Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies.
Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans.
Berraondo
Pedro
P
Laboratory of Gene Therapy of Viral Hepatitis, Division of Hepatology and Gene Therapy, Center for Applied Medical Research (CIMA), Clínica Universitaria/School of Medicine, University of Navarra, 31080 Pamplona, Navarra, Spain.
Crettaz
Julien
J
Ochoa
Laura
L
Vales
Africa
A
Ruiz
Juan
J
Prieto
Jesús
J
Martinez-Ansó
Eduardo
E
González-Aseguinolaza
Gloria
G
D015088:2',5'-Oligoadenylate Synthetase / Q000187:drug effects* / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004195:Disease Models, Animal; D006509:Hepatitis B / Q000276:immunology*; D006510:Hepatitis B Antibodies / Q000096:biosynthesis* / Q000276:immunology / Q000494:pharmacology; D016898:Interferon-alpha / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology; D008392:Marmota; D010766:Phosphorylation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D050796:STAT3 Transcription Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006510:Hepatitis B Antibodies; D016898:Interferon-alpha; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; D015088:2',5'-Oligoadenylate Synthetase