-
Foot-and-mouth disease virus (FMDV) is a highly infective agent of the Picornaviridae family, affecting all cloven-hoofed animals. FMDV contains a single-stranded, positive sense RNA encoding a single, long open-reading frame protein. Outbreaks of foot-and-mouth disease (FMD) in various countries in recent years have had severe economic impact on the agricultural industry worldwide[10].
In recent years, one of the most important developments in recombinant vaccines is the DNA vaccine, allowing a safe and efficient alternative to conventional vaccination. DNA vaccine technology facilitates the use of cytokines as modulators in vaccination to improve immune responses[14]. In FMD, the virus carrier state is always accompanied by FMD virus antibodies in serum and esophageal-pharyngeal fluid. Vaccinated animals with inactivated vaccine also become virus carriers after FMDV infection, to the same extent as unvaccinated animals[21]. Even in non-persistently infected cattle, viral RNA exists in the soft palate and pharynx over the normal time of virus clearance[25]. These findings indicate that the immune response induced by viral infection or traditional vaccination is not efficient for complete clearance of virus. Recent studies showed that some DNA vaccines could elicit complete protection against the challenge of FMDV[7, 22, 23], which provides us with another choice that is distinct from the traditional inactivated vaccine[24]. The aim of this study is the evaluation of a DNA immunization system using the pcDNA3.1+ plasmid containing the FMDV type O/IRN/1/2007 VP1 gene in mice and comparison with the conventional inactivated vaccine. FMDV type O/IRN/1/2007 causes severe symptoms in Iranian cattle and the goal of this research was to seek improved vaccination against this subtype. Also pCMV-SPORT vector containing the GMCSF gene (Granolocyte-Monocyte colony stimulating factor) was used as molecular adjuvant along with DNA vaccination.
HTML
-
Serotyping of the isolated FMDV antigen was done by ELISA which subsequently showed serotype O. The plasmid DNA, pcDNA3.1+-VP1 cassette, was constructed and confirmed by sequencing and digestion with restriction enzymes Kpn I and BamH I.
-
There are 672 nucleotides and 224 amino acid residues in the VP1 coding region. The nucleotide sequence data was deposited in GenBank with accession number: JF288761.
-
The transfected BHKT7 cells with sub-cloned pEGFP-N1-VP1 vector were expressed with GFP-VP1 fusion protein and displayed more green fluorescence spots than the transfected BHKT7 cells with pEGFP-N1 vector as shown in Fig. 1. The expression of VP1 protein was detected by specific bands in Western blotting analysis, shown in Fig. 2.
-
FMDV type O/IRN/1/2007 titration was calculated by the Read and Munch method and reported 108.5 TCID50 per mL. The Cytopathic Effect was not observed on IBRS-2 cells after three times blind culture of inactivated FMDV type O/IRN/1/2007 samples, therefore the inactivated vaccine was safe. The result of the CF test was also showed the antigenicity of the inactivated virus was not changed.
-
The anti FMDV type O/IRN/1/2007 sera titration of the vaccinated mice 10 and 70 days after last vaccination are shown in Tables 1 and 2, respectively. The mice were immunized subcutaneously using plasmids DNA to express FMDV type O/IRN/1/2007VP1 and GMCSF genes and showed significant differences compared with the negative control groups (the groups which were immunized by PBs, pcDNA3.1+ and PCMV-SPORT-GMCSF) (P < 0.01).
Table 1. The anti FMDV type O/IRN/1/2007 sera titration, MTT assay, IFN-γ, IL-4 and IL-10 concentrations of the vaccinated mice 10 days after last vaccination
Table 2. The anti FMDV type O/IRN/1/2007 sera titration, MTT assay, IFN-γ, IL-4 and IL-10 concentrations of the vaccinated mice 70 days after last vaccination
The results of Tables 1 and 2 indicate that both the mice immunized using co-administered DNA Vaccine with pCMV-SPORT-GMCSF vector and immunized with the inactivated vaccine show protective neutralizing antibody titer against FMDV type O/IRN/1/2007.However the response of vaccinated mice with PBS, pcDNA3.1+, PCMV-SPORT-GMCSF vector and DNA Vaccine were not protective.
-
The results of the MTT assay, IFN-γ, IL-4 and IL-10 concentrations in six groups of mice 10 days after last vaccination are shown in Table 1. Table 2 shows the results of the MTT assay and IFN-γ concentration after 70 days. The SI values of the vaccinated mice groups; DNA Vaccine, DNA Vaccine with PCMV-SPORT-GMCSF vector and conventional vaccine, were significantly higher than control groups (PBS and pcDNA3.1+ vector) at 10 and 70 days after the last vaccination (P < 0.05). The concentration of IFN-γ in group 5 (DNA Vaccine with PCMV-SPORT-GMCSF vector) was significantly higher than the other groups. Also, the IL-4 and IL-10 concentrations in group 6 were significantly higher than the other groups (P < 0.05).