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MZC Gel Inhibits SHIV-RT and HSV-2 in Macaque Vaginal Mucosa and SHIV-RT in Rectal Mucosa

Calenda, Giulia PhD; Villegas, Guillermo MS; Barnable, Patrick BS; Litterst, Claudia PhD; Levendosky, Keith BS; Gettie, Agegnehu MS; Cooney, Michael L. MS; Blanchard, James PhD; Fernández-Romero, José A. PhD; Zydowsky, Thomas M. PhD; Teleshova, Natalia MD, PhD

JAIDS Journal of Acquired Immune Deficiency Syndromes: March 1st, 2017 - Volume 74 - Issue 3 - p e67–e74
doi: 10.1097/QAI.0000000000001167
Basic Science
Free

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, herpes simplex virus (HSV), and human papillomavirus. Previous data show antiviral activity against these viruses in cell-based assays, prevention of vaginal and rectal simian–human immunodeficiency virus reverse transcriptase (SHIV-RT) infection, and reduction of vaginal HSV shedding in rhesus macaques and also excellent antiviral activity against HSV and human papillomavirus 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 coinfection 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 quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay readouts for monitoring SHIV-RT infection. MZC (at nontoxic dilutions) significantly inhibited SHIV-RT in vaginal and rectal mucosas and HSV-2 in vaginal mucosa when present during viral challenge. Analysis of SHIV-RT infection and MZC activity by 1-step simian immunodeficiency virus gag quantitative RT-PCR and p27 enzyme-linked immunosorbent assay demonstrated similar virus growth dynamics and MZC activity by both methods and higher sensitivity of quantitative RT-PCR. Our data provide more evidence that MZC is a promising dual compartment multipurpose prevention technology candidate.

*Population Council, New York, NY;

BioRad Laboratories, Hercules, CA;

Aaron Diamond AIDS Research Center, Rockefeller University, New York, NY; and

§Tulane National Primate Research Center, Tulane University, Covington, LA.

Correspondence to: Natalia Teleshova, MD, PhD, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, NY 10065 (e-mail: nteleshova@popcouncil.org).

Supported by the US President's Emergency Plan for AIDS Relief (PEPFAR) through the US 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).

The authors have no funding or conflicts of interest to disclose.

G. C. and G. V. contributed equally.

Received March 07, 2016

Accepted July 15, 2016

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INTRODUCTION

More than 2 decades ago, an “epidemiological synergy” between HIV-1 and other sexually transmitted infections (STIs) increasing the risk of HIV-1 acquisition was suggested.1 Among STIs affecting HIV transmission and pathogenesis, noncurable STIs like herpes simplex virus 2 (HSV-2) and human papillomavirus (HPV) deserve special attention. Recent studies reported up to 3- and 7-fold increased risk of HIV-1 transmission with prevalent and incident HSV-2 infection, respectively.2–5 HIV-1/HSV-2 coinfection affects the pathogenesis of both viruses, being associated with increased HIV-1 viral load6–8 and HSV-2 shedding quantity and frequency.9–11 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 infections.12 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 transmission.13 Specifically, frequency of vaginal CD4+ T cells expressing high level of α4β7, a gut-homing integrin that binds gp12014 and facilitates HIV/simian immunodeficiency virus (SIV) infection,13–18 is increased in HSV-2–infected RM.13 An increase of α4β7highCD4+ T cells in rectal mucosa was also observed in rectal HSV-2 infection in RM.19 Similar 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 incidence.24,25

