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Contents lists available atScienceDirectFish and Shellfish Immunologyjournal length articleVaccination of carp against SVCV with an oral DNA vaccine or an insectcells-based subunit vaccineC.W.E. Embregtsa, D. Rigaudeaub, L. Tacchia, G.P. Pijlmanc, L. Kampersa,c, T. Veselýd,D. Pokorovád, P. Boudinote, G.F. Wiegertjesa, M. Forlenzaa,aCell Biology and Immunology Group, Wageningen University, The NetherlandsbINRA, Infectiologie Expérimentale Rongeurs Poissons, Université Paris-Saclay, Jouy-en-Josas, FrancecLaboratory of Virology, Wageningen University, The NetherlandsdVeterinary Research Institute, Brno, Czech RepubliceINRA, Virologie et Immunologie Moléculaires, Université Paris-Saclay, Jouy-en-Josas, FranceARTICLE INFOKeywords:SVCV glycoproteinAlginate encapsulationInsect cellsBaculovirusDNA vaccineABSTRACTWe recently reported on a successful vaccine for carp against SVCV based on the intramuscular injection of aDNA plasmid encoding the SVCV glycoprotein (SVCV-G). This shows that the intramuscular (i.m.) route ofvaccination is suitable to trigger protective responses against SVCV, and that the SVCV G-protein is a suitablevaccine antigen. Yet, despite the general success of DNA vaccines, especially againstfish rhabdoviruses, theirpractical implementation still faces legislative as well as consumer's acceptance concerns. Furthermore, the i.m.route of plasmid administration is not easily combined with most of the current vaccination regimes largelybased on intraperitoneal or immersion vaccination. For this reason, in the current study we evaluated possiblealternatives to a DNA-based i.m. injectable vaccine using the SVCV-G protein as the vaccine antigen. To this end,we tested two parallel approaches: thefirst based on the optimization of an alginate encapsulation method fororal delivery of DNA and protein antigens; the second based on the baculovirus recombinant expression oftransmembrane SVCV-G protein in insect cells, administered as whole-cell subunit vaccine through the oral andinjection route. In addition, in the case of the oral DNA vaccine, we also investigated the potential benefits of themucosal adjuvantsEscherichia colilymphotoxin subunit B (LTB). Despite the use of various vaccine types, doses,regimes, and administration routes, no protection was observed, contrary to the full protection obtained with ourreference i.m. DNA vaccine. The limited protection observed under the various conditions used in this study, thenature of the host, of the pathogen, the type of vaccine and encapsulation method, will therefore be discussed indetails to provide an outlook for future vaccination strategies against SVCV.1. IntroductionSpring Viremia of Carp Virus (SVCV) is a cytopathic virus belongingto the genusSprivivirusof the familyRhabdoviridaeand is one of themain viruses affecting carp production. Outbreaks occur mainly duringSpring, causing an acute systemic infection in several cyprinid speciesleading to mortality rates of up to 90%, mainly in juvenilefish [ 2, 3].SVCV virions contain a single, linear, negative-sense, single-strandedRNA molecule, coding forfive structural proteins. The glycoprotein (G)is the only one present on the virion surface forming trimeric peplomersthat bind to cellular receptors to induce viral endocytosis. For thesereasons, the SVCV-G protein is the likely target of protective neu-tralizing antibodies [3, 9, 42].Recently we reported on an experimental DNA vaccine for Europeancommon carp (Cyprinus carpio carpio) against SVCV able to confer up to100% protection upon bath challenge with SVCV 2.5 months aftervaccination [13]. This protection was achieved by a single i.m. injectionof 0.1μg DNA/g offish of a pcDNA3 vector encoding the SVCV-Gprotein. We also showed that besides the local inflammation triggeredat the site of injection, the DNA vaccine triggered the production ofserum neutralizing antibodies and that SVCV-specific T cells were de-tectable in the blood of vaccinatedfish for at least 3 months aftervaccination. Thus, similar to the