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Synthesis and Structure Optimization of Star Copolymers as Tunable Macromolecular Carriers for Minimal Immunogen Vaccine Delivery
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Synthesis and Structure Optimization of Star Copolymers as Tunable Macromolecular Carriers for Minimal Immunogen Vaccine Delivery
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  • Gabriela Mixová
    Gabriela Mixová
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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  • Eva Tihlaříková
    Eva Tihlaříková
    Institute of Scientific Instruments, Czech Academy of Sciences, Královopolská 147, Brno 612 64, Czech Republic
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    Orcidhttps://orcid.org/0000-0002-7983-2971
  • Yaling Zhu
    Yaling Zhu
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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  • Lucie Schindler
    Lucie Schindler
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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  • Ladislav Androvič
    Ladislav Androvič
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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  • Lucie Kracíková
    Lucie Kracíková
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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    Eliška Hrdá
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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  • Bedřich Porsch
    Bedřich Porsch
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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    Michal Pechar
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
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    Orcidhttps://orcid.org/0000-0002-4507-1801
  • Christopher M. Garliss
    Christopher M. Garliss
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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  • David Wilson
    David Wilson
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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  • Hugh C. Welles
    Hugh C. Welles
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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    Orcidhttps://orcid.org/0000-0002-3336-1203
  • Jake Holechek
    Jake Holechek
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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  • Qiuyin Ren
    Qiuyin Ren
    Vaccine Research Center, National Institutes of Health, Rockville, Maryland 20892, United States
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  • Geoffrey M. Lynn
    Geoffrey M. Lynn
    Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
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  • Vilém Neděla
    Vilém Neděla
    Institute of Scientific Instruments, Czech Academy of Sciences, Královopolská 147, Brno 612 64, Czech Republic
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  • Richard Laga*
    Richard Laga
    Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    *Email: [email protected]. Tel.: +420-325 873 806. Fax: +420-296 809 410.
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    Orcidhttps://orcid.org/0000-0001-5420-2407
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2024, 35, 8, 1218–1232
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https://doi.org/10.1021/acs.bioconjchem.4c00273
Published July 31, 2024

Copyright © 2024 The Authors. Published by American Chemical Society. This publication is licensed under

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Abstract

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Minimal immunogen vaccines are being developed to focus antibody responses against otherwise challenging targets, including human immunodeficiency virus (HIV), but multimerization of the minimal peptide immunogen on a carrier platform is required for activity. Star copolymers comprising multiple hydrophilic polymer chains (“arms”) radiating from a central dendrimer unit (“core”) were recently reported to be an effective platform for arraying minimal immunogens for inducing antibody responses in mice and primates. However, the impact of different parameters of the star copolymer (e.g., minimal immunogen density and hydrodynamic size) on antibody responses and the optimal synthetic route for controlling those parameters remains to be fully explored. We synthesized a library of star copolymers composed of poly[N-(2-hydroxypropyl)methacrylamide] hydrophilic arms extending from poly(amidoamine) dendrimer cores with the aim of identifying the optimal composition for use as minimal immunogen vaccines. Our results show that the length of the polymer arms has a crucial impact on the star copolymer hydrodynamic size and is precisely tunable over a range of 20–50 nm diameter, while the dendrimer generation affects the maximum number of arms (and therefore minimal immunogens) that can be attached to the surface of the dendrimer. In addition, high-resolution images of selected star copolymer taken by a custom-modified environmental scanning electron microscope enabled the acquisition of high-resolution images, providing new insights into the star copolymer structure. Finally, in vivo studies assessing a star copolymer vaccine comprising an HIV minimal immunogen showed the criticality of polymer arm length in promoting antibody responses and highlighting the importance of composition tunability to yield the desired biological effect.

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Introduction

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Most modern vaccines for infectious disease prevention use full-length protein immunogens derived from a pathogenic organism (e.g., hemagglutinin from influenza virus or SARS-CoV-2 spike protein from corona virus) to induce antibodies that can prevent infection. (1−3) While this strategy has been highly effective in preventing many diseases, full-length proteins have two key limitations. First, use of full-length proteins that are typically composed of hundreds of amino acids can lead to antibody responses against highly variable sites of the pathogen that distracts antibody responses away from conserved sites needed for efficacy. (4) Second, antibodies generated against certain sites of full-length protein have been observed to exacerbate disease after infection in a process called antibody-mediated disease enhancement (ADE). (5−7)
An emerging strategy to overcome these limitations is to use only an active part of the full-length protein, referred to as a minimal immunogen, which allows antibody responses to be focused against key sites while avoiding those that could lead to ADE. (8,9) However, minimal immunogens, in isolation, are poorly immunogenic. (10) This is due to their relatively low molecular weight (<10 kg·mol–1) resulting in rapid clearance and poor drainage to lymph nodes (LNs) (11) and the inability of soluble, minimal immunogens to sufficiently cross-link B cell receptors needed for antibody induction. (12) To address these challenges, nanoparticles and other macromolecular carriers have been introduced to increase size as a means for reducing elimination half-life and scaffolding the immunogen to promote B cell receptor cross-linking. (13−15)
Some of the most advanced minimal immunogen vaccines in development have relied on recombinant nanoparticles that fuse the minimal immunogen to a structural protein that self-assembles into a multimer displaying multiple copies of the minimal immunogen. (16) Approaches under development include those using bacteriophages, (17) keyhole limpet hemocyanin (KLH, a naturally occurring multimeric protein nanoparticle), (18) ferritin nanoparticles, and various virus-like particles. (19,20) While these recombinant approaches have shown promise, certain minimal immunogens contain post-translation modifications (PTMs), including glycosylation sites key for antibody recognition that cannot be readily encoded using recombinant approaches; (21) additionally, the number and density of immunogens that can affect antibody responses cannot be readily controlled. (22,23)
While various synthetic nanocarriers based on lipids, amphiphilic polymers, and peptides or inorganic metal-based nanoparticles have been introduced for delivering minimal peptide immunogens, most were developed for delivering full-length protein or inducing T cell responses and are often limited in controlling minimal immunogen density and orientation in a stable array. (24−26)
As an alternative, fully synthetic star copolymer-based vaccines comprising minimal immunogens linked to hydrophilic polymer arms radiating from dendrimer cores provide the potential advantages that minimal immunogen density, orientation, and scaffold size can be precisely tuned to modulate antibody responses. Earlier work has established the utility of star copolymers as tunable platforms for a range of medicinal applications including delivery of chemotherapeutics for cancer treatment as well as prevention or treatment of genitourinary tract infections, providing proof of concept for use in humans. (27−32) More recently, star copolymer-based minimal immunogen vaccines were shown to be safe for administration to mice and primates for inducing neutralizing antibody responses; however, the impact of various structural parameters (minimal immunogen density and hydrodynamic diameter) on antibody responses and the synthetic approach to optimize such parameters needs further investigation. (33) Indeed, ligand density, linker length, and hydrodynamic size have been shown to impact antibody responses with other platforms. (12−14) Furthermore, optimizing these parameters for glycosylated minimal immunogens, which are notoriously poorly immunogenic may be even more critical to ensuring effective antibody responses. (34)
Therefore, the objectives of this work were to investigate how various parameters of star copolymer vaccines (polymer arm length, minimal peptide immunogen density, etc.) affect star copolymer physicochemical properties (e.g., hydrodynamic size) and capacity for inducing antibody responses against a glycosylated minimal immunogen (“Man9 V3”) derived from human immunodeficiency virus (HIV). (35) A custom-modified environmental scanning electron microscope (ESEM) equipped with a scanning transmission electron microscopy (STEM) detector was used to provide unique ultrahigh-resolution images of selected star copolymers that document their size and shape in the unsolvated state. After optimizing synthetic conditions for controlling polymer arm loading and star copolymer vaccine size, we examined how minimal immunogen density and hydrodynamic size impact antibody responses. The results show how the synthetic approach can be used to control the minimal peptide immunogen density and hydrodynamic size of star copolymer vaccines for promoting antibody responses.

Results and Discussion

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Synthesis and Characterization of Star Copolymers

The preparation of the star copolymer was performed in two synthetic steps, including the synthesis of heterobifunctional polymer arms, followed by conjugation of the arms to the dendrimer cores (for the reaction scheme, see Scheme 1).

Scheme 1

Scheme 1. Cartoon Depiction (A) and Reaction Scheme (B) for the Synthesis of Star Copolymers (II) Composed of the PAMAM Dendrimer Core and Heterobifunctional HPMA-Based Arms (I)
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The polymer arms P1–P5 were synthesized by the reversible addition–fragmentation chain-transfer (RAFT) polymerization of HPMA in the presence of different molar amounts of the thiazolidine-2-thione (TT)- (for polymers P1–P3) or azide group (N3)-functionalized (for polymers P4 and P5) chain-transfer agent and initiator. The polymers of number-average molecular weights (Mn) of ∼10–80 kg·mol–1 were distinguished by a narrow molecular weight distribution (Đ ≤ 1.1, Đ = Mw/Mn) and high functionalities (f ≥ 0.8, f is defined as the number of functional end groups per polymer chain calculated as the ratio between Mn obtained from the size exclusion chromatography (SEC) measurement and Mn calculated from the end group analysis) of either TT or azide end groups. The dithiobenzoate (DTB) groups of the heterobifunctional polymers were subsequently replaced with propargyl (for polymers P1–P3) or TT groups (for polymers P4 and P5) by the homolytic reaction with a high molar excess of the functionalized azoinitiators to introduce the reactive moieties to the ends of the polymer arms for the biorthogonal attachment of the peptide vaccine. No significant changes in Mn and Đ nor a decrease in the reactive groups at opposite ends of the chains were observed in this substitution reaction. The characteristics for heterobifunctional polymers P1–P5 are summarized in Table 1, and their SEC profiles are depicted in Figure S2.
Table 1. Characteristics of the Heterobifunctional PHPMA Polymer Arms with TT Groups at One Side and Propargyl (P1–P3) or Azide Groups (P4 and P5) on the Other Side of the Polymer Chain
polymer arm[M]0/[CTA]0/[I]0iMnii [kg·mol–1]ĐiiiRgiv[nm]f(TT)v
P1135:1:0.59.81.03n.d.0.97
P2200:1:0.515.81.083.90.91
P3698:1:0.541.11.056.10.86
P465:1:0.58.61.05n.d.0.92
P5700:1:0.571.31.108.00.81
i

Ratio of the molar concentrations of monomer (M), chain transfer agent (CTA), and initiator (I) in the polymerization feed.

ii

Number-average molecular weight of the polymer arm determined by SEC.

iii

Polymer arm dispersity defined as the ratio of weight-average (Mw) to number-average (Mn) molecular weight determined by SEC.

iv

Radius of gyration of the polymer arm determined by SEC.

v

Polymer arm functionality defined as the average number of TT groups per polymer chain.

The heterobifunctional polymer arms P1–P3 with TT and propargyl end groups were then grafted onto PAMAM dendrimers of different generations, varying in molecular weight and number of surface ∼NH2 groups (for PAMAM dendrimer characteristics, see Table S1), using three different molar ratios of the PAMAM surface ∼NH2 groups to the TT terminal group on the polymer arms (1:1, 2:1, and 3:1). The conjugation reaction was carried out in dry methanol to prevent competitive hydrolysis of the TT groups and hence lower conjugation recovery. Based on the reaction conditions and the composition of the reactants, the resulting product was a mixture of star copolymers of various physicochemical parameters (S1–S35) and unreacted heterobifunctional polymer. Although the resulting product consisted of a binary mixture of two polymers, suitably selected chromatographic conditions in combination with light scattering (LS) and refractive index (RI) detectors (for details, see Materials and Methods section) allowed precise characterization of both types of materials without the need for purification. Only the star copolymer S17, which was further used for covalent coupling with the electron microscopy (EM) contrast label and star copolymers S28–S31, which were further used for minimal peptide immunogen binding, were purified from unreacted linear polymer using centrifugal filter units (for SEC chromatograms of the star copolymer S17 before and after the purification, see Figure S1). All star copolymers, irrespective of the method of preparation, exhibited a narrow distribution of molecular weights (Đ ≤ 1.35), suggesting the preparation of defined materials. The influence of polymer arm length, dendrimer generation, and reactant functional group ratio on the size, molecular weight, morphology, and yield of the star copolymer was investigated in detail (for star copolymer characteristics, see Table 2).
Table 2. Characteristics of Star Copolymers S1–S26 Composed of Polymer Arms P1–P3 Extending from PAMAM Dendrimers G3–G5, Prepared at Three Different Molar Ratios (1:1, 2:1, and 3:1) of the PAMAM Surface ̃NH2 Groups to the ̃TT Terminal Group on the Polymer Arms
star copolymerpolymer armPAMAM generationn(̃NH2)/n(̃TT)iMnii [kg·mol–1]ĐiiiNiv(polymer arms)yieldv[%]Rgvi[nm]
S1P1G31:1246.61.122448.79.6
S2P1G32:1215.61.192173.910.3
S3P1G33:1157.01.171576.79.5
S4P1G41:1311.61.193046.19.6
S5P1G42:1242.31.302370.410.8
S6P1G43:1182.81.271772.99.2
S7P1G51:1405.11.213840.710.2
S8P1G52:1332.31.223163.010.2
S9P1G53:1235.31.312171.99.9
S10P2G31:1488.21.163048.912.6
S11P2G32:1336.51.222068.813.0
S12P2G33:1204.71.141670.710.4
S13P2G41:1452.61.222843.212.5
S14P2G42:1344.51.302167.413.1
S15P2G43:1251.01.291569.212.1
S16P2G51:1589.21.183628.512.6
S17P2G52:1501.71.353062.414.5
S18P2G53:1364.71.352167.013.6
S19P3G31:1709.91.031752.117.0
S20P3G33:1425.71.061064.915.0
S21P3G41:1889.41.062165.618.4
S22P3G42:1649.51.071560.517.6
S23P3G43:1500.51.081264.116.1
S24P3G51:11272.01.043034.818.6
S25P3G52:11045.01.062553.919.0
S26P3G53:1776.61.081857.917.0
i

Molar ratio of PAMAM∼NH2 groups to PHPMA∼TT groups in the reaction mixture.

ii

Number-average molecular weight of the star copolymer determined by SEC.

iii

Star copolymer dispersity defined as the ratio of weight-average (Mw) to number-average (Mn) molecular weight determined by SEC.

iv

Number of polymer arms attached to the PAMAM dendrimer core evaluated by SEC.

v

Yield of the conjugation reaction evaluated by SEC.

vi

Radius of gyration of the star copolymer determined by SEC.

Polymer Arm Length Is a Key Determinant of Star Copolymer Size

The size of the carrier used to deliver minimal immunogens has been shown to be a key factor for influencing immune responses. Therefore, we sought to investigate whether polymer arm length could be used to modulate the star copolymer hydrodynamic size.
Our results showed that the length of the polymer arm was found to play a key role in designing star copolymers of a certain size and the number of grafts that can be linked to the dendrimer core. In general, the gyration radius (Rg) and the number-average molecular weight (Mn) of the star copolymers increase with an increasing Mn of the polymer arm using a dendrimer of the same generation and the same molar ratio of reactants (see Table 2 and Figure 1). For example, the star copolymer S8 consisting of the arms of polymer P1 (9.8 kg·mol–1) has Rg and Mn values of 10.2 nm and 332.3 kg·mol–1, respectively; Rg and Mn of the star copolymer S17 with the P2 arms (15.8 kg·mol–1) are 14.5 nm and 501.7 kg·mol–1; and the star copolymer S26 formed by the P3 arms (41.1 kg·mol–1) has Rg and Mn of 19.0 nm and 1045.0 kg·mol–1. These data show that the star copolymer size can be precisely tuned by controlling the polymer arm Mn.

