Crocker, W. E. M.; Cunningham, C. J.; Damratoski, B. E.; Dando, L.; Datoo, M. S.; Davies, H.; De Graaf, H.; Demissie, T.; Di Maso, C.; Dietrich, I.; Dong, T.; Donnellan, F. R.; Douglas, N.; Downing, C.; Drake, J.; Drake-Brockman, R.; Drury, R. E.; Dunachie, S. J.; Edwards, N. J.; Edwards, F. D. L.; Edwards, C. J.; Elias, S. C.; Elmore, M. J.; Emary, K. R. W.; English, M. R.; Fagerbrink, S.; Felle, S.; Feng, S.; Field, S.; Fixmer, C.; Fletcher, C.; Ford, K. J.; Fowler, J.; Fox, P.; Francis, E.; Frater, J.; Furze, J.; Fuskova, M.; Galiza, E.; Gbesemete, D.; Gilbride, C.; Godwin, K.; Gorini, G.; Goulston, L.; Grabau, C.; Gracie, L.; Gray, Z.; Guthrie, L. B.; Hackett, M.; Halwe, S.; Hamilton, E.; Hamlyn, J.; Hanumunthadu, B.; Harding, I.; Harris, S. A.; Harris, A.; Harrison, D.; Harrison, C.; Hart, T. C.; Haskell, L.; Hawkins, S.; Head, I.; Henry, J. A.; Hill, J.; Hodgson, S. H. C.; Hou, M. M.; Howe, E.; Howell, N.; Hutlin, C.; Ikram, S.; Isitt, C.; Iveson, P.; Jackson, S.; Jackson, F.; James, S. W.; Jenkins, M.; Jones, E.; Jones, K.; Jones, C. E.; Jones, B.; Kailath, R.; Karampatsas, K.; Keen, J.; Kelly, S.; Kelly, D.; Kerr, D.; Kerridge, S.; Khan, L.; Khan, U.; Killen, A.; Kinch, J.; King, T. B.; King, L.; King, J.; Kingham-Page, L.; Klenerman, P.; Knapper, F.; Knight, J. C.; Knott, D.; Koleva, S.; Kupke, A.; Larkworthy, C. W.; Larwood, J. P. J.; Laskey, A.; Lawrie, A. M.; Lee, A.; Ngan Lee, K. Y.; Lees, E. A.; Legge, H.; Lelliott, A.; Lemm, N.-M.; Lias, A. M.; Linder, A.; Lipworth, S.; Liu, X.; Liu, S.; Lopez Ramon, R.; Lwin, M.; Mabesa, F.; Madhavan, M.; Mallett, G.; Mansatta, K.; Marcal, I.; Marinou, S.; Marlow, E.; Marshall, J. L.; Martin, J.; McEwan, J.; McInroy, L.; Meddaugh, G.; Mentzer, A. J.; Mirtorabi, N.; Moore, M.; Moran, E.; Morey, E.; Morgan, V.; Morris, S. J.; Morrison, H.; Morshead, G.; Morter, R.; Mujadidi, Y. F.; Muller, J.; Munera-Huertas, T.; Munro, C.; Munro, A.; Murphy, S.; Munster, V. J.; Mweu, P.; Noé, A.; Nugent, F. L.; Nuthall, E.; O’Brien, K.; O’Connor, D.; Oguti, B.; Oliver, J. L.; Oliveira, C.; O’Reilly, P. J.; Osborn, M.; Osborne, P.; Owen, C.; Owens, D.; Owino, N.; Pacurar, M.; Parker, K.; Parracho, H.; Patrick-Smith, M.; Payne, V.; Pearce, J.; Peng, Y.; Peralta Alvarez, M. P.; Perring, J.; Pfafferott, K.; Pipini, D.; Plested, E.; Pluess-Hall, H.; Pollock, K.; Poulton, I.; Presland, L.; Provstgaard-Morys, S.; Pulido, D.; Radia, K.; Ramos Lopez, F.; Rand, J.; Ratcliffe, H.; Rawlinson, T.; Rhead, S.; Riddell, A.; Ritchie, A. J.; Roberts, H.; Robson, J.; Roche, S.; Rohde, C.; Rollier, C. S.; Romani, R.; Rudiansyah, I.; Saich, S.; Sajjad, S.; Salvador, S.; Sanchez Riera, L.; Sanders, H.; Sanders, K.; Sapaun, S.; Sayce, C.; Schofield, E.; Screaton, G.; Selby, B.; Semple, C.; Sharpe, H. R.; Shaik, I.; Shea, A.; Shelton, H.; Silk, S.; Silva-Reyes, L.; Skelly, D. T.; Smee, H.; Smith, C. C.; Smith, D. J.; Song, R.; Spencer, A. J.; Stafford, E.; Steele, A.; Stefanova, E.; Stockdale, L.; Szigeti, A.; Tahiri-Alaoui, A.; Tait, M.; Talbot, H.; Tanner, R.; Taylor, I. J.; Taylor, V.; Te Water Naude, R.; Thakur, N.; Themistocleous, Y.; Themistocleous, A.; Thomas, M.; Thomas, T. M.; Thompson, A.; Thomson-Hill, S.; Tomlins, J.; Tonks, S.; Towner, J.; Tran, N.; Tree, J. A.; Truby, A.; Turkentine, K.; Turner, C.; Turner, N.; Turner, S.; Tuthill, T.; Ulaszewska, M.; Varughese, R.; Van Doremalen, N.; Veighey, K.; Verheul, M. K.; Vichos, I.; Vitale, E.; Walker, L.; Watson, M. E. E.; Welham, B.; Wheat, J.; White, C.; White, R.; Worth, A. T.; Wright, D.; Wright, S.; Yao, X. L.; Yau, Y. Safety and Immunogenicity of the ChAdOx1 NCoV-19 Vaccine against SARS-CoV-2: A Preliminary Report of a Phase 1/2, Single-Blind, Randomised Controlled Trial. Lancet 2020, 396 (10249), 467– 478, DOI: 10.1016/S0140-6736(20)31604-4[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVaku7fE&md5=4edd57b9d55d8a7c71ee84c8f62f7ca0Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trialFolegatti, Pedro M.; Ewer, Katie J.; Aley, Parvinder K.; Angus, Brian; Becker, Stephan; Belij-Rammerstorfer, Sandra; Bellamy, Duncan; Bibi, Sagida; Bittaye, Mustapha; Clutterbuck, Elizabeth A.; Dold, Christina; Faust, Saul N.; Finn, Adam; Flaxman, Amy L.; Hallis, Bassam; Heath, Paul; Jenkin, Daniel; Lazarus, Rajeka; Makinson, Rebecca; Minassian, Angela M.; Pollock, Katrina M.; Ramasamy, Maheshi; Robinson, Hannah; Snape, Matthew; Tarrant, Richard; Voysey, Merryn; Green, Catherine; Douglas, Alexander D.; Hill, Adrian V. S.; Lambe, Teresa; Gilbert, Sarah C.; Pollard, Andrew J.Lancet (2020), 396 (10249), 467-478CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of lab. confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 x 1010 viral particles or MenACWY as a single i.m. injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque redn. neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virol. confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Between Apr. 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493-1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96-317; n=127), and were boosted following a second dose (639 EU, 360-792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001). ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 program. UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Center, Thames Valley and South Midland's NIHR Clin. Research Network, and the German Center for Infection Research (DZIF), Partner site Giessen-Marburg-Langen.
- 17Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z.; Gao, H.; Ge, X.; Kan, B.; Hu, Y.; Liu, J.; Cai, F.; Jiang, D.; Yin, Y.; Qin, C.; Li, J.; Gong, X.; Lou, X.; Shi, W.; Wu, D.; Zhang, H.; Zhu, L.; Deng, W.; Li, Y.; Lu, J.; Li, C.; Wang, X.; Yin, W.; Zhang, Y.; Qin, C. Development of an Inactivated Vaccine Candidate for SARS-CoV-2. Science 2020, 369 (6499), 77– 81, DOI: 10.1126/science.abc1932[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlCmtL3P&md5=674788246758fd31fa8bb54c936be83aDevelopment of an inactivated vaccine candidate for SARS-CoV-2Gao, Qiang; Bao, Linlin; Mao, Haiyan; Wang, Lin; Xu, Kangwei; Yang, Minnan; Li, Yajing; Zhu, Ling; Wang, Nan; Lv, Zhe; Gao, Hong; Ge, Xiaoqin; Kan, Biao; Hu, Yaling; Liu, Jiangning; Cai, Fang; Jiang, Deyu; Yin, Yanhui; Qin, Chengfeng; Li, Jing; Gong, Xuejie; Lou, Xiuyu; Shi, Wen; Wu, Dongdong; Zhang, Hengming; Zhu, Lang; Deng, Wei; Li, Yurong; Lu, Jinxing; Li, Changgui; Wang, Xiangxi; Yin, Weidong; Zhang, Yanjun; Qin, ChuanScience (Washington, DC, United States) (2020), 369 (6499), 77-81CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale prodn. of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6μg per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, resp., without observable antibody-dependent enhancement of infection. These data support the clin. development and testing of PiCoVacc for use in humans.
- 18Graham, S. P.; McLean, R. K.; Spencer, A. J.; Belij-Rammerstorfer, S.; Wright, D.; Ulaszewska, M.; Edwards, J. C.; Hayes, J. W. P.; Martini, V.; Thakur, N.; Conceicao, C.; Dietrich, I.; Shelton, H.; Waters, R.; Ludi, A.; Wilsden, G.; Browning, C.; Bialy, D.; Bhat, S.; Stevenson-Leggett, P.; Hollinghurst, P.; Gilbride, C.; Pulido, D.; Moffat, K.; Sharpe, H.; Allen, E.; Mioulet, V.; Chiu, C.; Newman, J.; Asfor, A. S.; Burman, A.; Crossley, S.; Huo, J.; Owens, R. J.; Carroll, M.; Hammond, J. A.; Tchilian, E.; Bailey, D.; Charleston, B.; Gilbert, S. C.; Tuthill, T. J.; Lambe, T. Evaluation of the Immunogenicity of Prime-Boost Vaccination with the Replication-Deficient Viral Vectored COVID-19 Vaccine Candidate ChAdOx1 NCoV-19. npj Vaccines 2020, 5 (1), 69, DOI: 10.1038/s41541-020-00221-3[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVyltLbP&md5=b7dc7fdc2101fce4d607275b25bbfde9Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19Graham, Simon P.; McLean, Rebecca K.; Spencer, Alexandra J.; Belij-Rammerstorfer, Sandra; Wright, Daniel; Ulaszewska, Marta; Edwards, Jane C.; Hayes, Jack W. P.; Martini, Veronica; Thakur, Nazia; Conceicao, Carina; Dietrich, Isabelle; Shelton, Holly; Waters, Ryan; Ludi, Anna; Wilsden, Ginette; Browning, Clare; Bialy, Dagmara; Bhat, Sushant; Stevenson-Leggett, Phoebe; Hollinghurst, Philippa; Gilbride, Ciaran; Pulido, David; Moffat, Katy; Sharpe, Hannah; Allen, Elizabeth; Mioulet, Valerie; Chiu, Chris; Newman, Joseph; Asfor, Amin S.; Burman, Alison; Crossley, Sylvia; Huo, Jiandong; Owens, Raymond J.; Carroll, Miles; Hammond, John A.; Tchilian, Elma; Bailey, Dalan; Charleston, Bryan; Gilbert, Sarah C.; Tuthill, Tobias J.; Lambe, Teresanpj Vaccines (2020), 5 (1), 69CODEN: VACCBC; ISSN:2059-0105. (Nature Research)Abstr.: Clin. development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in Apr. 2020 following non-human primate studies using a single immunization. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. While a single dose induced antigen-specific antibody and T cells responses, a booster immunization enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralizing titers.
- 19Walsh, E. E.; Frenck, R. W.; Falsey, A. R.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Mulligan, M. J.; Bailey, R.; Swanson, K. A.; Li, P.; Koury, K.; Kalina, W.; Cooper, D.; Fontes-Garfias, C.; Shi, P.-Y.; Türeci, Ö.; Tompkins, K. R.; Lyke, K. E.; Raabe, V.; Dormitzer, P. R.; Jansen, K. U.; Şahin, U.; Gruber, W. C. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N. Engl. J. Med. 2020, 383, 2439, DOI: 10.1056/NEJMoa2027906[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1yksLrM&md5=71d40c71a24ff57a525dbaef791a320aSafety and immunogenicity of two RNA-based Covid-19 vaccine candidatesWalsh, Edward E.; Frenck, Robert W. Jr.; Falsey, Ann R.; Kitchin, Nicholas; Absalon, Judith; Gurtman, Alejandra; Lockhart, Stephen; Neuzil, Kathleen; Mulligan, Mark J.; Bailey, Ruth; Swanson, Kena A.; Li, Ping; Koury, Kenneth; Kalina, Warren; Cooper, David; Fontes-Garfias, Camila; Shi, Pei-Yong; Tuereci, Oezlem; Tompkins, Kristin R.; Lyke, Kirsten E.; Raabe, Vanessa; Dormitzer, Philip R.; Jansen, Kathrin U.; Sahin, Ugur; Gruber, William C.New England Journal of Medicine (2020), 383 (25), 2439-2450CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and the resulting disease, coronavirus disease 2019 (Covid-19), have spread to millions of persons worldwide. Multiple vaccine candidates are under development, but no vaccine is currently available. Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States. METHODS: In an ongoing, placebo-controlled, observer-blinded, dose-escalation, phase 1 trial conducted in the United States, we randomly assigned healthy adults 18 to 55 years of age and those 65 to 85 years of age to receive either placebo or one of two lipid nanoparticle-formulated, nucleoside-modified RNA vaccine candidates: BNT162b1, which encodes a secreted trimerized SARS-CoV-2 receptor-binding domain; or BNT162b2, which encodes a membrane-anchored SARS-CoV-2 fulllength spike, stabilized in the prefusion conformation. The primary outcome was safety (e.g., local and systemic reactions and adverse events); immunogenicity was a secondary outcome. Trial groups were defined according to vaccine candidate, age of the participants, and vaccine dose level (10μg, 20μg, 30μg, and 100μg). In all groups but one, participants received two doses, with a 21-day interval between doses; in one group (100μg of BNT162b1), participants received one dose. RESULTS: A total of 195 participants underwent randomization. In each of 13 groups of 15 participants, 12 participants received vaccine and 3 received placebo. BNT162b2 was assocd. with a lower incidence and severity of systemic reactions than BNT162b1, particularly in older adults. In both younger and older adults, the two vaccine candidates elicited similar dose-dependent SARS-CoV-2-neutralizing geometric mean titers, which were similar to or higher than the geometric mean titer of a panel of SARS-CoV-2 convalescent serum samples. CONCLUSIONS The safety and immunogenicity data from this U.S. phase 1 trial of two vaccine candidates in younger and older adults, added to earlier interim safety and immunogenicity data regarding BNT162b1 in younger adults from trials in Germany and the United States, support the selection of BNT162b2 for advancement to a pivotal phase 2-3 safety and efficacy evaluation.
- 20Funk, C. D.; Laferrière, C.; Ardakani, A. A Snapshot of the Global Race for Vaccines Targeting SARS-CoV-2 and the COVID-19 Pandemic. Front. Pharmacol. 2020, 11, 11, DOI: 10.3389/fphar.2020.00937
- 21Krammer, F. SARS-CoV-2 Vaccines in Development. Nature 2020, 586 (7830), 516– 527, DOI: 10.1038/s41586-020-2798-3[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyhs73N&md5=05f069b7aa258af442f848415a24c865SARS-CoV-2 vaccines in developmentKrammer, FlorianNature (London, United Kingdom) (2020), 586 (7830), 516-527CODEN: NATUAS; ISSN:0028-0836. (Nature Research)A review. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in late 2019 in China and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. To mitigate the effects of the virus on public health, the economy and society, a vaccine is urgently needed. Here, the development of vaccines against SARS-CoV-2 is reviewed. Development was initiated when the genetic sequence of the virus became available in early Jan. 2020, and has moved at an unprecedented speed: a phase I trial started in March 2020 and there are currently more than 180 vaccines at various stages of development. Data from phase I and phase II trials are already available for several vaccine candidates, and many have moved into phase III trials. The data available so far suggest that effective and safe vaccines might become available within months, rather than years.
- 22Thanh Le, T.; Andreadakis, Z.; Kumar, A.; Gómez Román, R.; Tollefsen, S.; Saville, M.; Mayhew, S. The COVID-19 Vaccine Development Landscape. Nat. Rev. Drug Discovery 2020, 19 (5), 305– 306, DOI: 10.1038/d41573-020-00073-5[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslOht7g%253D&md5=4be48c76fe66c0c69d24bebf2137eac3The COVID-19 vaccine development landscapeThanh Le, Tung; Andreadakis, Zacharias; Kumar, Arun; Gomez Roman, Raul; Tollefsen, Stig; Saville, Melanie; Mayhew, StephenNature Reviews Drug Discovery (2020), 19 (5), 305-306CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review on the development of vaccines for SARS-CoV-2. Topics include: the type of vaccines under development; the current stage of development; and the current no. of vaccine projects worldwide.
- 23Fausther-Bovendo, H.; Kobinger, G. P. Pre-Existing Immunity against Ad Vectors. Hum. Vaccines Immunother. 2014, 10 (10), 2875– 2884, DOI: 10.4161/hv.29594
- 24Thacker, E. E.; Timares, L.; Matthews, Q. L. Strategies to Overcome Host Immunity to Adenovirus Vectors in Vaccine Development. Expert Rev. Vaccines 2009, 8 (6), 761– 777, DOI: 10.1586/erv.09.29[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXms1Ort7g%253D&md5=9e29ff9e46cb488e385d1014f7946e02Strategies to overcome host immunity to adenovirus vectors in vaccine developmentThacker, Erin E.; Timares, Laura; Matthews, Qiana L.Expert Review of Vaccines (2009), 8 (6), 761-777CODEN: ERVXAX; ISSN:1476-0584. (Expert Reviews Ltd.)A review. The first clin. evaluations of adenovirus (Ad)-based vectors for gene therapy were initiated in the mid-1990s and led to great anticipation for future utility. However, excitement surrounding gene therapy, particularly Ad-based therapy, was diminished upon the death of Jesse Gelsinger, and recent discouraging results from the HIV vaccine STEP trial have brought efficacy and safety issues to the forefront again. Even so, Ad vectors are still considered among the safest and most effective vaccine vectors. Innate and pre-existing immunity to Ad mediate much of the acute toxicities and reduced therapeutic efficacies obsd. following vaccination with this vector. Thus, innovative strategies must continue to be developed to reduce Ad-specific antigenicity and immune recognition. This review provides an overview and critique of the most promising strategies, including results from preclin. trials in mice and nonhuman primates, which aim to revive the future of Ad-based vaccines.
- 25Moyle, P. M.; Toth, I. Modern Subunit Vaccines: Development, Components, and Research Opportunities. ChemMedChem 2013, 8 (3), 360– 376, DOI: 10.1002/cmdc.201200487[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntFyquw%253D%253D&md5=1a572bf2e8b4f53bac91a6b687a3cff4Modern Subunit Vaccines: Development, Components, and Research OpportunitiesMoyle, Peter Michael; Toth, IstvanChemMedChem (2013), 8 (3), 360-376CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Traditional vaccines, based on the administration of killed or attenuated microorganisms, have proven to be among the most effective methods for disease prevention. Safety issues related to administering these complex mixts., however, prevent their universal application. Through identification of the microbial components responsible for protective immunity, vaccine formulations can be simplified, enabling mol.-level vaccine characterization, improved safety profiles, prospects to develop new high-priority vaccines (e.g. for HIV, tuberculosis, and malaria), and the opportunity for extensive vaccine component optimization. This subunit approach, however, comes at the expense of decreased immunity, requiring the addn. of immunostimulatory agents (adjuvants). As few adjuvants are currently used in licensed vaccines, adjuvant development represents an exciting area for medicinal chemists to play a role in the future of vaccine development. In addn., immune responses can be further customized though optimization of delivery systems, tuning the size of particulate vaccines, targeting specific cells of the immune system (e.g. dendritic cells), and adding components to aid vaccine efficacy in whole immunized populations (e.g. promiscuous T-helper epitopes). Herein the authors review the current state of the art and future direction in subunit vaccine development, with a focus on the described components and their potential to steer the immune response toward a desired response.
- 26Wang, N.; Shang, J.; Jiang, S.; Du, L. Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses. Front. Microbiol. 2020, 11, 11, DOI: 10.3389/fmicb.2020.00298
- 27Chattopadhyay, S.; Chen, J.-Y.; Chen, H.-W.; Hu, C.-M. J. Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation. Nanotheranostics 2017, 1 (3), 244– 260, DOI: 10.7150/ntno.19796[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7ks1SgsA%253D%253D&md5=b2fc2f2c624f586c8bd909a64a00501dNanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune PotentiationChattopadhyay Saborni; Chen Jui-Yi; Hu Che-Ming Jack; Chattopadhyay Saborni; Chen Hui-Wen; Chen Hui-Wen; Hu Che-Ming JackNanotheranostics (2017), 1 (3), 244-260 ISSN:.Synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. These nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. As a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. This review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. It provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. As understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. These nanoformulations are poised to provide solutions towards pressing and emerging human diseases.
- 28López-Sagaseta, J.; Malito, E.; Rappuoli, R.; Bottomley, M. J. Self-Assembling Protein Nanoparticles in the Design of Vaccines. Comput. Struct. Biotechnol. J. 2016, 14, 58– 68, DOI: 10.1016/j.csbj.2015.11.001[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFWhs7vP&md5=0e1c8a6716b294cc150e08baf6e2333eSelf-assembling protein nanoparticles in the design of vaccinesLopez-Sagaseta, Jacinto; Malito, Enrico; Rappuoli, Rino; Bottomley, Matthew J.Computational and Structural Biotechnology Journal (2016), 14 (), 58-68CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estd. to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines contg. isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclin. evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a crit. selection of data that demonstrate the potential of this field. In addn., we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinol. for max. impact in the rational design of vaccine antigens.
- 29Zhao, L.; Seth, A.; Wibowo, N.; Zhao, C.-X.; Mitter, N.; Yu, C.; Middelberg, A. P. J. Nanoparticle Vaccines. Vaccine 2014, 32 (3), 327– 337, DOI: 10.1016/j.vaccine.2013.11.069[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3isFagug%253D%253D&md5=1e95159344066200fb35657c68795561Nanoparticle vaccinesZhao Liang; Seth Arjun; Wibowo Nani; Zhao Chun-Xia; Yu Chengzhong; Mitter Neena; Middelberg Anton P JVaccine (2014), 32 (3), 327-37 ISSN:.Nanotechnology increasingly plays a significant role in vaccine development. As vaccine development orientates toward less immunogenic "minimalist" compositions, formulations that boost antigen effectiveness are increasingly needed. The use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release. A number of nanoparticle vaccines varying in composition, size, shape, and surface properties have been approved for human use and the number of candidates is increasing. However, challenges remain due to a lack of fundamental understanding regarding the in vivo behavior of nanoparticles, which can operate as either a delivery system to enhance antigen processing and/or as an immunostimulant adjuvant to activate or enhance immunity. This review provides a broad overview of recent advances in prophylactic nanovaccinology. Types of nanoparticles used are outlined and their interaction with immune cells and the biosystem are discussed. Increased knowledge and fundamental understanding of nanoparticle mechanism of action in both immunostimulatory and delivery modes, and better understanding of in vivo biodistribution and fate, are urgently required, and will accelerate the rational design of nanoparticle-containing vaccines.
- 30Kanekiyo, M.; Bu, W.; Joyce, M. G.; Meng, G.; Whittle, J. R. R.; Baxa, U.; Yamamoto, T.; Narpala, S.; Todd, J.-P.; Rao, S. S.; McDermott, A. B.; Koup, R. A.; Rossmann, M. G.; Mascola, J. R.; Graham, B. S.; Cohen, J. I.; Nabel, G. J. Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell 2015, 162 (5), 1090– 1100, DOI: 10.1016/j.cell.2015.07.043[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlCis7zO&md5=a83f8141bfe7a23f5a5950adffca0f59Rational design of an Epstein-Barr virus vaccine targeting the receptor-binding siteKanekiyo, Masaru; Bu, Wei; Joyce, M. Gordon; Meng, Geng; Whittle, James R. R.; Baxa, Ulrich; Yamamoto, Takuya; Narpala, Sandeep; Todd, John-Paul; Rao, Srinivas S.; McDermott, Adrian B.; Koup, Richard A.; Rossmann, Michael G.; Mascola, John R.; Graham, Barney S.; Cohen, Jeffrey I.; Nabel, Gary J.Cell (Cambridge, MA, United States) (2015), 162 (5), 1090-1100CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Epstein-Barr virus (EBV) represents a major global health problem. Though it is assocd. with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a sym. array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to sol. gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.
- 31Walls, A. C.; Fiala, B.; Schäfer, A.; Wrenn, S.; Pham, M. N.; Murphy, M.; Tse, L. V.; Shehata, L.; O’Connor, M. A.; Chen, C.; Navarro, M. J.; Miranda, M. C.; Pettie, D.; Ravichandran, R.; Kraft, J. C.; Ogohara, C.; Palser, A.; Chalk, S.; Lee, E. C.; Guerriero, K.; Kepl, E.; Chow, C. M.; Sydeman, C.; Hodge, E. A.; Brown, B.; Fuller, J. T.; Dinnon, K. H.; Gralinski, L. E.; Leist, S. R.; Gully, K. L.; Lewis, T. B.; Guttman, M.; Chu, H. Y.; Lee, K. K.; Fuller, D. H.; Baric, R. S.; Kellam, P.; Carter, L.; Pepper, M.; Sheahan, T. P.; Veesler, D.; King, N. P. Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell 2020, 183, 1367, DOI: 10.1016/j.cell.2020.10.043[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit12ks7jL&md5=ba75e5c3a28f919f69dc5d3bf06c024fElicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2Walls, Alexandra C.; Fiala, Brooke; Schafer, Alexandra; Wrenn, Samuel; Pham, Minh N.; Murphy, Michael; Tse, Longping V.; Shehata, Laila; O'Connor, Megan A.; Chen, Chengbo; Navarro, Mary Jane; Miranda, Marcos C.; Pettie, Deleah; Ravichandran, Rashmi; Kraft, John C.; Ogohara, Cassandra; Palser, Anne; Chalk, Sara; Lee, E-Chiang; Guerriero, Kathryn; Kepl, Elizabeth; Chow, Cameron M.; Sydeman, Claire; Hodge, Edgar A.; Brown, Brieann; Fuller, Jim T.; Dinnon, Kenneth H., III; Gralinski, Lisa E.; Leist, Sarah R.; Gully, Kendra L.; Lewis, Thomas B.; Guttman, Miklos; Chu, Helen Y.; Lee, Kelly K.; Fuller, Deborah H.; Baric, Ralph S.; Kellam, Paul; Carter, Lauren; Pepper, Marion; Sheahan, Timothy P.; Veesler, David; King, Neil P.Cell (Cambridge, MA, United States) (2020), 183 (5), 1367-1382.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-assocd. enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manuf. of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufg. efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.
- 32Zhang, B.; Chao, C. W.; Tsybovsky, Y.; Abiona, O. M.; Hutchinson, G. B.; Moliva, J. I.; Olia, A. S.; Pegu, A.; Phung, E.; Stewart-Jones, G. B. E.; Verardi, R.; Wang, L.; Wang, S.; Werner, A.; Yang, E. S.; Yap, C.; Zhou, T.; Mascola, J. R.; Sullivan, N. J.; Graham, B. S.; Corbett, K. S.; Kwong, P. D. A Platform Incorporating Trimeric Antigens into Self-Assembling Nanoparticles Reveals SARS-CoV-2-Spike Nanoparticles to Elicit Substantially Higher Neutralizing Responses than Spike Alone. Sci. Rep. 2020, in press. DOI: 10.1038/s41598-020-74949-2 .
- 33Kanekiyo, M.; Wei, C.-J.; Yassine, H. M.; McTamney, P. M.; Boyington, J. C.; Whittle, J. R. R.; Rao, S. S.; Kong, W.-P.; Wang, L.; Nabel, G. J. Self-Assembling Influenza Nanoparticle Vaccines Elicit Broadly Neutralizing H1N1 Antibodies. Nature 2013, 499 (7456), 102– 106, DOI: 10.1038/nature12202[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotFSmtrw%253D&md5=473f68a2ad8d50bc94f8e3cba5f10110Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodiesKanekiyo, Masaru; Wei, Chih-Jen; Yassine, Hadi M.; McTamney, Patrick M.; Boyington, Jeffrey C.; Whittle, James R. R.; Rao, Srinivas S.; Kong, Wing-Pui; Wang, Lingshu; Nabel, Gary J.Nature (London, United Kingdom) (2013), 499 (7456), 102-106CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technol. and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here the authors use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited hemagglutination inhibition antibody titers more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
- 34Yassine, H. M.; Boyington, J. C.; McTamney, P. M.; Wei, C.-J.; Kanekiyo, M.; Kong, W.-P.; Gallagher, J. R.; Wang, L.; Zhang, Y.; Joyce, M. G.; Lingwood, D.; Moin, S. M.; Andersen, H.; Okuno, Y.; Rao, S. S.; Harris, A. K.; Kwong, P. D.; Mascola, J. R.; Nabel, G. J.; Graham, B. S. Hemagglutinin-Stem Nanoparticles Generate Heterosubtypic Influenza Protection. Nat. Med. 2015, 21 (9), 1065– 1070, DOI: 10.1038/nm.3927[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlynsL7N&md5=1a68e5764008fddbe28cada259ea5eb1Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protectionYassine, Hadi M.; Boyington, Jeffrey C.; McTamney, Patrick M.; Wei, Chih-Jen; Kanekiyo, Masaru; Kong, Wing-Pui; Gallagher, John R.; Wang, Lingshu; Zhang, Yi; Joyce, M. Gordon; Lingwood, Daniel; Moin, Syed M.; Andersen, Hanne; Okuno, Yoshinobu; Rao, Srinivas S.; Harris, Audray K.; Kwong, Peter D.; Mascola, John R.; Nabel, Gary J.; Graham, Barney S.Nature Medicine (New York, NY, United States) (2015), 21 (9), 1065-1070CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, detn. of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy anal. of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of Ig from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.
- 35Sliepen, K.; Ozorowski, G.; Burger, J. A.; van Montfort, T.; Stunnenberg, M.; LaBranche, C.; Montefiori, D. C.; Moore, J. P.; Ward, A. B.; Sanders, R. W. Presenting Native-like HIV-1 Envelope Trimers on Ferritin Nanoparticles Improves Their Immunogenicity. Retrovirology 2015, 12 (1), 82, DOI: 10.1186/s12977-015-0210-4[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtFKjsLc%253D&md5=f80be2e5c8bb712e06b42162f72a5fe8Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicitySliepen, Kwinten; Ozorowski, Gabriel; Burger, Judith A.; van Montfort, Thijs; Stunnenberg, Melissa; La Branche, Celia; Montefiori, David C.; Moore, John P.; Ward, Andrew B.; Sanders, Rogier W.Retrovirology (2015), 12 (), 82/1-82/5CODEN: RETRBO; ISSN:1742-4690. (BioMed Central Ltd.)Background: Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation. Findings: We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-bearing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses (but not significantly), to several tier 1B viruses and the autologous tier 2 virus than when the same trimers were delivered as sol. proteins. Conclusions: This or other nanoparticle designs may be practical ways to improve the immunogenicity of envelope glycoprotein trimers.
