Experimental design.

(A) Phylogenetic analysis of longitudinal env sequences isolated from four acutely infected participants in the RV217 study. (B) Selection of V1V2-specific escape variants and their fusion to the C-terminus of bacteriophage T4 small terminase protein, gp16, to generate gp16-V1V2 nanoscaffolds representing various escape mutations. (C) Expression of gp16-V1V2 escape mutants in GnTi mammalian cells to produce dodecameric nanoscaffolds displaying variant immunogens. (D) Immunization of mice with V1V2 variant nanoscaffolds and evaluation of immunogenicity. (E) Design of combinatorial V1V2 mutant immunogens and evaluation of their immunogenicity and breadth of immune responses in a successive immunization study.

Viral escape mutations in RV217 participants during the acute stage of HIV infection.

(A) Mutations observed in the RV217 participants – 7, 61, 94, and 100 are placed on HIV-1 BG505 pre-fusion trimer structure (PDB ID: 4TVP). Surface model showing dominant mutations (blue) falling on the solvent-exposed regions of the Env (gray) but not on the non-exposed or buried region (red). Modeling was done with PyMol (ver. 1.74) molecular visualization software and surface-exposed residues were defined as all residues that had >5Å2 exposure to the solvent. (B) Mutational hotspots in the env sequence based on the genetic diversification of T/F viruses from all four participants. Total number of mutations in a particular region is plotted on the y-axis against residue positions on the env sequence (x-axis), with reference to HXB2 strain. The Env regions (labeled on top) with gray background showed high frequency of mutations. (C) Phylogenetic tree displaying T/F07 virus evolution in participant_7. The evolutionary tree was constructed by the neighbor-joining method, rooted to the T/F07 virus sequence. The viruses are designated corresponding to the time post-infection; 1 wk, 1 month (4-wks)) or 6-month (24 wks) at the nodes of the branches. Prominent diverging mutations are labeled on the respective branches of the tree in red (V1V2 region) and gray (another region). (D) Dominant mutations that occurred until 24-wk post-infection in participant_7 are modeled on a ribbon model of the T/F07 trimer, generated through homology modeling using BG505 trimer (PDB ID: 4TVP) as a template. The zoomed-in image of V1V2 domain is shown to highlight the positions of V2-specific mutations, H173Y (red) and 3-residue deletion, DSV (blue). Deletion in the variable, V4 region is depicted in bright green and mutation in the conserved C2 region, K236T substitution, is shown in magenta. (E) A color-coded 4-5 β-stranded (A-D) conserved Greek-key motif structure of V1V2 domain is represented showing residue 173 on the C β-strand. (F) Snapshot of V1V2 sequences of T/F viruses from each RV217 participant under study. Major structural features of V1V2 region; semi-conserved four β-strands (A, B, C and D) and hypervariable V1 and V2 loops are labeled on top of the sequence. Prominent V1V2-specific mutation sites (observed in >50% of circulating viruses) until 24-wks are highlighted in red in the respective T/F virus sequence isolated from participants – 7, 61, 94 and 100.

A H173Y mutation in T/F07 HIV-1 Env led to virus escape in the participant 7.

(A) SPR binding curves showing reactivity of purified recombinant gp16-T/F07 (green), gp16-H173Y (red), gp16-ΔDSV (magenta) and gp16-H173Y+ΔDSV (blue) V1V2 proteins with a series of longitudinal plasma samples collected from participant 7 (shown on x-axis). The samples from wk 0 (pre-infection) until wk 144 (post-infection) were tested. The amount of binding is proportional to the response units (RU) plotted on the y-axis. The corresponding time points for each peak of antibodies are indicated. An early wave of V2-specific antibody represented by first peak at 6-month time-point recognized the gp16-T/F07 and gp16-ΔDSV variants [H173 variants] but not the gp16-H173Y and gp16-H173Y+ΔDSV variants [Y173 variants]. The second and third peaks of antibodies were found to be reactive to both the variants. (B-D) H173Y mutants poorly react to CH59 and CH58 antibodies. Immunoblot of the purified gp16 T/F07 V1V2 mutants (labeled on the top) showing respective binding with V2 mAbs, CH59 and CH58. Recognition by Strep-tag (purification tag) antibody served as a protein loading control (B). Binding curves of gp16-T/F07 (green), gp16-H173Y (red), gp16-ΔDSV (magenta), gp16-H173Y+ΔDSV (blue) V1V2-proteins, and gp16 scaffold only (negative control) (black) showing reactivity to CH59 (C) and CH58 (D) mAbs, as determined by ELISA.

