Individual Armor Against HIV

Individuals who have an apparent resistance to HIV infection may hold important clues for AIDS vaccine research, but solving the mystery of this resistance is extremely complex

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 offer 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 papers, according to Blake Ball, an immunogeneticist at the University of Manitoba involved with one of the longest-running and best-characterized of the cohorts, a group of commercial sex workers (CSWs) from Nairobi, Kenya. Ball works with one of the pioneers of ESN research, Francis Plummer, who began noticing early on in the epidemic that some women inexplicably resisted HIV infection despite participating in commercial sex work (Lancet 348, 1347, 1996). Moreover, HIV incidence among these women actually decreased with an increasing duration of potential exposure to the virus—each year these women participated in sex work, their risk of acquiring HIV decreased.

Other cohorts of ESNs have been identified as well, including men who have sex with men (MSM), the uninfected partners of serodiscordant couples, 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 within this rare segment of high-risk populations, it is remarkably unclear how these individuals continue to dodge HIV. Is it luck or nature? Good genes that provide grade-A immunity? Most scientists ruled out mere coincidence after Plummer’s landmark study in 1996. At the conclusion of this study the researchers suggested that this highly-exposed subset of HIV-uninfected CSWs in Kenya may possess immunological traits absent in most of the population, which allowed them to fend off HIV or clear it before the virus could establish an infection. 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 immunological mechanisms of protection in these individuals has become an exhaustive enterprise that has yielded conflicting, inconclusive, and sometimes controversial results. Some researchers remain deeply skeptical that ESNs mount unique immune responses to HIV and instead favor the theory that these individuals possess some other factor that confers protection. Even those who remain committed to studying ESNs are unsure whether any valuable immunological clues will be unearthed that could contribute to the development of a preventive AIDS vaccine. Still Barbara Shacklett, a microbiologist at the University of California in Davis, thinks it’s important to continue searching for answers. “My view is that there are such individuals who are highly exposed and remain uninfected by our classical definition.”

What’s driving ESNs?

During the roughly two decades since epidemiologists began identifying and tracking ESNs, studies have looked extensively at both innate and adaptive immune responses against HIV in these individuals. These studies have uncovered numerous factors that could play a role in their apparent HIV resistance.

The innate factors have ranged from mutations on the CCR5 coreceptor that prevent HIV from entering cells; upregulation of chemokine production, including overexpression of RANTES, MIP-1α, and MIP-1β; polymorphisms in human leukocyte antigen (HLA) haplotypes that interfere with efficiency of antigen presentation and can figure in either increased susceptibility or resistance to infectious diseases; and autoimmune responses to CD4 or CCR5, which induce antibodies to these coreceptors that neutralize their function and therefore obstruct HIV’s ability to infect cells.

Studies have also looked at adaptive immune responses such as HIV-specific helper T cells, cytotoxic T lymphocytes (CTLs), and humoral immune responses, including mucosally available antibodies (AIDS Rev. 5, 87, 2003). Researchers have also analyzed the infecting virus in both hemophiliac and serodiscordant couple cohorts to determine if any viral mutations may be responsible for impeding transmission. The general belief is that the rare individuals who can demonstrate long-term resistance to HIV must draw on multiple mechanisms—both innate and adaptive immune responses.

“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 also 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.”

But so far no evidence exists to suggest that any one of the known factors, or any combination of them, is actually responsible for the ability of some individuals to resist persistent HIV infection. Many of the associations between immune responses and HIV resistance that have been observed in ESNs have eventually been refuted. Among researchers who study these individuals there is still not even a widely accepted definition for ESNs or a standardized way of quantifying exposure in most high-risk groups. 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.

Identifying the factors that may play a protective role in ESNs is complex and the interpretation of studies involving these individuals has been hampered by several limitations. Previous studies have been criticized for the limited size of the study population, for omitting appropriate control groups, or for being unblinded. Other times investigators conducting the studies lacked the technological tools to detect low-level immune responses. As a result many of the studies involving ESNs have not been reproducible and the findings have therefore not been validated.

Researchers at the US Centers for Disease Control and Prevention conducted a meta-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 enzyme immunoassays—and found that some of the most compelling evidence points to an association between CTL activity and resistance to HIV infection in cohorts of CSWs and serodiscordant couples. There is also strong evidence for an association between chemokine receptor mutations, including the CCR5Δ32 mutation, and HIV resistance. This well-documented and studied mutation prevents the expression of CCR5, HIV’s preferred coreceptor, on the surface of cells and therefore can block the virus from infecting cells unless it mutates to gain entry via the CXCR4 coreceptor. But studies have shown that only a small proportion of individuals in the CSW or serodiscordant couple cohorts in Africa and Asia actually have the CCR5Δ32 mutation, suggesting this is not the only answer. Other receptor mutations have also been studied with regard to the HEPS phenotype, but according to the CDC’s analysis, none of them reached the level of being “strongly associated” with resistance to HIV infection. The CDC’s review also found that HIV-specific CD8+ T-cell responses against the Nef, Gag, Pol, and Env proteins of HIV have been detected in a proportion of ESNs from different cohorts, yet absolutely no evidence points to any of these responses as the explanation for an ESN’s apparent resistance to HIV.

