PROJECT SUMMARY Developing universal vaccines to influenza and HIV-1 is an urgent global goal. A critical challenge is that immune responses to native HIV-1 envelope (Env) and influenza hemagglutinin (HA) are dominated by non- neutralizing and highly strain-specific antibodies. Discoveries that some individuals produce broadly neutralizing antibodies (bnAbs) invigorated hope that, while not naturally dominant, broadly protective antibody responses are possible. Antibodies mature during through somatic hypermutation (SHM) and affinity-based selection in germinal centers (GCs) in competition with other antibodies that recognize different parts of the same virus. It is widely believed that a prime and boost vaccine tactic can effectively elicit bnAb precursors and strategically guide SHM trajectory can produce bnAbs. Challenges to this process are that native envelope proteins may not bind well to the bnAb precursor antibodies and may be poorly represented in the antibody repertoire. A strategic prime and boost strategy requires generation of designer viral envelope variants that bind well to bnAb ancestor antibodies acting as a primer, followed by modified variants to function as boosting immunogen(s) to shepherd bnAb maturation. This promising approach is hindered by time and effort required to identify Env or HA variants as immunogens, which traditionally require mutation library generation, in vitro static selection, cloning, expression, and validation testing. This extensive hands-on trial and error process greatly hinders the pace of progress. Here a new technology is proposed with power to explosively accelerate the pace of immunogen discovery by creatively harnessing the full spectrum of automated mutation and selection inherent in one of nature’s innovations in hyperevolution—namely the GC SHM and affinity maturation system—an automated in vivo dynamic mutation process coupled to parallel selection activity that dynamically shuttles superior binding variants back for further diversification and selection. In addition to dramatically improving binding affinity, the GC system can be engineered to generate new recognition. The objective is to create flipped GC systems in which antibody genes are replaced with viral envelope proteins— and deploy them for immunogen design. In contrast to dynamic antibody evolution to viral envelop protein in normal GCs, flipped GCs dynamically evolve viral envelop protein toward user-defined antibodies (e.g. select bnAb precursors and intermediates). The overall hypothesis is that, in the context of key modifications, the GC/affinity maturation system is sufficiently flexible to permit bioengineered viral envelope proteins to affinity mature toward user-defined bnAb precursors and intermediates. The objective will be pursued with two aims: 1) to establish parameters to engineer GCs as a platform for non-Ig protein evolution. And 2) to generate HIV-1 and influenza envelop variants from flipped GC mice. Completion...