Characteristics of HVTN 502 and 503 trial participants.

<p>Data shown are median values (interquartile range) unless otherwise indicated.</p><p>* At time of sampling</p><p>^§ HLA-B*27, B51, B*5701/03, B*5801 and B*8101</p><p>Characteristics of HVTN 502 and 503 trial participants.</p

CD8+ T cell responses to PTE peptides based on to the entire HIV proteome were measured in HIV-positive HVTN participants (502—vaccinees, black closed circles; placebos, black open circles) and (503—vaccinees, grey closed circles; placebos, grey open circles) at week 5 post-infection by intracellular staining for IFN-γ.

<p><b>A.</b><b>B.</b> Correlation between CD8+ T cell inhibition (CD8+/CD4+ cell ratio = 2:1) of a clade-matched virus and magnitude of CD8+ T cell response to the entire HIV proteome, colour-coded as indicated in A. Five subjects are not shown in B, due to lack of viral inhibition data for the 2:1 ratio.</p

Characteristics of Viremic Controller subjects.

<p>* At time of sampling</p><p>^§ HLA-B*27, B51, B*5701/03, B*5801 and B*8101</p><p>^# Presumed clade due to geographical location</p><p>Characteristics of Viremic Controller subjects.</p

Multivariate linear regression models to investigate associations between beneficial responses, entropy, HLA alleles and % inhibition (dependent variable).

<p>In each model, magnitude = total beneficial response (sum of responses to beneficial peptide pools)</p><p>Ratio of protective / total response = total beneficial response / total proteome response (both defined as IFN-γ+ cells/million CD8+ T cells)</p><p>* HLA-B*27, B*51, B*5701, B*5801, B*81</p><p>^§ HLA-B*35 (Py), HLA-B*53</p><p>Multivariate linear regression models to investigate associations between beneficial responses, entropy, HLA alleles and % inhibition (dependent variable).</p

Distribution of responses to Gag peptides in HIV-positive MVA.HIVA vaccinees.

<p>Distribution of responses to Gag peptides in HIV-positive MVA.HIVA vaccinees.</p

CD8+ T cell inhibitory activity and targeting of beneficial regions and conserved elements within the HIV proteome.

<p>CD8+ T cell responses to peptides based on (<b>A</b>) clade-specific ‘beneficial’ regions and (<b>B</b>) Gag ‘conserved elements’ were measured by IFN-γ Elispot assays. Net responses (background subtracted) are shown; values for negative controls were median (IQR)– 10 (0–15) SFU/million CD8+ T cells. Horizontal lines indicate median values. HVTN vaccinees and placebos are shown as closed and open symbols respectively in <b>A</b>. In <b>B</b>, HVTN subjects are grouped together and represented as follows: HVTN 502—vaccinees, black closed circles, placebos, black open circles; HVTN 503—vaccinees, grey closed circles, placebos, grey open circles. VC are shown as triangles in <b>A & B</b>. Six HVTN 503 subjects were excluded as viral subtype data were not confirmed at the time of the analysis. One VC subject was excluded as no sample was available for Elispot assay. <b>C.</b> Correlation between CD8+ T cell inhibition of a clade-matched virus (CD8+/CD4+ cell ratio = 2:1) and magnitude of CD8+ T cell responses to beneficial peptides (summed) in 26 HVTN subjects. <b>D.</b> The analysis was repeated after removal of subjects with protective HLA class I alleles and (<b>E</b>) with short-term cell lines expanded from CD8+ T cells recovered from Elispot assays in 15 subjects that were then tested with individual peptides from the pools which elicited a response in the ex vivo Elispot assay. <b>For C-F</b>: closed circles—502 and 503 vaccinees; open circles—502 and 503 placebos. <b>F.</b> Correlation between CD8+ T cell inhibition (2:1 ratio) of a clade-matched virus and magnitude of CD8+ T cell responses to conserved elements peptides in 27 HVTN subjects (left panel—sum of all CE peptides, middle—CE pool A, right—CE pool B).</p

CD8+ T cell antiviral inhibitory activity in HIV-positive HVTN subjects and HIV viremic controllers.

<p><b>A.</b> CD8+ T cell mediated-inhibition of a clade-matched virus was measured in 28 HIV-positive HVTN 502 & 503 subjects at a CD8+/CD4+ cell ratio of 2:1. Eight subjects are not shown as cell recoveries allowed testing at a CD8+/CD4+ cell ratio of 1:1 only (n = 6, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004658#ppat.1004658.s003" target="_blank">S1 Table</a>) or CD4+ cells did not survive HIV superinfection (n = 2). Data are stratified by vaccine or placebo allocation. <b>B-D.</b> CD8+ T cells from 16 HVTN 502 participants and 14 VC (all infected with clade B viruses) were tested for inhibition of a clade B HIV isolate at CD8+/CD4+ cell ratios of 2:1 (<b>B</b>) and 1:10 (<b>C</b>) or inhibition of a clade C isolate, ES X-1936 (CD8+/CD4+ = 2:1) (<b>D</b>). Placebos are indicated by open circles. In all graphs, horizontal lines indicate medians. <b>E.</b> Heatmap showing potency and breadth of CD8+ T cell-mediated inhibition (CD8+/CD4+ cells = 2:1) among 14 viremic controllers and 14 HIV-positive HVTN 502 and 503 participants for whom at least 3 virus isolates were tested. Viral inhibition was measured on day 6 for all viruses except A2 (RW93024), for which it was measured on day 3 due its different replication kinetics (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004658#sec008" target="_blank">Methods</a>). Darker colour indicates higher inhibition; scale 0–100%, grading—20%.</p

Vaccines based on full-length protein immunogens elicit subdominant responses to beneficial regions and / or conserved elements within the HIV proteome.

<p><b>A.</b> Post-vaccination, pre-infection CD8+ T cell responses to overlapping peptides spanning the MRK Ad5 Gag/Pol/Nef immunogen determined by IFN-γ Elispot assay in 13 HVTN 502 participants (dark blue bars) (4 weeks post-second vaccination). Responses to beneficial regions and Gag conserved elements within the immunogen are indicated by magenta and turquoise bars respectively. <b>B.</b> Pre- and post-vaccination responses to beneficial and non-beneficial regions within Gag determined by IFN-γ Elispot assay in 9 HIV-positive subjects after therapeutic vaccination with MVA.HIVA.</p

Univariate linear regression models to investigate associations between entropy, beneficial and conserved elements responses and % inhibition (dependent variable).

<p>Values shown are parameter estimates [95% confidence intervals] for each model.</p><p>All models: entropy refers to Shannon entropy score for beneficial peptides</p><p>Model 2: total beneficial response = sum of responses to beneficial peptide pools</p><p>Model 3: beneficial Gag response = sum of responses to beneficial Gag pools only</p><p>Model 4: conserved elements (CE) response = sum of responses to CE pools</p><p>Univariate linear regression models to investigate associations between entropy, beneficial and conserved elements responses and % inhibition (dependent variable).</p