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B: Biophysical and Biochemical Systems and Processes

Biophysical Correlates of Enhanced Immunogenicity of a Stabilized Variant of the Receptor Binding Domain of SARS-CoV-2
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  • Kawkab Kanjo
    Kawkab Kanjo
    Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
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  • Gopinath Chattopadhyay
    Gopinath Chattopadhyay
    Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
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    Orcidhttps://orcid.org/0000-0002-6408-1968
  • Sameer Kumar Malladi
    Sameer Kumar Malladi
    Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
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  • Randhir Singh
    Randhir Singh
    Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India
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  • Sowrabha Jayatheertha
    Sowrabha Jayatheertha
    Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India
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  • Raghavan Varadarajan*
    Raghavan Varadarajan
    Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
    Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India
    *E-mail: [email protected]. Phone: +91-80-22932612. Fax: +91-80-23600535.
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    Orcidhttps://orcid.org/0000-0002-0823-7577
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The Journal of Physical Chemistry B

Cite this: J. Phys. Chem. B 2023, 127, 8, 1704–1714
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https://doi.org/10.1021/acs.jpcb.2c07262
Published February 15, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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The receptor binding domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We have previously reported the design and characterization of a mammalian cell expressed RBD derivative, mRBD1-3.2, that has higher thermal stability and greatly enhanced immunogenicity relative to the wild type mRBD. The protein is highly thermotolerant and immunogenic and is being explored for use in room temperature stable Covid-19 vaccine formulations. In the current study, we have investigated the folding pathway of both WT and stabilized RBD. It was found that chemical denaturation of RBD proceeds through a stable equilibrium intermediate. Thermal and chemical denaturation is reversible, as assayed by binding to the receptor ACE2. Unusually, in its native state, RBD binds to the hydrophobic probe ANS, and enhanced ANS binding is observed for the equilibrium intermediate state. Further characterization of the folding of mRBD1-3.2, both in solution and after reconstitution of lyophilized protein stored for a month at 37 °C, revealed a higher stability represented by higher Cm, faster refolding, slower unfolding, and enhanced resistance to proteolytic cleavage relative to WT. In contrast to WT RBD, the mutant showed decreased interaction with the hydrophobic moiety linoleic acid. Collectively, these data suggest that the enhanced immunogenicity results from reduced conformational fluctuations that likely enhance in vivo half-life as well as reduce the exposure of irrelevant non-neutralizing epitopes to the immune system.

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Published as part of The Journal of Physical Chemistry virtual special issue “Steven G. Boxer Festschrift”.

Introduction

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Coronaviruses are the causative agent of several zoonotic outbreaks that have occurred since 2002. The recent ongoing pandemic caused by SARS-CoV-2 has, as of October 2022, resulted in ∼620 million confirmed cases, and 6.5 million deaths have been reported globally. (1)
SARS-CoV-2 is an enveloped RNA virus that uses its spike glycoprotein (S) to facilitate entry into the host cell. The spike protein is a homotrimeric class I fusion glycoprotein which is comprised of two subunits: S1, which contains the N-terminal domain (NTD) and the receptor binding domain (RBD), and S2, which contains the heptad repeats HR1 and HR2. The virus enters host cells through the binding of the viral RBD with the host cell surface receptor, angiotensin converting enzyme (ACE2). This process triggers conformational changes in the spike protein, leading to membrane fusion, mediated by the S2 subunit. (2) Therefore, the RBD plays a critical role in virus attachment and infectivity. Further, several serological studies and clinical trials have shown that most of the potent neutralizing antibodies elicited through vaccination or natural infection are targeted toward the RBD of the spike glycoprotein. (3−7)
The present study probes the folding pathway of the SARS-CoV-2 RBD, using mammalian cell expressed RBD-based subunit vaccine candidates, which were previously shown to be more tolerant to thermal stresses than a stabilized spike ectodomain. (8) The relevance of protein folding studies for biological function and molecular evolution is widely recognized. Despite its crucial role in vaccine design, little is known about the folding pathway of the RBD. Here, we report thermodynamic and kinetic characterization of the SARS-CoV-2 RBD, primarily using nanodifferential scanning fluorimetry (nanoDSF). Many proteins are known to populate intermediate states, which may aid folding through restricting the number of accessible conformations. (9,10) In the current study using nanoDSF coupled with ANS binding studies, we found an equilibrium intermediate state, populated at intermediate denaturant concentrations, which is stable at 25 °C. An RBD variant, mRBD1-3.2, which was isolated and characterized in a previous study, exhibited higher thermal stability (ΔTm = 7 °C), enhanced thermal tolerance (stable in adjuvanted formulations at 45 °C for over a week), and elicited sera with an over 100-fold increase in neutralizing antibody titers relative to the corresponding WT RBD. (11) The sera neutralized diverse pseudoviruses as well as replicative virus from variants of concern including Alpha, Beta, Delta, and Omicron BA.1. (11,12) Hamsters immunized with mRBD1-3.2 were protected from lethal challenge with a high dose of SARS-CoV-2 virus. Upon challenge, hamsters exhibited minimal weight loss and showed lower pathology scores compared to the control unimmunized animals. (11)
mRBD1-3.2 contains three mutations (A348P, Y365W, and P527L). (11) To further probe the stabilizing effect of these mutations and rationalize the enhanced immunogenicity, we investigated the folding pathway and proteolytic sensitivity of mRBD1-3.2. We observed faster refolding rates and slower unfolding rates of the stabilized variant compared to the WT protein as well as higher resistance to chemical denaturation and tryptic digestion. We also showed that the stabilized RBD has reduced sensitivity to destabilization by the hydrophobic ligand, linoleic acid, which has previously been suggested to bind to the SARS-CoV-2 spike. (13)

Materials and Methods

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Reagents

Ultrapure MB grade guanidium chloride (GdnCl) and hen egg white lysozyme (HEWL) were obtained from USB Corporation (Cleveland, OH). HEWL was diluted in 1 × PBS at pH 7.4 and used without any further purification. Linoleic acid was obtained from Sigma-Aldrich, cat no. L1012.

mRBD Protein Expression and Purification

The mRBD1-WT sequence consists of residues 331–532 of the SARS-CoV-2 RBD fused to a C-terminal cleavable His tag, while the stabilized RBD (mRBD1-3.2) sequence is the same as that of mRBD1-WT, residues 331–532, with incorporation of three stabilizing mutations, A348P, Y365W, and P527L, identified by YSD. (11) The purifications of mRBD1-WT and mRBD1-3.2 were carried out as described previously. (8,11) Briefly, transfections were performed with 1 μg of plasmid per 1 mL of Expi293F cells, complexed with ExpiFectamine293 and transiently transfected into Expi293F cells, followed by the addition of Enhancer 1 and Enhancer 2 post 16 h of transfection, according to the manufacturer’s protocol. Five days post transfection, culture supernatant was collected and 2-fold diluted with 1 × PBS (pH 7.4) and bound to a Ni-NTA column pre-equilibrated with 1 × PBS (pH 7.4). Proteins were affinity purified using Ni-NTA affinity chromatography by mixing 2 mL of Ni Sepharose 6 Fast Flow (GE Healthcare) with the supernatant. The unbound fraction was removed, and the resin was washed with a 10-column volume wash of wash buffer (1 × PBS, 25 mM imidazole, pH 7.4). The bound proteins were eluted with 300 mM imidazole in 1 × PBS (pH 7.4). The eluted fractions were pooled and dialyzed thrice using a 3–5 kDa (MWCO) dialysis membrane (Spectrum Laboratories) against 1 × PBS, at pH 7.4. The eluted fractions were subjected to 15% Tricine SDS-PAGE, and the protein concentration was determined.

Isothermal Denaturation

Equilibrium unfolding experiments of mRBD1-WT and mRBD1-3.2 were carried out by nanoDSF (Prometheus NT.48) as described previously. (14,15) Briefly, the changes in the fluorescence ratio (F350/F330) were monitored, after overnight incubation of 10 μM of protein at 25 °C in 1 × PBS containing various concentrations of the denaturant, guanidium chloride (GdnCl), to determine the stability parameters for chemical denaturation. GdnCl concentrations were calculated from measurements of the refractive index using a refractometer. The data were analyzed using Sigmaplot for Windows scientific graphing software, and the plots were fitted to a three-state unfolding model (N3 → 3D). mRBD is a monomer in the native state, while ACE2-hFc is present in a dimeric form. The fraction unfolded for all mRBD mutants was calculated as described previously. (15)

Refolding and Unfolding Kinetics

Refolding and unfolding kinetics for mRBD1-WT and mRBD1-3.2 (both in solution and the lyophilized form kept at 37 °C, for one month followed by reconstitution in 1 × PBS) were monitored by nanoDSF (F350/F330) using PR.Time Control software (Prometheus NT.48) at 25 °C as described previously. (14,15) To measure the rates of refolding from U → N, protein in 1 × PBS, at pH 7.4, was denatured in 4 M GdnCl and subsequently diluted to final concentrations of 0.4, 0.5, 0.6, 0.8, and 1.0 M GdnCl. The changes in signal were monitored as a function of time. To measure the unfolding kinetics from N → U, protein in a native buffer (1 × PBS, pH 7.4) was diluted into the same buffer containing 7 M GdnCl to a final concentration of GdnCl varying from 3 to 4 M, and the changes in the fluorescence ratio (F350/F330) were monitored as a function of time. To measure the unfolding kinetics from N → I, protein in native buffer (1 × PBS, pH 7.4) was diluted into the same buffer containing 4 M GdnCl to a final concentration of GdnCl varying from 1.8 to 2.2 M, and the changes in the fluorescence ratio (F350/F330) were monitored as a function of time. Refolding and unfolding kinetic traces of fluorescence intensity in all cases, as a function of time for RBD, were normalized from 0 to 1 between native and denatured baselines as described previously. (14−17) The data for the mRBD proteins were analyzed using Sigmaplot for Windows scientific graphing software, and data were fit to a three parameter equation for exponential decay for refolding (y = a0 + a1 exp(−kf1x)) and a five parameter exponential for unfolding (y = A0 + A1 exp(−ku1x) + a2 exp(−ku2x)), yielding slow and fast phase rate constants as described previously, where x is the time of refolding/unfolding.

Thermal Stability Measurement by nanoDSF of the mRBD Proteins

nanoDSF (Prometheus NT.48) was also used to probe the thermal unfolding of the mRBD proteins. The assays were carried out with 10 μM of each protein, and the apparent thermal stability (Tm) was determined by monitoring the changes in the fluorescence ratio (F350/F330) as a function of temperature as described previously. (14,15,18) Samples were heated from 20 to 95 °C with a ramp rate of 1 °C/min. For mRBD proteins, refolding was carried out in 0.5, 0.6, 0.8, and 1.0 M GdnCl, and refolded protein was subjected to thermal denaturation with native protein in 0.5, 0.6, 0.8, and 1.0 M GdnCl as controls. To probe the activity of the refolded mRBD proteins, the refolded proteins along with native proteins in the same GdnCl concentrations were incubated with ACE2 and subjected to thermal denaturation.

Unfolding and Refolding Fluorescence Studies

Experiments on equilibrium GdnCl induced unfolding were carried out using a FluoroMax-3 (Horiba Jobin Yvon) spectrofluorometer. Measurements were carried out with 5 μM of protein in 1 × PBS buffer, at pH 7.4 and 25 °C. The proteins were unfolded by incubating with different concentrations of GdnCl (3.0 to 4.0 M) for 4 h at 25 °C prior to the fluorescence measurements. Refolding was carried out with a fixed concentration of the protein (5 μM) in 1 × PBS buffer at pH 7.4 denatured in 3.0 M GdnCl and subsequently diluted to the final denaturant concentrations. The samples were excited at 280 nm, and the emission was measured between 300 and 450 nm. For each experiment, a cuvette containing only buffer and the appropriate GdnCl concentration were used to calculate blank values, which were then subtracted from the sample values.

ANS Binding

To investigate protein surface hydrophobicity, ANS binding studies were carried out. The fluorescence signal of ANS increases and is blue-shifted upon its binding to hydrophobic pockets or patches. (19) The samples were incubated overnight with different GdnCl concentrations in a range from 0.0 to 4.4 M, followed by the addition of ANS (1:10 protein to ANS molar ratio) and further incubated for 30 min. Samples incubated with ANS were then excited at 390 nm, and the emission was recorded from 400 to 600 nm. A native hen egg white lysozyme (HEWL) negative control in 1 × PBS at pH 7.4 was used. For all of the samples, fluorescence intensities were corrected by subtracting the blank containing the corresponding GdnCl concentration and ANS in 1 × PBS at pH 7.4. The protein was at a fixed concentration of 5 μM in all cases.

Trypsin Digestion

The proteins were dialyzed in autoclaved Milli-Q water and reconstituted in 50 mM Tris at pH 7.5 with 1 mM CaCl2 and incubated with TPCK treated trypsin (protein: TPCK trypsin = 50:1) at 37 °C for different time points. The reaction was stopped by adding SDS dye and boiling the samples for 10 min at 95 °C followed by SDS-PAGE.

Linoleic Acid Binding

Aliquots of 10 μM mRBD proteins were incubated with different concentrations of linoleic acid in 1 × PBS at pH 7.4 and subjected to thermal denaturation from 20 to 95 °C with a ramp rate of 1 °C/min using nanoDSF (Prometheus NT.48). The apparent thermal stability (Tm) was determined by monitoring the changes in the fluorescence ratio (F350/F330) as a function of temperature.

Results

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mRBD1 Unfolds Through an Equilibrium Intermediate

The equilibrium unfolding experiment of mRBD1-WT and mRBD1-3.2 was carried out as explained in the Materials and Methods. The unfolded fraction of mRBD1-WT and mRBD1-3.2 in the presence of different concentrations of GdnCl was plotted as a function of denaturant concentration (Figure 1a). An equilibrium intermediate state was observed for mRBD1-WT. The experiment was performed in triplicate. The stabilized variant mRBD1-3.2 was 2.5 kcal/mol more stable than mRBD1-WT, for the intermediate to unfolded state (I → U) transition, and 2.0 kcal/mol more stable than the mRBD1-WT, for the native to intermediate state (N → I) transition (Figure 1b, Table S1). The midpoint of chemical denaturation (Cm), ΔG0, and m values and are listed in Table S1. The equilibrium denaturation experiment was also performed in reverse, by unfolding then refolding the protein back to different final GdnCl concentrations ranging from 4.4 to 0.3 M, and the same profile, as in equilibrium unfolding Cm, was obtained (Figure S1a,b).

