New Member of the V1V2-Directed CAP256-VRC26 Lineage That Shows Increased Breadth and Exceptional Potency

Abstract

The epitopes defined by HIV-1 broadly neutralizing antibodies (bNAbs) are valuable templates for vaccine design, and studies of the immunological development of these antibodies are providing insights for vaccination strategies. In addition, the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of 12 V1V2-directed neutralizing antibodies, CAP256-VRC26, isolated from an HIV-1 clade C-infected donor at years 1, 2, and 4 of infection (N. A. Doria-Rose et al., Nature 509:55-62, 2014, http://dx.doi.org/10.1038/nature13036). Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. Thirteen antibodies were isolated from B cell culture, and eight were isolated using trimeric envelope probes for differential single B cell sorting. One of the new antibodies displayed a 10-fold greater neutralization potency than previously published lineage members. This antibody, CAP256-VRC26.25, neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency. Among the viruses neutralized, the median 50% inhibitory concentration was 0.001 μg/ml. All 33 lineage members targeted a quaternary epitope focused on V2. While all known bNAbs targeting the V1V2 region interact with the N160 glycan, the CAP256-VRC26 antibodies showed an inverse correlation of neutralization potency with dependence on this glycan. Overall, our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

Importance: Studies of HIV-1 broadly neutralizing antibodies (bNAbs) provide valuable information for vaccine design, and the most potent and broadly reactive of these bNAbs have potential for clinical use. We previously described a family of V1V2-directed neutralizing antibodies from an HIV-1 clade C-infected donor. Here, we report on the isolation and characterization of new members of the family mostly obtained at time points of peak serum neutralization breadth and potency. One of the new antibodies, CAP256-VRC26.25, displayed a 10-fold greater neutralization potency than previously described lineage members. It neutralized 57% of diverse clade viral isolates and 70% of clade C isolates with remarkable potency: the median 50% inhibitory concentration was 0.001 μg/ml. Our results highlight the ongoing evolution within a single antibody lineage and describe more potent and broadly neutralizing members with potential clinical utility, particularly in areas where clade C is prevalent.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Figures

FIG 1

Schematics of CAP256-VRC26 antibody isolation. (A) Timeline of antibody isolation. Numbers above the line indicate the week postinfection. The names of the antibodies isolated at each time point are shown below the line. (B) Schematic of high-throughput B cell culture method. Sorted IgD− IgM− B cells were plated at a density of ∼2 cells per well into 384-well plates, followed by assessment by a microneutralization assay (Microneut) on day 14. (C) Schematic of probe sorting method. IgG+ B cells bound to APC-labeled BG505 SOSIP were sorted into 96-well plates, followed by reverse transcription-PCR to recover the IgG genes.

FIG 2

Isolation of CAP256-VRC26 antibodies. (A) Class switch occurs under B cell culture conditions. Identical VDJ regions appear in both IgA and IgG forms in the same well. The red box highlights sequences that differ between IgG and IgA. (B to D) The BG505 trimer with a mutation in V2 selects for CAP256-VRC26-lineage B cells. (B) Effect of N160K and K169E mutations on neutralization of HIV BG505 by V1V2-directed antibodies and CAP256 plasma. wt, wild type; ID50, 50% inhibitory dilution. (C) Quality and specificity of probes. BG505 SOSIP-APC, BG505 SOSIP.K169E-PE, or gp140F-PE probes were used to stain beads coated with the PGT128, F105, or CAP256-VRC26.09 (CAP256.09) antibody. Histograms of anti-HIV antibodies (red) are overlaid on those for the anti-influenza virus control (gray). (D) Probe-specific B cell sorting. CAP256 PBMCs were stained with B cell markers and probes. Sorted IgG+ BG505 SOSIP-APC+ BG505-SOSIP-K169E-PE− B cells (purple) are shown overlaid on all live, IgG+ B cells (gray).

FIG 3

CAP256-VRC26 antibodies interact with N160 glycan in a potency-dependent manner. Wild-type Env pseudoviruses and mutants lacking the N160 glycan (dN160) were tested in a TZM-bl neutralization assay. Each pair of dots shows the IC50s for one virus pair, with the results for the wild type being shown on the left and those for the mutant lacking the N160 glycan being shown on the right. Each graph shows data for one antibody. (A) Results for four representative PG9-like antibodies; (B) results for the four most potent CAP256-VRC26 antibodies.

