Immunogenicities For Nucleocapsid Protein DNA Vaccines Of Severe Fever With Thrombocytopenia Syndrome Virus

Objective:To study the immunogenicities for nuclear protein of severe fever with thrombocytopenia sydrome virus by DNA immunization.Methods:1.According to the nucleocapsid protein coding sequence of SFTS virus HB29（GenBank:HM745932.1）,OptimumGene^TM software was used to analyse the sequence of SFTSV nucleocapsid protein and determine the codon differences between SFTSV and mammals and E.coli then replaced the codons from wild type to new codons having the highest usage by both mammals and E.coli gene translation maintaining the amino acid sequence unchanged.The wide type sequence and optimized sequence were chemically synthesized and cloned into vector pJW4303,constructing plasmids pJW4303-N and pJW4303-N-opt.Meanwhile,the introduction of Nhe I restriction site by PCR linked the optimized sequence with the tPA signal sequence located in pJW4303 contructing plasmids pJW4303-tPA-N-opt.2.In order to investigate the epitopes of SFTSV nucleoprotein to study the CTL immune response,the CTL epitopes were predicted by computer technology and bioinformation.After comparison of multiple parameters assay results,4 CTL epitopes were predicted and selected as possible epitopes,which are SN-1,SN-2,SN-3,SN-4.3.Western blot was used to detect the expressing nucleoprotein in the supernates and lysates harvesting of 293T cells after transient transfection with constructed plasmids,and pJW4303 used as a control.4.BALB/c mice were immunized with the constructed plasmids by intramuscular injection followed by electroporation at week 0,2,4 and 8.Sera were collected prior to the immunization and week 6 and week 10 after immunization.Sera specific IgG,IgG1,IgG2a antibodies were detected by ELISA.5.Meanwhile,BALB/c mice were immunized with the same method at week 0,2 and4.Mice spleen cells were analyzed to determine the lymphocyte subsets by flow cytometer and IFN-gamma positive CD8⁺T cells were assayed by Elispot.Results:1.Plasmids and expression in vitro:The plasmids pJW4303-N,pJW4303-N-opt,pJW4303-tPA-N-opt were successfully constructed and they could properly express nucleoprotein in 293T cells after transient transfection.The nucleoproteins expressing by pJW4303-N,pJW4303-N-opt were detected only in the 293T cell lysates,while pJW4303-tPA-N-opt highly expressed products in supernate and lysate.2.Antibodies responses:In week 2 after the first immunization,specific IgG antibody can be detected in BALB/c mice sera in the pJW4303-N,pJW4303-N-opt and pJW4303-tPA-N-opt group.With the booster immunization,the levels of IgG antibody increased and reached the highest levels in week 10.pJW4303-tPA-N-opt group IgG level was higher than pJW4303-N and pJW4303-N-opt group,there were statistical significant differences between them（p（27）0.05）.But there were no statistical significant difference between pJW4303-N and pJW4303-N-opt group（p（29）0.05）.IgG1 antibody weakly responsed in pJW4303-N and pJW4303-N-opt group,two mice were not detected IgG1 in week 10 after booster immunization in pJW4303-N group.All of mice detected high levels of IgG1 antibody from week 2 to week 10 in pJW4303-tPA-N-opt group,there were statistical significant differences between pJW4303-tPA-N-opt group and pJW4303-N and pJW4303-N-opt group（p（27）0.05）.In the three groups,IgG2a was detected in week 2 after immunization.pJW4303-tPA-N-opt group has the highest level of IgG2a among the three groups（p（27）0.05）,and pJW4303-N group IgG2a level higher than pJW4303-N-opt group（p（27）0.05）.IgG2a/IgG1 ratio had significantly differences among the three groups（p（27）0.05）,the ratio in the three groups was 3.81±0.51、2.56±1.03、0.93±0.086,respectively.There were no IgG,IgG1,IgG2a responsed in the pJW4303 group.3.Mice spleen lymphocyte subsets:Day 7:pJW4303-N-opt group had significantly evaluated CD3⁺T,CD8⁺T,NK cells than pJW4303-tPA-N-opt group（p（27）0.05）;and CD3⁺T cell showed significantly difference compared with pJW4303 group（p（27）0.05）;while NK cell had significantly difference compared with pJW4303-N-opt group（p（27）0.05）.But it indicated no significant changes in pJW4303-N group and pJW4303-tPA-N-opt group（p（29）0.05）,which also showed no significant differences that the two groups compared with pJW4304 group（p>0.05）.Day 17,pJW4303-N-opt group spleen lymphocyte cells increased significantly and showed statistical difference with pJW4303-tPA-N-opt group（p（27）0.05）;CD4⁺T,CD8⁺T cells in pJW4303-N-opt group showed differences compared with pJW4303 group（p（27）0.05）,among which CD8⁺T cell had difference with pJW4303-N group（p（27）0.05）.There was no more difference among other cells（p（29）0.05）.Day 31:pJW4303-N-opt group spleen NKT cells decreased significantly than pJW4303 geoup（p（27）0.05）,but there were no difference among other groups’cells（p（29）0.05）.4.CTL respone:Elispot assay identified that SN-4 had antigenicity,which can stimulate splenocytes secreting IFN-γafter immunization of three DNA vaccines.pJW4303-N-opt group had robust antigen-specific IFN-γresponses in day 17 and day31 in the splenocytes of vaccinated mice,there were statistical difference comparing with other groups（p（27）0.05）.More modest numbers of antigen-induced IFN-γspots were detected in pJW4303-N group in day 31 and in pJW4303-tPA-N-opt group in day 17 and day 31,there was no statistical difference between the two groups（p（29）0.05）.In fact,no IFN-γSFUs in pJW4303-N group in day 17,and the splenocytes stimulating by predicted peptides SN-1,SN-2,SN-3 had no IFN-γSFUs identifing by Elispot assay.Conclusions:1.SFTSV nucleoprotein DNA vaccines had substantial immunogenicities;2.SN-4 is a CTL epitope of SFTSV nucleoprotein.