Structural requirements of the major protective antibody to Haemophilus influenzae type b

Protective antibodies to the important childhood pathogen Haemophilus influ enzae type b (Hib) are directed against the capsular polysaccharide (HibCP) , Most of the antibody is encoded by a well-defined set of ("canonical") im munoglobulin genes, including the V-kappa A2 gene, and expresses an idiotyp ic marker (HibId-1). In comparison to noncanonical antibodies, the canonica l antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats. Using site-di rected mutagenesis, we here characterize canonical HibCP antibodies express ed as antigen-binding fragments (Fabs) in Escherichia coli, define amino ac ids involved in antigen binding and idiotype expression, and propose a thre e-dimensional structure for the variable domains. We found that canonical F abs, unlike a noncanonical Fab, bound effectively to HibCP in the absence o f somatic mutations. Nevertheless, pronounced mutation-based affinity matur ation was demonstrated in vivo. An almost perfect correlation was found bet ween unmutated gene segments that mediated binding in vitro and those encod ing canonical HibCP antibodies in vivo, Thus, the V-kappa, A2a gene could b e replaced by the A2c gene but not by the highly homologous sister gene, A1 8b, corresponding to the demonstrated usage of A2c but not of A18b in vivo. Similarly, only J(kappa)1 and J(kappa)3, which predominate in the response in vivo, were able to facilitate binding in vitro, These findings suggest that the restricted immunoglobulin gene usage in HibCP antibodies reflects strict structural demands ensuring relatively high affinity prior to somati c mutations-requirements met by only a limited spectrum of immunoglobulin g ene combinations.