Acquired Immunodeficiency Syndrome (AIDS)

Key Discoveries

The National Cancer Institute has assumed a leading role in Acquired Immunodeficiency Syndrome (AIDS) research since the disease was first recognized in 1981. Because of the research programs and administrative mechanisms already in place, investigators were able to rapidly apply existing methods in drug screening and advances in cancer virus research technology to the study of AIDS. The large scale preparation of HIV-1 in permanent cell lines led to the development of a serological test for AIDS which enabled the detection of AIDS in our nation's blood supply. Detection of the virus in latent form has been established through the in situ hybridization method which allowed scientists to detect the virus in brain and blood cells, T lymphocytes and macrophages. Selected recent key discoveries, by NCI investigators include:

Development, testing and successful clinical trials of the drugs azidothymidine (AZT), dideoxyinosine (ddI) and dideoxycytidine (ddC), confirming their effectiveness as anti-retroviral agents against AIDS.

Progress in treating children with AIDS has occurred through the rapid introduction of antiretroviral agents into clinical trials. The studies performed by the Pediatric Branch contributed to the licensure of AZT for children in May of 1990 and dideoxyinosine (ddI) in October 1991. The latter, based solely on Pediatric Branch Studies, occurred simultaneously with licensure for adults, a historical event. The Pediatric Branch is currently completing studies of combination regimens to optimize activity (e.g., AZT plus ddI) as well as to offset toxicity (e.g., AZT plus G-CSF and erythropoietin).

Viral particles are detectable in plasma throughout the early stages of primary infection. The number of viral particles in plasma decrease by up to 200-fold following the initial viremia of primary infection, but increases again as the infection moves from the asymptomatic phase in AIDS-related complex (ARC) to full blown AIDS. The increase in viral particles bears an inverse relationship to CD4 count. Scientists at NCI and elsewhere have adapted Polymerase Chain Reaction (PCR) technology to detect and quantitate the amount of HIV-1 RNA present in plasma. A new adaptation of PCR technology, designated "immuno-PCR", is capable of detecting minute amounts of HIV regulatory proteins (e.g. Tat, Rev) in solution or in tissue. "Immuno-PCR" can be applied to the in situ analysis of certain clinical specimens, such as lymph node. Thus, PCR technologies provide the opportunity to correlate HIV protein expression with virulence and pathogenicity in both the laboratory and clinical settings, and may serve as sensitive markers by which to measure response to therapy.

The ability to provide effective long-term anti-retroviral therapy using single agents for HIV infection is complicated by the emergence of drug-resistant HIV strains. Longitudinal studies of the phenotypic and genotypic changes of HIV strains isolated before and after prolonged therapy with either an alternating regimen of AZT and ddC or ddI alone demonstrate that HIV develops reduced susceptibility to AZT more readily than to ddC and with ddI. A new acyclic purine analog, PMEA, inhibits both HIV RT and DNA polymerase-alpha from CMV and other herpes viruses and may inhibit latent as well as replicating HIV, with perhaps a special activity against the HIV reservoir in monocytes/macrophages. NCI scientists began the Phase I clinical testing of PMEA in combination with AZT in January 1994, in conjunction with assessment of intrinsic or induced HIV resistance to PMEA.

A recent clinical trial by NCI investigators suggests that the simultaneous administration of AZT and ddI results in higher and more sustained elevations of CD4+ cells over a one year period than alternating drug administration. This difference in clinical activity may relate to differential intracellular drug activation. AZT is preferentially phosphorylated to the active triphosphate form (AZT-TP) in proliferating cells. In contrast, the phosphorylation of ddI (to ddATP) and ddC (to ddC-TP) occurs preferentially in resting cells. Thus, the data suggest that AZT, ddC and ddI may exert their antiviral effects depending on the activation state of the target cells; i.e., ddI and ddC likely exert antiviral activity against resting cells, while AZT protects actively growing cells against HIV infection. The combination of AZT+ddI may target both latent and actively replicating pools of virus, providing complementary and possibly synergistic anti-HIV activity.

