Fish and Shellfish Immunology 131 (2022) 1051–1062 Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi Full length article How modular protein nanoparticles may expand the ability of subunit anti-viral vaccines: The spring viremia carp virus (SVCV) case Mauricio Rojas-Peña a, 1, Patricia Aceituno a, 1, Maria E. Salvador c, Marlid Garcia-Ordoñez a, Mariana Teles a, b, Maria del Mar Ortega-Villaizan c, Luis Perez c, **, Nerea Roher a, b, d, * a Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain b Department of Cell Biology, Animal Physiology and Immunology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain c Instituto de Investigación, Desarrollo e Innovación en Biotecnología Sanitaria de Elche (IDiBE), Universidad Miguel Hernández (UMH), Elche, Spain d CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Spain A R T I C L E I N F O A B S T R A C T Keywords: Spring viremia of carp (SVC) remains as a vaccine orphan disease mostly affecting juvenile specimens. Young fish Protein nanoparticles are especially difficult to vaccinate and oral administration of vaccine combined with food would be the election SVCV system to minimise stress and the vaccination costs associated to injection. However, administration of pro­ Vaccine phylactics with food pellets faces off several drawbacks mainly related with vaccine degradation and weak IFNγ Zebrafish protection correlates of oral vaccines. Here we present a platform based on recombinant proteins (subunit vaccines) manufactured as highly resistant nanostructured materials, and providing excellent levels of protection against SVC virus in a preliminary i.p injection challenge. The G3 domain of SVCV glycoprotein G was over­ expressed in E. coli together with IFNγ and the modular protein was purified from bacterial aggregates (inclusion bodies) as highly organised nanostructured biomaterial (nanopellets, NP). These SVCV-IFNNP were taken up by zebrafish cells leading to the enhanced expression of different antiviral and IFN markers (e.g vig1, mx, lmp2 or ifngr1 among others) in zebrafish liver cells (ZFL). To monitor if SVCVNP and SVCV-IFNNP can be taken up by intestinal epithelia and can induce antiviral response we performed experiments with SVCVNP and SVCV-IFNNP in 3 days post fertilization (dpf) zebrafish larvae. Both, SVCVNP and SVCV-IFNNP were taken up and accumulated in the intestine without signs of toxicity. The antiviral response in larvae showed a different induction pattern: SVCV-IFNNP did not induce an antiviral response while SVCVNP showed a good antiviral induction. Interestingly ZF4, an embryonic derived cell line, showed an antiviral response like ZFL cells, although the lmp2 and ifngr1 (markers of the IFNγ response) were not overexpressed. Experiments with adult zebrafish indicated an excellent level of protection against a SVCV model infection where SVCV-IFNNP vaccinated fish reached 20% cumulative mortality while control fish reached over 80% cumulative mortality. 1. Introduction proteins—nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent polymerase (L). The G protein is Spring viremia of carp (SVC) is an infectious viral disease common to the most important viral antigenic protein determining the infectivity carp (C. carpio) and other cyprinid fish species (including zebrafish and serological properties of the virus [1]. (Danio rerio), koi (Cyprinus carpio koi), silver carp (Hypophthalmichthys Therapeutic or prophylactic approaches to combat SVCV have been molitrix), goldfish (Carassius auratus) or grass carp (Ctenopharyngodon ineffective and no commercial vaccine is currently available. The idella) among others, caused by the SVC virus (SVCV). SVCV is a member expansion of the geographical and host infection range of SVCV poses a of the family Rhabdoviridae, with negative-sense ssRNA encoding five major threat not only to the fish farms and ornamental fish industries, Abbreviations: IBs, Bacterial inclusion bodies; NPs, Nanopellets; SVCV, Spring Viremia of Carp Virus; iRFP, near-infrared fluorescent protein. * Corresponding author. Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain ** Corresponding author. E-mail addresses: luis.perez@umh.es (L. Perez), nerea.roher@uab.cat (N. Roher). 1 Both authors contributed equally. https://doi.org/10.1016/j.fsi.2022.10.067 Received 7 June 2022; Received in revised form 8 October 2022; Accepted 31 October 2022 Available online 9 November 2022 1050-4648/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 but also to wild fish populations [2]. SVC outbreaks can reach 90% of correlated with significant protection against SVCV challenge. mortality in juvenile fish and although there are no approved vaccines for SVC, inactivated, live-attenuated, DNA or other vaccines have been 2. Materials and methods tested in experimental trials [1,3,4]. However, attenuated vaccines pose a slight risk as there were very rare cases where attenuated strains may 2.1. Design, production, and characterization of nanostructured viral revert to pathogenic and may cause disease [5], and in the case of DNA antigenic proteins vaccines the security concerns are related to the use of DNA vectors. In this context, subunit vaccines overcome these security issues since do 2.1.1. Viral strains and plasmids not contain exogenous genetic material. Drawbacks of subunit vaccines Recombinant SVCV