Immunological Effect Of A Inclusion Of PT8A Peptide To Human Recombinant Rotavirus VP8* Subunit Vaccine

RV(rotavirus)is one of the main pathogens of diarrhea in infants and young children with more than a half of million annual motality.Up to date,in order to eradicate of rotavirus and improve the survival rate of infants and young children,it has developed a number of safe and effective rotavirus vaccines,such as Rotarix(G1P[8],monovalent,Glaxo SmithKline Co),Rotateq(G1-G4P[8],pentavalent,Merck Co)and LLR(type G10P[15],Lanzhou,China).However,Rotarix and Rotateq were not worked well in some under-developing countries,such as Africa,Asia and other places where demanded for RV vaccines.In addition,the two rotavirus vaccines(Rotarix[RV1] and Rota Teq [RV5])were attenuated live vaccines.Although live attenuated vaccine have stronger immunogenicity and longer effect than that of inactived vaccines,they also have potentially danger of intussusception and virulent recovery,etc.;And LLR,licensed only in China,is a attenuated lamb rotavirus strain,it's G10 and P [15] serotypes are neither the global popular G1,G4 or P [8] nor the popular serotypes in China.Therefore,it is necessary to develop a safe and efficient next generation RV vaccine.In this study,we screened the LTB antigen epitopes and select one epitope,named PT8 A,integrated into the C and N terminus of the RV VP8* gene to obtain a safer and more effective candidate VP8* subunit vaccine containing intramolecular adjuvant.Objective: To evaluate the PT8 A adjuvant activity to VP8* subunit vaccine and Preliminary to explore the mechanism of the PT8 A adjuvant activity.Methods: 1.PCR amplification of target gene fragment PT8A-VP8 and VP8-PT8 A.2.Construction of recombinant plasmid pET32a-PT8A-VP8 and pET32a-VP8-PT8A;3.Expression and purification of fusion proteins,and removal of fusion proteins endotoxin;4.Animal vaccination;5.Collection of serum and tissue samples;6.Detection of antibodies,cytokines and some target proteins with ELISA and immunohistochemistry.Results: 1.Recombinants of pET32a-PT8A-VP8 and pET32a-VP8-PT8 A were successfully constructed.2.The fusion proteins expressed as inclusion bodies and were purified using Nickel magnetic beads.3.IgG and s IgA titers: The IgG titers(OD values)in groups of VP8-PT8 A and VP8*+LTB were enhanced 4 times than that of in groups of PT8A-VP8 and VP8*,and about 256 times than that in non-immunized mice(p<0.05).That meant,VP8-PT8 A had adjuvant activity and,conversely,PT8A-VP8 had no adjuvant activity.Similarly,s IgA titers were also higher in groups of VP8-PT8 A and VP8*+LTB than that of in groups of VP8* and PT8A-VP8.The results suggested that the adjuvant activity of VP8-PT8 A fusion protein was stronger than that of VP8*+LTB mixture.4.Immunohistochemistry assay:(1)The TLR2 expression was increased significant only in group of VP8*+LTB in the spleen(p<0.05)and was no significant differences in the other three groups(p>0.05).However,there were no significant differences in both nasal and intestinal tissues between the four groups(p>0.05).The results indicated that LTB might function through TLR2 pathway in central immune organs.However,both the LTB and PT8 A could not function adjuvant through TLR2 activation in mucosal immune system.(2)JNK1/2 expression was significant enhanced in spleen in group of VP8-PT8 A and there were no significant differences in groups of PT8A-VP8,VP8*+LTB,and VP8*.Interestingly,JNK1/2 expression was increased significantly in group of VP8-PT8 A than that of in group of VP8*+LTB in both nasal and intestinal tissues.However,JNK1/2 expression was also increased significantly in PT8A-VP8 group.That meant,PT8 A and LTB had different mechanisms.(3)MAP2K3 expression was significant decreased in groups of VP8*+LTB and VP8-PT8 A in both spleen and nasal tissue than that of in groups VP8*.That meant,MAP2K3 might inhibit the adjuvant of VP8-PT8 A and LTB.Conclusions: 1.The peptide PT8 A fused at the C terminal of the VP8* gene can significantly enhance the immune effect of recombinant rotavirus VP8* subunit vaccine.However,the N terminal of PT8A-VP8 has no adjuvant activity.2.LTB adjuvant activity is associated with LTR2 pathway,but PT8 A is not.3.The adjuvant activity mechanism of PT8A(at C terminus)and LTB are different.4.MAP2K3 activaion may inhibit the adjuvant activity of LTB and PT8A(at C terminus).5.In central immune organs,the adjuvant activity of PT8A(at C terminus)may be related to the JNK1/2 signaling pathway.6.In the mucosal immune system,the adjuvant activity of both the LTB and LTB-PT8 A may have no direct relationship with the JNK1/2 activation.