The development of multipurpose prevention technologies active against HIV-1, HSV-2, and HPV vaginally and rectally could significantly improve global public health.20,26–28 The Population Council's gel containing 50 μM of the nonnucleoside reverse transcriptase inhibitor MIV-150 and 14 mM zinc acetate dihydrate (ZA) in carrageenan (CG) (MZC) demonstrated activity against vaginal SHIV reverse transcriptase (RT) (RM), vaginal HSV-2 (RM, mice), anorectal HSV-2 (mice), and 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 explants (In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa. Villegas G, Calenda G, Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep). A recently completed phase I clinical trial demonstrated a favorable vaginal safety profile of MZC38 (First-in-Human Trial of MIV-150 and Zinc Acetate Coformulated in a Carrageenan Gel: Safety, Pharmacokinetics, Acceptability, Adherence, and Pharmacodynamics. Friedland BA, Hoesley CJ, Plagianos M, Hoskin E, Zhang S, Teleshova N, Alami M, Novak L, Kleinbeck KR, Katzen LL, Zydowsky TM, Fernández-Romero JA, Creasy GW. J Acquir Immune Defic Syndr. 2016 Dec 15;73(5):489–496). The MZC gel is the only multipurpose prevention technology product currently in clinical testing that demonstrates activity against HIV and two other noncurable STIs that increase the risk of HIV-1 transmission, HSV-2, and HPV.26

Here we aimed to establish vaginal and rectal explant SHIV-RT/HSV-2 coinfection 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 enzyme-linked immunosorbent assays (ELISAs).36,39–42 Here we explored whether analysis of accumulation of viral RNA can be used as an alternative for p27 ELISA.

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MATERIALS AND METHODS

Macaques

Naive, SHIV-RT–, SIV-, and HSV-1–exposed uninfected/infected Chinese and Indian RMs (Macaca mulatta) were used. 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 no. 000594). The use of macaques was approved by the Animal Care and Use Committee of the TNPRC (OLAW assurance no. A4499-01), and animal care complied with the regulations in the Animal Welfare Act43 and the Guide for the Care and Use of Laboratory Animals.44 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 before and during all procedures, and analgesics were provided afterward as previously described.29,45 Necropsy tissues were available from 9 animals. These animals were euthanized using methods consistent with recommendations of the American Veterinary Medical Association Guidelines for Euthanasia. The animals were anesthetized with tiletamine–zolazepam (each at 4 mg/kg intramuscularly) and given buprenorphine (0.01 mg/kg intramuscularly) followed by an overdose of pentobarbital sodium. Death was confirmed by auscultation of the heart and pupillary dilation.

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Gels

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

TABLE 1

TABLE 1

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Viral Stocks

SHIV-RT was generated from the original stock provided by Disa Böttiger (Medivir AB, Sweden)46 using phytohaemagglutinin (PHA)/interleukin-2 (IL-2)-activated macaque peripheral blood mononuclear cells and titered in CEMx174 cells before use.29

HSV-2 strain G was generated as described previously.47 Briefly, HSV-2 strain G was propagated in Vero cells American Type Culture Collection (ATCC)48 as described earlier,49 and the viral titer in plaque-forming units (pfu)/mL was obtained by plaque formation assay on monolayer cultures of Vero cells.50

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Macaque Tissue Processing

Vaginal mucosa (n = 2, 3 × 5 mm biopsies procured at each collection time; or necropsy tissues) was obtained from RM. Tissues were transported overnight and cut into 3 × 3 mm explants as described previously 36,51 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.

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Tissue Viability

To assess tissue viability after overnight (∼18 hours) exposure to MZC or CG gels, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed as previously described.36,51

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Comparison of SHIV-RT Growth Kinetics Using Quantitative RT Polymerase Chain Reaction and p27 ELISA

To compare SHIV-RT infection readout methods in vaginal explants, tissues were processed and challenged with SHIV-RT as previously described.36 Briefly, vaginal explants were stimulated [5 μg/mL PHA (Sigma Aldrich) and 100 U/mL IL-2 (NCI BRB Preclinical Repository, Frederick, MD)] for 48 hours and then challenged with 104 TCID50 SHIV-RT per explant for ∼18 hours. After washout, explants were cultured in the presence of IL-2 for 14 days.36 Supernatants were collected on days 0, 3, 7, 11, and 14, and infection levels were analyzed by SIV gag one-step quantitative RT polymerase chain reaction (qRT-PCR) and p27 ELISA. To compare SHIV-RT infection readout methods in rectal biopsies, supernatants from SHIV-RT/HSV-2 cochallenge experiments (below) were used. Controls included 10 μM 3TC or 10 μM 3TC/100 μg/mL Acyclovir.

