Expression, Purification, Immunogenicity Of The Recombinant Antigen Of The Hepatitis A And Hepatitis E Viruses And Comparison With The Traditional Vaccine Of HAV

Hepatitis A virus (HAV) and hepatitis E virus (HEV) are two known enterically transmitted viral hepatitis agents. In addition, hepatitis A and E share a lot of similar epidemiologic and clinical profiles, and cannot be discriminated from each other without laboratory tests. For instance both of them are small, non-enveloped, positive strand RNA virus; fecal-oral transmission. HAV infection mainly affects children. However, severity of the disease increases with age. Meanwhile HEV is a kind of severe infection. In some countries more than 50%of the acute liver damage and the oxyhepatitis are caused by HEV. The high mortality rates, up to 25%, occur in infected pregnant women. Numerous HEV outbreaks have occurred in many developing countries, resulting in tens of thousands of people being infected. China is one of HEV rampant regions. Co-infection of HAV and HEV or simultaneous outbreaks due to a contaminated water supply by both HAV and HEV was previously reported. There are already kinds of vaccines for proghylaxis of HAV but no vaccine for HEV. Due to the similar characteristic of the two kinds of hepatitis virus, it will be feasible to research on the combined vaccine.In this study, to investigate the immunogenicity of chimeric virus-like particle (VLP) bearing important antigenic epitopes of HAV vp1 and HEV ORF2. The vpl fragment(aa 24～171) was linked to the ORF2(aa 431～615) N-terminal domain (AEAg342) or C-terminal domain (EAAg342). The gene fragment encoding chimeric antigenic AEAg342 or EAAg342 was inserted into expression vector pBV220. Both of the recombinant subunit antigens (AEAg342 and EAAg342) could be highly expressed in Escherichia coli (E. coli). The molecular mass of the expressed recombinant antigen is about 36 KD. But only the recombinant EAAg342 can assemble into VLP of 10-20 nm radius. It indicated that the two kinds of fusion proteins can respectively produce specific reaction with HAV and HEV positive sera by Western-blot.This paper evaluated the specific humoral immune response induced by recombinant proteins (AEAg342 and EAAg342) in Kunming mice. Four groups, each of six mice, two of the groups were immunized with 10μg recombinant protein, respectively, antigen absorbed by complete Freund's adjuvant. The other two groups were immunized with the traditional hepatitis A vaccine respectively (the inactivated vaccine & the attenuated vaccine (live), freeze-dried). Three weeks and five weeks later each group was immunized again; the recombinant antigen absorbed by incomplete Freund's adjuvant. The fifth set of six mice was used as an antibody-negative control. At the 2nd,4th,6th,8th, 10th and 12th weeks, after the first vaccinations, level of anti-HAV &HEV IgG antibody were determined. Result showed that geometric mean titer (GMT) of anti-HEV induced by EAAg342 was 1:1000 000 at the 8th week, which was higher than that of AEAg342 vaccinated mice. In the anti-HAV side, the level from high to low is:the inactivated vaccine; the attenuated vaccine (live), freeze-dried; EAAg342; AEAg342. This result indicated that recombinant EAAg342 could induce good HAV&HEV-specific humoral immune response in Kunming mice.The data demonstrated that the chimeric VLP EAAg342 could induce satisfactory immune response against HAV and HEV in mice compared with the AEAg342. Moreover, the HAV vp1 fragment, which is inserted into chimeirc VLP, could increase the immunogenicity of VLP against HEV, and the recombinant VLP protein also had positive effects on the immunogenicity of the HAV vp1. The data provided experimental confirmation for development of bivalent vaccine and serologically diagnostic kit of HAV and HEV.