Epitope specificity of T cells induced by chemo-vaccination.

<p><b>A</b>: T cell specificities in PrEP-protected macaques 4284 and 35451 change rapidly during SHIV exposures and PrEP, while they remain more consistent in infected control macaque AG94, and in 4284 after infection. T cell specificities were determined by IFNγ-ELISPOT with 14 peptide pools represented by the indicated colors. The pie charts depict percentages of contributions to the T cell response. Arrows indicate time point of virus exposures, adjacent numbers indicate how many exposures were given. Seroconversion is recorded by “Y”; numbers indicates the study week of seroconversion. <b>B</b>: T cells induced by chemo-vaccination appear focused on epitopes derived from the <i>pol</i> region. IFNγ-ELISPOT responses to 14 peptide pools were combined for the indicated gene products; their contribution to the response (all 14 peptide pools) was calculated. All IFNγ-ELISPOT results from week 1–41 are depicted. N refers to number of macaques in the 4 specified groups. P-values were obtained by unpaired, two-sided student's t-tests comparing all results before infection (12 time-points from 3 chemo-vaccinated macaques, filled circles and open diamonds combined) to those after infection (56 time-points from 5 macaques in control or PrEP-infected groups, open circles and filled triangles combined).</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

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

Rectal Application of a Highly Osmolar Personal Lubricant in a Macaque Model Induces Acute Cytotoxicity but Does Not Increase Risk of SHIV Infection

<div><p>Background</p><p>Personal lubricant use is common during anal intercourse. Some water-based products with high osmolality and low pH can damage genital and rectal tissues, and the polymer polyquaternium 15 (PQ15) can enhance HIV replication <i>in vitro</i>. This has raised concerns that lubricants with such properties may increase STD/HIV infection risk, although <i>in vivo</i> evidence is scarce. We use a macaque model to evaluate rectal cytotoxicity and SHIV infection risk after use of a highly osmolar (>8,000 mOsm/kg) water-based lubricant with pH of 4.4, and containing PQ15.</p><p>Methods</p><p>Cytotoxicity was documented by measuring inflammatory cytokines and epithelial tissue sloughing during six weeks of repeated, non-traumatic lubricant or control buffer applications to rectum and anus. We measured susceptibility to SHIV_SF162P3 infection by comparing virus doses needed for rectal infection in twenty-one macaques treated with lubricant or control buffer 30 minutes prior to virus exposure.</p><p>Results</p><p>Lubricant increased pro-inflammatory cytokines and tissue sloughing while control buffer (phosphate buffered saline; PBS) did not. However, the estimated AID₅₀ (50% animal infectious dose) was not different in lubricant- and control buffer-treated macaques (p = 0.4467; logistic regression models).</p><p>Conclusions</p><p>Although the test lubricant caused acute cytotoxicity in rectal tissues, it did not increase susceptibility to infection in this macaque model. Thus neither the lubricant-induced type/extent of inflammation nor the presence of PQ15 affected infection risk. This study constitutes a first step in the <i>in vivo</i> evaluation of lubricants with regards to HIV transmission.</p></div

SHIV162p3 challenge doses and infection.

<p>‘+’ = animal infected at the indicated dose;</p><p>‘0’ = animal not infected;</p><p>PBS = phosphate buffered saline; TCID₅₀ = tissue culture 50% infectious dose. The challenges were performed in six sets of macaques; sets1 and 2 were phosphate buffered saline (PBS)-treated controls; sets 3, 4, 5 and 6 were lubricant-treated.</p><p>¹Historical data from 5 uninfected and 1 infected animals were included at 250 TCID₅₀, and 4 uninfected and 1 infected animals at 50 TCID₅₀; these animals were non-PBS-treated</p><p>SHIV162p3 challenge doses and infection.</p

Cytotoxicity study design (Phase I) and induction of pro-inflammatory cytokines.

<p>Study design showing the cytotoxicity phase of the study (A); black rectangles = lubricant application; grey triangles = sample collections immediately prior to each product application (longitudinal time points); black triangles = samples taken 15 minutes to 48 hours after product application (acute time points); open hexagons = rectal biopsies, taken from one animal at 30 minutes post lubricant-application, and from one animal a week after last lubricant application; m = minutes; h = hours; B. Induction of pro-inflammatory cytokine TNF-α at acute time points (15 or 30 m, and 2, 4, 24, or 48 h post-product application); circles represent individual macaques; C. Induction of pro-inflammatory cytokine TNF-α at all time points during 6 weeks of product application; medians and ranges are graphed.</p

Cytokine concentration in rectal lavages at acute time points post lubricant/control buffer application.

<p>The p-values were calculated using unpaired t-tests (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#sec006" target="_blank">Materials & Methods</a> for details); the statistically significant ones are indicated in bold; CI = confidence interval.</p><p>¹We calculated the geometric means (GMs) of cytokines concentrations as shown, combining the measurements at 15m, 30m, 2- and 4-h acute time points. Levels of eight other cytokines were determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#sec006" target="_blank">Methods</a>) but many of the data points were below the assay limit of detection, and not suitable for accurate statistical analyses</p><p>Cytokine concentration in rectal lavages at acute time points post lubricant/control buffer application.</p

Epithelial sloughing and blood.

<p>A. Lubricant induces rectal shedding of epithelial cells. Examples of epithelial sloughing in a control- (top left) and lubricant-treated animal (top right). The lower panel shows a representative H&E stain of sloughed rectal epithelial cells (20x); B. Blood associated with rectal washes; photographs of microfuges containing rectal lavages; C,D. Epithelial sloughing measured at acute time points collected after the 2nd weekly lubricant application (C), and those measured over the entire study (D). The panel D in this figure shows three collections per week (day 1, pre-lubricant; day 2 pre-lubricant; day 2 post-lubricant), as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120021#pone.0120021.g001" target="_blank">Fig. 1</a>.</p

Hematoxylin and eosin stain (20x) of rectal biopsies.

<p>Showing biopsies from one animal (ID: 604962) collected before lubricant application (A) and 30 minutes after (B) product application; B shows focal infiltrates of inflammatory cells (square box), predominately mononuclear, seen in the lamina propria; there is no disruption of architecture. C is a magnified section (30x) of the square box with the green arrows showing mononuclear cells.</p