G protein was designed based on the glycopro­ are: a high susceptibility to degradation of the protein (labile system) tein G sequence (G3 region, AA 254–381, Uniprot acc. no: Q91DS0) and lack of pathogen associated molecular patterns (PAMPS) that the reported in Zhu et al., 2019 as the most antigenic [20]. Recombinant immune system utilizes to recognize pathogens via pattern recognition SVCV G antigen with the interferon gamma module (SVCV-IFN) was receptors (PRRs) thus needing adjuvants with co-stimulatory activity to based on the sequence of the G3 region (254–381) linked to the sequence enhance the magnitude of the immune response [5]. of the IFN gamma 1 from D. rerio (27–175) (NCBI GenBank In this study, we have designed a vaccination platform based on NP_998029.1). Clones were designed using the ORF and pET22b highly stable protein antigenic protein module (G protein fragment) removing the periplasmic location signal and including a C terminal with in-vaccine included adjuvant properties (IFNγ protein module). In polyHis-tag. Clones were codon optimized for expression in E. coli, 1957, the IFNs were originally described as molecules that “interfere” synthesized by GeneScript and subcloned into pET22b. Plasmids were with viral replication [6]. Since then, two types of IFNs, (type I and type transformed into E. coli BL21 (DE3) (Novagen). The red fluorescent II) have been characterized in several fish species. Within the fish type II protein (RFP), iRFPHis cloned into pET22b (Genscript), was used as a IFNs only two members, IFN-γ and IFN-γrel have been reported that non-relevant control protein. signal through CRFB13 and CRFB17 receptors (IFNGR1-2 and IFNGR1-1 respectively) [7]. IFN-γ and IFN-γrel differs on the number of N-glyco­ 2.2. Production of NPs, purification, quantification, and fluorescent sylation sites that would provide glycosylated IFN-γ with an extended labeling half-life [8]. Upon activation by the IFN-γ, the IFNGR1 and IFNGR2 recruit Janus Kinase (JAK) 1 and JAK2 to the cytosolic domain respec­ Methods for recombinant protein production and characterization tively, leading to phosphorylation and activation of STAT1 that migrates were previously reported [21]. Briefly, some modifications were intro­ into the nucleus and binds to the IFN-γ activation sites (GAS) and, ini­ duced for these particular constructs: E. coli were cultured overnight tiates the gene expression of IFN-γ dependent genes [9]. In zebrafish also (O/N) in LB medium (Gibco) with ampicillin (100 μg/ml, 2 isoforms of IFNγ (IFN-γ and IFNγ-rel) have been reported [8], however Sigma-Aldrich) and recombinant protein expression in E. coli was treatment of adult zebrafish with IFN-γrel failed to mediate resistance induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Pan­ against viral and bacterial infections [10]. We focused our attention on reac) when OD550 nm reached 0.5–0.8. Growth was carried out for 4,5 h IFNγ because in mammals is a highly versatile molecule with different at 37 ◦ C. For NPs purification, the cell cultures were subjected to an functions such as regulation of Th1 responses and clearance of intra­ enzymatic and mechanical disruption process [18]. Lysozyme (1,5 cellular pathogen, regulation of macrophage activation and function μg/ml, Roche), protease inhibitor cOmplete EDTA-free Tablets (Roche) [11,12] or promotion of antigen presentation to inhibit viral replication and phenyl-methanesulfonyl fluoride (PMSF, Roche) (0.4 mM) were among others [13]. In teleosts the expression of IFNγ is induced by added and incubated at 37 ◦ C for 2 h at 250 rpm. Cultures were then different virus including SVCV, VHSV, RGNNV, IHNV and also by syn­ frozen at − 80 ◦ C overnight (O/N). After thawing, Triton X-100 (Roche) thetic mimics such as Poly(I:C) [13]. At the same time IFNγ is able to was added (0.2% v/v) and stirred for 1 h at room temperature (RT). induce expression of antiviral genes and can inhibit virus replication at Then, DNase I (1 μg/ml) and MgSO4 (1 M) were added, and the cultures least in vitro [8,9]. IFNγ may also have antibacterial activity by were incubated for 1 h at 37 ◦ C with shaking. NPs were harvested by increasing NOS, ROS production and the phagocytic activity of macro­ centrifugation at 15,000×g, 15 min at 4 ◦ C and resuspended in lysis phages [8]. buffer (50 mM Tris HCl pH 8, 100 mM NaCl, 1 mM EDTA) at 1/10 the On the other hand, toxic effects of IFNγ in zebrafish larvae have been original culture volume. Finally, several freeze thaw cycles were per­ described probably associated to an excessive activation of inflamma­ formed to remove any remaining viable bacteria. Pellets of purified NPs, tion [14]. The zebrafish embryo is also able to fight viral infections, such named SVCVNP and SVCV-IFNNP were stored at -− 80 ◦ C until use. Pro­ as SVCV, snakehead rhabdovirus and nervous necrosis virus [15,16] and tein was semi-quantified by western blot using an anti-His-tag antibody during the early phase of viral infections, pathogens induce tlr3, tnfa, (Genscript A00186-100), and the protein concentration was calculated mxa-c, viperin [17] whereas bacterial infection led to the induction of using Image Lab 6.0.1 software (Bio-Rad). For flow cytometry or pro-inflammatory cytokines such as tnfa [17]. All together led us to confocal microscopy, NPs were conjugated with Atto-488 