""""""""s abstract): The research described in this proposal represents a continuation of our efforts to delineate the genetic and structural bases of the variable (V) regions encoding the human antibody (Ab) repertoire specific for the type b (Hib) capsular polysaccharide (PS), and to understand the dynamics of this protective Ab repertoire. The different Hib PS conjugate vaccines induce Hib PS-specific Ab in infants that differ profoundly in functional activity against Hib, and these differences correlate with usage of particular idiotypes/variable (V) regions. In addition, V repertoire modification occurs as a consequence of aging since infants and adults differentially utilize particular anti-Hib PS regions. The investigator's research seeks to define the molecular mechanisms underlying these phenomena. A central hypothesis of this proposal is that hyper mutation of Ab V regions, variably driven by different Hib PS vaccines and by exposure to naturally-occurring antigens as a consequence of aging, plays a central role in determining both V region/idiotypic expression and Ab functional quality. To define and manipulate the anti-Hib PS V region repertoires of infants and adults, two experimental methodologies will be used, idiotypic analysis and expression of human Ab Fab fragments in bacteriophage combinatorial libraries. An extensive anti-Hib PS V region cDNA sequence analysis will be performed on B lymphocytes obtained from vaccinated infants and adults. This approach will allow us to determine the V gene elements encoding the infant anti-Hib PS repertoire, which heretofore have been inaccessible to molecular definition. In addition, this analysis will permit assessment of the extent to which V region mutation has been induced by different vaccines and by the aging process. Serological reagents reactive with new idiotypes/V regions will be prepared and they will be used to extend our analysis of V region expression in populations defined by vaccine type and age. The relationship between autoantibody reactivity and specificity for Hib PS will also be studied since the V gene segments used anti-Hib PS antibodies also are commonly used by anti-self Ab. By manipulating V gene components using phage combinatorial libraries, V region recombination experiments can be performed to assess the specificity potential of individual V regions, the relative roles that the heavy and light chain V regions play in deterring Ab specificity and function, and structural determinants of self and non-self reactivity. Avidity and specificity analyses will be performed on sequence-defined, phage-derived Fabs to correlate V usage and function. The long term objectives of this week are to understand the evolutionary and somatic forces that shape human Ab repertoires to PS antigens. Delineating the rules governing the inheritance, the developmental control and the molecular basis for the expression of these important Ab specificities will help us to understand how different PS vaccines generate antibodies with such markedly different functional capabilities, and thus will enable the rational design of more effective future PS vaccines for use in infants as well as the elderly.
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