Antibody elicited by HIV-1 immunogen vaccination in macaques displaces Env fusion peptide and destroys a neutralizing epitope

Abstract

HIV-1 vaccine design aims to develop an immunogen that elicits broadly neutralizing antibodies against a desired epitope, while eliminating responses to off-target regions of HIV-1 Env. We report characterization of Ab1245, an off-target antibody against the Env gp120-gp41 interface, from V3-glycan patch immunogen-primed and boosted macaques. A 3.7 Å cryo-EM structure of an Ab1245-Env complex reveals one Ab1245 Fab binding asymmetrically to Env trimer at the gp120-gp41 interface using its long CDRH3 to mimic regions of gp41. The mimicry includes positioning of a CDRH3 methionine into the gp41 tryptophan clasp, resulting in displacement of the fusion peptide and fusion peptide-proximal region. Despite fusion peptide displacement, Ab1245 is non-neutralizing even at high concentrations, raising the possibility that only two fusion peptides per trimer are required for viral-host membrane fusion. These structural analyses facilitate immunogen design to prevent elicitation of Ab1245-like antibodies that block neutralizing antibodies against the fusion peptide.

Fig. 1. Characterization of Ab1245 elicited in macaques by sequential immunization.

a Sequential immunization protocol for the macaque that produced Ab1245 over the course of 40 weeks. b Sequence alignment of Ab1245 heavy and light chains with their germline precursors. Contacts with BG505 Env are indicated by a colored box around the residue (cyan box of CDRH3 contact residues; dark pink and light pink boxes for heavy chain and light chain contact residues, respectively). Residues within CDRs are indicated; residues between CDRs are within framework regions (FWRs). CDRH3 residues derived from VDJ joining are shown as dashes in the top germline sequence, and changes from the germline precursors are denoted a different residue in the mature Ab1245 sequence. Residues are numbered using the Kabat convention. c SEC-MALS profiles for BG505 SOSIP.664 Env trimer alone and complexed with a threefold molar excess of Ab1245 and 8ANC195 Fabs. Left: absorbance at 280 nm (left y-axis) plotted against elution volume from a Superdex 200 10/300 GL gel filtration column overlaid with the molar mass determined for each peak (right y-axis). Right: Table showing predicted and calculated molecular masses. d Mass photometry results. Derived molecular masses (MW) are listed for Env trimers (either BG505 or BG505_N611A) incubated without an added Fab or with the indicated Fab (Ab1245, BG1, or 8ANC195) as mean and standard deviation for the indicated number of independent measurements. The Fabs/trimer row shows the expected number of Fabs for each Fab/Env trimer complex. The predicted mass row shows the mass calculated assuming 310 kDa for BG505 trimer (derived by SEC-MALS, (c)) plus 50 kDa per bound Fab.

Fig. 2. Ab1245 binds the gp120-gp41 interface.

a Representation of Ab1245-BG505-8ANC195 structure. Fabs are shown in cartoon, BG505 is shown as surface, and glycans are shown as sticks. Within BG505, gp120 is light gray and gp41 is dark gray. The Ab1245 V_H-V_L is dark pink (heavy chain) and light pink (light chain); 8ANC195 Fabs are dark green (heavy chain) and light green (light chain). b Side view (left) and view looking up from the trimer base (right) of density map for 3.7 Å Ab1245-BG505-8ANC195 complex. Colors as in (a). c Close-up view of Ab1245 V_H-V_L domains (cartoon) interacting with gp120-gp41 interface with highlighted CDRH3 (cyan). N-linked glycans are light gray (gp120), dark gray (gp41), or yellow (N88_gp120 and N241_gp120 glycans) spheres. d Cartoon representation of VRC34-AMC011 SOSIP structure (PDB 6NC3) from the same view as c (left) and a different view from above the trimer (right). VRC34 heavy and light chains (PDB 6NC3) are dark and light purple, respectively.

Fig. 3. Ab1245 CDRH3 makes the majority of contacts to BG505 Env.

a Surface representation of BG505 trimer with colored highlights showing the epitopes of Ab1245 (light and dark pink with CDRH3 contacts highlighted in cyan and pink sticks for glycan contacts) and 8ANC195 (green, with green sticks representing glycans within the epitope). Glycans represented that are not part of an epitope are shown as gray sticks. b Cartoon representation of Ab1245 with paratope residue atoms shown as colored spheres. c gp120 interactions with Ab1245 CDRH1 and CDRH2 loops (dark pink spheres). gp120 is gray with contacts to Ab1245 highlighted in orange. Sidechains of gp120 contacts are shown as sticks. The Ab1245 paratope is represented as in (b). d Interactions of Ab1245 CDRH3 (cyan spheres) with gp120 (light gray) and gp41 (dark gray). Contacting residues are orange, and side chains discussed in the text are shown. e Ab1245 light chain (light pink spheres) contacts with the terminal helix of an adjacent gp41. Contacting residues are orange with side chains shown.

Fig. 4. Ab1245 CDRH3 mimics gp41 interactions with the tryptophan clasp.

a Cartoon representation of the interactions between Ab1245 V_H-V_L domains (pink with CDRH3 in cyan) and the tryptophan clasp of gp41 (gp41 in dark gray with Trp residues 623_gp41, 628_gp41, and 631_gp41 in dark blue) with gp120 in light gray. Interacting residues between M100c_HC and the tryptophan clasp shown as sticks. b Cartoon representation of the same view of an unbound protomer of gp41 with the portion of gp41 containing the fusion peptide (red) and fusion peptide proximal region (FPPR, green) interacting with the gp41 tryptophan clasp (same coloring as in (a)). Interactions between M530_gp41 and the tryptophan clasp are shown as sticks. c Surface representation of gp41 (dark gray) and gp120 (light gray) with tryptophan clasp residues in dark blue. Ab1245 CDRH3 is cyan with a stick representation for the Met100c_HC sidechain. d In vitro neutralization assays using IgGs Ab1245 or N6 (positive control for neutralization) against the indicated viral strains. In addition to those listed, Ab1245 was tested in-house against pseudoviruses from strains CE0217, CNE55, JRCSF, Du422, T250-4, Tro, X1632, 246F3, CH119, CE1176, BJOX002000_03_02, 25710, X2278, CNE8, and 398F1 at a top concentration of 500 µg/mL or 1000 µg/mL along with IgG N6 (positive control at a top concentration of 10 µg/mL). N6 was not evaluated in against the strains indicated by a dash because its neutralization potencies were previously published. Whereas N6 exhibited expected neutralization potencies against evaluated strains, Ab1245 exhibited no neutralization activity.