BACKGROUND: Pig-tailed macaques (PTMs) are commonly used as preclinical models to assess antiretroviral drugs for HIV prevention research. Drug toxicities and disease pathologies are often preceded by changes in blood hematology. To better assess the safety profile of pharmaceuticals, we defined normal ranges of hematological values in PTMs using an Isolation Forest (iForest) algorithm. METHODS: Eighteen female PTMs were evaluated. Blood was collected 1-24 times per animal for a total of 159 samples. Complete blood counts were performed, and iForest was used to analyze the hematology data to detect outliers. RESULTS: Median, IQR, and ranges were calculated for 13 hematology parameters. From all samples, 22 outliers were detected. These outliers were excluded from the reference index. CONCLUSIONS: Using iForest, we defined a normal range for hematology parameters in female PTMs. This reference index can be a valuable tool for future studies evaluating drug toxicities in PTMs.

Pre-exposure prophylaxis (PrEP) with a weekly oral regimen of antiretroviral drugs could be a suitable preventative option for individuals who struggle with daily PrEP or prefer not to use long-acting injectables. We assessed in macaques the efficacy of weekly oral tenofovir alafenamide (TAF) at doses of 13.7 or 27.4 mg/kg. Macaques received weekly oral TAF for six weeks and were exposed twice-weekly to SHIV vaginally or rectally on day 3 and 6 after each dose. Median TFV-DP levels in PBMCs following the 13.7 mg/kg dose were 3110 and 1137 fmols/10(6) cells on day 3 and 6, respectively. With the 27.4 mg/kg dose, TFV-DP levels were increased (~2-fold) on day 3 and 6 (6095 and 3290 fmols/10(6) cells, respectively). Both TAF doses (13.7 and 27.4 mg/kg) conferred high efficacy (94.1% and 93.9%, respectively) against vaginal SHIV infection. Efficacy of the 27.4 mg/kg dose against rectal SHIV infection was 80.7%. We estimate that macaque doses of 13.7 and 27.4 mg/kg are equivalent to approximately 230 and 450 mg of TAF in humans, respectively. Our findings demonstrate the effectiveness of a weekly oral PrEP regimen and suggest that a clinically achievable oral TAF dose could be a promising option for non-daily PrEP.

The prevention of HIV and unintended pregnancies is a public health priority. Multi-purpose prevention technologies capable of long-acting HIV and pregnancy prevention are desirable for women. Here, we utilized a preclinical macaque model to evaluate the pharmacokinetics of biodegradable ε-polycaprolactone implants delivering the antiretroviral islatravir (ISL) and the contraceptive etonogestrel (ENG). Three implants were tested: ISL-62 mg, ISL-98 mg, and ENG-33 mg. Animals received one or two ISL-eluting implants, with doses of 42, 66, or 108 µg of ISL/day with or without an additional ENG-33 mg implant (31 µg/day). Drug release increased linearly with dose with median [range] plasma ISL levels of 1.3 [1.0-2.5], 1.9 [1.2-6.3] and 2.8 [2.3-11.6], respectively. The ISL-62 and 98 mg implants demonstrated stable drug release over three months with ISL-triphosphate (ISL-TP) concentr54ations in PBMCs above levels predicted to be efficacious for PrEP. Similarly, ENG implants demonstrated sustained drug release with median [range] plasma ENG levels of 495 [229-1110] pg/mL, which suppressed progesterone within two weeks and showed no evidence of altering ISL pharmacokinetics. Two of the six ISL-98 mg implants broke during the study and induced implant-site reactions, whereas no reactions were observed with intact implants. We show that ISL and ENG biodegradable implants are safe and yield sufficient drug levels to achieve prevention targets. The evaluation of optimized implants with increased mechanical robustness is underway for improved durability and vaginal efficacy in a SHIV challenge model.

Ultra-long-acting delivery platforms for HIV pre-exposure prophylaxis (PrEP) may increase adherence and maximize public health benefit. We report on an injectable, biodegradable, and removable in-situ forming implant (ISFI) that is administered subcutaneously and can release the integrase inhibitor cabotegravir (CAB) above protective benchmarks for more than 6 months. CAB ISFIs are well-tolerated in female mice and female macaques showing no signs of toxicity or chronic inflammation. In macaques, median plasma CAB concentrations exceed established PrEP protection benchmarks within 3 weeks and confer complete protection against repeated rectal SHIV challenges. Implant removal via a small incision in 2 macaques at week 12 results in a 7- to 48-fold decrease in plasma CAB levels within 72 hours. Modeling to translate CAB ISFI dosing suggests that a 3 mL injection would exceed protective benchmarks in humans for over 5 months post administration. Our results support the clinical advancement of CAB ISFIs for ultra-long-acting PrEP in humans.

