Development of vaccines and mouse models for chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is a major causative agent for severe liver disease and cancer worldwide. Globally, it is estimated that approximately 185 million people are infected with HCV and 130-170 million of these are chronic carriers of the virus (1, 2). The HCV infection is one of the major causes of liver disease and the infection is characterized by a slow and silent progression. Patients infected with HCV have an increased risk of developing fibrosis, cirrhosis and hepatocellular carcinoma. Importantly, this infection is today considered curable in the majority of individuals receiving the recently introduced direct-acting antiviral (DAA) therapy (3-5). Therefore the urgency for a HCV vaccine has reduced. However, only 10% of all chronic HCV carriers receive any treatment due to high cost of treatment and the majority of the chronic carriers live in resource-poor countries. Hence, there is still an urgent need to prevent the spread of HCV through a vaccine. Today, no prophylactic or therapeutic vaccines are available. However, numerous vaccines have been developed and tested for efficacy in clinical trials (6-9). A few HCV vaccines are currently being tested for both protective and therapeutic effects (10). A key feature of patients with chronic HCV is their lack of functional T cell responses to HCV (11-13). Interestingly, a recent study showed that DAA induced cure of HCV rapidly restores at least partly HCV-specific T cell responses. However, these responses do not seem to protect against reinfection (14, 15). Thus, it may be of importance to broaden the post-cure T cell responses through vaccination to reduce the risk of reinfection. This will save significant costs and reduce HCV associated morbidity and mortality.



In this thesis we have evaluated our in-house therapeutic DNA vaccine in 12 patients with chronic HCV infection. The phase I clinical trial was performed in treatment naive HCV genotype 1 patients, receiving four monthly vaccinations in the deltoid muscles with 167, 500, or 1,500 μg codon-optimized HCV nonstructural (NS) 3/4A-expressing DNA vaccine delivered by in vivo electroporation (EP). This first-in-man therapeutic HCV DNA vaccine study with a DNA vaccine delivered by in vivo EP showed a good tolerability and safety with no severe adverse events. In addition, a transient immune activation and transient reductions in serum HCV RNA levels was seen in a few patients at the time of the vaccinations. Thus, DNA-based vaccines may be explored as a therapeutic or prophylactic tool in hepatitis C.



There are many ways to make a DNA vaccine more potent to get a good effect and fully utilize the vaccines effect. One approach is to use molecular adjuvants to obtain the most potent immune modulatory effect of a DNA vaccination. Other methods to increase the immunogenicity of the DNA vaccine can also be to make the delivery method more effective, like EP and in vivo intracellular injection (IVIN) device. The importance here is to be able to activate and reactivate the dysfunctional T-cells. Therefore have we made our DNA vaccine more potent where we have included a new molecular adjuvant for genetic vaccines based on sequences from the non-human stork hepatitis B virus core genes. It has previously been shown that HBcAg can act as an adjuvant and can be expressed as a recombinant protein. We here used avian stork HBcAg because avian HBcAg and human HBcAg only share around 40% sequence homology. Full-length and fragmented stork HBcAg gene-sequences were added to an HCV non-structural (NS) 3/4A gene (NS3/4A-stork-HBcAg). This addition enhanced priming of HCV-specific IFN-γ and IL-2 responses in wild-type (wt)- and NS3/4A-transgenic (Tg) mice, the latter with dysfunctional NS3/4A-specific T cells. In addition, the NS3/4A-stork-HBcAg vaccine also primed NS3/4A-specific T cells in human hepatitis B e antigen (HBeAg)-Tg mice with dysfunctional T cells to HBcAg and HBeAg. We also found that repeated immunizations boosted expansion of IFN-γ and IL-2-producing NS3/4A-specific T cells in wt- and NS3/4A-Tg mice. Importantly, NS3/4A-stork-HBcAg-DNA induced in vivo long-term functional memory T cell responses, whose maintenance required CD4+ T cells.



To study therapeutic vaccines and therapies for HCV there is a need for a simple small animal model. Currently there is no immuno-competent small animal model supporting HCV RNA replication, which has hampered studies of HCV-specific immune responses. We therefore aimed at developing an immuno-competent mouse model that allows in vivo growth of a syngeneic mouse hepatoma cell line harboring an autonomously replicating sub genomic HCV replicon of the genotype (gt) 2 JFH1 isolate (16). In this tumor model we can by different approaches study the functional immunity to HCV. By challenging the mice with the hepatoma cells we can measure the tumor growth and compare the volumes between vaccinated and naïve mice. Even though no virus particles are generated in this model, we have been able to show presence of HCV RNA through quantitative PCR and by in situ hybridization. To characterize a protective T cell response we first mapped cytotoxic T lymphocyte (CTL) epitopes within HCV NS3/4A gt2a. Using these epitopes we were able to quantify the number HCV-specific CTLs post immunization. We found that a NS3/4A-gt2a-based DNA vaccination protects against tumor growth, although this was dependent on an optimal vaccination. Importantly, challenge of naive or vaccinated mice with the HCV replicon resulted in a poor activation, or boosting of HCV-specific T cells. In contrast, challenge with a stably NS3/4A-expressing hepatoma cells resulted in a potent T cell activation and boosting in naive and vaccinated mice, respectively. Thus, the presence of HCV RNA replication seems to impair immunogenicity of HCV antigens. This mimics the human infection.



In conclusion, we have in a phase I clinical trial shown that DNA vaccine can be immunogenic in humans. However, the study also suggested that the immunogenicity needed improvement. We therefore developed new molecular adjuvants that greatly improved immunogenicity in a host with a dysfunctional immune response to HCV. Finally, we developed a new mouse model with replication of a subgenomic HCV RNA replicon. This model should be useful for evaluation of new HCV vaccines