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Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine. The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level., (Copyright © 2019. Published by Elsevier Ltd.)

The existence of a resident population of intrahepatic immune cells (IHICs) is well documented for mammalian vertebrates, however, it is uncertain whether IHICs are present in the liver of teleostean fish. In the present study we investigated whether trout liver contains an IHIC population, and if so, what the relative cellular composition of this population is. The results provide clear evidence for the existence of an IHIC population in trout liver, which constitutes 15-29% of the non-hepatocytes in the liver, and with a cellular composition different to that of the blood leukocyte population. We also analyzed the response of IHICs to a non-infectious liver challenge with the hepatotoxic and immunotoxic chemical, benzo[a]pyrene (BaP). Juvenile trout were treated with BaP (25 or 100mg/kgbw) at levels sufficient to induce the molecular pathway of BaP metabolism while not causing pathological and inflammatory liver changes. The IHIC population responded to the BaP treatments in a way that differed from the responses of the leukocyte populations in trout blood and spleen, suggesting that IHICs are an independently regulated immune cell population., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In mammals, CCR7 is the chemokine receptor for the CCL19 and CCL21 chemokines, molecules with a major role in the recruitment of lymphocytes to lymph nodes and Peyer's patches in the intestinal mucosa, especially naïve T lymphocytes. In the current work, we have identified a CCR7 orthologue in rainbow trout (Oncorhynchus mykiss) that shares many of the conserved features of mammalian CCR7. The receptor is constitutively transcribed in the gills, hindgut, spleen, thymus and gonad. When leukocyte populations were isolated, IgM(+) cells, T cells and myeloid cells from head kidney transcribed the CCR7 gene. In blood, both IgM(+) and IgT(+) B cells and myeloid cells but not T lymphocytes were transcribing CCR7, whereas in the spleen, CCR7 mRNA expression was strongly detected in T lymphocytes. In response to infection with viral hemorrhagic septicemia virus (VHSV), CCR7 transcription was down-regulated in spleen and head kidney upon intraperitoneal infection, whereas upon bath infection, CCR7 was up-regulated in gills but remained undetected in the fin bases, the main site of virus entry. Concerning its regulation in the intestinal mucosa, the ex vivo stimulation of hindgut segments with Poly I:C or inactivated bacteria significantly increased CCR7 transcription, while in the context of an infection with Ceratomyxa shasta, the levels of transcription of CCR7 in both IgM(+) and IgT(+) cells from the gut were dramatically increased. All these data suggest that CCR7 plays an important role in lymphocyte trafficking during rainbow trout infections, in which CCR7 appears to be implicated in the recruitment of B lymphocytes into the gut., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

CD8, belonging to the TCR complex, is the main marker molecule of CTLs. Although CD8 genes have been detected in many fish species, the analysis of teleost CD8+ cells has been limited because of the lack of antibodies. Using newly established mAbs against rainbow trout CD8α, we found high ratios of CD8α+ cells in trout thymus, gill and intestine, but relatively low abundance in pronephros, spleen and blood. Accordingly, tissue sections revealed many CD8α+ cells in thymus, numerous intra- and subepithelial CD8α+ cells in intestine and gill and few scattered CD8α+ cells in spleen and pronephros. In secondary lymphoid tissues, CD8α+ lymphocytes, which did not react with anti-thrombocyte or anti-IgM mAbs, expressed CD8α, CD8β and TCRα, while Ig and CD4 transcripts were found in CD8α⁻ lymphocytes. In contrast, considerable CD4 expression in CD8α+ thymocytes suggests the presence of double-positive early T cells. Highly expressed TCRγ, LAG3 and CTLA4 in CD8α+ lymphocytes imply that they constitute a heterogeneous population different from found in non-mucosal tissues. PHA stimulation resulted in an up-regulation of CTL effector genes (perforin, granulysin and IFN-γ) in CD8α+ pronephrocytes, while both Th1 (IFN-γ) and Th2 (IL-4/13A) cytokines were up-regulated in CD8α⁻ pronephrocytes. Although the basic characteristics of CD8α+ lymphocytes seem similar in teleost and mammals, features such as the low proportion of teleost CD8α+ lymphocytes in blood and their high abundance in respiratory tissue reveal a unique dynamics and distribution., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the European Union Viral Haemorrhagic Septicaemia (VHS) eradication is still based on stamping out. Due to the lack of effective low cost vaccines immune prophylaxis is currently not used to combat VHS. This paper describes a new oral delivery method for immunisation of trout with attenuated virus. The vaccine consists of lyophilised virus surrounded by polyethylene glycol (PEG) and was extruded under low temperature. In the stomach of trout, the use of additional neutralising and adsorbing bases resulted in a neutral pH around the vaccine pellets, thus protecting the antigen against gastric acid. The in vivo efficacy of this delivery method was examined in three animal challenge experiments using an attenuated VHS virus (VHSV) strain as a vaccine. After vaccination, VHSV mRNA in gut, heart, kidney, spleen and blood was amplified by semi-nested PCR after RT-PCR. Indirect immune fluorescence test detected VHS vaccine virus in the gut. The expression of MHC class II, CD4 and CD8alpha mRNAs after oral vaccination was measured in gut using real-time RT-PCR. Antibody levels were measured by ELISA one week before vaccination and five weeks after vaccination. Animals were challenged six weeks after vaccination with highly virulent VHSV and mortality was recorded. The experiments showed that orally delivered vaccine virus was released from the vaccine preparation, penetrated the gut mucosa and led to higher expression levels of MHC class II and CD4 mRNAs when compared to control guts. VHSV antibodies were detected after oral vaccination. Immunisation with this new vaccine formulation was followed by a significant protection against VHSV. While the cumulative mortality in the non-vaccinated control group reached 70%, more than 75% of the orally vaccinated fish were protected upon challenge.

