Two bicistronic DNA vaccines against Vibrio anguillarum and the immune effects on flounder Paralichthys olivaceus

Abstract

Chemokines are cytokines that can promote the activation and migration of immune cells, and increase the recognition of antigen by antigen-presenting cells (APC). Previous studies showed that a DNA vaccine can induce humoral and cellular immune responses of fl ounder after immunization. To explore the improvement of chemokines on the effi ciency of OmpK vaccine, two bicistronic DNA candidate vaccines were constructed and the immune responses they induced in the fl ounder were investigated by reverse transcription polymerase chain reaction (RT-PCR), indirect immunofl uorescent assay (IFA), H&E staining, fl ow cytometry (FCM), and quantifi cational real-time polymerase chain reaction (qRT-PCR). pBudCE4.1 plasmid as an expression vector, bicistronic DNA vaccines encoding OmpK gene and CC-motif ligand 4 gene (p-OmpK-CCL4), or Ompk gene and CC-motif ligand 19 gene (p-OmpK-CCL19) were successfully constructed. The results showed that two bicistronic DNA vaccines expressed Ompk protein of Vibrio anguillarum and CCL4/ CCL19 proteins of fl ounder both in vitro and in vivo. After immunization, a large number of leucocytes in muscle were recruited at the injection site in treatment groups. The constructed vaccines induced signifi cant increases in CD4-1 + and CD4-2 + T lymphocytes, and sIgM + B lymphocytes in peripheral blood, spleen, and head kidney. The percentage of T lymphocytes peaked on the 14 th post-vaccination day whereas that of B lymphocytes peaked in the 6 th post-vaccination week. Moreover, the expression profi les of 10 immune-related genes increased in muscles around the injection site, spleen, and head kidney. After the challenge, p-OmpK-CCL4 and p-OmpK-CCL19 conferred a relative percentage survival (RPS) of 74.1% and 63.3%, respectively, higher than p-OmpK alone (40.8%). In conclusion, both CCL4 and CCL19 can improve the protection of p-OmpK via evoking local immune response and then humoral and cellular immunity. CCL4 and CCL19 will be potential molecular adjuvants for use in DNA vaccines.

Characterization of Co-Stimulatory Ligand CD80/86 and Its Effect as a Molecular Adjuvant on DNA Vaccine Against Vibrio anguillarum in Flounder (Paralichthys olivaceus)

“…To enhance the protective effect of OmpK as a DNA vaccine candidate, adjuvants were co-administered with the vaccine. The previous study has shown that two chemokines, CCL4 and CCL19, were inserted into the OmpK-based DNA vaccine ( 39 ) and provided RPS of 74.1% and 63.3%, respectively. Furthermore, the leukocytes were recruited to the injection site after immunization.…”

“…The rabbit against OmpK antibodies were produced by Li et al. ( 39 ). The secondary antibodies included Dylight 488 (Abbkine; diluted 1:1,000) Rabbit or mouse IgG, Dylight 649 (Abbkine; diluted 1:1,000) Rabbit or mouse IgG, alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (H + L) (Abbkine; diluted 1:5,000), and HRP-conjugated goat anti-mouse IgG (Abbkine; diluted 1:5,000).…”

Characterization of myeloperoxidase and its contribution to antimicrobial effect on extracellular traps in flounder (Paralichthys olivaceus)

“…Escherichia coli ( E. coli ) , Staphylococcus aureus ( S. aureus ), Edwardsiella tarda ( E. tarda ), Vibrio anguillarum ( V. anguillarum ), and hirame rhabdovirus (HIRRV) strain were previously isolated from diseased flounder and stored in our laborotory ( 23 , 24 ). Bacteria were cultured in Luria-Bertani (LB) medium up to an OD600 of 0.5 and harvested by centrifugation before use.…”

Strategies for Prevention and Control of Vibriosis in Asian Fish Culture

“…It was reported that a DNA vaccine based on the zinc metalloprotease EmpA already displayed gratifying results against V. anguillarum early in 2009 [ 65 ]. In recent years, several DNA and subunit vaccines based on V. anguillarum outer membrane proteins and their recombinant proteins have shown effective and long-lasting immunity [ 66 , 67 , 68 , 69 ]. It has also been proven in the study that particularly high protection against V. anguillarum infection exists when related proteins are formulated as bivalent vaccines [ 68 ].…”