The African Connection
Sex workers provide clues for a vaccine.
By Anne-christine d'Adesky & Richard Jefferys

For the past decade, most of the spotlight on Africa has been negative when it comes to the subject of AIDS and HIV. Today, sub-Saharan Africa remains the epicenter of the epidemic, with rates of HIV infection climbing to 25 percent in some countries. Just behind Africa is Asia. The sheer scope of the devastation has tended to obscure what remains one of the most exciting and promising avenues of HIV research, both on the prevention and treatment fronts. Perhaps ironically, the people most socially maligned in this epidemic are now playing a key role in the fight: prostitutes.

The setting is Pumwani, Kenya, a sprawling slum area of the capital, Nairobi, where, like many African cities, HIV has rewritten the human map. The virus appeared there in the early 1980s, seemingly overnight, slipping in on the heels of other deadly diseases, including the great social one, hunger. Like elsewhere, poverty is the main force driving young Africans into prostitution. Not suprisingly, many have quickly contracted HIV and died, as have their sexual partners.

What surprised many researchers were the first reports, in 1993, that some Kenyan female sex workers remained uninfected, despite repeated exposure to the virus. For the first time, here was evidence that people might be immune to HIV, a finding that had-and still has-enormous implications for a vaccine. In fact, the idea ran so contrary to general thinking six years ago that Francis Plummer, the man who made the discovery, was reluctant to talk about it.

"I was a little tentative in the beginning because I thought people were gonna think I'm crazy," admits Plummer, looking back. "In retrospect, I think I would have gone after this harder."

In 1985, Plummer was a young infectious disease specialist from the University of Manitoba, Canada, with a degree in medical microbiology when a colleague suggested a brief turn in Kenya to study sexually-transmitted diseases. What began as a "detour" in an otherwise straight forward career path quickly blossomed into a life's work. Today, Plummer is at home in Pumwani, having carved out a critical niche in AIDS research with his groundbreaking studies of HIV genes and resistance. "Just about everything you find with HIV is here," he explains, "There's no reason to look further."

Plummer's HIV work began as a spinoff project of a larger study of STDs among female sex workers when it was discovered that two-thirds had HIV. "It quickly became a priority," Plummer says. For a year, his group followed a group of 600 women, taking medical histories, studying rates of STDs and other factors and interviewing their families and sexual partners. The study was later opened up, and as of December 1998, 2,000 female sex workers were being followed. Very quickly, Plummer found that a small percentage of women were testing negative for HIV antibodies, despite years of unprotected sexual contact with male partners.

Plummer provided his first description of apparent immunity to HIV at the World AIDS Conference in Berlin. By then, 29 commercial sex workers from the Nairobi cohort remained HIV negative. A similar report followed a year later by Sarah Rowland-Jones and colleagues at John Radcliffe Hospital in Oxford, England, who presented data on sex workers in Gambia who also appear immune to HIV. The British group recently reported that five out of six Gambian female sex workers they are following were repeatedly exposed to HIV and remain uninfected. They also had robust cellular immune, or CTL, responses that appeared to abort the infection before it could take hold.

At last year's World AIDS Conference in Geneva, Plummer updated his results with 90 women who continue to test antibody negative after repeated exposure to HIV. Of them, a half-dozen have been uninfected since 1985, when they first came to the clinic. The average woman has four to five sex partners a day, and uses a condom 75 percent of the time. Based on surveys, 25 percent of their sexual partners are HIV infected. That spells hundreds of exposures to HIV over a decade. The women have been exposed to a wide variety of viral strains prevalent in the local population, based on laboratory analyses of their immune responses.

The Nairobi women range in age from young girls up to age 50, with the average age of 30. Most are from the local area around Nairobi, but some are from Tanzania, and a few from Uganda, where HIV rates are also catastrophically high. As expected, Plummer's group found that STD rates are very high among these sex workers and may operate as a co-factor in HIV disease, an observation made by others (see "HIV's Silent Partner," page 13.). Among the Kenyan women, chancroid, a bacterial STD, is the biggest co-factor risk.

Cellular Immunity
Like many long-term HIV non-progressors, a small percentage of these resistant sex workers carry mutated genes that are linked to full or partial resistance to HIV (see main story). Many, however, don't. At Geneva, Plummer reported that two-thirds of the Nairobi women have strong HIV-specific CTL responses. A similar report regarding the role of HIV-specfic CTL was presented at Geneva by Busarawan Sriwanthana from the U.S. Centers for Disease Control who is studying a group of exposed-but-uninfected sex workers from northern Thailand. Another study by researchers at Dartmouth Medical School recently found that exposed-but-uninfected partners of people with HIV also have strong specific CTL responses that appear to protect them from active infection.

