Sundarasamy Mahalingam

Modulation of amplitude and direction of in vivo immune responses by co-administration of cytokine gene expression cassettes with DNA immunogens

Immunization with nucleic acids has been shown to induce both antigen‐specific cellular and humoral immune responses in vivo. We hypothesize that immunization with DNA could be enhanced by directing specific immune responses induced by the vaccine based on the differential correlates of protection known for a particular pathogen. Recently we and others reported that specific immune responses generated by DNA vaccine could be modulated by co‐delivery of gene expression cassettes encoding for IL‐12, granulocyte‐macrophage colony‐stimulating factor and the co‐stimulatory molecule CD86. To further engineer the immune response in vivo, we investigated the induction and regulation of immune responses following the co‐delivery of pro‐inflammatory cytokine (IL‐1α, TNF‐α, and TNF‐β), Th1 cytokine (IL‐2, IL‐12, IL‐15, and IL‐18), and Th2 cytokine (IL‐4, IL‐5 and IL‐10) genes. We observed enhancement of antigen‐specific humoral response with the co‐delivery of Th2 cytokine genes IL‐4, IL‐5, and IL‐10 as well as those of IL‐2 and IL‐18. A dramatic increase in antigen‐specific T helper cell proliferation was seen with IL‐2 and TNF‐α gene co‐injections. In addition, we observed a significant enhancement of the cytotoxic response with the co‐administration of TNF‐α and IL‐15 genes with HIV‐1 DNA immunogens. These increases in CTL response were both MHC class I restricted and CD8+ T cell dependent. Together with earlier reports on the utility of co‐immunizing using immunologically important molecules together with DNA immunogens, we demonstrate the potential of this strategy as an important tool for the development of more rationally designed vaccines.

Molecular and immunological analysis of genetic prostate specific antigen (PSA) vaccine

Nucleic acid immunization has been investigated as immunotherapy for infectious diseases as well as for treating specific types of cancers. In this approach, nucleic acid expression cassettes are directly inoculated into the host, whose transfected cells become the production source of novel and possibly immunologically foreign protein. We have developed a DNA vaccine construct which encodes for PSA by cloning a cDNA for PSA into a mammalian expression vector under control of a CMV promoter. We investigated and characterized the immunogenicity of PSA DNA expression cassettes in mice. PSA-specific immune responses induced in vivo by immunization were characterized by enzyme-linked immunosorbent assay (ELISA), T helper proliferation cytotoxic T lymphocyte (CTL), and flow cytometry assays. We observed a strong and persistent antibody response against PSA for at least 180 days following immunization. In addition, a significant T helper cell proliferation was observed against PSA protein. Using synthetic peptides spanning the PSA open frame, we identified four dominant T helper epitopes of PSA. Furthermore, immunization with PSA plasmid induced MHC Class I CD8+ T cell-restricted cytotoxic T lymphocyte response against tumor cell targets expressing PSA. The prostate represents a very specific functional organ critical for reproduction but not for the health and survival of the individual. Understanding the immunogenicity of PSA DNA immunization cassettes offers insight into the possible use of this tumor-associated antigen as a target for immunotherapy. These results demonstrate the ability of the genetic PSA to serve as a specific immune target capable of generating both humoral and cellular immune responses in vivo.

HIV-1 viral protein R (Vpr) regulates viral replication and cellular proliferation in T cells and monocytoid cells in vitro

Among the putative accessory genes of HIV‐1, the 96‐amino‐acid virion‐associated vpr gene product has been described to have several novel biological activities. These include cytoplasmic‐to‐nuclear translocation, which empowers HIV to infect and replicate in non‐dividing cells and to increase viral replication, particularly in macrophages. Along with these viral effects, we found that HIV‐1 Vpr induces dramatic biological changes in the target cells of HIV infection, including induction of changes in transcriptional patterns, morphological changes, and complete inhibition of proliferation, which collectively was termed differentiation. These changes occur in the absence of other viral gene products, suggesting that Vpr mediates its proviral effects partially or perhaps solely through modulation of the state of the target cell rather than directly on the virus. The inhibition of proliferation in T cell lines has been extended by several groups to demonstrate that the inhibition of proliferation is through G2 cell cycle arrest, further supporting the idea that Vpr acts directly on cellular targets. We have recently described a role for Vpr in modulating the glucocorticoid pathway, which is involved in the regulation of the state of the cell, in cytoplasmic‐to‐nuclear translocation, and in modulation of host cell transcription. It is important to note that certain anti‐glucocorticoid compounds modulate Vpr activity in vitro. These results support the idea that the host cell contains specific receptor molecule(s) through which Vpr mediates its activity. Consequently, Vpr represents a unique target for anti‐HIV drug development and has significance for HIV‐1 disease progression. J. Leukoc. Biol. 62: 93–99; 1997.