Suresh K. Arya

Identification of RANTES, MIP-1α, and MIP-1β as the Major HIV-Suppressive Factors Produced by CD8+ T Cells

Evidence suggests that CD8+ T lymphocytes are involved in the control of human immunodeficiency virus (HIV) infection in vivo, either by cytolytic mechanisms or by the release of HIV-suppressive factors (HIV-SF). The chemokines RANTES, MIP-1α, and MIP-1β were identified as the major HIV-SF produced by CD8+ T cells. Two active proteins purified from the culture supernatant of an immortalized CD8+ T cell clone revealed sequence identity with human RANTES and MIP-1α. RANTES, MIP-1α, and MIP-1β were released by both immortalized and primary CD8+ T cells. HIV-SF activity produced by these cells was completely blocked by a combination of neutralizing antibodies against RANTES, MIP-1α, and MIP-1β. Recombinant human RANTES, MIP-1α, and MIP-1β induced a dose-dependent inhibition of different strains of HIV-1, HIV-2, and simian immunodeficiency virus (SIV). These data may have relevance for the prevention and therapy of AIDS.

Control of viral rebound through therapeutic immunization with DermaVir.

Objective:To reconstitute immune responses capable of eliminating infected cells and suppressing viral load during chronic retroviral infection. Design:A topical, DNA-based therapeutic immunization (DermaVir) was designed to express most of the regulatory and structural viral genes in dendritic cells. Methods:DermaVir alone and in combination with antiretroviral drugs was tested in chronically SIV-infected macaques. Results:DermaVir provided virological, immunological and clinical benefit for SIV-infected macaques during chronic infection and AIDS. In combination with antiretroviral drugs, DermaVir augmented SIV-specific T-cell responses and enhanced control of viral load rebound during treatment interruptions. Conclusions:The results indicate the feasibility of therapeutic immunization even in immune compromised hosts, and suggest that DermaVir can complement antiretroviral drugs to sustain suppression of HIV-1 replication.

Induction of Potent Human Immunodeficiency Virus Type 1-Specific T-Cell-Restricted Immunity by Genetically Modified Dendritic Cells

ABSTRACT A novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4+ and CD8+ HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.

Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation.

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)-Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 10(5) transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1-5x10(7) TU/ml.

Towards developing HIV-2 lentivirus-based retroviral vectors for gene therapy: Dual gene expression in the context of HIV-2 LTR and Tat

Because of the distinct ability of retroviruses to integrate into the target cell genome and thus achieve long‐term expression, retrovirus vectors hold great promise for stable gene transfer. Such vectors derived from human immunodeficiency retroviruses (HIVs) and other lentiviruses are envisioned to possess several advantages, especially for in vivo gene therapy of HIV infection and acquired immunodeficiency syndrome (AIDS) where targeting CD4+ T cells/macrophages and pluripotent non‐dividing stem cells would be required. Among these is the ability of HIVs to transduce nondividing cells in contrast to the murine retroviruses which require target cell mitosis. The advantages of the lentivirus vectors will be further enhanced by the development of multigenic vectors carrying more than one gene in a dependent or independent transcriptional unit. Separate from the issue of transduction efficiency, information is needed about the impact of the configuration of the genes in a multigenic vector on their expression. Towards this end, we investigated the expression of genes specifically directed by the HIV‐2 LTR and Tat in a prototypic minimal transfer vector. We found that the expression of a gene in a dual gene configuration depended upon its position in the transcriptional unit and that the insertion of an internal translational initiation mechanism improved the expression of the downstream gene. Apparently not sufficiently appreciated previously, these effects were promoter and cell‐type dependent. Our data also suggest that the commonly used cellular or viral promoters may be orders of magnitude less effective than HIV‐2 LTR in the presence of Tat, and thus may not be useful as internal promoters in the context of the HIV‐2 LTR:Tat regulatory loop. J. Med. Virol. 54:118–128, 1998.

