Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates.

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide the maturation of other bnAb classes. To compare antigens designed to maintain this conformation, we evaluated apex-specific responses in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding the improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

Electron-Microscopy-Based Epitope Mapping Defines Specificities of Polyclonal Antibodies Elicited during HIV-1 BG505 Envelope Trimer Immunization.

Characterizing polyclonal antibody responses via currently available methods is inherently complex and difficult. Mapping epitopes in an immune response is typically incomplete, which creates a barrier to fully understanding the humoral response to antigens and hinders rational vaccine design efforts. Here, we describe a method of characterizing polyclonal responses by using electron microscopy, and we applied this method to the immunization of rabbits with an HIV-1 envelope glycoprotein vaccine candidate, BG505 SOSIP.664. We detected known epitopes within the polyclonal sera and revealed how antibody responses evolved during the prime-boosting strategy to ultimately result in a neutralizing antibody response. We uncovered previously unidentified epitopes, including an epitope proximal to one recognized by human broadly neutralizing antibodies as well as potentially distracting non-neutralizing epitopes. Our method provides an efficient and semiquantitative map of epitopes that are targeted in a polyclonal antibody response and should be of widespread utility in vaccine and infection studies.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

Elicitation of Robust Tier 2 Neutralizing Antibody Responses in Nonhuman Primates by HIV Envelope Trimer Immunization Using Optimized Approaches.

The development of stabilized recombinant HIV envelope trimers that mimic the virion surface molecule has increased enthusiasm for a neutralizing antibody (nAb)-based HIV vaccine. However, there is limited experience with recombinant trimers as immunogens in nonhuman primates, which are typically used as a model for humans. Here, we tested multiple immunogens and immunization strategies head-to-head to determine their impact on the quantity, quality, and kinetics of autologous tier 2 nAb development. A bilateral, adjuvanted, subcutaneous immunization protocol induced reproducible tier 2 nAb responses after only two immunizations 8 weeks apart, and these were further enhanced by a third immunization with BG505 SOSIP trimer. We identified immunogens that minimized non-neutralizing V3 responses and demonstrated that continuous immunogen delivery could enhance nAb responses. nAb responses were strongly associated with germinal center reactions, as assessed by lymph node fine needle aspiration. This study provides a framework for preclinical and clinical vaccine studies targeting nAb elicitation.

Antigenic analysis of the HIV-1 envelope trimer implies small differences between structural states 1 and 2.

The conformationally dynamic HIV-1 envelope trimer (Env) is the target of broadly neutralizing antibodies (bnAbs) that block viral entry. Single-molecule Förster resonance energy transfer (smFRET) has revealed that HIV-1 Env exists in at least three conformational states on the virion. Prior to complete host-receptor engagement (State 3), Env resides most prevalently in the smFRET-defined State 1, which is preferentially recognized by most bnAbs that are elicited by natural infection. smFRET has also revealed that soluble trimers containing prefusion-stabilizing disulfide and isoleucine-to-proline substitutions reside primarily in State 2, which is a required intermediate between States 1 and 3. While high-resolution Env structures have been determined for States 2 and 3, the structure of these trimers in State 1 is unknown. To provide insight into the State 1 structure, here we characterized antigenic differences between smFRET-defined states and then correlated these differences with known structural differences between States 2 and 3. We found that cell surface-expressed Env was enriched in each state using state-enriching antibody fragments or small-molecule virus entry inhibitors and then assessed binding to HIV-1 bnAbs preferentially binding different states. We observed small but consistent differences in binding between Env enriched in States 1 and 2, and a more than 10-fold difference in binding to Env enriched in these states versus Env enriched in State 3. We conclude that structural differences between HIV-1 Env States 1 and 3 are likely more than 10-fold greater than those between States 1 and 2, providing important insight into State 1.

The Glycan Hole Area of HIV-1 Envelope Trimers Contributes Prominently to the Induction of Autologous Neutralization.

