Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens

Chemical cross-linking is used to stabilise protein structure with additional benefits of pathogen and toxin inactivation for vaccine use, but its use is restricted by potential induction of local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for the purposes of understanding function, or for inducing a biological outcome such as elicitation of antibodies to conformationally-sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its functional analysis and use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent EDC to cross-link two soluble Env trimers, selected well-folded trimers using an antibody affinity column, and transferred this process to good manufacturing practice (GMP) for clinical trial use. Cross-linking enhanced GMP trimer stability to biophysical and enzyme attack, and had broadly beneficial effects on morphology, antigenicity and immunogenicity. Cryo-EM analysis revealed that cross-linking essentially completely retained overall structure with RMSDs between unmodified and cross-linked Env trimers of 0.4 - 0.5 Å. Despite this negligible distortion of global trimer structure we identified individual inter-subunit, intra-subunit and intra-protomer cross-links. Thus, EDC cross-linking maintains protein folding, improves stability, and is readily transferred to GMP, consistent with use of this approach in probing protein structure/function relationships and in the design of vaccines.

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Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens

Journal of Virology ◽

10.1128/jvi.01942-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 813-828 ◽

Cited By ~ 21

Author(s):

Torben Schiffner ◽

Natalia de Val ◽

Rebecca A. Russell ◽

Steven W. de Taeye ◽

Alba Torrents de la Peña ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Morphological Characteristics ◽

Neutralizing Antibody ◽

Structural Instability ◽

Cross Linking ◽

Public Health Importance ◽

Protective Antibodies ◽

Hiv 1 ◽

Chemical Cross Linking ◽

Selection Of

ABSTRACTMajor neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens.IMPORTANCEThe development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.

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Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1516618113 ◽

2015 ◽

Vol 113 (2) ◽

pp. E182-E190 ◽

Cited By ~ 38

Author(s):

Philip R. Tedbury ◽

Mariia Novikova ◽

Sherimay D. Ablan ◽

Eric O. Freed

Keyword(s):

Envelope Glycoprotein ◽

Drug Target ◽

Cytoplasmic Tail ◽

Cross Linking ◽

Biochemical Evidence ◽

The Matrix ◽

Novel Drug ◽

Hiv 1

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.

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The Presence of Chemical Cross-Linking Stabilises HIV-1 Envelope Glycoprotein Trimer Antigens in a Model of Intramuscular Immunisation

10.7244/cmj.2021.03.001.4 ◽

2021 ◽

Author(s):

Luiza Farache Trajano ◽

Rebecca Moore ◽

Quentin Sattentau

Keyword(s):

Human Serum ◽

Envelope Glycoprotein ◽

Proteolytic Enzymes ◽

Neutrophil Infiltration ◽

Inhibitory Effect ◽

Cross Linking ◽

The Stability ◽

Antibody Epitopes ◽

Hiv 1

Background: The HIV-1 envelope glycoprotein (Env) is the target of antigen design for antibody- based vaccination. In 2019, four trimeric Env vaccines entered an experimental trial: ConM, ConS, and their cross-linked counterparts. The trimers were formulated with MPLA adjuvant. Studies have demonstrated that adjuvants trigger neutrophil infiltration. Neutrophils activate and degranulate releasing proteases, namely elastase and cathepsinG. Aims: To assess the stability and immunogenicity of these vaccines in the presence of adjuvant- recruited neutrophils and their proteolytic enzymes. Methods: Trimers were incubated with commercially-sourced proteases. To analyse stability, samples were reduced, denatured and separated using gel electrophoresis. To assess antibody binding, a trimer-protease incubation was followed by an ELISA. To establish more physiologically relevant conditions, harvested neutrophils were exposed to various adjuvants. The supernatant, shown to contain elastase, was incubated alongside the vaccines. The reducing and denaturing gels, as well as the ELISA, was repeated. Results: Gel analysis revealed that un-crosslinked trimers underwent significant digestion whereas cross-linking conferred enhanced stability. In the presence of neutrophil-sourced protease-containing-supernatant, trimers displayed resistance to digestion. The differential stability profile of Env trimers when exposed to commercially sourced compared to supernatant- derived proteases may be due to the inhibitory effect of human serum on elastase. Antibody epitopes were maintained in vitro. Conclusion: The vaccine antigens are sensitive to enzymatic degradation. This is reduced by cross-linking and human serum.