NHS ester speculate that inclusion of IFNγ as an in-vaccine adjuvant may poten­ (Sigma-Aldrich) according to the manufacturer’s instructions. Labeling tiate the performance of the novel recombinant vaccine system. efficiency was determined with the Nanodrop ND-1000 (Thermo Another important issue previously mentioned is the instability of Scientific). recombinant proteins when expressed and purified in its native form. To overcome this issue, we use recombinant protein purified from inclusion 2.3. Characterization of viral recombinant protein NPs bodies (IB). These IBs have a specific nanostructured conformation and contain a mixture of aggregate scaffolds and native protein, and are Electron Microscopy was used to determine the external morphology extremely resistant to harsh chemical and biological conditions [18]. and physical dimensions of the NPs. Samples were prepared by resus­ These structural features make our vaccination platform also fit very pending NPs at 100 μg/ml in mQ water, pipetting 20 μl onto silicon well with oral vaccination approaches, for those situations where oral chips, and air-dried O/N. NPs were exposed to different in vitro condi­ administration together with food pellets would be the choice in fish tions. For pH, NPs were incubated under gentle stirring at pH 2 and 10 vaccinology a non-stressful and inexpensive administration method is for 3 h together with a control sample. For temperature, NPs were highly desirable, and administration of vaccines mixed with feed would incubated for 5 min at 100 ◦ C. After incubation all NPs were centrifuged be the election for juveniles [19]. In the present study, a proof of concept at 10.000×g for 5 min at 4 ◦ C and resuspended at 10 and 50 μg/ml in of vaccination with recombinant nanostructured SVCV G attached to phosphate buffered saline (PBS, Sigma-Aldrich) and finally 20 μl were IFN, induced a strong innate immune response in zebrafish that deposited onto silicon chips, and air-dried O/N. Electron microscopy 1052 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 images were taken with a FESEM (Zeiss Merlin) and analyzed using Fiji system (Bio-Rad) using iTaq Universal SYBR Green Supermix kit (Bio- package (National Institute of Health, USA). At least 60 particles were Rad). Each PCR mixture consisted of 5 μl SYBR green supermix, 0.4 μM measured, and size distribution histograms were generated using Past specific primers, 2 μl diluted cDNA and 2.6 μl water (Sigma-Aldrich) in a 4.04 software (University of Oslo). final volume of 10 μl. The ef1-a gene was used as a reference gene. The dilution factor for all the genes tested was 1:10 (vig1, mx, ccl4, lmp2, 2.4. Cell culture and virus ifngr1, il1b, tnfa, il10, prf19b, gzma and ef1-α) (Supplementary Table 1). Amplification efficiencies for new primers lmp2 and ifngr1 were calcu­ Zebrafish ZFL cells (CRL-2643, ATCC) were cultured according to lated (Supplementary Table 1). All the samples (n = 6 per treatment) Thwaite et al. [21] at 28 ◦ C and 5% CO2 in DMEM + GlutaMAX (Gibco), were run in triplicate, and data were analyzed for individual replicates 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 0.01 mg/ml using the Livak method [23]. insulin (Sigma-Aldrich), 50 ng/ml epidermal growth factor (Sigma-Al­ For total RNA isolation of ZF4 cells, the E.Z.N.A.® Total RNA Kit drich), 2% (v/v) antibiotic/antimycotic (Gibco), and 0.5% (v/v) trout (Omega Bio-Tek Inc., Norcross, GA, USA) was used according to the serum which had been filtered (0.20 μm filter Corning) and heat inac­ manufacturer’s instructions. Then, RNA was quantified with a Nano­ tivated for 30 min at 56 ◦ C. Drop Spectrophotometer (Nanodrop Technologies, Wilmington, DE, Zebrafish embryonic fibroblast (ZF4 cells) were cultivated at 28 ◦ C USA), and M-MLV reverse transcriptase (Invitrogen, Thermo Fisher and 5% CO2 in RPMI medium (Gibco) supplemented with 10% FBS Scientific) was used for cDNA synthesis from RNA, as previously (Cultek), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 μg/ml described [24]. To evaluate the gene expression in ZF4 cells, RT-qPCR gentamicin (Gibco) and 2 μg/ml amphotericin B (Gibco). was carried out in 20 μl reactions containing 24 ng of cDNA, 10 μl of Fathead minnow (Pimephales promelas) EPC cells were maintained at SYBR green PCR master mix (Thermo Fischer Scientific), and a 900 nM 28 ◦ C and 5% CO2 in medium RPMI-1640 supplemented with 10% FBS, final concentration of each primer using the QUANTSTUDIO 3 System 1 mM pyruvate, 2 mM L-glutamine, 50 μg/ml gentamicin and 2 μg/ml (Applied Biosystems, Thermo Fisher Scientific Inc.). The ef1-a gene was amphotericin B. used as a reference gene. SVCV (strain 56/70) originally isolated from carp (Fijan, 1971) was propagated in EPC cells at 28 ◦ C until a cytopathic effect was observed. 