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Cytokines have the potential as adjuvants for the application of vaccines in mammals. However, the adjuvant potential of teleost cytokines was limited. In the present work, the adjuvant effects of four recombinant cytokines including IL-1β, IL-8, TNF-α and G-CSF on E. tarda subunit vaccine rOmpV were comparatively investigated in flounder ( Paralichthys olivaceus ). Compared with control, the levels of specific serum antibodies and IgM + B lymphocytes were significantly enhanced by rIL-1β, rIL-8 and rG-CSF, whereas rIL-1β and rIL-8 induced significantly higher levels than rG-CSF. All four cytokines enhanced the expression of genes involved in humoral and/or cellular immunities, whereas rIL-1β and rIL-8 induced highest levels of genes involved in humoral immunities and cellular immunities, respectively. Compared to the relative percent survivals (RPS) of control group (40%) and rOmpV plus rG-CSF group (54%), rOmpV plus rIL-1β or rIL-8 produced higher RPS of 75% and 68%, respectively. Our results indicated that rIL-1β and rIL-8 are promising adjuvants for subunit vaccines against E. tarda . [ABSTRACT FROM AUTHOR]

In our previous study, we cloned and explored the biological functions of flounder (Paralichthys olivaceus) interleukin-2 (poIL-2), and showed that poIL-2 might have adjuvant potential for fish vaccines. In this study, the adjuvant effects of recombinant and molecular forms of poIL-2 (rIL-2 and pcIL-2) were comparatively analyzed and evaluated in flounder from several aspects by co-vaccination with the recombinant E. tarda OmpV (rOmpV). The results showed that co-vaccination with rOmpV plus rIL-2 or pcIL-2 resulted in a relative percent survival of 71% and 57% respectively, which was significantly higher than the control groups, rOmpV plus rHis (40%) or pcN3 (36%). Immunological analysis showed that: (1) the levels of specific serum antibodies and sIg + lymphocytes in head kidney, spleen and peripheral blood induced by rOmpV plus rIL-2 or pcIL-2 were significantly higher than that in the two control groups; (2) Compared to the two control groups, CD4-1, CD4-2, CD8α, CD8β, MHCIα, MHCIIα, IgM and IFN-γ mRNA levels were also significantly induced by rOmpV plus rIL-2 or pcIL-2; (3) the rOmpV plus rIL-2 could induce higher levels of sIg + lymphocytes, specific serum antibodies and the expressions of all investigated genes than rOmpV plus pcIL-2. These results demonstrated that co-vaccination with rOmpV with rIL-2 or pcIL-2 could induce stronger humoral and cellular immune responses, and evoked higher immune protective efficacy against E. tarda infection, suggesting that poIL-2 could be served as a promising candidate adjuvant and have a potential application in the control of flounder diseases. • Recombinant and plasmid forms of flounder IL-2 (rIL-2 and pcIL-2) were constructed as adjuvant candidates. • rIL-2 and pcIL-2 enhanced both the humoral and cellular responses and evoked higher protection against E. tarda infection. • The immune response and protection efficacy enhanced by rIL-2 was higher than pcIL-2. [ABSTRACT FROM AUTHOR]

Outer membrane protein (Omp) K, OmpR and VAA have been identified with good immunogenicity from Vibrio anguillarum , and their recombinant proteins showed variable relative percent survival (RPS) in previous study. In order to develop effective bivalent vaccine candidates, recombinant (r) VAA + rOmpK (AK), rVAA + rOmpR (AR), rOmpK + rOmpR (KR) among VAA, OmpK and OmpR, or formalin-killed cells (FKC) of V. anguillarum were immunized in flounder, respectively. Results revealed that AK, AR, KR and FKC could induce the proliferation of surface membrane immunoglobulin-positive B lymphocytes or CD3 + T lymphocytes in peripheral blood lymphocytes, and significantly enhance the total antibodies, specific antibodies and immune-related gene than those of control group. AK, AR, KR or FKC showed RPS of 74.92%, 78.49%, 82.09% and 56.99%, respectively. These results indicated that three bivalent vaccines AK, AR and KR could induce strong cellular and humoral immunity, and had high protection against V. anguillarum infection in flounders. [ABSTRACT FROM AUTHOR]

