Herpes simplex virus-2 (HSV-2) infection increases HIV susceptibility. We previously established a rhesus macaque model of vaginal HSV-2 preexposure followed by cochallenge with HSV-2 and simian/human immunodeficiency virus-reverse transcriptase (SHIV-RT). Using this model, we showed that a gel containing the nonnucleoside reverse transcriptase inhibitor (NNRTI) MIV-150 in carrageenan (CG) reduced SHIV-RT infection. To evaluate the efficacy of new generation microbicides against both viruses, we first established dual infection after single vaginal cochallenge with SHIV-RT and HSV-2 in HSV-2-naive macaques. All animals (6/6) became HSV-2 infected, with 4/6 coinfected with SHIV-RT. In a control group cochallenged with SHIV-RT and UV-inactivated HSV-2, 2/4 became SHIV-RT infected, and none had detectable HSV-2. Low-level HSV-2-specific antibody and T cell responses were detected in some HSV-2-infected animals. To test a CG gel containing MIV-150 and zinc acetate (MZC), which provided naive animals full protection from SHIV-RT for at least 8 h, MZC (vs. CG) was applied daily for 14 days followed by cochallenge 8 h later. MZC prevented SHIV-RT infection (0/9 infected, p=0.04 vs. 3/6 in CG controls), but only reduced HSV-2 infection by 20% (6/9 infected vs. 5/6 in CG, p=0.6). In HSV-2-infected animals, none of the gel-treated animals seroconverted, and only the CG controls had measurable HSV-2-specific T cell responses. This study shows the promise of MZC to prevent immunodeficiency virus infection (even in the presence of HSV-2) and reduce HSV-2 infection after exposure to a high-dose inoculum. Additionally, it demonstrates the potential of a macaque coinfection model to evaluate broad-spectrum microbicides.

We previously showed that a carrageenan (CG) gel containing 50 μM MIV-150 (MIV-150/CG) reduced vaginal simian/human immunodeficiency virus (SHIV)-RT infection of macaques (56%, p>0.05) when administered daily for 2 weeks with the last dose given 8 h before challenge. Additionally, when 100 mg of MIV-150 was loaded into an intravaginal ring (IVR) inserted 24 h before challenge and removed 2 weeks after challenge, >80% protection was observed (p<0.03). MIV-160 is a related NNRTI with a similar IC(50), greater aqueous solubility, and a shorter synthesis. To objectively compare MIV-160 with MIV-150, herein we evaluated the antiviral effects of unformulated MIV-160 in vitro as well as the in vivo protection afforded by MIV-160 delivered in CG (MIV-160/CG gel) and in an IVR under regimens used with MIV-150 in earlier studies. Like MIV-150, MIV-160 exhibited potent antiviral activity against SHIV-RT in macaque vaginal explants. However, formulated MIV-160 exhibited divergent effects in vivo. The MIV-160/CG gel offered no protection compared to CG alone, whereas the MIV-160 IVRs protected significantly. Importantly, the results of in vitro release studies of the MIV-160/CG gel and the MIV-160 IVR suggested that in vivo efficacy paralleled the amount of MIV-160 released in vitro. Hundreds of micrograms of MIV-160 were released daily from IVRs while undetectable amounts of MIV-160 were released from the CG gel. Our findings highlight the importance of testing different modalities of microbicide delivery to identify the optimal formulation for efficacy in vivo.

Previously we showed that repeated vaginal application of a MIV-150/zinc acetate carrageenan (MIV-150/ZA/CG) gel and a zinc acetate carrageenan (ZA/CG) gel significantly protected macaques from vaginal simian human immunodeficiency virus reverse transcriptase (SHIV-RT) infection. Gels were applied either daily for 2 weeks or every other day for 4 weeks, and the animals were challenged 4-24 h later. Herein, we examined the effects of a single vaginal dose administered either before or after virus challenge. Encouraged by the vaginal protection seen with MIV-150/ZA/CG, we also tested it rectally. Vaginal applications of MIV-150/ZA/CG, ZA/CG, and CG gel were performed once 8-24 h before, 1 h after, or 24 h before and 1 h after vaginal challenge. Rectal applications of MIV-150/ZA/CG and CG gel were performed once 8 or 24 h before rectal challenge. While vaginal pre-challenge and pre/post-challenge application of MIV-150/ZA/CG gel offered significant protection (88%, p<0.002), post-challenge application alone did not significantly protect. ZA/CG gel reduced infection prechallenge, but not significantly, and the effect was completely lost post-challenge. Rectal application of MIV-150/ZA/CG gel afforded limited protection against rectal challenge when applied 8-24 h before challenge. Thus, MIV-150/ZA/CG gel is a highly effective vaginal microbicide that demonstrates 24 h of protection from vaginal infection and may demonstrate efficacy against rectal infection when given close to the time of HIV exposure.

Epidemiological studies suggest that prevalent herpes simplex virus type 2 (HSV-2) infection increases the risk of HIV acquisition, underscoring the need to develop coinfection models to evaluate promising prevention strategies. We previously established a single high-dose vaginal coinfection model of simian human immunodeficiency virus (SHIV)/HSV-2 in Depo-Provera (DP)-treated macaques. However, this model does not appropriately mimic women's exposure. Repeated limiting dose SHIV challenge models are now used routinely to test prevention strategies, yet, at present, there are no reports of a repeated limiting dose cochallenge model in which to evaluate products targeting HIV and HSV-2. Herein, we show that 20 weekly cochallenges with 2-50 TCID50 simian human immunodeficiency virus reverse transcriptase (SHIV-RT) and 10(7) pfu HSV-2 results in infection with both viruses (4/6 SHIV-RT, 6/6 HSV-2). The frequency and level of vaginal HSV-2 shedding were significantly greater in the repeated exposure model compared to the single high-dose model (p<0.0001). We used this new model to test the Council's on-demand microbicide gel, MZC, which is active against SHIV-RT in DP-treated macaques and HSV-2 and human papillomavirus (HPV) in mice. While MZC reduced SHIV and HSV-2 infections in our repeated limiting dose model when cochallenging 8 h after each gel application, a barrier effect of carrageenan (CG) that was not seen in DP-treated animals precluded evaluation of the significance of the antiviral activity of MZC. Both MZC and CG significantly (p<0.0001) reduced the frequency and level of vaginal HSV-2 shedding compared to no gel treatment. This validates the use of this repeated limiting dose cochallenge model for testing products targeting HIV and HSV-2.