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Improving the Expression and Purification of Soluble, Recombinant Native-Like HIV-1 Envelope Glycoprotein Trimers by Targeted Sequence Changes

Journal of Virology ◽

10.1128/jvi.00264-17 ◽

2017 ◽

Vol 91 (12) ◽

Cited By ~ 14

Author(s):

Rajesh P. Ringe ◽

Gabriel Ozorowski ◽

Anila Yasmeen ◽

Albert Cupo ◽

Victor M. Cruz Portillo ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Affinity Column ◽

Tier 2 ◽

Original Design ◽

Virus Particles ◽

Vaccine Trials ◽

Clade C ◽

Immunogen Design ◽

Hiv 1

ABSTRACT Soluble, recombinant native-like envelope glycoprotein (Env) trimers of various human immunodeficiency virus type 1 (HIV-1) genotypes are being developed for structural studies and as vaccine candidates aimed at the induction of broadly neutralizing antibodies (bNAbs). The prototypic design is designated SOSIP.664, but many HIV-1 env genes do not yield fully native-like trimers efficiently. One such env gene is CZA97.012 from a neutralization-resistant (tier 2) clade C virus. As appropriately purified, native-like CZA97.012 SOSIP.664 trimers induce autologous neutralizing antibodies (NAbs) efficiently in immunized rabbits, we sought to improve the efficiency with which they can be produced and to better understand the limitations to the original design. By using structure- and antigenicity-guided mutagenesis strategies focused on the V2 and V3 regions and the gp120-gp41 interface, we developed the CZA97 SOSIP.v4.2-M6.IT construct. Fully native-like, stable trimers that display multiple bNAb epitopes could be expressed from this construct in a stable CHO cell line and purified at an acceptable yield using either a PGT145 or a 2G12 bNAb affinity column. We also show that similar mutagenesis strategies can be used to improve the yields and properties of SOSIP.664 trimers of the DU422, 426c, and 92UG037 genotypes. IMPORTANCE Recombinant trimeric proteins based on HIV-1 env genes are being developed for future vaccine trials in humans. A feature of these proteins is their mimicry of the envelope glycoprotein (Env) structure on virus particles that is targeted by neutralizing antibodies, i.e., antibodies that prevent cells from becoming infected. The vaccine concept under exploration is that recombinant trimers may be able to elicit virus-neutralizing antibodies when delivered as immunogens. Because HIV-1 is extremely variable, a practical vaccine may need to incorporate Env trimers derived from multiple different virus sequences. Accordingly, we need to understand how to make recombinant trimers from many different env genes. Here, we show how to produce trimers from a clade C virus, CZA97.012, by using an array of protein engineering techniques to improve a prototypic construct. We also show that the methods may have wider utility for other env genes, thereby further guiding immunogen design.

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Tightening the Cross-Linking Distance Restraints for Better Resolution of Protein Structure and Dynamics

SSRN Electronic Journal ◽

10.2139/ssrn.3650053 ◽

2020 ◽

Author(s):

Zhou Gong ◽

Shang-Xiang Ye ◽

Chun Tang

Keyword(s):

Protein Structure ◽

Cross Linking ◽

Structure And Dynamics ◽

Distance Restraints ◽

The Cross ◽

Protein Structure And Dynamics

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HIV-1 Accessory Proteins: Which one is Potentially Effective in Diagnosis and Vaccine Development?

Protein and Peptide Letters ◽

10.2174/0929866528999201231213610 ◽

2020 ◽

Vol 28 ◽

Author(s):

Alireza Milani ◽

Kazem Baesi ◽

Elnaz Agi ◽

Ghazal Marouf ◽

Maryam Ahmadi ◽

...

Keyword(s):

Immune Response ◽

Vaccine Development ◽

Treated Group ◽

Vaccine Antigen ◽

Accessory Proteins ◽

Serological Method ◽

Th2 Immune Response ◽

Untreated Individuals ◽

Immunogenic Properties ◽

Hiv 1

Background:: The combination antiretroviral therapy (cART) could increase the number of circulating naive CD4 T lymphocytes, but was not able to eradicate human immunodeficiency virus-1 (HIV-1) infection. Objective:: Thus, induction of strong immune responses is important for control of HIV-1 infection. Furthermore, a simple and perfect serological method is required to detect virus in untreated-, treated- and drug resistant- HIV-1 infected individuals. Methods:: This study was conducted to assess and compare immunogenic properties of Nef, Vif, Vpr and Vpu accessory proteins as an antigen candidate in mice and their diagnostic importance in human as a biomarker. Results:: Our data showed that in mice, all heterologous prime/ boost regimens were more potent than homologous prime/ boost regimens in eliciting Th1 response and Granzyme B secretion as CTL activity. Moreover, the Nef, Vpu and Vif proteins could significantly increase Th1 immune response. In contrast, the Vpr protein could considerably induce Th2 immune response. On the other hand, among four accessory proteins, HIV-1 Vpu could significantly detect treated group from untreated group as a possible biomarker in human. Conclusion:: Generally, among accessory proteins, Nef, Vpu and Vif antigens were potentially more suitable vaccine antigen candidates than Vpr antigen. Human antibodies against all these proteins were higher in HIV-1 different groups than healthy group. Among them, Vpu was known as a potent antigen in diagnosis of treated from untreated individuals. The potency of accessory proteins as an antigen candidate in an animal model and a human cohort study are underway.

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