Immunogenicity of a Prefusion HIV-1 Envelope Trimer in Complex with a Quaternary-Structure-Specific Antibody

ABSTRACTThe HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies and is being explored as a vaccine candidate to elicit protective antibodies. One of the most promising antigenic and structural mimics of HIV-1 Env is the SOSIP.664-stabilized soluble trimer from the clade A strain BG505, which is preferentially recognized by broadly neutralizing antibodies. Trimer immunization elicits high-titer neutralization of the autologous tier 2 BG505 strain; however, breadth is limited, and substantial interest has focused on understanding and improving trimer immunogenicity. We sought to improve the antigenic specificity of BG505 SOSIP.664 by reducing recognition of the variable loop 3 (V3) region, which elicits only weakly neutralizing antibodies. To stabilize the trimer in its prefusion closed conformation, we complexed trimeric BG505 SOSIP.664 with the antigen-binding fragment (Fab) of PGT145, a broadly neutralizing quaternary-structure-specific antibody. Compared to the ligand-free trimer, the PGT145 Fab-BG505 SOSIP.664 complex displayed increased melting temperature stability and reduced V3 recognition. In guinea pigs, immunization with the PGT145 Fab-BG505 SOSIP.664 complex elicited ∼100-fold lower V3-directed binding and neutralization titers than those obtained with ligand-free BG505 SOSIP.664. Both complexed and ligand-free BG505 SOSIP.664 elicited comparable neutralization of the autologous BG505 virus, and in both cases, BG505 neutralization mapped to the outer domain of gp120 for some guinea pigs. Our results indicate that it is possible to reduce immune recognition of the V3 region of the trimer while maintaining the antigenic profile needed to induce autologous neutralizing antibodies. These data suggest that appropriate modifications of trimer immunogens could further focus the immune response on key neutralization epitopes.IMPORTANCEHIV-1 Env trimers have been proposed as preferred HIV-1 vaccine immunogens. One version, BG505 SOSIP.664, a soluble stabilized trimer, was recently shown to elicit high-titer autologous neutralizing antibodies (NAbs) in rabbits. Here we compared two immunogens: the ligand-free BG505 SOSIP.664 trimer and the same trimer bound to the antigen-binding fragment (Fab) of the PGT145 antibody, a broadly neutralizing antibody which recognizes the trimer at its membrane-distal apex. We hypothesized that the Fab-bound complex would stabilize BG505 SOSIP.664 in its prefusion closed conformation and limit reactivity to weakly neutralizing antibodies targeting the variable loop 3 (V3) region. In guinea pigs, the Fab-complexed trimer induced 100-fold lower responses to the V3 region, while both ligand-free and Fab-complexed trimers elicited similar levels of autologous NAbs. Our findings demonstrate the potential to reduce “off-target” immunogenicity while maintaining the capacity to generate autologous NAbs.

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Antibody-Induced Internalization of HIV-1 Env Proteins Limits Surface Expression of the Closed Conformation of Env

Journal of Virology ◽

10.1128/jvi.00293-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 10

Author(s):

Sai Priya Anand ◽

Jonathan R. Grover ◽

William D. Tolbert ◽

Jérémie Prévost ◽

Jonathan Richard ◽

...

Keyword(s):

Immune Responses ◽

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Surface Expression ◽

Cellular Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Broadly Neutralizing Antibodies ◽

Closed Conformation ◽

Infected Cells ◽

Hiv 1

ABSTRACT To minimize immune responses against infected cells, HIV-1 limits the surface expression of its envelope glycoprotein (Env). Here, we demonstrate that this mechanism is specific for the Env conformation and affects the efficiency of antibody-dependent cellular cytotoxicity (ADCC). Using flow cytometry and confocal microscopy, we show that broadly neutralizing antibodies (bNAbs) targeting the “closed” conformation of Env induce its internalization from the surface. In contrast, non-neutralizing antibodies (nNAbs) are displayed on the cell surface for prolonged period of times. The bNAb-induced Env internalization can be decreased by blocking dynamin function, which translates into higher susceptibilities of infected cells to ADCC. Our results suggest that antibody-mediated Env internalization is a mechanism used by HIV-1 to evade immune responses against the “closed” conformation of Env expressed on HIV-1-infected cells. IMPORTANCE HIV-1 has evolved to acquire several strategies to limit the exposure of its envelope glycoproteins (Env) on the surface of infected cells. In this study, we show that antibody-induced Env internalization is conformation specific and reduces the susceptibility of infected cells to antibody-dependent cellular cytotoxicity (ADCC). Thus, a better understanding of this mechanism might help develop antibodies with improved capacities to mediate ADCC.