- 36He, L.; de Val, N.; Morris, C. D.; Vora, N.; Thinnes, T. C.; Kong, L.; Azadnia, P.; Sok, D.; Zhou, B.; Burton, D. R.; Wilson, I. A.; Nemazee, D.; Ward, A. B.; Zhu, J. Presenting Native-like Trimeric HIV-1 Antigens with Self-Assembling Nanoparticles. Nat. Commun. 2016, 7 (1), 12041, DOI: 10.1038/ncomms12041[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFSqsbzL&md5=ceccd03283a87943cc13ea2ca9a13a8aPresenting native-like trimeric HIV-1 antigens with self-assembling nanoparticlesHe, Linling; de Val, Natalia; Morris, Charles D.; Vora, Nemil; Thinnes, Therese C.; Kong, Leopold; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Wilson, Ian A.; Nemazee, David; Ward, Andrew B.; Zhu, JiangNature Communications (2016), 7 (), 12041CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Structures of BG505 SOSIP.664 trimer in complex with broadly neutralizing antibodies (bNAbs) have revealed the crit. role of trimeric context for immune recognition of HIV-1. Presentation of trimeric HIV-1 antigens on nanoparticles may thus provide promising vaccine candidates. Here we report the rational design, structural anal. and antigenic evaluation of HIV-1 trimer-presenting nanoparticles. We first demonstrate that both V1V2 and gp120 can be presented in native-like trimeric conformations on nanoparticles. We then design nanoparticles presenting various forms of stabilized gp140 trimer based on ferritin and a large, 60-meric E2p that displays 20 spikes mimicking virus-like particles (VLPs). Particle assembly is confirmed by electron microscopy (EM), while antigenic profiles are generated using representative bNAbs and non-NAbs. Lastly, we demonstrate high-yield gp140 nanoparticle prodn. and robust stimulation of B cells carrying cognate VRC01 receptors by gp120 and gp140 nanoparticles. Together, our study provides an arsenal of multivalent immunogens for HIV-1 vaccine development.
- 37Kamp, H. D.; Swanson, K. A.; Wei, R. R.; Dhal, P. K.; Dharanipragada, R.; Kern, A.; Sharma, B.; Sima, R.; Hajdusek, O.; Hu, L. T.; Wei, C.-J.; Nabel, G. J. Design of a Broadly Reactive Lyme Disease Vaccine. npj Vaccines 2020, 5 (1), 33, DOI: 10.1038/s41541-020-0183-8[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXos1Sgtbc%253D&md5=7cce8606c3451ba35cb08da52e45697dDesign of a broadly reactive Lyme disease vaccineKamp, Heather D.; Swanson, Kurt A.; Wei, Ronnie R.; Dhal, Pradeep K.; Dharanipragada, Ram; Kern, Aurelie; Sharma, Bijaya; Sima, Radek; Hajdusek, Ondrej; Hu, Linden T.; Wei, Chih-Jen; Nabel, Gary J.npj Vaccines (2020), 5 (1), 33CODEN: VACCBC; ISSN:2059-0105. (Nature Research)Abstr.: A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 mo. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.
- 38Swanson, K. A.; Rainho-Tomko, J. N.; Williams, Z. P.; Lanza, L.; Peredelchuk, M.; Kishko, M.; Pavot, V.; Alamares-Sapuay, J.; Adhikarla, H.; Gupta, S.; Chivukula, S.; Gallichan, S.; Zhang, L.; Jackson, N.; Yoon, H.; Edwards, D.; Wei, C.-J.; Nabel, G. J. A Respiratory Syncytial Virus (RSV) F Protein Nanoparticle Vaccine Focuses Antibody Responses to a Conserved Neutralization Domain. Sci. Immunol. 2020, 5 (47), eaba6466 DOI: 10.1126/sciimmunol.aba6466
- 39Cho, K. J.; Shin, H. J.; Lee, J.-H.; Kim, K.-J.; Park, S. S.; Lee, Y.; Lee, C.; Park, S. S.; Kim, K. H. The Crystal Structure of Ferritin from Helicobacter Pylori Reveals Unusual Conformational Changes for Iron Uptake. J. Mol. Biol. 2009, 390 (1), 83– 98, DOI: 10.1016/j.jmb.2009.04.078[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnt1Gksr8%253D&md5=b6c553c09b67386cc27350f1994aadeaThe Crystal Structure of Ferritin from Helicobacter pylori Reveals Unusual Conformational Changes for Iron UptakeCho, Ki Joon; Shin, Hye Jeong; Lee, Ji-Hye; Kim, Kyung-Jin; Park, Sarah S.; Lee, Youngmi; Lee, Cheolju; Park, Sung Soo; Kim, Kyung HyunJournal of Molecular Biology (2009), 390 (1), 83-98CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The crystal structure of recombinant ferritin from Helicobacter pylori has been detd. in its apo, low-iron-bound, intermediate, and high-iron-bound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-α-helix bundle. Significant conformational changes were obsd. at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surface-dominated process during biomineralization.
- 40Biswas, P.; Trozado, C.; Lee, J.; Schwartz, R. M. Development of a Mammalian Cell Culture Process for Rapid Clinical-Scale Production of Novel Influenza Nanoparticle Vaccines. BMC Proc. 2015, 9 (S9), O12, DOI: 10.1186/1753-6561-9-S9-O12
- 41Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zivanov, J.; Neufeldt, C. J.; Cerikan, B.; Lu, J. M.; Peukes, J.; Xiong, X.; Kräusslich, H.-G.; Scheres, S. H. W.; Bartenschlager, R.; Briggs, J. A. G. Structures and Distributions of SARS-CoV-2 Spike Proteins on Intact Virions. Nature 2020, 588, 498, DOI: 10.1038/s41586-020-2665-2[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Ggt7jM&md5=fa6e31604ece80a7c0934d689642a5c0Structures and distributions of SARS-CoV-2 spike proteins on intact virionsKe, Zunlong; Oton, Joaquin; Qu, Kun; Cortese, Mirko; Zila, Vojtech; McKeane, Lesley; Nakane, Takanori; Zivanov, Jasenko; Neufeldt, Christopher J.; Cerikan, Berati; Lu, John M.; Peukes, Julia; Xiong, Xiaoli; Krausslich, Hans-Georg; Scheres, Sjors H. W.; Bartenschlager, Ralf; Briggs, John A. G.Nature (London, United Kingdom) (2020), 588 (7838), 498-502CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of sol., overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. We applied cryo-electron microscopy and tomog. to image intact SARS-CoV-2 virions and det. the high-resoln. structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
- 42Turoňová, B.; Sikora, M.; Schürmann, C.; Hagen, W. J. H.; Welsch, S.; Blanc, F. E. C.; von Bülow, S.; Gecht, M.; Bagola, K.; Hörner, C.; van Zandbergen, G.; Landry, J.; de Azevedo, N. T. D.; Mosalaganti, S.; Schwarz, A.; Covino, R.; Mühlebach, M. D.; Hummer, G.; Krijnse Locker, J.; Beck, M. In Situ Structural Analysis of SARS-CoV-2 Spike Reveals Flexibility Mediated by Three Hinges. Science 2020, 370, 203 DOI: 10.1126/science.abd5223[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVCrsbbO&md5=6464149155235d90f7a9d0972a50f4fbIn situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hingesTuronova, Beata; Sikora, Mateusz; Schuermann, Christoph; Hagen, Wim J. H.; Welsch, Sonja; Blanc, Florian E. C.; von Buelow, Soeren; Gecht, Michael; Bagola, Katrin; Hoerner, Cindy; van Zandbergen, Ger; Landry, Jonathan; de Azevedo, Nayara Trevisan Doimo; Mosalaganti, Shyamal; Schwarz, Andre; Covino, Roberto; Muehlebach, Michael D.; Hummer, Gerhard; Krijnse Locker, Jacomine; Beck, MartinScience (Washington, DC, United States) (2020), 370 (6513), 203-208CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomog., subtomogram averaging, and mol. dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. The stalk domain of S contains 3 hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
- 43Klein, S.; Cortese, M.; Winter, S. L.; Wachsmuth-Melm, M.; Neufeldt, C. J.; Cerikan, B.; Stanifer, M. L.; Boulant, S.; Bartenschlager, R.; Chlanda, P. SARS-CoV-2 Structure and Replication Characterized by in Situ Cryo-Electron Tomography. Nat. Commun. 2020, 5885, DOI: 10.1038/s41467-020-19619-7[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVSgt7fL&md5=1f434e5bc92d9453d6b7f7b719e00a44SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomographyKlein, Steffen; Cortese, Mirko; Winter, Sophie L.; Wachsmuth-Melm, Moritz; Neufeldt, Christopher J.; Cerikan, Berati; Stanifer, Megan L.; Boulant, Steeve; Bartenschlager, Ralf; Chlanda, PetrNature Communications (2020), 11 (1), 5885CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report crit. insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomog. and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments assocd. with viral replication. The RNA filaments show a diam. consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple sep. vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.
- 44Zamecnik, C. R.; Rajan, J. V.; Yamauchi, K. A.; Mann, S. A.; Loudermilk, R. P.; Sowa, G. M.; Zorn, K. C.; Alvarenga, B. D.; Gaebler, C.; Caskey, M.; Stone, M.; Norris, P. J.; Gu, W.; Chiu, C. Y.; Ng, D.; Byrnes, J. R.; Zhou, X. X.; Wells, J. A.; Robbiani, D. F.; Nussenzweig, M. C.; DeRisi, J. L.; Wilson, M. R. ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Reports Med. 2020, in press. DOI: 10.1016/j.xcrm.2020.100123 .
- 45Li, Y.; Ma, M.; Lei, Q.; Wang, F.; Sun, Z.; Fan, X.; Tao, S. Linear Epitope Landscape of SARS-CoV-2 Spike Protein Constructed from 1,051 COVID-19 Patients. medRxiv 2020. DOI: 10.1101/2020.07.13.20152587 .Google ScholarThere is no corresponding record for this reference.
- 46Eckert, D. M.; Malashkevich, V. N.; Kim, P. S. Crystal Structure of GCN4-PIQI, a Trimeric Coiled Coil with Buried Polar Residues. J. Mol. Biol. 1998, 284 (4), 859– 865, DOI: 10.1006/jmbi.1998.2214[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXivFansg%253D%253D&md5=34feaf7ec906e7091998011bca277feeCrystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residuesEckert, Debra M.; Malashkevich, Vladimir N.; Kim, Peter S.Journal of Molecular Biology (1998), 284 (4), 859-865CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)Coiled coils consist of two or more α-helixes wrapped around each other with a superhelical twist. The interfaces between helixes of a coiled coil are formed by hydrophobic amino acid residues packed in a "knobs-into-holes" arrangement. Most naturally occurring coiled coils, however, also contain buried polar residues, as do the cores of the majority of naturally occurring globular proteins. Two common buried polar residues in both dimeric and trimeric coiled coils are asparagine and glutamine. In dimeric coiled coils, buried asparagine, but not glutamine, residues have been shown to confer specificity of oligomerization. We have placed a glutamine residue in the otherwise hydrophobic interior of a stable trimeric coiled coil, GCN4-pII, to study the effect of this buried polar residue in a trimeric coiled-coil environment. The resulting peptide, GCN4-pIQI, is a discrete, trimeric coiled coil with a lower stability than GCN4-pII. The crystal structure detd. to 1.8 Å shows that GCN4-pIQI is a trimeric coiled coil with a chloride ion coordinated by one buried glutamine residue from each monomer. (c) 1998 Academic Press.
- 47Crawford, K. H. D.; Eguia, R.; Dingens, A. S.; Loes, A. N.; Malone, K. D.; Wolf, C. R.; Chu, H. Y.; Tortorici, M. A.; Veesler, D.; Murphy, M.; Pettie, D.; King, N. P.; Balazs, A. B.; Bloom, J. D. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 2020, 12 (5), 513, DOI: 10.3390/v12050513[Crossref], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2gtLnF&md5=da4f1a5b2f0a2e4e52432c3d2a0798f1Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assaysCrawford, Katharine H. D.; Eguia, Rachel; Dingens, Adam S.; Loes, Andrea N.; Malone, Keara D.; Wolf, Caitlin R.; Chu, Helen Y.; Tortorici, M. Alejandra; Veesler, David; Murphy, Michael; Pettie, Deleah; King, Neil P.; Balazs, Alejandro B.; Bloom, Jesse D.Viruses (2020), 12 (5), 513CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key exptl. reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.
- 48Rogers, T. F.; Zhao, F.; Huang, D.; Beutler, N.; Burns, A.; He, W.; Limbo, O.; Smith, C.; Song, G.; Woehl, J.; Yang, L.; Abbott, R. K.; Callaghan, S.; Garcia, E.; Hurtado, J.; Parren, M.; Peng, L.; Ramirez, S.; Ricketts, J.; Ricciardi, M. J.; Rawlings, S. A.; Wu, N. C.; Yuan, M.; Smith, D. M.; Nemazee, D.; Teijaro, J. R.; Voss, J. E.; Wilson, I. A.; Andrabi, R.; Briney, B.; Landais, E.; Sok, D.; Jardine, J. G.; Burton, D. R. Isolation of Potent SARS-CoV-2 Neutralizing Antibodies and Protection from Disease in a Small Animal Model. Science 2020, 369, 956 DOI: 10.1126/science.abc7520[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1GrsLjF&md5=8684025692cbe1934cef2bfee369f3d1Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal modelRogers, Thomas F.; Zhao, Fangzhu; Huang, Deli; Beutler, Nathan; Burns, Alison; He, Wan-ting; Limbo, Oliver; Smith, Chloe; Song, Ge; Woehl, Jordan; Yang, Linlin; Abbott, Robert K.; Callaghan, Sean; Garcia, Elijah; Hurtado, Jonathan; Parren, Mara; Peng, Linghang; Ramirez, Sydney; Ricketts, James; Ricciardi, Michael J.; Rawlings, Stephen A.; Wu, Nicholas C.; Yuan, Meng; Smith, Davey M.; Nemazee, David; Teijaro, John R.; Voss, James E.; Wilson, Ian A.; Andrabi, Raiees; Briney, Bryan; Landais, Elise; Sok, Devin; Jardine, Joseph G.; Burton, Dennis R.Science (Washington, DC, United States) (2020), 369 (6506), 956-963CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained wt. and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.
- 49Amanat, F.; Stadlbauer, D.; Strohmeier, S.; Nguyen, T. H. O.; Chromikova, V.; McMahon, M.; Jiang, K.; Arunkumar, G. A.; Jurczyszak, D.; Polanco, J.; Bermudez-Gonzalez, M.; Kleiner, G.; Aydillo, T.; Miorin, L.; Fierer, D. S.; Lugo, L. A.; Kojic, E. M.; Stoever, J.; Liu, S. T. H.; Cunningham-Rundles, C.; Felgner, P. L.; Moran, T.; García-Sastre, A.; Caplivski, D.; Cheng, A. C.; Kedzierska, K.; Vapalahti, O.; Hepojoki, J. M.; Simon, V.; Krammer, F. A Serological Assay to Detect SARS-CoV-2 Seroconversion in Humans. Nat. Med. 2020, 26 (7), 1033– 1036, DOI: 10.1038/s41591-020-0913-5[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptFyqsbY%253D&md5=6f5df9742dec5a6bdb94ac5ccaad043dA serological assay to detect SARS-CoV-2 seroconversion in humansAmanat, Fatima; Stadlbauer, Daniel; Strohmeier, Shirin; Nguyen, Thi H. O.; Chromikova, Veronika; McMahon, Meagan; Jiang, Kaijun; Arunkumar, Guha Asthagiri; Jurczyszak, Denise; Polanco, Jose; Bermudez-Gonzalez, Maria; Kleiner, Giulio; Aydillo, Teresa; Miorin, Lisa; Fierer, Daniel S.; Lugo, Luz Amarilis; Kojic, Erna Milunka; Stoever, Jonathan; Liu, Sean T. H.; Cunningham-Rundles, Charlotte; Felgner, Philip L.; Moran, Thomas; Garcia-Sastre, Adolfo; Caplivski, Daniel; Cheng, Allen C.; Kedzierska, Katherine; Vapalahti, Olli; Hepojoki, Jussi M.; Simon, Viviana; Krammer, FlorianNature Medicine (New York, NY, United States) (2020), 26 (7), 1033-1036CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We describe a serol. ELISA for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma, and is amenable to scaling. Serol. assays are of crit. importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy, and investigate correlates of protection.
- 50Pallesen, J.; Wang, N.; Corbett, K. S.; Wrapp, D.; Kirchdoerfer, R. N.; Turner, H. L.; Cottrell, C. A.; Becker, M. M.; Wang, L.; Shi, W.; Kong, W.-P.; Andres, E. L.; Kettenbach, A. N.; Denison, M. R.; Chappell, J. D.; Graham, B. S.; Ward, A. B.; McLellan, J. S. Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (35), E7348– E7357, DOI: 10.1073/pnas.1707304114[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlWmsrfI&md5=3054f8054d852ed4192fd8f1fb6c870dImmunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigenPallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.; McLellan, Jason S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), E7348-E7357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also detd. high-resoln. structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.
- 51Kirchdoerfer, R. N.; Cottrell, C. A.; Wang, N.; Pallesen, J.; Yassine, H. M.; Turner, H. L.; Corbett, K. S.; Graham, B. S.; McLellan, J. S.; Ward, A. B. Pre-Fusion Structure of a Human Coronavirus Spike Protein. Nature 2016, 531 (7592), 118– 121, DOI: 10.1038/nature17200[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1emtb8%253D&md5=f7c2b90e8963b9519816be6c40d0b756Pre-fusion structure of a human coronavirus spike proteinKirchdoerfer, Robert N.; Cottrell, Christopher A.; Wang, Nianshuang; Pallesen, Jesper; Yassine, Hadi M.; Turner, Hannah L.; Corbett, Kizzmekia S.; Graham, Barney S.; McLellan, Jason S.; Ward, Andrew B.Nature (London, United Kingdom) (2016), 531 (7592), 118-121CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is detd. in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a mol. understanding of its function and limiting development of effective interventions. Here, the authors present the 4.0-Å resoln. structure of the trimeric HKU1 S protein detd. using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the 2 protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.
- 52Chen, W.-H.; Hotez, P. J.; Bottazzi, M. E. Potential for Developing a SARS-CoV Receptor-Binding Domain (RBD) Recombinant Protein as a Heterologous Human Vaccine against Coronavirus Infectious Disease (COVID)-19. Hum. Vaccines Immunother. 2020, 16 (6), 1239– 1242, DOI: 10.1080/21645515.2020.1740560[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosVegt74%253D&md5=27bfefa651ca8d65bad514f0caf4b4e6Potential for developing a SARS-CoV receptor-binding domain (RBD) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (COVID)-19Chen, Wen-Hsiang; Hotez, Peter J.; Bottazzi, Maria ElenaHuman Vaccines & Immunotherapeutics (2020), 16 (6), 1239-1242CODEN: HVIUAK; ISSN:2164-554X. (Taylor & Francis Ltd.)A SARS-CoV receptor-binding domain (RBD) recombinant protein was developed and manufd. under current good manufg. practices in 2016. The protein, known as RBD219-N1 when formulated on Alhydrogel, induced high-level neutralizing antibodies and protective immunity with minimal immunopathol. in mice after a homologous virus challenge with SARS-CoV (MA15 strain). We examd. published evidence in support of whether the SARS-CoV RBD219-N1 could be repurposed as a heterologous vaccine against Coronavirus Infectious Disease (COVID)-19. Our findings include evidence that convalescent serum from SARS-CoV patients can neutralize SARS-CoV-2. Addnl., a review of published studies using monoclonal antibodies (mAbs) raised against SARS-CoV RBD and that neutralizes the SARS-CoV virus in vitro finds that some of these mAbs bind to the receptor-binding motif (RBM) within the RBD, while others bind to domains outside this region within RBD. This information is relevant and supports the possibility of developing a heterologous SARS-CoV RBD vaccine against COVID-19, esp. due to the finding that the overall high amino acid similarity (82%) between SARS-CoV and SARS-CoV-2 spike and RBD domains is not reflected in RBM amino acid similarity (59%). However, the high sequence similarity (94%) in the region outside of RBM offers the potential of conserved neutralizing epitopes between both viruses.
- 53Mulligan, M. J.; Lyke, K. E.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Raabe, V.; Bailey, R.; Swanson, K. A.; Li, P.; Koury, K.; Kalina, W.; Cooper, D.; Fontes-Garfias, C.; Shi, P.-Y.; Türeci, Ö.; Tompkins, K. R.; Walsh, E. E.; Frenck, R.; Falsey, A. R.; Dormitzer, P. R.; Gruber, W. C.; Şahin, U.; Jansen, K. U. Phase I/II Study of COVID-19 RNA Vaccine BNT162b1 in Adults. Nature 2020, 586 (7830), 589– 593, DOI: 10.1038/s41586-020-2639-4[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVWjsr%252FM&md5=b0b354ee45f7171577edeec833a13c55Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adultsMulligan, Mark J.; Lyke, Kirsten E.; Kitchin, Nicholas; Absalon, Judith; Gurtman, Alejandra; Lockhart, Stephen; Neuzil, Kathleen; Raabe, Vanessa; Bailey, Ruth; Swanson, Kena A.; Li, Ping; Koury, Kenneth; Kalina, Warren; Cooper, David; Fontes-Garfias, Camila; Shi, Pei-Yong; Tureci, Ozlem; Tompkins, Kristin R.; Walsh, Edward E.; Frenck, Robert; Falsey, Ann R.; Dormitzer, Philip R.; Gruber, William C.; Sahin, Ugur; Jansen, Kathrin U.Nature (London, United Kingdom) (2020), 586 (7830), 589-593CODEN: NATUAS; ISSN:0028-0836. (Nature Research)In March 2020, the World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a pandemic. With rapidly accumulating nos. of cases and deaths reported globally, a vaccine is urgently needed. We report the available safety, tolerability, and immunogenicity data from an ongoing placebo-controlled, observer-blinded dose-escalation study (ClinicalTrials.gov identifier NCT04368728) among 45 healthy adults (18-55 yr), who were randomized to receive 2 doses-sepd. by 21 days-of 10μg, 30μg, or 100μg of BNT162b1. BNT162b1 is a lipid-nanoparticle-formulated, nucleoside-modified mRNA vaccine that encodes the trimerized receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. Local reactions and systemic events were dose-dependent, generally mild to moderate, and transient. A 2nd vaccination with 100μg was not administered because of the increased reactogenicity and a lack of meaningfully increased immunogenicity after a single dose compared with the 30-μg dose. RBD-binding IgG concns. and SARS-CoV-2 neutralizing titers in sera increased with dose level and after a 2nd dose. Geometric mean neutralizing titers reached 1.9-4.6-fold that of a panel of COVID-19 convalescent human sera, which were obtained ≥14 days after a pos. SARS-CoV-2 PCR. These results support further evaluation of this mRNA vaccine candidate.
- 54Watanabe, Y.; Allen, J. D.; Wrapp, D.; McLellan, J. S.; Crispin, M. Site-Specific Glycan Analysis of the SARS-CoV-2 Spike. Science 2020, eabb9983 DOI: 10.1126/science.abb9983
- 55Yuan, M.; Wu, N. C.; Zhu, X.; Lee, C. C. D.; So, R. T. Y.; Lv, H.; Mok, C. K. P.; Wilson, I. A. A Highly Conserved Cryptic Epitope in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV. Science 2020, 368 (6491), 630– 633, DOI: 10.1126/science.abb7269[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovFCrt7Y%253D&md5=54554781f340bf0361a96d62cb98b3d3A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoVYuan, Meng; Wu, Nicholas C.; Zhu, Xueyong; Lee, Chang-Chun D.; So, Ray T. Y.; Lv, Huibin; Mok, Chris K. P.; Wilson, Ian A.Science (Washington, DC, United States) (2020), 368 (6491), 630-633CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore detd. the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein at 3.1-angstrom resoln. CR3022 targets a highly conserved epitope, distal from the receptor binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBDs on the trimeric S protein are in the "up" conformation and slightly rotated. These results provide mol. insights into antibody recognition of SARS-CoV-2.
- 56Tian, X.; Li, C.; Huang, A.; Xia, S.; Lu, S.; Shi, Z.; Lu, L.; Jiang, S.; Yang, Z.; Wu, Y.; Ying, T. Potent Binding of 2019 Novel Coronavirus Spike Protein by a SARS Coronavirus-Specific Human Monoclonal Antibody. Emerging Microbes Infect. 2020, 9 (1), 382– 385, DOI: 10.1080/22221751.2020.1729069[Crossref], [PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVGks78%253D&md5=6d73882867e561b2056895c525136512Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibodyTian, Xiaolong; Li, Cheng; Huang, Ailing; Xia, Shuai; Lu, Sicong; Shi, Zhengli; Lu, Lu; Jiang, Shibo; Yang, Zhenlin; Wu, Yanling; Ying, TianleiEmerging Microbes & Infections (2020), 9 (1), 382-385CODEN: EMIMC4; ISSN:2222-1751. (Taylor & Francis Ltd.)The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 lab.-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a crit. impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.
- 57ter Meulen, J.; van den Brink, E. N.; Poon, L. L. M.; Marissen, W. E.; Leung, C. S. W.; Cox, F.; Cheung, C. Y.; Bakker, A. Q.; Bogaards, J. A.; van Deventer, E.; Preiser, W.; Doerr, H. W.; Chow, V. T.; de Kruif, J.; Peiris, J. S. M.; Goudsmit, J. Human Monoclonal Antibody Combination against SARS Coronavirus: Synergy and Coverage of Escape Mutants. PLoS Med. 2006, 3 (7), e237 DOI: 10.1371/journal.pmed.0030237
- 58Shi, R.; Shan, C.; Duan, X.; Chen, Z.; Liu, P.; Song, J.; Song, T.; Bi, X.; Han, C.; Wu, L.; Gao, G.; Hu, X.; Zhang, Y.; Tong, Z.; Huang, W.; Liu, W. J.; Wu, G.; Zhang, B.; Wang, L.; Qi, J.; Feng, H.; Wang, F.-S.; Wang, Q.; Gao, G. F.; Yuan, Z.; Yan, J. A Human Neutralizing Antibody Targets the Receptor-Binding Site of SARS-CoV-2. Nature 2020, 584 (7819), 120– 124, DOI: 10.1038/s41586-020-2381-y[Crossref], [PubMed], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVygtLjM&md5=55a93b450dd445483547b5f69cab393eA human neutralizing antibody targets the receptor-binding site of SARS-CoV-2Shi, Rui; Shan, Chao; Duan, Xiaomin; Chen, Zhihai; Liu, Peipei; Song, Jinwen; Song, Tao; Bi, Xiaoshan; Han, Chao; Wu, Lianao; Gao, Ge; Hu, Xue; Zhang, Yanan; Tong, Zhou; Huang, Weijin; Liu, William Jun; Wu, Guizhen; Zhang, Bo; Wang, Lan; Qi, Jianxun; Feng, Hui; Wang, Fu-Sheng; Wang, Qihui; Gao, George Fu; Yuan, Zhiming; Yan, JinghuaNature (London, United Kingdom) (2020), 584 (7819), 120-124CODEN: NATUAS; ISSN:0028-0836. (Nature Research)An outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addn., CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme 2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.
- 59Heinicke, E.; Kumar, U.; Munoz, D. G. Quantitative Dot-Blot Assay for Proteins Using Enhanced Chemiluminescence. J. Immunol. Methods 1992, 152 (2), 227– 236, DOI: 10.1016/0022-1759(92)90144-I[Crossref], [PubMed], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XmsVyjsLo%253D&md5=97273c9b337cfb1fe90b831048d48ba8Quantitative dot-blot assay for proteins using enhanced chemiluminescenceHeinicke, E.; Kumar, U.; Munoz, D. G.Journal of Immunological Methods (1992), 152 (2), 227-36CODEN: JIMMBG; ISSN:0022-1759.A sensitive nonradioactive method for detection of specific proteins on Western blots is com. available. The protein is immobilized on nitrocellulose membrane and immunolabeled with HRP-conjugated secondary antibody. HRP catalyzes the oxidn. of luminol, a cyclic diacylhydrazide, resulting in the emission of light which is recorded on film. By using dot blot, it was shown that the signal generated by this system is proportional to the amt. of protein loaded onto the membrane. Std. curves were linear (r2 > 0.95) over a 10-50-fold range. Linearity was also achieved with tissue exts. probed for a specific antigen. The sensitivity of the method is such that <10 fmol protein can be measured. The sensitivity and range are comparable to a previously reported dot-blotting assay based on the use of 125I-protein A, but the method does not require the handling of radioactive compds. This method was used to est. the level of chromogranin A in a mixt. of proteins extd. from human brain.
- 60Steppert, P.; Burgstaller, D.; Klausberger, M.; Tover, A.; Berger, E.; Jungbauer, A. Quantification and Characterization of Virus-like Particles by Size-Exclusion Chromatography and Nanoparticle Tracking Analysis. J. Chromatogr. A 2017, 1487, 89– 99, DOI: 10.1016/j.chroma.2016.12.085[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVKju7w%253D&md5=24338c5203ff0301f25843c8ef655af6Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysisSteppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Tover, Andres; Berger, Eva; Jungbauer, AloisJournal of Chromatography A (2017), 1487 (), 89-99CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking anal. (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multiangle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle no. is obtained with a lower limit of detection (LLOD) of 1.7 × 107 part/mL and lower limit of quantification (LLOQ) of 3.4 × 108 part/mL. SE-HPLC with UV detection was calibrated with stds. characterized by NTA, and a LLOD of 6.9 × 109 part/mL and LLOQ of 2.1 × 1010 part/mL were found. SE-HPLC with MALS does not require a precalibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approxn., the particle concn. can be directly deduced from the scattered light. A LLOD of 4.8 × 108 part/mL and LLOQ of 2.1 × 109 part/mL were measured and substantially lower compared to the UV method. The abs. particle concn. measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.
- 61Šagi, D.; Kienz, P.; Denecke, J.; Marquardt, T.; Peter-Katalinić, J. Glycoproteomics OfN-Glycosylation by in-Gel Deglycosylation and Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry Mapping: Application to Congenital Disorders of Glycosylation. Proteomics 2005, 5 (10), 2689– 2701, DOI: 10.1002/pmic.200401312[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmtF2gtb8%253D&md5=2c6d7fd83c626b4e7ffda029f360c825Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylationSagi, Dijana; Kienz, Petra; Denecke, Jonas; Marquardt, Thorsten; Peter-Katalinic, JasnaProteomics (2005), 5 (10), 2689-2701CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-sepd. proteins, their isolation, purifn. and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) anal. of their mixts. were optimized. Since many glycoproteins are available only at low quantities from SDS-PAGE or two-dimensional gels, high attention was paid to obtain N-glycan mixts. representing their actual compn. in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrixes for MS anal. of acidic N-glycans was compared. The most favorable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammonium citrate as a matrix. This matrix provided good results for both neutral and acidic mixts. as well as for methylated N-glycans. In the second part of this paper the potential of such an optimized MS strategy alone or in combination with high pH anion-exchange chromatog. profiling for the clin. diagnosis of congenital disorders of glycosylation is presented.
- 62Herrera, N. G.; Morano, N. C.; Celikgil, A.; Georgiev, G. I.; Malonis, R. J.; Lee, J. H.; Tong, K.; Vergnolle, O.; Massimi, A. B.; Yen, L. Y.; Noble, A. J.; Kopylov, M.; Bonanno, J. B.; Garrett-Thomson, S. C.; Hayes, D. B.; Bortz, R. H.; Wirchnianski, A. S.; Florez, C.; Laudermilch, E.; Haslwanter, D.; Fels, J. M.; Dieterle, M. E.; Jangra, R. K.; Barnhill, J.; Mengotto, A.; Kimmel, D.; Daily, J. P.; Pirofski, L.; Chandran, K.; Brenowitz, M.; Garforth, S. J.; Eng, E. T.; Lai, J. R.; Almo, S. C. Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis. ACS Omega 2020, in press. DOI: 10.1021/acsomega.0c03512 .
