Immunogenic Enhancement Of Interleukin-2 Expression Plasmids For Canine Parvovirus VP2 DNA Vaccine

Canine parvovirus disease is an acute infectious disease caused by canine parvovirus(CPV). CPV is a member of parvoviridae, and was first found in 1978。CPV can cause haemorrhagic gastroenteritis and myocarditis in dogs. Canine parvovirus (CPV) VP2 protein is the major antigenic protein. It was extensively used as a DNA vaccine to stimulate the immune responses against CPV. To improve the VP2 DNA vaccine potency, we tested to use the canine interleukin-2 (cIL-2) gene as an adjuvant to enhance its immunogenicity. Briefly, the canine IL-2 cDNA fragments with stop codon and without stop codon were amplified from canine lymphocytes by RT-PCR, and then the cIL-2 genes were inserted into pcDNA3.1A eukaryotic expression vector to construct the non-fused and the Myc/His-fused cIL-2 expression vectors, pcDNA-cIL-2 and pcDNA-cIL-2/MH, respectively. To determine whether the expression vector can mediate cIL-2 secretory expression in eukaryotic cells, the pcDNA-cIL-2/MH plasmids were transfected into HEK293FT cells mediated by calcium phosphate. The mice were separately immunized with VP2 vector (pcDNA-CD5sp-VP2 constructed previously in our laboratory) and VP2/cIL-2 combined vectors (pcDNA3.1 vector as a negative control). After immunization, the serum levels of VP2 antibodies were measured by ELISA, the spleen lymphocyte proliferation response was measured by lymphocyte proliferation assay, and the interferon-γexpression activity of the mouse lymphocytes was measured by ELISA.The results showed that the amplified cIL-2 gene sequence was identical with that published in GenBank, and the recombinant cIL-2 could be secretively expressed in HEK293FT cells. Immunization results showed that the antibody level of the mice co-immunized with VP2 and cIL-2 vectors was up to 1:5120 at 35d after immunization, significantly higher than that immunized with VP2 vector alone (P <0.01). Lymphocyte proliferation assay showed that the lymphocyte stimulation index of the two groups of VP2 gene-immunized mice were significantly higher than that of the negative control group (P <0.01), moreover, the index of VP2/cIL-2 co-immunized mice was significantly higher than that of VP2 immunized mice (P<0.05). The IFN-γlevels in the medium of lymphocytes isolated from co-immunized mice was significantly higher than that of the lymphocytes from VP2-immunized and the negative control mice (P <0.01). In conclusion, IL-2 gene expression vector can significantly improve the CPV VP2 DNA vaccine immunogenecity.