title: Structural requirements of the major protective antibody to Haemophilus influenzae type b.
abstract: Protective antibodies to the important childhood pathogen Haemophilus influenzae type b (Hib) are directed against the capsular polysaccharide (HibCP). Most of the antibody is encoded by a well-defined set of ("canonical") immunoglobulin genes, including the Vkappa A2 gene, and expresses an idiotypic marker (HibId-1). In comparison to noncanonical antibodies, the canonical antibody is generally of higher avidity, shows higher levels of in vitro bactericidal activity, and is more protective in infant rats. Using site-directed mutagenesis, we here characterize canonical HibCP antibodies expressed as antigen-binding fragments (Fabs) in Escherichia coli, define amino acids involved in antigen binding and idiotype expression, and propose a three-dimensional structure for the variable domains. We found that canonical Fabs, unlike a noncanonical Fab, bound effectively to HibCP in the absence of somatic mutations. Nevertheless, pronounced mutation-based affinity maturation was demonstrated in vivo. An almost perfect correlation was found between unmutated gene segments that mediated binding in vitro and those encoding canonical HibCP antibodies in vivo. Thus, the Vkappa A2a gene could be replaced by the A2c gene but not by the highly homologous sister gene, A18b, corresponding to the demonstrated usage of A2c but not of A18b in vivo. Similarly, only Jkappa1 and Jkappa3, 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 somatic mutations-requirements met by only a limited spectrum of immunoglobulin gene combinations.
authors.affiliation: Department of Clinical Immunology, The National University Hospital, Rigshospitalet, Copenhagen, Denmark. hougs@biobase.dk
authors.forename: L
authors.initials: L
authors.lastname: Hougs
authors.forename: L
authors.initials: L
authors.lastname: Juul
authors.forename: A
authors.initials: A
authors.lastname: Svejgaard
authors.forename: T
authors.initials: T
authors.lastname: Barington
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D001483:Base Sequence; D002648:Child; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005803:Genes, Immunoglobulin; D006192:Haemophilus Infections / Q000235:genetics / Q000276:immunology / Q000517:prevention & control; D019741:Haemophilus influenzae type b / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011135:Polysaccharides, Bacterial / Q000276:immunology; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins