The remarkable diversity of the B cell antibody repertoire is generated through the combinatorial assortment of distinct groups of immunoglobulin (Ig) germline elements. Further diversification of rearranged Ig genes is provided by a unique process of somatic hypermutation. The individual contribution of each of these mechanisms to the functional antibody repertoire is unknown. The antibody response to the capsular polysaccharide (PS) of a major childhood bacterial pathogen, Haemophilus influenzae type b (Hib), is restricted to a limited number of antibody species (cl notypes). Therefore it should serve as an excellent model to examine the relative contribution of different germline variable (V) regions and somatic mutation to the functional immune response. Furthermore, susceptibility to this infection is a function of age and may be related to the ontogeny of V gene usage. The overall goal of this proposal is to study the expression of the human V region gene repertoire as a function of age and the form of antigenic exposure. In this study we propose 1) To isolate human antiHib PS B cells in the form of hybridomas from infants, children and adults at various times following immunization with T-independent (TI) and T-dependent (TD) forms of Hib PS vaccine, 2) To determine the IgV region repertoire by sequence analysis of cDNA clones of hybridomas and by anti-idiotype analysis of serum antibodies and peripheral B cells, 3) To examine the relationship between V region sequence or Ig isotype, and functional activity of anti-Hib PS antibodies, and 4) To examine the contribution of Ig gene somatic hypermutation to IgV region diversity in response to both TD and TI vaccines. The V genes of the B cell hybridoma antibodies can be readily sequenced. In addition, the development of V region specific anti-idiotype antibodies will allow the analysis of a large number of serum samples without the need to isolate, clone and sequence a large number of hybridomas. Comparison of V genes captured at different timepoints following immunization will determine the contribution of V gene usage and somatic mutation to maturation of the antibody response. Determining the impact of somatic mutation in both TI and TD responses will clarify the role of T cells in this unique process. Finally, knowledge of V region usage at different ages will determine the ontogeny of V gene usage and may provide clues to the susceptibility of certain patients to this infection.
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