Relationship between amplicon length and PCR coverage.

<p>Percentage of sequences in each alignment that would be detectable by PCR, averaged over all amplicons of a specified length spanning HXB2 positions 2000 through 8000 at 10 bp intervals. Results are shown for 31 subjects and grouped by sample type (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005689#ppat.1005689.t001" target="_blank">Table 1</a>).</p

Sources and characteristics of sequence alignments analyzed to generate clonal prediction scores.

<p>Sources and characteristics of sequence alignments analyzed to generate clonal prediction scores.</p

Clonal prediction scores of 1 kb amplicons spanning the HIV-1 genome.

<p>(<b>a</b>) Schematic of algorithm to calculate clonal prediction score (CPS), which quantifies the proportion of unique sequences in an alignment that are correctly identified as unique using the amplicons produced by a specific primer set. (<b>b</b>) CPS of 1 kb-wide amplicons spanning the HIV-1 genome for six Acute treated–DNA subjects. (<b>c</b>) CPS of 1 kb-wide amplicons spanning the HIV-1 genome, averaged over all subjects in five different sample categories (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005689#ppat.1005689.t001" target="_blank">Table 1</a>). (<b>d</b>) Overlay of the five plots in part <b>c</b>. Schematics of the HIV-1 genome are aligned to the charts in parts <b>b</b> through <b>d</b> to highlight viral gene locations. The amplicons in parts <b>b</b> through <b>d</b> are defined with reference to the HXB2 genome.</p

Relationship between amplicon length and CPS.

<p>Summary statistics describing all amplicons of a given length, spanning the HXB2 reference genome at 10 bp intervals. Amplicons with undefined CPS were not included in these summary statistic calculations. (<b>a</b>) Average, median, and minimum of the CPS values for every amplicon of a specified length spanning the viral genome. Summary statistics are shown for each subject and grouped by sample type. (<b>b</b>) Proportion of all of the amplicons of a specified length that have CPS values above 80.</p

KK10 and KK10-cross-reactive microbial peptides differentially expand KK10-specific CD8⁺ T cells.

<p>(<b>A</b>) IFN-y release by ELISpot from HIV-positive HLA-B*27⁺ subject PBMCs stimulated with dilutions of KK10 or KK10CR peptides. (<b>B</b>) Characterization of KK10-specific TCR repertoires after PBMC stimulation with KK10 or KK10CR peptides. PBMCs from HIV-1-positive HLA-B*27⁺ subjects were stimulated with peptides for six days, and KK10-specific CD8⁺ T cells were sorted from fresh (unstimulated), KK10-stimulated, or KK10CR-stimulated PBMCs. Frequencies of unique TCR clones as measured by diversity in the TCR-β CDR3 region were quantified by ImmunoSEQ deep sequencing. Each dot shows the frequency of a single TCR clone in the specified KK10-specific CD8⁺ T cell populations. TCR clones that have the same frequency in both populations compared on a plot fall on the diagonal line. Magenta dots indicate TCR clones that are present at significantly different frequencies (minimum 20 reads in either condition, p<0.005, Fisher’s Exact test with Benjamini-Hochberg correction. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192098#pone.0192098.s001" target="_blank">S1 Table</a> shows all associated p-values).</p

PBMCs stimulated with cross-reactive microbial peptides can suppress HIV-1 infection.

<p>(<b>A-B</b>) Results of PBMC suppression assay. PBMCs from HIV-1-positive HLA-B*27⁺ subjects were stimulated for five days with no peptide, CEF pooled peptides, KK10 peptide, or KK10CR peptide and then infected with highly concentrated IIIB virus. After 40 hours, infection of CD3⁺/CD8^- cells was measured by staining for intracellular HIV-1 Gag. Values below 20% (in grey) fall below the limit of quantification. Part <b>A</b> shows flow cytometric results for subject ES31; part <b>B</b> shows summarized results for five subjects with CEF pooled peptides, KK10 peptide, and individual KK10CR peptides (<b>C-D</b>) PBMCs from HIV-1-positive HLA-A2⁺ subjects were stimulated for five days with no peptide, CEF pooled peptides, SL9 peptide, or SL9CR peptide and then infected with highly concentrated IIIB virus. After 40 hours, infection of CD3⁺/CD8^- cells was measured by staining for intracellular HIV-1 Gag. Part <b>C</b> shows flow cytometric results for subject CP37; part <b>D</b> shows summarized results for seven subjects with CEF pooled peptides, SL9 peptide, and individual SL9CR peptides.</p

SL9 and SL9-cross-reactive microbial peptides differentially expand SL9-specific CD8⁺ T cells.

<p>Characterization of SL9-specific TCR repertoires after PBMC stimulation with SL9 or SL9CR peptides. PBMCs from HIV-1-positive HLA-A2⁺ subjects were stimulated with peptides for six days, and SL9-specific CD8⁺ T cells were sorted from fresh (unstimulated), SL9-stimulated, or SL9CR-stimulated PBMCs. Frequencies of unique TCR clones as measured by diversity in the TCR-β CDR3 region were quantified by ImmunoSEQ deep sequencing. Each dot shows the frequency of a single TCR clone in the specified SL9-specific CD8⁺ T cell populations. TCR clones that have the same frequency in both populations compared on a plot fall on the diagonal line. Magenta dots indicate TCR clones that are present at significantly different frequencies (minimum 20 reads in either condition, p<0.005, Fisher’s Exact test with Benjamini-Hochberg correction. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192098#pone.0192098.s002" target="_blank">S2 Table</a> shows all associated p-values).</p

Panel of potential SL9 cross-reactive peptides.

<p>Panel of potential SL9 cross-reactive peptides.</p

CD8⁺ T cells stimulated with cross-reactive microbial peptides can suppress HIV-1 infection.

<p>Purified CD4⁺ T cells from three HIV-1-positive HLA-B*27⁺ subjects were spinoculated with single-round NL4-3 GFP reporter virus and then co-cultured with peptide-stimulated or unstimulated (fresh <i>ex vivo</i>) autologous CD8⁺ T cells. Viral suppression was measured at four different effector-to-target cell ratios. Left panels show suppression of wild-type (WT) NL4-3 reporter virus, and right panels show suppression of NL4-3 harboring the R264K and L268 mutations in KK10, which prevent KK10 presentation on HLA-B*27 MHC molecules.</p

Characteristics of HIV-infected patients.

<p>Characteristics of HIV-infected patients.</p