To identify viral proteins that induce cell-mediated cytotoxicity (CMC) against viral hemorrhagic septicemia virus (VHSV)-infected cells, rainbow trout were immunized with DNA vectors encoding the glycoprotein G or the nucleocapsid protein N of VHSV. The G protein was a more potent trigger of cytotoxic cells than the N protein. Peripheral blood leukocytes (PBL) isolated from trout immunized against the G protein killed both VHSV-infected MHC class I matched (RTG-2) and VHSV-infected xenogeneic (EPC) target cells, suggesting the involvement of both cytotoxic T lymphocytes (CTL) and NK cells, respectively. In contrast, PBL from trout that were immunized against the N protein only killed VHSV-infected RTG-2 cells, indicating that this protein only elicits a CTL response. Further, a significant killing capacity of these PBL was only observed during summer months. PBL from fish that were immunized against the VHSV G protein significantly killed VHSV-infected but not infectious hematopoietic necrosis virus (IHNV)-infected targets indicating antigen specificity. Thus, this is the first report on cytotoxic immune responses after DNA vaccination in fish. Furthermore, cells isolated from the inflamed site of DNA injection were stained and transferred to isogeneic DNA-vaccinated recipients. Most of the stained donor leukocytes accumulated at the recipients' DNA injection site showing, for the first time, leukocyte homing in fish. Transferred donor leukocytes mainly migrated to the homologous vaccine injection site rather than to injection sites of heterologous vaccines, suggesting the antigen specificity of homing. By demonstrating CMC responses to distinct viral proteins and homing in rainbow trout, these results substantially contribute to the understanding of the teleost immune system.

Structure and function of factors contributing to the innate immune system of lower vertebrates, including fish are only sparsely characterized. We retrieved with RT-PCR cDNA copies of two closely related Toll-like receptors (TLR) from liver RNA of the rainbow trout (Oncorhynchus mykiss). The cDNA sequences are homologous to 95.6%. The phylogenetic analysis of their deduced amino acid sequences places these twin factors closely to other known TLRs from fish. The twin factors are equally expressed in all tissues analysed, most abundantly in spleen and head kidney and lowest in adipose tissue. Formalin-inactivated Aeromonas salmonicida pathogens induce their expression up to eight-fold in vitro in peripheral blood lymphocytes and in tissues from spleen and head kidney. Our sequence information will be useful to establish expression constructs for these factors necessary to analyse the pathogen specific signal transduction activating the innate immune defence in fish.

The functional relationship between fish and mammalian thrombocytes is relatively unknown. In this study, a panel of monoclonal antibodies (mAbs) was used to investigate the functional properties of rainbow trout thrombocytes. The mAbs recognize cell-surface molecules on thrombocytes with molecular weights ranging from 17 to 160 kDa. Flow cytometric and immuno-electron microscopic analyses demonstrate that these molecules are expressed at different levels and that surface expression increased upon activation with bovine collagen. Two of these cell-surface molecules (17 and 21 kDa) were directly involved in collagen-induced aggregation of thrombocytes since aggregation was blocked upon pre-treatment with mAbs that recognize the two surface markers. Interestingly, the percentage of thrombocytes in blood increased after stimulation using different antigens. The transcriptional profile of trout thrombocytes was then examined after immuno-magnetic enrichment using the described mAbs to assess potential roles of trout thrombocytes in immune functions. Trout thrombocytes express components of the MHC class Ia pathway, IL1beta, TNFalpha, TGFbeta, the interleukin receptor common gamma chain as well as CXC and CC chemokines. MHC class IIB and TNFalpha were expressed at low levels in resting thrombocytes. No evidence was found for the expression of TCRalphabeta, Ig heavy chain, CD8alpha or CK1 mRNA. Taken together, these results suggest that rainbow trout thrombocytes express molecules involved in activation, aggregation and genes encoding proteins, that are involved in antigen presentation and immune regulation.

Rainbow trout surface-(s)IgM(-) leukocytes exhibited cell-mediated cytotoxicity (CMC) against allogeneic cells. This is described in concordance with a characterization of gene expression in the effector cells. Peripheral blood leukocytes (PBL) isolated from trout grafted with allogeneic tissue lysed allogeneic target cells (erythrocytes or cells of the RTG-2 cell line) in in vitro assays. The PBL were magnetically separated into different subpopulations using monoclonal antibodies (mabs) specific to thrombocytes, IgM, granulocytes and monocytes. Of the isolated subpopulations only the sIgM(-) lymphocytes were capable of lysing allogeneic targets. The separated PBL fractions were characterized by RT-PCR analysis using specific primers for the amplification of trout IgM heavy chain constant region (CH1), T cell receptor alpha chain (TCRalpha), CD8alpha and major histocompatibility complex (MHC) class I gene fragments. Most importantly, CD8alpha was expressed only by the sIgM(-) population. Combined with the requirement for sensitization to detect CMC, this strongly suggests T cell involvement in fish as in higher vertebrates. The involvement of CD8alpha-positive cytotoxic T cells in allograft rejection was supported by additional in vivo and in vitro observations. CD8alpha expression was barely detectable in the blood of unsensitized trout or trout that received xenografts, but was easily detected in the blood of allogeneically stimulated trout. Furthermore, CD8alpha expression in sIgM(-) lymphocytes from immunized trout was secondarily enhanced by addition of allogeneic targets in vitro. Collectively, these functional and genetic data suggest that fish possess specific cytotoxic cells with phenotype and gene expression pattern similar to those of cytotoxic T cells in higher vertebrates.