- 63Bornholdt, Z. A.; Turner, H. L.; Murin, C. D.; Li, W.; Sok, D.; Souders, C. A.; Piper, A. E.; Goff, A.; Shamblin, J. D.; Wollen, S. E.; Sprague, T. R.; Fusco, M. L.; Pommert, K. B. J.; Cavacini, L. A.; Smith, H. L.; Klempner, M.; Reimann, K. A.; Krauland, E.; Gerngross, T. U.; Wittrup, K. D.; Saphire, E. O.; Burton, D. R.; Glass, P. J.; Ward, A. B.; Walker, L. M. Isolation of Potent Neutralizing Antibodies from a Survivor of the 2014 Ebola Virus Outbreak. Science 2016, 351 (6277), 1078– 1083, DOI: 10.1126/science.aad5788[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjsVagtbc%253D&md5=55ef46697b99197ac8ac53efecbf1e09Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreakBornholdt, Zachary A.; Turner, Hannah L.; Murin, Charles D.; Li, Wen; Sok, Devin; Souders, Colby A.; Piper, Ashley E.; Goff, Arthur; Shamblin, Joshua D.; Wollen, Suzanne E.; Sprague, Thomas R.; Fusco, Marnie L.; Pommert, Kathleen B. J.; Cavacini, Lisa A.; Smith, Heidi L.; Klempner, Mark; Reimann, Keith A.; Krauland, Eric; Gerngross, Tillman U.; Wittrup, Karl D.; Saphire, Erica Ollmann; Burton, Dennis R.; Glass, Pamela J.; Ward, Andrew B.; Walker, Laura M.Science (Washington, DC, United States) (2016), 351 (6277), 1078-1083CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.
- 64Vandepapelière, P.; Horsmans, Y.; Moris, P.; Van Mechelen, M.; Janssens, M.; Koutsoukos, M.; Van Belle, P.; Clement, F.; Hanon, E.; Wettendorff, M.; Garçon, N.; Leroux-Roels, G. Vaccine Adjuvant Systems Containing Monophosphoryl Lipid A and QS21 Induce Strong and Persistent Humoral and T Cell Responses against Hepatitis B Surface Antigen in Healthy Adult Volunteers. Vaccine 2008, 26 (10), 1375– 1386, DOI: 10.1016/j.vaccine.2007.12.038[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXitlemt7s%253D&md5=8812f21501e9d8b1afabac9201e2eb10Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS21 induce strong and persistent humoral and T cell responses against hepatitis B surface antigen in healthy adult volunteersVandepapeliere, Pierre; Horsmans, Yves; Moris, Philippe; Van Mechelen, Marcelle; Janssens, Michel; Koutsoukos, Marguerite; Van Belle, Pascale; Clement, Frederic; Hanon, Emmanuel; Wettendorff, Martine; Garcon, Nathalie; Leroux-Roels, GeertVaccine (2008), 26 (10), 1375-1386CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)A randomized, double-blind study assessing the potential of four adjuvants in combination with recombinant hepatitis B surface antigen has been conducted to evaluate humoral and cell-mediated immune responses in healthy adults after three vaccine doses at months 0, 1 and 10. Three Adjuvant Systems (AS) contained 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21, formulated either with an oil-in-water emulsion (AS02B and AS02V) or with liposomes (AS01B). The fourth adjuvant was CpG oligonucleotide. High levels of antibodies were induced by all adjuvants, whereas cell-mediated immune responses, including cytolytic T cells and strong and persistent CD4+ T cell response were mainly obsd. with the three MPL/QS21-contg. Adjuvant Systems. The CD4+ T cell response was characterized in vitro by vigorous lymphoproliferation, high IFN-γ and moderate IL-5 prodn. Antigen-specific T cell immune response was further confirmed ex vivo by detection of IL-2- and IFN-γ-producing CD4+ T cells, and in vivo by measuring increased levels of IFN-γ in the serum and delayed-type hypersensitivity (DTH) responses. The CpG adjuvanted vaccine induced consistently lower immune responses for all parameters. All vaccine adjuvants were shown to be safe with acceptable reactogenicity profiles. The majority of subjects reported local reactions at the injection site after vaccination while general reactions were recorded less frequently. No vaccine-related serious adverse event was reported. Importantly, no increase in markers of auto-immunity and allergy was detected over the whole study course. In conclusion, the Adjuvant Systems contg. MPL/QS21, in combination with hepatitis B surface antigen, induced very strong humoral and cellular immune responses in healthy adults. The AS01B-adjuvanted vaccine induced the strongest and most durable specific cellular immune responses after two doses. These Adjuvant Systems, when added to recombinant protein antigens, can be fundamental to develop effective prophylactic vaccines against complex pathogens, e.g. malaria, HIV infection and tuberculosis, and for special target populations such as subjects with an impaired immune response, due to age or medical conditions.
- 65Leroux-Roels, G.; Van Belle, P.; Vandepapeliere, P.; Horsmans, Y.; Janssens, M.; Carletti, I.; Garçon, N.; Wettendorff, M.; Van Mechelen, M. Vaccine Adjuvant Systems Containing Monophosphoryl Lipid A and QS-21 Induce Strong Humoral and Cellular Immune Responses against Hepatitis B Surface Antigen Which Persist for at Least 4 Years after Vaccination. Vaccine 2015, 33 (8), 1084– 1091, DOI: 10.1016/j.vaccine.2014.10.078[Crossref], [PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSmsrnM&md5=d18386239bced8881c1e6d17f39a1005Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS-21 induce strong humoral and cellular immune responses against hepatitis B surface antigen which persist for at least 4 years after vaccinationLeroux-Roels, Geert; Van Belle, Pascale; Vandepapeliere, Pierre; Horsmans, Yves; Janssens, Michel; Carletti, Isabelle; Garcon, Nathalie; Wettendorff, Martine; Van Mechelen, MarcelleVaccine (2015), 33 (8), 1084-1091CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Recombinant hepatitis B surface antigen (HBsAg) was used as a model antigen to evaluate persistence of cellular and humoral immune responses when formulated with three different Adjuvant Systems contg. 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS-21, in an oil-in-water emulsion (AS02B and AS02V), or with liposomes (AS01B). This is an open, 4-yr follow-up of a previous randomised, double-blind study. Healthy subjects aged 18-40 years received three vaccine doses on a month 0, 1, 10 schedule and were initially followed for 18 mo. A total of 93 subjects (AS02B: n = 30; AS02V: n = 28; AS01B: n = 35) were enrolled in this follow-up and had an addnl. blood sample taken at Year 4 (NCT02153320). The primary endpoint was the frequency of HBsAg-specific CD4+ and CD8+ T-cells expressing cytokines upon short-term in vitro stimulation of peripheral blood mononuclear cells with HBsAg-derived peptides. Secondary endpoints were anti-HBs antibody titers and frequency of HBsAg-specific memory B-cells. A strong and persistent specific CD4+ T-cell response was obsd. at Year 4 in all groups. HBsAg-specific CD4+ T-cells expressed mainly CD40L and IL-2, and to a lesser extent TNF-α and IFN-γ. HBsAg-specific CD8+ T-cells were not detected in any group. A high, persistent HBsAg-specific humoral immune response was obsd. in all groups, with all subjects seroprotected (antibody titer ≥10 mIU/mL) at Year 4. The geometric mean antibody titer at Year 4 was above 100,000 mIU/mL in all groups. A strong memory B-cell response was obsd. post-dose 2, which tended to increase post-dose 3 and persisted at Year 4 in all groups. The MPL/QS-21/HBsAg vaccine formulations induced persistent immune responses up to 4 years after first vaccination. These Adjuvant Systems offer potential for combination with recombinant, synthetic or highly purified subunit vaccines, particularly for vaccination against challenging diseases, or in specific populations, although addnl. studies are needed.
- 66Marty-Roix, R.; Vladimer, G. I.; Pouliot, K.; Weng, D.; Buglione-Corbett, R.; West, K.; MacMicking, J. D.; Chee, J. D.; Wang, S.; Lu, S.; Lien, E. Identification of QS-21 as an Inflammasome-Activating Molecular Component of Saponin Adjuvants. J. Biol. Chem. 2016, 291 (3), 1123– 1136, DOI: 10.1074/jbc.M115.683011[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVCrtw%253D%253D&md5=0ed38ac6d2f804607d89167206328ed7Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin AdjuvantsMarty-Roix, Robyn; Vladimer, Gregory I.; Pouliot, Kimberly; Weng, Dan; Buglione-Corbett, Rachel; West, Kim; MacMicking, John D.; Chee, Jonathan D.; Wang, Shixia; Lu, Shan; Lien, EgilJournal of Biological Chemistry (2016), 291 (3), 1123-1136CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific mols. contribute to the stimulatory activity. QS-21 is a defined, highly purified, and sol. saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. The authors obsd. QS-21 to elicit caspase-1-dependent IL-1β and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, the authors' data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1β/IL-18 release. At higher concns., QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixt. of saponins also induced IL-1β in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, the authors have identified QS-21 as a nonparticulate single mol. saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigen-specific responses in vivo.
- 67Case, J. B.; Rothlauf, P. W.; Chen, R. E.; Liu, Z.; Zhao, H.; Kim, A. S.; Bloyet, L.; Zeng, Q.; Tahan, S.; Droit, L.; Ilagan, M. X. G.; Tartell, M. A.; Amarasinghe, G.; Henderson, J. P.; Miersch, S.; Ustav, M.; Sidhu, S.; Virgin, H. W.; Wang, D.; Ding, S.; Corti, D.; Theel, E. S.; Fremont, D. H.; Diamond, M. S.; Whelan, S. P. J. Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2. Cell Host Microbe 2020, 28, 475, DOI: 10.1016/j.chom.2020.06.021[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVeqsrjF&md5=ef59069d2200e4800c0830d7c0055050Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2Case, James Brett; Rothlauf, Paul W.; Chen, Rita E.; Liu, Zhuoming; Zhao, Haiyan; Kim, Arthur S.; Bloyet, Louis-Marie; Zeng, Qiru; Tahan, Stephen; Droit, Lindsay; Ilagan, Ma. Xenia G.; Tartell, Michael A.; Amarasinghe, Gaya; Henderson, Jeffrey P.; Miersch, Shane; Ustav, Mart; Sidhu, Sachdev; Virgin, Herbert W.; Wang, David; Ding, Siyuan; Corti, Davide; Theel, Elitza S.; Fremont, Daved H.; Diamond, Michael S.; Whelan, Sean P. J.Cell Host & Microbe (2020), 28 (3), 475-485.e5CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious mol. clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-redn. neutralization test with a clin. isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc sol. decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Addnl., replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
- 68Mandolesi, M.; Sheward, D. J.; Hanke, L.; Ma, J.; Pushparaj, P.; Vidakovics, L. P.; Kim, C.; Loré, K.; Dopico, X. C.; Coquet, J. M.; McInerney, G.; Karlsson Hedestam, G. B.; Murrell, B. SARS-CoV-2 Protein Subunit Vaccination Elicits Potent Neutralizing Antibody Responses. bioRxiv, 2020. DOI: 10.1101/2020.07.31.228486 .Google ScholarThere is no corresponding record for this reference.
- 69Chi, X.; Yan, R.; Zhang, J.; Zhang, G.; Zhang, Y.; Hao, M.; Zhang, Z.; Fan, P.; Dong, Y.; Yang, Y.; Chen, Z.; Guo, Y.; Zhang, J.; Li, Y.; Song, X.; Chen, Y.; Xia, L.; Fu, L.; Hou, L.; Xu, J.; Yu, C.; Li, J.; Zhou, Q.; Chen, W. A Neutralizing Human Antibody Binds to the N-Terminal Domain of the Spike Protein of SARS-CoV-2. Science 2020, 369 (6504), 650– 655, DOI: 10.1126/science.abc6952[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGms77F&md5=398d9f74ea0873b1e083d6ec457167c1A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2Chi, Xiangyang; Yan, Renhong; Zhang, Jun; Zhang, Guanying; Zhang, Yuanyuan; Hao, Meng; Zhang, Zhe; Fan, Pengfei; Dong, Yunzhu; Yang, Yilong; Chen, Zhengshan; Guo, Yingying; Zhang, Jinlong; Li, Yaning; Song, Xiaohong; Chen, Yi; Xia, Lu; Fu, Ling; Hou, Lihua; Xu, Junjie; Yu, Changming; Li, Jianmin; Zhou, Qiang; Chen, WeiScience (Washington, DC, United States) (2020), 369 (6504), 650-655CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by detg. with cryo-electron microscopy its structure in complex with the S protein to an overall resoln. of 3.1 angstroms and local resoln. of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.
- 70Lefeber, D. J.; Benaissa-Trouw, B.; Vliegenthart, J. F. G.; Kamerling, J. P.; Jansen, W. T. M.; Kraaijeveld, K.; Snippe, H. Th1-Directing Adjuvants Increase the Immunogenicity of Oligosaccharide-Protein Conjugate Vaccines Related to Streptococcus Pneumoniae Type 3. Infect. Immun. 2003, 71, 6915, DOI: 10.1128/IAI.71.12.6915-6920.2003[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpsFGntrk%253D&md5=2b4ada5a4d983fed83364bd0afe4f1b2Th1-directing adjuvants increase the immunogenicity of oligosaccharide-protein conjugate vaccines related to Streptococcus pneumoniae type 3Lefeber, Dirk J.; Benaissa-trouw, Barry; Vliegenthart, Johannes F. G.; Kamerling, Johannis P.; Jansen, Wouter T. M.; Kraaijeveld, Kees; Snippe, HarmInfection and Immunity (2003), 71 (12), 6915-6920CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)Oligosaccharide (OS)-protein conjugates are promising candidate vaccines against encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope of Streptococcus pneumoniae type 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an ELISA and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and di-Me dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related IgG subclasses. Phagocytic capacity correlated strongly with Th1-assocd. IgG2a and IgG2b levels, to a lesser extent with Th2-assocd. IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.
- 71Visciano, M. L.; Tagliamonte, M.; Tornesello, M. L.; Buonaguro, F. M.; Buonaguro, L. Effects of Adjuvants on IgG Subclasses Elicited by Virus-like Particles. J. Transl. Med. 2012, 10 (1), 4, DOI: 10.1186/1479-5876-10-4[Crossref], [PubMed], [CAS], Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XksFGntLk%253D&md5=9efd7b5ffca2426a0389e1275ee41f03Effects of adjuvants on IgG subclasses elicited by Virus-Like particlesVisciano, Maria Luisa; Tagliamonte, Maria; Tornesello, Maria Lina; Buonaguro, Franco M.; Buonaguro, LuigiJournal of Translational Medicine (2012), 10 (), 4CODEN: JTMOBV; ISSN:1479-5876. (BioMed Central Ltd.)Background: Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN 1826 or poly(I:C) adjuvants, has been evaluated in an animal model. Results: Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TH2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3), with poly(I:C) more potent than CpG ODN1826. Conclusions: The results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen.
- 72Bos, R.; Rutten, L.; van der Lubbe, J. E. M.; Bakkers, M. J. G.; Hardenberg, G.; Wegmann, F.; Zuijdgeest, D.; de Wilde, A. H.; Koornneef, A.; Verwilligen, A.; van Manen, D.; Kwaks, T.; Vogels, R.; Dalebout, T. J.; Myeni, S. K.; Kikkert, M.; Snijder, E. J.; Li, Z.; Barouch, D. H.; Vellinga, J.; Langedijk, J. P. M.; Zahn, R. C.; Custers, J.; Schuitemaker, H. Ad26 Vector-Based COVID-19 Vaccine Encoding a Prefusion-Stabilized SARS-CoV-2 Spike Immunogen Induces Potent Humoral and Cellular Immune Responses. npj Vaccines 2020, in press. DOI: 10.1038/s41541-020-00243-x .
- 73Lederer, K.; Castaño, D.; Atria, D. G.; Oguin, T. H.; Wang, S.; Manzoni, T. B.; Muramatsu, H.; Hogan, M. J.; Amanat, F.; Cherubin, P.; Lundgreen, K. A.; Tam, Y. K.; Fan, S. H. Y.; Eisenlohr, L. C.; Maillard, I.; Weissman, D.; Bates, P.; Krammer, F.; Sempowski, G. D.; Pardi, N.; Locci, M. SARS-CoV-2 MRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation. Immunity 2020, 53, 1281, DOI: 10.1016/j.immuni.2020.11.009[Crossref], [PubMed], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFeltL3J&md5=58bc06e6e9f1b7b33c0f05178b2b008bSARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody GenerationLederer, Katlyn; Castano, Diana; Gomez Atria, Daniela; Oguin, Thomas H. III; Wang, Sidney; Manzoni, Tomaz B.; Muramatsu, Hiromi; Hogan, Michael J.; Amanat, Fatima; Cherubin, Patrick; Lundgreen, Kendall A.; Tam, Ying K.; Fan, Steven H. Y.; Eisenlohr, Laurence C.; Maillard, Ivan; Weissman, Drew; Bates, Paul; Krammer, Florian; Sempowski, Gregory D.; Pardi, Norbert; Locci, MichelaImmunity (2020), 53 (6), 1281-1295.e5CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crit. to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quant. and qual. shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody prodn. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
- 74Dong, Y.; Dai, T.; Wei, Y.; Zhang, L.; Zheng, M.; Zhou, F. A Systematic Review of SARS-CoV-2 Vaccine Candidates. Signal Transduct. Target. Ther. 2020, 5 (1), 237, DOI: 10.1038/s41392-020-00352-y[Crossref], [PubMed], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7gvFKqsQ%253D%253D&md5=cd1dc82d2ffe985e958f86d51378ab84A systematic review of SARS-CoV-2 vaccine candidatesDong Yetian; Zheng Min; Dong Yetian; Zhang Long; Dai Tong; Zhou Fangfang; Wei YujunSignal transduction and targeted therapy (2020), 5 (1), 237 ISSN:.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that is highly pathogenic and has caused the recent worldwide pandemic officially named coronavirus disease (COVID-19). Currently, considerable efforts have been put into developing effective and safe drugs and vaccines against SARS-CoV-2. Vaccines, such as inactivated vaccines, nucleic acid-based vaccines, and vector vaccines, have already entered clinical trials. In this review, we provide an overview of the experimental and clinical data obtained from recent SARS-CoV-2 vaccines trials, and highlight certain potential safety issues that require consideration when developing vaccines. Furthermore, we summarize several strategies utilized in the development of vaccines against other infectious viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), with the aim of aiding in the design of effective therapeutic approaches against SARS-CoV-2.
- 75Iwata-Yoshikawa, N.; Uda, A.; Suzuki, T.; Tsunetsugu-Yokota, Y.; Sato, Y.; Morikawa, S.; Tashiro, M.; Sata, T.; Hasegawa, H.; Nagata, N. Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine. J. Virol. 2014, 88 (15), 8597– 8614, DOI: 10.1128/JVI.00983-14[Crossref], [PubMed], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFehu7jE&md5=d7b4e910c70ed7f89dc3becd656c30cfEffects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccineIwata-Yoshikawa, Naoko; Uda, Akihiko; Suzuki, Tadaki; Tsunetsugu-Yokota, Yasuko; Sato, Yuko; Morikawa, Shigeru; Tashiro, Masato; Sata, Tetsutaro; Hasegawa, Hideki; Nagata, NoriyoJournal of Virology (2014), 88 (15), 8597-8614, 19 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-mo-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from LDs of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was obsd. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U) and poly(I C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Addnl., microarray anal. showed that genes assocd. with chemotaxis, eosinophil migration, eosinophilia and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes assocd. with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathol. in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants. Importance: Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathol. in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray anal. revealed that genes assocd. with chemotaxis, eosinophil migration, eosinophilia and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.
- 76Honda-Okubo, Y.; Barnard, D.; Ong, C. H.; Peng, B.-H.; Tseng, C.-T. K.; Petrovsky, N. Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection While Ameliorating Lung Eosinophilic Immunopathology. J. Virol. 2015, 89 (6), 2995– 3007, DOI: 10.1128/JVI.02980-14[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktF2gu78%253D&md5=1e32c80e6d6a79ba1143d44a41e277cdSevere acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathologyHonda-Okubo, Yoshikazu; Barnard, Dale; Ong, Chun Hao; Peng, Bi-Hung; Tseng, Chien-Te Kent; Petrovsky, NikolaiJournal of Virology (2015), 89 (6), 2995-3007CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Although the severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathol. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addn. of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were assocd. with significantly increased lung eosinophilic immunopathol. on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathol. by vaccines contg. delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathol. predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the crit. importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.
- 77Tseng, C.-T.; Sbrana, E.; Iwata-Yoshikawa, N.; Newman, P. C.; Garron, T.; Atmar, R. L.; Peters, C. J.; Couch, R. B. Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus. PLoS One 2012, 7 (4), e35421 DOI: 10.1371/journal.pone.0035421[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslOksbs%253D&md5=36adfc4192c520b40f66f4a89d16448fImmunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virusTseng, Chien-Te; Sbrana, Elena; Iwata-Yoshikawa, Naoko; Newman, Patrick C.; Garron, Tania; Atmar, Robert L.; Peters, Clarence J.; Couch, Robert B.PLoS One (2012), 7 (4), e35421CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Background: Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathol.-type lung disease. Design: Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathol. Results: All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant redns. of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathol. changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathol. seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathol. with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathol. changes seen in all control groups lacked the eosinophil prominence. Conclusions: These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathol. suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.
- 78Vartak, A.; Sucheck, S. Recent Advances in Subunit Vaccine Carriers. Vaccines 2016, 4 (2), 12, DOI: 10.3390/vaccines4020012[Crossref], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnvF2mtbg%253D&md5=16f5079047f303f38533eca9e0e2e653Recent advances in subunit vaccine carriersVartak, Abhishek; Sucheck, Steven J.Vaccines (Basel, Switzerland) (2016), 4 (2), 12/1-12/18CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chem. properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technol., improved anal. methods, and use of computational methodol. have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorg. nanoparticles.
- 79Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the Receptor-Binding Domain (RBD) of 2019 Novel Coronavirus: Implication for Development of RBD Protein as a Viral Attachment Inhibitor and Vaccine. Cell. Mol. Immunol. 2020, 17 (6), 613– 620, DOI: 10.1038/s41423-020-0400-4[Crossref], [PubMed], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Chsrw%253D&md5=87bc49d070c84e78b01230518aaa465aCharacterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccineTai, Wanbo; He, Lei; Zhang, Xiujuan; Pu, Jing; Voronin, Denis; Jiang, Shibo; Zhou, Yusen; Du, LanyingCellular & Molecular Immunology (2020), 17 (6), 613-620CODEN: CMIEAO; ISSN:1672-7681. (Nature Research)The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.
- 80Tai, W.; Zhang, X.; Drelich, A.; Shi, J.; Hsu, J. C.; Luchsinger, L.; Hillyer, C. D.; Tseng, C.-T. K.; Jiang, S.; Du, L. A Novel Receptor-Binding Domain (RBD)-Based MRNA Vaccine against SARS-CoV-2. Cell Res. 2020, 30, 932, DOI: 10.1038/s41422-020-0387-5[Crossref], [PubMed], [CAS], Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGiu7fJ&md5=b5a93d5ac1c3a015ee131dd40a685544A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2Tai, Wanbo; Zhang, Xiujuan; Drelich, Aleksandra; Shi, Juan; Hsu, Jason C.; Luchsinger, Larry; Hillyer, Christopher D.; Tseng, Chien-Te K.; Jiang, Shibo; Du, LanyingCell Research (2020), 30 (10), 932-935CODEN: CREEB6; ISSN:1001-0602. (Nature Research)The pandemic of coronavirus disease 2019 (COVID-19) causedby severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)highlights the need to develop effective and safe vaccines. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as receptor for host cell entry. To identify an mRNA candidate vaccine, we initially designed two mRNA constructs expressing S1 and RBD, resp.,of SARS-CoV-2 S protein. To detect whether S1 and RBD mRNAs durably express antigens in multiple cell types, we constructed N-terminal mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs, encapsulated them with lipid nanoparticles. Overall, this study identifies RBD as a key antigen to design effective vaccines against SARS-CoV-2, indicating great potential of RBD-based mRNA vaccine for mitigation of the COVID-19 pandemic and possible SARS-related epidemics in the future. More studies will be needed to investigate vaccine-assocd. immunopathol. in addn. to evaluate their protective efficacy.
- 81Misasi, J.; Gilman, M. S. A.; Kanekiyo, M.; Gui, M.; Cagigi, A.; Mulangu, S.; Corti, D.; Ledgerwood, J. E.; Lanzavecchia, A.; Cunningham, J.; Muyembe-Tamfun, J. J.; Baxa, U.; Graham, B. S.; Xiang, Y.; Sullivan, N. J.; McLellan, J. S. Structural and Molecular Basis for Ebola Virus Neutralization by Protective Human Antibodies. Science 2016, 351 (6279), 1343– 1346, DOI: 10.1126/science.aad6117[Crossref], [PubMed], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktFarsLY%253D&md5=2481659874654aa9addb68ba996a19cbStructural and molecular basis for Ebola virus neutralization by protective human antibodiesMisasi, John; Gilman, Morgan S. A.; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E.; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S.; Xiang, Ye; Sullivan, Nancy J.; McLellan, Jason S.Science (Washington, DC, United States) (2016), 351 (6279), 1343-1346CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains assocd. after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.
- 82Scheres, S. H. W. RELION: Implementation of a Bayesian Approach to Cryo-EM Structure Determination. J. Struct. Biol. 2012, 180 (3), 519– 530, DOI: 10.1016/j.jsb.2012.09.006[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs12jsLvO&md5=e40ffab245ef16ca7385fce394a4484aRELION: Implementation of a Bayesian approach to cryo-EM structure determinationScheres, Sjors H. W.Journal of Structural Biology (2012), 180 (3), 519-530CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromol. structures by single-particle anal. of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying max. a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be detd. A so-called gold-std. Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resoln. ests. with minimal user intervention and at acceptable computational costs.
- 83Zheng, S. Q.; Palovcak, E.; Armache, J.-P.; Verba, K. A.; Cheng, Y.; Agard, D. A. MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods 2017, 14 (4), 331– 332, DOI: 10.1038/nmeth.4193[Crossref], [PubMed], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjt1ags7g%253D&md5=5f4e225ef8123dacd8475d526175e1d2MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopyZheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul; Verba, Kliment A.; Cheng, Yifan; Agard, David A.Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.
- 84Rohou, A.; Grigorieff, N. CTFFIND4: Fast and Accurate Defocus Estimation from Electron Micrographs. J. Struct. Biol. 2015, 192 (2), 216– 221, DOI: 10.1016/j.jsb.2015.08.008[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC287js1Whsg%253D%253D&md5=8500953ad4898ae82de6f8cdc95832cfCTFFIND4: Fast and accurate defocus estimation from electron micrographsRohou Alexis; Grigorieff NikolausJournal of structural biology (2015), 192 (2), 216-21 ISSN:.CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.
- 85Tang, G.; Peng, L.; Baldwin, P. R.; Mann, D. S.; Jiang, W.; Rees, I.; Ludtke, S. J. EMAN2: An Extensible Image Processing Suite for Electron Microscopy. J. Struct. Biol. 2007, 157 (1), 38– 46, DOI: 10.1016/j.jsb.2006.05.009[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD28jjt1elsQ%253D%253D&md5=792e9e052f5233faa3180d330513f532EMAN2: an extensible image processing suite for electron microscopyTang Guang; Peng Liwei; Baldwin Philip R; Mann Deepinder S; Jiang Wen; Rees Ian; Ludtke Steven JJournal of structural biology (2007), 157 (1), 38-46 ISSN:1047-8477.EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.
- 86Punjani, A.; Rubinstein, J. L.; Fleet, D. J.; Brubaker, M. A. CryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination. Nat. Methods 2017, 14 (3), 290– 296, DOI: 10.1038/nmeth.4169[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitlGisbs%253D&md5=95d468147707707e70ac0ad38dd6ebf6cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determinationPunjani, Ali; Rubinstein, John L.; Fleet, David J.; Brubaker, Marcus A.Nature Methods (2017), 14 (3), 290-296CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for detg. the structures of biol. macromols. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resoln. frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calcns. on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound max. likelihood optimization algorithms permit the major steps in cryo-EM structure detn. to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated anal. and discovery of unexpected structures without bias from a ref. map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).
- 87Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera?A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612, DOI: 10.1002/jcc.20084[Crossref], [PubMed], [CAS], Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvVOhsbs%253D&md5=944b175f440c1ff323705987cf937ee7UCSF Chimera-A visualization system for exploratory research and analysisPettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
Cited By
This article is cited by 7 publications.
- Yingzhu Li, Rumiana Tenchov, Jeffrey Smoot, Cynthia Liu, Steven Watkins, Qiongqiong Zhou. A Comprehensive Review of the Global Efforts on COVID-19 Vaccine Development. ACS Central Science 2021, 7 (4) , 512-533. https://doi.org/10.1021/acscentsci.1c00120
- Hani Nasser Abdelhamid, Gamal Badr. Nanobiotechnology as a platform for the diagnosis of COVID-19: a review. Nanotechnology for Environmental Engineering 2021, 6 (1) https://doi.org/10.1007/s41204-021-00109-0
- Maruthi Prasanna, Malgorzata Podsiadla-Bialoskorska, Damian Mielecki, Nicolas Ruffier, Amina Fateh, Annie Lambert, Mathieu Fanuel, Emilie Camberlein, Ewa Szolajska, Cyrille Grandjean. On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines. Glycoconjugate Journal 2021, 38 https://doi.org/10.1007/s10719-021-09999-3
- Qian He, Qunying Mao, Jialu Zhang, Lianlian Bian, Fan Gao, Junzhi Wang, Miao Xu, Zhenglun Liang. COVID-19 Vaccines: Current Understanding on Immunogenicity, Safety, and Further Considerations. Frontiers in Immunology 2021, 12 https://doi.org/10.3389/fimmu.2021.669339
- Megan A. Yamoah, Phung N. Thai, Xiao-Dong Zhang. Transgene Delivery to Human Induced Pluripotent Stem Cells Using Nanoparticles. Pharmaceuticals 2021, 14 (4) , 334. https://doi.org/10.3390/ph14040334
- Benjamin N. Bell, Abigail E. Powell, Carlos Rodriguez, Jennifer R. Cochran, Peter S. Kim. Neutralizing antibodies targeting the SARS‐CoV‐2 receptor binding domain isolated from a naïve human antibody library. Protein Science 2021, 30 (4) , 716-727. https://doi.org/10.1002/pro.4044
- Philip J.M. Brouwer, Mitch Brinkkemper, Pauline Maisonnasse, Nathalie Dereuddre-Bosquet, Marloes Grobben, Mathieu Claireaux, Marlon de Gast, Romain Marlin, Virginie Chesnais, Ségolène Diry, Joel D. Allen, Yasunori Watanabe, Julia M. Giezen, Gius Kerster, Hannah L. Turner, Karlijn van der Straten, Cynthia A. van der Linden, Yoann Aldon, Thibaut Naninck, Ilja Bontjer, Judith A. Burger, Meliawati Poniman, Anna Z. Mykytyn, Nisreen M.A. Okba, Edith E. Schermer, Marielle J. van Breemen, Rashmi Ravichandran, Tom G. Caniels, Jelle van Schooten, Nidhal Kahlaoui, Vanessa Contreras, Julien Lemaître, Catherine Chapon, Raphaël Ho Tsong Fang, Julien Villaudy, Kwinten Sliepen, Yme U. van der Velden, Bart L. Haagmans, Godelieve J. de Bree, Eric Ginoux, Andrew B. Ward, Max Crispin, Neil P. King, Sylvie van der Werf, Marit J. van Gils, Roger Le Grand, Rogier W. Sanders. Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection. Cell 2021, 184 (5) , 1188-1200.e19. https://doi.org/10.1016/j.cell.2021.01.035
Abstract
Figure 1
Figure 1. Construct design for SARS-CoV-2 spike-functionalized ferritin nanoparticles. All constructs are based on the Wuhan-Hu-1 amino acid sequence (GenBank MN9089473) of SARS-CoV-2 spike. Spike-functionalized ferritin constructs were made by fusing spike ectodomain (residues 1–1213) or spikeΔC (residues 1–1143) to the H. pylori ferritin subunit separated by an SGG linker. A structural representation based on the spike trimer cryo-EM structure (PDB 6VXX) and the H. pylori ferritin crystal structure (PDB 3BVE) depicts the 24-subunit particle displaying spike or spikeΔC on the surface. The estimated size of the spike-functionalized ferritin particles based on structural data is ∼300 Å. The S-GCN4 and SΔC-GCN4 trimer constructs were made by fusing either the full-length spike residues (1–1213) or spikeΔC (1–1137) to a modified GCN4 trimerization domain followed by a hexahistidine tag. A structural representation of the spike trimers based on the cryo-EM structure (PDB 6VXX) is shown with an estimate length of ∼100 Å. The RBD spans residues 319–541 of the spike protein and is preceded by the native signal peptide (not shown) and followed by a hexahistidine tag.