Figure 1

Figure 1. Influence of PAMAM dendrimer generation and molar ratio of the PAMAM surface. ̃NH2 groups to the ̃TT terminal group on the polymer arms on the gyration radius (Rg) and number (#) of polymer arms of star copolymers.

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Perhaps not unexpectedly, the number of arms that can be attached to the dendrimer core (N) decreases with increasing polymer arm Mn (see Table 2 and Figure 1). Accordingly, for star copolymers S4, S13, and S22 comprising polymer arms P1, P2, and P3 with increasing molecular weight, the number of polymer arms attached (N) is 30, 28, and 21. As size and minimal immunogen density can impact biological activity, these competing factors must therefore be balanced in the design of star copolymers for use as minimal vaccines.

Dendrimer Core Generation Impacts Arm Density but not Star Copolymer Size

The dendrimer core generation (G3–G5) was another parameter assessed for its impact on size, molecular weight, and number of polymer arms attached. Our results showed that the Rg values of star copolymers were almost independent of the dendrimer generation, while the number of attached polymer arms and hence the Mn of the star copolymers increased linearly (see Table 2 and Figure 1). For example, attaching polymer arms P1 to PAMAM, G3 resulted in a 9.6 nm star copolymer S1 with 24 arms, whereas the star copolymer S4 composed of PAMAM, G4 had a Rg of 9.6 nm and 30 arms, and the star copolymer S7 measured 10.2 nm and contained 38 arms. These results suggest that the PAMAM dendrimer generation (i.e., degree of dendrimer branching) affects the density of the arms that can be attached, but minimally impact the size of the star copolymer, and that increasing the dendrimer generation can be used as a means for accommodating higher densities of polymers chains. Given the higher density of arm loading that can be achieved with higher generations of PAMAM dendrimers, G5 PAMAM dendrimers were selected as the preferred core for further evaluation as vaccines later. Though, as the studies herein only evaluated G3-G5 dendrimers, further assessment may be warranted to understand if the observed trends apply to lower and higher dendrimer generations (<G3, > G5).

Impact of Polymer to Dendrimer Molar Ratio on Star Copolymer Size and Yield

We also assessed whether the synthetic scheme had an impact on the star copolymer properties. Our results showed that star copolymer size and yield are impacted by the reactant molar ratio. The data showed that both the N and the Mn of the star copolymers decreased linearly with increasing molar ratio of PAMAM ̃NH2 groups to PHPMA ̃TT groups, regardless of the polymer arm length and PAMAM generation used (see Table 2 and Figure 1). For example, star copolymers S13, S14, and S15 were synthesized by mixing PAMAM, G4 with polymer arm P2 at functional group ratios (n(̃NH2)/n(̃TT)) of 1:1, 2:1, and 3:1, respectively. The resulting star copolymer S13 had an Mn of 452.6 kg·mol–1 and 28 polymer arms; the copolymer S14 had 344.5 kg·mol–1 and 21 polymer arms; and the copolymer S15 had 251.0 kg·mol–1 and 15 polymer arms. This suggests that at lower ratios (n(̃NH2)/n(̃TT)) when there are fewer amines available on the dendrimer core, there is steric hindrance that results in competition for binding, which is also consistent observed yields of 43, 67, and 69% for S13, S14, and S15, respectively. Notably, increasing the n(̃NH2)/n(̃TT) ratio has minimal to no impact on the size of the resulting star copolymers, with S13, S14, and S15, all having Rg values between ∼12 and 13 nm. This can be partly attributed to the low dependence of the star copolymer size on the number of polymer arms and partly to the different sensitivity of the characterization technique (static light scattering) to changes in the monitored quantities (while the weight-average molecular weight (Mw), according to the Rayleigh’s approximation for the spherical particle, increases linearly with the scattering intensity (ILS), the diameter is proportional to the sixth root of the ILS). Based on these data, the reaction scheme using a 2:1 ratio of core and polymer chain functional groups, n(̃NH2)/n(̃TT), was selected as the preferred route as it affords star copolymers with high polymer arm density at an acceptable yield.

Ultrahigh-Resolution Imaging of Star Copolymers

To our knowledge, the size and shape of star copolymer vaccines have not been assessed in detail. Therefore, ultrahigh-resolution images of selected star copolymers were provided to document their size and shape. For these purposes, the purified star copolymer S17 was chosen as a representative carrier with satisfactory size (Rg = 29 nm) and a high number of polymer arms (N = 30). To increase the signal-to-noise ratio and improve the contrast of the image from the STEM detector, the copolymer was labeled with Au-based nanoparticles (mono-sulfo-NHS-undecagold, Au11). In this work, the Au11 nanoparticles were attached to either (i) the surface of the PAMAM core, (ii) the end groups of the PHPMA arms, or (iii) both the PAMAM core and the ends of the PHPMA arms. We found that the best imaging was provided by a copolymer that was labeled only at the ends of the polymer arms, with the contrast of the micrographs also being affected by the number of attached Au11 nanoparticles. From the group of star copolymers decorated with approximately 30, 15, and 5 contrast agent molecules, the one with the smallest number of Au11 nanoparticles (5) proved to be the most suitable for STEM analysis. Several examples of micrographs of star copolymer S17 containing ∼5 Au11 nanoparticles at the end of its PHPMA arms are shown in Figure 2.

Figure 2

Figure 2. Examples of ultrahigh-resolution images of star copolymers S17: (A–E) STEM analysis of star copolymers. Indication of the inner arrangement is viewed. The differences between the images are due to the different distribution of Au11 nanoparticles in the sample. (F) Cluster of 8 free Au-based nanoparticles, each with a diameter of 0.8 nm, corresponding to the Au11.

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The observed local decrease in the intensity of the detected signal (or the contrast, respectively) in the analyzed sample (Figure 2A–E) can be attributed to the absence of Au11 nanoparticles on PHPMA arms (only ∼17% of the arms are modified with Au11) or the spatial arrangement of individual PHPMA arms (some ends of the arms may be hidden inside the polymer coils). In Figure 2 D (white arrow), it can be seen – due to in situ freeze-drying sample preparation – that there is local change of shape or length of the polymer arms. In contrast, a sample with an intact structure and a homogeneous distribution of arms is shown in Figure 2E. The more contrasting area around the star copolymer core (compared to the outer edges) may indicate that the dense dendrimer core (∼6 nm in diameter for G5) is surrounded by expanded chains of hydrophilic polymer arms (Figure 2B,C). Although the obtained micrographs of star copolymers are two-dimensional projections into the X and Y planes, based on changes in intensity in image contrast, star copolymers can be described as spatially symmetric structures of approximately a spherical shape. The most frequently observed star copolymers of a spherical shape with a typical radius of about 7.5 nm are depicted in Figure 2A–D. The difference between the radius of the samples measured by EM and dynamic light scattering (DLS) (Rh ∼ 15 nm) is about 50%. This can be attributed to the different measurement conditions of the two methods; while in the case of electron microscopy, the sample is measured in a completely dried state (using the freeze-drying method), with DLS, the sample is analyzed in a fully solvated state (aqueous solution), which causes the expansion (swelling) of the arms. In addition, the DLS provides a z-average size that is strongly influenced by the presence (even of low populations) of large particles, while the resulting particle size from the EM measurement is calculated by arithmetic averaging, giving the number-average size. Spatially asymmetric formations (Figure 2E) were observed very rarely.
Since the resolution required for convincing imaging of samples is at the limit of the possibilities of the microscope used, the obtained micrographs had to be modified by postprocessing methods. Brightness and contrast were optimized, in particular. The effect of micrograph modification can be seen in Figure 2A, where the background of the micrograph is the original raw micrograph, whereas the postprocessed micrograph is depicted in a white frame with bar. During the detailed analysis of the sample, it was possible to find also an area in which a cluster of free (unbound or released) Au11 nanoparticles can be seen (Figure 2 F). The measured size of Au nanoparticles in the cluster (0.6–0.8 nm) corresponds to the size of the undecagold (Au11) contrast agent, which is reported by the manufacturer (Nanoprobes Inc.). Star copolymer micrographs confirm a notably uneven surface structure (Figure 2A–E), which is consistent with the intended application of these materials as carriers of minimal peptide immunogens mimicking viruses.

Synthesis and Characterization of Star Copolymer Vaccines

After having optimized a synthetic route for modulating polymer arm density and particle size, we next sought to investigate how these parameters impact biological activity of a star copolymer vaccine delivering a representative minimal immunogen consisting of a glycopeptide derived from the V3 loop of HIV Envelope protein, referred to as Man9V3.
Star copolymer vaccines V1–V4 were synthesized by reacting purified star copolymers S27–S30 having different lengths and densities of azide-terminated polymer arms with the dibenzocyclooctyne (DBCO)-functionalized V3 glycopeptide (Man9V3-DBCO) via a strain-promoted cycloaddition reaction (see Scheme 2). The coupling of (macro)molecules mediated by the interaction between azides and DBCO groups was chosen because of their biorthogonality, fast reaction kinetics, and lack of catalyst and byproducts simplifying purification. The conjugation of Man9V3-DBCO to the polymer platform was manifested by an increase in the Mn, from which the number of bound glycopeptide units was calculated. The degree of substitution of the polymer arms by glycopeptide units ranged from ∼66 to 100%, with higher conjugation efficiencies achieved for star copolymers with longer polymer arms. Binding of the glycopeptide also led to an increase in the hydrodynamic radii (Rh), although it should be noted that the radii of gyration (Rg) of the star copolymer vaccines were similar to those of the unmodified star copolymers within experimental measurement error, which is likely due to the lower sensitivity of static light scattering used to determine Rg. Changes in the slope of the Zimm plot, from which Rg is calculated, are very small for particles <30 nm, while changes in the diffusive motion of the particles, from which Rh is calculated, are more sensitive to be reliably detected. The Đ values of the star copolymer vaccines were very low (Đ < 1.3), indicating that conjugation was not accompanied by cross-linking or other undesirable side reactions (see Table 3).

Scheme 2

Scheme 2. Cartoon Depiction (A) and Reaction Scheme (B) for the Preparation of Star Copolymer Vaccines (II) Synthesized by Conjugation of Man9V3 Glycopeptide to Star Copolymers (I)
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Table 3. Characteristics of Star Copolymers S27–S30 (A) and Their Conjugates with the Man9V3 Glycopeptide and the Star Copolymer Vaccines V1–V4 (B)
A       
 star copolymerpolymer armMni [kg·mol–1]ĐiiRgiii[nm]Rhiv[nm]Nv(polymer arms)
 S27P4275.71.1110.212.228.5
 S28P51890.71.1631.833.726.1
 S29P484.81.108.79.79.0
 S30P5559.11.0721.622.97.7
B       
 star copolymer vaccinestar copolymerMni [kg·mol–1]ĐiiRgiii[nm]Rhiv[nm]Nvi(Man9V3 units)
 V1S27419.81.176.216.418.9
 V2S282107.11.2438.434.428.4
 V3S29135.91.18n.d.11.86.7
 V4S30609.71.1418.027.06.6
i

Number-average molecular weight of the star copolymer/star copolymer vaccine determined by SEC.

ii

Star copolymer/star copolymer vaccine dispersity defined as the ratio of weight-average (Mw) to number-average (Mn) molecular weight determined by SEC.

iii

Radius of gyration of the star copolymer/star copolymer vaccine determined by SEC.

iv

Hydrodynamic radius of the star copolymer/star copolymer vaccine determined by DLS.

v

Number of polymer arms attached to the PAMAM dendrimer core evaluated by SEC.

vi

Number of minimal peptide immunogen units attached to the PHPMA arms of the star copolymer evaluated by SEC.

Ultrahigh-Resolution Imaging of Star Copolymer Vaccines

To document the size and shape of the star copolymer vaccines not only in solution but also in the dry state, we subjected them to detailed EM analysis. However, while in the case of unmodified star copolymers, it was possible to decorate their surfaces with gold nanoparticles (Au11) to enhance the image contrast, this strategy could not be implemented in the case of vaccines because the end groups of the polymer arms were occupied by minimal peptide immunogen molecules. For this reason, the direct visualization of unlabeled star copolymer vaccines by EM was a major challenge. However, as can be seen from the acquired micrographs of a representative star copolymer vaccine with ∼70 kg·mol–1 polymer arms (Figure 3), by using a suitable method and optimizing the measurement conditions, it was possible to reliably capture the analyzed subjects. The analyzed vaccine nanoparticles had a spherical shape with a radius of ∼25 nm, which is in close agreement with the values obtained from the DLS measurement.

Figure 3

Figure 3. Ultrahigh-resolution micrographs of unlabeled star copolymer vaccine construct with ∼70 kg·mol–1 polymer arms obtained by ESEM. The overall image at the top left shows the arrangement of star copolymer vaccine of similar shapes and sizes, while the remaining images show a zoomed-in view of individual ∼50 nm star copolymer vaccines emphasizing their spherical shape and entangled structure of the polymer coils.

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Impact of Star Copolymer Arm Density and Molecular Weight on Vaccine Activity

We next assessed whether modular properties of the star copolymer could be tuned to impact immune responses when used as a vaccine delivering the minimal immunogen, Man9V3. Star copolymers with two different arm lengths (Mn target ∼10 or 80 kg·mol–1) and number of polymer arms (N ∼ 10 or 30) were prepared to generate star copolymer vaccines with varying size and minimal immunogen density as summarized in Table 3 and then assessed for the capacity to induce antibody responses in mice following vaccination.
Whereas vaccination with the soluble monomeric Man9V3 (“Free Man9V3”) did not induce antibodies above background levels in naïve, untreated animals, all the star copolymer compositions induced detectable antibody responses (Figure 4). Furthermore, there was a clear trend between increasing arm Mn and increased antibody responses with the star copolymers with 80 kg·mol–1 arms (V2 and V4) inducing antibody titers that were greater than 10-fold higher than the star copolymers with 10 kg·mol–1 arms (V1 and V3) and nearly 100-fold and statistically significantly higher than background levels observed in naïve animals. No differences in antibody responses were observed with star copolymers having 10 or 30 arms, which suggests that the star copolymer size may be more critical than the polymer arm density (number of polymer arms) in promoting antibody responses. Of note, the responses observed at 2 weeks after the final vaccination (day 70), corresponding to the peak of the antibody response, are comparable to the levels observed against Man9V3 in other studies that were considered biologically meaningful. (33,36)

Figure 4

Figure 4. Antibody responses following vaccination with different star copolymer vaccine compositions, free (unformulated Man9V3) or naïve control. Balb/c mice (n = 5/group) were immunized at days 0, 28, and 56 and serum was collected on day 70 and assessed for anti-Man9V3 IgG antibodies by enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± standard deviation end point titer. Differences between each experimental group and the naïve control were assessed for statistical significance using one-way ANOVA with Bonferroni correction for multiple comparisons; asterisks (*) indicate statistical significance (P < 0.05) between the indicated group and naïve.

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While these preliminary data highlight some potential advantages of the star copolymer as a tunable platform for delivering minimal immunogens to modulate antibody responses, further immunological assessment characterizing how such parameters impact pharmacokinetics and biodistribution as well as uptake and processing by innate immune cell populations to influence antibody and T cell responses will be needed. For instance, prior studies have established that increasing the hydrodynamic size of particulate vaccines can lead to greater uptake by immune cell subsets in lymph nodes associated with enhanced antibody and T cell responses. (13,37−39) Therefore, future studies assessing how star copolymer arm length (and hydrodynamic size) impacts distribution to and uptake by immune cells in lymph nodes may be helpful for understanding the mechanistic basis for the results presented herein.

