Antiretroviral Drug Activity in Macaques Infected during Pre-Exposure Prophylaxis Has a Transient Effect on Cell-Associated SHIV DNA Reservoirs

<div><p>Background</p><p>Pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) is a novel HIV prevention strategy. Suboptimal PrEP adherence and HIV infection creates an opportunity for continued antiretroviral drug activity during undiagnosed infection. We previously showed that macaques infected with SHIV during PrEP with FTC/TDF display reduced acute plasma viremias and limited virus diversity. We investigated the effect of PrEP on acute SHIV DNA dynamics and on the size of the persistent virus reservoir in lymphoid tissues.</p><p>Design</p><p>Cell-associated SHIV DNA levels in PBMCs were measured in 8 macaques infected during PrEP with FTC/TDF or single-agent TAF and was compared to those seen in untreated infections (n = 10). PrEP breakthrough infections continued treatment with 1–2 weekly drug doses to model suboptimal drug exposure during undiagnosed HIV infection in humans. SHIV DNA was also measured in lymphoid tissues collected from FTC/TDF PrEP breakthroughs after 1 year of infection.</p><p>Results</p><p>Compared to untreated controls, PrEP infections had reduced plasma RNA viremias both at peak and throughout weeks 1–12 (p<0.005). SHIV DNA levels were also reduced at peak and during the first 12 weeks of infection (p<0.043) but not throughout weeks 12–20. At 1 year, SHIV DNA reservoirs in lymphoid tissues were similar in size among macaques that received PrEP or placebo.</p><p>Conclusions</p><p>Antiviral drug activity due to PrEP limits acute SHIV replication but has only a transient effect on cell-associated SHIV DNA levels. Our model suggests that suboptimal drug exposure in persons that are taking PrEP and become infected with HIV may not be sufficient to reduce the pool of HIV-infected cells, and that treatment intensification may be needed to sustain potential virological benefits from the PrEP regimen.</p></div

Acute plasma RNA and cell-associated SHIV DNA levels in macaques infected with SHIV_162P3.

<p>Time 0 denotes peak plasma viremia. SHIV RNA levels in plasma are shown in red. Cell-associated SHIV DNA levels in PBMCs are shown in black. Small panels indicate the correlation between plasma RNA and DNA levels seen between peak viremia and week 5. Pearson correlation coefficients are also shown.</p

SHIV_162P3 DNA levels in lymphoid tissues from PrEP breakthrough and placebo infections.

<p>SHIV_162P3 DNA levels in lymphoid tissues from PrEP breakthrough and placebo infections.</p

Acute plasma RNA and cell-associated SHIV DNA levels in macaques infected with SHIV_162P3 while receiving PrEP.

<p>Time 0 denotes peak plasma viremia. SHIV RNA levels in plasma are shown in red. Cell-associated SHIV DNA levels in PBMCs are shown in black. The shaded areas denote the length of treatment (green = FTC/TDF; orange = TAF). Small panels indicate the correlation between plasma RNA and DNA levels seen between peak viremia and week 5. Pearson correlation coefficients are also shown.</p

SHIV_162P3 plasma RNA and cell-associated DNA in PrEP breakthroughs and placebo infections.

<p>SHIV_162P3 plasma RNA and cell-associated DNA in PrEP breakthroughs and placebo infections.</p

Treatment dosing schedule and duration of treatment in PrEP breakthrough infections.

<p>Treatment dosing schedule and duration of treatment in PrEP breakthrough infections.</p

Acute viral RNA and cell-associated DNA levels in macaques infected with SHIV_162P3 while receiving PrEP of placebo.

<p>The two top panels show plasma RNA levels in placebo and PrEP breakthrough infections, and the two panels at the bottom show cell-associated DNA levels in PBMCs. Time 0 denotes peak viremia. The green and red lines indicate median RNA or DNA levels, respectively. Horizontal dotted line denotes the limit of detection of the SHIV RNA or DNA assays.</p

Cytokine production of CD4⁺ and CD8⁺ T cells induced by chemo-vaccination.

<p>Intracellular production of IFNγ, IL-2, MIP-1β, or TNFα was measured by flow cytometry after in-vitro incubation of freeze-thawed cells with the two dominant peptide pools as determined by previous IFNγ-ELISPOT. We gated on CD3⁺ and CD69⁺ (<b>A, B</b>), or on CD3⁺, CD69⁺, and CD4⁺ or CD8⁺ (<b>C</b>), and determined the number of cells with intracellular production of any of the factors, regardless of whether they simultaneously produced the remaining 3 factors. “Any” refers to cells producing any of the indicated factors, not necessarily all simultaneously. Samples from infected controls or infected PrEP-treated macaques were from peak viremia or 6 weeks thereafter, whenever available. Such samples are not shown for CD4/CD8 analysis, because CD4⁺ cells significantly decline depending on the stage of SHIV infection. (<b>B</b>) Representative example of results obtained with cells from macaque 34912 before its infection, without stimulation (“no stim.”), with the two dominant peptide pools, or with SEB for polyclonal stimulation.</p

Experimental Design.

<p>SHIV-specific T cells were measured during the indicated experimental procedures. Arrows indicate repeated viral exposures, horizontal lines depict intermittent, oral PrEP. PrEP consisted of human-equivalent doses of oral Truvada. Each virus exposure was flanked by a waning drug dose of 7 days prior, and one drug dose administered 2 hours after exposure, as a model for intermittent PrEP use in humans. Bolded rectangles highlight final outcomes of SHIV challenges. Numbers in lower right corners refer to macaque identifications (IDs).</p

Chemo-vaccination effect.

<p>SHIV-specific T cells are induced in two PrEP-protected macaques during PrEP and virus exposures. PrEP protected four macaques from infection during 14 SHIV exposures in weeks 1–14 (<b>A</b> and <b>B</b>), while two became infected despite PrEP (<b>C</b>); three macaques were controls (<b>D</b>). SHIV-specific T cells were determined by IFNγ-ELISPOT. The black bars represent the number of specific T cells as a sum of responses to 14 SHIV-derived peptide pools for antigenic simulation, measured in SFU (spot forming units, left axis). Grey lines depict plasma viremia (right axes). The graphs represent data from individual macaques, their identification codes are bolded. The dotted lines are cut-off values for positive T cell responses. Two PrEP-protected macaques (35451 (<b>A</b>), 4284 (<b>B</b>)) showed signs of chemo-vaccination. During re-challenge with 14 SHIV exposures in weeks 42–55, additional PrEP protected macaques 35451 and 33756 (<b>A</b>). Chemo-vaccinated macaques 4284 and 33246 were not protected from SHIV infection (<b>B</b>). Throughout the study, anti-SHIV antibodies were determined every 6 weeks (weeks 1–41) or 4 weeks (weeks 42–69). “Y” indicates time of seroconversion.</p