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SHIV-RT and HSV-2 Cochallenge of Vaginal and Rectal Mucosae and Antiviral Activity of MZC

PHA/IL-2–stimulated vaginal explants and unstimulated rectal biopsies were cochallenged with 104 TCID50 SHIV-RT and 106 pfu HSV-2 per explant for ∼18 (vaginal) or 4 hours (rectal). To test the antiviral activity of MZC, viral challenge was done in the presence of 1:300 and/or 1:100 diluted MZC and CG gels vs. untreated (medium) and 3TC/Acyclovir controls. After washout, vaginal explants were cultured in the presence of IL-2 for 14 days.36 Rectal biopsies (n = 4) were placed on 12-mm diameter Gelfoam sponges (Ethicon, Somerville, NJ) presoaked in complete DMEM (cDMEM) (DMEM Cellgro Mediatech) containing 10% fetal bovine serum (Gibco; Life Technologies, Grand Island, NY), 100 U/mL penicillin, 100 μg/mL streptomycin (Cellgro Mediatech), and 100 μM Eagle's minimum essential medium (MEM) nonessential amino acids (Irvine Scientific, Santa Ana, CA) at 37°C, 5% CO2, for at least 30 minutes and cultured for 14 days (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.

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qPCR and qRT-PCR

Five microliters 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 National Institutes of Health AIDS Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health,52 as the standard (37; A Novel Microbicide/Contraceptive Intravaginal Ring Protects Macaque Genital Mucosa against SHIV-RT Infection Ex Vivo. Villegas G, Calenda G, Ugaonkar S, Zhang S, Kizima L, Mizenina O, Gettie A, Blanchard J, Cooney ML, Robbiani M, Fernández-Romero JA, Zydowsky TM, Teleshova N. PLoS One. 2016 Jul 18;11(7):e0159332. doi: 10.1371/journal.pone.0159332. eCollection 2016).

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 2.6-kb fragment of the HSV-2 UL30 gene was amplified by PCR using the iProof High-Fidelity DNA Polymerase (BioRad, 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.

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p27 ELISA

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

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Statistical Analyses

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

Analysis of tissue viability (MTT assay) was performed as described earlier36 using a log-normal generalized linear mixed model predicting the weight-normalized optical density (OD)570 of each replicate.

Analysis of gel activity against SHIV-RT and HSV-2 was performed as described previously36,37,39,40,54 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 experiments, a random intercept was included for each biopsy, noting that 2 animals contributed 2 biopsies.

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

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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 postchallenge (days 0, 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 were collected. In vaginal tissue culture supernatants, 8/45 samples had a positive readout (values ≥ Lower Limit of Quantification (LLOQ)) by ELISA, whereas 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 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 and 0.22 for rectal supernatants, indicating poor and fair strength of agreement, respectively (Table 2). These results further emphasize higher sensitivity of qRT-PCR vs. ELISA.

FIGURE 1

FIGURE 1

TABLE 2

TABLE 2

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MZC Protects Macaque Vaginal Mucosa Against SHIV-RT and HSV-2 Coinfection

Stimulated vaginal explants were cochallenged 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 cochallenged tissues cultured in the presence of 3TC and Acyclovir. Figure 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 with SHIV-RT alone.

FIGURE 2

FIGURE 2

We previously demonstrated that MZC gel is active against single SHIV-RT infection in PHA/IL-2–stimulated vaginal explants.36 In this study, we aimed to determine whether MZC is active against SHIV and HSV-2 in a high-dose SHIV-RT/HSV-2 cochallenge model. Stimulated tissues were exposed to SHIV-RT and HSV-2 in the presence of nontoxic (Fig. 3A) 1:100 or 1:300 diluted MZC or CG (placebo) gel. Of note, one outlier in 1:100 MZC 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.

FIGURE 3

FIGURE 3

To allow a direct comparison with our previous work on MZC in the single infection model,36 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.

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MZC Protects Rectal Mucosa Against SHIV-RT Infection

Rectal biopsies were cochallenged 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 in 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) cochallenge settings to mimic possible real-life HIV-1/HSV-2 coexposure scenario.

