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. Data show the importance of aromatic amino acids in the CDRs of anti-PS antibodies. In our analysis of the regulation of diversity in the anti-In response we have mapped the Sr1 diversity gene to mouse chromosome 14. Recent studies in TCR KO mice have shown that the response to In is not only TI, but is not influenced by T cells when added back. Previous analyses of anti-MCPS and anti-MCPS TT mAb reveal that VH gene family usage is dominated by VHJ558. Sequence analysis of anti MCPS mAb shaows that several different germline genes are used in this response, even by mAb of the same fine specificity. One particular VH:VL pait, VH3609/Vk23, correlates with a fine specificity of reactivity with native MCPS and not non-O-acetylated MCPS. Few somatic mutations are seen in response to MCPS, but when observed, correlate with an increase in affinity. 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. Recent studies demonstrate that there is a developmental increase in lineage -specific markers on both B cells and DC and that the increase in MHC class II on B cells and CD11C on DC correlates with the development of their functional capacities. 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. One of the goals of the Children's Vaccine Initiative is to reduce the number of contacts required to immunize a child fully. To meet this goal several combined vaccines have been tested for their safety and immunogenicity in infants. Initial studies conducted in Chile showed a decrease in antibody response to Haemophilus influenzae b (Hib) polysaccharide (PRP; polyribosylribitolphosphate)and to the pertussis component of a DTP vaccine combined with an Hib conjugate (PRP-T) vaccine when the preparation was administered to infants in the same syringe or separately. We have developed a model in SW outbred mice to examine the effects of combining vaccines on the response to individual components. We have observed that DTaP interferes with the anti-Hib response to Hib- TT and we will examine the basis for this interference in the coming year. We also observed that Hib-TT interfered with the response to all three types of polio, given as IPV. The basis for this will also be examined in the coming year.
