SR-2P Vaginal Microbicide Gel Provides Protection against Herpes Simplex Virus 2 When Administered as a Combined Prophylactic and Postexposure Therapeutic

Abstract

Previously, we demonstrated that a single prophylactic dose of SR-2P, a novel dual-component microbicide gel comprising acyclovir and tenofovir, led to a modest increase in mouse survival following a lethal challenge of herpes simplex virus 2 (HSV-2). Here, we show that a dose of SR-2P administered 24 h prior to infection provides some protection against the virus, but to a lesser degree than SR-2P administered either once a day for 2 days or 1 h prior to infection. None of the prophylactic doses blocked infection by the virus, and all resulted in 80 to 100% lethality. However, given that a prophylactic dose still provided a significant reduction in overall clinical score, reduced rate of body weight loss, and increased median survival of the mice, we examined whether a repetitive dose regimen (postinfection) in addition to the prophylactic dose could prevent death and reduce the levels of virus in mice. Nearly all (9 of 10 in each group) of the mice that received SR-2P for 2 days prior to infection or that received SR-2P 1 h prior to infection and were administered SR-2P once a day for 10 days after infection showed no clinical symptoms of infection and no viral loads in vaginal swabs and survived for 28 days postinfection. Conversely, mice receiving no treatment or an identical vehicle treatment demonstrated advanced clinical signs and did not survive past day 9 postinfection. We conclude that SR-2P is an effective anti-HSV-2 agent in mice.

INTRODUCTION

There are approximately 500 million people infected with herpes simplex virus 2 (HSV-2) worldwide, and there are 20 million new cases every year (1). Genital HSV-2 infections result in lesions that typically resolve in 2 to 21 days, but the virus has a latent phase that can be reactivated, resulting in a cycle of lesion manifestations and clearance (2–4). HSV-2 and HIV have a reinforcing relationship in which prior infection with either virus appears to make an individual more susceptible to the other virus. Prior HSV-2 infection has been shown to lead to a 3- to 4-fold increase in the risk of acquiring HIV (5–7), while coinfection with HIV seems to lead to an increase in the frequency of reactivation of HSV-2 (8–10). Logically, if one of the viruses could be eliminated from the population, the prevalence of the other could be dramatically reduced. It is estimated that the incidence of HIV worldwide could be reduced by up to 40% in 20 years if a viable HSV-2 vaccine could be produced and widely distributed today (11). Unfortunately, no approval of an HSV-2 or HIV vaccine appears to be imminent, although there are a number of drugs with demonstrated efficacy against these agents that either have been approved or are in clinical trials (12). These include two drugs, acyclovir and tenofovir, which have demonstrated clinical benefits and are nucleoside analogue inhibitors that block the herpes viral polymerase and the HIV reverse transcriptase, respectively, effectively blocking replication of the virus (13–15). While these drugs are usually administered as a postexposure therapeutic (usually as part of a multidrug cocktail), there is a great deal of interest in pursuing them as prophylactic inhibitors of early infection to prevent dissemination of the viruses throughout the body.

Topical vaginal microbicides against viruses such as HIV and HSV-2 have been evaluated in clinical trials, with mixed results. Candidates such as nonoxynol-9, cellulose sulfate, and C31G not only failed to protect against HIV or HSV-2 but actually led to an increased risk of acquiring the viruses (16–19). In contrast, tenofovir microbicide gel led to 31% protection against HIV and surprisingly showed 51% protection against HSV-2 (20). This was an unexpected benefit, considering that tenofovir demonstrated no protection in cell-based assays, but suggests that, in vivo, tenofovir could be a dual antiviral therapeutic. Acyclovir is currently being pursued as a vaginal microbicide candidate (21), but it may have to be used in conjunction with tenofovir to be effective against both HIV and HSV-2. More recently, a phase III clinical trial of tenofovir gel alone, where the gel was to be administered 12 h prior to sex and 12 h after sex, did not show any benefit versus placebo in protecting women from HIV (http://www.conrad.org/news-pressreleases-107.html). This appears to be due to a lack of adherence to the study protocol of a twice-a-day application.

