Recent studies have shown that broad neutralizing antibody responses are found in about 20% of HIV-1 infected individuals and develop in most cases during the first two to three years following infecfion. In this program, we seek to study the dynamics and mechanism of development of broad neutralizing antibody responses through access to two large and well-characterized primary infection cohorts, Protocol C (donors from sub-Saharan Africa), and the First Choice Program (donors from Southern California). Our focus in this project will be to characterize the neutralizing specificifies as they emerge and evolve in individuals who develop broad and potent serum neutralizafion. Our specific aims are: Aim 1. To map the neutralizing specificities in the sera of individuals with broad and potent neutralizing Ab responses. This characterization will allow the selecfion of the most appropriate donors for isolafion of broadly neutralizing human monoclonal Abs (bNmAbs) in Aim 2 and the design of suitable baits for the isolafion of bNmAbs in collaboration with Project 2. A detailed analysis of the specificities responsible for broad serum neutralization will be carried out on the top 5% of neutralizers in the 2 cohorts. Aim 2. To isolate and characterize bNmAbs from individuals selected in Aim 1. This informafion will be used to trace the evolutionary history of the broadly neutralizing responses in combination with Project 2. We will use the recently successful high-throughput direct functional screening strategy to isolate bNmAbs from four selected individuals from procedures in Aim 1. We will precisely map the epitopes recognized by the bNmABs. This informafion will be used to design baits to follow the evolufion of the B cell receptor specificity associated with broad neutralization. The studies will yield a panel of new broadly neutralizing antibodies and reveal for the first fime the evolutionary pathway leading to the development of broad neutralizing specificities in interplay with viral escape. The results will have important consequences for vaccine design by showing for example whether high levels of somafic mutations are required for broad neutralization, which would signify the need of immunization strategies favoring prolonged antigen exposure, or whether particular pathways of Ab-Env interacfion and viral escape may favor the development of broad specificities, which will lead to the development of new immunogens and immunizafion protocols.