An Efficiently Cleaved HIV-1 Subtype C Env that Is Selectively Recognized by Neutralizing Antibodies: A Platform for Immunogen Design

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.

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Neutralizing Antibody Responses in Acute Human Immunodeficiency Virus Type 1 Subtype C Infection

Journal of Virology ◽

10.1128/jvi.00239-07 ◽

2007 ◽

Vol 81 (12) ◽

pp. 6187-6196 ◽

Cited By ~ 209

Author(s):

E. S. Gray ◽

P. L. Moore ◽

I. A. Choge ◽

J. M. Decker ◽

F. Bibollet-Ruche ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Subtype C ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The study of the evolution and specificities of neutralizing antibodies during the course of human immunodeficiency virus type 1 (HIV-1) infection may be important in the discovery of possible targets for vaccine design. In this study, we assessed the autologous and heterologous neutralization responses of 14 HIV-1 subtype C-infected individuals, using envelope clones obtained within the first 2 months postinfection. Our data show that potent but relatively strain-specific neutralizing antibodies develop within 3 to 12 months of HIV-1 infection. The magnitude of this response was associated with shorter V1-to-V5 envelope lengths and fewer glycosylation sites, particularly in the V1-V2 region. Anti-MPER antibodies were detected in 4 of 14 individuals within a year of infection, while antibodies to CD4-induced (CD4i) epitopes developed to high titers in 12 participants, in most cases before the development of autologous neutralizing antibodies. However, neither anti-MPER nor anti-CD4i antibody specificity conferred neutralization breadth. These data provide insights into the kinetics, potency, breadth, and epitope specificity of neutralizing antibody responses in acute HIV-1 subtype C infection.

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Reducing V3 Antigenicity and Immunogenicity on Soluble, Native-Like HIV-1 Env SOSIP Trimers

Journal of Virology ◽

10.1128/jvi.00677-17 ◽

2017 ◽

Vol 91 (15) ◽

Cited By ~ 23

Author(s):

Rajesh P. Ringe ◽

Gabriel Ozorowski ◽

Kimmo Rantalainen ◽

Weston B. Struwe ◽

Katie Matthews ◽

...

Keyword(s):

Animal Models ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Tier 2 ◽

Present Generation ◽

Coreceptor Binding ◽

Tier 1 ◽

Immunogen Design ◽

V3 Region ◽

Hiv 1

ABSTRACT Native-like trimers of the SOSIP design are being developed as immunogens in human immunodeficiency virus type 1 (HIV-1) vaccine development programs. These trimers display the epitopes for multiple broadly neutralizing antibodies (bNAbs) but can also expose binding sites for some types of nonneutralizing antibodies (non-NAbs). Among the latter are epitopes in the gp120 V3 region that are highly immunogenic when SOSIP trimers are evaluated in animal models. It is presently uncertain whether antibodies against V3 can interfere with the induction of NAbs, but there are good arguments in favor of suppressing such “off-target” immune responses. Accordingly, we have assessed how to minimize the exposure of V3 non-NAb epitopes and thereby reduce their immunogenicity by introducing N-glycans within the V3 region of BG505 SOSIP trimers. We found that inserting glycans at positions 306 and 314 (termed M1 and M7) markedly reduced V3 antigenicity while improving the presentation of trimer apex bNAb epitopes. Both added glycans were shown to be predominantly of the Man6GlcNAc2 form. The additional introduction of the E64K ground-state stabilizing substitution markedly reduced or ablated soluble CD4 (sCD4) induction of non-NAb epitopes in V3 and/or associated with the coreceptor binding site. When a V3 glycan- and E64K-modified trimer variant, BG505 SOSIP.664-E64K.M1M7, was tested in rabbits, V3 immunogenicity was eliminated while the autologous NAb response was unchanged. IMPORTANCE Trimeric proteins are being developed for future HIV-1 vaccine trials in humans, with the goal of eliciting broadly active neutralizing antibodies (NAbs) that are active against a wide variety of circulating strains. In animal models, the present generation of native-like trimer immunogens, exemplified by the BG505 SOSIP.664 construct, induces narrow-specificity antibodies against the neutralization-resistant (tier-2), sequence-matched virus and more broadly active antibodies against sequence-divergent atypically neutralization-sensitive (tier-1) viruses. A concern in the trimer immunogen design field has been whether the latter off-target antibodies might interfere with the induction of the more desired responses to tier-2 epitopes. Here, we have inserted two glycans into the dominant site for tier-1 NAbs, the gp120 V3 region, to block the induction of off-target antibodies. We characterized the new trimers, tested them as immunogens in rabbits, and found that the blocking glycans eliminated the induction of tier-1 NAbs to V3-epitopes.

