Ron A. Otten

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.

Efficacy of Postexposure Prophylaxis after Intravaginal Exposure of Pig-Tailed Macaques to a Human-Derived Retrovirus (Human Immunodeficiency Virus Type 2)

ABSTRACT Postexposure prophylaxis (PEP) after intravaginal exposure to human immunodeficiency virus (HIV) was investigated using the HIV type 2 (HIV-2)/pig-tailed macaque transmission model. PEP for 28 days with the reverse transcriptase inhibitor (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA; tenofovir) was initiated 12 to 72 h following HIV-2 exposure. Systemic infection was not evident in the 12- and 36-h groups, as defined by plasma viremia, cell-associated provirus, antibody responses, and lymph node virus. Breakthrough infection in the 72-h group was detected at week 16 post-virus exposure. These results demonstrate for the first time using a vaginal transmission model that early intervention after high-risk sexual exposures may prevent infection.

Complete Protection from Repeated Vaginal Simian-Human Immunodeficiency Virus Exposures in Macaques by a Topical Gel Containing Tenofovir Alone or with Emtricitabine

ABSTRACT 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.

Intermittent Prophylaxis with Oral Truvada Protects Macaques from Rectal SHIV Infection

Treating monkeys with single doses of an antiretroviral drug before and after exposure to SHIV provides protection against infection, a schedule that may prove practical in humans. Rearranging Retroviral Regimens for HIV Antiretroviral drugs have transformed the lives of HIV-infected people by preventing progression to full-blown AIDS. These drugs also dramatically reduce HIV transmission from mothers to infants during pregnancy and breastfeeding, and work in monkeys suggests that daily doses can also reduce transmission from unprotected sex. But prophylactic treatment with antiretroviral drugs is costly and impractical—even if confined to a high-risk population. García-Lerma et al. now show that in monkeys a more realistic medication schedule may work just as well as daily doses. To simulate how people are likely to be infected with HIV, the authors exposed macaque monkeys rectally to 14 weekly doses of simian-human immunodeficiency virus (SHIV) engineered to resemble the human virus. Control macaques treated in this way became infected within the first five exposures to SHIV. Researchers then assessed whether oral, human-equivalent doses of antiretroviral agents could prevent infection in monkeys. The best protection—equivalent to that provided by daily antivirals—occurred when the drug Truvada was given 1, 3, or 7 days before virus exposure followed by a second dose 2 hours after exposure. Less effective, but still better than no treatment at all, was a schedule in which the drug was given 2 hours before or after exposure and then again 24 hours later. Drugs given only 24 or 48 hours after exposure did not safeguard against infection. The results of this study are preliminary, largely because each of the groups had only six macaques, but they are nevertheless promising. If ongoing clinical trials in healthy people show that daily antiretroviral therapy can diminish the chances of acquiring HIV after exposure, a reasonable next step would be to evaluate more practical, less costly drug schedules in humans. For example, a weekly dose followed by a second dose after a possible exposure could prove both effective and tractable. It will also be important to evaluate treatments based solely on exposure, as these would not require ongoing prophylactic drug treatment and would minimize any drug toxicity. If one or more of these therapeutic regimens is successful, antiretroviral drugs may expand the transformation they have already engendered by preventing many more new infections as well as controlling existing ones. 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.

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.

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 SHIVSF162P3 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.

A molecular epidemiologic survey of HIV in Uganda.

Objective:Previous data, based on a small sampling of convenience, reported subtypes A, B, C, D, and G in Uganda, but neither the extent nor the proportion of these subtypes could be evaluated. To establish correctly the prevalence and distribution of HIV-1 subtypes, we analysed viral clades in 739 HIV-1-seropositive specimens from different areas of Uganda. Methods:Blood specimens from 1100 patients were collected in five districts of Uganda. Within this collection, 929 HIV-1-seroreactive samples underwent analysis of viral DNA, and 739 were selected for further subtyping in env or pol regions. Results:Using a combination of subtype A- and D-specific probes to C2–V3 region and DNA sequencing, HIV-1 env subtypes were determined in 594 specimens: 341 were of subtype A (57.4%), 250 of subtype D (42.1%), and three of subtype C (0.5%). Sixty-two samples showed reactivity with both probes, suggesting potential mixed infections, cross-reactivity to probes, or possibly other subtypes. Subsequent sequence analysis of 19 randomly selected specimens revealed subtypes A (n = 4), D (n = 12), and C (n = 3). Sequence analysis of the 27 samples chosen from the remaining 83 samples, which could be amplified only with viral gp41 or protease gene primers, classified them as subtypes A (n = 13) and D (n = 14). No significant clinical, demographic, or geographic differences were found between HIV-1 infections with viruses of subtypes A and D, despite considerable genetic diversity within these clades. Conclusions:This is the first major population-based study of the prevalent HIV-1 strains in an African country selected for vaccine trials. The subtyping methods we describe should be of use to investigators seeking to conduct large-scale screening for HIV variants in other populations.

Identification of a Window Period for Susceptibility to Dual Infection with Two Distinct Human Immunodeficiency Virus Type 2 Isolates in a Macaca nemestrina (Pig-tailed Macaque) Model

The potential to establish dual retroviral infections was investigated in this study. Groups of macaques infected with human immunodeficiency virus type 2 (HIV-2) isolate (either GB122 or CDC77618) were exposed to the other virus at 2, 4, 8, 12, 14, or 72 weeks after primary inoculation. Dual infections were established in macaques simultaneously exposed to both viruses. In other groups, secondary infections were observed only if challenge occurred at early intervals after primary infection but before a full seroconversion. Polymerase chain reaction and virus-isolation data demonstrated that challenges at 8, 12, 14, or 72 weeks after infection with the initial isolate failed to result in a dual infection. Anti-HIV-2 serologic titers, CD4 levels, virus burden, and the ability to superinfect peripheral blood mononuclear cells in vitro were not correlated with susceptibility to or protection from secondary challenges in this investigation. These findings demonstrate a window period for susceptibility to dual infection and indicate that protection from retroviral infection may be achievable.

Safe and Sustained Vaginal Delivery of Pyrimidinedione HIV-1 Inhibitors from Polyurethane Intravaginal Rings

ABSTRACT 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% [PYD10.5wt%], PYD11wt%, PYD24wt%, and PYD214wt%) 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 PYD214wt% 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.

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.