Star Copolymer Vaccines Have Excellent Recovery Following Sterile Filtration

Sterile filtration to ensure product sterility is a key step in vaccine manufacturing. A potential advantage of star copolymers is that their size can be tuned and tightly controlled between about 10 and 50 nm diameter, which is well below most sterile filter pore sizes of about 0.2 μm. To assess the capacity of the star copolymer vaccines to undergo sterile filtration, a representative star-Man9V3 vaccine with 30 fully conjugated ∼10 kg·mol–1 polymer arms was filtered through 0.2 μm pore filters comprising either polytetrafluoroethylene (PTFE), nylon, polyethlysulfone (PES), or cellulose acetate (CA) and then assessed for material properties and recovery by SEC and high-performance liquid chromatography (HPLC). Notably, the star copolymer vaccine showed excellent recovery with no impact on particle size and molecular weight following filtration from PTFE, nylon, and PES, whereas no star copolymer vaccine was recoverable following filtration through CA (see Table S2). These data show that star copolymer vaccines can be efficiently and nondestructively filtered through conventional filtration membranes (except CA), thereby ensuring sterility required for injectables intended for human use.

Stability of Star Copolymers and Star Copolymer Vaccines in Aqueous Buffer

For commercial utility, star copolymer vaccines should have sufficient stability in aqueous buffers during manufacturing, storage, and solutions prior to administration in the clinical or hospital setting. First, we assessed the stability of a representative star copolymer with ∼10 PHPMA arms of Mn ∼ 30 kg·mol–1 placed at 37 °C in a temperature-controlled environment and then sampled at up to 16 weeks to assess for degradation by DLS and SEC. Notably, the star copolymer showed excellent stability at 37 °C; 10% was degraded through 2 weeks, which increased to 15% degraded by 4 weeks and more than 40% degraded by 16 weeks (Table S3A and Figure S4). The gradual degradation of the star copolymer was probably due to the retro-Michael reaction occurring in the PAMAM dendrimers, which is especially favored in aqueous solutions at elevated temperatures. As a consequence, a branching methacrylamide chain (conjugated with PHPMA arm) is cleaved, and a defective dendrimer with one missing branch is formed. (40) We further evaluated the stability of the representative star copolymer vaccine comprising Man9V3 glycopeptide (∼10 units) stored under various conditions, including 25 °C for 5 days and 4 °C and −20 °C for up to 16 weeks either as a lyophilized solid or in PBS solution. The results of the DLS and SEC analyses showed that the values of all monitored quantities (Rh, Mw, and Đ) remained within the experimental error practically unchanged (Table S3B and Figure S5). Overall, these data clearly demonstrate that the star copolymers based on PAMAM and PHPMA as well as their conjugates with Man9V3 have excellent stability under manufacturing, handling, and storage conditions, which bodes well for their potential use in commercial applications.

Conclusions

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The presented article discusses the synthesis and optimization of the structure of star copolymers as tunable nanoparticle carriers of minimal immunogens for use as vaccines. Specifically, we studied how the length of PHPMA arms, dendrimer generation, and molar ratio of arms to dendrimer functional groups impact star copolymer size, arm density, and yield. In addition, ultrahigh-resolution images of a selected star copolymer were provided to document its size and shape in the unsolvated state. Three important conclusions can be drawn from the measured data: (i) the polymer arms with a higher Mn form larger-sized star copolymers, but they attach to the surface of the dendrimer to a lower extent than the polymer arms with lower Mn; (ii) the branching degree (generation) of the dendrimer can control the density of the arms extending from the dendrimer core, but not the size of the star copolymer; and (iii) the use of a higher molar ratio of PAMAM ̃NH2 groups to PHPMA ̃TT groups leads to a lower polymer arm density but higher star copolymer yield, regardless of the polymer arm length and PAMAM generation used. Finally, with an eye to clinical translation, we investigated how the modular parameters of star copolymers impact their activity for use as minimal immunogen vaccines using a glycopeptide derived from the envelope protein of the HIV-1 virus as a representative minimal immunogen. The results showed that all star copolymer vaccine compositions elicited a detectable antibody response, with the effect being more pronounced for the larger star copolymers with longer arms, while the number of polymer arms had little effect on the antibody titer. Considering the long-term storage stability as refrigerated (4 °C) or frozen (−20 °C) solutions or even better as lyophilized solids and excellent yields after sterile filtration, star copolymer vaccines represent a very promising platform for minimal immunogen vaccines.

Experimental Procedures

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Chemicals

Acetic anhydride, (RS)-1-aminopropan-2-ol, l-ascorbic acid, 3-azidopropylamine, 4,4′-azobis(4-cyanovaleric acid) (ACVA), copper(I) bromide (CuBr), copper(II) sulfate pentahydrate (CuSO4·5H2O), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPP), N,N′-dicyclohexylcarbodiimide (DCC), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), 4-(dimethylamino)pyridine (DMAP), 8-hydroxyquinoline, methacryloyl chloride, sodium carbonate, thiazolidine-2-thione (TT), and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) were purchased from Sigma-Aldrich, Czech Republic and used as received. Polyamidoamine dendrimers, ethylendiamine core, generation 3.0–5.0 (PAMAM, G3–5) were purchased from Sigma-Aldrich, Czech Republic. Mono-Sulfo-NHS-Undecagold (Au11) was obtained from Nanoprobes, NY, USA. The Man9V3-DBCO glycopeptide was kindly provided by Chemitope Glycopeptide, NY, USA. All compounds are >95% pure by HPLC analysis. All solvents were of HPLC grade and dried over a layer of activated molecular sieves (4 Åm) before use.

Synthesis of Monomer, Functionalized Chain Transfer Agent, and Initiators

N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by reacting methacryloyl chloride with (RS)-1-aminopropan-2-ol in dichloromethane in the presence of sodium carbonate as described in reference. (41)
Dithiobenzoic acid 1-cyano-1-methyl-4-oxo-4-(2-thioxothiazolidin-3-yl)butyl ester (CPP-TT) was prepared by the reaction of CPP with TT in dichloromethane in the presence of DCC and DMAP. (42)
Dithiobenzoic acid 3-(3-azidopropylcarbamoyl)-1-cyano-1-methylpropyl ester (CPP-N3) was produced by the reaction of CPP with 3-azidopropylamine in ethyl acetate in the presence of EDC. (43)
2-[1-Cyano-1-methyl-4-oxo-4-(2-thioxo-thiazolidin-3-yl)-butylazo]-2-methyl-5-oxo-5-(2-thioxothiazolidin-3-yl)-pentanenitrile (ACVA-(TT)2) was prepared by the reaction of ACVA with TT in tetrahydrofuran in the presence of DCC and DMAP. (44)
4-Cyano-4-(1-cyano-3-ethynylcarbamoyl-1-methylpropylazo)-N-ethynyl-4-methylbutyramide (ACVA-(Pg)2) was synthesized by reacting ACVA with propargylamine in dichloromethane in the presence of EDC and DMAP. (43)
N-(3-Azidopropyl)-4-[3-(3-azidopropylcarbamoyl)-1-cyano-1-methylpropylazo]-4-cyano-4-methylbutyramide (ACVA-(N3)2) was synthesized by reacting ACVA with 3-azidopropylamine in dichloromethane in the presence of EDC and DMAP. (45)

Synthesis of Functionalized Immunostimulant and Minimal Peptide Immunogen

1-(4-(Aminomethyl)benzyl)-2-butyl-1H-imidazo[4,5-c]quinolin-4-amine (2Bxy) was prepared by a multistep reaction starting from quinoline-2,4-diol as previously described. (11)
Man9V3, a glycosylated HIV V3 minimal peptide immunogen of the composition EINCTRPNNNTRPGEIIGDIRQAHCNISRA with a C-terminal PEG3-DBCO linker, was synthesized following a procedure similar to that described in ref (21), except that the C-terminus of the glycopeptide was linked to a Boc-amine-PEG3 linker instead of biotin as shown in Scheme S1. Briefly, Man9GlcNAc2–NH2 (5) was originally synthesized using complex glycosyl coupling chemistry while N-terminal fragment (4) and Boc-amine-PEG3-linked C-terminal fragment (1) were acquired through solid-phase peptide synthesis. Man9GlcNAc2 glycosyl amine (5) was attached to the free carboxylic acid side chain at position 301 on the N-terminal fragment (4) and at position 332 on the Boc-amine-PEG3 linked C-terminal fragment (1) to provide glycopeptide thioester (7) and N-terminal cysteinyl glycopeptide (8), respectively. These two fragments were then coupled using native chemical ligation, immediately followed by cyclization via disulfide formation to yield the Man9V3-PEG3-amine. DBCO-NHS ester was then introduced to react with the amine group, affording the final product Man9V3-DBCO (11).

Synthesis of Heterobifunctional Polymer Arms

Polymer arms P1–P3 were synthesized by RAFT polymerization of HPMA in a tert-butyl alcohol/DMSO mixture in the presence of various amounts of CPP-TT and ACVA-(TT)2 (see Table 1). Polymer arms P4 and P5 were prepared under the same conditions but in the presence of CPP-N3 and ACVA-(N3)2. In the second step, the dithiobenzoate (DTB) end groups of the polymers were capped by the homolytic reaction with an excess of functionalized initiators, ACVA-(Pg)2 in the case of polymer arms P1–P3, and ACVA-(TT)2 in the case of polymer arms P4 and P5, to form propargyl (Pg) or carbonylthiazolidine-2-thione (TT) groups, respectively. Below is given a typical procedure for the synthesis of heterobifunctional polymer P1.
A mixture of CPP-TT (39.4 mg, 103.0 μmol) and ACVA-(TT)2 (24.9 mg, 51.5 μmol) was dissolved in 1.6 mL of DMSO and added to a solution of HPMA (2.0 g, 14.0 mmol) in 14.1 mL of tert-butanol. The reaction mixture was thoroughly bubbled with argon and polymerized in a sealed glass ampule at 70 °C for 16 h. Then, the mixture was precipitated into 300 mL of acetone/diethyl ether (3:1), and the precipitate formed was filtered, redissolved in methanol, and precipitated again into the same precipitant. After drying under vacuum, 828 mg (41%) of the product was obtained as an orange, amorphous powder. The number-average molecular weight (Mn) and the dispersity (Đ) of the polymer precursor were 9.2 kg·mol–1 and 1.03, respectively.
A mixture of polymer precursor (200.0 mg, 21.7 μmol of DTB gr.) and ACVA-(Pg)2 (231.1 mg, 652.2 μmol) was dissolved in DMSO (2 mL), thoroughly bubbled with argon, and incubated at 80 °C for 3 h. Then, the mixture was precipitated into 40 mL of acetone/diethyl ether (3:1), the precipitate formed was filtered, redissolved in methanol, and precipitated again into the same precipitant. After drying under vacuum, 154 mg (77%) of the P1 polymer was obtained as a pale-yellow amorphous powder. The Mn and Đ of the P1 polymer were 9.8 kg·mol–1 and 1.03, respectively. The molar content of the thiazolidine-2-thione (TT) end group of the polymer was 98.9 μmol/g, corresponding to an average functionality of 0.97.

Synthesis of Star Copolymers

Star copolymers were synthesized by acylation of the PAMAM dendrimer of various generations (G3, G4, and G5) with a heterobifunctional polymer arm of different molecular weights (10, 16, 41, and 96 kDa) at different molar ratios of the PAMAM amino groups to the TT terminal group on the polymer arms (1:1, 2:1, and 3:1). Below is given a typical procedure for the synthesis of star copolymer S1.
Methanolic solution of PAMAM, G3 (1.1 μL of 20 wt %, 30.3 nmol of ̃NH2 gr.) was added to a solution of linear polymer P1 (9.8 mg, 0.97 μmol of TT gr.) in 100 μL of methanol, and the reaction mixture was shaken at 25 °C for 16 h. Then, the mixture was precipitated into 2.0 mL of diethyl ether; the precipitate formed was collected by centrifugation, redissolved in water, and freeze-dried to give 9.5 mg of a mixture of star copolymer S1 and linear polymer P1. The Mn, Đ, radius of gyration (Rg) and weight fraction of star copolymer S1 in the isolated product were 246.6 kg·mol–1, 1.12, 9.6 nm, and 48.7%, respectively.
A selected star copolymer S17 was further purified from the unreacted linear polymers by membrane filtration using RC centrifugal filter units with molecular weight cutoff (MWCO) 100 kg·mol–1 (Sigma-Aldrich, Czech Republic) in PBS (4×) and in H2O (2×) and isolated by lyophilization. For SEC chromatograms of star copolymer S17 before and after the purification, see Figure S1.

Labeling of Star Copolymers with the Contrast Agent

The purified star copolymer S17 was labeled by reaction with an Au-based contrast agent (Au11). The Au11 nanoparticles were covalently linked to either (i) the surface of the PAMAM core, (ii) the end groups of the PHPMA arms, or (iii) both the PAMAM core and the ends of the PHPMA arms. In addition, in the case of approach (i), different numbers (30, 15, and 5) of Au11 nanoparticles were attached to the ends of the polymer arms. An example of the preparation of a star copolymer labeled with approximately 5 Au11 nanoparticles attached to the ends of PHPMA arms is described below:
The purified star copolymer S17 (171.8 mg, 47 μmol of −NH2 gr.) was dissolved in 3.436 mL of N,N’-dimethylacetamide (DMAc) and mixed with 45 μL of acetic anhydride (470 μmol), and the solution was shaken at 25 °C for 2 h. The reaction mixture was diluted with methanol (1:1) and separated on a column filled with Sephadex LH-20 (Sigma-Aldrich, Czech Republic) in methanol. The polymer fraction was precipitated into diethyl ether yielding 165 mg of the star copolymer with acetylated amino groups on the surface of PAMAM.
Afterward, the copolymer (155.0 mg, 11 μmol of Pg gr.) was dissolved in 3.2 mL of DMAc, 3-azidopropylamine (2.2 μL, 22 μmol), TBTA (5.9 mg, 11 μmol), and CuBr (1.6 mg, 11 μmol) were added, and the reaction mixture was thoroughly bubbled with Ar. After 24 h, the reaction mixture was diluted by the addition of 8-hydroxyquinoline and separated on a Sephadex LH-20 column in methanol. The polymer fraction was precipitated into diethyl ether, yielding 147 mg of the star copolymer with the primary amino groups at the ends of the PHPMA arms.
Finally, an aqueous solution of the star copolymer (0.64 mg, 46 nmol of ̃NH2 gr.) was mixed with the Au11 nanoparticles (8 nmol) dissolved in 1 mL of 10% isopropanol in water and the reaction mixture was shaken overnight at 25 °C. The resulting product was purified on a PD-10 column (Sigma-Aldrich, Czech Republic) in H2O and lyophilized to give 0.58 mg of the star copolymer with approximately 5 Au11 molecules attached to the ends of the PHPMA arms.

Synthesis of Star Copolymer Vaccines

Star copolymer vaccines comprising a G5 PAMAM dendrimer core with different lengths and densities of PHPMA polymer arms were prepared as described above, except the polymer arms (P4 and P5) were terminated with an azide group used to link the HIV minimal peptide immunogen, Man9V3 bearing a dibenzylcyclooctyl (DBCO) group, to the star copolymer via strain-promoted azide–alkyne cycloaddition. For example, star copolymer vaccine V3 was prepared by mixing 1.09 mg of star copolymer S29 (0.116 μmol of ̃N3 group) dissolved in 1.1 μL of DMSO/DMF (1/1 v/v) and 0.91 mg of Man9V3-DBCO (0.119 μmol) in 18.3 μL of DMSO/DMF (1/1 v/v). The reaction of star copolymer with a slight, 3 mol % excess of Man9V3 (or 1:1.03 molar ratio) was allowed to proceed at 25 °C overnight. An aliquot was then injected onto the HPLC instrument, and conversion was evaluated by comparing the areas under the curve of unreacted Man9V3-DBCO before and after the reaction. If free Man9V3-DBCO (>3%) was present in the reaction mixture, additional star copolymer was added to consume the remaining glycopeptide until its content was below 3%. The star copolymer vaccine was lyophilized, redissolved in 1× PBS, and stored at −20 °C before use for animal studies. The Mn and Đ of star copolymer vaccine V3 were 135.9 kg·mol–1 and 1.18, respectively.

