TAGline/Volume 4 Issue 11

Contents:

1. The Worldwide Epidemic: Global AIDS Picture Worsens
2. Global Infectious Causes of Death (Table)
3. The Future of AIDS (Table)
4. The Twilight of Eradication: Infectious HIV Persists After Up to 2 1/2 Years of Triple Drug Therapy

As people in the North America, Western Europe and Australia begin to live longer with HIV and AIDS (and even begin to savor the first wafts of what might turn out to be a kind of cure), the bitter contrast between the haves and the have nots grows all the more stark. The United Nations reports that a staggering 16,000 people are newly infected with HIV each day, and a total of 30 million people are now estimated to be living with HIV worldwide. TAGs Spencer Cox has transcribed the dire statistics straight from the source. His report from this past autumns ICAAC plenary reminds us how far weve yet to go -- and offers the first etchings of a road map, even while those living the nightmare scenario see only political posturing and empty promises.

Despite last years wistful pronouncement of "The End of AIDS," observers world-wide report that the global HIV pandemic continues to pick up steam. In September, Dr. Peter Piot of the United Nations Joint Program on HIV/AIDS told delegates to the 37th Interscience Conference on Anti-microbial Agents and Chemotherapy (ICAAC) of the toll which HIV continues to extract world-wide.

Piot began his talk with the startling statistic that worldwide there are an estimated 16,000 new HIV infections each day (1,600 of them children); 90% of cases occur in developing countries; 40% among women and greater than 50% in persons between the ages of 15 and 25 years of age.

The front line of the epidemic today is quite clearly Asia. For example, there are four million HIV-infected persons in India (most of these individuals infected within the last two to three years). Seroprevalence among Bombay sex workers is estimated to be 51%; STD clinic patients, 40%; pregnant women, 3%. In Sub-Saharan Africa, AIDS has been concentrated primarily in the Southern, East Coast and Central African countries, with pockets of disease in the Ivory Coast. HIV prevalence among pregnant women ranged from 17%-40% in these countries, the majority of mothers infected between the ages of 15 and 25.

Among Latin America countries, Brazil has the greatest number of HIV-infected persons; as many as 23% of gay men, 27% of sex workers and 60% of injection drug users being HIV-infected. In the state of Sao Paulo, the economic powerhouse of Brazil, AIDS is now the leading cause of death -- even though Sao Paulo state now pays for triple combination therapy for "Paulistas" with an AIDS diagnosis.

The projected life expectancy has been reduced by as much as 25 years in countries in Sub-Saharan Africa where HIV is endemic. The economic and social impact of the epidemic in these countries will be devastating. AIDS is reversing any gains in human development made in this part of the world prior to the onset of the epidemic.

While Africa continues to have the most severe epidemic in the world, Piot warned that we are poised for a "new epidemic explosion" in Eastern Europe and parts of the former Soviet Union, where needle use and heterosexual transmission are the most frequent routes for the disease. A recent series in Newsday by Pulitzer-prize winning journalist Laurie Garrett noted that as a result of the collapsed public health system in nations of the former Soviet Union, infectious disease experts were forced to stand by as HIV infection rates increased by up to 300% annually. In addition, Piot warned, Asia -- and particularly India -- comprise the "front line of the epidemic," with skyrocketing rates of new infections.

Amidst all the dire statistics, Dr. Piot explained that significant gains had been made in the prevention of new infections in several parts of the world. In Switzerland, Thailand and Uganda, aggressive sex education (including information on sexually transmitted diseases and condom use) has been met with initial success. In Uganda, for example, rigorous prevention campaigns in prenatal clinics had reduced the HIV seroprevalence rate in pregnant women from 21% in 1990-1993 to 15% in 1994-1995. In Thailand, an aggressive behavioral modification among pregnant women and 21-year old military conscripts from 1989-1995 has led to a significant reduction in the rate of new seroconversions in these populations.

