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. Author manuscript; available in PMC: 2018 Mar 1.
Published in final edited form as: J Acquir Immune Defic Syndr. 2017 Mar 1;74(3):e67–e74. doi: 10.1097/QAI.0000000000001167

MZC gel inhibits SHIV-RT and HSV-2 in macaque vaginal mucosa and SHIV-RT in rectal mucosa

Giulia Calenda 1,*, Guillermo Villegas 1,*, Patrick Barnable 1, Claudia Litterst 2, Keith Levendosky 1, Agegnehu Gettie 3, Michael L Cooney 1, James Blanchard 4, José A Fernández-Romero 1, Thomas M Zydowsky 1, Natalia Teleshova 1,§
PMCID: PMC5303173  NIHMSID: NIHMS810276  PMID: 27552154

Abstract

The Population Council’s microbicide gel MZC (also known as PC-1005) containing MIV-150 and zinc acetate dihydrate (ZA) in carrageenan (CG) has shown promise as a broad spectrum microbicide against HIV, HSV and HPV. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal SHIV-RT infection and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and HPV in murine models. Recently we demonstrated that MZC is safe and effective against SHIV-RT in macaque vaginal explants. Here we established models of ex vivo SHIV-RT/HSV-2 co-infection of vaginal mucosa and SHIV-RT infection of rectal mucosa in macaques (challenge of rectal mucosa with HSV-2 did not result in reproducible tissue infection), evaluated antiviral activity of MZC and compared qPCR and ELISA readouts for monitoring SHIV-RT infection. MZC (at non-toxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosa and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by one-step SIV gag qRT-PCR and p27 ELISA demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of qRT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

INTRODUCTION

More than two decades ago, an “epidemiological synergy” between HIV-1 and other sexually transmitted infections (STIs) increasing the risk of HIV-1 acquisition was suggested1. Among STIs affecting HIV transmission and pathogenesis, non-curable STIs like HSV-2 and HPV deserve special attention. Recent studies reported up to 3-fold and 7-fold increased risk of HIV-1 transmission with prevalent and incident HSV-2 infection, respectively2–5. HIV-1/HSV-2 co-infection affects the pathogenesis of both viruses, being associated with increased HIV-1 viral load6–8 and HSV-2 shedding quantity and frequency9–11. HSV-2 plays a significant role promoting HIV transmission and acquisition in Sub-Saharan Africa, where HSV-2 may account for 25% – 35% of incident HIV infections12. Importantly, studies in rhesus macaques (RM) showed that vaginal HSV-2 infection is associated with increased susceptibility to the simian/human immunodeficiency virus SHIV SF162P3 and provided some insights into possible mechanisms of increased transmission13. Specifically, frequency of vaginal CD4+ T cells expressing high level of α4β7, a gut-homing integrin that binds gp12014 and facilitates HIV/SIV infection13–18, is increased in HSV-2 infected RM13. An increase of α4β7highCD4+ T cells in rectal mucosa was also observed in rectal HSV-2 infection in RM19. Similarly to HSV-2, HPV infection is associated with an increase of HIV-1 acquisition20 and HIV-1 positivity is associated with increased HPV prevalence21–23 and incidence24,25.

The development of multipurpose prevention technologies (MPTs) active against HIV-1, HSV-2 and HPV vaginally and rectally could significantly improve global public health20,26–28. The Population Council (PC) gel containing 50μM of the NNRTI MIV-150 and 14mM zinc acetate dihydrate (ZA) in carrageenan (CG) demonstrated activity against vaginal SHIV-RT (RM), vaginal HSV-2 (RM, mice), anorectal HSV-2 (mice), vaginal and anorectal HPV (mice)29–35. MZC protects against SHIV-RT in RM vaginal explants36,37 and against HIV and HSV-2 in human cervical explants64. A recently completed Phase I clinical trial demonstrated a favorable vaginal safety profile of MZC38,66. The MZC gel is the only MPT product currently in clinical testing that demonstrates activity against HIV and two other non-curable STIs that increase the risk of HIV-1 transmission, HSV-2 and HPV26.

Here we aimed to establish vaginal and rectal explant SHIV-RT/HSV-2 co-infection models for microbicide testing and assess the activity of MZC against both viruses in these models. Traditionally, infection with HIV and SIV/SHIV in explants is monitored by p24 and p27 ELISAs36,39–42. Here we explored whether analysis of accumulation of viral RNA can be used as an alternative for p27 ELISA.