Figure 2
Figure 2. Spike ferritin nanoparticles can be expressed in mammalian cell culture and purified to homogeneity. (A) Scheme for expressing and purifying spike ferritin nanoparticle antigens in mammalian cells. Spike ferritin particle subunits are encoded in a single plasmid that is transfected into the Expi293F suspension human cell line. Expi293F cells are harvested, and culture supernatant is buffer exchanged and purified via anion exchange chromatography. Protein-containing fractions are identified via Western blot, pooled, and purified by size-exclusion chromatography (SRT SEC-1000). Purified nanoparticles are assessed using biophysical characterization methods including SDS-PAGE, analytical size-exclusion chromatography, and BLI follow by in vivo characterization of the immune responses elicited in mice. (B) SEC-MALS UV A280 (left) and light scattering signals (right) from analysis of spike-based ferritin antigens using an SRT SEC-1000 size-exclusion column. A single prominent peak in both the UV and light-scattering traces confirms that spike ferritin nanoparticle preparations are homogeneous and do not aggregate.
Figure 3
Figure 3. Cryo-EM and BLI confirm that spike proteins are presented on the particle surface with mAb epitopes intact. (A) Representative motion-corrected cryo-EM micrograph of the SΔC-Fer nanoparticles. Circles indicate representative particles that were picked for further analysis. Micrographs demonstrate that particles are approximately 300 Å. (B) Reference-free 2D class averages of SΔC-Fer. 2D class averages confirm the presence of both ferritin particles and the display of spike on the surface seen as density surrounding the particles. (C) Reconstructed cryo-EM map of the SΔC-Fer nanoparticle in two views. A single spike trimer on the surface is highlighted with each protomer of the trimer shown in a different color. (D) BLI binding of SARS-CoV-2 mAbs to purified spike antigens. Antigens were diluted to 100 nM monomer concentration (100 nM RBD, 33.3 nM S-GCN4 and SΔC-GCN4 trimer, and 4.2 nM S-Fer and SΔC-Fer 24-mer ferritin particle). Binding of all antigens to three SARS-CoV-2 reactive mAbs indicates that spike ferritin nanoparticles display epitopes similarly to the RBD and spike trimers. Curves were fitted with an association/dissociation nonlinear regression, and fits are represented with dashed black lines; kon values for each binding reaction are shown in Figure S4A. Both S-Fer and SΔC-Fer exhibited a slight increase in signal during the dissociation step, perhaps due to rearrangements of the particles on the BLI sensor tip due to the extensive avidity present on the multimerized particles. Lack of binding to an off-target Ebola-specific antibody (ADI-15731) is presented in Figure S4B. Binding experiments were performed in at least duplicate; a representative trace and fit are shown from one replicate.
Figure 4
Figure 4. Immunization with SΔC-Fer nanoparticles elicits a stronger neutralizing response than immunization with nonferritin groups in mice. (A) Immunization schedule including a priming dose with 10 μg of antigen at day 0 and a boost with 10 μg of antigen at day 21. Serum was collected on days 0, 21, and 28. Both doses were adjuvanted with 10 μg of Quil-A and 10 μg of MPLA in a total volume of 100 μL per mouse administered via subcutaneous injection. (B) ELISA binding titers to both the RBD and full-length spike ectodomain after a single dose of antigen demonstrate that all groups elicited a SARS-CoV-2-directed antibody response following immunization. Each point represents the EC50 titer from a single animal; each bar represents the mean EC50 titer from the group (n = 10 mice per group). Error bars represent standard deviation. Points with signal less than EC50 1:100 dilution are placed at the limit of quantitation for the assay. (C) S-Fer and SΔC-Fer antigens elicit stronger neutralizing antibody responses than spike trimers alone or RBD, as indicated by spike-pseudotyped lentivirus neutralizing titers after a single dose of antigen. Immunization with a single dose of S-Fer or SΔC-Fer elicits neutralizing responses that are at least 2-fold greater on average than those found in plasma from 20 convalescent COVID-19 patients (CCP). Each point represents the IC50 titer from a single animal or patient; each bar represents the mean IC50 titer from each group (n = 10 per group, with the exception of CCP which is n = 20). Error bars represent standard deviation. Samples with neutralizing activity that was undetectable at 1:50 dilution or with an IC50 less than 1:100 dilution are placed at the limit of quantitation. (D) ELISA binding titers to the RBD and spike after two doses of antigen show that the SARS-CoV-2-specific response against both antigens was boosted in all groups. Groups and error are as defined in part B. (E) Spike-pseudotyped lentivirus neutralization following two doses of antigen indicates that although all groups had a neutralizing response following two doses, animals immunized with SΔC-Fer have the highest neutralizing titers, and these are significantly greater than S-GCN4 and SΔC-GCN4. Groups and error are as defined in part C. Statistical comparisons for panels B–E were performed using Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons. All p values are represented as follows: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. Mean titers with standard deviation and values from pairwise comparisons between groups can be found in Tables S1–S3.
References
ARTICLE SECTIONSThis article references 87 other publications.
- 1World Health Organization. WHO Coronavirus Disease (COVID-19) Dashboard. https://covid19.who.int/ (accessed 02 December 2020).Google ScholarThere is no corresponding record for this reference.
- 2Xia, S.; Liu, M.; Wang, C.; Xu, W.; Lan, Q.; Feng, S.; Qi, F.; Bao, L.; Du, L.; Liu, S.; Qin, C.; Sun, F.; Shi, Z.; Zhu, Y.; Jiang, S.; Lu, L. Inhibition of SARS-CoV-2 (Previously 2019-NCoV) Infection by a Highly Potent Pan-Coronavirus Fusion Inhibitor Targeting Its Spike Protein That Harbors a High Capacity to Mediate Membrane Fusion. Cell Res. 2020, 30 (4), 343– 355, DOI: 10.1038/s41422-020-0305-x[Crossref], [PubMed], [CAS], Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlvFKhtL0%253D&md5=00a4a7fb87b1053fb1033973f3734af3Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusionXia, Shuai; Liu, Meiqin; Wang, Chao; Xu, Wei; Lan, Qiaoshuai; Feng, Siliang; Qi, Feifei; Bao, Linlin; Du, Lanying; Liu, Shuwen; Qin, Chuan; Sun, Fei; Shi, Zhengli; Zhu, Yun; Jiang, Shibo; Lu, LuCell Research (2020), 30 (4), 343-355CODEN: CREEB6; ISSN:1001-0602. (Nature Research)The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the mol. mechanism that underlies viral infection must first be defined. Therefore, we herein established a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed a superior plasma membrane fusion capacity compared to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in the SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be assocd. with enhanced interactions with the HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted the HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. Here we generated a series of lipopeptides derived from EK1 and found that EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than the original EK1 peptide, resp. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, and potently inhibited the replication of 5 live human coronaviruses examd., including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by the currently circulating SARS-CoV-2 and other emerging SARSr-CoVs.
- 3Wrapp, D.; Wang, N.; Corbett, K. S.; Goldsmith, J. A.; Hsieh, C. L.; Abiona, O.; Graham, B. S.; McLellan, J. S. Cryo-EM Structure of the 2019-NCoV Spike in the Prefusion Conformation. Science 2020, 367 (6483), 1260– 1263, DOI: 10.1126/science.abb2507[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvFemt70%253D&md5=27d08cbb9a43d1da051a8a92a9f68aa5Cryo-EM structure of the 2019-nCoV spike in the prefusion conformationWrapp, Daniel; Wang, Nianshuang; Corbett, Kizzmekia S.; Goldsmith, Jory A.; Hsieh, Ching-Lin; Abiona, Olubukola; Graham, Barney S.; McLellan, Jason S.Science (Washington, DC, United States) (2020), 367 (6483), 1260-1263CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we detd. a 3.5-angstrom-resoln. cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophys. and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Addnl., we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
- 4Robbiani, D. F.; Gaebler, C.; Muecksch, F.; Lorenzi, J. C. C.; Wang, Z.; Cho, A.; Agudelo, M.; Barnes, C. O.; Gazumyan, A.; Finkin, S.; Hägglöf, T.; Oliveira, T. Y.; Viant, C.; Hurley, A.; Hoffmann, H.-H.; Millard, K. G.; Kost, R. G.; Cipolla, M.; Gordon, K.; Bianchini, F.; Chen, S. T.; Ramos, V.; Patel, R.; Dizon, J.; Shimeliovich, I.; Mendoza, P.; Hartweger, H.; Nogueira, L.; Pack, M.; Horowitz, J.; Schmidt, F.; Weisblum, Y.; Michailidis, E.; Ashbrook, A. W.; Waltari, E.; Pak, J. E.; Huey-Tubman, K. E.; Koranda, N.; Hoffman, P. R.; West, A. P.; Rice, C. M.; Hatziioannou, T.; Bjorkman, P. J.; Bieniasz, P. D.; Caskey, M.; Nussenzweig, M. C. Convergent Antibody Responses to SARS-CoV-2 in Convalescent Individuals. Nature 2020, 584 (7821), 437– 442, DOI: 10.1038/s41586-020-2456-9[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFegtb3P&md5=952577f69ffe8282d0b2451b17a5ecadConvergent antibody responses to SARS-CoV-2 in convalescent individualsRobbiani, Davide F.; Gaebler, Christian; Muecksch, Frauke; Lorenzi, Julio C. C.; Wang, Zijun; Cho, Alice; Agudelo, Marianna; Barnes, Christopher O.; Gazumyan, Anna; Finkin, Shlomo; Hagglof, Thomas; Oliveira, Thiago Y.; Viant, Charlotte; Hurley, Arlene; Hoffmann, Hans-Heinrich; Millard, Katrina G.; Kost, Rhonda G.; Cipolla, Melissa; Gordon, Kristie; Bianchini, Filippo; Chen, Spencer T.; Ramos, Victor; Patel, Roshni; Dizon, Juan; Shimeliovich, Irina; Mendoza, Pilar; Hartweger, Harald; Nogueira, Lilian; Pack, Maggi; Horowitz, Jill; Schmidt, Fabian; Weisblum, Yiska; Michailidis, Eleftherios; Ashbrook, Alison W.; Waltari, Eric; Pak, John E.; Huey-Tubman, Kathryn E.; Koranda, Nicholas; Hoffman, Pauline R.; West Jr, Anthony P.; Rice, Charles M.; Hatziioannou, Theodora; Bjorkman, Pamela J.; Bieniasz, Paul D.; Caskey, Marina; Nussenzweig, Michel C.Nature (London, United Kingdom) (2020), 584 (7821), 437-442CODEN: NATUAS; ISSN:0028-0836. (Nature Research)During the coronavirus disease-2019 (COVID-19) pandemic, severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has led to the infection of millions of people and has claimed hundreds of thousands of lives. The entry of the virus into cells depends on the receptor-binding domain (RBD) of the spike (S) protein of SARS-CoV-2. Although there is currently no vaccine, it is likely that antibodies will be essential for protection. However, little is known about the human antibody response to SARS-CoV-2. We report on 149 COVID-19-convalescent individuals. Plasma samples collected an av. of 39 days after the onset of symptoms had variable half-maximal pseudovirus neutralizing titers; titers were <50 in 33% of samples, <1000 in 79% of samples, and only 1% of samples had titers >5000. Antibody sequencing revealed the expansion of clones of RBD-specific memory B cells that expressed closely related antibodies in different individuals. Despite low plasma titers, antibodies to 3 distinct epitopes on the RBD neutralized the virus with half-maximal inhibitory concns. (IC50 values) as low as 2 ng/mL. In conclusion, most convalescent plasma samples obtained from individuals who recover from COVID-19 do not contain high levels of neutralizing activity. Nevertheless, rare but recurring RBD-specific antibodies with potent antiviral activity were found in all individuals tested, suggesting that a vaccine designed to elicit such antibodies could be broadly effective.
- 5Brouwer, P. J. M.; Caniels, T. G.; van der Straten, K.; Snitselaar, J. L.; Aldon, Y.; Bangaru, S.; Torres, J. L.; Okba, N. M. A.; Claireaux, M.; Kerster, G.; Bentlage, A. E. H.; van Haaren, M. M.; Guerra, D.; Burger, J. A.; Schermer, E. E.; Verheul, K. D.; van der Velde, N.; van der Kooi, A.; van Schooten, J.; van Breemen, M. J.; Bijl, T. P. L.; Sliepen, K.; Aartse, A.; Derking, R.; Bontjer, I.; Kootstra, N. A.; Wiersinga, W. J.; Vidarsson, G.; Haagmans, B. L.; Ward, A. B.; de Bree, G. J.; Sanders, R. W.; van Gils, M. J. Potent Neutralizing Antibodies from COVID-19 Patients Define Multiple Targets of Vulnerability. Science 2020, 369 (6504), 643– 650, DOI: 10.1126/science.abc5902[Crossref], [PubMed], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGms7vJ&md5=59e9b7db3e2d86923e5ecbb880aa24ccPotent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerabilityBrouwer, Philip J. M.; Caniels, Tom G.; van der Straten, Karlijn; Snitselaar, Jonne L.; Aldon, Yoann; Bangaru, Sandhya; Torres, Jonathan L.; Okba, Nisreen M. A.; Claireaux, Mathieu; Kerster, Gius; Bentlage, Arthur E. H.; van Haaren, Marlies M.; Guerra, Denise; Burger, Judith A.; Schermer, Edith E.; Verheul, Kirsten D.; van der Velde, Niels; van der Kooi, Alex; van Schooten, Jelle; van Breemen, Marielle J.; Bijl, Tom P. L.; Sliepen, Kwinten; Aartse, Aafke; Derking, Ronald; Bontjer, Ilja; Kootstra, Neeltje A.; Wiersinga, W. Joost; Vidarsson, Gestur; Haagmans, Bart L.; Ward, Andrew B.; de Bree, Godelieve J.; Sanders, Rogier W.; van Gils, Marit J.Science (Washington, DC, United States) (2020), 369 (6504), 643-650CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a large impact on global health, travel, and economy. Therefore, preventative and therapeutic measures are urgently needed. We isolated monoclonal antibodies from 3 convalescent coronavirus disease 2019 (COVID-19) patients using a SARS-CoV-2 stabilized prefusion spike protein. These antibodies had low levels of somatic hypermutation and showed a strong enrichment in VH1-69, VH3-30-3, and VH1-24 gene usage. A subset of the antibodies was able to potently inhibit authentic SARS-CoV-2 infection at a concn. as low as 0.007μg/mL. Competition and electron microscopy studies illustrate that the SARS-CoV-2 spike protein contains multiple distinct antigenic sites, including several receptor-binding domain (RBD) epitopes as well as non-RBD epitopes. In addn. to providing guidance for vaccine design, the antibodies described are promising candidates for COVID-19 treatment and prevention.
- 6Hoffmann, M.; Kleine-Weber, H.; Pöhlmann, S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol. Cell 2020, 78 (4), 779– 784, DOI: 10.1016/j.molcel.2020.04.022[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovVCgs7o%253D&md5=18c626bd22d3d9c280d8b5e19c116db8A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung CellsHoffmann, Markus; Kleine-Weber, Hannah; Poehlmann, StefanMolecular Cell (2020), 78 (4), 779-784.e5CODEN: MOCEFL; ISSN:1097-2765. (Elsevier Inc.)The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site contg. multiple arginine residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
- 7Walls, A. C.; Park, Y.-J.; Tortorici, M. A.; Wall, A.; McGuire, A. T.; Veesler, D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 2020, 181 (2), 281– 292, DOI: 10.1016/j.cell.2020.02.058[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXkvVejsLk%253D&md5=ac8a8a208d9c26f88f702fb7634ab1abStructure, Function, and Antigenicity of the SARS-CoV-2 Spike GlycoproteinWalls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, DavidCell (Cambridge, MA, United States) (2020), 181 (2), 281-292.e6CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We detd. cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
- 8Yan, R.; Zhang, Y.; Li, Y.; Xia, L.; Guo, Y.; Zhou, Q. Structural Basis for the Recognition of SARS-CoV-2 by Full-Length Human ACE2. Science 2020, 367 (6485), 1444– 1448, DOI: 10.1126/science.abb2762[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlslymsLo%253D&md5=ff4dfdfc646ea878cfb325019160e94aStructural basis for the recognition of SARS-CoV-2 by full-length human ACE2Yan, Renhong; Zhang, Yuanyuan; Li, Yaning; Xia, Lu; Guo, Yingying; Zhou, QiangScience (Washington, DC, United States) (2020), 367 (6485), 1444-1448CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for severe acute respiratory syndrome coronavirus (SARS-CoV) and the new coronavirus (SARS-CoV-2) that is causing the serious coronavirus disease 2019 (COVID-19) epidemic. Here, we present cryo-electron microscopy structures of full-length human ACE2 in the presence of the neutral amino acid transporter B0AT1 with or without the receptor binding domain (RBD) of the surface spike glycoprotein (S protein) of SARS-CoV-2, both at an overall resoln. of 2.9 angstroms, with a local resoln. of 3.5 angstroms at the ACE2-RBD interface. The ACE2-B0AT1 complex is assembled as a dimer of heterodimers, with the collectrin-like domain of ACE2 mediating homodimerization. The RBD is recognized by the extracellular peptidase domain of ACE2 mainly through polar residues. These findings provide important insights into the mol. basis for coronavirus recognition and infection.
- 9Shang, J.; Ye, G.; Shi, K.; Wan, Y.; Luo, C.; Aihara, H.; Geng, Q.; Auerbach, A.; Li, F. Structural Basis of Receptor Recognition by SARS-CoV-2. Nature 2020, 581 (7807), 221– 224, DOI: 10.1038/s41586-020-2179-y[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtbs%253D&md5=33bc9151641b2adcfb0dbf446621a1dcStructural basis of receptor recognition by SARS-CoV-2Shang, Jian; Ye, Gang; Shi, Ke; Wan, Yushun; Luo, Chuming; Aihara, Hideki; Geng, Qibin; Auerbach, Ashley; Li, FangNature (London, United Kingdom) (2020), 581 (7807), 221-224CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystn.) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Addnl., we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.
- 10Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L.; Wang, X. Structure of the SARS-CoV-2 Spike Receptor-Binding Domain Bound to the ACE2 Receptor. Nature 2020, 581 (7807), 215– 220, DOI: 10.1038/s41586-020-2180-5[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXoslOqtL8%253D&md5=279c60143e8e5eb505457e0778baa8efStructure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptorLan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, XinquanNature (London, United Kingdom) (2020), 581 (7807), 215-220CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Abstr.: A new and highly pathogenic coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) caused an outbreak in Wuhan city, Hubei province, China, starting from Dec. 2019 that quickly spread nationwide and to other countries around the world1-3. Here, to better understand the initial step of infection at an at. level, we detd. the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 bound to the cell receptor ACE2. The overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also uses ACE2 as the cell receptor4. Structural anal. identified residues in the SARS-CoV-2 RBD that are essential for ACE2 binding, the majority of which either are highly conserved or share similar side chain properties with those in the SARS-CoV RBD. Such similarity in structure and sequence strongly indicate convergent evolution between the SARS-CoV-2 and SARS-CoV RBDs for improved binding to ACE2, although SARS-CoV-2 does not cluster within SARS and SARS-related coronaviruses1-3,5. The epitopes of two SARS-CoV antibodies that target the RBD are also analyzed for binding to the SARS-CoV-2 RBD, providing insights into the future identification of cross-reactive antibodies.
- 11Corbett, K. S.; Flynn, B.; Foulds, K. E.; Francica, J. R.; Boyoglu-Barnum, S.; Werner, A. P.; Flach, B.; O’Connell, S.; Bock, K. W.; Minai, M.; Nagata, B. M.; Andersen, H.; Martinez, D. R.; Noe, A. T.; Douek, N.; Donaldson, M. M.; Nji, N. N.; Alvarado, G. S.; Edwards, D. K.; Flebbe, D. R.; Lamb, E.; Doria-Rose, N. A.; Lin, B. C.; Louder, M. K.; O’Dell, S.; Schmidt, S. D.; Phung, E.; Chang, L. A.; Yap, C.; Todd, J.-P. M.; Pessaint, L.; Van Ry, A.; Browne, S.; Greenhouse, J.; Putman-Taylor, T.; Strasbaugh, A.; Campbell, T.-A.; Cook, A.; Dodson, A.; Steingrebe, K.; Shi, W.; Zhang, Y.; Abiona, O. M.; Wang, L.; Pegu, A.; Yang, E. S.; Leung, K.; Zhou, T.; Teng, I.-T.; Widge, A.; Gordon, I.; Novik, L.; Gillespie, R. A.; Loomis, R. J.; Moliva, J. I.; Stewart-Jones, G.; Himansu, S.; Kong, W.-P.; Nason, M. C.; Morabito, K. M.; Ruckwardt, T. J.; Ledgerwood, J. E.; Gaudinski, M. R.; Kwong, P. D.; Mascola, J. R.; Carfi, A.; Lewis, M. G.; Baric, R. S.; McDermott, A.; Moore, I. N.; Sullivan, N. J.; Roederer, M.; Seder, R. A.; Graham, B. S. Evaluation of the MRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N. Engl. J. Med. 2020, 383, 1544, DOI: 10.1056/NEJMoa2024671[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFWmsr7I&md5=8ecba6b18bc615d886ca5016b7c85496Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primatesCorbett, K. S.; Flynn, B.; Foulds, K. E.; Francica, J. R.; Boyoglu-Barnum, S.; Werner, A. P.; Flach, B.; O'Connell, S.; Bock, K. W.; Minai, M.; Nagata, B. M.; Andersen, H.; Martinez, D. R.; Noe, A. T.; Douek, N.; Donaldson, M. M.; Nji, N. N.; Alvarado, G. S.; Edwards, D. K.; Flebbe, D. R.; Lamb, E.; Doria-Rose, N. A.; Lin, B. C.; Louder, M. K.; O'Dell, S.; Schmidt, S. D.; Phung, E.; Chang, L. A.; Yap, C.; Todd, J.-P. M.; Pessaint, L.; Van Ry, A.; Browne, S.; Greenhouse, J.; Putman-Taylor, T.; Strasbaugh, A.; Campbell, T.-A.; Cook, A.; Dodson, A.; Steingrebe, K.; Shi, W.; Zhang, Y.; Abiona, O. M.; Wang, L.; Pegu, A.; Yang, E. S.; Leung, K.; Zhou, T.; Teng, I-T.; Widge, A.; Gordon, I.; Novik, L.; Gillespie, R. A.; Loomis, R. J.; Moliva, J. I.; Stewart-Jones, G.; Himansu, S.; Kong, W.-P.; Nason, M. C.; Morabito, K. M.; Ruckwardt, T. J.; Ledgerwood, J. E.; Gaudinski, M. R.; Kwong, P. D.; Mascola, J. R.; Carfi, A.; Lewis, M. G.; Baric, R. S.; McDermott, A.; Moore, I. N.; Sullivan, N. J.; Roederer, M.; Seder, R. A.; Graham, B. S.New England Journal of Medicine (2020), 383 (16), 1544-1555CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)Background: Vaccines to prevent coronavirus disease 2019 (Covid-19) are urgently needed. The effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines on viral replication in both upper and lower airways is important to evaluate in nonhuman primates. Methods: Nonhuman primates received 10 or 100μg of mRNA-1273, a vaccine encoding the prefusion-stabilized spike protein of SARS-CoV-2, or no vaccine. Antibody and T-cell responses were assessed before upper- and lower-airway challenge with SARS-CoV-2. Active viral replication and viral genomes in bronchoalveolar-lavage (BAL) fluid and nasal swab specimens were assessed by polymerase chain reaction, and histopathol. anal. and viral quantification were performed on lung-tissue specimens. Results The mRNA-1273 vaccine candidate induced antibody levels exceeding those in human convalescent-phase serum, with live-virus reciprocal 50% inhibitory diln. (ID50) geometric mean titers of 501 in the 10-μg dose group and 3481 in the 100-μg dose group. Vaccination induced type 1 helper T-cell (Th1)-biased CD4 T-cell responses and low or undetectable Th2 or CD8 T-cell responses. Viral replication was not detectable in BAL fluid by day 2 after challenge in seven of eight animals in both vaccinated groups. No viral replication was detectable in the nose of any of the eight animals in the 100-μg dose group by day 2 after challenge, and limited inflammation or detectable viral genome or antigen was noted in lungs of animals in either vaccine group. conclusions Vaccination of nonhuman primates with mRNA-1273 induced robust SARS-CoV-2 neutralizing activity, rapid protection in the upper and lower airways, and no pathol. changes in the lung.
- 12Corbett, K. S.; Edwards, D.; Leist, S. R.; Abiona, O. M.; Boyoglu-Barnum, S.; Gillespie, R. A.; Himansu, S.; Schäfer, A.; Ziwawo, C. T.; DiPiazza, A. T.; Dinnon, K. H.; Elbashir, S. M.; Shaw, C. A.; Woods, A.; Fritch, E. J.; Martinez, D. R.; Bock, K. W.; Minai, M.; Nagata, B. M.; Hutchinson, G. B.; Bahl, K.; Garcia-Dominguez, D.; Ma, L.; Renzi, I.; Kong, W.-P.; Schmidt, S. D.; Wang, L.; Zhang, Y.; Stevens, L. J.; Phung, E.; Chang, L. A.; Loomis, R. J.; Altaras, N. E.; Narayanan, E.; Metkar, M.; Presnyak, V.; Liu, C.; Louder, M. K.; Shi, W.; Leung, K.; Yang, E. S.; West, A.; Gully, K. L.; Wang, N.; Wrapp, D.; Doria-Rose, N. A.; Stewart-Jones, G.; Bennett, H.; Nason, M. C.; Ruckwardt, T. J.; McLellan, J. S.; Denison, M. R.; Chappell, J. D.; Moore, I. N.; Morabito, K. M.; Mascola, J. R.; Baric, R. S.; Carfi, A.; Graham, B. S. SARS-CoV-2 MRNA Vaccine Development Enabled by Prototype Pathogen Preparedness. Nature 2020, 586, 567, DOI: 10.1038/s41586-020-2622-0[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1ejtLbO&md5=fa0483e26f66c6a899486917083d257aSARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparednessCorbett, Kizzmekia S.; Edwards, Darin K.; Leist, Sarah R.; Abiona, Olubukola M.; Boyoglu-Barnum, Seyhan; Gillespie, Rebecca A.; Himansu, Sunny; Schafer, Alexandra; Ziwawo, Cynthia T.; DiPiazza, Anthony T.; Dinnon, Kenneth H.; Elbashir, Sayda M.; Shaw, Christine A.; Woods, Angela; Fritch, Ethan J.; Martinez, David R.; Bock, Kevin W.; Minai, Mahnaz; Nagata, Bianca M.; Hutchinson, Geoffrey B.; Wu, Kai; Henry, Carole; Bahl, Kapil; Garcia-Dominguez, Dario; Ma, LingZhi; Renzi, Isabella; Kong, Wing-Pui; Schmidt, Stephen D.; Wang, Lingshu; Zhang, Yi; Phung, Emily; Chang, Lauren A.; Loomis, Rebecca J.; Altaras, Nedim Emil; Narayanan, Elisabeth; Metkar, Mihir; Presnyak, Vlad; Liu, Cuiping; Louder, Mark K.; Shi, Wei; Leung, Kwanyee; Yang, Eun Sung; West, Ande; Gully, Kendra L.; Stevens, Laura J.; Wang, Nianshuang; Wrapp, Daniel; Doria-Rose, Nicole A.; Stewart-Jones, Guillaume; Bennett, Hamilton; Alvarado, Gabriela S.; Nason, Martha C.; Ruckwardt, Tracy J.; McLellan, Jason S.; Denison, Mark R.; Chappell, James D.; Moore, Ian N.; Morabito, Kaitlyn M.; Mascola, John R.; Baric, Ralph S.; Carfi, Andrea; Graham, Barney S.Nature (London, United Kingdom) (2020), 586 (7830), 567-571CODEN: NATUAS; ISSN:0028-0836. (Nature Research)A vaccine for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is needed to control the coronavirus disease 2019 (COVID-19) global pandemic. Structural studies have led to the development of mutations that stabilize Betacoronavirus spike proteins in the prefusion state, improving their expression and increasing immunogenicity1. This principle has been applied to design mRNA-1273, an mRNA vaccine that encodes a SARS-CoV-2 spike protein that is stabilized in the prefusion conformation. Here we show that mRNA-1273 induces potent neutralizing antibody responses to both wild-type (D614) and D614G mutant SARS-CoV-2 as well as CD8+ T cell responses, and protects against SARS-CoV-2 infection in the lungs and noses of mice without evidence of immunopathol. MRNA-1273 is currently in a phase III trial to evaluate its efficacy.