2.7. Zebrafish husbandry Then, the cell culture medium was harvested, centrifuged at 4000×g for 30 min, and virus aliquots were stored at − 80 ◦ C. Wild type zebrafish were kept in a re-circulating aquarium with water temperature at 28 ± 1 ◦ C. The lighting conditions were 14:10 h 2.5. Uptake of nanostructured viral antigens by ZFL cells (light:dark) and adult fish were fed twice a day at a rate of 2% body­ weight. Ammonia, nitrite, nitrate and pH levels were measured once a To test cellular uptake, fluorescently labeled NPs were added to ZFL week and were kept below toxic levels and pH at 7.5 ± 0.5. For in-tank cells cultures at 70–80% confluence after 2–3 h incubation in minimal breeding, two females and four males were transferred in groups to a media (0–0.5% FBS) at the doses and times indicated below. For dos­ breeding tank in the late afternoon. The next morning after 3 h of the e–response assays, SVCVNP and SVCV-IFNNP were added at 2.5, 5, 10, 20 onset of light embryos were collected and cultured in embryo medium and 40 μg/m L and cultures were then incubated O/N (12–14 h). For (E3 medium) in a Petri dish (Deltalab). Fertilized eggs were separated time-course assays, SVCV NPs were added at 5 and 10 μg/ml and cul­ from unfertilized eggs using a plastic pipette (Deltalab). All experiments tures were simultaneously incubated for 12 and 24 h. For comparative involving zebrafish were performed following International Guiding assays between SVCVNP and SVCV-IFNNP were added at 5 and 10 μg/ml Principles for Research Involving Animals (EU 2010/63) and the Ethics and cultures were then incubated O/N (12–14 h). All experiments were Committees of the Universitat Autònoma de Barcelona (UAB, CEEH) and performed in duplicate. Post treatment, ZFL cells were washed in PBS Generalitat Valenciana (2019/VSC/PEA/0203) and incubated at 28 ◦ C and 5% CO2 with 1 mg/ml Trypsin (Gibco) for 15 min. This strong trypsinization step aimed to remove NPs attached to the 2.8. In vivo uptake in zebrafish larvae cell surface [22]. Then, two volumes of complete medium were added, and cells were pelleted by centrifugation at 300×g for 5 min at RT. Groups of five larvae per treatment were distributed on 6-well plates Pellets were resuspended in PBS for flow cytometry (FACSCalibur BD), (Thermo Fisher) in duplicate and immersed in 1 ml of E3 medium with and 10,000 events were counted. Data was analyzed using FlowJo 10.4 PBS, fluorescent SVCVNP and SVCV-IFNNP (10 μg/ml), GFP (10 μg/ml) (Leland Stanford University) and plotted with Prism 8.01 (GraphPad and LPS (10 μg/ml). Biodistribution after 24 and 48 h zebrafish larvae Prism). To confirm the fluorescent NPs were inside the cells, we per­ was evaluated using a fluorescence stereomicroscope (Nikon SMZ800) formed confocal microscopy (Zeiss LSM 700). ZFL cells were seeded on coupled with a camera (Nikon DS-Fi2). In parallel groups of n = 5 larvae Ibidi plates (μ-Dish 35 mm) and incubated at 28 ◦ C and 5% CO2. The were treated as indicated, washed and homogenized in TriReagent for next day cells at approximately 50–60% confluence were placed in gene expression analysis as indicated in RNA extraction and Q-PCR minimal media. SVCVNP and SVCV-IFNNP at 20 μg/ml were added 2 h section. later and cells were incubated for 16 h at 28 ◦ C. The cells were stained with Hoechst (nuclei) and CellMask Deep Red (membrane) (Life Tech­ 2.9. SVCV infection challenge nologies). Images were analyzed using Imaris software v8.2.1 (Bitplane) and ImageJ (National Institute of Health, USA). Zebrafish, average size 35 mm and 0.4 g, were purchased from a local pet store. Fish were acclimatized in 20 L tanks at 21 ◦ C for 14 days before 2.6. RNA extraction and Q-PCR the vaccination trial. Zebrafish were divided into five groups with n = 12 animals each: the non-infected control, the SVCV-infected control, the Total RNA of ZFL cells was extracted using TriReagent (Sigma- iRFPNP control group, SVCVNP group, and SVCV-IFNNP group. Each in­ Aldrich) following the manufacturer’s instructions and RNA yield and dividual was anesthetized with tricaine (tricaine methanesulfonate, quality was determined on Nanodrop ND-1000 (Thermo Fisher Scien­ Sigma-Aldrich) (40 mg/l) and intraperitoneally (i.p.) injected with 30 μl tific) and integrity assessed on the Agilent 2100 Bioanalyzer using RNA of PBS containing 5 μg of each antigen using 23G syringe-needle (Ter­ 6000 Nano Lab-Chip kit (Agilent Technologies). Then, cDNA was syn­ umo Europe N.V.). At 30 days post-vaccination (dpv), fish were infected thesized from 1 μg high quality total RNA using iScript cDNA systhesis by bath-immersion for 2 h in aquarium water containing SVCV (1.4 × kit (Bio-Rad). Quantitative real-time PCR (qPCR) was performed at 107 SVCV infectious particles). Individuals were maintained at 21 ◦ C 60 ◦ C annealing temperature in a CFX384 touch real-time PCR detection over the course of the challenge, and the mortality rate of each group 1053 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 was recorded daily. and SVCV-IFNNP 3.6 mg/l (Supplementary Fig. 1). Expression of re­ combinant proteins is an empiric process with several experimental constraints that affect the production yield. SVCVNP and SVCV-IFNNP 2.10. Statistics production yields ranged from moderate to good compared with other NPs such as VHSVNP and VHSV-IFNNP where we obtained 11.7 and 14.2 Graphs and analyses were performed with Prism 8.01 software mg/l, respectively (unpublished data). (GraphPad Prism). Comparisons of means for each experimental group The size and morphology of both NPs were evaluated by FESEM versus control and between treatments (NPs) were analyzed by one-way (Fig. 1). SVCV-IFNNP is generally oviform (elliptical shaped) while ANOVA, followed by Dunnett’s multiple comparisons test or unpair non SVCVNP is rounder and smaller. We have observed similar morphologies parametric Mann-Whitney test. Data