Immune escape mutations selected by neutralizing antibodies in natural HIV-1 infection can alter coreceptor usage repertoire of the transmitted/founder virus

The ability of HIV-1 to evade neutralizing antibodies (NAbs) in vivo is well demonstrated, but the impact of NAb escape mutations on HIV-1 phenotype other than immune escape itself has rarely been studied. Here, we show that immune escape mutations selected by V3-glycan specific NAbs in vivo can alter the coreceptor usage repertoire of the transmitted/founder (T/F) HIV-1. In a participant developed V3-glycan NAb response, naturally selected mutations at the V3 N301 and N332 glycan sites abrogated CCR8 usage while conferred APJ usage on the cognate T/F strain. Mutations at the N301 glycan also impaired CCR3 usage and partially compromised the efficiency in using CCR5, which could be fully restored by a single escape mutation at the N332 glycan site. Our study demonstrates the link between NAb escape and coreceptor usage alteration in natural HIV-1 infection and indicates that NAb response could drive virus entry tropism evolution in vivo.

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

ABSTRACTIn 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.IMPORTANCEEngineered 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.

Trispecific broadly neutralizing HIV antibodies mediate potent SHIV protection in macaques

The development of an effective AIDS vaccine has been challenging because of viral genetic diversity and the difficulty of generating broadly neutralizing antibodies (bnAbs). We engineered trispecific antibodies (Abs) that allow a single molecule to interact with three independent HIV-1 envelope determinants: the CD4 binding site, the membrane-proximal external region (MPER), and the V1V2 glycan site. Trispecific Abs exhibited higher potency and breadth than any previously described single bnAb, showed pharmacokinetics similar to those of human bnAbs, and conferred complete immunity against a mixture of simian-human immunodeficiency viruses (SHIVs) in nonhuman primates, in contrast to single bnAbs. Trispecific Abs thus constitute a platform to engage multiple therapeutic targets through a single protein, and they may be applicable for treatment of diverse diseases, including infections, cancer, and autoimmunity.

HIV-1 Glycan Density Drives the Persistence of the Mannose Patch within an Infected Individual

ABSTRACTThe HIV envelope glycoprotein (Env) is extensively modified with host-derived N-linked glycans. The high density of glycosylation on the viral spike limits enzymatic processing, resulting in numerous underprocessed oligomannose-type glycans. This extensive glycosylation not only shields conserved regions of the protein from the immune system but also acts as a target for anti-HIV broadly neutralizing antibodies (bnAbs). In response to the host immune system, the HIV glycan shield is constantly evolving through mutations affecting both the positions and numbers of potential N-linked glycosylation sites (PNGSs). Here, using longitudinal Env sequences from a clade C-infected individual (CAP256), we measured the impact of the shifting glycan shield during HIV infection on the abundance of oligomannose-type glycans. By analyzing the intrinsic mannose patch from a panel of recombinant CAP256 gp120s displaying high protein sequence variability and changes in PNGS number and positioning, we show that the intrinsic mannose patch persists throughout the course of HIV infection and correlates with the number of PNGSs. This effect of the glycan density on the processing state was also supported by the analysis of a cross-clade panel of recombinant gp120 glycoproteins. Together, these observations underscore the importance of glycan clustering for the generation of carbohydrate epitopes for anti-HIV bnAbs. The persistence of the intrinsic mannose patch over the course of HIV infection further highlights this epitope as an important target for HIV vaccine strategies.IMPORTANCEDevelopment of an HIV vaccine is critical for control of the HIV pandemic, and elicitation of broadly neutralizing antibodies (bnAbs) is likely to be a key component of a successful vaccine response. The HIV envelope glycoprotein (Env) is covered in an array of host-derived N-linked glycans often referred to as the glycan shield. This glycan shield is a target for many of the recently isolated anti-HIV bnAbs and is therefore under constant pressure from the host immune system, leading to changes in both glycan site frequency and location. This study aimed to determine whether these genetic changes impacted the eventual processing of glycans on the HIV Env and the susceptibility of the virus to neutralization. We show that despite this variation in glycan site positioning and frequency over the course of HIV infection, the mannose patch is a conserved feature throughout, making it a stable target for HIV vaccine design.