- 13Smith, T. R. F.; Patel, A.; Ramos, S.; Elwood, D.; Zhu, X.; Yan, J.; Gary, E. N.; Walker, S. N.; Schultheis, K.; Purwar, M.; Xu, Z.; Walters, J.; Bhojnagarwala, P.; Yang, M.; Chokkalingam, N.; Pezzoli, P.; Parzych, E.; Reuschel, E. L.; Doan, A.; Tursi, N.; Vasquez, M.; Choi, J.; Tello-Ruiz, E.; Maricic, I.; Bah, M. A.; Wu, Y.; Amante, D.; Park, D. H.; Dia, Y.; Ali, A. R.; Zaidi, F. I.; Generotti, A.; Kim, K. Y.; Herring, T. A.; Reeder, S.; Andrade, V. M.; Buttigieg, K.; Zhao, G.; Wu, J. M.; Li, D.; Bao, L.; Liu, J.; Deng, W.; Qin, C.; Brown, A. S.; Khoshnejad, M.; Wang, N.; Chu, J.; Wrapp, D.; McLellan, J. S.; Muthumani, K.; Wang, B.; Carroll, M. W.; Kim, J. J.; Boyer, J.; Kulp, D. W.; Humeau, L. M. P. F.; Weiner, D. B.; Broderick, K. E. Immunogenicity of a DNA Vaccine Candidate for COVID-19. Nat. Commun. 2020, 11 (1), 1– 13, DOI: 10.1038/s41467-020-16505-0
- 14Wang, H.; Zhang, Y.; Huang, B.; Deng, W.; Quan, Y.; Wang, W.; Xu, W.; Zhao, Y.; Li, N.; Zhang, J.; Liang, H.; Bao, L.; Xu, Y.; Ding, L.; Zhou, W.; Gao, H.; Liu, J.; Niu, P.; Zhao, L.; Zhen, W.; Fu, H.; Yu, S.; Zhang, Z.; Xu, G.; Li, C.; Lou, Z.; Xu, M.; Qin, C.; Wu, G.; Gao, G. F.; Tan, W.; Yang, X. Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2. Cell 2020, 182 (3), 713– 721, DOI: 10.1016/j.cell.2020.06.008[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtFylur7P&md5=057a1910bb9c65887d8647a1b1d8c310Development of an Inactivated Vaccine Candidate, BBIBP-CorV, with Potent Protection against SARS-CoV-2Wang, Hui; Zhang, Yuntao; Huang, Baoying; Deng, Wei; Quan, Yaru; Wang, Wenling; Xu, Wenbo; Zhao, Yuxiu; Li, Na; Zhang, Jin; Liang, Hongyang; Bao, Linlin; Xu, Yanfeng; Ding, Ling; Zhou, Weimin; Gao, Hong; Liu, Jiangning; Niu, Peihua; Zhao, Li; Zhen, Wei; Fu, Hui; Yu, Shouzhi; Zhang, Zhengli; Xu, Guangxue; Li, Changgui; Lou, Zhiyong; Xu, Miao; Qin, Chuan; Wu, Guizhen; Gao, George Fu; Tan, Wenjie; Yang, XiaomingCell (Cambridge, MA, United States) (2020), 182 (3), 713-721.e9CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) threatens global public health. The development of a vaccine is urgently needed for the prevention and control of COVID-19. We report the pilot-scale prodn. of an inactivated SARS-CoV-2 vaccine candidate (BBIBP-CorV) that induces high levels of neutralizing antibodies titers in mice, rats, guinea pigs, rabbits, and nonhuman primates (cynomolgus monkeys and rhesus macaques) to provide protection against SARS-CoV-2. Two-dose immunizations using 2μg/dose of BBIBP-CorV provided highly efficient protection against SARS-CoV-2 intratracheal challenge in rhesus macaques, without detectable antibody-dependent enhancement of infection. In addn., BBIBP-CorV exhibits efficient productivity and good genetic stability for vaccine manuf. These results support the further evaluation of BBIBP-CorV in a clin. trial.
- 15van Doremalen, N.; Lambe, T.; Spencer, A.; Belij-Rammerstorfer, S.; Purushotham, J. N.; Port, J. R.; Avanzato, V. A.; Bushmaker, T.; Flaxman, A.; Ulaszewska, M.; Feldmann, F.; Allen, E. R.; Sharpe, H.; Schulz, J.; Holbrook, M.; Okumura, A.; Meade-White, K.; Pérez-Pérez, L.; Edwards, N. J.; Wright, D.; Bissett, C.; Gilbride, C.; Williamson, B. N.; Rosenke, R.; Long, D.; Ishwarbhai, A.; Kailath, R.; Rose, L.; Morris, S.; Powers, C.; Lovaglio, J.; Hanley, P. W.; Scott, D.; Saturday, G.; de Wit, E.; Gilbert, S. C.; Munster, V. J. ChAdOx1 NCoV-19 Vaccine Prevents SARS-CoV-2 Pneumonia in Rhesus Macaques. Nature 2020, 586, 578, DOI: 10.1038/s41586-020-2608-y[Crossref], [PubMed], [CAS], Google ScholarThere is no corresponding record for this reference.https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=&md5=9874b665cc7a056b8e2f928dd3112440
- 16Folegatti, P. M.; Ewer, K. J.; Aley, P. K.; Angus, B.; Becker, S.; Belij-Rammerstorfer, S.; Bellamy, D.; Bibi, S.; Bittaye, M.; Clutterbuck, E. A.; Dold, C.; Faust, S. N.; Finn, A.; Flaxman, A. L.; Hallis, B.; Heath, P.; Jenkin, D.; Lazarus, R.; Makinson, R.; Minassian, A. M.; Pollock, K. M.; Ramasamy, M.; Robinson, H.; Snape, M.; Tarrant, R.; Voysey, M.; Green, C.; Douglas, A. D.; Hill, A. V. S.; Lambe, T.; Gilbert, S. C.; Pollard, A. J.; Aboagye, J.; Adams, K.; Ali, A.; Allen, E.; Allison, J. L.; Anslow, R.; Arbe-Barnes, E. H.; Babbage, G.; Baillie, K.; Baker, M.; Baker, N.; Baker, P.; Baleanu, I.; Ballaminut, J.; Barnes, E.; Barrett, J.; Bates, L.; Batten, A.; Beadon, K.; Beckley, R.; Berrie, E.; Berry, L.; Beveridge, A.; Bewley, K. R.; Bijker, E. M.; Bingham, T.; Blackwell, L.; Blundell, C. L.; Bolam, E.; Boland, E.; Borthwick, N.; Bower, T.; Boyd, A.; Brenner, T.; Bright, P. D.; Brown-O’Sullivan, C.; Brunt, E.; Burbage, J.; Burge, S.; Buttigieg, K. R.; Byard, N.; Cabera Puig, I.; Calvert, A.; Camara, S.; Cao, M.; Cappuccini, F.; Carr, M.; Carroll, M. W.; Carter, V.; Cathie, K.; Challis, R. J.; Charlton, S.; Chelysheva, I.; Cho, J.-S.; Cicconi, P.; Cifuentes, L.; Clark, H.; Clark, E.; Cole, T.; Colin-Jones, R.; Conlon, C. P.; Cook, A.; Coombes, N. S.; Cooper, R.; Cosgrove, C. A.; Coy, K.; Crocker, W. E. M.; Cunningham, C. J.; Damratoski, B. E.; Dando, L.; Datoo, M. S.; Davies, H.; De Graaf, H.; Demissie, T.; Di Maso, C.; Dietrich, I.; Dong, T.; Donnellan, F. R.; Douglas, N.; Downing, C.; Drake, J.; Drake-Brockman, R.; Drury, R. E.; Dunachie, S. J.; Edwards, N. J.; Edwards, F. D. L.; Edwards, C. J.; Elias, S. C.; Elmore, M. J.; Emary, K. R. W.; English, M. R.; Fagerbrink, S.; Felle, S.; Feng, S.; Field, S.; Fixmer, C.; Fletcher, C.; Ford, K. J.; Fowler, J.; Fox, P.; Francis, E.; Frater, J.; Furze, J.; Fuskova, M.; Galiza, E.; Gbesemete, D.; Gilbride, C.; Godwin, K.; Gorini, G.; Goulston, L.; Grabau, C.; Gracie, L.; Gray, Z.; Guthrie, L. B.; Hackett, M.; Halwe, S.; Hamilton, E.; Hamlyn, J.; Hanumunthadu, B.; Harding, I.; Harris, S. A.; Harris, A.; Harrison, D.; Harrison, C.; Hart, T. C.; Haskell, L.; Hawkins, S.; Head, I.; Henry, J. A.; Hill, J.; Hodgson, S. H. C.; Hou, M. M.; Howe, E.; Howell, N.; Hutlin, C.; Ikram, S.; Isitt, C.; Iveson, P.; Jackson, S.; Jackson, F.; James, S. W.; Jenkins, M.; Jones, E.; Jones, K.; Jones, C. E.; Jones, B.; Kailath, R.; Karampatsas, K.; Keen, J.; Kelly, S.; Kelly, D.; Kerr, D.; Kerridge, S.; Khan, L.; Khan, U.; Killen, A.; Kinch, J.; King, T. B.; King, L.; King, J.; Kingham-Page, L.; Klenerman, P.; Knapper, F.; Knight, J. C.; Knott, D.; Koleva, S.; Kupke, A.; Larkworthy, C. W.; Larwood, J. P. J.; Laskey, A.; Lawrie, A. M.; Lee, A.; Ngan Lee, K. Y.; Lees, E. A.; Legge, H.; Lelliott, A.; Lemm, N.-M.; Lias, A. M.; Linder, A.; Lipworth, S.; Liu, X.; Liu, S.; Lopez Ramon, R.; Lwin, M.; Mabesa, F.; Madhavan, M.; Mallett, G.; Mansatta, K.; Marcal, I.; Marinou, S.; Marlow, E.; Marshall, J. L.; Martin, J.; McEwan, J.; McInroy, L.; Meddaugh, G.; Mentzer, A. J.; Mirtorabi, N.; Moore, M.; Moran, E.; Morey, E.; Morgan, V.; Morris, S. J.; Morrison, H.; Morshead, G.; Morter, R.; Mujadidi, Y. F.; Muller, J.; Munera-Huertas, T.; Munro, C.; Munro, A.; Murphy, S.; Munster, V. J.; Mweu, P.; Noé, A.; Nugent, F. L.; Nuthall, E.; O’Brien, K.; O’Connor, D.; Oguti, B.; Oliver, J. L.; Oliveira, C.; O’Reilly, P. J.; Osborn, M.; Osborne, P.; Owen, C.; Owens, D.; Owino, N.; Pacurar, M.; Parker, K.; Parracho, H.; Patrick-Smith, M.; Payne, V.; Pearce, J.; Peng, Y.; Peralta Alvarez, M. P.; Perring, J.; Pfafferott, K.; Pipini, D.; Plested, E.; Pluess-Hall, H.; Pollock, K.; Poulton, I.; Presland, L.; Provstgaard-Morys, S.; Pulido, D.; Radia, K.; Ramos Lopez, F.; Rand, J.; Ratcliffe, H.; Rawlinson, T.; Rhead, S.; Riddell, A.; Ritchie, A. J.; Roberts, H.; Robson, J.; Roche, S.; Rohde, C.; Rollier, C. S.; Romani, R.; Rudiansyah, I.; Saich, S.; Sajjad, S.; Salvador, S.; Sanchez Riera, L.; Sanders, H.; Sanders, K.; Sapaun, S.; Sayce, C.; Schofield, E.; Screaton, G.; Selby, B.; Semple, C.; Sharpe, H. R.; Shaik, I.; Shea, A.; Shelton, H.; Silk, S.; Silva-Reyes, L.; Skelly, D. T.; Smee, H.; Smith, C. C.; Smith, D. J.; Song, R.; Spencer, A. J.; Stafford, E.; Steele, A.; Stefanova, E.; Stockdale, L.; Szigeti, A.; Tahiri-Alaoui, A.; Tait, M.; Talbot, H.; Tanner, R.; Taylor, I. J.; Taylor, V.; Te Water Naude, R.; Thakur, N.; Themistocleous, Y.; Themistocleous, A.; Thomas, M.; Thomas, T. M.; Thompson, A.; Thomson-Hill, S.; Tomlins, J.; Tonks, S.; Towner, J.; Tran, N.; Tree, J. A.; Truby, A.; Turkentine, K.; Turner, C.; Turner, N.; Turner, S.; Tuthill, T.; Ulaszewska, M.; Varughese, R.; Van Doremalen, N.; Veighey, K.; Verheul, M. K.; Vichos, I.; Vitale, E.; Walker, L.; Watson, M. E. E.; Welham, B.; Wheat, J.; White, C.; White, R.; Worth, A. T.; Wright, D.; Wright, S.; Yao, X. L.; Yau, Y. Safety and Immunogenicity of the ChAdOx1 NCoV-19 Vaccine against SARS-CoV-2: A Preliminary Report of a Phase 1/2, Single-Blind, Randomised Controlled Trial. Lancet 2020, 396 (10249), 467– 478, DOI: 10.1016/S0140-6736(20)31604-4[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVaku7fE&md5=4edd57b9d55d8a7c71ee84c8f62f7ca0Safety and immunogenicity of the ChAdOx1 nCoV-19 vaccine against SARS-CoV-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trialFolegatti, Pedro M.; Ewer, Katie J.; Aley, Parvinder K.; Angus, Brian; Becker, Stephan; Belij-Rammerstorfer, Sandra; Bellamy, Duncan; Bibi, Sagida; Bittaye, Mustapha; Clutterbuck, Elizabeth A.; Dold, Christina; Faust, Saul N.; Finn, Adam; Flaxman, Amy L.; Hallis, Bassam; Heath, Paul; Jenkin, Daniel; Lazarus, Rajeka; Makinson, Rebecca; Minassian, Angela M.; Pollock, Katrina M.; Ramasamy, Maheshi; Robinson, Hannah; Snape, Matthew; Tarrant, Richard; Voysey, Merryn; Green, Catherine; Douglas, Alexander D.; Hill, Adrian V. S.; Lambe, Teresa; Gilbert, Sarah C.; Pollard, Andrew J.Lancet (2020), 396 (10249), 467-478CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might be curtailed by vaccination. We assessed the safety, reactogenicity, and immunogenicity of a viral vectored coronavirus vaccine that expresses the spike protein of SARS-CoV-2. We did a phase 1/2, single-blind, randomised controlled trial in five trial sites in the UK of a chimpanzee adenovirus-vectored vaccine (ChAdOx1 nCoV-19) expressing the SARS-CoV-2 spike protein compared with a meningococcal conjugate vaccine (MenACWY) as control. Healthy adults aged 18-55 years with no history of lab. confirmed SARS-CoV-2 infection or of COVID-19-like symptoms were randomly assigned (1:1) to receive ChAdOx1 nCoV-19 at a dose of 5 x 1010 viral particles or MenACWY as a single i.m. injection. A protocol amendment in two of the five sites allowed prophylactic paracetamol to be administered before vaccination. Ten participants assigned to a non-randomised, unblinded ChAdOx1 nCoV-19 prime-boost group received a two-dose schedule, with the booster vaccine administered 28 days after the first dose. Humoral responses at baseline and following vaccination were assessed using a standardised total IgG ELISA against trimeric SARS-CoV-2 spike protein, a muliplexed immunoassay, three live SARS-CoV-2 neutralisation assays (a 50% plaque redn. neutralisation assay [PRNT50]; a microneutralisation assay [MNA50, MNA80, and MNA90]; and Marburg VN), and a pseudovirus neutralisation assay. Cellular responses were assessed using an ex-vivo interferon-γ enzyme-linked immunospot assay. The co-primary outcomes are to assess efficacy, as measured by cases of symptomatic virol. confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were done by group allocation in participants who received the vaccine. Safety was assessed over 28 days after vaccination. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. The study is ongoing, and was registered at ISRCTN, 15281137, and ClinicalTrials.gov, NCT04324606. Between Apr. 23 and May 21, 2020, 1077 participants were enrolled and assigned to receive either ChAdOx1 nCoV-19 (n=543) or MenACWY (n=534), ten of whom were enrolled in the non-randomised ChAdOx1 nCoV-19 prime-boost group. Local and systemic reactions were more common in the ChAdOx1 nCoV-19 group and many were reduced by use of prophylactic paracetamol, including pain, feeling feverish, chills, muscle ache, headache, and malaise (all p<0·05). There were no serious adverse events related to ChAdOx1 nCoV-19. In the ChAdOx1 nCoV-19 group, spike-specific T-cell responses peaked on day 14 (median 856 spot-forming cells per million peripheral blood mononuclear cells, IQR 493-1802; n=43). Anti-spike IgG responses rose by day 28 (median 157 ELISA units [EU], 96-317; n=127), and were boosted following a second dose (639 EU, 360-792; n=10). Neutralising antibody responses against SARS-CoV-2 were detected in 32 (91%) of 35 participants after a single dose when measured in MNA80 and in 35 (100%) participants when measured in PRNT50. After a booster dose, all participants had neutralising activity (nine of nine in MNA80 at day 42 and ten of ten in Marburg VN on day 56). Neutralising antibody responses correlated strongly with antibody levels measured by ELISA (R2=0·67 by Marburg VN; p<0·001). ChAdOx1 nCoV-19 showed an acceptable safety profile, and homologous boosting increased antibody responses. These results, together with the induction of both humoral and cellular immune responses, support large-scale evaluation of this candidate vaccine in an ongoing phase 3 program. UK Research and Innovation, Coalition for Epidemic Preparedness Innovations, National Institute for Health Research (NIHR), NIHR Oxford Biomedical Research Center, Thames Valley and South Midland's NIHR Clin. Research Network, and the German Center for Infection Research (DZIF), Partner site Giessen-Marburg-Langen.
- 17Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z.; Gao, H.; Ge, X.; Kan, B.; Hu, Y.; Liu, J.; Cai, F.; Jiang, D.; Yin, Y.; Qin, C.; Li, J.; Gong, X.; Lou, X.; Shi, W.; Wu, D.; Zhang, H.; Zhu, L.; Deng, W.; Li, Y.; Lu, J.; Li, C.; Wang, X.; Yin, W.; Zhang, Y.; Qin, C. Development of an Inactivated Vaccine Candidate for SARS-CoV-2. Science 2020, 369 (6499), 77– 81, DOI: 10.1126/science.abc1932[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtlCmtL3P&md5=674788246758fd31fa8bb54c936be83aDevelopment of an inactivated vaccine candidate for SARS-CoV-2Gao, Qiang; Bao, Linlin; Mao, Haiyan; Wang, Lin; Xu, Kangwei; Yang, Minnan; Li, Yajing; Zhu, Ling; Wang, Nan; Lv, Zhe; Gao, Hong; Ge, Xiaoqin; Kan, Biao; Hu, Yaling; Liu, Jiangning; Cai, Fang; Jiang, Deyu; Yin, Yanhui; Qin, Chengfeng; Li, Jing; Gong, Xuejie; Lou, Xiuyu; Shi, Wen; Wu, Dongdong; Zhang, Hengming; Zhu, Lang; Deng, Wei; Li, Yurong; Lu, Jinxing; Li, Changgui; Wang, Xiangxi; Yin, Weidong; Zhang, Yanjun; Qin, ChuanScience (Washington, DC, United States) (2020), 369 (6499), 77-81CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale prodn. of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6μg per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, resp., without observable antibody-dependent enhancement of infection. These data support the clin. development and testing of PiCoVacc for use in humans.
- 18Graham, S. P.; McLean, R. K.; Spencer, A. J.; Belij-Rammerstorfer, S.; Wright, D.; Ulaszewska, M.; Edwards, J. C.; Hayes, J. W. P.; Martini, V.; Thakur, N.; Conceicao, C.; Dietrich, I.; Shelton, H.; Waters, R.; Ludi, A.; Wilsden, G.; Browning, C.; Bialy, D.; Bhat, S.; Stevenson-Leggett, P.; Hollinghurst, P.; Gilbride, C.; Pulido, D.; Moffat, K.; Sharpe, H.; Allen, E.; Mioulet, V.; Chiu, C.; Newman, J.; Asfor, A. S.; Burman, A.; Crossley, S.; Huo, J.; Owens, R. J.; Carroll, M.; Hammond, J. A.; Tchilian, E.; Bailey, D.; Charleston, B.; Gilbert, S. C.; Tuthill, T. J.; Lambe, T. Evaluation of the Immunogenicity of Prime-Boost Vaccination with the Replication-Deficient Viral Vectored COVID-19 Vaccine Candidate ChAdOx1 NCoV-19. npj Vaccines 2020, 5 (1), 69, DOI: 10.1038/s41541-020-00221-3[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVyltLbP&md5=b7dc7fdc2101fce4d607275b25bbfde9Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19Graham, Simon P.; McLean, Rebecca K.; Spencer, Alexandra J.; Belij-Rammerstorfer, Sandra; Wright, Daniel; Ulaszewska, Marta; Edwards, Jane C.; Hayes, Jack W. P.; Martini, Veronica; Thakur, Nazia; Conceicao, Carina; Dietrich, Isabelle; Shelton, Holly; Waters, Ryan; Ludi, Anna; Wilsden, Ginette; Browning, Clare; Bialy, Dagmara; Bhat, Sushant; Stevenson-Leggett, Phoebe; Hollinghurst, Philippa; Gilbride, Ciaran; Pulido, David; Moffat, Katy; Sharpe, Hannah; Allen, Elizabeth; Mioulet, Valerie; Chiu, Chris; Newman, Joseph; Asfor, Amin S.; Burman, Alison; Crossley, Sylvia; Huo, Jiandong; Owens, Raymond J.; Carroll, Miles; Hammond, John A.; Tchilian, Elma; Bailey, Dalan; Charleston, Bryan; Gilbert, Sarah C.; Tuthill, Tobias J.; Lambe, Teresanpj Vaccines (2020), 5 (1), 69CODEN: VACCBC; ISSN:2059-0105. (Nature Research)Abstr.: Clin. development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in Apr. 2020 following non-human primate studies using a single immunization. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. While a single dose induced antigen-specific antibody and T cells responses, a booster immunization enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralizing titers.
- 19Walsh, E. E.; Frenck, R. W.; Falsey, A. R.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Mulligan, M. J.; Bailey, R.; Swanson, K. A.; Li, P.; Koury, K.; Kalina, W.; Cooper, D.; Fontes-Garfias, C.; Shi, P.-Y.; Türeci, Ö.; Tompkins, K. R.; Lyke, K. E.; Raabe, V.; Dormitzer, P. R.; Jansen, K. U.; Şahin, U.; Gruber, W. C. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. N. Engl. J. Med. 2020, 383, 2439, DOI: 10.1056/NEJMoa2027906[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXis1yksLrM&md5=71d40c71a24ff57a525dbaef791a320aSafety and immunogenicity of two RNA-based Covid-19 vaccine candidatesWalsh, Edward E.; Frenck, Robert W. Jr.; Falsey, Ann R.; Kitchin, Nicholas; Absalon, Judith; Gurtman, Alejandra; Lockhart, Stephen; Neuzil, Kathleen; Mulligan, Mark J.; Bailey, Ruth; Swanson, Kena A.; Li, Ping; Koury, Kenneth; Kalina, Warren; Cooper, David; Fontes-Garfias, Camila; Shi, Pei-Yong; Tuereci, Oezlem; Tompkins, Kristin R.; Lyke, Kirsten E.; Raabe, Vanessa; Dormitzer, Philip R.; Jansen, Kathrin U.; Sahin, Ugur; Gruber, William C.New England Journal of Medicine (2020), 383 (25), 2439-2450CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and the resulting disease, coronavirus disease 2019 (Covid-19), have spread to millions of persons worldwide. Multiple vaccine candidates are under development, but no vaccine is currently available. Interim safety and immunogenicity data about the vaccine candidate BNT162b1 in younger adults have been reported previously from trials in Germany and the United States. METHODS: In an ongoing, placebo-controlled, observer-blinded, dose-escalation, phase 1 trial conducted in the United States, we randomly assigned healthy adults 18 to 55 years of age and those 65 to 85 years of age to receive either placebo or one of two lipid nanoparticle-formulated, nucleoside-modified RNA vaccine candidates: BNT162b1, which encodes a secreted trimerized SARS-CoV-2 receptor-binding domain; or BNT162b2, which encodes a membrane-anchored SARS-CoV-2 fulllength spike, stabilized in the prefusion conformation. The primary outcome was safety (e.g., local and systemic reactions and adverse events); immunogenicity was a secondary outcome. Trial groups were defined according to vaccine candidate, age of the participants, and vaccine dose level (10μg, 20μg, 30μg, and 100μg). In all groups but one, participants received two doses, with a 21-day interval between doses; in one group (100μg of BNT162b1), participants received one dose. RESULTS: A total of 195 participants underwent randomization. In each of 13 groups of 15 participants, 12 participants received vaccine and 3 received placebo. BNT162b2 was assocd. with a lower incidence and severity of systemic reactions than BNT162b1, particularly in older adults. In both younger and older adults, the two vaccine candidates elicited similar dose-dependent SARS-CoV-2-neutralizing geometric mean titers, which were similar to or higher than the geometric mean titer of a panel of SARS-CoV-2 convalescent serum samples. CONCLUSIONS The safety and immunogenicity data from this U.S. phase 1 trial of two vaccine candidates in younger and older adults, added to earlier interim safety and immunogenicity data regarding BNT162b1 in younger adults from trials in Germany and the United States, support the selection of BNT162b2 for advancement to a pivotal phase 2-3 safety and efficacy evaluation.
- 20Funk, C. D.; Laferrière, C.; Ardakani, A. A Snapshot of the Global Race for Vaccines Targeting SARS-CoV-2 and the COVID-19 Pandemic. Front. Pharmacol. 2020, 11, 11, DOI: 10.3389/fphar.2020.00937
- 21Krammer, F. SARS-CoV-2 Vaccines in Development. Nature 2020, 586 (7830), 516– 527, DOI: 10.1038/s41586-020-2798-3[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVyhs73N&md5=05f069b7aa258af442f848415a24c865SARS-CoV-2 vaccines in developmentKrammer, FlorianNature (London, United Kingdom) (2020), 586 (7830), 516-527CODEN: NATUAS; ISSN:0028-0836. (Nature Research)A review. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in late 2019 in China and is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. To mitigate the effects of the virus on public health, the economy and society, a vaccine is urgently needed. Here, the development of vaccines against SARS-CoV-2 is reviewed. Development was initiated when the genetic sequence of the virus became available in early Jan. 2020, and has moved at an unprecedented speed: a phase I trial started in March 2020 and there are currently more than 180 vaccines at various stages of development. Data from phase I and phase II trials are already available for several vaccine candidates, and many have moved into phase III trials. The data available so far suggest that effective and safe vaccines might become available within months, rather than years.
- 22Thanh Le, T.; Andreadakis, Z.; Kumar, A.; Gómez Román, R.; Tollefsen, S.; Saville, M.; Mayhew, S. The COVID-19 Vaccine Development Landscape. Nat. Rev. Drug Discovery 2020, 19 (5), 305– 306, DOI: 10.1038/d41573-020-00073-5[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXnslOht7g%253D&md5=4be48c76fe66c0c69d24bebf2137eac3The COVID-19 vaccine development landscapeThanh Le, Tung; Andreadakis, Zacharias; Kumar, Arun; Gomez Roman, Raul; Tollefsen, Stig; Saville, Melanie; Mayhew, StephenNature Reviews Drug Discovery (2020), 19 (5), 305-306CODEN: NRDDAG; ISSN:1474-1776. (Nature Research)A review on the development of vaccines for SARS-CoV-2. Topics include: the type of vaccines under development; the current stage of development; and the current no. of vaccine projects worldwide.
- 23Fausther-Bovendo, H.; Kobinger, G. P. Pre-Existing Immunity against Ad Vectors. Hum. Vaccines Immunother. 2014, 10 (10), 2875– 2884, DOI: 10.4161/hv.29594
- 24Thacker, E. E.; Timares, L.; Matthews, Q. L. Strategies to Overcome Host Immunity to Adenovirus Vectors in Vaccine Development. Expert Rev. Vaccines 2009, 8 (6), 761– 777, DOI: 10.1586/erv.09.29[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXms1Ort7g%253D&md5=9e29ff9e46cb488e385d1014f7946e02Strategies to overcome host immunity to adenovirus vectors in vaccine developmentThacker, Erin E.; Timares, Laura; Matthews, Qiana L.Expert Review of Vaccines (2009), 8 (6), 761-777CODEN: ERVXAX; ISSN:1476-0584. (Expert Reviews Ltd.)A review. The first clin. evaluations of adenovirus (Ad)-based vectors for gene therapy were initiated in the mid-1990s and led to great anticipation for future utility. However, excitement surrounding gene therapy, particularly Ad-based therapy, was diminished upon the death of Jesse Gelsinger, and recent discouraging results from the HIV vaccine STEP trial have brought efficacy and safety issues to the forefront again. Even so, Ad vectors are still considered among the safest and most effective vaccine vectors. Innate and pre-existing immunity to Ad mediate much of the acute toxicities and reduced therapeutic efficacies obsd. following vaccination with this vector. Thus, innovative strategies must continue to be developed to reduce Ad-specific antigenicity and immune recognition. This review provides an overview and critique of the most promising strategies, including results from preclin. trials in mice and nonhuman primates, which aim to revive the future of Ad-based vaccines.
- 25Moyle, P. M.; Toth, I. Modern Subunit Vaccines: Development, Components, and Research Opportunities. ChemMedChem 2013, 8 (3), 360– 376, DOI: 10.1002/cmdc.201200487[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXntFyquw%253D%253D&md5=1a572bf2e8b4f53bac91a6b687a3cff4Modern Subunit Vaccines: Development, Components, and Research OpportunitiesMoyle, Peter Michael; Toth, IstvanChemMedChem (2013), 8 (3), 360-376CODEN: CHEMGX; ISSN:1860-7179. (Wiley-VCH Verlag GmbH & Co. KGaA)A review. Traditional vaccines, based on the administration of killed or attenuated microorganisms, have proven to be among the most effective methods for disease prevention. Safety issues related to administering these complex mixts., however, prevent their universal application. Through identification of the microbial components responsible for protective immunity, vaccine formulations can be simplified, enabling mol.-level vaccine characterization, improved safety profiles, prospects to develop new high-priority vaccines (e.g. for HIV, tuberculosis, and malaria), and the opportunity for extensive vaccine component optimization. This subunit approach, however, comes at the expense of decreased immunity, requiring the addn. of immunostimulatory agents (adjuvants). As few adjuvants are currently used in licensed vaccines, adjuvant development represents an exciting area for medicinal chemists to play a role in the future of vaccine development. In addn., immune responses can be further customized though optimization of delivery systems, tuning the size of particulate vaccines, targeting specific cells of the immune system (e.g. dendritic cells), and adding components to aid vaccine efficacy in whole immunized populations (e.g. promiscuous T-helper epitopes). Herein the authors review the current state of the art and future direction in subunit vaccine development, with a focus on the described components and their potential to steer the immune response toward a desired response.
- 26Wang, N.; Shang, J.; Jiang, S.; Du, L. Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses. Front. Microbiol. 2020, 11, 11, DOI: 10.3389/fmicb.2020.00298
- 27Chattopadhyay, S.; Chen, J.-Y.; Chen, H.-W.; Hu, C.-M. J. Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation. Nanotheranostics 2017, 1 (3), 244– 260, DOI: 10.7150/ntno.19796[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC1M7ks1SgsA%253D%253D&md5=b2fc2f2c624f586c8bd909a64a00501dNanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune PotentiationChattopadhyay Saborni; Chen Jui-Yi; Hu Che-Ming Jack; Chattopadhyay Saborni; Chen Hui-Wen; Chen Hui-Wen; Hu Che-Ming JackNanotheranostics (2017), 1 (3), 244-260 ISSN:.Synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. These nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. As a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. This review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. It provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. As understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. These nanoformulations are poised to provide solutions towards pressing and emerging human diseases.
- 28López-Sagaseta, J.; Malito, E.; Rappuoli, R.; Bottomley, M. J. Self-Assembling Protein Nanoparticles in the Design of Vaccines. Comput. Struct. Biotechnol. J. 2016, 14, 58– 68, DOI: 10.1016/j.csbj.2015.11.001[Crossref], [PubMed], [CAS], Google Scholar28https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFWhs7vP&md5=0e1c8a6716b294cc150e08baf6e2333eSelf-assembling protein nanoparticles in the design of vaccinesLopez-Sagaseta, Jacinto; Malito, Enrico; Rappuoli, Rino; Bottomley, Matthew J.Computational and Structural Biotechnology Journal (2016), 14 (), 58-68CODEN: CSBJAC; ISSN:2001-0370. (Elsevier B.V.)For over 100 years, vaccines have been one of the most effective medical interventions for reducing infectious disease, and are estd. to save millions of lives globally each year. Nevertheless, many diseases are not yet preventable by vaccination. This large unmet medical need demands further research and the development of novel vaccines with high efficacy and safety. Compared to the 19th and early 20th century vaccines that were made of killed, inactivated, or live-attenuated pathogens, modern vaccines contg. isolated, highly purified antigenic protein subunits are safer but tend to induce lower levels of protective immunity. One strategy to overcome the latter is to design antigen nanoparticles: assemblies of polypeptides that present multiple copies of subunit antigens in well-ordered arrays with defined orientations that can potentially mimic the repetitiveness, geometry, size, and shape of the natural host-pathogen surface interactions. Such nanoparticles offer a collective strength of multiple binding sites (avidity) and can provide improved antigen stability and immunogenicity. Several exciting advances have emerged lately, including preclin. evidence that this strategy may be applicable for the development of innovative new vaccines, for example, protecting against influenza, human immunodeficiency virus, and respiratory syncytial virus. Here, we provide a concise review of a crit. selection of data that demonstrate the potential of this field. In addn., we highlight how the use of self-assembling protein nanoparticles can be effectively combined with the emerging discipline of structural vaccinol. for max. impact in the rational design of vaccine antigens.
