Important note: Information in this article was accurate in May 2001. The state of the art may have changed since the publication date.
American Foundation for AIDS Research, May 2001
Jo Ann Berg
This year's annual Keystone Symposium on HIV, held March 28 to April 3 in Keystone, Colorado, featured concurrent sessions on "AIDS Vaccines in the New Millennium" and "HIV Pathogenesis" (how the microbe causes disease). Previous meetings on pathogenesis have emphasized the adaptive immune response: antibodies and CD8+ cytotoxic T lymphocytes (CTLs). The antibodies are specifically shaped to attach to and help eliminate free HIV particles in the body while the CTLs are specially selected and activated to kill HIV-infected cells. Antibodies to HIV have generally proven ineffective, and the CTL response wanes as HIV infection progresses.
AIDS scientists are taking a renewed interest in the body's first line of defense — the nonspecific, or innate, immune system. Fifteen years ago, Jay Levy's laboratory at the University of California San Francisco observed that certain CD8+ T-cells active during HIV infection do not kill HIV-infected cells.1 They instead release a soluble factor or factors that render nearby CD4+ T-helper cells resistant to destruction by HIV.
Although scientists still disagree on the identification of such factors, they now distinguish the cytotoxic (or killing) from the noncytotoxic control of an HIV-infected cell. The latter arises very early after infection, is not specific to any particular retrovirus, and does not target a specific HIV strain or the cells it infects. Both cytotoxic and noncytotoxic functions are important to study, especially since the innate immune system interacts with and influences the nature of the adaptive immune system. For example, Francis Chisari 2 of the Scripps Research Institute recently revealed how a strong innate response helps the adaptive CTLs control acute hepatitis B virus infection in chimpanzees.
Levy, in the course of long years working on the HIV-suppressing factor, has dubbed it the cellular antiviral factor, or CAF. CAF's exact makeup remains undetermined, and considerable disagreement has arisen over its nature.
Many different nonspecific factors — such as the "acute phase proteins" that directly attack larger pathogens like bacteria and parasitic worms — are released from several cell types during the innate response. But the agents that protect against HIV infection are most likely a type of soluble factor known as cytokines. Cytokines are signaling molecules that cells use to activate or deactivate immune cells. Each cytokine fits into particular cell surface receptors. This docking activates a chain of metabolic events, eventually causing certain regulatory genes in the nucleus to turn on and change the cell's overall function. Cytokines can be broadly divided into three categories: (1) pro-inflammatories, which cause the expression of proteins that activate immune cells; (2) anti-inflammatories, which cause immune cells to shut down; and (3) chemokines, whose functions include attracting immune cells to the site of infection.
At the Keystone Symposium, Alfredo Garzino-Demo 3 , representing Robert Gallo of the University of Maryland, said his group believes that a mixture of chemokines suppresses HIV activity. They believe people genetically programmed or perhaps environmentally induced to produce more of these chemokines should be more resistant to HIV infection or fare better if infected. He pointed out several papers showing an association between higher chemokine levels and a better HIV disease status or protection. But he cited as well a few papers finding no such correlation. Two Keystone posters 4, 5 also found no correlation. Garzino-Demo argued that the latter studies are wrongly based on plasma chemokine levels. "Since," he reasoned, "chemokines are not circulating molecules," researchers should not look at the plasma as "chemokines are not supposed to be there."
Gallo's lab originally correlated the major activity of CAF to three chemokines: MIP-1a, MIP-1b, and RANTES. The receptor for these chemokines (called CCR5) is the same one that most HIV variants - including nearly all the variants that are transmitted from person to person — must also latch onto before they can enter their target cells. The Gallo team believes that these chemokines function as the cellular antiviral factor by blocking HIV entry. This lab has also reported the anti-HIV activity of a fourth CD8+ T-cell-secreted chemokine, MDC (macrophage-derived chemokine). Garzino-Demo said MDC has inhibitory effects against HIV isolates that latch onto either CCR5 or CXCR4 (the latter the more virulent variant that can emerge in late HIV disease), but its mechanism of action is not via receptor blocking and remains largely uncertain.
He then announced that his lab has just identified a fifth chemokine with anti-HIV activity, derived from an HIV+ long-term nonprogressor. Garzino-Demo said this chemokine is already commercially available, but referred to it as "X." Chemokine "X" does not attach to either the CCR5 or CXCR4 receptors, and its antiviral activity is weaker than the other chemokines. His lab found good synergistic action against both CCR5 and CXCR4 HIV variants when "X" was added to MDC. He said the most potent suppression of HIV came from "Y," a mixture of all five chemokines. But upon questioning, he conceded that since high levels of these chemokines can accompany high viral loads, there must be other important factors driving a decrease in viral load.
Jay Levy 6 said in his presentation that although he has not yet isolated CAF, he has ruled out dozens of soluble factors, including the chemokines. Levy still believes CAF is a single factor, although he has found some antiviral activity from several CD8+ T-cell-secreted cytokines, especially interleukin-8, IL-16, and transforming growth factor-b (TGF-b). He also mentioned the antiviral activity of interferon-a and interferon-b, but these cytokines are not secreted from CD8+ T-cells. Levy's research leads him to indicate that CAF works by somehow blocking the HIV genome imbedded in an infected cell's genes. The factor makes it impossible for that genome to turn on and produce new virus particles that would escape and infect new cells.
