Despite the availability of antibiotics, Haemophilus influenzae b (Hib) remains a significant human pathogen: it is the leading cause of meningitis in children in North America, and is the etiologic agent in other serious invasive diseases such as epiglottitis, septic arthritis, cellulitis and pneumonia. The Hib capsular polysaccharide (Hib PS) is the primary virulence factor, and antibodies to Hib PS confer protection from invasive Hib disease. Children less than two years of age synthesize little or no antibody to Hib PS and this accounts for this group having the highest susceptibility to infection. The experiments described here will examine the diversity of the neonatal and adult Hib PS antibody variable (V) region repertoire. Our objective is to determine the extent to which particular, germ-line encoded V domains dominate the antibody response to Hib PS, and to determine whether the age-dependent development of immunocompetence, is explicable in terms of limited and/or differential V region utilization. Idiotypic and sequencing analyses will be used to delineate V region expression. Hybridomas, secreting Hib-PS specific monoclonal antibody (mAb) will be prepared from adult peripheral blood mononuclear cells (MNC) and from hyperimmunized scid mice engrafted with cord blood MNC. This panel of human mAb's will be tested for expression of cross-reactive idiotype (CRI) using polyclonal and monoclonal antibodies specific for a recurrent and predominant CRI associated with antibodies to Hib PS. V region sequences of these mAbs will be determined by sequencing cloned, polymerase chain reaction amplified, hybridoma cDNA. Comparison between V region sequence and CRI expression should help elucidate the structural correlates of CRI and will delineate the molecular diversity of the neonatal and adult V region repertoires to Hib PS. To further localize CRI determinants, antibodies will be prepared against synthetic peptides which encode conserved hypervariable regions of mAb anti-Hib PS heavy (H) and light (L) chains. These anti-peptide antibodies will be tested for idiotypic specificity against the panel of human Hib PS-specific mAb's and their isolated H and L chains. This approach should allow for identification of particular hypervariable regions which encode CRI determinants, as well as delineate the individual roles of H and L chain V regions in CRI expression. The relationship between CRI/V region expression and Hib PS antibody affinity and functional activity will also be examined. MAb's reactive with the major CRI of Hib PS antibodies will be administered to scid-hu mice to determine whether they might function as a surrogate Hib vaccine. These studies should provide insight into the ontogeny of V region expression and the relationship between V region structure, idiotypes and antibody function.
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