are shown as mean ± standard in other IBs produced in E. coli using the same strain BL21(DE3) and in deviation (SD) and p < 0.05 was considered statistically significant. M15(pREP4) [21]. The size range is shown in Fig. 1 with average width and length being 420 ± 67.5 nm and 693 ± 154 nm for SVCVNP and 624 3. Results ± 104.9 nm and 982 ± 216.9 nm for SVCV-IFNNP, respectively. The morphological features of the nanostructured immunologically irrele­ 3.1. Characterization of modular nanostructured protein antigens vant control protein iRFPNP were previously reported in Torrealba et al. SVCVNP and SVCV-IFNNP [25]. Morphological and structural integrity of NPs were evaluated under different environmental conditions of temperature and pH to We produced two different recombinant viral proteins in E. coli as assess the resistance of the NPs under industrial extrusion procedures bacterial IBs (i.e., NPs) with moderate to good yields: SVCVNP 0.3 mg/l Fig. 1. Characterization of nanostructured SVCVNP and SVCV-IFNNP proteins. Field Emission Scanning Electron Microscopy images (FESEM) (i) of SVCVNP (A) and SVCV-IFNNP (B). Size distribution histograms (n = 60) of length (ii) and width (iii) measurements of SVCVNP and SVCV-IFNNP. 1054 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 and gastrointestinal pH conditions. SVCVNP (Fig. 2A) and SVCV-IFNNP ml versus ~90% fluorescent positive cells at 10 μg/ml, respectively (Fig. 2B) were subjected to high temperature (100 ◦ C) and extreme pH (Fig. 3A and C). In both cases, the mean fluorescence intensity (MFI) conditions (pH 2 and 10) and we did not observe significant changes in increased with dose until 5–10 μg/ml (Fig. 3A and C) and at higher doses size or morphology. dropped significatively. This decrease was not related with toxicity or apoptosis since cell survival was 100% across doses and time (data not shown). Maybe, the MFI dropped indicating cells were not able to take 3.2. Uptake of modular nanostructured antigens by ZFL cells up more NP (100% of cells were fluorescent) and NP had started to be metabolized. For time course experiments, a 10 μg/ml dose was chosen. Modular SVCV NPs were taken up by ZFL cells. In dose–response In the time course experiments, at 12 and 24 h ZFL cells treated either experiments, uptake of SVCVNP was found to be slightly more efficient with SVCVNP or SVCV-IFNNP reached the same percentage of than SVCV-IFNNP, achieving ~100% fluorescent positive cells at 10 μg/ Fig. 2. Characterization of SVCVNP and SVCV-IFNNP under different conditions pH and Temperature. FESEM images (i) of SVCVNP (A) and SVCV-IFNNP (B) submitted to pH 2, pH 10 and high temperature (5 min at 100 ◦ C). Size distribution histograms (n = 60) of length (ii) and width (iii) measurements of SVCVNP and SVCV-IFNNP. 1055 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 Fig. 3. Uptake of SVCVNP and SVCV-IFNNP nanopellets by ZFL cells. (A) SVCVNP dose response; (B) SVCVNP time course; (C) SVCV-IFNNP dose response; (D) SVCV- IFNNP time course and (E) Comparative uptake of SVCVNP and SVCV-IFNNP at 10 μg/ml for 12 h. For dose response assays 2,5–40 μg/ml of fluorescently labeled SVCVNP and SVCV-IFNNP were added to ZFL cells and incubated for 12 h and the uptake evaluated by cytometry. For time course 10 μg/ml of fluorescently labeled SVCVNP and SVCV-IFNNP were added to ZFL cells and incubated for 12 and 24 h. Differences between means were analyzed by a one-way ANOVA with Dunnett’s multiple comparisons test, treatments versus control. Significance levels ns: no significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. 1056 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 fluorescence and the same MFI (Fig. 3B and D). No differences in ZFL stimulate an antiviral response showing that IFNγ could act as a potent uptake at 5 or 10 μg/ml at 12 h incubation were observed between adjuvant when included within the same NP (Fig. 5). Induction of the SVCVNP and SVCV-IFNNP when both NPs were assayed in parallel expression of IFNγ receptor (ifngr) and low molecular mass polypeptide (Fig. 3E). In addition, these results (Fig. 3E) were highly consistent with (lmp)-2 were observed after SVCV-IFNNP indicating a parallel induction the previous ones when the NPs were assayed separately (Fig. 3A–D). of INFγ signalling pathways presumably after IFNγ binds to its receptor. The confocal microscopy images of SVCVNP and SVCV-IFNNP treated Pro-inflammatory (tnfa) and anti-inflammatory cytokines (il10) also ZFL cells showed that both NPs were internalized and accumulated in­ were up-regulated in ZFL but no significant differences between SVCVNP side the cytosol. The 3D images demonstrated the complete internali­ and SVCV-IFNNP were observed. zation of both SVCVNP and SVCV-IFNNP in ZFL cells (Fig. 4). Confocal microscopy assays were performed at 20 μg/ml of NPs at 24 h and the cytometry uptake data showed that 100% and 90% of the cells were 3.4. Uptake of SVCVNP and SVCV-IFNNP by zebrafish larvae fluorescent at 20 μg/ml. However, the Mean Fluorescent Intensity (MFI) in SVCV-IFNNP treated cells (Fig. 3C) was lower than in SVCVNP treated Hatched zebrafish larvae (approximately at 72 h post fertilization cells (Fig. 3A). Apparently, confocal microscopy images did not show (hpf)) were incubated with 10 and 20 μg/ml of fluorescent SVCVNP and any difference between