The human immunodeficiency virus type 1 (HIV-1) trimeric envelope glycoprotein (Env) is heavily glycosylated, creating a dense glycan shield that protects the underlying peptidic surface from antibody recognition. The absence of conserved glycans, due to missing potential N-linked glycosylation sites (PNGS), can result in strain-specific, autologous neutralizing antibody (NAb) responses. Here, we sought to gain a deeper understanding of the autologous neutralization by introducing holes in the otherwise dense glycan shields of the AMC011 and AMC016 SOSIP trimers. Specifically, when we knocked out the N130 and N289 glycans, which are absent from the well-characterized B41 SOSIP trimer, we observed stronger autologous NAb responses. We also analyzed the highly variable NAb responses induced in rabbits by diverse SOSIP trimers from subtypes A, B, and C. Statistical analysis, using linear regression, revealed that the cumulative area exposed on a trimer by glycan holes correlates with the magnitude of the autologous NAb response. IMPORTANCE Forty years after the first description of HIV-1, the search for a protective vaccine is still ongoing. The sole target for antibodies that can neutralize the virus are the trimeric envelope glycoproteins (Envs) located on the viral surface. The glycoprotein surface is covered with glycans that shield off the underlying protein components from recognition by the immune system. However, the Env trimers of some viral strains have holes in the glycan shield. Immunized animals developed antibodies against such glycan holes. These antibodies are generally strain specific. Here, we sought to gain a deeper understanding of what drives these specific immune responses. First, we show that strain-specific neutralizing antibody responses can be increased by creating artificial holes in the glycan shield. Second, when studying a diverse set of Env trimers with different characteristics, we found that the surface area of the glycan holes contributes prominently to the induction of strain-specific neutralizing antibodies.

SOSIP Trimer-Specific Antibodies Isolated from a Simian-Human Immunodeficiency Virus-Infected Monkey with versus without a Pre-blocking Step with gp41.

BG505 SOSIP.664 (hereafter referred to as SOSIP), a stabilized trimeric mimic of the HIV-1 envelope spike resembling the native viral spike, is a useful tool for isolating anti-HIV-1 neutralizing antibodies. We screened long-term SHIV-AD8 infected rhesus monkeys for potency and breadth of serum neutralizing activity against autologous and heterologous viruses: SHIV-AD8, HIV-1 YU2, HIV-1 JR-CSF, and HIV-1 NL4-3. Monkey rh2436 neutralized all viruses tested and showed strong reactivity to the SOSIP trimer, suggesting this was a promising candidate for attempts at monoclonal antibody (MAb) isolation. MAbs were isolated by performing single B-cell sorts from peripheral blood mononuclear cells (PBMC) by FACS using the SOSIP trimer as a probe. An initial round of sorted cells revealed the majority of isolated MAbs were directed to the gp41 external domain portion of the SOSIP trimer and were mostly non-neutralizing against tested isolates. A second sort was performed, introducing a gp41 blocking step prior to PBMC staining and FACS sorting. These isolated MAbs bound SOSIP trimer but were no longer directed to the gp41 external domain portion. A significantly higher proportion of MAbs with neutralizing activity were obtained with this strategy. Our data show this pre-blocking step with gp41 greatly increases the yield of non-gp41-reactive, SOSIP-specific MAbs and increases the likelihood of isolating MAbs with neutralizing activity. IMPORTANCE Recent advancements in the field have focused on the isolation and use of broadly neutralizing antibodies for both prophylaxis and therapy. Finding a useful probe to isolate broad potent neutralizing antibodies while avoiding non-neutralizing antibodies is important. The SOSIP trimer has been shown to be a great tool for this purpose because it binds known broadly neutralizing antibodies. However, the SOSIP trimer can isolate non-neutralizing antibodies as well, including gp41-specific MAbs. Introducing a pre-blocking step with gp41 recombinant protein decreased the percent of gp41-specific antibodies isolated with SOSIP probe, as well as increased the number of neutralizing antibodies isolated. This method can be used as a tool to increase the chances of isolating neutralizing antibodies.

Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B cell reprograming may be scientifically and therapeutically useful, but current approaches limit B cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identify more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and PG16. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B cell repertoires recognizing a key HIV-1 neutralizing epitope.

Covalent binding of human two-domain CD4 to an HIV-1 subtype C SOSIP.664 trimer modulates its structural dynamics.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates host cell infection by binding to the cellular receptor CD4. Recombinant Env bound to CD4 has been explored for its potential as an HIV vaccine immunogen as receptor binding exposes otherwise shielded, conserved functional sites. Previous preclinical studies showed an interchain disulphide linkage facilitated between Env and 2dCD4S60C generates an immunogenic complex that elicits potent, broadly neutralizing antibodies (bNAbs) against clinically relevant HIV-1. This study investigated conformational dynamics of 2dCD4WT and 2dCD4S60C bound to an HIV-1C SOSIP.664 Env trimer using hydrogen-deuterium exchange mass spectrometry. The Env:2dCD4S60C complex maintains key contact residues required for MHCII and Env/gp120 binding and the residues encompassing Ibalizumab's epitope. Important residues remaining anchored, with an increased flexibility in surrounding regions, evidenced by the higher exchange seen in flanking residues compared to Env:2dCD4WT. While changes in Env:2dCD4S60C dynamics in domain 1 were moderate, domain 2 exhibited greater variation. Lack of stability-inducing H-bonds in these allosteric sites suggest the improved immunogenicity of Env:2dCD4S60C result from exposed CD4 residues providing diverse/novel antigenic targets for the development of potent, broadly neutralizing Ibalizumab-like antibodies.

Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

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

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.

Autologous Antibody Responses to an HIV Envelope Glycan Hole Are Not Easily Broadened in Rabbits.

Extensive studies with subtype A BG505-derived HIV envelope glycoprotein (Env) immunogens have revealed that the dominant autologous neutralizing epitope in rabbits is located in an exposed region of the heavily glycosylated trimer that lacks potential N-linked glycosylation sites at positions 230, 241, and 289. The Env derived from B41, a subtype B virus, shares a glycan hole centered on positions 230 and 289. To test whether broader neutralization to the common glycan hole can be achieved, we immunized rabbits with B41 SOSIP (gp120-gp41 disulfide [SOS] with an isoleucine-to-proline mutation [IP] in gp41) alone, as well as B41 and BG505 coimmunization. We isolated autologous neutralizing antibodies (nAbs) and described their structure in complex with the B41 Env. Our data suggest that distinct autologous nAb lineages are induced by BG505 and B41 immunogens, even when both were administered together. In contrast to previously described BG505 glycan hole antibodies, the B41-specific nAbs accommodate the >97% conserved N241 glycan, which is present in B41. Single-particle cryo-electron microscopy studies confirmed that B41- and BG505-specific nAbs bind to overlapping glycan hole epitopes. We then used our high-resolution data to guide mutations in the BG505 glycan hole epitope in an attempt to broaden the reactivity of a B41-specific nAb, but we recovered only partial binding. Our data demonstrate that the lack of cross-reactivity in glycan hole antibodies is due to amino acid differences within the epitope, and our attempts to rationally design cross-reactive trimers resulted in only limited success. Thus, even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.IMPORTANCE A glycan hole is one of the most dominant autologous neutralizing epitopes targeted on BG505 and B41 SOSIP trimer-immunized rabbits. Our high-resolution cryo-electron microscopy (cryoEM) studies of B41 in complex with a B41-specific antibody complex elucidate the molecular basis of this strain-specific glycan hole response. We conclude that even for the immunodominant glycan hole shared between BG505 and B41, the prospect of designing prime-boost immunogens remains difficult.

Neutralizing Antibody Induction by HIV-1 Envelope Glycoprotein SOSIP Trimers on Iron Oxide Nanoparticles May Be Impaired by Mannose Binding Lectin.

