Characterizing antibody responses to germline targeting HIV-1 immunogens in human antibody transgenic mice

The development of an effective HIV-1 vaccine has focused on eliciting broadly neutralizing antibodies (bnAbs) that target conserved regions on the viral envelope glycoprotein (Env). One approach, germline targeting, aims to activate naïve B-cell precursors capable of maturing into bnAb-producing cells. VRC01-class bnAbs bind the conserved CD4-binding site (CD4bs) of HIV-1 Env but require key somatic hypermutations to reach their mature forms. Engagement of VRC01-class precursors is sterically hindered by the N276 glycan near the CD4bs, explaining its omission from immunogens targeting VRC01-class precursors. However, mature VRC01-class bnAbs can accommodate this glycan, indicating that booster immunogens need to guide precursor B cells toward glycan accommodation. This study characterizes monoclonal antibodies (mAbs) isolated from human-antibody transgenic mice immunized with three regimens based on eOD-GT8 (GT8), a VRC01-class germline-targeting immunogen designed without the canonical N276 glycan. Specifically, animals were immunized with GT8 and modified variants hypothesized to heighten CD4bs-directed antibody responses: GT8 prime/GT8 boost, GT8 prime/eOD-GT8-NVT (NVT) boost, and eODGT8-NIK prime/NVT boost. Single antigen-specific memory B cells (GT8⁺/KO⁻) were sorted and antibody genes recovered by RT-PCR. Cloned mAbs were evaluated for binding to GT8, NVT, and KO by ELISA and biolayer interferometry (BLI). Sequence analysis was used to examine heavy-chain gene usage, HCDR3 features, light-chain usage, and sequence similarities among binding antibodies. ELISA analysis showed that all immunization regimens elicited antibodies that bound the tested antigens, although regimen-level differences in binding frequency and ELISA AUC values were not statistically significant. BLI analysis using monomeric antigens showed reduced overall binding, with binding retained for GT8 but lost for KO. VH gene analysis showed that VRC01-class antibodies, defined by IGHV1-2 usage, were not elicited; instead, IGHV3- 23 was enriched among ELISA-binding antibodies. Light-chain analysis did not show enrichment of a specific κ or λ gene, and neither HCDR3 nor LCDR3 length distinguished binding from non-binding antibodies. These data enlighten potential alternative strategies for guiding CD4bs-directed antibodies toward bnAb development.