Mikulas Popovic

SEROEPIDEMIOLOGICAL STUDIES OF HUMAN T-LYMPHOTROPIC RETROVIRUS TYPE III IN ACQUIRED IMMUNODEFICIENCY SYNDROME

In a double-blind study, sera of 34 patients with acquired immunodeficiency syndrome (AIDS), 19 patients with lymphadenopathy syndrome, and 14 homosexual men with an increased risk of AIDS were screened for antibodies to proteins of the novel human T-lymphotropic retrovirus (leukaemia virus), HTLV-III, recently isolated from cultured T cells of AIDS patients. On a combination of a convenient and rapid enzyme-linked immunosorbent assay and a more sensitive electroblot (Western) assay, 100% of the AIDS sera were scored positive. Similarly, 84% of the lymphadenopathy patients were found to have serum antibodies to HTLV-III. A lower, but significant, proportion (21%) of healthy homosexual men with an increased risk of AIDS were also positive. No heterosexual controls, including those with heterophile antibodies during the course of infectious mononucleosis and patients with T-cell or B-cell lymphoma, had antibodies to HTLV-III. The results strongly indicate that the antibodies to HTLV-III are diagnostic of AIDS or indicate significant risk of the disease, and suggest that HTLV-III is the primary cause of human AIDS.

Serological detection of antibodies to HTLV-III in sera of patients with AIDS and pre-AIDS conditions

This invention relates to the detection of antibodies in sera of AIDS and pre-AIDS patients and describes the biochemical and immunological analysis of antigens associated with the virus HTLV-III Human T-Cell Leukemia Virus. It is shown that antigens associated with the infection of human cells by this virus are specifically recognized by antibodies from AIDS patients. Specifically, HTLV-III isolated from AIDS patients and transmitted by cocultivation with an HT cell line is specifically detected by antibodies from human sera taken from AIDS patients. The method of detection of antibodies preferred is a strip radioimmunoassay (RIA) based on the Western Blot technique or an ELISA (an enzyme-linked immunosorbent assay) or an indirect immunofluorescence assay.

Antibodies to HTLV-III in Swiss Patients with AIDS and Pre-AIDS and in Groups at Risk for AIDS

We tested serum samples from Swiss subjects by three different assays based on enzyme-linked immunosorbent assay (ELISA) and Western blot techniques for antibodies to proteins associated with the recently discovered human T-cell leukemia/lymphoma virus HTLV-III, the putative etiologic agent for the acquired immunodeficiency syndrome (AIDS). Of 10 patients with AIDS and 10 with pre-AIDS, all were antibody-positive. Furthermore, 37 of 103 intravenous-drug addicts (36 per cent), 4 of 40 healthy homosexual men (10 per cent), 7 of 83 patients with various types of hepatitis (8.4 per cent), but none of 83 healthy blood donors or 10 other controls were antibody-positive. Antibodies to the major viral protein p24 were found consistently and at high titers in the seropositive members of the groups at risk and in those with pre-AIDS but were dramatically reduced in patients with AIDS. In contrast, antibodies to another virus-associated protein, p41, were present in all cases of AIDS and pre-AIDS but were absent in nearly 10 per cent of seropositive persons at risk. Whereas p41 and p24 thus appear to be the targets of choice for future screening tests, the ELISA test that is currently available is a useful screening tool.

Langerhans cells in HIV-1 infection

The skin-specific immune surveillance system protects against invading microorganisms and transformed cells expressing tumor-specific neoantigens. This system includes antigen-presenting Langerhans cells, dermal and epidermal T lymphocytes, cytokine-producing keratinocytes, and draining peripheral lymph nodes. In patients infected with human immunodeficiency virus-1 (HIV-1), this surveillance system appears to be compromised, as evidenced by a reduction in the epidermal Langerhans cell population. Because human epidermal Langerhans cell express surface-bound CD4 antigens, HLA-DR antigens, and Fc-IgG receptors, all of which are involved in HIV-1 binding to, or entry into, the target cell, the reduction in Langerhans cells in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) may be a direct consequence of HIV-1 infection and subsequent injury to Langerhans cells. Detailed ultrastructural studies have confirmed moderate to severe morphologic damage in some Langerhans cells of such patients and the presence of HIV-1-like particles on Langerhans cell surface membranes and in the extracellular spaces. The biologic consequences of Langerhans cell infection by HIV-1 could be either impaired antigen presentation function of viable Langerhans cells or possible transmission of the retrovirus to the T-cell compartment in skin or lymph nodes, with subsequent depletion of CD4+ T cells via widespread syncytia formation between HIV-1-infected and noninfected cells. The facts that herpes simplex virus, specific cytokines, and ultraviolet B radiation can activate signals for HIV-1 expression and that epidermal cells can elaborate large amounts of cytokines, particularly with enhanced ultraviolet B exposure, may have important clinical implications for HIV-1-infected patients.

