SCIENCE
Research on AIDS Moving Quietly Forward Not all advances are comforting, but scientists' understanding of the disease is increasing, raising hope for ways to treat and prevent it Rudy M. Baum, C&EN San Francisco
Amidst bickering government officials who can't agree on what to tell teenagers about condoms, AIDS basic research quietly continues. Amidst hysterical pronouncements based on dubious science about the spread of the AIDS virus, the research continues. Research on AIDS (acquired immune deficiency syndrome), on related diseases of species other than humans, on the AIDS virus itself— h u m a n immunodeficiency virus (HIV)—and on related retroviruses is advancing scientists' understanding of the etiology of these devas-
Levy: bowel mucosa
tating diseases. With increased understanding comes increased hope for treatment and prevention of AIDS. However, not all research advances are comforting—to people with AIDS, people infected with HIV, or scientists trying to sort out the details of the remarkably complex disease. Recently, scientists have reported that: • At least half of those infected with HIV will develop AIDS within nine years and another one fourth will develop AIDS-related complex (ARC). • HIV infects cells of the intestine and likely is a direct cause of the chronic and debilitating diarrhea often found in AIDS patients. • Studies with feline leukemia virus (FeLV), which causes an AIDSlike condition in cats, suggest that the most pathogenic variants of HIV may be missed when current isolation techniques are used to screen for the virus. On a more positive note, scientists at a number of institutions | working independently have shown | that a soluble version of the CD4 g antigen, which appears to be the ^ primary target of HIV in its infection of susceptible cells, can block the infectivity of the virus in vitro. The protein might be able to slow the course of an HIV infection. In another positive development of sorts, epidemiologists at the Centers for Disease Control, Atlanta, and the New York City Department of Health report that HIV infection continues to be confined primarily within the traditional high-risk groups (male homosexuals and intravenous drug users and their sexual partners) and that the modes of HIV transmission have remained stable. FeLV is a retrovirus that is associated with a variety of fatal diseases
Capon: soluble CD4 decoy in domestic cats including an immunodeficiency disease that resembles AIDS in some respects. Although FeLV and HIV belong to different subfamilies of retroviruses, James I. Mullins, associate professor of virology at the Harvard School of Public Health; Edward A. Hoover, professor of pathology at Colorado State University, Fort Collins; and coworkers are studying the molecular mechanisms of induction of FeLV-associated immunodeficiency in cats to gain insights into the process of induction of AIDS in humans. The research was supported by the American Foundation for AIDS Research, the National Institutes of Health, and the Massachusetts AIDS Research Council. Unlike HIV, which is difficult to detect in the tissue of AIDS patients, FeLV antigens and DNA are relatively easy to detect in vivo, Mullins says. As a consequence, direct molecular analysis of FeLV without initial propagation of the virus in cell March 28, 1988 C&EN
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Science culture is fairly straightforward. HIV, by contrast, is generally prop agated in a culture of immortalized Τ lymphocytes. Such propagation represents a form of biological se lection, Mullins points out. The scientists cloned and se quenced a replication-competent strain of FeLV obtained directly from cats with immunodeficiency syndrome [Science, 239, 906 (1988)]. They found that the virus infects cats but does not induce immuno deficiency. However, they also iden tified variants of the FeLV strain that do induce an acute, fatal im munodeficiency disease. These var iants are replication defective and require the replication-competent vi rus as a helper for transmission. The scientists find that the genet ic differences responsible for the pathogenicity of the FeLV variants are localized in the region that en codes the FeLV envelope glycopro tein, gp70, and/or the long termi nal repeat (LTR) regulatory region. The differences are not that exten sive: three nucleotides in the LTR; and changes in the viral RNA that result in 11 amino acid changes, a six-amino acid insertion, and a sixamino acid deletion in gp70. Ac cording to Mullins, efforts to char acterize these differences in terms of the structure and function of gp70 and the LTR are under way. The results dovetail to a degree with results from Mullins' labora tory and two other laboratories working with simian immunodefi ciency virus (SIV), a very close rela tive of HIV that causes an AIDSlike disease in macaque monkeys. SIV, like HIV, is difficult to obtain directly from the tissues of infected animals. Mullins and coworkers have innoculated macaques with a cloned, replication-competent SIV. Although the monkeys have become persistently infected with the SIV strain, after 12 months none have developed symptoms of immunode ficiency. Other labs report attenu ated SIV pathogenicity after tissue culture propagation of the virus, Mullins says. The results with FeLV and SIV suggest, Mullins says, that the tech niques used to isolate HIV may be selecting strains of low pathogenic ity. The HIV isolated from AIDS 30
