Coreceptor tropism of early SHIV stocks.

<p>(A-B) TZM-bl cells were incubated for 1 h with different concentrations of the CCR5 inhibitor TAK-779 or the CXCR4 inhibitor AMD-3100 and subsequently were infected with 100 TCID₅₀ of the indicated SHIV stocks. The luciferase activity was quantified after 48 h. (C) GHOST cell lines expressing CXCR4 (X4) and/or CCR5 (R5) coreceptors were used, and inoculated with 100-TCID₅₀ SHIV stocks. Cell culture supernatant was collected for SIV p27 determination after 4 days of infection. All assays were done in triplicate. The means with standard deviation are shown.</p

i.r. challenge with SHIV-AE6, SHIV-AE6RM, SHIV-AE16 stocks in rhesus monkeys.

<p>Twelve animals were challenged once with 1 ml of undiluted (A) SHIV-AE6 (n = 4), (B) SHIV-AE6RM (n = 4), and (C) SHIV-AE16 (n = 4) stocks by the i.r. route. The upper panel shows plasma viral loads, and the lower panel shows the percentage of CD4⁺ T cells in peripheral blood. The dotted line reflected the limit of detection of the assay (50 RNA copies/ml).</p

HIV-1 ENV sequence characteristics.

<p>HIV-1 ENV sequence characteristics.</p

Summary of large-scale SHIV stocks^a.

<p>Summary of large-scale SHIV stocks<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005431#t003fn001" target="_blank">^a</a>.</p

Neutralization properties of SHIV stocks in TZM-bl assays^a.

<p>Neutralization properties of SHIV stocks in TZM-bl assays<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005431#t004fn001" target="_blank">^a</a>.</p

Summary of in vivo infectivity of SHIVs -AE6, -AE6RM, and -AE16 stocks in rhesus monkeys.

<p>Summary of in vivo infectivity of SHIVs -AE6, -AE6RM, and -AE16 stocks in rhesus monkeys.</p

Log viral DNA copies/10⁶ cells from week 12 PBMC, LMNC, and colorectal samples obtained from rhesus monkeys infected with SHIV-AE16 stock at (A) 1:1 and (B) 1:10 dilution.

<p>The dotted line depicts the limit of detection of the assay (3 copies/10⁶ cells).</p

Cloning and replication of 293T-derived SHIVs in human and rhesus PBMC^a.

<p>Cloning and replication of 293T-derived SHIVs in human and rhesus PBMC<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005431#t002fn001" target="_blank">^a</a>.</p

Recognition of latently-infected primary CD4⁺ T-cells by virus-specific CD8⁺ T-cell clones following exposure to candidate LRAs.

<p>HIV-, CMV- and HERV-K-specific CD8⁺ T-cell clones were derived from the HIV-infected participant OM9. Latently-infected and productively-infected target cells were prepared and characterized as in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.g001" target="_blank">Fig 1A</a></b>. The designated CD8⁺ T-cell clones were co-cultured with the indicated target CD4⁺ T-cells (autologous) immediately after the depletion of activated cells. Cells were stained and fixed after 16 hour co-cultures. <b>A.</b> Shown are flow cytometry data gated on CD3⁺CD8⁺ lymphocytes and depicting CD107a (degranulation)–y-axis, by IFN-γ –x-axis. Latently-infected cells did not induce CD107a exposure. <b>B.</b> Summary flow cytometry data of the same experiment depicted in <b>A</b>. P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with latent-mock). All conditions/replicates tested are shown. Latent-mock and latent-HIV conditions of MHC-I mismatch were omitted from the HIV-Nef-spec CD8⁺ T-cells due to insufficient cell numbers, as were replicates of MHC-I mismatch conditions for the HERV-K-Env-specific CD8⁺ T-cell clone. CD107a exposure by an HIV-specific CD8⁺ T-cell clone was only induced by productive HIV infection. <b>C</b>. In a separate experiment, latently-infected target cells were prepared in the same manner as <b>A,</b> rested for 72 hours, and then combined with an autologous HIV-Gag-specific CD8⁺ T-cell clone (upper panels) or an autologous CMV-pp65-specific CD8⁺ T-cell clone (lower panels) for a 24 hour co-culture period. Candidate latency-reversing drugs were added as indicated above the corresponding panels, and left in for the duration of co-cultures. Shown are flow cytometry data gated on CD3⁺CD8⁺ lymphocytes and depicting CD137 (4-1BB)–y-axis by CD8 x-axis. CD137 expression by an HIV-specific CD8⁺ T-cell clone was induced following treatment with IL-15, IL-2, and prostratin, but not SAHA.</p

Induction of HIV by LRAs in a primary CD4+ T-cell direct infection latency model.

<p>CD4⁺ T-cells were isolated from PBMC by negative selection, cultured with CCL19 and then magnetofected with HIV, or mock infected (no virus). Two days later, cells were depleted of activated cells. We defined activated cells as those expressing at least one of CD69, HLA-DR, or CD25 based on a study that defined CD4+ T-cells with the triple-negative phenotype as quiescent[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005545#ppat.1005545.ref029" target="_blank">29</a>]. In parallel, CD4⁺ T-cells were activated using anti-CD3/anti-CD28 antibodies and infected with HIV (productively infected). <b>A</b>. Depletion of activated cells by anti-PE microbeads. Shown are flow cytometry data gated on CD3⁺CD4⁺ lymphocytes (95% pure) and depicting CD25/CD69/HLA-DR staining (all pooled on PE channel)–y-axis, by HIV-Gag–x-axis. The left and middle panels represent pre- and post-depletion of activated cells, respectively to generate latently-infected cells. The right panel shows productively-infected target cells. <b>B</b>. Latently infected or mock-infected resting CD4⁺ T-cells were prepared as in <b>A</b> and then either stimulated with 1.5 nM IL-15, with 2.6 μM prostratin, or left unstimulated for 36 hours. Shown are flow cytometry data, gated on lymphocytes (SSC/FSC) and depicting CD4 –y-axis, by HIV-Gag–x-axis. <b>C</b>. In a separate experiment analogous to <b>B</b> latently-infected or mock-infected cells were treated with the indicated concentrations of IL-15 for 16 hours. The percentage of HIV-Gag⁺ cells (gated as in <b>B</b>) is plotted against the concentration of IL-15 (red circles = infected, green squares = uninfected). <b>D.</b> Latently-infected cells were stimulated with the indicated LRAs for 36 hours and HIV p24 in supernatants was quantified by ELISA. Shown are background-subtracted mean ± SEM values (duplicates). P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with no treatment [No Tx]) * p < 0.05, ** p < 0.01.</p