ADCC responses in longitudinal plasma samples of participant 7.

(A-C) ADCC responses at wk 0 (A, pre-infection), 4-wks post-infection (B), 24-wks post-infection (C) of participant 7 plasma samples measured as percentage Granzyme activity (y-axis), against gp120-T/F07 (pink), gp120-H173Y (teal), gp120-H173Y+ΔDSV (orange) and control antigen, gp120-92TH023 (violet) coated target cells. (D-F) The corresponding area under curve (AUC) values for A-C graphs are shown for each curve. (G-J) V2-specific CH58 (positive control antibody, G-H) and Flu-specific CH65 (negative control antibody, I-J) mediated ADCC responses are shown with corresponding AUC values. For CH65 and CH58 mAbs, 4-fold serial dilutions starting at 12.5 µg/ml concentration were used as shown on the x-axis. Participant 7 plasma samples from each visit were 5-fold serially diluted to determine the ADCC responses. N.D is not detected.

Binding patterns of V1V2 variant-immunized mice sera with overlapping V2-peptides.

(A) Immunization scheme. Prime plus 3-boost immunizations were performed at an interval of three wks. Pre-bleed sera were collected prior to immunizations. (B) V1V2-specific responses in different immunized groups after the last immunization (terminal bleed). Binding curves for each group are color-coded, as shown on the right side of the graph. (C) Schematic showing T/F07-V1V2 sequence labelled on top for A-D β-strands and hypervariable V1 and V2 loops. The overlapping V2 peptides used for SPR binding analysis with immunized mice sera are shown in different colors. (D-G) SPR binding responses shown for gp16-T/F07 (D), gp16-H173Y (E), gp16-ΔDSV (F), gp16-H173Y+ΔDSV (G) groups determined two weeks post-immunization (post) of the last boost with specific V2 peptides represented by the corresponding color of the peptides shown in (C). Pre-bleed sera prior to immunization sera were used as negative controls. The assay was performed with four technical replicates. Each dot represents individual mouse sera. The binding signal is estimated as Response Units (RU) shown on the y-axis. P-value was determined through unpaired t-test, *p value = 0.0252.

The H173 and Y173 V2-variants induce distinct antibody responses.

(A) Sequence alignment of V1V2 region of all the antigens used in the cross-reactivity ELISA experiment. The sequences are compared with T/F07 V1V2 (top-most) sequence. T/F07 matching residues are shown as dots and different residues are highlighted in red. Difference in the lengths of hypervariable loops is shown by dashed lines (gaps). Degree of conservation is depicted graphically at the bottom of the sequence alignment. (B) Heat-map showing cross-reactivity for each immunized group. The map is generated based on AUC values calculated from the binding curves shown in Fig S5 for each immunized group. The antigens used in the binding experiment are labelled horizontally at the bottom of the heat-map. The immunization groups are labelled vertically on the left-side of the heat map. The color gradient scale on the right side shows the degree of reactivity with corresponding numerical values. (C) Cumulative AUC values are plotted to display the summarized or total response of each immunization group towards all the antigens, calculated from the binding curves shown in fig.S5. Each antigen is depicted as a small rectangle colored with respect to the key provided at the side of the graph. Binding to each antigen corresponds to the area of rectangle in the bar graph. (D-E) In an antibody blocking assay, significant reduction in sera reactivity was observed in gp140 T/F07 coated wells pre-incubated/blocked with purified mAbs, CH59 (D, orange), and CH58 antibodies (E, teal), for gp16-T/F07 or ΔDSV (H173 variants) but not gp16-H173Y or H173Y+ΔDSV (Y173 variants) immunized mice groups, compared with the unblocked wells (gray). Titrated and optimized sera dilution was used for each group in this assay. (F-I) Sensitivity to D180A mutation. Binding curves showing reactivity of gp16-T/F07 (F), H173Y (G), ΔDSV (H) and H173Y+ΔDSV (I) immunized sera to respective autologous T/F07-gp140 (with matching V2 mutations) coating antigens (color-coded curves) versus T/F07 gp140-D180A mutant (blue curves). Binding is determined by ELISA. Triplicate absorbance (OD 650 nm) readings were used to generate binding curves. P-values were determined through unpaired t-test, **** = p < 0.00001, *** = p < 0.0001, ** = p < 0.001 and * = p < 0.01.

