Epitope-based vaccine design yields fusion peptide-directed antibodies that neutralize diverse strains of HIV-1

Abstract

A central goal of HIV-1 vaccine research is the elicitation of antibodies capable of neutralizing diverse primary isolates of HIV-1. Here we show that focusing the immune response to exposed N-terminal residues of the fusion peptide, a critical component of the viral entry machinery and the epitope of antibodies elicited by HIV-1 infection, through immunization with fusion peptide-coupled carriers and prefusion stabilized envelope trimers, induces cross-clade neutralizing responses. In mice, these immunogens elicited monoclonal antibodies capable of neutralizing up to 31% of a cross-clade panel of 208 HIV-1 strains. Crystal and cryoelectron microscopy structures of these antibodies revealed fusion peptide conformational diversity as a molecular explanation for the cross-clade neutralization. Immunization of guinea pigs and rhesus macaques induced similarly broad fusion peptide-directed neutralizing responses, suggesting translatability. The N terminus of the HIV-1 fusion peptide is thus a promising target of vaccine efforts aimed at eliciting broadly neutralizing antibodies.

Figure 1

Design, properties, and immunogenicity of FP immunogens based on the epitope of antibody VRC34.01. (a) Structure-based design, antigenic characteristics, and EM structure of FP immunogens. The glycosylated structure of the HIV-1 Env trimer is shown at far left, with exposed N-terminus of FP highlighted in red. Subsequent images show site recognized by VRC34.01 antibody, schematics and antigenicity of FP immunogens, and negative stain EM of FP-KLH (see Supplementary Fig. 1 for details of FP antigenicity). For EM study, n=3 experiments were performed independent with similar results. (b) Immunization regimen 1. At day 52, mouse spleens were harvested and hybridomas created. (c) ELISA and neutralization of serum from regimen 1-immunized mice. Protein probes used for ELISA are defined in top row and include BG505 SOSIP.664 (green), FP-epitope scaffold based on PDB 1M6T (red), and 1M6T scaffold with no FP (blue). Column 1 defines mouse identification number and subsequent columns show ELISA and neutralization. ELISAs are shown as a function of serum dilution for pre-bleed, days 21, 35, and 52 (ELISA curves colored according to probe, with sera mostly unreactive with IM6T scaffold with no FP). Neutralization (ID₅₀, ID₈₀) values provided for day 52 serum; see Supplementary Fig. 2a for neutralization details. (d) Immunization regimen 2. At day 38, mouse spleens were harvested and hybridomas created. (e) ELISA and serum neutralization of serum from regimen 2-immunized mice, displayed as in c.

Figure 2

FP assumes disparate antibody-bound conformations, with neutralization restricted to a select angle of trimer approach. (a) Structural definition of vFP1.01 recognition. Top panels, cryo-EM reconstruction at 8.6 Å resolution (density shown in gray) of Fab vFP1.01 in complex with BG505 DS-SOSIP trimer. Expanded view, crystal structure of Env trimer and FP-bound Fab vFP1.01 at 2.0 Å resolution, as modeled into the cryo-EM map by rigid-body docking. Env trimer in green for gp120 and gray for gp41, Fab vFP1.01 in cyan for heavy chain and in yellow for light chain, and FP N-terminus in purple. Surface areas provided for N-terminal region of FP. (b) Same as a, but for vFP5.01 with FP in pink. Note that the angle of the lower right Fab differs from the angles of the other two. (c) Comparison of FP bound by vFP1.01 versus VRC34.01, with antibody shown in ribbons and FP in stick representation. (d) Same as c, but for vFP5.01. (e) Angle of recognition and Fv-domain overlap for vFP1.01, vFP5.01, and VRC34.01. Measured angles (red) are between antibody angle of approach and Env-trimer axis (left, with viral membrane located below trimer) and between antibody angle of approach parallel to viral membrane and Env protomer (right, looking down trimer axis towards viral membrane).