SVCVNP and SVCV-IFNNP treated cells in terms of SVCV-IFNNP in E3 medium. After 24 and 48 h living larvae were anes­ size or qualitative intensity of the fluorescent agglomerates (Fig. 4). thetised and imaged using a stereoscopic microscope (Fig. 6A). Uptake of SVCVNP at 24 h was almost undetectable (n = 6 larvae) while we observed high levels of fluorescence at 48 h in around 100% of larvae 3.3. Gene expression analysis in ZFL stimulated with NPs mainly showing accumulation in the gastrointestinal tract and pancreas (Fig. 6A). Importantly, SVCVNP and SVCV-IFNNP treated larvae died To see whether the antigenic NPs could induce an antiviral response after 24 h incubation at the highest dose, 20 μg/ml. However, at 10 μg/ in line with that provoked by viral infection, ZFL cells were stimulated ml the whole set of larvae survived after immersion in the SVCVNP and with the viral NPs overnight (16 h) at 10 μg/ml. We selected the time SVCV-IFNNP solution. and dose from the uptake data shown in section 3.2: those conditions We also tested the antiviral response in zebrafish larvae after SVCVNP which the highest percentage of fluorescent positive cells (SVCVNP and SVCV-IFNNP treatments. To try to understand the in vivo response to ~90% and SVCV-IFNNP ~100%), with a significant intensity of fluo­ SVCVNP and SVCV-IFNNP modular proteins, we selected a panel of genes rescence (MFI) and no differences among time. We also used Poly(I:C) potentially representative of differential immune responses: direct virus (10 μg/ml), as a viral dsRNA mimic and LPS as a bacterial mimic. iRFPNP response, macrophage response, Th1 or cytotoxic T cell responses. (10 μg/ml) was the control of a NP made with immunologically irrele­ However, as is shown in Fig. 6B almost all the selected genes were vant protein but with all the potential E. coli contaminants washed down stimulated in vivo by SVCVNP but not by SVCV-IFNNP. To better explore during NPs preparation. The expression of genes, markers of the immune why SVCV-IFNNP treatments failed to induce the IFNγ response in larvae, response to viral infection was tested by qPCR (Fig. 5). In ZFL cells a we used the zebrafish embryonic cell line ZF4 to assess whether this typical strong antiviral response after Poly(I:C) treatment was previ­ pattern was common in cells from same origin and developmental stage. ously reported [26]. iRFPNP protein without a relevant immune role ZF4 cells were tested for gene expression after treatment with SVCVNP, does not induce the expression of antiviral genes while SVCVNP and SVCV-IFNNP, Poly(I:C) (10 μg/ml), LPS (10 μg/ml), and iRFPNP. As is SVCV-IFNNP were able to stimulate ZFL cells by increasing the gene shown in Fig. 6C embryonic derived cells ZF4 treated with SVCVNP and expression of vig1, mx, lmp2, irf1, ccl4 or ifngr1. Importantly, the inclu­ SVCV-IFNNP stimulated a canonical antiviral response at similar levels of sion of the IFNγ protein module enhanced the ability of SVCVNP to those observed in ZFL cells (Fig. 5) although not showing difference in Fig. 4. Confocal microscopy and digitalized image (z-stacks) of ZFL cells with SVCVNP and SVCV-IFNNP. NPs are green, cell membrane red, and nuclei blue. SVCVNP; (a, b) confocal microscopy and (c) digitalized image (z-stacks), and SVCV-IFNNP treated ZFL cells; (d, e) confocal microscopy and (f) digitalized image (z-stacks). Cells were incubated for 16 h with SVCVNP and SVCV-IFNNP at 20 μg/ml. 1057 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 Fig. 5. Gene expression analysis of ZFL treated with SVCVNP and SVCV-IFNNP. (A) ZFL cells were incubated as follow: unstimulated control cells (black), 10 μg/ml of PolyI:C and LPS (red and blue respectively), 10 μg/ml of SVCVNP (purple), 10 μg/ml of SVCV-IFNNP(orange) and 10 μg/ml of iRFPNP as immunogenically irrelevant control (green). Data are mean ± SD (n = 6). Statistical differences between treatments and controls were analyzed by unpair non parametric Mann-Whitney test. Significance levels *p < 0.05; **p < 0.01. stimulation levels between SVCVNP and SVCV-IFNNP. However, ZF4 virus in the diseased fish was confirmed by RT-qPCR (data not shown). failed to up-regulate lmp-2 and ifngr. 4. Discussion 3.5. Protective effect of the vaccines against SVCV infection in zebrafish Finding cost effective, safe and efficient therapeutic and prophylactic A preliminary test of NPs toxicity on adult animals was run after i.p methods for fish viral diseases is still a challenge. The main issue when injection of 5 and 10 μg of SVCVNP and SVCV-IFNNP and no signs of using subunit vaccines is the short lifetime of soluble proteins. This is macrotoxicity were observed (data not shown). A preliminary survival particularly important for oral administration methods. To overcome rate assay was performed on SVCV-infected zebrafish after different degradability, encapsulation using different biocompatible materials nanoparticle vaccine treatments. Unvaccinated zebrafish had 83.3% cu­ have been investigated. Among others, liposomes, poly-lactic-co-gly­ mulative percentage mortality after 16 days of infection with SVCV. colic acid (PLGA) or carbon nanotubes (CNTs) have shown to be efficient Mortality rates in the iRFPNP, SVCVNP and SVCV-IFNNP were 41.7%, for therapeutics and prophylactics delivery (see Ref. [27] for a review). 