There is a serious need for a smallpox vaccine alternative because of the significant incidence of adverse events to the current vaccine (Dryvax). Large groups in the American population are not qualified to receive the current smallpox vaccine due to immunodeficiency (from genetic causes, HIV, or immunosuppressive drugs), old age, skin disorders, young age (< 1 yr), or pregnancy. These groups are major populations and must be accounted for in any reasonable national smallpox vaccination strategy. Therefore, assessment of the immunogenicity of alternative smallpox vaccines such as modified vaccinia Ankara (MVA) must be done accurately and expeditiously. Our access to samples from ongoing clinical trials of Dryvax and MVA, our expertise in quantitating both cellular and humoral immunity, and our immunologic proteomics approach places our research group in a unique position to address this important issue. Long term protective immunity to smallpox is likely provided by three arms of the immune system: circulating neutralizing antibodies, memory B cells, and memory T cells. Virtually none of the vaccinia protein targets responsible for these T cell, B cell, and neutralizing antibody responses have been identified. The experiments in this proposal are designed to determine the dominant anti-smallpox immune responses and to compare the magnitude and breadth of the T and B cell responses induced by Dryvax versus MVA. This valuable information will not only allow an accurate assessment of the quality of the immune responses elicited by the alternative smallpox vaccine MVA but can also then be immediately parlayed into additional areas of proposed research such as: 1) diagnostic tools for measuring long term smallpox immunity in vaccinees; 2) development of simple, rapid, and sensitive immunological tools for assessing recent smallpox (variola major) exposure/infection; and 3) development of therapeutic anti-smallpox neutralizing monoclonal human antibodies. Finally, the knowledge acquired from the detailed studies described herein regarding the immunodominant protein targets of human vaccinia-specific T and B cells will lay the foundation for developing a safe and effective smallpox subunit vaccine.