Size-Exclusion Chromatography

The number- and weight-average molecular weights (Mn and Mw), dispersities (Đ), and gyration radii (Rg) of all linear polymer arms as well as the star copolymers were determined by size-exclusion chromatography (SEC) on an HPLC system (Shimadzu Corp., Japan) equipped with internal UV–VIS diode array detector (SPD-M20A) and external differential refractometer (Optilab T-rEX) and multiangle light scattering detector (DAWN HELEOS II, both Wyatt Technology Corp., CA, USA). TSKgel SuperAW3000 and SuperAW4000 columns (Tosoh Bioscience, PA, USA) in series were used to analyze samples in a mobile phase of 80% methanol/20% sodium acetate buffer (0.3 M, pH 6.5) at a flow rate of 0.6 mL·min–1. The dn/dc values of 0.168 and 0.176 mL·g–1 were used to calculate the molecular weights of linear/star copolymers and star copolymer vaccines, respectively.

High-Performance Liquid Chromatography

The purity of low-molecular-weight compounds, including monomer, chain transfer agents, initiators, and immunostimulant, was verified on a high-performance liquid chromatography (HPLC) system (Shimadzu, Japan) equipped with an internal UV–vis diode array (SPD-M20A) and ELSD (LTII) detectors using the Chromolith HighResolution RP-18e reverse-phase column (Merck, USA), with a linear gradient (0–100%) of water–acetonitrile mixture containing 0.1% TFA at a flow rate of 2.5 mL·min–1. The course of conjugation of Man9V3-DBCO glycopeptide to the star copolymers was monitored by an HPLC system (Agilent 1260 Infinity II, CA, USA) equipped with an internal UV–vis diode array detector using an Agilent InfinityLab Poroshell 120 EC-C18 reverse-phase column, with a linear gradient (5–65%) of water–acetonitrile mixture containing 0.05% TFA at a flow rate of 1.25 mL·min–1.

UV–vis Spectrophotometry

The spectrophotometric analyses of functionalized linear polymers were performed in quartz glass cuvettes on a Specord Plus UV–vis spectrophotometer (Analytik Jena, Jena, Germany). The molar content of the terminal DTB and TT groups in the polymers was determined at 302 and 305 nm in methanol using the molar absorption coefficient of 12,100 and 10,300 L·mol–1·cm–1, respectively.

Dynamic Light Scattering

The hydrodynamic sizes of purified star copolymers were determined by the dynamic light scattering (DLS) technique at a scattering angle of 173° using a Nano-ZS instrument (Malvern Instruments, UK) equipped with a 4 mW, 633 nm laser. Measurements were performed at a sample concentration of 1.0 mg·mL–1 in PBS buffers (0.15 M, pH 7.4) at 37 °C. For the evaluation of the dynamic light scattering data, the DTS (Nano) program was used. The resulting hydrodynamic sizes were arithmetic means of at least 10 independent measurements.

Electron Microscopy

Ultrahigh-resolution electron microscopy imaging of Au-labeled star copolymers and unlabeled star copolymer vaccines was performed on a custom-modified environmental scanning electron microscope (ESEM) Quanta 650 FEG (Thermo Fisher Scientific, MA, USA) (46) equipped with a detector for scanning transmission electron microscopy (STEM). The lyophilized samples were dissolved in distilled water and applied to a lacey carbon film on a copper grid. The solution of star copolymers in water (2 μL) was applied on a TEM grid covered with holey carbon film. (47,48) Then, the samples were in situ freeze-dried at 20 °C and 10 Pa in the ESEM specimen chamber (49) (operated under environmental mode). Observation was performed at a beam energy of 30 keV, beam current of 5 pA, and working distance of 5.3 mm in high vacuum mode using a dark-field STEM detector. Micrographs were postprocessed using MountainsSEM software (Digital Surf, France).

In Vivo Vaccination

Animals were housed and cared for in accordance with the American Association for Accreditation of Laboratory Animal Care standards in accredited facilities at the Vaccine Research Center, and all animal procedures were performed according to a protocol approved by the Institutional Animal Care and Use Committees of the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Female Balb/c mice, 8–12 weeks of age, were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained at the Vaccine Research Center’s (VRC) Animal Care Facility (Bethesda, MD, USA) under pathogen-free conditions. Star copolymers for vaccination were prepared as 25 μg of Man9V3 minimal peptide immunogen equivalent star copolymer in PBS buffer with adjuvant comprising 5 μg PADRE and 5 nmol of the TLR-7/8 agonist, 2BXy, as previously described. (33) Immunizations were given intramuscularly at days 0, 28, and 56 and blood was sampled at day 70 to isolate serum for assessment of antibody responses.

Antibody Measurements

Pierce Streptavidin Coated Plates (Thermo Scientific) were coated overnight at 4 °C with a Man9V3-biotin probe in PBS buffer. Plates were then blocked with PBS + FCS, and serum was applied in serial 10-fold dilutions and incubated at 37 °C. Detection was performed at room temperature with total antimouse IgG HRP-conjugated secondary antibodies (1:6000, Southern Biotech) followed by TMB + substrate–chromogen (Dako) and a 2N sulfuric acid stop solution. Washing was performed between steps with PBS + 0.05% Tween 20. Plates were read on a spectrophotometer (and data were analyzed in Prism (GraphPad). End point titers were determined by fitting data using a four-parameter dose–response curve.

Data Availability

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The data underlying this study are available in the published article and its Supporting Information.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.4c00273.

  • Reaction scheme for the preparation of Man9V3-DBCO (Scheme S1), characteristics of PAMAM dendrimers (Table S1), SEC chromatograms of unpurified and purified star copolymer S17 (Figure S1), SEC chromatograms of heterobifunctional polymer arms P1–P3 (Figure S2), Rh-distribution function of purified star copolymer S17 (Figure S3), size parameters and mass recoveries of star copolymer vaccine solutions before and after filtration (Table S2), size parameters of star copolymers and star copolymer vaccines stored under different conditions (Table S3), SEC chromatograms of star copolymers stored under different conditions (Figure S4), and SEC chromatograms of star copolymer vaccines stored under different conditions (Figure S5) (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • Gabriela Mixová - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Eva Tihlaříková - Institute of Scientific Instruments, Czech Academy of Sciences, Královopolská 147, Brno 612 64, Czech Republic;  Orcidhttps://orcid.org/0000-0002-7983-2971
    • Yaling Zhu - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
    • Lucie Schindler - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Ladislav Androvič - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Lucie Kracíková - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Eliška Hrdá - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Bedřich Porsch - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic
    • Michal Pechar - Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, Prague 162 06, Czech Republic;  Orcidhttps://orcid.org/0000-0002-4507-1801
    • Christopher M. Garliss - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
    • David Wilson - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
    • Hugh C. Welles - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States;  Orcidhttps://orcid.org/0000-0002-3336-1203
    • Jake Holechek - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
    • Qiuyin Ren - Vaccine Research Center, National Institutes of Health, Rockville, Maryland 20892, United States
    • Geoffrey M. Lynn - Barinthus Biotherapeutics North America, Inc. (formerly Avidea Technologies, Inc.), 20400 Century Boulevard, Germantown, Maryland 20874, United States
    • Vilém Neděla - Institute of Scientific Instruments, Czech Academy of Sciences, Královopolská 147, Brno 612 64, Czech Republic
  • Author Contributions

    The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This research was financially supported by the project National Institute for Cancer Research (Programme EXCELES, Project No. LX22NPO5102) – Funded by the European Union – Next Generation EU and by the Czech Science Foundation (Project No. 24-10980S).

