The Screening Of CpG ODNs And Enhancing Efficacy On Inactived PR Vaccine In Minks

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs is a novel vaccine adjuvant. It can combine the Toll-like receptor 9(TLR-9) present in the ER membrane of immune cells, and stimulate the body to produce innate immune response. By promoting Th1-type immune responses and secreting proinflammatory cytokines to enhance the non-specific adaptive immune response, it plays a role of immunity protection against the invasion of pathogens and anti-tumor. CpG ODN is a vaccine adjuvant with the advantages of high efficience, safety and can be used with other adjuvants together. But its immune stimulating activity is complex and species specific and this specificity are related to its own molecular structures and the nucleotide sequences. Currently, effective CpG ODNs have been found in humans, pigs, cattle, chickens, dogs, cats and other animals, at the same time it was also researched deeply in human vaccines as an immune adjuvant. However the research developments of CpG ODN as an immune adjuvant in veterinary vaccines especially in fur animal vaccines are slow, and to date, the report about the effective CpG ODN in mink vaccine has not been published.In this study, twenty-five pieces of CpG ODNs refered to canine effective CpG motifs were designed and synthesized. The effective CpG ODNs were chosen through the mink peripheral blood mononuclear cells(PBMC) proliferation test. In the test, the CpG-23 was obtained by contrasting the CpG ODNs effects that stimulate mink PBMC proliferation and detecting the antiviral activity of the PBMC supernatants. After that, CpG-23 was verified as an adjuvant with pseudo-rabies inactivated vaccine. In the preliminary test, on the conditions of mink peripheral blood mononuclear cells of 4 × 105/well, CpG ODN working concentration of 10μg/ml and cultivated for 48 h with 37 ℃, 5% CO2, PBMC could be detected the most obvious proliferation by MTS assay. According to the above criteria, CpG ODNs were screened through the test of the stimulation proliferation of mink peripheral blood mononuclear cells(PBMC), then picked out CpG-2, CpG-7, CpG-10, CpG-21, CpG-23 and CpG-24(p<0.01) with obvious stimulation proliferation effect. Wherein the CpG-23, CpG-24 stimulation index(SI) were up to 14.874 and 14.261 respectively. In the following test, the mink PBMC supernatants respectively stimulated by CpG-23 and CpG-24 were used to co-infect mink lung(ML) cells with pseudo-rabies virus(PRV), only mink PBMC supernatants with CpG-23 was found that could inhibit PRV from infecting ML cells to a certain degree and showed some antiviral activity. Then 20μg, 40μg, 80μg of CpG-23 combined with pseudo-rabies inactivated vaccine were respectively used to immunize mink to observe the impact of CpG-23 to influence pseudo-rabies vaccine effect. In this experiment, 25, 2-month-old minks were randomly divided into five groups: PBS group, the vaccine group, 20μg CpG ODN combined with immunization group, 40μg CpG ODN combined with immunization group, 80μg CpG ODN combined immunization group.14 days later, second immunization with the first immunization dose were inoculated.7, 14, 21 and 28 days after the first immunization, mink blood of five groups were collected and blood serums were separated to detect serum PRV neutralizing antibody titers. The results showed that during the entire immune process, neutralizing antibody titers of CpG-23 with different dose were higher than the control vaccine group, meanwhile neutralizing antibody levels were rising along with the dose of CpG-23 increased. These results suggested that CpG-23 could effectively assist pseudo-rabies inactivated vaccine in producing higher levels of neutralizing antibodies in mink.In summary, in the whole study, the CpG-23 was demonstrated has good immunostimulatory activity in vitro and in vivo and it can be a candidate adjuvant for mink vaccine. This study laid the foundation of the use of CpG ODN in mink vaccine and provided experiment basis.