Safe and sustained vaginal delivery of pyrimidinedione HIV-1 inhibitors from polyurethane intravaginal rings.

The potent antiretroviral pyrimidinediones IQP-0528 (PYD1) and IQP-0532 (PYD2) were formulated in polyurethane intravaginal rings (IVRs) as prophylactic drug delivery systems to prevent the sexual transmission of HIV-1. To aid in the selection of a pyrimidinedione candidate and the optimal loading of the drug in the IVR delivery system, four pyrimidinedione IVR formulations (PYD1 at 0.5 wt% [PYD1(0.5 wt%)], PYD1(1 wt%), PYD2(4 wt%), and PYD2(14 wt%)) were evaluated in pigtail macaques over 28 days for safety and pyrimidinedione vaginal biodistribution. Kinetic analysis of vaginal proinflammatory cytokines, native microflora, and drug levels suggested that all formulations were safe, but only the high-loaded PYD2(14 wt%) IVR demonstrated consistently high pyrimidinedione vaginal fluid and tissue levels over the 28-day study. This formulation delivered drug in excess of 10 µg/ml to vaginal fluid and 1 µg/g to vaginal tissue, a level over 1,000 times the in vitro 50% effective concentration. The in vitro release of PYD1 and PYD2 under nonsink conditions correlated well with in vivo release, both in amount and in kinetic profile, and therefore may serve as a more biologically relevant means of evaluating release in vitro than typically employed sink conditions. Lastly, the pyrimidinediones in the IVR formulation were chemically stable after 90 days of storage at elevated temperature, and the potent nanomolar-level antiviral activity of both molecules was retained after in vitro release. Altogether, these results point to the successful IVR formulation and vaginal biodistribution of the pyrimidinediones and demonstrate the usefulness of the pigtail macaque model in evaluating and screening antiretroviral IVR formulations prior to preclinical and clinical evaluation.

Complete protection from repeated vaginal simian-human immunodeficiency virus exposures in macaques by a topical gel containing tenofovir alone or with emtricitabine.

New-generation gels that deliver potent antiretroviral drugs against human immunodeficiency virus type 1 have renewed hopes for topical prophylaxis as a prevention strategy. Previous preclinical research with monkey models suggested that high concentrations and drug combinations are needed for high efficacy. We evaluated two long-acting reverse transcriptase inhibitors, tenofovir (TFV) and emtricitabine (FTC), by using a twice-weekly repeat challenge macaque model and showed that a preexposure vaginal application of gel with 1% TFV alone or in combination with 5% FTC fully protected macaques from a total of 20 exposures to simian-human immunodeficiency virus SF162p3. FTC and TFV were detected in plasma 30 min after vaginal application, suggesting rapid absorption. FTC was detected more frequently than TFV and showed higher levels, reflecting the fivefold-higher concentration of this drug than of TFV. Two of 12 repeatedly exposed but protected macaques showed limited T-cell priming, which did not induce resistance to infection when macaques were rechallenged. Thus, single drugs with durable antiviral activity can provide highly effective topical prophylaxis and overcome the need for noncoital use or for drug combinations which are more complex and costly to formulate and approve.

Generation of a dual RT Env SHIV that is infectious in rhesus macaques.

