RUSSELL found out he was infected with HIV in 1988. The news came as no great surprise – his boyfriend had recently been diagnosed HIV-positive and they had often had unprotected sex. Russell recalls that he “went into a little bit of a spiral, with drink and drugs”. But eventually Russell came to terms with what seemed at the time to be a death sentence. He began to prepare for his eventual demise from AIDS.
As the years went by, Russell, who now lives in San Francisco, watched HIV-positive friends fall ill and die. Yet he remained free from AIDS symptoms, and his CD4 cells, the immune cells that HIV targets, stayed at healthy levels. Eventually, Russell began to wonder what was so special about him. “I started putting it all together,” he says. “I’ve been HIV-positive for nearly 15 years. I have never been on HIV drugs, never sick, and my blood counts are good. Why are they getting sick and I’m not?”
The explanation lies in the fact that Russell’s immune system is a little bit different from most people’s. All this time it has been keeping him alive when by rights he should be dead. And Russell is not the only one. Without drugs the vast majority of people infected with HIV die within 10 years, but a fortunate few, called “long-term non-progressors”, somehow keep the virus at bay. Equally intriguing are the tiny minority of people who never become infected at all despite repeatedly having unprotected sex with HIV-positive partners. They are known as “exposed-uninfecteds”.
Advertisement
Not surprisingly, scientists around the world are fascinated by these two groups. Natural genetic variation in people’s immune systems has meant that these lucky individuals are safe from HIV, for a variety of reasons. And if we can work out what makes them so special, we may be able to translate that knowledge into better anti-HIV drugs, or even a vaccine.
One of the first protective mechanisms to be well understood was discovered in 1996, in two men who were HIV-negative despite frequent unprotected anal sex (Cell, vol 86, p 367). Bill Paxton, a young molecular biologist at the Aaron Diamond AIDS Research Center in New York City, showed that both men had the same mutation in an immune cell molecule called CCR5. This protein normally resides on the surface of CD4 cells, along with the CD4 protein after which they are named. HIV must bind first to CD4 and then to CCR5 before it can get into the cell (see Diagram). Paxton’s two patients produced shorter versions of CCR5, with 32 amino acids missing from the middle. As a result, the defective CCR5 molecules never made it to the cell surface. Could the missing protein be what was protecting the two men?
It is now known that about 1 per cent of Caucasians are “homozygous” for this particular mutation (in other words, both their two copies of the CCR5 gene have the mutation), and 18 per cent are “heterozygous” (with one mutation and one normal copy). To be protected against HIV you need two copies of the mutation – there were no homozygous carriers of the mutation in a subsequent study of 2000 HIV-infected people. Since then a few infected homozygous carriers have been found, but they seem to carry rare virus strains that don’t need to bind to CCR5 to infect cells.
In its normal role, CCR5 acts as the receptor for several immune chemical messengers known as chemokines. Yet surprisingly, neither heterozygous nor homozygous carriers of the CCR5 mutation have health problems stemming from their lack of this molecule. This suggests that blocking CCR5 could be a useful strategy for new anti-HIV therapies. There are several such drugs being developed, including Tak-779 from the Japanese company Takeda, due to start clinical trials soon. Schering-Plough tried out another such compound called SCH-C in clinical trials, but it caused irregular heartbeats in some volunteers. The company is now working on a related agent, called SCH-D.
Intriguing though the CCR5 research is, it cannot explain most cases of exposed-uninfecteds or long-term non-progressors. The mutation is almost never found in Africans or Asians, and many Caucasians who resist HIV have two normal copies of the CCR5 gene. What’s going on with all these other people?
Scientists have made a lot of progress studying prostitutes in the slums of Nairobi in Kenya. These women frequently have unprotected sex with HIV-infected clients – an average of 60 to 150 times a year, it’s been calculated. Frank Plummer, a microbiologist at the University of Manitoba in Canada, found that between 15 and 20 per cent of them were HIV-negative, and those who had been in their profession for five years or more were much more likely to be HIV-negative than women new to the job. The veterans had survived the grisly forces of natural selection. As the majority succumbed to AIDS and died, only those resistant to infection remained.
Plummer first announced his findings in 1993 at the biennial world AIDS congress in Berlin, triggering a surge of global interest in the Nairobi survivors.
