Efficacy of antitat gene therapy in the presence of high multiplicity infection and inflammatory cytokines

Because human immunodeficiency virus type 1 (HIV-1) infection is characterized by a large number of viral replication cycles and rapid cell turnover in vivo, successful gene therapy requires an approach effective under these conditions. The antitat gene has been proposed for gene therapy because it effectively blocks Tat function and the replication of HIV-1. However, neither antitat nor any other antiviral gene has been shown to inhibit HIV in the presence of high viral load and inflammatory cytokines, a condition closer to the in vivo situation. We show that cells transduced with antitat retrovirus vector are resistant to high multiplicity of HIV infection. In the presence of inflammatory cytokines, including interleukin-1 and tumor necrosis factor, both known to activate viral gene expression independently of Tat, antitat suppressed virus replication. HIV-1 inhibition was observed when cell were treated with a mixture of inflammatory cytokines able to induce acquired immunodeficiency syndrome (AIDS) Kaposi's sarcoma cell growth. These molecules have been shown to be increased in HIV-1-infected individuals, and it is suggested they play a role in the pathogenesis of AIDS. Our results suggest that antitat is effective under conditions present in vivo and therefore a primary candidate for HIV-1 gene therapy.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

Inhibition of apoptosis in chlamydia-infected cells: blockade of mitochondrial cytochrome c release and caspase activation

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.     

Inhibition of HIV-1 expression by HIV-2

HIV-1 and HIV-2 are co-endemic in certain geographic areas. HIV-2 is more weakly pathogenic than HIV-1, and progression to AIDS occurs less frequently and over a longer period of time. Recent epidemiologic studies suggest that individuals infected with HIV-2 have a lower risk of HIV-1 infection. Both immune mechanisms and various modes of viral interference have been proposed to account for these results. Our findings, described in this paper, suggest that HIV-2 inhibits HIV-1 replication. To study the molecular interactions between HIV-1 and HIV-2, proviral clones were transfected alone or in combination into the human T cell line CEM. LTR-CAT indicator constructs were included for the purpose of monitoring viral promoter activity. Viral replication in transfected cells was monitored by p24 antigen capture assay of cell culture supernatants and Western blot analysis of cell extracts. HIV-2 inhibited HIV-1 replication as determined by intracellular and extracellular p24 antigen levels. Similar results were obtained with simultaneous virus infection using HIV-1 and HIV-2, rather than transfections of proviral DNA. Using cotransfection of HIV-1 and HIV-2 LTR indicator gene constructs, the mechanism of inhibition was found to be suppression of the HIV-1 LTR by HIV-2. The inhibitory effect of HIV-2 is not due to Tat-2, but appears to discriminate between the HIV-1 and HIV-2 LTRs based on differences in the Tat activation response element, TAR. These results suggest both a molecular mechanism for HIV-2 interference with HIV-1 replication and a potential molecular approach to therapy.     

The X-ray structure of a divergent cyclophilin from the nematode parasite Brugia malayi

The following is a review of an emerging topic in the literature which has led to new hypotheses regarding the mechanisms of pathogenesis of the various tissue specific AIDS associated syndromes. The fundamental hypothesis in this review proposes that HIV-1 is able to increase lymphocyte and monocyte localization in tissues where released HIV-1 proteins cause local tissue damage leading to any one of the various AIDS associated syndromes. It is also hypothesized here that syndromes associated with other lymphotrophic viruses result from the ability of these viruses to direct leukocyte extravasation of blood vessel walls and to initiate tissue specific pathogenesis. Further, it is suggested here that new concepts and strategies for delivering gene therapy to specific tissues can be derived from our understanding of the mechanisms through which lymphotrophic viruses localize in specific tissues. HIV-1 infection of lymphocytes and monocytes leads to increased adhesion of these cells to vascular endothelium and extracellular matrix molecules. In addition, HIV-1 infection of various leukocytes leads to increased secretion of extracellular matrix degrading matrix metalloproteinases. Increases in leukocyte adhesion and matrix metalloproteinase secretion are associated with the normal mechanisms through which leukocytes localize in tissues during inflammation. The ability of HIV-1 to activate leukocyte adhesion and matrix metalloproteinase secretion suggests that HIV-1 has evolved a way to take advantage of leukocyte inflammatory mechanisms in order to exit the blood stream and gain access to body tissues. The ability of HIV-1 to use infected cells to localize in various tissues may lead to the establishment of HIV-1 reservoirs in tissues. Such viral reservoirs may cause the various tissue specific AIDS associated syndromes. AIDS patients have been found to have elevated adhesion molecules (integrins, and cell adhesion molecules or CAMs) on their peripheral blood lymphocytes (PBLs). While there is little clinical evidence that the tissue localization of HIV-1 infected leukocytes are the cause of the HIV-1 related syndromes, studies in vitro and with animal models have shown that the HIV-1 gene products Tat, Rev and gp120 are potent neurotoxins. It has also been shown that Tat can contribute to the growth of cells from Kaposi's sarcoma lesions. Further, HIV-1 infected cells have been shown to secrete cytotoxic levels of a variety of growth factors and small molecules. Thus, it is likely that the localization of HIV-1 infected cells in specific tissues could contribute to the HIV-1 associated syndromes such as AIDS dementia, HIV-1 related interstitial lung disease, HIV-1 associated nephropathy, the HIV-1 wasting syndrome and perhaps AIDS associated Kaposi's sarcoma and hyperproliferative skin disorders. This review will examine studies in the literature which demonstrate that HIV-1 infection increases leukocyte adhesion and matrix metalloproteinase secretion. Clinical reports of AIDS patient's leukocyte integrin levels will also be reviewed and evidence that tissue localized HIV-1 infected cells could contribute to a variety of HIV-1 associated syndromes will be presented.     

