We have spent substantial effort demonstrating that the smallpox vaccine elicits lifelong immunological memory in human (JI 2003, Clin Vaccine Imm 2007) and the human neutralizing antibody response is multifaceted, targeting several viral proteins and utilizing several different mechanisms of action to provide protecive immunity (JV 2005, JV 2008, Immunity 2008, JV 2009a, JV 2009b, Vaccine 2009, Antiviral Therapy 2010). Our analysis of antigen-specific CD4 T cell and B cell responses to VACV in mice discovered that nearly half of the CD4 T cell responses targeted matched viral virion proteins of the antibody response, suggesting a deterministic linkage between the specificities. A series of experiments revealed that help between CD4 T cells and B cells of matched viral virion protein specificity is non-transferable to other viral proteins, including other viral particle surface or core proteins (Immunity 2008). This study demonstrated that individual protein antigens-not the virion-are the primary unit of immunological recognition for a complex pathogen. Therefore MHC restriction is a critical selective event for humoral immune responses and protective immunity to complex pathogens, an issue of great relevance for vaccine design. Furthermore, we recently demonstrated that follicular helper CD4 T cell (TFH) differentiation is controlled by Bcl6 and Blimp-1 as reciprocal and antagonistic master regulator transcription factors (Science 2009), and that germinal centers and long term antibody responses are completely dependent on TFH. That work solidly established TFH as a true and biologically important CD4 T cell differentiation subset. We additionally demonstrated that CD4 T cell TFH differentiation is heavily dependent on B cells (Science 2009), yet another powerful indication of the co-dependence and linkage of B and CD4 T cell immunity (Nature Immunology 2010). In the present application we describe our plans to further these studies to determine the underlying immunological mechanisms of B-T bidirectional interrelationships and explore new aspects of the mechanisms of neutralizing antibody function.) PUBLIC HEALTH RELEVANCE: Antibody responses are critical components of protective immune responses to many viruses, but it remains unclear what parameters determine which pathogen proteins are targeted by the host antibody response. Vaccines are one of the most cost-effective medical treatments in modern civilization. The smallpox vaccine (vaccinia virus, VACV) has been extraordinarily effective, having brought about the worldwide eradication of smallpox disease. Elucidating the immunobiology underlying the protection provided by the smallpox vaccine will continue to reveal critical vaccinology principles that can be applied to future vaccine development against other infectious scourges. We study mechanisms of humoral immunity development and function to the smallpox vaccine virus to understand this gold standard of human vaccine efficacy.