In 1995 a team led by Sarah Rowland-Jones, an immunologist at the University of Oxford, began working with Plummer. Their research suggested that a type of immune cell called cytotoxic T lymphocytes (CTLs) could hold the answer (see Diagram). The role of CTLs is to kill cells that have “foreign” proteins on their surface, such as cells that are cancerous or infected by viruses. All HIV patients initially produce CTLs that recognise and kill infected cells, although their numbers ultimately wane.
The researchers showed that despite having no virus or HIV antibodies in their blood, over half the uninfected prostitutes had CTLs that recognised the virus (The Journal of Clinical Investigation, vol 102, p 1758). Somehow their CTLs, unlike everyone else’s, were succeeding in defeating the virus. The women are not invulnerable, though. If they take a break from sex work their CTL numbers dip, leaving them susceptible to infection.
These women are not the only group who seem to owe their lives to CTLs. Rowland-Jones, along with workers at the Universities of Nairobi and Washington, has also been studying 500 uninfected babies of HIV-positive women in Nairobi. The women took the anti-HIV drug zidovudine (AZT) in their last month of pregnancy, but could not be dissuaded from breast-feeding despite the risk they would pass on the virus to their babies. In an unpublished study, the researchers found that after birth, 20 per cent of the uninfected babies had CTLs that recognised HIV.
Yet a third group of exposed-uninfecteds has provided backing for the CTL theory. Rowland-Jones has been studying 22 British couples where one partner is HIV-positive but the other is not, despite having had regular unprotected sex for three years or more. A study submitted for publication has shown that every one of the uninfected partners has CTLs that recognise the virus.
So why do these lucky few people have CTLs capable of keeping HIV at bay? The reason seems to involve the interactions between CTLs and virus-infected cells. CTLs don’t take an interest in any old cell-surface proteins, only those “presented” to them – cut up, and bound to special molecules on the virus-infected cell called the major histocompatibility complex or MHC (see Diagram). Different people have different MHC molecules, depending on their genes, and an individual’s MHC type is thought to dictate which bits of which viral proteins are shown to their CTLs.
Rowland-Jones’s team found that most of the uninfected Nairobi prostitutes have at least one of a special group of five MHC genes. These MHC molecules seem to do well at triggering a good CTL response. It might be because they are expert at flagging up proteins for the CTLs’ attention or alternatively that the proteins the MHCs select for presentation are particularly stimulating. Evidence for this last theory comes from recent research showing that the surviving prostitutes’ CTLs target a viral enzyme called reverse transcriptase (The Journal of Clinical Investigation, vol 107, p 1303). In contrast, most HIV-infected people’s CTLs recognise viral structural proteins. Rowland-Jones says, “We think this means there are some responses that are better able to protect than other CTL responses.”
These findings have not yet been replicated in other groups of exposed-uninfecteds, so no one yet knows whether they are the main factor responsible for resistance to HIV. But they are of enormous interest to vaccine researchers. In the mid-1990s, scientists began to think that a successful HIV vaccine would have to be able to stimulate a CTL response. This idea was partly driven by the findings about the Nairobi prostitutes, and partly by the lack of success with more traditional, antibody-based vaccines. Because of her findings, Rowland-Jones thinks that the most effective vaccine would trigger CTLs against reverse transcriptase. Several such vaccines are currently in small-scale human trials, and researchers are eagerly awaiting their results, due in the next few years, although most now believe that CTL vaccines will have to be used in conjunction with antibody vaccines.
Another researcher studying the Nairobi sex workers, Rupert Kaul of the University of Toronto, believes that as well as CTLs, a different part of the immune system is important. He has found that the uninfected women have HIV antibodies in the mucous membrane lining their vagina and cervix. Researchers have long known that HIV-infected people have antibodies to the virus in their blood that cannot defeat the virus – indeed, the standard test for HIV infection actually looks for these antibodies. Mucosal antibodies, on the other hand, seem to present a powerful barrier to infection, at least in the Nairobi women. As a result, several vaccine researchers now think that future vaccines should aim to generate mucosal antibodies.
One of these groups is testing a strategy that combines two separate lessons learned from exposed-uninfecteds. Thomas Lehner, an immunologist at Guy’s Hospital in London, has a team working on a vaginal vaccine that triggers mucosal antibodies, not against the virus, but against the CCR5 molecule so crucial for the infection of CD4 cells. The vaccine also includes fragments of a bacterial protein called hsp70, which triggers the release of the chemokines that normally bind to CCR5. So both antibodies and chemokines get in the way of CCR5 binding to HIV. In June 2002, Lehner presented a study at the Experimental and Cellular AIDS Research Conference in Genoa, in which this strategy seemed to protect five out of eight macaque monkeys from infection. This compared with none out of four in the control group.
