Characterization of a new chimeric marker vaccine candidate with a mutated antigenic E2-epitope.

A new chimeric pestivirus "CP7_E1E2alf_TLA", based on the infectious cDNA of bovine viral diarrhea virus (BVDV) strain CP7, was constructed. The substitution of BVDV E1 and E2 with the respective proteins of classical swine fever (CSF) strain Alfort 187 allows an optimal heterodimerization of E1 and E2 in the chimeric virus, which is beneficial for efficient and authentic virus assembly and growth. In addition, for implementation of E2-based marker diagnostics, the previously described antigenic CSFV-specific TAVSPTTLR epitope was exchanged with the corresponding E2-epitope of BVDV strain CP7. Recombinant virus CP7_E1E2alf_TLA displayed a growth defect, and was not reacting with monoclonal antibodies used in commercial E2 antibody blocking ELISAs. Therefore, efficacy as well as marker properties of CP7_E1E2alf_TLA were investigated in an animal experiment with both a high dose and a low dose vaccine preparation. All CP7_E1E2alf_TLA-vaccinated animals seroconverted until day 28 post-vaccination with neutralizing antibodies. Furthermore, at the day of challenge infection CP7_E1E2alf_TLA-immunized animals showed distinct lower ELISA values in a commercial CSFV E2 antibody test in comparison to the C-strain vaccinated controls. However, E2-ELISA reactivity as well as neutralizing titers were directly connected to the dosage used for vaccination, and only the low dose group had E2-ELISA values below threshold until challenge infection. Following challenge infection with highly virulent CSFV strain Koslov, all vaccinees were protected, however, short-term fever episodes and very limited CSFV genome detection with very low copy numbers could be observed. In conclusion, manipulation of the TAVSPTTLR-epitope within the tested chimeric virus resulted in an slightly reduced efficacy, but the E2 marker properties unexpectedly did not allow a clear differentiation of infected from vaccinated animals in some cases.

Cell-mediated immune responses in rainbow trout after DNA immunization against the viral hemorrhagic septicemia virus.

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.

Cell-mediated cytotoxicity in rainbow trout, Oncorhynchus mykiss, infected with viral haemorrhagic septicaemia virus.

Mammalian cytotoxic T cells as part of the adaptive immune system recognize virus-infected target cells by binding of their T-cell receptors (TCR) to classical MHC class I molecules loaded with viral peptides. Our previous studies have shown that the allele of the single dominant polymorphic classical MHC class I locus Onmy-UBA is identical in the rainbow trout clone C25 and in the permanent rainbow trout cell line RTG-2. This enabled us to develop an assay to measure antiviral cytotoxicity in rainbow trout using a system of MHC class I-matched effector and target cells. Peripheral blood leucocytes (PBL) isolated from low dose viral haemorrhagic septicaemia virus (VHSV)-infected rainbow trout killed MHC class I-matched and later also xenogeneic MHC class I-mismatched VHSV-infected cells. When compared to PBL from uninfected control fish PBL from infected fish showed a higher transcriptional level of the CD8alpha gene which is a typical marker for mammalian cytotoxic T cells. Concurrently, the expression of the natural killer cell enhancement factor (NKEF)-like gene was enhanced as measured by real-time RT - PCR. Taken together, these results suggest that both innate and adaptive cell-mediated immune responses represented by NK and cytotoxic T cells, respectively, are triggered after VHSV infection. PBL that were able to kill VHSV-infected MHC class I-mismatched xenogeneic cells were generated later during infection than PBL capable of lysing VHSV-infected MHC class I-matched targets. This is contradictory to the generally accepted rule that innate immune mechanisms represent the first line of defence after viral infections.

Cytotoxic activities of fish leucocytes.

Like mammalian leucocytes, white blood cells of fish are able to kill altered (e.g. virus-infected) and foreign (allogeneic or xenogeneic) cells. The existence of natural killer (NK)-like and specific cytotoxic cells in fish was first shown using allogeneic and xenogeneic effector/target cell systems. In addition to in vivo and ex vivo studies, very important contributions were made by in vitro analysis using a number of different long-term cytotoxic cell lines established from channel catfish. In mammals, specific cell-mediated cytotoxicity (CMC) as part of the adaptive immune response requires a number of key molecules expressed on effector leucocytes and target cells. CD8+ T lymphocytes kill infected cells only, if their antigen receptor (TCR) matches the MHC class I with bound peptide of the target cell. Expression patterns of the fish gene homologues for TCR, CD8 and MHC class I, as well as related genes, are in agreement with similar function. Convenient systems for the analysis of specific CMC have only recently become available for fish with the combination of clonal fish with syngeneic or allogeneic but MHC class I matching cell lines. It was demonstrated that both, NK- and cytotoxic T (Tc) cells are involved in the killing of virus infected MHC class I matching and mismatching target cells. Analysis of these lymphocyte subsets is only starting for fish. There is also evidence that the different viral proteins trigger different subsets of killer cells. This review further discusses findings on fish CMC with regard to temperature/seasons and ontogeny.

Adaptive cell-mediated cytotoxicity against allogeneic targets by CD8-positive lymphocytes of rainbow trout (Oncorhynchus mykiss).

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.