Figure 3

Second-generation vaccine-elicited antibodies neutralize up to 31% of HIV-1. Neutralization dendrograms display the diversity of tested viral strains, with branches colored according to neutralization potency (non-neutralized branches shown in gray). Top row: 208-strain panel for vaccine-elicited antibodies vFP16.02 and vFP20.01. Bottom left, 58-strain panel, displaying only branches with FP8=AVGIGAVF. Bottom right, comparison of breadth on 58- and 208-strain panels shown with Pearson correlation (n=7 antibodies).

Figure 4

Substantial glycan contacts by 2^nd-generation FP-directed antibodies. (a) CryoEM map of quaternary complex with antibody 2712-vFP16.02, segmented by components at a contour level that allowed visualization of Fv domains of antibodies (also see Supplementary Figs. 9 and 10). (b) Same as a, but with 2716-vFP20.01. (c) Details of vFP16.02 interaction, with right panels showing experimental EM density in blue mesh, with contour level adjusted to allow visualizing of partially ordered glycan. Residues altered by SHM highlighted in cyan. Antibody vFP16.02 contacts both FP and neighboring glycans to achieve 31% breadth. (d) Same as c, but for vFP20.01. (e) Sequence alignment of vaccine-elicited FP-directed antibodies and origin genes. FP contacts (red highlight), glycan contacts (green rectangle) and SHM (cyan font) are highlighted. Additional Env contacts are indicated by double underlining. Because the density from the cryo-EM reconstructions was not always sufficient to allow for atomic-level fitting, contacts shown with dotted green rectangles were inferred.

Figure 5

Immunization of guinea pigs and NHPs with FP-coupled carriers and DS-SOSIP trimer elicits heterologous neutralizing responses. (a) Elicitation of serum neutralizing responses in guinea pigs. Immunization regimen and week 28 serum ID₅₀ titers as measured on a 10-wildtype strain panel, 5 with complete glycans around FP, and 5 naturally missing glycans at sites defined in the figure. Also shown are titers for Δ88, Δ611, and Δ88+611 glycan-deleted variants of BG505. (b) Plot comparing guinea pig-serum neutralization breadth for FP-KLH prime and DS-SOSIP-trimer boost regimen versus DS-SOSIP alone regimen (one-tailed Mann-Whitney; see Supplementary Fig. 7 for DS-SOSIP alone immunizations at 0, 4 and 16 weeks); n=5 animals for each group. (c) Elicitation of heterologous neutralizing responses in rhesus macaques. Immunization scheme, and week 46 serum titers assessed and displayed as in a. (d) Plot comparing NHP-serum neutralization breadth for immunization regimens, displayed as described in b for guinea pigs; n=5 animals for each group.

Figure 6

Patterns of neutralization indicate FP-directed responses in mice and NHP are related. (a) Neutralization on a 58-strain panel, comprising strains from the 208-strain panel with the FP sequence, AVGIGAVF, matching both FP8-KLH and BG505 trimer immunogens. Top panel, IC₅₀ values for FP-directed antibodies. Bottom panel ID₅₀ titers for NHP plasma: top rows, FP-KLH prime and DS-SOSIP-trimer boost regimen at week 46, 2 weeks after third trimer boost; bottom row, DS-SOSIP alone regimen at week 18, 2 weeks after third trimer boost. Number of neutralized strains and neutralization breadths are shown. (IC₈₀ and ID₈₀ provided in Supplementary Table 3c and Supplementary Fig. 7.) (b) Neutralization curves of NHP plasma (DF1W week 46) and control monoclonal antibodies (VRC34.01 and VRC01) on four representative strains in the presence of no peptide (black), FP (red) or an irrelevant Flag peptide (blue). Mean and standard deviation of results from triplicated experiments shown (n=3 independent experiments). Location of representative tested strains are labeled and shown on dendrogram in c. (c) Neutralization dendrogram (ID₅₀) for NHP DF1W week 46 plasma on 58-strain panel. (d) Neutralization-fingerprint dendrogram calculated from 58-strain panel. Vaccine-elicited vFP antibodies (highlight with green background) and three NHP week 46 plasma (highlighted with forest green background for three of five NHP with sufficient neutralization to yield accurate fingerprint analysis) clustered next to each other (see also fingerprint dendrogram on 132-curated strains shown in Supplementary Fig. 11a).