So could any of these mechanisms be keeping long-term non-progressors like Russell alive, too? Bruce Walker, a physician and AIDS researcher at the Massachusetts General Hospital in Boston, believes that CTLs could be important. Walker, who was one of the first to show that all HIV-infected people have a strong CTL response, and his colleague Marylyn Addo, have been gathering data on 75 HIV-positive individuals who have stayed healthy without drug therapy. Walker has not yet found that their CTLs target different viral proteins from those of most HIV patients (unlike the exposed-uninfected Nairobi prostitutes). He thinks this could be because their CTLs are more effective for other reasons, or perhaps his studies were simply flawed.
The CCR5 mutation could be playing a role in this group too. People who are heterozygous for the mutation have fewer than normal CCR5 molecules on their CD4 cells, and this could explain why those who do become HIV-infected take longer to develop AIDS. But this mutation does not seem to protect against AIDS indefinitely, as in Russell’s case.
For most long-term non-progressors, researchers believe another type of immune cell is more important, the CD8 cell. When CD8 cells encounter virus-infected or cancerous cells, they mature into cytotoxic CTLs, and it is this later stage in their development that has traditionally been thought the most important. But the CD8 cells themselves may be saving the lives of people like Russell. They seem to secrete a mystery molecule, the identity of which is hotly debated by three of the world’s most prominent HIV experts.
Jay Levy, a high-profile AIDS researcher at the University of California, San Francisco, has been on the trail of the elusive protein for nearly 15 years. He found that CD8 cells from long-term non-progressors could block virus replication in CD4 cells without killing them. The CD8 cells seemed to be switching off virus replication by releasing a soluble substance that Levy dubbed CD8 anti-viral factor, or CAF.
In an effort to identify the molecule Levy has studied about 60 volunteers who have remained healthy for more than 10 years, despite taking no anti-HIV drugs. By growing their CD8 cells in the lab, Levy’s team showed that CAF appears to bind to part of the HIV genome that triggers replication (Proceedings of the National Academy of Sciences, vol 92, p 2308). They have narrowed down the search to 44 proteins, and Levy hopes to pin down the factor “soon”. His aim is to use CAF as the basis for new anti-HIV therapies that are more potent or less toxic than existing ones.
But last September Levy appeared to have been pipped to the post by David Ho, director of the Aaron Diamond AIDS Research Center (named Time magazine’s 1996 Man of the Year, for helping develop anti-HIV drug cocktails). Ho claimed in Science (vol 298, p 995) that CAF was a mix of three proteins called defensins, already known as natural antibiotics produced by another class of immune cell called neutrophils. His team had isolated the compounds from three non-progressors’ CD8 cells, cultured in the lab with HIV.
The press said Ho had solved the mystery of long-term non-progressors, but the reality may be more complex. Levy, for one, is adamant that Ho is wrong. Several groups including his own had tested defensins for CAF activity, and drawn a blank. Levy says defensins do not bind to and block the same region of the HIV genome as CAF, nor are they potent enough to explain all his patients’ CAF activity. “Defensins are not very effective,” he says. “They work if you put in a small amount of virus, but not a large amount. Our CAF works against 10 times as much virus as the Ho group used.”
Robert Gallo, the co-discoverer of HIV, claims both Levy and Ho are wrong. He says non-progressors’ CD8 cells secrete many different chemicals that cause CAF activity, several of which have already been identified. His team is preparing a paper that will reveal at least another two compounds. “There is no one magic molecule,” he says.
It looks like we’ll have to wait for Levy and Gallo to announce their results before the confusion is cleared up. If several compounds are responsible for CAF activity, perhaps all of them could provide leads for developing new and better anti-HIV drugs.
So where does this leave patients? Russell joined Levy’s register of long-term non-progressors two years ago. The research team found that the secret of his self-defence seemed to lie in CAF-secreting CD8 cells, rather than unusual CTLs or CCR5 receptors.
The exact identity of CAF may still be unclear, but that doesn’t bother Russell. He has started to tentatively make future plans. He says: “Having Dr Levy tell me ‘Yes, you have this protein in your body’ really strengthened my hope for a long-term future. I have health insurance, I started putting money into a retirement account. I’m amazingly lucky.”
