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

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

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

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

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

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

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

Plasma and rectal SHIV RNA.

<p>A. Plasma SHIV RNA levels measured in control buffer-treated (black symbols; solid lines) and lubricant-treated (grey symbols; broken lines) animals. B. SHIV RNA levels measured in rectal secretions, in control buffer-treated (open symbols) and lubricant-treated (closed symbols) animals; 0 = time of peak plasma viremia.</p