T and B lymphocytes immune responses in flounder (Paralichthys olivaceus) induced by two forms of outer membrane protein K from Vibrio anguillarum: Subunit vaccine and DNA vaccine
Introduction
T and B lymphocytes are significant components of the immune system of fish, playing a critical role in the adaptive immune response (Scapigliati, 2013) and used as indexes for evaluating the immune response of the body after vaccination (Khanolkar et al., 2002). Recently, two distinct CD4 molecules (CD4-1 and CD4-2) and a CD8 molecule on the surface of teleost T lymphocytes have been identified (Castro et al., 2011; Laing and Hansen, 2011). CD4+ T lymphocytes mainly facilitate a secondary humoral immune response similar to that of mammalian T help cells (Kono and Korenaga, 2013; Yamaguchi et al., 2013; Somamoto et al., 2014a). CD8+ T lymphocytes are involved in antiviral adaptive immunity (Somamoto et al., 2014b). Previous studies have found that CD4+ T lymphocytes responded rapidly in flounder (Paralichthys olivaceus) after immunisation with subunit vaccine against Edwardsiella tarda, whereas CD8+ T lymphocytes varied dramatically after Hirame novirhabdovirus infection (Liu et al., 2017a; Xing et al., 2018a). Cyclosporine A (CsA), a cyclic polypeptide composed of 11 amino acids, is a lymphocyte immunosuppressant found in mammals (Zhang et al., 2013a). The inhibition on T lymphocytes by CsA has been verified in teleosts both in vitro and in vivo (Park et al., 2002; Xing et al., 2017c). The immune response of B lymphocytes has been shown to be inhibited after T lymphocytes were inhibited by CsA against a thymus-dependent antigen (Xing et al., 2017c Xing et al., 2019). These findings suggest that the CD4+ and CD8+ T lymphocytes may play different roles in the immune response of fish.
Vibrio anguillarum, a gram-negative and rod-shaped bacterium, imperils a wide range of fish species and causes huge economic losses in aquaculture (Myhre and Steen, 1977; Westerdahl et al., 1991; Mo et al., 2001; Sepulcre et al., 2007; Tang et al., 2008a). Various vaccines against V. anguillarum have been developed for disease prevention (Sun et al., 2011a; Hamod et al., 2012a; Zhang et al., 2013b; Kwon and Kang, 2016). Among these, outer membrane proteins (Omps) possess good immunogenicity and protective efficacy and are considered vaccine candidates (Lin et al., 2002 Vazquez-Juarez et al., 2004 Ellis and Kuehn, 2010). Previous studies have shown that OmpK recombinant protein and plasmid DNA of V. anguillarum (rOmpK and pOmpK) induced the production of specific antibodies, with a relative percent survival of 62.16 % and 50.00 % in flounder, respectively (Xing et al., 2017a; Xu et al., 2019). Studies in mammalian models have shown that subunit vaccines mainly provoke humoral immunity, whereas both humoral and cellular immunities are aroused by DNA vaccines (Coban et al., 2008). However, due to the absence of specific antibodies against lymphocytes, the cellular immune response to vaccines has only been evaluated at the genetic level (Munang’andu et al., 2013; Liu et al., 2016, 2017b; Ahmadivand et al., 2018).
In our previous studies, specific antibodies against flounder CD3, CD4-1, CD4-2, and CD8 were produced and different subpopulations of T lymphocytes were identified (Tang et al., 2017a; Xing et al., 2017b). A monoclonal antibody against flounder immunoglobulin M (IgM) was developed to analyse B lymphocytes and specific antibodies (Li et al., 2007). In the present study, rOmpK and pOmpK plus CsA were immunised to flounder and the variations of CD4-1+, CD4-2+, and CD8+ T lymphocytes and IgM+ B lymphocytes and specific antibody production were investigated. The aim was to elucidate the immune response of T and B lymphocytes induced by two different types of vaccine.
Section snippets
Fish
Healthy flounder with a mean body weight of 35 g were obtained from a marine farm (Rizhao, Shandong, China) and maintained in tanks with aerated seawater at 21 ± 1 °C and fed commercial dry food pellets daily. Prior to the vaccination experiments, the fish were acclimated to laboratory conditions for 2 weeks and then samples were randomly taken from the liver, kidney, and spleen to confirm for V. anguillarum negative detected by 16S DNA.
The study was conducted following the procedures in the
Gating strategy for FCM
After being labelled with FIgM-Mab, FCD4-1-Pab, FCD4-2-Pab, or FCD8β-Pab, the leukocytes from the peripheral blood, spleen, and head kidney were analysed using FCM. The cell granularity and size were indicated by side-scatter (SSC) and forward-scatter (FSC) parameters, respectively, and the major cell populations containing 10 000 cells were gated to analyse the positive percentage of lymphocytes. FITC- and Alexa Fluor® 647-labelled cells were measured with fluorescent light (FL)-1 and FL-4,
Discussion
In the present study, the indexes of CD4-1+, CD4-2+, CD8β+ T lymphocytes subpopulations at the cell level were added to the recombinant subunit vaccine evaluation system for the first time. Pooling lymphocytes from different individuals could lead to autoreactivity; therefore, several measures were taken to ensure the authenticity and reliability of our data. For example, lymphocytes were disposed with anticoagulants and PBS to remove cytokines and other immune factors, and the same treatment
Authors statement
Jing Xing, Zhiqi Zhang and Keke Luo contributed to the conception and design of this study, performed most of experiments and statistical analysis, drafted and revised the manuscript. Xiaoqian Tang and Xiuzhen Sheng participated in the design of the study, helped analyzed experiments and data. Wenbin Zhan designed the study, edited the manuscript, and provided reagentsand experiment space. All the authors read and approved this version of the final manuscript and confirm the integrity of this
Acknowledgements
This work was funded by the National Key Research and Development Program of China (2018YFD0900503), the National Natural Science Foundation of China (31730101; 31672684 and 31672685), the Fundamental Research Funds for the Central Universities (201822015), the Director Foundation of Functional Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology (2018MFSD-01), NBRPC (2012CB114406), Key Research and Development
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