Gene combination raises broad human immunodeficiency virus-specific cytotoxicity.

DNA plasmid immunization has the important advantage over traditional vaccines of making it possible to combine selected genes into one vaccine. The efficacy of a combination of DNA plasmids encoding the nef, rev, and tat HIV-1 regulatory genes in inducing cellular immune responses was analyzed in asymptomatic HIV-1-infected patients. Patients initially selected for having low or no detectable immune responses to Nef, Rev, or Tat antigens developed MHC class I-restricted cytolytic activities as well as enhanced bystander effects. The induction of memory cells against target cells infected with the whole HIV-1 genome was analyzed by using a pseudovirus HIV-1/murine leukemia virus (MuLV), and target cells infected with vaccinia virus carrying the respective gene. The most remarkable change observed after immunization with the gene combination was an increase in cytotoxic T lymphocyte (CTL) precursors to target cells infected with the whole HIV-1 genome. Infection by the pseudotype HIV-1/MuLV virus should result in a multitude of HIV-1 peptides presented on the target cell surface, representative of the in vivo situation. An in vitro assessment of the expression of the single and combined gene products showed that this was consistent with the induction of CTL responses in vivo. No clinical advantage or adverse effects were noted. Therapeutic effects of such immunization may become measurable by structured therapy interruption.

Gene therapy for the treatment of AIDS: animal models and human clinical experience.

Although antiretroviral drug therapy has had a significant impact on the natural history of HIV infection, complete virus eradication still remains an unattainable goal. Drug-mediated virological control only occurs transiently, in part as a result of the development of drug resistance. Gene therapy for the treatment of AIDS is a promising area of research that has as its goal the replacement of the HIV-infected cellular pool with cells engineered to resist virus replication. A variety of anti-HIV genes have been designed and tested in laboratory systems, and available results from pilot clinical trials demonstrate the safety and feasibility of this approach. Obstacles to effective application of this technology include partial protection of HIV resistance genes, lack of effective vectoring systems, and unregulated gene expression. Herein, we review recent advances in transduction methods, data from in vivo preclinical studies in relevant animal models, and emerging results derived from pilot clinical gene therapy studies.

Enhanced T cell engraftment after retroviral delivery of an antiviral gene in HIV-infected individuals.

Intracellular expression of gene products that inhibit viral replication have the potential to complement current antiviral approaches to the treatment of AIDS. We previously have shown that a mutant inhibitory form of an essential viral protein, Rev M10, prolongs the survival of T cells transduced with a nonviral vector in HIV-infected individuals. Because these gene-modified cells were not observed in patients beyond 8 weeks, efforts were made to improve the duration of engraftment. In this study, we used retroviral vector delivery of Rev M10 to CD4(+) cells and analyzed relevant immune responses in a pilot study of three HIV-seropositive patients. DNA and RNA PCR analyses revealed that cells transduced with Rev M10 retroviral vectors survived and expressed the recombinant gene for significantly longer time periods than those transduced with a negative control vector in all three patients. Immune responses were not detected either to Rev M10 or to Moloney murine leukemia virus gp70 envelope protein. Rev M10-transduced cells were detected for an average of 6 months after retroviral gene transfer compared with approximately 3 weeks for the previously reported nonviral vector delivery. These findings suggest that retroviral delivery of an antiviral gene may potentially contribute to immune reconstitution in AIDS and could provide a more effective vector to prolong survival of CD4(+) cells in HIV infection.

Analysis of trans-dominant mutants of the HIV type 1 Rev protein for their ability to inhibit Rev function, HIV type 1 replication, and their use as anti-HIV gene therapeutics.

The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.

Therapeutic approaches to HIV infection based on virus structure and the host pathogen interaction.

The HIV-1 infection of central nervous system, with attendant neuropathy and dementia, poses a unique challenge for antiviral therapy. For practical considerations, it is important to define carefully the precise therapeutic objectives. (1) Is it necessary to inhibit spreading HIV-1 infection in the central nervous system? (2) What is the role of inflammatory responses in central nervous system disease during HIV-1 infection? (3) Is there a correlation between pathology and dementia? (4) Are virions or virus gene products toxic in the central nervous system? (5) Is there a role for immune suppression and opportunistic pathogens in AIDS dementia? The development of therapeutic agents for HIV-1 infection is guided by our knowledge of virus structure, the function of viral proteins, the interactions with host components, and detailed features of the virus life cycle. In each case, unique features of the virus can be identified and established as targets for unique antiviral compounds. Drugs acting as inhibitors of virus enzymatic functions are plagued by the rapid development in vivo of drug-resistant virus variants, although combination or alternating chemotherapeutic regimens may obviate some of these concerns. Novel approaches to inhibiting virus are flourishing. In vitro studies show the value of agents as diverse as molecular decoys for tat activity to efforts to mutagenize integrated proviruses by modified oligonucleotides that form triple helices with chromosomal genes. As each particular clinical situation is better defined, the design and application of these agents can be refined to inhibit HIV-1 replication and reduce the associated morbidity.