MATERIALS AND METHODS

Macaques

Naïve, SHIV-RT, SIV and HSV-1 exposed uninfected/infected Chinese and Indian RMs (Macaca mulatta) were utilized. Macaques were housed at the Tulane National Primate Research Center (TNPRC; Covington, LA), accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC#000594). The use of macaques was approved by the Animal Care and Use Committee of the TNPRC (OLAW assurance #A4499-01), and animal care complied with the regulations in the Animal Welfare Act43 and the Guide for the Care and Use of Laboratory Animals44. All vaginal and rectal biopsy procedures were performed by Board Certified veterinarians (American College of Laboratory Animal Medicine). The biopsies were collected not more often than every 4 weeks. Anesthesia was administered prior to and during all procedures, and analgesics were provided afterwards as previously described29,45. Necropsy tissues were available from 9 animals. These animals were euthanized using methods consistent with recommendations of the American Veterinary Medical Association (AVMA) Guidelines for Euthanasia. The animals were anesthetized with tiletamine-zolazepam (each at 4 mg/kg intramuscularly [i.m.]) and given buprenorphine (0.01 mg/kg i.m.) followed by an overdose of pentobarbital sodium. Death was confirmed by auscultation of the heart and pupillary dilation.

Gels

The components of MZC and CG gels are summarized in Table 1.

Table 1.

Components of MZC and CG gels

Components Gel formulation
MZC (batch # 130605A1005TR) CG (batch # 130613A525TR)
MIV-150 0.00184 wt.% (50 μM) –
ZA 0.3 wt.% (14 mM) –
CG 2.8 wt.% (28 mg/mL) 2.6 wt.% (26 mg/mL)
Propylene glycol 2 wt.% 2 wt.%
Methylparaben 0.2 wt.% 0.2 wt.%
Sodium Acetate 0.131 wt.% (10mM) 0.131 wt.% (10mM)
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Viral stocks

SHIV-RT was generated from the original stock provided by Disa Böttiger (Medivir AB, Sweden)46 using PHA/IL-2-activated macaque PBMCs and titered in CEMx174 cells before use29. HSV-2 strain G was generated as in47. Briefly, HSV-2 strain G was propagated in Vero cells (ATCC)48 as described in49 and the viral titer in pfu/ml was obtained by plaque formation assay on monolayer cultures of Vero cells50.

Macaque tissue processing

Vaginal mucosa (n = 2, 3×5mm biopsies procured at each collection time; or necropsy tissues) was obtained from RM. Tissues were transported overnight and cut into 3×3mm explants as described in36,51 before viral challenge (below). Rectal mucosa (n=15–20, 1.5×1.5 mm biopsies procured at each collection time) was processed for viral challenge (below) at TNPRC immediately after collection.

Tissue viability

To assess tissue viability after overnight (~18h) exposure to MZC or CG gels, MTT assay was performed as previously described36,51.

Comparison of SHIV-RT growth kinetics using qRT-PCR and p27 ELISA

To compare SHIV-RT infection readout methods in vaginal explants, tissues were processed and challenged with SHIV-RT as in36. Briefly, vaginal explants were stimulated (5 μg/ml PHA (Sigma Aldrich) and 100 U/ml IL-2 (NCI BRB Preclinical Repository, Frederick, MD) for 48h and then challenged with 104 TCID50 SHIV-RT per explant for ~18h. Following washout, explants were cultured in cDMEM (DMEM (Cellgro Mediatech) containing 10% FBS (Gibco, Life Technologies, Grand Island, NY), 100 U/ml penicillin, 100 μg/ml streptomycin (Cellgro Mediatech), 100μM MEM non-essential amino acids (Irvine Scientific, Santa Ana, CA) in the presence of IL-2 for 14d36. Supernatants were collected on days 0, 3, 7, 11, and 14 and infections levels were analyzed by SIV gag one-step qRT-PCR and p27 ELISA. To compare SHIV-RT infection readout methods in rectal biopsies, supernatants from SHIV-RT/HSV-2 co-challenge experiments (below) were used. Controls included 10 μM 3TC or 10 μM 3TC/100 μg/ml Acyclovir.