We covalently attached human immunodeficiency virus type 1 (HIV-1) Env SOSIP trimers to iron oxide nanoparticles (IO-NPs) to create a particulate immunogen for neutralizing antibody (NAb) induction. The attached trimers, ∼20 per particle, retained native-like antigenicity, judged by reactivity with NAbs and non-NAbs. Bivalent (BG505 and B41) trimer IO-NPs were made, as were IO-NPs displaying B41 trimers carrying a PADRE T-cell helper epitope (TCHE). We immunized mice with B41 soluble or IO-NP trimers after PADRE peptide priming. After two immunizations, IO-NP presentation and the TCHE tag independently and substantially increased anti-trimer antibody responses, but titer differences waned after two further doses. Notable and unexpected findings were that autologous NAbs to the N289 glycan hole epitope were consistently induced in mice given soluble but not IO-NP trimers. Various recombinant mannose binding lectins (MBLs) and MBLs in sera of both murine and human origin bound to soluble and IO-NP trimers. MBL binding occluded the autologous NAb epitope on the B41 IO-NP trimers, which may contribute to its poor immunogenicity. The exposure of a subset of broadly active NAb epitopes was also impaired by MBL binding, which could have substantial implications for the utility of trimer-bearing nanoparticles in general and perhaps also for soluble Env proteins.IMPORTANCE Recombinant trimeric SOSIP proteins are vaccine components intended to induce neutralizing antibodies (NAbs) that prevent cells from infection by human immunodeficiency virus type 1 (HIV-1). A way to increase the strength of antibody responses to these proteins is to present them on the surface of nanoparticles (NPs). We chemically attached about 20 SOSIP trimers to NPs made of iron oxide (IO). The resulting IO-NP trimers had appropriate properties when we studied them in the laboratory but, unexpectedly, were less able to induce NAbs than nonattached trimers when used to immunize mice. We found that mannose binding lectins, proteins naturally present in the serum of mice and other animals, bound strongly to the soluble and IO-NP trimers, blocking access to antibody epitopes in a way that may impede the development of NAb responses. These findings should influence how trimer-bearing NPs of various designs are made and used.

Antigenicity and Immunogenicity of HIV-1 Envelope Trimers Complexed to a Small-Molecule Viral Entry Inhibitor.

Small-molecule viral entry inhibitors, such as BMS-626529 (BMS-529), allosterically block CD4 binding to HIV-1 envelope (Env) and inhibit CD4-induced structural changes in Env trimers. Here, we show that the binding of BMS-529 to clade C soluble chimeric gp140 SOSIP (ch.SOSIP) and membrane-bound trimers with intact transmembrane domain (gp150) prevented trimer conformational transitions and enhanced their immunogenicity. When complexed to BMS-529, ch.SOSIP trimers retained their binding to broadly neutralizing antibodies (bNAbs) and to their unmutated common ancestor (UCA) antibodies, while exposure of CD4-induced (CD4i) non-bNAb epitopes was inhibited. BMS-529-complexed gp150 trimers in detergent micelles, which were isolated from CHO cells, bound to bNAbs, including UCA and intermediates of the CD4 binding site (bs) CH103 bNAb lineage, and showed limited exposure of CD4i epitopes and a glycosylation pattern with a preponderance of high-mannose glycans. In rabbits, BMS-529-complexed V3 glycan-targeting ch.SOSIP immunogen induced in the majority of immunized animals higher neutralization titers against both autologous and select high mannose-bearing heterologous tier 2 pseudoviruses than those immunized with the noncomplexed ch.SOSIP. In rhesus macaques, BMS-529 complexed to CD4 bs-targeting ch.SOSIP immunogen induced stronger neutralization against tier 2 pseudoviruses bearing high-mannose glycans than noncomplexed ch.SOSIP trimer immunogen. When immunized with gp150 complexed to BMS-529, rhesus macaques showed neutralization against tier 2 pseudoviruses with targeted glycan deletion and high-mannose glycan enrichment. These results demonstrated that stabilization of Env trimer conformation with BMS-529 improved the immunogenicity of select chimeric SOSIP trimers and elicited tier 2 neutralizing antibodies of higher potency than noncomplexed trimers.IMPORTANCE Soluble forms of HIV-1 envelope trimers exhibit conformational heterogeneity and undergo CD4-induced (CD4i) exposure of epitopes of non-neutralizing antibodies that can potentially hinder induction of broad neutralizing antibody responses. These limitations have been mitigated through recent structure-guided approaches and include trimer-stabilizing mutations that resist trimer conformational transition and exposure of CD4i epitopes. The use of small-molecule viral inhibitors that allosterically block CD4 binding represents an alternative strategy for stabilizing Env trimer in the pre-CD4-triggered state of both soluble and membrane-bound trimers. In this study, we report that the viral entry inhibitor BMS-626529 restricts trimer conformational transition and improves the immunogenicity of select Env trimer immunogens.

