ANALYSIS OF THE MURINE IMMUNOGLOBULIN GENES IN THE IMMUNE RESPONSE TO HEPATITIS B SURFACE ANTIGEN AND HUMAN LEUKOCYTE ANTIGEN

Abstract

In this study, the antibody (Ab) heavy chain variable region (VH) gene utilization by B cells in a murine immune response to complex or multi-epitope antigens was investigated. Hybridomas were selected in this study because the VH genes expressed will approximately resemble the B cell antibody repertoire in an immune response to complex antigens. Two panels of hybridomas producing monoclonal antibodies (mAbs) specific for the hepatitis B surface antigen (HBsAg) and the human leukocyte antigen (HLA) were employed in this study. The method of RNA dot blot hybridization was chosen for the study of VH gene expression. Hybridization was performed with a panel of VH gene family-specific probes labelled with the radiolabel ?-32P dCTP by random priming. The results of ribonucleic acid (RNA) dot blot hybridization indicated that hybridomas producing anti-HBsAg mAbs expressed VH genes in a manner that approximately resembled the normalized B cell antibody repertoire, where the larger VH gene family VHJ558 was more frequently expressed than the smaller VH gene families. In contrast, hybridomas producing anti-HLA mAbs showed preferential VH gene expression, in particular VH genes from the VHQ52 gene family. These results suggested that the pattern of VH gene utilization by B cells in an immune response to complex antigens could be dependent on the immunizing antigen and not on the multiepitope nature of the antigens. The hybridoma 3S10/1 was selected for further VH gene analysis because the VH gene expressed could not be determined in the RNA dot blot hybridization. Furthermore, the mAb is specific for the a determinant of the HBsAg and thus has potential in prophylactic applications. The VH gene VH 11.1 expressed by the mAb 3S 10/1 was found to belong to the VHJ606 gene family. Nucleotide sequence comparison study between VH 11.1 and VH 22.1, a prototype VHJ606 family gene, indicated that VH 11.1 could be derived from a new germline gene. The mAb 3S10/1 has potential in prophylactic applications. Thus, the possibility of generating a functional humanized antibody heavy chain gene containing the murine VH gene VH 11.1 was evaluated. The human gamma I heavy chain constant region gene designated IgGICH23 was cloned and sequenced. Nucleotide sequence analysis of VH 11.1 and IgG1CH23 revealed that the generation of a 39 base pair (bp) linker deoxyribonucleic acid (DNA) with BanI and EcoO109 sites at the 5' and 3' ends respectively would be able to join the VH 11.1 and IgG1CH23 gene segments together to form a functional antibody heavy chain gene. The human kappa light chain constant region gene designated IgkCL42 was cloned and sequenced. Upon cloning of the mAb 3S 10/1 light chain variable region gene (VL), similar modification can be performed to produce a functional humanized antibody molecule.