Figure 1

Figure 1. Chemical denaturation and refolding kinetics of wildtype (WT) and stabilized mRBD1. (a) Equilibrium denaturation profile of mRBD1-WT and stabilized mutant, mRBD1-3.2, with 10 μM protein in 1 × PBS, at pH 7.4 and 25 °C monitored using nanoDSF. The experimental data are shown in blue and red circles for mRBD1-WT and mRBD-1-3.2, respectively, while the fit is shown in blue and red lines for mRBD1-WT and mRBD-1-3.2, respectively. The theoretical curves were obtained by fitting all the melts with three-state unfolding models. (b) The estimated values of ΔG° for different transitions at 25 °C for mRBD1-WT (blue) and mRBD1-3.2 (red). (c,d) Refolding of mRBD1-WT and mRBD1-3.2 from U → N follows single exponential kinetics. Representative refolding kinetic traces of (c) mRBD1-WT and (d) mRBD1-3.2 at 10 μM protein concentration obtained at 0.4, 0.5, and 0.6 M final GdnCl concentrations are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated refolding rate constants on denaturant concentration for the U → N transition for mRBD1-WT (blue circles) and mRBD1-3.2 (red circles). The estimated refolding rate constant and the refolding m values at zero denaturant concentration are determined to be 0.067 s–1 and −0.07 M–1 s–1, respectively, for mRBD1-WT and 0.096 s–1 and −0.11 M–1 s–1, respectively, for mRBD1-3.2. (f) The amplitudes A0 (triangles) and A1 (circles) of burst and fast phases as a function of GdnCl concentration. Data for mRBD1-WT and mRBD1-3.2 are shown in blue and red colors, respectively. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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RBD Refolding and Unfolding Kinetics

Refolding kinetics of mRBD1-WT and mRBD1-3.2 were also monitored by time-course fluorescence spectroscopy at 25 °C using differential scanning fluorimetry (DSF). The refolding of mRBD1-WT and mRBD1-3.2 follow single exponential kinetics. Refolding reactions were carried out at three different GdnCl concentrations for mRBD1-WT and mRBD1-3.2 (Figure1, Table S2). The estimated refolding rate constants showed high dependence on GdnCl concentration (Figure 1e, Table S2). The estimated refolding rate constant and the refolding m values extrapolated to zero denaturant concentration are determined to be 0.067 s–1 and −0.07 M–1 s–1, respectively, for mRBD1-WT and 0.096 s–1 and −0.11 M–1 s–1, respectively, for mRBD1-3.2 (Table S2). With an increase in GdnCl concentration, there is an increase in the refolding phase amplitude at the expense of the burst phase (Figure 1f, Table S2). The unfolding from the native to the unfolded state of mRBD1-WT follows biphasic exponential kinetics (Figure S2). The unfolding was carried out at three different GdnCl concentrations, and representative kinetic traces obtained at 3.2, 3.4, and 3.6 M are shown in Figure S2a,c. Since unfolding was rapid under these conditions, the rate constants of unfolding of both mRBD1-WT and mRBD1-3.2 for N → U could not be accurately determined.

Biphasic Unfolding Kinetics for mRBD1 Between Native and Intermediate States

The unfolding kinetics of mRBD1-WT and mRBD1-3.2 from N → I follow biphasic exponential kinetics, with a burst, fast, and a slow phase (Figure 2, Table S3), suggestive either of native state heterogeneity that might arise from multiple glycoforms or of parallel unfolding pathways. Unfolding reactions were carried out at four different GdnCl concentrations for mRBD1-WT and three different GdnCl concentrations for mRBD1-3.2. Representative unfolding kinetic traces of mRBD1-WT and mRBD1-3.2 obtained at 1.8, 2.0, and 2.2 M final GdnCl concentrations are shown in Figure 3a and d, respectively. The estimated unfolding rate constants and amplitudes were plotted as a function of GdnCl concentration. The unfolding of mRBD1-WT is fast, with an estimated rate constant of 0.06 s–1 for the fast phase at zero denaturant concentration, while that for the slow phase is 0.008 s–1 (Table S3). The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.16 M–1 s–1 and 0.10 M–1 s–1, respectively, for mRBD-1 WT (Table S3). The unfolding of mRBD1-3.2 is relatively slower, with an estimated rate constant of 0.012 s–1 for the fast phase at zero denaturant concentration, while that for the slow phase is 0.003 s–1 (Table S3). The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.20 M–1 s–1 and 0.13 M–1 s–1, respectively, for mRBD1-3.2 (Table S3). The amplitudes of the burst phase increase, while that of the slow phase decreases with an increase in GdnCl concentration, as shown in Figure 3c for mRBD1-WT and Figure 3f for mRBD1-3.2 (Table S3).

Figure 2

Figure 2. Unfolding kinetics of WT and stabilized mRBD1 (N → I). (a) Biphasic unfolding kinetics of mRBD1-WT from N→I, with a burst, fast, and a slow phase. Representative unfolding kinetic traces of mRBD1-WT at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (b) The dependence of the estimated unfolding rate constants on denaturant concentration for the N→I transition. The estimated rate constant for mRBD1-WT of the fast phase at zero denaturant concentration is determined to be 0.06 s–1, while that of the slow phase is 0.008 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.16 M–1 s–1 and 0.10 M–1 s–1, respectively. (c) The amplitudes of the burst (pink triangles) and slow (red circles) phases as a function of GdnCl concentration. (d) Biphasic unfolding kinetics of mRBD1-3.2 from N→I, with a burst, fast, and a slow phase. Representative unfolding kinetic traces of mRBD1-3.2 at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated unfolding rate constants for the N→I transition on the denaturant concentration. The estimated rate constant for mRBD1-3.2 of the fast phase at zero denaturant concentration is determined to be 0.012 s–1, while that of the slow phase is 0.003 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.20 M–1 s–1 and 0.13 M–1 s–1, respectively. (f) The amplitudes of the burst (pink triangles) and slow (red circles) phases as a function of GdnCl concentration are shown. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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Figure 3

Figure 3. Refolding and unfolding kinetics following resolubilization of lyophilized, stabilized mRBD1-3.2 after storage at 37 °C for one month. (a) Refolding kinetics of mRBD1-3.2 for the U→N transition at 10 μM protein concentration. The experimental refolding kinetic traces obtained at 0.4, 0.5, and 0.6 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (b) The dependence of the estimated refolding rate constants on denaturant concentration for the U→N transition. The estimated refolding rate constant and the refolding m values at zero denaturant concentration are determined to be 0.094 s–1 and −0.11 M–1 s–1, respectively. (c) The amplitudes of the A0 (pink triangles) and A1 (red circles) phases as a function of GdnCl concentration. (d) Biphasic unfolding kinetics of mRBD1-3.2 from N→I, with a burst, fast, and slow phase. Representative unfolding kinetic traces of mRBD1-3.2 at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentrations are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated unfolding rate constants for the N→I transition on denaturant concentration. The estimated unfolding rate constant for mRBD1-3.2 of the fast phase at zero denaturant concentration is determined to be 0.035 s–1, while that of the slow phase is 0.004 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.10 M–1 s–1 and 0.07 M–1 s–1, respectively. (f) The amplitudes of the burst (pink triangles) and slow (red circles) phases with an increase in GdnCl concentration. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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mRBD1-3.2 Folding Kinetics is Unchanged Even after Storage for One Month in the Lyophilized State at 37 °C

mRBD1-3.2 protein was dialyzed against 1 × PBS, lyophilized, and then incubated for one month at 37 °C. After sample reconstitution in water, unfolding and refolding kinetics were carried out to compare the integrity of 37 °C stored lyophilized protein with that of freshly prepared, unlyophilized protein. The refolding of lyophilized mRBD1-3.2 protein that had been stored at 37 °C was found to follow single exponential kinetics. All of the refolding reactions were carried out at three different GdnCl concentrations, of 0.4, 0.5, and 0.6 M. Representative refolding kinetic traces of mRBD1-3.2 at 37 °C (lyophilized and incubated for 1 month at 37 °C prior to reconstitution) at 10 μM protein concentration are shown in Figure 3a. The estimated refolding rate constants and amplitudes were plotted as a function of GdnCl concentration (Figure 3b,c, Table S4). The estimated refolding rate constant and the refolding m values at zero denaturant concentration for mRBD1-3.2 at 37 °C for the U→N transition are determined to be 0.094 s–1 and −0.11 M–1 s–1, respectively. Increasing GdnCl concentration led to an increase in the slow phase amplitude and decrease in the burst phase amplitude (Figure 3c, Table S4).
The unfolding kinetics of mRBD1-3.2 at 37 °C from N→I follow biphasic exponential kinetics, with a burst, fast, and slow phase (Figure 3, Table S4). Three different GdnCl concentrations were used for the unfolding experiment of lyophilized and reconstituted mRBD1-3.2 (Figure 3d). The estimated unfolding rate constants and amplitudes from N→I were also plotted as a function of GdnCl concentration. The estimated rate constant for mRBD1-3.2 at 37 °C for the fast phase at zero denaturant concentration was found to be 0.035 s–1, while that of the slow phase was found to be 0.004 s–1 (Table S4). The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.10 M–1 s–1 and 0.07 M–1 s–1, respectively (Table S4). The amplitudes of the burst phase increase, while that of the slow phase decreases with an increase in GdnCl concentration, as shown in Figure 3f and Table S4.

Refolded mRBD Proteins Bind ACE2 in Vitro

The refolded and the native mRBD1 proteins in the presence of different concentrations of GdnCl were also subjected to thermal denaturation, and the apparent Tm was calculated for mRBD1-WT (Figure 4a) and for stabilized RBD (Figure 5a). mRBD1 proteins showed clear thermal transitions even at 1.0 M GdnCl, confirming that they were in a folded conformation in the presence of GdnCl. Further, the ability of the refolded mRBD proteins and the native proteins in the presence of different concentrations of GdnCl to bind ACE2 was also qualitatively assessed using nanoDSF for mRBD1-WT (Figure 4b–d) and for mRBD1-3.2 (Figure 5b–d). In all cases, both the refolded and native mRBD1 proteins in the presence of GdnCl were able to bind to ACE2, indicating that the functional integrity of the proteins is still maintained after refolding.

Figure 4

Figure 4. Thermal stability and ACE2 binding of native and refolded mRBD1-WT protein. (a) Thermal unfolding traces of 10 μM of refolded mRBD1-WT protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) and native mRBD1-WT protein in the same concentrations of GdnCl (solid lines). (b) Thermal unfolding in the presence of ACE2 of 10 μM of native mRBD1-WT protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) or refolded mRBD1-WT in the same concentrations of GdnCl (solid lines). In all cases, refolded and native proteins show a similar shift in the Tm in the presence of ACE2. Thermal unfolding of native protein in the absence of GdnCl (solid black lines). (c and d) Comparison of Tm values for native and refolded proteins in the absence or presence of ACE2, respectively.

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Figure 5

Figure 5. Thermal stability and ACE2 binding of native and refolded stabilized mRBD1 protein. (a) Thermal unfolding traces of 10 μM of refolded mRBD1-3.2 protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) and native mRBD1-3.2 protein in the same concentrations of GdnCl (solid lines). (b) Thermal unfolding in the presence of ACE2 of 10 μM of native mRBD1-3.2 protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) or refolded mRBD1-3.2 in the same concentrations of GdnCl (solid lines). In all cases, refolded and native proteins show a similar shift in the Tm in the presence of ACE2. Thermal unfolding of native protein in the absence of GdnCl (solid black lines). (c and d) Comparison of absolute Tm values for native and refolded proteins in the absence or presence of ACE2, respectively.

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Characterization of the Native, Intermediate, and Unfolded States of mRBD1 Using Fluorescence Spectroscopy

We compared the Tm of both mRBD1 and its stabilized version using nanoDSF. mRBD1-3.2 exhibited a higher Tm than WT by ∼7 °C (Figure 6a). The refolded mRBD1 proteins and the native proteins in 1.0 M GdnCl, display a similar fluorescence profile to that of the native protein in the absence of any GdnCl (Figure 6b for mRBD1-WT and Figure 6c for mRBD1-3.2). Increasing GdnCl concentration to 2.1 and 2.3 M where the intermediate state is populated for mRBD1-WT and mRBD1-3.2, respectively, resulted in a shift of the fluorescence peak to 350 nm. Upon complete unfolding of the mRBD1-WT protein in GdnCl concentrations ranging from 3.0 to 4.0 M, there is a further shift of the peak maximum to about 380 nm.

Figure 6

Figure 6. Thermal and chemical denaturation of WT and stabilized mRBD1, probed by fluorescence spectroscopy. (a) Tm of WT and mRBD1-3.2 measured by nanoDSF. Fluorescence profiles of mRBD1-WT (b) and mRBD1-3.2 (c) with 5 μM protein in 1 × PBS at pH 7.4 at 25 °C. Solid and dashed lines represent native and refolded mRBD1 protein incubated at various GdnCl concentrations. The samples were unfolded for 4 h in 4.0 M GdnCl before refolding. Refolding was initiated by diluting the samples with 1 × PBS to 0.5 M GdnCl. For accessing the intermediate state, samples were unfolded for 4 h before refolding by diluting the samples with 1 × PBS to a 2.1 M GdnCl concentration for mRBD1-WT and to a 2.3 M GdnCl concentration for mRBD1-3.2. The unfolded state fluorescence profile of mRBD1 at a GdnCl concentration of 4.0 M is shown as a blue solid line.

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mRBD1-3.2 Exhibits Higher Stability and Resistance to Proteolytic Cleavage than mRBD1-WT and Both Proteins Exhibit Binding to ANS in the Native State

The three introduced mutations in mRBD1-3.2 render it more proteolysis-resistant than mRBD1. SDS-PAGE shows that mRBD1-3.2 protein is stable even after 1 h of incubation with trypsin at 37 °C (Figure 7a) in contrast to mRBD1, which is significantly degraded in 20 min.

Figure 7

Figure 7. Proteolytic resistance and ANS binding of WT and stabilized mRBD1. (a) mRBD1 and mRBD1-3.2 proteins were incubated with trypsin at a ratio of 50:1 protein to trypsin at 37 °C for different time points and subjected to SDS-PAGE. (b) ANS fluorescence intensity at 465 nm following incubation with 5 μM of mRBD1 proteins at GdnCl concentrations ranging from 0.0 to 4.4 M as indicated above. The samples were incubated with GdnCl overnight at 25 °C before the addition of ANS at a final concentration of 50 μM and further incubation for 30 min. The samples were then excited at 390 nm, and the emission was monitored from 300 to 600 nm. Each point represents the fluorescence intensity at 465 nm of ANS bound to mRBD1 at a specific GdnCl concentration.

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We next investigated the ability of mRBD1-WT and mRBD1-3.2 proteins to bind to ANS, a hydrophobic, fluorescent molecular probe. ANS is used to probe the surface hydrophobicity of proteins. Hen egg white lysozyme (HEWL) was used as a control because, like most folded proteins, it does not exhibit binding to ANS in its native state. Relative to lysozyme, both mRBD1 proteins showed significant binding to ANS in their native state, suggesting that both proteins have exposed hydrophobic patches even in the absence of any denaturant (Figure 7b). Interestingly, ANS bound to mRBD1, relative to mRBD1-3.2, showed a larger blue shift, suggesting that the dye is bound in a more hydrophobic environment in WT mRBD1. With increasing GdnCl concentration, ANS binding increases until it displays a maximum fluorescence signal when the intermediate state is well populated (Figure 7b). Increasing the denaturant concentration further leads to a decrease in ANS binding until the protein is completely unfolded.

mRBD1-3.2 Shows Reduced Binding to Destabilizing Fatty Acid, Linoleic Acid, Compared to WT

It has previously been suggested that linoleic acid (LA) binds to a hydrophobic pocket located in the RBD of the SARS-CoV-2 spike. (13) We therefore examined the binding of linoleic acid to both WT and stabilized RBD by measuring the thermal stability in the presence and absence of linoleic acid, with the expectation that the RBD thermal stability would be enhanced in the presence of LA. Surprisingly (Figure 8), the thermal stability decreased in the presence of LA, suggesting that LA binds preferentially to denatured or partially unfolded RBD relative to the native state. WT mRBD1 exhibited a large decrease in Tm at all concentrations of LA (Figure 8a). mRBD1-3.2 exhibited a small amount of destabilization in the presence of LA in a manner that depended on the LA concentration (Figure 8b). This difference in binding of LA between the two protein variants is likely due to the mutation Y365W present in mRBD1-3.2 proximal to the hydrophobic binding pocket of LA, which might obstruct LA binding (20) as well as enhanced global stability, resulting in decreased conformational fluctuations, thus inhibiting LA binding.

Figure 8

Figure 8. Interaction of WT and stabilized mRBD1 with linoleic acid assayed by nanodifferential scanning fluorimetry (nanoDSF). Thermal denaturation profiles of (a) mRBD1-WT and (b) mRBD1-3.2 protein without and with different concentrations of linoleic acid (LA), measured by nanoDSF.

