The Immunogenic Evaluation Of Porcine Rotavirus Gottfried And SB-1A Strains ?VP8~*Proteins And The Role Of Combination Adjuvants

Porcine rotavirus?Porcine rotavirus,PRV?is a major cause of diarrhea in piglets,which endanger domestic pig husbandry,responsible for great economic losses annually.The efficacy of current vaccines which control the occurrence and spread of the disease is unsatistified.Therefore,developing the novel vaccine against porcine rotavirus is one of top priority facing veterinary workers.VP4 is the major antigen protein of rotavirus,defining the viral P type.It induces neutralizing antibodies independently,as well as glycoprotein VP7.Rotavirus VP8*protein is the minor trypsin cleavage fragment of the VP4,which covers the main neutralizing antigen epitopes that are linear and conservative.In theory,the rotavirus vaccines which share the same P type can induce heterotypic immunity to fight against the rotaviruses which confer different G type.The P type of rotaviruses isolated from diarrheal piglet in the world is categorized Gottfried-like?P[6]?or OSU-like?P[7]?.So we aim to develop subunit vaccine candidate to cover much more G types porcine rotaviruses by the generation of two recombinant VP8*proteins that bearring P[6]or P[7]genotype.Additionally,the T cell epitope P2?830-844 amino acid?of tetanus toxin was introduced into the upstream of?VP8*and P2-?VP8*recombinant proteins were expressed in prokaryotic system.Immobilized Ni⁺-ion affinity chromatography was used to purify the soluble part of four recombinant proteins.Female Balb/c mice at 6 to 8 weeks of age were immunized intramuscularly with 10?g of recombinant proteins with aluminum hydroxide adjuvant three times at a 2-week interval.We compared the immunogenicity of four recombinant proteins and evaluated the effect of T cell epitope P2 on immunogenicity upon the level of antibody by ELISA in an attempt to develop a porcine rotavirus subunit vaccine candidate.Recombinant protein P2-SB-1A?VP8*demonstrated the highest immunogenicity based on test above,followed by the evaluation of combination adjuvants on the selected recombinant protein.We generated LTK63 recombinant protein which is a non-toxic mutant of Enterotoxigenic Escherichia coli heat-labile enterotoxin.Female Balb/c mice at 6 to 8 weeks of age were immunized subcutaneously with recombinant protein P2-SB-1A?VP8*formulated with Al?OH?_3,plus either CpG ODN2006 or LTK63,or plus the both.The serum IgG and IgG subclasses?IgG1,IgG2a?antibody from mice were measured by ELISA.The spleen T-cell proliferation responses were quantified by MTT test.The GMT of control group which immunized P2-SB-1A?VP8*formulated with Al?OH?₃ was only 1:8444.9.The data in this study showed that P2-SB-1A?VP8*formulated with Al?OH?₃ plus CpG ODN2006 induced the highest titer of IgG,up to 1:55153.5?Geometric serum antibody titer,GMT?,Significantly higher than control group.The GMT of mice which immunized P2-SB-1A?VP8*formulated with Al?OH?₃ plus CpG ODN2006 and LTK63 is 1:14367.5,and P2-SB-1A?VP8*formulated with Al?OH?₃ plus LTK63 induced GMT of 1:11403.5.The serum IgG subclasses antibody in Al?OH?₃plus CpG ODN2006 group was mainly IgG2a,the ratio of IgG2a/IgG1 was 2.15.But the serum IgG subclasses antibody in control group was mainly IgG1?Ig G2a/IgG1=0.21?.And the ratio of IgG2a/IgG1 in other two groups was approximately 1.0.The stimulation index?SI?of spleen T lymphocyte proliferation in Al?OH?₃ plus CpG ODN2006 group was 2.65,Significantly higher than control group?1.503?.The SI of other groups was approximately 2.0.So combination adjuvants CpG ODN2006 plus Al?OH?₃ improved the humoral immunity and cell immunity responses overally.They showed obvious synergistic effect.