BACKGROUND: The best current animal model for HIV infection and evaluation of antiviral compounds is the Simian-human immunodeficiency virus (SHIV)/macaque system. There are multiple recombinant SHIVs available, but these viruses have limitations in evaluating combination drug strategies for prevention. Drug combinations that target reverse transcriptase (RT, either nRTI or nnRTI) and envelope (entry or fusion inhibitors) have to be tested separately, which does not permit the assessment of additive, synergistic, or antagonistic effects of ARV combinations. We describe construction of a dual SHIV containing both HIV RT and a CCR5-specific HIV envelope gene in a simian immunodeficiency virus backbone. METHODS: The RT Env SHIV molecular clone was constructed using RT SHIV and SHIV162p3 sequences as templates to generate RT Env SHIV. RT Env SHIV was expanded in vitro in CD8-depleted macaque peripheral blood mononuclear cells (PBMC). Recombinant virus was used to infect a rhesus macaque (4.3 x 10(4) tissue culture infectious dose [TCID(50)], intravenously [IV]). A second passage in a macaque by IV transfer of 10 ml of blood obtained from the first infection was also done. The in vivo adapted virus stock from these macaques was used to produce high titer stocks in vitro and used to rectally infect an additional macaque. RESULTS: Peak viral load reached 6 x 10(5) vRNA copies/ml in plasma in both IV-exposed macaques and remained detectable in the one animal for 16 weeks after infection. A viral stock (1.68 x 10(4) TCID(50)) derived from the second macaque passage has been produced in CD8-depleted rhesus PBMC and was successfully used to demonstrate mucosal transmission. The resulting RT Env SHIV retained the sensitivity to HIV RT and entry inhibitors of its parental viruses. CONCLUSIONS: The objective of this study was to develop and characterize a SHIV recombinant virus for evaluating the efficacy of ART and microbicide products that target both HIV RT and/or Env-mediated entry. RT Env SHIV can productively infect macaques by both the IV and mucosal route, making it a valuable tool for transmission studies.

Evaluation of the lymphocyte trafficking drug FTY720 in SHIVSF162P3-infected rhesus macaques.

OBJECTIVES: FTY720 causes retention of lymphocytes in lymphatic tissues. Previous studies revealed that FTY720 can decrease or eliminate chronic viral infections of mice. We address here whether therapeutic use of FTY720 in simian human immunodeficiency virus (SHIV)-infected rhesus macaques could also decrease viraemia. METHODS: FTY720 was administered intravenously to three SHIV(SF162P3)-infected macaques at 39, 7 or 6 weeks of infection; three control macaques (47, 48 or 6 weeks of infection) did not receive drug. FTY720 was given at 0.004 mg/kg on days 0, 1, 2, 14, 15 and 16, followed by 0.1 mg/kg on days 28, 29, 30, 42, 43 and 44. Blood was collected seven times throughout and four times during 47 days of follow-up. RESULTS: Only the 0.1 mg/kg dose resulted in a reduction in mean blood CD4+ T cells and B cells (to 33% and 27% of pre-drug levels, P=0.0024 and 0.003, respectively). FTY720 treatment did not lead to significant deviations from the natural pattern of viral control. Plasma viraemia progressed from a range of 10(4)-10(2) copies/mL before treatment to 10(4)-temporarily undetectable levels on the last day of treatment. SHIV(SF162P3) was not eliminated, however, as plasma viraemia and proviral DNA persisted during the follow-up. No significant alterations in T cell activity were noted throughout the drug course. CONCLUSIONS: FTY720 administration had no detectable therapeutic effect at the doses and schedules outlined here, although blood CD4+ T cells and B cells were effectively reduced. Future work might reveal whether FTY720 could be beneficial in more pathogenic SHIV, simian immunodeficiency virus or HIV infections.

Development and evaluation of a vaginal ring device for sustained delivery of HIV microbicides to non-human primates.

BACKGROUND: There is considerable interest in developing coitally independent, sustained release formulations for long-term administration of HIV microbicides. Vaginal ring devices are at the forefront of this formulation strategy. METHODS: Non-medicated silicone elastomer vaginal rings were prepared having a range of appropriate dimensions for testing vaginal fit in pig-tailed and Chinese rhesus macaques. Cervicovaginal proinflammatory markers were evaluated. Compression testing was performed to compare the relative flexibility of various macaque and commercial human rings. RESULTS: All rings remained in place during the study period and no tissue irritation or significant induction of cervicovaginal proinflammatory markers or signs of physical discomfort were observed during the 8-week study period. CONCLUSIONS: Qualitative evaluation suggests that the 25 x 5-mm ring provided optimal fit in both macaque species. Based on the results presented here, low-consistency silicone elastomers do not cause irritation in macaques and are proposed as suitable materials for the manufacture of microbicide-loaded vaginal rings.

Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir.

BACKGROUND: In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission. METHODS AND FINDINGS: We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected. CONCLUSIONS: This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.

Lack of in vitro-in vivo correlation for a UC781-releasing vaginal ring in macaques.

This study describes the preclinical development of a matrix-type silicone elastomer vaginal ring device designed to provide controlled release of UC781, a non-nucleoside reverse transcriptase inhibitor. Testing of both human- and macaque-sized rings in a sink condition in vitro release model demonstrated continuous UC781 release in quantities considered sufficient to maintain vaginal fluid concentrations at levels 82-860-fold higher than the in vitro IC50 (2.0 to 10.4 nM) and therefore potentially protect against mucosal transmission of HIV. The 100-mg UC781 rings were well tolerated in pig-tailed macaques, did not induce local inflammation as determined by cytokine analysis and maintained median concentrations in vaginal fluids of UC781 in the range of 0.27 to 5.18 mM during the course of the 28-day study. Analysis of residual UC781 content in rings after completion of both the in vitro release and macaque pharmacokinetic studies revealed that 57 and 5 mg of UC781 was released, respectively. The pharmacokinetic analysis of a 100-mg UC781 vaginal ring in pig-tailed macaques showed poor in vivo-in vitro correlation, attributed to the very poor solubility of UC781 in vaginal fluid and resulting in a dissolution-controlled drug release mechanism rather than the expected diffusion-controlled mechanism.

Chronic HIV-2 infection protects against total CD4+ cell depletion and rapid disease progression induced by SHIV89.6p challenge.

OBJECTIVE: To better understand HIV-1 sexual transmission risk, we have studied the susceptibility of HIV-2-exposed, uninfected (EU) female pig-tailed macaques to intravaginal (IVAG) re-challenge with the homologous HIV-2 strain, followed by heterologous SHIV89.6p. METHODS: Nine female macaques, previously protected by a post-exposure prophylaxis (PEP) regimen, along with one mock-treated EU animal, were re-exposed to HIV-2 by the IVAG route approximately 1.5 years later. A single follow-up challenge was performed approximately 1 year later with SHIV89.6p to assess susceptibility of chronic HIV-2-infected animals to further re-infection and pathogenic effects with a heterologous virus, somewhat mimicking HIV-1. RESULTS: Eight of ten macaques (80%) became infected systemically with HIV-2, and plasma or cervicovaginal vRNA levels did not appreciably differ from prior historic non-PEP control macaques. Interestingly, all eight HIV-2-infected females were susceptible to SHIV89.6p infection by either intravenous (n = 4) or IVAG exposure (n = 4) after one inoculation. Plasma vRNA levels in these groups were controlled by week 8 and there were no decrease in CD4+ T cells > 50%. The remaining two HIV-2 EU macaques, inoculated intrarectally with SHIV89.6p, were unable to control virus replication and succumbed to disease by week 25 or week 61. CONCLUSIONS: Our findings demonstrate that successful PEP regimens to prevent an initial infection do not have any lasting protective effects. The observed lack of cross-protection against SHIV89.6p transmission among chronic HIV-2-infected macaques provides modeling support for limited epidemiologic data indicating that human HIV-2 infection does not protect against HIV-1 infection, but may serve to alter overt clinical outcome.

Antiretroviral therapy in HIV-2-infected patients: changes in plasma viral load, CD4+ cell counts, and drug resistance profiles of patients treated in Abidjan, Côte d'Ivoire.

