On Friday, October 26, the U.S. Food and Drug Administration granted accelerated approval to Gilead's nucleotide analogue reverse transcriptase inhibitor, tenofovir disoproxil fumarate (brand name, Viread). The labeling requested was for the "treatment of HIV-1 infection in combination with other antiretroviral medicines." Tenofovir is the first nucleotide analog approved for the treatment of HIV infection, and Gilead's second such product. Adefovir (trade name Preveon) was turned down by the FDA in late 1999 due to safety concerns and lackluster clinical results. Nucleotide analogue reverse transcriptase inhibitors (RTIs) are similar to nucleoside analogue RTIs and block HIV replication in the same manner. The difference is that nucleotide analogues already have one phosphate group attached.

The FDA based its approval of tenofovir on the results of two clinical studies, study 907 and study 902, involving more than 700 patients who had previously been treated with antiretroviral agents but showed signs of treatment failure. Study 907 is a 552-patient, 48-week placebo-controlled, treatment intensification study. Twenty-four week results were presented to the FDA. Study 902 is a smaller study, also in treatment experienced individuals, that compares treatment intensification with three different doses of tenofovir or placebo. Study volunteers who received tenofovir showed a mean viral load reduction of 0.62 logs compared to individuals who received a placebo with the standard antiretroviral regimen. Tenofovir is available as a 300 mg tablet to be taken orally, with a meal.

Because the approval of tenofovir was based on clinical trials involving individuals previously treated with antiretrovirals, the risk-benefit ratio for untreated individuals has yet to be determined. Furthermore, there are no study results to show long-term inhibition of the clinical progression of HIV by tenofovir. Additional studies under way are expected to address these issues: Study 903 enrolled nearly 600 treatment naïve individuals and is expected to produce results by mid-2002; Study 910, a roll-over extension study of individuals from studies 902 and 907, will examine long-term efficacy and safety issues. ¤

Identifying 'resistance' epitopes

In the United States, long-term studies of HIV-infected and high-risk people have mainly involved gay men–the group most heavily affected in the epidemic's early years. But a continent away, in the Pumwani district of Nairobi, a group of just over 100 women have become well-known to HIV researchers around the world by offering tantalizing evidence that the immune system can, in rare cases, fight off HIV. Richard Jeffreys prepared this report. Two related articles, "Holding HIV At Bay: What Keeps Exposed Babies Uninfected?" and ""HLA Genes and Immunity" appeared in the September issue of IAVI Report and can also be found at www.iavi.org.

The evidence derives from a cohort of female sex workers, established in 1984 by Elizabeth Ngugi and colleagues from the University of Nairobi and the University of Manitoba for the purpose of studying STDs. Despite an estimated 60 or more unprotected exposures to HIV every year–one of the highest documented exposure rates in the world–over 100 of the 2000 women enrolled in the cohort have tested negative for HIV infection for at least three years, and in some cases up to 15. Studies of these "highly exposed persistently seronegative" (HEPS, also sometimes referred to as "exposed seronegative" or ESN) women convinced many skeptics that immunological resistance to HIV–and by extension, an HIV vaccine–is possible.

Since the first description of this phenomenon by Canadian researcher Frank Plummer (at the 1993 International AIDS Conference in Berlin), the Human Immunology Unit of Oxford University in the UK has joined the Manitoba and Nairobi teams to conduct detailed immunological studies of these women. Their goal: to identify which immune responses protect the women against HIV, and to use that information to guide the design of preventive HIV vaccines.

Over the past few years, the Nairobi studies–along with those on other HEPS cohorts (see table) and on HIV-infected, long-term non-progressors–have been suggesting some answers. In the late 1990s they helped focus the AIDS vaccine field's attention on the importance of cellular immune responses in protection, especially the CD8+ killer T-cells (also called cytotoxic lymphocytes, or CTLs). These days, emphasis is on identifying the precise regions of HIV (called epitopes) that stimulate what appear to be protective responses, and on elucidating the roles of less well-characterized immune players, including CD4+ T-helper cells and mucosal responses, in resistance to HIV.