We are developing a multiviral (HSV-2 and HIV) vaginal microbicide gel formulation using a two-component system. One component is 10% acyclovir (wt/wt) suspended in a 2% (wt/wt) solution of Pluronic F-127 (a thermoresponsive polymer), and the second component is 2% tenofovir (wt/wt) suspended in a 2% (wt/wt) solution of Noveon AA-1 (a pH-responsive polycarbophil). The novelty of this formulation (referred to here as SR-2P) is that the two components can be mixed together prior to being administered to the vaginal cavity. Once the microbicide is in the vagina, the low-pH environment coupled with the increase in temperature provided by the body causes the mixture to form an adherent gel that sticks to the vaginal epithelium (22–24). When bound to the vaginal mucosa, the drugs are slowly released into the tissues where the viruses replicate.

We previously demonstrated that the SR-2P antiviral placebo formulation is well tolerated in a mouse vaginal irritation model (24) and that both acyclovir and tenofovir were released into the vaginal tissues at therapeutic levels up to 24 h after treatment (G. Bhatia, S. A. Fields, J. M. Fong, M. Liu, and G. N. Shankar, submitted for publication). In addition, we demonstrated in preliminary studies the efficacy of SR-2P against HSV-2 infection in mice, where 30% of mice pretreated with SR-2P at 1 h prior to infection exhibited survival against a lethal challenge of the virus (24). Here we show that either repeat or single prophylactic doses of SR-2P provide marginal protection against HSV-2, while a regimen of one dose a day for 10 days postinfection combined with the prophylactic dose provides nearly complete protection from the virus in mice.

MATERIALS AND METHODS

Cell lines and viruses.

African green monkey kidney cells (Vero) and HSV-2 G strain virus were obtained from ATCC. To propagate the virus, Vero cells were cultured in Vero medium (modified essential medium [MEM] plus 5% fetal bovine serum [FBS] and l-glutamine) to 70 to 80% confluence in T175 culture flasks grown at 37°C and 5% CO₂. The medium was removed, 1 ml of virus was added, and the flask was incubated at 37°C and 5% CO₂ for 1 h with a gentle rocking of the flask every 15 min. At the end of the hour of incubation, 15 ml Vero medium was added, and the cells were returned to the incubator; when 80 to 90% of the cells exhibited virally induced cytopathic effect (CPE), the flask was moved to a −80°C freezer for 15 min, and the frozen medium was allowed to warm to room temperature. The freeze-thaw was repeated twice, the resulting slurry was harvested and cleared by centrifugation, and the viral suspension was aliquoted and stored at −80°C. To determine the viral titer, Vero cells were plated in wells of a 6-well plate to a confluence of 70 to 80%; 150 μl of viral serial dilutions (10⁻¹ to 10⁻⁶) was added to the appropriate wells in duplicate, and the plate was incubated at 37°C and 5% CO₂ for 1 h with agitation every 15 min. Following incubation, a 2-ml overlay of 2× MEM (Lonza, Basel, Switzerland) and 2% agarose (Genesee, San Diego, CA) was added to the cells, and the plate was returned to the incubator. After 3 days, the titer was evaluated by removing the agar overlay, washing once with 1× phosphate-buffered saline (PBS; pH 7.4), and staining with 1% crystal violet in 20% ethanol for 10 min at room temperature. The crystal violet was removed, the plates were washed twice with 1× PBS, and the wells with 10 to 100 plaques per well were counted to determine PFU/ml with the lower limit of detection for the assay equal to 50 PFU/ml. Virus was passaged three times to obtain a titer of 1.125 × 10⁷ PFU/ml.

Preparation of SR-2P.

Poloxamer 407 NF and acyclovir were purchased from Spectrum Chemicals Manufacturing Company (Gardena, CA), tenofovir was purchased from Hangzhou Starshine Pharmaceutical (Shanghai, China), and polycarbophil USP was received as a gift from Lubrizol (Wickliffe, OH, USA). SR-2P containing 5% acyclovir and 1% tenofovir, with and without preservatives, was prepared as described previously (24). In brief, compounds were prepared in aqueous solutions containing poloxamer 407 NF (1.0% [wt/wt]) and polycarbophil USP (1.0% [wt/wt]). Tenofovir was included at 2% (wt/wt) in the poloxamer 407 NF component, and acyclovir was included at 10% (wt/wt) in the polycarbophil USP component. The individual components were fed into two separate 10-ml syringes, the syringes were connected, and the two components were mixed by pushing and retracting the syringes until the mixture was homogeneous. The combined SR-2P was stored in a single syringe at 4°C for up to 5 days prior to use.