SHIV-RT and HSV-2 co-challenge of vaginal and rectal mucosa and antiviral activity of MZC

PHA/IL-2 stimulated vaginal explants and unstimulated rectal biopsies were co-challenged with 104 TCID50 SHIV-RT and 106 pfu HSV-2 per explant for ~18 (vaginal) or 4h (rectal). To test the antiviral activity of MZC, viral challenge was done in the presence 1:300 and/or 1:100 diluted MZC and CG gels vs. untreated (Medium) and 3TC/Acyclovir controls. Following washout, vaginal explants were cultured in cDMEM in the presence of IL-2 for 14d36. Rectal biopsies (n=4) were placed on 12mm diameter Gelfoam sponges (Ethicon, Somerville, NJ) presoaked in cDMEM at 37°C, 5% CO2 for at least 30 minutes and cultured for 14d (no IL-2). Rectal biopsies were cultured in cDMEM to which 80 μg/ml gentamicin had been added (Gibco). Supernatants from both vaginal and rectal tissues were collected on days 0, 3, 7, 11, and 14 and levels of infections were analyzed by qPCR and/or ELISA.

qPCR and qRT-PCR

5 μl of tissue culture supernatants were analyzed using the KAPA SYBR FAST Universal One- Step qRT-PCR kit (Kapa Biosystems, Wilmington, MA) for the quantification of SIV gag copies and the KAPA SYBR FAST Universal qPCR assay (Kapa Biosystems) for the quantification of HSV pol copies with the Viia 7 real time PCR system (Applied Biosystems, Carlsbad, CA). Primers for SIV were 5′-GGTTGCACCCCCTATGACAT-3′ (SIV667gag Fwd) and 5′-TGCATAGCCGCTTGATGGT-3′ (SIV731gag Rev). Results were analyzed by the standard curve method, using SIVmac1A11 DNA obtained from Dr. Paul Luciw through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH52 as the standard (37,65. Primers for HSV-2 pol were 5′-GCTCGAGTGCGAAAAAACGTTC-3′ (HSV-2pol Fwd) and 5′-TGCGGTTGATAAACGCGCAGT-3′ (HSV-2pol Rev)13,53. For the generation of HSV-2 positive control plasmid, a 2.6kb fragment of the HSV-2 UL30 gene was amplified by PCR using the iProof™ High-Fidelity DNA Polymerase (Bio-Rad, Hercules, CA). Genomic DNA template was prepared from HSV-2 stock and primers 5′-GACGAGCGCGACGTCCTC-3’ (Fwd) and 5′-TCGTCGTAAAACAGCAGGTC-3’ (Rev) were used. The PCR product was cloned into the pCR® Blunt II TOPO® vector (Life Technologies) and the construct verified by sequencing.

p27 ELISA

p27 content in tissue culture supernatants was measured by RETRO-TEK SIV p27 Antigen ELISA kit (ZeptoMetrix, Buffalo, NY).

Statistical analysis

The sensitivity of paired p27 ELISA and SIV gag qRT-PCR results was compared using McNemar’s test and inter-rater agreement (Kappa) statistics using GraphPad calculators available online (graphpad.com).

Analysis of tissue viability (MTT assay) was performed as described in36, using a log-normal generalized linear mixed model predicting the weight-normalized OD570 of each replicate.

Analysis of gel activity against SHIV-RT and HSV-2 was performed as described in36,37,39,40,54 using a log-normal generalized linear mixed model with SOFT or CUM as the response and gel treatment as the predictor. For the experiments using rectal biopsies, a random intercept for each animal was included. For the vaginal tissue experiments, a random intercept was included for each biopsy, noting that two animals contributed two sets of biopsies.

All analyses were performed with SAS V9.4, SAS/STAT V13.1 with α=0.05. Significant p-values of <0.05 (*), <0.01 (**), and ≤0.001 (***) are indicated.

RESULTS

SIV gag qRT-PCR and p27 ELISA demonstrate similar SHIV-RT growth kinetics in explants with qRT-PCR being a more sensitive infection readout

