Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.     

Macrophages and CD4+ T lymphocytes from two multiply exposed, uninfected individuals resist infection with primary non-syncytium-inducing isolates of human immunodeficiency virus type 1.

Despite multiple, high-risk sexual exposures, some individuals remain uninfected with human immunodeficiency virus type 1 (HIV-1). CD4+ lymphocytes from these individuals are less susceptible to infection in vitro with some strains of HIV-1, suggesting that the phenotype of the virus may influence its ability to interact with certain CD4+ cells. In the present study, we examined the susceptibility of CD4+ T lymphocytes and macrophages from two exposed uninfected individuals (EU2 and EU3) to infection with a panel of biologically cloned isolates of HIV-1 having either a non-syncytium-inducing (NSI) or a syncytium-inducing (SI) phenotype. Our results indicate that CD4+ T lymphocytes from EU2 and EU3 are resistant to infection with NSI isolates of HIV-1 but are susceptible to infection with primary SI isolates. In addition, we found that macrophages from EU2 and EU3 are resistant to infection with both NSI and SI isolates. The latter finding was confirmed by using several uncloned NSI and SI isolates obtained from patients during acute HIV-1 infection. In further experiments, env clones encoding glycoproteins characteristic of NSI or SI viruses were used in single-cycle infectivity assays to evaluate infection of CD4+ lymphocytes and macrophages from EU2 and EU3. Consistent with our previous results, we found that macrophages from these individuals are resistant to infection with NSI and SI env-pseudotyped viruses, while CD4+ T lymphocytes are resistant to NSI, but not SI, pseudotyped viruses. Overall, our results demonstrate that CD4+ cells from two exposed uninfected individuals resist infection in vitro with primary, macrophage-tropic, NSI isolates of HIV-1, which is the predominant viral phenotype found following HIV-1 transmission. Furthermore, infection with NSI isolates was blocked in both CD4+ T lymphocytes and macrophages from these individuals, suggesting that there may be a common mechanism for resistance in both cell types.     

Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection

We have analyzed a number of biological features of HTLV-IV, a retrovirus indistinguishable from a macaque isolate of simian immunodeficiency virus (SIV), and compared this virus with several strains of human immunodeficiency virus type 1 (HIV-1). Although HTLV-IV was found to be similar to HIV-1 in its tropism for CD4+ lymphocytes, its effects on CD4 expression and the ability of its externalized envelope molecule to form a complex directly with the CD4 molecule, a number of striking differences were noted. Unlike with HIV-1, the range of cells susceptible to HTLV-IV infection and syncytia formation was restricted to a subset of CD4+ cell lines, particularly those that coexpressed CD4 with human leukocyte antigen (HLA) class II antigens. An analysis of the patterns of HTLV-IV infection with B x T somatic cell hybrids indicated that for this virus, molecules in addition to CD4 were probably required to facilitate infection and cell fusion. Additional studies of HTLV-IV infection of Sup-T1 cells, which are exquisitely sensitive to cytopathic effects induced by HIV-1, demonstrated that HTLV-IV infection could occur in the absence of cytopathic effects and, remarkably, with minimal or no downmodulation of the CD4 molecule from the cell surface. The failure of HTLV-IV infection to reduce the expression of several CD4 epitopes suggested that the HTLV-IV envelope produced by Sup-T1 cells was altered in its ability to interact with or bind to CD4. Additional differences were also noted in the size of the transmembrane envelope molecule of HTLV-IV produced by Sup-T1 cells, indicating that cell-specific alterations in processing of the HTLV-IV envelope occurred during the production of virus in this cell line. Understanding the basis for these biological differences between HTLV-IV and the HIV-1 viruses may help to elucidate more general mechanisms for pathogenesis of other members of the SIV and HIV families of retroviruses.     

The anti-idiotypic antibody 1F7 selectively inhibits cytotoxic T cells activated in HIV-1 infection

Circulating CD8+ T lymphocyte numbers rise substantially following infection with HIV-1. This expanded CD8+ T cell population includes HIV-specific CTL and CTL that kill activated uninfected CD4+ lymphocytes. Experimental, epidemiological and clinical evidence supports the possibility that expansion of CD8+ CTL contributes to CD4+ T cell depletion and disease progression in human HIV infection. Therefore, modulation of CD8+ T cell numbers or of certain CD8+ CTL activated in HIV-infected individuals may be beneficial. It was found that 1F7, a mAb against an idiotype common to anti-HIV and anti-simian immunodeficiency virus (SIV) antibodies, selectively inhibited both anti-HIV CTL and CTL against uninfected CD4+ T cells. Alloantigen-specific CTL and NK cells from either HIV-infected individuals or controls were unaffected by 1F7. Prolonged incubation of CD8+ T cells from HIV-infected individuals with 1F7 induces apoptosis, which was shown to be reflected functionally in reduced total CTL activity and in especially reduced CTL activity against uninfected CD4+ lymphocytes. The selective reactivity of 1F7 with certain CD8+ CTL could be applied towards the modulation of CD8+ T cell responses involved in AIDS pathogenesis.     

CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses.

T lymphocytes expressing the CD8 surface antigen block HIV replication in CD4+ peripheral blood cells from HIV-infected individuals. We report here that CD4+ cells from HIV seronegative donors, when infected in vitro with HIV, also do not replicate virus when cocultured with CD8+ T cells from HIV-infected individuals. CD8+ cells from HIV-uninfected donors did not show this effect on virus replication. HLA-restriction of the antiviral response was not observed, and virus-containing cells were not eliminated from culture. The antiviral activity was broadly cross-reactive, as CD8+ cells from individuals infected only with HIV-1 suppressed the replication of diverse strains of HIV-1 and HIV-2, as well as the simian immunodeficiency virus. This ability of CD8+ cells to control HIV replication could play an important role in the maintenance of an asymptomatic state in HIV-infected individuals.     

A human recombinant Fab identifies a human immunodeficiency virus type 1-induced conformational change in cell surface-expressed CD4.

To explore the role of the CD4 molecule in human immunodeficiency virus (HIV) infection following initial virus-CD4 binding, we have characterized CD4-specific antibodies raised by immunizing an HIV-1-infected human with human recombinant soluble CD4 (rsCD4). Fabs were selected from a human recombinant Fab library constructed from the bone marrow of this immunized individual. Here, we describe a human rsCD4-specific recombinant Fab clone selected by panning the library over complexes of human rsCD4 and recombinant HIV-1 envelope protein. While this Fab does not bind to CD4-positive T-cell lines or to human T lymphocytes, it recognizes cell surface-expressed CD4 following the incubation of these cells with a recombinant form of HIV-1 gp120 or with HIV-1 virions. The Fab is not HIV-1 envelope specific, since it does not bind to recombinant gp120 or to native cell surface-expressed HIV-1 envelope proteins. As confirmation of its CD4 specificity, we show that this Fab immunoprecipitates a 55-kDa protein, corresponding to the molecular mass of cellular CD4, from an H9 cell lysate. The specificity of this human Fab provides evidence for a virus-induced conformational change in cell surface-expressed on CD4. The characterization of this altered CD4 conformation and its effects on the host cell will be important in defining postbinding events in HIV infection.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.     

Contact of human immunodeficiency virus type 1-infected and uninfected CD4+ T lymphocytes is highly cytolytic for both cells.

Individuals infected with the human immunodeficiency virus (HIV) experience a marked loss of CD4+ T lymphocytes, leading to fatal immunodeficiency. The mechanisms causing the depletion of these cells are not yet understood. In this study, we observed that CD4+ T lymphocytes from HIV type 1 (HIV-1)-infected and uninfected individuals rapidly lysed B lymphoblasts expressing the HIV-1 envelope glycoprotein on the cell surface and Jurkat cells expressing the complete virus. Contact of uninfected CD4+ T cells with envelope glycoprotein-expressing cells also resulted in the lysis of the uninfected CD4+ T cells. Cytolysis did not require priming or in vitro stimulation of the CD4+ T cells and was not restricted by major histocompatibility complex molecules. Cytotoxicity was inhibited by soluble CD4 and anti-CD4 monoclonal antibodies that block binding of CD4 to gp120. In addition, neutralizing anti-CD4 and anti-gp120 monoclonal antibodies which block postbinding membrane fusion events and syncytium formation also inhibited cell lysis, suggesting that identical mechanisms in HIV-infected cultures underlie cell-cell fusion and the cytolysis observed. However, cytotoxicity was not always accompanied by the formation of visible syncytia. Rapid cell lysis after contact of uninfected and HIV-1-infected CD4+ T cells may explain CD4+ T-cell depletion in the absence of detectable syncytia in infected individuals. Moreover, because of its vigor, lysis of envelope-expressing targets by contact with unprimed CD4+ T lymphocytes may at first glance resemble antigen-specific immune responses and should be excluded when cytotoxic T-lymphocyte responses in infected individuals and vaccinees are evaluated.     

