ABSTRACT VRC01 is prototypical broadly neutralizing antibody (bnAb), capable of neutralizing diverse HIV strains. Vaccine-based shepherding of VRC01 germline-reverted B-cells to the mature phenotype has proven difficult in mouse models. The overall goal of the proposed research is to design and test novel immunogens for their ability to guide the evolution of the VRC01 B-cell lineage towards a mature phenotype capable of producing bNAbs. We have used existing structural information to aid in the design and construction of prototypes of these immunogens, and we propose to use artificial intelligence-based protein design tools to engineer new immunogens. Engineered immunogens will be tested for a defined set of biochemical attributes, fulfillment of which will lead to testing in an animal model containing the VRC01 lineage. Specific aim 1 is to perform in vitro testing of existing prototype immunogens and proposed immunogens. Binding assays will be performed to determine the affinity of our immunogens for germline- reverted VRC01 mutants using surface plasmon resonance and cell-based methods. Additionally, we will assay immunogens for their ability to activate B-cells in vitro using a flow cytometry-based method. Specific aim 2 is to test the engineered vaccines in a knock-in mouse model. We will test our immunogens in a mouse model producing an overabundance of the VRC01 germline. We will monitor populations of VRC01 germline B-cells using flow cytometry. We will also determine the complementary determining region (CDR) sequences of flow-sorted VRC01-class B-cells to monitor affinity maturation in the population. Lastly, we will determine neutralizing potency of sera from immunized mice during the course of their vaccine regimen.