Study On The Immune Effects Of The Asia1FMDV Multi-epitope Immunogen

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating viral diseaseof cloven-hoofed animals worldwide. Thus, controlling the disease especially caused by the O, A andAsia1foot and mouth disease virus (FMDV) in China through regular vaccination and slaughter ofinfected and contact animals are very important. Currently, the conventional FMD inactivated vaccinehas played an important role in controlling the disease.Humoral immune response of neutralizing antibodies plays an important role in the anti-FMDVprocess. Production of high titres of neutralizing antibodies induced by the inactivated vaccine is notalways protective in livestock. Stimulating CD4+T-cell responses is important for the induction ofoptimum antibody responses to inactivated vaccine. Therefore, it is essential to consider the efficacy andpractical use of vaccines in order to induce an effective humoral immune response and cell-mediatedimmunity. The epitope vaccines combined with the T cell and B cell epitopes, which reduced theingredients independent within the vaccines is the ideal choice to achieve high humoral and cellularimmune response. The spread of FMD is mainly based on the close contact and the air. The mucosalimmunity induces the mucous membrane to produce many sIgA, which not only can neutralize the virusat the first time, but also induce system immune response. Therefore, effective in inducing the mucousmembranes sIgA is the new direction of development of the foot-and-mouth disease vaccine.In this study, a multi-epitope peptide was designed, fused to HBcAg and Bovine IgG2a Fc,individually. The three multi-epitope immunogens pET28a-HBc-EB, pET22b-EB-BIg andrGS115-pPICZA-HBc-EB were expressed in E.coli and Pichia pastoris. The three multi-epitopeimmunogens were used to immune mice by intraperitoneal injection, oral and intranasal, then theimmune effects were evaluated by humoral immunity, cellular immunity and mucosal immunity.(1) Design and synthesis of Asia1FMDV multi-epitope immunogenIn this study, two B cell epitopes (141-160aa and200-213aa) in VP1protein and the T cell epitopes(21-40aa in VP1and21-35aa in3A protein) were linked by the suitable linker sequence between themand synthesized as the candidate contents of the vaccine (the multi-epitopes designated as the EB). ThisEB sequences were inserted to the78to79amino acids of the HBcAg sequence with the linker GSGSG(spike position), and the multi-epitopes designated as the HBC-EB. Based on the software analysis, thelinker sequence formed the flexible regions and corners between the epitopes, especially the sequence(G4S)3guaranteed the two B cell epitopes exist as the independent role. The HBc-EB had a high antigenicity and most of the multi-epitope expressing in the HBc spike position, which was possible insurface. Then, the HBC-EB with its optimized gene sequences was optimized and synthesized.(2) Generation of multi-epitope immunogen and effects of immunity by intraperitoneal injection1) Generation of multi-epitope immunogen in E.coliThe target gene was amplified by the PCR and the recombinant expression plasmidpET28a-HBc-EB was constructed. Based on the synthesized pUC57-simple-BIg plasmid as the template,the BIg gene was amplified and fused by the EB gene to generate the recombinant expression plasmidpET22b-EB-BIg. The recombinant plasmids were expressed in E.coli. Electron microscopy showed thatthe refolded protein pET28a-HBc-EB complex formed as virus-like particles. Western blot and DotELISA results confirmed that the recombinant proteins had great antigenicity, and the recombinantprotein pET22b-EB-BIg reacted directly with Horseradish peroxidase-conjugated rabbit anti-bovine IgGantibody. The immunization strategy was done using the antigens based on the Asia1FMDVmulti-epitope immunogen.2) Effects of immunity by intraperitoneal injectionThe test was taken at28d after the mice immunized and the results showed that the antibody levelsof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01or P<0.05), the antibody levels of pET28a-HBc-EB group were significantlyhigher than the pET22b-EB-BIg group (P<0.05); The liquid phase blocking antibody titer of thepET22b-EB-BIg group was near vaccine group while pET28a-HBc-EB group higher than inactivatedvaccine group, which was from1:256to1:512. T-lymphocyte proliferation assay was performed usingthe Cell Titer96aqueous one solution assay (MTS assay). The results showed that the stimulation indexof the pET28a-HBc-EB group and pET22b-EB-BIg group were significantly higher than the inactivatedvaccine group (P<0.01). The CD4+and CD8+T cell were detected by