A macaque model to study vaginal HSV-2/immunodeficiency virus co-infection and the impact of HSV-2 on microbicide efficacy

Abstract

Background: Herpes simplex virus type-2 (HSV-2) infection enhances the transmission and acquisition of human immunodeficiency virus (HIV). This occurs in symptomatic and asymptomatic stages of HSV-2 infection, suggesting that obvious herpetic lesions are not required to increase HIV spread. An animal model to investigate the underlying causes of the synergistic action of the two viruses and where preventative strategies can be tested under such complex physiological conditions is currently unavailable.

Methodology/principal findings: We set out to establish a rhesus macaque model in which HSV-2 infection increases the susceptibility to vaginal infection with a model immunodeficiency virus (simian-human immunodeficiency virus, SHIV-RT), and to more stringently test promising microbicides. HSV-2 exposure significantly increased the frequency of vaginal SHIV-RT infection (n = 6). Although cervical lesions were detected in only approximately 10% of the animals, long term HSV-2 DNA shedding was detected (in 50% of animals followed for 2 years). Vaginal HSV-2 exposure elicited local cytokine/chemokine (n = 12) and systemic low-level HSV-2-specific adaptive responses in all animals (n = 8), involving CD4(+) and CD8(+) HSV-specific T cells (n = 5). Local cytokine/chemokine responses were lower in co-infected animals, while simian immunodeficiency virus (SIV)-specific adaptive responses were comparable in naïve and HSV-2-infected animals (n = 6). Despite the increased frequency of SHIV-RT infection, a new generation microbicide gel, comprised of Carraguard(R) and a non-nucleoside reverse transcriptase inhibitor MIV-150 (PC-817), blocked vaginal SHIV-RT infection in HSV-2-exposed animals (n = 8), just as in naïve animals.

Conclusions/significance: We established a unique HSV-2 macaque model that will likely facilitate research to define how HSV-2 increases HIV transmission, and enable more rigorous evaluation of candidate anti-viral approaches in vivo.

Figure 1. HSV-2 shedding after vaginal exposure.

(A) Vaginal swabs were collected at the indicated times after HSV-2 exposure and HSV-2 DNA shedding was determined by measuring the presence of HSV-2 UL30 DNA by PCR. Each sample's integrity was verified by amplifying the GAPDH gene. The percentage of samples positive for HSV-2 UL30 DNA at the indicated time points is shown relative to the primary exposure to HSV-2. The numbers above the bars indicate the number of animals tested at each time point. (B) The DNA shedding is shown for each animal, where the numbers above the bars denote the number of times that animal was sampled during the study. (C,D) 3–6 (Biopsy 1) or 9–12 (Biopsy 2) months after co-challenge vaginal and cervical biopsies were taken from 8 animals and vaginal swabs were collected 4 hours, 1, 3 and 7 days later. (C) Representative gels resolving the amplified 146 bp HSV-2 UL30 gene and the 226 bp GAPDH gene control are shown for a set of samples from one animal. (D) The percentage of HSV-2 DNA positive animals for the indicated samples are provided (n = 8 for each biopsy).

Figure 2. HSV-2-exposed macaques are more susceptible to vaginal SHIV-RT infection.

(A) Naïve or HSV-2-exposed macaques were challenged with the indicated TCID₅₀ of SHIV-RT or SHIV-RT and HSV-2 (respectively), after receiving one (1X) or two (2X) doses of Depo-Provera (Fig. S1) and the percentage of SHIV-RT-infected animals is shown (numbers above the bars indicate animal numbers per group). The asterisk marks the statistically significant (P<0.05) difference between 1X Depo-Provera-treated naïve vs HSV-2-infected animals receiving 10³ TCID₅₀ of SHIV-RT. (B) Plasma viremia (mean RNA copies/ml±SEM) in SHIV-RT-infected naïve (left panel) and HSV-2-exposed (right panel) macaques post challenge with 10³ TCID₅₀ after 2 doses of Depo (10³ 2X Depo - Naïve n = 4; HSV-2 n = 2) or after 1 dose of Depo (10³ 1X Depo - Naïve n = 6; HSV-2 n = 6) and 200 TCID₅₀ after 1 dose of Depo (200 1X Depo - HSV-2 n = 3).

