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

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

Two-step bead depletion procedure yields highly purified resting CD4⁺ T cells from HIV-1 infected patients.

<p>A two-step negative selection strategy to purify resting CD4⁺ T cells from patient PBMC. (<b>A</b>) Representative FSC/SSC plot indicating live cell population after the two-step bead depletion procedure. (<b>B</b>) Representative dot plot indicating purity of resting CD4⁺ T cells. Purified cells were stained with antibodies to CD4 and HLA-DR.</p

Accurate measurement of IUPM at day 7 using HIV-1 specific RT-PCR.

<p>(<b>A</b>) Using the rapid MOLT-4/CCR5 viral outgrowth assay, the frequency of latently infected cells was measured for HAART patients S1–S14 and viremic patients V1–V3 at day 7 with the HIV-1 specific RT-PCR assay and at both days 7 and 14 with HIV-1 p24 antigen ELISA. Statistical significance of the differences in IUPM values was assessed by Wilcoxon rank sum test. (<b>B</b>) Correlation of the IUPM measured at day 7 using HIV-1 p24 antigen ELISA with the IUPM measured at day 14 using HIV-1 p24 antigen ELISA (Pearson's correlation coefficient, r). (<b>C</b>) Correlation of the IUPM measured at day 7 using HIV-1 specific RT-PCR with the IUPM measured at day 14 using HIV-1 p24 antigen ELISA (Pearson's correlation coefficient, r). (<b>D</b>) Correlation of the IUPM measured at day 7 using the rapid MOLT-4/CCR5 outgrowth assay with the IUPM measured at day 14 using the standard outgrowth assay (Pearson's correlation coefficient, r).</p

The standard and the MOLT-4/CCR5 viral outgrowth assays.

<p>The frequency of HIV-1 latent infection of resting CD4⁺ T cells can be measured using a viral outgrowth assay. PBMC are collected from HIV-1 infected individuals and resting CD4⁺ T cells (CD25⁻, CD69⁻, HLA-DR⁻) are purified. Resting T cells are plated in 5-fold serial dilutions in duplicate, such that the input number of patient cells ranges from 1,000,000 to 320 cells per well. To reverse latency in the cells that harbor a replication-competent HIV-1 provirus, patient cells are activated with PHA and a 10-fold excess of irradiated PBMC from healthy donors. The next day, target cells for HIV-1 infection are added to allow outgrowth of replication-competent HIV-1 released from infected cells in which latency has been reversed. In the standard viral outgrowth assay, CD4⁺ lymphoblasts from healthy donors are added on days 2 and 7 of the assay. In the MOLT-4/CCR5 viral outgrowth assay, MOLT-4/CCR5 cells are added on day 2 only. For the standard assay, HIV-1 p24 antigen ELISA is used to identify wells positive for HIV-1 outgrowth at 14 days. For the MOLT-4/CCR5 assay, RT-PCR is used to identify wells positive for outgrowth at 7 days. The frequency of latently infected cells can be determined using limiting dilution statistics based on the input number of patient cells in the wells positive for outgrowth. This frequency is reported in infectious units per million (IUPM).</p

Kinetics of HIV-1 outgrowth from latently infected CD4⁺ T cells measured by HIV-1 p24 antigen ELISA and HIV-1 specific RT-PCR.

<p>Resting CD4⁺ T cells were isolated from HAART patient S15, whose latent reservoir was previously measured to be 3.25 IUPM. Twenty-nine replicate wells were plated in which 200,000 resting cells were activated with PHA and irradiated PBMC from a healthy donor and subsequently cultured with MOLT-4/CCR5 cells. Outgrowth of reactivated HIV-1 was measured in positive wells over 14 days using both (<b>A</b>) HIV-1 p24 antigen ELISA and (<b>B</b>) HIV-1 specific RT-PCR. (<b>C</b>) The difference between the day on which a particular well becomes positive by RT-PCR versus p24 ELISA. (<b>D</b>) The day of detection of HIV-1 outgrowth from the latent reservoir is shown for HIV-1 p24 antigen ELISA and HIV-1 specific RT-PCR.</p

Characteristics of study patients.

†<p>Race abbreviations: W, white, non-Hispanic; H, Hispanic; B, black.</p>§<p>Time of documented continuous suppression of plasma viremia to <50 copies/mL on HAART.</p>‡<p>Drug abbreviations: DRV/r, darunavir boosted with ritonavir; FTC, emtricitabine; TDF, tenofovir disoproxil fumarate; EFV, efavirenz; RAL, raltegravir; RAL/r, raltegravir boosted with ritonavir; ATV/r, atazanavir boosted with ritonavir; 3TC, lamivudine; MVC, maraviroc; FPV/r, fosamprenavir boosted with ritonavir; NVP, nevirapine.</p

Silencing of TLR2 expression attenuates the increased susceptibility to HIV.

<p>Expression of TLR2 increases at the transcription level in cells stimulated with <i>M. bovis</i> BCG when compared to cells stimulated with <i>M. smegmatis</i> (A). The expression levels of TLR4 and TLR9 do not show a significant difference in cells stimulated with different strains of mycobacteria as evidenced by RT-PCR. Comparison of HIV infectivity of CD4+ cells transfected with scrambled siRNA and subsequently stimulated with <i>M. bovis</i> BCG to CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (B) **p<0.005. TLR2 expression on CD4+ cells after stimulation with <i>M. bovis</i> BCG, <i>M. tuberculosis</i>, and <i>M. smegmatis</i> compared to TLR2 expression on CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (C). The expression showed similar trends for the four samples tested. Dot plots comparing the percentage of surface expression of TLR2 on unstimulated CD4+ cells and CD4+ cells stimulated with <i>M. bovis</i> BCG, <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155), and CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (D).</p