Development Of Chimeric DNA Vaccine Against Classical Swine Fever And TRIF-induced Enhanced Immune Responses Of DNA Vaccination

Classical swine fever (CSFV) is a contagious disease of swine and leads to severe economic losses worldwide. According to "Animal Health Code", CSF was an OIE (Office International des Epizooties) listed disease. CSF was identified as class one contagious animal diseases in our country. From 1950s, hog cholera lapinized virus (Chinese "C" strain), a live attenuated CSF vaccine, was developed by our country and used to control CSFV spread. Though a live attenuated CSF vaccine is effective to control outbreak of CSF, a lot of new features of CSF such as sporadic epidemics, chronic infection, sustainable contagion and pregnancy with CSFV were recently observed. So, it is necessary to develop a new marker vaccine.In this study, E2 and Ems encoded sequences were amplified by RT-PCR using genomic RNA of CSFV strain Shimen as template, and then fused with nature E2 or tissue plasminogen activator (tPA) signal sequences to construct recombinant DNA vaccines, including ptPAs/△E2, ptPAs/△E2/△Erns, pE2s/△E2/△Erns and pE2s/E2. PK15 cells were transfected with the four plasmids to detect the expression of the foreign genes, respectively. The female BALB/c mice immunized with the recombinant plasmids for 3 times with 2 weeks interval. Antibody titers were tested by indirect ELISA using the purified E2 or Ems protein by prokaryotic expression as the coating antigen. CSFV or ConcanavalinA (ConA) was used as stimulus to detect splenocyte proliferation by MTT assay or to detect IFN-y secreting by Elispot assay. The results showed that the serum antibody titers in the four immunization groups were significantly increased compared to pcDNA3.1(+) group (p<0.05). Two weeks after the last inoculation mice immunized with plasmid fused with tPA signal exhibited significantly higher OD492nm than those immunized with nature E2 signal (p<0.05). After CSFV or protein stimulation, splenocyte isolated from mice immunized with chimeric DNA vaccine induced more IFN-γsecreting cells and enhanced spleen cell proliferation than that immunized with the single gene vaccine. Mice immunized with DNA vaccines fused with tPA signals induced better immune responses compared to that immunized with DNA vaccines with E2 signal.Serum-negative pigs were immunized with ptPAs/△E2/△Erns, pE2s/E2 and pcDNA3.1(+) separately with 3 times at a 2 week interval, for ptPAs/△E2/AEms has induced a better immune responses in mice model and pE2s/E2 and pcDNA3.1(+) were used as control group. Two weeks after the last immunization, all the pigs were intramuscularly challenged with 3×105 TCID50 Shimen strain CSFV. The temperature and clinical symptom was monitored and recorded daily and the blood and nasal swab samples were collected on days 0,4,8,12 post-challenge. After challenge neutralizing antibody titers was evaluated by virus neutralization assay, IgG antibody titers were detected by ELISA. CSFV genomic RNA in tissue or serum samples was tested by RT-PCR. Our results demonstrated that the pigs vaccinated with chimeric DNA vaccine fused with tPA signal induced higher titer of neutralizing antibody than those vaccinated with native E2 vaccine. The pigs inoculated with pcDNA3.1 (+) were all dead, pigs immunized with pE2s/E2 were partly dead and pigs immunized with ptPAs/△E2/AErns were all survived.Adaptor protein TRIF as molecular adjuvant of DNA vaccines was investigated. Female BALB/c mice immunized with pRK-TRIF plasmid for 3 times at 2-week interval, and pRK was used as negative control. Antibody titers were detected by indirect ELISA using the purified E2 or Erns protein as the coating antigen. CSFV or ConA was used as stimulus to induce IFN-γsecreting. The results showed that the serum antibody titers were not significantly increased with TRIF. After CSFV or protein stimulation, splenocyte isolated from mice immunized with the mixture of DNA vaccine and TRIF induced more IFN-γsecreting cells and higher level of spleen cell proliferation than non-adjuvant DNA immunized mice.Serum-negative pigs were immunized with ptPAs△E2AErns and TRIF or pE2s/E2 and TRIF separately, with 3 times at a 2 week interval. Two weeks after the last immunization, all the pigs were intramuscularly challenged with 3×105 TCID50 Shimen strain CSFV. The temperature and clinlcal symptom was monitored and recorded daily. The blood and nasal swab samples were collected on days 0,4,8,12 post-challenge. After challenge neutralizing antibody titers were evaluated by virus neutralization assay, and IgG antibody titers were detected by ELISA and and CSFV genomic RNA in tissue or serum samples was tested by RT-PCR. The results showed that the pigs co-vaccinated with DNA vaccine and TRIF were more white cell numbers than non-TRIF vaccine. But pigs immunized with DNA vaccine and TRIF revealed similar neutralizing antibody titers compared to that immunized with DNA vaccine alone. All of pigs co-immunized with DNA vaccine and TRIF survived.In summary, we first constructed chimeric DNA vaccines encoded E2 and Ems, and examined their efficacies in mice and pigs. We also investigated the immune responses in mice and pigs co-immunized with DNA vaccine and TRIF. Our results demonstrated that chimeric DNA vaccine could induce higher humoral and cellular immune responses in mice and protection against CSFV in pigs. TRIF as molecular adjuvant could enhance the cellular immune responses of DNA vaccines. These results will be contributed to develop a novel DNA vaccine against CSFV.