Anti-V3/Glycan and Anti-MPER Neutralizing Antibodies, but Not Anti-V2/Glycan Site Antibodies, Are Strongly Associated with Greater Anti-HIV-1 Neutralization Breadth and Potency

ABSTRACTThe membrane-proximal external region (MPER), the V2/glycan site (initially defined by PG9 and PG16 antibodies), and the V3/glycans (initially defined by PGT121–128 antibodies) are targets of broadly neutralizing antibodies and potential targets for anti-HIV-1 antibody-based vaccines. Recent evidence shows that antibodies with moderate neutralization breadth are frequently attainable, with 50% of sera from chronically infected individuals neutralizing ≥50% of a large, diverse set of viruses. Nonetheless, there is little systematic information addressing which specificities are preferentially targeted among such commonly found, moderately broadly neutralizing sera. We explored associations between neutralization breadth and potency and the presence of neutralizing antibodies targeting the MPER, V2/glycan site, and V3/glycans in sera from 177 antiretroviral-naive HIV-1-infected (>1 year) individuals. Recognition of both MPER and V3/glycans was associated with increased breadth and potency. MPER-recognizing sera neutralized 4.62 more panel viruses than MPER-negative sera (95% prediction interval [95% PI], 4.41 to 5.20), and V3/glycan-recognizing sera neutralized 3.24 more panel viruses than V3/glycan-negative sera (95% PI, 3.15 to 3.52). In contrast, V2/glycan site-recognizing sera neutralized only 0.38 more panel viruses (95% PI, 0.20 to 0.45) than V2/glycan site-negative sera and no association between V2/glycan site recognition and breadth or potency was observed. Despite autoreactivity of many neutralizing antibodies recognizing MPER and V3/glycans, antibodies to these sites are major contributors to neutralization breadth and potency in this cohort. It may therefore be appropriate to focus on developing immunogens based upon the MPER and V3/glycans.IMPORTANCEPrevious candidate HIV vaccines have failed either to induce wide-coverage neutralizing antibodies or to substantially protect vaccinees. Therefore, current efforts focus on novel approaches never before successfully used in vaccine design, including modeling epitopes. Candidate immunogen models identified by broadly neutralizing antibodies include the membrane-proximal external region (MPER), V3/glycans, and the V2/glycan site. Autoreactivity and polyreactivity of anti-MPER and anti-V3/glycan antibodies are thought to pose both direct and indirect barriers to achieving neutralization breadth. We found that antibodies to the MPER and the V3/glycans contribute substantially to neutralization breadth and potency. In contrast, antibodies to the V2/glycan site were not associated with neutralization breadth/potency. This suggests that the autoreactivity effect is not critical and that the MPER and the V3/glycans should remain high-priority vaccine candidates. The V2/glycan site result is surprising because broadly neutralizing antibodies to this site have been repeatedly observed. Vaccine design priorities should shift toward the MPER and V3/glycans.

Two Classes of Broadly Neutralizing Antibodies within a Single Lineage Directed to the High-Mannose Patch of HIV Envelope

ABSTRACTThe high-mannose patch of human immunodeficiency virus (HIV) envelope (Env) elicits broadly neutralizing antibodies (bnAbs) during natural infection relatively frequently, and consequently, this region has become a major target of vaccine design. However, it has also become clear that antibody recognition of the region is complex due, at least in part, to variability in neighboring loops and glycans critical to the epitopes. bnAbs against this region have some shared features and some distinguishing features that are crucial to understand in order to design optimal immunogens that can induce different classes of bnAbs against this region. Here, we compare two branches of a single antibody lineage, in which all members recognize the high-mannose patch. One branch (prototype bnAb PGT128) has a 6-amino-acid insertion in CDRH2 that is crucial for broad neutralization. Antibodies in this branch appear to favor a glycan site at N332 on gp120, and somatic hypermutation is required to accommodate the neighboring V1 loop glycans and glycan heterogeneity. The other branch (prototype bnAb PGT130) lacks the CDRH2 insertion. Antibodies in this branch are noticeably effective at neutralizing viruses with an alternate N334 glycan site but are less able to accommodate glycan heterogeneity. We identify a new somatic variant within this branch that is predominantly dependent on N334. The crystal structure of PGT130 offers insight into differences from PGT128. We conclude that different immunogens may be required to elicit bnAbs that have the optimal characteristics of the two branches of the lineage described.IMPORTANCEDevelopment of an HIV vaccine is of vital importance for prevention of new infections, and it is thought that elicitation of HIV bnAbs will be an important component of an effective vaccine. Increasingly, bnAbs that bind to the cluster of high-mannose glycans on the HIV envelope glycoprotein, gp120, are being highlighted as important templates for vaccine design. In particular, bnAbs from IAVI donor 36 (PGT125 to PGT131) have been shown to be extremely broad and potent. Combination of these bnAbs enhanced neutralization breadth considerably, suggesting that an optimal immunogen should elicit several antibodies from this family. Here we study the evolution of this antibody family to inform immunogen design. We identify two classes of bnAbs that differ in their recognition of the high-mannose patch and show that different immunogens may be required to elicit these different classes.