References

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    Li, Y.-D.; Chi, W.-Y.; Su, J.-H.; Ferrall, L.; Hung, C.-F.; Wu, T.-C. Coronavirus vaccine development: from SARS and MERS to COVID-19. J. Biomed. Sci. 2020, 27, 104,  DOI: 10.1186/s12929-020-00695-2
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    Plotkin, S. A. Correlates of Protection Induced by Vaccination. Clin Vaccine Immunol 2010, 17, 1055– 1065,  DOI: 10.1128/CVI.00131-10
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    Correlates of protection induced by vaccination
    Plotkin, Stanley A.
    Clinical and Vaccine Immunology (2010), 17 (7), 1055-1065CODEN: CVILA6; ISSN:1556-679X. (American Society for Microbiology)
    A review. This paper attempts to summarize current knowledge about immune responses to vaccines that correlate with protection. Although the immune system is redundant, almost all current vaccines work through antibodies in serum or on mucosa that block infection or bacteremia/viremia and thus provide a correlate of protection. The functional characteristics of antibodies, as well as quantity, are important. Antibody may be highly correlated with protection or synergistic with other functions. Immune memory is a crit. correlate: effector memory for short-incubation diseases and central memory for long-incubation diseases. Cellular immunity acts to kill or suppress intracellular pathogens and may also synergize with antibody. For some vaccines, we have no true correlates, but only useful surrogates, for an unknown protective response.
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    de Taeye, S. W.; de la Peña, A. T.; Vecchione, A.; Scutigliani, E.; Sliepen, K.; Burger, J. A.; van der Woude, P.; Schorcht, A.; Schermer, E. E.; van Gils, M. J. Stabilization of the gp120 V3 loop through hydrophobic interactions reduces the immunodominant V3-directed non-neutralizing response to HIV-1 envelope trimers. J. Biol. Chem. 2018, 293, 1688– 1701,  DOI: 10.1074/jbc.RA117.000709
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    Arvin, A. M.; Fink, K.; Schmid, M. A.; Cathcart, A.; Spreafico, R.; Havenar-Daughton, C.; Lanzavecchia, A.; Corti, D.; Virgin, H. W. A perspective on potential antibody-dependent enhancement of SARS-CoV-2. Nature 2020, 584, 353– 363,  DOI: 10.1038/s41586-020-2538-8
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    A perspective on potential antibody-dependent enhancement of SARS-CoV-2
    Arvin, Ann M.; Fink, Katja; Schmid, Michael A.; Cathcart, Andrea; Spreafico, Roberto; Havenar-Daughton, Colin; Lanzavecchia, Antonio; Corti, Davide; Virgin, Herbert W.
    Nature (London, United Kingdom) (2020), 584 (7821), 353-363CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    A review. Antibody-dependent enhancement (ADE) of disease is a general concern for the development of vaccines and antibody therapies because the mechanisms that underlie antibody protection against any virus have a theor. potential to amplify the infection or trigger harmful immunopathol. This possibility requires careful consideration at this crit. point in the pandemic of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We review observations relevant to the risks of ADE of disease, and their potential implications for SARS-CoV-2 infection. At present, there are no known clin. findings, immunol. assays or biomarkers that can differentiate any severe viral infection from immune-enhanced disease, whether by measuring antibodies, T cells, or intrinsic host responses. In vitro systems and animal models do not predict the risk of ADE of disease, in part because protective and potentially detrimental antibody-mediated mechanisms are the same and designing animal models depends on understanding how antiviral host responses may become harmful in humans. The implications of our lack of knowledge are 2-fold. First, comprehensive studies are urgently needed to define clin. correlates of protective immunity against SARS-CoV-2. Second, because ADE of disease cannot be reliably predicted after either vaccination or treatment with antibodies-regardless of what virus is the causative agent-it will be essential to depend on careful anal. of safety in humans as immune interventions for COVID-19 move forward.
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    Kozak, M.; Hu, J. The Integrated Consideration of Vaccine Platforms, Adjuvants, and Delivery Routes for Successful Vaccine Development. Vaccines 2023, 11, 695,  DOI: 10.3390/vaccines11030695
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    Pollard, A. J.; Bijker, E. M. A guide to vaccinology: from basic principles to new developments. Nat. Rev. Immunol 2021, 21, 83– 100,  DOI: 10.1038/s41577-020-00479-7
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    A guide to vaccinology: from basic principles to new developments
    Pollard, Andrew J.; Bijker, Else M.
    Nature Reviews Immunology (2021), 21 (2), 83-100CODEN: NRIABX; ISSN:1474-1733. (Nature Research)
    A review. Abstr.: Immunization is a cornerstone of public health policy and is demonstrably highly cost-effective when used to protect child health. Although it could be argued that immunol. has not thus far contributed much to vaccine development, in that most of the vaccines we use today were developed and tested empirically, it is clear that there are major challenges ahead to develop new vaccines for difficult-to-target pathogens, for which we urgently need a better understanding of protective immunity. Moreover, recognition of the huge potential and challenges for vaccines to control disease outbreaks and protect the older population, together with the availability of an array of new technologies, make it the perfect time for immunologists to be involved in designing the next generation of powerful immunogens. This Review provides an introductory overview of vaccines, immunization and related issues and thereby aims to inform a broad scientific audience about the underlying immunol. concepts.
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    Irvine, D. J.; Read, B. J. Shaping humoral immunity to vaccines through antigen-displaying nanoparticles. Curr. Opin Immunol 2020, 65, 1– 6,  DOI: 10.1016/j.coi.2020.01.007
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    Shaping humoral immunity to vaccines through antigen-displaying nanoparticles
    Irvine, Darrell J.; Read, Benjamin J.
    Current Opinion in Immunology (2020), 65 (), 1-6CODEN: COPIEL; ISSN:0952-7915. (Elsevier Ltd.)
    A review. Strategies to qual. and quant. enhance the humoral response to immunizations with protein and polysaccharide antigens are of broad interest for development of new and more effective vaccines. A strategy of increasing importance is the formulation of antigens into a particulate format, mimicking the phys. form of viruses. The potential benefits of enhanced B cell receptor engagement by nanoparticles have been long been appreciated, but recent studies are defining addnl. important factors governing how nanoparticle immunogens interact with the immune system in the context of lymphoid organs. This review will discuss findings about how nanoparticles enhance humoral immunity in vivo and factors governing the fate of nanoparticle immunogens in lymph nodes.
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    Purcell, A. W.; McCluskey, J.; Rossjohn, J. More than one reason to rethink the use of peptides in vaccine design. Nat. Rev. Drug Discovery 2007, 6, 404– 414,  DOI: 10.1038/nrd2224
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    More than one reason to rethink the use of peptides in vaccine design
    Purcell, Anthony W.; McCluskey, James; Rossjohn, Jamie
    Nature Reviews Drug Discovery (2007), 6 (5), 404-414CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
    A review. The use of peptides as therapeutics is experiencing renewed enthusiasm owing to advances in delivery, stability and design. Moreover, there is a growing emphasis on the use of peptides in vaccine design as insights into tissue-specific processing of the immunogenic epitopes of proteins and the discovery of unusually long cytotoxic T-lymphocyte epitopes broaden the range of targets and give clues to enhancing peptide immunogenicity. Peptides can also be synthesized with known post-translational modifications and/or deliberately introduced protease-resistant peptide bonds to regulate their processing independent of tissue-specific proteolysis and to stabilize these compds. in vivo. We discuss the potential of peptide-based vaccines for the treatment of chronic viral diseases and cancer, and review recent developments in the field of peptide-based vaccines.
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    Malonis, R. J.; Lai, J. R.; Vergnolle, O. Peptide-Based Vaccines: Current Progress and Future Challenges. Chem. Rev. 2020, 120, 3210– 3229,  DOI: 10.1021/acs.chemrev.9b00472
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    Peptide-Based Vaccines: Current Progress and Future Challenges
    Malonis, Ryan J.; Lai, Jonathan R.; Vergnolle, Olivia
    Chemical Reviews (Washington, DC, United States) (2020), 120 (6), 3210-3229CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Vaccines have had a profound impact on the management and prevention of infectious disease. In addn., the development of vaccines against chronic diseases has attracted considerable interest as an approach to prevent, rather than treat, conditions such as cancer, Alzheimer's disease, and others. Subunit vaccines consist of nongenetic components of the infectious agent or disease-related epitope. In this Review, we discuss peptide-based vaccines and their potential in three therapeutic areas: infectious disease, Alzheimer's disease, and cancer. We discuss factors that contribute to vaccine efficacy and how these parameters may potentially be modulated by design. We examine both clin. tested vaccines as well as nascent approaches and explore current challenges and potential remedies. While peptide vaccines hold substantial promise in the prevention of human disease, many obstacles remain that have hampered their clin. use; thus, continued research efforts to address these challenges are warranted.
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    Lynn, G. M.; Laga, R.; Darrah, P. A.; Ishizuka, A. S.; Balaci, A. J.; Dulcey, A. E.; Pechar, M.; Pola, R.; Gerner, M. Y.; Yamamoto, A. In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity. Nat. Biotechnol. 2015, 33, 1201,  DOI: 10.1038/nbt.3371
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    In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity
    Lynn, Geoffrey M.; Laga, Richard; Darrah, Patricia A.; Ishizuka, Andrew S.; Balaci, Alexandra J.; Dulcey, Andres E.; Pechar, Michal; Pola, Robert; Gerner, Michael Y.; Yamamoto, Ayako; Buechler, Connor R.; Quinn, Kylie M.; Smelkinson, Margery G.; Vanek, Ondrej; Cawood, Ryan; Hills, Thomas; Vasalatiy, Olga; Kastenmuller, Kathrin; Francica, Joseph R.; Stutts, Lalisa; Tom, Janine K.; Ryu, Keun Ah; Esser-Kahn, Aaron P.; Etrych, Tomas; Fisher, Kerry D.; Seymour, Leonard W.; Seder, Robert A.
    Nature Biotechnology (2015), 33 (11), 1201-1210CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
    The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small mol. TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochem. properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer-TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer-TLR-7/8a was also assocd. with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temp.-responsive polymer-TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiol. temps. in vivo. Our findings provide a chem. and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.
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    Dintzis, H. M.; Dintzis, R. Z.; Vogelstein, B. Molecular determinants of immunogenicity: the immunon model of immune response. Proc. Natl. Acad. Sci. U. S. A. 1976, 73, 3671– 5,  DOI: 10.1073/pnas.73.10.3671
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    Molecular determinants of immunogenicity: The immunon model of immune response
    Dintzis, H. M.; Dintzis, R. Z.; Vogelstein, B.
    Proceedings of the National Academy of Sciences of the United States of America (1976), 73 (10), 3671-5CODEN: PNASA6; ISSN:0027-8424.
    The immunol. response in vivo to a series of size-fractionated linear polymers of acrylamide substituted with hapten was measured in mice. A sharp threshold was obsd. in immunogenic response elicited by various polymer prepns. All polymers with ≤12-16 appropriately spaced hapten groups per mol. were nonimmunogenic, whereas those polymers with greater than this no. were fully immunogenic. Thus, the immunol. response at its most elementary level is quantized, i.e., a min. specific no. of antigen receptors (∼12-16) must be connected together as a spatially continuous cluster, an immunon, before an immunogenic signal is delivered to the responding cell.
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    Bachmann, M. F.; Jennings, G. T. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nature Reviews Immunology 2010, 10, 787– 796,  DOI: 10.1038/nri2868
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    Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns
    Bachmann, Martin F.; Jennings, Gary T.
    Nature Reviews Immunology (2010), 10 (11), 787-796CODEN: NRIABX; ISSN:1474-1733. (Nature Publishing Group)
    A review. Researchers working on the development of vaccines face an inherent dilemma: to maximize immunogenicity without compromising safety and tolerability. Early vaccines often induced long-lived protective immune responses, but tolerability was a major problem. Newer vaccines have very few side effects but can be of limited immunogenicity. One way to tackle this problem is to design vaccines that have all the properties of pathogens with the exception of causing disease. Key features of pathogens that can be mimicked by vaccine delivery systems are their size, shape and surface mol. organization. In addn., pathogen-assocd. mol. patterns can be used to induce innate immune responses that promote adaptive immunity. In this review, the authors discuss the approaches currently being used to optimize the delivery of antigens and enhance vaccine efficacy.
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    Brito, L. A.; O’Hagan, D. T. Designing and building the next generation of improved vaccine adjuvants. J. Controlled Release 2014, 190, 563– 579,  DOI: 10.1016/j.jconrel.2014.06.027
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    Designing and building the next generation of improved vaccine adjuvants
    Brito, Luis A.; O'Hagan, Derek T.
    Journal of Controlled Release (2014), 190 (), 563-579CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    A review. Vaccine adjuvants interact with the immune system, to increase the potency of vaccine antigens. Many of the adjuvants currently available were developed with little understanding of how they worked. Highly pure recombinant antigens are typically very poorly immunogenic due to a lack of exogenous immune activating components such as nucleic acids, lipids, and cell membrane components. In this review we discuss the role of adjuvants and their role as 'delivery systems' or 'immune potentiators'. We also highlight the need for appropriate delivery of immune potentiators with several 'delivery system' adjuvants such as alum, emulsions, liposomes, and polymeric particles. The challenges faced by vaccinologists to create the next generation of vaccines can be solved in-part by developing a greater understanding of the impact of delivery, and an appreciation of the key role of pharmaceutical sciences.
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    Irvine, D. J.; Hanson, M. C.; Rakhra, K.; Tokatlian, T. Synthetic Nanoparticles for Vaccines and Immunotherapy. Chem. Rev. 2015, 115, 11109– 11146,  DOI: 10.1021/acs.chemrev.5b00109
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    Synthetic Nanoparticles for Vaccines and Immunotherapy
    Irvine, Darrell J.; Hanson, Melissa C.; Rakhra, Kavya; Tokatlian, Talar
    Chemical Reviews (Washington, DC, United States) (2015), 115 (19), 11109-11146CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
    A review. Topics reviewed include immune targeting and delivery of vaccines and antigens by nanoparticles; and nanoparticles as adjuvants.
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    Morales-Hernández, S.; Ugidos-Damboriena, N.; López-Sagaseta, J. Self-Assembling Protein Nanoparticles in the Design of Vaccines: 2022 Update. Vaccines 2022, 10, 1447,  DOI: 10.3390/vaccines10091447
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    Lamontagne, F.; Khatri, V.; St-Louis, P.; Bourgault, S.; Archambault, D. Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds. Vaccines 2022, 10, 1920,  DOI: 10.3390/vaccines10111920
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    Vaccination Strategies Based on Bacterial Self-Assembling Proteins as Antigen Delivery Nanoscaffolds
    Lamontagne, Felix; Khatri, Vinay; St-Louis, Philippe; Bourgault, Steve; Archambault, Denis
    Vaccines (Basel, Switzerland) (2022), 10 (11), 1920CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)
    Vaccination has saved billions of human lives and has considerably reduced the economic burden assocd. with pandemic and endemic infectious diseases. Notwithstanding major advancements in recent decades, multitude diseases remain with no available effective vaccine. While subunit-based vaccines have shown great potential to address the safety concerns of live-attenuated vaccines, their limited immunogenicity remains a major drawback that still needs to be addressed for their use fighting infectious illnesses, autoimmune disorders, and/or cancer. Among the adjuvants and delivery systems for antigens, bacterial proteinaceous supramol. structures have recently received considerable attention. The use of bacterial proteins with self-assembling properties to deliver antigens offers several advantages, including biocompatibility, stability, mol. specificity, sym. organization, and multivalency. Bacterial protein nanoassemblies closely simulate most invading pathogens, acting as an alarm signal for the immune system to mount an effective adaptive immune response. Their nanoscale architecture can be precisely controlled at the at. level to produce a variety of nanostructures, allowing for infinite possibilities of organized antigen display. For the bottom-up design of the proteinaceous antigen delivery scaffolds, it is essential to understand how the structural and physicochem. properties of the nanoassemblies modulate the strength and polarization of the immune responses. The present review first describes the relationships between structure and the generated immune responses, before discussing potential and current clin. applications.
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    Montégut, L.; Chen, H.; Bravo-San Pedro, J. M.; Motiño, O.; Martins, I.; Kroemer, G. Immunization of mice with the self-peptide ACBP coupled to keyhole limpet hemocyanin. Star Protoc 2022, 3, 101095  DOI: 10.1016/j.xpro.2021.101095
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    Doucet, M.; El-Turabi, A.; Zabel, F.; Hunn, B. H. M.; Bengoa-Vergniory, N.; Cioroch, M.; Ramm, M.; Smith, A. M.; Gomes, A. C.; Cabral de Miranda, G.; Wade-Martins, R.; Bachmann, M. F.; Kahle, P. J. Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles. PLoS One 2017, 12, e0181844  DOI: 10.1371/journal.pone.0181844
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    Kanekiyo, M.; Wei, C. J.; Yassine, H. M.; McTamney, P. M.; Boyington, J. C.; Whittle, J. R. R.; Rao, S. S.; Kong, W. P.; Wang, L. S.; Nabel, G. J. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013, 499, 102,  DOI: 10.1038/nature12202
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    Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies
    Kanekiyo, Masaru; Wei, Chih-Jen; Yassine, Hadi M.; McTamney, Patrick M.; Boyington, Jeffrey C.; Whittle, James R. R.; Rao, Srinivas S.; Kong, Wing-Pui; Wang, Lingshu; Nabel, Gary J.
    Nature (London, United Kingdom) (2013), 499 (7456), 102-106CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technol. and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here the authors use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited hemagglutination inhibition antibody titers more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
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    Alam, S. M.; Aussedat, B.; Vohra, Y.; Meyerhoff, R. R.; Cale, E. M.; Walkowicz, W. E.; Radakovich, N. A.; Anasti, K.; Armand, L.; Parks, R.; Sutherland, L.; Scearce, R.; Joyce, M. G.; Pancera, M.; Druz, A.; Georgiev, I. S.; Von Holle, T.; Eaton, A.; Fox, C.; Reed, S. G.; Louder, M.; Bailer, R. T.; Morris, L.; Abdool-Karim, S. S.; Cohen, M.; Liao, H. X.; Montefiori, D. C.; Park, P. K.; Fernández-Tejada, A.; Wiehe, K.; Santra, S.; Kepler, T. B.; Saunders, K. O.; Sodroski, J.; Kwong, P. D.; Mascola, J. R.; Bonsignori, M.; Moody, M. A.; Danishefsky, S.; Haynes, B. F. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide. Sci. Transl Med. 2017, 9, eaai7521  DOI: 10.1126/scitranslmed.aai7521
