PROJECT SUMMARY A central goal in HIV/AIDS vaccine research is the elicitation of broadly neutralizing antibodies (bNAbs). Here, we propose to leverage three recent discoveries from our groups to generate novel envelope (Env) immunogens that target the V1V2 region of the trimer apex. By studying the evolution of the HIV-1 Env glycan shield, we discovered that unshielded regions (“glycan holes”) in transmitted founder (TF) Envs are negatively associated with bNAb development, suggesting that strain-specific glycan holes delay or subvert bNAb development (1). Generating bNAb sensitivity signatures to design novel Signature-based Epitope Targeted (SET) vaccines, we found that V2-SET immunogens induced broader and more potent tier 2 heterologous NAbs in guinea pigs than wild-type Envs, indicating that inclusion of bNAb signatures significantly improved vaccine performance (2). Studying 20 novel SHIVs in ~100 rhesus macaques (RMs) (3), we found that ~15% of animals developed varying degrees of heterologous breadth by 6-24 months, with the most common bNAb specificity targeting the V2 apex (Table 1). However, bNAb induction in SHIV infection is still infrequent, thus providing a unique experimental setting to test iterative Env design improvements in a manner that is faster and less costly than human trials. Our hypothesis is that by (i) minimizing distracting glycan hole epitopes, (ii) increasing Env affinity for V2 apex bNAb precursors, (iii) increasing relevant epitope diversity in vaccine boosts, and (iv) incorporating B cell lineage immunogen designs, we will improve V2 bNAb germline engagement and bNAb lineage maturation. We have selected the CRF.AG.T250 (T250) and CAP256SU (CAP256) Envs as baseline immunogens, because both have generated V2 apex bNAbs in SHIV infected RMs (Table 1). In Aim #1, we will optimize the Env glycan shield and V2 germline targeting properties of T250 and CAP256 Envs, test their replication potential and tier 2 antigenicity in SHIV vectors, and down-select the best performing set for subsequent infection of RMs. In Aim #2, we will compare the bNAb induction capacity of SHIVs expressing wildtype (WT), glycan-optimized (GLY- OPT), and glycan and germline-optimized (GLY/UCA-OPT) versions of the same Env in RMs and determine the envelope-antibody (Env-Ab) coevolution pathways in all animals that develop neutralization breadth. In Aim #3, we will rationally design new V2 apex directed immunogens using Env-Ab co-evolution data and the V2-SET strategy as a guide, and deliver them using nucleoside-modified mRNA containing lipid nanoparticles (mRNA/LNPs) that express stabilized, membrane bound gp160s. We will prime RMs with the best performing Env from Aim #2, and then compare the bNAb induction capacity of this Env with...