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Discussion

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It is clear that SARS-CoV-2 and its variants are firmly entrenched in humans as well as multiple other animal hosts. While several efficacious Covid-19 vaccines exist, they all require low temperature or ultra-low temperature storage, which are barriers to deployment in many parts of the world. There is a continued need for efficacious vaccines that do not require a cold chain, and this has led to isolation of stabilized forms of the spike protein and its constituent RBD domain.
Yeast surface display (YSD) coupled to second-site saturation suppressor mutagenesis (SSSM) in which incorporation of a single destabilizing mutation into each member of a single-site saturation mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. (23,24) This method was used to identify three stabilizing mutations (A348P, Y365W, and P527L) in the RBD that was displayed on the yeast surface. These mutations significantly improved RBD expression, thermal stability, thermal tolerance, and immunogenicity. (11)
In the present study, we have compared the unfolding and refolding kinetics, chemical denaturation, and LA binding of the mRBD1-WT with that of the stabilized mRBD1-3.2. The goal of these studies was to obtain insights into the reasons for enhanced immunogenicity of the stabilized RBD, and its ability to withstand prolonged exposure to high temperatures in both a lyophilized state as well as in solution.
Faster refolding and slower unfolding rates were observed for the stabilized RBD compared to mRBD1-WT. Upon lyophilization and incubation at 37 °C for one month, following resolubilization, mRBD1-3.2 exhibited similar refolding and unfolding kinetics to fresh protein in solution, consistent with its unchanged ACE2 binding observed previously. (11) The equilibrium GdnCl denaturation profile of stabilized RBD showed an unusual three-state profile with higher stability than WT, represented by shifts in the position of both the N → I and I → U transitions to higher GdnCl concentration than that of mRBD1-WT. A shift was also observed in the ANS binding fluorescence profile, which interestingly showed that both WT and stabilized RBD show a high ANS binding fluorescence signal in their native states, compared to that of the lysozyme control. This suggests the presence of exposed hydrophobic patches on the RBD surface in its native state, which contains several loops. The positions of regions that are disordered in the absence of ACE2 but become ordered upon ACE2 binding are highlighted in Figure 9a. These differences are localized to an important region of RBD called the receptor binding motif (RBM), which consists of residues 438–505. The RBM, as the name suggests, is the region that contacts the ACE2 receptor. The majority of neutralizing antibodies also contact the RBM. The dynamic stability of mRBD1-3.2 was also probed by examining its resistance to proteolytic digestion by TPCK trypsin. The protein was resistant to digestion at 37 °C even after 60 min of incubation, in contrast to WT RBD, which showed significant degradation after 20 min. These stability results in vitro suggest that the stabilized protein may have a longer half-life in vivo, contributing to enhanced immunogenicity. mRBD1 harbors four pairs of disulfide bonds, of which three are in the core of RBD, thereby stabilizing the protein. (21) In its native state, mRBD1 binds to ANS, a well-known probe for hydrophobic surfaces, and this binding reaches a maximum at the equilibrium intermediate state. The intermediate state did not bind to ACE2 at 2.0 and 2.2 M GdnCl as assessed by ELISA and nanoDSF, possibly because of the partial unfolding of ACE2 at these denaturant concentrations (Figure S3). The stabilized variant mRBD1-3.2 was 2.5 kcal/mol more stable than mRBD1-WT, for the intermediate to unfolded state (I → U) transition and 2.0 kcal/mol more stable than the mRBD1-WT, for the native to intermediate state (N → I) transition (Figure 1b, Table S1). At the present time, we do not have detailed structural information on the equilibrium intermediate. However, examination of the structure of the native, folded protein reveals that RBD has five twisted antiparallel beta strands connected via loops and short helices to form a well packed core, while the RBM is comprised of extended helices and loops which form the contact surface with ACE2. We hypothesize that the RBM unfolds first at low concentrations of the denaturant to reach a stable intermediate state that retains the tightly packed core, which is resistant to unfolding under denaturant concentrations less than 3.0 M. The locations of three stabilizing mutations A348P, Y365W, and P527L are highlighted in the structure of RBD shown in Figure 9. The A348P substitution is found in multiple other sarbecovirus RBDs including SARS-CoV-1. It likely stabilizes the protein through decreasing the conformational entropy of the unfolded state, thereby increasing the unfolded state free energy. (25) The Y365W mutation partially fills in a cavity in the RBD structure, while the origin of the stabilizing effect of the P527L substitution is currently unclear, as this residue is disordered in the crystal structure of RBD bound to ACE2 (PDB ID 6m0j). (21) None of these residues is part of the RBM. However, if RBM unfolding is coupled to structural destabilization of the core, it might explain why the native to intermediate transition is shifted to higher denaturant concentration for the stabilized protein. Destabilization of the unfolded state by the A348P substitution would also explain why the intermediate to unfolded state transition is shifted to higher denaturant concentration for the stabilized triple mutant RBD. Y365W was also identified as a stabilizing mutation in another study, (20) which examined mutations previously identified by deep mutational scanning (26) in conjunction with computational modeling. In the stabilized RBD, the lower red shift upon ANS binding (Figure 7b), resistance to proteolysis (Figure 7a), increase in Cm of the equilibrium intermediate, and reduced destabilization by linoleic acid (Figure 8a) all suggest that the stabilized RBD shows lower dynamic flexibility than WT RBD. In vivo, this probably results in increased half-life as well as lower exposure of irrelevant non-neutralizing epitopes, thus resulting in improved elicitation of neutralizing antibodies. The relationship between protein stability, protein dynamic fluctuations, and immunogenicity is poorly understood, and there are very few studies that have explored this systematically. Although we do not have definitive explanations for the increased immunogenicity of the stabilized RBD relative to WT, we have identified some biophysical correlates. In the future, we will attempt to image both WT and stabilized immunogens within B-cell follicles in vivo (27) to better understand the origins of the enhanced immunogenicity.

Figure 9

Figure 9. The structure of SARS-CoV-2 RBD bound to ACE2 and linoleic acid (a) RBD bound to ACE2 with the RBM regions that are disordered in the unbound structure but become ordered upon ACE2 binding highlighted in red, ordered RBM region shown in blue, disulfides shown in yellow sticks. Coordinates for bound and unbound RBD from the RBD:ACE2 crystal structure 6M0J (21) and the unliganded spike Cryo-EM structure 6VYB, (22) respectively. (b) Linoleic acid bound SARS-CoV-2 RBD. Linoleic acid (LA) moiety is indicated in red sticks and accommodated in a hydrophobic pocket gated by residue Y365, which is shown in magenta color. Y365, along with A348 and P527, are the three residues that are mutated in the stabilized mRBD1-3.2 and are highlighted in magenta with residue name and number. Coordinates taken from Cryo-EM structure of LA bound spike PDB ID: 6ZB4. (13)

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Statistical Analysis

All the experiments are carried out in biological replicates (n = 3), and the listed errors are the standard errors derived from the values obtained for individual replicates. For the nanoDSF measurements, each experiment has been carried out thrice (n = 3), and the listed errors are the standard errors derived from the values obtained for individual replicates.

Data Availability

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The data relevant to the figures in the paper have been made available within the article and in the Supporting Information section. All unique/stable reagents generated in this study are available from the Lead Contact Raghavan Varadarajan ([email protected]).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.2c07262.

  • Thermodynamic parameters (Cm, ΔG0, m) and kinetic parameters for unfolding and refolding of mRBD proteins; unfolding kinetics for mRBD-WT from the native to unfolded state and equilibrium denaturation profile of ACE2-Fc (PDF)

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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

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  • Corresponding Author
    • Raghavan Varadarajan - Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India;  Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India;  Orcidhttps://orcid.org/0000-0002-0823-7577;  Email: [email protected]
  • Authors
    • Kawkab Kanjo - Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
    • Gopinath Chattopadhyay - Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India;  Orcidhttps://orcid.org/0000-0002-6408-1968
    • Sameer Kumar Malladi - Molecular Biophysics Unit (MBU), Indian Institute of Science, Bengaluru 560012, India
    • Randhir Singh - Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India
    • Sowrabha Jayatheertha - Mynvax Private Limited, Fourth Floor, Brigade MLR Center, 50, Vanivilas Rd, Gandhi Bazaar, Basavanagudi, Bangalore, Karnataka 560004, India
  • Author Contributions

    R.V., K.K., and G.C. designed most of the experiments. K.K. performed all the experiments. G.C. assisted in some of the experiments. G.C. and K.K. analyzed the data for all the experiments. S.K.M performed the initial studies of RBD denaturation and made several useful suggestions. R.S. and S.J. carried out protein expression and purification. R.V., K.K., and G.C. wrote most of the manuscript with critical input and review from all other authors.

  • Notes
    The authors declare the following competing financial interest(s): R.V. and S.K.M. are inventors on a patent application describing the stabilized RBD. The other authors have no competing interests.

Acknowledgments

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This work was funded in part by a grant to R.V. from the Bill and Melinda Gates Foundation (INV-005948) and by the Biotechnology Industry Research and Assistance Council, Government of India. Funding for infrastructural support was from DST FIST, UGC Centre for Advanced study, MHRD, and the DBT IISc Partnership Program. S.K.M. acknowledges the support of an MHRD-IISc doctoral fellowship. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. K.K. and G.C. acknowledge MHRD for their fellowships. We also thank all the members of the RV lab for their valuable suggestions.

References

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This article references 27 other publications.