According to Piot, the UN program is also attempting to improve access to generic drugs in the developing world, and to assist in bringing antiretroviral therapy to those countries whose public health infrastructures and economies would permit their use. (This claim, however, sparks fury in the eyes of our Venezuelan ex-patriate translator -- who is also the co-founder of a group (Aid for AIDS, 212.358.9715) that collects and distributes AIDS medicines to South American and the Caribbean PWHIVs. So apparently, alot of this is still just politically correct posturing and hollow lip service.) Piot also says that by working with the World Bank, the UN is making an effort to improve the public health infrastructure in other countries. The World Bank, it seems, is increasingly concerned that the AIDS epidemic is posing a significant barrier to economic development of "third-world" countries.

In his closing summary, Dr. Piot summed up the current situation with AIDS as being "very, very grim." "None of us could have predicted this 15 years ago," he said. "The AIDS epidemic," he continued, "is far, far from over. And we will have to cope with it for many generations to come."

Many observers believe that the only way that the world-wide epidemic can be brought under control is through the development of a vaccine to prevent infection. Dr. David Baltimore, soon to leave MITs Whitehead Institute to become the president of California Institute of Technology (Cal Tech) and chairman of the NIH AIDS Vaccine Research Committee (AVRC), reviewed the current approaches to vaccine development and the hurdles which still need to be overcome before a safe and effective candidate vaccine can move forward into large-scale human trials.

In a quick retrospective homage to effective vaccines strategies, Baltimore noted that some were successful because they stimulated T-cell memory; while others were successful because they induced an antibody-mediated response. The best historical models for antiviral vaccines, Baltimore explained, are the live, attenuated vaccine (à la the Sabin polio vaccine) and the whole, killed vaccine (à la the polio vaccine of Jonas Salk). Of the seven current approaches to the development of an HIV vaccine, the best results to date have been achieved with a live attenuated vaccine using the simian immunodeficiency virus (SIV) in monkeys. Baltimore was quick to point out, however, that even though some of these animals resisted infection with a highly virulent strain of SIV subsequent to vaccination, the vaccine itself caused disease in some of the animals.

Earlier approaches to HIV vaccines, which used recombinant subunit proteins of HIV (e.g., gp120 and gp160) have not proved effective in producing neutralizing antibodies to wild-type HIV. Other approaches to vaccine constructs include live vectors (pox and non-pox), pseudovirions, naked DNA, peptide epitopes and whole, killed viruses.

At this point in the presentation, Dr. Baltimore departed from the specifics of vaccine constructs to raise the practical question of "What could a vaccine do?" Specifically, if the gold standard -- sterilizing immunity -- is not currently a realistic expectation with an HIV vaccine, would a vaccine that reduced the initial multiplication of HIV (and resulted in a lower viral "set point") enable the maintenance of a long-term low viral load which would lead to more long-term non-progressors -- and a significant reduction in new transmissions?

In summary, Dr. Baltimore enumerated the requirements for a successful vaccine program; he especially emphasized the need to integrate the latest knowledge of HIV into the current vaccine development programs and that human testing of vaccine candidates "is integral." Baltimore also pressed for better organization among government, university and industry entities. He then ended with a final plea for "creative, new ideas."

In addition, Dr. Baltimore noted that the current government funding for the United States HIV vaccine effort is in excess of $100 million a year, and that the President of the United States and Secretary of Health and Human Services, Donna Shalala, have promised that "more funds could be found if they were needed." At the present time the National Institute of Allergy and Infectious Disease and National Cancer Institute are committed to constructing a joint vaccine laboratory which is expected to be housed under a single roof at a vaccine-dedicated site by the year 2000. A search for a Director of the program is currently underway. Baltimore closed by stating that if a preventive vaccine is not developed soon, "it will not be a pretty future" for the world and we must "redouble our efforts to make vaccine efforts work."

Can HIV be eradicated from a human host? Can currently available combination therapy approaches eliminate HIV from every last cell within a reasonable amount of time (short of the lifetime of the host)? This provocative question was first raised before a global audience by David Ho at Vancouver in July 1996. After months of rumors and an accumulation of presentations at St. Petersburg in June, at Istanbul and Toronto in September, and at Hamburg in October, three papers published in Science and the Proceedings of the National Academy of Sciences (U.S.A.) in November 1997 definitively laid to rest the idea that HIV could be eradicated within three years using currently available triple combination antiretroviral therapy. Mark Harrington helps us make sense of it all with this sobering and long feared year-end report.