Cytopathic killing of peripheral blood CD4(+) T lymphocytes by human immunodeficiency virus type 1 appears necrotic rather than apoptotic and does not require env

An important unresolved issue of AIDS pathogenesis is the mechanism of human immunodeficiency virus (HIV)-induced CD4(+) T-lymphocyte destruction. We show here that HIV type 1 (HIV-1) exerts a profound cytopathic effect upon peripheral blood CD4(+) T lymphocytes that resembles necrosis rather than apoptosis. Necrotic cytopathology was found with both laboratory-adapted strains and primary isolates of HIV-1. We carefully investigated the role of env, which has been previously implicated in HIV cytopathicity. HIV-1 stocks with equivalent infectivity were prepared from constructs with either an intact or mutated env coding region and pseudotyped with the glycoprotein of vesicular stomatitis virus (VSV-G) so that the HIV envelope was not rate-limiting for infection. Infected Jurkat T cells died whether or not env was intact; however, the expression of env accelerated death significantly. The accelerated death was blocked by protease inhibitors, indicating that it was due to reinfection by newly produced virus in env(+) cultures. Accordingly, we found no disparity in kinetics in CD4(lo) Jurkat cells. In highly infected peripheral blood T cells, profound necrosis occurred equivalently with both env(+) and env(-) stocks of HIV-1. We also found that HIV-1 cytopathicity was undiminished by the absence of nef. However, viral stocks made by complementation or packaging of HIV-1 genomes with the natural protein-coding sequences replaced by the green fluorescent protein were highly infectious but not cytopathic. Thus, env can accelerate cell death chiefly as an entry function, but one or more viral functions other than env or nef is essential for necrosis of CD4(+) T cells induced by HIV-1.     

Inhibition of CD4+ T cell function by the HIV envelope protein, gp120

The CD4 molecule is functionally involved in the class II MHC-restricted T cell response to Ag. CD4 is also the receptor for HIV-1, the major etiologic agent of AIDS. We have assessed whether the interaction of the HIV-1 envelope protein with the CD4 molecule might interfere with the normal function of CD4, thereby contributing to the immunosuppression observed after HIV infection. Using a murine T cell hybridoma which expresses the human CD4 protein and exhibits a CD4-dependent response to Ag, we demonstrate that the HIV envelope protein gp120 can specifically inhibit this response.     

Infection of CD127+ (interleukin-7 receptor+) CD4+ cells and overexpression of CTLA-4 are linked to loss of antigen-specific CD4 T cells during primary human immunodeficiency virus type 1 infection

We recently found that human immunodeficiency virus (HIV)-specific CD4+ T cells express coreceptor CCR5 and activation antigen CD38 during early primary HIV-1 infection (PHI) but then rapidly disappear from the circulation. This cell loss may be due to susceptibility to infection with HIV-1 but could also be due to inappropriate apoptosis, an expansion of T regulatory cells, trafficking out of the circulation, or dysfunction. We purified CD38+++CD4+ T cells from peripheral blood mononuclear cells, measured their level of HIV-1 DNA by PCR, and found that about 10% of this population was infected. However, a small subset of HIV-specific CD4+) T cells also expressed CD127, a marker of long-term memory cells. Purified CD127+CD4+ lymphocytes contained fivefold more copies of HIV-1 DNA per cell than did CD127-negative CD4+ cells, suggesting preferential infection of long-term memory cells. We observed no apoptosis of antigen-specific CD4+ T cells in vitro and only a small increase in CD45RO+CD25+CD127dimCD4+ T regulatory cells during PHI. However, 40% of CCR5+CD38+++ CD4+ T cells expressed gut-homing integrins, suggesting trafficking through gut-associated lymphoid tissue (GALT). Furthermore, 80% of HIV-specific CD4+ T cells expressed high levels of the negative regulator CTLA-4 in response to antigen stimulation in vitro, which was probably contributing to their inability to produce interleukin-2 and proliferate. Taken together, the loss of HIV-specific CD4+ T cells is associated with a combination of an infection of CCR5+ CD127+ memory CD4+ T cells, possibly in GALT, and a high expression of the inhibitory receptor CTLA-4.     

High CCR5 Density on Central Memory CD4+ T Cells in Acute HIV-1 Infection Is Mostly Associated with Rapid Disease Progression

CD4+ central memory T cells play a critical role in the pathogenesis of simian immunodeficiency virus disease, and the CCR5 density on the surface of CD4 T cells is an important factor in human immunodeficiency virus (HIV)-1 disease progression. We hypothesized that quantifying central memory cells and CCR5 expression in the early stages of HIV-infection could provide useful prognostic information. We enrolled two different groups of acute HIV-infected subjects. One group progressed to CD4 T cell numbers below 250 cells/µl within 2 years (CD4 Low group), while the other group maintained CD4 cell counts above 450 cells/µl over 2 years (CD4 High group). We compared the CCR5 levels and percentage of CD4 subsets between the two groups during the 1st year of HIV infection. We found no differences between the two groups regarding the percentage of naïve, central memory and effector memory subsets of CD4 cells during the 1st year of HIV-1 infection. CCR5 levels on CD4+ CM subset was higher in the CD4 Low group compared with the CD4 High group during the 1st year of HIV-1 infection. High CCR5 levels on CD4 central memory cells in acute HIV infection are mostly associated with rapid disease progression. Our data suggest that low CCR5 expression on CD4 central memory cells protects CD4 cells from direct virus infection and favors the preservation of CD4⁺ T cell homeostasis.