  1. 1
    World Health Organization. WHO Coronavirus Disease (Covid-19) Dashboard. Available at https://Covid19.Who.Int/ (accessed October 13, 2022).
    Google Scholar
    There is no corresponding record for this reference.
  2. 2
    Yang, Y.; Du, L. SARS-CoV-2 Spike Protein: A Key Target for Eliciting Persistent Neutralizing Antibodies. Signal Transduct. Target. Ther. 2021, 6, 95,  DOI: 10.1038/s41392-021-00523-5
    Google Scholar
    2
    SARS-CoV-2 spike protein: a key target for eliciting persistent neutralizing antibodies
    Yang Yang; Yang Yang; Yang Yang; Du Lanying
    Signal transduction and targeted therapy (2021), 6 (1), 95 ISSN:.
    There is no expanded citation for this reference.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BB3snptlKgsw%253D%253D&md5=36abc1048e4725b46a3e0469471e4354
  3. 3
    Zhu, F.-C.; Li, Y.-H.; Guan, X.-H.; Hou, L.-H.; Wang, W.-J.; Li, J.-X.; Wu, S.-P.; Wang, B.-S.; Wang, Z.; Wang, L. Safety, Tolerability, and Immunogenicity of a Recombinant Adenovirus Type-5 Vectored COVID-19 Vaccine: A Dose-Escalation, Open-Label, Non-Randomised, First-in-Human Trial. Lancet 2020, 395, 1845– 1854,  DOI: 10.1016/S0140-6736(20)31208-3
    Google Scholar
    3
    Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial
    Zhu, Feng-Cai; Li, Yu-Hua; Guan, Xu-Hua; Hou, Li-Hua; Wang, Wen-Juan; Li, Jing-Xin; Wu, Shi-Po; Wang, Bu-Sen; Wang, Zhao; Wang, Lei; Jia, Si-Yue; Jiang, Hu-Dachuan; Wang, Ling; Jiang, Tao; Hu, Yi; Gou, Jin-Bo; Xu, Sha-Bei; Xu, Jun-Jie; Wang, Xue-Wen; Wang, Wei; Chen, Wei
    Lancet (2020), 395 (10240), 1845-1854CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
    A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. We did a dose-escalation, single-center, open-label, non-randomized, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults 18-60 yr were sequentially enrolled and allocated to 1 of 3 dose groups (5 × 1010, 1 × 1011, and 1.5 × 1011 viral particles) to receive an i.m. injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralizing antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralization and pseudovirus neutralization tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36.3 yr) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the anal. At least 1 adverse reaction within the 1st 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]). Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralizing antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhtVSjtrvO&md5=a14ff6459d359bd1743e3549191b3508
  4. 4
    Corbett, 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. Evaluation of the MRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N. Engl. J. Med. 2020, 383, 1544– 1555,  DOI: 10.1056/NEJMoa2024671
    Google Scholar
    4
    Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates
    Corbett, 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.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFWmsr7I&md5=8ecba6b18bc615d886ca5016b7c85496
  5. 5
    Lau, E. H. Y.; Tsang, O. T. Y.; Hui, D. S. C.; Kwan, M. Y. W.; Chan, W.-h.; Chiu, S. S.; Ko, R. L. W.; Chan, K. H.; Cheng, S. M. S.; Perera, R. A. P. M.; Cowling, B. J.; Poon, L. L. M.; Peiris, M. Neutralizing Antibody Titres in SARS-CoV-2 Infections. Nat. Commun. 2021, 12, 63,  DOI: 10.1038/s41467-020-20247-4
    Google Scholar
    5
    Neutralizing antibody titers in SARS-CoV-2 infections
    Lau, Eric H. Y.; Tsang, Owen T. Y.; Hui, David S. C.; Kwan, Mike Y. W.; Chan, Wai-hung; Chiu, Susan S.; Ko, Ronald L. W.; Chan, Kin H.; Cheng, Samuel M. S.; Perera, Ranawaka A. P. M.; Cowling, Benjamin J.; Poon, Leo L. M.; Peiris, Malik
    Nature Communications (2021), 12 (1), 63CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
    The SARS-CoV-2 pandemic poses the greatest global public health challenge in a century. Neutralizing antibody is a correlate of protection and data on kinetics of virus neutralizing antibody responses are needed. We tested 293 sera from an observational cohort of 195 reverse transcription polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections collected from 0 to 209 days after onset of symptoms. Of 115 sera collected ≥61 days after onset of illness tested using plaque redn. neutralization (PRNT) assays, 99.1% remained seropos. for both 90% (PRNT90) and 50% (PRNT50) neutralization endpoints. We est. that it takes at least 372, 416 and 133 days for PRNT50 titers to drop to the detection limit of a titer of 1:10 for severe, mild and asymptomatic patients, resp. At day 90 after onset of symptoms (or initial RT-PCR detection in asymptomatic infections), it took 69, 87 and 31 days for PRNT50 antibody titers to decrease by half (T1/2) in severe, mild and asymptomatic infections, resp. Patients with severe disease had higher peak PRNT90 and PRNT50 antibody titers than patients with mild or asymptomatic infections. Age did not appear to compromise antibody responses, even after accounting for severity. We conclude that SARS-CoV-2 infection elicits robust neutralizing antibody titers in most individuals.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXnsVKmtQ%253D%253D&md5=fbb3dd051d862ea8b21f57e22e4c26c6
  6. 6
    Anderson, E. J.; Rouphael, N. G.; Widge, A. T.; Jackson, L. A.; Roberts, P. C.; Makhene, M.; Chappell, J. D.; Denison, M. R.; Stevens, L. J.; Pruijssers, A. J. Safety and Immunogenicity of SARS-CoV-2 MRNA-1273 Vaccine in Older Adults. N. Engl. J. Med. 2020, 383, 2427– 2438,  DOI: 10.1056/NEJMoa2028436
    Google Scholar
    6
    Safety and immunogenicity of SARS-CoV-2mRNA-1273 vaccine in older adults
    Anderson, E. J.; Rouphael, N. G.; Widge, A. T.; Jackson, L. A.; Roberts, P. C.; Makhene, M.; Chappell, J. D.; Denison, M. R.; Stevens, L. J.; Pruijssers, A. J.; McDermott, A. B.; Flach, B.; Lin, B. C.; Doria-Rose, N. A.; O'Dell, S.; Schmidt, S. D.; Corbett, K. S.; Swanson, P. A.; Padilla, M.; Neuzil, K. M.; Bennett, H.; Leav, B.; Makowski, M.; Albert, J.; Cross, K.; Edara, V. V.; Floyd, K.; Suthar, M. S.; Martinez, D. R.; Baric, R.; Buchanan, W.; Luke, C. J.; Phadke, V. K.; Rostad, C. A.; Ledgerwood, J. E.; Graham, B. S.; Beigel, J. H.
    New England Journal of Medicine (2020), 383 (25), 2427-2438CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
    Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been assocd. with an older age. We conducted a phase 1, dose-escalation, open-label trial of a mRNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or 71 years). All the participants were assigned sequentially to receive two doses of either 25μg or 100μg of vaccine administered 28 days apart. Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-μ g dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-μ g dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, resp. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Bindingand neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. In this small study involving older adults, adverse events assocd. with the mRNA-1273 vaccine were mainly mild or moderate. The 100-μ g dose induced higher binding- and neutralizing-antibody titers than the 25-μ g dose, which supports the use of the 100-μ g dose in a phase 3 vaccine trial.
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  7. 7
    Ramasamy, M. N.; Minassian, A. M.; Ewer, K. J.; Flaxman, A. L.; Folegatti, P. M.; Owens, D. R.; Voysey, M.; Aley, P. K.; Angus, B.; Babbage, G. Safety and Immunogenicity of ChAdOx1 NCoV-19 Vaccine Administered in a Prime-Boost Regimen in Young and Old Adults (COV002): A Single-Blind, Randomised, Controlled, Phase 2/3 Trial. Lancet 2020, 396, 1979– 1993,  DOI: 10.1016/S0140-6736(20)32466-1
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    7
    Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial
    Ramasamy, Maheshi N.; Minassian, Angela M.; Ewer, Katie J.; Flaxman, Amy L.; Folegatti, Pedro M.; Owens, Daniel R.; Voysey, Merryn; Aley, Parvinder K.; Angus, Brian; Babbage, Gavin; Belij-Rammerstorfer, Sandra; Berry, Lisa; Bibi, Sagida; Bittaye, Mustapha; Cathie, Katrina; Chappell, Harry; Charlton, Sue; Cicconi, Paola; Clutterbuck, Elizabeth A.; Colin-Jones, Rachel; Dold, Christina; Emary, Katherine R. W.; Fedosyuk, Sofiyah; Fuskova, Michelle; Gbesemete, Diane; Green, Catherine; Hallis, Bassam; Hou, Mimi M.; Jenkin, Daniel; Joe, Carina C. D.; Kelly, Elizabeth J.; Kerridge, Simon; Lawrie, Alison M.; Lelliott, Alice; Lwin, May N.; Makinson, Rebecca; Marchevsky, Natalie G.; Mujadidi, Yama; Munro, Alasdair P. S.; Pacurar, Mihaela; Plested, Emma; Rand, Jade; Rawlinson, Thomas; Rhead, Sarah; Robinson, Hannah; Ritchie, Adam J.; Ross-Russell, Amy L.; Saich, Stephen; Singh, Nisha; Smith, Catherine C.; Snape, Matthew D.; Song, Rinn; Tarrant, Richard; Themistocleous, Yrene; Thomas, Kelly M.; Villafana, Tonya L.; Warren, Sarah C.; Watson, Marion E. E.; Douglas, Alexander D.; Hill, Adrian V. S.; Lambe, Teresa; Gilbert, Sarah C.; Faust, Saul N.; Pollard, Andrew J.
    Lancet (2020), 396 (10267), 1979-1993CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
    Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunization should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19, in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clin. research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either i.m. ChAdOx1 nCoV-19 (2·2 x 1010 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the std.-dose cohort (3·5-6·5 x 1010 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 yr after the booster were assessed using an inhouse standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA80). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the no. of cases of symptomatic, virol. confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labeled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two std. doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (std.-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titers after a boost dose were similar across all age groups (median MNA80 at day 42 in the std.-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single std. dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Center, Thames Valley and South Midlands NIHR Clin. Research Network, and AstraZeneca.
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    Malladi, S. K.; Singh, R.; Pandey, S.; Gayathri, S.; Kanjo, K.; Ahmed, S.; Khan, M. S.; Kalita, P.; Girish, N.; Upadhyaya, A. Design of a Highly Thermotolerant, Immunogenic SARS-CoV-2 Spike Fragment. J. Biol. Chem. 2021, 296, 100025,  DOI: 10.1074/jbc.RA120.016284
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    Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment
    Malladi, Sameer Kumar; Singh, Randhir; Pandey, Suman; Gayathri, Savitha; Kanjo, Kawkab; Ahmed, Shahbaz; Khan, Mohammad Suhail; Kalita, Parismita; Girish, Nidhi; Upadhyaya, Aditya; Reddy, Poorvi; Pramanick, Ishika; Bhasin, Munmun; Mani, Shailendra; Bhattacharyya, Sankar; Joseph, Jeswin; Thankamani, Karthika; Raj, V. Stalin; Dutta, Somnath; Singh, Ramandeep; Nadig, Gautham; Varadarajan, Raghavan
    Journal of Biological Chemistry (2021), 296 (), 100025CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
    Virtually all SARS-CoV-2 vaccines currently in clin. testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufg. expertise for these nucleic-acid-based modalities, esp. in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temps. as high as 100°C when lyophilized, up to 70°C in soln. and stable for over 4 wk at 37°C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD deriv. elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
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  9. 9
    Connell, K. B.; Miller, E. J.; Marqusee, S. The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants That Fold via Two-State and Three-State Mechanisms. J. Mol. Biol. 2009, 391, 450– 460,  DOI: 10.1016/j.jmb.2009.05.085
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    9
    The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms
    Connell, Katelyn B.; Miller, Erik J.; Marqusee, Susan
    Journal of Molecular Biology (2009), 391 (2), 450-460CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
    Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli RNase H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. The folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topol. but altered regional stability, were compared using a protein engineering approach. The data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. The results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.
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  10. 10
    Brockwell, D. J.; Radford, S. E. Intermediates: Ubiquitous Species on Folding Energy Landscapes?. Curr. Opin. Struct. Biol. 2007, 17, 30– 37,  DOI: 10.1016/j.sbi.2007.01.003
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    10
    Intermediates: ubiquitous species on folding energy landscapes?
    Brockwell, David J.; Radford, Sheena E.
    Current Opinion in Structural Biology (2007), 17 (1), 30-37CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
    A review. Although intermediates have long been recognized as fascinating species that form during the folding of large proteins, the role that intermediates play in the folding of small, single-domain proteins has been widely debated. Recent discoveries using new, sensitive methods of detection and studies combining simulation and expt. have now converged on a common vision for folding, involving intermediates as ubiquitous stepping stones en route to the native state. The results suggest that the folding energy landscapes of even the smallest proteins possess significant ruggedness in which intermediates stabilized by both native and non-native interactions are common features.
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  11. 11
    Ahmed, S.; Khan, M. S.; Gayathri, S.; Singh, R.; Kumar, S.; Patel, U. R.; Malladi, S. K.; Rajmani, R. S.; van Vuren, P. J.; Riddell, S. A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern. Front. Immunol. 2021, 12, 765211,  DOI: 10.3389/fimmu.2021.765211
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    11
    A stabilized, monomeric, receptor binding domain elicits high-titer neutralizing antibodies against all SARS-CoV-2 variants of concern
    Ahmed, Shahbaz; Khan, Mohammad Suhail; Gayathri, Savitha; Singh, Randhir; Kumar, Sahil; Patel, Unnatiben Rajeshbhai; Malladi, Sameer Kumar; Rajmani, Raju S.; van Vuren, Petrus Jansen; Riddell, Shane; Goldie, Sarah; Girish, Nidhi; Reddy, Poorvi; Upadhyaya, Aditya; Pandey, Suman; Siddiqui, Samreen; Tyagi, Akansha; Jha, Sujeet; Pandey, Rajesh; Khatun, Oyahida; Narayan, Rohan; Tripathi, Shashank; McAuley, Alexander J.; Singanallur, Nagendrakumar Balasubramanian; Vasan, Seshadri S.; Ringe, Rajesh P.; Varadarajan, Raghavan
    Frontiers in Immunology (2021), 12 (), 765211CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
    Satn. suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temp. of ~ 7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An addnl. deriv. with three addnl. mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temp. exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 deriv. showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qual. improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or addnl. components, and can be stored at room temp. They are a useful modality to combat COVID-19, esp. in remote and low-resource settings.
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  12. 12
    Jansen van Vuren, P.; McAuley, A. J.; Kuiper, M. J.; Singanallur, N. B.; Bruce, M. P.; Riddell, S.; Goldie, S.; Mangalaganesh, S.; Chahal, S.; Drew, T. W. Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice. Viruses 2022, 14, 800,  DOI: 10.3390/v14040800
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    12
    Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice
    Jansen van Vuren, Petrus; McAuley, Alexander J.; Kuiper, Michael J.; Singanallur, Nagendrakumar Balasubramanian; Bruce, Matthew P.; Riddell, Shane; Goldie, Sarah; Mangalaganesh, Shruthi; Chahal, Simran; Drew, Trevor W.; Blasdell, Kim R.; Tachedjian, Mary; Caly, Leon; Druce, Julian D.; Ahmed, Shahbaz; Khan, Mohammad Suhail; Malladi, Sameer Kumar; Singh, Randhir; Pandey, Suman; Varadarajan, Raghavan; Vasan, Seshadri S.
    Viruses (2022), 14 (4), 800CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivs. that can withstand 100°C for 90 min and 37°C for four weeks and help eliminate cold-chain requirements. We show that mice immunized with these vaccine formulations elicit high titers of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an av. 14.4-fold redn. in neutralization against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold redn. for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clin. trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modeling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerization domain.
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    Toelzer, C.; Gupta, K.; Yadav, S. K. N.; Borucu, U.; Davidson, A. D.; Kavanagh Williamson, M.; Shoemark, D. K.; Garzoni, F.; Staufer, O.; Milligan, R. Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein. Science 2020, 370, 725– 730,  DOI: 10.1126/science.abd3255
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    13
    Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein
    Toelzer, Christine; Gupta, Kapil; Yadav, Sathish K. N.; Borucu, Ufuk; Davidson, Andrew D.; Kavanagh Williamson, Maia; Shoemark, Deborah K.; Garzoni, Frederic; Staufer, Oskar; Milligan, Rachel; Capin, Julien; Mulholland, Adrian J.; Spatz, Joachim; Fitzgerald, Daniel; Berger, Imre; Schaffitzel, Christiane
    Science (Washington, DC, United States) (2020), 370 (6517), 725-730CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathol. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor-binding domains tightly bind the essential free fatty acid linoleic acid (LA) in 3 composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.
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  14. 14
    Chattopadhyay, G.; Varadarajan, R. Facile Measurement of Protein Stability and Folding Kinetics Using a Nano Differential Scanning Fluorimeter. Protein Sci. 2019, 28, 1127– 1134,  DOI: 10.1002/pro.3622
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    14
    Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter
    Chattopadhyay, Gopinath; Varadarajan, Raghavan
    Protein Science (2019), 28 (6), 1127-1134CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
    With advancements in high-throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chem. denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample vols. 10-50-fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodn. and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.
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  15. 15
    Chattopadhyay, G.; Bhowmick, J.; Manjunath, K.; Ahmed, S.; Goyal, P.; Varadarajan, R. Mechanistic Insights into Global Suppressors of Protein Folding Defects. PLoS Genet. 2022, 18, e1010334  DOI: 10.1371/journal.pgen.1010334
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    15
    Mechanistic insights into global suppressors of protein folding defects
    Chattopadhyay, Gopinath; Bhowmick, Jayantika; Manjunath, Kavyashree; Ahmed, Shahbaz; Goyal, Parveen; Varadarajan, Raghavan
    PLoS Genetics (2022), 18 (8), e1010334CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)
    Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examd. six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we detd. the crystal structures of three such suppressors to obtain insight into the specific mol. interactions responsible for the obsd. effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodn. stabilization is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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  16. 16
    Baliga, C.; Varadarajan, R.; Aghera, N. Homodimeric Escherichia Coli Toxin CcdB (Controller of Cell Division or Death B Protein) Folds via Parallel Pathways. Biochemistry 2016, 55, 6019– 6031,  DOI: 10.1021/acs.biochem.6b00726
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    16
    Homodimeric Escherichia coli Toxin CcdB (Controller of Cell Division or Death B Protein) Folds via Parallel Pathways
    Baliga, Chetana; Varadarajan, Raghavan; Aghera, Nilesh
    Biochemistry (2016), 55 (43), 6019-6031CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
    The existence of parallel pathways in the folding of proteins seems intuitive, yet remains controversial. We explore the folding kinetics of the homodimeric E. coli toxin CcdB using multiple optical probes and approaches. Kinetic studies performed as a function of protein and denaturant concns. demonstrate that the folding of CcdB is a four-state process. The two intermediates populated during folding are present on parallel pathways. Both form by rapid assocn. of the monomers in a diffusion limited manner and appear to be largely unstructured, as they are silent to the optical probes employed in the current study. The existence of parallel pathways is supported by the insensitivity of the amplitudes of the refolding kinetic phases to the different probes used in the study. More importantly, interrupted refolding studies and ligand binding studies clearly demonstrate that the native state forms in a bi-exponential manner, implying the presence of at least two pathways. Our studies indicate that the CcdA antitoxin binds only to the folded CcdB dimer and not to any earlier folding intermediates. Thus, despite being part of the same operon, the antitoxin does not appear to modulate the folding pathway of the toxin encoded by the downstream cistron. This study highlights the utility of ligand binding in distinguishing between sequential and parallel pathways in protein folding studies, while also providing insights into mol. interactions during folding in Type II TA systems.
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  17. 17
    Tripathi, A.; Gupta, K.; Khare, S.; Jain, P. C.; Patel, S.; Kumar, P.; Pulianmackal, A. J.; Aghera, N.; Varadarajan, R. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data. Mol. Biol. Evol. 2016, 33, 2960– 2975,  DOI: 10.1093/molbev/msw182
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    17
    Molecular determinants of mutant phenotypes, inferred from saturation mutagenesis data
    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan
    Molecular Biology and Evolution (2016), 33 (11), 2960-2975CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used satn. mutagenesis combined with deep sequencing to det. mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliph. and charged amino acid substitutions. At nonactive-site positions, the av. mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were obsd. for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and soly. The inability to refold reversibly, as well as a decreased folding rate in vitro, is assocd. with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most delete riousmutants showed an increased in vivo activity and soly. only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.
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  18. 18
    Chattopadhyay, G.; Bhasin, M.; Ahmed, S.; Gosain, T. P.; Ganesan, S.; Das, S.; Thakur, C.; Chandra, N.; Singh, R.; Varadarajan, R. Functional and Biochemical Characterization of the MazEF6 Toxin-Antitoxin System of Mycobacterium Tuberculosis. J. Bacteriol. 2022, 204, e0005822  DOI: 10.1128/jb.00058-22
    Google Scholar
    There is no corresponding record for this reference.
  19. 19
    Acharya, P.; Madhusudhana Rao, N. Stability Studies on a Lipase from Bacillus Subtilis in Guanidinium Chloride. J. Protein Chem. 2003, 22, 51– 60,  DOI: 10.1023/A:1023067827678
    Google Scholar
    19
    Stability studies on a lipase from Bacillus subtilis in guanidinium chloride
    Acharya, Priyamvada; Madhusudhana Rao, N.
    Journal of Protein Chemistry (2003), 22 (1), 51-60CODEN: JPCHD2; ISSN:0277-8033. (Kluwer Academic/Plenum Publishers)
    Lipase from B. subtilis is a "lidless" lipase that does not show interfacial activation. Due to exposure of the active site to solvent, the lipase tends to aggregate. Here, the authors investigated the soln. properties and unfolding of B. subtilis lipase in guanidinium chloride (GdmCl) to understand its aggregation behavior and stability. Dynamic light scattering (DLS), near- and far-UV CD, activity, and intrinsic fluorescence of lipase suggest that the protein undergoes unfolding between 1M and 2M GdmCl. The polarity-sensitive dye, 1,1',-bis-(4-anilino)naphthalene-5,5''-disulfonic acid (bis-ANS), a probe for hydrophobic pockets, bound cooperatively to the native lipase. An intermediate populated in 1.75M GdmCl that strongly bound bis-ANS was identified. The tendency of the native protein to aggregate in soln. and specific binding to bis-ANS confirmed that the lipase has exposed hydrophobic pockets and that this surface hydrophobicity strongly influences the unfolding pathway of the lipase in GdmCl.
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  20. 20
    Ellis, D.; Brunette, N.; Crawford, K. H. D.; Walls, A. C.; Pham, M. N.; Chen, C.; Herpoldt, K.-L.; Fiala, B.; Murphy, M.; Pettie, D. Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design. Front. Immunol. 2021, 12, 710263,  DOI: 10.3389/fimmu.2021.710263
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    20
    Stabilization of the SARS-CoV-2 spike receptor-binding domain using deep mutational scanning and structure-based design
    Ellis, Daniel; Brunette, Natalie; Crawford, Katharine H. D.; Walls, Alexandra C.; Pham, Minh N.; Chen, Chengbo; Herpoldt, Karla-Luise; Fiala, Brooke; Murphy, Michael; Pettie, Deleah; Kraft, John C.; Malone, Keara D.; Navarro, Mary Jane; Ogohara, Cassandra; Kepl, Elizabeth; Ravichandran, Rashmi; Sydeman, Claire; Ahlrichs, Maggie; Johnson, Max; Blackstone, Alyssa; Carter, Lauren; Starr, Tyler N.; Greaney, Allison J.; Lee, Kelly K.; Veesler, David; Bloom, Jesse D.; King, Neil P.
    Frontiers in Immunology (2021), 12 (), 710263CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
    The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufg. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as 2nd-generation vaccine candidates, as they feature robust manufg. characteristics and have shown promising immunogenicity in preclin. models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid-binding pocket. Screening of several designs led to the selection of 2 lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clin. trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.
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  21. 21
    Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L. Structure of the SARS-CoV-2 Spike Receptor-Binding Domain Bound to the ACE2 Receptor. Nature 2020, 581, 215– 220,  DOI: 10.1038/s41586-020-2180-5
    Google Scholar
    21
    Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
    Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
    Nature (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.
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  22. 22
    Walls, 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, 281– 292.e6,  DOI: 10.1016/j.cell.2020.02.058
    Google Scholar
    22
    Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein
    Walls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, David
    Cell (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.
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  23. 23
    Ahmed, S.; Manjunath, K.; Chattopadhyay, G.; Varadarajan, R. Identification of Stabilizing Point Mutations through Mutagenesis of Destabilized Protein Libraries. J. Biol. Chem. 2022, 298, 101785,  DOI: 10.1016/j.jbc.2022.101785
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    23
    Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries
    Ahmed, Shahbaz; Manjunath, Kavyashree; Chattopadhyay, Gopinath; Varadarajan, Raghavan
    Journal of Biological Chemistry (2022), 298 (4), 101785CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
    Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, esp. for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site satn. mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, satn. suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addn., multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temp. of about 20°C, and that displayed enhanced tolerance to high temp. exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.
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  24. 24
    Ahmed, S.; Bhasin, M.; Manjunath, K.; Varadarajan, R. Prediction of Residue-Specific Contributions to Binding and Thermal Stability Using Yeast Surface Display. Front. Mol. Biosci. 2022, 8, 800819,  DOI: 10.3389/fmolb.2021.800819
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    There is no corresponding record for this reference.
  25. 25
    Prajapati, R. S.; Das, M.; Sreeramulu, S.; Sirajuddin, M.; Srinivasan, S.; Krishnamurthy, V.; Ranjani, R.; Ramakrishnan, C.; Varadarajan, R. Thermodynamic Effects of Proline Introduction on Protein Stability. Proteins 2007, 66, 480– 491,  DOI: 10.1002/prot.21215
    Google Scholar
    25
    Thermodynamic effects of proline introduction on protein stability
    Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C.; Varadarajan, Raghavan
    Proteins: Structure, Function, and Bioinformatics (2007), 66 (2), 480-491CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
    The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodn. effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucinevaline binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by x-ray crystallog. to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chem. denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chem. denaturation at room temp. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the redn. in entropy of unfolding was typically several fold larger than the theor. est. of -4 cal K-1 mol-1 derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, resp. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.
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  26. 26
    Starr, T. N.; Greaney, A. J.; Hilton, S. K.; Ellis, D.; Crawford, K. H.D.; Dingens, A. S.; Navarro, M. J.; Bowen, J. E.; Tortorici, M. A.; Walls, A. C.; King, N. P.; Veesler, D.; Bloom, J. D. Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 2020, 182, 1295– 1310.e20,  DOI: 10.1016/j.cell.2020.08.012
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    26
    Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding
    Starr, Tyler N.; Greaney, Allison J.; Hilton, Sarah K.; Ellis, Daniel; Crawford, Katharine H. D.; Dingens, Adam S.; Navarro, Mary Jane; Bowen, John E.; Tortorici, M. Alejandra; Walls, Alexandra C.; King, Neil P.; Veesler, David; Bloom, Jesse D.
    Cell (Cambridge, MA, United States) (2020), 182 (5), 1295-1310.e20CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we exptl. measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial no. of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open anal. pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations obsd. during viral surveillance.
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  27. 27
    Tokatlian, T.; Read, B. J.; Jones, C. A.; Kulp, D. W.; Menis, S.; Chang, J. Y. H.; Steichen, J. M.; Kumari, S.; Allen, J. D.; Dane, E. L. Innate Immune Recognition of Glycans Targets HIV Nanoparticle Immunogens to Germinal Centers. Science 2019, 363, 649– 654,  DOI: 10.1126/science.aat9120
    Google Scholar
    27
    Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers
    Tokatlian, Talar; Read, Benjamin J.; Jones, Christopher A.; Kulp, Daniel W.; Menis, Sergey; Chang, Jason Y. H.; Steichen, Jon M.; Kumari, Sudha; Allen, Joel D.; Dane, Eric L.; Liguori, Alessia; Sangesland, Maya; Lingwood, Daniel; Crispin, Max; Schief, William R.; Irvine, Darrell J.
    Science (Washington, DC, United States) (2019), 363 (6427), 649-654CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    In vaccine design, antigens are often arrayed in a multi-valent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or "free" forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concd. in germinal centers in a complement-, mannose-binding lectin (MBL)-, and immunogen glycan-dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune-mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.
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Cite this: J. Phys. Chem. B 2023, 127, 8, 1704–1714
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  • Abstract

