title: Ex vivo transfection of trout pronephros leukocytes, a model for cell culture screening of fish DNA vaccine candidates.
abstract: DNA vaccination opened a new era in controlling and preventing viral diseases since DNA vaccines have shown to be very efficacious where some conventional vaccines have failed, as it occurs in the case of the vaccines against fish novirhabdoviruses. However, there is a big lack of in vitro model assays with immune-related cells for preliminary screening of in vivo DNA vaccine candidates. In an attempt to solve this problem, rainbow trout pronephros cells in early primary culture were transfected with two plasmid DNA constructions, one encoding the green fluorescent protein (GFP) and another encoding the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV)) - the only viral antigen which has conferred in vivo protection. After assessing the presence of GFP- and G(VHSV)-expressing cells, at transcription and protein levels, the immune response in transfected pronephros cells was evaluated. At 24h post-transfection, G(VHSV) up-regulated migm and tcr transcripts expression, suggesting activation of B and T cells, as well, a high up-regulation of tnfα gene was observed. Seventy-two hours post-transfection, we detected the up-regulation of mx and tnfα genes transcripts and Mx protein which correlated with the induction of an anti-VHSV state. All together we have gathered evidence for successful transfection of pronephros cells with pAE6G, which correlates with in vivo protection results, and is less time-consuming and more rapid than in vivo assays. Therefore, this outcome opens the possibility to use pronephros cells in early primary culture for preliminary screening fish DNA vaccines as well as to further investigate the function that these cells perform in fish immune response orchestration after DNA immunisation.
authors: IBMC, Miguel Hernandez University, 03202-Elche, Spain.
authors: M
authors: M
authors: Ortega-Villaizan
authors: A
authors: A
authors: Martinez-Lopez
authors: P
authors: P
authors: Garcia-Valtanen
authors: V
authors: V
authors: Chico
authors: L
authors: L
authors: Perez
authors: J M
authors: JM
authors: Coll
authors: A
authors: A
authors: Estepa
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology; D018929:Cell Culture Techniques; D002460:Cell Line; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology; D031941:Hemorrhagic Septicemia, Viral / Q000276:immunology / Q000517:prevention & control*; D007962:Leukocytes / Q000276:immunology*; D029141:Novirhabdovirus / Q000472:pathogenicity; D010957:Plasmids / Q000235:genetics; D060910:Pronephros / Q000166:cytology* / Q000276:immunology; D017422:Sequence Analysis, DNA; D014162:Transfection; D014337:Trout / Q000276:immunology; D015854:Up-Regulation; D014611:Vaccination; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; C096089:G protein, viral hemorrhagic septicemia virus; D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; D049452:Green Fluorescent Proteins