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    Jefferis, R. Posttranslational Modifications and the Immunogenicity of Biotherapeutics. J. Immunol. Res. 2016, 2016, 5358272  DOI: 10.1155/2016/5358272
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    Posttranslational Modifications and the Immunogenicity of Biotherapeutics
    Jefferis Roy
    Journal of immunology research (2016), 2016 (), 5358272 ISSN:.
    Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.
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    Li, W.; Li, F.; Zhang, X.; Lin, H.-K.; Xu, C. Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduction Targeted Ther. 2021, 6, 422,  DOI: 10.1038/s41392-021-00825-8
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    Viegas, C.; Seck, F.; Fonte, P. An insight on lipid nanoparticles for therapeutic proteins delivery. J. Drug Delivery Sci. Technol. 2022, 77, 103839  DOI: 10.1016/j.jddst.2022.103839
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    Gouveia, M. G.; Wesseler, J. P.; Ramaekers, J.; Weder, C.; Scholten, P. B. V.; Bruns, N. Polymersome-based protein drug delivery – quo vadis?. Chem. Soc. Rev. 2023, 52, 728– 778,  DOI: 10.1039/D2CS00106C
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    Scaletti, F.; Hardie, J.; Lee, Y. W.; Luther, D. C.; Ray, M.; Rotello, V. M. Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies. Chem. Soc. Rev. 2018, 47, 3421– 3432,  DOI: 10.1039/C8CS00008E
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    Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies
    Scaletti, Federica; Hardie, Joseph; Lee, Yi-Wei; Luther, David C.; Ray, Moumita; Rotello, Vincent M.
    Chemical Society Reviews (2018), 47 (10), 3421-3432CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
    The delivery of proteins into cells is a potential game changer for a wide array of therapeutic purposes, including cancer therapy, immunomodulation and treatment of inherited diseases. In this review, we present recently developed nanoassemblies for protein delivery that utilize strategies that range from direct assembly, encapsulation and composite formation. We will discuss factors that affect the efficacy of nanoassemblies for delivery from the perspective of both nanoparticles and proteins. Challenges in the field, particularly achieving effective cytosolar protein delivery through endosomal escape or evasion are discussed.
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  27. 27
    Skoulas, D.; Fattah, S.; Wang, D.; Cryan, S.; Heise, A. Systematic Study of Enzymatic Degradation and Plasmid DNA Complexation of Mucus Penetrating Star-Shaped Lysine/Sarcosine Polypept(o)ides with Different Block Arrangements. Macromol. Biosci. 2022, 22, 2200175  DOI: 10.1002/mabi.202200175
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    England, R. M.; Moss, J. I.; Gunnarsson, A.; Parker, J. S.; Ashford, M. B. Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications. Biomacromolecules 2020, 21, 3332– 3341,  DOI: 10.1021/acs.biomac.0c00768
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    28
    Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications
    England, Richard M.; Moss, Jennifer I.; Gunnarsson, Anders; Parker, Jeremy S.; Ashford, Marianne B.
    Biomacromolecules (2020), 21 (8), 3332-3341CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymn. of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were obsd., representing ideal properties for biophys. targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic mol. SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body wt. loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chem. moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufg. perspective and report characterization of the polymers with a variety of techniques.
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  29. 29
    Mehta, D.; Leong, N.; McLeod, V. M.; Kelly, B. D.; Pathak, R.; Owen, D. J.; Porter, C. J. H.; Kaminskas, L. M. Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker. Mol. Pharmaceut 2018, 15, 4568– 4576,  DOI: 10.1021/acs.molpharmaceut.8b00581
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    Chavoustie, S. E.; Carter, B. A.; Waldbaum, A. S.; Donders, G. G. G.; Peters, K. H.; Schwebke, J. R.; Paull, J. R. A.; Price, C. F.; Castellarnau, A.; McCloud, P. Two phase 3, double-blind, placebo-controlled studies of the efficacy and safety of Astodrimer 1% Gel for the treatment of bacterial vaginosis. Eur. J. Obstet Gynecol Reprod Biol. 2020, 245, 13– 18,  DOI: 10.1016/j.ejogrb.2019.11.032
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    Moscicki, A. B.; Kaul, R.; Ma, Y.; Scott, M. E.; Daud, I. I.; Bukusi, E. A.; Shiboski, S.; Rebbapragada, A.; Huibner, S.; Cohen, C. R. Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% StarPharma LTD 7013 gel (VivaGel) to assess expanded safety. J. Acquir Immune Defic Syndr 2012, 59, 134– 40,  DOI: 10.1097/QAI.0b013e31823f2aeb
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    Measurement of Mucosal Biomarkers in a Phase 1 Trial of Intravaginal 3% StarPharma LTD 7013 Gel (VivaGel) to Assess Expanded Safety
    Moscicki, Anna-Barbara; Kaul, Rupert; Ma, Yifei; Scott, Mark E.; Daud, Ibrahim I.; Bukusi, Elizabeth A.; Shiboski, Stephen; Rebbapragada, Anuradha; Huibner, Sanja; Cohen, Craig R.
    JAIDS, Journal of Acquired Immune Deficiency Syndromes (2012), 59 (2), 134-140CODEN: JJASFJ; ISSN:1525-4135. (Lippincott Williams & Wilkins)
    Objective: The aim of this study was to examine the effect of the 3% StarPharma LTD 7013 gel (VivaGel) on mucosal immune markers hypothesized to be assocd. with HIV-1 acquisition. Design: Phase 1, placebo-controlled, randomized, double-blind clin. trial was performed in 54 young women in the United States and Kenya. Participants used carbopol gel with and without (placebo) StarPharma LTD 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T cells, and dendritic cells at days 0, 7, 14, and 21. A neg. binomial regression model was used to assess differences between study arms. Results: Several mucosal immune parameters were increased in the VivaGel arm compared with placebo. For cytokines D7, IL-6 (P = 0.05); D 14, interferon gamma (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.001) were increased. On D7, CD8+/CD69+ T cells tended to be increased (P < 0.08); limiting anal. to visits without blood or bacterial vaginosis, these findings were stronger as follows: at D7, CD8+/CD69+ T cells were increased in the VivaGel arm (P < 0.005), as were CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01). The changes described for D7 and 14 were no longer seen at D21. Conclusions: Markers assocd. with inflammation and epithelial damage were reversibly elevated in the VivaGel arm compared with the placebo arm after 7-14 days of twice daily product use.
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  32. 32
    Mignani, S.; Shi, X.; Rodrigues, J.; Tomas, H.; Karpus, A.; Majoral, J. P. First-in-class and best-in-class dendrimer nanoplatforms from concept to clinic: Lessons learned moving forward. Eur. J. Med. Chem. 2021, 219, 113456  DOI: 10.1016/j.ejmech.2021.113456
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    First-in-class and best-in-class dendrimer nanoplatforms from concept to clinic: Lessons learned moving forward
    Mignani, Serge; Shi, Xangyang; Rodrigues, Joao; Tomas, Helena; Karpus, Andrii; Majoral, Jean-Pierre
    European Journal of Medicinal Chemistry (2021), 219 (), 113456CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
    A review. Research to develop active dendrimers by themselves or as nanocarriers represents a promising approach to discover new biol. active entities that can be used to tackle unmet medical needs including difficult diseases. These developments are possible due to the exceptional physicochem. properties of dendrimers, including their biocompatibility, as well as their therapeutic activity as nanocarriers and drugs themselves. Despite a large no. of academic studies, very few dendrimers have crossed the 'valley of death' between. Only a few no. of pharmaceutical companies have succeeded in this way. In fact, only Starpharma (Australia) and Orpheris, Inc. (USA), an Ashvattha Therapeutics subsidiary, can fill all the clinic requirements to have in the market dendrimers based drugs/nancocarriers. After evaluating the main physicochem. properties related to the resp. biol. activity of dendrimers classified as first-in-class or best-in-class in nanomedicine, this original review analyzes the advantages and disadvantages of these two strategies as well the concerns to step in clin. phases. Various solns. are proposed to advance the use of dendrimers in human health.
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  33. 33
    Francica, J. R.; Laga, R.; Lynn, G. M.; Mužíková, G.; Androvič, L.; Aussedat, B.; Walkowicz, W. E.; Padhan, K.; Ramirez-Valdez, R. A.; Parks, R.; Schmidt, S. D.; Flynn, B. J.; Tsybovsky, Y.; Stewart-Jones, G. B. E.; Saunders, K. O.; Baharom, F.; Petrovas, C.; Haynes, B. F.; Seder, R. A.; Moon, J. J. Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates. Plos Biol. 2019, 17, e3000328  DOI: 10.1371/journal.pbio.3000328
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    Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates
    Francica, Joseph R.; Lagaid, Richard; Lynn, Geoffrey M.; Muzikova, Gabriela; Androvicid, Ladislav; Aussedatid, Baptiste; Walkowiczid, William E.; Padhan, Kartika; Ramirez-Valdez, Ramiro Andrei; Parks, Robert; Schmidt, Stephen D.; Flynn, Barbara J.; Tsybovsky, Yaroslav; Stewart-Jones, Guillaume B. E.; Saunders, Kevin O.; Baharomid, Faezzah; Petrovas, Constantinos; Haynes, Barton F.; Seder, Robert A.
    PLoS Biology (2019), 17 (6), e3000328CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)
    Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the phys. characteristics of peptide immunogens can limit their pharmacokinetic and immunol. properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 h following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen d. with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in sep. nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/ glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not obsd. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
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    Tong, W. Y.; Maira, M.; Roychoudhury, R.; Galan, A.; Brahimi, F.; Gilbert, M.; Cunningham, A. M.; Josephy, S.; Pirvulescu, I.; Moffett, S. Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy. Cell Chem. Biol. 2019, 26, 1013,  DOI: 10.1016/j.chembiol.2019.03.018
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    Fera, D.; Lee, M. S.; Wiehe, K.; Meyerhoff, R. R.; Piai, A.; Bonsignori, M.; Aussedat, B.; Walkowicz, W. E.; Ton, T.; Zhou, J. O.; Danishefsky, S.; Haynes, B. F.; Harrison, S. C. HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope. Nat. Commun. 2018, 9, 1111,  DOI: 10.1038/s41467-018-03565-6
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    HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope
    Fera Daniela; Lee Matthew S; Harrison Stephen C; Wiehe Kevin; Bonsignori Mattia; Haynes Barton F; Wiehe Kevin; Meyerhoff R Ryan; Bonsignori Mattia; Haynes Barton F; Meyerhoff R Ryan; Piai Alessandro; Aussedat Baptiste; Walkowicz William E; Danishefsky Samuel; Ton Therese; Zhou Jeffrey O; Harrison Stephen C
    Nature communications (2018), 9 (1), 1111 ISSN:.
    HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.
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  36. 36
    Williams, W. B.; Meyerhoff, R. R.; Edwards, R. J.; Li, H.; Manne, K.; Nicely, N. I.; Henderson, R.; Zhou, Y.; Janowska, K.; Mansouri, K. Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies. Cell 2021, 184, 2955,  DOI: 10.1016/j.cell.2021.04.042
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    Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies
    Williams, Wilton B.; Meyerhoff, R. Ryan; Edwards, R. J.; Li, Hui; Manne, Kartik; Nicely, Nathan I.; Henderson, Rory; Zhou, Ye; Janowska, Katarzyna; Mansouri, Katayoun; Gobeil, Sophie; Evangelous, Tyler; Hora, Bhavna; Berry, Madison; Abuahmad, A. Yousef; Sprenz, Jordan; Deyton, Margaret; Stalls, Victoria; Kopp, Megan; Hsu, Allen L.; Borgnia, Mario J.; Stewart-Jones, Guillaume B. E.; Lee, Matthew S.; Bronkema, Naomi; Moody, M. Anthony; Wiehe, Kevin; Bradley, Todd; Alam, S. Munir; Parks, Robert J.; Foulger, Andrew; Oguin, Thomas; Sempowski, Gregory D.; Bonsignori, Mattia; LaBranche, Celia C.; Montefiori, David C.; Seaman, Michael; Santra, Sampa; Perfect, John; Francica, Joseph R.; Lynn, Geoffrey M.; Aussedat, Baptiste; Walkowicz, William E.; Laga, Richard; Kelsoe, Garnett; Saunders, Kevin O.; Fera, Daniela; Kwong, Peter D.; Seder, Robert A.; Bartesaghi, Alberto; Shaw, George M.; Acharya, Priyamvada; Haynes, Barton F.
    Cell (Cambridge, MA, United States) (2021), 184 (11), 2955-2972.e25CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geog. diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
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    Manolova, V.; Flace, A.; Bauer, M.; Schwarz, K.; Saudan, P.; Bachmann, M. F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008, 38, 1404– 1413,  DOI: 10.1002/eji.200737984
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    Nanoparticles target distinct dendritic cell populations according to their size
    Manolova, Vania; Flace, Anna; Bauer, Monika; Schwarz, Katrin; Saudan, Philippe; Bachmann, Martin F.
    European Journal of Immunology (2008), 38 (5), 1404-1413CODEN: EJIMAF; ISSN:0014-2980. (Wiley-VCH Verlag GmbH & Co. KGaA)
    The efficiency of a vaccine largely depends on the appropriate targeting of the innate immune system, mainly through prolonged delivery of antigens and immunomodulatory substances to professional antigen-presenting cells in the lymphoid environment. Particulate antigens, such as virus-like particles (VLP) induce potent immune responses. However, little is known about the relative importance of direct drainage of free antigen to lymph nodes (LN) vs. cellular transport and the impact of particle size on the process. Here, we show that nanoparticles traffic to the draining LN in a size-dependent manner. Whereas large particles (500-2000 nm) were mostly assocd. with dendritic cells (DC) from the injection site, small (20-200 nm) nanoparticles and VLP (30 nm) were also found in LN-resident DC and macrophages, suggesting free drainage of these particles to the LN. In vivo imaging studies in mice conditionally depleted of DC confirmed the capacity of small but not large particles to drain freely to the LN and demonstrated that DC are strictly required for transport of large particles from the injection site to the LN. These data provide evidence that particle size dets. the mechanism of trafficking to the LN and show that only small nanoparticles can specifically target LN-resident cells.
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    Reddy, S. T.; Rehor, A.; Schmoekel, H. G.; Hubbell, J. A.; Swartz, M. A. In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles. J. Controlled Release 2006, 112, 26– 34,  DOI: 10.1016/j.jconrel.2006.01.006
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    In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles
    Reddy, Sai T.; Rehor, Annemie; Schmoekel, Hugo G.; Hubbell, Jeffrey A.; Swartz, Melody A.
    Journal of Controlled Release (2006), 112 (1), 26-34CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
    Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100 nm diam. poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 Nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-contg. DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.
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    Stano, A.; Nembrini, C.; Swartz, M. A.; Hubbell, J. A.; Simeoni, E. Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization. Vaccine 2012, 30, 7541– 7546,  DOI: 10.1016/j.vaccine.2012.10.050
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    Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization
    Stano, Armando; Nembrini, Chiara; Swartz, Melody A.; Hubbell, Jeffrey A.; Simeoni, Eleonora
    Vaccine (2012), 30 (52), 7541-7546CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)
    The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinol., being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants.To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice.We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4+ T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4+ T cells as compared to 30 nm NPs.The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4+ T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.
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    Lyu, Z.; Ding, L.; Huang, A. Y. T.; Kao, C. L.; Peng, L. Poly(amidoamine) dendrimers: covalent and supramolecular synthesis. Mater. Today Chem. 2019, 13, 34– 48,  DOI: 10.1016/j.mtchem.2019.04.004
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    Poly(amidoamine) dendrimers: covalent and supramolecular synthesis
    Lyu, Z.; Ding, L.; Huang, A. Y. T.; Kao, C.-L.; Peng, L.
    Materials Today Chemistry (2019), 13 (), 34-48CODEN: MTCAD8; ISSN:2468-5194. (Elsevier Ltd.)
    A review. Dendrimers, a class of synthetic macromols. which are notable for their well-defined ramified structures and unique multivalent cooperativity, hold great promise for developing various functional materials. Among all the reported dendrimers, poly(amidoamine) (PAMAM) dendrimers are the most extensively studied by virtue of their readily availability via robust synthesis as well as their dendritic structure and peptide/protein mimic features. Since the seminal report by Tomalia et al., various strategies have been made available for PAMAM dendrimers, including divergent and/or convergent synthesis alongside click chem. Nevertheless, prepn. of high-generation and defect-free PAMAM dendrimers on a large scale remains challenging. To overcome the limitations, an alternative strategy based on self-assembling approach has emerged for dendrimer synthesis, where small dendritic components form large non-covalent supramol. structures that mimic high-generation covalent dendrimers. This approach is easy to implement in practice and requires much less synthetic effort. Here, we present a brief overview of the different approaches established for PAMAM dendrimer synthesis. We start with a general introduction to dendrimers and the common strategies for dendrimer synthesis, and then we illustrate the specific approaches for PAMAM dendrimer synthesis and highlight the related advantages and limitations using representative examples. Although various strategies have been established for PAMAM dendrimer synthesis, innovative concepts and approaches are still in high demand for reliably prepg. defect-free and high-generation dendrimers in large quantity.
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    Ulbrich, K.; Subr, V.; Strohalm, J.; Plocová, D.; Jelínková, M.; Ríhová, B. Polymeric drugs based on conjugates of synthetic and natural macromolecules I.: Synthesis and physico-chemical characterisation. J. Controlled Release 2000, 64, 63– 79,  DOI: 10.1016/S0168-3659(99)00141-8
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    Tao, L.; Liu, J. Q.; Xu, J. T.; Davis, T. P. Synthesis and bioactivity of poly(HPMA)-lysozyme conjugates: the use of novel thiazolidine-2-thione coupling chemistry. Org. Biomol Chem. 2009, 7, 3481– 3485,  DOI: 10.1039/b907061c
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    Subr, V.; Kostka, L.; Strohalm, J.; Etrych, T.; Ulbrich, K. Synthesis of Well-Defined Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] Polymers with Functional Group at the α-End of the Polymer Chain by RAFT Polymerization. Macromolecules 2013, 46, 2100– 2108,  DOI: 10.1021/ma400042u
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    Synthesis of Well-Defined Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] Polymers with Functional Group at the α-End of the Polymer Chain by RAFT Polymerization
    Subr, V.; Kostka, L.; Strohalm, J.; Etrych, T.; Ulbrich, K.
    Macromolecules (Washington, DC, United States) (2013), 46 (6), 2100-2108CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
    N-(2-Hydroxypropyl)methacrylamide polymer precursors (pHPMA) with narrow distribution of mol. wts. and polymer chain terminating in a single reactive group have big potential in the synthesis of various polymer drug and gene delivery systems. This paper shows that pHPMA can be prepd. by reversible addn.-fragmentation chain transfer (RAFT) polymn.; the pHPMAs prepd. by RAFT polymn. conducted to high conversions contained at the α-end of the polymer chain a single functional group originated from both, the chain transfer agent (CTA) and initiator (INI) in ratio depending on polymn. conditions. The well-defined monofunctional pHPMAs with narrow mol. wt. distribution and with single reactive group situated at the α-polymer chain end can be prepd. in one step by RAFT polymn. initiated by the tailor-made CTA and INI, both contg. the same functional group in their structure. Synthesis of pHPMAs terminating in various reactive groups (azide, propargyl, thiazolidin-2-thione, amino, hydrazide) was also described.
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  • Abstract