OBJECTIVE: To describe changes in plasma viral load, CD4+ cell counts, and drug resistance profiles of HIV-2-infected patients receiving antiretroviral (ARV) therapy in Abidjan, Côte d'Ivoire. METHODS: Consecutive blood samples were collected from 18 HIV-2-infected ARV-naive patients who had received ARV therapy in the UNAIDS drug access initiative (UNAIDS-DAI) in Abidjan between August 1998 and July 2000. Changes in HIV-2 plasma viral load, CD4+ cell counts, and genotypic and phenotypic drug resistance testing were determined. RESULTS: At baseline, 11 (61%) of the 18 patients initiated highly active antiretroviral therapy (HAART) and seven (39%) received dual therapy. No significant change in median viral load was observed at 2 months (P = 0.09), at 6 months (P = 0.06), and at 12 months of therapy (P = 0.26). No significant increase in CD4+ cell counts was observed at 12 months (P = 0.10). All four patients on indinavir-containing HAART had undetectable viral loads at 2-4 months of therapy. However, none of seven patients on nelfinavir-containing HAART had a substantial decrease in viral load. Viruses from 14 patients were analyzed, 12 of which (86%) had at least one primary resistance mutation that is known to confer resistance to HIV-1 virus. Three patients had the multi-drug-resistant mutation, Q151M, two of whom showed reduced susceptibility to zidovudine, didanosine, stavudine and zalcitabine. CONCLUSION: Our limited findings show that nelfinavir-containing regimens may have limited virologic benefit to HIV-2-infected patients.

High susceptibility to repeated, low-dose, vaginal SHIV exposure late in the luteal phase of the menstrual cycle of pigtail macaques.

Fluctuations in susceptibility to HIV or SHIV during the menstrual cycle are currently not fully documented. To address this, the time point of infection was determined in 19 adult female pigtail macaques vaginally challenged during their undisturbed menstrual cycles with repeated, low-dose SHIV(SF162P3) exposures. Eighteen macaques (95%) first displayed viremia in the follicular phase, as compared with 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 and 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 the findings of higher susceptibility to HIV in women during progesterone-dominated periods including pregnancy and contraceptive use.

T cell chemo-vaccination effects after repeated mucosal SHIV exposures and oral pre-exposure prophylaxis.

Pre-exposure prophylaxis (PrEP) with anti-viral drugs is currently in clinical trials for the prevention of HIV infection. Induction of adaptive immune responses to virus exposures during anti-viral drug administration, i.e., a "chemo-vaccination" effect, could contribute to PrEP efficacy. To study possible chemo-vaccination, we monitored humoral and cellular immune responses in nine rhesus macaques undergoing up to 14 weekly, low-dose SHIV(SF162P3) rectal exposures. Six macaques concurrently received PrEP with intermittent, oral Truvada; three were no-PrEP controls. PrEP protected 4 macaques from infection. Two of the four showed evidence of chemo-vaccination, because they developed anti-SHIV CD4(+) and CD8(+) T cells; SHIV-specific antibodies were not detected. Control macaques showed no anti-SHIV immune responses before infection. Chemo-vaccination-induced T cell responses were robust (up to 3,940 SFU/10(6) PBMCs), predominantly central memory cells, short-lived (≤22 weeks), and appeared intermittently and with changing specificities. The two chemo-vaccinated macaques were virus-challenged again after 28 weeks of rest, after T cell responses had waned. One macaque was not protected from infection. The other macaque concurrently received additional PrEP. It remained uninfected and T cell responses were boosted during the additional virus exposures. In summary, we document and characterize PrEP-induced T cell chemo-vaccination. Although not protective after subsiding in one macaque, chemo-vaccination-induced T cells warrant more comprehensive analysis during peak responses for their ability to prevent or to control infections after additional exposures. Our findings highlight the importance of monitoring these responses in clinical PrEP trials and suggest that a combination of vaccines and PrEP potentially might enhance efficacy.

Resistance to Simian HIV infection is associated with high plasma interleukin-8, RANTES and Eotaxin in a macaque model of repeated virus challenges.

