The long term objective of this project is to understand the structural and genetic bases for the shift n antibody variable (V) region gene usage during the course of the BALB/c response to the Sb antigenic site of the hemagglutinin molecule (HA(SB)) of influenza virus A/PR/8/34. Analysis of the mutations in secondary (2 degrees) antibodies suggests that somatic mutation is a significant factor in this shift, and in the specificity of 2 degrees antibodies. The working hypothesis for the experiments proposed here is that unmutated antibodies encoded by V region genes characteristic of the 2 degrees response cannot bind HA, and that they acquire this specificity as a result mutation. This model suggests that the effect of somatic mutation is not to simply increase the affinity of antibodies contributing to the primary (1 degrees) response as is the case in responses to other antigens, but to shift the B cell repertoire to one which expresses different and more V region genes. To test this hypothesis, unmutated rearranged V region genes characteristic of the 2 degrees response will be cloned, inserted into expression vectors, and transfected into the murine B cell hybridoma cell Sp2/0 to obtain antibodies for functional analysis. The ability of unmutated transfectoma antibodies to blind HA will be assessed by a competition ELISA. To test whether somatic mutation can generate this specificity, or improve the ability of antibody to bind HA (Sb) (consistent with antigen selection of mutant antibodies), we will introduce one, two, or more mutations into the germline V genes by site directed mutagenesis. The mutations to be introduced will be those that appear to have been selected for in vivo. The effect of these mutations on HA (Sb) biding will be assessed by a competition ELISA.