In the Nairobi group, there are no signs of active infection based on PCR viral-load tests of peripheral blood and genital mucosa. But Plummer admits he hasn't yet looked for HIV particles (provirus) that may be lingering inside dormant T-cells in the genital tract or lymphoid tissue reservoirs-and won't rule it out. That would tell us if these women are really HIV-free. In a recent interview with Ashley Haase, a leading expert on latent HIV infection, Haase said that he thought the Nairobi sex workers were probably harboring a latent infection, albeit one that may never cause them to get sick. Other vaccine researchers voice the same opinion.

For now, Plummer remains hopeful: "My opinion-and obviously we don't have proof of this-is that these women are immune to HIV, that they have aquired protective immunity. They have cellular immune responses in the blood and probably in the genital tract and also antibody responses in the genital tract that are protecting them. Why these responses develop is a very hot question."

As Plummer notes, along with strong CTL responses, many of the resistant women also have strong HIV-specific antibody responses. These include high levels of IgA (interferon alpha) antibodies and low levels of IgG (interferon gamma) antibodies in the genital mucosa, but no levels of IgG in the peripheral blood. These data suggest that secondary humoral, or antibody responses, may also play a role in fighting the virus, a finding that contrasts somewhat with earlier vaccine studies that suggest HIV antibodies play little role in blocking HIV.

At the National Cancer Institute, Mario Clerici's team has found HIV- specific IgA antibody responses, but not IgG, in exposed-but-uninfected heterosexual partners of people with HIV. In another Geneva paper, Timothy Flan-igan of the Miriam Hospital in Pro-vidence, Rhode Island, also found a minority of HIV-positive women had HIV-specific IgA antibodies in their genital mucosa and blood.

Role of Repeated Exposure
We're hypothesizing that this is a gene involved in the governance of the immune response," says Plummer, who thinks there are probably two main explanations for the specific immune responses seen in the Nairobi women and others, including the Gambian sex worker group. "One is that there is something unique about them and their immune system. The other is the way they were exposed to HIV."

Regarding the second possibility, repeated sexual exposure to small amounts of HIV or not very strong viruses might be akin to what happens when someone receives low doses of a vaccine. Plummer says other studies have shown that when you expose a person to a small dose of a vaccine, you get a primary type-1 CTL response; using bigger doses, you get a secondary type-2 antibody response. "In animals, you can manipulate the immune response, and this is also true in humans, by the amount of antigen you give them," he explains. "Where IgA antibody comes in is another thing, because IgA responses are not classically thought of as a Type-1 immune (CTL) response; they're more thought of as Type-2 (antibody) response and how all that fits in we're not really sure."

The bottom line is that if the route of exposure plays an important role to protect these women, it could serve as a model for a vaccine. Backing the theory of low-dose exposure is a December report in the Journal of Virology by McChesney and colleagues who found they could induce a temporary viral infection and cellular immune responses to SIV in 31 adult female macaques by exposing them, through intravaginal innoculation, to low doses of a deadly SIV virus (the sister simian virus to HIV). What they found next is startling: 10 of 10 animals who were later dissected showed no signs of active SIV infection in their blood, lymphoid tissue, or vaginal mucosa. But the researchers did find SIV particles (provirus) inside dormant T-cells in their lymphoid tissue.

The researchers conclude that repeated exposure to low doses of the virus didn't completely protect the macaques, but caused either a latent or very low-level SIV infection. This could explain what we see in long-term non-progressors. What about the Nairobi group? Are they actually latently infected? We won't know until further studies are done. "I hope that's not true," Plummer says.

More heartening news comes from another study in the same Journal of Virology issue, this one by an Australian team led by vaccine researcher Stephen Kent, who's reported initial success in boosting HIV-specific cellular immune resposes in mice and four macaques by repeatedly immunizing them with a candidate DNA virus. So far, it's too early to tell how protective these cellular immune responses will be, but the CTL levels approached those seen in people with acute HIV infection. Related studies with other retroviruses in animals also support this approach.

In the October issue of Nature Medicine, another Australian vaccine group tested a vaccine in sheep that induced a "retrovirus-specific CTL" response that was protective against bovine leukemia virus. "CTLs alone, in the absence of antibody or CD4 responses, can prevent the establishment of a latent infection," they concluded, a finding that has implications for HIV vaccines. Of course there are inherent limitations in applying what happens in animals to humans, but it's another step.

So far, Plummer hasn't thought much about how his work might be expanded therapeutically, for example, to study mucosal immunity or to develop a preventive intravaginal vaccine or microbicide. "It potentially could," he says of the idea, adding that he's open to collaboration. "I think what [Bruce] Walker says is a very positive message overall which is that the immune system can control HIV. We need to learn to imitate nature. I think all the human data is pointing to it as well. There is protective immunity, so you must be able to do it."