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HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes

Journal of Virology ◽

10.1128/jvi.00878-18 ◽

2018 ◽

Vol 92 (17) ◽

Cited By ~ 18

Author(s):

Zanele Ditse ◽

Maximilian Muenchhoff ◽

Emily Adland ◽

Pieter Jooste ◽

Philip Goulder ◽

...

Keyword(s):

Immune System ◽

Neutralizing Antibodies ◽

High Titer ◽

Neutralizing Antibody ◽

Subtype C ◽

Child Pair ◽

Intact Immune System ◽

Antibody Specificities ◽

Hiv Envelope ◽

Hiv 1

ABSTRACT We have previously shown that HIV-1-infected children develop broader and more potent neutralizing antibody responses than adults. This study aimed to determine the antibody specificities in 16 HIV-1 subtype C-infected children who displayed exceptional neutralization breadth on a 22-multisubtype virus panel. All children were antiretroviral treatment (ART) naive with normal CD4 counts despite being infected for a median of 10.1 years with high viral loads. The specificity of broadly neutralizing antibodies (bNAbs) was determined using epitope-ablating mutants, chimeric constructs, and depletion or inhibition of activity with peptides and glycoproteins. We found that bNAbs in children largely targeted previously defined epitopes, including the V2-glycan, V3-glycan, CD4bs, and gp120-gp41 interface. Remarkably, 63% of children had antibodies targeting 2 or 3 and, in one case, 4 of these bNAb epitopes. Longitudinal analysis of plasma from a mother-child pair over 9 years showed that while they both had similar neutralization profiles, the antibody specificities differed. The mother developed antibodies targeting the V2-glycan and CD4bs, whereas bNAb specificities in the child could not be mapped until 6 years, when a minor V2-glycan response appeared. The child also developed high-titer membrane-proximal external region (MPER) binding antibodies not seen in the mother, although these were not a major bNAb specificity. Overall, exceptional neutralization breadth in this group of children may be the result of extended exposure to high antigenic load in the context of an intact immune system, which allowed for the activation of multiple B cell lineages and the generation of polyclonal responses targeting several bNAb epitopes. IMPORTANCE An HIV vaccine is likely to require bNAbs, which have been shown to prevent HIV acquisition in nonhuman primates. Recent evidence suggests that HIV-infected children are inherently better at generating bNAbs than adults. Here, we show that exceptional neutralization breadth in a group of viremic HIV-1 subtype C-infected children was due to the presence of polyclonal bNAb responses. These bNAbs targeted multiple epitopes on the HIV envelope glycoprotein previously defined in adult infection, suggesting that the immature immune system recognizes HIV antigens similarly. Since elicitation of a polyclonal bNAb response is the basis of next-generation HIV envelope vaccines, further studies of how bNAb lineages are stimulated in children is warranted. Furthermore, our findings suggest that children may respond particularly well to vaccines designed to elicit antibodies to multiple bNAb epitopes.

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Recapitulation of HIV-1 Env-Antibody Coevolution in Macaques Leading to Neutralization Breadth

10.1101/2020.08.05.237693 ◽

2020 ◽

Cited By ~ 1

Author(s):

Ryan S. Roark ◽

Hui Li ◽

Wilton B. Williams ◽

Hema Chug ◽

Rosemarie D. Mason ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Virus Antibody ◽

