Characteristics of the study population.

<p>Groups of participants with genital viral load (GVL) < 40 HIV RNA copies/mL (N = 132) versus GVL≥40 HIV RNA copies/mL (N = 111) at baseline are compared. Comparison is made using t-test for continuous and Chi-square for dichotomous variables. Significant differences are highlighted with*.</p><p>Characteristics of the study population.</p

Changes in proportion of detectable cytokine levels from baseline to month 12 in participants receiving ART,

<p>Only cytokines analyzed as dichotomous variable are included in this table. Changes of proportion of women with detectable cytokines are compared between baseline and month 12 using McNemar. No significant differences were found. Results on cytokines analyzed as continuous variables are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127201#pone.0127201.g002" target="_blank">Fig 2</a>.</p><p>Changes in proportion of detectable cytokine levels from baseline to month 12 in participants receiving ART,</p

Cohort profile.

<p>This flow chart provides information on the number of patients recruited, eligible for antiretroviral therapy and who had viral load and CD4 data available at baseline and at month 12. Patients were classified in groups of genital viral load (GVL) <40 RNA copies/ml and GVL ≥40 copies /mL.</p

Predictors of GVL at baseline and at month 12 among participants receiving ART.

<p>Predictors of genital viral load ≥40 copies/mL at baseline and at month 12 were determined. Ninety six women initiating ART were included in the analysis at baseline. Forty-nine women receiving ART had viral load data available at month 12 and were included in the analysis at month 12. The variables included were:</p><p>At baseline: Plasma viral load, CD4, age, IL-8, IP-10, MIP-1b, VEGF, IL-6, GCSF, IL-1β, <i>N</i>. <i>gonorrhoea</i>, <i>Chlamydia trachomatis</i>, <i>Trichomonas vaginalis</i>, HSV2</p><p>At month 12: Use of family planning method, marital status, <i>Trichomonas vaginalis</i> at baseline & month 12, IL-8 at baseline & month 12, IP-10 at baseline & month 12, MIP-1b at month 12, IL-1β at month 12, IL-1RA at month 12, IL-6 at month 12, Genital VL at baseline, plasma VL at month 12.</p><p>AIC: Aikaike Information criteria.</p><p>Predictors of GVL at baseline and at month 12 among participants receiving ART.</p

Changes of biological marker levels in the genital compartments between baseline and month 12 among study participants receiving anti-retroviral therapy.

<p>Changes of genital (GVL) and plasma viral loads (PVL), CD4 counts and cytokines levels analysed as continuous variables were compared between baseline and month 12 in patients receiving antiretroviral therapy. Thin broken lines represent the change in immune parameter of a study participant between the two measurement times and the thick solid line in red is the average change in the parameter between the two time points. P values were calculated using the paired t-test and are displayed inside each graph. GVL, PVL and CD4 counts changes were significant, whereas cytokines levels remained comparable at baseline and month 12.</p

Diversification in the HIV-1 Envelope Hyper-variable Domains V2, V4, and V5 and Higher Probability of Transmitted/Founder Envelope Glycosylation Favor the Development of Heterologous Neutralization Breadth

<div><p>A recent study of plasma neutralization breadth in HIV-1 infected individuals at nine International AIDS Vaccine Initiative (IAVI) sites reported that viral load, HLA-A*03 genotype, and subtype C infection were strongly associated with the development of neutralization breadth. Here, we refine the findings of that study by analyzing the impact of the transmitted/founder (T/F) envelope (Env), early Env diversification, and autologous neutralization on the development of plasma neutralization breadth in 21 participants identified during recent infection at two of those sites: Kigali, Rwanda (n = 9) and Lusaka, Zambia (n = 12). Single-genome analysis of full-length T/F Env sequences revealed that all 21 individuals were infected with a highly homogeneous population of viral variants, which were categorized as subtype C (n = 12), A1 (n = 7), or recombinant AC (n = 2). An extensive amino acid sequence-based analysis of variable loop lengths and glycosylation patterns in the T/F Envs revealed that a lower ratio of NXS to NXT-encoded glycan motifs correlated with neutralization breadth. Further analysis comparing amino acid sequence changes, insertions/deletions, and glycan motif alterations between the T/F Env and autologous early Env variants revealed that extensive diversification focused in the V2, V4, and V5 regions of gp120, accompanied by contemporaneous viral escape, significantly favored the development of breadth. These results suggest that more efficient glycosylation of subtype A and C T/F Envs through fewer NXS-encoded glycan sites is more likely to elicit antibodies that can transition from autologous to heterologous neutralizing activity following exposure to gp120 diversification. This initiates an Env-antibody co-evolution cycle that increases neutralization breadth, and is further augmented over time by additional viral and host factors. These findings suggest that understanding how variation in the efficiency of site-specific glycosylation influences neutralizing antibody elicitation and targeting could advance the design of immunogens aimed at inducing antibodies that can transition from autologous to heterologous neutralizing activity.</p></div