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    Scheme 1

    Scheme 1. Cartoon Depiction (A) and Reaction Scheme (B) for the Synthesis of Star Copolymers (II) Composed of the PAMAM Dendrimer Core and Heterobifunctional HPMA-Based Arms (I)
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    Figure 1

    Figure 1. Influence of PAMAM dendrimer generation and molar ratio of the PAMAM surface. ̃NH2 groups to the ̃TT terminal group on the polymer arms on the gyration radius (Rg) and number (#) of polymer arms of star copolymers.

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    Figure 2

    Figure 2. Examples of ultrahigh-resolution images of star copolymers S17: (A–E) STEM analysis of star copolymers. Indication of the inner arrangement is viewed. The differences between the images are due to the different distribution of Au11 nanoparticles in the sample. (F) Cluster of 8 free Au-based nanoparticles, each with a diameter of 0.8 nm, corresponding to the Au11.

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    Scheme 2

    Scheme 2. Cartoon Depiction (A) and Reaction Scheme (B) for the Preparation of Star Copolymer Vaccines (II) Synthesized by Conjugation of Man9V3 Glycopeptide to Star Copolymers (I)
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    Figure 3

    Figure 3. Ultrahigh-resolution micrographs of unlabeled star copolymer vaccine construct with ∼70 kg·mol–1 polymer arms obtained by ESEM. The overall image at the top left shows the arrangement of star copolymer vaccine of similar shapes and sizes, while the remaining images show a zoomed-in view of individual ∼50 nm star copolymer vaccines emphasizing their spherical shape and entangled structure of the polymer coils.

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    Figure 4

    Figure 4. Antibody responses following vaccination with different star copolymer vaccine compositions, free (unformulated Man9V3) or naïve control. Balb/c mice (n = 5/group) were immunized at days 0, 28, and 56 and serum was collected on day 70 and assessed for anti-Man9V3 IgG antibodies by enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± standard deviation end point titer. Differences between each experimental group and the naïve control were assessed for statistical significance using one-way ANOVA with Bonferroni correction for multiple comparisons; asterisks (*) indicate statistical significance (P < 0.05) between the indicated group and naïve.