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Neutralizing Antibody Responses following Long-Term Vaccination with HIV-1 Env gp140 in Guinea Pigs

Journal of Virology ◽

10.1128/jvi.00369-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00369-18 ◽

Cited By ~ 7

Author(s):

Christine A. Bricault ◽

James M. Kovacs ◽

Alexander Badamchi-Zadeh ◽

Krisha McKee ◽

Jennifer L. Shields ◽

...

Keyword(s):

Guinea Pigs ◽

Neutralizing Antibodies ◽

Antibody Responses ◽

Tier 2 ◽

Broadly Neutralizing Antibodies ◽

Vaccination Regimen ◽

Tier 1 ◽

Clade C ◽

Hiv 1

ABSTRACTA vaccination regimen capable of eliciting potent and broadly neutralizing antibodies (bNAbs) remains an unachieved goal of the HIV-1 vaccine field. Here, we report the immunogenicity of longitudinal prime/boost vaccination regimens with a panel of HIV-1 envelope (Env) gp140 protein immunogens over a period of 200 weeks in guinea pigs. We assessed vaccine regimens that included a monovalent clade C gp140 (C97ZA012 [C97]), a tetravalent regimen consisting of four clade C gp140s (C97ZA012, 459C, 405C, and 939C [4C]), and a tetravalent regimen consisting of clade A, B, C, and mosaic gp140s (92UG037, PVO.4, C97ZA012, and Mosaic 3.1, respectively [ABCM]). We found that the 4C and ABCM prime/boost regimens were capable of eliciting greater magnitude and breadth of binding antibody responses targeting variable loop 2 (V2) over time than the monovalent C97-only regimen. The longitudinal boosting regimen conducted over more than 2 years increased the magnitude of certain tier 1 NAb responses but did not increase the magnitude or breadth of heterologous tier 2 NAb responses. These data suggest that additional immunogen design strategies are needed to induce broad, high-titer tier 2 NAb responses.IMPORTANCEThe elicitation of potent, broadly neutralizing antibodies (bNAbs) remains an elusive goal for the HIV-1 vaccine field. In this study, we explored the use of a long-term vaccination regimen with different immunogens to determine if we could elicit bNAbs in guinea pigs. We found that longitudinal boosting over more than 2 years increased tier 1 NAb responses but did not increase the magnitude and breadth of tier 2 NAb responses. These data suggest that additional immunogen designs and vaccination strategies will be necessary to induce broad tier 2 NAb responses.

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A site of vulnerability at V3 crown defined by HIV-1 bNAb M4008_N1

Nature Communications ◽

10.1038/s41467-021-26846-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kun-Wei Chan ◽

Christina C. Luo ◽

Hong Lu ◽

Xueling Wu ◽

Xiang-Peng Kong

Keyword(s):

Neutralizing Antibodies ◽

Vaccine Design ◽

Tier 2 ◽

Broadly Neutralizing Antibodies ◽

Closed Conformation ◽

Immunogen Design ◽

A Site ◽

Β Sheet ◽

Hiv 1

AbstractIdentification of vulnerable sites defined by broadly neutralizing antibodies (bNAbs) on HIV-1 envelope (Env) is crucial for vaccine design, and we present here a vulnerable site defined by bNAb M4008_N1, which neutralizes about 40% of a tier-2 virus panel. A 3.2 Å resolution cryo-EM structure of M4008_N1 in complex with BG505 DS-SOSIP reveals a large, shallow protein epitope surface centered at the V3 crown of gp120 and surrounded by key glycans. M4008_N1 interacts with gp120 primarily through its hammerhead CDR H3 to form a β-sheet interaction with the V3 crown hairpin. This makes M4008_N1 compatible with the closed conformation of the prefusion Env trimer, and thus distinct from other known V3 crown mAbs. This mode of bNAb approaching the immunogenic V3 crown in the native Env trimer suggests a strategy for immunogen design targeting this site of vulnerability.