Animal models for research on susceptibility to HIV are currently not available. Here we explore whether a macaque model of repeated low-dose rectal or vaginal virus challenges could be employed. We tested the hypothesis that susceptibility to Simian HIV is not merely stochastic in this model but rather is associated with identifiable host factors. Forty macaques required a median of 3.5 SHIVSF162P3 challenges for infection. We studied the association of their susceptibility with 13 predisposing plasma cytokines/chemokines (RANTES, Eotaxin, monocyte chemoattractant protein (MCP)-1, IL-7, MIP-1beta, TNF-alpha, MIP-1alpha, granulocyte colony-stimulating factor, IL-8, interferon-gamma, IL-17, IL-1beta, IL-6). Higher plasma RANTES, IL-8, and Eotaxin were associated with lower susceptibility, that is, higher resistance to infection. In a group of macaques with low IL-8 and RANTES, a median 3 exposures were required to infect; whereas, when either IL-8 or RANTES were high, a median 12 exposures were required. Thus, susceptibility was associated with identifiable discrete host factors and was not stochastic. In addition, the macaque model identified key human resistance factors (RANTES, Eotaxin), but also revealed a novel association with resistance (IL-8). Future direct evaluation of these or other factors in the animal model may be beneficial for developing new immunomodulation strategies for HIV prevention.

Intermittent prophylaxis with oral truvada protects macaques from rectal SHIV infection.

HIV continues to spread globally, mainly through sexual contact. Despite advances in treatment and care, preventing transmission with vaccines or microbicides has proven difficult. A promising strategy to avoid transmission is prophylactic treatment with antiretroviral drugs before exposure to HIV. Clinical trials evaluating the efficacy of daily treatment with the reverse transcriptase inhibitors tenofovir disoproxil fumarate (TDF) or Truvada (TDF plus emtricitabine) are under way. We hypothesized that intermittent prophylactic treatment with long-acting antiviral drugs would be as effective as daily dosing in blocking the earliest stages of viral replication and preventing mucosal transmission. We tested this hypothesis by intermittently giving prophylactic Truvada to macaque monkeys and then exposing them rectally to simian-human immunodeficiency virus (SHIV) once a week for 14 weeks. A simple regimen with an oral dose of Truvada given 1, 3, or 7 days before exposure followed by a second dose 2 hours after exposure was as protective as daily drug administration, possibly because of the long intracellular persistence of the drugs. In addition, a two-dose regimen initiated 2 hours before or after virus exposure was effective, and full protection was obtained by doubling the Truvada concentration in both doses. We saw no protection if the first dose was delayed until 24 hours after exposure, underscoring the importance of blocking initial replication in the mucosa. Our results show that intermittent prophylactic treatment with an antiviral drug can be highly effective in preventing SHIV infection, with a wide window of protection. They strengthen the possibility of developing feasible, cost-effective strategies to prevent HIV transmission in humans.

Repeated rectal SHIVSF162P3 exposures do not consistently induce sustained T cell responses prior to systemic infection in the repeat-low dose preclinical macaque model.

The macaque model of repeated SHIV exposures is increasingly used as a preclinical tool to evaluate biomedical HIV intervention strategies. It is unclear whether multiple virus exposures induce immune responses in macaques, as documented in uninfected individuals repeatedly exposed to HIV. We here address whether repeated, rectal SHIV(SF162P3) exposures lead to systemic T cell activation in 12 rhesus macaques, and whether this is associated with increased infection resistance. Eight macaques became systemically infected after 2-7 exposures, three macaques were less susceptible (infection after 10-12 exposures), and one macaque remained uninfected after 14 exposures. PBMCs were retrospectively monitored for increases in T cell activation by analyzing the proportion of CD8(+) T cells, recently activated or proliferated T cells (markers CD38, Ki67), a marker for cytotoxicity (granzyme B), or T cell-produced plasma cytokines (IFN-gamma, RANTES, IL-2). Repeated virus exposures did not induce sustained, potent, or diverse T cell responses prior to systemic infection. Some changes occurred in the analyzed parameters during repeated virus exposures, but similar T cell activities were also observed in five SHIV-unexposed control macaques. Thus, we found no evidence that delayed infection or resistance to infection was associated with systemic, long-lasting, protective T cell responses to repeated rectal virus exposures. Our results provide further insights into the repeat exposure macaque model. We find that this model can be used for testing biomedical prevention strategies without concern of eliciting a systemic vaccination effect.