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      There is no corresponding record for this reference.
    19. 19
      Doucet, M.; El-Turabi, A.; Zabel, F.; Hunn, B. H. M.; Bengoa-Vergniory, N.; Cioroch, M.; Ramm, M.; Smith, A. M.; Gomes, A. C.; Cabral de Miranda, G.; Wade-Martins, R.; Bachmann, M. F.; Kahle, P. J. Preclinical development of a vaccine against oligomeric alpha-synuclein based on virus-like particles. PLoS One 2017, 12, e0181844  DOI: 10.1371/journal.pone.0181844
      There is no corresponding record for this reference.
    20. 20
      Kanekiyo, M.; Wei, C. J.; Yassine, H. M.; McTamney, P. M.; Boyington, J. C.; Whittle, J. R. R.; Rao, S. S.; Kong, W. P.; Wang, L. S.; Nabel, G. J. Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies. Nature 2013, 499, 102,  DOI: 10.1038/nature12202
      20
      Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies
      Kanekiyo, Masaru; Wei, Chih-Jen; Yassine, Hadi M.; McTamney, Patrick M.; Boyington, Jeffrey C.; Whittle, James R. R.; Rao, Srinivas S.; Kong, Wing-Pui; Wang, Lingshu; Nabel, Gary J.
      Nature (London, United Kingdom) (2013), 499 (7456), 102-106CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technol. and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here the authors use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited hemagglutination inhibition antibody titers more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.
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    21. 21
      Alam, S. M.; Aussedat, B.; Vohra, Y.; Meyerhoff, R. R.; Cale, E. M.; Walkowicz, W. E.; Radakovich, N. A.; Anasti, K.; Armand, L.; Parks, R.; Sutherland, L.; Scearce, R.; Joyce, M. G.; Pancera, M.; Druz, A.; Georgiev, I. S.; Von Holle, T.; Eaton, A.; Fox, C.; Reed, S. G.; Louder, M.; Bailer, R. T.; Morris, L.; Abdool-Karim, S. S.; Cohen, M.; Liao, H. X.; Montefiori, D. C.; Park, P. K.; Fernández-Tejada, A.; Wiehe, K.; Santra, S.; Kepler, T. B.; Saunders, K. O.; Sodroski, J.; Kwong, P. D.; Mascola, J. R.; Bonsignori, M.; Moody, M. A.; Danishefsky, S.; Haynes, B. F. Mimicry of an HIV broadly neutralizing antibody epitope with a synthetic glycopeptide. Sci. Transl Med. 2017, 9, eaai7521  DOI: 10.1126/scitranslmed.aai7521
      There is no corresponding record for this reference.
    22. 22
      Jefferis, R. Posttranslational Modifications and the Immunogenicity of Biotherapeutics. J. Immunol. Res. 2016, 2016, 5358272  DOI: 10.1155/2016/5358272
      22
      Posttranslational Modifications and the Immunogenicity of Biotherapeutics
      Jefferis Roy
      Journal of immunology research (2016), 2016 (), 5358272 ISSN:.
      Whilst the amino acid sequence of a protein is determined by its gene sequence, the final structure and function are determined by posttranslational modifications (PTMs), including quality control (QC) in the endoplasmic reticulum (ER) and during passage through the Golgi apparatus. These processes are species and cell specific and challenge the biopharmaceutical industry when developing a production platform for the generation of recombinant biologic therapeutics. Proteins and glycoproteins are also subject to chemical modifications (CMs) both in vivo and in vitro. The individual is naturally tolerant to molecular forms of self-molecules but nonself variants can provoke an immune response with the generation of anti-drug antibodies (ADA); aggregated forms can exhibit enhanced immunogenicity and QC procedures are developed to avoid or remove them. Monoclonal antibody therapeutics (mAbs) are a special case because their purpose is to bind the target, with the formation of immune complexes (ICs), a particular form of aggregate. Such ICs may be removed by phagocytic cells that have antigen presenting capacity. These considerations may frustrate the possibility of ameliorating the immunogenicity of mAbs by rigorous exclusion of aggregates from drug product. Alternate strategies for inducing immunosuppression or tolerance are discussed.
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    23. 23
      Li, W.; Li, F.; Zhang, X.; Lin, H.-K.; Xu, C. Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduction Targeted Ther. 2021, 6, 422,  DOI: 10.1038/s41392-021-00825-8
      There is no corresponding record for this reference.
    24. 24
      Viegas, C.; Seck, F.; Fonte, P. An insight on lipid nanoparticles for therapeutic proteins delivery. J. Drug Delivery Sci. Technol. 2022, 77, 103839  DOI: 10.1016/j.jddst.2022.103839
      There is no corresponding record for this reference.
    25. 25
      Gouveia, M. G.; Wesseler, J. P.; Ramaekers, J.; Weder, C.; Scholten, P. B. V.; Bruns, N. Polymersome-based protein drug delivery – quo vadis?. Chem. Soc. Rev. 2023, 52, 728– 778,  DOI: 10.1039/D2CS00106C
      There is no corresponding record for this reference.
    26. 26
      Scaletti, F.; Hardie, J.; Lee, Y. W.; Luther, D. C.; Ray, M.; Rotello, V. M. Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies. Chem. Soc. Rev. 2018, 47, 3421– 3432,  DOI: 10.1039/C8CS00008E
      26
      Protein delivery into cells using inorganic nanoparticle-protein supramolecular assemblies
      Scaletti, Federica; Hardie, Joseph; Lee, Yi-Wei; Luther, David C.; Ray, Moumita; Rotello, Vincent M.
      Chemical Society Reviews (2018), 47 (10), 3421-3432CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)
      The delivery of proteins into cells is a potential game changer for a wide array of therapeutic purposes, including cancer therapy, immunomodulation and treatment of inherited diseases. In this review, we present recently developed nanoassemblies for protein delivery that utilize strategies that range from direct assembly, encapsulation and composite formation. We will discuss factors that affect the efficacy of nanoassemblies for delivery from the perspective of both nanoparticles and proteins. Challenges in the field, particularly achieving effective cytosolar protein delivery through endosomal escape or evasion are discussed.
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    27. 27
      Skoulas, D.; Fattah, S.; Wang, D.; Cryan, S.; Heise, A. Systematic Study of Enzymatic Degradation and Plasmid DNA Complexation of Mucus Penetrating Star-Shaped Lysine/Sarcosine Polypept(o)ides with Different Block Arrangements. Macromol. Biosci. 2022, 22, 2200175  DOI: 10.1002/mabi.202200175
      There is no corresponding record for this reference.
    28. 28
      England, R. M.; Moss, J. I.; Gunnarsson, A.; Parker, J. S.; Ashford, M. B. Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications. Biomacromolecules 2020, 21, 3332– 3341,  DOI: 10.1021/acs.biomac.0c00768
      28
      Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications
      England, Richard M.; Moss, Jennifer I.; Gunnarsson, Anders; Parker, Jeremy S.; Ashford, Marianne B.
      Biomacromolecules (2020), 21 (8), 3332-3341CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymn. of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were obsd., representing ideal properties for biophys. targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic mol. SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body wt. loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chem. moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufg. perspective and report characterization of the polymers with a variety of techniques.
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    29. 29
      Mehta, D.; Leong, N.; McLeod, V. M.; Kelly, B. D.; Pathak, R.; Owen, D. J.; Porter, C. J. H.; Kaminskas, L. M. Reducing Dendrimer Generation and PEG Chain Length Increases Drug Release and Promotes Anticancer Activity of PEGylated Polylysine Dendrimers Conjugated with Doxorubicin via a Cathepsin-Cleavable Peptide Linker. Mol. Pharmaceut 2018, 15, 4568– 4576,  DOI: 10.1021/acs.molpharmaceut.8b00581
      There is no corresponding record for this reference.
    30. 30
      Chavoustie, S. E.; Carter, B. A.; Waldbaum, A. S.; Donders, G. G. G.; Peters, K. H.; Schwebke, J. R.; Paull, J. R. A.; Price, C. F.; Castellarnau, A.; McCloud, P. Two phase 3, double-blind, placebo-controlled studies of the efficacy and safety of Astodrimer 1% Gel for the treatment of bacterial vaginosis. Eur. J. Obstet Gynecol Reprod Biol. 2020, 245, 13– 18,  DOI: 10.1016/j.ejogrb.2019.11.032
      There is no corresponding record for this reference.
    31. 31
      Moscicki, A. B.; Kaul, R.; Ma, Y.; Scott, M. E.; Daud, I. I.; Bukusi, E. A.; Shiboski, S.; Rebbapragada, A.; Huibner, S.; Cohen, C. R. Measurement of mucosal biomarkers in a phase 1 trial of intravaginal 3% StarPharma LTD 7013 gel (VivaGel) to assess expanded safety. J. Acquir Immune Defic Syndr 2012, 59, 134– 40,  DOI: 10.1097/QAI.0b013e31823f2aeb
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      Measurement of Mucosal Biomarkers in a Phase 1 Trial of Intravaginal 3% StarPharma LTD 7013 Gel (VivaGel) to Assess Expanded Safety
      Moscicki, Anna-Barbara; Kaul, Rupert; Ma, Yifei; Scott, Mark E.; Daud, Ibrahim I.; Bukusi, Elizabeth A.; Shiboski, Stephen; Rebbapragada, Anuradha; Huibner, Sanja; Cohen, Craig R.
      JAIDS, Journal of Acquired Immune Deficiency Syndromes (2012), 59 (2), 134-140CODEN: JJASFJ; ISSN:1525-4135. (Lippincott Williams & Wilkins)
      Objective: The aim of this study was to examine the effect of the 3% StarPharma LTD 7013 gel (VivaGel) on mucosal immune markers hypothesized to be assocd. with HIV-1 acquisition. Design: Phase 1, placebo-controlled, randomized, double-blind clin. trial was performed in 54 young women in the United States and Kenya. Participants used carbopol gel with and without (placebo) StarPharma LTD 7013 twice daily over 14 days. Cervical specimens were collected for cytokines, chemokines, T cells, and dendritic cells at days 0, 7, 14, and 21. A neg. binomial regression model was used to assess differences between study arms. Results: Several mucosal immune parameters were increased in the VivaGel arm compared with placebo. For cytokines D7, IL-6 (P = 0.05); D 14, interferon gamma (P = 0.03), IL-2 (P = 0.04), IL-5 (P = 0.003), and IL-10 (P = 0.001) were increased. On D7, CD8+/CD69+ T cells tended to be increased (P < 0.08); limiting anal. to visits without blood or bacterial vaginosis, these findings were stronger as follows: at D7, CD8+/CD69+ T cells were increased in the VivaGel arm (P < 0.005), as were CD4+/CD69+ cells (P = 0.001) and CD4+/CCR5+ T cells (P = 0.01). The changes described for D7 and 14 were no longer seen at D21. Conclusions: Markers assocd. with inflammation and epithelial damage were reversibly elevated in the VivaGel arm compared with the placebo arm after 7-14 days of twice daily product use.
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    32. 32
      Mignani, S.; Shi, X.; Rodrigues, J.; Tomas, H.; Karpus, A.; Majoral, J. P. First-in-class and best-in-class dendrimer nanoplatforms from concept to clinic: Lessons learned moving forward. Eur. J. Med. Chem. 2021, 219, 113456  DOI: 10.1016/j.ejmech.2021.113456
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      First-in-class and best-in-class dendrimer nanoplatforms from concept to clinic: Lessons learned moving forward
      Mignani, Serge; Shi, Xangyang; Rodrigues, Joao; Tomas, Helena; Karpus, Andrii; Majoral, Jean-Pierre
      European Journal of Medicinal Chemistry (2021), 219 (), 113456CODEN: EJMCA5; ISSN:0223-5234. (Elsevier Masson SAS)
      A review. Research to develop active dendrimers by themselves or as nanocarriers represents a promising approach to discover new biol. active entities that can be used to tackle unmet medical needs including difficult diseases. These developments are possible due to the exceptional physicochem. properties of dendrimers, including their biocompatibility, as well as their therapeutic activity as nanocarriers and drugs themselves. Despite a large no. of academic studies, very few dendrimers have crossed the 'valley of death' between. Only a few no. of pharmaceutical companies have succeeded in this way. In fact, only Starpharma (Australia) and Orpheris, Inc. (USA), an Ashvattha Therapeutics subsidiary, can fill all the clinic requirements to have in the market dendrimers based drugs/nancocarriers. After evaluating the main physicochem. properties related to the resp. biol. activity of dendrimers classified as first-in-class or best-in-class in nanomedicine, this original review analyzes the advantages and disadvantages of these two strategies as well the concerns to step in clin. phases. Various solns. are proposed to advance the use of dendrimers in human health.
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    33. 33
      Francica, J. R.; Laga, R.; Lynn, G. M.; Mužíková, G.; Androvič, L.; Aussedat, B.; Walkowicz, W. E.; Padhan, K.; Ramirez-Valdez, R. A.; Parks, R.; Schmidt, S. D.; Flynn, B. J.; Tsybovsky, Y.; Stewart-Jones, G. B. E.; Saunders, K. O.; Baharom, F.; Petrovas, C.; Haynes, B. F.; Seder, R. A.; Moon, J. J. Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates. Plos Biol. 2019, 17, e3000328  DOI: 10.1371/journal.pbio.3000328
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      Star nanoparticles delivering HIV-1 peptide minimal immunogens elicit near-native envelope antibody responses in nonhuman primates
      Francica, Joseph R.; Lagaid, Richard; Lynn, Geoffrey M.; Muzikova, Gabriela; Androvicid, Ladislav; Aussedatid, Baptiste; Walkowiczid, William E.; Padhan, Kartika; Ramirez-Valdez, Ramiro Andrei; Parks, Robert; Schmidt, Stephen D.; Flynn, Barbara J.; Tsybovsky, Yaroslav; Stewart-Jones, Guillaume B. E.; Saunders, Kevin O.; Baharomid, Faezzah; Petrovas, Constantinos; Haynes, Barton F.; Seder, Robert A.
      PLoS Biology (2019), 17 (6), e3000328CODEN: PBLIBG; ISSN:1545-7885. (Public Library of Science)
      Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the phys. characteristics of peptide immunogens can limit their pharmacokinetic and immunol. properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 h following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen d. with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in sep. nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/ glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not obsd. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
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    34. 34
      Tong, W. Y.; Maira, M.; Roychoudhury, R.; Galan, A.; Brahimi, F.; Gilbert, M.; Cunningham, A. M.; Josephy, S.; Pirvulescu, I.; Moffett, S. Vaccination with Tumor-Ganglioside Glycomimetics Activates a Selective Immunity that Affords Cancer Therapy. Cell Chem. Biol. 2019, 26, 1013,  DOI: 10.1016/j.chembiol.2019.03.018
      There is no corresponding record for this reference.
    35. 35
      Fera, D.; Lee, M. S.; Wiehe, K.; Meyerhoff, R. R.; Piai, A.; Bonsignori, M.; Aussedat, B.; Walkowicz, W. E.; Ton, T.; Zhou, J. O.; Danishefsky, S.; Haynes, B. F.; Harrison, S. C. HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope. Nat. Commun. 2018, 9, 1111,  DOI: 10.1038/s41467-018-03565-6
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      HIV envelope V3 region mimic embodies key features of a broadly neutralizing antibody lineage epitope
      Fera Daniela; Lee Matthew S; Harrison Stephen C; Wiehe Kevin; Bonsignori Mattia; Haynes Barton F; Wiehe Kevin; Meyerhoff R Ryan; Bonsignori Mattia; Haynes Barton F; Meyerhoff R Ryan; Piai Alessandro; Aussedat Baptiste; Walkowicz William E; Danishefsky Samuel; Ton Therese; Zhou Jeffrey O; Harrison Stephen C
      Nature communications (2018), 9 (1), 1111 ISSN:.
      HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.
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    36. 36
      Williams, W. B.; Meyerhoff, R. R.; Edwards, R. J.; Li, H.; Manne, K.; Nicely, N. I.; Henderson, R.; Zhou, Y.; Janowska, K.; Mansouri, K. Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies. Cell 2021, 184, 2955,  DOI: 10.1016/j.cell.2021.04.042
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      Fab-dimerized glycan-reactive antibodies are a structural category of natural antibodies
      Williams, Wilton B.; Meyerhoff, R. Ryan; Edwards, R. J.; Li, Hui; Manne, Kartik; Nicely, Nathan I.; Henderson, Rory; Zhou, Ye; Janowska, Katarzyna; Mansouri, Katayoun; Gobeil, Sophie; Evangelous, Tyler; Hora, Bhavna; Berry, Madison; Abuahmad, A. Yousef; Sprenz, Jordan; Deyton, Margaret; Stalls, Victoria; Kopp, Megan; Hsu, Allen L.; Borgnia, Mario J.; Stewart-Jones, Guillaume B. E.; Lee, Matthew S.; Bronkema, Naomi; Moody, M. Anthony; Wiehe, Kevin; Bradley, Todd; Alam, S. Munir; Parks, Robert J.; Foulger, Andrew; Oguin, Thomas; Sempowski, Gregory D.; Bonsignori, Mattia; LaBranche, Celia C.; Montefiori, David C.; Seaman, Michael; Santra, Sampa; Perfect, John; Francica, Joseph R.; Lynn, Geoffrey M.; Aussedat, Baptiste; Walkowicz, William E.; Laga, Richard; Kelsoe, Garnett; Saunders, Kevin O.; Fera, Daniela; Kwong, Peter D.; Seder, Robert A.; Bartesaghi, Alberto; Shaw, George M.; Acharya, Priyamvada; Haynes, Barton F.
      Cell (Cambridge, MA, United States) (2021), 184 (11), 2955-2972.e25CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geog. diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
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    37. 37
      Manolova, V.; Flace, A.; Bauer, M.; Schwarz, K.; Saudan, P.; Bachmann, M. F. Nanoparticles target distinct dendritic cell populations according to their size. Eur. J. Immunol. 2008, 38, 1404– 1413,  DOI: 10.1002/eji.200737984
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      Nanoparticles target distinct dendritic cell populations according to their size
      Manolova, Vania; Flace, Anna; Bauer, Monika; Schwarz, Katrin; Saudan, Philippe; Bachmann, Martin F.
      European Journal of Immunology (2008), 38 (5), 1404-1413CODEN: EJIMAF; ISSN:0014-2980. (Wiley-VCH Verlag GmbH & Co. KGaA)
      The efficiency of a vaccine largely depends on the appropriate targeting of the innate immune system, mainly through prolonged delivery of antigens and immunomodulatory substances to professional antigen-presenting cells in the lymphoid environment. Particulate antigens, such as virus-like particles (VLP) induce potent immune responses. However, little is known about the relative importance of direct drainage of free antigen to lymph nodes (LN) vs. cellular transport and the impact of particle size on the process. Here, we show that nanoparticles traffic to the draining LN in a size-dependent manner. Whereas large particles (500-2000 nm) were mostly assocd. with dendritic cells (DC) from the injection site, small (20-200 nm) nanoparticles and VLP (30 nm) were also found in LN-resident DC and macrophages, suggesting free drainage of these particles to the LN. In vivo imaging studies in mice conditionally depleted of DC confirmed the capacity of small but not large particles to drain freely to the LN and demonstrated that DC are strictly required for transport of large particles from the injection site to the LN. These data provide evidence that particle size dets. the mechanism of trafficking to the LN and show that only small nanoparticles can specifically target LN-resident cells.
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    38. 38
      Reddy, S. T.; Rehor, A.; Schmoekel, H. G.; Hubbell, J. A.; Swartz, M. A. In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles. J. Controlled Release 2006, 112, 26– 34,  DOI: 10.1016/j.jconrel.2006.01.006
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      In vivo targeting of dendritic cells in lymph nodes with poly(propylene sulfide) nanoparticles
      Reddy, Sai T.; Rehor, Annemie; Schmoekel, Hugo G.; Hubbell, Jeffrey A.; Swartz, Melody A.
      Journal of Controlled Release (2006), 112 (1), 26-34CODEN: JCREEC; ISSN:0168-3659. (Elsevier B.V.)
      Delivery of biodegradable nanoparticles to antigen-presenting cells (APCs), specifically dendritic cells (DCs), has potential for immunotherapy. This study investigates the delivery of 20, 45, and 100 nm diam. poly(ethylene glycol)-stabilized poly(propylene sulfide) (PPS) nanoparticles to DCs in the lymph nodes. These nanoparticles consist of a cross-linked rubbery core of PPS surrounded by a hydrophilic corona of poly(ethylene glycol). The PPS domain is capable of carrying hydrophobic drugs and degrades within oxidative environments. 20 Nm particles were most readily taken up into lymphatics following interstitial injection, while both 20 and 45 nm nanoparticles showed significant retention in lymph nodes, displaying a consistent and strong presence at 24, 72, 96 and 120 h post-injection. Nanoparticles were internalized by up to 40-50% of lymph node DCs (and APCs) without the use of a targeting ligand, and the site of internalization was in the lymph nodes rather than at the injection site. Finally, an increase in nanoparticle-contg. DCs (and other APCs) was seen at 96 h vs. 24 h, suggesting an infiltration of these cells to lymph nodes. Thus, PPS nanoparticles of 20-45 nm have the potential for immunotherapeutic applications that specifically target DCs in lymph nodes.
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    39. 39
      Stano, A.; Nembrini, C.; Swartz, M. A.; Hubbell, J. A.; Simeoni, E. Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization. Vaccine 2012, 30, 7541– 7546,  DOI: 10.1016/j.vaccine.2012.10.050
      39
      Nanoparticle size influences the magnitude and quality of mucosal immune responses after intranasal immunization
      Stano, Armando; Nembrini, Chiara; Swartz, Melody A.; Hubbell, Jeffrey A.; Simeoni, Eleonora
      Vaccine (2012), 30 (52), 7541-7546CODEN: VACCDE; ISSN:0264-410X. (Elsevier Ltd.)
      The development of nanoparticulate antigen-delivery systems is an important emerging area of vaccinol., being sought to amplify immune responses to recombinant antigens that are poorly immunogenic. Nanoparticle size may play an important role in influencing the activity of such particulate-based adjuvants.To explore how the size of nanoparticles that are in the range of many common viruses can modulate the magnitude and quality of mucosal immune responses, the model antigen ovalbumin (OVA) was conjugated to 30 nm or 200 nm polypropylene sulfide nanoparticles (NPs) and administered intranasally to C57BL/6 mice.We show that by increasing the size of the NPs from 30 to 200 nm, OVA was more effectively delivered into both MHC class I and MHC class II-presentation pathways. Intranasal immunization with the 200 nm NPs increased the magnitude of CD4+ T cell responses in the lungs, as well as systemic and mucosal humoral responses. Most importantly, 200 nm NPs increased the proportion of antigen-specific polyfunctional CD4+ T cells as compared to 30 nm NPs.The 200 nm NPs are a very interesting antigen nanocarrier for prophylactic vaccines against mucosal pathogens that require multifunctional CD4+ T cells for protection. These results contribute to our understanding of how the size of an antigen-conjugated nanoparticle modulates mucosal immune responses to a protein antigen and may be useful to engineer subunit vaccines able to elicit appropriate mucosal immune responses that correlate with protection.
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    40. 40
      Lyu, Z.; Ding, L.; Huang, A. Y. T.; Kao, C. L.; Peng, L. Poly(amidoamine) dendrimers: covalent and supramolecular synthesis. Mater. Today Chem. 2019, 13, 34– 48,  DOI: 10.1016/j.mtchem.2019.04.004
      40
      Poly(amidoamine) dendrimers: covalent and supramolecular synthesis
      Lyu, Z.; Ding, L.; Huang, A. Y. T.; Kao, C.-L.; Peng, L.
      Materials Today Chemistry (2019), 13 (), 34-48CODEN: MTCAD8; ISSN:2468-5194. (Elsevier Ltd.)
      A review. Dendrimers, a class of synthetic macromols. which are notable for their well-defined ramified structures and unique multivalent cooperativity, hold great promise for developing various functional materials. Among all the reported dendrimers, poly(amidoamine) (PAMAM) dendrimers are the most extensively studied by virtue of their readily availability via robust synthesis as well as their dendritic structure and peptide/protein mimic features. Since the seminal report by Tomalia et al., various strategies have been made available for PAMAM dendrimers, including divergent and/or convergent synthesis alongside click chem. Nevertheless, prepn. of high-generation and defect-free PAMAM dendrimers on a large scale remains challenging. To overcome the limitations, an alternative strategy based on self-assembling approach has emerged for dendrimer synthesis, where small dendritic components form large non-covalent supramol. structures that mimic high-generation covalent dendrimers. This approach is easy to implement in practice and requires much less synthetic effort. Here, we present a brief overview of the different approaches established for PAMAM dendrimer synthesis. We start with a general introduction to dendrimers and the common strategies for dendrimer synthesis, and then we illustrate the specific approaches for PAMAM dendrimer synthesis and highlight the related advantages and limitations using representative examples. Although various strategies have been established for PAMAM dendrimer synthesis, innovative concepts and approaches are still in high demand for reliably prepg. defect-free and high-generation dendrimers in large quantity.
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      Ulbrich, K.; Subr, V.; Strohalm, J.; Plocová, D.; Jelínková, M.; Ríhová, B. Polymeric drugs based on conjugates of synthetic and natural macromolecules I.: Synthesis and physico-chemical characterisation. J. Controlled Release 2000, 64, 63– 79,  DOI: 10.1016/S0168-3659(99)00141-8
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      Tao, L.; Liu, J. Q.; Xu, J. T.; Davis, T. P. Synthesis and bioactivity of poly(HPMA)-lysozyme conjugates: the use of novel thiazolidine-2-thione coupling chemistry. Org. Biomol Chem. 2009, 7, 3481– 3485,  DOI: 10.1039/b907061c
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      Subr, V.; Kostka, L.; Strohalm, J.; Etrych, T.; Ulbrich, K. Synthesis of Well-Defined Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] Polymers with Functional Group at the α-End of the Polymer Chain by RAFT Polymerization. Macromolecules 2013, 46, 2100– 2108,  DOI: 10.1021/ma400042u
      43
      Synthesis of Well-Defined Semitelechelic Poly[N-(2-hydroxypropyl)methacrylamide] Polymers with Functional Group at the α-End of the Polymer Chain by RAFT Polymerization
      Subr, V.; Kostka, L.; Strohalm, J.; Etrych, T.; Ulbrich, K.
      Macromolecules (Washington, DC, United States) (2013), 46 (6), 2100-2108CODEN: MAMOBX; ISSN:0024-9297. (American Chemical Society)
      N-(2-Hydroxypropyl)methacrylamide polymer precursors (pHPMA) with narrow distribution of mol. wts. and polymer chain terminating in a single reactive group have big potential in the synthesis of various polymer drug and gene delivery systems. This paper shows that pHPMA can be prepd. by reversible addn.-fragmentation chain transfer (RAFT) polymn.; the pHPMAs prepd. by RAFT polymn. conducted to high conversions contained at the α-end of the polymer chain a single functional group originated from both, the chain transfer agent (CTA) and initiator (INI) in ratio depending on polymn. conditions. The well-defined monofunctional pHPMAs with narrow mol. wt. distribution and with single reactive group situated at the α-polymer chain end can be prepd. in one step by RAFT polymn. initiated by the tailor-made CTA and INI, both contg. the same functional group in their structure. Synthesis of pHPMAs terminating in various reactive groups (azide, propargyl, thiazolidin-2-thione, amino, hydrazide) was also described.
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      Subr, V.; Konák, C.; Laga, R.; Ulbrich, K. Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[ N-(2-hydroxypropyl)methacrylamide]. Biomacromolecules 2006, 7, 122– 130,  DOI: 10.1021/bm050524x
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      Androvic, L.; Woldrichová, L.; Jozefjaková, K.; Pechar, M.; Lynn, G. M.; Kanková, D.; Malinová, L.; Laga, R. Cyclotriphosphazene-Based Star Copolymers as Structurally Tunable Nanocarriers with Programmable Biodegradability. Macromolecules 2021, 54, 3139– 3157,  DOI: 10.1021/acs.macromol.0c02889
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      Neděla, V.; Tihlaříková, E.; Maxa, J.; Imrichová, K.; Bučko, M.; Gemeiner, P. Simulation-based optimization of thermodynamic conditions in the ESEM for dynamical in-situ study of spherical polyelectrolyte complex particles in their native state. Ultramicroscopy 2020, 211, 112954  DOI: 10.1016/j.ultramic.2020.112954
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      Rimankova, L.; Cernocka, H.; Tihlarikova, E.; Nedela, V.; Ostatna, V. Chronopotentiometric sensing of native, oligomeric, denatured and aggregated serum albumin at charged surfaces. Bioelectrochemistry 2022, 145, 108100  DOI: 10.1016/j.bioelechem.2022.108100
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      Lobaz, V.; Liscakova, V.; Sedlak, F.; Musil, D.; Petrova, S. L.; Sedenkova, I.; Panek, J.; Kucka, J.; Konefal, R.; Tihlarikova Tuning polymer-blood and polymer-cytoplasm membrane interactions by manipulating the architecture of poly(2-oxazoline) triblock copolymers. Colloids Surf. B Biointerfaces 2023, 231, 113564  DOI: 10.1016/j.colsurfb.2023.113564
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      Tihlaříková, E.; Neděla, V.; Đorđević, B. In-situ preparation of plant samples in ESEM for energy dispersive x-ray microanalysis and repetitive observation in SEM and ESEM. Sci. Rep-Uk 2019, 9, 2300,  DOI: 10.1038/s41598-019-38835-w
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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.bioconjchem.4c00273.

    • Reaction scheme for the preparation of Man9V3-DBCO (Scheme S1), characteristics of PAMAM dendrimers (Table S1), SEC chromatograms of unpurified and purified star copolymer S17 (Figure S1), SEC chromatograms of heterobifunctional polymer arms P1–P3 (Figure S2), Rh-distribution function of purified star copolymer S17 (Figure S3), size parameters and mass recoveries of star copolymer vaccine solutions before and after filtration (Table S2), size parameters of star copolymers and star copolymer vaccines stored under different conditions (Table S3), SEC chromatograms of star copolymers stored under different conditions (Figure S4), and SEC chromatograms of star copolymer vaccines stored under different conditions (Figure S5) (PDF)


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