Animals.

All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at SRI International and were performed in accordance with relevant guidelines and regulations in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

Determination of the ID₅₀ of HSV-2 in mice.

Female BALB/c mice were purchased from Harlan Laboratories (Livermore, CA) and were housed in microisolator cages with hardwood chip bedding; Purina rodent chow 5002 and reverse-osmosis-purified water were provided ad libitum. All mice (7 to 9 weeks of age) were hormonally synchronized with a subcutaneous dose of 3 mg of medroxyprogesterone acetate on day −8. The mice were randomized into treatment groups consisting of 10 mice per group per study and were dosed intravaginally using a p200 pipette tip with 30 μl of neat virus or of a 1:5 dilution, a 1:25 dilution, or a 1:125 dilution of virus. Mice were evaluated for changes in body weight and clinical observations (redness and swelling of the genitalia, discharge, ruffled fur, hunched posture, or loss of limb functionality). Mice that lost more than 20% of their initial body weight or exhibited extreme clinical symptoms were humanely euthanized and counted as moribund sacrifices the following day. A 1:5 dilution of our stock virus (6.75 × 10⁴ total PFU) resulted in full lethality (data not shown) and was used for all the studies described below.

Efficacy testing of SR-2P in mice.

Female BALB/c mice were purchased from Harlan Laboratories (Livermore, CA) and were housed in hanging polypropylene cages with hardwood chip bedding; Purina rodent chow 5002 and reverse osmosis purified water were provided ad libitum. All mice (7 to 9 weeks of age) were hormonally synchronized with a subcutaneous dose of 3 mg of medroxyprogesterone acetate on day −8. The mice were randomized into treatment groups consisting of 10 animals per group (Table 1). Individual 1-ml Luer lock syringes were filled with SR-2P and warmed to room temperature just prior to use. Mice were pretreated with either placebo (both component vehicles lacking drug) or 20 μl of SR-2P using a Luer lock syringe attached to a 20-gauge applicator inserted 0.8 cm into the vagina and challenged with 30 μl of diluted HSV-2 virus using a p200 pipette tip. Following dose administration, the blunt tip on the syringe was moved in a back-and-forth motion 10 times to simulate sexual intercourse and more evenly distribute the compound gel. For prophylactic studies, controls included mice infected with virus without treatment (group 1), mice infected 1 h after placebo treatment (group 2), mice infected 1 h after SR-2P dose treatment (group 3); mice infected 24 h after SR-2P treatment (group 4), and mice dosed once a day with 20 μl of SR-2P at day −2, day −1, and 1 h prior to infection on day 0 (group 5).

TABLE 1.

Details of study design and treatment groups for SR-2P efficacy experiments^a

Study and group	Treatment	Preinfection treatment	Day of HSV-2 infection	Postinfection treatment(s)
				First day	Repeat days
Prophylactic-only study
1	No gel/uninfected	NT	NT	NT	NT
2	Placebo	Day 0 (1 h prior to infection)	Day 0	NT	NT
3	SR-2P	Day 0 (1 h prior to infection)	Day 0	NT	NT
4	SR2-P	Day −1 (24 h prior to infection)	Day 0	NT	NT
5	SR-2P	Day −2, day −1, day 0 (1 h prior to infection)	Day 0	NT	NT
Prophylactic/postexposure study
6	No gel/uninfected	NT	Day 0	NT	NT
7	No gel/infected	NT	Day 0	NT	NT
8	Placebo	Day −2, day −1, day 0 (1 h prior to infection)	Day 0	Day 1	Day 2 through day 10, once daily
9	SR-2P	Day −2, day −1, day 0 (1 h prior to infection)	Day 0	Day 1	Day 2 through day 10, once daily
10	Placebo	Day 0 (1 h prior to infection)	Day 0	Day 1	Day 2 through day 10, once daily
11	SR-2P	Day 0 (1 h prior to infection)	Day 0	Day 1	Day 2 through day 10, once daily

^aNT, no treatment.