Identification through the high-capacity AIDS drug screen of many new compounds which are active against the AIDS virus in tissue culture experiments. These compounds include both synthetic drugs and natural products. Several of these are in the initial phases of development.

NCI's AIDS Drug Screen has recently uncovered an active compound with an unprecedented mechanism of action, namely the disruption of the highly conserved zinc finger regions of the HIV nucleocapsid protein, p7. The nucleocapsid protein is necessary for the incorporation of the viral RNA genome into intact viral particles and the ultimate packaging of the infectious virions. The ability to block viral reproduction and dissemination by inhibiting the structural and functional integrity of p7 will serve two critical goals: a heightened molecular understanding of the late stages of the viral life cycle, and a template for rational drug design based upon the protein sequence and structure.

The HIV-1 enzyme reverse transcriptase (RT) is the target for inhibition by many of the currently available anti-retroviral agents, in particular the nucleosides (AZT, ddI, ddC, d4T). Unfortunately, RT is able to undergo mutations that confer resistance to the inhibitory effects of these drugs. Scientists at NCI's Frederick Cancer Research and Development Center have both structural and biochemical data to suggest that the Leu74Val mutation, which causes resistance to ddI, affects the interaction between RT and its nucleic acid substrate. In addition, these scientists continue to define the structure of HIV-1 RT and the complexes formed between RT and its nucleic acid substrates.

Integration of RT-transcribed HIV DNA into the host genome is facilitated by HIV integrase, an enzyme encoded by the HIV pol gene. NCI scientists have devised rapid fluorescent assays that identify both the DNA cleavage reaction and the subsequent integration process in response to purified HIV integrase. The ability to quantitate integrase activity by measuring cleavage-induced changes in fluorescence will also identify agents that block integrase action and thus present a novel molecular target for therapy.

Determination of the first crystal structure of retroviral protease and its successful use to predict the structure of the HIV protease and substrate using supercomputer methodology. HIV protease is an enzyme whose action is required in the processing of HIV proteins and production of infectious virions. NCI scientists have identified several inhibitors of the HIV protease including KNI-272 which has exhibited potent anti-HIV activity and favorable pharmacokinetics in test animals. In March 1994 NCI scientists began Phase 1 clinical trials of KNI-272.

Individuals infected with HIV may be asymptomatic for years before progressing to overt AIDS. Since monocytes possess surface CD4 molecules, they can bind and act as a reservoir for HIV in infected individuals. Thus, monocytes in AIDS patients can harbor latent HIV inducible by T cells during an immune response. HIV produced by such monocytes infects T cells leading to viral-induced pathology. In addition to monocytes, NIAID scientists determined that follicular dendritic cells (FDC) also serve as reservoirs for latent HIV infection, sequestering HIV for eventual transmission to CD4+ cells.

IL-12, produced by macrophages and B cells in response to diverse infectious pathogens, is a natural killer (NK) cell stimulatory factor which activates NK cells in vitro and appears to have a significant antitumor effect in tumor-bearing animals. IL-12 drives the differentiation of naive CD4+ T cells into TH1 cells, thereby promoting cell-mediated immunity. IL-12 may have a special role as an immunostimulant in HIV infection, where both NK cell and TH1 cell functions are defective. IL-12 may also be able to restore the HIV-related imbalance between TH1 and TH2 cells which, in turn, leads to defects in cellular immunity and excessive humoral (antibody) responses.

The magnitude of CNS disease is often more prominent and the latency period which precedes HIV- related encephalopathy shorter in children than in adults, suggesting that fetal or developing brain cells (in particular, glial cells) may release cytokines capable of activating expression of latent HIV. To address the pathogenesis of neurologic disorders in HIV-1 infected children, NCI scientists have developed an in vitro model using a normal fetal olfactory neuroblast cell line, to investigate the potential contributions of direct viral infection and virally-induced cytokines in glial (and perhaps other accessory) cells to neurodevelopmental impairment.

