Complementary Roles of Antibody Heavy and Light Chain Somatic Hypermutation in Conferring Breadth and Potency to the HIV-1-Specific CAP256-VRC26 bNAb Lineage

Some HIV-infected people develop broadly neutralizing antibodies (bNAbs) that block many diverse, unrelated strains of HIV from infecting target cells and, through passive immunization, protect animals and humans from infection. Therefore, understanding the development of bNAbs and their neutralization can inform the design of an HIV vaccine. Here, we extend our previous studies of the ontogeny of the CAP256-VRC26 V2-targeting bNAb lineage by defining the mutations that confer neutralization to the unmutated common ancestor (CAP256.UCA). Analysis of the sequence of the CAP256.UCA showed that many improbable mutations were located in the third complementarity-determining region of the heavy chain (CDRH3) and the heavy chain framework 3 (FR3). Transferring the CDRH3 from bNAb CAP256.25 (63% breadth and 0.003 μg/mL potency) into the CAP256.UCA introduced breadth and the ability to neutralize emerging viral variants. In addition, we showed that the framework and light chain contributed to potency and that the second CDR of the light chain forms part of the paratope of CAP256.25. Notably, a minimally mutated CAP256 antibody, with 41% of the mutations compared to bNAb CAP256.25, was broader (64% breadth) and more potent (0.39 μg/mL geometric potency) than many unrelated bNAbs. Together, we have identified key regions and mutations that confer breadth and potency in a V2-specific bNAb lineage. These data indicate that immunogens that target affinity maturation to key sites in CAP256-VRC26-like precursors, including the CDRHs and light chain, could rapidly elicit breadth through vaccination. A major focus in the search for an HIV vaccine is elucidating the ontogeny of broadly neutralizing antibodies (bNAbs), which prevent HIV infection and . The unmutated common ancestors (UCAs) of bNAbs are generally strain specific and acquire breadth through extensive, and sometimes redundant, somatic hypermutation during affinity maturation. We investigated which mutations in the CAP256-VRC26 bNAb lineage conferred neutralization capacity to the UCA. We found that mutations in the antibody heavy and light chains had complementary roles in neutralization breadth and potency, respectively. The heavy chain, particularly the third complementarity-determining region, was responsible for conferring breadth. In addition, previously uninvestigated mutations in the framework also contributed to breadth. Together, approximately half of the mutations in CAP256.25 were necessary for broader and more potent neutralization than many unrelated neutralizing antibodies. Vaccine approaches that promote affinity maturation at key sites could therefore more rapidly produce antibodies with neutralization breadth.