Expression, Purification And Immunogenicity Of The Recombinant Antigen Of The Hepatitis A And Hepatitis E Viruses

Hepatitis A virus(HAV) and hepatitis E virus(HEV) are two known enterically transmitted viral hepatitis agents.Both of them are small,non-enveloped,positive strand RNA virus.HAV infection mainly affects children.However,severity of the disease increases with age.Numerous HEV outbreaks have occurred in many developing countries,resulting in tens of thousands of people being infected.The high mortality rates,up to 20%,occur in infected pregnant women.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.Co-infection of HAV and HEV or simultaneous outbreaks due to a contaminated water supply by both HAV and HEV was previously reported.In this study,to investigate the immunogenicity of chimeric virus-like particle (VLP) bearing important antigenic epitopes of HAV vp1 and HEV ORF2.The vp1 fragrnent(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).But only the recombinant EAAg342 can assemble into VLP of 10～20 nm radius.Recombinant protein EAAg342 migrates predominantly as dimmer in SDS-PAGE,and could be recognized by HAV and HEV reactive human serum.This paper evaluated the specific humoral immune response induced by recombinant proteins(AEAg342 and EAAg342) in Kunming mice.Two groups,each of five mice,were immunized with 10μg recombinant protein,antigen absorbed by complete Freund's adjuvant.Three weeks later each group was immunized again.The third set of five mice was used as an antibody-negative control.At the 2^nd,4^th,6^th,8^th and 10^th weeks,after the first vaccinations,level of anti-HAV & HEV IgG antibody were determined.Result showed that geometric mean titer(GMT) ofanti-HEV induced by EAAg342 was 1:80 000 at the 8^th week,which was higher than that of AEAg342 vaccinated mice.The EAAg342 group had a high anti-HAV titer of 1:1 700, which was also higher than AEAg342 group.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.