SCANNING THE VACCINIA PROTEOME FOR ANTISMALLPOX ANTIGENS

DESCRIPTION (provided by applicant): A technology developed by Gene Therapy Systems Inc. under a previously funded Phase I SBIR grant will be applied to producing the complete proteome from vaccinia virus and the proteome will be applied to the general problem of identifying potent vaccine antigens affective against smallpox. The technology called Transciptionally Active PCR (TAP) is a method for generating functional PCR fragments that can be used directly in in vitro transfection assays, and in vivo. TAP fragments can also be used as templates in cell free in vitro transcription/translation reactions generating >20 micrograms of protein/50 microliter reaction volume, and the TAP system has been placed onto a robotics workstation enabling 384 different purified proteins to be produced and purified in 1 day.This system will be used to amplify and purify all 266 proteins encoded by vaccinia virus and the proteins will be used to scan humoral and cellular immune responses in vaccinia virus vaccinated mice. In this way humoral and cellular "vaccine antigen potency indexes" will be generated for each antigen. These potency indexes will be used together with other immunological and bioinformatics criteria to identify likely candidate antigens for a DNA vaccine. Plasmids encoding the antigens identified in this way, will be injected into mice and the mice will be challenged with an infectious dose of virus to determine DNA vaccine efficacy of the different antigens. The outcome of these challenge studies will be used to validate and refine this approach for identifying effective vaccine antigen candidates.The assays developed in mice will be adapted to human tissue samples, and the humoral aid cellular vaccine antigen potency indexes will be determined in a small group of vaccinia virus vaccinated human volunteers. Since vaccinia virus is substantially homologous to variola virus and since the vaccinia virus vaccine is known to be effective against variola virus infection, the responsive antigens identified from this assay will be candidates for use in a DNA or subunit subunit vaccine against smallpox. This vaccine will have no risk of producing vaccinia virus disease or Eczema vaccinatum.