Tissues were challenged in the presence of nontoxic (Fig. 4A) 1:100 dilution of MZC for 4 hours vs. untreated (medium), CG, and 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).

FIGURE 4

FIGURE 4

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DISCUSSION

In this study, we introduced ex vivo vaginal SHIV-RT/HSV-2 coinfection 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 the 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 group (In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa. Villegas G, Calenda G, Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep) demonstrated enhancement of ex vivo cervical HIV-1 infection in the HSV-2 coinfection 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 cochallenged with the same viral doses (not shown). It is worth pointing out that in this study and in the human ectocervical tissue coinfection model (In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa. Villegas G, Calenda G, Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep), 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 the genital fluids of HSV-2–positive patients.56,57 Infection with or exposure to HSV-2 can induce apoptosis and impair dendritic cells and T cells,58–60 potentially affecting tissue susceptibility to SHIV-RT. Although we cannot exclude these effects of HSV-2 in our tissue models, SHIV-RT infection after cochallenge of vaginal and rectal tissues was robust and reproducible. In our human cervical tissue explant model, HIV-1BaL/HSV-2 cochallenge results in ∼60% frequency of productive HIV-1BaL vs. 100% after HIV-1BaL only challenge (In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa. Villegas G, Calenda G, Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep). A more physiologically relevant, low-dose SHIV-RT/HSV-2 cochallenge model would be needed to explore whether and how coinfection 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-2.19 However, in naive 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 1-step SIV gag qRT-PCR using 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 1-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 infection.62 Also in agreement with this report,62 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) and 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-2.47 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 infection.31,47,63 The use of undiluted formulations in a mouse model allowed to appreciate the advantage of the CG/zinc combination when compared with 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 coinfection 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 mucosa.36,37 These results are also consistent with the potent activity of MZC against coinfection with HIV-1BaL and HSV-2 in human cervical mucosa (In Vitro Exposure to PC-1005 and Cervicovaginal Lavage Fluid from Women Vaginally Administered PC-1005 Inhibits HIV-1 and HSV-2 Infection in Human Cervical Mucosa. Villegas G, Calenda G, Zhang S, Mizenina O, Kleinbeck K, Cooney ML, Hoesley CJ, Creasy GW, Friedland B, Fernández-Romero JA, Zydowsky TM, Teleshova N. Antimicrob Agents Chemother. 2016 Aug 22;60(9):5459–5466. doi: 10.1128/AAC.00392-16. Print 2016 Sep), 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.