glycoprotein of otherfish rhabdo-viruses (i.e. viral haemorrhagic septicaemia virus (VHSV) and in-fectious haematopoietic necrosis virus (IHNV)) (Reviewed in Ref. [26],the glycoprotein of SVCV is an excellent vaccine antigen, at least whenadministered through i.m. DNA vaccination.Although to date i.m or intraperitoneal (i.p.), injection vaccination 4 December 2017; Received in revised form 11 March 2018; Accepted 17 March 2018Corresponding author. Wageningen University and Research, De Elst 1, Wageningen, The Netherlands.E-mail Forlenza).Fish and Shellfish Immunology 85 (2019) 66–77Available online 19 March 20181050-4648/ © 2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (
is most effective, oral vaccination would be the ideal delivery methodfrom an animal welfare and handling costs' point of view, (reviewed byRef. [12]. However, owing to the limited efficacy of the current ex-perimental oral formulations, mass vaccination offish via the oral routeis not common practice. The difficulties in the development of effectiveoral vaccines are linked to the need to use relatively high vaccine doses,the necessity to protect the antigen against intestinal degradation, aswell as the challenges infinding optimal conditions to overcome oraltolerance (Reviewed in Refs. [12, 29, 30, 41]. To date, a strong interest inthe use of DNA-based or subunit vaccines also for oral delivery is cur-rently increasing. For example, oral delivery of alginate encapsulatedDNA vaccines against infectious haemorrhagic necrosis virus (IHNV) oragainst infectious pancreatic necrosis virus (IPNV) was shown to confervarious degrees of protection in brown trout (Salmo truttaL.) andrainbow trout (Oncorhynchus mykiss)[4,10], showing the potential oforal DNA vaccination offish against viruses. Interestingly, high pro-tection against IPNV was achieved not only after oral administration ofthe vaccine by oral gavage, but also after mixing the alginate-en-capsulated DNA vaccine [5], or the chitosan-triphosphate (CS-TPP)nanoparticles containing the DNA vaccine, in feed pellets [1]. Mostrecently, CLYNAV was thefirst DNA vaccine to receive a positive re-commendation for marketing authorization in the European Union forvaccination of salmon against Salmon Alphavirus 3 (SAV3) [11]. Al-though this is a major breakthrough in the European legislation, theimplementation of DNA vaccines in the daily practice is far from beingcomplete. Alternatives to DNA-based vaccines remain of interest andinclude inactivated pathogens or subunit vaccines.While successful oral vaccines against SVCV have not been reportedthus far, one study showed the potential of oral vaccination of commoncarp and koi carp (Cyprinus carpio koi) against SVCV and Koi HerpesVirus (KHV), using recombinantLactococcus plantarum(L. plantarum)expressing both the SVCV-G protein and the KHV-ORF81 protein [8].Based on the assessed potential of oral vaccination against SVCV, andon the efficacy of the SVCV-G-based i.m. DNA vaccine [13], in thecurrent study we used two parallel approaches to vaccinate carp againstSVCV: one based on the oral administration of the SVCV-G DNA vac-cine, and the other based on the use of the SVCV-G protein as subunitvaccine for i.m., i.p. or oral delivery. In thefirst approach we examinedthe efficacy of alginate microspheres in assuring intact delivery ofprotein antigens or DNA plasmid to the carp intestine. Next, we com-pared various vaccination regimes and antigen doses, either or not incombination with the potent mucosal adjuvantEscherichia colilym-photoxin-beta (LTB) [32]. Furthermore, we analysed local as well assystemicimmune responses, based on the expression analysis of im-mune-relevant genes and the distribution of Igm+B cells, mucosal Tcells, neutrophilic granulocytes and macrophages in the spleen andintestine of carp vaccinated orally with the SVCV-G DNA plasmid. Forthe second approach, we generated two recombinant Autographa cali-fornica multicapsid nucleopolyhedrovirus (AcMNPV) baculoviruses fortransmembrane SVCV-G expression in insect cells, based on the provenefficiency