ABSTRACT-PROJECT 1 Previous vaccines have been unable to elicit HIV-1 broadly neutralizing antibodies (bnAbs) in humans. Two of the reasons for this failure are that bnAb precursor B cells are often rare and that the B cell receptors on those rare B cells require extensive somatic mutation to evolve into high-affinity bnAbs. A subset of these somatic mutations—termed improbable mutations—have a low probability of being made by the somatic mutation machinery due to codon bias and positioning of somatic mutation recognition motifs. Our central hypothesis for eliciting bnAbs is that immunogens must have a higher affinity for Abs with the required improbable mutations than for Abs lacking these key mutations. Moreover, we hypothesize that mutation-guided vaccine design will need to engage low-affinity rare bnAb precursors and, then, through immunization with sequential immunogens, select for Abs acquiring the somatic changes requisite for a broad neutralization phenotype. We and others have observed that multimerizing HIV-1 envelope (Env) immunogens on nanoparticles (NPs) can increase Ab titers and affinity maturation of HIV-1 bnAb lineages. However, Env is unstable and can adopt non-native conformations on the surface of NPs, leading to undesired Ab responses. Additionally, NPs bearing HIV-1 envelope can be of low protein yield. To overcome these pitfalls, we have developed an innovative, rapid platform that uses the enzyme sortase A to covalently link well-folded, cleaved Env trimers to self- assembling ferritin protein NPs. These sortase A-conjugated NPs (scNPs) maintain near-native Env trimer conformation, allowing avid selection of improbable mutations in evolving bnAb lineages. In humanized mice, we used a scNP displaying an engineered Env trimer called CH505.M5 G458Y to select for a key improbable mutation required for maturation of the 8ANC131/CH235 class of CD4 binding site (CD4bs) bnAbs. The same scNP elicited CD4bs monoclonal neutralizing Abs and serum neutralizing Abs in nonhuman primates (NHPs). To further guide these Abs to develop neutralization breadth, we have generated a boosting Env trimer scNP immunogen called CH505.TF scNP and have a third boosting Env under development. In CH505.M5 G458Y scNP-primed NHPs, CH505 TF scNP immunization boosts neutralizing Ab titers against multiple CH505 viruses. This IPCAVD will generate a CH505 TF scNP boosting immunogen and a second sequential Env trimer scNP boosting immunogen for a Phase I trial. In Specific Aim 1, Project 1 will determine an optimal NP boosting regimen to broaden the neutralization capacity of current CD4bs B cell lineages in humanized mice. In Specific Aim 2, we will compare conjugation efficiency and immunogenicity of scNPs assembled from research-grade or development-run ferritin and sortase A components from Project 2. Lastly, Specific Aim 3 will demonstrate the induction of CD4bs lineages in NHPs using the prime-boost strategy and cGMP scNPs proposed for the Phase I trial...