The problem is that current combination therapies, no matter how potent, only act against the virus when it is replicating. If there is a significant number of cells in which the virus is resting dormantly -- integrated into the nucleus -- the drugs will be unable to target these infected cells which, when activated, will be able to reignite the infection -- no matter how long someone has suppressed new virus production with combination therapy. Thus, the last sixteen months have seen an intense search for a cell population -- the so-called "third compartment" -- in which HIV might persist in dormancy, unresponsive to combination therapy. These experiments built on work first shown by David Ho at Vancouver in July 1996, and followed up by Ho and Los Alamos Alan Perelson at the Fourth Retrovirus Conference in January 1997, which estimated the time it might take to eradicate HIV from an infected human host. These were the first rational, quantitative estimates based on in vivo data on viral and host cell kinetics, and they assumed that there were just two cellular compartments in which HIV dwelled:

1. A primary compartment consisting of activated, HIV-infected CD4 T cells actively producing virus and rapidly being killed either by HIV or by the immune response, which turn over rapidly in the body with a half-life of a 1.4 days, and produce over 90% of the new virus population;

2. A second compartment -- possibly consisting of latently infected CD4 T lymphocytes or macrophages -- which, when activated, produce virus, turning over with a slower half-life of one to four weeks, and producing less than 10% of the new virus population.

Regardless of the number of HIV-infected cells in the body, if these were the only two compartments in which HIV dwelt, the time-to-eradication could be estimated. If the half-life of the second compartment was four weeks, the time-to-eradication would range from two to three years, depending on the number of HIV-infected cells. The entire model, however, would break down if there proved to be a third compartment. If one existed, it would be important to measure whether it, like the first two compartments, was reduced in size by triple combination antiretroviral therapy and, if so, by how much, and how fast. Only then could one reliably estimate the time-to-eradication using current therapies. Now, it turns out that there is a third compartment. It consists of latently infected, resting, previously activated memory CD4 T lymphocytes with integrated proviral HIV DNA in their nucleus. These cells are a small but crucial reservoir for HIV and, at least after two years of triple therapy, do not appear to diminish over time.

In other words, the third compartment does not have a half-life of decay, because it does not appear to decay at all. If this holds true, then we can no longer expect the current therapeutic approaches by themselves to lead to HIVs eradication -- which is really just a fancy word for cure. New approaches, however, could be developed to target this third compartment, possibly speeding up the turnover of these cells and leading to their elimination. Such approaches remain speculative, but some are rapidly moving towards preliminary clinical trials. In the meantime, however, no one should begin antiretroviral therapy with the belief that triple (or even quadruple) antiretroviral therapy is likely to eradicate HIV infection. The best we can hope for, at this time, is chronic suppression of HIV and thereby preventing disease progression, protecting the immune system from further damage, and avoiding the development of drug resistant HIV strains.

HIV Infection, Reverse Transcription, CD4 Cell Activation, DNA Integration

HIV DNA can be detected either in an unintegrated form within the cell cytoplasm, or integrated within the cells DNA in the nucleus. Robert Siliciano, of Johns Hopkins University, and colleagues developed an assay earlier this year to distinguish the two types of HIV DNA. They found that if a cell is infected while in a resting state, reverse transcription may occur, but the new HIV DNA will not be transported into the nucleus for integration. If the cell is kept in a resting state for six days, cellular proteins will degrade the HIV DNA and integration will be aborted. Thus, cellular activation is required for integration and productive infection of a cell.

Most cells which experience activation and integration then undergo mitosis (cell division), in the process of which HIV copies are made. The cell is either killed by direct cytolytic effects of the virus or by antiviral effector mechanisms of the immune system (cytotoxic T lymphocytes ("CTLs"), antibodies, macrophages). However, a small minority of infected cells may survive with integrated HIV DNA. Of these, only a small subset are replication competent. Of one million white blood cells taken from an infected individual, 1,000-100,000 cells may have detectable HIV DNA provirus, but only 10-100 of these cells will have integrated provirus and only 1-10 of these will be replication competent. (Other cells, however, may remain targets for the immune mechanisms mentioned above if any viral proteins, even defective ones, are made.)

