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 2,000 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 "Studies on Exposed Seronegative Cohorts" in this issue) 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.
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.
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.
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."
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."
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.
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.
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