Correlation of T/F Env features with neutralization breadth.

<p>T/F Env amino acid sequence features for 21 subjects were defined using the LANL N-GlycoSite and Variable Region Characteristics online tools. A) Spearman’s correlations were performed between neutralization breadth AUC and variable loop lengths, number of total, (NXS + NXT), NXS, and NXT glycan motifs (excluding X = proline) in gp120, and the ratio of NXS motifs divided by NXT motifs. Spearman’s r values and p values are shown for each comparison. P values < 0.05 are considered significant. B) Scatter plot of median 3-year breadth AUC value vs ratio of NXS:NXT sites in the transmitted/founder Env. Spearman correlation r = 0.56, p = 0.008.</p

Validation of the Immunotype Diversity Index approach to quantify Env diversification using R880F.

<p>Subject R880F was chosen to validate the Sequence Harmony approach because the earliest neutralizing antibody response was previously mapped to an epitope located at the base of the V3 loop [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005989#ppat.1005989.ref011" target="_blank">11</a>]. Positions indicated with an asterisk were identified and evaluated in the previous study. Thirty-three SGA derived T/F Env amino acid sequences from R880F (Group 1) were aligned with 10 sequences from 3-months post-infection <b>(A)</b>, or 12 sequences from 6-months post-infection <b>(B)</b> (Group 2). In (A), Sequence Harmony analysis identified four positions, highlighted yellow, that were significantly different (Z<-3) between the two populations at 3-months. Two of these positions were determined to be autologous neutralizing antibody escape mutations at 3-months via mutagenesis (295 and 338), whereas 456 could not be directly attributed to neutralizing antibody or cytotoxic T lymphocyte selective pressure. In (B), Sequence Harmony analysis identified two positions that remained significant from 3 months (highlighted yellow), six positions that became significant at 6 months (highlighted green), and two positions that lost significance at 6 months (not highlighted). Positions 335, 338, and 341 were all determined to be neutralizing antibody escape mutations at 6 months.</p

Reference <i>pol</i> sequences from the Los Alamos database included in the phylogenetic analyses.

<p>1480 n long references sequences of the subtype identified in the Rwandan cohorts were selected from the Los Alamos database. The table indicates the HIV-1 subtypes, respective number of sequences, country of origin and year of sampling. In earlier years subtype A was not yet subdivided into sub-subtype A1. The phylogenetic analysis was done in three separate trees involving subtype A or A1 and C (analysis 1 also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042557#pone-0042557-g004" target="_blank">figure 4</a>), subtype D (analysis 2) and A1/C recombinant (analysis 3).</p

Autologous early neutralization of T/F Envs vs. contemporaneous Envs.

<p>For 11 patients, sampling allowed neutralization assays to be performed on T/F Envs, and Envs from an early time-point (range 2 to 12 months, median 5 months), with the early time-point plasma listed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005989#ppat.1005989.g008" target="_blank">Fig 8</a>. Wilcoxon matched-pairs signed rank test revealed a significant difference between neutralization IC50 titer of the early plasma vs. T/F Envs, compared to the same plasma against the contemporaneous Envs (p = 0.002).</p