While worldwide eradication of smallpox represents a major accomplishment of medicine in the 20th century, use of this virus as a bioterrorism agent against our largely disease-susceptible civilian population could result in unprecedented mortality. Individuals at risk for live-virus vaccine complications, including those with cancer and eczema, comprise a large percentage of the US population, mandating against massive large-scale vaccination. Recent developments in immunology, both with regard to mechanistic understanding of adaptive and innate immune responses now allow for evaluation of the cellular and humoral bases of protective immunity against orthopox and other classes of viruses. These advances include details of immune recognition at a structural level, antigen presentation, cell migration and T cell memory. Here, four groups of investigators will utilize their considerable talents in vaccinology, virology, immunology, cutaneous biology, structure and bioinformatics to identify critical orthopox epitopes affording protective human immunity. Project 1 will examine protective immunity to vaccinia virus in normal and high-risk patients elicited during virus vaccination trials based on parameters identified in Project 2. Project 2 will identify T cell epitopes shared by vaccinia, MVA and smallpox by genome-wide comparison using bioinformatics and position-specific scoring matrices, and confirmed by T cell functional assays and mass spectrometry. Antigen-specific T memory cells elicited through vaccination will be assessed by pMHC tetramers, conventional and new biomarkers of T cell memory and molecularly detailed T cell memory repertoires as examined by single cell PCR at different times post-vaccination. Likewise, targets and biophysical parameters of human neutralizing antibodies to vaccinia and variola, the latter in conjunction with CDC, will be identified using recombinant orthopox proteins, BIAcore, ELISA and neutralization studies. In Project 3, investigators from the Harvard Skin Disease Research Center will examine human skin elements of orthopox vaccinated normals or atopic dermatitis patients for productive viral infection, and compare and contrast the nature of central memory and skin homing effector T cells therein. Murine models using biologic response modifiers and transgenic mice will be exploited to examine how manipulation of the cutaneous environment alters vaccination efficacy. Project 4 will use contemporary molecular genetics to mutate vaccinia virus-Wyeth strain to lower virulence by deleting immune escape functions but maintaining host range, replication and immunogenicity. Pathogenicity and immunogenicity assessment will be in C57BL/6, transgenic or mutant mice using systematic, mucosal and dermal scarification infectious routes. An Educational Component, Pilot Project Component and Research Resource Technical Development Component are proposed for rapid dissemination of methods and reagents resulting from this Center's effort.