Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in ∼20% of HIV-1–infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man₅GlcNAc₂ glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.It is widely believed that a key characteristic of an effective HIV-1 vaccine would be its ability to induce broadly neutralizing antibodies (BnAbs). Known BnAbs have been shown to target conserved HIV-1 envelope (Env) regions including glycans, the glycoprotein 41 (gp41) membrane-proximal region, the gp120 variable loop 1/2 (V1V2), and the CD4 binding site (CD4bs) (1–7). Most mature BnAbs have one or more unusual features such as long heavy-chain third complementarity-determining regions, polyreactivity for non–HIV-1 antigens, and high levels of somatic mutations (2, 7, 8). In particular, CD4bs BnAbs have extremely high levels of somatic mutations, suggesting complex or prolonged maturation pathways (2–5). Adding to the challenge has been the difficulty in achieving binding of proposed antigens to germ-line or unmutated common ancestors (UCAs). Binding to BnAb UCAs would be a desirable characteristic for putative immunogens intended to induce BnAbs (5, 9–13).Immunization of humans with Env proteins has not resulted in high plasma titers of BnAbs (14, 15). Rather, dominant strain-specific neutralizing epitopes have selectively been induced. This was most clearly seen in the ALVAC/AIDSVAX RV144 HIV-1 vaccine efficacy trial, in which Env immunogens 92TH023 and A244 CRFAE_01 gp120s both expressed a dominant linear V2 epitope and bound with high-nanomolar affinity to the glycan-dependent V1V2 BnAbs PG9 and CH01 (16). Although both linear and glycan-dependent V2 epitopes were expressed on the A244 immunogen, the dominant V2 plasma antibody responses in this trial were targeted to linear V2 epitopes and not to the glycan-dependent BnAb epitope (14–16). A series of mAbs, the prototype of which is the mAb CH58, has been isolated from RV144 vaccines and shown to bind to linear V2 epitopes that include lysine 169 (16). However, they are strain-specific and only neutralize laboratory-adapted but not primary isolate HIV-1 strains (16). Although PG9 and CH01 V1V2 BnAbs also bind to V2 K169 and surrounding amino acids, they also bind to high-mannose glycans at N¹⁵⁶ and N¹⁶⁰ (17). Crystal structures of the CH58 antibody bound to V2 peptides demonstrated the V2 structure around K169 to be helical (16), whereas the crystal structure of the PG9 antibody with a V1V2 scaffold showed the same polypeptide region in a β-strand conformation (17).The rationale that undergirded the studies described below envisioned that an optimal immunogen for the V1V2 BnAb peptide–glycan envelope region would be one that presented a chemically homogeneous entity that binds to V1V2 BnAbs with high affinity. In addition, an optimal immunogen for the V1V2 BnAb site would be one that binds with high affinity to V1V2 BnAb UCAs. Recently, in a preliminary disclosure, we described chemically synthesized glycopeptides of the HIV-1 Env V1V2 148–184 aa region with Man₃GlcNAc₂ or Man₅GlcNAc₂ glycan units at N¹⁵⁶ and N¹⁶⁰ (18). It was found that these homogeneous glycopeptide constructs with oligomannose units bound avidly to the V1V2 BnAb PG9. In this study, we report that the disulfide-linked dimeric forms of these glycopeptides bound preferentially to V1V2 BnAb mature antibodies (PG9 and CH01) over the V2 strain-specific mAb CH58, to which the binding was minimal. Importantly, the V1V2 peptide–glycans also bound to both PG9 and CH01 V1V2 BnAb UCAs, thus providing a strong rationale for their evaluation as experimental immunogens.