A lucky few

With only between one in 700 and one in 1,000 estimated sexual acts between discordant couples resulting in HIV infection, it’s difficult to determine precisely what is special about high-risk individuals who remain HIV uninfected.

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.”

Within the spectrum of ESNs, Shacklett says there doesn’t appear to be one overwhelming mechanism that protects individuals. Take for example the association between the presence of the mucosal antibody immunoglobulin A (IgA) and resistance to HIV. Separate studies, using different methodologies, have shown that HIV-specific IgA may be present in the genital tracts of ESNs (AIDS 13, 23, 1999; J Immunol. 165, 5170, 2000).

Recently researchers conducted the first prospective, controlled study to look for IgA with direct HIV-neutralization capacity in a Kenyan cohort of CSWs and correlate its presence or absence with subsequent HIV acquisition (AIDS 22, 727, 2008). All immunologic assays in this trial were performed on blinded samples, which the study’s authors argue adds robustness to their data that has been absent from other studies characterizing the role of IgA in ESNs. This study showed that HIV-neutralizing activity in IgA from the genital tract secretions, as well as HIV-specific cellular immune responses in the blood, were significantly associated with HIV protection, says Rupert Kaul, University of Toronto’s research chair in HIV, who was a coauthor of the study.

But this association is 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 promulgated different ideas and there is no consensus that any are correct,” he adds.

This has been an ongoing controversy in the field for some time. “There is a very active debate over the issues,” Shacklett says. “I remember heated talks at Keystone meetings, almost arguments, over the validity and reproducibility of findings from one cohort to another.” In different cohorts the amount of possible HIV exposure or the route of transmission is variable, and Shacklett says this has made it hard to replicate findings or draw conclusions. Studies of discordant couples allow scientists to sequence the virus the HIV-uninfected partner is exposed to, which helps with the interpretation of HIV-specific T-cell or antibody responses. The HLA types of both partners are also known. But the level of HIV exposure among CSWs in high HIV-prevalence countries is usually greater because they have more sexual partners and use condoms less. This means that the HIV-resistant phenotype found in CSWs is more trusted than it is in HIV-serodiscordant couples and therefore more likely to have resulted from immunological factors within the exposed seronegative individual.

Collaborative study of ESNs

“I do think more collaboration is needed in the field and a consensus definition, as well as a consensus core set of assays, to define ESNs,” says Corey. “That would be helpful.” 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 viral replication at undetectable levels—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 whole genome association studies in these individuals.

“The challenge with exposed seronegatives is defining the phenotype,” says Walker. “Different people have defined exposed seronegatives in different ways and there are fewer quantifiable parameters that are available to define the group than with elite controllers.” In Walker’s opinion, the best group of ESNs to study would be hemophiliacs because only in that case is there clear documentation of exposure to HIV. “That is the purest phenotype,” he says (see Turning to Hemophiliacs for Answers, below).

There are now a few collaborations—some in full swing and others in early stages—that are trying to bring much needed clarity to the study of ESNs by conducting case-control studies in larger, more well-defined cohorts, applying more sensitive methods of laboratory evaluation, and developing a clearer definition of what qualifies an individual as an ESN.

At the same time, the role of innate immunity in HIV infection is now a growing field of study that could ultimately redefine the current understanding of how the immune system operates with regard to HIV. Researchers are now focusing more on the role of innate immune responses in acute HIV infection, with considerable attention being paid to natural killer (NK) cells and their receptors that suggest a prominent role in the progression and perhaps inhibition of HIV. “There is increasing activity in this area in the HIV field, fueled by exciting genetic findings and discoveries about NK cells in other systems,” says Andrew McMichael, an Oxford University immunologist and investigator with the Center for HIV/AIDS Vaccine Immunology (CHAVI).

CHAVI launched a study last year to address many of the conflicting or inconclusive data surrounding ESNs. The project is using more sensitive detection assays to try and nail down whether ESNs from serodiscordant-couple cohorts in Uganda and the UK harbor any detectable levels of HIV. This prospective study will enroll up to 702 participants, making it one of the largest cohorts of ESNs ever evaluated. Identifying extremely low levels of HIV by ultra-sensitive assays might suggest that there is some immune response, either innate or adaptive, against HIV that is capable of preventing the virus from establishing a productive infection.

If traces of HIV are discovered, the CHAVI investigators will then sequence the viruses found in the individuals who were exposed to HIV but remain uninfected, by the traditional definition, and compare it to the virus in the infected partner. Analyses will hone in on the cellular immune responses in the ESNs in particular. Additionally, researchers will conduct genetic analyses of the cohort to see which individuals may have polymorphisms known to be associated with HIV resistance.

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 McMichael is cautiously optimistic. He 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.”