Closing and Opening Holes in the Glycan Shield of HIV-1 Envelope Glycoprotein SOSIP Trimers Can Redirect the Neutralizing Antibody Response to the Newly Unmasked Epitopes.

In HIV-1 vaccine research, native-like, soluble envelope glycoprotein SOSIP trimers are widely used for immunizing animals. The epitopes of autologous neutralizing antibodies (NAbs) induced by the BG505 and B41 SOSIP trimers in rabbits and macaques have been mapped to a few holes in the glycan shields that cover most of the protein surfaces. For BG505 trimers, the dominant autologous NAb epitope in rabbits involves residues that line a cavity caused by the absence of a glycan at residue 241. Here, we blocked this epitope in BG505 SOSIPv4.1 trimer immunogens by knocking in an N-linked glycan at residue 241. We then opened holes elsewhere on the trimer by knocking out single N-linked glycans at residues 197, 234, 276, 332, and 355 and found that NAb responses induced by the 241-glycan-bearing BG505 trimers were frequently redirected to the newly opened sites. The strongest evidence for redirection of the NAb response to neoepitopes, through the opening and closing of glycan holes, was obtained from trimer immunogen groups with the highest occupancy of the N241 site. We also attempted to knock in the N289-glycan to block the sole autologous NAb epitope on the B41 SOSIP.v4.1 trimer. Although a retrospective analysis showed that the new N289-glycan site was substantially underoccupied, we found some evidence for redirection of the NAb response to a neoepitope when this site was knocked in and the N356-glycan site knocked out. In neither study, however, was redirection associated with increased neutralization of heterologous tier 2 viruses.IMPORTANCE Engineered SOSIP trimers mimic envelope-glycoprotein spikes, which stud the surface of HIV-1 particles and mediate viral entry into cells. When used for immunizing test animals, they elicit antibodies that neutralize resistant sequence-matched HIV-1 isolates. These neutralizing antibodies recognize epitopes in holes in the glycan shield that covers the trimer. Here, we added glycans to block the most immunogenic neutralization epitopes on BG505 and B41 SOSIP trimers. In addition, we removed selected other glycans to open new holes that might expose new immunogenic epitopes. We immunized rabbits with the various glycan-modified trimers and then dissected the specificities of the antibody responses. Thus, in principle, the antibody response might be diverted from one site to a more cross-reactive one, which would help in the induction of broadly neutralizing antibodies by HIV-1 vaccines based on envelope glycoproteins.

Antibody Responses Elicited by Immunization with BG505 Trimer Immune Complexes.