Human Antibodies ◽

Immunogen Design ◽

Cd4 Binding Site ◽

Human Immunodeficiency Viruses ◽

Chemical Solutions ◽

Developmental Features ◽

Hiv 1

ABSTRACTNeutralizing antibodies elicited by HIV-1 coevolve with viral Envs in distinctive patterns, in some cases acquiring substantial breadth. Here we show that primary HIV-1 Envs, when expressed by simian-human immunodeficiency viruses in rhesus macaques, elicited patterns of Env-antibody coevolution strikingly similar to those in humans. This included conserved immunogenetic, structural and chemical solutions to epitope recognition and precise Env-amino acid substitutions, insertions and deletions leading to virus persistence. The structure of one rhesus antibody, capable of neutralizing 49% of a 208-strain panel, revealed a V2-apex mode of recognition like that of human bNAbs PGT145/PCT64-35M. Another rhesus antibody bound the CD4-binding site by CD4 mimicry mirroring human bNAbs 8ANC131/CH235/VRC01. Virus-antibody coevolution in macaques can thus recapitulate developmental features of human bNAbs, thereby guiding HIV-1 immunogen design.One sentence summaryVirus-antibody coevolution in rhesus macaques recapitulates developmental features of human antibodies.

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Networks of HIV-1 envelope glycans maintain antibody epitopes in the face of glycan additions and deletions

10.1101/2020.02.21.959981 ◽

2020 ◽

Cited By ~ 2

Author(s):

Gemma E. Seabright ◽

Christopher A. Cottrell ◽

Marit J. van Gils ◽

Alessio D’addabbo ◽

David J. Harvey ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Broadly Neutralizing Antibodies ◽

Site Specific ◽

Glycan Analysis ◽

Cryo Electron Microscopy ◽

Immunogen Design ◽

The Face ◽

Antibody Epitopes ◽

Hiv 1

SUMMARYNumerous broadly neutralizing antibodies (bnAbs) have been identified that target the glycans of the HIV-1 envelope spike. Neutralization breadth is notable given that glycan processing can be substantially influenced by the presence or absence of neighboring glycans. Here, using a stabilized recombinant envelope trimer, we investigate the degree to which mutations in the glycan network surrounding an epitope impact the fine glycan processing of antibody targets. Using cryo-electron microscopy and site-specific glycan analysis, we reveal the hierarchy of importance of glycans in the formation of the 2G12 bnAb epitope, and show that the epitope is only subtly impacted by variations in the glycan network. In contrast, we show that the PG9 and PG16 glycan-based epitopes at the trimer apex are dependent on the presence of the highly conserved surrounding glycans. Glycan networks underpin the conservation of bnAb epitopes and are an important parameter in immunogen design.

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Neutralizing antibodies induced in immunized macaques recognize the CD4-binding site on an occluded-open HIV-1 envelope trimer

10.1101/2021.11.02.466200 ◽

2021 ◽

Author(s):

Zhi Yang ◽

Kim-Marie A. Dam ◽

Michael D. Bridges ◽

Magnus A.G. Hoffmann ◽

Andrew T. DeLaitsch ◽

...

Keyword(s):

Binding Site ◽

Neutralizing Antibodies ◽

Resonance Spectroscopy ◽

Broadly Neutralizing Antibodies ◽

Receptor Binding Site ◽

Double Electron ◽

Immunogen Design ◽

Cd4 Binding Site ◽

Double Electron Electron Resonance ◽

Hiv 1

Broadly-neutralizing antibodies (bNAbs) against HIV-1 Env can protect from infection. We characterized Ab1303 and Ab1573, neutralizing CD4-binding site (CD4bs) antibodies, isolated from sequentially-immunized macaques. Ab1303/Ab1573 binding was observed only when Env trimers were not constrained in the closed, prefusion conformation. Fab-Env cryo-EM structures showed that both antibodies recognized the CD4bs on Env trimer with an occluded-open conformation between closed, as targeted by bNAbs, and fully-open, as recognized by CD4. The occluded-open Env trimer conformation included outwardly-rotated gp120 subunits, but unlike CD4-bound Envs, did not exhibit V1V2 displacement, co-receptor binding site exposure, or a 4-stranded gp120 bridging sheet. Inter-protomer distances within trimers measured by double electron-electron resonance spectroscopy suggested an equilibrium between occluded-open and closed Env conformations, consistent with Ab1303/Ab1573 binding stabilizing an existing conformation. Studies of Ab1303/Ab1573 demonstrate that CD4bs neutralizing antibodies that bind open Env trimers can be raised by immunization, thereby informing immunogen design and antibody therapeutic efforts.