In our previous study, the differentially expressed proteins have been identified by proteomic analysis in total haemocytes of shrimp ( Fenneropenaeus chinensis ) after white spot syndrome virus (WSSV) infection. To further investigate the differential response of haemocyte subpopulations to WSSV infection, granulocytes and hyalinocytes were separated from healthy and WSSV-infected shrimp by immunomagnetic bead (IMB) method, respectively. Then two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) were used to analyze the differentially expressed proteins in haemocyte subpopulations between healthy and WSSV-infected shrimp. The results of flow cytometry (FCM) showed that about 98% of granulocytes and about 96% of hyalinocytes in purity were obtained. Quantitative intensity analysis revealed that 26 protein spots in granulocytes and 24 spots in hyalinocytes were significantly changed post WSSV infection. Among them, 24 proteins in granulocytes and 23 proteins in hyalinocytes were identified by MS analysis, which could be divided into eight categories according to Gene Ontology. The identification of prophenoloxidase (proPO), proPO 2 and peroxiredoxin in WSSV-infected granulocytes was consistent with the facts that the proPO-activating system and peroxiredoxin were mainly existed in granulocytes. The phagocytosis of hyalinocytes seemed to be enhanced during the infection, because several proteins that involved in phagocytosis, including clathrin heavy chain, ADP ribosylation factor 4 and Alpha2 macroglobulin were up-regulated in hyalinocytes upon WSSV infection. Our results also reflected the vital biological significance of calcium ion binding proteins in granulocytes and ATPase/GTPase in hyalinocytes during WSSV infection. The data in this study verified the roles of granulocytes and hyalinocytes involved in WSSV infection, and differentially expressed proteins identified in granulocytes and hyalinocytes had a close correlation with their function characteristics. [ABSTRACT FROM AUTHOR]

Cytokines, a class of small molecular proteins with a wide range of biological activities, are secreted mainly by immune cells and function by binding to the corresponding receptors to regulate cell growth, differentiation and effects. CD4+ T cells can be defined into different lineages based on the unique set of signature cytokines and transcription factors, including helper T cells (Th1, Th2, Th17) and regulatory T cells (Treg). In teleost, CD4+ T cells have been identified in a variety of fish species, thought to play roles as Th cells, and shown to be involved in the immune response following specific antigen stimulation. With the update of sequencing technologies, a variety of cytokines and transcription factors capable of characterizing CD4+ T cell subsets also have been described in fish, including hallmark cytokines such as IFN-γ, TNF-α, IL-4, IL-17, IL-10, TGF-β and unique transcription factors such as T-bet, GATA3, RORγt, and Foxp3. Hence, there is increasing evidence that the subpopulation of Th and Treg cells present in mammals may also exist in teleost fish. However, the differentiation, plasticity and precise roles of Th cell subsets in mammals remain controversial. Research on the identification and differentiation of fish Th cells is still in its infancy and requires more significant effort. Here we will review recent research advances in characterizing the differentiation of fish CD4+ T cells by cytokines and transcription factors, mainly including the identification of Th and Treg cell hallmark cytokines and transcription factors, the regulatory role of cytokines on Th cell differentiation, and the function of Th and Treg cells in the immune response. The primary purpose of this review is to deepen our understanding of cytokine networks in characterizing the differentiation of CD4+ T cells in teleost. • Teleost CD4+ T cells can be divided into four subpopulations, including Th1, Th2, Th17 and T regulatory (Treg) cells. • The cytokines and transcription factors in characterizing the differentiation of CD4+ T cells were reviewed in teleost fish. • The recent advances on the functions of Th and Treg cells were reviewed in teleost fish. [ABSTRACT FROM AUTHOR]

