Classification of a Novel Subgroup of Shared Hepatitis B Virus Core and e Antigen Epitopes and Their Antibody Reactivity in Clinical Immunoassay Formats

摘要

An estimated 3.5% of the world's population is chronically infected with hepatitis B virus (HBV), leading to over 600,000 related deaths annually. The HBV core-related antigen (HBcrAg) is an emerging clinical biomarker in the management of HBV infection. HBcrAg is defined as a 149 amino acid region, shared by multiple proteins expressed by the HBV precore/core gene including HBV core antigen (HBc) and e antigen (HBe). Recent clinical studies have shown that elevated HBcrAg can predict increased viral reactivation that can lead to major causes of HBV mortality including cirrhosis and hepatocellular carcinoma.  To develop a novel lateral flow immunoassay (LFI) capable of rapid detection of elevated HBcrAg from serum, mice were immunized with recombinant HBc leading to the establishment of a hybridoma library producing over 20 unique monoclonal antibodies (mAbs), of which eight were found to be highly HBc/HBe cross reactive. While the existing HBcrAg assay uses antibodies targeting fully linear epitopes, the mAbs isolated for this project were found to be specific to epitopes that appear to have linear properties while also being mediated by dimeric conformational factors. Utilizing mAbs targeting these distinct epitopes was found to have both advantages and disadvantages. One advantage was that they enabled the optimization of pretreatment protocols more suited for the point-of-care (POC) than current protocols. In this dissertation, proof of concept is achieved for a novel POC LFI capable of detecting HBc in human serum samples using a modified acidification pretreatment protocol. The same LFI can also detect HBe without pretreatment allowing for a novel HBc/HBe dual detection format that could serve to cost-effectively screen large populations to identify the chronic HBV patients most in need of anti-viral therapy or closer monitoring protocols. The HBV precore/core gene plays a multifunctional yet still enigmatic role during HBV infection. Unexpected findings during assay development provided evidence that HBc dimers have the potential to exert immunomodulatory functions previously attributed to only HBe, such as IgG1/2b isotype switching. This led to novel hypotheses that, if proven correct, may provide new insights to explain the origin of HBe and the evolutionary factors that strongly select for HBe competent strains of HBV.