Studies Of Genetic Marker Vaccines Against Classical Swine Fever And Swine Vesicular Disease

Both classical swine fever (CSF) and swine vesicular disease (SVD) are highly contagious viral diseases of pigs, and are included in the list A by the Office International des Epizooties (OIE). Vaccination is the major method to prevent them. However a major shortcoming of the existing vaccines is lack of marker status, and vaccinated pigs cannot be distinguished from animals infected with field virus. Therefore, marker vaccines have been developed in combination with the appropriate diagnostic teses based on serology. Genetic vaccination would potentially offers an elegant and cost effective means of providing marker vaccine for the livestock industry. Such vaccines would provide valuable tools for incorporation current and future eradication programs on CSF and SVD. In the present study, by using of reverse transcription (RT), nest polymerase chain reaction (PCR) and subclone technology, the genetic vaccines of SVD or/and CSF were developed. The in vitro expression of each recombinant plasmids were analyzed, then they were inoculated rabbits and pigs respectively to analyze both humoral and cellular immunity, and finally challenged with viruses to investigate the protection effect of the genetic vaccines.1) Recombinant plasmids of pcDNA Esl-11, which has a copy of CSFV E2 gene;pBudCE Es1-11/Es2-22, which has dual copy of CSFV E2 gene;pBudCE lacZ/Es2-22, which has a copy of CSFV E2 gene & a copy of reporter gene lacZ;pBudCE Esl-11/ IFN-y, which has a copy of CSFV E2 gene & a copy of porcine Interferon y gene;and pBudCE Esl-11/ 1L-18, which has a copy of CSFV E2 gene & a copy of porcine Interlukine 18 gene, were constructed. The expression of CSFV E2 protein was confirmed in the transfected supernants and BHK-21 cells with a CSFV Antigen Kit. The expression of reporter gene lacZ was showed in situ staining by (3-Gal Staining Kit, and many blue cells were observed by a microscope. The mRNA of porcine Interferon y or Interlukine 18 was tested by RT-PCR. Results showed that all recombinant plasmids could express foreign genes in vitro.2) All CSF genetic vaccines were inoculated in rabbits twice or three times, 3 or 4 weeks apart, 4 weeks after the last immunization, the rabbits were challenged with CSF live vaccine (II). Results showed that 3/4 rabbits produced specific antibodies to CSFV E2 in recombinant plasmid pcDNA Esl-11 immunized group, and 4/4 rabbits' rectal temperature remained normal, fully protected against challenge. Of rabbits immunized with pBudCE Esl-1 l/Es2-22 plasmid, 1/4 of which had CSFV E2 antibody, and were completely protected;but 3/4 of which absence of E2 antibody, two of them developing classical fever and one of them developing mild fever (partially protected). Rabbits immunized with either pBudCE Esl-11/ IFN-y or pBudCE Esl-11/ IL-18 were 4/4 negative to E2 antibody, and only 1/4 get partially protected following challenge. In recombinant plasmid pBudCE lacZ/Es2-22 immunized group, all (3/3) lacked detectable CSFV E2 antibody, but all got partially protected following challenge. While the blank plasmid controls immunized with either pcDNA3.1 or pBudCE4.1, 4/4 remained negative to E2 antibody, and 4/4 developed classical fever following challenge. T lymphocyte proliferation response of rabbits was elicited after inoculation with ail CSFgenetic vaccines.3) The dual-antigen-expression system pBudCE E2/P1-1I , which contains both the PI coding region of swine vesicular disease virusC SVDV)and E2 gene of classical swine fever virus Shimen strain, was constructed by RT-PCR, nest PCR and subclone. To explore the in vitro expression by pBudCE E2/P1-11 in BHK-21cells, in situ staining by the indirect immunofluorescence assay was used, and many cells with bright green were observed by a fluorescence microscope. The expression of CSFV E2 protein in the transfected supernants and cells was determined with a CSFV Antigen Kit. Rabbits immunized with expression plasmid pBudCE E2/ Pl-11 didn't development of specific antibody to CSFV E2, while 1/4 got completely protected following challenge with CSF live vaccine (II). And T lymphocyte proliferation response of rabbits was elicited after inoculation. SVDV neutralization antibodies titers of all rabbits were below 1:4 by new-born-mice neutralization test.4) Genetic vaccine pcDNA PI against SVD was constructed. To test the transient expression in cultured BHK-21 cells, in situ staining by the indirect immunofluorescence assay was used, and many bright green cells were observed by a fluorescence microscope. Subsequently, to evaluate the in vivo immune response induced by recombinant plasmid pcDNA PI and blank plasmid control pcDNA3.1 with or without pcDNA3.1 IFN-y, 13 pigs were used. Anti-SVDV antibodies were detected by ELISA and neutralization antibodies titers were analyzed by new-born-mice neutralization test. Low level of SVDV special antibodies were detected in two out of four (2/4) pigs by ELISA in Group vaccinated with pcDNA PI or in Group pcDNA PI plus pcDNA3.1 IFN-y. And higher lever of SVDV special antibodies were observed upon challenge. The other result shows that only two out of four (2/4) pigs developed lower neutralization antibodies (1:4) in the group vaccinated with pcDNA PI plus pcDNA3.1 IFN-y, while SVDV neutralization antibodies titers in the other groups were below 1:4 by new-born-mice neutralization test. Three mouths after the third immunization, half of pigs were challenged with 5mL 104 LD50/0.1 mL of SVDV (strain HK) on neck intramuscular/ (3 mL) and at the junction of heel (2 mL), and the other half were challenged with 5mL 10" LD5()/0.1 mL of same SVDV strain. One out of four in pcDNA PI group achieved total protection upon challenge (the one challenge with5mL 104 LD50/0.I mLof SVDV), while one out of four in pcDNA PI pluspcDNA3.1 IFN-y group achieved partially protection upon challenge (the one challenge with 5mL 10" LD50/0.1 mL of SVDV). While the blank plasmid control pcDNA3.1 with (3 pigs) or without (2 pigs) pcDNA3.1 IFN-y immunized groups, 5/5 developed SVD following challenge.In conclusion, the researches opened the possibility of exploring the potential of the DNA vaccine approach used to develop marker vaccine against CSF and SVD.