Cooperation between Strain-Specific and Broadly Neutralizing Responses Limited Viral Escape and Prolonged the Exposure of the Broadly Neutralizing Epitope

Abstract

V3-glycan-targeting broadly neutralizing antibodies (bNAbs) are a focus of HIV-1 vaccine development. Understanding the viral dynamics that stimulate the development of these antibodies can provide insights for immunogen design. We used a deep-sequencing approach, together with neutralization phenotyping, to investigate the rate and complexity of escape from V3-glycan-directed bNAbs compared to overlapping early strain-specific neutralizing antibody (ssNAb) responses to the V3/C3 region in donor CAP177. Escape from the ssNAb response occurred rapidly via an N334-to-N332 glycan switch, which took just 7.5 weeks to reach >50% frequency. In contrast, escape from the bNAbs was mediated via multiple pathways and took longer, with escape first occurring through an increase in V1 loop length, which took 46 weeks to reach 50% frequency, followed by an N332-to-N334 reversion, which took 66 weeks. Importantly, bNAb escape was incomplete, with contemporaneous neutralization observed up to 3 years postinfection. Both the ssNAb response and the bNAb response were modulated by the presence/absence of the N332 glycan, indicating an overlap between the two epitopes. Thus, selective pressure by ssNAbs to maintain the N332 glycan may have constrained the bNAb escape pathway. This slower and incomplete viral escape resulted in prolonged exposure of the bNAb epitope, which may in turn have aided the maturation of the bNAb lineage.IMPORTANCE The development of an HIV-1 vaccine is of paramount importance, and broadly neutralizing antibodies are likely to be a key component of a protective vaccine. The V3-glycan-targeting bNAb responses are among the most promising vaccine targets, as they are commonly elicited during infection. Understanding the interplay between viral evolution and the development of these antibodies provides insights that may guide immunogen design. Our work contrasted the dynamics of the early strain-specific antibodies and the later broadly neutralizing responses to a common Env target (V3C3), showing slower and more complex escape from bNAbs. Constrained bNAb escape, together with evidence of contemporaneous autologous virus neutralization, supports the proposal that prolonged exposure of the bNAb epitope enabled the maturation of the bNAb lineage.

Keywords: N332 glycan; V3-glycan supersite; broadly neutralizing antibodies; deep sequencing; glycan holes; glycan shield; helper/cooperating NAb responses; neutralization escape; viral escape.

FIG 1

Dynamics of gross changes in the V1 and V3/C3 regions of CAP177 env. (A) Phylogeny of the V3/C3 regions, generated using the maximum likelihood approach. Colors indicate time from 2 wpi (red) to 172 wpi (purple). Multiple-variant transmission is indicated (arrows), with one major variant detected at 2 wpi and two distinct variants identified at 4 wpi. Bubble sizes are proportional to the frequency of the given sequence. (B) Heatmap showing positional entropy over env V3C3 regions. Time points corresponding to the emergence of ssNAb and bNAb responses are indicated (dotted lines). (C) The frequencies of glycosylation at positions N334 (blue) and N332 (red) are indicated on the left y axis, with changes in viral load (black) shown on the right y axis. (D) Changes in V1 loop length and glycosylation content over time. Bubbles indicate the proportion of viruses with a given V1 loop length (y axis) and number of glycan sequons (color). Bubble sizes were normalized for sequencing depth and scaled by viral load. The emergence of ssNAbs at 19 wpi is indicated by a dotted line, with time points corresponding to the emergence of the breadth response shaded gray.

FIG 2

Predicted epitopes for the early ssNAb and bNAb responses. A trimer model of CAP177 gp160 (white), with sequence entropy mapped onto the structure, for the regions covered by the NGS data (light gray), highlighted using a white-to-red gradient. Predictions of the approximated epitopes for the ssNAb (blue circle) and bNAb (orange circle) responses are indicated. Glycosylation at position N332 is shown (green spheres).

FIG 3

Effect of an N332-to-N334 glycan shift and V1 loop length on escape from bNAb activity. (A) CAP177 plasma neutralization titers (ID₅₀) for N332 (red) and N334 (blue) glycan variants of CAP177 clone 5D. (B) Sensitivity of the N332 and N334 glycan variants to a panel of V3-glycan supersite MAbs (IC₅₀, in μg/ml). (C) CAP177 plasma neutralization titers (ID₅₀) for N332 (red) and N334 (blue) glycan variants of CAP177 clone 4C. (D) Sensitivity of the N332 and N334 glycan variants to a panel of V3-glycan supersite MAbs (IC₅₀, in μg/ml). (E) Sequence alignment for the V1 loop of several clones, with key V1 loop glycans indicated (red). Clones with either sensitive or resistant neutralization phenotypes are indicated, with those from the resistant group having V1 loops which were between 5 and 9 aa longer. (F) CAP177 plasma neutralization titers (ID₅₀) to a resistant clone (clone 4C) from 107 wpi (blue), as well as to a variant of this clone with a shorter V1 loop (red) taken from a sensitive clone (clone 5D). The dotted line indicates the plasma sampling point (107 wpi) from which the clone was isolated. (G) Sensitivity of V1 loop length variants to a panel of V3-glycan supersite MAbs (IC₅₀, in μg/ml). The dotted line indicates the plasma sampling point (107 wpi) from which the clones were isolated.

FIG 4

Effect of V1 loop length and glycosylation on V3-glycan supersite accessibility. Modeled trimeric structures of CAP177 neutralization-resistant (A) and -sensitive (B) clones (cartoon representation), with Man-9 glycans (colored spheres) attached to potential N-linked glycan sites (with clash resolution). The key V3-glycan bNAb ³²⁴GDIR³²⁷ motif is indicated (dark blue). An overlay of several modeled trimeric structures of CAP177 neutralization-resistant (C) and -sensitive (D) clones (cartoon representation) is shown.

FIG 5

Effect of N332/N334 glycosylation and V1 loop length on viral entry efficiency. (A) Differences in entry efficiency for N332 glycan and matched N334 glycan IECs are shown, with the Wilcoxon matched-pairs test P value indicated. (B) Differences in entry efficiency for unmatched IECs with long (31 or 32 amino acids) or short (23 or 26 amino acids) V1 loops are shown, with the Mann-Whitney test P value indicated.

FIG 6

Kinetics of escape from ssNAb and bNAb responses. (A) Rate of escape from early ssNAb responses via the N334-to-N332 glycan shift (black) is shown, with 50% escape reached 11 weeks after the emergence of the ssNAb response (red). (B) Rates of escape from the bNAb response, including escape via the V1 loop (green), and reversion to the N334 glycan (blue). The incremental development of neutralization breadth is indicated (red).

FIG 7

Contemporaneous neutralization and changes in viral load associated with the development of the bNAb response. (A) Longitudinal autologous neutralization ID₅₀ titers over the development of bNAbs are depicted for several viral clones, sampled from 80 wpi (green), 107 wpi (purple), 133 wpi (blue), and 155 wpi (red), showing contemporaneous neutralization at all time points. (B) The increase in neutralization for longitudinal CAP177 plasma to a panel of 18 heterologous, cross-clade viruses (black lines) overlaps a 40-week period (gray shading) of a sustained, greater-than-1-log decrease in viral load (red). Time points corresponding to the emergence of ssNAb and bNAb responses are indicated (dotted line).