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    Figure 1

    Figure 1. Chemical denaturation and refolding kinetics of wildtype (WT) and stabilized mRBD1. (a) Equilibrium denaturation profile of mRBD1-WT and stabilized mutant, mRBD1-3.2, with 10 μM protein in 1 × PBS, at pH 7.4 and 25 °C monitored using nanoDSF. The experimental data are shown in blue and red circles for mRBD1-WT and mRBD-1-3.2, respectively, while the fit is shown in blue and red lines for mRBD1-WT and mRBD-1-3.2, respectively. The theoretical curves were obtained by fitting all the melts with three-state unfolding models. (b) The estimated values of ΔG° for different transitions at 25 °C for mRBD1-WT (blue) and mRBD1-3.2 (red). (c,d) Refolding of mRBD1-WT and mRBD1-3.2 from U → N follows single exponential kinetics. Representative refolding kinetic traces of (c) mRBD1-WT and (d) mRBD1-3.2 at 10 μM protein concentration obtained at 0.4, 0.5, and 0.6 M final GdnCl concentrations are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated refolding rate constants on denaturant concentration for the U → N transition for mRBD1-WT (blue circles) and mRBD1-3.2 (red circles). The estimated refolding rate constant and the refolding m values at zero denaturant concentration are determined to be 0.067 s–1 and −0.07 M–1 s–1, respectively, for mRBD1-WT and 0.096 s–1 and −0.11 M–1 s–1, respectively, for mRBD1-3.2. (f) The amplitudes A0 (triangles) and A1 (circles) of burst and fast phases as a function of GdnCl concentration. Data for mRBD1-WT and mRBD1-3.2 are shown in blue and red colors, respectively. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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    Figure 2

    Figure 2. Unfolding kinetics of WT and stabilized mRBD1 (N → I). (a) Biphasic unfolding kinetics of mRBD1-WT from N→I, with a burst, fast, and a slow phase. Representative unfolding kinetic traces of mRBD1-WT at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (b) The dependence of the estimated unfolding rate constants on denaturant concentration for the N→I transition. The estimated rate constant for mRBD1-WT of the fast phase at zero denaturant concentration is determined to be 0.06 s–1, while that of the slow phase is 0.008 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.16 M–1 s–1 and 0.10 M–1 s–1, respectively. (c) The amplitudes of the burst (pink triangles) and slow (red circles) phases as a function of GdnCl concentration. (d) Biphasic unfolding kinetics of mRBD1-3.2 from N→I, with a burst, fast, and a slow phase. Representative unfolding kinetic traces of mRBD1-3.2 at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated unfolding rate constants for the N→I transition on the denaturant concentration. The estimated rate constant for mRBD1-3.2 of the fast phase at zero denaturant concentration is determined to be 0.012 s–1, while that of the slow phase is 0.003 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.20 M–1 s–1 and 0.13 M–1 s–1, respectively. (f) The amplitudes of the burst (pink triangles) and slow (red circles) phases as a function of GdnCl concentration are shown. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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    Figure 3

    Figure 3. Refolding and unfolding kinetics following resolubilization of lyophilized, stabilized mRBD1-3.2 after storage at 37 °C for one month. (a) Refolding kinetics of mRBD1-3.2 for the U→N transition at 10 μM protein concentration. The experimental refolding kinetic traces obtained at 0.4, 0.5, and 0.6 M final GdnCl concentration are shown in black, green, and blue, respectively, while the fits are shown in red. (b) The dependence of the estimated refolding rate constants on denaturant concentration for the U→N transition. The estimated refolding rate constant and the refolding m values at zero denaturant concentration are determined to be 0.094 s–1 and −0.11 M–1 s–1, respectively. (c) The amplitudes of the A0 (pink triangles) and A1 (red circles) phases as a function of GdnCl concentration. (d) Biphasic unfolding kinetics of mRBD1-3.2 from N→I, with a burst, fast, and slow phase. Representative unfolding kinetic traces of mRBD1-3.2 at 10 μM protein concentration are shown. The experimental unfolding kinetic traces obtained at 1.8, 2.0, and 2.2 M final GdnCl concentrations are shown in black, green, and blue, respectively, while the fits are shown in red. (e) The dependence of the estimated unfolding rate constants for the N→I transition on denaturant concentration. The estimated unfolding rate constant for mRBD1-3.2 of the fast phase at zero denaturant concentration is determined to be 0.035 s–1, while that of the slow phase is 0.004 s–1. The unfolding m values of the transition states of the fast and the slow phases were calculated to be 0.10 M–1 s–1 and 0.07 M–1 s–1, respectively. (f) The amplitudes of the burst (pink triangles) and slow (red circles) phases with an increase in GdnCl concentration. The error bars, wherever shown, represent the standard deviation from two independent experiments.

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    Figure 4

    Figure 4. Thermal stability and ACE2 binding of native and refolded mRBD1-WT protein. (a) Thermal unfolding traces of 10 μM of refolded mRBD1-WT protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) and native mRBD1-WT protein in the same concentrations of GdnCl (solid lines). (b) Thermal unfolding in the presence of ACE2 of 10 μM of native mRBD1-WT protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) or refolded mRBD1-WT in the same concentrations of GdnCl (solid lines). In all cases, refolded and native proteins show a similar shift in the Tm in the presence of ACE2. Thermal unfolding of native protein in the absence of GdnCl (solid black lines). (c and d) Comparison of Tm values for native and refolded proteins in the absence or presence of ACE2, respectively.

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    Figure 5

    Figure 5. Thermal stability and ACE2 binding of native and refolded stabilized mRBD1 protein. (a) Thermal unfolding traces of 10 μM of refolded mRBD1-3.2 protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) and native mRBD1-3.2 protein in the same concentrations of GdnCl (solid lines). (b) Thermal unfolding in the presence of ACE2 of 10 μM of native mRBD1-3.2 protein in 0.5 M, 0.6 M, 0.8 M, and 1.0 M GdnCl (dashed lines) or refolded mRBD1-3.2 in the same concentrations of GdnCl (solid lines). In all cases, refolded and native proteins show a similar shift in the Tm in the presence of ACE2. Thermal unfolding of native protein in the absence of GdnCl (solid black lines). (c and d) Comparison of absolute Tm values for native and refolded proteins in the absence or presence of ACE2, respectively.

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    Figure 6

    Figure 6. Thermal and chemical denaturation of WT and stabilized mRBD1, probed by fluorescence spectroscopy. (a) Tm of WT and mRBD1-3.2 measured by nanoDSF. Fluorescence profiles of mRBD1-WT (b) and mRBD1-3.2 (c) with 5 μM protein in 1 × PBS at pH 7.4 at 25 °C. Solid and dashed lines represent native and refolded mRBD1 protein incubated at various GdnCl concentrations. The samples were unfolded for 4 h in 4.0 M GdnCl before refolding. Refolding was initiated by diluting the samples with 1 × PBS to 0.5 M GdnCl. For accessing the intermediate state, samples were unfolded for 4 h before refolding by diluting the samples with 1 × PBS to a 2.1 M GdnCl concentration for mRBD1-WT and to a 2.3 M GdnCl concentration for mRBD1-3.2. The unfolded state fluorescence profile of mRBD1 at a GdnCl concentration of 4.0 M is shown as a blue solid line.

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    Figure 7

    Figure 7. Proteolytic resistance and ANS binding of WT and stabilized mRBD1. (a) mRBD1 and mRBD1-3.2 proteins were incubated with trypsin at a ratio of 50:1 protein to trypsin at 37 °C for different time points and subjected to SDS-PAGE. (b) ANS fluorescence intensity at 465 nm following incubation with 5 μM of mRBD1 proteins at GdnCl concentrations ranging from 0.0 to 4.4 M as indicated above. The samples were incubated with GdnCl overnight at 25 °C before the addition of ANS at a final concentration of 50 μM and further incubation for 30 min. The samples were then excited at 390 nm, and the emission was monitored from 300 to 600 nm. Each point represents the fluorescence intensity at 465 nm of ANS bound to mRBD1 at a specific GdnCl concentration.

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    Figure 8

    Figure 8. Interaction of WT and stabilized mRBD1 with linoleic acid assayed by nanodifferential scanning fluorimetry (nanoDSF). Thermal denaturation profiles of (a) mRBD1-WT and (b) mRBD1-3.2 protein without and with different concentrations of linoleic acid (LA), measured by nanoDSF.

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    Figure 9

    Figure 9. The structure of SARS-CoV-2 RBD bound to ACE2 and linoleic acid (a) RBD bound to ACE2 with the RBM regions that are disordered in the unbound structure but become ordered upon ACE2 binding highlighted in red, ordered RBM region shown in blue, disulfides shown in yellow sticks. Coordinates for bound and unbound RBD from the RBD:ACE2 crystal structure 6M0J (21) and the unliganded spike Cryo-EM structure 6VYB, (22) respectively. (b) Linoleic acid bound SARS-CoV-2 RBD. Linoleic acid (LA) moiety is indicated in red sticks and accommodated in a hydrophobic pocket gated by residue Y365, which is shown in magenta color. Y365, along with A348 and P527, are the three residues that are mutated in the stabilized mRBD1-3.2 and are highlighted in magenta with residue name and number. Coordinates taken from Cryo-EM structure of LA bound spike PDB ID: 6ZB4. (13)

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  • References

    This article references 27 other publications.

