The humoral immune response to a pathogen is complex. First exposure generates a primary response of secreted antibodies as well as initiates a poorly understood differentiation cascade that results in the generation of memory B cells. These cells play a critical role upon re- exposure to pathogen by providing a rapid, high affinity antibody response that is protective. Our long term goals are to understand how B cells are selected to enter the memory pool. This problem has important implications for the production of effective vaccines to pathogens with multiple epitopes, because for some pathogens, it may be key that protective immunity is generated immediately to relevant epitopes (primary response), as well as upon memory recall. We use the polypeptide (Tyr,Glu)-Ala--Lys as a model multideterminant antigen; its primary and memory antibody responses differ significantly in epitope recognition. The primary response is directed toward side- chain epitopes (GT+) and dominated by B cells using the H10/Vkappa1 gene combination. The memory response recognizes side-chain epitopes through diversified V gene usage; in addition, about 30% of the memory B cells produce antibody to backbone epitopes (A-L). Our hypothesis for the H10/Vkappa1+primary dominance and for this epitope-specific repertoire shift, is that primary B cells can differentiate either to secrete antibody or to mutate and form memory cells; and that the primary B cell's affinity for antigen is critical in determining which pathway is taken. In this model, H10/Vkappa1+primary B cells are predicted to have good affinity for antigen, and they may exist in high frequency in the pre-immune repertoire relative to (T,G)-A--L-specific B cells using other V genes. We propose that A-L+primary B cells are of low affinity, thus are silent in terms of antibody production. However, they can mutate and form memory cells. Somatic mutation and antigen selection of high affinity variants enables their expression during memory recall. To test this model, the affinities of unmutated and mutated antibodies specific for side-chain and backbone epitopes will be measured with defined peptides, using hybridoma and transfectoma antibodies, and antibodies produced in splenic focus cultures of cloned B cells. The hypothesis will be further explored using mice that express the mutated (memory) vs unmutated (primary) Ig genes that encode a memory A- L+antibody, or that express the unmutated Ig genes for a primary GT+antibody. Mice who germline JH locus is replaced with the VDJ rearrangement by homologous recombination will be bred to light chain transgenic mice. In addition to measuring their primary and memory antibody responses, the three sets of "transgenics" will be compared for their kinetics of germinal center formation, proliferation, antibody secretion, and mutation after priming. Since the dominant GT+ response and repertoire shift may also reflect frequency differences in the pre- immune repertoire of individual VH gene usage among GT+ and A-L+ B cells, the frequencies of H10 and two other VH genes used by GT+ and A-L+ B cells will be quantitated. These studies will address the fundamental question of whether a B cell's affinity for antigen influences the differentiation pathway that results in either antibody secretion or memory cell formation. Such mechanisms are central to the rational design of effective vaccines, where it is important that protective immunity be generated to multideterminant pathogens.