FIG 4

Development of the CAP256-VRC26 lineage. Maximum likelihood trees of heavy chain (left) and lambda chain (right) sequences. Labeled branches show heavy chains of antibodies from B cell cultures or probe sorts; unlabeled branches represent sequences from 454 pyrosequencing. The color coding indicates the time of sampling. The circle indicates the first node in which the signature Cys-Cys motif appears in the CDRH3. Scale, rate of nucleotide change (per site) between nodes.

FIG 5

Structural characteristics of CAP256-VRC26.25. (A) Crystal structure of the antigen-binding fragment (Fab) of CAP256-VRC26.25 shown in ribbon diagram representation. CDRs are highlighted. (B) The CDRH3 of CAP256-VRC26.25 contains an inserted Gly and is rotated 79 degrees compared to the orientation of the other lineage members without the insertion. Gray, CAP256-VRC26.03; red, the location of the insertion in the structure and the sequence alignment. (Bottom left) The electrostatics of the highly anionic CDRH3 (charge, −7) in the same orientation in the image above. The CDRH3 contains two sulfated tyrosines and a disulfide bond (blue mesh, 2Fo-Fc at 1 sigma). (Bottom right) Electron density of the disulfide bond acquired through affinity maturation. (C) The CDRH3s bend in different directions. All CAP256-VRC26 structures are shown, with CDRH3s projecting up from the top of the antibody. Loops are colored by the week of isolation: black, week 34, CAP256-VRC26.UCA; green, week 59, CAP256-VRC26.01; cyan, week 119, CAP256-VRC26.03, CAP256-VRC26.04, CAP256-VRC26.06, and CAP256-VRC26.07; yellow, week 193, CAP256-VRC26.25; purple, week 206, CAP256-VRC26.10. Disordered residues in the crystal structures were modeled using the Loopy program. These consist of 19, 14, 3, 11, and 10 residues for CAP256-VRC26.UCA, CAP256-VRC26.01, CAP256-VRC26.06, CAP256-VRC26.07, and CAP256-VRC26.10, respectively. All CDRH3 residues are well defined for CAP256-VRC26.03, CAP256-VRC26.04, and CAP256-VRC26.25. (D) The orientation at the base of the protruding CDRH3 loop is partially stabilized through interactions with residues in the CDRH1. Shown are interactions for CAP256-VRC26.01, CAP256-VRC26.03, and CAP256-VRC26.25. Modeled residues of CAP256-VRC26.01 are transparent.

FIG 6

Neutralization breadth and potency of CAP256-VRC26.08, CAP256-VRC26.25, and selected broadly neutralizing antibodies. The neutralization of a multiclade virus panel (n = 183) was assessed by a TZM-bl pseudovirus assay. (A) Neutralization breadth-potency curves for V1V2-directed bNAbs. Curves show the percentage of virus neutralized at any given IC50 or IC80. (B, C) Neutralization by bNAbs directed to diverse epitopes. Each dot shows the value for a single virus. Bars, median value of viruses that are neutralized. (B) IC50s; (C) IC80s.

FIG 7

Neutralization breadth, potency, and clade dependency of CAP256-VRC26 antibodies. The neutralization of large virus panels was assessed by a TZM-bl pseudovirus assay. (Left) IC50s; (right) IC80s. (A) Values indicate the percentage of viruses (n = 198) neutralized at the given cutoff; (B) values indicate the percentage of viruses neutralized within each virus clade.

FIG 8

CAP256-VRC26.25 resistance analysis of amino acids in Env V2. (A) (Top) Amino acid frequency analysis. A total of 198 sequences were analyzed (see Fig. S6 in the supplemental material). The resistance score for each possible amino acid at a given residue position was defined as the ratio of its number of occurrences in sequences from CAP256-VRC26.25-resistant viruses to its overall number of occurrences. A higher score indicates that the amino acid was preferentially found among sequences from resistant viruses, with a score of 1 indicating that the amino was found only among sequences from resistant viruses. Amino acids that occurred at least 3 times at the given position are shown. (Bottom) Logo plot showing the frequency of all amino acids at each position for clade B (n = 40) and non-clade B (n = 158) sequences. Red, amino acids associated with resistance; green, amino acids associated with sensitivity. (B) Frequency of amino acids at positions associated with resistance or sensitivity. Values indicate the percentage of sequences in a given category that bear the indicated amino acids. For example, 84% of Env sequences from sensitive strains have D or E at position 164. A total of 125 sensitive strains and 73 resistant strains were analyzed. P was <0.001 for each comparison (Fisher's exact test with the Bonferroni correction).