Prophylactic/postexposure studies included mice that were untreated and uninfected (group 6) or mice infected but untreated (group 7). In addition, two groups consisted of mice administered prophylactic treatment at day −2, day −1 and 1 h prior to infection followed by 10 days of treatment after infection with placebo or SR-2P (groups 8 and 9, respectively). The final two groups were mice treated 1 h prior to infection and once daily postinfection for 10 days with placebo or SR-2P (groups 10 and 11, respectively).

Mice were evaluated daily for changes in body weight and clinical observations (redness and swelling of the genitalia, discharge, ruffled fur, hunched posture, or loss of limb functionality). Redness and swelling were scored on a scale from 0 to 4, where 0 indicated no symptoms and 4 indicated severe symptoms. Presence of discharge, ruffled fur, hunched posture, and loss of limb functionality did not allow for gradation and were given a score of 1 each. Mice that lost more than 20% of their initial body weight or exhibited extreme clinical symptoms were humanely euthanized, counted as moribund sacrifices, recorded as dead the following day, and assigned a clinical observation score of 12 for that day. Survival graphs, percent body weight change graphs, and total clinical score graphs were generated using GraphPad Pro 4 software.

Vaginal swab collection for viral titer analysis.

Mice used for the solely prophylactic arms of this study were not swabbed for viral titers because we were concerned about removing any active compound from our single dose of SR-2P. However, 4 randomly chosen mice from each group were swabbed for viral titer analysis in the prophylactic/therapeutic treatment groups. Briefly, sterile cotton swabs (Puritan Medical, Guilford, ME) were dipped in sterile PBS, inserted approximately 0.8 cm into the vaginal cavity, and moved in a circular motion to swab the vaginal mucosa. The tip of the swab was placed into 200 μl of sterile 1× MEM, vortexed, and frozen at −80°C until used in a plaque assay which was conducted as described above.

Statistical analysis.

P values were calculated using ordinary one-way analysis of variance (ANOVA) multiple comparison scoring for each of the treatment groups versus placebo controls. P values of ≤0.05 were considered significant.

RESULTS

Efficacy of SR-2P prophylactic treatment against intravaginal HSV-2 infection in mice.

To evaluate the prophylactic protection provided by SR-2P against HSV-2 infection, mice were dosed intravaginally with 20 μl of SR-2P gel prior to intravaginal infection with HSV-2. Arms of this study included infection with virus following a 1-h pretreatment with SR-2P (group 3) to evaluate infection caused by simulated coitus occurring shortly after administration of drug, infection with virus following treatment with SR-2P 24 h prior to simulated coitus (group 4), and treatment with SR-2P at day −2, day −1, and 1 h prior to infection on day 0 to evaluate whether drug could accumulate in the vaginal tissues and provide added protection prior to simulated coitus (group 5). We observed extreme symptoms in placebo-treated mice infected with HSV-2 manifested within 7 days postinfection (group 2), but treatment with SR-2P (in all treatment groups) appeared to delay onset of the extreme symptoms by approximately 1 to 3 days (Fig. 1). We observed that the overall clinical scores for postinfection days 6 to 10 in the SR-2P 1 h pretreatment group (group 3) were significantly lower than those in placebo controls (group 2) (Fig. 1), which was supported by our measurements of individual body weights over the same period (Fig. 2). This suggests that even a single prophylactic dose of SR-2P is effective in reducing or delaying the onset of symptoms caused by HSV-2 infection in mice. This is particularly evident in the day 7 clinical observation scores, where all of the placebo control mice (group 2) demonstrated a score over 5 while 80% of the mice in the 1 h pretreatment group (group 3) had a score below 5. Even mice treated 24 h prior to infection (group 4) had significantly reduced clinical scores at days 6 and 7 postinfection compared to placebo controls. However, while the clinical scores for group 4 were still improved on days 8 to 10, they did not reach statistical significance, suggesting that treatment 24 h prior to infection may be at the limit of the prophylactic treatment window. Treatment of mice for 2 days prior to infection (group 5) did not lead to significantly improved clinical observation scores or body weights compared to the 1 h prophylactic dose (group 3), suggesting that additional days of pretreatment will not provide additional protection from HSV-2 infection. In addition, the survival of the SR-2P-treated mice was extended by several days in all SR-2P treatment groups (Fig. 3) with 50% of the 1 h pretreatment group (group 3), 30% of the 24 h pretreatment (group 4), and 60% of the 2-day pretreatment group (group 5) surviving to day 10 postinfection, compared with the placebo controls (group 2). Only 30% of the placebo control mice were alive on day 9, and all of them were deceased by day 10. About 20% and 10% of the mice in the 1-h and 2-day preinfection treatment groups, respectively, survived until the end of the study (day 21), while 20% of the mice in the 24-h preinfection treatment group survived until day 14 before succumbing to the infection. For the prophylactic-only study, vaginal swabs to determine viral loads were not collected because of concerns that any active SR-2P remaining from a single application prior to infection would be removed. However, we expect that viral titers in the vaginal cavity would be high in all mice that succumbed to infection. We concluded that SR-2P has limited potential as a prophylactic therapy against intravaginal HSV-2 infection, but given the improved clinical outcomes, we believed that if SR-2P was administered continuously following infection, it could provide a much better clinical outcome and perhaps prevent the establishment of infection.

