A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV

Abstract

Prevalent infection with human herpes simplex 2 (HSV-2) or human papillomavirus (HPV) is associated with increased human immunodeficiency virus (HIV) acquisition. Microbicides that target HIV as well as these sexually transmitted infections (STIs) may more effectively limit HIV incidence. Previously, we showed that a microbicide gel (MZC) containing MIV-150, zinc acetate (ZA) and carrageenan (CG) protected macaques against simian-human immunodeficiency virus (SHIV-RT) infection and that a ZC gel protected mice against HSV-2 infection. Here we evaluated a modified MZC gel (containing different buffers, co-solvents, and preservatives suitable for clinical testing) against both vaginal and rectal challenge of animals with SHIV-RT, HSV-2 or HPV. MZC was stable and safe in vitro (cell viability and monolayer integrity) and in vivo (histology). MZC protected macaques against vaginal (p<0.0001) SHIV-RT infection when applied up to 8 hours (h) prior to challenge. When used close to the time of challenge, MZC prevented rectal SHIV-RT infection of macaques similar to the CG control. MZC significantly reduced vaginal (p<0.0001) and anorectal (p = 0.0187) infection of mice when 10(6) pfu HSV-2 were applied immediately after vaginal challenge and also when 5×10(3) pfu were applied between 8 h before and 4 h after vaginal challenge (p<0.0248). Protection of mice against 8×10(6) HPV16 pseudovirus particles (HPV16 PsV) was significant for MZC applied up to 24 h before and 2 h after vaginal challenge (p<0.0001) and also if applied 2 h before or after anorectal challenge (p<0.0006). MZC provides a durable window of protection against vaginal infection with these three viruses and, against HSV-2 and HPV making it an excellent candidate microbicide for clinical use.

Figure 1. Modified MZC is safe in vitro.

A) Caco-2 cell monolayer integrity. TEER was measured in differentiated Caco-2 cell monolayers after treatment with 1∶10 diluted formulations for 0-6 h. Means ± SD are shown from two independent experiments performed in triplicate. B) Lactobacilli in vitro viability. MZC toxicity was measured by treating L. jensenii (empty bars) and L. crispatus (filled bars) with 1∶10 diluted MZC vs. P/S for 30 min. The number of colonies (mean ± SD from three independent experiments) relative to saline 7.5% FBS, is shown as a percent. C) C. albicans in vitro viability. MZC toxicity was measured by incubating C. albicans yeasts with 1∶10 diluted MZC vs. Amph.B for 2 h. Left axis (filled bars) represents the numbers of colonies counted for each condition and expressed as % colonies relative to the Sabouraud dextrose broth (SB) control (set as 100%). Right axis (empty bars) is the number of viable yeasts counted and shown as the percentage relative to the SB-treated controls. Means ± SD of 5 independent experiments are summarized.

Figure 2. In vivo application of MZC does not compromise epithelial integrity or increase HSV-2 susceptibility.

A) HSV-2 enhancement model. Balb/C mice (n = 40 per group) were depo-treated 7 d before starting vaginal application of each formulation daily for 7 d, followed by HSV-2 G challenge 12 h post-last gel. Percent of uninfected animals over time is shown (*P<0.05 vs. D-PBS, Fisher's exact test). B) Epithelial integrity testing. Depo-treated (vaginal dosing) and fasted (rectal dosing) mice were treated with MZC, D-PBS, or Gynol II. At 1, 6 and 24 h post-application, mice were euthanized, and the entire reproductive or rectal tract was surgically excised for morphological analysis. Pictures (20× magnification) are representative of six sections from two to three animals per group.

Figure 3. MIV-150 local and systemic levels after vaginal and rectal application.

Macaques were treated vaginally with MZC daily for 2 wks or once rectally. A) MIV-150 was measured by LCMS/MS in the plasma of vaginally (n = 6, filled circles) and rectally (n = 6, filled squares) treated macaques at various time points after the last gel application. B) Tissue biopsies (n = 12 per time point/type of tissue) and C) swabs (n = 6 per condition) were collected at the indicated times following the last gel and MIV-150 was measured (by LCMS/MS for tissues and RIA for swabs). The mean (± SD) concentrations of MIV-150 for each treatment group are shown (rectal, R; vaginal, V; cervical, C).

Figure 4. MZC completely protects against SHIV-RT infection vaginally for up to 8 h and rectally for 1 h.

A) MZC or CG was administrated vaginally daily for 2 wks followed by vaginal challenge 8 or 24 h after the last gel application. The number of animals in each treatment group is indicated. For CG, this includes 8 real time and 14 historical controls. Plasma viral loads for each animal are shown over time. B) Mean (± SEM) plasma viral load of infected animals from each group in (A). C) The percent infection in each of the different treatment groups. D) MZC or CG was administrated once rectally followed by rectal challenge 1 h later. Plasma viral loads are shown for each animal over time. The number of animals in each treatment group is indicated.

Figure 5. MZC protects mice against vaginal and anorectal HSV-2 infection.

A) Depo-treated (vaginal) or untreated (anorectal) Balb/C mice were challenged with 10⁶ pfu HSV-2 10 min after applying the indicated formulations (n = 20/formulation). The percentages of uninfected animals over time, based on symptoms, are shown for each treatment group (*P<0.05 vs. D-PBS and CG, Fisher's exact test). B) Depo-treated Balb/C mice were treated with 10 µl of the indicated formulations intravaginally at different time points before or after HSV-2 challenge with 5×10³ pfu (n = 20/formulation). The percentages of uninfected animals are shown for each time of gel dosing relative to challenge (*P<0.05 vs. D-PBS, Fisher's exact test).

Figure 6. MZC has potent anti-HPV activity in vitro and prevents HPV-16 PsV vaginal infection in mice.

A) The anti-HPV-16 (open circles), 18 (open trianbles) and 45 (open squares) IC₅₀ values (shown as a vertical dotted line within the 95% confidence interval) were estimated using the luciferase assay in HeLa cells. All gel dilutions were tested in triplicate. B) Depo-treated Balb/C mice were given the indicated formulations (HEC placebo or MZC) intravaginally at different time points before HPV-16 PsV challenge (15 animals/treatment). In vivo luciferase expression was measured 24 h after challenge using the IVIS Lumina and is expressed as mean luminescence in photons per second per centimeter square per steridian ± SD (*P<0.05 vs. HEC, Mann Whitney U test). C) CG levels (mean μg/ml ± SD) in vaginal washes from mice treated intravaginally with MZC were measured by ELISA at 1, 2, 4, 8 and 24 h post-gel (n = 6 per time point).

Figure 7. MZC prevents HPV-16 PsV anorectal infection in mice.

A) Ketamine/xylazine-anesthetized Balb/C mice were treated in the anorectal area with Conceptrol or D-PBS (no Conceptrol). Mice were challenged with HPV-16 PsV to determine the optimal time for conceptrol treatment that gives the best luminescence signal. B) Ketamine/xylazine-anesthetized Balb/C mice received Conceptrol in the anorectal area and were treated with the indicated formulations (HEC placebo for HPV-16 PsV control or MZC) in the anorectal area at different time points before and after HPV-16 PsV challenge (15 animals/treatment). (A and B) In vivo luciferase expression was measured 48 h after challenge using the IVIS Lumina and is expressed as mean luminescence in photons per second per centimeter square per steridian + SD (*P<0.05 vs. HEC, Mann Whitney U test). C) CG PK in rectal washes from mice treated with MZC formulation was determined at 0.5, 2, and 8 h (n = 6 or 8 per time point) after MZC application. The graph shows CG concentration (mean μg/ml ± SD) per time point.