Reservoir Dogs
We've stopped active HIV infection. Can we control latent infection? The odds are long, but promising. By Anne-christine d'Adesky

HIV has often been compared to a kaleidoscope: every time we learn something new about this clever virus, the picture shifts to reveal a new pattern. For the past year, the spotlight has been hotly focused on the lastest frontier in HIV: latent infection. A growing number of studies show that people taking potent HIV drug cocktails (also called HAART: highly active antiretroviral therapy) who have "undetectable" levels of the virus in their blood aren't really HIV-free; instead, virus particles (called provirus) can be found lying inside dormant or latently-infected immune cells. These "sanctuary sites" include different long-lived cell types found in the lymphoid tissue, the genital tract, semen, lymph nodes, gut, thymus, spleen, eye, cerebrospinal fluid and brain.

The exact size of the latent HIV proviral pool isn't yet known, but it is thought to be only a fraction of the total amount of cells in the body. Studies show this pool is established during the first days of HIV infection and persists as a low-level infection. Unfortunately, several studies show that in people taking HAART therapy, some of these latently-infected tissue reservoirs contain virus particles that may be infectious, meaning that the virus can reproduce and infect other cells. Latent reservoirs like semen, the female genital tract or the brain can also contain different strains than those found in the blood. Some of these strains contain drug-resistant mutations.

There's more. In February, new research showed that some people who have tested HIV-negative to date on standard antibody tests actually have a latent HIV infection. Some of them have been repeatedly exposed to the virus through sexual contact with HIV-positive partners, but remain healthy today. At the recent Sixth Conference on Retroviruses and Opportunistic Infections in Chicago, a University of Washington team reported on 37 "exposed seronegative" individuals with what appears to be latent HIV infections that are kept in check by their immune systems. In some of them, the dormant cells harbor infectious virus particles.

The public health implications of these findings are important. Like genital herpes, HIV is a sexually-transmitted and blood-borne virus. If it lies dormant in people, can it be passed on to others? A latently-infected HIV reservoir could also serve as a continual source of reinfection for people on therapy, say experts.

How many HIV-negative people may really be latently infected? We don't know yet. Nor do we know the extent of latent infection in other groups of long-term non-progressors with HIV who have so-called "undetectable" or very low-level HIV infections based on tests of the amount of virus circulating in their blood. What is the status of hemophiliacs, infants born to HIV-positive mothers, and sex workers who appear to be genetically resistant to the virus (see "Designer Genes")?

While the news about latency has thrown up a roadblock to eradication of the virus, it's also revealed a promising new weapon: the immune system. A number of studies presented in Chicago showed that HIV-specific immune responses appear capable of controlling a dormant or low-level infection (see "Thanks for the Memories"). A subset of immune cells called CD8 T-cells, as well as CD4 T-cells, are the main players involved in this cellular immune control. A variety of immune-based therapies and vaccines appear to induce this protective immune response, although it's too early to tell how well or how long this immune control will last. Another question that remains is whether a combination of drugs and immune -boosters can actually attack or eradicate latently-infected cells and here, too, the news is cautiously optimistic.

Among the most exciting reports presented at Chicago was the first evidence of eradication of latent infection in three patients who recently stopped taking any HIV drugs. The study was conducted by a team from the laboratory of Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases (NIAID). They compared a group of chronically-infected people who were given a three-drug HIV antiretroviral (called HAART) cocktail for 20 months to a group that also took HAART plus intermittent doses of an experimental immune-boosting substance called interleukin-2 (IL-2) for 21.9 months. In the HAART-only group, the NIAID team found that all of them harbored infectious virus particles (provirus) inside latently-infected resting or "memory" CD4 T-cells taken from peripheral blood cells and lymph node samples. But in six of 14 people receiving HAART and IL-2, no infectious virus particles could be detected. In three of these six people, no traces of infectious virus could be found even after screening up to 360 million resting CD4 T-cells. Further tests of lymphoid tissue samples taken of the lymph nodes of two of these three people also failed to detect any infectious viral particles. After three weeks off all drugs, the virus had not rebounded in these three individuals.

"We cannot conclude that we have eradicated all HIV in the body," cautioned NIAID's Tae-Wook Chun, the lead author of the study, adding, "It has not been ruled out that other body compartments may contain virus." But he said the results were very encouraging. Although the mechanism of eradication of latent HIV is still unclear, prior NIAID studies showed that IL-2 can flush dormant virus from resting T-cells, where it can then be killed by HIV antiviral drugs. Another molecule, IL-12 can boost memory T-cells.

