Development and evaluation of new therapeutic interventions for chronic hepatitis C virus infection

The hepatitis C virus (HCV) is a major global health problem with around 130-150 million individuals chronically infected and it is estimated that 2 millions are newly infected each year. 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.

The effectiveness of new treatments has drastically improved during the last years and the cure rate is today around 90 %. However, several obstacles remain to be solved. Firstly, the treatment is associated with high costs. Secondly, there are still uncertainties whether patient groups such as children, pregnant women and treatment-experienced patients will be eligible for this treatment. Finally, the direct-acting antiviral (DAA) treatment does not protect against a re-infection. Also, only around 10 % of all chronic HCV carriers have access to treatment and the highest prevalence of HCV are seen in developing countries, which highlights the need of alternative less-expensive treatment strategies.

In this thesis we developed and characterized new treatments strategies for HCV based on genetic vaccines and re-direction of T cells. We utilized the non-structural (NS) 3/4A and 5A proteins of HCV as vaccine antigens and as targets for re-directed T cells. Both the NS3/4A and NS5A proteins have essential functions in HCV life cycle.

We generated an NS5A-based DNA vaccine and performed detailed characterization of its in vivo immunogenicity. We have previously developed a DNA vaccine based on NS3/4A that effectively primes immune responses in vivo. A codon optimized NS5A vaccine sequence was delivered intramuscularly in combination with in vivo electroporation for efficient uptake. The DNA vaccine activated NS5A-specific immune responses in both wild type and NS5A-transgenic (Tg) mice, with dysfunctional HCV-specific T cells. The vaccine primed responses were functional in vivo as evidenced by protection against in vivo tumor growth of NS5A-expressing cells. Furthermore, the vaccines were also used to develop NS3- and NS5A-specific T cell receptors (TCRs). Both the NS3/4A and NS5A TCRs were able to inhibit HCV RNA replication in vitro albeit using different mechanisms, possibly related to differences in the T cell avidity. The NS3/4A and the NS5A vaccines activated distinct HCV-specific immune responses. The NS3/4A vaccine was dependent on IFNγ and CD8+ T cells but not CD4+ T cells, whereas the NS5A vaccine was dependent on both CD4+ and CD8+ T cells but not on IFNγ. This is consistent with high-avidity NS3-specific TCRs inducing polyfunctional T cells, as compared to the low-avidity NS5A TCRs inducing monofunctional T cells. Finally, we analyzed the possibility to improve the intrinsic immunogenicity of the NS5A vaccine. We have previously showed that the immunogenicity of a NS3/4A-based vaccine could be significantly increased by co-expression with IL-12 and the addition of heterologous sequences from hepatitis B virus core antigen (HBcAg). However, the immunogenicity of NS5A was enhanced by addition of HBcAg sequences, but not co-expression of IL-12.

In conclusion, we have developed and evaluated new potential therapeutic interventions for the treatment of chronic HCV infection. Although the effective DAA treatment is available, it is clear that alternative treatment strategies are needed. These should be associated with lower costs and preferentially induce a post cure immunity that protect against re-infection.

List of scientific papers

I. Fredrik Holmström, Anna Pasetto, Veronica Nähr, Anette Brass, Malte Kriegs, Eberhard Hildt, Kate E. Broderick, Margaret Chen, Gustaf Ahlén, and Lars Frelin. A synthetic codon-optimized hepatitis C virus nonstructural 5A DNA vaccine primes polyfunctional CD8+ T cell responses in wild-type and NS5A-transgenic mice. J Immunol. 2013 Feb 1;190(3):1113-24.
https://doi.org/10.4049/jimmunol.1201497

II. Anna Pasetto, Lars Frelin, Soo Aleman, Fredrik Holmström, Anette Brass, Gustaf Ahlén, Erwin D. Brenndörfer, Volker Lohmann, Ralph Bartenschlager, Matti Sällberg, Antonio Bertoletti, and Margaret Chen. TCR-redirected human T cells inhibit hepatitis C virus replication: hepatotoxic potential is linked to antigen specificity and functional avidity. J Immunol. 2012 Nov 1;189(9):4510-9.
https://doi.org/10.4049/jimmunol.1201613

III. Antony Chen, Gustaf Ahlén Erwin D. Brenndörfer, Anette Brass, Fredrik Holmström, Margaret Chen, Jonas Söderholm, David R. Milich, Lars Frelin, and Matti Sällberg. Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination. J Immunol. 2011 May 1;186(9):5107-18.
https://doi.org/10.4049/jimmunol.1001790

IV. Fredrik Holmström, Margaret Chen, Anangi Balasiddaiah, Gustaf Ahlén, and Lars Frelin. Functional differences in hepatitis C virus nonstructural (NS) 3/4A- and 5A-specific T cell responses. [Manuscript]