Date Published: August 5, 2008
Publisher: Public Library of Science
Author(s): Martin Cranage, Sally Sharpe, Carolina Herrera, Alethea Cope, Mike Dennis, Neil Berry, Claire Ham, Jonathan Heeney, Naser Rezk, Angela Kashuba, Peter Anton, Ian McGowan, Robin Shattock, Florian Hladik
Abstract: BackgroundThe rectum is particularly vulnerable to HIV transmission having only a single protective layer of columnar epithelium overlying tissue rich in activated lymphoid cells; thus, unprotected anal intercourse in both women and men carries a higher risk of infection than other sexual routes. In the absence of effective prophylactic vaccines, increasing attention is being given to the use of microbicides and preventative antiretroviral (ARV) drugs. To prevent mucosal transmission of HIV, a microbicide/ARV should ideally act locally at and near the virus portal of entry. As part of an integrated rectal microbicide development programme, we have evaluated rectal application of the nucleotide reverse transcriptase (RT) inhibitor tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy) propyl] adenine monohydrate), a drug licensed for therapeutic use, for protective efficacy against rectal challenge with simian immunodeficiency virus (SIV) in a well-established and standardised macaque model.Methods and FindingsA total of 20 purpose-bred Indian rhesus macaques were used to evaluate the protective efficacy of topical tenofovir. Nine animals received 1% tenofovir gel per rectum up to 2 h prior to virus challenge, four macaques received placebo gel, and four macaques remained untreated. In addition, three macaques were given tenofovir gel 2 h after virus challenge. Following intrarectal instillation of 20 median rectal infectious doses (MID50) of a noncloned, virulent stock of SIVmac251/32H, all animals were analysed for virus infection, by virus isolation from peripheral blood mononuclear cells (PBMC), quantitative proviral DNA load in PBMC, plasma viral RNA (vRNA) load by sensitive quantitative competitive (qc) RT-PCR, and presence of SIV-specific serum antibodies by ELISA. We report here a significant protective effect (p = 0.003; Fisher exact probability test) wherein eight of nine macaques given tenofovir per rectum up to 2 h prior to virus challenge were protected from infection (n = 6) or had modified virus outcomes (n = 2), while all untreated macaques and three of four macaques given placebo gel were infected, as were two of three animals receiving tenofovir gel after challenge. Moreover, analysis of lymphoid tissues post mortem failed to reveal sequestration of SIV in the protected animals. We found a strong positive association between the concentration of tenofovir in the plasma 15 min after rectal application of gel and the degree of protection in the six animals challenged with virus at this time point. Moreover, colorectal explants from non-SIV challenged tenofovir-treated macaques were resistant to infection ex vivo, whereas no inhibition was seen in explants from the small intestine. Tissue-specific inhibition of infection was associated with the intracellular detection of tenofovir. Intriguingly, in the absence of seroconversion, Gag-specific gamma interferon (IFN-γ)-secreting T cells were detected in the blood of four of seven protected animals tested, with frequencies ranging from 144 spot forming cells (SFC)/106 PBMC to 261 spot forming cells (SFC)/106 PBMC.ConclusionsThese results indicate that colorectal pretreatment with ARV drugs, such as tenofovir, has potential as a clinically relevant strategy for the prevention of HIV transmission. We conclude that plasma tenofovir concentration measured 15 min after rectal administration may serve as a surrogate indicator of protective efficacy. This may prove to be useful in the design of clinical studies. Furthermore, in vitro intestinal explants served as a model for drug distribution in vivo and susceptibility to virus infection. The finding of T cell priming following exposure to virus in the absence of overt infection is provocative. Further studies would reveal if a combined modality microbicide and vaccination strategy is feasible by determining the full extent of local immune responses induced and their protective potential.
Partial Text: The development of an effective vaccine against HIV is still thought to be a long-term endeavour; meanwhile the HIV pandemic continues relentlessly, fuelled primarily by sexual transmission. The relative ease by which HIV is transmitted rectally [1–4] makes this a particularly important, although relatively neglected, route to target with prevention strategies. Unlike the vagina, the rectal canal has only a single layer of columnar epithelium overlying tissue rich in activated lymphoid cells [5,6] and therefore for reasons of both anatomy and immunological status presents a particular challenge for preventative modalities. It is difficult to estimate the prevalence of anal intercourse (AI) in the heterosexual population, but recent studies have indicated that it may be far more common than had been thought. In a population-based study of 2,547 Northern Californian women aged 18–29 y, AI was reported in 21.7% of sexually active individuals . Higher rates of AI have been described in women at particular risk of HIV infection through drug use, forced intercourse, and prostitution [8–11]. Data from Africa are very limited, but levels in excess of 40% incidence have been described in commercial sex workers in South Africa [12,13]. These figures alone demonstrate the need for the development of rectal microbicides, a focus catalyzed by the more visible need of the population of men who have sex with men, which still accounts for the majority of new infections in North America, South America, and Europe. Moreover, many of these men who have sex with men are having unprotected AI irrespective of their HIV status . In the absence of vaccines, increasing effort is being applied to the parallel approach of preventative microbicides/pre-exposure use of antiretrovirals (ARVs). To date preclinical studies of tenofovir in the macaque rectal challenge model using either simian immunodeficiency virus (SIV) or recombinant chimeric simian HIV (SHIV) have focussed on the use of the orally bio-available prodrug tenofovir disoproxil fumarate (TDF) [15,16]. Although partial protection was observed against multiple low dose mucosal virus challenge, it appeared that the dose of available drug may have been suboptimal. We have taken a different approach, reasoning that the optimal route to protect cells of the rectum and possibly beyond may be through local application of tenofovir given, in effect, as a rectal microbicide. As well as testing this hypothesis, we sought to determine if protective efficacy correlated with drug uptake and whether exposure to virus in the absence of overt infection stimulated virus-specific T cell responses.