In other news, Kendall Smith of NYU/Cornell found that the CD4 T-cells of people with chronic HIV infection have a defect in IL-2 production. New studies are underway there to see if a daily replacement dose of IL-2 will work as post-Haart maintenance therapy for latent infection.

Other individuals are testing the idea of "drug holidays" and at least one--the famous "Berlin patient"--quit his HIV drug cocktail over two years and remains healthy, with a broad, but specific immune response to HIV that appears to be controlling any active infection. But the Berlin patient is not completely HIV-free: infectious traces of HIV particles have been found, indicating that he is still latently infected. Before going off therapy, he was taking the cancer drug hydroxyurea as part of his drug regimen. It is a drug that acts differently from other anti-HIV drugs and can target resting T-cells. Research is ongoing to measure hydroxyurea's effectiveness against latently-infected cells.

HIV's First Steps
Much of the groundbreaking work on HIV reservoirs was done in the early 90s by Ashley Haase, a leading virologist at the University of Minnesota, who has pioneered new techniques to measure latent HIV infection in tissue compartments. Haase and other researchers are working with SIV, the simian (primate) sister virus to HIV, to track where the virus goes during the first days of acute (primary) infection and how it actually infects cells in various tissue reservoirs. As predicted, the virus infects the cells that establish latent SIV reservoirs within a matter of hours and days. This work is particularly relevent since primates like chimps are natural hosts for SIV (see, "Lucy, Meet Marilyn"), but they don't get sick. By studying how the immune responses of chimps control SIV, we might mimic that protection in humans.

"The central issues are: How can these viruses persist in the face of the immune system?" asked Haase, who recently shifted his focus to developing an HIV vaccine. "Why do they eventually cause disease? Why does it manifest so slowly over months and years? And now, of course, the 64-million-dollar question is: How can you prevent it and how can you treat it?"

In a recent meeting in Delaware, Haase presented new data on sexual transmission of SIV during acute infection. In one study, the virus first targeted the endocervical region of the female genital tract in primates, and by day three, had infected lymphocytes and other cells in the lamina propia, a layer of cells underneath the epithelial cell layer of the cervix and vagina. This is where star-shaped dendritic cells can be found; these cells then pass the virus on to lymphocytes which carry it to the lymph nodes. "This is the first evidence of productive viral infection [during the acute phase]," Haase said. In his study, Haase found that by day four, more scattered cells were infected, including macrophage cells. Local lesions had also developed in the genital mucosa. By day seven, there was up to a seven-fold increase in the infection of cells in the lamina propia and, by day 12, an 800-fold increase.

By then, the vagina, cervix, uterus and draining lymph nodes were productively infected. So were the thymus, spleen, liver, and bone marrow, although there was less sign of infection in these tissues. In Haase's study, the cerebrospinal fluid was not infected by day 12. However, another researcher, Sean Clarke, has found signs of brain tissue inflammation at day seven in his studies of acute SIV infection. Others have found early signs of the virus in lymphoid cells in the gut.

Haase also thinks HIV can infect unactivated T-cells which make up part of the latent pool. But he's encouraged by the latest reports regarding immune control of the virus and the prospects for a vaccine in the future. "I think we're on the right track. We just have to keep pushing ahead."

Drug Alerts IF YOU'RE HIV-POSITIVE AND ON METHADONE, HEADS UP! A new study has found that the anti-HIV drug Viramune (nevirapine) may cause methadone withdrawal. In a recent study, physicians from the Yale University AIDS Program reported seven cases of methadone withdrawal that occurred within four to eight days after people started taking Viramune. Symptoms included nausea, chills, sweats and hypertension. When doctors upped the methadone dosage-usually to around 150 mg. per day-the symptoms went away. Methadone and Viramune are both processed in the body by a liver pathway called CYP3A4. Viramune is a potent inducer of the CYP3A4 pathway that causes the metabolism of methadone if the drugs are taken together. So higher methadone doses are needed to maintain adequate levels of the drug in the blood. Drug-drug interactions could take place with other drugs, and the Yale group is already enrolling a study of Sustiva-methadone interactions. For now, methadone users and their doctors should monitor symptoms carefully, especially during the first weeks of a new drug regimen. "Drug treatment programs need to be prepared to provide rapid escalation of methadone doses," says Yale researcher Frederick Altice. FINALLY, SOME GOOD NEWS ABOUT NORVIR: Abbott Laboratories has developed a new, problem-free Norvir (ritonavir) capsule that does not require refrigeration. At press time, the company was waiting for a green light from the Food and Drug Administration to start marketing the drug. -Emily Bass