Alongside the science, the project was set up from the beginning to provide medical services for the women and frequent exchange with the research team. "The cohort is a partnership between the sex workers and the researchers," says Joshua Kimani, part of the team from the Department of Medical Microbiology at the University of Nairobi. "The partnership has worked over the years due to the monthly meetings we have with the sex workers' peer leaders. In these meetings issues related to poor follow-up or any unhappiness with the service providers are ironed out." There is also an annual meeting between the researchers and the entire cohort. The research team provides free medical services, free condoms, covers hospitalization at the Kenyatta National Hospital and can assist with bus fares and other expenses. Treatments available include those for STDs and the more easily managed opportunistic infections, but do not at present include expensive brand-name drugs such as the antifungal Diflucan and antiretrovirals.

The Origin of the Cohort
HIV testing in the Pumwani cohort began in 1985, when infectious disease specialist Plummer took what was intended to be a brief detour from Manitoba to join the STD project in Pumwani. Out of 600 women enrolled at the time, Plummer was dismayed to find that two-thirds tested positive for HIV. Shifting the focus of his work, he began to assess the factors associated with both seroconversion and, presciently, lack of seroconversion in the one-third of the women who tested HIV-negative. The startling observation reported in Berlin was that women remaining negative two years after starting sex work had only one-tenth the risk of subsequent seroconversion (over the following two years) compared to HIV-negative women newly joining the cohort. Furthermore, this apparent resistance to HIV infection was associated with certain class I HLA genes, suggesting a link to CTL responses.

Plummer's data caught the attention of Sarah Rowland-Jones, who had previously seen some cases of persistently seronegative women among sex workers in the Gambia. (Gambia is that west-east finger-like protrusion of formerly British ruled territory which extends into the lower third of Senegal, sheathing the eponymous river Gambia.) Joining up with the Manitoba and Nairobi investigators, Rowland-Jones and colleagues Tao Dong and Andrew McMichael analyzed blood samples from the HEPS women for evidence of HIV-specific CD8+ T-cell activity. Their results, published in late 1998, showed a strong association between the HEPS phenomenon and the presence of HIV-specific CTLs directed against a broad range of HIV epitopes. This association was strengthened by later studies, while various explanations based on non-immune-factors, such as mutations in the CCR5 co-receptor gene, were excluded.

But then came a finding that initially seemed somewhat counter-intuitive, according to Rowland-Jones: the level of the CTL responses in the HEPS women was as much as ten times lower than in uninfected women. "That means that it's not enough to simply count T-cells," she says. Instead, it pointed the researchers towards a more qualitative analysis of the responding cells, for example in terms of their epitope specificity, breadth and functional properties.

Mucosal Responses
After the initial description of HIV-specific CTLs in blood, the researchers turned to analyzing the mucosal immune responses in the HEPS women. The first published study, led by new team member Rupert Kaul, reported the presence of HIV-specific IgA antibody in the genital tract of 16 out of 21 HEPS women compared to 5/19 HIV-infected women. Conversely, HIV-specific IgG antibody was absent from HEPS and present in all infected women. Working with Italian immunologist Mario Clerici to assess blood T-helper responses, the paper also reported evidence of Env-specific T-cells in 11/20 HEPS, but there was no correlation with mucosal IgA production.

The role of IgA was explored further in another collaboration, this time with Claudia DeVito and colleagues from the Karolinska Institute in Stockholm. The investigators designed a system to model the transfer of HIV across the human mucosal epithelium, then tested the ability of IgA isolated from the cervico-vaginal fluid of HEPS women to block the transfer process (called transcytosis). Samples from six women were examined, and 3/6 reduced transcytosis of a primary clade B HIV isolate by more than two-thirds. The work suggests a mechanism by which IgA could contribute to protection at the mucosal surface, although the authors emphasize that other factors are probably also at play.