Meanwhile other research groups continue the hunt for different factors that may be responsible for conferring protection against HIV. Ball is exploring the role of interferon regulatory factor 1 (IRF-1), a gene that belongs to a cluster of immunoregulatory genes thought to increase susceptibility to HIV by stimulating HIV transcription. However a recent study of 687 CSWs from the Kenyan cohort that Plummer’s laboratory has been tracking since the mid-1980s located a number of polymorphisms in IRF-1. It was the first report suggesting a viral transcriptional regulator, which is necessary for viral replication, might also contribute to HIV resistance (AIDS 21, 1091, 2007).

Everyone has slight variations of IRF-1 that may or may not impact the triggering of cellular immune responses, but with ESNs, the variations appear to be more complicated. Ball says the initial hypothesis was that ESNs would have higher concentrations of IRF-1 and therefore higher levels of cellular immune responses, but instead they found lower levels of IRF-1. Somehow, Ball says, even with reduced IRF-1 levels these ESN women still develop cellular immune responses against HIV.

IRF-1 seems to be one of these multifunctional proteins, almost a double-edged sword,” says Ball. “In one case, it is important in triggering cellular immune responses. Secondly it is exploited by HIV, like many other host factors, to help it replicate.” Ball’s lab is now trying to define the precise mechanism of IRF-1 in ESN women.

Meanwhile Jennes and colleagues conducted an extensive genetic analysis of a small cohort of CSWs from Cote d’Ivoire and found unusual interactions between killer Ig-like receptors (KIR) and human leukocyte antigen (HLA) molecules in exposed seronegative CSWs. The group focused on this piece of the genetic puzzle because KIR and HLA genes function as reins on the functions of NK cells. The findings suggest that KIR/HLA interactions, which previous studies associate with slow disease progression, may also influence viral transmission (J. Immunol. 177, 6588, 2006).

In the study, the women were compared to HIV-infected CSWs and HIV-uninfected female blood donors from the same west African cohort. The analysis revealed that CSWs in the ESN group more often lacked the HLA ligand genes for their inhibitory KIR genes, compared to the HIV-infected CSWs. Also, ESNs more frequently possessed a B KIR haplotype, which contains a high number of activating KIR genes, than HIV-infected CSWs.

Jennes suggests that when HLA molecules are subtracted from the immunological equation, a chain reaction occurs that ultimately frees the NK cells to eliminate HIV-infected cells before a systemic infection is established. But scientists have been unable to determine the roles activating and inhibitory NK cells play against HIV, and efforts to replicate results observed in the cohort from Cote d’Ivoire have been limited by sample size and even political tensions. Researchers left the site in Cote d’Ivoire in 2004 because of ongoing civil unrest and now Jennes’ group is tracking an ESN cohort of discordant couples in Senegal.

Jennes says if researchers can better identify the mechanisms that drive NK cells, it might be possible to manipulate them to block the KIR/HLA interaction. “It is important to continue with these studies,” he adds. “Knowing that HIV protection is probably multifactorial, different ESN classes may reveal different protective factors,” says Jennes. “Every new finding may provide a new mechanism.”

Turning to hemophiliacs for answers  

While researchers have primarily concentrated on commercial sex workers and discordant couples in their research of exposed seronegatives, hemophiliacs may provide the most interesting clues about what may be responsible for resistance to HIV.

An alarming number of hemophiliacs were exposed to HIV during the early 1980s after receiving transfusions of HIV-contaminated blood plasma to control their bleeding disorder. 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.

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 in 1984 with HIV and by his death in 1991 had already become an international symbol in the fight against AIDS.

But despite receiving regular injections of HIV-tainted blood products, not all of the hemophiliacs who were accidentally exposed to HIV became infected. This small number of individuals, classified as highly-exposed seronegatives, 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 are in the process of assembling a cohort of roughly 800 HIV-exposed, yet uninfected, hemophiliacs from the US, UK, Canada, Spain, and Germany who received Factor VIII concentrates between 1979 and 1984. The Factor VIII concentrates were derived from large pools of blood plasma collected from donors, some of whom were infected with HIV.

Managing to evade HIV infection is even more remarkable given the incredibly high risk of infection associated with this route of transmission. Jacques Fellay, a research associate at the IGSP Center for Population Genomics & Pharmacogenetics at Duke University, who is working on the CHAVI study, 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.

Researchers affiliated with this CHAVI study will conduct whole-genome analyses of 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 genotyping for this study will employ technology that offers an unprecedented level of genetic information—around 1 million single nucleotide polymorphisms—to locate variants of higher frequency associated with resistance to HIV in the uninfected hemophiliacs.

Andrew McMichael of Oxford University, and principal investigator on this CHAVI study, thinks the genetic research on HIV-uninfected hemophiliacs holds the most promise for solving the ESN puzzle. Goldstein hopes this work will translate into better interventions for preventing HIV transmission, as well as better therapies. “The more we understand how people naturally vary in resistance to HIV, the more information we have to try and develop therapies,” says Goldstein. —RM