Haemophilus influenzae b (Hib) is the most common cause of meningitis and childhood sepsis in the US. The capsular polysaccharide (CP) is a major virulence factor of Hib. Vaccines composed of the purified CP prevent Hib disease in children older than l8 mo., but have proven poorly immunogenic in infants, the age-group with the highest attack rate. The delayed ontogeny of the antibody (Ab) response to the Hib CP in man may mimic the delayed ontogeny of the response to certain polysaccharides in mice. In addition to being delayed in ontogeny, the murine Ab response to certain polysaccharides is restricted in diversity and expresses cross-reactive idiotypes (IdX) that are found on a high proportion of Ab molecules in most individuals of a strain. We have found that the human Ab response to the Hib CP is also highly limited in diversity, with restriction in light chain type and Ab charge heterogeneity as detected on isoelectric focusing (IEF) gels. The limited diversity, the finding of identical Ab IEF patterns in unrelated individuals, and the ability to immunize older humans to induce high levels of an Ab that is directed to a clinically relevant antigen, makes it possible to define the structural basis, regulation, and ontogeny of the diversity of the human anti-Hib CP Ab response.
The aims of the proposed research are to: l) determine the relative contribution of the Ab light (L) and heavy (H) chains to human Ab diversity and to binding of the Hib CP. Both human serum and hybridoma Ab will be prepared and purified. The L and H chains will be isolated and we will determine L chain charge heterogeneity by IEF, L and H chain subgroups by amino acid sequencing, and L and H chain contribution to antigen binding; 2) define the existence of IdX and its molecular basis by amino acid sequencing; 3) define the molecular basis of the Ab repertoire by amino acid sequencing; 4) determine the relative protective capacity of Ab with different V-regions using an animal model of the Hib disease; 5) define the normal ontogeny of Ab V-region expression and determine whether it is altered by passive immunization of the infant with Ab through maternal immunization or by active immunization with the Hib CP linked to a protein carrier; 6) determine whether autologous anit-idiotypic Ab is produced after immunization; and 7) determine whether Ab V-region expression is inherited and linked to immunoglobulin allotype markers. The long-range goal will be to apply the knowledge acquired of Ab V-region structure and regulation to develop an effective vaccine strategy to prevent Hib disease in man.
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