Individual armor against HIV

Individuals who have an apparent resistance to HIV infection may hold important clues for AIDS vaccine research

By Regina McEnery

For legions of scientists trying to unravel the mysteries of HIV and figure out ways to protect against infection, the notion that certain people possess a natural resistance to the virus represents both an intriguing hypothesis and a conundrum.

Two decades of research offers ample evidence that the phenomenon exists. More than 30 different high-risk cohorts have revealed individuals known generally as exposed seronegatives (ESNs), who have evaded infection despite known, sometimes repeated, exposure to HIV. Research involving these individuals has resulted in publication of more than 100 scientific articles, beginning with a landmark study involving a cohort of commercial sex workers (CSWs) from Nairobi, Kenya. This early study conducted by Frances Plummer, a University of Manitoba immunologist, showed that some CSWs in this cohort seemed to be resistant to HIV infection and that the rate of HIV infection in these women actually seemed to decrease with each year they participated in sex work.

Other cohorts of ESNs have been identified as well, including men who have sex with men (MSM), the uninfected partners of discordant couples in which one partner is known to be HIV infected, hemophiliacs who received HIV-contaminated blood products in the early 1980s before blood screening was implemented, and injection-drug users. Still, after many years of research, it is remarkably unclear how these individuals continue to dodge HIV.

Most scientists ruled out mere coincidence after Plummer’s study and instead think these rare individuals may possess some genetic traits that are absent in most of the population. The chase has been on ever since to discover what those traits are and why only certain individuals have them.

But identifying and characterizing the possible mechanisms of protection in these individuals has become an exhaustive enterprise that has yielded conflicting, inconclusive, and sometimes controversial results, leaving some researchers deeply skeptical. If it were possible to identify unique immune responses to HIV in these individuals it could inform AIDS vaccine research, but even those who remain committed to studying ESNs are unsure whether any valuable immunological clues will be unearthed from these individuals. Still, most everyone agrees that the study of ESNs is worth continuing.

What’s driving ESNs?

Epidemiologists have been tracking ESNs for nearly two decades, yet even among researchers there is still not a widely accepted definition of what qualifies an individual as an ESN. One difficulty is quantifying a person’s exposure to HIV. In different cohorts the route of transmission is variable and this influences the amount of HIV a person is exposed to, says Barbara Shacklett, a microbiologist at the University of California in Davis. This has made it hard to replicate findings or draw conclusions from different studies, she adds.

Studies of discordant couples allow scientists to closely analyze the precise virus the HIV-uninfected partner is exposed to, which helps researchers interpret their immune responses against HIV. But the level of HIV exposure among CSWs in high HIV-prevalence countries is often greater because they have more sexual partners and use condoms less, making these women a more trusted cohort for studying the ESN phenomenon.

Studies in both of these cohorts have looked extensively at a variety of immune responses against HIV in ESNs and have uncovered numerous factors that could play a role in their apparent HIV resistance. The findings have looked at their innate immune responses—the first defense against any invading virus—as well as their adaptive immune responses, which are generated against a specific pathogen such as HIV.

But so far there is no evidence to suggest that any one or combination of these factors is actually responsible for the ability of some individuals to resist persistent HIV infection. Many of the associations between different types of immune responses observed in ESNs and HIV resistance have eventually been refuted. Further complicating matters is the fact that several of these reputed protective factors are also observed in HIV-infected individuals; however, it is unclear if they are present at the time of HIV infection or only subsequently.

“Protection against HIV transmission is probably going to be multifactorial,” says Wim Jennes, a microbiologist at the Institute of Tropical Medicine in Belgium, who has been studying ESNs in Africa for nearly a decade. “There probably will be several different factors playing a role. Some of them may have a minor impact or occur in a small number of people, while others occur in a higher percentage. That’s why it is important to continue these studies.”

The interpretation of studies involving ESNs has been hampered by several limitations, including the limited size of the study population or omitting appropriate control groups. Other times investigators conducting the studies lacked the technological tools to detect immune responses present at lower levels, which may actually be contributing to resistance to HIV. As a result, many of the studies involving ESNs have not been reproducible and the findings have not been validated.

Researchers at the US Centers for Disease Control and Prevention conducted an analysis in 2003 of all published articles relating to the study of highly-exposed persistently seronegatives (HEPS)—individuals repeatedly exposed to HIV who have undetectable levels of HIV antibodies by standard tests and therefore are considered uninfected—and found that some of the most compelling evidence points to an association between cellular immune responses in these individuals and resistance to HIV infection. The responses in particular that are most strongly associated with apparent HIV resistance in cohorts of CSWs and serodiscordant couples are cytotoxic T lymphocytes (CTLs), or killer T cells, which can kill virus-infected human cells. Still, there is little agreement in the field that this is the key to unlocking the mysteries of ESNs.

