Potent broadly neutralizing antibodies (bnAbs) to HIV have been very challenging to elicit by vaccination in wild-type animals. Here, by x-ray crystallography, cryo–electron microscopy, and site-directed mutagenesis, we structurally and functionally elucidate the mode of binding of a potent bnAb (NC-Cow1) elicited in cows by immunization with the HIV envelope (Env) trimer BG505 SOSIP.664. The exceptionally long (60 residues) third complementarity-determining region of the heavy chain (CDR H3) of NC-Cow1 forms a mini domain (knob) on an extended stalk that navigates through the dense glycan shield on Env to target a small footprint on the gp120 CD4 receptor binding site with no contact of the other CDRs to the rest of the Env trimer. These findings illustrate, in molecular detail, how an unusual vaccine-induced cow bnAb to HIV Env can neutralize with high potency and breadth.

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Ab diversity in most vertebrates results from the assortment of amino acid side chains on CDR loops formed through V(D)J recombination. Cows (Bos taurus) have a low combinatorial diversity potential because of a small number of highly homologous V, D, and J gene segments. Despite this, a subset of the Ab repertoire (;10%) contains exceptionally long CDR H chain (HC) 3 (H3) regions with a rich diversity of cysteines and disulfide-bonded loops that diversify through a single V-D-J recombination event followed by massive somatic hypermutation. However, the much larger portion of the repertoire, encoding shorter CDR H3s, has not been examined in detail. Analysis of germline gene segments reveals noncanonical cysteines in the HC V regions and significant cysteine content in the HC D regions. Deep sequencing analysis of naturally occurring shorter CDR H3 (,40 aa) Ab genes shows that HC V and HC D regions preferentially combine to form a functional gene with an even number of total cysteines in the final V region, suggesting that disulfide bonds contribute to diversity not only in ultralong CDR H3 bovine Abs but in shorter CDR H3 bovine Abs as well. In addition to germline "hard-coded" cysteines, the bovine Ab repertoire can produce additional cysteine codons through somatic hypermutation, further diversifying the repertoire. Given the limited combinatorial diversity at the bovine Ig loci, this helps to explain how diversity is created in shorter CDR H3 Abs and potentially provides novel structural paratopes in bovine Ab combining sites. ImmunoHorizons, 2019, 3: 478-487.