Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies

Abstract

Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Extended Data Figure 1. Sequences of CAP256-VRC26 heavy and light chains

Sequences of the 12 B-cell culture derived antibodies, inferred germline V and J genes, and inferred intermediates are compared to the predicted UCA. a, heavy chain. b, lambda light chain.

Extended Data Figure 2. Neutralization breadth and potency of CAP256-VRC26 antibodies

a, Neutralization of autologous (CAP256 PI and SU) and 47 heterologous viruses by CAP256-VRC26 antibodies. Neutralization was measured using a TZM-bl assay with Env-pseudoviruses. Geometric mean was calculated for values <50 mg/ml. b, Breadth-potency curves. Neutralization of a 194-virus panel was measured for VRC26.08, PG9, PGT145, and CH01. The curves show the percent of viruses neutralized at any given IC50.

Extended Data Figure 3. CAP256-VRC26 antibodies recognize a quaternary epitope

a, All 12 CAP256-VRC26 mAbs were tested by ELISA against gp120 from ZM53 and CAP210. Positive control antibody PG9 bound to both gp120s (not shown). b, 23 proteins and scaffolded V1V2 constructs were tested by ELISA for binding of CAP256-VRC26.03 and CAP256-VRC26.08. PG9 bound to several of these (not shown). Similar data were observed for CAP256-VRC26.06, .07 and .09. c, Binding of CAP256-VRC26.03 and CAP256-VRC26.08 to virus-like particles (VLP). VLP expressing ZM53, ZM53.K169E, CAP210, or no Env were concentrated by pelleting and used to coat ELISA plates; assays were performed without detergent to preserve the trimer spikes. Similar data were observed for CAP256-VRC26.06, .07 and .09.

Extended Data Figure 4. Visualization of CAP256-VRC26.09 bound to Env trimers by negative-stain electron microscopy

a, Raw micrograph and corresponding reference free 2D class averages of VRC26.09 in complex with cleaved soluble BG505 SOSIP.664 gp140 trimers. b, Projection matching of 3D model refinement and FSC curve used to calculate resolution. Resolution, 28 Å at FSC=0.5. c, 3D reconstruction of VRC26.09:BG505 SOSIP.664 complex (green surface) alone and overlayed with PG9:SOSIP (purple mesh). The reconstructions are nearly identical in the trimer portion while displaying small differences in the Fab angles.

Extended Data Figure 5. Effects of V2 mutations on neutralization activity of CAP256-VRC26 antibodies

a, Each panel shows neutralization of wild-type and N160 glycan mutant CAP210.E8, ConC, KER2018.11, and ZM53.12 viruses. CAP256-VRC26 mAbs are partially and variably affected by loss of N160 glycan, in a virus-strain specific manner. In contrast, PG9-class antibodies PG9, PGT142, and CH01 are uniformly knocked out by N160 mutation. b, CAP256-VRC26 mAbs are partially and variably affected by changes in V2 glycans. Neutralization by each antibody was measured against wild-type ZM32.12, mutants N156A and N160K, and ZM53.12 grown in the presence of kifunensine, an inhibitor of glycan processing. In contrast to CAP256-VRC26 antibodies, PG9 activity is knocked out by the mutations and by kifunensine. c, HIV-6405 wild type is resistant to PG9 and CAP256-VRC26 antibodies, while a PG9-sensitive mutant is also sensitive to CAP256-VRC26 antibodies. d, Sequences of wild type and mutant HIV-6405.