BACKGROUND: Vaginal products for HIV prevention that can be used on-demand before or after sex may be a preferable option for women with low frequency or unplanned sexual activity or who prefer not to use daily or long-acting pre-exposure prophylaxis (PrEP). We performed dose ranging pharmacokinetics (PK) and efficacy studies of a vaginally applied insert containing tenofovir alafenamide fumarate (TAF) and elvitegravir (EVG) in macaques under PrEP or post-exposure prophylaxis (PEP) modalities. METHODS: PK studies were performed in 3 groups of pigtailed macaques receiving inserts with different fixed-dose combinations of TAF and EVG (10/8, 20/16 and 40/24 mg). PrEP and PEP efficacy of a selected insert was investigated in a repeat exposure vaginal SHIV transmission model. Inserts were administered 4 h before (n = 6) or after (n = 6) repeated weekly SHIV exposures. Infection outcome was compared with macaques receiving placebo inserts (n = 12). FINDINGS: Dose ranging studies showed rapid and sustained high drug concentrations in vaginal fluids and tissues across insert formulations with minimal dose proportionality. TAF/EVG (20/16 mg) inserts were selected for efficacy evaluation. Five of the 6 animals receiving these inserts 4 h before and 6/6 animals receiving inserts 4 h after SHIV exposure were protected after 13 challenges (p = 0.0088 and 0.0077 compared to placebo, respectively). The calculated PrEP and PEP efficacy was 91.0% (95% CI = 32.2%-98.8%) and 100% (95% CI = undefined), respectively. INTERPRETATION: Inserts containing TAF/EVG provided high protection against vaginal SHIV infection when administered within a 4 h window before or after SHIV exposure. Our results support the clinical development of TAF/EVG inserts for on-demand PrEP and PEP in women. FUNDING: Funded by CDC intramural funds, an interagency agreement between CDC and USAID (USAID/CDC IAA AID-GH-T-15-00002), and by the U.S. President's Emergency Plan for AIDS Relief (PEPFAR) through the U.S. Agency for International Development (USAID) under a Cooperative Agreement (AID-OAA-A-14-00010) with CONRAD/Eastern Virginia Medical School.

The administration of antiretrovirals (ARVs) for HIV pre-exposure prophylaxis (PrEP) is highly efficacious and may benefit from new long-acting (LA) drug delivery approaches. This paper describes a subcutaneous, reservoir-style implant for the LA delivery of tenofovir alafenamide (TAF) and documents the preclinical assessment of implant safety and pharmacokinetics (PK) in New Zealand White (NZW) rabbits (3 groups of n = 5), beagle dogs (2 groups of n = 6), and rhesus macaques (2 groups of n = 3). Placebo implants were placed in rabbits (n = 10) and dogs (n = 12). Implant parameters, including selection of the TAF form, choice of excipient, and PCL formulation were tuned to achieve targeted concentrations of the active anabolite of TAF, tenofovir diphosphate (TFV-DP), within peripheral blood mononuclear cells (PBMCs) and mucosal tissues relevant to HIV transmission. Sustained concentrations of TFV-DP in PBMCs over 100fmol/10(6) cells were achieved in all animal species indicating that the implants effectively delivered TAF for 3-6months. Unlike placebo implants without TAF, all active implants resulted in local adverse events (AEs) proximal to the implant ranging in severity from mild to moderate and included dermal inflammation and necrosis across all species. Despite these AEs, the implant performed as designed and achieved a constant drug release profile, supporting the continued development of this drug delivery platform.

OBJECTIVES: To advance the initiative of ending the global epidemic, long-lasting HIV protection is needed through sustained release of antiretroviral drugs for months to years. We investigated in macaques the safety and efficacy of biodegradable polycaprolactone implants releasing tenofovir alafenamide for HIV pre-exposure prophylaxis (PrEP). METHODS: Implants were administered subcutaneously in the arm using a contraceptive trocar. Efficacy against vaginal simian-HIV (SHIV) infection was investigated in six pigtailed macaques that received two tenofovir alafenamide implants (0.35 mg/day), one in each arm, for a total release rate of tenofovir alafenamide at 0.7 mg/day. Macaques were exposed to SHIV twice weekly for 6 weeks. Statistical analyses were used to compare outcome with eight untreated controls. Histological assessments were performed on skin biopsies collected near implantation sites. RESULTS: Median (range) tenofovir diphosphate level in PBMCs was 1519 (1068-1898) fmol/106 cells. All macaques with tenofovir alafenamide implants were protected against vaginal SHIV infection. In contrast, 7/8 controls were infected after a median of 4 SHIV exposures (P = 0.0047). Histological assessment of tissues near tenofovir alafenamide implant sites showed inflammation and necrosis in 5/6 animals, which were not evident by visual inspection. CONCLUSIONS: We demonstrated complete protection against vaginal SHIV infection with two implants releasing a total of 0.7 mg of tenofovir alafenamide per day. We also identified tenofovir diphosphate concentrations in PBMCs associated with complete vaginal protection. Consistent with previous findings, we observed adverse local toxicity and necrosis near the tenofovir alafenamide implant site. Improved tenofovir alafenamide implants that are safe and maintain high efficacy have the potential to provide long-lasting protection against vaginal HIV infection.

