Yeast Display Reveals Plentiful Mutations that Improve Fusion-Peptide Vaccine-Elicited Antibodies Beyond 59% HIV-1-Neutralization Breadth

Background/Objectives: Vaccine elicitation of antibodies with high HIV-1-neutralization breadth is a long-standing goal. Recently, induction of such antibodies has been achieved at the fusion peptide-site of vulnerability; questions remain, however, as to whether their neutralization breadth and potency were sufficient to prevent HIV-1 infection. Methods: Here, we use yeast display coupled with deep-mutational screening, biochemical and structural analysis to study improvement of the best fusion-peptide-directed, vaccine-elicited antibody, DFPH_a.01, with initial 59% breadth. Results: Yeast display identified both single and double mutations that improved recognition of HIV-1-envelope trimers. We characterized two paratope-distal light chain (LC) mutations, S10R and S59P, which together increased breadth to 63%. Biochemical analysis demonstrated DFPH-a.01_10R59P-LC, and its component mutations, to have increased affinity and stability. Cryo-EM structural analysis revealed elbow-angle influencing by S10RLC and isosteric positioning by S59PLC as explanations for enhanced breadth, affinity, and stability. Conclusions: These results, along with another antibody with enhanced performance (DFPH-a.01_1G10A56K-LC with 64% breadth), suggest possible mutations that improve DFPH_a.01 are plentiful, an important vaccine insight.