The breadth of HIV-1 neutralizing antibodies depends on the conservation of key sites in their epitopes.
ABSTRACT: Developing HIV-1 vaccines that trigger broadly neutralizing antibodies (bnAbs) is a priority as bnAbs are considered key to elicitation of a protective immune response. To investigate whether the breadth of a neutralizing antibody (nAb) depended on the conservation of its epitope among circulating viruses, we examined Antibody:Envelope (Ab:Env) interactions and worldwide Env diversity. We found that sites corresponding to bnAb epitopes were as variable as other accessible, non-hypervariable Env sites (p = 0.50, Mann-Whitney U-test) with no significant relationship between epitope conservation and neutralization breadth (Spearman's ? = -0.44, adjusted p = 0.079). However, when accounting for key sites in the Ab:Env interaction, we showed that the broadest bnAbs targeted more conserved epitopes (Spearman's ? = -0.70, adjusted p = 5.0e-5). Neutralization breadth did not stem from the overall conservation of Ab epitopes but depended instead on the conservation of key sites of the Ab:Env interaction, revealing a mechanistic basis for neutralization breadth that could be exploited for vaccine design.
Project description:The development of HIV broadly neutralizing antibodies (bNAbs) has previously been shown to be associated with viral evolution and high levels of genetic diversity in the HIV envelope (Env) glycoprotein. However, few studies have examined Env evolution in those who fail to develop neutralization breadth in order to assess whether bNAbs result from distinct evolutionary pathways. We compared Env evolution in eight HIV-1-infected participants who developed bNAbs to six donors with similar viral loads who did not develop bNAbs over three years of infection. We focused on Env V1V2 and C3V4, as these are major targets for both strain-specific neutralizing antibodies (nAbs) and bNAbs. Overall evolutionary rates (ranging from 9.92?×?10-3 to 4.1?×?10-2 substitutions/site/year) and viral diversity (from 1.1% to 6.5%) across Env, and within targeted epitopes, did not distinguish bNAb donors from non-bNAb donors. However, bNAb participants had more positively selected residues within epitopes than those without bNAbs, and several of these were common among bNAb donors. A comparison of the kinetics of strain-specific nAbs and bNAbs indicated that selection pressure at these residues increased with the onset of breadth. These data suggest that highly targeted viral evolution rather than overall envelope diversity is associated with neutralization breadth. The association of shared positively selected sites with the onset of breadth highlights the importance of diversity at specific positions in these epitopes for bNAb development, with implications for the development of sequential and cocktail immunization strategies.IMPORTANCE Millions of people are still being infected with HIV decades after the first recognition of the virus. Currently, no vaccine is able to elicit bNAbs that will prevent infection by global HIV strains. Several studies have implicated HIV Env diversity in the development of breadth. However, Env evolution in individuals who fail to develop breadth despite mounting potent strain-specific neutralizing responses has not been well defined. Using longitudinal neutralization, epitope mapping, and sequence data from 14 participants, we found that overall measures of viral diversity were similar in all donors. However, the number of positively selected sites within Env epitopes was higher in bNAb participants than in strain-specific donors. We further identified common sites that were positively selected as bNAbs developed. These data indicate that while viral diversity is required for breadth, this should be highly targeted to specific residues to shape the elicitation of bNAbs by vaccination.
Project description:HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a "single" agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs). The optimal Bi-ScFv crosslinks adjacent protomers within one HIV-1 Env spike and has greater neutralization breadth than its parental bNAbs. Furthermore, the combination of this Bi-ScFv with a third bNAb recognizing the Env membrane proximal external region (MPER) results in a trispecific bNAb, which has nearly pan-isolate neutralization breadth and high potency. Thus, multispecific antibodies combining functional moieties of bNAbs could achieve outstanding neutralization capacity with augmented avidity.
