Construction Of Porcine Circovirus Type 2 DNA Vaccine And Its Immune Efficacy In Mice

Porcine circovirus (PCV) belongs to to the viral family Circoviridae and to the genus Circovirus, which can be divided into two genotypes: PCV1 and PCV2. PCV2 has pathogenic, and closely related to postweaning multisystemic wasting syndrome (PMWS). Apart from PMWS, porcine dermatologic and nephropathy syndrome (PDNS), reproductive disorders, enteritis, piglets congenital tremor (PCT), and proliferative and necrotising pneumonia(PNP)and other diseases were linked to PCV2 infection. Besides original infection, it has been strongly suggested that PCV2 causes secondary immunodeficiency in pigs due to lymphopenia and severe damage to lymphoid tissues in the affected pigs. Secondary infection viral infectious diseases, such as porcine reproductive and respiratory syndrome (PRRS), swine influenza, Hog cholera (HC), and Pseudorabies (PR), exacerbate clinical signs of the disease in pigs. Therefore, PCV2 infection has been caused serious economic losses in the porcine industry worldwide. At home and abroad, there is no effective vaccines to prevent this disease. Thus, developing a vaccine to control this disease with higher efficacy and fewer side effects is highly desirable.Since gene immunization was found from 1990, the protective antigen genes of various viruses have been delivered into different animals and good results have been obtained. DNA vaccines now become the focus of the vaccine field. However, DNA vaccines induce animals to produce low antibody levels which partially protect animals from a lethal dose pathogen challenge. Therefore, it is an urgent need how to enhance the effect of immunization protection. Many studies showed that immunogenicity of an antigen could be enhanced by cytokines, including IL-2, IFN-γ, IL-6, and IL-18 etc.The pIRES bicistronic plasmid was constructed and identified previously, which was used to construct monocistronic and bicistronic DNA vaccines. This plasmid contained two MCS (A and B)which were inserted into the multiple cloning sites (MCS) located on either side of the internal ribosome entry site from the encephalomyocarditis virus (ECMV). A pair of the specific primers for amplifying ORF2 gene of PCV2 was designed and synthesized according to the pIRES bicistronic plasmid and nucleotide sequence of complete genome from PCV2 LY strain. ORF2 gene was amplified by PCR, and cloned into MCS A of pIRES between MluⅠand XhoⅠrestriction sites to generate the recombinant plasmid pIRES-ORF2. Two pairs of the specific primers for amplifying ORF2 gene of PCV2 were designed and synthesized according to the pIRES bicistronic plasmid, nucleotide sequence of porcine IL-18 (PIL-18) and porcine IL-2 (PIL-2). PIL-18 and PIL-2 genes were amplified by PCR, and cloned into MCS B of pIRES and pIRES-ORF2 between SalⅠand NotⅠrestriction sites, respectively, resulting in the recombinant plasmid pIRES-PIL18, pIRES-PIL2, pIRES-ORF2/PIL18, and pIRES-ORF2/PIL2.To evaluate the immune efficacy of DNA vaccines, six-week-old female Kunming mice were immunized intramuscularly into the quadriceps muscle with pIRES-ORF2, pIRES-PIL18, pIRES-ORF2/PIL18, pIRES-PIL2 and pIRES-ORF2/ PIL2, and boosted with an equivalent dose at 2 weeks after the initial inoculation. After the second immunization, peripheral blood lymphocytes were detected by flow cytometry and the sera were collected to examine antibodies of mice each week by ELISA. The mice were challenged with the virulent PCV2 LY strain in the first week after the second immunization.The results indicated that pIRES-ORF2, pIRES-ORF2/PIL18, and pIRES-ORF2/PIL2 could induce mice to produce specific humoral and cellular immune responses. The level of ELISA antibody and the percentage of CD3+,CD3+CD4+,CD3+CD8+ subgroups of peripheral blood in mice vaccinated with pIRES-ORF2/PIL18 and pIRES-ORF2/PIL2 were higher than those in mice immunized with pIRES-ORF2, pIRES-PIL18, pIRES-PIL2. Mice vaccinated with pIRES-ORF2/PIL18, and pIRES-ORF2/PIL2 were protected against challenge with PCV2. Therefore, co-expression of an adjuvant cytokine from a bicistronic DNA vaccine may be a potent strategy for enhancing the protective efficacy of vaccines against PCV2.