We generated a panel of cell lines secreting monoclonal human anti-Hib PS Ab from subjects immunized with Hib oligosaccharide-CRM197 protein conjugate vaccine (HbOC). Whereas the affinity of monoclonal Abs from adults immunized with HbOC, as measured by inhibition ELISA, is comparable to that of pooled immune serum, 30% of monclonal Ab from infants are low affinity(non-inhibitable). Most non-inhibitable Ab from infants cross-reacts with other bacterial or self antigens but inhibitable Ab does not. To identify the molecular basis for these differences in affinity and reactivity we cloned and sequenced variable region genes encoding both inhibitable and non-inhibitable Abs.

We previously showed that the adult anti-Hib PS repertoire is highly restricted and conserved. Two inhibitable anti-Hib PS Ab obtained from infants are encoded by variable region genes (V3-23 and VkII A2) that encode typical adult anti-Hib PS Ab and their light chains have an arginine residue at the Vk-Jk junction that is also characteristic of the adult anti-Hib PS response. In contrast, 8 non-inhibitable anti-Hib PS Ab from infants are encoded by a very diverse group of heavy and light chain variable region genes. Although some low affinity Ab are encoded by genes expressed in the adult repertoire, they differ in the use of long D segments and unusual VH-VL pairing. Light chains of non-inhibitable anti-Hib PS Ab do not feature the adult invariant arginine residue. Genes encoding these anti-Hib PS Ab also encode other anti-bacterial Ab and autoantibodies. Use of these genes and relatively less mutation than is observed in adult anti-Hib PS Ab may contribute to the polyreactivity of the infant response. We conclude that the repertoire of infants immunized with HbOC vaccine is comprised of two distinct antibody populations. It is likely that high affinity adult anti-Hib PS Ab is recruited from the early diverse and polyreactive repertoire elicited by immunization of infants with HbOC. Although readily detected by ELISA the contribution of low-affinity anti-Hib PS Ab to protective immunity is not clear.