data_sheet_1.DOCX

<p>Strain-specific neutralizing antibodies develop in all human immunodeficiency virus type 1 (HIV-1)-infected individuals. However, only 10–30% of infected individuals produce broadly neutralizing antibodies (bNAbs). Identification and characterization of these bNAbs and understanding their evolution dynamics are critical for obtaining useful clues for the development of an effective HIV vaccine. Very recently, we published a study in which we identified 12 HIV-1 subtype C-infected individuals from India whose plasma showed potent and broad cross-clade neutralization (BCN) ability (1). In the present study, we report our findings on the evolution of host bNAb response over a period of 4 years in a subset of these individuals. Three of the five individuals (NAB033, NAB059, and NAB065) demonstrated a significant increase (p < 0.05) in potency. Interestingly, two of the three samples also showed a significant increase in CD4 binding site-specific antibody response, maintained stable CD4+ T cell counts (>350 cells/mm³) and continued to remain ART-naïve for more than 10 years after initial diagnosis, implying a strong clinical correlation with the development and evolution of broadly neutralizing antibody response against HIV-1.</p

Frequency of IFN- γ ELISPOT T-cell response.

<p>na: Not Applicable, nd: not done.</p

T-cell immune responses as assessed by IFN-γ secretory ELISPOT assays against ADVAX matched peptides after 1^st and 2^nd DNA vaccinations in group A and TBC-M4 matched peptides at the other time points in group A and at all the time points in group B participants are shown.

<p>Average magnitude of IFN-γ ELISPOT responses in SFC/10⁶ cells (Y-axis) at each time point, by Groups A and B volunteers (X-axis) against different antigens are represented by different colours. 7d and 14d indicate 7 and 14 days after every vaccination, respectively. Values above the bars represent the percent of volunteers with positive responses to any or at least one peptide at that visit. The black lines and the corresponding p values showed comparison between the responses in group A and group B at days 7 and 14 after the first and second MVA vaccinations.</p

Flow chart of screened and enrolled volunteers.

<p>Flow chart of screened and enrolled volunteers.</p

Frequency of grade 1, 2 and 3 unsolicited adverse events recorded within 28 days post-vaccinations.

<p>Bars represent the maximum severity per volunteers within 28 days after either 2 vaccinations in Group A (separately for ADVAX and TBC-M4) or 3 vaccinations in Group B. Relative frequency of adverse events is plotted on the Y axis and Groups A, B vaccine or placebo recipients are plotted on the X-axis.</p

Spectrum of HIV-specific antibodies as determined by Western blot among Groups A and B vaccine recipients.

<p>Antigens recognized by HIV-specific antibodies as determined by HIV Western blot assay by group and visit after the last vaccination. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055831#pone-0055831-g002" target="_blank">Figure 2a</a> shows the frequency of volunteers recognizing each HIV antigen (Env: gp160, gp120 and gp41, Pol: p65, Gag: p55, p24 and p40) by the presence of bands in Western blot. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055831#pone-0055831-g002" target="_blank">Figure 2b</a> shows the distribution of the spectrum of HIV-specific antibodies (number of HIV antigens identified) by Western blot. median, inter-quartile and minimum-maximum ranges are presented in the Box-Whiskers plots.</p

HIV neutralizing antibody titres (expressed as ID₅₀ values) of Group A and Group B volunteers after last vaccination.

<p>ID₅₀ values were determined by TZM-bl assay from serum samples of Group A (red in colour) and Group B (blue in colour) volunteers at 14 days (indicated as circles) and 3 months (indicated as squares) after last vaccination against a panel of pseudoviruses (X-axis). The pseudoviruses shown in the graph are MW965.26 (Tier-1 subtype C), SF162.LS (Tier-1 subtype B), IVC 5–41 (recently transmitted Indian strain) and TV1.21 (Tier-2 subtype C). No neutralization response was seen against HIV 001428-2.42 (Tier-2 subtype C) (not shown). The neutralizing antibody titres from Group B volunteers were found to be higher than in Group A volunteers at 14 days and 3 months following last vaccination. P-values were calculated by the Mann Whitney test. The vertical bars represent median and inter-quartile range.</p

HIV binding ELISA antibody response rates.

<p>HIV binding ELISA antibody response rates.</p