Conditional regulatory elements of human immunodeficiency virus type 2 long terminal repeat

Mutational analysis of the human immunodeficiency virus type 2 (HIV-2) long terminal repeat (LTR) revealed a novel cis-acting positive and a negative regulatory element in the U3 region, located upstream of the enhancer-promoter region. These elements acted in a cell type-specific manner, being most active in human lymphocytic CEM cells, more active in Jurkat cells than in human monocytic U937 cells and least active in epithelioid HeLa cells. The down-modulatory effect of the negative regulatory element was abolished by HIV-2 Tat, suggesting the involvement of upstream DNA elements in optimal Tat-mediated trans-activation. The sequence elements that respond to T cell activation signals were also located in the upstream U3 region. Notably, the magnitude of the effect of the upstream regulatory elements depended on the basal activity of the LTR, which was also cell type-dependent. This emphasizes the importance of the cell-specific transcriptional factors and other effectors in regulating HIV gene expression. These observations may be relevant to the cell type-specific restriction of virus replication in vivo.

Organization of polyoma virus DNA in mammary tumours of athymic mice.

Restriction mapping of polyoma virus DNA in mammary tumours of athymic mice gave patterns that varied with the tumour examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. All tumours contained tandemly integrated full-length and defective virus genomes. Some tumours also contained unintegrated virus DNA molecules, some full-length and others defective. The deletions were localized in the virus genomic sequences coding for the distal part of the large T antigen. After the first transplantation, the organization of polyoma virus genomes in tumours remained essentially unchanged through four successive transplantations. The tumour cells that initially contained free virus DNA molecules continued to possess such molecules during serial transplantations. The virus DNA molecules in transplanted tumours lacking unintegrated virus genomes were more methylated than in tumours containing unintegrated virus genomes.

Mouse mammary tumour virus and polyoma virus information in mammary tumours of athymic mice inoculated with polyoma virus.

Polyoma virus inoculation of athymic mice results in the development of mammary tumours with a much higher incidence than the development of salivary gland tumours, the latter being the most common for immunocompetent normal mice. The possibility existed that polyoma virus might act as a co-carcinogen in activating the expression of mouse mammary tumour virus (MMTV). Molecular hybridization studies, however, showed that the mammary tumour development was accompanied by neither the amplification of MMTV genomic sequences nor by their more extensive transcription. In contrast, tumour tissue contained about 60 to 100 copies of polyoma virus genome equivalents per cell and some of these sequences were apparently transcribed into RNA. While these results do not rule out the transient involvement of MMTV expression in mammary tumour development, it appeared that the mammary gland cells were directly transformed by polyoma virus. Apparently, polyoma virus displayed a tropism in athymic mice that was different from that in normal mice.

The Family of Human T-Cell Leukemia Viruses and Their Role in the Cause of T-Cell Leukemia and AIDS

HTLV is the generic name we gave the first human retroviruses. The majority of isolates are very closely related; we call them human T-cell leukemia virus type I (HTLV-I). HTLV-I is endemic (at low rates) in the Caribbean, South and Central America, southeast U.S., southern Japan, and especially Africa. Viruses closely related to HTLV-I, but distinct from it, have been isolated from Old World monkeys. This and other facts led us to propose that the ancestral origin of HTLV is in Africa. Evidence indicates that HTLV-I is the direct cause of an aggressive form of adult T-cell leukemia and lymphoma. The mechanisms involved in the in vitro immortalization and in vivo malignancy are not yet clear but apparently do not involve any visible consistent chromosomal change, consistent virus expression, or known onc genes. Whichever the mechanism for growth induction by HTLV-I, its efficiency in causing malignancy may be because it has dual major effects on infected cells: (1) immortalization of some T cells, and (2) interference with function and cytopathic changes in many others. In collaboration with D. Golde and colleagues, we also discovered a second class of human T-lymphotropic retroviruses (HTLV-II). It shares many features with HTLV-I but has major genomic differences.

Sequence complexity of polyadenylated RNA in human breast carcinoma cells.

Abstract 1. 1. The sequence complexity of total cellular and polysomal poly(A)-containing RNA in human breast carcinoma cells was estimated to be respectively about 200,000 and 38.000 kilobases based on kinetics of RNA: cDNA hybridization. 2. 2. Prolactin treatment of these cells moderately altered the apparent distribution of polysomal RNA sequences among the frequency classes. 3. 3. Total cellular and polysomal poly(A)-containing RNA corresponded to about 4.8 and 1.6% of the single-copy DNA in these cells, respectively.