Extended Data Figure 6. Origins of long CDRH3s in donor CAP256

a, Week 38 sequences from 454 that support the calculation of the UCA. Unique amino acid sequences with 2–5 residue changes in the CDR H3 are compared to the calculated UCA sequence. Each contained fewer than 3 combined nucleotide mutations in VH and JH. Parentheses, number of corresponding reads in the raw 454 data. b, c, Lack of autoreactivity. b, ELISA for binding to cardiolipin. 4E10 was strongly positive, CAP256-VRC26.03 was weakly positive, and the other 11 CAP256-VRC26 mAbs and the UCA were negative along with control antibody VRC01. c, Staining on Hep2 cells was assessed at 50 and 25 mg/ml. Only the positive control, mAb 4E10, showed positive staining. d, Distribution of CDRH3 lengths among 454 sequencing reads of B cell transcripts. The percentage of high-quality NGS reads that have CDR H3≥24 or ≥28 are shown for three HIV-1 uninfected donors (solid circles on both right and left plots) and for donor CAP256 (week 176) amplified with all-VH primers donor, and CAP256 (week 30) amplified with VH3 primers. High-quality reads are defined as successful V and J assignments and a continuous open reading frame. CDRH3 lengths use the IMGT definitions.

Extended Data Figure 7. Loss of flexibility at the base of the CDR H3

a, Top, logograms of CDR H3 sequences extracted from the heavy chain phylogenetic tree from weeks 59 and 119. The height of each letter is proportional its frequency in the population. Sequences that lack a disulfide bond contain a highly conserved glycine at the 3rd position of the CDR H3 (residue 97, Kabat definition). The appearance of the two cysteines that form the disulfide bond coincides with a glycine to arginine mutation at this site. (Bottom) Overlay and close-up of crystal structures from Supplementary Figure 6A. Loss of the glycine limits flexibility at the base of the CDR H3 and is shown in the crystal structures to be the initial site of divergence in the CDR H3 loops between the antibodies without the disulfide bond (UCA and CAP256-VRC26.01) and those with it (CAP256-VRC26.03, .04, .06, .07, .10). This mutation may contribute to the conserved trajectory of the CDR H3 protrusion towards the heavy chain that is seen in the more mature antibody structures. b, CDRH3 and flanking sequences for VRC26.01, VRC26.03, and a mutant VRC26.03 in which the conserved cysteines are changed to the corresponding amino acids found in VRC26.01. c, neutralization activity of VRC26.03 and the mutant shown in panel b. The mutant shows reduced activity against CAP256 SU and complete loss of heterologous activity.

Extended Data Figure 8. Viral polymorphisms and escape mutations

a, Frequency of CAP256 PI and SU polymorphisms at positions 160-162 (glycosylation sequon), 165, and 169. Colored slices on pie charts and percentages indicate prevalence of these polymorphisms within global circulating viruses in the Los Alamos Sequence Database (n=3,990). b, Distribution of net charge of the V2 epitope, defined as residues 160 – 171, within global circulating viruses (n=3,990). The charge of the PI, SU and 176 week clones are indicated. c, CAP256-VRC26 mAb neutralization of the SU and PI viruses, and of the SU virus mutated to contain PI polymorphisms 162I, 165V or 169Q. d, CAP256-VRC26 mAb neutralization of the SU virus mutated to contain known CAP256 escape mutations in the V2 epitope. e. CAP256-VRC26 mAb neutralization of 34 week clone (designated wildtype, wt) with an SU-like V1V2, compared to the I169K back mutant. (c–e) The V2 epitope sequence, with mutated residues in red is shown on the left, IC50 values in the middle, and the time point when mutations were first detected in Env sequences on the right (weeks post-infection).

Extended Data Figure 9. Longitudinal changes in CAP256 V1V2

a, Variation in the V1V2 sequence of six Env clones. Amino acid mutations from residues 160-171 are highlighted and corresponding changes in neutralization for the six Env clones by CAP256-VRC26.01-.12 and the UCA are shown. The charge of the displayed sequences that make up the central region of the trimer are shown on the right. b, Residue changes highlighted in a were mapped onto the V1V2 domain in the crystal structure of the HIV-1 BG505.664 SOSIP Env trimer. The structure is viewed looking towards the viral membrane along the trimer axis. Mutations are colored as in panel a and represented as spheres (amino acids) or stick and surface (glycan). c, Electrostatic surface representations of (top row) the full V1V2 region for each Env clone, (bottom row) Fabs. Timeline of infection is shown in the middle. V1V2 sequences were modeled with SWISS-MODEL using the BG505.664 SOSIP as a template. Escape mutations R166S, K171N, and K169E resulted in a net charge change in the V2 epitope from +3 (SU) to a rare 0. Antibody CDR H3s became less negatively charged over time, suggesting co-evolution of the viral epitope and the antibody paratope.