Project description:The high-mannose patch on HIV Env is a preferred target for broadly neutralizing antibodies (bnAbs), but to date, no vaccination regimen has elicited bnAbs against this region. Here, we present the development of a bnAb lineage targeting the high-mannose patch in an HIV-1 subtype-C-infected donor from sub-Saharan Africa. The Abs first acquired autologous neutralization, then gradually matured to achieve breadth. One Ab neutralized >47% of HIV-1 strains with only ?11% somatic hypermutation and no insertions or deletions. By sequencing autologous env, we determined key residues that triggered the lineage and participated in Ab-Env coevolution. Next-generation sequencing of the Ab repertoire showed an early expansive diversification of the lineage followed by independent maturation of individual limbs, several of them developing notable breadth and potency. Overall, the findings are encouraging from a vaccine standpoint and suggest immunization strategies mimicking the evolution of the entire high-mannose patch and promoting maturation of multiple diverse Ab pathways.
Project description:Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.
Project description:Broadly neutralizing antibodies (bNAbs) can prevent and control an HIV-1 infection, but their breadth is invariably too limited for use as monotherapy. To address this problem, bi- and trispecific antibody-like constructs have been developed. These engineered antibodies typically have greater breadth than the native bNAbs from which they were derived, but they are not more potent because they do not, in most cases, simultaneously engage more than a single epitope of the HIV-1 envelope glycoprotein (Env). Here, we describe a new class of bispecific antibodies targeting the V2-glycan (apex) and V3-glycan regions of the HIV-1 envelope glycoprotein (Env). Specifically, bispecific antibodies with a single-chain (scFv) form of the CAP256.VRC26.25 V2-glycan (apex) antibody on one antibody arm and a full V3-glycan Fab on the other arm neutralizes more HIV-1 isolates than the bNAbs from which they were derived. Moreover, these bispecific antibodies are markedly more potent than their parental bNAbs, likely because they simultaneously engage both the apex and V3-glycan epitopes of Env. Our data show that simultaneous engagement of two critical epitopes of a single Env trimer can markedly increase the potency of a bispecific antibody.IMPORTANCE Broadly neutralizing antibodies (bNAbs) can prevent a new HIV-1 infection and can at least temporarily suppress an established infection. However, antibody-resistant viruses rapidly emerge in infected persons treated with any single bNAb. Several bispecific antibodies have been developed to increase the breadth of these antibodies, but typically only one arm of these bispecific constructs binds the HIV-1 envelope glycoprotein trimer (Env). Here, we develop and characterize bispecific constructs based on well-characterized V2-glycan and V3-glycan bNAbs and show that at least one member of this class is more potent than its parental antibodies, indicating that they can simultaneously bind both of these epitopes of a single Env trimer. These data show that bispecific antibody-like proteins can achieve greater neutralization potency than the bNAbs from which they were derived.
Project description:Broadly neutralizing antibodies (bNAbs) are rarely elicited by current human immunodeficiency virus type 1 (HIV-1) vaccine designs, but the presence of bNAbs in naturally infected individuals may be associated with high plasma viral loads, suggesting that the magnitude, duration, and diversity of viral exposure may contribute to the development of bNAbs. Here, we report the isolation and characterization of a panel of human monoclonal antibodies (mAbs) from two subjects who developed broadly neutralizing autologous antibody responses during HIV-1 infection. In both subjects, we identified collections of mAbs that exhibited specificity only to a few autologous envelopes (Envs), with some mAbs exhibiting specificity only to a subset of Envs within the quasispecies of a particular sample at one time point. Neutralizing antibodies (NAbs) isolated from these subjects mapped mostly to epitopes in the Env V3 loop region and the CD4 binding site. None of the individual neutralizing mAbs recovered exhibited the cumulative breadth of neutralization present in the serum of the subjects. Surprisingly, however, the activity of polyclonal mixtures comprising individual mAbs that each possessed limited neutralizing activity, could achieve increased breadth of neutralizing activity against autologous isolates. While a single broadly neutralizing antibody targeting one epitope can mediate neutralization breadth, the findings presented here suggest that a cooperative polyclonal process mediated by diverse antibodies with more limited breadth targeting multiple epitopes also can achieve neutralization breadth against HIV-1.