Immune complex (IC) vaccines have been successfully used to increase immune responses against various pathogens, including HIV-1. Additionally, IC vaccines can induce qualitatively different antibody responses, with distinct antigenic specificities compared to the same antigens used alone. Here we measured the HIV-1-specific antibody responses in female New Zealand White rabbits after immunization with ICs made from BG505 SOSIP.664 trimers (BG505 trimers) and three rabbit monoclonal antibodies (MAbs) with different neutralization profiles. Two of the MAbs were specific for a hole in the glycan shield of the BG505 trimer, while the third, which bound less avidly, was specific for determinants at the gp41-gp120 interface. We found that immunization with one of the glycan-hole-specific ICs resulted in lower levels of trimer-binding antibodies compared to vaccination with the uncomplexed trimer, and that ICs made using either of the glycan-hole-specific MAbs resulted in lower rates of anti-trimer antibody decay. We concluded that ICs based on MAbs that bound to the immunodominant glycan hole epitope likely diverted antibody responses, to some extent, away from this site and to other regions of the trimer. However, this outcome was not accompanied by a widening of the breadth or an increase in the potency of neutralizing antibody responses compared with uncomplexed trimers.IMPORTANCE Immunodominant epitopes may suppress immune responses to more desirable determinants, such as those that elicit potentially protective neutralizing antibody responses. To overcome this problem, we attempted to mask immunodominant glycan holes by immunizing rabbits with ICs consisting of the BG505 SOSIP.664 gp140 trimer and MAbs that targeted the glycan holes. We found that IC vaccination likely diverted antibody responses, to some extent, away from the glycan holes and toward other regions of the trimer. IC vaccination resulted in slower decay of HIV-1-specific antibodies than did immunization with uncomplexed trimer. We did not observe a widening of the breadth or an increase in the potency of neutralizing antibody responses compared to uncomplexed trimers. Our results suggest that selective epitope dampening of BG505 trimers by ICs is rather ineffective. However, IC vaccination may represent a novel means of increasing the duration of vaccine-induced antibody responses.

An HIV-1 Broadly Neutralizing Antibody from a Clade C-Infected Pediatric Elite Neutralizer Potently Neutralizes the Contemporaneous and Autologous Evolving Viruses.

Broadly neutralizing antibodies (bNAbs) have demonstrated protective effects against HIV-1 in primate studies and recent human clinical trials. Elite neutralizers are potential candidates for isolation of HIV-1 bNAbs. The coexistence of bNAbs such as BG18 with neutralization-susceptible autologous viruses in an HIV-1-infected adult elite controller has been suggested to control viremia. Disease progression is faster in HIV-1-infected children than in adults. Plasma bNAbs with multiple epitope specificities are developed in HIV-1 chronically infected children with more potency and breadth than in adults. Therefore, we evaluated the specificity of plasma neutralizing antibodies of an antiretroviral-naive HIV-1 clade C chronically infected pediatric elite neutralizer, AIIMS_330. The plasma antibodies showed broad and potent HIV-1 neutralizing activity with >87% (29/33) breadth, a median inhibitory dilution (ID50) value of 1,246, and presence of N160 and N332 supersite-dependent HIV-1 bNAbs. The sorting of BG505.SOSIP.664.C2 T332N gp140 HIV-1 antigen-specific single B cells of AIIMS_330 resulted in the isolation of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01. The AIIMS-P01 neutralized 67% of HIV-1 cross-clade viruses, exhibited substantial indels despite limited somatic hypermutations, interacted with native-like HIV-1 trimer as observed in negative stain electron microscopy, and demonstrated high binding affinity. In addition, AIIMS-P01 neutralized the coexisting and evolving autologous viruses, suggesting the coexistence of vulnerable autologous viruses and HIV-1 bNAbs in the AIIMS_330 pediatric elite neutralizer. Such pediatric elite neutralizers can serve as potential candidates for isolation of novel HIV-1 pediatric bNAbs and for understanding the coevolution of virus and host immune response.IMPORTANCE More than 50% of the HIV-1 infections globally are caused by clade C viruses. To date, there is no effective vaccine to prevent HIV-1 infection. Based on the structural information of the currently available HIV-1 bNAbs, attempts are under way to design immunogens that can elicit correlates of protection upon vaccination. Here, we report the isolation and characterization of an HIV-1 N332 supersite-dependent bNAb, AIIMS-P01, from a clade C chronically infected pediatric elite neutralizer. The N332 supersite is an important epitope and is one of the current HIV-1 vaccine targets. AIIMS-P01 potently neutralized the contemporaneous and autologous evolving viruses and exhibited substantial indels despite low somatic hypermutations. Taken together with the information on infant bNAbs, further isolation and characterization of bNAbs contributing to the plasma breadth in HIV-1 chronically infected children may help provide a better understanding of their role in controlling HIV-1 infection.