In parallel to the IgA studies, the MRC group found evidence of HIV-specific CTL in the mucosa. Examining cervical and blood samples, they found responses in 11/16 HEPS and 8/11 HIV-infected women using an ELISpot assay for interferon-gamma production. They also found that the HEPS women tended to have slightly higher responses in the cervix compared to blood, whereas infected women had significantly more HIV-specific CTL in blood than cervix. This apparent enrichment of mucosal CTLs in the resistant women supports the idea that they play a role in protection from HIV.

Late Seroconversions
But as these studies were going on, unexpected findings were emerging: between 1996 and 2000, 11 of the 114 women who had met the working definition of "resistance" (>3 years sex work without seroconversion or a positive PCR) became HIV-infected and seroconverted. It took the researchers by surprise, since long-term seronegativity had appeared to be closely associated with a decreasing risk of new infection. And it triggered an intense effort to find out what was going on.

It soon became clear that there was no obvious correlation between this "late seroconversion" and the presence or absence of CTLs in previous tests. "Half of the women who seroconverted had CTL [at earlier time points]," says Kaul. "We had looked at a couple of those women repeatedly and seen CTLs many times. So we were quite surprised and disappointed to see them seroconvert." An obvious possibility–that infecting viruses had "escape" mutations in regions targeted by the women's CTL–was quickly ruled out.

An answer began to unfold when the search turned to the amount or type of the women's recent exposure to HIV. Their analysis showed that a reduction in sex work–either stopping for over two months or reducing the number of clients by more than two per day–was strongly, but not absolutely, associated with subsequent infection: 10 of the 11 seroconverters had reduced their exposure by these criteria, compared to 10 of the 22 persistently seronegative women. Analysis of six women in the latter group found that–rather than seroconverting when they resumed sex work–they showed a boosting of their HIV-specific CTL responses. "In those women we saw a general trend that when you take a break from sex work, the immune responses go away," says Kaul. "If you start sex work again, these responses often come back." But it's unclear why the responses return in some women while others become infected. Some possibilities: persistence of HIV-specific CTL below detectable levels in the HEPS women, differences between blood and mucosal responses, the precise nature of the HIV exposure after a break and immune responses not analyzed in the initial study, such as HIV-specific T-helper cells and/or HIV-specific IgA antibody.

The implication of these results, widely reported in the mainstream press, was that continuous exposure to HIV may be important to maintain resistance in at least some HEPS women. Whether this would also apply to vaccines is unclear. The Oxford group point out two possibilities. One is that ongoing stimulation with HIV antigens is required, either through periodic vaccine boosters or through the use of vaccine strategies employing persistent antigen. Alternatively, vaccine-induced responses established prior to any HIV exposure (as opposed to immunity induced by live virus) might show a very different dynamic.

To look more closely for correlates of late seroconversion versus continued resistance, Kaul is now involved in a prospective study, which will monitor a broad range of immune parameters. "We'll try and get women to come see us before they go on a break, so that we can look for HIV-specific responses at that time. Then we'll try to get them to see us as soon as they return, before they've started sex work again, so we can see what's happened to those immune responses." In addition to monitoring CTL from the blood, the researchers will also follow mucosal responses, while Keith Fowke from the University of Manitoba will study the CD4+ T-helper responses.

The nature of the infecting virus in late seroconverters is also coming under the microscope. Some scientists have hypothesized that the resistant women have a latent, undetectable HIV infection, and that the late overt infections could represent an escape of this virus from immune control. "It would not at all surprise me," says Rowland-Jones. She's enlisted the help of Bette Korber from the Los Alamos National Laboratory and Harold Burger from the University of Albany to apply "molecular clock" techniques to date the viral isolates found in late seroconverters. "They plan to sequence virus to try and find out if it is an old Nairobi virus," reports Rowland-Jones. "Although this can't answer the question definitively, it might provide suggestive evidence of a latent infection."