FIG 1.

Clinical observation scores for the prophylactic study in mice. Mice were treated with 20 μl of placebo or SR-2P prior to infection with 6.75 × 10⁴ total PFU of HSV-2. Mouse groups are as follows: group 2, placebo controls; group 3, SR-2P pretreated mice that were infected 1 h following drug administration; group 4, mice that were pretreated for 24 h prior to infection; group 5, mice that were pretreated at day −2, day −1, and 1 h prior to infection on day 0. Mice were observed daily for clinical symptoms, including redness and swelling (each graded on a scale of 0 for no symptoms to 4 for extreme symptoms), vaginal discharge, ruffled fur, hunched posture, and loss of hind limb functionality (each assigned a score of 1 if present). Mice that either were found dead or were moribund sacrifices were assigned a score of 12 (solid line). P values represent ordinary one-way ANOVA multiple comparison scores for each of the treatment groups versus placebo controls. P values of ≤0.05 are considered significant. NS, not significant.

FIG 2.

Body weight changes for the prophylactic study in mice. Mouse groups were placebo controls that received treatment 1 h prior to infection with HSV-2 (group 2), were pretreated with SR-2P for 1 h prior to infection (group 3), were pretreated with SR-2P for 24 h prior to infection (group 4), or were pretreated at day −2, day −1, and 1 h prior to infection on day 0 (group 5). Body weights were tracked for 21 days in mice surviving until the end of the study.

FIG 3.

Survival curve for the prophylactic study in mice. Mice were either uninfected (group 1), placebo controls treated 1 h prior to infection with HSV-2 (group 2), pretreated with SR-2P for 1 h prior to infection (group 3), pretreated with SR-2P for 24 h prior to infection (group 4), or pretreated at day −2, day −1, and 1 h prior to infection on day 0 (group 5). P values were calculated using the log rank (Mantel-Cox) test.

Efficacy of SR-2P prophylactic/postexposure therapy against intravaginal HSV-2 infection in mice.