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Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

10.1101/2020.09.15.297697 ◽

2020 ◽

Author(s):

Rajesh Kumar ◽

Suprit Deshpande ◽

Leigh M. Sewall ◽

Gabriel Ozorowski ◽

Christopher A. Cottrell ◽

...

Keyword(s):

Electron Microscopy ◽

Neutralizing Antibodies ◽

Natural Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Clade C ◽

Immunogen Design ◽

Env Protein ◽

Immunogenic Properties ◽

Hiv 1

AbstractEvaluating the structure-function relationship of viral envelope (Env) evolution and the development of broadly cross-neutralizing antibodies (bnAbs) in natural infection can inform rational immunogen design. In the present study, we examined the magnitude and specificity of autologous neutralizing antibodies induced in rabbits by a novel HIV-1 clade C Env protein (1PGE-THIVC) vis-à-vis those developed in an elite neutralizer from whom the env sequence was obtained that was used to prepare the soluble Env protein. The thermostable 1PGE-THIVC Env displayed a native like pre-fusion closed conformation in solution as determined by small angle X-ray scattering (SAXS) and negative stain electron microscopy (EM). This closed spike conformation of 1PGE-THIVC Env trimers was correlated with weak or undetectable binding of non-neutralizing monoclonal antibodies (mAbs) compared to neutralizing mAbs. Furthermore, 1PGE-THIVC SOSIP induced potent neutralizing antibodies in rabbits to autologous virus variants. The autologous neutralizing antibody specificity induced in rabbits by 1PGE-THIVC was mapped to the C3/V4 region (T362/P401) of viral Env. This observation agreed with electron microscopy polyclonal epitope mapping (EMPEM) of the Env trimer complexed with IgG Fab prepared from the immunized rabbit sera. While the specificity of antibodies elicited in rabbits associated with neutralizing autologous viruses were distinct to those developed in the elite neutralizer, EMPEM analysis demonstrated significant changes to Env conformations when incubated with polyclonal antibody sera from the elite neutralizer, suggesting these antibodies lead to the destabilization of Env trimers. Our study not only shows distinct mechanisms associated with potent neutralization of sequence matched and unmatched autologous viruses by antibodies induced in rabbits and in the elite neutralizer, but also highlights how neutralizing antibodies developed during the course of natural infection can impact viral Env conformations.Author SummaryThe interplay between circulating virus variants and broadly cross neutralizing polyclonal antibodies developed in a subset of elite neutralizers is widely believed to provide strategies for rational immunogen design. In the present study, we studied the structural, antigenic and immunogenic properties of a thermostable soluble trimeric protein with near native pre-fusion conformation prepared using the primary sequence of an HIV-1 clade C env isolated from the broadly cross neutralizing plasma of an elite neutralizer. This novel SOSIP Env trimer demonstrated comparable antigenic, structural and immunogenic properties that favoured several ongoing subunit vaccine design efforts. The novel clade C SOSIP induced polyclonal neutralizing antibody response developed in rabbits not only differed in its epitope specificity compared to that elicited in natural infection in presence of pool of viral quasispecies but also showed how they differ in their ability to influence Env structure and conformation. A better understanding of how vaccine-induced polyclonal neutralizing antibody responses compares to responses that developed in natural infection will improve our knowledge in designing better vaccine design strategies.

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Efficiently cleaved HIV-1 envelopes: can they be important for vaccine immunogen development?

Therapeutic Advances in Vaccines and Immunotherapy ◽

10.1177/2515135520957763 ◽

2020 ◽

Vol 8 ◽

pp. 251513552095776

Author(s):