- 29Zhao, L.; Seth, A.; Wibowo, N.; Zhao, C.-X.; Mitter, N.; Yu, C.; Middelberg, A. P. J. Nanoparticle Vaccines. Vaccine 2014, 32 (3), 327– 337, DOI: 10.1016/j.vaccine.2013.11.069[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2c3isFagug%253D%253D&md5=1e95159344066200fb35657c68795561Nanoparticle vaccinesZhao Liang; Seth Arjun; Wibowo Nani; Zhao Chun-Xia; Yu Chengzhong; Mitter Neena; Middelberg Anton P JVaccine (2014), 32 (3), 327-37 ISSN:.Nanotechnology increasingly plays a significant role in vaccine development. As vaccine development orientates toward less immunogenic "minimalist" compositions, formulations that boost antigen effectiveness are increasingly needed. The use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release. A number of nanoparticle vaccines varying in composition, size, shape, and surface properties have been approved for human use and the number of candidates is increasing. However, challenges remain due to a lack of fundamental understanding regarding the in vivo behavior of nanoparticles, which can operate as either a delivery system to enhance antigen processing and/or as an immunostimulant adjuvant to activate or enhance immunity. This review provides a broad overview of recent advances in prophylactic nanovaccinology. Types of nanoparticles used are outlined and their interaction with immune cells and the biosystem are discussed. Increased knowledge and fundamental understanding of nanoparticle mechanism of action in both immunostimulatory and delivery modes, and better understanding of in vivo biodistribution and fate, are urgently required, and will accelerate the rational design of nanoparticle-containing vaccines.
- 30Kanekiyo, M.; Bu, W.; Joyce, M. G.; Meng, G.; Whittle, J. R. R.; Baxa, U.; Yamamoto, T.; Narpala, S.; Todd, J.-P.; Rao, S. S.; McDermott, A. B.; Koup, R. A.; Rossmann, M. G.; Mascola, J. R.; Graham, B. S.; Cohen, J. I.; Nabel, G. J. Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site. Cell 2015, 162 (5), 1090– 1100, DOI: 10.1016/j.cell.2015.07.043[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlCis7zO&md5=a83f8141bfe7a23f5a5950adffca0f59Rational design of an Epstein-Barr virus vaccine targeting the receptor-binding siteKanekiyo, Masaru; Bu, Wei; Joyce, M. Gordon; Meng, Geng; Whittle, James R. R.; Baxa, Ulrich; Yamamoto, Takuya; Narpala, Sandeep; Todd, John-Paul; Rao, Srinivas S.; McDermott, Adrian B.; Koup, Richard A.; Rossmann, Michael G.; Mascola, John R.; Graham, Barney S.; Cohen, Jeffrey I.; Nabel, Gary J.Cell (Cambridge, MA, United States) (2015), 162 (5), 1090-1100CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Epstein-Barr virus (EBV) represents a major global health problem. Though it is assocd. with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a sym. array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to sol. gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.
- 31Walls, A. C.; Fiala, B.; Schäfer, A.; Wrenn, S.; Pham, M. N.; Murphy, M.; Tse, L. V.; Shehata, L.; O’Connor, M. A.; Chen, C.; Navarro, M. J.; Miranda, M. C.; Pettie, D.; Ravichandran, R.; Kraft, J. C.; Ogohara, C.; Palser, A.; Chalk, S.; Lee, E. C.; Guerriero, K.; Kepl, E.; Chow, C. M.; Sydeman, C.; Hodge, E. A.; Brown, B.; Fuller, J. T.; Dinnon, K. H.; Gralinski, L. E.; Leist, S. R.; Gully, K. L.; Lewis, T. B.; Guttman, M.; Chu, H. Y.; Lee, K. K.; Fuller, D. H.; Baric, R. S.; Kellam, P.; Carter, L.; Pepper, M.; Sheahan, T. P.; Veesler, D.; King, N. P. Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell 2020, 183, 1367, DOI: 10.1016/j.cell.2020.10.043[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit12ks7jL&md5=ba75e5c3a28f919f69dc5d3bf06c024fElicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2Walls, Alexandra C.; Fiala, Brooke; Schafer, Alexandra; Wrenn, Samuel; Pham, Minh N.; Murphy, Michael; Tse, Longping V.; Shehata, Laila; O'Connor, Megan A.; Chen, Chengbo; Navarro, Mary Jane; Miranda, Marcos C.; Pettie, Deleah; Ravichandran, Rashmi; Kraft, John C.; Ogohara, Cassandra; Palser, Anne; Chalk, Sara; Lee, E-Chiang; Guerriero, Kathryn; Kepl, Elizabeth; Chow, Cameron M.; Sydeman, Claire; Hodge, Edgar A.; Brown, Brieann; Fuller, Jim T.; Dinnon, Kenneth H., III; Gralinski, Lisa E.; Leist, Sarah R.; Gully, Kendra L.; Lewis, Thomas B.; Guttman, Miklos; Chu, Helen Y.; Lee, Kelly K.; Fuller, Deborah H.; Baric, Ralph S.; Kellam, Paul; Carter, Lauren; Pepper, Marion; Sheahan, Timothy P.; Veesler, David; King, Neil P.Cell (Cambridge, MA, United States) (2020), 183 (5), 1367-1382.e17CODEN: CELLB5; ISSN:0092-8674. (Cell Press)A safe, effective, and scalable vaccine is needed to halt the ongoing SARS-CoV-2 pandemic. We describe the structure-based design of self-assembling protein nanoparticle immunogens that elicit potent and protective antibody responses against SARS-CoV-2 in mice. The nanoparticle vaccines display 60 SARS-CoV-2 spike receptor-binding domains (RBDs) in a highly immunogenic array and induce neutralizing antibody titers 10-fold higher than the prefusion-stabilized spike despite a 5-fold lower dose. Antibodies elicited by the RBD nanoparticles target multiple distinct epitopes, suggesting they may not be easily susceptible to escape mutations, and exhibit a lower binding:neutralizing ratio than convalescent human sera, which may minimize the risk of vaccine-assocd. enhanced respiratory disease. The high yield and stability of the assembled nanoparticles suggest that manuf. of the nanoparticle vaccines will be highly scalable. These results highlight the utility of robust antigen display platforms and have launched cGMP manufg. efforts to advance the SARS-CoV-2-RBD nanoparticle vaccine into the clinic.
- 32Zhang, B.; Chao, C. W.; Tsybovsky, Y.; Abiona, O. M.; Hutchinson, G. B.; Moliva, J. I.; Olia, A. S.; Pegu, A.; Phung, E.; Stewart-Jones, G. B. E.; Verardi, R.; Wang, L.; Wang, S.; Werner, A.; Yang, E. S.; Yap, C.; Zhou, T.; Mascola, J. R.; Sullivan, N. J.; Graham, B. S.; Corbett, K. S.; Kwong, P. D. A Platform Incorporating Trimeric Antigens into Self-Assembling Nanoparticles Reveals SARS-CoV-2-Spike Nanoparticles to Elicit Substantially Higher Neutralizing Responses than Spike Alone. Sci. Rep. 2020, in press. DOI: 10.1038/s41598-020-74949-2 .
- 33Kanekiyo, M.; Wei, C.-J.; Yassine, H. M.; McTamney, P. M.; Boyington, J. C.; Whittle, J. R. R.; Rao, S. S.; Kong, W.-P.; Wang, L.; Nabel, G. J. Self-Assembling Influenza Nanoparticle Vaccines Elicit Broadly Neutralizing H1N1 Antibodies. Nature 2013, 499 (7456), 102– 106, DOI: 10.1038/nature12202[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXotFSmtrw%253D&md5=473f68a2ad8d50bc94f8e3cba5f10110Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodiesKanekiyo, Masaru; Wei, Chih-Jen; Yassine, Hadi M.; McTamney, Patrick M.; Boyington, Jeffrey C.; Whittle, James R. R.; Rao, Srinivas S.; Kong, Wing-Pui; Wang, Lingshu; Nabel, Gary J.Nature (London, United Kingdom) (2013), 499 (7456), 102-106CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technol. and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here the authors use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited hemagglutination inhibition antibody titers more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
- 34Yassine, H. M.; Boyington, J. C.; McTamney, P. M.; Wei, C.-J.; Kanekiyo, M.; Kong, W.-P.; Gallagher, J. R.; Wang, L.; Zhang, Y.; Joyce, M. G.; Lingwood, D.; Moin, S. M.; Andersen, H.; Okuno, Y.; Rao, S. S.; Harris, A. K.; Kwong, P. D.; Mascola, J. R.; Nabel, G. J.; Graham, B. S. Hemagglutinin-Stem Nanoparticles Generate Heterosubtypic Influenza Protection. Nat. Med. 2015, 21 (9), 1065– 1070, DOI: 10.1038/nm.3927[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtlynsL7N&md5=1a68e5764008fddbe28cada259ea5eb1Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protectionYassine, Hadi M.; Boyington, Jeffrey C.; McTamney, Patrick M.; Wei, Chih-Jen; Kanekiyo, Masaru; Kong, Wing-Pui; Gallagher, John R.; Wang, Lingshu; Zhang, Yi; Joyce, M. Gordon; Lingwood, Daniel; Moin, Syed M.; Andersen, Hanne; Okuno, Yoshinobu; Rao, Srinivas S.; Harris, Audray K.; Kwong, Peter D.; Mascola, John R.; Nabel, Gary J.; Graham, Barney S.Nature Medicine (New York, NY, United States) (2015), 21 (9), 1065-1070CODEN: NAMEFI; ISSN:1078-8956. (Nature Publishing Group)The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, detn. of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy anal. of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of Ig from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.
- 35Sliepen, K.; Ozorowski, G.; Burger, J. A.; van Montfort, T.; Stunnenberg, M.; LaBranche, C.; Montefiori, D. C.; Moore, J. P.; Ward, A. B.; Sanders, R. W. Presenting Native-like HIV-1 Envelope Trimers on Ferritin Nanoparticles Improves Their Immunogenicity. Retrovirology 2015, 12 (1), 82, DOI: 10.1186/s12977-015-0210-4[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XmtFKjsLc%253D&md5=f80be2e5c8bb712e06b42162f72a5fe8Presenting native-like HIV-1 envelope trimers on ferritin nanoparticles improves their immunogenicitySliepen, Kwinten; Ozorowski, Gabriel; Burger, Judith A.; van Montfort, Thijs; Stunnenberg, Melissa; La Branche, Celia; Montefiori, David C.; Moore, John P.; Ward, Andrew B.; Sanders, Rogier W.Retrovirology (2015), 12 (), 82/1-82/5CODEN: RETRBO; ISSN:1742-4690. (BioMed Central Ltd.)Background: Presenting vaccine antigens in particulate form can improve their immunogenicity by enhancing B cell activation. Findings: We describe ferritin-based protein nanoparticles that display multiple copies of native-like HIV-1 envelope glycoprotein trimers (BG505 SOSIP.664). Trimer-bearing nanoparticles were significantly more immunogenic than trimers in both mice and rabbits. Furthermore, rabbits immunized with the trimer-bearing nanoparticles induced significantly higher neutralizing antibody responses against most tier 1A viruses, and higher responses (but not significantly), to several tier 1B viruses and the autologous tier 2 virus than when the same trimers were delivered as sol. proteins. Conclusions: This or other nanoparticle designs may be practical ways to improve the immunogenicity of envelope glycoprotein trimers.
- 36He, L.; de Val, N.; Morris, C. D.; Vora, N.; Thinnes, T. C.; Kong, L.; Azadnia, P.; Sok, D.; Zhou, B.; Burton, D. R.; Wilson, I. A.; Nemazee, D.; Ward, A. B.; Zhu, J. Presenting Native-like Trimeric HIV-1 Antigens with Self-Assembling Nanoparticles. Nat. Commun. 2016, 7 (1), 12041, DOI: 10.1038/ncomms12041[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XhtFSqsbzL&md5=ceccd03283a87943cc13ea2ca9a13a8aPresenting native-like trimeric HIV-1 antigens with self-assembling nanoparticlesHe, Linling; de Val, Natalia; Morris, Charles D.; Vora, Nemil; Thinnes, Therese C.; Kong, Leopold; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Wilson, Ian A.; Nemazee, David; Ward, Andrew B.; Zhu, JiangNature Communications (2016), 7 (), 12041CODEN: NCAOBW; ISSN:2041-1723. (Nature Publishing Group)Structures of BG505 SOSIP.664 trimer in complex with broadly neutralizing antibodies (bNAbs) have revealed the crit. role of trimeric context for immune recognition of HIV-1. Presentation of trimeric HIV-1 antigens on nanoparticles may thus provide promising vaccine candidates. Here we report the rational design, structural anal. and antigenic evaluation of HIV-1 trimer-presenting nanoparticles. We first demonstrate that both V1V2 and gp120 can be presented in native-like trimeric conformations on nanoparticles. We then design nanoparticles presenting various forms of stabilized gp140 trimer based on ferritin and a large, 60-meric E2p that displays 20 spikes mimicking virus-like particles (VLPs). Particle assembly is confirmed by electron microscopy (EM), while antigenic profiles are generated using representative bNAbs and non-NAbs. Lastly, we demonstrate high-yield gp140 nanoparticle prodn. and robust stimulation of B cells carrying cognate VRC01 receptors by gp120 and gp140 nanoparticles. Together, our study provides an arsenal of multivalent immunogens for HIV-1 vaccine development.
- 37Kamp, H. D.; Swanson, K. A.; Wei, R. R.; Dhal, P. K.; Dharanipragada, R.; Kern, A.; Sharma, B.; Sima, R.; Hajdusek, O.; Hu, L. T.; Wei, C.-J.; Nabel, G. J. Design of a Broadly Reactive Lyme Disease Vaccine. npj Vaccines 2020, 5 (1), 33, DOI: 10.1038/s41541-020-0183-8[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXos1Sgtbc%253D&md5=7cce8606c3451ba35cb08da52e45697dDesign of a broadly reactive Lyme disease vaccineKamp, Heather D.; Swanson, Kurt A.; Wei, Ronnie R.; Dhal, Pradeep K.; Dharanipragada, Ram; Kern, Aurelie; Sharma, Bijaya; Sima, Radek; Hajdusek, Ondrej; Hu, Linden T.; Wei, Chih-Jen; Nabel, Gary J.npj Vaccines (2020), 5 (1), 33CODEN: VACCBC; ISSN:2059-0105. (Nature Research)Abstr.: A growing global health concern, Lyme disease has become the most common tick-borne disease in the United States and Europe. Caused by the bacterial spirochete Borrelia burgdorferi sensu lato (sl), this disease can be debilitating if not treated promptly. Because diagnosis is challenging, prevention remains a priority; however, a previously licensed vaccine is no longer available to the public. Here, we designed a six component vaccine that elicits antibody (Ab) responses against all Borrelia strains that commonly cause Lyme disease in humans. The outer surface protein A (OspA) of Borrelia was fused to a bacterial ferritin to generate self-assembling nanoparticles. OspA-ferritin nanoparticles elicited durable high titer Ab responses to the seven major serotypes in mice and non-human primates at titers higher than a previously licensed vaccine. This response was durable in rhesus macaques for more than 6 mo. Vaccination with adjuvanted OspA-ferritin nanoparticles stimulated protective immunity from both B. burgdorferi and B. afzelii infection in a tick-fed murine challenge model. This multivalent Lyme vaccine offers the potential to limit the spread of Lyme disease.
- 38Swanson, K. A.; Rainho-Tomko, J. N.; Williams, Z. P.; Lanza, L.; Peredelchuk, M.; Kishko, M.; Pavot, V.; Alamares-Sapuay, J.; Adhikarla, H.; Gupta, S.; Chivukula, S.; Gallichan, S.; Zhang, L.; Jackson, N.; Yoon, H.; Edwards, D.; Wei, C.-J.; Nabel, G. J. A Respiratory Syncytial Virus (RSV) F Protein Nanoparticle Vaccine Focuses Antibody Responses to a Conserved Neutralization Domain. Sci. Immunol. 2020, 5 (47), eaba6466 DOI: 10.1126/sciimmunol.aba6466
- 39Cho, K. J.; Shin, H. J.; Lee, J.-H.; Kim, K.-J.; Park, S. S.; Lee, Y.; Lee, C.; Park, S. S.; Kim, K. H. The Crystal Structure of Ferritin from Helicobacter Pylori Reveals Unusual Conformational Changes for Iron Uptake. J. Mol. Biol. 2009, 390 (1), 83– 98, DOI: 10.1016/j.jmb.2009.04.078[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXnt1Gksr8%253D&md5=b6c553c09b67386cc27350f1994aadeaThe Crystal Structure of Ferritin from Helicobacter pylori Reveals Unusual Conformational Changes for Iron UptakeCho, Ki Joon; Shin, Hye Jeong; Lee, Ji-Hye; Kim, Kyung-Jin; Park, Sarah S.; Lee, Youngmi; Lee, Cheolju; Park, Sung Soo; Kim, Kyung HyunJournal of Molecular Biology (2009), 390 (1), 83-98CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)The crystal structure of recombinant ferritin from Helicobacter pylori has been detd. in its apo, low-iron-bound, intermediate, and high-iron-bound states. Similar to other members of the ferritin family, the bacterial ferritin assembles as a spherical protein shell of 24 subunits, each of which folds into a four-α-helix bundle. Significant conformational changes were obsd. at the BC loop and the entrance of the 4-fold symmetry channel in the intermediate and high-iron-bound states, whereas no change was found in the apo and low-iron-bound states. The imidazole rings of His149 at the channel entrance undergo conformational changes that bear resemblance to heme configuration and are directly coupled to axial translocation of Fe ions through the 4-fold channel. Our results provide the first structural evidence of the translocation of Fe ions through the 4-fold channel in prokaryotes and the transition from a protein-dominated process to a mineral-surface-dominated process during biomineralization.
- 40Biswas, P.; Trozado, C.; Lee, J.; Schwartz, R. M. Development of a Mammalian Cell Culture Process for Rapid Clinical-Scale Production of Novel Influenza Nanoparticle Vaccines. BMC Proc. 2015, 9 (S9), O12, DOI: 10.1186/1753-6561-9-S9-O12
- 41Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zivanov, J.; Neufeldt, C. J.; Cerikan, B.; Lu, J. M.; Peukes, J.; Xiong, X.; Kräusslich, H.-G.; Scheres, S. H. W.; Bartenschlager, R.; Briggs, J. A. G. Structures and Distributions of SARS-CoV-2 Spike Proteins on Intact Virions. Nature 2020, 588, 498, DOI: 10.1038/s41586-020-2665-2[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXit1Ggt7jM&md5=fa6e31604ece80a7c0934d689642a5c0Structures and distributions of SARS-CoV-2 spike proteins on intact virionsKe, Zunlong; Oton, Joaquin; Qu, Kun; Cortese, Mirko; Zila, Vojtech; McKeane, Lesley; Nakane, Takanori; Zivanov, Jasenko; Neufeldt, Christopher J.; Cerikan, Berati; Lu, John M.; Peukes, Julia; Xiong, Xiaoli; Krausslich, Hans-Georg; Scheres, Sjors H. W.; Bartenschlager, Ralf; Briggs, John A. G.Nature (London, United Kingdom) (2020), 588 (7838), 498-502CODEN: NATUAS; ISSN:0028-0836. (Nature Research)Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of sol., overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. We applied cryo-electron microscopy and tomog. to image intact SARS-CoV-2 virions and det. the high-resoln. structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
- 42Turoňová, B.; Sikora, M.; Schürmann, C.; Hagen, W. J. H.; Welsch, S.; Blanc, F. E. C.; von Bülow, S.; Gecht, M.; Bagola, K.; Hörner, C.; van Zandbergen, G.; Landry, J.; de Azevedo, N. T. D.; Mosalaganti, S.; Schwarz, A.; Covino, R.; Mühlebach, M. D.; Hummer, G.; Krijnse Locker, J.; Beck, M. In Situ Structural Analysis of SARS-CoV-2 Spike Reveals Flexibility Mediated by Three Hinges. Science 2020, 370, 203 DOI: 10.1126/science.abd5223[Crossref], [PubMed], [CAS], Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVCrsbbO&md5=6464149155235d90f7a9d0972a50f4fbIn situ structural analysis of SARS-CoV-2 spike reveals flexibility mediated by three hingesTuronova, Beata; Sikora, Mateusz; Schuermann, Christoph; Hagen, Wim J. H.; Welsch, Sonja; Blanc, Florian E. C.; von Buelow, Soeren; Gecht, Michael; Bagola, Katrin; Hoerner, Cindy; van Zandbergen, Ger; Landry, Jonathan; de Azevedo, Nayara Trevisan Doimo; Mosalaganti, Shyamal; Schwarz, Andre; Covino, Roberto; Muehlebach, Michael D.; Hummer, Gerhard; Krijnse Locker, Jacomine; Beck, MartinScience (Washington, DC, United States) (2020), 370 (6513), 203-208CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo-electron tomog., subtomogram averaging, and mol. dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. The stalk domain of S contains 3 hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
- 43Klein, S.; Cortese, M.; Winter, S. L.; Wachsmuth-Melm, M.; Neufeldt, C. J.; Cerikan, B.; Stanifer, M. L.; Boulant, S.; Bartenschlager, R.; Chlanda, P. SARS-CoV-2 Structure and Replication Characterized by in Situ Cryo-Electron Tomography. Nat. Commun. 2020, 5885, DOI: 10.1038/s41467-020-19619-7[Crossref], [PubMed], [CAS], Google Scholar43https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisVSgt7fL&md5=1f434e5bc92d9453d6b7f7b719e00a44SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomographyKlein, Steffen; Cortese, Mirko; Winter, Sophie L.; Wachsmuth-Melm, Moritz; Neufeldt, Christopher J.; Cerikan, Berati; Stanifer, Megan L.; Boulant, Steeve; Bartenschlager, Ralf; Chlanda, PetrNature Communications (2020), 11 (1), 5885CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)Abstr.: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic β-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report crit. insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomog. and subtomogram averaging. We directly visualize RNA filaments inside the double membrane vesicles, compartments assocd. with viral replication. The RNA filaments show a diam. consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We report that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple sep. vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.
- 44Zamecnik, C. R.; Rajan, J. V.; Yamauchi, K. A.; Mann, S. A.; Loudermilk, R. P.; Sowa, G. M.; Zorn, K. C.; Alvarenga, B. D.; Gaebler, C.; Caskey, M.; Stone, M.; Norris, P. J.; Gu, W.; Chiu, C. Y.; Ng, D.; Byrnes, J. R.; Zhou, X. X.; Wells, J. A.; Robbiani, D. F.; Nussenzweig, M. C.; DeRisi, J. L.; Wilson, M. R. ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Reports Med. 2020, in press. DOI: 10.1016/j.xcrm.2020.100123 .
- 45Li, Y.; Ma, M.; Lei, Q.; Wang, F.; Sun, Z.; Fan, X.; Tao, S. Linear Epitope Landscape of SARS-CoV-2 Spike Protein Constructed from 1,051 COVID-19 Patients. medRxiv 2020. DOI: 10.1101/2020.07.13.20152587 .Google ScholarThere is no corresponding record for this reference.
- 46Eckert, D. M.; Malashkevich, V. N.; Kim, P. S. Crystal Structure of GCN4-PIQI, a Trimeric Coiled Coil with Buried Polar Residues. J. Mol. Biol. 1998, 284 (4), 859– 865, DOI: 10.1006/jmbi.1998.2214[Crossref], [PubMed], [CAS], Google Scholar46https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1MXivFansg%253D%253D&md5=34feaf7ec906e7091998011bca277feeCrystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residuesEckert, Debra M.; Malashkevich, Vladimir N.; Kim, Peter S.Journal of Molecular Biology (1998), 284 (4), 859-865CODEN: JMOBAK; ISSN:0022-2836. (Academic Press)Coiled coils consist of two or more α-helixes wrapped around each other with a superhelical twist. The interfaces between helixes of a coiled coil are formed by hydrophobic amino acid residues packed in a "knobs-into-holes" arrangement. Most naturally occurring coiled coils, however, also contain buried polar residues, as do the cores of the majority of naturally occurring globular proteins. Two common buried polar residues in both dimeric and trimeric coiled coils are asparagine and glutamine. In dimeric coiled coils, buried asparagine, but not glutamine, residues have been shown to confer specificity of oligomerization. We have placed a glutamine residue in the otherwise hydrophobic interior of a stable trimeric coiled coil, GCN4-pII, to study the effect of this buried polar residue in a trimeric coiled-coil environment. The resulting peptide, GCN4-pIQI, is a discrete, trimeric coiled coil with a lower stability than GCN4-pII. The crystal structure detd. to 1.8 Å shows that GCN4-pIQI is a trimeric coiled coil with a chloride ion coordinated by one buried glutamine residue from each monomer. (c) 1998 Academic Press.
- 47Crawford, K. H. D.; Eguia, R.; Dingens, A. S.; Loes, A. N.; Malone, K. D.; Wolf, C. R.; Chu, H. Y.; Tortorici, M. A.; Veesler, D.; Murphy, M.; Pettie, D.; King, N. P.; Balazs, A. B.; Bloom, J. D. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 2020, 12 (5), 513, DOI: 10.3390/v12050513[Crossref], [CAS], Google Scholar47https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtF2gtLnF&md5=da4f1a5b2f0a2e4e52432c3d2a0798f1Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 spike protein for neutralization assaysCrawford, Katharine H. D.; Eguia, Rachel; Dingens, Adam S.; Loes, Andrea N.; Malone, Keara D.; Wolf, Caitlin R.; Chu, Helen Y.; Tortorici, M. Alejandra; Veesler, David; Murphy, Michael; Pettie, Deleah; King, Neil P.; Balazs, Alejandro B.; Bloom, Jesse D.Viruses (2020), 12 (5), 513CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key exptl. reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.
- 48Rogers, T. F.; Zhao, F.; Huang, D.; Beutler, N.; Burns, A.; He, W.; Limbo, O.; Smith, C.; Song, G.; Woehl, J.; Yang, L.; Abbott, R. K.; Callaghan, S.; Garcia, E.; Hurtado, J.; Parren, M.; Peng, L.; Ramirez, S.; Ricketts, J.; Ricciardi, M. J.; Rawlings, S. A.; Wu, N. C.; Yuan, M.; Smith, D. M.; Nemazee, D.; Teijaro, J. R.; Voss, J. E.; Wilson, I. A.; Andrabi, R.; Briney, B.; Landais, E.; Sok, D.; Jardine, J. G.; Burton, D. R. Isolation of Potent SARS-CoV-2 Neutralizing Antibodies and Protection from Disease in a Small Animal Model. Science 2020, 369, 956 DOI: 10.1126/science.abc7520[Crossref], [PubMed], [CAS], Google Scholar48https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1GrsLjF&md5=8684025692cbe1934cef2bfee369f3d1Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal modelRogers, Thomas F.; Zhao, Fangzhu; Huang, Deli; Beutler, Nathan; Burns, Alison; He, Wan-ting; Limbo, Oliver; Smith, Chloe; Song, Ge; Woehl, Jordan; Yang, Linlin; Abbott, Robert K.; Callaghan, Sean; Garcia, Elijah; Hurtado, Jonathan; Parren, Mara; Peng, Linghang; Ramirez, Sydney; Ricketts, James; Ricciardi, Michael J.; Rawlings, Stephen A.; Wu, Nicholas C.; Yuan, Meng; Smith, Davey M.; Nemazee, David; Teijaro, John R.; Voss, James E.; Wilson, Ian A.; Andrabi, Raiees; Briney, Bryan; Landais, Elise; Sok, Devin; Jardine, Joseph G.; Burton, Dennis R.Science (Washington, DC, United States) (2020), 369 (6506), 956-963CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Countermeasures to prevent and treat coronavirus disease 2019 (COVID-19) are a global health priority. We enrolled a cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-recovered participants, developed neutralization assays to investigate antibody responses, adapted our high-throughput antibody generation pipeline to rapidly screen more than 1800 antibodies, and established an animal model to test protection. We isolated potent neutralizing antibodies (nAbs) to two epitopes on the receptor binding domain (RBD) and to distinct non-RBD epitopes on the spike (S) protein. As indicated by maintained wt. and low lung viral titers in treated animals, the passive transfer of a nAb provides protection against disease in high-dose SARS-CoV-2 challenge in Syrian hamsters. The study suggests a role for nAbs in prophylaxis, and potentially therapy, of COVID-19. The nAbs also define protective epitopes to guide vaccine design.
- 49Amanat, F.; Stadlbauer, D.; Strohmeier, S.; Nguyen, T. H. O.; Chromikova, V.; McMahon, M.; Jiang, K.; Arunkumar, G. A.; Jurczyszak, D.; Polanco, J.; Bermudez-Gonzalez, M.; Kleiner, G.; Aydillo, T.; Miorin, L.; Fierer, D. S.; Lugo, L. A.; Kojic, E. M.; Stoever, J.; Liu, S. T. H.; Cunningham-Rundles, C.; Felgner, P. L.; Moran, T.; García-Sastre, A.; Caplivski, D.; Cheng, A. C.; Kedzierska, K.; Vapalahti, O.; Hepojoki, J. M.; Simon, V.; Krammer, F. A Serological Assay to Detect SARS-CoV-2 Seroconversion in Humans. Nat. Med. 2020, 26 (7), 1033– 1036, DOI: 10.1038/s41591-020-0913-5[Crossref], [PubMed], [CAS], Google Scholar49https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXptFyqsbY%253D&md5=6f5df9742dec5a6bdb94ac5ccaad043dA serological assay to detect SARS-CoV-2 seroconversion in humansAmanat, Fatima; Stadlbauer, Daniel; Strohmeier, Shirin; Nguyen, Thi H. O.; Chromikova, Veronika; McMahon, Meagan; Jiang, Kaijun; Arunkumar, Guha Asthagiri; Jurczyszak, Denise; Polanco, Jose; Bermudez-Gonzalez, Maria; Kleiner, Giulio; Aydillo, Teresa; Miorin, Lisa; Fierer, Daniel S.; Lugo, Luz Amarilis; Kojic, Erna Milunka; Stoever, Jonathan; Liu, Sean T. H.; Cunningham-Rundles, Charlotte; Felgner, Philip L.; Moran, Thomas; Garcia-Sastre, Adolfo; Caplivski, Daniel; Cheng, Allen C.; Kedzierska, Katherine; Vapalahti, Olli; Hepojoki, Jussi M.; Simon, Viviana; Krammer, FlorianNature Medicine (New York, NY, United States) (2020), 26 (7), 1033-1036CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)We describe a serol. ELISA for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma, and is amenable to scaling. Serol. assays are of crit. importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy, and investigate correlates of protection.