HIV-1 gene expression and replication in neuronal and glial cell lines with immature phenotype: effects of nerve growth factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.     

Potent inhibition of HIV type 1 replication by an antiinflammatory alkaloid, cepharanthine, in chronically infected monocytic cells

Cepharanthine is a biscoclaurine alkaloid isolated from Stephania cepharantha Hayata and has been shown to have antiinflammatory, antiallergic, and immunomodulatory activities in vivo. As several inflammatory cytokines and oxidative stresses are involved in the pathogenesis of HIV-1 infection, we investigated the inhibitory effects of cepharanthine on tumor necrosis factor alpha (TNF-alpha)- and phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 replication in chronically infected cell lines. Two chronically HIV-1-infected cell lines, U1 (monocytic) and ACH-2 (T lymphocytic), were stimulated with TNF-alpha or PMA and cultured in the presence of various concentrations of the compound. HIV-1 replication was determined by p24 antigen level. The inhibitory effects of cepharanthine on HIV-1 long terminal repeat (LTR)-driven gene expression and nuclear factor kappaB (NF-kappaB) activation were also examined. Cepharanthine dose dependently inhibited HIV-1 replication in TNF-alpha- and PMA-stimulated U1 cells but not in ACH-2 cells. Its 50% effective and cytotoxic concentrations were 0.016 and 2.2 microg/ml in PMA-stimulated U1 cells, respectively. Cepharanthine was found to suppress HIV-1 LTR-driven gene expression through the inhibition of NF-kappaB activation. These results indicate that cepharanthine is a highly potent inhibitor of HIV-1 replication in a chronically infected monocytic cell line. Since biscoclaurine alkaloids, containing cepharanthine as a major component, are widely used for the treatment of patients with various inflammatory diseases in Japan, cepharanthine should be further pursued for its chemotherapeutic potential in HIV-1-infected patients.     

A prophylactic and therapeutic AIDS vaccine containing as a component the innocuous Tat toxoid

Extracellular Tat can act as a viral toxin on uninfected cells of different tissues, including the CNS and the immune system, thus in order to immunize humans against Tat we have prepared a biologically inactivated but immunogenic Tat (Tat Toxoid). Tat Toxoid is not toxic in mice even at high doses. It triggers high levels of specific Tat Abs in the mouse and rabbit. Furthermore, in humans Tat Toxoid immunization was safe and induced in seronegatives persistent high levels of Tat Abs and in immunodeficient patients a significant rise of these specific Abs. Facing acute HIV-1 infection, the presence of high level of circulating Tat Abs promoted by Tat Toxoid vaccine should prevent Tat-induced immunosuppression and allow anti-HIV-1 cellular response to develop. As a consequence, early release of beta-chemokines could enhance host resistance towards HIV-1, and, in infected people, inhibit viral replication and evolution towards AIDS.     

An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses

Inhibitors of the protease of HIV-1 have been used successfully for the treatment of HIV-1-infected patients and AIDS disease. We tested whether these protease inhibitory drugs exerted effects in addition to their antiviral activity. Here, we show in mice infected with lymphocytic choriomeningitis virus and treated with the HIV-1 protease inhibitor ritonavir a marked inhibition of antiviral cytotoxic T lymphocyte (CTL) activity and impaired major histocompatibility complex class I-restricted epitope presentation in the absence of direct effects on lymphocytic choriomeningitis virus replication. A potential molecular target was found: ritonavir selectively inhibited the chymotrypsin-like activity of the 20S proteasome. In view of the possible role of T cell-mediated immunopathology in AIDS pathogenesis, the two mechanisms of action (i.e., reduction of HIV replication and impairment of CTL responses) may complement each other beneficially. Thus, the surprising ability of ritonavir to block the presentation of antigen to CTLs may possibly contribute to therapy of HIV infections but potentially also to the therapy of virally induced immunopathology, autoimmune diseases, and transplantation reactions.     

