MC, SS, and RS designed the experiments; SS and MD were responsible for the in vivo work; C. Herrera performed the explant experiments; AC performed the ELISpot experiments; NB, C. Ham, and JH performed the quantitative PCR analyses; NR and AK were responsible for the assay of tenofovir; PA coordinated the overall Microbicide Development Programme; IMcG had oversight of the preclinical testing project; and MC wrote the manuscript.
¤ Current address: Magee Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
The authors declare no competing financial interests.
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.
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 (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 (
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.
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.
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.
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.
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.
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.
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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 [
A well-established macaque rectal challenge model using uncloned SIVmac251/32H; a virus that results in high cell-associated and plasma viral RNA (vRNA) loads shortly after a single application to naïve macaques [
Assignment of Macaques to Experimental Groups
All macaques were challenged with 20 median rectal infectious doses MID50 of SIVmac 251/32H 11/88 stock equivalent to 18,974 50% tissue culture doses (TCID50) as determined by previous titration [
Two other SIV-naïve rhesus macaques were used to obtain gut tissues for explant studies of tenofovir antiviral activity in vitro (see below). A further four SIV-naïve macaques were dosed with tenofovir gel as described above 3 h prior to necropsy, and gut tissues used for ex vivo analysis of intracellular drug concentration and viral inhibitory activity (
At each time point of sampling, 2 × 106 peripheral blood mononuclear cells (PBMC) were cocultured with C8166 cells essentially as described previously [
Plasma vRNA concentrations were determined by quantitative competitive (qc) real time reverse transcriptase (RT)-PCR with a cut-off sensitivity of 40 RNA equivalents/ml [
Levels of proviral SIV
Plasma samples were assayed for tenofovir concentration by the Clinical Pharmacology and Analytical Chemistry Core of the University of North Carolina Center for AIDS Research. Drug concentrations in plasma were determined by a validated high pressure, liquid chromatography (HPLC) method with ultraviolet detection [
Freshly isolated tissue-derived MNC [
Full-length, replication and infection competent proviral HIV-1 clones pYU2 [
TZM-bl cells, a CXCR4-positive HeLa cell clone engineered to express CD4 and CCR5 and to contain integrated reporter genes for firefly luciferase and
Colorectum and ileum–jejenum explants were prepared and cultivated at an air-media interface as described previously for human tissue [
The significance of the protection data was determined using the two-tailed Fisher exact probability test by manual calculation. Drug activity titration curves were compared by cross-sectional time series analysis using Stata v10 (Stata Corp).
Having shown that tenofovir had no evidence of toxicity on TZM-bl cells, as assessed by MTT viability assay at the highest dose tested (unpublished data), we initially compared the activity of tenofovir, formulated as a solution from PMPA powder or formulated as a gel, as supplied by Gilead Sciences, against a panel of HIV-1 isolates with alternative secondary receptor usage (CXCR4-using RF, IIIB, and NL4.3; CCR5-using BaL, YU.2, and R8BaL) and against SIVmac251/32H; the latter being used for the macaque challenge experiments. There was no evidence of any difference in the mean average level of inhibition of SIVmac251/32H replication with either formulation at any dose (
Infection of TZM-bl indicator cells in the presence and absence of tenofovir was compared by luminescence analysis of cell lysates and the results expressed as percent inhibition. The graph shows the full titration of drug formulations on infection with SIVmac251/32H; the virus stock used in subsequent challenge experiments in vivo. Each point represents the mean of three independent experiments performed in triplicate +/− standard deviation. The results for a panel of HIV-1 strains in comparison to SIVmac251/32H are shown as IC50 values in the inset.
We used a 1% tenofovir gel formulation made and supplied by Gilead Sciences as part of a programme on the development of a vaginal microbicide. Although this formulation had not been optimised for rectal use, we considered that it would provide a suitable starting point for preclinical evaluation. No adverse effects were seen following rectal gel administration. Virological analysis over a 20-wk period after rectal challenge with 20 MID50 showed that four of six animals in group A (tenofovir 15 min before challenge) and two of three animals in group D (tenofovir 2 h before challenge) were protected from systemic infection on the basis of failure to (a) recover virus from PBMC, (b) to detect proviral DNA in PBMC, (c) to detect vRNA in plasma, and (d) to detect SIV-specific antibodies in serum. All the naïve/not-dosed animals and three of four animals receiving placebo gel were infected and had high levels of circulating vRNA, and proviral DNA and virus was recoverable from the PBMC at all times of testing (
The results of VI from PBMC are shown as + or − for each animal. The temporal profiles of plasma vRNA concentration (red dot) and frequency of PMBC-associated proviral DNA (blue triangle) are shown for each animal in the study.
Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for “Complete” Protection (i.e., No Virus Detection) Associated with Tenofovir Gel Administered Intrarectally Prior to Virus Challenge;
Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for Modified Outcome (i.e., “Complete” Protection or Reduced Frequency of Virus Detection) Associated with Tenofovir Gel Administered Intrarectally Prior to Virus Challenge;
Contingency Table for Dosing and Outcome of Virus Challenge: Analysis for “Complete” Protection, Including Historical Controls Challenged with the Same Virus Stock Intrarectally, Associated with Tenofovir Gel Administered Intrarectally Prior to Virus Challenge;
Of the eight animals that remained VI/PCR negative, seven were clinically normal throughout the study, and one, D68, which had received placebo gel, whilst clinically normal at necropsy had enlarged axillary and inguinal nodes at week 12. In contrast, all of the animals in which virus was recovered at high frequency had some clinical signs and/or necropsy findings consistent with SIV infection, such as enlarged iliac, axillary and inguinal lymph nodes, and splenomegaly. One animal, D83, had evidence of progression to AIDS including loss of weight, tenting of skin, lung abnormalities including, grey discolouration, numerous petechial haemorrhages on the surface, and adhesions to the thoracic wall. Animal D43, which had been given tenofovir 15 min prior to virus challenge and showed only a weak and transient viraemia, remained clinically normal throughout the study.
To determine if virus was sequestered in tissue associated with the virus challenge, quantitative proviral PCR was used to examine MNC isolated from rectum and ileum, as well as iliac, inguinal, and mesenteric lymph nodes. In the apparently protected animals, there was no evidence of infection whereas in animal E81, a naïve challenged control, proviral loads of 26, 5, 130, 2,100, and 105 proviral DNA copies/105 MNC equivalents were detected in each tissue respectively.
To determine if mucosal exposure to virus in the absence of overt infection had stimulated T cell immunity, SIV-specific IFN-γ ELISpot analysis was performed on PBMC from protected animals and one infected animal (E81) taken 20 wk after challenge. Four of seven protected animals had IFN-γ-secreting Gag-specific T cells at frequencies ranging from 144 to 261 SFU/106 cells, whereas the infected animal E81 had both Gag and Tat-specific circulating T cells (
(A) IFN-γ secreting T cell frequencies in PBMC from protected animals (D68–D14) compared to those in an SIV-infected animal (E81) measured 20 wk after virus exposure measured by ex vivo ELISpot. The mean frequencies of three replicate determinations plus one standard deviation are shown for each peptide pool used.
(B) SIV-specific IFN-γ secreting T cell frequencies in MNC isolated from ileum–jejunum tissue of four protected animals measured post mortem at 21 wk after virus challenge by ex vivo ELISpot.
(C) The group mean +/− standard deviation profile of anti-SIV Gag p27 binding antibody titres (measured by ELISA) from animals infected with SIV (○) and the individual profile for an SIV-infected macaque E81 (○), in which T cell ELISpot was analysed was compared with animals from which no virus was detected following challenge (▴).
Analysis of plasma tenofovir concentration at the time of virus challenge, 15 min after gel administration, revealed a strong association with protective efficacy. The lowest concentration of plasma tenofovir associated with protection as defined by failure to isolate and/or detect virus in PBMC and plasma and lack of seroconversion, was 119.9 ng/ml (
Plasma Tenofovir Levels and Association with Protection from Infection
To further address the possible mechanism of the protection observed we utilised our recently described in vitro colorectal explant model [
(A) Replication dynamics of SIVmac251/32H in explants from two untreated animals (group F: M3, M6) in the presence or absence of exogenously added tenofovir at 100 μg/ml. A total of 104 TCID50 of virus was added to each well containing three explants in a total volume of 200 μl of medium. Virus replication was assayed by SIV Gag p27 production and mean values +/− standard deviations are shown for four replicates of each tissue.
(B) Colorectal explants from four animals (group G: M1, M38, M5, M32) dosed in vivo with tenofovir per rectum 3 h before tissue removal were exposed to virus in vitro (as described above) and culture supernatants assayed for Gag p27. Mean percent inhibition of SIV Gag p27 production plus standard deviations are shown.
Intracellular Concentrations of Base and Phosphorylated Tenofovir in Colorectal and Ileum–Jejunum of Macaques Given Tenofovir Gel Rectally
The preclinical study reported here using the SIV-macaque challenge model, showing a statistically significant protective effect, strongly supports the notion that local application of an ARV agent can efficiently protect against subsequent intrarectal challenge with virus. In addition, the study revealed a potential metric of protective efficacy wherein, above a critical threshold, the concentration of tenofovir detectable in the plasma 15 min after rectal application was positively associated with protection. Topical use of tenofovir prior to virus exposure also facilitated the priming of SIV-specific T cell responses in a proportion of macaques, opening up the possibility that this type of prophylaxis may be able to prime and/or boost immune responses elicited with experimental vaccines.
