Construction Of Recombinant Hepatitis A Virus With Chimeric Hepatitis E Antigen Gene And Evaluation Of Its Immune Effect

HAV and HEV are two known viral acute hepatitis agents.HAV is a small,non-enveloped,positive strand RNA virus in the family Picomaviridae[13].HAV infection is a common infection responsible for about 1.5 million new infections worldwide each year.But serologic evidence suggests that the rate of infection is probably as much as ten times higher[2].HEV is a non-enveloped virus with a single-stranded positive-sense RNA in the genus Hepevirus of the family Hepeviridae[14].A global burden of disease study estimated that HEV accounts for approximately 20.1 million HEV infections,70000 deaths,and 3000 stillbirths annually[3].Around 20%HEV infected pregnant women die because of liver failure[4].Both HAV and HEV are transmitted via the fecal-oral route and share many similar clinical symptoms,fulminant forms and epidemiological features.It is not possible to discriminate from each other by clinical or biochemical parameters.Co-infection of HAV and HEV or simultaneous outbreaks due to a contaminated water supply by both HAV and HEV was previously reported[5,6].Hepatitis A can be prevented by vaccination.In 1979,Provost and Hilleman propagated HAV in cell culture,which laid foundation for developing a hepatitis A vaccine(HA vaccine)[15].Both inactivated and live attenuated HA vaccines showed high efficacy in preventing hepatitis A[7,8].Attenuated live vaccine has shown promising results in effectively preventing HAV infection in a hepatitis A outbreak[8,16].The first licensed recombinant hepatitis E vaccine(HE vaccine)Hecolin is derived from immunogenic region of amino facid(aa)452-617 of HEV open reading frame 2(ORF2)encoded capsid protein[9,11].A phase ? clinical trial published in 2010 showed that it was highly effective in preventing infection among almost 100,000 healthy participants[9].Most conformation-dependent McAbs recognized the surface epitopes of native HEV,located at aa459-606 of HEV ORF2[12].Combined vaccines against two or more diseases have simplified the immunization procedures,improved acceptability and reduced the cost of vaccination.Some studies have tested the possibility of developing a combined vaccine against hepatitis A and E.The inactivated HA vaccine increased the immunogenicity of the recombinant HEV protein,and the recombinant HEV protein had no adverse effects on the immunogenicity of the inactivated HAV component in the combined HA and E vaccine[17].Similar results have also been reported in studies regarding recombinant subunit vaccine based on HAV and HEV[18,19].Viable HAV particles can package full length viral genomes containing insertions of approximately 600 nt at the 2A-2B junction and maintain infectivity[10].So HAV could be used as an expression vector for the development of combination vaccines at lower cost.To verify this hypothesis,in this study,we first created a recombinant chimeric HAV(HAV-HEp148)expressing the HEV neutralization epitope located at aa 459-606 of HEV ORF2(HEp148).RT-PCR and IF analyses showed that recombinant HAV HAV-HEp148 could proliferate in HAV-susceptible cells and express the HEp148 protein.Western blot analysis showed that HEpl48 formed partial dimmers.IEM test confirmed HAV particles.The HEp148 gene was stably expressed for 5 passages.After inoculation with the HAV-HEp148 viruses at passage 3,the anti-HAV and HEV IgG titers in the HAV-HEp148 group increased significantly from week 4.The data demonstrated that the 18f-E148 could induce similar antibodies response against HAV and HEV in immunized mice compared with the monovalent vaccines.To Study the splicing mechanism of heterologous gene,genes with different length were inserted into the 2A-2B junction of HAV.The result show that secondary structure in RNA determines the splice site,and free energy result in an probability of splice.The result also show that HAV could tolerated insertions of 180 nt at the 2A-2B junction.The HAV recombinant containing 153-nt insertion coding for aa 408-458 of HEV ORF2(HEp51)was stable for at least 10 passages.Immunization with the HAV-HEp51 induced strong HEp51-specific antibody responses in mice.This result indicated that HAV has ability to express foreign small peptides and present them to immune system.Our results show a new idea for development of combined vaccines against different diseases.Moreover splicing mechanism and stability factors of foreign gene are discussed.These work could lead to a better understanding of HAV replication and development of HAV vector.