Development of vaccines and experimental models for chronic infections caused by the hepatitis C virus

Abstract: The hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. It is estimated that HCV affects approximately 170 million people around the world. One feature of HCV infection is the high rate of viral persistence. The mechanism of viral persistence is largely unknown, although the high genetic variability is thought to play a key role. Today, no vaccine is available to prevent or cure HCV infections, albeit antiviral therapy is used quite effectively. This study aimed at developing new vaccines and new model systems to study HCV. We studied the HCV NS3 protein in detail since it performs key functions in the viral life cycle. These are unwinding and strand separation of the viral RNA and proteolytic processing of the precursor polyprotein. To obtain the complete protease we included the NS4A co-factor in our NS3-based vaccines. NS4A has been shown to enhance the stability of NS3 and to target the NS3/4A complex to intracellular membranes. The latter is most likely of importance for the formation of the replication complex. Also, the NS3 region has a limited genetic variability and several studies have now demonstrated that NS3-specific CD4+ and CD8+ T-cell responses are crucial for the resolution of HCV infections. Thus, several factors suggest that the NS3 region should be well suited for vaccine development. We could show that HCV NS3-based genetic vaccines effectively primed both humoral and cellular immune responses in mice. NS3/4A was shown to prime a Th1 CD4+ T-cell responses. The inclusion of NS4A in NS3-based vaccines primed antibody, CD4+, and CD8+ T-cell responses that were superior to those primed by NS3-gene alone. Thus, NS4A enhanced the immunogenicity of NS3. We could show that enhancement of the immunogenicity was most probably a result of the higher expression levels of NS3 generated by the inclusion of NS4A. We next tested if the overall immunogenicity of NS3/4A could be further enhanced by codon optimization or by mRNA amplification using the Semliki forest virus (SFV) replicon. The NS3 protein expression levels were further improved by either codon optimization and mRNA amplification. Subsequently, both these modifications enhanced the NS3-specific immune responses. One concern in development of genetic vaccines is that the gene displays unwanted properties when expressed in vivo. We therefore, generated a new transgenic mouse expressing the HCV NS3/4Aprotein in the liver. The protein expression was restricted to the liver to mimic the in vivo situation during a HCV infection. Protein expression was localized to the cytoplasm of the hepatocytes and displayed a similar staining pattern as seen in hepatocytes from HCV infected individuals. The intrahepatic protein expression did not cause overt liver damage, except for a slight enlargement of the liver. However, the NS3/4A-transgenic mice displayed less spontaneously appearing intrahepatic inflammatory foci, which are commonly found in laboratory mice. Thus, expression of NS3/4A-protein may affect the distribution of immune cells within the liver. The present studies demonstrate that NS3-based genetic vaccines that contain NS4A more effectively prime humoral and cellular immune responses against NS3. Intra-hepatic expression of NS3/4A did not cause any spontaneous liver disease or overt pathology suggesting that it safely can be used in genetic vaccines. Thus, the NS3/4A gene can safely activate immune responses that are similar to those found in humans who can clear HCV. The NS3/4A should therefore be a potential vaccine candidate against chronic HCV infections.