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Multiple Antibody Lineages in One Donor Target the Glycan-V3 Supersite of the HIV-1 Envelope Glycoprotein and Display a Preference for Quaternary Binding

Journal of Virology ◽

10.1128/jvi.01012-16 ◽

2016 ◽

Vol 90 (23) ◽

pp. 10574-10586 ◽

Cited By ~ 16

Author(s):

Nancy S. Longo ◽

Matthew S. Sutton ◽

Andrea R. Shiakolas ◽

Javier Guenaga ◽

Marissa C. Jarosinski ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Culture Method ◽

High Serum ◽

Broadly Neutralizing Antibodies ◽

Monomeric Gp120 ◽

Antibody Specificities ◽

V3 Region ◽

Hiv 1

ABSTRACT One of the goals of HIV-1 vaccine development is the elicitation of neutralizing antibodies against vulnerable regions on the envelope glycoprotein (Env) viral spike. Broadly neutralizing antibodies targeting the Env glycan-V3 region (also called the N332 glycan supersite) have been described previously, with several single lineages each derived from different individual donors. We used a high-throughput B-cell culture method to isolate neutralizing antibodies from an HIV-1-infected donor with high serum neutralization breadth. Clonal relatives from three distinct antibody lineages were isolated. Each of these antibody lineages displayed modest breadth and potency but shared several characteristics with the well-characterized glycan-V3 antibodies, including dependence on glycans N332 and N301, VH4 family gene utilization, a heavy chain complementarity-determining region 2 (CDRH2) insertion, and a longer-than-average CDRH3. In contrast to previously described glycan-V3 antibodies, these antibodies preferentially recognized the native Env trimer compared to monomeric gp120. These data indicate the diversity of antibody specificities that target the glycan-V3 site. The quaternary binding preference of these antibodies suggests that that their elicitation likely requires the presentation of a native-like trimeric Env immunogen. IMPORTANCE Broadly neutralizing antibodies targeting the HIV-1 glycan-V3 region with single lineages from individual donors have been described previously. Here we describe three lineages from a single donor, each of which targets glycan-V3. Unlike previously described glycan-V3 antibodies, these mature antibodies bind preferentially to the native Env trimer and weakly to the gp120 monomer. These data extend our knowledge of the immune response recognition of the N332 supersite region and suggest that the mode of epitope recognition is more complex than previously anticipated.

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Neutralizing Antibody Responses Induced by HIV-1 Envelope Glycoprotein SOSIP Trimers Derived from Elite Neutralizers

Journal of Virology ◽

10.1128/jvi.01214-20 ◽

2020 ◽

Vol 94 (24) ◽

Author(s):

Anna Schorcht ◽

Tom L. G. M. van den Kerkhof ◽

Christopher A. Cottrell ◽

Joel D. Allen ◽

Jonathan L. Torres ◽

...

Keyword(s):