NCI epidemiologists have played a major role in uncovering the emergence of a new peak of tuberculosis (TB)-associated death in young individuals (ages 20-49) that appears linked to AIDS.

Recent studies of vaginally-delivered multiple birth cohorts in HIV-infected women demonstrate that HIV transmission is greatest for the first-born infant, suggesting that some component of HIV transmission occurs at the time of the delivery in the cervix or vagina.

Indeed, about 60 percent of mother-to-infant HIV transmission occurs at the time of birth. On this basis, scientists are designing a clinical trial of inexpensive viricidal solution to cleanse the birth canal to lower the risk of HIV transmission in this setting.

NCI has established a multi-state AIDS/cancer match registry linking AIDS and cancer registries in five areas of NCI's Surveillance, Epidemiology and End Results (SEER) program (San Francisco, Los Angeles, Atlanta, Detroit and Connecticut) and 10 other sites (New York City and state, New Jersey, Puerto Rico, San Diego, Sacramento, Florida, Illinois, Colorado and Massachusetts). The Registry encompasses about 75 percent of all reported AIDS cases and involves approximately 85,000 matches of individuals with AIDS to cancer registries. This very large data base allows for the first time quantitative estimates of rare as well as common malignancies and provides a framework for determining the role of HIV as a cofactor in the development of diverse malignancies. The registry will also serve to identify patients with concomitant AIDS and cancer from whom tumor tissue and other biologic specimens can be obtained for molecular epidemiologic studies.

NCI scientists have developed prototype synthetic vaccines consisting of broadly-recognized histocompatibility determinants of T helper cells (so-called "cluster peptides") and a combined site constructed to elicit both cytotoxic T lymphocytes (CTL) and neutralizing antibody. Clinical trials of these constructs are now being launched.

NCI scientist have constructed novel vaccines comprised of various recombinant and live vectors carrying HIV-1, HIV-2 and SIV antigenic proteins or protein units. These constructs are now being tested in rhesus macaques for their efficacy as initial immunogens, followed by "boosters" using purified native or viral antigens, in eliciting protective immune responses. Recombinant constructs coupling vaccinia virus (poxvirus) vectors to various HIV antigens induce virus-specific cellular and humoral responses in primates. Vaccine constructs coupling adenovirus with HIV-1 MN or IIIB env genes have been shown to elicit both T cell and neutralizing antibody responses, and will be examined in chimpanzees for their protective effects against viral challenge. Finally, influenza recombinants, designed such that the V3 loop is located within a region of the hemagglutinin molecule that is conformationally accessible to antibodies, are being developed as a booster immunogen.

NCI investigators have put the poly Tat activation region (TAR) which binds to the viral regulatory protein, Tat, into the HIV promoter, thereby inhibiting viral replication. Since binding of Tat to TAR is necessary for RNA expression and viral replication, the polymeric TAR (poly TAR) provides a molecular decoy which inhibits viral replication. Cultures containing the protected cells show a gradual decline in virus production that reaches 90 percent in two months. Six months after infection the protected cell cultures express little detectable virus and are resistant to reinfection. Poly TAR appears to be an effective antiviral gene that may have eventual clinical application as a gene therapy modality.

NCI investigators have detected large numbers of KS-like spindle cells in cultures of circulating peripheral blood of HIV+ patients with active KS or at high risk for development of KS. These cells have spindle-shaped morphology and immunophenotypic characteristics of activated endothelial cells, and produce angiogenic factors. The numbers of peripheral blood spindle cells from HIV+ patients with active KS and from those at high risk are increased 78-fold and 18-fold, respectively, over the numbers detectable in HIV- or HIV+ low-risk individuals. The ability to detect such cells may predict susceptibility to develop KS and could serve to monitor the impact of therapeutic and prevention interventions.

NCI scientists have developed spindle cell strains that provide models of KS for the exploration of new therapeutic approaches. Most recently, a unique KS patient-derived cell line exhibits unlimited cellular life span in vitro and aggressive, metastatic behavior in vivo in immunosuppressed mice, with formation of highly vascularized tumor nodules. This model mimics KS progression in the human setting and thus may provide an excellent model for dissection of KS pathophysiology and development of targeted antitumor modalities.