To determine the feasibility of one-step SIV gag qRT-PCR for analysis of SHIV-RT infection in vaginal and rectal tissue cultures, a comparison with p27 ELISA was carried out. Supernatants from vaginal (n=7 experiments) and rectal (n=8 experiments) tissues challenged with SHIV-RT (vaginal) or SHIV-RT and HSV-2 (rectal) were analyzed by both ELISA and qRT-PCR at 5 time points post challenge (D0, 3, 7, 11 and 14). 3TC and 3TC/Acyclovir controls were available in n=2 vaginal tissue experiments and in all n=8 rectal tissue experiments, respectively. The results show similar SHIV-RT growth kinetics by both methods (Fig. 1). A total of n=45 (vaginal) and n=80 (rectal) data points was collected. In vaginal tissue culture supernatants, 8/45 samples had a positive readout (values ≥ LLOQ) by ELISA, while 42/45 samples had a positive readout by qRT-PCR. In rectal tissue supernatants, positive readout was obtained in 28/80 samples by ELISA, and in 65/80 by qRT-PCR. McNemar’s test for matched pairs showed higher sensitivity of qRT-PCR vs. ELISA (p< 0.0001, both vaginal and rectal tissue experiments). Examining statistical agreement by Kappa analysis we found a Kappa coefficient of 0.03 for vaginal supernatants and 0.22 for rectal, indicating poor and fair strength of agreement, respectively (Table 2). These results further emphasize higher sensitivity of qRT-PCR vs. ELISA.

Fig 1. SIV gag qRT-PCR and p27 ELISA demonstrate similar SHIV-RT growth kinetic in explants.

Fig 1

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PHA/IL-2 stimulated macaque vaginal explants were challenged with 104 TCID50 SHIV-RT ~18h. After washout, tissues were cultured (three explants per condition) for 14d. A summary of n=7 SHIV-RT challenge experiments (Mean±SEM) is shown. 3TC controls were included in 2 of 7 experiments. Rectal explants were challenged with 104 TCID50 SHIV-RT and 106 pfu HSV-2 vs. 3TC/Acyclovir control for 4h. After washout, tissues were cultured (4 biopsies/well; single well per condition) for 14d. A summary of n=8 experiments (Mean±SEM) is shown. SHIV-RT infection kinetics were followed by one-step SIV gag qRT-PCR and p27 ELISA.

Table 2.

Readouts obtained in vaginal and rectal tissue supernatants by ELISA and PCR assay. Matched pairs were analyzed by McNemar’s test and Kappa analysis.

Vaginal Positive(PCR) Negative(PCR)
Positive (ELISA) 8 0
Negative (ELISA) 34 3
Rectal
Positive (ELISA) 28 0
Negative (ELISA) 37 15
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MZC protects macaque vaginal mucosa against SHIV-RT and HSV-2 co-infection

Stimulated vaginal explants were co-challenged with SHIV-RT (104 TCID50/explant) based on our published data36,40,51 and with HSV-2 (106 pfu/explant) based on titration experiments demonstrating robust infection with this challenge dose (not shown). Controls included co-challenged tissues cultured in the presence of 3TC and Acyclovir. Fig. 2A provides representative examples of SHIV-RT and HSV-2 growth curves. Reproducible tissue infection was achieved with both viruses (Fig. 2B). In this system, no enhancement of SHIV-RT infection was detected in the presence of HSV-2 compared to SHIV-RT alone.

Fig 2. Macaque vaginal tissue is susceptible to SHIV-RT/HSV-2 infections after co-challenge.

Fig 2

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PHA/IL-2 stimulated macaque vaginal explants were challenged with 104 TCID50 SHIV-RT and/or 104 TCID50 HSV-2 (three explants per condition) vs. 3TC/Acy controls for ~18h. After washout, tissues were cultured for 14d in the presence of IL-2. (A) Representative examples of SHIV-RT and HSV-2 growth after single challenge or co-challenge are shown. Infections were monitored by one-step SIV gag qRT-PCR and HSV-2 pol qPCR. (B) Summaries of 7 experiments (SOFT and CUM analyses of SHIV-RT and HSV-2 infection) are shown. Dotted lines represent input virus (Mean) post washout at D0.

We previously demonstrated that MZC gel is active against single SHIV-RT infection in PHA/IL-2 stimulated vaginal explants36. In this study we aimed to determine whether MZC is active against SHIV and HSV-2 in a high-dose SHIV-RT/HSV-2 co-challenge model. Stimulated tissues were exposed to SHIV-RT and HSV-2 in the presence of non-toxic (Fig. 3A) 1:100 or 1:300 diluted MZC or CG (placebo) gels. Of note, one outlier in MZC 1:100 group was detected by MTT assay (Fig. 3A). The outlier was not excluded from the analysis as the data from explants in medium and CG 1:100 groups from the same donor were within the viable range.

Fig 3. MZC under non-toxic dilutions inhibits SHIV-RT/HSV-2 co-infection in macaque vaginal explants.