the Flow cytometry. The resultsshowed that the CD4+/CD8+ratio of the pET28a-HBc-EB group and the pET22b-EB-Big group wassignificantly higher than the inactivated vaccine group (P<0.01and <0.05). The cytokine ELISA assayshowed that the mouse spleen cells stimulated IL-2and IFN-gamma levels of the pET28a-HBc-EBgroup and pET22b-EB-BIg were significantly higher than those in the inactivated vaccine group(P<0.01); and the IL-4and IL-10levels of the pET28a-HBc-EB group were significantly higher than theinactivated vaccine group (P<0.05), while the difference between the pET22b-EB-BIg group and theinactivated vaccine was not significant. In conclusion, the Asia1FMDV type multi-epitopeimmunogenic vaccines had the good immune effect, which stimulate the mice to generate high levels ofhumoral and cellular immunity, especially the pET28a-HBc-EB immunogen.(3) Production of multi-epitope immunogen in Pichia pastoris and effects of immunity by oral1) Production of multi-epitope immunogen in Pichia pastorisIn this study, as the Pichia pastoris has the characteristics of the eukaryotic cell expression systemmodification, we select Pichia pastoris to generate multi-epitope immunogen that has the natural structure. The multi-epitopes gene EB was digested from pET28a-HBc-EB and cloned into pPICZA, toconstruct the recombinant expression plasmid pPICZA-HBc-EB. Using the plasmids containing Asia1FMDV gene prepared in our lab as the template, the genes of the three proteins were cloned into Pichiapastoris expression vector-pPICZA. Then the restriction enzyme sites were introduced into theexpression cassette individually by PCR, which was ligated into pPICZA orderly to construct thepPICZA-vp031. Western blot and Dot ELISA showed that the recombinant expression products hadgreat antigenicity.2) Effects of immunity by oralThe mice were immunized by oral with recombinant Pichia pastoris at the dose of5×108. Theresults showed that the antibody levels of the experimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than pPICZA control group at28d post immunization (P<0.01), butsignificantly lower than inactivated vaccine group (P<0.01); the liquid phase blocking antibody titer was1:8to1:16. The mucosal sIgA levels of flushing fluid of the mice immunized with Pichia pastoris weresignificantly higher than inactivated vaccine group (P<0.01); and the mucosa-specific sIgA of theexperimental group pPICZA-HBc-EB and pPICZA-vp031were significantly higher than inactivatedvaccine group, pPICZA group (P<0.01). The stimulation index of pPICZA-HBc-EB group andpPICZA-vp031group were significantly higher than the inactivated vaccine group (P<0.01) and thepPICZA-HBc-EB group was higher than pPICZA-vp031group (P<0.05). The CD4+/CD8+ratio of theinactivated vaccine group were significantly higher than pPICZA-HBc-EB group and pPICZA-vp031group (P <0.01). The cytokine ELISA assay showed that the mouse spleen cells stimulated IL-2andIFN-gamma levels of pPICZA-HBc-EB group were significantly higher than those in the vaccine group(P<0.01), while the differences among the pPICZA-vp031group the inactivated vaccine group were notsignificant (P>0.05); the IL-4and IL-10levels of pPICZA-HBc-EB group and pPICZA-vp031grouphad no significant difference with the inactivated vaccine group (P>0.05). Taken together, the resultsindicated that the oral immunization with the Asia1oral immunogen could not only stimulate theproduction of humoral and cellular immunity, but also significantly induce mucosal immunity.(4) Effects of immunity by intranasalThe mice were immunized i.n. with the recombinant protein pET28a-HBc-EB andpET22b-EB-BIg.28d past first immunization, results showed that the antibody levels of thepET22b-EB-BIg group were significantly higher than the pET28a-HBc-EB group (P<0.01),significantly lower than the inactivated vaccine group (P<0.01), with lower the antibody grown trend;The liquid phase blocking antibody titer was down to1:8; However, the level of total sIgA and specificsIgA of the pET22b-EB-BIg group were significantly higher than the other groups (P<0.01); Thestimulation index of the pET22b-EB-BIg group was significantly higher than the other groups (P<0.05);While, its CD4+/CD8+ratio was significantly lower than inactivated vaccine group (P<0.01). Theseresults indicate that Bovine IgG2a Fc fragment fusion protein could stimulate both the humoral and cellular immune responses, which can induce mucosal immunity, although the overall strength is nothigh, while pET28a-HBc-EB did not, our work confirmed for the first time the feasibility of targetingthe FcRn stimulate mucosal immune, which may provide a new way for preparing the FMD mucosalvaccine.