OBJECTIVES: We conducted a detailed pharmacokinetic assessment in macaques treated with vaginal gels formulated with HIV integrase strand transfer inhibitors (INSTIs) to better understand drug distribution and identify INSTI concentrations associated with previously demonstrated in vivo protection against vaginal simian HIV challenge. METHODS: Six macaques received vaginal gel containing 1% raltegravir (30 mg) once-weekly over 6 weeks. Following a washout period, five macaques received once-weekly gel containing 0.23% L-870,812 (7 mg). Drug concentrations were measured in plasma, mucosal fluids and vaginal tissues at baseline and 2, 5 and 24 h post-dosing. RESULTS: The median maximum concentration (Cmax) for raltegravir and L-870,812 in plasma was below the limit of quantification and 41.1 ng/mL, respectively. The Cmax in vaginal fluids (1441 and 1250 μg/mL) and tissues (266.7 and 368.4 μg/g) was achieved 2-5 h after dosing, respectively. A similar half-life was observed for raltegravir and L-870,812 in vaginal fluids (8-10 h) and remained 3-4 orders of magnitude above the protein-adjusted IC95 (0.016 and 0.106 μg/mL, respectively) at 24 h. Drug concentrations in vaginal fluids correlated well with those in vaginal tissues (Pearson r ≥ 0.788). Both drugs were consistently detected in rectal fluids 2 h after vaginal dosing, albeit at much lower levels (31-92-fold) than those in vaginal fluids. CONCLUSIONS: To the best of our knowledge, this study provides the first data on INSTI levels in vaginal tissues associated with in vivo protection and demonstrates rectal drug distribution of INSTIs after vaginal dosing. These findings may inform dose selection for topical products with INSTIs for HIV prevention.

Receptive anal intercourse (RAI) contributes significantly to HIV acquisition underscoring the need to develop HIV prevention options for populations engaging in RAI practices. We explored the feasibility of formulating rectal suppositories with potent antiviral drugs for on-demand use. A fixed-dose combination of tenofovir (TFV) and elvitegravir (EVG) (40 mg each) was co-formulated in six different suppository bases (three fat-and three water-soluble). Fat-soluble witepsol H15 and water-soluble polyethylene glycol (PEG) based suppositories demonstrated favorable in vitro release and were advanced to assess in vivo pharmacokinetics following rectal administration in macaques. In vivo drug release profiles were similar for both suppository bases. Median concentrations of TFV and EVG detected in rectal fluids at 2 h were 1-and 2-logs higher than the in vitro IC50, respectively; TFV-diphosphate levels in rectal tissues met or exceeded those associated with high efficacy against rectal simian HIV (SHIV) exposure in macaques. Leveraging on these findings, a PEG-based suppository with a lower dose combination of tenofovir alafenamide (TAF) and EVG (8 mg each) was developed and found to achieve similar rectal drug exposures in macaques. This study establishes the utility of rectal suppositories as a promising on-demand strategy for HIV PrEP and supports their clinical development. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

The ability to successfully develop a safe and effective vaccine for the prevention of HIV infection has proven challenging. Consequently, alternative approaches to HIV infection prevention have been pursued, and there have been a number of successes with differing levels of efficacy. Currently, only two oral pre-exposure prophylaxis (PrEP) products are available, Truvada and Descovy. Descovy is a newer product not yet indicated in individuals at risk of HIV-1 infection from receptive vaginal sex, since it still needs to be evaluated in this population. A topical dapivirine vaginal ring is currently under regulatory review, and a long acting (LA) injectable cabotegravir product shows strong promise. Although demonstrably effective, daily oral PrEP presents adherence challenges for many users, particularly adolescent girls and young women, key target populations. This limitation has triggered development efforts in LA HIV prevention options. This article reviews efforts supported by the Bill & Melinda Gates Foundation, as well as similar work by other groups, to identify and develop optimal LA HIV prevention products. Specifically, this article is a summary review of a meeting convened by the foundation in early 2020 that focused on the development of LA products designed for extended delivery of tenofovir alafenamide (TAF) for HIV prevention. The review broadly serves as technical guidance for preclinical development of LA HIV prevention products. The meeting examined the technical feasibility of multiple delivery technologies, in vivo pharmacokinetics, and safety of subcutaneous delivery of TAF in animal models. Ultimately, the foundation concluded that there are technologies available for long-term delivery of TAF. However, due to potentially limited efficacy and possible toxicity issues with subcutaneous (SC) delivery, the foundation will not continue investing in the development of LA, SC delivery of TAF products for HIV prevention.

