ABSTRACT – OVERALL HIV-1 broadly neutralizing antibodies (bnAbs) are protective in animal models of HIV-1 infection, but are not elicited in humans by current vaccine regimens. To elicit bnAbs, the B cell lineage vaccine design approach aims to administer multiple immunogens in a specific sequence to shepherd bnAb maturation through immunologic roadblocks that typically halt bnAb development. One roadblock we recently identified are somatic mutations that encode key amino acids for antibody function but that are rarely made by the somatic mutation enzyme activation-induced cytidine deaminase. Our central vaccine design hypothesis is that antibodies (Abs) encoding these improbable mutations, will be rare; thus, vaccine immunogens will need to have higher affinity for Abs with these desired amino acid changes than Abs without the amino acid changes in order to select for them. The problem facing this strategy is that the only antigen for HIV-1 bnAbs is HIV-1 envelope (Env), which is poorly immunogenic and for which bnAb precursors generally have low affinity. We and others have found these two obstacles can be overcome by designing Envs with high affinity for bnAb precursors and by multimerizing these Envs on nanoparticles (NPs) to provide avidity and improved antigen trafficking to germinal centers. However, Env trimer NPs can have low expression and present misfolded Env trimers that elicit undesired non-neutralizing Abs. This application is significant because it will establish a cGMP-compliant vaccine platform that rapidly generates higher quality HIV-1 Env trimer NP vaccines without time-consuming iterative immunogen design. This platform uses the sortase A enzyme to site-specifically, covalently-link well- folded HIV-1 Env trimers to intact Helicobacter pylori ferritin NPs. The resultant HIV-1 Env trimer sortase A- conjugated NPs (scNPs) display only well-folded Env trimers, and in preliminary studies, have successfully initiated CD4 binding site bnAb lineages in human bnAb precursor knock-in mice and CD4bs nAbs in rhesus macaques. The scNP platform is universal in nature since it can incorporate diverse viral type I fusion proteins by simply adding a 6-amino acid sortase A tag to their C-terminus. In Specific Aim 1, we will compare the ability of monovalent and bivalent HIV-1 Env scNPs to guide affinity maturation of CD4 binding site bnAbs in humanized mice and rhesus macaques. In Specific Aim 2, we will produce and assemble two CD4 binding site-bnAb- targeting HIV-1 Env trimer scNPs (CH505 TF scNP and a second sequential Env trimer scNP) under cGMP conditions. This program will deliver an optimized cGMP process for making scNPs, two cGMP-produced Env trimer scNPs, and additional ferritin and sortase A components for the manufacture of future immunogens. The CH505 TF Env trimer scNPs will be used in a Phase I trial through the HIV Vaccine Trial Network. Ultimately, the impact of this platform is that it will enable multiple Env trimer scNPs ...