Development of a therapeutic vaccine against the hepatitis C virus

Sammanfattning: The hepatitis C virus (HCV) infection is a major cause of liver disease and it is estimated that 170 million people worldwide are chronically infected by HCV. There are no protective or curable vaccines available for HCV, however treatment consisting of interferon-α and ribavirin is curative in 45-75% of the chronic infected patients, depending on the viral genotype. The antiviral treatment has the lowest efficacy for patients infected by genotype 1. Today’s treatment regimens are associated with many side effects. Thus, new antiviral treatment regimens and/or vaccines are in urgent need. The majority of infected individuals develop a chronic disease and the reason for the high rate of persistence is in part explained by the highly genetic variability of HCV. Thus, development of a therapeutic vaccine against HCV should therefore be targeted against a region of the HCV genome with a limited genetic variability. We have based our development of a genetic vaccine on the non-structural (NS) 3 and NS4A proteins. The NS3 protein performs essential functions in the viral life cycle including protease and helicase activities. The NS3 co-factor, NS4A, is important for NS3 to stabilize the protein complex and to fully utilize its functions. Previous studies have shown that NS3/4A when delivered as a genetic vaccine induce both humoral and cellular immune responses in mice. We now investigated if we could further enhance the immunogenicity of the NS3/4A DNA vaccine. Codon optimization (co) of the NS3/4A DNA gene resulted in an enhanced immunogenicity explained by the increase of NS3/4A-protein expression. Due to the lack of small animal models to study HCV, we have generated a mouse model with transient expression of HCV proteins in the liver. By using this model we could show that peripherally vaccine-primed T cells could enter the liver, recognize and eradicate NS3/4A expressing hepatocytes, a prerequisite for a functional therapeutic vaccine against HCV. The NS3/4A protein has recently been shown to interfere with the innate immunity through cleavage of Cardif (also known as IPS-1, MAVS, VISA), resulting in suppression of the interferon response within the infected cell. We found that cleavage of Cardif by NS3/4A also occur in murine cells making it possible to study the effect of this interaction also in mouse models with hepatic expressing the NS3/4Aprotein. The effects that NS3/4A exert on the innate immunity do not seem to affect the adaptive immunity, since NS3/4A-protein expression did not prevent clearance of transiently transfected hepatocytes in vivo. This helps to explain why escaping the adaptive immunity through mutations should be beneficial for HCV. To better understand the relationship between immune escape and viral fitness we studied the immunodominant human HLA-A2-restricted epitope at residues 1073-1081 of NS3. Despite that the epitope is immunodominant, only a limited number of mutations occur within this epitope. We now show that the absence of mutations at some of these positions can be explained by a reduced protease activity and viral replication, which reduce the viral fitness. DNA vaccines have been shown to induce promising immune responses in animal models. However, so far DNA vaccines have not been found to prime effective immune responses when tested in primates / humans. The explanation for this is at least partly explained by the poor uptake of the plasmid DNA in humans. We therefore evaluated different delivery methods of our DNA vaccine using transdermal delivery by the gene gun or by intramuscular delivery in combination with in vivo electroporation (EP). These studies revealed that the coNS3/4A vaccine primed the broadest immune responses when delivered with in vivo EP. Importantly, although NS3/4A can block the response to dsRNA, these signal pathways are not activated during DNA immunizations which helps to explain the effectiveness of the DNA-based coNS3/4A vaccine. The coNS3/4A vaccine was evaluated in a toxicological study in rabbits which showed that the vaccine had an acceptable safety profile and biodistribution when administred using in vivo EP. Finally, the current coNS3/4A DNA vaccine delivered using in vivo electroporation was recently approved by the Swedish Medical Products Agency to enter a clinical trial, which will be the first DNA vaccine delivered in combination with in vivo electroporation against an infectious disease in humans.