Prevention of HIV infection and unintended pregnancies are public health priorities. In sub-Saharan Africa, where HIV prevalence is highest, depot-medroxyprogesterone acetate (DMPA) is widely used as contraception. Therefore, understanding potential interactions between DMPA and antiretrovirals is critical. Here, we use a macaque model to investigate the effect of DMPA on the pharmacology of the antiretroviral tenofovir alafenamide (TAF). Female rhesus macaques received 30 mg of DMPA (n=9) or were untreated (n=9). Macaques received a human equivalent dose of TAF (1.5 mg/kg) orally by gavage. Tenofovir (TFV) and TFV-diphosphate (TFV-DP) were measured in blood, secretions, and tissues over 72 hours. The median area under the curve (AUC(0-72h)) values for TFV-DP in peripheral blood mononuclear cells were similar in DMPA-treated (6,991 fmol*h/10(6) cells) and untreated controls (5,256 fmol*h/10(6) cells) (P=0.174). Rectal tissue TFV-DP concentrations from DMPA+ animals [median: 20.23 fmol/mg of tissue (range: 4.94-107.95)] were higher than the DMPA-group [median: below the limit of quantification (BLOQ-11.92)], (P=0.019). TFV-DP was not detectable in vaginal tissue from either group. A high-dose DMPA treatment in macaques was associated with increased rectal TFV-DP levels, indicating a potential tissue-specific drug-drug interaction. The lack of detectable TFV-DP in the vaginal tissue warrants further investigation of PrEP efficacy with single-agent TAF products. DMPA did not affect systemic TAF metabolism, with similar PBMC TFV-DP in both groups, suggesting that DMPA use should not alter the antiviral activity of TAF.

We used a novel penile simian HIV (SHIV) transmission model to investigate if cabotegravir long-acting (CAB LA) prevents penile SHIV acquisition in macaques. Twenty-two macaques were exposed to SHIV via the foreskin and urethra once-weekly for 12 weeks. Of these, six received human-equivalent doses of CAB LA, six received oral FTC/TDF, and 10 were untreated. The efficacy of CAB LA was high (94.4% [95%CI=58.2%-99.3%]) and similar to that seen with oral FTC/TDF (94.0% [95%CI=55.1%-99.2%]). The high efficacy of CAB LA in the penile transmission model supports extending the clinical advancement of CAB LA PrEP to heterosexual men.

Vaginal microbicides containing antiretrovirals (ARV) have shown to prevent vaginally acquired HIV but these products may not protect women who engage in anal sex. Intravaginal dosing with ARVs have shown to result in drug exposures in rectal tissues, thus raising the possibility of dual compartment protection. To test this concept, we investigated whether intravaginal dosing with emtricitabine (FTC)-tenofovir (TFV) gel, which fully protected macaques against repeated vaginal exposures to simian human immunodeficiency virus (SHIV), protects against rectal SHIV exposures. Pharmacokinetic (PK) studies revealed rapid distribution of FTC and TFV to rectal tissues and luminal fluids, albeit at concentrations 1-2 log10 lower than those in the vaginal compartment. Efficacy measurements against repeated rectal SHIV challenges demonstrated a 4.5-fold reduction in risk of infection in macaques that received intravaginal FTC/TFV compared to placebo gel (p=0.047; log-rank test). These data support the concept of dual compartment protection by vaginal dosing and warrants developing ARV-based vaginal products with improved bidirectional dosing.

Penile acquisition of HIV infection contributes substantially to the global epidemic. Our goal was to establish a preclinical macaque model of penile HIV infection for evaluating the efficacy of new HIV prevention modalities. Rhesus macaques were challenged once or twice weekly with consistent doses of SHIVsf162P3 (a chimeric simian-human immunodeficiency virus containing HIV env) ranging from 4-600 TCID50 (50% tissue culture infective dose), via two penile routes, until systemic SHIV infection was confirmed. One route exposed the inner foreskin, glans and urethral os to virus following deposition into the prepuce (foreskin) pouch. The second route introduced the virus non-traumatically into the distal urethra only. Single-route challenges resulted in dose-dependent rates of SHIV acquisition informing selection of optimal SHIV dosing. Concurrent SHIV challenges via the prepuce pouch (200 TCID50) and urethra (16 TCID50) resulted in infection of 100% (10/10) animals following a median of 2.5 virus exposures (range, 1-12). We describe the first rhesus macaque repeat-exposure SHIV challenge model of penile HIV acquisition. Utilization of the model should further our understanding of penile HIV infection and facilitate the development of new HIV prevention strategies for men.