Multiple cytokines (growth factors) with inflammatory, growth-promoting and immunostimulating activities make pivotal contributions to the molecular pathogenesis and clinical phenotype of AIDS-KS. The HIV Tat protein, in particular the biologically active form that is released extracellularly, augments both viral and host gene expression. Basic fibroblast growth factor (b-FGF), an inflammatory cytokine produced by AIDS-KS cells as well as stromal cells, promotes new blood vessel formation (angiogenesis) and wound healing. It has now been shown that b-FGF and Tat interact synergistically to induce proliferation of normal vascular cells and produce KS-like angiogenic lesions in mice in vivo. This cooperation is magnified in the HIV+ setting, where b-FGF, extracellular Tat and Tat receptors are present to drive the emergence and aggressive progression of KS.

A glycoprotein growth factor known as Oncostatin M, derived from activated T-cells, is a potent growth stimulator for AIDS-KS cells. This growth factor is distinct from other important cytokines in AIDS-KS, namely IL-6 and the HIV Tat protein, but binds to the active subunit of the IL-6 receptor. Oncostatin M appears to cause AIDS-KS cell proliferation both directly and in part by enhancing the expression of IL- 6 by vascular endothelial cells, and further induces morphologic changes in AIDS-KS cells, namely to the spindle configuration of smooth muscle cells.

NCI scientists have found a non-cytotoxic bacterial product, a sulfated polysaccharide-peptidoglycan compound (SP-PG) which inhibits the growth and vascular responses, in particular the induction of angiogenesis and hyperpermeability, of AIDS-KS spindle cells in vitro and in a nude mouse model.

The striking production of autostimulatory and angiogenic growth factors by KS cells suggest that these factors should be an important target for therapy. Phase I clinical trials of angiogenesis inhibitors are underway.

NCI scientists are investigating the antitumor effects of the cytotoxic natural product taxol, a unique tubulin-binding agent, in AIDS-related Kaposi's sarcoma (KS). Of 17 patients treated to date, 50 percent have achieved objective partial responses with roughly 50 percent decreases in number, size and/or nodularity of KS lesions and an additional 40 percent have had stabilization of disease.

Profound cellular immunodeficiency plays a central role in lymphomagenesis. NCI investigators have found that the most important risk factor determinant for both the AZT- and ddI-treated cohort/s is a CD4 count below the critical level of 50/mm3. In addition, elevated serum levels of IL-6 predict a high risk for NHL development.

The remarkable occurrence of high-grade B-cell, non-Hodgkin's lymphomas (NHL) has recently emerged as a major sequela of HIV infection, especially in patients who survive other consequences of AIDS in a protracted state of profound immunosuppression. NHLs develop in approximately 10 percent of AIDS patients treated with dideoxynucleosides. NCI investigators have developed a "lymphoma subpanel" comprised of two AIDS lymphoma cell lines including an EBV+ Burkitt's lymphoma, and eight non-AIDS lymphoma cell lines for screening potential therapeutic compounds.

The severe combined immunodeficiency (SCID) mouse provides a unique model for the study of AIDS- related lymphoma biology and anti-lymphoma drug development. To date, about 500 agents have been examined for in vitro antitumor activity against an EBV+ Burkitt's lymphoma derived from an HIV-infected patient. This human tumor cell line has been established as a reproducible in vivo model within the SCID mouse. Approximately 18 agents have been evaluated in the in vivo model with 3 showing antitumor activity. Further, the CNS involvement of the SCID mouse with this lymphoma provides an opportunity to predict agents that have access to this frequently involved sanctuary in patients. The lymphoma subpanel is being expanded to establish and characterize new AIDS-related lymphoma cell lines, develop "mechanism of action" assays (e.g. IL-6 inhibition, induction of programmed cell death, antiviral effects targeting EBV or other viral cofactors) and define the differential drug sensitivity testing in the in vivo SCID model.

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