The immune response to polysaccharide (PS) antigens is highly regulated and has several distinguishing features including restricted subclass, variable region gene usage, fine specificity, and avidity. Simple PS not conjugated to protein (such as bacterial Levan (BL, Neisseria meningitidis group C (MCPS)) elicit a thymus-independent (TI) response. PS conjugated to proteins (such as MCPS coupled to tetanus toxoid, (MCPS-TT)), on the other hand, elicit a different type of response, termed thymus-dependent (TD). Our earlier analysis of anti-BL antibodies had shown a germline primary response to the inulin (In) branch determinants. Two injections of BL, however, elicited IgM mAb with somatic mutations and higher affinity. We have now performed site-directed mutagenesis of the germline antibodies and identified specific sites resulting in higher or lower affinity. Analysis of the regulation of diversity in the anti-In response has led us to map the Sr1 diversity gene. Our previous data indicated linkage between Sr1 and markers on mouse chromosome 14 and this has been supported by studies using CXB recombinant inbred (RI) lines and more recently by phenotype analysis of (BALB/c x B6.C-H8)F1 mice. We are in the process of completing phenotype and genotype analysis of additional BALB/c x (BALB/c x C57BL/6)F1 backcross mice. Previous analyses of anti-MCPS and anti-MCPS TT mAb reveal that VH gene family usage is dominated by VHJ558. IgG, IgA and IgM mAbs from 3 panels were purified and tested for avidity. MAbs from mice immunized with MCPS-TT conjugate 2X(C2) and immunized with MCPS-TT and boosted with MCPS(CP), in general, had 2 orders of magnitude lower concentrations for 50% binding than anti _MCPS mAb, indicating that the TD mAb were of significantly higher avidity (10-100 fold higher) than the anti-MCPS. Sequence analysis in progress will determine if somatic mutation or different VH gene usage with in J558 or other families accounts for the increased avidity. Earlier mouse model studies in our laboratory indicated a developmental delay in the immune response to MCPS even when it was administered as TD MCPS-TT conjugates. Therefore, studies were undertaken to examine whether the delay in response TD conjugates in neonatal mice is due to defective antigen presentation. we have found that adult B cells were much more effective in presenting TT or MCPS-TT to T cells than total spleen cells or macrophages and that adult dendritic cells were the most effective antigen presenting cells in this system,the efficiency being about 2-fold higher than B cells. Of significance, both neonatal B cells and neonatal dendritic cells were defective in their ability to present antigen. The data suggest that factors that inprove the ability of neonatal cells to present antigen might be useful in stimulating neonatal responses to conjugate vaccines.