Figure 3. Local immune responses following HSV-2 infection.

(A) Naïve Depo-Provera-treated monkeys (n = 12) were challenged i.vag. with 2×10⁸ pfu HSV-2. Cytokine/chemokine levels were measured in the vaginal swabs during the first days after challenge using the 14-plex Luminex assay. The mean concentration ng/ml (±SEM) of each factor is shown. (B) Vaginal swabs were collected after HSV-2/SHIV-RT co-challenge of HSV-2-infected animals (n = 5, closed symbols) or SHIV-RT challenge of naïve animals (n = 6, open symbols). The cytokine/chemokine levels (mean±SEM ng/ml) were measured by Luminex assay on a weekly basis.

Figure 4. HSV-2 infection induces adaptive immunity and does not impede SIV-specific adaptive responses.

(A) HSV-2- and SIV-specific T-cell responses were monitored over time by IFN-γ ELISPOT and the data are expressed as the mean (±SEM) HSV-2- (left panel) or SIV-specific (right panel) spot-forming cells (SFC) per 2×10⁵ PBMCs. (HSV-2/SHIV-RT co-infected closed symbols, n = 8; SHIV-RT-infected open symbols, n = 10). (B) The mean (±SEM) numbers of SIV-specific SFC per 2×10⁵ cells are plotted for the blood (left panel) and for the LNs (right panel) collected upon euthanization of HSV-2/SHIV-RT co-infected animals (n = 8, 10–13 months post co-challenge) vs animals only infected with SHIV-RT (n = 3, 10–12 months post challenge).

Figure 5. HSV-2-specific CD4⁺ and CD8⁺ T cell responses are detected in HSV-2/SHIV-RT co-infected animals.

(A) HSV-2- (left panel) and SIV-specific (right panel) CD4⁺ (black bars) and CD8⁺ (grey bars) T cells were detected in the blood of co-infected animals (∼12 months after co-challenge, n = 8) by multicolor ICS. The percent positive cells producing the indicated cytokines in response to each antigen (mean±SEM; single vs double or triple producers) are shown after subtracting the respective background signals of the IgG and no-virus MV controls. (B,C) SIV-specific T-cell responses were compared between SHIV-RT-infected (n = 5) and HSV-2/SHIV-RT co-infected (n = 5) animals ∼12 months post co-challenge, by IFNγ ELISPOT (B) or ICS (C). (B) The mean (±SEM) SIV-specific SFC/2×10⁵ cells are shown. (C) The percent positive cells producing the indicated cytokines in response to SIV (mean±SEM; single vs double or triple producers) are shown after subtracting the respective background signals of the IgG and no-virus MV control. (D) Approximately 12 months after challenge with SHIV-RT or co-challenge with HSV-2/SHIV-RT, the fold decreases in CD4 counts (mean±SEM, relative to pre-exposure levels) (left axis, stars) and plasma viral loads (mean RNA copies/ml±SEM, right axis, circles) are shown (n = 5 for both).

Figure 6. PC-817 protects against vaginal SHIV-RT infection when given just prior to HSV-2/SHIV-RT co-challenge.

(A) PC-817 or MC was applied 30 minutes (30 min) and 24 hours (24 h) prior to challenge of HSV-2-exposed animals with 10³ TCID₅₀ SHIV-RT and 2×10⁸ pfu HSV-2. Plasma viral loads are shown for control animals (MC) (n = 5) in the left panel, for animals that received PC-817 30 min before the challenge (n = 8) in the middle panel, and for animals that received PC-817 24 h prior to challenge (n = 5) in the right panel. (B) Mean (±SEM) plasma viremia of the 3 treatment groups from (A) are shown. (C) PC-817 or MC was applied 24 h prior to challenge with 200 TCID₅₀ of SHIV-RT and 2×10⁸ pfu HSV-2. The plasma viral loads are provided for the MC control animals (n = 2) in the left panel and the PC-817-treated animals (n = 6) in the right panel. (D) Data from panels (A) and (C) are summarized to show the frequency of infection for the differently treated groups. The asterisk highlights the statistically significant (P<0.001) difference in the numbers of infected animals receiving PC-817 (vs MC) 30 min prior to challenge with 10³ TCID₅₀ of SHIV-RT and HSV-2.