INDUCING APOPTOSIS TO AUGMENT CROSS-PRIMING FOR CTL

DESCRIPTION: Naive CTL precursors can only be activated to proliferate and differentiate into effector and memory CTLs by antigen presented by dendritic cells (DCs). DCs can acquire antigen by phagocytosing apoptotic cells and transferring antigen present in the apoptotic cells to the MHC class I antigen-processing pathway of the DC. The process whereby antigen from one cell (undergoing apoptosis) is presented by another (the DC) is known as cross-presentation. DC presentation of antigen is responsible for CTL priming by DNA vaccination, and it is likely that this is largely due to cross-presentation of antigen from transfected non-hematopoietic cells. We propose to augment this process by linking a pro-apoptotic gene to antigen in plasmid DNA vaccination. SIV-nef will be expressed behind the CMV promoter, and the pro-apoptotic gene driven off the same promoter from an internal ribosome entry site (IRES). We will test two apoptosis inducing genes: the adenovirus E4 ORF4, and caspase 3. Transiently transfected cells will be assessed for antigen expression and apoptosis induction. The CTL response will be assessed initially in mice, using ELISPOT to detect SIV-nef-specific CTL. We will compare constructs, doses and numbers of injections in mice to determine regimen yielding the greatest number of nef-specific CTL. The optimized regimen will be modified for use in rhesus monkeys, and compared with a protocol using the same injection strategy without apoptosis-inducing genes. The monkeys will be assessed for CTL numbers after DNA vaccination; they will then be boosted with a poxvirus expressing nef and challenged with virulent SIV.