The effects of CCL3, CCL4, CCL19 and CCL21 as molecular adjuvants on the immune response to VAA DNA vaccine in flounder (Paralichthys olivaceus)

The magnitude of the immune response induced by DNA vaccines depends on the amount and type of antigen-presenting cells attracted to the injection site. In our previous study, a DNA plasmid encoding the VAA gene of Vibrio anguillarum was constructed and shown to confer moderate protection against V. anguillarum challenge. To augment the protective efficacy of the VAA DNA vaccine and compare the adjuvant effects of CCL3, CCL4, CCL19 and CCL21, four bicistronic DNA plasmids containing the VAA gene of V. anguillarum together with the gene encoding the CCL3/CCL4/CCL19/CCL21 chemokines of flounder were successfully constructed and administered to fish, and the immune response of the animals and the enhancement of immunoprotection by the four chemokines were investigated. Vaccinated with pCCL3-VAA, pCCL4-VAA, pCCL19-VAA and pCCL21-VAA, flounder showed relative percent survivals of 62.16%, 83.78%, 78.38% and 72.97%, respectively, higher than the relative survival of flounder immunized with pVAA (40.54%). Compared with the pVAA group, the percentages of sIgM(+), CD4-1(+), and CD4-2(+) lymphocytes and the levels of specific antibodies increased in pCCL3-VAA, pCCL4-VAA, pCCL19-VAA and pCCL21-VAA injection groups; CCL4 and CCL19 induced significantly higher levels of these parameters than CCL3 and CCL21 did. The amount of V. anguillarum in liver, spleen and kidney of pCCL3-VAA-, pCCL4-VAA-, pCCL19-VAA- and pCCL21-VAA-immunized flounder after V. anguillarum challenge was reduced compared to that in the pVAA group. Moreover, the co-expression of CCL3/CCL4/CCL19/CCL21 up-regulated immune-related gene expression associated with the local immune response. Our results indicate that CCL4 and CCL19 are promising adjuvants for use in VAA DNA vaccine against V. anguillarum.