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REFERENCES

1. Wasserheit JN. Epidemiological synergy. Interrelationships between human immunodeficiency virus infection and other sexually transmitted diseases. Sex Transm Dis. 1992;19:61–77.
2. Barnabas RV, Celum C. Infectious co-factors in HIV-1 transmission herpes simplex virus type-2 and HIV-1: new insights and interventions. Curr HIV Res. 2012;10:228–237.
3. Freeman EE, Weiss HA, Glynn JR, et al. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS. 2006;20:73–83.
4. Sobngwi-Tambekou J, Taljaard D, Lissouba P, et al. Effect of HSV-2 serostatus on acquisition of HIV by young men: results of a longitudinal study in Orange Farm, South Africa. J Infect Dis. 2009;199:958–964.
5. Glynn JR, Carael M, Auvert B, et al. Why do young women have a much higher prevalence of HIV than young men? A study in Kisumu, Kenya and Ndola, Zambia. AIDS. 2001;15(suppl 4):S51–S60.
6. Duffus WA, Mermin J, Bunnell R, et al. Chronic herpes simplex virus type-2 infection and HIV viral load. Int J STD AIDS. 2005;16:733–735.
7. Gray RH, Li X, Wawer MJ, et al. Determinants of HIV-1 load in subjects with early and later HIV infections, in a general-population cohort of Rakai, Uganda. J Infect Dis. 2004;189:1209–1215.
8. Mole L, Ripich S, Margolis D, et al. The impact of active herpes simplex virus infection on human immunodeficiency virus load. J Infect Dis. 1997;176:766–770.
9. Augenbraun M, Feldman J, Chirgwin K, et al. Increased genital shedding of herpes simplex virus type 2 in HIV-seropositive women. Ann Intern Med. 1995;123:845–847.
10. Wright PW, Hoesley CJ, Squires KE, et al. A prospective study of genital herpes simplex virus type 2 infection in human immunodeficiency virus type 1 (HIV-1)-seropositive women: correlations with CD4 cell count and plasma HIV-1 RNA level. Clin Infect Dis. 2003;36:207–211.
11. Corey L, Wald A, Celum CL, et al. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. J Acquir Immune Defic Syndr. 2004;35:435–445.
12. Abu-Raddad LJ, Magaret AS, Celum C, et al. Genital herpes has played a more important role than any other sexually transmitted infection in driving HIV prevalence in Africa. PLoS One. 2008;3:e2230.
13. Goode D, Truong R, Villegas G, et al. HSV-2-driven increase in the expression of alpha4beta7 correlates with increased susceptibility to vaginal SHIV(SF162P3) infection. PLoS Pathog. 2014;10:e1004567.
14. Arthos J, Cicala C, Martinelli E, et al. HIV-1 envelope protein binds to and signals through integrin alpha4beta7, the gut mucosal homing receptor for peripheral T cells. Nat Immunol. 2008;9:301–309.
15. Cicala C, Martinelli E, McNally JP, et al. The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1. Proc Natl Acad Sci U S A. 2009;106:20877–20882.
16. Kader M, Wang X, Piatak M, et al. Alpha4(+)beta7(hi)CD4(+) memory T cells harbor most Th-17 cells and are preferentially infected during acute SIV infection. Mucosal Immunol. 2009;2:439–449.
17. Martinelli E, Veglia F, Goode D, et al. The frequency of alpha(4)beta(7)(high) memory CD4(+) T cells correlates with susceptibility to rectal simian immunodeficiency virus infection. J Acquir Immune Defic Syndr. 2013;64:325–331.
18. Ansari AA, Reimann KA, Mayne AE, et al. Blocking of alpha4beta7 gut-homing integrin during acute infection leads to decreased plasma and gastrointestinal tissue viral loads in simian immunodeficiency virus-infected rhesus macaques. J Immunol. 2011;186:1044–1059.
19. Martinelli E, Tharinger H, Frank I, et al. HSV-2 infection of dendritic cells amplifies a highly susceptible HIV-1 cell target. PLoS Pathog. 2011;7:e1002109.
20. Houlihan CF, Larke NL, Watson-Jones D, et al. Human papillomavirus infection and increased risk of HIV acquisition. A systematic review and meta-analysis. AIDS. 2012;26:2211–2222.
21. Ng'ayo MO, Bukusi E, Rowhani-Rahbar A, et al. Epidemiology of human papillomavirus infection among fishermen along lake Victoria Shore in the Kisumu District, Kenya. Sex Transm Infect. 2008;84:62–66.
22. Marais DJ, Passmore JA, Denny L, et al. Cervical and oral human papillomavirus types in HIV-1 positive and negative women with cervical disease in South Africa. J Med Virol. 2008;80:953–959.