Figure 1. Development of broad neutralization by donor CAP256 and isolation of neutralizing antibodies

(a) Timing of antibody isolation in relation to plasma neutralization titers against the primary infecting virus (PI), the superinfecting virus (SU), and a panel of 40 heterologous viruses (geometric mean titer shown). Percentage breadth (gray area), % of viruses neutralized with plasma ID50 >45. (b) Genetic characteristics and neutralization breadth and potency of the 12 isolated antibodies. Week of antibody isolation and V-gene mutation rates are indicated. Residues flanking the Kabat-defined CDR H3 sequences are shown in gray. Neutralization was assessed against a panel of 47 heterologous viruses. (c) Breadth and potency of antibody CAP256-VRC26.08 on a panel of 194 Env-pseudoviruses. Dendrogram shows phylogenetic relatedness of Env sequences in the panel.

Figure 2. Mapping of CAP256-VRC26 epitope on the HIV-1 Env spike

(a) Left, correlations between neutralization fingerprints (see detailed methods) of CAP256-VRC26 antibodies and CAP256 plasma. Darker gray indicates stronger correlation. Right, correlations between neutralization fingerprints of CAP256-VRC26 antibodies and representative antibodies targeting the major HIV-1 neutralization epitopes. Correlations are color-coded by antibody; darker shades indicate stronger correlations. (b) Competition assay. Binding to ZM53-Env-expressing 293T cells by labeled CAP256-VRC26.08 and unlabeled competitor antibodies measured by flow cytometry. Assay shown is representative of three experiments. (c) Left, negative stain electron microscopy (EM) 3D reconstruction of CAP256-VRC26.09 Fab in complex with soluble cleaved BG505 SOSIP.664 trimer; right, 2D-class averages of VRC26.09 and PG9 in complex with BG505 SOSIP.664 trimer. (d) Neutralization of Env-pseudoviruses with HIV-ConC and V2 point mutants. Sequence shows amino acids 160-175. (e) Location of HIV-1 epitopes. Left, EM density of viral spike, with viral membrane at top and major sites of vulnerability shown as determined by structural mapping of antibody interactions. The gp41 membrane proximal external region (MPER) is shown schematically. Right, model of V1V2 based on EM reconstruction of PG9 with BG505 SOSIP.664 trimer,, viewed looking towards the viral membrane along the trimer axis. Green ribbon, strand C. V2 mutations from panel d are shown with surface representation; brighter green indicates more potent effects on neutralization.

Figure 3. Maturation of the CAP256-VRC26 lineage revealed by NGS and VH:VL paired sequencing of B cell transcripts

(a) Timeline of longitudinal peripheral blood samples with quantification of total NGS sequence reads (total), and CAP256-VRC26 lineage-related reads (total and unique). Arrows below the line indicate time points of 454 pyrosequencing for heavy and light chain sequences. Circles indicate time points of paired sequencing of sorted B cells (see detailed methods). PCR amplifications for pyrosequencing used primers specific for VH3 family sequences (heavy chain) and V lambda sequences (light chain), with the exception of the week 176 sample (asterisk), which was amplified using all-VH gene primers, resulting in fewer CAP256-VRC26 specific reads. (b) Maturation time course for CAP256-VRC26.01 (top panels) and CAP256-VRC26.08 (bottom panels). Heat map plots show sequence identity (vertical axis) versus germline divergence (horizontal axis) for NGS data. The 12 isolated antibodies are displayed as red ‘x’s for reference, with the exception of the CAP256-VRC26.01 and 08 antibodies which are shown as black dots. Numbers between the top and bottom panels correspond to the number of raw reads with at least 85% identity to the indicated antibody (top: VRC26.01, bottom: VRC26.08). (c) Phylogenetic trees of the CAP256-VRC26 clonal lineage for heavy chain (left) and light chain (right) were constructed by maximum likelihood using the 454 sequences and the isolated antibodies (black dots, labeled with antibody name). Branches are colored by time point when NGS sequences were first detected. The orange and blue circles indicate linked heavy and light chain sequences from the paired sequencing data. Scale, rate of nucleotide change (per site) between nodes.

