Construction Of Multi-epitope-based Recombinant Proteins Of Canine Distemper Virus,Canine Parvovirus And Rabies Virus And Study On Their Immunogenicity In Mice

Canine distemper virus(CDV),Canine parvovirus(CPV)and Rabies virus(RV)are common pathogens of carnivores with a worldwide distribution.All three viruses can cause serious diseases in dogs.Infection with the viruses can only be controlled by vaccination.Live attenuated and inactivated vaccines are both currently used;however,these vaccines have several disadvantages.Epitope-based vaccines present a preferable substitution with many advantages.The purpose of this research was to construct multi-epitope-based recombinant proteins of Canine parvovirus,Canine distemper virus and Rabies virus and study their immunogenicity in mice.Meanwhile the adjuvanticities of polyethyleneimine(PEI)or CpG oligodeoxynucleotides coformulated PEI for the multi-epitope-based recombinant proteins were studied.These researches provided the basis for development of combination multi-epitope-based canine vaccines.Numerous B cell and T cell epitopes of CDV,CPV and RV have been identified and tested in previous studies.Our strategy was to select appropriates epitopes from the previous studies under certain basic criteria.A set of thirteen epitopes was finally selected,including two B-cell epitopes,two Th epitopes and one CTL epitope of CDV,two B-cell epitopes and one Th epitope of CPV and two B-cell epitopes,two Th epitopes,one CTL epitope and one mimotope of RV,to construct the muti-epitope-based proteins.The epitopes of recombinant proteins were assembled in different sequences with linker sequences(GGGGS)or(KK)between each epitope.Six sequences were selected based on results of analyzing of hydrophilicity,antigenicity,flexibility,surface accessibility,secondary structure and third structure of candidate sequences.After codon optimization and synthesis,rP1,rP2 and rP3 were cloned into expression vector pET-28a(+)respectively.Meanwhile rP4,rP5 and rP6 were cloned into expression vector pET-30a(+)respectively.After induction the six expressing plasmids transformed BL-21(DE3)with IPTG,target proteins were successfully expressed and identified by SDS-PAGE.The expressed proteins existed mainly as inclusion bodies and were purified by Ni-NTA agarose resin column.Finally,denatured proteins were refolded by dialysis against a linear gradient urea buffer.Western blotting against anti-His-tag monoclonal antibody identified them as the target proteins.9 groups of 4-week-old BALB/c mice were injected with Freund’s adjuvant emulsified 6 rP proteins and 3 inactivated viruses respectively and bled 14 days after the third dose.ELISA and Western blotting results indicated that injection of mice with each rP proteins generated high antibody titers and all the 6 proteins were high antigenicity in mice.All the 6 anti-rP serums could react with the native proteins of the viruses.These data showed that at least one of the epitopes of rP for each virus could elicit an immune response and produce antibody against a target epitope.Antiserums against the 3 viruses individually also strongly reacted with the 6 rP proteins and these results indicated that the epitopes displayed independently on the recombinant proteins as they did on the original protein sequences of the three viruses.Neutralizing antibodies against CDV,CPV and RV in the serums of the proteins immunized mice were identified by virus neutralization assay,hemagglutination inhibition assay and rapid fluorescence inhibition test respectively and the results demonstrated that the 6 proteins all can simultaneously induce humoral responses against all 3 viruses in mice although the HI and NA titers were significantly lower than the CDV,CPV and RV virus vaccinated mice.Recombinant rP1 and rP4 could induce higher HI and NA titers than other 4 proteins.There was no statistically significant difference between rP4 and rP1 in the results of all three experiments.Meanwhile,a side-by-side comparison study of adjuvanticity among Freund’s adjuvant,PEI + CpG,PEI,alum adjuvant and CpG was carried out.Protein rP4 was used as antigen and emulsified with the five different adjuvants respectively.Then 6 groups of 4-week-old BALB/c mice were injected with the 5 emulsified proteins and proteins alone respectively and bled 2 weeks,4 weeks,6 weeks and 8 weeks after the third dose.The antibody titers were tested by ELISA and the results demonstrated that all the 5 adjuvants enhanced antigen-specific serum antbodies production compared to the no-adjuvant control group.The potency order from strong to weak among each adjuvant was Freund’s,PEI + CpG,PEI,alum and CpG.Protein rP4-specifc IgGl and IgG2a titers of 2-week-bleed seums were also determined by ELISA as indicators of humoral or cellular biased mimune responses,respectively.The PEI-adjuvanted immune response was characterized by an intermediate IgG1/IgG2a titer ratio for PEI between that of Freund’s(cellular)and alum(humoral)adjuvants,indicating a mixed humoral or cellular immune response.When coformulated with CpG oligodeoxynucleotides,PEI adjuvant potency was biased toward a cellular immune profile.In summary,as a first step in developing a multi-epitope-based vaccine,we succefully constructed,expressed and purified 6 novel multi-epitope recombinant proteins containing 13 different B-cell and T-cell epitopes of CDV,CPV and RV in E.coli.All the 6 proteins could simultaneously induce humoral responses against the three viruses in mice.And this study also identifies that PEI and PEI-CpG are potential adjuvants for multi-epitope recombinant proteins.In conclusion,this study provides the basis for development of combination multi-epitope-based canine vaccines.