SOSIP Changes Affect Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Conformation and CD4 Engagement.

The entry of human immunodeficiency virus into host cells is mediated by the envelope glycoprotein (Env) trimeric spike, which consists of three exterior gp120 subunits and three transmembrane gp41 subunits. The trimeric Env undergoes extensive conformational rearrangement upon interaction with the CD4 receptor, transitioning from the unliganded, "closed" State 1 to more-open downstream State 2 and State 3 conformations. Changes in "restraining" amino acid residues, such as leucine 193 and isoleucine 423, destabilize State 1 Env, which then assumes entry-competent, downstream conformations. The introduction of an artificial disulfide bond linking the gp120 and gp41 subunits (SOS) in combination with the I559P (IP) change has allowed structural characterization of soluble gp140 (sgp140) trimers. The conformation of these SOSIP-stabilized sgp140 trimers has been suggested to represent the closed native State 1 conformation. Here we compare the impact on the membrane Env conformation of the SOSIP changes with that of the well-characterized changes (L193R and I423A) that shift Env to downstream States 2 and 3. The results presented here suggest that the SOSIP changes stabilize Env in a conformation that differs from State 1 but also from the downstream Env conformations stabilized by L193R or I423A.IMPORTANCE The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer is triggered by receptor binding to mediate the entry of the virus into cells. Most structural studies of Env trimers have utilized truncated soluble gp140 Envs stabilized with the I559P and SOS changes. Here we present evidence indicating that these stabilizing changes have a profound impact on the conformation of Env, moving Env away from the native pretriggered Env conformation. Our studies underscore the need to acquire structural information on the pretriggered Env conformation, which is recognized by most broadly reactive neutralizing antibodies.

Characterization of a stable HIV-1 B/C recombinant, soluble, and trimeric envelope glycoprotein (Env) highly resistant to CD4-induced conformational changes.

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.

cGMP production and analysis of BG505 SOSIP.664, an extensively glycosylated, trimeric HIV-1 envelope glycoprotein vaccine candidate.

We describe the properties of BG505 SOSIP.664 HIV-1 envelope glycoprotein trimers produced under current Good Manufacturing Practice (cGMP) conditions. These proteins are the first of a new generation of native-like trimers that are the basis for many structure-guided immunogen development programs aimed at devising how to induce broadly neutralizing antibodies (bNAbs) to HIV-1 by vaccination. The successful translation of this prototype demonstrates the feasibility of producing similar immunogens on an appropriate scale and of an acceptable quality for Phase I experimental medicine clinical trials. BG505 SOSIP.664 trimers are extensively glycosylated, contain numerous disulfide bonds and require proteolytic cleavage, all properties that pose a substantial challenge to cGMP production. Our strategy involved creating a stable CHO cell line that was adapted to serum-free culture conditions to produce envelope glycoproteins. The trimers were then purified by chromatographic methods using a 2G12 bNAb affinity column and size-exclusion chromatography. The chosen procedures allowed any adventitious viruses to be cleared from the final product to the required extent of >12 log10 . The final cGMP production run yielded 3.52 g (peptidic mass) of fully purified trimers (Drug Substance) from a 200 L bioreactor, a notable yield for such a complex glycoprotein. The purified trimers were fully native-like as judged by negative-stain electron microscopy, and were stable over a multi-month period at room temperature or below and for at least 1 week at 50°C. Their antigenicity, disulfide bond patterns, and glycan composition were consistent with trimers produced on a research laboratory scale. The methods reported here should pave the way for the cGMP production of other native-like Env glycoprotein trimers of various designs and genotypes.