Molecular Characterization of Murine Monoclonal Antibody Variable Regions Specific for Hepatitis B Surface Antigen

Background: Hepatitis B virus (HBV) surface antigen (HBsAg) induces a vigorous neutralizing antibody response, which causes effective protection against HBV infection. Little is known about the profile of variable region genes of immunoglobuline heavy (VH) and light (VL) chains rearranged in anti-HBs antibodies, and also the possible association of this profile with specificity pattern of these antibodies to mutant forms of HBsAg. Aims: The present study determined the nucleotide sequence of VH and VL genes of mouse monoclonal antibodies (MAbs) generated against HBsAg. Methods: Hybridoma clones secreting anti-HBsAg MAbs were developed from hyperimmunized Balb/c mice. VH and VL gene sequences of all MAbs were determined by amplifying the genes using a panel of VH and VL family specific primers by reverse transcription polymerase chain reaction. The reactivity pattern of anti-HBs MAbs with different mutant forms of HBsAg was evaluated by enzyme-linked immunosorbent assay, and then the profile of antigen specificity and its association to VH/VL family expression was analyzed. Results: Twenty-three murine hybridomas producing anti-HBs MAbs were generated. Nucleotide sequence analysis revealed that heavy chains of these MAbs were encoded by IGHV genes from the HV1 (52%), HV6 (22%), HV5 (17%), and HV3 (9%) families in combination with IGHJ2 (57%), HJ1 (26%), and HJ4 (17%). Besides, 56% of MAbs used IGHD1 genes in their VDJ rearrangements. Concerning the IGKV gene, 26% and 22% of clones used KV4 and KV10 gene families, while the rest of the clones used KV8, KV6, KV1, KV12, and KV14 gene families. Besides, the IGKJ2 gene was the most represented KJ gene (43%). No association was found between the specificity pattern of MAbs to mutant forms of HBsAg with their preferential V, D, and J genes usage for most of MAbs. Conclusion: The data suggest that heavy chains of anti-HBs MAbs preferentially use genes derived from the IGHV1, IGHV6, IGHJ2, and IGHD1 families. In contrast to heavy chains, which predominantly use four families of IGHV genes, light chains use more diverse IGKV gene families.