It has become increasingly evident that current experimental approaches to the development of an HIV vaccine, largely based upon the generation of T cell immunity, are, at best, likely only to reduce the level of viraemia following exposure to virus. Although this may have partial benefit, in the longer term, as shown in the SIV model, virus frequently escapes from immune control following the selection of variants mutated in T cell epitopes [
The long intracellular half-life of tenofovir diphosphate, the pharmacologically active metabolite of tenofovir [
Another critical consideration is whether protective efficacy can be increased to 100%. Of particular interest in the present study was the observation of a positive association between the concentration of tenofovir measured in the circulation 15 min after topical application and the outcome of virus challenge. Our working hypothesis is that this metric provides a surrogate measure of local uptake of tenofovir; thus only in animals where the local tissue concentration is beyond a critical threshold is protection achieved. Using the newly developed assay for intracellular tenofovir it should now be possible to test this hypothesis directly. If indeed the rate limiting step to 100% protective efficacy is local uptake of tenofovir, several possibilities require further investigation, including: is efficacy increased (a) at higher concentrations of administered tenofovir gel; (b) with larger volumes of administered gel; (c) with optimised gel formulations; (d) with combinations of ARTs having differing pharmacokinetics; (e) with prior washing to remove residual faecal matter? Thus, the quantification of plasma tenofovir following rectal administration could be used to inform the optimisation of rectal dosing in terms of concentration, volume, formulation, and timing to accelerate the progression of this approach to clinical trials.
Our study has also further validated the use of the ex-vivo rectal explant system by, for the first time, extending its use to macaques. Not only is this system useful for the evaluation of antiviral compounds and combinations in vitro; as we have shown here, it may also be used to measure antiviral activity following application of drug in vivo. Detailed pharmacokinetic studies are required to determine when the peak concentration of tenofovir appears in the plasma, but our results from this preliminary study suggest that the peak is early. Interestingly ileum/jejunum tissue taken from dosed macaques remained susceptible to infection. Indeed, the lack of drug in the small intestine was confirmed by analysis of intracellular tenofovir concentration, suggesting that secondary adsorption at this site is insignificant. The relatively low rectal dose of tenofovir applied, equating to an average of 10 μg/kg, of which a maximum of 0.19% was detected in plasma 15 min later, was far below the dose used in oral pre-exposure prophylaxis [
At the doses used in the present study it is predicted that the protective effects will be confined to the rectal portal of entry; however, the high degree of protection against rectal challenge seen in the present study, including the absence of SIV detection by highly sensitive qPCR in regional lymph nodes was perhaps surprising given that virus may translocate rapidly in dendritic cells [
Although our study was not powered to allow statistical analysis of rectal postexposure efficacy, in the group of animals receiving gel 2 h after virus exposure only one of three animals was protected. Given that the virus replication cycle is approximately 24 h, this preliminary observation is in accord with the hypothesis that virus is translocated rapidly from the portal of entry and is sequestered either in a cell in which tenofovir is metabolised only slowly and/or that the virus may be held in trans in a nonreplicating state, as has been proposed for dendritic cell-mediated infection [
The priming of SIV-specific T cells detectable in the circulation in the absence of seroconversion is reminiscent of similar responses seen in so-called highly exposed, persistently seronegative individuals (reviewed in [
The study reported here has several limitations. Firstly, the ARV-gel was delivered controllably under idealised conditions. Ensuring that this occurs in practice will require careful development. Secondly, finding an acceptable form of administration may require different strategies for different target populations. For some users application of a rectal gel may be acceptable, for others, formulation of the ARV into a suppository may be more acceptable. Compatibility with other products that may be used before AI is a further consideration. Thirdly, cell-free challenge virus was applied in a controlled, atraumatic manner in the absence of semen. Fourthly, the predictive value of the SIV-macaque model requires further investigation. This issue was highlighted recently by the much publicized failure of two large clinical trials in HIV prevention (the Merck STEP vaccine trial, and a trial of the vaginal microbicide cellulose sulphate), using modalities that had apparently shown promise in the macaque model. However, in the case of the recombinant replication deficient adenovirus vaccine (STEP trial), reduction in virus load was originally demonstrated following immunization with a construct expressing SIV Gag and challenge with simian HIV (SHIV)-89.6P [
Given the caveats discussed above, our data nonetheless suggest that rectal mucosal dosing with tenofovir shows promise for protection against rectal transmission of an immunodeficiency virus. Moreover, given the functional IFN-γ T cell responses induced, there may be potential for synergy between topical ARV/microbicide use and vaccination as a two-pronged strategy for preventing infection with HIV.
We thank Jim Rooney, Gilead Sciences for providing tenofovir gel; Carol Fraser (St. George's University of London [SGUL]) for serological analysis; Sharon Leach and Stuart Dowall (Centre for Emergency Preparedness and Response [CEPR]) for virus isolation and specimen dispatch; and Zara Fagrouch (Biomedical Primate Research Centre [BPRC]) for quantitative competitive (qc)-real time RT- PCR. We are grateful to Neil Almond (National Institute for Biological Standards & Control [NIBSC]) for making laboratory facilities and training available; to Harvey Holmes and the staff of the Centralised Facility for AIDS Reagents, NIBSC, for SIV peptide pools and recombinantly expressed p27 antigen; and to Jim Turpin (NIH) for useful discussion. We thank Alicja Rudnicka (SGUL) for statistical advice and analysis.
anal intercourse
antiretroviral
gamma interferon
mononuclear cells
spot forming unit
simian immunodeficiency virus
peripheral blood mononuclear cells
reverse transcriptase
virus isolation
viral RNA