of this system to produce membrane-bound and solubleglycoproteins from other rhabdoviruses [14,21]. Indeed, analysis re-vealed high expression of SVCV-G protein on the membrane of re-combinant baculovirus-infected insect cells, which allowed us to usewhole-cell preparations as SVCV-G subunit vaccine, using various de-livery routes.Despite the various approaches adopted in this study with regard tovaccine design, dose, vaccination regime, the vaccines did not lead tosufficient protection. These factors, as well as the nature of the pa-thogen, of the host and of the encapsulation method will be discussed indetails with the aim to provide an outlook for future vaccination stra-tegies.2. Materials and methods2.1. AnimalsEuropean common carp (Cyrpinus carpio carpio) R3xR8, originatedfrom cross-breeding of the Hungarian R8 strain and the Polish R3 strain[22], were used in all experiments. In this study we will refer to carp asthe European common carp subspecies, unless stated otherwise. Carpwere bred in the Aquatic Research Facility Carus of the animal facilityat Wageningen University, the Netherlands. Carp eggs were either keptand raised at the local facility or transported to the Institut National dela Recherche Agronomique (INRA, Paris, France) for viral challengeexperiments. Carp were raised at 2023 °C in recirculating UV-treatedwater and fed pelleted carp food (Skretting, Nutreco) twice daily. Allanimals were handled in accordance with good animal practice as de-fined by the European Union guidelines for the handling of laboratoryanimals ( All vaccination and challenge studies were performedat INRA. All animal work at INRA was approved by the Direction of theVeterinary Services of Versailles and COMETHEA (authorizationnumber 7828, project authorization #27072016011318282761), aswell asfish facilities (authorization number B78-720). Animal work inWageningen University was approved by the local animal committee(DEC number 2015098).2.2. SVCVThe reference SVCV strain VR-1390 (isolate stock of the INRA la-boratory [6, 15]), was propagated in Epithelioma Papulosum Cyprinid(EPC) cells grown in Glasgow's modified Eagle's medium(GMEM)25mMHEPES (Eurobio), supplemented with 10% foetal calfserum (FCS; Eurobio), 1% tryptose phosphate broth (Eurobio), 2 mML-glutamine (PAA), 100μg/mL penicillin (Biovalley) and 100μg/mLstreptomycin (Biovalley). Virus titers were determined by the methodof Reed and Muench [35] and were given as plaque-forming units (pfu).2.3. Insect cellsSpodoptera frugiperda21 (Sf21) cells were used for the constructionof the recombinant baculoviruses and initial validation of the con-structs;S. frugiperda9 (Sf9) cells were used for the preparation of theSVCV-G subunit vaccine forin vivovaccination experiments.Sf21 cells were cultured in Grace's insect medium (Gibco) supple-mented with 10% foetal calf serum (FCS) (Gibco) and 10μg/mLGentamycin at 27 °C. Sf9 cells were cultured in Sf-900 II SFM (ThermoFisher) supplemented with 5% FCS and 10μg/mL Gentamycin. Forinfection of both cell lines, medium without addition of Gentamycinwas used.2.4. Construction of recombinant AcMnPV baculoviruses expressing SVCV-GThree recombinant AcMnPV baculoviruses were constructed: oneencoding the SVCV-G protein under the control of the polyhedrin (PH)promotor and the reporter gene greenfluorescent protein (GFP) underthe control of the p10 promotor (bAc-GFP-SVCV-G); the second en-coding the SVCV-G protein alone under the PH promotor (bAc-SVCV-G); and the third encoding the GFP protein alone under the p10 pro-motor (Fig. 1). The SVCV-G coding sequence was obtained by PstI-BamHI (NEB) digestion of the pcDNA3-SVCV-G vector [13, 43], fol-lowed by ligation in the PstI-BamHI restriction sites of the pFastBacDual-GFP/Polyhedrin vector (Invitrogen), thereby replacing the poly-hedrin gene. The pFastBac Dual vectors were then used to transformcompetent DH10Bac cells (Thermo Fisher) for subsequent bacmid iso-lation.For construction of bAc-SVCV-G, the SVCV G gene was amplifiedC.W.E. Embregts et al.Fish and Shellfish Immunology 85 (2019) 66–7767