Impact of repeated vaccination on the effectiveness of seasonal influenza vaccines Summary Influenza viruses cause pandemic and seasonal outbreaks that lead to the loss of thousands to millions of human lives. Vaccination is the best option for preventing influenza outbreaks and minimizing their effects on health. In the United States, annual influenza vaccination has been recommended since 2010 for persons 6 months of age and older. However, vaccine performance varies significantly between influenza seasons, and reduced vaccine effectiveness has been observed. Studies have reported that persons vaccinated during two consecutive influenza seasons had lower vaccine effectiveness during the second season than persons who had not been vaccinated during the prior season. These findings have caused profound confusion among the public regarding the potential benefit of annual influenza vaccination. Thus, there is a critical need to address the effect of repeated vaccination?associated pre-existing immunity on influenza vaccine performance. The objective of this project is to characterize the effects of repeated influenza vaccination on the specificity and magnitude of cross-reactive antibodies and on the effectiveness of seasonal influenza vaccines. Two specific aims are proposed: 1) determine the specificity, magnitude, and longitudinal patterns of humoral responses in humans with repeated seasonal vaccination, and 2) test the effect of repeated vaccination? associated pre-existing immunity on influenza vaccine performance in ferrets. By comparing antibodies in persons with and without repeated influenza vaccination, we expect to show whether and how pre-existing immunity achieved through repeated influenza vaccination affects the specificity and magnitude of the cross-reactivity for the resulting antibodies and, thus, vaccine effectiveness. From our studies in ferrets, we expect to show whether and how variations in repeated vaccination?associated pre- existing immunity affect influenza vaccine performance. This study will expand our understanding of molecular mechanisms that may influence how repeated vaccination affects influenza vaccine performance. Thus, this study will provide basic knowledge for evaluating the need for annual influenza vaccination and for optimizing influenza vaccine performance.