A number of white spot syndrome virus (WSSV)-binding proteins have been identified previously in the hemocytes of Fenneropenaeus chinensis. In order to further investigate the differential WSSV-binding proteins in hemocyte subpopulations, granular hemocytes and hyalinocytes were sorted from WSSV-infected shrimp by immunomagnetic bead (IMB) method. The results of ELISA and immuno-dot blot assay showed that the WSSV-binding activity of granular hemocytes proteins was much stronger than that of hyalinocytes proteins. And the percentage of WSSV-positive granular hemocytes was significantly higher than that of hyalinocytes post WSSV infection, indicating that granular hemocytes were more susceptible to WSSV infection. Moreover, a total of 9 WSSV-binding proteins were successfully identified in granular hemocytes and hyalinocytes by two-dimensional virus overlay protein binding assay (2D-VOPBA) and MALDI-TOF MS analysis, of which 3 binding proteins (arginine kinase, protease 1 and transglutaminase) existing in both hyalinocytes and granular hemocytes and 6 proteins (F 1 ATP synthase β-chain, hnRNPs, GAPDH, RACK1, β-actin and cellular retinoic acid) detected only in granular hemocytes. Among these identified WSSV-binding proteins, the transglutaminase (TG) was further recombinantly expressed, and the recombinant TG could be bound with WSSV. Subsequently, quantitative real-time PCR analysis showed that differential expression levels of WSSV-binding proteins were observed in granular hemocytes and hyalinocytes. The results of this study revealed that the WSSV-binding proteins were differentially expressed in granular hemocytes and hyalinocytes, which provided a deeper insight into the interaction between WSSV and hemocyte subpopulations. • Granulocytes proteins exhibited higher WSSV-binding activity than that of hyalinocytes. • A total of 9 WSSV-binding proteins were identified in granulocytes and hyalinocytes. • 3 WSSV-binding proteins were identified in both granulocytes and hyalinocytes. • 6 WSSV-binding proteins were detected in only granulocytes. [ABSTRACT FROM AUTHOR]

IFN-γ plays a key role in T-cell activation and the establishment of the adaptive immune response, which has a potential as a cytokine adjuvant in the context of vaccination. In this study, we evaluated the immune adjuvant effects of two forms of flounder (Paralichthys olivaceus) IFN-γ, including pcDNA3.1-IFN-γ (pcIFN-γ) and recombinant IFN-γ (rIFN-γ), and comparatively analyzed the immune responses of flounder to E. tarda subunit vaccine rOmpV. The results showed that vaccination with rOmpV plus pcIFN-γ or rIFN-γ produced a relative percent survival of 57% and 71%, respectively, which were significantly higher than that of the control groups, rOmpV plus pcN3 (36%) or rHis (40%). Compared with the two control groups, vaccination with rOmpV plus pcIFN-γ or rIFN-γ could induce significantly higher levels of specific serum antibodies and sIg + lymphocytes in peripheral blood, spleen and head kidney, and significantly higher upregulated expressions of CD4-1, CD8α, IgM, MHC Ⅰα, MHC Ⅱα, IL-1β and TNF-α were also detected in rOmpV plus pcIFN-γ or rIFN-γ vaccinated fish. In addition, compared with pcIFN-γ, rOmpV co-vaccination with rIFN-γ elicited higher levels of sIg + lymphocytes, specific serum antibodies and several immune-related genes expressions in vaccinated flounder. These results demonstrated that rOmpV co-vaccination with rIFN-γ or pcIFN-γ could both boost the immune responses and evoke highly protective effects against E. tarda , indicating that flounder IFN-γ is a promising adjuvant candidate for fish vaccination via an injection administering route. • Flounder IFN-γ displayed strong adjuvant stimulatory activity. • The rIFN-γ and pcIFN-γ both can enhance the immune protective efficacy of subunit vaccines rOmpV. • Recombinant IFN-γ combined with rOmpV can elicit stronger immune response and confer higher RPS. [ABSTRACT FROM AUTHOR]

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. • Four bicistronic DNA plasmids were constructed successfully. • Plasmid chemokines enhanced the percentages of CD4-1+, CD4-2+ and sIgM+ lymphocytes. • Plasmid chemokines significantly increased the level of anti-VAA antibody. • Plasmid chemokines provided higher protection for flounder against challenge. • Plasmids CCL4 and CCL19 are promising adjuvants for VAA DNA vaccine. [ABSTRACT FROM AUTHOR]