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor α and IFN-γ released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-γ and tumor necrosis factor α, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH2-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

A Tat subunit vaccine confers protective immunity against the immune-modulating activity of the human immunodeficiency virus type-1 Tat protein in mice

The rational design of new therapies against HIV-1 necessitates an improved understanding of the mechanisms underlying the production of ineffective immune responses to HIV-1 in most infected individuals. This report shows that the CD8+ T cell responses to gp120 were greatly diminished in mice vaccinated with a bicistronic gp120-Tat DNA vaccine, compared with those induced by a DNA vaccine encoding gp120 alone. The CD8+ T cell responses induced by the latter included strong gp120-specific IFN-γ secretion and protective antiviral immunity against challenge by a vaccinia-env pseudotype. The degree to which Tat influenced CD8+ T cell responses depended on the bioactivity of Tat. Thus, a bicistronic DNA vaccine that expresses gp120 and a truncated Tat defective for LTR activation elicited strong IFN-γ -secreting CD8+ T cell responses to gp120 but conferred only marginal protection against the vaccinia-env challenge. The effect of Tat was completely blocked, however, by immunization with inactivated Tat protein before vaccination with the bicistronic gp120-Tat DNA vaccine.

Biological characterization of paired human immunodeficiency virus type 1 isolates from blood and cerebrospinal fluid

Virus has been isolated from the blood and cerebrospinal fluid (CSF) of eight subjects with varying severity of human immunodeficiency virus type 1 (HIV-1) infection and from the frontal lobe of one patient with AIDS. The five patients with AIDS-related complex (ARC) and AIDS also showed neurological/psychiatric complications. With the exception of one isolate from the CSF of an asymptomatic carrier, all isolates replicated in peripheral blood mononuclear cells and monocytes after cell-free transmission. Isolates obtained from the blood of patients in late stages of HIV infection replicated in 3 (of 4) cases in H9 cells, whereas none of the blood isolates from patients in the early stages did so. The capacity of CSF isolates to replicate in H9 cells was low (only 2 of 12). Paired virus isolates from blood and CSF of the same patient could be distinguished by their replicative capacity in different cell lines, type of cytopathic effect, and protein profile as tested by radioimmunoprecipitation. The results indicate that variant viruses with distinct biological characteristics may be isolated from the blood and CSF of the same patient.

Relatedness by nucleic acid hybridization of new isolates of human T-cellleukemia-lymphoma virus (HTLV) and demonstration of provirus in uncultured leukemic blood cells

Human T-cell leukemia-lymphoma virus (HTLV) has now been isolated from many different patients with cutaneous T-cell lymphoma and leukemia, as judged by detection of media reverse transcriptase and virus particles and of antigenic determinants related to those of viral structural proteins p24 and p19. Molecular hybridization experiments with HTLV cDNA to viral mRNA or proviral DNA to ascertain the relatedness of four of these new isolates to the first HTLV isolate have been used. By these assays, three appear virtually indistinguishable from the original isolate, HTLV-I(CR), the second U.S. isolate (HTLV-I[MB]), and the Japanese ATLV isolates. Proviral sequences indistinguishable from those of HTLV-I(CR) were also detected in uncultured leukemic blood leukocytes from a patient of Japanese origin with adult T-cell leukemia. These viral isolates thus form a closely related virus group, HTLV-I. In contrast, however, RNA and DNA from one cell line derived from a patient with a T-cell variant of hairy cell leukemia, which expresses media reverse transcriptase and antigenic determinants related to but distinguishable from HTLV p24, did not hybridize substantially with HTLV cDNA. This latter virus appears to represent a second type of HTLV (HTLV-II), related to but substantially different from HTLV-I.

Soluble factors from T cells inhibiting X4 strains of HIV are a mixture of β chemokines and RNases

T-cell-derived soluble factors that inhibit both X4 and R5 HIV are recognized as important in controlling HIV. Whereas three β chemokines, regulated-on-activation normal T-cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1α, and MIP-1β, account for the suppression of R5 HIV by blockade of HIV entry, the major components responsible for the inhibition of X4 HIV strains have not been identified previously. We identify these factors primarily as a mixture of three β chemokines [macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), and I-309] and two RNases (angiogenin and RNase 4) of lesser potency and show that in a clade B population, some correlate with clinical status and are produced by both CD4+ and CD8+ T cells (chemokines, angiogenin) or only by CD8+ T cells (RNase 4). The antiviral mechanisms of these HIV X4-suppressive factors differ from those of the previously described HIV R5-suppressive β chemokines.

Molecular and immunologic analysis of a chronic lymphocytic leukemia case with antibodies against human T-cell leukemia virus

The human T‐cell leukemia virus type‐I (HTLV‐I) is a unique, exogenous, horizontally transmitted retrovirus which is T‐cell tropic, and has been associated with a specific type of aggressive leukemia/lymphoma of mature T‐cell origin. In a survey of lymphoid malignancies in Jamaica, antibodies to HTLV‐I were also found in 6 of 17 patients with chronic lymphocytic leukemia (CLL), raising the possibility of an etiologic relationship. Further studies were undertaken on one of these patients to clarify the nature of the disease and possible virus relationship. Cell surface marker analysis of her peripheral blood cells documented that the majority of circulating lymphocytes were B‐cells. DNA‐cloned probe analysis with a complete HTLV‐I proviral genome of these peripheral malignant B‐cells, was negative for integrated virus. A T‐cell line was established in culture from her peripheral blood. The presence of HTLV‐I in the cultured T‐cell line was established by the detection of expressed viral specific gag protein p‐19 and proviral DNA. Thus, a B‐cell lymphoid malignancy can occur in the presence of HTLV‐I infected T‐cells, suggesting the possibility of an indirect leukemogenic mechanism.