March 28, 1988 C&EN
Scientist pooh-poohs current AIDS theories Duesberg presented his ideas first in Peter Duesberg is getting a lot of ink lately. Katie Leishman, a writer who is a long literature survey that appeared making something of a career of giving last year [Cancer Research, 47, 1199 serious treatment to questionable and (1987)]. He cites numerous deficien sometimes mutually contradictory theo cies in the theory that HIV causes AIDS, ries about AIDS, recently wrote up but essentially he maintains that the Duesberg on the editorial page of the virus is latent and inactive even in peo Wall Street Journal. A number of promi ple dying of AIDS and that HIV infects nent newspapers, for which controver too few susceptible T4 lymphocytes to sy is an irresistible magnet, have written account for the immunosuppression seen stories about his ideas. Genetic Engi in AIDS. He also argues that HIV can be neering News did a piece on him, and isolated only from 50% of people with Duesberg himself presented his ideas in AIDS, and only using methods originally Bio/Technology's "The Last Word" designed to activate latent virus. Dues berg believes this indicates that people column. Duesberg, a professor of molecular develop a successful immune response biology at the University of California, to HIV. Berkeley, stirs controversy easily. He Duesberg also argues that the long does not believe that HIV causes AIDS. latent period between infection with HIV He does not, in fact, believe that AIDS and onset of AIDS makes no sense, and is a communicable disease. He doesn't that no likely mechanism for HIVbelieve that a large group of retroviruses, mediated cell killing has been devel ones lacking what is known as an one oped. He maintains that no other patho gene, cause disease; if true, that would gen infects predominantly members of mean that SIV does not cause AIDS in only one sex, and that the claim that monkeys, and FeLV does not cause an HIV behaves in this way is silly. AIDS-like disease in cats, and so on. Duesberg is a charming individual, a
patients clearly is capable of killing f T4 cells in vitro. T4 cells are the 1 immune system cells depleted in g> people with AIDS and ARC. How- ξ ever, Mullins says, "I think it is f likely that there are viruses in peo- | pie that are even more pathogenic 1 than the ones we are able to grow | in the lab." That could result from f loss of a replication-defective vari- j ant as in the case of FeLV or simply I because more highly pathogenic strains kill all the cells they infect in culture. In either case, Mullins says, the issue needs to be investigated. If the monkeys infected with the apparently nonpathogenic strain of SIV eventually come down with dis ease, the researchers will redone the virus to compare it to the origi nal strain. The researchers also are seeking to isolate SIV strains with demonstrated acute pathogenicity directly from tissue. "We are not going to do any further tissue cul ture work," Mullins says. The types of differences seen be Rhesus macaque monkeys develop an tween the pathogenic and nonpatho AIDS-like disease when infected with genic strains of FeLV, Mullins says, simian immunodeficiency virus (SIV)
Peter Duesberg highly competent scientist—he is a member of the National Academy of Sciences and a codiscoverer of oncogenes—and a persuasive conversationalist. The problem with responding to him is that he likes things his own way. When it is pointed out that researchers
"are the same types of differences you observe between different HIV isolates." Thus a subtle change in an existing virus might have given rise to the virus that causes AIDS. Another feature of FeLV reported by Mullins and Hoover is that it directly infects and disrupts the intestinal epithelium of cats. A symptom of HIV infection in humans is chronic diarrhea, which often leads to severe weight loss, malnutrition, and sometimes death among AIDS patients. In many cases, the diarrhea is caused by opportunistic infections by organisms such as giardia, salmonella, and cytomegalovirus (CMV). However, in numerous cases no such infection can be detected. Researchers at the University of California, San Francisco; the University of California, San Diego; and the Research Institute of Scripps Clinic, La Jolla, Calif., described in the Feb. 6 issue of Lancet an investigation into whether HIV might infect intestinal cells and directly disrupt bowel function. In their work, supported by NIH and the California State Task Force on AIDS, the
