Week 48 resistance analysis of Elvitegravir/Cobicistat/Emtricitabine/Tenofovir DF versus Atazanavir + Ritonavir + Emtricitabine/Tenofovir DF in HIV-1 infected women (WAVES study GS-US-236-0128)

<p>Women and those with non-B subtype HIV-1 are typically underrepresented in clinical trials. WAVES (GS-US-236-0128) was a double-blind phase 3b study among treatment-naïve HIV-1-infected women that demonstrated that elvitegravir/cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF; <i>N</i> = 289) was superior to atazanavir + ritonavir + FTC/TDF (ATV + RTV + FTC/TDF; <i>N</i> = 286) for HIV-1 RNA < 50 copies/mL by FDA snapshot analysis at week 48. Here, we describe resistance development through week 48 in women with virologic failure and determine the impact of pre-existing mutations and HIV-1 subtype on viral suppression.</p> <p>Genotypic analyses (population and deep sequencing) and phenotypic analyses of HIV-1 protease, reverse transcriptase (RT), and integrase (IN) were performed. The resistance analysis population (participants with HIV-1 RNA ≥ 400 copies/mL at confirmed virologic failure, at discontinuation ≥ week 8, or at week 48) had genotypic and phenotypic analyses at failure and baseline.</p> <p>The proportion of women qualifying for resistance analyses was similar between treatment groups (6.2% EVG/COBI/FTC/TDF; 7.3% ATV + RTV + FTC/TDF). Emergent resistance was rare (0% EVG/COBI/FTC/TDF; 1% ATV + RTV + FTC/TDF – 3 with M184V/I in RT). Deep sequencing of HIV-1 did not detect additional resistance development. Pre-existing mutations did not lead to virologic failure; most with the polymorphic primary IN substitution T97A (92%), or with substitutions in RT (i.e. A62V, V90I, K103N, or E138A/G/K/Q; 68–82%) demonstrated virologic suppression at week 48, with no resistance development except for one patient with M184V and pre-existing K103N in the ATV + RTV + FTC/TDF group. Most participants (74%) had non-B HIV-1, and subtype did not affect outcome.</p> <p>Emergent resistance to study drugs was rare in this study of women, with no resistance observed among EVG/COBI/FTC/TDF-treated participants, despite a high proportion of participants with natural or transmitted viral mutations and non-B HIV-1 subtypes.</p

CTLA-4 blockade decreases TGF-β and IL-10 expression by HIV-specific CD8+ T Cells.

<p>PBMC (n = 6) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-IL-10 APC, anti-CD3 AmCyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. (A) Representative plots of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. (B-D) Dashed line represents the cutoff for significant TGF-β (B), IL-10 (C), and IFN-γ (D) expression. Percentages in between brackets are median values. The two dots joined by a line represent the values obtained from the same individual and analysis was performed by paired <i>t-</i>test.</p

Analysis of regulatory surface markers expression by HIV-specific TGF-β positive CD8+ T cells.

<p>PBMC were stimulated with HIV peptides, then stained for various memory and regulatory markers and the percentage of TGF-β positive CD8+ T cells was determined by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population and then the percentages of TGF-β positive cells were determined and the extent of FOXP3, CD127, CD25, and CD27 expression was also examined. Gating was performed using the fluorescence-minus-one (FMO) control for each marker. Representative plots of the phenotype of the HIV-specific CD8+ T cells expressing TGF-β. The values marked with an asterisk represent the fraction of TGF-β positive cells that express FOXP3, CD127, CD25, or CD27 over the total number of TGF-β positive cells (equivalent to 100%).</p

Removal of CD4+ T cells abrogates the effects of CTLA-4 blockade on HIV-specific CD8+ T Cells.

<p>PBMC (or CD4 negative PBMC) were stimulated with HIV peptides in the presence of anti-CTLA4 (or isotype control), then stained with anti-IFN-γ FITC, anti-TGF-β PE, IL-10 APC, anti-CD3 Am Cyan, anti-CD4 PerCP CY5.5, anti-CD8 PE CY7, and analyzed by flow cytomerty. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IL-10, and IFN-γ positive cells were determined. Results were expressed as percent of HIV-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ after subtraction of the back ground. Representative plots of (A) Gag and (B) Nef-specific CD8+ T cells expressing TGF-β, IL-10, or IFN-γ in the presence or absence of anti-CTLA-4. Data plots shown are representative of three volunteers examined in three independent experiments yielding similar results.</p

TGF-β positive, IL-10 positive, and IFN-γ positive HIV-specific CD8+ T cell populations are distinct.

<p>PBMC were stimulated with HIV peptides then stained with anti-IFN-γ FITC, anti-TGF-β PE, anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, and anti-CD8 PE Cy7, anti-IL-10 APC, and analyzed by flow cytometry. Samples were first gated on the CD3+/CD8+ lymphocyte population then the percent of TGF-β, IFN-γ, and IL-10 positive CD8+ T cells were determined. (A) Data from individuals with significant cytokine expression and analysis were performed by Mann-Whitney <i>U</i> test. (B) Representative plots of the number of HIV-specific CD8+ T cells expressing TGF-β, IFN-γ, and IL-10 after subtraction of the back ground values.</p

HIV-specific TGF-β and IL-10 positive CD8+ T cells are CTLA-4 negative.

<p>PBMC were stimulated with HIV peptides then stained with anti-TGF-β PE (or IL-10 PE), anti-CD3 AmCyan, anti-CD4 PerCP Cy5.5, anti-CD8 PE Cy7, anti-CTLA-4 APC, and analyzed by flow cytometry. Gating on the CTLA-4 positive cells was performed using the fluorescence-minus-one (FMO) control for CTLA-4. (A) Representative plots of samples that were first gated on the CD3+/CD4+ and CD3+/CD8+ lymphocyte population and then the percentages of CTLA-4 positive cells were determined. (B) Representative plots of samples that were first gated on the CD3+/CD8+ lymphocyte population and then the percent of TGF-β and IL-10 positive cells that express CTLA-4 was determined after subtraction of the back ground values. The values marked with an asterisk represent the fraction of TGF-β (or IL-10) positive cells that express CTLA-4 over the total number of TGF-β (or IL-10) positive cells (equivalent to 100%). Plots are from three independent experiments yielding similar results.</p

Diagnostic accuracy of peripheral blood IGRA.

*<p> <b>% (95% confidence interval); indeterminate IGRA results excluded.</b></p><p><b><u>Abbreviations:</u></b> IGRA (interferon-gamma release assay), TB (tuberculosis), PPV (positive predictive value), NPV (negative predictive value).</p

Demographic and clinical characteristics.

*<p>Continuous variables presented as medians (interquartile range).</p><p><b><u>Abbreviations:</u></b> TB (tuberculosis).</p

Study flow.

<p>Of 110 smear-negative tuberculosis suspects referred for bronchoscopy, 104 (95%) underwent the procedure and 94 (85%) were included in the study. Bronchoalevolar lavage TSPOT and peripheral blood TSPOT provided interpretable results in 62 (66%) and 81 (86%) patients, respectively.</p

BAL and blood IGRA results (N = 94).

<p><b><u>Abbreviations:</u></b> BAL (bronchoalveolar lavage), IGRA (interferon-gamma release assay).</p