The Search for "Resistant" CTL Epitopes
Another major focus of the current work is to identify the CTL epitopes associated with resistance. In the first set of data to emerge from this work, the researchers report some striking differences. Looking at CTL responses to a panel of 54 known epitopes (restricted by 21 different HLA molecules), they found that HEPS women showed strong responses to four epitopes that were very rarely immunodominant in infected women–two in Pol and two in p24-Gag. They also found that infected women responded most strongly to epitopes recognized only rarely, or not at all, by the HEPS group. Of the seven late seroconverters evaluated in the study, five showed a switch from the HEPS pattern of epitope responses towards that of infected women and/or the complete loss of responses to the "resistant" epitope.

Another striking observation was that all four epitopes showing differences between HEPS and infected women are restricted by HLA alleles known to be associated epidemiologically with HIV resistance in the Nairobi cohort (A2, A24, A*6802, B14 and B18), suggesting that the effect of these HLA types is related to their greater likelihood of generating CTL responses to a repertoire of more protective epitopes.

The study represents a first step in identifying "resistance" epitopes, but there is more work ahead–particularly given the "information gap" revealed when the researchers use whole HIV proteins, rather than known epitopes, to measure T-cell responses. "We see a number of women who don't respond to a panel of CTL epitopes, but do respond to Env or Gag," says Kaul. "So there are probably some epitopes within those genes that haven't been mapped yet."

T-helper Responses in ESN Women
Not all the HLA alleles associated with protection in HEPS women belong to class I, the system for presenting epitopes to CD8+ T-cells. A comprehensive analysis by Kelly MacDonald's group from the University of Toronto revealed a highly significant link with the class II allele HLA DRB*01, suggesting an important role for CD4+ T-helper responses in mediating resistance. "This points to the fact that there's a multifactorial immune response," says Keith Fowke, who has taken on the task of analyzing helper responses in the Nairobi cohort. "To ignore the T-helper response would be a mistake."

Fowke was recently lead author on the first published report to look at both HIV-specific T-helper and CTL responses in the HEPS women from the Nairobi cohort. This study detected T-helper responses in 7/17 HEPS women using an assay for IL-2 production in response to five Env peptides. Fowke's team then carried out both helper and CTL assays on samples from 15 women, and found a statistically significant link between the presence of T-helper responses and CTL. "The data is suggesting that it's important to have not only CTL but good 'help,'" notes Fowke. This observation is consistent with basic immunology work in animal models, demonstrating a key role for virus-specific T-helper cells in generating and maintaining effective CTL responses.

To clarify the role of CD4+ T-cells in protection, Fowke's group is using ELISpot assays to conduct a broader analysis of responses in the HEPS women. Although a significant amount of CTL epitope data is available, there is a dearth of defined class II-restricted T-helper epitopes, one that Fowke aims to address by mapping the responses using clade A and clade A/D recombinant viruses. Another priority for the Manitoba team is investigating HIV-specific T-helper activity in the mucosa, which has never been studied in the cohort (or any other exposed seronegative individuals to date), due to the difficulty of obtaining samples with sufficient numbers of cells.

Shaping Vaccine Design
The presence of apparent immunity in the HEPS women, and its association with T-cell responses in the absence of antibody, have strongly influenced the thinking of AIDS vaccine designers-an influence readily evidenced by a new crop of candidates that aim to induce cellular immune responses to HIV. Several vaccines based on this strategy have shown promise in recent monkey studies, including those of Emory University researcher Harriet Robinson, Harvard's Norman Letvin and Merck. As more is learned about the protective responses in the women of Pumwani, that knowledge is likely to continue guiding vaccine developers towards the types of responses to target and the HIV epitopes that can best induce them.

Results from the collaborative studies of the Oxford, Manitoba and Nairobi teams are also being passed on to vaccine designers Tomas Hanke and Andrew McMichael in Oxford, whose first generation DNA/MVA constructs are currently in Phase I human trials in Oxford and Nairobi. Later generations of this vaccine will draw on information gleaned from the continuing work with these women. ¤