To evaluate the prophylactic/postexposure therapy using SR-2P, mice were administered the prophylactic dose regimen described above, with the exception that we did not include a 24-h preinfection group. On the day of infection, all mice were administered either placebo or SR-2P 1 h prior to infection with HSV-2. Placebo or SR-2P was then administered once a day for 10 days after infection, and the mice were evaluated for clinical observations, body weights, and survival. In addition, for all surviving mice, we measured the presence of viral shedding at days 5 and 15 (5 days after the last dose of SR-2P) and on the final day of the study (day 28). Unlike in our prophylactic-only study, mice that continued to receive SR-2P after infection demonstrated few clinical signs of viral infection. This was strikingly clear in both the clinical observation scores (Fig. 4) and body weight scores (Fig. 5), where there was virtually no change for either endpoint in mice receiving the SR-2P treatment (compare groups 9 and 11 with groups 8 and 10). Interestingly, mice that received placebo (gel without acyclovir or tenofovir) in the 2 days prior to infection (group 8) appeared to obtain some benefit from the gel, since there was a delay of onset of severe symptoms compared to the untreated group and to the placebo group, which received only a single prophylactic dose of the gel 1 h prior to infection (groups 7 and 10, respectively). This could be due to saturation of the vaginal mucosa over time, allowing a more evenly maintained, low-pH environment, which is inhibitory to the virus. Our previous work characterizing the base gel supports this conclusion (22). The improvement in clinical scores and body weights for the SR-2P-treated groups translated into vastly improved survival of the mice (Fig. 6A), with at least 9 out of 10 mice surviving until the end of the study. One mouse in the group receiving 1-h preinfection treatment only (group 11) succumbed to the anesthesia and could not be revived on day 9. One mouse from the 2-day SR-2P preinfection treatment group (group 9) demonstrated no clinical signs of infection until 3 days after the last dose of SR-2P; it then rapidly lost weight (Fig. 5), developed hind-limb paralysis, and was euthanized. This mouse demonstrated no classical signs of HSV-2 infection in the vagina (redness, swelling, or discharge). We hypothesize that for this one mouse, the acyclovir in SR-2P failed to inhibit viral replication in the vaginal epithelium and the virus was able to gain access to the ganglia, which reactivated once SR-2P was cleared from its system. To evaluate viral shedding in the vagina, we collected vaginal swabs from a number (4 or 5) of surviving animals at days 5 (Fig. 6B), 15 (data not shown), and 28 (data not shown) postinfection and determined the number of PFU. We could find no evidence of PFU in vaginal swab samples from mice treated with SR-2P, even when testing undiluted swab material (lower limit of detection for the plaque assay was 50 PFU/ml). In contrast, mice receiving no treatment (group 7), placebo 2 days prior to infection (group 8), or placebo 1 h prior to infection (group 10) demonstrated averages of 3.04 × 10⁴, 1.27 × 10⁴, and 2.41 × 10⁴ PFU/ml, respectively. The group of mice receiving placebo 2 days prior to infection (group 8) had a trend toward lower values (but not to a statistically significant level) than the untreated mice (group 7) and the mice receiving placebo treatment only 1 h prior to infection (group 10), suggesting that the improvement in clinical score may have been a result of lower viral titers in the vagina. No PFU were observed in swabs harvested at either day 15 or day 28 postinfection (days 5 and 24 after the last dose of SR-2P, respectively) in mice from the SR-2P treatment groups, including the one mouse that died following the last dose of SR-2P (day 15 collection [data not shown]). We conclude that when administered as a prophylactic/postexposure therapeutic, SR-2P provides nearly complete protection against HSV-2 in mice.

FIG 4.

Clinical observation scores for the prophylactic/postexposure therapeutic study in mice. Mice were administered drug as described in Table 1. Mouse groups are as follows: group 7, infected but untreated controls; group 8, mice that received placebo at day −2, day −1, and 1 h prior to infection on day 0; group 9, mice that received SR-2P at day −2, day −1, and 1 h prior to infection on day 0; group 10, mice that received placebo 1 h prior to infection on day 0; and group 11, mice that received SR-2P 1 h prior to infection on day 0. Groups 8 to 11 continued to receive placebo or SR-2P for 10 days following infection. Mice were observed daily for clinical symptoms, including redness and swelling (each graded on a scale of 0 for no symptoms to 4 for extreme symptoms), vaginal discharge, ruffled fur, hunched posture, and loss of hind limb functionality (each assigned a score of 1 if present) for 10 days following infection. Mice that either were found dead or were moribund sacrifices were assigned a score of 12 (solid line). Note that the death of the mouse in the SR-2P 1-h-prior dose group on day 9 was a result of an anesthesia error where the mouse did not recover and could not be resuscitated. P values represent ordinary one-way ANOVA multiple comparison scores for each of the treatment groups versus placebo controls. P values of ≤0.05 are considered significant. NS, not significant.

FIG 5.

Body weight changes for the prophylactic/postexposure therapeutic study in mice. Mouse groups are as follows: group 7, infected but untreated controls; group 8, mice that received placebo at day −2, day −1, and 1 h prior to infection on day 0; group 9, mice that received SR-2P at day −2, day −1, and 1 h prior to infection on day 0; group 10, mice that received placebo 1 h prior to infection on day 0; group 11, mice that received SR-2P 1 h prior to infection on day 0. Groups 8 to 11 continued to receive placebo or SR-2P for 10 days following infection.

FIG 6.