BACKGROUND: Chlamydia trachomatis (CT) and Trichomonas vaginalis (TV), two prevalent sexual transmitted infections, are known to increase HIV risk in women and could potentially diminish pre-exposure prophylaxis (PrEP) efficacy, particularly for topical interventions that rely on local protection. We investigated in macaques whether co-infection with CT/TV reduces protection by vaginal TFV gel. METHODS: Vaginal TFV gel dosing previously shown to provide 100% or 74% protection when applied either 30 minutes or 3 days before SHIV challenge was assessed in pigtailed macaques co-infected with CT/TV and challenged twice-weekly with SHIV162p3 for up to 10 weeks (2 menstrual cycles). Three groups of six macaques received either placebo or 1% TFV gel 30 minutes or 3 days before each SHIV challenge. We additionally assessed TFV and TFV-diphosphate (TFV-DP) concentrations in plasma and vaginal tissues in CT/TV co-infected (n = 4) and uninfected (n = 4) macaques. RESULTS: CT/TV co-infections were maintained during the SHIV challenge period. All macaques that received placebo gel were SHIV-infected after a median of 7 challenges (1 menstrual cycle). In contrast, no infections were observed in macaques treated with TFV gel 30 minutes before SHIV challenge (p < 0.001). Efficacy was reduced to 60% when TFV gel was applied 3 days before SHIV challenge (p = 0.07). Plasma TFV and TFV-DP concentrations in tissues and vaginal lymphocytes were significantly higher in CT/TV co-infected compared to CT/TV uninfected macaques. CONCLUSIONS: Our findings in this model suggest that CT/TV co-infection may have little or no impact on the efficacy of highly effective topical TFV modalities and highlight a significant modulation of TFV pharmacokinetics.

BACKGROUND: An established macaque model to assess HIV interventions against penile transmission is currently not available. Physiological changes during penile erections may affect susceptibility to infection and drug pharmacokinetics (PK). Here, we identify methods to establish erections in macaques to evaluate penile transmission, PK, and efficacy under physiologic conditions. METHODS: Penile rigidity and length were evaluated in eight rhesus macaques following rectal electrostimulation (RES), vibratory stimulation (VS), or pharmacological treatment with Sildenafil Citrate (Viagra) or Alprostadil. RESULTS: Rectal electrostimulation treatment increased penile rigidity (>82%) and length (2.5 +/- 0.58 cm), albeit the response was transient. In contrast, VS alone or coupled with Viagra or Alprostadil failed to elicit an erection response. CONCLUSION: Rectal electrostimulation treatment elicits transient but consistent penile erections in macaques. High rigidity following RES treatment demonstrates increased blood flow and may provide a functional model for penile PK evaluations and possibly simian immunodeficiency virus (SIV) transmission under erect conditions.

BACKGROUND: Topically delivered tenofovir (TFV) from intravaginal rings, tablets, or gels is being evaluated for HIV prevention. We previously demonstrated that TFV delivered vaginally by gel protected macaques from vaginal infection with SHIV. Here we investigated efficacy of the TFV gel against vaginal transmission of a TFV-resistant SHIV containing the K65R mutation (SHIV162P3K65R) and its relationship to drug levels in vaginal tissues. RESULTS: SHIV162P3K65R shows approximately a 5-fold reduction in susceptibility to TFV compared to wild-type SHIV. Efficacy was evaluated in pig-tailed macaques exposed vaginally twice-weekly (up to 10 weeks) to SHIV162P3K65R 30 min after receiving placebo (n = 6) or 1% TFV (n = 6) gel. Four of the six controls were infected after a median of 5 exposures. In contrast, five of six macaques that received TFV gel remained uninfected after 20 vaginal SHIV162P3K65R exposures, resulting in an estimated efficacy of 75%. The mean intracellular TFV-diphosphate (TFV-DP) concentrations in vaginal lymphocytes 4 h after a single gel dose were found to be high (1,631 fmol/10(6) cells, range 492-3,847) and within the in vitro IC75 range (1,206 fmol/10(6) cells) for SHIV162P3K65R. CONCLUSION: Both the modest resistance conferred by K65R and the high TFV-DP exposure in vaginal lymphocytes, likely explain the observed protection. The findings in this model do not predict complete loss of protection by topical TFV against vaginal exposure to HIV-1K65R viruses and provide a tissue drug target for high efficacy. These data will facilitate the development of TFV delivery platforms that have high activity on both wild-type and TFV-resistant viruses.