- 50Pallesen, J.; Wang, N.; Corbett, K. S.; Wrapp, D.; Kirchdoerfer, R. N.; Turner, H. L.; Cottrell, C. A.; Becker, M. M.; Wang, L.; Shi, W.; Kong, W.-P.; Andres, E. L.; Kettenbach, A. N.; Denison, M. R.; Chappell, J. D.; Graham, B. S.; Ward, A. B.; McLellan, J. S. Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (35), E7348– E7357, DOI: 10.1073/pnas.1707304114[Crossref], [PubMed], [CAS], Google Scholar50https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlWmsrfI&md5=3054f8054d852ed4192fd8f1fb6c870dImmunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigenPallesen, Jesper; Wang, Nianshuang; Corbett, Kizzmekia S.; Wrapp, Daniel; Kirchdoerfer, Robert N.; Turner, Hannah L.; Cottrell, Christopher A.; Becker, Michelle M.; Wang, Lingshu; Shi, Wei; Kong, Wing-Pui; Andres, Erica L.; Kettenbach, Arminja N.; Denison, Mark R.; Chappell, James D.; Graham, Barney S.; Ward, Andrew B.; McLellan, Jason S.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (35), E7348-E7357CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus that since its emergence in 2012 has caused outbreaks in human populations with case-fatality rates of ∼36%. As in other coronaviruses, the spike (S) glycoprotein of MERS-CoV mediates receptor recognition and membrane fusion and is the primary target of the humoral immune response during infection. Here we use structure-based design to develop a generalizable strategy for retaining coronavirus S proteins in the antigenically optimal prefusion conformation and demonstrate that our engineered immunogen is able to elicit high neutralizing antibody titers against MERS-CoV. We also detd. high-resoln. structures of the trimeric MERS-CoV S ectodomain in complex with G4, a stem-directed neutralizing antibody. The structures reveal that G4 recognizes a glycosylated loop that is variable among coronaviruses and they define four conformational states of the trimer wherein each receptor-binding domain is either tightly packed at the membrane-distal apex or rotated into a receptor-accessible conformation. Our studies suggest a potential mechanism for fusion initiation through sequential receptor-binding events and provide a foundation for the structure-based design of coronavirus vaccines.
- 51Kirchdoerfer, R. N.; Cottrell, C. A.; Wang, N.; Pallesen, J.; Yassine, H. M.; Turner, H. L.; Corbett, K. S.; Graham, B. S.; McLellan, J. S.; Ward, A. B. Pre-Fusion Structure of a Human Coronavirus Spike Protein. Nature 2016, 531 (7592), 118– 121, DOI: 10.1038/nature17200[Crossref], [PubMed], [CAS], Google Scholar51https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xjs1emtb8%253D&md5=f7c2b90e8963b9519816be6c40d0b756Pre-fusion structure of a human coronavirus spike proteinKirchdoerfer, Robert N.; Cottrell, Christopher A.; Wang, Nianshuang; Pallesen, Jesper; Yassine, Hadi M.; Turner, Hannah L.; Corbett, Kizzmekia S.; Graham, Barney S.; McLellan, Jason S.; Ward, Andrew B.Nature (London, United Kingdom) (2016), 531 (7592), 118-121CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is detd. in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a mol. understanding of its function and limiting development of effective interventions. Here, the authors present the 4.0-Å resoln. structure of the trimeric HKU1 S protein detd. using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the 2 protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.
- 52Chen, W.-H.; Hotez, P. J.; Bottazzi, M. E. Potential for Developing a SARS-CoV Receptor-Binding Domain (RBD) Recombinant Protein as a Heterologous Human Vaccine against Coronavirus Infectious Disease (COVID)-19. Hum. Vaccines Immunother. 2020, 16 (6), 1239– 1242, DOI: 10.1080/21645515.2020.1740560[Crossref], [PubMed], [CAS], Google Scholar52https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXosVegt74%253D&md5=27bfefa651ca8d65bad514f0caf4b4e6Potential for developing a SARS-CoV receptor-binding domain (RBD) recombinant protein as a heterologous human vaccine against coronavirus infectious disease (COVID)-19Chen, Wen-Hsiang; Hotez, Peter J.; Bottazzi, Maria ElenaHuman Vaccines & Immunotherapeutics (2020), 16 (6), 1239-1242CODEN: HVIUAK; ISSN:2164-554X. (Taylor & Francis Ltd.)A SARS-CoV receptor-binding domain (RBD) recombinant protein was developed and manufd. under current good manufg. practices in 2016. The protein, known as RBD219-N1 when formulated on Alhydrogel, induced high-level neutralizing antibodies and protective immunity with minimal immunopathol. in mice after a homologous virus challenge with SARS-CoV (MA15 strain). We examd. published evidence in support of whether the SARS-CoV RBD219-N1 could be repurposed as a heterologous vaccine against Coronavirus Infectious Disease (COVID)-19. Our findings include evidence that convalescent serum from SARS-CoV patients can neutralize SARS-CoV-2. Addnl., a review of published studies using monoclonal antibodies (mAbs) raised against SARS-CoV RBD and that neutralizes the SARS-CoV virus in vitro finds that some of these mAbs bind to the receptor-binding motif (RBM) within the RBD, while others bind to domains outside this region within RBD. This information is relevant and supports the possibility of developing a heterologous SARS-CoV RBD vaccine against COVID-19, esp. due to the finding that the overall high amino acid similarity (82%) between SARS-CoV and SARS-CoV-2 spike and RBD domains is not reflected in RBM amino acid similarity (59%). However, the high sequence similarity (94%) in the region outside of RBM offers the potential of conserved neutralizing epitopes between both viruses.
- 53Mulligan, M. J.; Lyke, K. E.; Kitchin, N.; Absalon, J.; Gurtman, A.; Lockhart, S.; Neuzil, K.; Raabe, V.; Bailey, R.; Swanson, K. A.; Li, P.; Koury, K.; Kalina, W.; Cooper, D.; Fontes-Garfias, C.; Shi, P.-Y.; Türeci, Ö.; Tompkins, K. R.; Walsh, E. E.; Frenck, R.; Falsey, A. R.; Dormitzer, P. R.; Gruber, W. C.; Şahin, U.; Jansen, K. U. Phase I/II Study of COVID-19 RNA Vaccine BNT162b1 in Adults. Nature 2020, 586 (7830), 589– 593, DOI: 10.1038/s41586-020-2639-4[Crossref], [PubMed], [CAS], Google Scholar53https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitVWjsr%252FM&md5=b0b354ee45f7171577edeec833a13c55Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adultsMulligan, Mark J.; Lyke, Kirsten E.; Kitchin, Nicholas; Absalon, Judith; Gurtman, Alejandra; Lockhart, Stephen; Neuzil, Kathleen; Raabe, Vanessa; Bailey, Ruth; Swanson, Kena A.; Li, Ping; Koury, Kenneth; Kalina, Warren; Cooper, David; Fontes-Garfias, Camila; Shi, Pei-Yong; Tureci, Ozlem; Tompkins, Kristin R.; Walsh, Edward E.; Frenck, Robert; Falsey, Ann R.; Dormitzer, Philip R.; Gruber, William C.; Sahin, Ugur; Jansen, Kathrin U.Nature (London, United Kingdom) (2020), 586 (7830), 589-593CODEN: NATUAS; ISSN:0028-0836. (Nature Research)In March 2020, the World Health Organization (WHO) declared coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a pandemic. With rapidly accumulating nos. of cases and deaths reported globally, a vaccine is urgently needed. We report the available safety, tolerability, and immunogenicity data from an ongoing placebo-controlled, observer-blinded dose-escalation study (ClinicalTrials.gov identifier NCT04368728) among 45 healthy adults (18-55 yr), who were randomized to receive 2 doses-sepd. by 21 days-of 10μg, 30μg, or 100μg of BNT162b1. BNT162b1 is a lipid-nanoparticle-formulated, nucleoside-modified mRNA vaccine that encodes the trimerized receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2. Local reactions and systemic events were dose-dependent, generally mild to moderate, and transient. A 2nd vaccination with 100μg was not administered because of the increased reactogenicity and a lack of meaningfully increased immunogenicity after a single dose compared with the 30-μg dose. RBD-binding IgG concns. and SARS-CoV-2 neutralizing titers in sera increased with dose level and after a 2nd dose. Geometric mean neutralizing titers reached 1.9-4.6-fold that of a panel of COVID-19 convalescent human sera, which were obtained ≥14 days after a pos. SARS-CoV-2 PCR. These results support further evaluation of this mRNA vaccine candidate.
- 54Watanabe, Y.; Allen, J. D.; Wrapp, D.; McLellan, J. S.; Crispin, M. Site-Specific Glycan Analysis of the SARS-CoV-2 Spike. Science 2020, eabb9983 DOI: 10.1126/science.abb9983
- 55Yuan, M.; Wu, N. C.; Zhu, X.; Lee, C. C. D.; So, R. T. Y.; Lv, H.; Mok, C. K. P.; Wilson, I. A. A Highly Conserved Cryptic Epitope in the Receptor Binding Domains of SARS-CoV-2 and SARS-CoV. Science 2020, 368 (6491), 630– 633, DOI: 10.1126/science.abb7269[Crossref], [PubMed], [CAS], Google Scholar55https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXovFCrt7Y%253D&md5=54554781f340bf0361a96d62cb98b3d3A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoVYuan, Meng; Wu, Nicholas C.; Zhu, Xueyong; Lee, Chang-Chun D.; So, Ray T. Y.; Lv, Huibin; Mok, Chris K. P.; Wilson, Ian A.Science (Washington, DC, United States) (2020), 368 (6491), 630-633CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has now become a pandemic, but there is currently very little understanding of the antigenicity of the virus. We therefore detd. the crystal structure of CR3022, a neutralizing antibody previously isolated from a convalescent SARS patient, in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein at 3.1-angstrom resoln. CR3022 targets a highly conserved epitope, distal from the receptor binding site, that enables cross-reactive binding between SARS-CoV-2 and SARS-CoV. Structural modeling further demonstrates that the binding epitope can only be accessed by CR3022 when at least two RBDs on the trimeric S protein are in the "up" conformation and slightly rotated. These results provide mol. insights into antibody recognition of SARS-CoV-2.
- 56Tian, X.; Li, C.; Huang, A.; Xia, S.; Lu, S.; Shi, Z.; Lu, L.; Jiang, S.; Yang, Z.; Wu, Y.; Ying, T. Potent Binding of 2019 Novel Coronavirus Spike Protein by a SARS Coronavirus-Specific Human Monoclonal Antibody. Emerging Microbes Infect. 2020, 9 (1), 382– 385, DOI: 10.1080/22221751.2020.1729069[Crossref], [PubMed], [CAS], Google Scholar56https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXmsVGks78%253D&md5=6d73882867e561b2056895c525136512Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibodyTian, Xiaolong; Li, Cheng; Huang, Ailing; Xia, Shuai; Lu, Sicong; Shi, Zhengli; Lu, Lu; Jiang, Shibo; Yang, Zhenlin; Wu, Yanling; Ying, TianleiEmerging Microbes & Infections (2020), 9 (1), 382-385CODEN: EMIMC4; ISSN:2222-1751. (Taylor & Francis Ltd.)The newly identified 2019 novel coronavirus (2019-nCoV) has caused more than 11,900 lab.-confirmed human infections, including 259 deaths, posing a serious threat to human health. Currently, however, there is no specific antiviral treatment or vaccine. Considering the relatively high identity of receptor-binding domain (RBD) in 2019-nCoV and SARS-CoV, it is urgent to assess the cross-reactivity of anti-SARS CoV antibodies with 2019-nCoV spike protein, which could have important implications for rapid development of vaccines and therapeutic antibodies against 2019-nCoV. Here, we report for the first time that a SARS-CoV-specific human monoclonal antibody, CR3022, could bind potently with 2019-nCoV RBD (KD of 6.3 nM). The epitope of CR3022 does not overlap with the ACE2 binding site within 2019-nCoV RBD. These results suggest that CR3022 may have the potential to be developed as candidate therapeutics, alone or in combination with other neutralizing antibodies, for the prevention and treatment of 2019-nCoV infections. Interestingly, some of the most potent SARS-CoV-specific neutralizing antibodies (e.g. m396, CR3014) that target the ACE2 binding site of SARS-CoV failed to bind 2019-nCoV spike protein, implying that the difference in the RBD of SARS-CoV and 2019-nCoV has a crit. impact for the cross-reactivity of neutralizing antibodies, and that it is still necessary to develop novel monoclonal antibodies that could bind specifically to 2019-nCoV RBD.
- 57ter Meulen, J.; van den Brink, E. N.; Poon, L. L. M.; Marissen, W. E.; Leung, C. S. W.; Cox, F.; Cheung, C. Y.; Bakker, A. Q.; Bogaards, J. A.; van Deventer, E.; Preiser, W.; Doerr, H. W.; Chow, V. T.; de Kruif, J.; Peiris, J. S. M.; Goudsmit, J. Human Monoclonal Antibody Combination against SARS Coronavirus: Synergy and Coverage of Escape Mutants. PLoS Med. 2006, 3 (7), e237 DOI: 10.1371/journal.pmed.0030237
- 58Shi, R.; Shan, C.; Duan, X.; Chen, Z.; Liu, P.; Song, J.; Song, T.; Bi, X.; Han, C.; Wu, L.; Gao, G.; Hu, X.; Zhang, Y.; Tong, Z.; Huang, W.; Liu, W. J.; Wu, G.; Zhang, B.; Wang, L.; Qi, J.; Feng, H.; Wang, F.-S.; Wang, Q.; Gao, G. F.; Yuan, Z.; Yan, J. A Human Neutralizing Antibody Targets the Receptor-Binding Site of SARS-CoV-2. Nature 2020, 584 (7819), 120– 124, DOI: 10.1038/s41586-020-2381-y[Crossref], [PubMed], [CAS], Google Scholar58https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVygtLjM&md5=55a93b450dd445483547b5f69cab393eA human neutralizing antibody targets the receptor-binding site of SARS-CoV-2Shi, Rui; Shan, Chao; Duan, Xiaomin; Chen, Zhihai; Liu, Peipei; Song, Jinwen; Song, Tao; Bi, Xiaoshan; Han, Chao; Wu, Lianao; Gao, Ge; Hu, Xue; Zhang, Yanan; Tong, Zhou; Huang, Weijin; Liu, William Jun; Wu, Guizhen; Zhang, Bo; Wang, Lan; Qi, Jianxun; Feng, Hui; Wang, Fu-Sheng; Wang, Qihui; Gao, George Fu; Yuan, Zhiming; Yan, JinghuaNature (London, United Kingdom) (2020), 584 (7819), 120-124CODEN: NATUAS; ISSN:0028-0836. (Nature Research)An outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally. Countermeasures are needed to treat and prevent further dissemination of the virus. Here we report the isolation of two specific human monoclonal antibodies (termed CA1 and CB6) from a patient convalescing from COVID-19. CA1 and CB6 demonstrated potent SARS-CoV-2-specific neutralization activity in vitro. In addn., CB6 inhibited infection with SARS-CoV-2 in rhesus monkeys in both prophylactic and treatment settings. We also performed structural studies, which revealed that CB6 recognizes an epitope that overlaps with angiotensin-converting enzyme 2 (ACE2)-binding sites in the SARS-CoV-2 receptor-binding domain, and thereby interferes with virus-receptor interactions by both steric hindrance and direct competition for interface residues. Our results suggest that CB6 deserves further study as a candidate for translation to the clinic.
- 59Heinicke, E.; Kumar, U.; Munoz, D. G. Quantitative Dot-Blot Assay for Proteins Using Enhanced Chemiluminescence. J. Immunol. Methods 1992, 152 (2), 227– 236, DOI: 10.1016/0022-1759(92)90144-I[Crossref], [PubMed], [CAS], Google Scholar59https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK38XmsVyjsLo%253D&md5=97273c9b337cfb1fe90b831048d48ba8Quantitative dot-blot assay for proteins using enhanced chemiluminescenceHeinicke, E.; Kumar, U.; Munoz, D. G.Journal of Immunological Methods (1992), 152 (2), 227-36CODEN: JIMMBG; ISSN:0022-1759.A sensitive nonradioactive method for detection of specific proteins on Western blots is com. available. The protein is immobilized on nitrocellulose membrane and immunolabeled with HRP-conjugated secondary antibody. HRP catalyzes the oxidn. of luminol, a cyclic diacylhydrazide, resulting in the emission of light which is recorded on film. By using dot blot, it was shown that the signal generated by this system is proportional to the amt. of protein loaded onto the membrane. Std. curves were linear (r2 > 0.95) over a 10-50-fold range. Linearity was also achieved with tissue exts. probed for a specific antigen. The sensitivity of the method is such that <10 fmol protein can be measured. The sensitivity and range are comparable to a previously reported dot-blotting assay based on the use of 125I-protein A, but the method does not require the handling of radioactive compds. This method was used to est. the level of chromogranin A in a mixt. of proteins extd. from human brain.
- 60Steppert, P.; Burgstaller, D.; Klausberger, M.; Tover, A.; Berger, E.; Jungbauer, A. Quantification and Characterization of Virus-like Particles by Size-Exclusion Chromatography and Nanoparticle Tracking Analysis. J. Chromatogr. A 2017, 1487, 89– 99, DOI: 10.1016/j.chroma.2016.12.085[Crossref], [PubMed], [CAS], Google Scholar60https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVKju7w%253D&md5=24338c5203ff0301f25843c8ef655af6Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysisSteppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Tover, Andres; Berger, Eva; Jungbauer, AloisJournal of Chromatography A (2017), 1487 (), 89-99CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)The rapid quantification of enveloped virus-like particles (VLPs) requires orthogonal methods to obtain reliable results. Three methods-nanoparticle tracking anal. (NTA), size-exclusion HPLC (SE-HPLC) with UV detection, and detection with multiangle light scattering (MALS)-for quantification of enveloped VLPs have been compared, and the lower and upper limits of detection and quantification have been evaluated. NTA directly counts the enveloped VLPs, and a particle no. is obtained with a lower limit of detection (LLOD) of 1.7 × 107 part/mL and lower limit of quantification (LLOQ) of 3.4 × 108 part/mL. SE-HPLC with UV detection was calibrated with stds. characterized by NTA, and a LLOD of 6.9 × 109 part/mL and LLOQ of 2.1 × 1010 part/mL were found. SE-HPLC with MALS does not require a precalibrated sample because with a spherical model based on the Rayleigh-Gans-Debye approxn., the particle concn. can be directly deduced from the scattered light. A LLOD of 4.8 × 108 part/mL and LLOQ of 2.1 × 109 part/mL were measured and substantially lower compared to the UV method. The abs. particle concn. measured by SE-HPLC-MALS is one order of magnitude lower compared to measurement by NTA, which is explained by the wide size distribution of an enveloped VLP suspension. The model used for evaluation of light scattering data assumes monodisperse, homogeneous, and spherical particles.
- 61Šagi, D.; Kienz, P.; Denecke, J.; Marquardt, T.; Peter-Katalinić, J. Glycoproteomics OfN-Glycosylation by in-Gel Deglycosylation and Matrix-Assisted Laser Desorption/Ionisation-Time of Flight Mass Spectrometry Mapping: Application to Congenital Disorders of Glycosylation. Proteomics 2005, 5 (10), 2689– 2701, DOI: 10.1002/pmic.200401312[Crossref], [PubMed], [CAS], Google Scholar61https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXmtF2gtb8%253D&md5=2c6d7fd83c626b4e7ffda029f360c825Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylationSagi, Dijana; Kienz, Petra; Denecke, Jonas; Marquardt, Thorsten; Peter-Katalinic, JasnaProteomics (2005), 5 (10), 2689-2701CODEN: PROTC7; ISSN:1615-9853. (Wiley-VCH Verlag GmbH & Co. KGaA)A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-sepd. proteins, their isolation, purifn. and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) anal. of their mixts. were optimized. Since many glycoproteins are available only at low quantities from SDS-PAGE or two-dimensional gels, high attention was paid to obtain N-glycan mixts. representing their actual compn. in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrixes for MS anal. of acidic N-glycans was compared. The most favorable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammonium citrate as a matrix. This matrix provided good results for both neutral and acidic mixts. as well as for methylated N-glycans. In the second part of this paper the potential of such an optimized MS strategy alone or in combination with high pH anion-exchange chromatog. profiling for the clin. diagnosis of congenital disorders of glycosylation is presented.
- 62Herrera, N. G.; Morano, N. C.; Celikgil, A.; Georgiev, G. I.; Malonis, R. J.; Lee, J. H.; Tong, K.; Vergnolle, O.; Massimi, A. B.; Yen, L. Y.; Noble, A. J.; Kopylov, M.; Bonanno, J. B.; Garrett-Thomson, S. C.; Hayes, D. B.; Bortz, R. H.; Wirchnianski, A. S.; Florez, C.; Laudermilch, E.; Haslwanter, D.; Fels, J. M.; Dieterle, M. E.; Jangra, R. K.; Barnhill, J.; Mengotto, A.; Kimmel, D.; Daily, J. P.; Pirofski, L.; Chandran, K.; Brenowitz, M.; Garforth, S. J.; Eng, E. T.; Lai, J. R.; Almo, S. C. Characterization of the SARS-CoV-2 S Protein: Biophysical, Biochemical, Structural, and Antigenic Analysis. ACS Omega 2020, in press. DOI: 10.1021/acsomega.0c03512 .
- 63Bornholdt, Z. A.; Turner, H. L.; Murin, C. D.; Li, W.; Sok, D.; Souders, C. A.; Piper, A. E.; Goff, A.; Shamblin, J. D.; Wollen, S. E.; Sprague, T. R.; Fusco, M. L.; Pommert, K. B. J.; Cavacini, L. A.; Smith, H. L.; Klempner, M.; Reimann, K. A.; Krauland, E.; Gerngross, T. U.; Wittrup, K. D.; Saphire, E. O.; Burton, D. R.; Glass, P. J.; Ward, A. B.; Walker, L. M. Isolation of Potent Neutralizing Antibodies from a Survivor of the 2014 Ebola Virus Outbreak. Science 2016, 351 (6277), 1078– 1083, DOI: 10.1126/science.aad5788[Crossref], [PubMed], [CAS], Google Scholar63https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XjsVagtbc%253D&md5=55ef46697b99197ac8ac53efecbf1e09Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreakBornholdt, Zachary A.; Turner, Hannah L.; Murin, Charles D.; Li, Wen; Sok, Devin; Souders, Colby A.; Piper, Ashley E.; Goff, Arthur; Shamblin, Joshua D.; Wollen, Suzanne E.; Sprague, Thomas R.; Fusco, Marnie L.; Pommert, Kathleen B. J.; Cavacini, Lisa A.; Smith, Heidi L.; Klempner, Mark; Reimann, Keith A.; Krauland, Eric; Gerngross, Tillman U.; Wittrup, Karl D.; Saphire, Erica Ollmann; Burton, Dennis R.; Glass, Pamela J.; Ward, Andrew B.; Walker, Laura M.Science (Washington, DC, United States) (2016), 351 (6277), 1078-1083CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.
- 64Vandepapelière, P.; Horsmans, Y.; Moris, P.; Van Mechelen, M.; Janssens, M.; Koutsoukos, M.; Van Belle, P.; Clement, F.; Hanon, E.; Wettendorff, M.; Garçon, N.; Leroux-Roels, G. Vaccine Adjuvant Systems Containing Monophosphoryl Lipid A and QS21 Induce Strong and Persistent Humoral and T Cell Responses against Hepatitis B Surface Antigen in Healthy Adult Volunteers. Vaccine 2008, 26 (10), 1375– 1386, DOI: 10.1016/j.vaccine.2007.12.038[Crossref], [PubMed], [CAS], Google Scholar64https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXitlemt7s%253D&md5=8812f21501e9d8b1afabac9201e2eb10Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS21 induce strong and persistent humoral and T cell responses against hepatitis B surface antigen in healthy adult volunteersVandepapeliere, Pierre; Horsmans, Yves; Moris, Philippe; Van Mechelen, Marcelle; Janssens, Michel; Koutsoukos, Marguerite; Van Belle, Pascale; Clement, Frederic; Hanon, Emmanuel; Wettendorff, Martine; Garcon, Nathalie; Leroux-Roels, GeertVaccine (2008), 26 (10), 1375-1386CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)A randomized, double-blind study assessing the potential of four adjuvants in combination with recombinant hepatitis B surface antigen has been conducted to evaluate humoral and cell-mediated immune responses in healthy adults after three vaccine doses at months 0, 1 and 10. Three Adjuvant Systems (AS) contained 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21, formulated either with an oil-in-water emulsion (AS02B and AS02V) or with liposomes (AS01B). The fourth adjuvant was CpG oligonucleotide. High levels of antibodies were induced by all adjuvants, whereas cell-mediated immune responses, including cytolytic T cells and strong and persistent CD4+ T cell response were mainly obsd. with the three MPL/QS21-contg. Adjuvant Systems. The CD4+ T cell response was characterized in vitro by vigorous lymphoproliferation, high IFN-γ and moderate IL-5 prodn. Antigen-specific T cell immune response was further confirmed ex vivo by detection of IL-2- and IFN-γ-producing CD4+ T cells, and in vivo by measuring increased levels of IFN-γ in the serum and delayed-type hypersensitivity (DTH) responses. The CpG adjuvanted vaccine induced consistently lower immune responses for all parameters. All vaccine adjuvants were shown to be safe with acceptable reactogenicity profiles. The majority of subjects reported local reactions at the injection site after vaccination while general reactions were recorded less frequently. No vaccine-related serious adverse event was reported. Importantly, no increase in markers of auto-immunity and allergy was detected over the whole study course. In conclusion, the Adjuvant Systems contg. MPL/QS21, in combination with hepatitis B surface antigen, induced very strong humoral and cellular immune responses in healthy adults. The AS01B-adjuvanted vaccine induced the strongest and most durable specific cellular immune responses after two doses. These Adjuvant Systems, when added to recombinant protein antigens, can be fundamental to develop effective prophylactic vaccines against complex pathogens, e.g. malaria, HIV infection and tuberculosis, and for special target populations such as subjects with an impaired immune response, due to age or medical conditions.
- 65Leroux-Roels, G.; Van Belle, P.; Vandepapeliere, P.; Horsmans, Y.; Janssens, M.; Carletti, I.; Garçon, N.; Wettendorff, M.; Van Mechelen, M. Vaccine Adjuvant Systems Containing Monophosphoryl Lipid A and QS-21 Induce Strong Humoral and Cellular Immune Responses against Hepatitis B Surface Antigen Which Persist for at Least 4 Years after Vaccination. Vaccine 2015, 33 (8), 1084– 1091, DOI: 10.1016/j.vaccine.2014.10.078[Crossref], [PubMed], [CAS], Google Scholar65https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhvFSmsrnM&md5=d18386239bced8881c1e6d17f39a1005Vaccine Adjuvant Systems containing monophosphoryl lipid A and QS-21 induce strong humoral and cellular immune responses against hepatitis B surface antigen which persist for at least 4 years after vaccinationLeroux-Roels, Geert; Van Belle, Pascale; Vandepapeliere, Pierre; Horsmans, Yves; Janssens, Michel; Carletti, Isabelle; Garcon, Nathalie; Wettendorff, Martine; Van Mechelen, MarcelleVaccine (2015), 33 (8), 1084-1091CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)Recombinant hepatitis B surface antigen (HBsAg) was used as a model antigen to evaluate persistence of cellular and humoral immune responses when formulated with three different Adjuvant Systems contg. 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS-21, in an oil-in-water emulsion (AS02B and AS02V), or with liposomes (AS01B). This is an open, 4-yr follow-up of a previous randomised, double-blind study. Healthy subjects aged 18-40 years received three vaccine doses on a month 0, 1, 10 schedule and were initially followed for 18 mo. A total of 93 subjects (AS02B: n = 30; AS02V: n = 28; AS01B: n = 35) were enrolled in this follow-up and had an addnl. blood sample taken at Year 4 (NCT02153320). The primary endpoint was the frequency of HBsAg-specific CD4+ and CD8+ T-cells expressing cytokines upon short-term in vitro stimulation of peripheral blood mononuclear cells with HBsAg-derived peptides. Secondary endpoints were anti-HBs antibody titers and frequency of HBsAg-specific memory B-cells. A strong and persistent specific CD4+ T-cell response was obsd. at Year 4 in all groups. HBsAg-specific CD4+ T-cells expressed mainly CD40L and IL-2, and to a lesser extent TNF-α and IFN-γ. HBsAg-specific CD8+ T-cells were not detected in any group. A high, persistent HBsAg-specific humoral immune response was obsd. in all groups, with all subjects seroprotected (antibody titer ≥10 mIU/mL) at Year 4. The geometric mean antibody titer at Year 4 was above 100,000 mIU/mL in all groups. A strong memory B-cell response was obsd. post-dose 2, which tended to increase post-dose 3 and persisted at Year 4 in all groups. The MPL/QS-21/HBsAg vaccine formulations induced persistent immune responses up to 4 years after first vaccination. These Adjuvant Systems offer potential for combination with recombinant, synthetic or highly purified subunit vaccines, particularly for vaccination against challenging diseases, or in specific populations, although addnl. studies are needed.
- 66Marty-Roix, R.; Vladimer, G. I.; Pouliot, K.; Weng, D.; Buglione-Corbett, R.; West, K.; MacMicking, J. D.; Chee, J. D.; Wang, S.; Lu, S.; Lien, E. Identification of QS-21 as an Inflammasome-Activating Molecular Component of Saponin Adjuvants. J. Biol. Chem. 2016, 291 (3), 1123– 1136, DOI: 10.1074/jbc.M115.683011[Crossref], [PubMed], [CAS], Google Scholar66https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XovVCrtw%253D%253D&md5=0ed38ac6d2f804607d89167206328ed7Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin AdjuvantsMarty-Roix, Robyn; Vladimer, Gregory I.; Pouliot, Kimberly; Weng, Dan; Buglione-Corbett, Rachel; West, Kim; MacMicking, John D.; Chee, Jonathan D.; Wang, Shixia; Lu, Shan; Lien, EgilJournal of Biological Chemistry (2016), 291 (3), 1123-1136CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific mols. contribute to the stimulatory activity. QS-21 is a defined, highly purified, and sol. saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. The authors obsd. QS-21 to elicit caspase-1-dependent IL-1β and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, the authors' data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1β/IL-18 release. At higher concns., QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixt. of saponins also induced IL-1β in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, the authors have identified QS-21 as a nonparticulate single mol. saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigen-specific responses in vivo.
- 67Case, J. B.; Rothlauf, P. W.; Chen, R. E.; Liu, Z.; Zhao, H.; Kim, A. S.; Bloyet, L.; Zeng, Q.; Tahan, S.; Droit, L.; Ilagan, M. X. G.; Tartell, M. A.; Amarasinghe, G.; Henderson, J. P.; Miersch, S.; Ustav, M.; Sidhu, S.; Virgin, H. W.; Wang, D.; Ding, S.; Corti, D.; Theel, E. S.; Fremont, D. H.; Diamond, M. S.; Whelan, S. P. J. Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2. Cell Host Microbe 2020, 28, 475, DOI: 10.1016/j.chom.2020.06.021[Crossref], [PubMed], [CAS], Google Scholar67https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsVeqsrjF&md5=ef59069d2200e4800c0830d7c0055050Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2Case, James Brett; Rothlauf, Paul W.; Chen, Rita E.; Liu, Zhuoming; Zhao, Haiyan; Kim, Arthur S.; Bloyet, Louis-Marie; Zeng, Qiru; Tahan, Stephen; Droit, Lindsay; Ilagan, Ma. Xenia G.; Tartell, Michael A.; Amarasinghe, Gaya; Henderson, Jeffrey P.; Miersch, Shane; Ustav, Mart; Sidhu, Sachdev; Virgin, Herbert W.; Wang, David; Ding, Siyuan; Corti, Davide; Theel, Elitza S.; Fremont, Daved H.; Diamond, Michael S.; Whelan, Sean P. J.Cell Host & Microbe (2020), 28 (3), 475-485.e5CODEN: CHMECB; ISSN:1931-3128. (Elsevier Inc.)Antibody-based interventions against SARS-CoV-2 could limit morbidity, mortality, and possibly transmission. An anticipated correlate of such countermeasures is the level of neutralizing antibodies against the SARS-CoV-2 spike protein, which engages with host ACE2 receptor for entry. Using an infectious mol. clone of vesicular stomatitis virus (VSV) expressing eGFP as a marker of infection, we replaced the glycoprotein gene (G) with the spike protein of SARS-CoV-2 (VSV-eGFP-SARS-CoV-2) and developed a high-throughput-imaging-based neutralization assay at biosafety level 2. We also developed a focus-redn. neutralization test with a clin. isolate of SARS-CoV-2 at biosafety level 3. Comparing the neutralizing activities of various antibodies and ACE2-Fc sol. decoy protein in both assays revealed a high degree of concordance. These assays will help define correlates of protection for antibody-based countermeasures and vaccines against SARS-CoV-2. Addnl., replication-competent VSV-eGFP-SARS-CoV-2 provides a tool for testing inhibitors of SARS-CoV-2 mediated entry under reduced biosafety containment.