    1. 1
      World Health Organization. WHO Coronavirus Disease (Covid-19) Dashboard. Available at https://Covid19.Who.Int/ (accessed October 13, 2022).
      There is no corresponding record for this reference.
    2. 2
      Yang, Y.; Du, L. SARS-CoV-2 Spike Protein: A Key Target for Eliciting Persistent Neutralizing Antibodies. Signal Transduct. Target. Ther. 2021, 6, 95,  DOI: 10.1038/s41392-021-00523-5
      2
      SARS-CoV-2 spike protein: a key target for eliciting persistent neutralizing antibodies
      Yang Yang; Yang Yang; Yang Yang; Du Lanying
      Signal transduction and targeted therapy (2021), 6 (1), 95 ISSN:.
      There is no expanded citation for this reference.
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    3. 3
      Zhu, F.-C.; Li, Y.-H.; Guan, X.-H.; Hou, L.-H.; Wang, W.-J.; Li, J.-X.; Wu, S.-P.; Wang, B.-S.; Wang, Z.; Wang, L. Safety, Tolerability, and Immunogenicity of a Recombinant Adenovirus Type-5 Vectored COVID-19 Vaccine: A Dose-Escalation, Open-Label, Non-Randomised, First-in-Human Trial. Lancet 2020, 395, 1845– 1854,  DOI: 10.1016/S0140-6736(20)31208-3
      3
      Safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial
      Zhu, Feng-Cai; Li, Yu-Hua; Guan, Xu-Hua; Hou, Li-Hua; Wang, Wen-Juan; Li, Jing-Xin; Wu, Shi-Po; Wang, Bu-Sen; Wang, Zhao; Wang, Lei; Jia, Si-Yue; Jiang, Hu-Dachuan; Wang, Ling; Jiang, Tao; Hu, Yi; Gou, Jin-Bo; Xu, Sha-Bei; Xu, Jun-Jie; Wang, Xue-Wen; Wang, Wei; Chen, Wei
      Lancet (2020), 395 (10240), 1845-1854CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      A vaccine to protect against COVID-19 is urgently needed. We aimed to assess the safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 (Ad5) vectored COVID-19 vaccine expressing the spike glycoprotein of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain. We did a dose-escalation, single-center, open-label, non-randomized, phase 1 trial of an Ad5 vectored COVID-19 vaccine in Wuhan, China. Healthy adults 18-60 yr were sequentially enrolled and allocated to 1 of 3 dose groups (5 × 1010, 1 × 1011, and 1.5 × 1011 viral particles) to receive an i.m. injection of vaccine. The primary outcome was adverse events in the 7 days post-vaccination. Safety was assessed over 28 days post-vaccination. Specific antibodies were measured with ELISA, and the neutralizing antibody responses induced by vaccination were detected with SARS-CoV-2 virus neutralization and pseudovirus neutralization tests. T-cell responses were assessed by enzyme-linked immunospot and flow-cytometry assays. This study is registered with ClinicalTrials.gov, NCT04313127. Between March 16 and March 27, 2020, we screened 195 individuals for eligibility. Of them, 108 participants (51% male, 49% female; mean age 36.3 yr) were recruited and received the low dose (n=36), middle dose (n=36), or high dose (n=36) of the vaccine. All enrolled participants were included in the anal. At least 1 adverse reaction within the 1st 7 days after the vaccination was reported in 30 (83%) participants in the low dose group, 30 (83%) participants in the middle dose group, and 27 (75%) participants in the high dose group. The most common injection site adverse reaction was pain, which was reported in 58 (54%) vaccine recipients, and the most commonly reported systematic adverse reactions were fever (50 [46%]), fatigue (47 [44%]), headache (42 [39%]), and muscle pain (18 [17%]). Most adverse reactions that were reported in all dose groups were mild or moderate in severity. No serious adverse event was noted within 28 days post-vaccination. ELISA antibodies and neutralizing antibodies increased significantly at day 14, and peaked 28 days post-vaccination. Specific T-cell response peaked at day 14 post-vaccination. The Ad5 vectored COVID-19 vaccine is tolerable and immunogenic at 28 days post-vaccination. Humoral responses against SARS-CoV-2 peaked at day 28 post-vaccination in healthy adults, and rapid specific T-cell responses were noted from day 14 post-vaccination. Our findings suggest that the Ad5 vectored COVID-19 vaccine warrants further investigation.
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    4. 4
      Corbett, 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. Evaluation of the MRNA-1273 Vaccine against SARS-CoV-2 in Nonhuman Primates. N. Engl. J. Med. 2020, 383, 1544– 1555,  DOI: 10.1056/NEJMoa2024671
      4
      Evaluation of the mRNA-1273 vaccine against SARS-CoV-2 in nonhuman primates
      Corbett, 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.
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    5. 5
      Lau, E. H. Y.; Tsang, O. T. Y.; Hui, D. S. C.; Kwan, M. Y. W.; Chan, W.-h.; Chiu, S. S.; Ko, R. L. W.; Chan, K. H.; Cheng, S. M. S.; Perera, R. A. P. M.; Cowling, B. J.; Poon, L. L. M.; Peiris, M. Neutralizing Antibody Titres in SARS-CoV-2 Infections. Nat. Commun. 2021, 12, 63,  DOI: 10.1038/s41467-020-20247-4
      5
      Neutralizing antibody titers in SARS-CoV-2 infections
      Lau, Eric H. Y.; Tsang, Owen T. Y.; Hui, David S. C.; Kwan, Mike Y. W.; Chan, Wai-hung; Chiu, Susan S.; Ko, Ronald L. W.; Chan, Kin H.; Cheng, Samuel M. S.; Perera, Ranawaka A. P. M.; Cowling, Benjamin J.; Poon, Leo L. M.; Peiris, Malik
      Nature Communications (2021), 12 (1), 63CODEN: NCAOBW; ISSN:2041-1723. (Nature Research)
      The SARS-CoV-2 pandemic poses the greatest global public health challenge in a century. Neutralizing antibody is a correlate of protection and data on kinetics of virus neutralizing antibody responses are needed. We tested 293 sera from an observational cohort of 195 reverse transcription polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 infections collected from 0 to 209 days after onset of symptoms. Of 115 sera collected ≥61 days after onset of illness tested using plaque redn. neutralization (PRNT) assays, 99.1% remained seropos. for both 90% (PRNT90) and 50% (PRNT50) neutralization endpoints. We est. that it takes at least 372, 416 and 133 days for PRNT50 titers to drop to the detection limit of a titer of 1:10 for severe, mild and asymptomatic patients, resp. At day 90 after onset of symptoms (or initial RT-PCR detection in asymptomatic infections), it took 69, 87 and 31 days for PRNT50 antibody titers to decrease by half (T1/2) in severe, mild and asymptomatic infections, resp. Patients with severe disease had higher peak PRNT90 and PRNT50 antibody titers than patients with mild or asymptomatic infections. Age did not appear to compromise antibody responses, even after accounting for severity. We conclude that SARS-CoV-2 infection elicits robust neutralizing antibody titers in most individuals.
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    6. 6
      Anderson, E. J.; Rouphael, N. G.; Widge, A. T.; Jackson, L. A.; Roberts, P. C.; Makhene, M.; Chappell, J. D.; Denison, M. R.; Stevens, L. J.; Pruijssers, A. J. Safety and Immunogenicity of SARS-CoV-2 MRNA-1273 Vaccine in Older Adults. N. Engl. J. Med. 2020, 383, 2427– 2438,  DOI: 10.1056/NEJMoa2028436
      6
      Safety and immunogenicity of SARS-CoV-2mRNA-1273 vaccine in older adults
      Anderson, E. J.; Rouphael, N. G.; Widge, A. T.; Jackson, L. A.; Roberts, P. C.; Makhene, M.; Chappell, J. D.; Denison, M. R.; Stevens, L. J.; Pruijssers, A. J.; McDermott, A. B.; Flach, B.; Lin, B. C.; Doria-Rose, N. A.; O'Dell, S.; Schmidt, S. D.; Corbett, K. S.; Swanson, P. A.; Padilla, M.; Neuzil, K. M.; Bennett, H.; Leav, B.; Makowski, M.; Albert, J.; Cross, K.; Edara, V. V.; Floyd, K.; Suthar, M. S.; Martinez, D. R.; Baric, R.; Buchanan, W.; Luke, C. J.; Phadke, V. K.; Rostad, C. A.; Ledgerwood, J. E.; Graham, B. S.; Beigel, J. H.
      New England Journal of Medicine (2020), 383 (25), 2427-2438CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      Testing of vaccine candidates to prevent infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in an older population is important, since increased incidences of illness and death from coronavirus disease 2019 (Covid-19) have been assocd. with an older age. We conducted a phase 1, dose-escalation, open-label trial of a mRNA vaccine, mRNA-1273, which encodes the stabilized prefusion SARS-CoV-2 spike protein (S-2P) in healthy adults. The trial was expanded to include 40 older adults, who were stratified according to age (56 to 70 years or 71 years). All the participants were assigned sequentially to receive two doses of either 25μg or 100μg of vaccine administered 28 days apart. Solicited adverse events were predominantly mild or moderate in severity and most frequently included fatigue, chills, headache, myalgia, and pain at the injection site. Such adverse events were dose-dependent and were more common after the second immunization. Binding-antibody responses increased rapidly after the first immunization. By day 57, among the participants who received the 25-μ g dose, the anti-S-2P geometric mean titer (GMT) was 323,945 among those between the ages of 56 and 70 years and 1,128,391 among those who were 71 years of age or older; among the participants who received the 100-μ g dose, the GMT in the two age subgroups was 1,183,066 and 3,638,522, resp. After the second immunization, serum neutralizing activity was detected in all the participants by multiple methods. Bindingand neutralizing-antibody responses appeared to be similar to those previously reported among vaccine recipients between the ages of 18 and 55 years and were above the median of a panel of controls who had donated convalescent serum. The vaccine elicited a strong CD4 cytokine response involving type 1 helper T cells. In this small study involving older adults, adverse events assocd. with the mRNA-1273 vaccine were mainly mild or moderate. The 100-μ g dose induced higher binding- and neutralizing-antibody titers than the 25-μ g dose, which supports the use of the 100-μ g dose in a phase 3 vaccine trial.
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    7. 7
      Ramasamy, M. N.; Minassian, A. M.; Ewer, K. J.; Flaxman, A. L.; Folegatti, P. M.; Owens, D. R.; Voysey, M.; Aley, P. K.; Angus, B.; Babbage, G. Safety and Immunogenicity of ChAdOx1 NCoV-19 Vaccine Administered in a Prime-Boost Regimen in Young and Old Adults (COV002): A Single-Blind, Randomised, Controlled, Phase 2/3 Trial. Lancet 2020, 396, 1979– 1993,  DOI: 10.1016/S0140-6736(20)32466-1
      7
      Safety and immunogenicity of ChAdOx1 nCoV-19 vaccine administered in a prime-boost regimen in young and old adults (COV002): a single-blind, randomised, controlled, phase 2/3 trial
      Ramasamy, Maheshi N.; Minassian, Angela M.; Ewer, Katie J.; Flaxman, Amy L.; Folegatti, Pedro M.; Owens, Daniel R.; Voysey, Merryn; Aley, Parvinder K.; Angus, Brian; Babbage, Gavin; Belij-Rammerstorfer, Sandra; Berry, Lisa; Bibi, Sagida; Bittaye, Mustapha; Cathie, Katrina; Chappell, Harry; Charlton, Sue; Cicconi, Paola; Clutterbuck, Elizabeth A.; Colin-Jones, Rachel; Dold, Christina; Emary, Katherine R. W.; Fedosyuk, Sofiyah; Fuskova, Michelle; Gbesemete, Diane; Green, Catherine; Hallis, Bassam; Hou, Mimi M.; Jenkin, Daniel; Joe, Carina C. D.; Kelly, Elizabeth J.; Kerridge, Simon; Lawrie, Alison M.; Lelliott, Alice; Lwin, May N.; Makinson, Rebecca; Marchevsky, Natalie G.; Mujadidi, Yama; Munro, Alasdair P. S.; Pacurar, Mihaela; Plested, Emma; Rand, Jade; Rawlinson, Thomas; Rhead, Sarah; Robinson, Hannah; Ritchie, Adam J.; Ross-Russell, Amy L.; Saich, Stephen; Singh, Nisha; Smith, Catherine C.; Snape, Matthew D.; Song, Rinn; Tarrant, Richard; Themistocleous, Yrene; Thomas, Kelly M.; Villafana, Tonya L.; Warren, Sarah C.; Watson, Marion E. E.; Douglas, Alexander D.; Hill, Adrian V. S.; Lambe, Teresa; Gilbert, Sarah C.; Faust, Saul N.; Pollard, Andrew J.
      Lancet (2020), 396 (10267), 1979-1993CODEN: LANCAO; ISSN:0140-6736. (Elsevier Ltd.)
      Older adults (aged ≥70 years) are at increased risk of severe disease and death if they develop COVID-19 and are therefore a priority for immunization should an efficacious vaccine be developed. Immunogenicity of vaccines is often worse in older adults as a result of immunosenescence. We have reported the immunogenicity of a novel chimpanzee adenovirus-vectored vaccine, ChAdOx1 nCoV-19, in young adults, and now describe the safety and immunogenicity of this vaccine in a wider range of participants, including adults aged 70 years and older. In this report of the phase 2 component of a single-blind, randomised, controlled, phase 2/3 trial (COV002), healthy adults aged 18 years and older were enrolled at two UK clin. research facilities, in an age-escalation manner, into 18-55 years, 56-69 years, and 70 years and older immunogenicity subgroups. Participants were eligible if they did not have severe or uncontrolled medical comorbidities or a high frailty score (if aged ≥65 years). First, participants were recruited to a low-dose cohort, and within each age group, participants were randomly assigned to receive either i.m. ChAdOx1 nCoV-19 (2·2 x 1010 virus particles) or a control vaccine, MenACWY, using block randomisation and stratified by age and dose group and study site, using the following ratios: in the 18-55 years group, 1:1 to either two doses of ChAdOx1 nCoV-19 or two doses of MenACWY; in the 56-69 years group, 3:1:3:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY; and in the 70 years and older, 5:1:5:1 to one dose of ChAdOx1 nCoV-19, one dose of MenACWY, two doses of ChAdOx1 nCoV-19, or two doses of MenACWY. Prime-booster regimens were given 28 days apart. Participants were then recruited to the std.-dose cohort (3·5-6·5 x 1010 virus particles of ChAdOx1 nCoV-19) and the same randomisation procedures were followed, except the 18-55 years group was assigned in a 5:1 ratio to two doses of ChAdOx1 nCoV-19 or two doses of MenACWY. Participants and investigators, but not staff administering the vaccine, were masked to vaccine allocation. The specific objectives of this report were to assess the safety and humoral and cellular immunogenicity of a single-dose and two-dose schedule in adults older than 55 years. Humoral responses at baseline and after each vaccination until 1 yr after the booster were assessed using an inhouse standardised ELISA, a multiplex immunoassay, and a live severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) microneutralisation assay (MNA80). Cellular responses were assessed using an ex-vivo IFN-γ enzyme-linked immunospot assay. The coprimary outcomes of the trial were efficacy, as measured by the no. of cases of symptomatic, virol. confirmed COVID-19, and safety, as measured by the occurrence of serious adverse events. Analyses were by group allocation in participants who received the vaccine. Here, we report the preliminary findings on safety, reactogenicity, and cellular and humoral immune responses. This study is ongoing and is registered with ClinicalTrials.gov, NCT04400838, and ISRCTN, 15281137. Between May 30 and Aug 8, 2020, 560 participants were enrolled: 160 aged 18-55 years (100 assigned to ChAdOx1 nCoV-19, 60 assigned to MenACWY), 160 aged 56-69 years (120 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY), and 240 aged 70 years and older (200 assigned to ChAdOx1 nCoV-19: 40 assigned to MenACWY). Seven participants did not receive the boost dose of their assigned two-dose regimen, one participant received the incorrect vaccine, and three were excluded from immunogenicity analyses due to incorrectly labeled samples. 280 (50%) of 552 analysable participants were female. Local and systemic reactions were more common in participants given ChAdOx1 nCoV-19 than in those given the control vaccine, and similar in nature to those previously reported (injection-site pain, feeling feverish, muscle ache, headache), but were less common in older adults (aged ≥56 years) than younger adults. In those receiving two std. doses of ChAdOx1 nCoV-19, after the prime vaccination local reactions were reported in 43 (88%) of 49 participants in the 18-55 years group, 22 (73%) of 30 in the 56-69 years group, and 30 (61%) of 49 in the 70 years and older group, and systemic reactions in 42 (86%) participants in the 18-55 years group, 23 (77%) in the 56-69 years group, and 32 (65%) in the 70 years and older group. As of Oct 26, 2020, 13 serious adverse events occurred during the study period, none of which were considered to be related to either study vaccine. In participants who received two doses of vaccine, median anti-spike SARS-CoV-2 IgG responses 28 days after the boost dose were similar across the three age cohorts (std.-dose groups: 18-55 years, 20 713 arbitrary units [AU]/mL [IQR 13 898-33 550], n=39; 56-69 years, 16 170 AU/mL [10 233-40 353], n=26; and ≥70 years 17 561 AU/mL [9705-37 796], n=47; p=0·68). Neutralising antibody titers after a boost dose were similar across all age groups (median MNA80 at day 42 in the std.-dose groups: 18-55 years, 193 [IQR 113-238], n=39; 56-69 years, 144 [119-347], n=20; and ≥70 years, 161 [73-323], n=47; p=0·40). By 14 days after the boost dose, 208 (>99%) of 209 boosted participants had neutralising antibody responses. T-cell responses peaked at day 14 after a single std. dose of ChAdOx1 nCoV-19 (18-55 years: median 1187 spot-forming cells [SFCs] per million peripheral blood mononuclear cells [IQR 841-2428], n=24; 56-69 years: 797 SFCs [383-1817], n=29; and ≥70 years: 977 SFCs [458-1914], n=48). ChAdOx1 nCoV-19 appears to be better tolerated in older adults than in younger adults and has similar immunogenicity across all age groups after a boost dose. Further assessment of the efficacy of this vaccine is warranted in all age groups and individuals with comorbidities. UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, NIHR Oxford Biomedical Research Center, Thames Valley and South Midlands NIHR Clin. Research Network, and AstraZeneca.