BACKGROUND: Vaginal SHIVSF162P3 acquisition in pigtail macaques (Macaca nemestrina) is dependent on time point during the menstrual cycle. Susceptibility is higher around menstruation and lower at ovulation in mid cycle. This complicates design of repeat low-dose (RLD) SHIV exposure studies because virus challenges given during low susceptibility periods have lower chances to infect. To account for fluctuating susceptibility, we analyzed menstrual cycles rather than exposures until infection following virus challenges. FINDINGS: We first re-analyzed infection data of 41 macaques receiving placebo or no treatment during once (n=18) or twice (n=23) weekly virus exposures. The same number of cycles was required for infection with either challenge frequency, while it took a median 4 or 6 challenges for once or twice weekly exposures, respectively. More virus challenges to infection likely reflect frequent unsuccessful exposures in frequently exposed animals. When re-analyzing two previously reported biomedical HIV intervention studies, we found 1% Tenofovir gel was 74 or 86 % efficacious based on cycles or exposures (p = 0.019 or = 0.003, respectively, Fisher's exact test), while 1% Raltegravir gel was 84 or 89 % efficacious, respectively (p = 0.047 or = 0.031). CONCLUSIONS: Evaluating number of menstrual cycles rather than exposures until infection can account for varying susceptibility during the menstrual cycle. Our observations have implications for future study designs such as planning frequency of virus exposures. Menstrual cycle analysis may also avoid potential over-estimation of efficacy against vaginal challenges during low susceptibility periods in the cycle that are unlikely to cause infection.

BACKGROUND: Rectal HIV transmission is an important driver of the HIV epidemic. Optimally formulated gels of antiretroviral drugs are under development for preventing rectally acquired HIV. We investigated in a macaque model the pharmacokinetics and efficacy of three rectal gel formulations METHODS: Single-dose pharmacokinetics of low-osmolar 1% maraviroc (MVC), 1% tenofovir (TFV), or 1%MVC/1%TFV combination gel were evaluated in blood, rectal fluids, colorectal biopsies, and rectal lymphocytes. Efficacy was evaluated over 10 twice-weekly rectal SHIV162p3 challenges in rhesus macaques that received either placebo (n=7), MVC (n=6), TFV (n=6), or MVC/TFV (n=6) gel 30 minutes before each challenge. RESULTS: MVC and TFV were detected in plasma 30 minutes after gel application and remained above IC95 levels in rectal fluids at 24h. MVC, TFV, and TFV-DP concentrations in colorectal tissues collected up to 30 cm from the anal margin were all high at 2h, demonstrating rapid and extended tissue dosing. TFV-DP concentrations in tissue homogenates and rectal lymphocytes were highly correlated (r2=0.82). All three gel formulations were highly protective (82% efficacy; log-rank test; p≤0.02). CONCLUSION: Desirable pharmacokinetic profiles and high efficacy in this macaque model support the clinical development of these gel formulations for preventing rectal HIV infection.

BACKGROUND: Pre-Exposure Prophylaxis (PrEP) for HIV prevention is a novel biomedical prevention method. We have previously modeled PrEP during rectal SHIV exposures in macaques and identified that SHIV-specific T cell responses were induced in the presence of antiretroviral drugs, an observation previously termed T cell chemo-vaccination. This report expands those initial findings by examining a larger group of macaques that were given oral or topical PrEP during repeated vaginal virus exposure. METHODS: Thirty-six female pigtail macaques received up to 20 repeat low-dose vaginal inoculations with wild type (WT) SHIVSF162P3 (n=24) or a clonal derivative with the tenofovir K65R drug resistant mutation (n=12). PrEP consisted of oral Truvada (n=6, WT), tenofovir vaginal gel (n=6, K65R), or tenofovir intra-vaginal ring (n=6, WT). The remaining animals were PrEP-inexperienced controls (n=12, WT; n=6, K65R). SHIV-specific T cells were identified and characterized using IFNgamma ELISPOT and multi-parameter flow cytometry. RESULTS: Of nine animals that were on PrEP and remained uninfected during WT SHIV vaginal challenges, eight (88.9%) developed virus-specific T cell responses. T cells were in CD4 and CD8 compartments, reached up to 4,900 IFNgamma producing cells per million PBMCs, and primarily pol directed. In contrast, the replication impaired K65R virus did not induce detectable T cell responses, likely reflecting the need for adequate replication. CONCLUSION: Virus-specific T cell responses occur frequently in oral or topical PrEP-protected pigtail macaques after vaginal exposure to WT SHIV virus. The contribution of such immune responses to protection from infection during and following PrEP warrants further investigation.

BACKGROUND AND METHODS: Cell isolation from macaque tissues involves laborious enzymatic digestion. The Medimachine provides a simpler, quicker nonenzymatic method, yielding 1.5-5 million cells/g of vaginal or rectal tissue from pigtailed macaques. RESULTS AND CONCLUSIONS: Flow cytometry analysis of the two methods revealed similar levels of cell viability and most major cell lineage and activation markers.