at Harvard University say they have published definitive proof that SIV causes AIDS in macaque monkeys, he responds, "That's just bad research." When it is pointed out that AIDS afflicts men and women equally in Africa, he responds, "I don't even want to talk about Africa. The data are too confused." But in his Cancer Research article, Duesberg himself cites 1985 data from Africa to argue that for some groups infection with HIV posed no risk for AIDS. It is now recognized that, for political reasons, AIDS was severely underreported in many African countries until very recently, a point Duesberg now chooses to ignore. This sort of selective perception permeates a conversation with the Berkeley scientist. AIDS researchers contacted by C&EN say that Duesberg is simply wrong. Harvard University assistant professor of pathology Joseph Sodroski says that Duesberg claims that "people get sick with no evidence of virus replication. That simply isn't true. There is a strong correlation between level of virus replication and the chance of going on to
researchers recovered infectious HIV from two out of four rectal mucosal biopsy specimens from AIDS patients with chronic diarrhea of unknown cause. In-situ hybridization of biopsy specimens from rectum, colon, and duodenum from 10 other AIDS patients detected viral nucleic acid in five specimens. Virus was present in both rectal and duodenal tissues, which suggests two routes of infection. Rectal cells could be infected directly by exposure to infected seminal fluid; duodenal cells likely are infected by spread of HIV through the blood. Enterochromaffin cells, which are neuroendocrine cells that play a role in regulating fluid balance in the intestine, appear to be infected with HIV. Other cell types also may be infected. Disruption by HIV of the function of enterochromaffin cells and other intestinal cells is probably the cause of the diarrhea seen in these AIDS patients, says Jay A. Levy, the professor of medicine who directed the effort at UCSF. Although demonstrating that HIV di-
develop clinically significant disease." Another researcher points out that research on HIV latency and activation in numerous laboratories is making significant progress in explaining these processes. Daniel J. Capon, a senior scientist with Genentech, points out that there are many unresolved questions about HIV and AIDS, but that the existence of such questions does not negate the overwhelming body of evidence that links HIV and AIDS. Most AIDS researchers have refused to respond to Duesberg's arguments, claiming that it is a waste of their time. That could be a mistake, since Duesberg and his ideas will not go away. Duesberg states that prominent AIDS researchers like Robert C. Gallo of the National Cancer Institute and William Haseltine of Harvard University have become millionaires on the basis of their work with HIV and that this accounts for their unshakable conviction that HIV is the cause of AIDS. The charge is distasteful, but it seems likely to be repeated so long as no major figure in the AIDS research establishment rebuts Duesberg point by point.
rectly causes intestinal dysfunction is difficult, "I assume that the virus is causing some pathology/' Levy says. Whether some therapeutic strategy can be developed to combat these symptoms of an HIV infection remains an open question. "I would hope that this provides even further evidence that anal sex is a major factor in HIV transmission/' Levy says. Receptive anal sexual intercourse has been recognized for some time as a particularly highrisk activity, and researchers have speculated that the trauma associated with anal intercourse could facilitate the spread of HIV. But as Levy points out, the research shows that "you don't need breaks in the mucosa of the bowel" for infection to occur. "All you need is for virus to come into contact with the bowel mucosa." A three-year study of 288 HIVinfected, initially asymptomatic individuals by scientists at UCSF; Abbott Laboratories (Abbott Park, 111.); and the University of California, Davis, holds grim news about the progression of the disease. After March 28, 1988 C&EN
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March 28, 1988 C&EN
three years, the scientists report in the March 12 issue of the British Medical Journal 22% of the individ uals had progressed to AIDS; 41% had progressed to AIDS or ARC. On average, the researchers estimate, the individuals in the study had been infected with HIV for three years prior to the start of the study, so the 22% progression rate likely reflects what occurs six years after infection. The research was sup ported by the state of California. The research, which was directed by Andrew R. Moss, UCSF associate professor of epidemiology and in ternational health, also found that five blood parameters—β 2 micro globulin concentration, presence of antigen to the HIV protein p24, he matocrit, proportion of T4 lympho cytes, and absolute number of T4 lymphocytes—were predictive of progression to AIDS. Of HIV sero positive men who were normal on all five variables at the beginning of the study, none progressed to AIDS before at least 17 months had passed. In contrast, two thirds of the men diagnosed for AIDS at two years or three years were abnormal for two or more variables at the beginning of the study. The best single predictor for pro gression to AIDS, the researchers