Project description:Here we report the construction, antigenicity and initial immunogenicity testing of DNA and modified vaccinia Ankara (MVA) vaccines expressing virus-like particles (VLPs) displaying sequential clade C Envelopes (Envs) that co-evolved with the elicitation of broadly neutralizing antibodies (bnAbs) to the CD4 binding site (CD4bs) in HIV-infected individual CH0505. The VLP-displayed Envs showed reactivity for conformational epitopes displayed on the receptor-binding form of Env. Two inoculations of the DNA-T/F vaccine, followed by 3 inoculations of the MVA-T/F vaccine and a final inoculation of the MVA-T/F plus a gp120-T/F protein vaccine elicited nAb to the T/F virus in 2 of 4 rhesus macaques (ID50 of ~175 and ~30). Neutralizing Ab plateaued at 100% neutralization and mapped to the CD4bs like the bnAbs elicited in CH0505. The nAb did not have breadth for other tier 2 viruses. Immunizations with T/F followed by directed-lineage vaccines, both with and without co-delivery of directed-lineage gp120 boosts, failed to elicit tier 2 neutralizing Ab for the CD4bs. Thus, pulsed exposures to DNA and MVA-expressed VLPs plus gp120 protein of a T/F Env can induce autologous tier 2 nAbs to the CD4bs.
Project description:Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape both delineate pathways of resistance to these fusion peptide-directed antibodies and provide a strategy to improve the breadth or potency of future vaccine-induced antibodies against Env's fusion peptide.
Project description:Broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) suppress viremia in animal models of HIV-1 and humans. To achieve potent activity without the emergence of viral escape mutants, co-administration of different bNAbs is necessary to target distinct epitopes essential for viral fitness. Here, we report the development of bispecific anti-Env neutralizing antibodies (biNAbs) with potent activity. Synergistic activity of biNAbs was achieved by combining an engineered hinge domain of IgG3 to increase Fab domain flexibility necessary for hetero-bivalent binding to the Env trimer while retaining the functional properties of the IgG1-Fc. Compared to unmodified biNAbs, hinge domain variants exhibited substantially improved neutralization activity, with particular combinations showing evidence of synergistic neutralization potency in vitro and enhanced in vivo therapeutic activity in HIV-1-infected humanized mice. These findings suggest innovative strategies for generating biNAbs with enhanced neutralization breadth and potency, representing ideal candidate molecules for the control of HIV-1 infection.
Project description:HIV-1 neutralization requires Ab accessibility to the functional envelope glycoprotein (Env) spike. We recently reported the isolation of previously unidentified vaccine-elicited, CD4 binding site (CD4bs)-directed mAbs from rhesus macaques immunized with soluble Env trimers, indicating that this region is immunogenic in the context of subunit vaccination. To elucidate the interaction of the trimer-elicited mAbs with gp120 and their insufficient interaction with the HIV-1 primary isolate spike, we crystallized the Fab fragments of two mAbs, GE136 and GE148. Alanine scanning of their complementarity-determining regions, coupled with epitope scanning of their epitopes on gp120, revealed putative contact residues at the Ab/gp120 interface. Docking of the GE136 and GE148 Fabs to gp120, coupled with EM reconstructions of these nonbroadly neutralizing mAbs (non-bNAbs) binding to gp120 monomers and EM modeling to well-ordered trimers, suggested Ab approach to the CD4bs by a vertical angle of access relative to the more lateral mode of interaction used by the CD4bs-directed bNAbs VRC01 and PGV04. Fitting the structures into the available cryo-EM native spike density indicated clashes between these two vaccine-elicited mAbs and the topside variable region spike cap, whereas the bNAbs duck under this quaternary shield to access the CD4bs effectively on primary HIV isolates. These results provide a structural basis for the limited neutralizing breadth observed by current vaccine-induced, CD4bs-directed Abs and highlight the need for better ordered trimer immunogens. The analysis presented here therefore provides valuable information to guide HIV-1 vaccine immunogen redesign.