Systemic and mucosal immunological responses during repeated mucosal SHIV(162P3) challenges prior to and following infection in pigtailed macaques.

Local and systemic immunological changes following vaginal HIV-1 exposures are poorly characterized and may influence susceptibility to infection. Therefore, we examined longitudinal mucosal, plasma cytokine profiles and viral-specific T-cell responses (vSTRs) before and during weekly repeated low-dose SHIV(SF162P3) viral challenges in six female pigtailed macaques, even in the absence of overt systemic infection. Following a single viral challenge, induction of several cytokines was detected consistently in cervico-vaginal lavages (CVL). With additional exposure and documented systemic infection, a hallmark of response profile was defined as peak levels in both CVL (MCP-1, MIP-1alpha, TNF-alpha, IL-1beta, IL-1RA and IL-8) and plasma cytokines (MCP-1, eotaxin and IL-1RA) in the macaques. In the periphery, vSTRs were observed within the first one or two viral challenges, but prior to the detection of systemic infection in 5/6 exposed pigtailed macaques. These findings provide valuable information regarding mucosal HIV-1 infection that may benefit microbicide research and development.

Short communication: no evidence of occult SHIV infection as demonstrated by CD8+ cell depletion after chemoprophylaxis-induced protection from mucosal infection in rhesus macaques.

Preexposure prophylaxis (PrEP) with antiretroviral drugs constitutes a promising strategy for HIV prevention. Potent PrEP regimens with reverse transcriptase inhibitors can prevent detectable SHIV infection in a repeated low-dose macaque model that resembles human transmission, supporting plans to quickly move this approach into human trials. However, the possibility remains that extremely low levels of virus replication could nonetheless occur during PrEP and seed viral reservoirs in tissues. Therefore, seemingly protected macaques may harbor occult virus that may be initially contained by cytotoxic T cells, but could emerge later. To explore this possibility, we studied whether CD8(+) cells suppress viremia in four rhesus macaques apparently protected by daily or intermittent Truvada (FTC and tenofovir) during 14 low-dose, rectal SHIV(SF162P3) challenges and during a subsequent drug washout period. CD8(+) cells were efficiently ablated with antibodies in these and two additional control macaques that were previously infected but had reached undetectable virus set points. During 4 weeks of follow-up, all four macaques remained free of plasma viremia and provirus in blood lymphocytes. In contrast, plasma viremia resurged to 10(6) to 10(7) copies per milliliter within 2 weeks in both control macaques. Thus, these results indicate that the undetectable viremia in the PrEP-protected macaques was not due to CD8(+) cells that were containing a low-level infection. Rather, the PrEP treatment created conditions in which infection was prevented, eliminated, or controlled by unknown mechanisms. These data provide important information for PrEP usage to prevent HIV transmission, and fully support the continued pursuit of PrEP prevention measures in humans.

Direct stringency comparison of two macaque models (single-high vs. repeat-low) for mucosal HIV transmission using an identical anti-HIV chemoprophylaxis intervention.

