A combination microbicide gel protects macaques against vaginal simian human immunodeficiency virus-reverse transcriptase infection, but only partially reduces herpes simplex virus-2 infection after a single high-dose cochallenge

Abstract

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.

FIG. 1.

Simian human immunodeficiency virus-reverse transcriptase (SHIV-RT) productively infects animals cochallenged with SHIV-RT/herpes simplex virus-2 (HSV-2). (A) Viral load of SHIV-RT [copies of simian immunodeficiency virus (SIV) gag RNA per ml of plasma] in animals cochallenged with either UV-inactivated HSV-2 and SHIV-RT (UV-HSV-2) or live HSV-2 and SHIV-RT (live HSV-2). The number of animals in each group is indicated. (B) Mean (±SEM) SHIV-RT viral loads for SHIV-RT-infected animals after cochallenge with UV-HSV-2 or live HSV-2 and SHIV-RT.

FIG. 2.

Detection of HSV-2-specific T cell responses in animals cochallenged with live HSV-2 and SHIV-RT. (A, B) Intracellular cytokine staining (ICS) of HSV-2-specific CD4⁺ or CD8⁺ T cells in peripheral blood mononuclear cells (PBMCs) (A) and lymph node mononuclear cells (LNMCs) (B) of animals cochallenged with UV HSV-2 (n=4) vs. live HSV-2 (n=6) and SHIV-RT. Shown are the means (±SEM) of the percent positive cells [calculated after subtracting background microvesicle (MV) control values from each] detected at the indicated weeks post cochallenge (weeks 15 and 23 for the PBMCs and weeks 4 and 24 for the LNMCs). (C) Polyreactive CD4⁺ and CD8⁺ lymphocytes from PBMCs or LNs relative to single cytokine-reactive cells.

FIG. 3.

Infection with SHIV-RT is blocked and HSV-2 reduced by MZC gel. (A) SHIV-RT viral loads of animals cochallenged with SHIV-RT/HSV-2 8 h after the last of 14 daily applications of either carrageenan (CG) control or MZC. The number of animals in each group is indicated. (B) Mean (±SEM) SHIV-RT viral loads of animals infected after application of CG (n=3) or no gel (n=4). (C) The percentage of animals infected by either SHIV-RT or HSV-2 after cochallenge without a gel or after application of CG or MZC. The numbers above each bar denote the number of infected animals over the total number cochallenged for each group. Fisher's exact test was performed to determine statistical significance.

FIG. 4.

Lower HSV-2-specific cellular response in animals infected after application of MZC compared to CG. (A) ICS staining of HSV-2-responsive CD4⁺ or CD8⁺ T cells in blood taken 23 weeks post-SHIV-RT/HSV-2 cochallenge from animals treated with MZC (n=6 HSV-2⁺ and n=2 HSV-2⁻) or CG (n=5 HSV-2⁺ and n=1 HSV-2⁻). Three animals were HSV-2⁻ in the MZC group, but a blood sample from one was unavailable at this time point. (B) Polyreactive CD4⁺ and CD8⁺ lymphocytes relative to single cytokine-reactive cells in blood from CG-treated HSV-2⁺ animals.