Functional role of T-cell activation in viral hepatitis

Hepatitis B and C (HBV, HCV) constitute global health problems and there are approximately 350 million chronically HBV infected people around the world, most of them are found in East and South East Asia and Africa. Worldwide there are 170 million HCV infected individuals with the highest prevalence seen in Egypt. Reported cases of HBV and HCV have decreased significantly due to improvements in screening of blood donors and in particular with respect to HBV due to the introduction of a preventive vaccine. HBV and HCV are transmitted mainly by blood-to-blood contact or by sexual transmission (less efficient for HCV).

There are great differences in the ability to clear the viral infection between HBV and HCV infected individuals. In HCV almost 75% develop a chronic disease, while the rate of chronicity caused by HBV is influenced by the age at infection. Thus, only 5% of adult-acquired infection leads to chronic hepatitis B (CHB) whereas the chronicity rate for perinatal infection is up to 90%. Failure to mount effective T cell responses may possibly cause both an inability to respond to HBV vaccination, as well as to CHB.

Protective immunity towards HBV can be achieved by vaccination; the protection is based on the emergence of antibodies to the hepatitis B surface antigen (anti-HBs). Though a small group of individuals i.e. non-responders (5 to 10%) fail to do this. We therefore designed a study where previously nonresponders to the HBV vaccine were re-vaccinated using a double dose of the combined hepatitis A virus (HAV) and HBV vaccine in hope to improve priming of a protective HBV response. Almost all non-responders developed protective levels of anti-HBs and approximately half of the patients developed a HBs antigen (HBsAg)-speific T-cell proliferation after re-vaccination. Thus, a nonresponse to the HBV vaccine is not absolute, it most often represent a low responder satus which can be circumvented with the appropriate stimulus.

We next investigated how HBV-specific T-cell responses emerged in children with different stages of CHB. In the majority of children an HBV core antigen (HBcAg) specific T-cell proliferation was detected, also in an anti-HBeAg positive group, indicating that a continuous T-cell proliferation is important to maintain HBe antigen (HBeAg) clearance. Exogenous HBcAg is well known to be a potent activator of both B cells and T helper (TH). An intrinsic feature of HBcAg it is the binding to naïve human and mouse B-cells. Its high immunogenicity suggests that it may be a good target for immunotherapy. Essential for this binding were the residue 76 to 80 on the tip of the spike of the HBcAg.We could also note that the induced CD8+ T-cell response after immunization with endogenously expressed HBcAg was not Bcell dependent whereas priming with exogenously expressed HBcAg was.

New treatment strategies are needed for both HBV and HCV infections. We have developed two DNA based vaccines, one based on HBcAg for HBV: and the other based on the non-structural protein 3/4A (NS3/4A) for HCV. Previous studies have shown that the HBcAg- and NS3/4A-vaccine induce both a strong humoral and cellular response in mice, but less is known if they are functional in the liver. We therefore developed a model to test if CD8+ T-cells could home to the liver and eliminate NS3/4A expressing hepatocytes. Transiently transgenic mice were generated using hydrodynamic injection of HBcAg- or NS3/4A-expressing plasmids. We could show that the primed peripheral CD8+ T cells indeed entered the liver and eliminated NS3/4A expressing hepatocytes.

We lastly characterized the ability of HBcAg-DNA to induce CTLs. We found that HBcAg-DNA was surprisingly poor in inducing CTLs at a low DNA level when compared to the NS3/4A gene. Despite delivery by in vivo electroporation and gene codon optimization, low levels of DNA still failed to effectively prime CTLs. This is an unexpected property of HBcAg. Overall, these studies show that a poor T cell response, or poor ability to activate T cells, can effectively be overcomed by the appropriate measures, which has implications for human vaccine design.

List of scientific papers

I. Fischler B, Nyström J, Björnsdottir T, Lindh G, Hultgren C (2007). Virus-specific T cell immune response in children and adolescents with chronic hepatitis B virus infection. J Pediatr Gastroenterol Nutr. 45(1): 75-83
https://pubmed.ncbi.nlm.nih.gov/17592368

II. Nyström J, Cardell K, Björnsdottir TB, Fryden A, Hultgren C, Sällberg M (2008). Improved cell mediated immune responses after successful re-vaccination of non-responders to the hepatitis B virus surface antigen (HBsAg) vaccine using the combined hepatitis A and B vaccine. Vaccine. 26(47): 5967-72. Epub 2008 Sep 17
https://pubmed.ncbi.nlm.nih.gov/18804140

III. Ahlen G, Nystrom J, Pult I, Frelin L, Hultgren C, Sallberg M (2005). In vivo clearance of hepatitis C virus nonstructural 3/4A-expressing hepatocytes by DNA vaccine-primed cytotoxic T lymphocytes. J Infect Dis. 192(12): 2112-6. Epub 2005 Nov 4
https://pubmed.ncbi.nlm.nih.gov/16288375

IV. Lazdina U, Alheim M, Nyström J, Hultgren C, Borisova G, Sominskaya I, Pumpens P, Peterson DL, Milich DR, Sällberg M (2003). Priming of cytotoxic T cell responses to exogenous hepatitis B virus core antigen is B cell dependent. J Gen Virol. 84(Pt 1): 139-46
https://pubmed.ncbi.nlm.nih.gov/12533710

V. Nyström J, Frelin L, Peterson DL, Milich DR, Hultgren C, Sällberg M (2009). Endogenously produced native hepatitis B core antigen is a surprisingly poor inducer of specific cytotoxic T lymphocytes. [Submitted]