50.0% and 20.0%, respectively (Fig. 7B). The main onset of mortality However, the cost of encapsulation is still an issue in fish health, occurred between 4 and 8 days post-infection (dpi), although this was particularly for those species with moderate market value and high delayed in the SVCV-IFNNP group where first mortalities were recorded at production such as common carp (FAO, 2020). These high levels of 7 dpi. Dead fish showed clinical signs of SVCV (Fig. 7A) and presence of production of common carp (C. carpio) and grass carp (C. idella) (a total 1058 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 Fig. 6. SVCVNP and SVCV-IFNNP biodistribution in vivo in zebrafish larvae. (A) Uptake in larvae. Zebrafish larvae (3 dpf) were immersed with 10 μg/ml of fluorescent SVCVNP and SVCV-IFNNP for 48 h. E3 medium with PBS were used as control. (i) Transmitted image, (ii) fluorescent image and (iii) merged image. (B) Gene expression in larvae. Groups of 5 larvae were inmersed for 24 h as follow: unstimulated control cells (black), 50 μg/ml of PolyI:C (red), 25 μg/ml of LPS (blue), 10 μg/ 1059 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 ml of SVCVNP (purple), 10 μg/ml of SVCV-IFNNP (orange) and 10 μg/ml of iRFPNP as immunogenically irrelevant control (green). Data are mean ± SD (n = 3). No differences were detected between treatments and control (unpair non parametric Mann-Whitney test). (C) ZF4 cells were incubated as follow: unstimulated control cells (black), 10 μg/ml of Poly(I:C) and LPS (red and blue respectively), 10 μg/ml of SVCVNP (purple), 10 μg/ml of SVCV-IFNNP and 10 μg/ml of iRFPNP as immunogenically irrelevant control (green). Data are mean ± SD (n = 6). Statistical differences between treatments and controls were analyzed by unpair non parametric Mann-Whitney test. Significance levels *p < 0.05; **p < 0.01. Fig. 6. (continued). Fig. 7. SVCVNP and SVCV-IFNNP administration protects from SVC virus in vivo infection. (A) Representative fish images of SVC virus clinical signs. (B) Cumulative mortality rate of zebrafish after SVCV challenge. Fish were vaccinated with 5 μg/fish of different antigens (iRFPNP, SVCVNP, SVCV-IFNNP) by intraperitoneal injection. Thirty days post-vaccination fish were challenged with SVCV (4 × 104 TCID50/ml). Number of dead fish in each group was counted daily. 1060 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 of 9.89 million tonnes/year) makes even more relevant to develop [35]. This means that during the first weeks of development the embry­ vaccines against SVC virus. o/larvae depend solely on innate immune system and could be that within Here we present a biocompatible, cheap and modular platform that this period the different antiviral response mechanisms mature at a allow the production of recombinant proteins in a highly stable different pace and only adult systems show the full repertoire of antiviral conformation, suitable for injection and for feed inclusion and oral mechanisms. Dios et al. characterized the progress of antiviral system administration. SVCVNP and SVCV-IFNNP exposed to high temperatures maturation in zebrafish between 2 and 29 dpf using IFNγ-1 expression and extreme pH did not show changes in shape or size. Previous works and they describe how IFNγ-1 started to be detectable at 14 dpf without demonstrated that nanostructured TNFαNP also remains structurally and viral stimuli, and at 8 dpf with viral stimuli. In contrast, IFN type I genes functionally intact when subjected to extreme temperature or pH [18, were overexpressed after viral stimuli at high levels at 2 dpf [36]. 21]. Moreover, high salinity has no impact either on structure or func­ Adult zebrafish vaccinated with SVCV-IFNNP and infected with SVC tion of NPs (data not shown). Although IFNs can be used for treatment of virus showed excellent levels of survival compared with non-vaccinated different human diseases its stability and plasma half-life remained the and control fish. Previous attempts at protecting carp from SVCV using a main obstacles that hindered the utilization of recombinant proteins as DNA vaccine engineered with a plasmid that drives the expression of the therapeutics [28]. IFNγ failed to show the expected bioactivity in vivo SVCV-G protein provided different degree of protection against SVC due to degradation, and also to the tendency to aggregate irreversibly virus [37,38], although some of these DNA vaccines achieved excellent under mild denaturing conditions [29]. protection levels even at low doses [39]. These experimental vaccines In the present study, nanostructured IFNγ combined with the SVCV involve the use of exogenous genetic material with the corresponding antigenic module remained stable and functional even under the biosecurity concerns. Other successful approaches that do not involve harshest conditions of pH and temperature. This modular approach is exogenous genetic material, engineered Lactobacilus to express SVCV-G highly versatile and could include other modules such as ligands for protein on the bacterial external surface [3,40] obtaining good levels of specific molecules or receptors, that would provide a plethora of extra protection against SVCV infection. Overall, our findings demonstrate the functions to the recombinant antigenic protein. The production yield of capacity of self-adjuvanted modular NP vaccines to confer efficient