HIV Hide and Seek

Caption: T cells killed by HIV that was hiding in the latent reservoir of a patient on antiviral treatment.

These findings were released in a series of journal articles this week. Reporting in Science, research teams from opposite coasts presented new data indicating patients receiving combination therapy with potent antiretroviral drugs can have all but a trace amount of HIV removed from their systems, with corresponding improvement in clinical condition, lasting as long as 30 months. However, the researchers discovered that some virus remains hiding in T-cells in a latent form. Moreover, this virus, if taken from the cells and introduced to other cells, can become active again.

The findings give hope for long-term survival with HIV infection. However, the results suggest that patients must continue combination drug therapy to prevent reactivation of the infection and active disease. Combination therapy utilizes "cocktails" of antiretroviral agents including protease inhibitors, zidovudine and didanosine.

"The bad news is we can't yet get rid of the virus entirely," says Robert F. Siliciano, M.D., associate professor of medicine at Johns Hopkins University, Baltimore, Md. "The number of immune system cells that remain infected with HIV declines only very slowly. But the good news is that as long as people infected with HIV keep taking the triple-drug cocktail, they have an excellent chance of surviving the infection for a long time, without developing symptoms of the disease."

The initial results of clinical trials with combination therapy showing that HIV levels could be reduced to barely detectable amounts breathed new optimism into the AIDS research community. Some researchers, including David Ho, M.D. of the Aaron Diamond Center, suggested that treatment might even stop active replication of the virus completely.

"Ho's idea of measuring the decline in the level of HIV in various reservoirs of the virus was a revolutionary way of looking at the disease," says Siliciano. "Our study identified a specific reservoir that may persist for a long time."

The Hopkins-led researchers studied 22 patients treated with triple-drug therapy for up to 30 months. The patients, who were treated at the Johns Hopkins AIDS Service, received close supervision to ensure they remained on their demanding treatment schedule. This prevented patients from suffering rebounds in their HIV levels and provided the team with a group of individuals with no detectable levels of HIV measurable by standard laboratory methods.

The researchers found that HIV was hiding in a type of  CD4+ lymphocyte, the same kind of cells targeted by HIV during the initial infection phase. Most CD4+ lymphocytes infected with HIV produce many copies of the virus before dying. But some of these infected cells survive, become inactive, and enter a resting phase. It is these resting CD4 cells that are a reservoir of  the virus that triple-drug therapy cannot eliminate, the researchers say.

"We studied patients whose viral loads had been undetectable for prolonged periods of time," says Joel Gallant,M.D., director of the AIDS outpatient clinic. "That kind of success in keeping levels so low was achieved by the combined efforts of our AIDS Service staff and highly motivated patients. That was important in our ability to study HIV in their resting CD4+ cells."

Detecting 'undetectable' amounts of HIV is no small trick. Hopkins researchers used a sophisticated cell-sorting technique called flow cytometry to produce very pure populations of resting CD4+ cells from the partially purified cells provided by Siliciano.

"Using the highly purified cells, we found that even after triple-drug therapy reduced HIV to undetectable levels, AIDS virus genetic material remained "hidden" inside "resting" CD4+ lymphocytes," says Diana Finzi, a graduate student working with Siliciano, and lead author of the study. "The team also showed that when the resting cells were stimulated to reproduce, the AIDS virus also replicated."

The multi-institutional study also found that "cocktail" therapy increased the numbers of healthy, uninfected CD4+ cells in all patients--strong evidence that the treatment was successful despite the potentially deadly reservoir of HIV in resting CD4+ cells.

"Fortunately, however, HIV in these resting cells doesn't appear to mutate into drug-resistant forms during triple-drug therapy," Siliciano says. "And because the virus mutates only when it's replicating, this lack of drug resistance suggests the virus is not replicating. So this is a strong argument for not taking these patients off triple-drug therapy."

Researchers from the National Institute of Allergy and Infectious Diseases (NIAID) reported related findings in the Proceedings of the National Academy of Science

"Our findings indicate that an inducible reservoir of HIV exists in infected patients despite prolonged treatment with highly active antiretroviral therapy (HAART), and suggest that the time required for eradication of HIV from the body, if indeed possible, may be considerably longer than previously predicted," says lead author Tae-Wook Chun, Ph.D., of NIAID's Laboratory of Immunoregulation (LIR).

The researchers isolated highly purified, resting CD4+ T cells from 18 patients, and used the polymerase chain reaction method to detect HIV DNA in an integrated form (i.e., inserted into the genes) in cells from each individual.

"Interestingly, levels of integrated HIV DNA were not significantly higher in untreated patients than in    HAART-treated individuals," notes Dr. Chun. "As others have postulated, this finding suggests that resting CD4+ T cells with integrated DNA do not decay rapidly in patients receiving HAART, and therefore may serve as a stable 'reservoir' of virus."

The NIAID team also found worrisome signs that that HIV was still reproducing, if slowly, even in treated patients. The investigators found unintegrated HIV DNA in cells from all patients.

"The presence of unintegrated HIV DNA suggests that a low level of viral replication may continue even in the setting of HAART. In HAART-treated patients, we found that levels of unintegrated HIV DNA were 28 times higher than integrated HIV DNA levels. This suggests that even when HIV is undetectable in the plasma, a low degree of viral replication contributes to the maintenance of a reservoir of HIV-infected CD4+ T cells," said Anthony S. Fauci, M.D., NIAID director and LIR chief.

"These results underscore the importance of developing more potent antiretroviral drugs, as well as treatment strategies that specifically target latently infected cells that serve as hiding places for the virus. Although our current armamentarium of antiretroviral drugs has served many patients well, at least in the short-term, more progress must be made in the area of HIV therapeutics if we are to speak of a cure for HIV disease," he said.

One new focus of research is likely to be aimed at finding ways to disable HIV-infected resting CD4 cells. The cells can live anywhere from 200 days to more than a decade.

The studies by Dr. Siliciano et al. appear in the November 14, 1997 issue of Science. The related research by Drs. Chun, Fauci and colleagues appear in the Nov. 25 issue of the Proceedings of the National Academy of Sciences (PNAS).