Development of strategies to enhance CTL responses against HIV antigens is critical for the development of effective HIV vaccines. Listeria monocytogenes has long been used as a model system for the study of CTL responses. Listeria has been used as a potent vector to generate strong CTL responses against viral or tumor antigens, and Listeriolysin 0 (LLO) plays a critical role in the ability to induce CTL responses. The ability of LLO to lyse the endosome membrane enables antigens to be released into the cytosol and presented to the MHC I-restricted antigen presentation pathway. Furthermore, LLO has been shown to have strong adjuvant activity for eliciting CTL responses possibly through the induction of IL-12. These properties of LLO will be used to enhance the CTL response against SHIV gag and env proteins. The hypothesis is that the use of DNA vaccines expressing chimeric proteins will confer two advantages; first, that the LLO sequence in the chimeric proteins will retain its membranelytic activity which will promote the entry of the chimeric proteins into the MHC I antigen presentation pathway; secondly, that the LLO sequence in the chimeric proteins will retain the ability to induce cytokines such as IL-12 and therefore serve as an adjuvant to enhance CTL responses against SHIV antigens.
Specific Aim 1 will involve construction of DNA vectors expressing SHIV Env/LLO and Gag/LLO chimeric proteins and characterization of their expression. A series of genes encoding SHIV 89.6 Gag/LLO and Env/LLO chimeric proteins will be constructed. Expression of proteins will be characterized before the DNA vectors are used for immunization studies.
Specific Aim 2 will involve immunization of mice using DNA vectors expressing SHIV Env/LLO and Gag/LLO chimeric proteins and characterization of CTL responses against SHIV antigens in comparison with immunizing mice using DNA vectors expressing SHIV Env or Gag protein alone or in combination with a DNA vector separately expressing LLO. DNA vectors that induce strong CTL responses against SHIV antigens will be advanced for future vaccine studies in rhesus macaques.