The repeat low-dose virus challenge model is commonly used in nonhuman primate studies of HIV transmission and biomedical preventions. For some viruses or challenge routes, it is uncertain whether the repeated exposure design might induce virus-directed innate or adaptive immunity that could affect infection or viremic outcomes. Retrospective cohorts of male Indian rhesus (n=40) and female pigtail (n=46) macaques enrolled in repeat low-dose rectal or vaginal SHIVSF162p3 challenge studies, respectively, were studied to compare the relationship between the number of previous exposures and peak plasma SHIV RNA levels or viral load area under the curve (AUC), surrogate markers of viral control. Repeated mucosal exposures of 10 or 50 TCID50 of virus for rectal and vaginal exposures, respectively, were performed. Virus levels were measured by quantitative reverse-transcriptase real-time PCR. The cumulative number of SHIVSF162p3 exposures did not correlate with observed peak virus levels or with AUC in rectally challenged rhesus macaques [peak: rho (rho)=0.04, p=0.8; AUC: rho=0.33, p=0.06] or vaginally challenged pigtail macaques (peak: rho=-0.09, p=0.7; AUC: rho=0.11, p=0.6). Infections in these models occur independently of exposure history and provide assurance that neither inoculation route nor number of exposures required for infection correlates with postinfection viremia. These data also indicate that both the vaginal and rectal repeated low-dose virus exposure models using SHIVSF162p3 provide a reliable system for nonhuman primate studies.

BACKGROUND: Hormonal changes during menstrual cycling may affect susceptibility to HIV. METHODS: We determined the simian human immunodeficiency virus (SHIV) acquisition time point in 43 cycling pigtail macaques infected by repeated vaginal virus exposures initiated randomly in the cycle. RESULTS: SHIV infection was first detected in the follicular phase in 38 macaques (88%), and in the luteal phase in five macaques (12%), indicating a statistically significant timing difference. Assuming a 7-day eclipse phase, most infections occurred during or following a high-progesterone period associated with menstruation, vaginal epithelium thinning, and suppressed mucosal immunity. CONCLUSIONS: This raises questions whether other high-progesterone conditions (pregnancy, hormonal contraception) similarly affect HIV risk.

Coitally delivered microbicide gels containing antiretroviral drugs are important for HIV prevention. However, to date, microbicides have contained entry or reverse transcriptase inhibitors that block early steps in virus infection and thus need to be given as a preexposure dose that interferes with sexual practices and may limit compliance. Integrase inhibitors block late steps after virus infection and therefore are more suitable for post-coital dosing. We first determined the kinetics of strand transfer in vitro and confirmed that integration begins about 6 hours after infection. We then used a repeat-challenge macaque model to assess efficacy of vaginal gels containing integrase strand transfer inhibitors when applied before or after simian/human immunodeficiency virus (SHIV) challenge. We showed that gel containing the strand transfer inhibitor L-870812 protected two of three macaques when applied 30 min before SHIV challenge. We next evaluated the efficacy of 1% raltegravir gel and demonstrated its ability to protect macaques when applied 3 hours after SHIV exposure (five of six protected; P < 0.05, Fisher's exact test). Breakthrough infections showed no evidence of drug resistance in plasma or vaginal secretions despite continued gel dosing after infection. We documented rapid vaginal absorption reflecting a short pharmacological lag time and noted that vaginal, but not plasma, virus load was substantially reduced in the breakthrough infection after raltegravir gel treatment. We provide a proof of concept that topically applied integrase inhibitors protect against vaginal SHIV infection when administered shortly before or 3 hours after virus exposure.

A vaginal gel containing 1% tenofovir (TFV) was found to be safe and effective in reducing HIV infection in women when used pericoitally. Because of the long intracellular half-life of TFV and high drug exposure in vaginal tissues, we hypothesized that a vaginal gel containing TFV may provide long-lasting protection. Here, we performed delayed-challenge experiments and showed that vaginal 1% TFV gel protected 4/6 macaques against vaginal simian-human immunodeficiency virus (SHIV) exposures occurring 3 days after gel application, demonstrating long-lasting protection. Despite continued gel dosing postinfection, neither breakthrough infection had evidence of drug resistance by ultrasensitive testing of SHIV in plasma and vaginal lavage. Analysis of the active intracellular tenofovir diphosphate (TFV-DP) in vaginal lymphocytes collected 4 h to 3 days after gel dosing persistently showed high TFV-DP levels (median, 1,810 fmol/10(6) cells) between 4 and 24 h that exceed the 95% inhibitory concentration (IC(95)), reflecting rapid accumulation and long persistence. In contrast to those in peripheral blood mononuclear cells (PBMCs) following oral dosing, TFV-DP levels in vaginal lymphocytes decreased approximately 7-fold by 3 days, exhibiting a much higher rate of decay. We observed a strong correlation between intracellular TFV-DP in vaginal lymphocytes, in vitro antiviral activity, and in vivo protection, suggesting that TFV-DP above the in vitro IC(95) in vaginal lymphocytes is a good predictor of high efficacy. Data from this model reveal an extended window of protection by TFV gel that supports coitus-independent use. The identification of protective TFV-DP concentrations in vaginal lymphocytes may facilitate the evaluation of improved delivery methods of topical TFV and inform clinical studies.