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    8. 8
      Malladi, S. K.; Singh, R.; Pandey, S.; Gayathri, S.; Kanjo, K.; Ahmed, S.; Khan, M. S.; Kalita, P.; Girish, N.; Upadhyaya, A. Design of a Highly Thermotolerant, Immunogenic SARS-CoV-2 Spike Fragment. J. Biol. Chem. 2021, 296, 100025,  DOI: 10.1074/jbc.RA120.016284
      8
      Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment
      Malladi, Sameer Kumar; Singh, Randhir; Pandey, Suman; Gayathri, Savitha; Kanjo, Kawkab; Ahmed, Shahbaz; Khan, Mohammad Suhail; Kalita, Parismita; Girish, Nidhi; Upadhyaya, Aditya; Reddy, Poorvi; Pramanick, Ishika; Bhasin, Munmun; Mani, Shailendra; Bhattacharyya, Sankar; Joseph, Jeswin; Thankamani, Karthika; Raj, V. Stalin; Dutta, Somnath; Singh, Ramandeep; Nadig, Gautham; Varadarajan, Raghavan
      Journal of Biological Chemistry (2021), 296 (), 100025CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
      Virtually all SARS-CoV-2 vaccines currently in clin. testing are stored in a refrigerated or frozen state prior to use. This is a major impediment to deployment in resource-poor settings. Furthermore, several of them use viral vectors or mRNA. In contrast to protein subunit vaccines, there is limited manufg. expertise for these nucleic-acid-based modalities, esp. in the developing world. Neutralizing antibodies, the clearest known correlate of protection against SARS-CoV-2, are primarily directed against the receptor-binding domain (RBD) of the viral spike protein, suggesting that a suitable RBD construct might serve as a more accessible vaccine ingredient. We describe a monomeric, glycan-engineered RBD protein fragment that is expressed at a purified yield of 214 mg/l in unoptimized, mammalian cell culture and, in contrast to a stabilized spike ectodomain, is tolerant of exposure to temps. as high as 100°C when lyophilized, up to 70°C in soln. and stable for over 4 wk at 37°C. In prime:boost guinea pig immunizations, when formulated with the MF59-like adjuvant AddaVax, the RBD deriv. elicited neutralizing antibodies with an endpoint geometric mean titer of ∼415 against replicative virus, comparing favorably with several vaccine formulations currently in the clinic. These features of high yield, extreme thermotolerance, and satisfactory immunogenicity suggest that such RBD subunit vaccine formulations hold great promise to combat COVID-19.
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    9. 9
      Connell, K. B.; Miller, E. J.; Marqusee, S. The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants That Fold via Two-State and Three-State Mechanisms. J. Mol. Biol. 2009, 391, 450– 460,  DOI: 10.1016/j.jmb.2009.05.085
      9
      The Folding Trajectory of RNase H Is Dominated by Its Topology and Not Local Stability: A Protein Engineering Study of Variants that Fold via Two-State and Three-State Mechanisms
      Connell, Katelyn B.; Miller, Erik J.; Marqusee, Susan
      Journal of Molecular Biology (2009), 391 (2), 450-460CODEN: JMOBAK; ISSN:0022-2836. (Elsevier Ltd.)
      Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. Some proteins never significantly populate these high-energy states and fold by an apparently two-state process. However, many proteins populate detectable, partially folded forms during the folding process. The role of such intermediates is a matter of considerable debate. A single amino acid change can convert Escherichia coli RNase H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. The folding trajectories of the three-state RNase H and the two-state RNase H, proteins with the same native-state topol. but altered regional stability, were compared using a protein engineering approach. The data suggest that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. Mutations in the core and the periphery of the protein affect similar aspects of folding for both variants, suggesting a common trajectory with folding of the core region followed by the folding of the periphery. The results suggest that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.
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    10. 10
      Brockwell, D. J.; Radford, S. E. Intermediates: Ubiquitous Species on Folding Energy Landscapes?. Curr. Opin. Struct. Biol. 2007, 17, 30– 37,  DOI: 10.1016/j.sbi.2007.01.003
      10
      Intermediates: ubiquitous species on folding energy landscapes?
      Brockwell, David J.; Radford, Sheena E.
      Current Opinion in Structural Biology (2007), 17 (1), 30-37CODEN: COSBEF; ISSN:0959-440X. (Elsevier Ltd.)
      A review. Although intermediates have long been recognized as fascinating species that form during the folding of large proteins, the role that intermediates play in the folding of small, single-domain proteins has been widely debated. Recent discoveries using new, sensitive methods of detection and studies combining simulation and expt. have now converged on a common vision for folding, involving intermediates as ubiquitous stepping stones en route to the native state. The results suggest that the folding energy landscapes of even the smallest proteins possess significant ruggedness in which intermediates stabilized by both native and non-native interactions are common features.
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    11. 11
      Ahmed, S.; Khan, M. S.; Gayathri, S.; Singh, R.; Kumar, S.; Patel, U. R.; Malladi, S. K.; Rajmani, R. S.; van Vuren, P. J.; Riddell, S. A Stabilized, Monomeric, Receptor Binding Domain Elicits High-Titer Neutralizing Antibodies Against All SARS-CoV-2 Variants of Concern. Front. Immunol. 2021, 12, 765211,  DOI: 10.3389/fimmu.2021.765211
      11
      A stabilized, monomeric, receptor binding domain elicits high-titer neutralizing antibodies against all SARS-CoV-2 variants of concern
      Ahmed, Shahbaz; Khan, Mohammad Suhail; Gayathri, Savitha; Singh, Randhir; Kumar, Sahil; Patel, Unnatiben Rajeshbhai; Malladi, Sameer Kumar; Rajmani, Raju S.; van Vuren, Petrus Jansen; Riddell, Shane; Goldie, Sarah; Girish, Nidhi; Reddy, Poorvi; Upadhyaya, Aditya; Pandey, Suman; Siddiqui, Samreen; Tyagi, Akansha; Jha, Sujeet; Pandey, Rajesh; Khatun, Oyahida; Narayan, Rohan; Tripathi, Shashank; McAuley, Alexander J.; Singanallur, Nagendrakumar Balasubramanian; Vasan, Seshadri S.; Ringe, Rajesh P.; Varadarajan, Raghavan
      Frontiers in Immunology (2021), 12 (), 765211CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
      Satn. suppressor mutagenesis was used to generate thermostable mutants of the SARS-CoV-2 spike receptor-binding domain (RBD). A triple mutant with an increase in thermal melting temp. of ~ 7°C with respect to the wild-type B.1 RBD and was expressed in high yield in both mammalian cells and the microbial host, Pichia pastoris, was downselected for immunogenicity studies. An addnl. deriv. with three addnl. mutations from the B.1.351 (beta) isolate was also introduced into this background. Lyophilized proteins were resistant to high-temp. exposure and could be stored for over a month at 37°C. In mice and hamsters, squalene-in-water emulsion (SWE) adjuvanted formulations of the B.1-stabilized RBD were considerably more immunogenic than RBD lacking the stabilizing mutations and elicited antibodies that neutralized all four current variants of concern with similar neutralization titers. However, sera from mice immunized with the stabilized B.1.351 deriv. showed significantly decreased neutralization titers exclusively against the B.1.617.2 (delta) VOC. A cocktail comprising stabilized B.1 and B.1.351 RBDs elicited antibodies with qual. improved neutralization titers and breadth relative to those immunized solely with either immunogen. Immunized hamsters were protected from high-dose viral challenge. Such vaccine formulations can be rapidly and cheaply produced, lack extraneous tags or addnl. components, and can be stored at room temp. They are a useful modality to combat COVID-19, esp. in remote and low-resource settings.
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    12. 12
      Jansen van Vuren, P.; McAuley, A. J.; Kuiper, M. J.; Singanallur, N. B.; Bruce, M. P.; Riddell, S.; Goldie, S.; Mangalaganesh, S.; Chahal, S.; Drew, T. W. Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice. Viruses 2022, 14, 800,  DOI: 10.3390/v14040800
      12
      Highly Thermotolerant SARS-CoV-2 Vaccine Elicits Neutralising Antibodies against Delta and Omicron in Mice
      Jansen van Vuren, Petrus; McAuley, Alexander J.; Kuiper, Michael J.; Singanallur, Nagendrakumar Balasubramanian; Bruce, Matthew P.; Riddell, Shane; Goldie, Sarah; Mangalaganesh, Shruthi; Chahal, Simran; Drew, Trevor W.; Blasdell, Kim R.; Tachedjian, Mary; Caly, Leon; Druce, Julian D.; Ahmed, Shahbaz; Khan, Mohammad Suhail; Malladi, Sameer Kumar; Singh, Randhir; Pandey, Suman; Varadarajan, Raghavan; Vasan, Seshadri S.
      Viruses (2022), 14 (4), 800CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      As existing vaccines fail to completely prevent COVID-19 infections or community transmission, there is an unmet need for vaccines that can better combat SARS-CoV-2 variants of concern (VOC). We previously developed highly thermo-tolerant monomeric and trimeric receptor-binding domain derivs. that can withstand 100°C for 90 min and 37°C for four weeks and help eliminate cold-chain requirements. We show that mice immunized with these vaccine formulations elicit high titers of antibodies that neutralise SARS-CoV-2 variants VIC31 (with Spike: D614G mutation), Delta and Omicron (BA.1.1) VOC. Compared to VIC31, there was an av. 14.4-fold redn. in neutralization against BA.1.1 for the three monomeric antigen-adjuvant combinations and a 16.5-fold redn. for the three trimeric antigen-adjuvant combinations; the corresponding values against Delta were 2.5 and 3.0. Our findings suggest that monomeric formulations are suitable for upcoming Phase I human clin. trials and that there is potential for increasing the efficacy with vaccine matching to improve the responses against emerging variants. These findings are consistent with in silico modeling and AlphaFold predictions, which show that, while oligomeric presentation can be generally beneficial, it can make important epitopes inaccessible and also carries the risk of eliciting unwanted antibodies against the oligomerization domain.
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    13. 13
      Toelzer, C.; Gupta, K.; Yadav, S. K. N.; Borucu, U.; Davidson, A. D.; Kavanagh Williamson, M.; Shoemark, D. K.; Garzoni, F.; Staufer, O.; Milligan, R. Free Fatty Acid Binding Pocket in the Locked Structure of SARS-CoV-2 Spike Protein. Science 2020, 370, 725– 730,  DOI: 10.1126/science.abd3255
      13
      Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein
      Toelzer, Christine; Gupta, Kapil; Yadav, Sathish K. N.; Borucu, Ufuk; Davidson, Andrew D.; Kavanagh Williamson, Maia; Shoemark, Deborah K.; Garzoni, Frederic; Staufer, Oskar; Milligan, Rachel; Capin, Julien; Mulholland, Adrian J.; Spatz, Joachim; Fitzgerald, Daniel; Berger, Imre; Schaffitzel, Christiane
      Science (Washington, DC, United States) (2020), 370 (6517), 725-730CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathol. Our 2.85-angstrom cryo-electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor-binding domains tightly bind the essential free fatty acid linoleic acid (LA) in 3 composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.
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    14. 14
      Chattopadhyay, G.; Varadarajan, R. Facile Measurement of Protein Stability and Folding Kinetics Using a Nano Differential Scanning Fluorimeter. Protein Sci. 2019, 28, 1127– 1134,  DOI: 10.1002/pro.3622
      14
      Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter
      Chattopadhyay, Gopinath; Varadarajan, Raghavan
      Protein Science (2019), 28 (6), 1127-1134CODEN: PRCIEI; ISSN:1469-896X. (Wiley-Blackwell)
      With advancements in high-throughput generation of phenotypic data on mutant proteins, it has become important to individually characterize different proteins or their variants rapidly and with minimal sample consumption. We have made use of a nano differential scanning fluorimetric device, from NanoTemper technologies, to rapidly carry out isothermal chem. denaturation and measure folding/unfolding kinetics of proteins and compared these to corresponding data obtained from conventional spectrofluorimetry. We show that using sample vols. 10-50-fold lower than with conventional fluorimetric techniques, one can rapidly and accurately measure thermodn. and kinetic stability, as well as folding/unfolding kinetics. This method also facilitates characterization of proteins that are difficult to express and purify.
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    15. 15
      Chattopadhyay, G.; Bhowmick, J.; Manjunath, K.; Ahmed, S.; Goyal, P.; Varadarajan, R. Mechanistic Insights into Global Suppressors of Protein Folding Defects. PLoS Genet. 2022, 18, e1010334  DOI: 10.1371/journal.pgen.1010334
      15
      Mechanistic insights into global suppressors of protein folding defects
      Chattopadhyay, Gopinath; Bhowmick, Jayantika; Manjunath, Kavyashree; Ahmed, Shahbaz; Goyal, Parveen; Varadarajan, Raghavan
      PLoS Genetics (2022), 18 (8), e1010334CODEN: PGLEB5; ISSN:1553-7404. (Public Library of Science)
      Most amino acid substitutions in a protein either lead to partial loss-of-function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue defects caused by diverse loss-of-function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. To address this, we characterized multiple suppressor mutations both in isolation and in combination with inactive mutants. We examd. six global suppressors of the bacterial toxin CcdB, the known M182T global suppressor of TEM-1 β-lactamase, the N239Y global suppressor of p53-DBD and three suppressors of the SARS-CoV-2 spike Receptor Binding Domain. When coupled to inactive mutants, they promote increased in-vivo solubilities as well as regain-of-function phenotypes. In the case of CcdB, where novel suppressors were isolated, we detd. the crystal structures of three such suppressors to obtain insight into the specific mol. interactions responsible for the obsd. effects. While most individual suppressors result in small stability enhancements relative to wildtype, which can be combined to yield significant stability increments, thermodn. stabilization is neither necessary nor sufficient for suppressor action. Instead, in diverse systems, we observe that individual global suppressors greatly enhance the foldability of buried site mutants, primarily through increase in refolding rate parameters measured in vitro. In the crowded intracellular environment, mutations that slow down folding likely facilitate off-pathway aggregation. We suggest that suppressor mutations that accelerate refolding can counteract this, enhancing the yield of properly folded, functional protein in vivo.