Fig 3

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(A) MTT assay was performed on tissues exposed to 1:100 and 1:300 diluted MZC and CG (two-three explants per condition) for ~18h. 1:10 diluted gynol served as a toxicity control. Each symbol indicates a donor and the Mean±SEM of the Log10(OD570/g) for each condition is shown. (B) PHA/IL-2 stimulated explants were challenged with 104 TCID50 SHIV-RT and 106 pfu HSV-2 in the presence of 1:100 and 1:300 diluted gels (three explants per condition) vs. untreated (Medium) and 3TC/Acy controls for ~18h. Then the tissues were washed and cultured for 14d in the presence of IL-2. Infections were monitored at 0, 3, 7, 11, 14d of culture by p27 ELISA and HSV-2 pol qPCR. Summary of 5–10 experiments (Mean±SEM of SOFT and CUM analyses) is shown. Dotted lines represent input virus (Mean) post washout at D0.

To allow a direct comparison with our previous work on MZC in the single infection model36, p27 ELISA was used as a readout. MZC (1:100 and 1:300 dilutions) strongly inhibited SHIV-RT infection relative to untreated (Medium) and CG controls (SOFT/CUM, 90–99% inhibition, p< 0.0001/0.05) (Fig. 3B). MZC and CG at 1:100 dilution (SOFT/CUM, 99% inhibition, p<0.0001) and CG at 1:300 dilution (SOFT/CUM, >90% inhibition, p< 0.05) inhibited HSV-2 vs. untreated control, pointing to CG-mediated activity of MZC against HSV-2.

MZC protects rectal mucosa against SHIV-RT infection

Rectal biopsies were co-challenged with 104 TCID50 SHIV-RT and HSV-2 103–106 pfu per biopsy vs. 3TC/Acyclovir controls. The SHIV-RT challenge dose was chosen based on studies in vaginal tissues (above) and resulted in reproducible SHIV-RT infection (Fig. 1). In contrast, no productive HSV-2 infection was detected in rectal mucosa as similar HSV pol copy numbers were detected in cultures with and without Acyclovir. A differential with Acyclovir control was observed only 1 out of 10 experiments using 106 pfu challenge dose (not shown). We chose to test gel activity in the SHIV-RT (104 TCID50) and HSV-2 (106 pfu) co-challenge settings to mimic possible real life HIV-1/HSV-2 co-exposure scenario.

Tissues were challenged in the presence of the non-toxic (Fig. 4A) 1:100 dilution of MZC for 4h vs. untreated (Medium), CG, 3TC/Acyclovir controls. MZC afforded significant protection against SHIV-RT relative to untreated and CG controls (SOFT/CUM, 92–98% inhibition, p<0.0001) (Fig. 4B). Similar anti-SHIV-RT activity (SOFT/CUM, 97–98% inhibition, p<0.0001) was detected by analysis of the same data set by qRT-PCR (not shown). As expected, HSV-2 failed to infect the rectal tissues (not shown).

Fig 4. MZC under non-toxic dilutions inhibits SHIV-RT infection in SHIV-RT/HSV-2 co-challenged macaque rectal explants.

Fig 4

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(A) MTT assay was performed on tissues exposed to 1:100 diluted MZC and CG (three explants per condition) for ~18h. 1:10 diluted gynol served as a toxicity control. Each symbol indicates a donor and the Mean±SEM of the log10(OD570/g) for each condition shown. (B) Explants were challenged with 104 TCID50 SHIV-RT and 106 pfu HSV-2 immersed in medium containing 1:100 diluted gels (three explants per condition) vs. untreated (Medium) and 3TC/Acy controls for 4h. Then the tissues were washed, transferred to Gelfoam sponges and cultured for 14d. SHIV-RT infection was measured at 0, 3, 7, 11, 14d of culture by p27 ELISA. Summary of 8 experiments (Mean±SEM of SOFT and CUM analyses) is shown. Dotted line represents input virus (Mean) post washout at D0.

DISCUSSION

In this study we introduced ex vivo vaginal SHIV-RT/HSV-2 co-infection and rectal SHIV-RT infection models. These models were used to test MZC’s activity. We also explored methodological aspects of monitoring tissue infection ex vivo. Here we compared sensitivity of SIV gag one-step qRT-PCR and p27 ELISA methods to monitor SHIV-RT infection in vaginal and rectal explants.

A recent study by Rollenhagen et al55 and data from our group64 demonstrated enhancement of ex vivo cervical HIV-1 infection in the HSV-2 co-infection settings. The elevated tissue HIV-1 infection coincided with increased numbers of CD4+CCR5+CD38+ T cells and reduced anti-HIV-1 activity of low dose Tenofovir (1 μg/ml)55.