these modular proteins is good although is highly dependent on the protection against viral challenge in a bio secure and cheap context. protein and the SVCVNP and SVCV-IFNNP showed moderate expression Future work should be done to adapt this promising vaccination levels compared to other viral antigens such as VHSVNP or VNNVNP [21]. platform to oral administration and to incorporate NPs into aquafeeds to At the functional level SVCVNP and SVCV-IFNNP were efficiently be tested as oral vaccines against SVC virus infection in aquaculture uptaken by ZFL cells and elicited a strong antiviral response. The IFNγ relevant species (e.g carp), paying special attention to survival and module within the SVCV-IFNNP conferred the NPs a stronger ability to correlates of protection and gut mucosal immune response. induce an antiviral response probably by stimulating the signalling pathways downstream of the IFN receptor, and leading for example to Ethics statement upregulation of lmp-2. Sieger et al. described in zebrafish larvae that except for some irg family members (immune-related GTPases) the only All methods were performed in accordance with the Spanish and genes that responded to IFNγ were ifn-phi1 and lmp-2 [17]. Lmp-2 is European regulations (RD53/2013 and EU Directive 2010/63/EU) for involved in degradation of intracellular proteins into antigenic peptides the protection of animals used for research experimentation and other and is a good marker of IFNγ activation. It is well described that IFNγ scientific purposes. Zebrafish procedures were conducted under Gen­ genes in the zebrafish are inducible by Poly(I:C) in cultured cells [30]. eralitat Valenciana permit 2019/VSC/PEA/0203. The SVCVNP and SVCV-IFNNP were even more efficient than the Poly(I: C) stimulating the antiviral response in ZFL cells, indicating excellent Funding immunostimulant properties compared with adjuvants such as Mon­ tanide, saponin or peptides [4,31–33]. This work was supported by grants from the Spanish Ministry of In ZF4 cells the picture is slightly different and although SVCVNP and Science, European commission and AGAUR funds to NR (RTI2018- SVCV-IFNNP stimulated a similar antiviral response and within the range 096957-B-C21 MINECO/FEDER and 2017-SGR-695 (AGAUR). The Eu­ of the Poly(I:C), the SVCV-IFNNP failed to induce the IFNγ response ropean Research Council fund to MO (ERC Starting Grant GA639249). observed in ZFL cells. Lopez-Muñoz et al., had previously tested the MR and PA were supported by a pre-doctoral scholarship from Agencia ability of HEK293-expressed zebrafish recombinant IFNγ stimulating the Nacional de Investigación y Desarrollo (ANID, Chile). University Miguel expression of anti-viral genes such as mx in ZF4, and preventing SVCV Hernandez (“Ayudas Impulso Divulgación de la Ciencia 2021”, Vice­ infection [10]. Worth to mention that recombinant protein production rrectorado de Investigación) is acknowledged for funding support of using a bacterial system or a mammalian heterologous system like publication charges. HEK293 are substantially different in terms of structure and bioactivity. The zebrafish larvae showed high gastrointestinal and pancreas up­ CRediT authorship contribution statement take of both SVCVNP and SVCV-IFNNP but again zebrafish larvae failed to respond to SVCV-IFNNP while could mount a good virus specific Mauricio Rojas: Formal analysis, Data curation. Patricia Aceituno: response and a proinflammatory response. Similar results in 4 dpf larvae Formal analysis, Data curation. Maria E. Salvador: Formal analysis, infected with SVCV were reported by Lopez-Muñoz et al. [34]. Not less Data curation. Marlid Garcia-Ordoñez: Formal analysis, Data curation. important, zebrafish larvae exposed to SVCVNP and SVCV-IFNNP died Maria del Mar Ortega-Villaizan: Formal analysis, Supervision. Luis after incubation (24 h) at higher dose (20 μg/ml). However, at dose of Perez: Formal analysis, Supervision. Nerea Roher: Formal analysis, 10 μg/ml – double of the dose used in the zebrafish challenge - all set of Conceptualization, Supervision, Writing - original draft. larvae survived in the SVCVNP and SVCV-IFNNP solution. Toxicity of IFNγ has been previously described by Lopez-Muñoz et al. in micro­ Declaration of competing interest injected zebrafish embryos overexpressing IFNγ [14]. Although it is difficult to compare bath immersion administration of IFNγ with The authors declare that the research was performed without any microinjection administration to zebrafish embryos. conflict of interest. Importantly, zebrafish larvae and ZF4 cells are from the same origin (embryo) and the antiviral responses against the recombinant antigen Data availability alone were consistent among them (vig1 and mx up regulation). The adaptive immune system of the zebrafish develops at about 4 weeks of age Data will be made available on request. 1061 M. Rojas-Peña et al. Fish and Shellfish Immunology 131 (2022) 1051–1062 Acknowledgements [19] S. Mutoloki, H.M. Munang’andu, Ø. Evensen, Oral vaccination of fish – antigen preparations, uptake, and immune induction, Front. Immunol. 6 (2015) 519, https://doi.org/10.3389/fimmu.2015.00519. We thank Dr. Manuela Costa and Servei de Microscopia (UAB) for [20] B. Zhu, C. Zhang, B. Yang, Z.-R. Guo, Y.-Y. Zheng, Y.-M. Gong, G.-X. 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