Envelope Glycoprotein ◽

Neutralizing Antibodies ◽

High Titer ◽

Vaccine Design ◽

Neutralizing Antibody ◽

Antibody Responses ◽

Tier 2 ◽

Broadly Neutralizing Antibodies ◽

Subtype B ◽

Hiv 1

ABSTRACT The induction of broadly neutralizing antibodies (bNAbs) is a major goal in vaccine research. HIV-1-infected individuals that develop exceptionally strong bNAb responses, termed elite neutralizers, can inform vaccine design by providing blueprints for the induction of similar bNAb responses. We describe a new recombinant native-like envelope glycoprotein (Env) SOSIP trimer, termed AMC009, based on the viral founder sequences of an elite neutralizer. The subtype B AMC009 SOSIP protein formed stable native-like trimers that displayed multiple bNAb epitopes. Overall, its structure at 4.3-Å resolution was similar to that of BG505 SOSIP.664. The AMC009 trimer resembled one from a second elite neutralizer, AMC011, in having a dense and complete glycan shield. When tested as immunogens in rabbits, the AMC009 trimers did not induce autologous neutralizing antibody (NAb) responses efficiently while the AMC011 trimers did so very weakly, outcomes that may reflect the completeness of their glycan shields. The AMC011 trimer induced antibodies that occasionally cross-neutralized heterologous tier 2 viruses, sometimes at high titer. Cross-neutralizing antibodies were more frequently elicited by a trivalent combination of AMC008, AMC009, and AMC011 trimers, all derived from subtype B viruses. Each of these three individual trimers could deplete the NAb activity from the rabbit sera. Mapping the polyclonal sera by electron microscopy revealed that antibodies of multiple specificities could bind to sites on both autologous and heterologous trimers. These results advance our understanding of how to use Env trimers in multivalent vaccination regimens and the immunogenicity of trimers derived from elite neutralizers. IMPORTANCE Elite neutralizers, i.e., individuals who developed unusually broad and potent neutralizing antibody responses, might serve as blueprints for HIV-1 vaccine design. Here, we studied the immunogenicity of native-like recombinant envelope glycoprotein (Env) trimers based on viral sequences from elite neutralizers. While immunization with single trimers from elite neutralization did not recapitulate the breadth and potency of neutralization observed in these infected individuals, a combination of three subtype B Env trimers from elite neutralizers resulted in some neutralization breadth within subtype B viruses. These results should guide future efforts to design vaccines to induce broadly neutralizing antibodies.

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Structure-Based Design of a Soluble Prefusion-Closed HIV-1 Env Trimer with Reduced CD4 Affinity and Improved Immunogenicity

Journal of Virology ◽

10.1128/jvi.02268-16 ◽

2017 ◽

Vol 91 (10) ◽

Cited By ~ 43

Author(s):

Gwo-Yu Chuang ◽

Hui Geng ◽

Marie Pancera ◽

Kai Xu ◽

Cheng Cheng ◽

...

Keyword(s):

Disulfide Bonds ◽

Neutralizing Antibodies ◽

Neutralizing Antibody ◽

Tier 2 ◽

Broadly Neutralizing Antibodies ◽

Closed Conformation ◽

Closed State ◽

Tier 1 ◽

Hiv 1 ◽

A Current

ABSTRACT The HIV-1 envelope (Env) trimer is a target for vaccine design as well as a conformational machine that facilitates virus entry by transitioning between prefusion-closed, CD4-bound, and coreceptor-bound conformations by transitioning into a postfusion state. Vaccine designers have sought to restrict the conformation of the HIV-1 Env trimer to its prefusion-closed state as this state is recognized by most broadly neutralizing, but not nonneutralizing, antibodies. We previously identified a disulfide bond, I201C-A433C (DS), which stabilizes Env in the vaccine-desired prefusion-closed state. When placed into the context of BG505 SOSIP.664, a soluble Env trimer mimic developed by Sanders, Moore, and colleagues, the engineered DS-SOSIP trimer showed reduced conformational triggering by CD4. Here, we further stabilize DS-SOSIP through a combination of structure-based design and 96-well-based expression and antigenic assessment. From 103 designs, we identified one, named DS-SOSIP.4mut, with four additional mutations at the interface of potentially mobile domains of the prefusion-closed structure. We also determined the crystal structures of DS-SOSIP.4mut at 4.1-Å resolution and of an additional DS-SOSIP.6mut variant at 4.3-Å resolution, and these confirmed the formation of engineered disulfide bonds. Notably, DS-SOSIP.4mut elicited a higher ratio of tier 2 autologous titers versus tier 1 V3-sensitive titers than BG505 SOSIP.664. DS-SOSIP.4mut also showed reduced recognition of CD4 and increased thermostability. The improved antigenicity, thermostability, and immunogenicity of DS-SOSIP.4mut suggest utility as an immunogen or a serologic probe; moreover, the specific four alterations identified here, M154, M300, M302, and L320 (4mut), can also be transferred to other HIV-1 Env trimers of interest to improve their properties. IMPORTANCE One approach to elicit broadly neutralizing antibodies against HIV-1 is to stabilize the structurally flexible HIV-1 envelope (Env) trimer in a conformation that displays predominantly broadly neutralizing epitopes and few to no nonneutralizing epitopes. The prefusion-closed conformation of HIV-1 Env has been identified as one such preferred conformation, and a current leading vaccine candidate is the BG505 DS-SOSIP variant, comprising two disulfides and an Ile-to-Pro mutation of Env from strain BG505. Here, we introduced additional mutations to further stabilize BG505 DS-SOSIP in the vaccine-preferred prefusion-closed conformation. In guinea pigs, our best mutant, DS-SOSIP.4mut, elicited a significantly higher ratio of autologous versus V3-directed neutralizing antibody responses than the SOSIP-stabilized form. We also observed an improvement in thermostability and a reduction in CD4 affinity. With improved antigenicity, stability, and immunogenicity, DS-SOSIP.4mut-stabilized trimers may have utility as HIV-1 immunogens or in other antigen-specific contexts, such as with B-cell probes.

