M. Fouchard

Persistent HIV-2 Infection of Rhesus Macaque, Baboon, and Mangabeys

Six monkeys of three different species (mangabey, macaque and baboon) were infected with human immunodeficiency type 2 (HIV-2) NIH-DZ using intraperitoneal or intravenous injections of cell-free HIV-2 or autologous HIV-2-infected cells with no prior immunostimulation. Viral expression was demonstrated by reverse transcriptase activity in cells after coculture with human peripheral blood lymphocytes or by electron microscopy. Serum was analyzed by western blot, enzyme-linked immunosorbent assay (detection of antigen and antibody), and neutralization assay carried out using immunofluorescence techniques. The 6 inoculated animals seroconverted during the 1st month after inoculation and remained persistently infected after 6-11 months. We also observed proviral DNA by genomic analysis in the six tested samples. No sign of immunodeficiency disease has been observed so far. The data suggest that HIV-2 infection of nonhuman primates provides an acceptable animal model to investigate vaccination or specific immunotherapeutic procedures.

Detection of infectious HTLV-III/LAV virus in cell-free plasma from AIDS patients.

The authors report data that confirm that plasma and plasma fractions derived from acquired immunodeficiency syndrome (AIDS) patients contain infectious human T-lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV). Whole heparinized blood was obtained from 5 HTLV-III-antibody-positive patients with AIDS and from 3 healthy heterosexuals who were negative for HTLV-III infection. Mononuclear cells treated with either whole plasma or filtered plasma from 4 out of 5 AIDS patients showed a significant level of reverse transcriptase and expression of HTLV-III p15 and p24 while culture fluids from cells treated with plasma from normal donors were negative for reverse transcriptase. Normal peripheral blood lymphocytes treated with reverse transcriptase-positive culture supernatants in the presence of interleukin-2 and hydrocortisone produced readily detectable virus. This suggests that HTLV-III is readily transmitted from the HTLV-III-positive plasma cultures to normal mononuclear cells. In addition the platelet-associated fraction from AIDS patients was shown to induce the release of reverse transcriptase activity. It is important to know that platelet fractions may contain HTLV-III since these fractions are also used in therapy. Overall these findings indicate that HTLV-III circulates as cell-free virus in the blood of AIDS patients as it does in saliva and seminal fluid. The detection of HTLV-III by a simple cell culture technique may prove useful in examining HTLV-III seronegative individuals at high risk and may also help determine whether donated blood found positive in HTLV-III antibody test systems contains free infectious virus.

Production of human T-lymphocyte clones. I. monoclonal culture and functional cytotoxic maturation.

Well-defined clones of human T-lymphocytes were produced and monoclonal T-cell cultures were maintained for long periods of time. Single T-lymphocytes were isolated with the help of a micropipette from PBL cultures prior to any cellular stimulation (MLC), collected separately at the bottom of a 200 microliter tissue culture microwell under the control of stereomicroscopic observation, and cultured with irradiated lymphoid cells in the presence of TCGF. After 12 days, 20-50% of the seeded wells exhibited clones of 3 x 104-105 T-lymphocytes, which were transferred to larger tissue culture wells (2 ml, LINBRO) for long-term culture. Recloning of the growing cloned cells under the same conditions as the primary culture was carried out successfully. In the preliminary cytotoxic assays performed in 11 clones (a) a marked activity directed against lectin-coated targets was observed in may clones; (b) an important NK-like activity was exhibited by the clone 45B9 (65% of the tested cells lysed K562 cell targets); (c) 2 clones did not demonstrate cytotoxic activity against either PHA-coated L-1210 cells or K562 cell targets. These results could be explained hypothetically by the difference of functional maturation of T-cells within each clone.

Repair of the in vitro HIV-1-induced immunosuppression and blockade of the generation of functional suppressive CD8 cells by anti-alpha interferon and anti-Tat antibodies.

The acute human immunodeficiency virus type 1 (HIV-1) infection of activated peripheral blood mononuclear cells (PBMCs) from normal donors results in inhibition of cell proliferation and generation of functional suppressive T cells. Cultured HIV-1 infected PBMCs but not uninfected PBMCs, following irradiation, can inhibit the proliferation of antigen-activated autologous T cells in a dose-dependent way. CD8+ cell subpopulation is responsible for this inhibition. The presence of anti-alpha interferon (IFN alpha) and anti-Tat antibodies in the culture medium counteracts the HIV-1-induced immunosuppression and prevents the generation of suppressive T cells by these PBMCs. The reported data should have major implications for strategies of AIDS treatment which, in association with antiviral drugs, aim at targetting immune disorders.

Enumeration of T-effector cells mediating direct and/or lectin-dependent lysis

Abstract A new method based on the association of single lymphocytes with appropriate allogeneic tumor target cells has been developed to determine CTL frequencies in murine alloimmune lymphoid populations. As assessed by this microassociation method, CTL frequencies in alloimmune populations generated either in vivo or in vitro are higher than those obtained previously using the conjugation method. Moreover, Con A-dependent CTL are even more numerous than directly cytotoxic effector cells, especially after in vitro immunization. Finally, direct evidence is provided that individual CTL can mediate both direct and Con A-dependent lysis.

Biological effect of active anti-IFNα immunization in HIV-infected patients

Circulating interferon (IFN) was investigated in HIV-1 seropositive patients by measuring the IFN alpha antiviral effect in the serum. While serum of healthy seronegative individuals exhibits an antiviral effect, not due to IFNs, considered as background, serum of seropositive patients showed an additional antiviral effect due to the abnormal presence of IFN alpha. Increased titers of IFN alpha were found in the course of the HIV infection and seemed to correlate with the evolution of AIDS disease. Furthermore, patients immunized against IFN alpha had both stabilized CD4 cell count and decreased IFN alpha in their serum. HIV-1-infected patients also exhibited higher titers of natural anti-IFN antibodies than seronegative controls and the level of specific antibodies (Abs) markedly increased in immunized patients. Finally, serum from immunized patients, when compared to seronegative controls, exhibits an interferon neutralizing capacity.

Effect of purified IgGs from HIV-1-infected and non infected individuals on immune activation

The purification and analysis of IgGs from sera of HIV-1-infected and non infected individuals are reported. The effect of antibodies purified from sera of infected individuals on antigen-induced T cell proliferation was investigated in relation to their possible involvement in an autoimmune reaction in AIDS, in view of the previously unravelled striking peptide similarities between HIV-1 gp120 and the immunoregulatory CD4 and Fas molecules. However, our data do not allow definite conclusions to be drawn. The necessity of purifying antibodies against specific peptides to show their direct effect on T-cell activation is further stressed.