23. Didelot-Rousseau MN, Nagot N, Costes-Martineau V, et al. Human papillomavirus genotype distribution and cervical squamous intraepithelial lesions among high-risk women with and without HIV-1 infection in Burkina Faso. Br J Cancer. 2006;95:355–362.
24. Safaeian M, Kiddugavu M, Gravitt PE, et al. Determinants of incidence and clearance of high-risk human papillomavirus infections in rural Rakai, Uganda. Cancer Epidemiol Biomarkers Prev. 2008;17:1300–1307.
25. Lissouba P, Van de Perre P, Auvert B. Association of genital human papillomavirus infection with HIV acquisition: a systematic review and meta-analysis. Sex Transm Infect. 2013;89:350–356.
26. Fernandez-Romero JA, Deal C, Herold BC, et al. Multipurpose prevention technologies: the future of HIV and STI protection. Trends Microbiol. 2015;23:429–436.
27. Fernandez-Romero JA, Teleshova N, Zydowsky TM, et al. Preclinical assessments of vaginal microbicide candidate safety and efficacy. Adv Drug Deliv Rev. 2015;92:27–38.
28. Malcolm RK, Boyd P, McCoy CF, et al. Beyond HIV microbicides: multipurpose prevention technology products. BJOG. 2014;121(suppl 5):62–69.
29. Kenney J, Aravantinou M, Singer R, et al. An antiretroviral/zinc combination gel provides 24 hours of complete protection against vaginal SHIV infection in macaques. PLoS One. 2011;6:e15835.
30. Kenney J, Singer R, Derby N, et al. A single dose of a MIV-150/Zinc acetate gel provides 24 h of protection against vaginal simian human immunodeficiency virus reverse transcriptase infection, with more limited protection rectally 8–24 h after gel use. AIDS Res Hum Retroviruses. 2012;28:1476–1484.
31. Kizima L, Rodriguez A, Kenney J, et al. A potent combination microbicide that targets SHIV-RT, HSV-2 and HPV. PLoS One. 2014;9:e94547.
32. Hsu M, Aravantinou M, Menon R, et al. 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. AIDS Res Hum Retroviruses. 2014;30:174–183.
33. Kenney J, Derby N, Aravantinou M, et al. Short communication: a repeated simian human immunodeficiency virus reverse transcriptase/herpes simplex virus type 2 cochallenge macaque model for the evaluation of microbicides. AIDS Res Hum Retroviruses. 2014;30:1117–1124.
34. Levendosky K, Mizenina O, Martinelli E, et al. Griffithsin and carrageenan combination to target HSV-2 and HPV. Antimicrob Agents Chemother. 2015.
35. Roberts JN, Buck CB, Thompson CD, et al. Genital transmission of HPV in a mouse model is potentiated by nonoxynol-9 and inhibited by carrageenan. Nat Med. 2007;13:857–861.
36. Barnable P, Calenda G, Ouattara L, et al. A MIV-150/zinc acetate gel inhibits SHIV-RT infection in macaque vaginal explants. PLos One. 2014;9:e108109.
37. Barnable P, Calenda G, Bonnaire T, et al. MIV-150/zinc acetate gel inhibits cell-associated simian-human immunodeficiency virus reverse transcriptase infection in a macaque vaginal explant model. Antimicrob Agents Chemother. 2015;59:3829–3837.
38. Friedland B, Plagianos, Zhang, et al. A First-in-Human Trial of PC-1005 (MIV-150 and Zinc Acetate in a Carrageenan Gel). CROI 2016; February 22-25, 2016; Boston, MA. Abstract 875.
39. Richardson-Harman N, Mauck C, McGowan I, et al. Dose-response relationship between tissue concentrations of UC781 and explant infectibility with HIV type 1 in the RMP-01 rectal safety study. AIDS Res Hum Retroviruses. 2012;28:1422–1433.
40. Ouattara LA, Barnable P, Mawson P, et al. MIV-150 containing intravaginal rings protect macaque vaginal explants against SHIV-RT infection. Antimicrob Agents Chemother. 2014.
41. Introini A, Vanpouille C, Grivel JC, et al. An ex vivo Model of HIV-1 Infection in Human Lymphoid Tissue and Cervico-vaginal Tissue. Bio Protoc. 2014;4.
42. Abner SR, Guenthner PC, Guarner J, et al. A human colorectal explant culture to evaluate topical microbicides for the prevention of HIV infection. J Infect Dis. 2005;192:1545–1556.
43. Animal Welfare Act and Regulation of 2001. ed. Code of Federal Regulations, t., Chapter 1, Subchapter A: Animals and Animal Products. U.S. Department of Agriculture, Beltsville, MD.
44. Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Guide for the Care and Use of Laboratory Animals. Vol 85-23. Bethesda, MD: U.S. Department of Health and Human Services, National Institutes of Health; 1985:1–83.
45. Singer R, Mawson P, Derby N, et al. An intravaginal ring that releases the NNRTI MIV-150 reduces SHIV transmission in macaques. Sci Transl Med. 2012;4:150ra123.
46. Turville SG, Aravantinou M, Miller T, et al. Efficacy of Carraguard-based microbicides in vivo despite variable in vitro activity. PLoS One. 2008;3:e3162.
47. Fernandez-Romero JA, Abraham CJ, Rodriguez A, et al. Zinc acetate/carrageenan gels exhibit potent activity in vivo against high-dose herpes simplex virus 2 vaginal and rectal challenge. Antimicrob Agents Chemother. 2012;56:358–368.
48. Ammerman NC, Beier-Sexton M, Azad AF. Growth and maintenance of Vero cell lines. Curr Protoc Microbiol. 2008; Appendix 4:Appendix 4E.
49. McDermott MR, Smiley JR, Leslie P, et al. Immunity in the female genital tract after intravaginal vaccination of mice with an attenuated strain of herpes simplex virus type 2. J Virol. 1984;51:747–753.
50. Ashley R. Chapter 22: diagnostic procedures for viral, rickettsial, and chlamydial infections. In: Schmidt NJ, Emmons RW, eds. Washington, DC: American Public Health Association; 1995.
51. Aravantinou M, Singer R, Derby N, et al. The nonnucleoside reverse transcription inhibitor MIV-160 delivered from an intravaginal ring, but not from a carrageenan gel, protects against simian/human immunodeficiency virus-RT Infection. AIDS Res Hum Retroviruses. 2012;28:1467–1475.
52. Luciw PA, Shaw KE, Unger RE, et al. Genetic and biological comparisons of pathogenic and nonpathogenic molecular clones of simian immunodeficiency virus (SIVmac). AIDS Res Hum Retroviruses. 1992;8:395–402.
53. Legoff J, Bouhlal H, Gresenguet G, et al. Real-time PCR quantification of genital shedding of herpes simplex virus (HSV) and human immunodeficiency virus (HIV) in women coinfected with HSV and HIV. J Clin Microbiol. 2006;44:423–432.
54. Richardson-Harman N, Lackman-Smith C, Fletcher PS, et al. Multisite comparison of anti-human immunodeficiency virus microbicide activity in explant assays using a novel endpoint analysis. J Clin Microbiol. 2009;47:3530–3539.
55. Rollenhagen C, Lathrop MJ, Macura SL, et al. Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides. Mucosal Immunol. 2014.
56. Tronstein E, Johnston C, Huang ML, et al. Genital shedding of herpes simplex virus among symptomatic and asymptomatic persons with HSV-2 infection. JAMA. 2011;305:1441–1449.
57. Mark KE, Wald A, Magaret AS, et al. Rapidly cleared episodes of oral and anogenital herpes simplex virus shedding in HIV-infected adults. J Acquir Immune Defic Syndr. 2010;54:482–488.
58. Vanden Oever MJ, Han JY. Caspase 9 is essential for herpes simplex virus type 2-induced apoptosis in T cells. J Virol. 2010;84:3116–3120.
59. Stefanidou M, Ramos I, Mas Casullo V, et al. Herpes simplex virus 2 (HSV-2) prevents dendritic cell maturation, induces apoptosis, and triggers release of proinflammatory cytokines: potential links to HSV-HIV synergy. J Virol. 2013;87:1443–1453.
60. Peretti S, Shaw A, Blanchard J, et al. Immunomodulatory effects of HSV-2 infection on immature macaque dendritic cells modify innate and adaptive responses. Blood. 2005;106:1305–1313.
61. Guerra-Perez N, Aravantinou M, Veglia F, et al. Rectal HSV-2 infection may increase rectal SIV acquisition even in the Context of SIVDeltanef vaccination. PLoS One. 2016;11:e0149491.
62. Janocko L, Althouse AD, Brand RM, et al. The molecular characterization of intestinal explant HIV infection using Polymerase Chain Reaction-Based Techniques. AIDS Res Hum Retroviruses. 2015;31:981–991.
63. Kenney J, Rodriguez A, Kizima L, et al. A modified zinc acetate gel, a potential nonantiretroviral microbicide, is safe and effective against simian-human immunodeficiency virus and herpes simplex virus 2 infection in vivo. Antimicrob Agents Chemother. 2013;57:4001–4009.
Keywords:

MIV-150; multipurpose prevention technology; SHIV-RT; HSV-2; vaginal; rectal

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