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Trimeric Glycosylphosphatidylinositol-Anchored HCDR3 of Broadly Neutralizing Antibody PG16 Is a Potent HIV-1 Entry Inhibitor

Journal of Virology ◽

10.1128/jvi.01038-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1899-1905 ◽

Cited By ~ 8

Author(s):

Lihong Liu ◽

Weiming Wang ◽

Lifei Yang ◽

Huanhuan Ren ◽

Jason T. Kimata ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Neutralizing Antibodies ◽

Quaternary Structure ◽

Neutralizing Antibody ◽

Entry Inhibitor ◽

Broadly Neutralizing Antibodies ◽

Broadly Neutralizing Antibody ◽

Anti Hiv ◽

Hiv 1

ABSTRACTPG9 and PG16 are two quaternary-structure-specific broadly neutralizing antibodies with unique HCDR3 subdomains. Previously, we showed that glycosylphosphatidylinositol (GPI)-anchored HCDR3 subdomains (GPI-HCDR3) can be targeted to lipid rafts of the plasma membrane, bind to the epitope recognized by HCDR3 of PG16, and neutralize diverse HIV-1 isolates. In this study, we further developed trimeric GPI-HCDR3s and demonstrated that trimeric GPI-HCDR3 (PG16) dramatically improves anti-HIV-1 neutralization, suggesting that a stoichiometry of recognition of 3 or 2 HCDR3 molecules (PG16) to 1 viral spike is possible.

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Broadly Neutralizing Bovine Antibodies: Highly Effective New Tools against Evasive Pathogens?

Viruses ◽

10.3390/v12040473 ◽

2020 ◽

Vol 12 (4) ◽

pp. 473

Author(s):

Matthew J. Burke ◽

Peter G. Stockley ◽

Joan Boyes

Keyword(s):

Neutralizing Antibodies ◽

Current Knowledge ◽

Antibody Repertoire ◽

Antigen Binding ◽

Broadly Neutralizing Antibodies ◽

Antigen Binding Site ◽

Vdj Recombination ◽

Vast Array ◽

Hiv 1 ◽

Potent Neutralization

Potent antibody-mediated neutralization is critical for an organism to combat the vast array of pathogens it will face during its lifetime. Due to the potential genetic diversity of some viruses, such as HIV-1 and influenza, standard neutralizing antibodies are often ineffective or easily evaded as their targets are masked or rapidly mutated. This has thwarted efforts to both prevent and treat HIV-1 infections and means that entirely new formulations are required to vaccinate against influenza each year. However, some rare antibodies isolated from infected individuals confer broad and potent neutralization. A subset of these broadly neutralizing antibodies possesses a long complementarity-determining 3 region of the immunoglobulin heavy chain (CDR H3). This feature generates unique antigen binding site configurations that can engage conserved but otherwise inaccessible epitope targets thus neutralizing many viral variants. Remarkably, ultralong CDR H3s are a common feature of the cow antibody repertoire and are encoded by a single variable, diversity, joining (VDJ) recombination that is extensively diversified prior to antigen exposure. Recently, it was shown that cows rapidly generate a broadly neutralizing response upon exposure to HIV-1 and this is primarily mediated by these novel ultralong antibody types. This review summarises the current knowledge of these unusual CDR H3 structures and discusses their known and potential future uses.

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