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    16. 16
      Baliga, C.; Varadarajan, R.; Aghera, N. Homodimeric Escherichia Coli Toxin CcdB (Controller of Cell Division or Death B Protein) Folds via Parallel Pathways. Biochemistry 2016, 55, 6019– 6031,  DOI: 10.1021/acs.biochem.6b00726
      16
      Homodimeric Escherichia coli Toxin CcdB (Controller of Cell Division or Death B Protein) Folds via Parallel Pathways
      Baliga, Chetana; Varadarajan, Raghavan; Aghera, Nilesh
      Biochemistry (2016), 55 (43), 6019-6031CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
      The existence of parallel pathways in the folding of proteins seems intuitive, yet remains controversial. We explore the folding kinetics of the homodimeric E. coli toxin CcdB using multiple optical probes and approaches. Kinetic studies performed as a function of protein and denaturant concns. demonstrate that the folding of CcdB is a four-state process. The two intermediates populated during folding are present on parallel pathways. Both form by rapid assocn. of the monomers in a diffusion limited manner and appear to be largely unstructured, as they are silent to the optical probes employed in the current study. The existence of parallel pathways is supported by the insensitivity of the amplitudes of the refolding kinetic phases to the different probes used in the study. More importantly, interrupted refolding studies and ligand binding studies clearly demonstrate that the native state forms in a bi-exponential manner, implying the presence of at least two pathways. Our studies indicate that the CcdA antitoxin binds only to the folded CcdB dimer and not to any earlier folding intermediates. Thus, despite being part of the same operon, the antitoxin does not appear to modulate the folding pathway of the toxin encoded by the downstream cistron. This study highlights the utility of ligand binding in distinguishing between sequential and parallel pathways in protein folding studies, while also providing insights into mol. interactions during folding in Type II TA systems.
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    17. 17
      Tripathi, A.; Gupta, K.; Khare, S.; Jain, P. C.; Patel, S.; Kumar, P.; Pulianmackal, A. J.; Aghera, N.; Varadarajan, R. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data. Mol. Biol. Evol. 2016, 33, 2960– 2975,  DOI: 10.1093/molbev/msw182
      17
      Molecular determinants of mutant phenotypes, inferred from saturation mutagenesis data
      Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan
      Molecular Biology and Evolution (2016), 33 (11), 2960-2975CODEN: MBEVEO; ISSN:0737-4038. (Oxford University Press)
      Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used satn. mutagenesis combined with deep sequencing to det. mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliph. and charged amino acid substitutions. At nonactive-site positions, the av. mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were obsd. for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and soly. The inability to refold reversibly, as well as a decreased folding rate in vitro, is assocd. with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most delete riousmutants showed an increased in vivo activity and soly. only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations.
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    18. 18
      Chattopadhyay, G.; Bhasin, M.; Ahmed, S.; Gosain, T. P.; Ganesan, S.; Das, S.; Thakur, C.; Chandra, N.; Singh, R.; Varadarajan, R. Functional and Biochemical Characterization of the MazEF6 Toxin-Antitoxin System of Mycobacterium Tuberculosis. J. Bacteriol. 2022, 204, e0005822  DOI: 10.1128/jb.00058-22
      There is no corresponding record for this reference.
    19. 19
      Acharya, P.; Madhusudhana Rao, N. Stability Studies on a Lipase from Bacillus Subtilis in Guanidinium Chloride. J. Protein Chem. 2003, 22, 51– 60,  DOI: 10.1023/A:1023067827678
      19
      Stability studies on a lipase from Bacillus subtilis in guanidinium chloride
      Acharya, Priyamvada; Madhusudhana Rao, N.
      Journal of Protein Chemistry (2003), 22 (1), 51-60CODEN: JPCHD2; ISSN:0277-8033. (Kluwer Academic/Plenum Publishers)
      Lipase from B. subtilis is a "lidless" lipase that does not show interfacial activation. Due to exposure of the active site to solvent, the lipase tends to aggregate. Here, the authors investigated the soln. properties and unfolding of B. subtilis lipase in guanidinium chloride (GdmCl) to understand its aggregation behavior and stability. Dynamic light scattering (DLS), near- and far-UV CD, activity, and intrinsic fluorescence of lipase suggest that the protein undergoes unfolding between 1M and 2M GdmCl. The polarity-sensitive dye, 1,1',-bis-(4-anilino)naphthalene-5,5''-disulfonic acid (bis-ANS), a probe for hydrophobic pockets, bound cooperatively to the native lipase. An intermediate populated in 1.75M GdmCl that strongly bound bis-ANS was identified. The tendency of the native protein to aggregate in soln. and specific binding to bis-ANS confirmed that the lipase has exposed hydrophobic pockets and that this surface hydrophobicity strongly influences the unfolding pathway of the lipase in GdmCl.
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    20. 20
      Ellis, D.; Brunette, N.; Crawford, K. H. D.; Walls, A. C.; Pham, M. N.; Chen, C.; Herpoldt, K.-L.; Fiala, B.; Murphy, M.; Pettie, D. Stabilization of the SARS-CoV-2 Spike Receptor-Binding Domain Using Deep Mutational Scanning and Structure-Based Design. Front. Immunol. 2021, 12, 710263,  DOI: 10.3389/fimmu.2021.710263
      20
      Stabilization of the SARS-CoV-2 spike receptor-binding domain using deep mutational scanning and structure-based design
      Ellis, Daniel; Brunette, Natalie; Crawford, Katharine H. D.; Walls, Alexandra C.; Pham, Minh N.; Chen, Chengbo; Herpoldt, Karla-Luise; Fiala, Brooke; Murphy, Michael; Pettie, Deleah; Kraft, John C.; Malone, Keara D.; Navarro, Mary Jane; Ogohara, Cassandra; Kepl, Elizabeth; Ravichandran, Rashmi; Sydeman, Claire; Ahlrichs, Maggie; Johnson, Max; Blackstone, Alyssa; Carter, Lauren; Starr, Tyler N.; Greaney, Allison J.; Lee, Kelly K.; Veesler, David; Bloom, Jesse D.; King, Neil P.
      Frontiers in Immunology (2021), 12 (), 710263CODEN: FIRMCW; ISSN:1664-3224. (Frontiers Media S.A.)
      The unprecedented global demand for SARS-CoV-2 vaccines has demonstrated the need for highly effective vaccine candidates that are thermostable and amenable to large-scale manufg. Nanoparticle immunogens presenting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein (S) in repetitive arrays are being advanced as 2nd-generation vaccine candidates, as they feature robust manufg. characteristics and have shown promising immunogenicity in preclin. models. Here, we used previously reported deep mutational scanning (DMS) data to guide the design of stabilized variants of the RBD. The selected mutations fill a cavity in the RBD that has been identified as a linoleic acid-binding pocket. Screening of several designs led to the selection of 2 lead candidates that expressed at higher yields than the wild-type RBD. These stabilized RBDs possess enhanced thermal stability and resistance to aggregation, particularly when incorporated into an icosahedral nanoparticle immunogen that maintained its integrity and antigenicity for 28 days at 35-40°, while corresponding immunogens displaying the wild-type RBD experienced aggregation and loss of antigenicity. The stabilized immunogens preserved the potent immunogenicity of the original nanoparticle immunogen, which is currently being evaluated in a Phase I/II clin. trial. Our findings may improve the scalability and stability of RBD-based coronavirus vaccines in any format and more generally highlight the utility of comprehensive DMS data in guiding vaccine design.
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    21. 21
      Lan, J.; Ge, J.; Yu, J.; Shan, S.; Zhou, H.; Fan, S.; Zhang, Q.; Shi, X.; Wang, Q.; Zhang, L. Structure of the SARS-CoV-2 Spike Receptor-Binding Domain Bound to the ACE2 Receptor. Nature 2020, 581, 215– 220,  DOI: 10.1038/s41586-020-2180-5
      21
      Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor
      Lan, Jun; Ge, Jiwan; Yu, Jinfang; Shan, Sisi; Zhou, Huan; Fan, Shilong; Zhang, Qi; Shi, Xuanling; Wang, Qisheng; Zhang, Linqi; Wang, Xinquan
      Nature (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.
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    22. 22
      Walls, 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, 281– 292.e6,  DOI: 10.1016/j.cell.2020.02.058
      22
      Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein
      Walls, Alexandra C.; Park, Young-Jun; Tortorici, M. Alejandra; Wall, Abigail; McGuire, Andrew T.; Veesler, David
      Cell (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.
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    23. 23
      Ahmed, S.; Manjunath, K.; Chattopadhyay, G.; Varadarajan, R. Identification of Stabilizing Point Mutations through Mutagenesis of Destabilized Protein Libraries. J. Biol. Chem. 2022, 298, 101785,  DOI: 10.1016/j.jbc.2022.101785
      23
      Identification of stabilizing point mutations through mutagenesis of destabilized protein libraries
      Ahmed, Shahbaz; Manjunath, Kavyashree; Chattopadhyay, Gopinath; Varadarajan, Raghavan
      Journal of Biological Chemistry (2022), 298 (4), 101785CODEN: JBCHA3; ISSN:1083-351X. (Elsevier Inc.)
      Although there have been recent transformative advances in the area of protein structure prediction, prediction of point mutations that improve protein stability remains challenging. It is possible to construct and screen large mutant libraries for improved activity or ligand binding. However, reliable screens for mutants that improve protein stability do not yet exist, esp. for proteins that are well folded and relatively stable. Here, we demonstrate that incorporation of a single, specific, destabilizing mutation termed parent inactivating mutation into each member of a single-site satn. mutagenesis library, followed by screening for suppressors, allows for robust and accurate identification of stabilizing mutations. We carried out fluorescence-activated cell sorting of such a yeast surface display, satn. suppressor library of the bacterial toxin CcdB, followed by deep sequencing of sorted populations. We found that multiple stabilizing mutations could be identified after a single round of sorting. In addn., multiple libraries with different parent inactivating mutations could be pooled and simultaneously screened to further enhance the accuracy of identification of stabilizing mutations. Finally, we show that individual stabilizing mutations could be combined to result in a multi-mutant that demonstrated an increase in thermal melting temp. of about 20°C, and that displayed enhanced tolerance to high temp. exposure. We conclude that as this method is robust and employs small library sizes, it can be readily extended to other display and screening formats to rapidly isolate stabilized protein mutants.
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    24. 24
      Ahmed, S.; Bhasin, M.; Manjunath, K.; Varadarajan, R. Prediction of Residue-Specific Contributions to Binding and Thermal Stability Using Yeast Surface Display. Front. Mol. Biosci. 2022, 8, 800819,  DOI: 10.3389/fmolb.2021.800819
      There is no corresponding record for this reference.
    25. 25
      Prajapati, R. S.; Das, M.; Sreeramulu, S.; Sirajuddin, M.; Srinivasan, S.; Krishnamurthy, V.; Ranjani, R.; Ramakrishnan, C.; Varadarajan, R. Thermodynamic Effects of Proline Introduction on Protein Stability. Proteins 2007, 66, 480– 491,  DOI: 10.1002/prot.21215
      25
      Thermodynamic effects of proline introduction on protein stability
      Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C.; Varadarajan, Raghavan
      Proteins: Structure, Function, and Bioinformatics (2007), 66 (2), 480-491CODEN: PSFBAF ISSN:. (Wiley-Liss, Inc.)
      The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodn. effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucinevaline binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by x-ray crystallog. to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chem. denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chem. denaturation at room temp. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the redn. in entropy of unfolding was typically several fold larger than the theor. est. of -4 cal K-1 mol-1 derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, resp. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive.
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    26. 26
      Starr, T. N.; Greaney, A. J.; Hilton, S. K.; Ellis, D.; Crawford, K. H.D.; Dingens, A. S.; Navarro, M. J.; Bowen, J. E.; Tortorici, M. A.; Walls, A. C.; King, N. P.; Veesler, D.; Bloom, J. D. Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding. Cell 2020, 182, 1295– 1310.e20,  DOI: 10.1016/j.cell.2020.08.012
      26
      Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding
      Starr, Tyler N.; Greaney, Allison J.; Hilton, Sarah K.; Ellis, Daniel; Crawford, Katharine H. D.; Dingens, Adam S.; Navarro, Mary Jane; Bowen, John E.; Tortorici, M. Alejandra; Walls, Alexandra C.; King, Neil P.; Veesler, David; Bloom, Jesse D.
      Cell (Cambridge, MA, United States) (2020), 182 (5), 1295-1310.e20CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we exptl. measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial no. of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open anal. pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations obsd. during viral surveillance.
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    27. 27
      Tokatlian, T.; Read, B. J.; Jones, C. A.; Kulp, D. W.; Menis, S.; Chang, J. Y. H.; Steichen, J. M.; Kumari, S.; Allen, J. D.; Dane, E. L. Innate Immune Recognition of Glycans Targets HIV Nanoparticle Immunogens to Germinal Centers. Science 2019, 363, 649– 654,  DOI: 10.1126/science.aat9120
      27
      Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers
      Tokatlian, Talar; Read, Benjamin J.; Jones, Christopher A.; Kulp, Daniel W.; Menis, Sergey; Chang, Jason Y. H.; Steichen, Jon M.; Kumari, Sudha; Allen, Joel D.; Dane, Eric L.; Liguori, Alessia; Sangesland, Maya; Lingwood, Daniel; Crispin, Max; Schief, William R.; Irvine, Darrell J.
      Science (Washington, DC, United States) (2019), 363 (6427), 649-654CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      In vaccine design, antigens are often arrayed in a multi-valent nanoparticle form, but in vivo mechanisms underlying the enhanced immunity elicited by such vaccines remain poorly understood. We compared the fates of two different heavily glycosylated HIV antigens, a gp120-derived mini-protein and a large, stabilized envelope trimer, in protein nanoparticle or "free" forms after primary immunization. Unlike monomeric antigens, nanoparticles were rapidly shuttled to the follicular dendritic cell (FDC) network and then concd. in germinal centers in a complement-, mannose-binding lectin (MBL)-, and immunogen glycan-dependent manner. Loss of FDC localization in MBL-deficient mice or via immunogen deglycosylation significantly affected antibody responses. These findings identify an innate immune-mediated recognition pathway promoting antibody responses to particulate antigens, with broad implications for humoral immunity and vaccine design.
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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.2c07262.

    • Thermodynamic parameters (Cm, ΔG0, m) and kinetic parameters for unfolding and refolding of mRBD proteins; unfolding kinetics for mRBD-WT from the native to unfolded state and equilibrium denaturation profile of ACE2-Fc (PDF)


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