Title: Production of recombinant woodchuck surface antigen and antibodies for use in ELISAs, ELISpots, and western blots

Michelle Tang1, Simmone D’souza1,2, Gerardo Balderas Figueroa1, Layla Al-Yasiri1, Trushar R Patel1,2 ,3 , and Carla S Coffin1,4

1 Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Alberta, T2N 4N1, Canada
2 Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, T1K 3M4, Canada
3 DiscoveryLab, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada
4 Department of Medicine, Cumming School of Medicine, Calgary, AB, Canada

Background: Hepatitis B virus (HBV) has established chronic infection in over 296 million people worldwide and is the primary cause for liver cancer and failure. North American woodchucks (Marmota Monax) are a natural host for the woodchuck hepatitis virus (WHV). WHV belongs to the same viral family as HBV (Hepadnaviridae). WHV share many similarities to HBV including mode of transmission, chronicity, genome size and organization, replication strategy, and disease pathologies), hence, the WHV-woodchuck model is an ideal platform for HBV (pre)clinical research. Currently there are only a handful of labs in the world that have access to these animals, and there is a limited supply of commercially available reagents to study the similarities between WHV and HBV.

Purpose: We aim to develop a pipeline that can produce recombinant woodchuck surface antigen (WHsAg) and antibodies to WHsAg (anti-WHs) to characterize WHV infection status diagnostic immunoassays (e.g., ELISA, ELISpot, and by Western blot).

Methods/Results: We have engineered a plasmid that contains the WHsAg gene. Using recombinant protein expression in E. coli and yeast cells, we will express and produce WHsAg, followed by an application of affinity and size-exclusion chromatography to purify WHsAg. Next, we will test antigenicity using circulating anti-WHs antibodies from the serum of woodchucks that have had acute WHV infection and developed self-limited acute hepatitis. The recombinantly purified WHsAg will then be used for commercial production of monoclonal antibodies to ultimately develop diagnostic immunoassays.

Significance: By producing WHsAg and monoclonal antibodies targeting the WHsAg, we will be able to develop assays to assess important markers of acute and chronic WHV infection in woodchucks.