RC-101 inhibits HIV-1 infection of vaginal tissues. (a) Vaginal tissues were treated with either HIV-1 BaL alone or with HIV-1 BaL in the presence of RC-101 (2 μg in 100 μl PBS) and then maintained for 9 days as described in the Materials and methods. Tissue DNA (200 ng) served as templates for real-time polymerase chain reaction using primers specific for HIV-1 BaL 5′ LTR, gag, or env, or for cellular β-actin (control). Results are presented as fold-decrease in HIV-1 BaL DNA for RC-101 versus vehicle-treated vaginal tissues (mean ± SEM, n = 3). (b–d) Quantitative analysis of HIV-1 reverse transcripts in RC-101 versus vehicle-treated vaginal tissues. Standard curves for HIV-1 transcript and β-globin levels were generated by plotting cycle threshold (Ct) values for known numbers of input templates (b), and Ct values for tissue DNA samples were fit to the standard curves and presented as copies of HIV-1 transcript per 10 000 copies of β-globin. (c) Vaginal tissues were treated with either 2 μg or 0·2 μg RC-101 at the time of HIV-1 BaL infection and then maintained for 9 days as described in the Materials and methods. (d) Levels of HIV-1 transcript in full-thickness tissues treated with HIV-1 BaL alone or in the presence of 2 μg RC-101. Bars represent mean ± SEM, n = 4. *P < 0·01.

Topically applied RC-101 is retained within or at the apical surface of vaginal tissues. (a) Quantitative dot blot demonstrating that rabbit polyclonal anti-RC-101 recognized RC-101 peptide but not the tissue maintenance media or untreated vaginal tissue. (b,c) Vaginal tissues were infected (day 0) with HIV-1 BaL in the presence of 200 ng RC-101 in 100 μl PBS (b) or 2000 ng RC-101 in 100 μl PBS (c) and then tissues were washed and re-applied with RC-101 at days 1, 3 and 6 postinfection (Input), as described in the Materials and methods. Collected apical surface wash fluids (Wash, W) and underlay media (Underlay, U), as well as harvested tissues (Tissue, T) were then assayed for RC-101 by dot blot. Values are displayed as stacked bars correlating to harvested tissue or collection day number. (d) Full-thickness vaginal tissues were treated and maintained over 6 days as described in the Materials and methods. Bars represent mean ± SEM, n = 4–6. (e–g) Tissues infected with HIV-1 BaL (25 ng/tissue) and treated with two applications of 400 ng RC-101 (in 50 μl PBS). Tissue sections were subjected to H&E staining (e) and immunohistochemistry with preimmune (f) and anti-RC-101 polyclonal antisera (g) Note the immuno-localization of RC-101 (red stain) primarily in the apical, suprabasal and basal layers.

RC-101 retains anti-HIV-1 activity while in the presence of vaginal fluid. HIV-1 entry indicator cells, TZM-bl, were infected either with HIV-1 BaL virus alone (V), virus in combination with pooled human vaginal fluid (VF, 1 : 20 final dilution), virus in combination with RC-101 (2 μg/ml), or virus combined with VF and RC-101 for 24 hr before luciferase measurement. Data are expressed as per cent infection and bars represent mean ± SEM, n = 4–6 for each treatment condition. *P < 0·001 versus V. **P < 0·01 versus V + VF.

RC-101 does not alter the viability or inflammatory profiles of human vaginal tissue. Full-thickness vaginal tissues were treated with 100 μl PBS (control) or PBS containing 2–32 μg/tissue RC-101 on day 0 and then washed and re-applied with 50 μl PBS or PBS containing half-doses of RC-101 (1–16 μg/tissue, respectively) at days 1 and 3 postinfection. Tissue viability was assessed by measuring MTT reduction at day 6 post-treatment (a) and LDH activity in the underlay media collected on days 1, 3 and 6 post-treatment (b) Media alone (negative control) and LDH-spiked media (positive control) values are indicated by a single and double asterisk, respectively (b). Underlay media collected on days 1, 3 and 6 post-treatment were also assayed for IL-6 (c) and IL-8 (d) by ELISA. Data are presented as mean ± SEM, n = 4.