BACKGROUND: In our previous work, oral chemoprophylaxis with tenofovir disoproxil fumarate (TDF) provided partial protection in rhesus macaques against repeated low-dose (RL) intrarectal SHIV162p3 exposure. METHODS: Here, we make a direct comparison of these previous findings with data generated using a single high (SH)-dose challenge strategy. RESULTS: All 5 (100%) control macaques were infected after a SH challenge and only three of five (60%) TDF-treated macaques became infected. The remaining two TDF-treated macaques remained virus-negative and were susceptible to virus infection upon re-challenge in the absence of oral TDF. Thus, two of five (40%) TDF-treated macaques were protected by the pre-exposure chemoprophylaxis regimen. By comparison with the RL challenge system, only one of four (25%) of TDF-treated macaques were protected from infection, whereas four of four (100%) control macaques became infected using RL challenges. CONCLUSION: Taken together, these findings indicate that the stringency of the RL challenge model for testing antiretroviral interventions is not lower and possibly greater than that of the SH challenge model.

Repetitive exposures with simian/human immunodeficiency viruses: strategy to study HIV pre-clinical interventions in non-human primates.

BACKGROUND: Non-human primate models for human immunodeficiency virus (HIV) infection represent a valuable pre-clinical tool to evaluate interventions (e.g., topical microbicides, vaccines, and chemoprophylaxis) designed to prevent transmission or slow disease progression after infection. Standard transmission models use a single-dose exposure with high, non-physiologic levels of virus to approach 100% infection rates of control animals. These single-exposure models do not represent the circumstances of mucosal HIV transmission in humans and may result in misleading data with regard to intervention efficacy. Therefore, we have developed a repetitive mucosal exposure model using doses of virus that better reflects human exposures. METHODS: The virus used for these evaluations was simian-human immunodeficiency virus [SHIVSF162P3 (R5-using, subtype B HIV-1 envelope)] and the virus dose used (approximately 10(5)-10(6) viral particle equivalents or approximately 10 tissue culture infectious doses per exposure) approximates viral loads observed in the semen during acute HIV-1 infection. Using the repeated mucosal exposure approach, we have evaluated a candidate vaginal microbicide (cellulose acetate phthalate, CAP) given 15 minutes prior to each weekly virus exposure. Pig-tailed macaques were exposed weekly by vaginal inoculations with and without microbicide until systemic viral RNA was detected. RESULTS: Groups of naïve control monkeys were infected after an average of three to four exposures for the vaginal route of inoculation. Data from the first application of this monkey model to evaluate the topical microbicide CAP suggested that protection from SHIV infection was possible with three of four CAP-treated monkeys remaining uninfected after 12 exposures (P = 0.015). CAP efficacy was markedly improved from 66% in a previous single-dose virus exposure study to 92% in this repeated exposure system. CONCLUSIONS: Our experience with using repetitive virus exposures to study topical microbicides and the findings to date from this study provides a basis to refine monkey models to more closely resemble human exposure during HIV transmission. This model may be highly relevant to pre-clinical evaluation for a variety of therapeutic interventions which is discussed here.

Multiple vaginal exposures to low doses of R5 simian-human immunodeficiency virus: strategy to study HIV preclinical interventions in nonhuman primates.

A nonhuman-primate model of human immunodeficiency virus type 1 (HIV-1) infection that more closely emulates human heterosexual transmission by use of multiple exposures to low doses of virus is critical to better evaluate intervention strategies that include microbicides or vaccines. In this report, we describe such a system that uses female pig-tailed macaques exposed vaginally to a CCR5-using simian-human immunodeficiency virus (SHIV(SF162P3)) at weekly intervals. Results of dose-titration experiments indicated that 3 once-weekly exposures to 10 tissue culture infectious doses of SHIV(SF162P3) resulted in consistent transmission of virus and establishment of systemic infection. The efficacy of cellulose acetate phthalate (CAP) as a vaginal microbicide was evaluated by applying it to the vaginal vault of macaques (n = 4) 15 min before each weekly exposure to SHIV(SF162P3). One conclusion that can be drawn from the data derived from multiple exposures to virus is that CAP prevented infection in 12 of 13 possible chances for infection, over the course of 39 total exposures. Our findings provide a basis to refine monkey models for transmission of HIV-1, which may be relevant to preclinical evaluation for therapeutic interventions.