title: Rational development of an attenuated recombinant cyprinid herpesvirus 3 vaccine using prokaryotic mutagenesis and in vivo bioluminescent imaging.
abstract: Cyprinid herpesvirus 3 (CyHV 3) is causing severe economic losses worldwide in common and koi carp industries, and a safe and efficacious attenuated vaccine compatible with mass vaccination is needed. We produced single deleted recombinants using prokaryotic mutagenesis. When producing a recombinant lacking open reading frame 134 (ORF134), we unexpectedly obtained a clone with additional deletion of ORF56 and ORF57. This triple deleted recombinant replicated efficiently in vitro and expressed an in vivo safety/efficacy profile compatible with use as an attenuated vaccine. To determine the role of the double ORF56-57 deletion in the phenotype and to improve further the quality of the vaccine candidate, a series of deleted recombinants was produced and tested in vivo. These experiments led to the selection of a double deleted recombinant lacking ORF56 and ORF57 as a vaccine candidate. The safety and efficacy of this strain were studied using an in vivo bioluminescent imaging system (IVIS), qPCR, and histopathological examination, which demonstrated that it enters fish via skin infection similar to the wild type strain. However, compared to the parental wild type strain, the vaccine candidate replicated at lower levels and spread less efficiently to secondary sites of infection. Transmission experiments allowing water contamination with or without additional physical contact between fish demonstrated that the vaccine candidate has a reduced ability to spread from vaccinated fish to naïve sentinel cohabitants. Finally, IVIS analyses demonstrated that the vaccine candidate induces a protective mucosal immune response at the portal of entry. Thus, the present study is the first to report the rational development of a recombinant attenuated vaccine against CyHV 3 for mass vaccination of carp. We also demonstrated the relevance of the CyHV 3 carp model for studying alloherpesvirus transmission and mucosal immunity in teleost skin.
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Maxime
authors.initials: M
authors.lastname: Boutier
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Maygane
authors.initials: M
authors.lastname: Ronsmans
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Ping
authors.initials: P
authors.lastname: Ouyang
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Fournier
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Anca
authors.initials: A
authors.lastname: Reschner
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Rakus
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Gavin S
authors.initials: GS
authors.lastname: Wilkie
authors.affiliation: Biostatistics and Bioinformatics, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Farnir
authors.affiliation: Pathology, Department of Morphology and Pathology, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Calixte
authors.initials: C
authors.lastname: Bayrou
authors.affiliation: Fish Pathology Lab, Department of Biotechnology, CER Groupe, Marloie, Belgium.
authors.forename: François
authors.initials: F
authors.lastname: Lieffrig
authors.affiliation: USDA-ARS-ADRU, Washington State University, Pullman, Pullman, Washington, United States of America.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Pathology, Department of Morphology and Pathology, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Daniel
authors.initials: D
authors.lastname: Desmecht
authors.affiliation: MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Davison
authors.affiliation: Immunology-Vaccinology, Department of Infectious and Parasitic Diseases, Fundamental and Applied Research for Animals & Health (FARAH), Faculty of Veterinary Medicine, University of Liège, Liège, Belgium.
authors.forename: Alain
authors.initials: A
authors.lastname: Vanderplasschen
mesh_terms: D000818:Animals; D002347:Carps; D005393:Fish Diseases / Q000276:immunology* / Q000821:virology; D006564:Herpesviridae / Q000235:genetics / Q000276:immunology*; D006566:Herpesviridae Infections / Q000276:immunology / Q000517:prevention & control / Q000662:veterinary*; D022283:Herpesvirus Vaccines / Q000009:adverse effects / Q000276:immunology*; D008163:Luminescent Measurements; D016366:Open Reading Frames / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014611:Vaccination / Q000379:methods; D014614:Vaccines, Synthetic / Q000009:adverse effects / Q000276:immunology*
chemical_list: D022283:Herpesvirus Vaccines; D012097:Repressor Proteins; D015534:Trans-Activators; D014614:Vaccines, Synthetic