A structurally conserved antibody combining site, encoded by the IGH V3-23 and kappa A2 variable (V) region gene segments, predominates the adult immune response to the Haemophilus influenzae type b (Hib) capsular polysaccharide (PS). This site has been elevated to canonical status based upon its relative molecular uniformity and prevalence in adults. To date, no studies have examined the primary structure of Hib PS-specific antibodies in young infants, who are the primary targets of Hib vaccination. In this study we show that canonical Hib PS-specific heavy (H) and light (L) chain V regions are present in 4-month-old infants following two vaccinations with Hib PS-protein conjugates. The infant V regions contain sequence polymorphisms that resemble those found in adult antibodies, as well as polymorphisms at position 95a of the A2 L chain not previously observed in adults. In vitro studies of Fab fragments and recombinant IgG2 antibodies using these V regions identify sequence polymorphisms that impact Hib PS binding affinity and bactericidal activity. These results demonstrate the establishment of canonical V regions in early ontogeny and provide a structural explanation of how canonical antibodies in the infant can vary in their affinity and protective activity against Hib.

Two licensed serogroup B meningococcal vaccines contain factor H binding protein (FHbp). The antigen specifically binds human FH, which downregulates complement. In wild-type mice whose mouse FH does not bind to FHbp vaccines, the serum anti-FHbp antibody response inhibited binding of human FH to FHbp. The inhibition was important for eliciting broad anti-FHbp serum bactericidal activity. In human FH transgenic mice and some nonhuman primates, FHbp was able to form a complex with FH and FHbp vaccination elicited anti-FHbp antibodies that did not inhibit FH binding. To investigate the human anti-FHbp repertoire, we cloned immunoglobulin heavy- and light-chain-variable-region genes of individual B cells from three adults immunized with FHbp vaccines and generated 10 sequence-distinct native anti-FHbp antibody fragments (Fabs). All 10 Fabs bound to live meningococci; only 1 slightly inhibited binding of human FH, while 4 enhanced FH binding. Affinity-purified anti-FHbp antibody from serum of a fourth immunized adult also enhanced binding of human FH to live meningococcal bacteria. Despite the bound FH, the affinity-purified serum anti-FHbp antibodies elicited human complement-mediated bactericidal activity that was amplified by the alternative pathway. The lack of FH inhibition by the human anti-FHbp Fabs and serum antibodies suggests that binding of human FH to the vaccine antigen skews the anti-FHbp antibody repertoire to epitopes outside the FH-binding site. Mutant FHbp vaccines with decreased FH binding may represent a means to redirect the human antibody repertoire to epitopes within the FH binding site, which can inhibit FH binding and, potentially, increase safety and protective activity.