Daily pre-exposure prophylaxis (PrEP) with Truvada (FTC and TDF) is a novel HIV prevention strategy recently found to reduce HIV incidence among men who have sex with men. We used a macaque model of HIV transmission to investigate if Truvada maintains prophylactic efficacy against an FTC-resistant isolate containing the M184V mutation. Five macaques received a dose of Truvada 3 days before exposing them rectally to SHIV162p3(M184V) followed by a second dose 2h afterwards. Five untreated animals were used as controls. Virus exposures were done weekly for up to 14 weeks. Despite the high (>100-fold) level of FTC resistance conferred by M184V, all five treated animals were protected from infection while the five untreated macaques were infected (p=0.0008). Our results show that Truvada maintains high prophylactic efficacy against an FTC-resistant isolate. Increased susceptibility to tenofovir due to M184V and other factors including residual antiviral activity by FTC and/or reduced virus fitness due to M184V may have all contributed to the observed protection.

Fluctuations in susceptibility to HIV or SHIV during the menstrual cycle are currently not fully documented. To address this, time point of infection was determined in 19 adult female pigtail macaques vaginally challenged during their undisturbed menstrual cycles with repeated, low-dose SHIVSF162P3 exposures. Eighteen macaques (95%) first displayed viremia in the follicular phase, as compared to 1 macaque (5%) in the luteal phase (p<0.0001). Due to a viral eclipse phase, we estimated a window of most frequent virus transmission between days 24-31 of the menstrual cycle, in the late luteal phase. Thus, susceptibility to vaginal SHIV infection is significantly elevated in the second half of the menstrual cycle when progesterone levels are high, and when local immunity may be low. Such susceptibility windows have been postulated before but not definitively documented. Our data support findings of higher susceptibility to HIV in women during progesterone-dominated periods including pregnancy and contraceptive use.

Transmission of drug-resistant HIV has been widely documented. We generated tenofovir (TFV)- and emtricitabine (FTC)-resistant SHIV162P3 mutants that can be used to investigate the transmission efficiency of drug-resistant viruses and their impact on the efficacy of pre-exposure prophylaxis. Both SHIV162p3(M184V) and SHIV162p3(K65R) replicated in vitro at high titers. Drug resistance profiles were similar to those seen in HIV. Virus infectivity to virion particle ratios were 4- and 10-fold lower in SHIV162p3(M184V) and SHIV162p3(K65R), compared to a concurrently generated WT SHIV162p3, respectively. Mucosal transmissibility studies using a repeat low-dose macaque model of rectal and vaginal transmission showed that both mutants were able to efficiently infect macaques only after the dose was increased to adjust for fitness reductions due to K65R and M184V. Our results in limited number of macaques suggest that the reduction in fitness due to M184V and K65R decreases virus transmissibility, and identify in vitro infectivity parameters that associate with mucosal transmissibility.

After membrane fusion with a target cell, the core of human immunodeficiency virus type 1 (HIV-1) enters into the cytoplasm, where uncoating occurs. The cone-shaped core is composed of the viral capsid protein (CA), which disassembles during uncoating. The underlying factors and mechanisms governing uncoating are poorly understood. Several CA mutations can cause changes in core stability and a block at reverse transcription, demonstrating the requirement for optimal core stability during viral replication. HIV-1 integrase (IN) catalyzes the insertion of the viral cDNA into the host genome, and certain IN mutations are pleiotropic. Similar to some CA mutants, two IN mutants, one with a complete deletion of IN (NL-DeltaIN) and the other with a Cys-to-Ser substitution (NL-C130S), were noninfectious, with a replication block at reverse transcription. Compared to the wild type (WT), the cytoplasmic CA levels of the IN mutants in infected cells were reduced, suggesting accelerated uncoating. The role of IN during uncoating was examined by isolating and characterizing cores from NL-DeltaIN and NL-C130S. Both IN mutants could form functional cores, but the core yield and stability were decreased. Also, virion incorporation of cyclophilin A (CypA), a cellular peptidyl-prolyl isomerase that binds specifically to CA, was decreased in the IN mutants. Cores isolated from WT virus depleted of CypA had an unstable-core phenotype, confirming a role of CypA in promoting optimal core stability. Taken together, our results indicate that IN is required during uncoating for maintaining CypA-CA interaction, which promotes optimal stability of the viral core.