Antibodies (Abs) against bacterial capsular polysaccharides (PS) provide protective immunity against many important bacterial pathogens. These TD immune responses are characterized by the absence of affinity maturation in secondary responses, attributed to a deficiency of mutation. However, we have noted sequences of heavy chain variable segments (VH) encoding anti-Haemophilus influenzae type b (Hib) PS Abs differ from putative germline genes when elicited by both TI (plain PS) and TD (PS-protein conjugate) formulations. Analysis of mutation rates of VH segments is complicated by the large number of closely homologous and polymorphic VH segments. The correct identification of VH germline genes may, therefore, be difficult. To precisely ascertain the frequency and nature of mutation in TI and TD responses, we cloned and sequenced rearranged JH4-5 introns from monoclonal anti-Hib PS Abs and compared these to germline genes.

Two JH4-5 alleles were identified. Rearranged JH4-5 introns are 94.9-98.4% homologous to these germline genes, comparable to the homology of the rearranged variable segments to previously identified candidate VH germline genes. The overall mutation rate of 3.8-4.1% does not differ between antibodies elicited by TI and TD antigen formulations. Mutation rates are highest in rearranged VH segments and diminish in frequency throughout the 3′ untranslated introns. Many mutations occur in previously identified hot spots and in a new motif, TGCY.

We conclude that somatic hypermutation occurs at a high and equal frequency in both TI and TD responses. The low affinity of Abs in secondary TI immune responses is not due to the absence of somatic hypermutation, but is more likely related to another mechanism, possibly the existence of rigorous molecular requirements for antigen-antibody binding.