Structural analyses of H173 and Y173 variant V1V2 domains.

(A) Secondary-structure content variation as a function of time in nanoseconds (ns) initiated with V2/strand conformation (left) and V2/helix conformation (right) of H173 and Y173 V1V2 models during MD simulation. The β-sheet content (purple) and α-helix content (red) changes are depicted over the 100 ns time-course of simulation. (B-C) Initial (left) and final (right) structures of the V2/helix trajectory for H173 (B) Y173 (C) V1V2 models. Histidine and tyrosine side chains are shown in the model with C strand highlighted in red. A portion of the helix remains stable (encircled by black dashed lines) at the end of 100 ns simulation time in H173/helix trajectory. (D-E) Ab initio structure modelling of H173 and Y173 variant V1V2 domains. Five models were generated through QUARK modelling tool for H173-V1V2 (D) Y173-V1V2 (E). C strand is colored in red with sidechain shown for residue 173. Sidechain for residue 180 (critical for helix/coil V2-conformation recognizing CH58/59 like antibodies) is shown in blue.

Combinatorial V2-immunogens induce broad antibody responses.

(A) Consensus logo of C β-strand (residue 166 to 178) of the V1V2 region generated using 100 env sequences each of clades A, B, C, D and AE. The size of the letter depicting the residue in the logo is proportional to its conservation across viral sequences under analysis. (B) Four highly variable residue positions (166, 169, 170 and 171) were selected for combinatorial mutagenesis with additional substitutions shown in blue to generate V2 combinatorial libraries. (C) Heat-map showing binding of sera from different immunized mice groups to diverse HIV-1 Env antigens. Percent AUC values derived from the binding curves and normalized to autologous antigen binding response were used to generate the heat-map. The antigens used in the binding experiment are labelled horizontally at the top of the heat-map. The immunized mice groups are labelled vertically on the left side of the heat-map. The color gradient scale on the right side shows the degree of reactivity with corresponding AUC percent values. (D) Effect of C strand sequence variation on the binding of T/F07 and H173Y immunized groups’ sera. Scatter plot showing significant difference in the reactivity of gp16-T/F07 but not H173Y immunized sera with Env antigens having C strand sequence closely matching (red circles) versus mismatching (blue squares) to T/F07. Normalized mean AUC (%) values are plotted on the y-axis estimated from the binding curve for each antigen represented as sphere or square in the graph (each circle or square represents different antigen). (E) Cumulative AUC values are plotted for T/F07 and H173Y groups to display the summarized or total response from all the diverse antigens in the library. Each antigen is depicted as a small rectangle colored with respect to the key provided at the bottom of the graph. Binding to each antigen corresponds to the area of rectangle in the bar graph. (F) CH58 antibody blocking assay data. Significant reduction in sera reactivity was observed for T/F07 and T/F07 library groups when gp140 T/F07 coated wells were pre-incubated/blocked with purified mAb, CH58 (grey) compared to unblocked (pink). No statistically significant inhibition of binding signal was observed for any H173Y-based immunogen groups. P values determined using unpaired t-test, ** = p < 0.01 and *** = p < 0.001 (significant difference).