Supratik Das ◽

Rajesh Kumar ◽

Shubbir Ahmed ◽

Hilal Ahmad Parray ◽

Sweety Samal

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Immunogen ◽

Naturally Occurring ◽

Tremendous Progress ◽

Antigenic Properties ◽

Immunogen Design ◽

Membrane Bound ◽

Work Done ◽

Hiv 1 ◽

Made In

The enormous diversity of HIV-1 is a significant impediment in selecting envelopes (Envs) that can be suitable for designing vaccine immunogens. While tremendous progress has been made in developing soluble, trimeric, native-like Env proteins, those that have elicited neutralizing antibodies (Abs) in animal models are relatively few. A strategy of selecting naturally occurring Envs suitable for immunogen design by studying the correlation between efficient cleavage on the cell surface and their selective binding to broadly neutralizing Abs (bNAbs) and not to non-neutralizing Abs (non-NAbs), properties essential in immunogens, may be useful. Here we discuss some of the challenges of developing an efficacious HIV-1 vaccine and the work done in generating soluble immunogens. We also discuss the study of naturally occurring, membrane-bound, efficiently cleaved (naturally more sensitive to furin) Envs and how they may positively add to the repertoire of HIV-1 Envs that can be used for vaccine immunogen design. However, even with such Envs, the challenges of developing well-folded, native-like trimers as soluble proteins or using other immunogen strategies such as virus-like particles with desirable antigenic properties remain, and are formidable. In spite of the progress that has been made in the HIV-1 vaccine field, an immunogen that elicits neutralizing Abs with significant breadth and potency in vaccines has still not been developed. Efficiently cleaved Envs may increase the number of available Envs suitable for immunogen design and should be studied further.

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Structure-Guided Redesign Increases the Propensity of HIV Env To Generate Highly Stable Soluble Trimers

Journal of Virology ◽

10.1128/jvi.02652-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 2806-2817 ◽

Cited By ~ 69

Author(s):

Javier Guenaga ◽

Viktoriya Dubrovskaya ◽

Natalia de Val ◽

Shailendra K. Sharma ◽

Barbara Carrette ◽

...

Keyword(s):

Neutralizing Antibodies ◽

Small Animal ◽

Subtype C ◽

Disulfide Linkage ◽

Vaccine Candidates ◽

Subtype B ◽

Subtype A ◽

Induced Changes ◽

Hiv 1 ◽

Hiv Diversity

ABSTRACTDue to high viral diversity, an effective HIV-1 vaccine will likely require Envs derived from multiple subtypes to generate broadly neutralizing antibodies (bNAbs). Soluble Env mimics, like the native flexibly linked (NFL) and SOSIP trimers, derived from the subtype A BG505 Env, form homogeneous, stable native-like trimers. However, other Env sequences, such as JRFL and 16055 from subtypes B and C, do so to a lesser degree. The high-resolution BG505 SOSIP crystal structures permit the identification and redesign of Env elements involved in trimer stability. Here, we identified structure trimer-derived (TD) residues that increased the propensity of the subtype B JRFL and subtype C 16055 Env sequences to form well-ordered, homogenous, and highly stable soluble trimers. The generation of these spike mimics no longer required antibody-based selection, positive or negative. Using the redesigned subtype B and C trimer representatives as respective foundations, we further stabilized the NFL TD trimers by engineering an intraprotomer disulfide linkage in the prebridging sheet, I201C-A433C (CC), that locks the gp120 in the receptor nontriggered state. We demonstrated that this disulfide pair prevented CD4 induced-conformational rearrangements in NFL trimers derived from the prototypic subtype A, B, and C representatives. Coupling the TD-based design with the engineered disulfide linkage, CC, increased the propensity of Env to form soluble highly stable spike mimics that are resistant to CD4-induced changes. These advances will allow testing of the hypothesis that such stabilized immunogens will more efficiently elicit neutralizing antibodies in small-animal models and primates.IMPORTANCEHIV-1 displays unprecedented global diversity circulating in the human population. Since the envelope glycoprotein (Env) is the target of neutralizing antibodies, Env-based vaccine candidates that address such diversity are needed. Soluble well-ordered Env mimics, typified by NFL and SOSIP trimers, are attractive vaccine candidates. However, the current designs do not allow most Envs to form well-ordered trimers. Here, we made design modifications to increase the propensity of representatives from two of the major HIV subtypes to form highly stable trimers. This approach should be applicable to other viral Envs, permitting the generation of a repertoire of homogeneous, highly stable trimers. The availability of such an array will allow us to assess if sequential or cocktail immune strategies can overcome some of the vaccine challenges presented by HIV diversity.

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