Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies

Abstract

The elicitation of broadly neutralizing antibodies (bNAbs) is likely to be essential for a preventative HIV-1 vaccine, but this has not yet been achieved by immunization. In contrast, some HIV-1-infected individuals naturally mount bNAb responses during chronic infection, suggesting that years of maturation may be required for neutralization breadth. Recent studies have shown that viral diversification precedes the emergence of bNAbs, but the significance of this observation is unknown. Here we delineate the key viral events that drove neutralization breadth within the CAP256-VRC26 family of 33 monoclonal antibodies (mAbs) isolated from a superinfected individual. First, we identified minority viral variants, termed bNAb-initiating envelopes, that were distinct from both of the transmitted/founder (T/F) viruses and that efficiently engaged the bNAb precursor. Second, deep sequencing revealed a pool of diverse epitope variants (immunotypes) that were preferentially neutralized by broader members of the antibody lineage. In contrast, a 'dead-end' antibody sublineage unable to neutralize these immunotypes showed limited evolution and failed to develop breadth. Thus, early viral escape at key antibody-virus contact sites selects for antibody sublineages that can tolerate these changes, thereby providing a mechanism for the generation of neutralization breadth within a developing antibody lineage.

Figure 1

Viral diversification over time in CAP256 HIV-1 Env V1V2. (a) BG505 SOSIP.664 trimer structure (PDB:4TVP) fitted into a 3D reconstruction of the CAP256-VRC26.09 Fab-trimer complex (EMD-5856, left panel). BG505 V1V2 cap with residues 166 and 169 shown as spheres (right panel) and the C-strand highlighted in green. (b) Hamming distance from the PI sequence (y-axis) versus weeks after infection (x-axis) for all next-generation V1V2 consensus sequences. Criteria of <0.03 and >0.23 were used to distinguish PI-like (blue) and SU-like (black) viruses respectively, while those sequences with an intermediate distance (0.05–0.21) were classified as PI-SU recombinant viruses (purple, REC). Hamming distances were normalized for sequence length, and the relative frequency of sequences with a given distance are indicated by the size of the circle (normalized for depth of sequencing, but not for viral load, see Supplementary Figure 1). CAP256-VRC26 next generation transcript detection in the blood is shown by gray shading. (c and d) The frequency of amino acids (y-axis) at positions 169 (c) and 166 (d) are shown as stacked bars, with each bar representing a single time-point from 6 to 206 weeks after infection (x-axis). Amino acids are colored as indicated in the key, with Del representing a deletion. The inset (*) in the 169 graph expands the 42 week time-point (when nine immunotypes are present), to highlight the seven minority immunotypes present at frequencies of <5%. The time of superinfection (SU), the emergence of CAP256-VRC26 NGS transcripts and onset of plasma breadth are shown.

Figure 2

Characterization of the CAP256 34-week Envs. (a) Frequency of 34-week next-generation V1V2 consensus sequences possessing a C-strand derived from the PI (blue, n = 862) or SU (black, n = 75) viruses. (b) Amino acid highlighter plot of single genome amplification (SGA)-derived Env, comparing six SU-like 34-week clones to the SU virus (designated as the master). Mismatches from the SU are shown as coloured ticks and the V1V2 region is boxed. (c) Amino acid sequence alignment of the V1V2 region (HXB2 residues 126–196) comparing the SU and the six SGA-derived SU-like 34-week sequences. Red font denotes clones with a wildtype K169, while gray font indicates sequences with 169I or E mutations. Amino acid identity to the SU virus is represented by dots, deletions are shown as dashes and glycosylation sequons are shaded in gray. (d) CAP256-VRC26 UCA neutralization (y-axis) of the SU virus (black) and the 34-week clones with 169K (red) and 169I or E (gray). UCA mAb concentration is shown on the x-axis. Bars indicate standard error of the mean (triplicate).

Figure 3

Relationship between bNAb phylogeny, neutralization breadth and tolerance of multiple immunotypes at positions 169 and 166. (a) A condensed heavy chain phylogenetic tree, rooted on the VH3-30*18 germline gene and displaying the positions of the 33 CAP256-VRC26 mAbs. Antibody transcripts and each mAb is colored according to whether it was isolated from 34–59 weeks (gray) or 119–206 weeks (black). The tree bifurcates into two sublineages (separated by a dashed line); one showing restricted evolution (left) contains only antibody transcripts from early time-points, while a continually evolving sublineage (right) contains transcripts from all time-points. Scale shows the rate of nucleotide change between nodes. (b) Dead-end mAbs in the sublineage with restricted evolution are ranked according to percentage neutralization breadth in the histogram. Neutralization of the SU virus, 169 and 166 mutant viruses by CAP256-VRC26.01 and .24 are shown as a heatmap, with shading showing neutralization potency, as indicated in the key. (c) Thirty mAbs in the continually evolving sublineage are ranked according to neutralization breadth in the histogram. Neutralization of the SU virus, 169 and 166 mutant viruses by CAP256-VRC26 mAbs are shown as a heatmap, with shading showing neutralization potency, as indicated in the key. Correlations (r values) between neutralization breadth and the IC₅₀ titres of each mutant virus are indicated on the right of the heatmap with P values shown as stars (*P < 0.05; **P < 0.005; ***P < 0.0001 calculated using a two-tailed non-parametric Spearman correlation).

Figure 4

Viral variants that shaped the maturation of broad and off-track antibodies. (a) Relationship between neutralization breadth of CAP256-VRC26 mAbs (%, y-axis) and mutations away from the UCA (%, x-axis). Broad mAbs with >40% neutralization breadth, off-track mAbs with limited neutralization breadth (<6%) and dead-end mAbs are shown in red, turquoise and gray respectively. (b) CAP256-VRC26 mAbs and longitudinal CAP256 viruses isolated between 6–59 weeks are hierarchically clustered by their neutralization profiles. Neutralization sensitivity is shown as a heatmap with potency indicated in the key. The sequence alignment shows viral C-strand amino acid residues between positions 160–172 (HXB2 numbering), shaded to designate sequences that are PI-derived (blue shading), PI-SU recombinants (purple shading) or SU-derived (no shading). Off-track (turquoise) and broad (red) mAbs are clustered separately, while longitudinal viral variants are clustered by neutralization sensitivity, largely segregated on the basis of an SU-derived V1V2 C-strand. (c) A condensed heavy chain phylogenetic tree (Figure 3) with a single clade expanded to illustrate the phylogenetic relatedness of an off-track mAb (turquoise) to seven broad mAbs (red). (d) CAP256 viral evolution results in the engagement of the CAP256-VRC26 UCA (light gray) and the subsequent development of two distinct mAb sublineages. The sublineage with restricted evolution contains dead-end mAbs (dark gray) which cannot tolerate viral escape mutations. The continually evolving sublineage contains bNAbs (red) and off-track mAbs (turquoise). While both bNAbs and off-track mAbs have high levels of SHM, only the former mature to recognise multiple immunotypes, and therefore acquire breadth.