Prevention of SIV Rectal Transmission and Priming of T Cell Responses in Macaques after Local Pre-exposure Application of Tenofovir Gel

Abstract

Background

The 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 Findings

A 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 (MID ₅₀) of a noncloned, virulent stock of SIV _mac251/32H, 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)/10 ⁶ PBMC to 261 spot forming cells (SFC)/10 ⁶ PBMC.

Conclusions

These 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.

Abstract

Martin Cranage and colleagues find that topical tenofovir gel can protect against rectal challenge with SIV in a macaque model, and can permit the induction of SIV-specific T cell responses.

Editors' Summary

Background.

About 33 million people are now infected with the human immunodeficiency virus (HIV), which causes AIDS by killing immune system cells. As yet, there is no cure for AIDS, although HIV infections can be held in check with antiretroviral drugs. Also, despite years of research, there is no vaccine available that effectively protects people against HIV infection. So, to halt the AIDS epidemic, other ways of preventing the spread of HIV are being sought. For example, pre-exposure treatment (prophylaxis) with antiretroviral drugs is being investigated as a way to prevent HIV transmission. In addition, because HIV is often spread through heterosexual penile-to-vaginal sex with an infected partner, several vaginal microbicides (compounds that protect against HIV when applied inside the vagina) are being developed, some of which contain antiretroviral drugs.

Why Was This Study Done?

Because HIV can cross the membranes that line the mouth and the rectum (the lower end of the large intestine that connects to the anus) in addition to the membrane that lines the vagina, HIV transmission can also occur during oral and anal sex. The lining of the rectum in particular is extremely thin and overlies tissues rich in activated T cells (the immune system cells that HIV targets), so unprotected anal intercourse carries a high risk of HIV infection. Anal intercourse is common among men who have sex with men but is also more common in heterosexual populations than is generally thought. Tenofovir (an antiretroviral drug that counteracts HIV after it has entered human cells) given by mouth partly protects macaques against rectal infection with simian immunodeficiency virus (SIV; a virus that induces AIDS in monkeys and apes) so the researchers wanted to know whether this drug might be effective against rectal SIV infection if applied at the site where the virus enters the body.

What Did the Researchers Do and Find?

To answer this question, the researchers rectally infected several macaques with SIV up to 2 h after rectal application of a gel containing tenofovir, after rectal application of a gel not containing the drug, or after no treatment. In addition, a few animals were treated with the tenofovir gel after the viral challenge. Most of the animals given the tenofovir gel before the viral challenge were partly or totally protected from SIV infection, whereas all the untreated animals and most of those treated with the placebo gel or with the drug-containing gel after the viral challenge became infected with SIV. High blood levels of tenofovir 15 min after its rectal application correlated with protection from viral infection. The researchers also collected rectal and small intestine samples from tenofovir-treated macaques that had not been exposed to SIV and asked which samples were resistant to SIV infection in laboratory dishes. They found that only the rectal samples were resistant to infection and only rectal cells contained tenofovir. Finally, activated T cells that recognized an SIV protein were present in the blood of some of the animals that were protected from SIV infection by the tenofovir gel.

What Do These Findings Mean?

These findings, although based on experiments in only a few animals, suggest that rectal treatment with antiretroviral drugs before rectal exposure to HIV might prevent rectal HIV transmission in people. However, results from animal experiments do not always reflect what happens in people. Indeed, clinical trials of a potential vaginal microbicide that worked well in macaques were halted recently because women using the microbicide had higher rates of HIV infection than those using a control preparation. The finding that immune-system activation can occur in the absence of overt infection in animals treated with the tenofovir gel additionally suggests that a combination of a local antiretroviral/microbicide and vaccination might be a particularly effective way to prevent HIV transmission. However, because HIV targets activated T cells, viral rechallenge experiments must be done to check that the activated T cells induced by the virus in the presence of tenofovir do not increase the likelihood of infection upon re-exposure to HIV before this potential microbicide is tried in people.

Additional Information

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050157.

• Read the accompanying PLoS Medicine Perspective by Florian Hladik

• An overview of HIV infection and AIDS is available from the US National Institute of Allergy and Infectious Diseases

• HIVInSite has comprehensive information on all aspects of HIV/AIDS, including an article on safer sex, which includes information on the risks associated with specific types of sex and on microbicides and other methods to prevent the sexual transmission of HIV

• Information on all aspects of HIV/AIDS is available from Avert, an international AIDS charity, including information on HIV prevention and on microbicides

• The World Health Organization has a fact sheet on microbicides

• The UK charity NAM also provides detailed information on microbicides

• PrEP Watch is a comprehensive information source on pre-exposure prophylaxis for HIV prevention

• Global Campaign for Microbicides is an international coalition of organisations dedicated to accelerating access to new HIV prevention options