Discovery of the Third Compartment

All three teams took a similar approach in searching for the resting, latently infected CD4 cells. First they eliminated B cells, macrophages, natural killer cells and CD8 cells. Activated CD4 cells were eliminated to make sure the sample cells were truly resting. Then they stimulated the resting T cells into activation thereby "turning on" any replication-competent HIV DNA which may reside within this cell population. They measured HIV within the activated, previously resting cells in several ways: by looking for proviral DNA in the nucleus, or unintegrated DNA in the cytoplasm; by amplifying proviral DNA with PCR; and -- importantly -- by culturing the cells to see if replication-competent HIV could be grown from the cells. It could.

In the largest study, led by Silicianos team, Finzi and colleagues examined cells from 22 individuals treated with tripe therapy for up to 30 months. Subjects had to 1) be on highly active antiretroviral therapy ("HAART"); 2) be highly adherent; 3) rapidly have achieved "undetectable" plasma HIV RNA levels (<200 RNA copies/mL by RT-PCR); and 4) have remained "undetectable" for the duration of the study. Replicating virus was detected in all 18 cases when sufficient resting CD4 cells were isolated (they could not be isolated from four individuals with low CD4 counts). Very few resting cells had integrated HIV DNA which could be stimulated into active replication -- just 0.2 to 16.4 infectious units per million resting cells. But what proved to be the most discouraging finding is that when, in a cross-sectional analysis, the number of infectious cells in individual patients were correlated with their time on HAART, there appeared to be no relationship between time on HAART and number of infectious cells -- and thus no apparent decay in the third compartment. Serial samples were taken from only one patient -- whose level of infectious cells actually rose slightly between months four and eight. Thus, while the third compartment appears to be small, it also appears to be very stable.

To see how old the provirus in these resting cells was -- and whether it had evolved resistance to combination therapy -- the investigators sequenced the protease genes. The good news is that (in patients with plasma RNA levels continually suppressed below 200 copies/mL), the proviral HIV did not display mutations associated with drug resistance. This indicates that the cells had likely been infected prior to the initiation of triple therapy, and that viral evolution appeared to have greatly slowed down -- if not stopped altogether -- after the initiation of triple therapy. (Some individuals had mutations from previous experience with monotherapy.) "Taken together," the authors explain, "these results demonstrate that purified resting CD4+ T cell populations from patients on HAART harbor replication-competent virus that in some cases is cytopathic." (Given the inherent vagaries of translating results from in vitro experiments to in vivo conditions, however, the question of whether these latently infected T cells can be reactivated to produce virus in treated individuals remains, for now, unanswerable. GMHCs treatment wonk Dave Gilden, writes in a second exhaustive analysis (Treatment Issues, November 97) of the 3 eradication papers, "It is not clear, though, that any particular therapy is needed for latent cells. It is possible that as the immune system recovers in the nearly HIV-free environment, anti-HIV cytotoxic lymphocytes will be able to quickly kill the few cells with active HIV infection and prevent spread of the virus;" to which Diamonds Ho replies matter-of-factly, "I wouldnt bet on it.") The Johns Hopkins investigators are understandably wary to come down on either side of this issue and warn only that "the existence of a small but relatively stable compartment of latently infected cells should be considered in deciding whether treatment should be stopped in patients with no other evidence of residual virus."

In the second paper, Joseph Wong, Doug Richman and colleagues conducted a similar but smaller study at U.C. San Diego. They isolated resting CD4 T lymphocytes from six individuals who had achieved undetectable (<50 copies/mL) viral load for up to two years on HAART. As in the previous study, infrequent cells with integrated proviral HIV DNA were found, their protease genes sequenced, and no genotypic evidence of protease resistance identified, suggesting that the third compartment is stable over up to two years of HAART and that HAART prevents viral evolution and the development of drug resistance. In summary, there is evidence for a third phase of viral persistence among latently infected CD4 cells with integrated provirus. These cells are predominantly CD45RO+, and their isolation requires CD8 cell depletion and CD4 cell activation.

