Improving immunogenicity of hepatitis B and C viral proteins for vaccine design
Sammanfattning: The hepatitis B and C virus (HBV, HCV) both presents a global health challenge, with an estimated 350 million individuals chronically infected with HBV and over 200 million infected with HCV. Reported cases of HBV and HCV have declined significantly due to screening of blood products and the availability of HBV vaccination. The chronic infection with HBV and HCV can lead to life threatening conditions such as cirrhosis, liver failure and hepatocellular carcinoma. Both HBV and HCV are blood-born diseases. Around 95% of the adults infected with HBV spontaneously clear the infection while 90% of children younger than one year develop a chronic infection. For HCV, 60-80% of all those infected fail to clear the virus and develops chronic infection. Treatment is available for chronic infections, with response varying between genotypes. HBV treatment stops viral replication but does not completely eradicate the infection. HCV treatment with IFN-alpha and ribavirin has an approximate 50% cure rate, with considerate side effects to the patient. The need for new therapeutic strategies against both diseases is of great importance. The immunogenic properties of the HBV core antigen (HBcAg) have been well documented and many of these may promote weak immunogens. The inherent immunogenicity of the native HBcAg B-cell epitopes could be successfully "transferred" by placement of heterologous epitope to the immunodominant regions of HBcAg. Preexisting immunity and poor assembly hampers the use of the human HBcAg-based platform. To address this, an alternative vaccine carrier based on the WHcAg platform was developed with a combinatorial capability. WHcAg appears to tolerate insertions of foreign epitopes better than human HBcAg. Importantly, additional C-terminal modifications allowed previously unsuccessful epitopes to be inserted. Many of the anti-epitope responses generated using the WHcAg platform clearly recognize the full-length proteins suggesting that they may also be functional in vivo. Thus, our data suggests that WHcAg may be a platform that is superior to human HBcAg. In human HBV infections, the HBcAg is thought to be the major target for specific cytotoxic T cells (CTLs). Using DNA immunization, we characterized the immunogenicity of endogenously expressed HBcAg. We immediately noted differences when comparing HBcAg DNA to HCV NS3/4A DNA. NS3/4A-DNA effectively primes CTL responses at both low (<1 mug) and high doses (100 mug) of DNA. In contrast, HBcAg only primed CTL responses when administered at high doses. A major factor that improved immunogenicity was to increase the expression levels of HBcAg. This was effectively achieved by combining codon optimization of HBcAg DNA with in vivo electroporation (EP). However, the effects of codon optimization and in vivo EP on HBcAg did still not help in priming an efficient CTL response at DNA doses of <5 mug. This suggests clear differences when compared to NS3/4A DNA. Thus, the efficiency of DNA immunization is highly dependent on the used protein. The HCV avoids immune recognition through several ways during chronic infection, resulting in an inability of T cells to eradicate infected cells. It has been shown that a strong T cell response is vital to clear acute HCV infections. We now wanted to apply our knowledge from HBcAg to improve on a therapeutic vaccine against HCV. The idea here was to target the priming of heterologous T cells to the same site as the activation/reactivation of impaired T cells. We generated a HCV NS3/4A-transgenic mouse simulating HCV-specific T cell dysfunction seen in human HCV-infection. Depletion of CD25+ regulatory T cells restored T cell fuction showing that the dysfunction, like in humans, is reversible and actively maintained. Paper I showed that presence of Th cells provided by HBcAg particle improved immunogenicity of an inserted epitope. We therefore tested various DNA constructs co-expressing coNS3/4A and coHBcAg DNA in mice. We first found that endogenous HBcAg antibody responses were TLR7 dependent. To determine the best way of transferring the adjuvant effect of HBcAg to NS3/4A, various fusion genes were generated consisting of different post-translational processing of HBcAg (paper III). Importantly, the immunization of NS3/4A-HLA-A2-Tg mice with coNS3/4A-HBcAg DNA fusion genes effectively restored NS3-specific T helper and CTL responses. Thus, recruiting healthy heterologous HBcAg-specific T cells can help restoring dysfunctional NS3-specific T cell responses in a transgenic mouse model representing the human infection. Overall, HBcAg is a highly immunogenic particle, which can be used in various vaccine combinations. The presence of HBcAg-specific T cells can improve the immunogenicity of inserted epitopes and help restoring a dysfunctional T cell response.
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