<i>In vivo</i> study protocol in pigtailed macaques.

<p>(<b>A</b>) Two studies were performed. The first included five macaques, in which each monkey received placebo films followed six weeks later with RC-101 films. The second study included the five original macaques plus an additional monkey, in which half received RC-101 films and half received placebo films. Six weeks later, each monkey receiving the opposite film type. (<b>B</b>) Baseline measurements for colposcopy, vaginal pH, and microflora were obtained at day 0. At days 1–4, RC-101- or placebo-containing films were instilled intravaginally. At days 5 and 8, cytobrushes, blood, and pinch biopsies were obtained, and at day 8 CVL fluids were obtained from each monkey.</p

Activity of formulated RC-101 is equivalent to unformulated peptide.

<p>Antiviral activities of RC-101-containing films and placebo films against HIV-1 strain BaL were compared against unformulated “pure” RC-101 peptide in TZM-bl reporter assays expressed as percent inhibition of HIV-1 infection (<b>A</b>) and p24^gag release assay in PM1 cells expressed in pg/ml of p24 released (<b>B</b>). p24^gag differed between formulated and unformulated RC-101 at days 5 and 7, <i>P</i><0.001 and <i>P</i> = 0.019, respectively. Error bars represent SEM; n = 2-5.</p

RC-101 was retained in the cervicovagina up to four days post-application.

<p>RC-101 was assessed by quantitative western blot from acetic acid-extracted samples of cervical biopsies, vaginal biopsies and cytobrushes at day 5, and cytobrushes and CVL fluid at day 8 (<b>A</b>). Note that CVL fluid was subdivided into both the cellular portion and the fluid portion. A representative western blot for RC-101 in cytobrushes at day 5 is shown in (<b>B</b>), with “M1”, “M2”, and “M3” signifying three individual monkeys. Note that the same monkey received both RC-101 films and placebo films six weeks apart, and that RC-101 was absent in cytobrushes obtained from monkeys that received placebo films. The <i>P</i>-values presented were adjusted for multiple comparisons using Tukey method.</p

Colposcopy of the cervicovagina reveals no adverse effects of RC-101 films.

<p>Images from colposcopic examination of cervicovaginal mucosa of a representative pigtailed macaque obtained prior to film insertion, at the time of insertion (Time 0), and 30 min and 24 hr after film insertion. Note the absence of mucosal aberrations at all time points with either RC-101 films or placebo films.</p

RC-101 films do not alter vaginal pH or microbiological profile of the cervicovagina.

<p>(<b>A</b>) Vaginal pH was measured prior to film instillation at days 1-4 (D1-D4) and at followup (D5 and D8). Each measurement represents one experimental condition in a pigtailed macaque. Horizontal lines represent the mean value for all 11 replicates. No differences were observed between monkeys that were receiving RC-101 films or placebo films. (<b>B</b>) represents the difference in vaginal pH between the time zero condition and 30 minutes after film instillation for each day films were applied (D1–D4). Horizontal lines represent the mean value for all 11 replicates. A significant difference was observed at Day 4 (<i>P</i> = 0.003), with the placebo films inducing a greater change in vaginal pH than the RC-101 films. (<b>C</b>) Four (of twenty) microbes are presented for both RC-101 films and placebo films. For each microbe, there are 10 vertical bars, representing the number of replicates (from a total of 11) that were positive for the microbe indicated. The ten bars for each microbe signify the following from left to right: Day 1 (D1) time zero (t0), D1, time 30 min (t30), D2 t0, D2 t30, D3 t0, D3 t30, D4 t0, D4 t30, D5 and D8.</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

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

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

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