The half-life of this third compartment is likely to be many months to years, but the virus isolated from these cells taken from patients with very low HIV RNA levels (<50/mL) after two years of treatment showed no evidence of viral evolution or the emergence of drug resistance. Thus, hopes for short term eradication of HIV from an individual within three to six years are "clearly unrealistic," according to Richman, unless we can figure out a way to shorten the third phase, perhaps by stimulating the latent infection out of the CD4 cells. "Conceivably," he continued, "one could eradicate in ten years or more." Richman joked that inducing toxic shock might be one way of shortening the third phase and concluded that, "It would be wrong to be discouraged about anything but the prospect for short term eradication; we have achieved suppression of viral evolution, which bodes well for maintenance."

The third paper, from Faucis lab published by Chun et al. in P.N.A.S., also found persistent low level RNA production despite triple therapy. Eighteen HIV-infected individuals on various antiretroviral regimens had leukopheresis (blood extraction) at the NIH, and their cells were analyzed for HIV RNA at baseline and, in thirteen patients, after a median of ten months of "HAART." They were able to isolate resting memory CD4 cells with integrated, replication-competent HIV provirus at low frequencies from 11 of the 13 patients on HAART. Some of these viruses (in cell culture) could induce syncytia, indicating virulent cytopathicity. They also found low levels of unintegrated DNA in some cells, indicating recent infection and reverse transcription. The amount of unintegrated DNA was higher in patients whose viral load was still detectable. In contrast to the Siliciano and Richman paper, the NIAID study "suggests persistent active virus replication in vivo," but this might simply reflect the fact that viral suppression in the NIAID study was less stringent ("undetectable" in the NIAID study was defined as <500 copies/ml) than in the other two studies (Siliciano, <200; Richman, <50/mL).

In addition, the NIAID team found that "levels of integrated HIV-1 DNA within resting CD4+ T cells in HAART naïve patients was not significantly higher... than in treated patients... [suggesting] that resting CD4+ T cells with integrated HIV-1 DNA do not decay rapidly in patients receiving HAART and thus represent a stable reservoir of HIV-1 DNA." In other words, the third compartment appears to lack a measurable decay curve despite potent combination antiretroviral therapy. Chun et al. concluded their paper by noting that "the time required for virus eradication -- if indeed this is possible -- will be considerably longer than previously predicted," and they call for the development of "strategies for eradicating those minor populations of infected cells that serve as reservoirs of inducible and replication-competent HIV."

What Can Be Done to Eliminate the Third Compartment?

ACTG 387 is a protocol in development which aims to determine more exactly the kinetics of viral decay with the three current classes of antiretroviral agents (nucleoside and non-nucleoside RTIs and protease inhibitors) by measuring their pharmacokinetics and viral kinetics in the first 72 hours of therapy. Participants will then begin four drug therapy with AZT, 3TC, indinavir and nevirapine. After six days they will be randomized to receive immune modulation with interleukin-12 (IL-12), GM-CSF, or nothing. IL-12 is a Th1-type cytokine which induces production of gamma interferon, and may inhibit CD4 cell apoptosis. Immune modulation will continue for fourteen weeks and quadruple antiretroviral therapy will continue for 48 weeks. The hypothesis here is that IL-12 will accelerate the clearance of HIV from long-lived CD4 cells and that GM-CSF will do the same for macrophages. This study may help to clarify whether it is possible to use immune stimulatory cytokines to speed up clearance of the third compartment.

Additional Questions

1. Can memory cells revert to naïve cells in humans? How frequent is this?
2. Can latently infected cells undergo mitosis into infected daughter cells without turning on viral replication?
3. Do memory cells that revert to naïve cells retain their capacity for further T cell receptor gene reshuffling?
4. Is there new thymic emigration in HIV-infected adults?
5. Is there extrathymic maturation of new naïve or memory CD4 cells?
6. As the likelihood of eradication recedes, does the argument for treatment of newly infected or asymptomatic individuals weaken?
7. What destroys the lymphoid tissue late in disease?