Other speakers referred to a recent finding 7 that shows influenza A virus can stimulate both CD4+ and CD8+ T-cells to secrete interferon-a as well as a novel anti-HIV factor. The study's authors ruled out three of the chemokines described by Gallo's laboratory and said this antiviral soluble factor is also not CAF, as it inhibits an earlier part of HIV's life cycle than Levy is positing.
Levy reported he has narrowed down CAF's activity to 106 genes that differ in activity between non-CAF producers and CAF producers, the latter being predominantly long-term nonprogressors and HIV-exposed but uninfected individuals (EUs). He said he finds CAF in almost all EUs up to six months after their last HIV exposure and that some of these individuals have no CTL responses to HIV. But if he waits until a year after their last HIV exposure, he sees a big drop-off in the CAF response. Since exposure to HIV is needed to maintain the response and CAF is active against all strains of HIV and SIV, Levy recently came to the conclusion that he has been studying an innate immune response all these years.
Levy added that although CAF blocks HIV reproduction within cells, it does not disturb cell function or proliferation (division). Further, he has found that CAF's production depends on the cytokine IL-2. IL-2 production in turn depends on the normal functioning of a type of cell called the dendritic cell. He has said that an unexpected benefit of IL-2 treatment (primarily used to increase the number of CD4+ T-cells) may be that it induces the secretion of CAF.
The most promising attempt to isolate human CAF came from Mary Klotman's 8 group at Mt. Sinai School of Medicine. Klotman ruled out CTL killing of infected cells because her lab tests only the supernatant from CD8+ T-cells, i.e., the mixture of secretions left in a lab dish after the cells have been removed. They divide this supernatant into fractions and test each fraction for anti-HIV activity. If a fraction shows no activity, they discard it. But if it has activity, they fractionate it further, hoping to eventually isolate the active agent. Klotman and her coworkers start with liters of fluid and must narrow down to nanograms of active substance. They have identified a nine amino acid sequence from a protein with "great" antiviral activity. With this step forward comes a new mystery, for Klotman knows only that her factor acts between the time HIV enters a cell and the time HIV reaches the cell's nucleus and integrates its genes with the cell's. This is later than Gallo's chemokines act and earlier than Levy's CAF. (She noted, though, that the impurified raw supernatant also stopped HIV reproduction the way Levy posits for CAF.) The answer to the "What is CAF?" debate, suggested Klotman, is that there are multiple factors acting in different ways.
Of course, unknown toxicities or side effects could hinder the identification and eventual development of therapies using CAF. Such problems are not insurmountable. Paolo Lusso 9 of the San Raffaele Scientific Institute described producing a RANTES derivative that could still block HIV entry although it was free of RANTES' potentially toxic cell-signaling activity.
Further, the effects of chemokines can vary depending on their context. For example, Levy suggested the cytokine TGF-b had antiviral effects, yet two posters found it either increased viral load 10 or promoted CD4+ T-cell depletion 11 . All three groups are probably right, their differing results due to different experimental conditions.
Another poster 12 described HIV resistance to noncytotoxic CD8+ T-cell-mediated suppression. Just as HIV reportedly can "escape" from antibody, CTL, and antiretroviral drug pressures, it may be able to mutate to escape from soluble suppressor factors. This resistance was observed so far only in the lab and not in human beings. The source of resistance was linked to changes in the "long terminal repeat," the zone at the end of the HIV genome that promotes the production of new copies of the entire HIV gene set. This would be in line with Jay Levy's conception of how CAF operates.
1. Walker CM, Moody DJ, Stites DP, Levy JA, "CD8+ lymphocytes can control HIV infection in vitro by suppressing virus replication", Science 1986 Dec 19;234(4783):1563-6
2. Chisari F, "Noncytopathic Control of Viral Infections by the Immune Response", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; oral presentation 020.
3. Garzino-Demo A, "New Biologic Approaches to Control HIV-1. HIV Pathogenesis", Keystone Symposium. March 28-April 3, 2001; oral presentation 041.
4. Eveson J et al, "Characterisation of Immune Mechanisms in a Cohort of HIV Exposed Uninfected (EU) Individuals", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; poster 118.
5. Jennes W et al, "Increased Intracellular Beta-Chemokine Expression Is a Consequence of Increased Cellular Immune Activation and Correlates with Plasma Viremia in HIV-1 Infected Subjects in Abidjan, Ivory Coast", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; poster 128.
6. Levy J, "CD8+ Cell Noncytotoxic Anti-HIV Response Can Prevent Virus Infection and Pathogenesis", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; oral presentation 007.
7. Pinto L et al., "Inhibition of Human Immunodeficiency Virus Type 1 Replication prior to Reverse Transcription by Influenza Virus Stimulation", J Virol 2000 May;74(10):4505-11
8. Klotman M., "Purification and Characterization of Soluble HIV Suppressive Factors from CD8+ Cells", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; oral presentation 013
9. Lusso P, "Chemokines and Derivatives as Potential Anti-HIV Agents", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001;oral presentation 008.
10. Bou-Habib D et al., "HIV-1 Replication in Macrophages is Enhanced After Phagocytosis of Apoptotic Cells.", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; poster 302
11. Wang J et al., "Synergistic Induction of Apoptosis in Primary CD4+ T Cells by Macrophage-tropic HIV-1 and TGF-beta1.", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; poster 444.
12. Tomaras G et al., "Selection of Human Immunodeficiency Viruses Resistant to Noncytolytic CD8+ Antiviral Activity", HIV Pathogenesis Keystone Symposium. March 28-April 3, 2001; poster 166.
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