In contrast to data in human cervical tissue, no enhancement of SHIV-RT infection by HSV-2 in stimulated macaque vaginal mucosa was seen in the current study. The same result was obtained when unstimulated tissues were co-challenged with the same viral doses (not shown). It is worth pointing out that that in this study and in the human ectocervical tissue co-infection model64 we resorted to a high HSV-2 viral challenge dose (106 pfu/explant) to assure reproducible HSV-2 infection and to test MZC’s activity under stringent conditions. Of note, 106 pfu (~107 copies of DNA) HSV-2 per explant highly exceeds the amount of HSV-2 shed in genital fluids of HSV-2 positive patients56,57. Infection with or exposure to HSV-2 can induce apoptosis and impair DCs and T cells58,59,60, potentially affecting tissue susceptibility to SHIV-RT. Although we cannot exclude these effects of HSV-2 in our tissue models, SHIV-RT infection after co-challenge of vaginal and rectal tissue was robust and reproducible. In our human cervical tissue explants model, HIV-1BaL/HSV-2 co-challenge results in ~60% frequency of productive HIV-1BaL vs. 100% following HIV-1BaL only challenge64. A more physiologically relevant, low-dose SHIV-RT/HSV-2 co-challenge model would be needed to explore whether and how co-infection drives mucosal SHIV-RT infection in macaques. We were unable to infect rectal mucosa with HSV-2 ex vivo. In vivo rectal HSV-2 infection was previously reported in 9 out of 11 SIV-infected macaques that were challenged rectally with 2×108 pfu HSV-219. However, in naïve animals, the frequency of infection after the same dose HSV-2 challenge is 55%61. As epithelial cells represent the primary target for HSV-2, loss of the single layer columnar epithelium during the culture period42 could have contributed to the lack of ex vivo infection in rectal biopsies.

Our side-by-side comparison indicates that one-step SIV gag qRT-PCR utilizing tissue culture supernatants can be used as an alternative to p27 ELISA to monitor tissue infection and product activity. The data indicate similar SHIV-RT growth kinetics and MZC activity as detected by both assays. The one-step SIV gag qRT-PCR has proven to be a more sensitive method than p27 ELISA. Our results are in agreement with the findings of Janocko et al.62, who demonstrated the feasibility of qRT-PCR as a readout of HIV infection62. Also in agreement with this report62, qRT-PCR did not shorten the time to detect evidence of infection. Overall, the qRT-PCR approach offers increased sensitivity and high dynamic range. The assay requires only a small volume of the supernatant (5 μl), is time and cost effective.

MZC gel protected against SHIV-RT at 1:100 (~0.18 μg/ml MIV-150) and 1:300 (~0.06 μg/ml MIV-150) dilutions and against HSV-2 at 1:100 dilution in vaginal mucosa. The activity against HSV-2 was CG-mediated. Similarly, the gel at 1:100 dilution also protected against SHIV-RT in rectal mucosa. It is important to note that previous studies demonstrated that the combination of CG and zinc acetate results in antiviral synergy against HSV-247. The HSV-2 mouse model has shown that under stringent conditions the combination of CG and zinc protects significantly while CG alone does not protect against HSV-2 infection31,47,63. The use of undiluted formulations in a mouse model allowed to appreciate the advantage of the CG/zinc combination when compared to CG alone. In our explant system CG alone provides strong inhibition (even after diluting the gel) that masks zinc’s contribution.

The results demonstrating potent activity of MZC against co-infection of vaginal mucosa with SHIV-RT and HSV-2 add to our previously published data showing potent MZC’s activity against single cell-free or cell-associated SHIV-RT challenge of macaque vaginal mucosa36,37. These results are also consistent with the potent activity of MZC against co-infection with HIV-1BaL and HSV-2 in human cervical mucosa64, suggesting that ex vivo activity testing in macaque mucosa may predict results in human mucosa. Overall, our data support further development of MZC as a potential broad-spectrum vaginal and rectal on-demand microbicide.

Acknowledgments

This work was supported by the United States President’s Emergency Plan for AIDS Relief (PEPFAR) through the United States Agency for International Development (USAID) Cooperative Agreement [GPO-A-00-04-00019-00, www.usaid.gov] and from the Tulane National Primate Research Center [Primate Center base grant P51 OD011104, http://tulane.edu/tnprc].

Footnotes

The authors have no conflicts to disclose.

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