Immunogenic Response Assessment of Hepatitis Delta Virus Antigen from Pakistani Isolate in Rabbits Using a Prokaryotic Expression System

Abstract

The hepatitis delta virus (HDV) is a defective and small, blood-borne viroid-like pathogen that coinfects with the hepatitis B virus (HBV) in about 5% of the infected individuals as it is a satellite virus of HBV. The treatment of HDV infection is quite challenging because there is no vaccine against HDV. Several commercial PCR and in-house assays have been developed, but there are greater variations in the results of these assays because they are not standardized properly. Studies are also delayed because of the unavailability of commercial HDV-specific antibodies for the diagnosis of HDV infection, even for the research devotions. To fill this gap, the recombinant antigenic HDAg protein of genotype I of HDV from the local isolate was successfully expressed and purified in the Escherichia coli (E. coli) system, followed by anti-HDAg antibodies production in rabbits. After determining and amplifying the antigenic region of HDAg of HDV, the fragment was cloned into the pET-28a vector and expressed in E. coli TOP10 cells. Following the successful expression of recombinant HDAg protein with a His-tag at its C-terminal end, we purified the HDAg protein using affinity chromatography. Both the expression and purification of HDAg-An protein were confirmed through SDS-PAGE and Western blot analysis. Through proper optimization, the HDAg-An protein was obtained with more than 90% purity. The next step involved immunizing Japanese White rabbits with the purified HDAg. The immunization protocol included 80 µg of HDAg-An in two subcutaneous priming doses and four 40 µg booster doses, followed by blood collection two weeks after each boost to monitor antibody production. The level of anti-HDAg antibodies in the rabbit serum was assessed using a quantitative ELISA technique. In the future, these antibodies could be used for the development of HDV-specific in-house assays as well as vaccines against the HDV genotype I that is locally predominant, potentially decreasing the burden of imported diagnostic tools and reagents.