- 68Mandolesi, M.; Sheward, D. J.; Hanke, L.; Ma, J.; Pushparaj, P.; Vidakovics, L. P.; Kim, C.; Loré, K.; Dopico, X. C.; Coquet, J. M.; McInerney, G.; Karlsson Hedestam, G. B.; Murrell, B. SARS-CoV-2 Protein Subunit Vaccination Elicits Potent Neutralizing Antibody Responses. bioRxiv, 2020. DOI: 10.1101/2020.07.31.228486 .Google ScholarThere is no corresponding record for this reference.
- 69Chi, X.; Yan, R.; Zhang, J.; Zhang, G.; Zhang, Y.; Hao, M.; Zhang, Z.; Fan, P.; Dong, Y.; Yang, Y.; Chen, Z.; Guo, Y.; Zhang, J.; Li, Y.; Song, X.; Chen, Y.; Xia, L.; Fu, L.; Hou, L.; Xu, J.; Yu, C.; Li, J.; Zhou, Q.; Chen, W. A Neutralizing Human Antibody Binds to the N-Terminal Domain of the Spike Protein of SARS-CoV-2. Science 2020, 369 (6504), 650– 655, DOI: 10.1126/science.abc6952[Crossref], [PubMed], [CAS], Google Scholar69https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGms77F&md5=398d9f74ea0873b1e083d6ec457167c1A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2Chi, Xiangyang; Yan, Renhong; Zhang, Jun; Zhang, Guanying; Zhang, Yuanyuan; Hao, Meng; Zhang, Zhe; Fan, Pengfei; Dong, Yunzhu; Yang, Yilong; Chen, Zhengshan; Guo, Yingying; Zhang, Jinlong; Li, Yaning; Song, Xiaohong; Chen, Yi; Xia, Lu; Fu, Ling; Hou, Lihua; Xu, Junjie; Yu, Changming; Li, Jianmin; Zhou, Qiang; Chen, WeiScience (Washington, DC, United States) (2020), 369 (6504), 650-655CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)Developing therapeutics against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could be guided by the distribution of epitopes, not only on the receptor binding domain (RBD) of the Spike (S) protein but also across the full Spike (S) protein. We isolated and characterized monoclonal antibodies (mAbs) from 10 convalescent COVID-19 patients. Three mAbs showed neutralizing activities against authentic SARS-CoV-2. One mAb, named 4A8, exhibits high neutralization potency against both authentic and pseudotyped SARS-CoV-2 but does not bind the RBD. We defined the epitope of 4A8 as the N-terminal domain (NTD) of the S protein by detg. with cryo-electron microscopy its structure in complex with the S protein to an overall resoln. of 3.1 angstroms and local resoln. of 3.3 angstroms for the 4A8-NTD interface. This points to the NTD as a promising target for therapeutic mAbs against COVID-19.
- 70Lefeber, D. J.; Benaissa-Trouw, B.; Vliegenthart, J. F. G.; Kamerling, J. P.; Jansen, W. T. M.; Kraaijeveld, K.; Snippe, H. Th1-Directing Adjuvants Increase the Immunogenicity of Oligosaccharide-Protein Conjugate Vaccines Related to Streptococcus Pneumoniae Type 3. Infect. Immun. 2003, 71, 6915, DOI: 10.1128/IAI.71.12.6915-6920.2003[Crossref], [PubMed], [CAS], Google Scholar70https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpsFGntrk%253D&md5=2b4ada5a4d983fed83364bd0afe4f1b2Th1-directing adjuvants increase the immunogenicity of oligosaccharide-protein conjugate vaccines related to Streptococcus pneumoniae type 3Lefeber, Dirk J.; Benaissa-trouw, Barry; Vliegenthart, Johannes F. G.; Kamerling, Johannis P.; Jansen, Wouter T. M.; Kraaijeveld, Kees; Snippe, HarmInfection and Immunity (2003), 71 (12), 6915-6920CODEN: INFIBR; ISSN:0019-9567. (American Society for Microbiology)Oligosaccharide (OS)-protein conjugates are promising candidate vaccines against encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, little is known about the influence of adjuvants on the immunogenicity of OS-protein conjugates. In this study, a minimal protective trisaccharide epitope of Streptococcus pneumoniae type 3 conjugated to the cross-reacting material of diphtheria toxin was used for immunization of BALB/c mice in the presence of different adjuvants. Subsequently, half of the mice received a booster immunization with conjugate alone. Independent of the use and type of adjuvant, all mice produced long-lasting anti-polysaccharide type 3 (PS3) antibody levels, which provided full protection against challenge with pneumococcal type 3 bacteria. All adjuvants tested increased the anti-PS3 antibody levels and opsonic capacities as measured by an ELISA and an in vitro phagocytosis assay. The use of QuilA or a combination of the adjuvants CpG and di-Me dioctadecyl ammonium bromide resulted in the highest phagocytic capacities and the highest levels of Th1-related IgG subclasses. Phagocytic capacity correlated strongly with Th1-assocd. IgG2a and IgG2b levels, to a lesser extent with Th2-assocd. IgG1 levels, and weakly with thiocyanate elution as a measure of avidity. Thus, the improved immunogenicity of OS-protein conjugates was most pronounced for Th1-directing adjuvants.
- 71Visciano, M. L.; Tagliamonte, M.; Tornesello, M. L.; Buonaguro, F. M.; Buonaguro, L. Effects of Adjuvants on IgG Subclasses Elicited by Virus-like Particles. J. Transl. Med. 2012, 10 (1), 4, DOI: 10.1186/1479-5876-10-4[Crossref], [PubMed], [CAS], Google Scholar71https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XksFGntLk%253D&md5=9efd7b5ffca2426a0389e1275ee41f03Effects of adjuvants on IgG subclasses elicited by Virus-Like particlesVisciano, Maria Luisa; Tagliamonte, Maria; Tornesello, Maria Lina; Buonaguro, Franco M.; Buonaguro, LuigiJournal of Translational Medicine (2012), 10 (), 4CODEN: JTMOBV; ISSN:1479-5876. (BioMed Central Ltd.)Background: Virus-Like Particles (VLPs) represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN 1826 or poly(I:C) adjuvants, has been evaluated in an animal model. Results: Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TH2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3), with poly(I:C) more potent than CpG ODN1826. Conclusions: The results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen.
- 72Bos, R.; Rutten, L.; van der Lubbe, J. E. M.; Bakkers, M. J. G.; Hardenberg, G.; Wegmann, F.; Zuijdgeest, D.; de Wilde, A. H.; Koornneef, A.; Verwilligen, A.; van Manen, D.; Kwaks, T.; Vogels, R.; Dalebout, T. J.; Myeni, S. K.; Kikkert, M.; Snijder, E. J.; Li, Z.; Barouch, D. H.; Vellinga, J.; Langedijk, J. P. M.; Zahn, R. C.; Custers, J.; Schuitemaker, H. Ad26 Vector-Based COVID-19 Vaccine Encoding a Prefusion-Stabilized SARS-CoV-2 Spike Immunogen Induces Potent Humoral and Cellular Immune Responses. npj Vaccines 2020, in press. DOI: 10.1038/s41541-020-00243-x .
- 73Lederer, K.; Castaño, D.; Atria, D. G.; Oguin, T. H.; Wang, S.; Manzoni, T. B.; Muramatsu, H.; Hogan, M. J.; Amanat, F.; Cherubin, P.; Lundgreen, K. A.; Tam, Y. K.; Fan, S. H. Y.; Eisenlohr, L. C.; Maillard, I.; Weissman, D.; Bates, P.; Krammer, F.; Sempowski, G. D.; Pardi, N.; Locci, M. SARS-CoV-2 MRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation. Immunity 2020, 53, 1281, DOI: 10.1016/j.immuni.2020.11.009[Crossref], [PubMed], [CAS], Google Scholar73https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXisFeltL3J&md5=58bc06e6e9f1b7b33c0f05178b2b008bSARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody GenerationLederer, Katlyn; Castano, Diana; Gomez Atria, Daniela; Oguin, Thomas H. III; Wang, Sidney; Manzoni, Tomaz B.; Muramatsu, Hiromi; Hogan, Michael J.; Amanat, Fatima; Cherubin, Patrick; Lundgreen, Kendall A.; Tam, Ying K.; Fan, Steven H. Y.; Eisenlohr, Laurence C.; Maillard, Ivan; Weissman, Drew; Bates, Paul; Krammer, Florian; Sempowski, Gregory D.; Pardi, Norbert; Locci, MichelaImmunity (2020), 53 (6), 1281-1295.e5CODEN: IUNIEH; ISSN:1074-7613. (Elsevier Inc.)The deployment of effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crit. to eradicate the coronavirus disease 2019 (COVID-19) pandemic. Many licensed vaccines confer protection by inducing long-lived plasma cells (LLPCs) and memory B cells (MBCs), cell types canonically generated during germinal center (GC) reactions. Here, we directly compared two vaccine platforms-mRNA vaccines and a recombinant protein formulated with an MF59-like adjuvant-looking for their abilities to quant. and qual. shape SARS-CoV-2-specific primary GC responses over time. We demonstrated that a single immunization with SARS-CoV-2 mRNA, but not with the recombinant protein vaccine, elicited potent SARS-CoV-2-specific GC B and T follicular helper (Tfh) cell responses as well as LLPCs and MBCs. Importantly, GC responses strongly correlated with neutralizing antibody prodn. mRNA vaccines more efficiently induced key regulators of the Tfh cell program and influenced the functional properties of Tfh cells. Overall, this study identifies SARS-CoV-2 mRNA vaccines as strong candidates for promoting robust GC-derived immune responses.
- 74Dong, Y.; Dai, T.; Wei, Y.; Zhang, L.; Zheng, M.; Zhou, F. A Systematic Review of SARS-CoV-2 Vaccine Candidates. Signal Transduct. Target. Ther. 2020, 5 (1), 237, DOI: 10.1038/s41392-020-00352-y[Crossref], [PubMed], [CAS], Google Scholar74https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3s7gvFKqsQ%253D%253D&md5=cd1dc82d2ffe985e958f86d51378ab84A systematic review of SARS-CoV-2 vaccine candidatesDong Yetian; Zheng Min; Dong Yetian; Zhang Long; Dai Tong; Zhou Fangfang; Wei YujunSignal transduction and targeted therapy (2020), 5 (1), 237 ISSN:.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging virus that is highly pathogenic and has caused the recent worldwide pandemic officially named coronavirus disease (COVID-19). Currently, considerable efforts have been put into developing effective and safe drugs and vaccines against SARS-CoV-2. Vaccines, such as inactivated vaccines, nucleic acid-based vaccines, and vector vaccines, have already entered clinical trials. In this review, we provide an overview of the experimental and clinical data obtained from recent SARS-CoV-2 vaccines trials, and highlight certain potential safety issues that require consideration when developing vaccines. Furthermore, we summarize several strategies utilized in the development of vaccines against other infectious viruses, such as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), with the aim of aiding in the design of effective therapeutic approaches against SARS-CoV-2.
- 75Iwata-Yoshikawa, N.; Uda, A.; Suzuki, T.; Tsunetsugu-Yokota, Y.; Sato, Y.; Morikawa, S.; Tashiro, M.; Sata, T.; Hasegawa, H.; Nagata, N. Effects of Toll-Like Receptor Stimulation on Eosinophilic Infiltration in Lungs of BALB/c Mice Immunized with UV-Inactivated Severe Acute Respiratory Syndrome-Related Coronavirus Vaccine. J. Virol. 2014, 88 (15), 8597– 8614, DOI: 10.1128/JVI.00983-14[Crossref], [PubMed], [CAS], Google Scholar75https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXhsFehu7jE&md5=d7b4e910c70ed7f89dc3becd656c30cfEffects of Toll-like receptor stimulation on eosinophilic infiltration in lungs of BALB/c mice immunized with UV-inactivated severe acute respiratory syndrome-related coronavirus vaccineIwata-Yoshikawa, Naoko; Uda, Akihiko; Suzuki, Tadaki; Tsunetsugu-Yokota, Yasuko; Sato, Yuko; Morikawa, Shigeru; Tashiro, Masato; Sata, Tetsutaro; Hasegawa, Hideki; Nagata, NoriyoJournal of Virology (2014), 88 (15), 8597-8614, 19 pp.CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Severe acute respiratory syndrome-related coronavirus (SARS-CoV) is an emerging pathogen that causes severe respiratory illness. Whole UV-inactivated SARS-CoV (UV-V), bearing multiple epitopes and proteins, is a candidate vaccine against this virus. However, whole inactivated SARS vaccine that includes nucleocapsid protein is reported to induce eosinophilic infiltration in mouse lungs after challenge with live SARS-CoV. In this study, an ability of Toll-like receptor (TLR) agonists to reduce the side effects of UV-V vaccination in a 6-mo-old adult BALB/c mouse model was investigated, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. Immunization of adult mice with UV-V, with or without alum, resulted in partial protection from LDs of SARS-CoV challenge, but extensive eosinophil infiltration in the lungs was obsd. In contrast, TLR agonists added to UV-V vaccine, including lipopolysaccharide, poly(U) and poly(I C) (UV-V+TLR), strikingly reduced excess eosinophilic infiltration in the lungs and induced lower levels of interleukin-4 and -13 and eotaxin in the lungs than UV-V-immunization alone. Addnl., microarray anal. showed that genes assocd. with chemotaxis, eosinophil migration, eosinophilia and cell movement and the polarization of Th2 cells were upregulated in UV-V-immunized but not in UV-V+TLR-immunized mice. In particular, CD11b+ cells in the lungs of UV-V-immunized mice showed the upregulation of genes assocd. with the induction of eosinophils after challenge. These findings suggest that vaccine-induced eosinophil immunopathol. in the lungs upon SARS-CoV infection could be avoided by the TLR agonist adjuvants. Importance: Inactivated whole severe acute respiratory syndrome-related coronavirus (SARS-CoV) vaccines induce neutralizing antibodies in mouse models; however, they also cause increased eosinophilic immunopathol. in the lungs upon SARS-CoV challenge. In this study, the ability of adjuvant Toll-like receptor (TLR) agonists to reduce the side effects of UV-inactivated SARS-CoV vaccination in a BALB/c mouse model was tested, using the mouse-passaged Frankfurt 1 isolate of SARS-CoV. We found that TLR stimulation reduced the high level of eosinophilic infiltration that occurred in the lungs of mice immunized with UV-inactivated SARS-CoV. Microarray anal. revealed that genes assocd. with chemotaxis, eosinophil migration, eosinophilia and cell movement and the polarization of Th2 cells were upregulated in UV-inactivated SARS-CoV-immunized mice. This study may be helpful for elucidating the pathogenesis underlying eosinophilic infiltration resulting from immunization with inactivated vaccine.
- 76Honda-Okubo, Y.; Barnard, D.; Ong, C. H.; Peng, B.-H.; Tseng, C.-T. K.; Petrovsky, N. Severe Acute Respiratory Syndrome-Associated Coronavirus Vaccines Formulated with Delta Inulin Adjuvants Provide Enhanced Protection While Ameliorating Lung Eosinophilic Immunopathology. J. Virol. 2015, 89 (6), 2995– 3007, DOI: 10.1128/JVI.02980-14[Crossref], [PubMed], [CAS], Google Scholar76https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXktF2gu78%253D&md5=1e32c80e6d6a79ba1143d44a41e277cdSevere acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathologyHonda-Okubo, Yoshikazu; Barnard, Dale; Ong, Chun Hao; Peng, Bi-Hung; Tseng, Chien-Te Kent; Petrovsky, NikolaiJournal of Virology (2015), 89 (6), 2995-3007CODEN: JOVIAM; ISSN:1098-5514. (American Society for Microbiology)Although the severe acute respiratory syndrome-assocd. coronavirus (SARS-CoV) epidemic was controlled by nonvaccine measures, coronaviruses remain a major threat to human health. The design of optimal coronavirus vaccines therefore remains a priority. Such vaccines present major challenges: coronavirus immunity often wanes rapidly, individuals needing to be protected include the elderly, and vaccines may exacerbate rather than prevent coronavirus lung immunopathol. To address these issues, we compared in a murine model a range of recombinant spike protein or inactivated whole-virus vaccine candidates alone or adjuvanted with either alum, CpG, or Advax, a new delta inulin-based polysaccharide adjuvant. While all vaccines protected against lethal infection, addn. of adjuvant significantly increased serum neutralizing-antibody titers and reduced lung virus titers on day 3 postchallenge. Whereas unadjuvanted or alum-formulated vaccines were assocd. with significantly increased lung eosinophilic immunopathol. on day 6 postchallenge, this was not seen in mice immunized with vaccines formulated with delta inulin adjuvant. Protection against eosinophilic immunopathol. by vaccines contg. delta inulin adjuvants correlated better with enhanced T-cell gamma interferon (IFN-γ) recall responses rather than reduced interleukin-4 (IL-4) responses, suggesting that immunopathol. predominantly reflects an inadequate vaccine-induced Th1 response. This study highlights the crit. importance for development of effective and safe coronavirus vaccines of selection of adjuvants based on the ability to induce durable IFN-γ responses.
- 77Tseng, C.-T.; Sbrana, E.; Iwata-Yoshikawa, N.; Newman, P. C.; Garron, T.; Atmar, R. L.; Peters, C. J.; Couch, R. B. Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus. PLoS One 2012, 7 (4), e35421 DOI: 10.1371/journal.pone.0035421[Crossref], [PubMed], [CAS], Google Scholar77https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XmslOksbs%253D&md5=36adfc4192c520b40f66f4a89d16448fImmunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virusTseng, Chien-Te; Sbrana, Elena; Iwata-Yoshikawa, Naoko; Newman, Patrick C.; Garron, Tania; Atmar, Robert L.; Peters, Clarence J.; Couch, Robert B.PLoS One (2012), 7 (4), e35421CODEN: POLNCL; ISSN:1932-6203. (Public Library of Science)Background: Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathol.-type lung disease. Design: Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathol. Results: All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant redns. of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathol. changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathol. seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathol. with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathol. changes seen in all control groups lacked the eosinophil prominence. Conclusions: These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathol. suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.
- 78Vartak, A.; Sucheck, S. Recent Advances in Subunit Vaccine Carriers. Vaccines 2016, 4 (2), 12, DOI: 10.3390/vaccines4020012[Crossref], [CAS], Google Scholar78https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXnvF2mtbg%253D&md5=16f5079047f303f38533eca9e0e2e653Recent advances in subunit vaccine carriersVartak, Abhishek; Sucheck, Steven J.Vaccines (Basel, Switzerland) (2016), 4 (2), 12/1-12/18CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)The lower immunogenicity of synthetic subunit antigens, compared to live attenuated vaccines, is being addressed with improved vaccine carriers. Recent reports indicate that the physio-chem. properties of these carriers can be altered to achieve optimal antigen presentation, endosomal escape, particle bio-distribution, and cellular trafficking. The carriers can be modified with various antigens and ligands for dendritic cells targeting. They can also be modified with adjuvants, either covalently or entrapped in the matrix, to improve cellular and humoral immune responses against the antigen. As a result, these multi-functional carrier systems are being explored for use in active immunotherapy against cancer and infectious diseases. Advancing technol., improved anal. methods, and use of computational methodol. have also contributed to the development of subunit vaccine carriers. This review details recent breakthroughs in the design of nano-particulate vaccine carriers, including liposomes, polymeric nanoparticles, and inorg. nanoparticles.
- 79Tai, W.; He, L.; Zhang, X.; Pu, J.; Voronin, D.; Jiang, S.; Zhou, Y.; Du, L. Characterization of the Receptor-Binding Domain (RBD) of 2019 Novel Coronavirus: Implication for Development of RBD Protein as a Viral Attachment Inhibitor and Vaccine. Cell. Mol. Immunol. 2020, 17 (6), 613– 620, DOI: 10.1038/s41423-020-0400-4[Crossref], [PubMed], [CAS], Google Scholar79https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXlt1Chsrw%253D&md5=87bc49d070c84e78b01230518aaa465aCharacterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccineTai, Wanbo; He, Lei; Zhang, Xiujuan; Pu, Jing; Voronin, Denis; Jiang, Shibo; Zhou, Yusen; Du, LanyingCellular & Molecular Immunology (2020), 17 (6), 613-620CODEN: CMIEAO; ISSN:1672-7681. (Nature Research)The outbreak of Coronavirus Disease 2019 (COVID-19) has posed a serious threat to global public health, calling for the development of safe and effective prophylactics and therapeutics against infection of its causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV). The CoV spike (S) protein plays the most important roles in viral attachment, fusion and entry, and serves as a target for development of antibodies, entry inhibitors and vaccines. Here, we identified the receptor-binding domain (RBD) in SARS-CoV-2 S protein and found that the RBD protein bound strongly to human and bat angiotensin-converting enzyme 2 (ACE2) receptors. SARS-CoV-2 RBD exhibited significantly higher binding affinity to ACE2 receptor than SARS-CoV RBD and could block the binding and, hence, attachment of SARS-CoV-2 RBD and SARS-CoV RBD to ACE2-expressing cells, thus inhibiting their infection to host cells. SARS-CoV RBD-specific antibodies could cross-react with SARS-CoV-2 RBD protein, and SARS-CoV RBD-induced antisera could cross-neutralize SARS-CoV-2, suggesting the potential to develop SARS-CoV RBD-based vaccines for prevention of SARS-CoV-2 and SARS-CoV infection.
- 80Tai, W.; Zhang, X.; Drelich, A.; Shi, J.; Hsu, J. C.; Luchsinger, L.; Hillyer, C. D.; Tseng, C.-T. K.; Jiang, S.; Du, L. A Novel Receptor-Binding Domain (RBD)-Based MRNA Vaccine against SARS-CoV-2. Cell Res. 2020, 30, 932, DOI: 10.1038/s41422-020-0387-5[Crossref], [PubMed], [CAS], Google Scholar80https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhsFGiu7fJ&md5=b5a93d5ac1c3a015ee131dd40a685544A novel receptor-binding domain (RBD)-based mRNA vaccine against SARS-CoV-2Tai, Wanbo; Zhang, Xiujuan; Drelich, Aleksandra; Shi, Juan; Hsu, Jason C.; Luchsinger, Larry; Hillyer, Christopher D.; Tseng, Chien-Te K.; Jiang, Shibo; Du, LanyingCell Research (2020), 30 (10), 932-935CODEN: CREEB6; ISSN:1001-0602. (Nature Research)The pandemic of coronavirus disease 2019 (COVID-19) causedby severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)highlights the need to develop effective and safe vaccines. Similar to SARS-CoV, SARS-CoV-2 recognizes angiotensin-converting enzyme 2 (ACE2) as receptor for host cell entry. To identify an mRNA candidate vaccine, we initially designed two mRNA constructs expressing S1 and RBD, resp.,of SARS-CoV-2 S protein. To detect whether S1 and RBD mRNAs durably express antigens in multiple cell types, we constructed N-terminal mCherry-tagged SARS-CoV-2 S1 and RBD mRNAs, encapsulated them with lipid nanoparticles. Overall, this study identifies RBD as a key antigen to design effective vaccines against SARS-CoV-2, indicating great potential of RBD-based mRNA vaccine for mitigation of the COVID-19 pandemic and possible SARS-related epidemics in the future. More studies will be needed to investigate vaccine-assocd. immunopathol. in addn. to evaluate their protective efficacy.
- 81Misasi, J.; Gilman, M. S. A.; Kanekiyo, M.; Gui, M.; Cagigi, A.; Mulangu, S.; Corti, D.; Ledgerwood, J. E.; Lanzavecchia, A.; Cunningham, J.; Muyembe-Tamfun, J. J.; Baxa, U.; Graham, B. S.; Xiang, Y.; Sullivan, N. J.; McLellan, J. S. Structural and Molecular Basis for Ebola Virus Neutralization by Protective Human Antibodies. Science 2016, 351 (6279), 1343– 1346, DOI: 10.1126/science.aad6117[Crossref], [PubMed], [CAS], Google Scholar81https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XktFarsLY%253D&md5=2481659874654aa9addb68ba996a19cbStructural and molecular basis for Ebola virus neutralization by protective human antibodiesMisasi, John; Gilman, Morgan S. A.; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E.; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S.; Xiang, Ye; Sullivan, Nancy J.; McLellan, Jason S.Science (Washington, DC, United States) (2016), 351 (6279), 1343-1346CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains assocd. after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.
- 82Scheres, S. H. W. RELION: Implementation of a Bayesian Approach to Cryo-EM Structure Determination. J. Struct. Biol. 2012, 180 (3), 519– 530, DOI: 10.1016/j.jsb.2012.09.006[Crossref], [PubMed], [CAS], Google Scholar82https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38Xhs12jsLvO&md5=e40ffab245ef16ca7385fce394a4484aRELION: Implementation of a Bayesian approach to cryo-EM structure determinationScheres, Sjors H. W.Journal of Structural Biology (2012), 180 (3), 519-530CODEN: JSBIEM; ISSN:1047-8477. (Elsevier Inc.)RELION, for REgularized LIkelihood OptimizatioN, is an open-source computer program for the refinement of macromol. structures by single-particle anal. of electron cryo-microscopy (cryo-EM) data. Whereas alternative approaches often rely on user expertise for the tuning of parameters, RELION uses a Bayesian approach to infer parameters of a statistical model from the data. This paper describes developments that reduce the computational costs of the underlying max. a posteriori (MAP) algorithm, as well as statistical considerations that yield new insights into the accuracy with which the relative orientations of individual particles may be detd. A so-called gold-std. Fourier shell correlation (FSC) procedure to prevent overfitting is also described. The resulting implementation yields high-quality reconstructions and reliable resoln. ests. with minimal user intervention and at acceptable computational costs.
- 83Zheng, S. Q.; Palovcak, E.; Armache, J.-P.; Verba, K. A.; Cheng, Y.; Agard, D. A. MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods 2017, 14 (4), 331– 332, DOI: 10.1038/nmeth.4193[Crossref], [PubMed], [CAS], Google Scholar83https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXjt1ags7g%253D&md5=5f4e225ef8123dacd8475d526175e1d2MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopyZheng, Shawn Q.; Palovcak, Eugene; Armache, Jean-Paul; Verba, Kliment A.; Cheng, Yifan; Agard, David A.Nature Methods (2017), 14 (4), 331-332CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)A review on anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Here we describe MotionCor2, a software tool for anisotropic correction of beam-induced motion. Overall, MotionCor2 is extremely robust and sufficiently accurate at correcting local motions so that the very time-consuming and computationally intensive particle polishing in RELION can be skipped, importantly, it also works on a wide range of data sets, including cryo tomog. tilt series.
- 84Rohou, A.; Grigorieff, N. CTFFIND4: Fast and Accurate Defocus Estimation from Electron Micrographs. J. Struct. Biol. 2015, 192 (2), 216– 221, DOI: 10.1016/j.jsb.2015.08.008[Crossref], [PubMed], [CAS], Google Scholar84https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC287js1Whsg%253D%253D&md5=8500953ad4898ae82de6f8cdc95832cfCTFFIND4: Fast and accurate defocus estimation from electron micrographsRohou Alexis; Grigorieff NikolausJournal of structural biology (2015), 192 (2), 216-21 ISSN:.CTFFIND is a widely-used program for the estimation of objective lens defocus parameters from transmission electron micrographs. Defocus parameters are estimated by fitting a model of the microscope's contrast transfer function (CTF) to an image's amplitude spectrum. Here we describe modifications to the algorithm which make it significantly faster and more suitable for use with images collected using modern technologies such as dose fractionation and phase plates. We show that this new version preserves the accuracy of the original algorithm while allowing for higher throughput. We also describe a measure of the quality of the fit as a function of spatial frequency and suggest this can be used to define the highest resolution at which CTF oscillations were successfully modeled.
- 85Tang, G.; Peng, L.; Baldwin, P. R.; Mann, D. S.; Jiang, W.; Rees, I.; Ludtke, S. J. EMAN2: An Extensible Image Processing Suite for Electron Microscopy. J. Struct. Biol. 2007, 157 (1), 38– 46, DOI: 10.1016/j.jsb.2006.05.009[Crossref], [PubMed], [CAS], Google Scholar85https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BD28jjt1elsQ%253D%253D&md5=792e9e052f5233faa3180d330513f532EMAN2: an extensible image processing suite for electron microscopyTang Guang; Peng Liwei; Baldwin Philip R; Mann Deepinder S; Jiang Wen; Rees Ian; Ludtke Steven JJournal of structural biology (2007), 157 (1), 38-46 ISSN:1047-8477.EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.
- 86Punjani, A.; Rubinstein, J. L.; Fleet, D. J.; Brubaker, M. A. CryoSPARC: Algorithms for Rapid Unsupervised Cryo-EM Structure Determination. Nat. Methods 2017, 14 (3), 290– 296, DOI: 10.1038/nmeth.4169[Crossref], [PubMed], [CAS], Google Scholar86https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXitlGisbs%253D&md5=95d468147707707e70ac0ad38dd6ebf6cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determinationPunjani, Ali; Rubinstein, John L.; Fleet, David J.; Brubaker, Marcus A.Nature Methods (2017), 14 (3), 290-296CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for detg. the structures of biol. macromols. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resoln. frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calcns. on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound max. likelihood optimization algorithms permit the major steps in cryo-EM structure detn. to be performed in hours or minutes on an inexpensive desktop computer. Furthermore, SGD with Bayesian marginalization allows ab initio 3D classification, enabling automated anal. and discovery of unexpected structures without bias from a ref. map. These algorithms are combined in a user-friendly computer program named cryoSPARC (http://www.cryosparc.com).
- 87Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera?A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25 (13), 1605– 1612, DOI: 10.1002/jcc.20084[Crossref], [PubMed], [CAS], Google Scholar87https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmvVOhsbs%253D&md5=944b175f440c1ff323705987cf937ee7UCSF Chimera-A visualization system for exploratory research and analysisPettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C.; Couch, Gregory S.; Greenblatt, Daniel M.; Meng, Elaine C.; Ferrin, Thomas E.Journal of Computational Chemistry (2004), 25 (13), 1605-1612CODEN: JCCHDD; ISSN:0192-8651. (John Wiley & Sons, Inc.)The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale mol. assemblies such as viral coats, and Collab., which allows researchers to share a Chimera session interactively despite being at sep. locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and assocd. structures; ViewDock, for screening docked ligand orientations; Movie, for replaying mol. dynamics trajectories; and Vol. Viewer, for display and anal. of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
Supporting Information
ARTICLE SECTIONSThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.0c01405.
Additional data and figures including expression levels, size-exclusion chromatography, cryo-EM, ELISA results, assay validation, and off-target antibody responses (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.