Characterizing mutant intermediate Fabs to probe affinity maturation in the HIV-1 CH103 broadly neutralizing antibody lineage

Understanding affinity maturation of antibodies of great breadth that can target many variants of HIV-1 is important for the development of potential vaccine strategies. To this end, we probed affinity maturation of the CH103 broadly neutralizing antibody (bnAb) clonal lineage using wild-type antigen-binding fragments (Fabs) and I3.2 mutant Fabs. Fabs are derived from the CH103 bnAb lineage, and have mutations found at the interface between the variable heavy chain (V_(H)) and the variable light chain (V_(L)) that likely contributed to the development of breadth. In this lineage, the variable light chain shifted away from gp120 over the course of affinity maturation in order to accommodate insertion mutations in a variable loop, called V5, of the HIV Env, a protein involved in membrane fusion and thus infection. We crystallized mutants to confirm that residues at the V_(H) – V_(L) interface contributed to the shift in orientation. In addition to the V_(H) – V_(L) shift observed, the lineage also had altered stability throughout affinity maturation. The results demonstrate the importance of residues located outside the antigen-binding site in affinity maturation.