The recent isolation of HIV broadly neutralizing antibodies (bNAbs) from HIV infected individuals has reinvigorated efforts to develop B cell-based vaccines. As the sole viral target for bNAbs, HIV envelope glycoprotein (Env) has been engineered as soluble trimers to recapitulate bNAbs responses via vaccination. However, Env-based immunogens thus far primarily induce vaccine-matched neutralizing antibody (nAb) responses. This thesis aims to understand the mechanisms restricting the neutralization breadth and to provide strategies for iterative improvements.

First, we have established an antigen-specific single B cell sorting and monoclonal antibody (mAb) cloning platform for guinea pigs, a small animal model desirable in the field for initial immunogenicity analysis. This method allowed us to dissect the antibody responses at the clonal level with high accuracy and efficiency.

Secondly, we have delineated the specificity of autologous neutralization elicited by the current generation HIV trimer mimicry, BG505 SOSIP.664. Our results reveal a prominent epitope in the C3/V4 region of the Env targeted by one nAb/B cell clonal lineage.

We demonstrate that the nAb responses to this neutralization determinant are prevalent in trimer-vaccinated guinea pigs, rabbits, and non-human primates. In addition, this defined nAb response shares a high degree of similarity with the early nAb response in an HIV- infected pediatric patient, who later developed a bNAb response. This study offers insights into re-designing Env immunogens in the highly immunogenic region to broaden nAb responses.

Lastly, we have engineered novel immunogens based on the Env sequence of a virus strain isolated from bNAb VRC01 donor, which can engage the VRC01 germline precursor in vitro. Sequential prime-boost immunizations in a VRC01-germline immunoglobulin (Ig) encoding genes knock-in mouse model with the designed immunogens induced focused VRC01-like serum antibody responses and clustered VRC01-class somatic mutations in the knock-in VRC01-germline Ig genes. In addition, the mAbs recovered from the immunized mice neutralize selected viruses containing the N276 glycan, a critical roadblock impeding the affinity maturation of VRC01-class bNAbs. Our findings demonstrate that, in the transgenic mouse model, our immunogens effectively activate bNAb precursor B cells and guide their affinity maturations required for bNAb function, which has important implications for HIV vaccine development.