Survival curve and plaque assay of vaginal swabs for mice in the prophylactic/postexposure therapeutic study. (A) Survival curve for mice that were either uninfected (group 6), infected but untreated (group 7), pretreated with placebo for 2 days and then 1 h prior to infection (group 8), pretreated with SR-2P for 2 days and then 1 h prior to infection (group 9), placebo for 1 h prior to infection (group 10), and SR-2P for 1 h prior to infection (group 11). Following infection, groups 8 to 10 received additional placebo or SR-2P once a day for 10 days postinfection. The asterisk represents one mouse that did not recover from anesthesia and did not die as a result of viral infection. P values are calculated using the log rank (Mantel-Cox) test. (B) Vaginal swab samples were collected on day 5 postinfection, and PFU per ml were measured using a plaque assay. Mouse groups are as follows: group 7, infected but untreated controls; group 8, mice that received placebo at day −2, day −1, and 1 h prior to infection on day 0; group 9, mice that received SR-2P at day −2, day −1, and 1 h prior to infection on day 0; group 10, mice that received placebo 1 h prior to infection on day 0; and group 11, mice that received SR-2P 1 h prior to infection on day 0. Groups 8 to 11 continued to receive placebo or SR-2P for 10 days following infection. ZP, zero plaques detected where the number of plaques fell below the lower limit of detection for the assay (50 PFU/ml).

DISCUSSION

In this study, we determined that either a single prophylactic, intravaginal dose of SR-2P or a repetitive dose of SR-2P prior to infection provides significant protection from HSV-2 via the vaginal route. This was observed not only by increased survival of the mice but also by a reduction in symptoms observed over the course of the study. Interestingly, we also observed significant protection from HSV-2 infection following a single, 24-h prophylactic pretreatment with SR-2P. While this formulation is not 100% effective as a one-time-use prophylactic, the data suggest that enough acyclovir and tenofovir enter the vaginal tissues to elicit some protection against the virus. We hypothesized that continued vaginal treatment with SR-2P after infection could be therapeutically beneficial and, when combined with the prophylactic benefits observed, should dramatically increase the survival of the mice and reduce the severity of the infection. In fact, when SR-2P was administered for an additional 10 days postinfection, we observed 90 to 100% efficacy against the virus. This was true of clinical observation scores, body weights, and survival. The additional treatment seemed to prevent the establishment of an infection, as we could find no evidence of virus in the vaginal cavity 5 days postinfection. HSV-2 first infects vaginal epithelium and then spreads to the dorsal root ganglia, where latency is established (25). Thus, stopping the virus in the epithelium should prevent latent HSV-2 infection.

Acyclovir, a component of SR-2P, is a nucleoside analogue inhibitor of HSV-2, which works only inside infected cells because it relies on a viral kinase to convert it to the triphosphate active form. Once active, it incorporates into the elongating viral DNA during replication and terminates chain elongation, effectively blocking spread of the virus (26). This makes SR-2P an ideal candidate for a vaginal microbicide, and it has the added benefit of also containing tenofovir, a potent HIV reverse transcriptase inhibitor. Future studies will include an examination of SR-2P efficacy against HSV-2 in the more clinically relevant guinea pig model and efficacy testing against HIV in the humanized immune system mouse model.

To date, most clinical trials of vaginal microbicide candidates have provided mixed results. The most promising candidate, a tenofovir gel formulation, has demonstrated up to 30% efficacy when administered correctly during the trial. The most recent trial (unpublished) of this microbicide (FACTS 001) showed that adherence to the protocol is critical to the success of the drug. Overall, the drug showed no benefit compared with placebo controls, but women who strictly adhered to the protocol guidelines for use were protected to some degree from HIV infection (http://www.conrad.org/news-pressreleases-107.html). Adherence is always going to be problematic in the microbicide field; that is why it is important to develop a long-acting microbicide addressing multiple viruses. Topical gels are being passed over in favor of long-lived treatments like vaginal rings, which are currently undergoing clinical trials; however, there is a clear market for gels, since we believe that women will prefer a variety of options. In addition, the use of a gel formulation will likely be the only option for homosexual males. Further, preclinical evaluation of such rings requires a delay between insertion and exposure to virus (typically 1 week) (27, 28), and the ring must be replaced every 14 to 30 days, which will also affect patient compliance. In addition, while rings may provide protection over the course of a month, drug is continually released into the body regardless of the sexual activity of the individual, therefore exposing the individual to toxicity due to prolonged use of these antivirals. We believe that a controlled gel that needs to be administered only once a day while an individual is sexually active, which can provide protection against both HSV-2 and HIV, would be ideal.