found, was β2 microglobulin con centration. This small protein is lo cated on the surface of all nucleated cells and is a subunit of the class I histocompatibility antigen. It is not clear whether the increased level of 02 microglobulin in men rapidly progressing to AIDS is a direct re sult of t h e HIV infection or a reactivated CMV infection, which is common in pre-AIDS cases, the scientists report. They suggest that for asymptomatic, HIV-infected in dividuals, "the combination of β2 microglobulin and p24 levels with T4 lymphocyte counts will identify much larger groups at high risk than lymphocyte counts alone/' The researchers write that the "most striking feature of our study was the change in the seropositive subjects who had not yet progressed to AIDS/' They noted progressive loss of T4 lymphocytes and increases in the proportions of individuals who were abnormal on all predic tive variables. "Clearly, the subjects
who have progressed to AIDS do not represent a simple selecting out of a subgroup at risk, leaving the rest of the cohort unaffected," the scientists write. "On the contrary, the prognosis for the rest of the cohort is worsening over time, two thirds showing AIDS or ARC or lab oratory results that are highly pre dictive of AIDS at the third year of the study." The results suggest that, conser vatively, half of those in the study will progress to AIDS after six years of followup, or nine years after prob able infection, and three quarters will progress to AIDS or ARC. "What we saw was that the number of those showing no effects from HIV infection is very small," Moss says. "This means that if you are infected with the AIDS virus, you will almost certainly go on to get AIDS." The scientists argue that their results strongly suggest that clinical trials of anti-AIDS drugs should rapidly be expanded to in clude asymptomatic HIV-positive individuals. On a more hopeful note, late last year, scientists at Genentech Inc., South San Francisco, Calif., and Harvard Medical School reported that a soluble version of the CD4 antigen blocks the infectivity of HIV particles in vitro [Science, 238, 1704 (1987)]. Shortly thereafter, four oth er independent research groups pub lished much the same results [Nature, 331,76,78, 82 and 84 (1988)]. The interaction between the CD4 antigen and HIV's envelope glyco protein, gpl20, is a key feature in the HIV life cycle and likely an important aspect in the development of AIDS (C&EN, Nov. 23, 1987, page 14). Binding of gpl20 to the CD4 molecule appears to be the first step in the process of HIV infection of T4 lymphocytes. HIV-infected T4 cells express a version of gpl20 on their surfaces, and experiments have shown that fusion of HIV-infected T4 cells with uninfected T4 cells to form multinucleated giant cells called syncytia is mediated by the interaction of these molecules with CD4 molecules on the non-HIVinfected cells. This process of cell fusion is thought to be a major mechanism of HIV-mediated deple tion of T4 cells, a hallmark of AIDS.
Thus, blocking the interaction of gpl20 and CD4 holds promise for stemming the spread of HIV through an infected individual's population of T4 cells and slowing the depletion of T4 cells. According to Daniel J. Capon, who directed the work on soluble CD4 at Genentech, a soluble CD4 "decoy" could have other advantages as a therapeutic as well. The HIV envelope has been shown to be very variable among different strains of the virus. Therefore, antibodies against gpl20, which can also block HIV infectivity, generally are HIV-strain specific. By contrast, binding to CD4 appears almost to define HIV; presumably all strains of the virus would be susceptible to treatment with soluble CD4. "The beauty of the decoy is that the virus can not make a change to avoid it," Capon says. "For an antibody, that isn't true." A potential problem with soluble CD4 as an AIDS therapeutic is that the protein plays an essential role in the immune function of T4 cells. It is possible that the soluble version could interfere with that function and exert an immunosuppressive effect. Harvard scientists Ellis L. Reinherz, Joseph Sodroski, and coworkers addressed that possibility in their Nature report. They showed that at least two in-vitro measures of T4 function are not disrupted by soluble CD4. According to Capon, Genentech scientists are pursuing similar studies of several measures of T4 function as part of the preclinical work needed to gain Food & Drug Administration approval of clinical trials of soluble CD4. The company expects to begin Phase I trials later this year in HIV-infected individuals. Capon points out that production of sufficient quantities of CD4 is not a trivial consideration. Unlike a vaccine, for example, where an immune response is the goal of the therapy and hence where some level of antigenic contaminants may be acceptable, purity is a major factor in producing a therapeutic version of CD4. If the soluble version of CD4 does find use as an AIDS therapeutic, it likely would have to be administered for the rest of a patient's life. D
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