Figure 5. Mutagenesis studies pinpoint key affinity-matured residues responsible for broad binding and neutralization activity of bnAbs 2411a and 2413a.

(A) List of revertant antibodies. Each mutated residue of 2411a and selected residues in 2413a were individually reverted to its germline residue, and mutant antibodies that contain only the specified reverted SHM point mutations were made.

(B) The impact of individual residues reverted to germline on 2411a binding and neutralization titers. Left, heat map showing the ELISA binding (OD450) of parental and revertant 2411a IgGs to various HIV-Env antigens. Right, the neutralization titers (IC₅₀) of parental 2411a and 2413a as well as their revertants against a panel of 10 tier II viruses. Middle, the mean fold changes in ELISA binding titers to each antigen and neutralization titers to each tested virus of every SHM revertant antibody compared to its parental antibody. Revertant antibodies with ELISA fold change >1.5 and/or neutralization fold change >2.0 were highlighted in red, both in A and B. The 2411a-H-V37M revertant with a neutralization fold change of 1.91 was labeled in dark red.

(C) Mutability analysis identifies the key mutated residues as rare mutations. The positional mutation frequency of IGHV1-2 and IGKV3-20 genes was estimated from 6883 and 21915 antibody clones from 293 non-HIV human antibody repertoires, respectively (see STAR Methods for detail). The logo graphs show the positional mutation frequency for all mutations on each position of the VH1-2 (top) and VK3-20 (bottom) genes. The 2411a and 2413a mutated residues with lower than 0.5% frequency are deemed rare and labeled in red. The germline residues of the identified key mutations as in (B) are labeled in teal for comparison.