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

Abstract

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.

Fig. 1.

V1V2 glycopeptides form disulfide-linked dimers. (A) SDS/PAGE analysis of V1V2 glycopeptides showing dimers under nonreducing and monomers under reducing conditions. Lanes from left to right are MW stds I, MW stds II (6.5, 14.2, 26.6 kDa), glycopeptides Man3 nonreduced, Man3 reduced, Man5 nonreduced, and Man5 reduced. Data are representative of at least three independent experiments. (B) Size-exclusion chromatography of oxidized Man₃ (B) and Man₅ (C) -derivatized glycopeptides showing a single dimeric peak. Molecular masses of protein standards are marked. The elution volume (10.87 mL) of the Man₃ C157A mutant, which does not form disulfide-linked dimers, is marked with an arrow and asterisk. mAU, milli absorbance units.

Fig. 2.

Selective binding of V1V2 BnAbs to mannose-derivatized V1V2 glycopeptides but not to aglycone or GlcNAc₂ V1V2 peptides. SPR curves showing preferential binding of PG9 and CH01 BnAbs to Man₅ (A) and Man₃ (B) GlcNAc₂ V1V2 glycopeptides but not to GlcNAc₂ (C) and aglycone (D) peptides. By contrast, V2 mAbs CH58 and CH59 bound to both GlcNAc₂ (C) and aglycone (D) V1V2 peptides. Each V1V2 peptide was oxidized by solubilization in DMSO and injected over the indicated mAb at 50 µg/mL. Data shown are after reference subtraction of nonspecific signal measured over the control mAb (Synagis). Binding data are representative of at least three experiments for Man₅ and Man₃ V1V2 peptides and two experiments for GlcNAc₂ and aglycone V1V2.

Fig. 3.

Circular dichroism analyses of the secondary structure of the synthetic V1V2 peptides. V1V2 peptides derivatized with oligomannose units Man₅ (A) or Man₃ (B) GlcNAc₂ V1V2 or only the proximal GlcNAc₂-V1V2 (C) peptides show predominantly ordered secondary structure with β-strand and helical conformation. In D, Man₃ and Man₅ V1V2 glycopeptides were oxidized by iodine treatment and CD analysis was performed as above. CD spectra of each of the V1V2 peptides were taken at least two times. V1V2 peptides were solubilized in DMSO and allowed to fully dimerize in 20% DMSO/phosphate buffer for about 20 h. The CD spectrum deconvolution analysis (K2D3) of the Man₅ glycopeptide gave an estimated 23% β-strand, Man₃ V1V2 glycopeptide gave 33% β-strand, and GlcNAc₂ V1V2 glycopeptide gave 17% β-strand.

Fig. 4.

Circular dichroism secondary structure and antigenicity of C157A mutant Man₃ V1V2 glycopeptide. (A) CD spectrum of C157A Man₃ GlcNAc₂ mutant showing glycopeptide in random-coil conformation and the lack of signature β-sheet features. (B) The CH58 mAb but not the V1V2 BnAbs (PG9 and CH01) bound to the C157A mutant V1V2 Man₃ GlcNAc₂ peptide (injected at 50 µg/mL). A second experiment in which Man₃ C157A glycopeptide was initially solubilized in 20% DMSO (as described in Materials and Methods) gave similar binding to the CH58 mAb but not to either PG9 or CH01 V1V2 BnAbs.

Fig. 5.

Surface plasmon resonance measurements of PG9 and CH01 BnAb binding to dimerized V1V2 glycopeptides. V1V2 BnAbs PG9 (A and B) and CH01 (C and D) binding to varying concentrations of Man₅ GlcNAc₂ (A and C) and Man₃ GlcNAc₂ V1V2 (B and D). CH58 mAb binding to Man₅ GlcNAc2 (E) and Man₃ GlcNAc2 (F). V1V2 glycopeptides were injected at concentrations ranging from 1 to 10 µg/mL for PG9 and CH01, and from 1 to 50 µg/mL for the CH58 mAb. Data are representative of at least three measurements for PG9 and CH01 binding to either the Man₅ or Man₃ V1V2 glycopeptides. V1V2 peptides were solubilized in 20% DMSO overnight to allow complete dimer formation.

Fig. 6.

Binding of BnAb UCAs and the CH58 UCA to synthetic V1V2 glycopeptides. Man₅ GlcNAc₂ V1V2 glycopeptide at concentrations ranging from 2 to 25 µg/mL binding to the PG9 UCA (A) or CH01 UCA (B). Man₃ GlcNAc₂ V1V2 at concentrations ranging from 1 to 8 µg/mL binding to the PG9 UCA (C) or CH01 UCA (D). Man₅ (E) and Man₃ (F) glycopeptides were injected at concentrations ranging from 1 to 10 µg/mL over the CH58 UCA captured on anti-IgG–immobilized surface as above. Both peptides were solubilized in 20% DMSO overnight to allow complete dimer formation as described in Materials and Methods.