Mucosal Immunogenicity Study Of Enterically Transmitted Hepatitis Virus

Hepatitis A virus (HAV) and Hepatitis E virus (HEV) are small, non-enveloped, positive strand RNA virus, and transmitted via the fecal-oral route and share many similar clinical symptoms, fulminant forms and epidemiological features, causing considerable economic loss in developing country. The devastating effects of simultaneous outbreaks or coinfection of HAV and HEV due to contaminated water supply has been reported previously. Although effective vaccines against HAV are available and an effective HEV vaccine is licensed recently. However, no mucosal vaccine that can protect against hepatitis A and E at the same time is available. The vaccines delivered by intramuscular injection were shown to produce IgG antibodies in serum which could not block viral infection timely in the mucosa tract. But mucosal immunizations can imitate mode of infection of HAV and HEV, and produce not only systemic IgG but also local secretory IgA. In addition, mucosal immunizations are relatively safer, cheaper, more acceptable to children and easier to administer than intramuscular injection, more simplified vaccination schedules. Mucosal vaccine is one of the most potential candidate vaccine.To enhance the antigenicity and immunogenicity of proteins, we choose the targeting gene-tuftsin. The product of the tuftsin gene is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. It can be recognized by specific receptors on macrophages and microglia, which express tuftsin receptors, and is capable of targeting proteins to these sites. Tuftsin also acts as a stimulatory factor to enhance cellular processes such as migration, chemotaxis and antigen presentation. To evaluate whether tuftsin could enhance the mucosal immune responses of the protein. We selected a partial HAV VP1 (aa 1-199) and partial HEV ORF2 (aa 368-607) to formulate the vaccine, which include the dominant structural protein among the capsid proteins and viral neutralization epitopes of HAV and HEV respectively. The experimental techniques mainly included codon optimization, PCR, gene cloning, prokaryotic expression, protein purification, protein identification. The tuftsin gene was conjugated to the N-terminal of HE-ORF2 and HA-VP1 by linker and inserted into the prokaryotic expression vector pET43a named pET43a-HA-VP1-tuftsin、 pET43a-HE-ORF2-tuftsin. Two genetic constructs were prepared for the expression of pET43a-HE-ORF2 or pET43a-HA-VP1 without tuftsin and linker as the control plasmids. All the four plasmids were expressed in E.coli BL21(DE3), purified by ion exchange chromatography and protein refolding to obtaine high purity and high concentration proteins:HA-VP1-tuftsin and HA-VP1, HE-ORF2-tuftsin, HE-ORF2. The molecular weights of the proteins were at the expected sizes of approximately 23 kDa,23 kDa,28 kDa and 28 kDa. The identity of the four recombinant proteins was confirmed by Western Blotting and ELISA. The results showed that HA-VP1-tuftsin and HA-VPl can combine the positive serum from patients infected with HEV; HE-ORF2-tuftsin and HE-ORF2 can combine the positive serum from patients infected with HEV.We confirmed the antigenicity and immunogenicity of HA-VP1-tuftsin and HE-ORF2-tuftsin in vitro and in vivo experiment. Firstly, we compared the immunogenicity of HE-ORF2-tuftsin、HA-VP1-tuftsin and the control protein HE-ORF2、HA-VP1. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally, the results demonstrated that the tuftsin can effectively activate the humoral immune response by intranasal immunization; IgA titers elicited by intramuscular administration in mice were not as strong as those by the intranasal route. In the ELISPOT assay, numbers of IFN-y spot-forming cells in the tuftsin groups were both higher than no-tuftsin groups with both intramuscular and intranasal immunization, the results suggest that tuftsin has a good ability to enhance the Thl response. Moreover, the number of CD4+T cells increased, while CD8+T cells decreased, causing the CD4+/CD8+T cell ratio to increase in the tuftsin group compared with the PBS and no-tuftsin groups with both intranasal and intramuscular inoculations, but no statistically significant difference was observed between intranasal and intramuscular administration. The results shown that the antigen fused to tuftsin may promote stronger immune responses in mice by improving CD4+T cell proliferation and inhibiting CD8+T cells. Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection.In addtion, future experiments were designed to explore the interactions between the two types of antigens in the combined HAV and HEV vaccine and determine whether the two antigens would have adverse effects on the immunogenicity after intranasal immunization. Moreover, comparing with the single HA-VP1-tuftsin group, the combined vaccine induced higher level of serum-specific IgG and mucosal IgA antibodies against HAV. In the ELISPOT assays, numbers of IFN-y spot-forming cells in the the combined vaccine was higher than single HA-VP1-tuftsin group, while no significantly different anti-HEV titers between combined vaccines and single HEV-ORF2-tuftsin vaccination. Summarizing, the two recombinant protein components play no adverse effects on the immunogenicity of each other and the immunogenicity of the HAV-tuftsin protein was increased after combined vaccinate.In summary, the aim protein which integrating HE-ORF2, HA-VP1 and immunostimulatory molecule tuftsin respectively was successfully expressed and purified using E.coli expression system. The efficacy and protective functions of immunization with the tuftsin group were analyzed and evaluated using the Balb/c mouse. The results demonstrated that linking tuftsin to targeted sequences could highly potentiate the immunogenicity of a protein antigen and facilitate the induction of humoral and cellular immune responses. Moreover, enhanced mucosal immune responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. Furthermore, the recombinant HA-VP1-tuftsin protein had no adverse effects on the immunogenicity of the recombinant HE-ORF2-tuftsin protein component of the combined protein vaccine. Significantly higher HAV antigen-specific antibody titers and Thl cellular immune response were elicited after combined vaccination compared with that induced by single vaccination. These results shown that the tuftsin was conducive to enhance the antigenicity and immunogenicity of proteins and provided the basis of further research into the development of a combined protein-based mucosal HA+HE vaccine.