Figure 4. Structural characteristics of the developing CAP256-VRC26 lineage

(a) Crystal structure of the antigen-binding fragment (Fab) of CAP256-VRC26.03 shown in ribbon diagram representation. (b) Left, the intra-loop disulfide bond and tyrosine sulfation are shown in stick representation, and enlarged to show electron density (blue mesh, 2Fo-Fc at 1σ). Right, molecular surface, with electrostatic potentials colored red for acidic and blue for basic. CDR H3 regions of broadly neutralizing V1V2-directed antibodies are shown for comparison, with the left image in ribbon representation (tyrosine sulfates highlighted) and the right image in electrostatic representation. (c) Left, a condensed heavy chain phylogenetic tree highlights the isolated antibodies. Scale, rate of nucleotide change between nodes. The number of mutations to the heavy chain (H) and light chain (L) relative to the UCA are shown. Middle, structures of the variable regions. Mutations from the UCA are represented as spheres colored according to the week of antibody isolation at which the mutations first appear. Right, CDR H3 details. Residues that are (or evolve to become) cysteines are labeled (gray dashes indicate modeled disordered regions). The position of tyrosines predicted to be sulfated (scores >1) are noted and were included in the formal charges shown for each CDR H3 and the electrostatic representations (far right).

Figure 5. HIV-1 Env evolution and the development of the CAP256-VRC26 lineage

(a) V1V2 sequences are shown in highlighter format with the primary infecting virus (PI) designated as master and V2 residues 160 to 171 boxed. Asterisk at week 15 denotes sequences amplified with strain-specific primers matching the SU virus. (b) Logogram of the V2 epitope for all CAP256 sequences, with mutations away from the PI (master sequence) in color. (c) SU-like V1V2 sequences are indicated by black (present) and grey (absent) boxes. Escape mutations (K169E or R166S/K) are indicated by brown boxes. The net charge of the V2 epitope (residues 160 to 171) is shown in purple/white, ranging from +3 to 0. White lines separate clones within a time point; black lines separate time points. (d) Neutralization by the 12 CAP256-VRC26 mAbs of representative longitudinal Env clones isolated between 6 and 176 weeks post infection (weeks shown at far right). The CAP256 mAbs are colored by time of isolation (as in Fig. 1). The development of the CAP256-VRC26 antibody lineage, V1V2-directed plasma neutralizing antibodies, and plasma heterologous neutralization, are indicated on the right.

Figure 6. Development from UCA to CAP256-VRC26.01

(a) Expanded view of the phylogenetic trees from Fig. 3c, highlighting the maturation pathway of CAP256-VRC26.01. Off-pathway branches were collapsed and are shown as dashed lines. Inferred intermediates VRC26-I1 and VRC26-I2 were expressed for functional analyses. (b–e) Binding and neutralization of antibodies UCA, VRC26-I1, VRC26-I2, VRC26.01. (b, d), Binding to cell-surface expressed Env (SU and ZM53). MFI, median fluorescence intensity. (c, e) Neutralization of (c) PI, SU and point mutants, and (e) seven heterologous viruses. Bars, standard error of the mean (triplicates). (f) Structural models of VRC26.01 lineage antibodies. Affinity matured residues are shown as spheres colored according to the intermediate at which they first appear: red, VRC26-I1; orange, VRC26-I2; green, VRC26.01. Grey dots, disordered residues in the CDR H3. The number of changes from the UCA to each intermediate are noted for V gene only (VH or VL), or from the full UCA (UCA-HC or UCA-LC).