Natural killer cells from HIV-1+ patients produce C-C chemokines and inhibit HIV-1 infection

Human NK cells have been shown to produce cytokines (e.g., IFN-gamma and TNF-alpha) and the chemokine macrophage inflammatory protein (MIP)-1alpha following stimulation with the combination of two monokines, IL-15 plus IL-12. The C-C chemokines MIP-1alpha, MIP-1beta, and RANTES have been identified as the major soluble macrophage-tropic HIV-1-suppressive factors produced by CD8+ T cells, which exert their action at the level of viral entry. Here, we demonstrate that monokine-activated NK cells, isolated from both normal and HIV-1+ donors, produce similar amounts of MIP-1alpha, MIP-1beta, and RANTES protein, in vitro. Further, supernatants of monokine-activated NK cells obtained from both normal donors and AIDS patients showed potent (routinely > or = 90%) suppressive activity against HIV-1 replication in vitro, compared with unstimulated control supernatants. NK cell supernatants inhibited both macrophage-tropic HIV-1(NFN-SX) and T cell-tropic HIV-1(NL4-3) replication in vitro, but not dual-tropic HIV-1(89.6). Importantly, the C-C chemokines MIP-1alpha, MIP-1beta, and RANTES were responsible only for a fraction of the HIV-1-suppressive activity exhibited by NK cell supernatants against macrophage-tropic HIV-1. Collectively these data indicate that NK cells from normal and HIV-1+ donors produce C-C chemokines and other unidentified factors that can inhibit both macrophage- and T cell-tropic HIV-1 replication in vitro. Since NK cells can be expanded in patients with HIV-1, AIDS, and AIDS malignancy in vivo, this cell type may have an important role in the in vivo regulation of HIV-1 infection.     

Interferon alpha and Tat involvement in the immunosuppression of uninfected T cells and C-C chemokine decline in AIDS

HIV type 1 (HIV-1) not only directly kills infected CD4(+) T cells but also induces immunosuppression of uninfected T cells. Two immunosuppressive proteins, interferon alpha (IFNalpha) and extracellular Tat, mediate this process because specific antibodies against these proteins prevent generation of suppressor cells in HIV-1-infected peripheral blood mononuclear cell cultures. Furthermore, the production of C-C chemokines in response to immune cell activation, initially enhanced by IFNalpha and Tat, ultimately is inhibited by these proteins in parallel with their induction of immunosuppression. The clinical corollary is the immunosuppression of uninfected T cells and the decline in C-C chemokine release found at advanced stages of HIV-1 infection paralleling rising levels of IFNalpha and extracellular Tat. We, therefore, suggest that IFNalpha and Tat may be critical targets for anti-AIDS strategies.     

Bovine immunodeficiency virus tat gene: cloning of two distinct cDNAs and identification, characterization, and immunolocalization of the tat gene products

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.     

In vitro characterization of adult primary human immunodeficiency virus type 1: demonstration of distinctive single-cell killing phenotypes in spite of similar levels of viral replication

Two primary human immunodeficiency virus (HIV)-1 biologic clones have been studied extensively in a system using CD4 T cell-enriched peripheral blood lymphocytes and anti-CD4 antibody to measure viral replication kinetics and single-cell cytopathicity. Biologic clones from a person with AIDS replicated to high levels and were cytopathic in the absence of syncytium formation. Unexpectedly, biologic clones from an adult long-term nonprogressor were noncytopathic in spite of similar levels of viral replication. A correlation has recently been demonstrated between reduced mitochondrial viability and cell death in HIV-1-infected cultures. Peripheral blood-derived CD4 T cells infected with the cytopathic clone showed a progressive reduction in mitochondrial viability, while those infected with the noncytopathic clone demonstrated functionally viable mitochondria. These studies demonstrate that primary HIV-1-induced cytopathicity is separable from syncytium formation and replication rate.     

The effect of commencing combination antiretroviral therapy soon after human immunodeficiency virus type 1 infection on viral replication and antiviral immune responses

Twelve subjects were treated with zidovudine, lamivudine, and ritonavir within 90 days of onset of symptoms of acute infection to determine whether human immunodeficiency virus type 1 (HIV-1) infection could be eradicated from an infected host. In adherent subjects, with or without modifications due to intolerance, viral replication was suppressed during the 24-month treatment period. Durable suppression reduced levels of HIV-1-specific antibodies and cytotoxic T lymphocyte responses in selected subjects. Proviral DNA in mononuclear cells uniformly persisted. The persistence of HIV-1 RNA expression in lymphoid tissues and peripheral blood mononuclear cells suggests that elimination of this residual pool of virus should be achieved before considering adjustments in antiretroviral therapeutic regimens. In addition, given the reduction in levels of virus-specific immune responses, it would seem prudent to consider enhancing these responses using vaccine strategies prior to the withdrawal of antiviral therapy.