Shacklett abandoned her research of ESNs mostly for logistical reasons, after relocating from the Aaron Diamond AIDS Research Center in New York City, but she also admits getting cold feet over whether the ESN research would lead to anything conclusive. Shacklett says the HIV transmission study she was working on found what appeared to be CTL responses in some of the 16 HIV-uninfected women who were characterized as ESNs. “But I did feel it was kind of a stretch,” she says, reflecting on the preliminary findings. “The magnitude and breadth of responses was not that compelling so it was hard to know if they were truly protective.”

Several research groups have also been studying a possible association between anti-HIV proteins known as antibodies that are found at mucosal tissues—such as those that line the genital tract, which is where HIV transmission occurs in female CSWs—and resistance to HIV infection. In a recent study, Rupert Kaul, University of Toronto’s research chair in HIV, and colleagues detected antibodies that could neutralize HIV in the genital tracts of women in a Kenyan cohort of CSWs who are considered ESNs.

But this study is still not likely to end the debate over what protects ESNs from HIV infection. “I don’t think there is a good strong theory,” says Larry Corey, principal investigator of the HIV Vaccine Trials Network. “Different people have different ideas and there is no consensus that any are correct,” he adds.

A lucky few

To better understand this phenomenon, some say an approach similar to the HIV Elite Controller study—a collaborative effort to study a subset of long-term nonprogressors who are HIV infected but are able to control the virus without the aid of antiretroviral therapy so that it is undetectable by conventional tests—is necessary. The Elite Controller study, led by Bruce Walker, director of the Partners AIDS Research Center at Massachusetts General Hospital, wants to recruit a cohort of 1,000 elite controllers to enable researchers to conduct a genetic analysis of these individuals in the hope of uncovering any possible factors that might be associated with their ability to control HIV infection.

In Walker’s opinion, the best group of ESNs to study would be hemophiliacs who were exposed to HIV through contaminated blood transfusions because only in that case is there clear documentation of exposure to HIV. Though the number of HIV-exposed hemophiliacs is small compared to other risk groups, their level of exposure was highest because HIV transmits most efficiently when injected directly into the bloodstream. Jacques Fellay, a research associate at the IGSP Center for Population Genomics & Pharmacogenetics at Duke University, says a single transfusion or infusion of a blood product containing HIV carries an estimated risk of infection of more than 95%, compared to only a 1-3% estimated risk of infection from exposure through an HIV-contaminated needle.

More than half of the 20,000 Americans with hemophilia were infected with HIV between 1979 and 1985, when an HIV test was finally developed and blood banks began safeguarding their supplies through routine screening of all blood. One of the most famous was US teenager Ryan White, who was diagnosed with HIV in 1984 and by his death in 1991 had already become an international symbol in the fight against AIDS.

Despite receiving regular injections of HIV-tainted blood products, some of the hemophiliacs who were accidentally exposed to HIV never actually became infected. A small number of these hemophiliacs are considered ESNs and this group is now the focus of a research study being conducted by the Center for HIV/AIDS Vaccine Immunology (CHAVI), to identify any key genetic determinants of their apparent resistance to HIV.

Researchers affiliated with the CHAVI study will conduct genetic analyses of roughly 800 HIV-exposed, uninfected hemophiliacs and will compare them to a control group of approximately 1,000 HIV-infected individuals, not hemophiliacs in particular, who were recruited for other CHAVI studies. David Goldstein, a Duke University immunologist heading up the CHAVI study, says the technology used in this study to sequence the genetic information of these ESNs will offer an unprecedented amount of data that can be used to look for genetic factors that may play a part in their ability to resist HIV infection.

Several other collaborations are also in process to try to bring some much-needed clarity to the study of ESNs by conducting case-control studies in larger cohorts, applying more sensitive methods of laboratory evaluation, and developing a clearer definition of what qualifies an individual as an ESN.

Along with the hemophiliac study cited above, CHAVI launched a study last year, involving one of the largest cohorts of ESNs ever evaluated, to address the conflicting and inconclusive data surrounding ESNs. This study is using more sensitive tests to try and nail down whether 700 ESNs from serodiscordant-couple cohorts in Uganda and the UK harbor any detectable amounts of HIV by ultra-sensitive tests. Although they aren’t classified as being HIV-infected by conventional measures, being able to detect extremely low quantities of HIV in these individuals might suggest there is something—possibly effective immune responses against the virus—that is preventing HIV from establishing a productive infection in these individuals. This could eventually offer clues for AIDS vaccine researchers about the types of responses a vaccine would ultimately have to induce.

If the CHAVI study ultimately finds little difference between unexposed, uninfected individuals and highly-exposed uninfected individuals, there would be a weaker argument for the role of HIV-specific immune responses in protecting these individuals from infection. But Andrew McMichael, an Oxford University immunologist and investigator with CHAVI, thinks results from the CHAVI study will help quell some of the skepticism about ESNs. “We are finding some positive responses,” he says. “There may be something there, but the study is still blinded.”