A novel chimeric vaccine protects pigs from infection with Japanese encephalitis virus

A novel chimeric vaccine protects pigs from infection with Japanese encephalitis virus | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A novel chimeric vaccine protects pigs from infection with Japanese encephalitis virus Paul M Hick, Henry de Malmanche, Jessica J Harrison, Gervais Habarugira, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8775364/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Widespread infection with a rare genotype 4 (GIV) strain of Japanese encephalitis virus (JEV) throughout eastern Australia in 2022 created new threats to animal and public health. Extensive production losses were incurred by the reproductive impacts of JEV infection at piggeries. Further, as an amplifying host for JEV, there are broader One Health advantages in minimising JEV infection of pigs. A candidate vaccine for pigs was developed using a chimeric virus with the structural glycoprotein genes (prM and E) of the outbreak JEV strain (JEV NSW/22 ) inserted into an insect-specific orthoflavivirus (Binjari virus - BinJV) genome backbone (BinJ/JEV NSW/22 ). Vaccines prepared with adjuvants using purified chimeric virus or a non-purified, formalin-inactivated formulation, induced JEV-specific antibody responses in mice and pigs. No adverse effects of vaccine administration were observed in either species for either vaccine formulation when used with a prime and boost delivery 28 days apart. Ninety-four percent (17/18) of weaner pigs that received the inactivated vaccine were protected from challenge with 1 x 10 5.0 TCID 50 of JEV NSW/22 , as determined by testing for JEV in blood and tonsils by RT-qPCR and virus isolation. In contrast, all control animals (n = 9) became viraemic and harboured JEV in the tonsils. When adult sows (1.5 to 3 years of age) (n = 9) were given a double-dose of the inactivated vaccine, there were no adverse effects, and each animal produced JEV-specific neutralising antibodies. Hence, BinJ/JEV NSW/22 appears to be a promising candidate vaccine for prevention of JEV infection in pigs and can be used live or inactivated at varying levels of purity to provide manufacturers with logistical options for commercial production of the vaccine. Health sciences/Diseases Biological sciences/Immunology Biological sciences/Microbiology Japanese encephalitis virus Binjari pig vaccine orthoflavivirus Figures Figure 1 Figure 2 Figure 3 Introduction Japanese encephalitis virus (JEV, Orthoflavivirus japonicum ) is a mosquito-borne orthoflavivirus that sometimes causes fatal encephalitis in humans, reproductive disease in pigs and occasionally neurological disease in horses 1–3 . There are five genotypes (GI to GV) of JEV with GI and GIII the most prevalent 4,5 . Prior to the southern hemisphere summer of 2021/2022, JEV was considered exotic to Australia, with isolated incursions limited to the islands of the Torres Strait 6–8 and Cape York Peninsula 9–11 , none of which led to the establishment of transmission on the Australian mainland. However, in early 2022, a large outbreak of the rarer GIV of JEV was documented across Queensland (Qld), New South Wales (NSW), Victoria (Vic) and South Australia (SA), involving greater than 80 piggeries and causing 42 cases of human disease, resulting in seven fatalities as reported by the Australian Department of Health and Aged Care 12–14 . The virus now appears to have a strong foothold on the Australian mainland, with detections in mosquito populations ( Culex species) during the 2024/2025 Australian summer, evidence for active infections in feral and farmed pigs, in addition to human infections with several deaths (8 cases, 3 fatal, Tables S1, S2). Thus, JEV is now likely endemic in Australia. The 2022 outbreak of JEV infection caused large scale economic and welfare impacts through reproductive losses for the pig industry in eastern Australia. In NSW, impacted piggeries saw up to 60% of porcine fetuses and neonates affected on farms during the summer of 2021/22 ( https://www.agriculture.gov.au/biosecurity-trade/pests-diseases-weeds/animal/japanese-encephalitis ; https://www.swinehealth.org/wp-content/uploads/2024/03/2024-JEV-Economic-Assessment-SHIC-White-Paper-Final.pdf ) Most of the losses in pigs were due to abortions and stillbirths often associated with severe congenital defects. Neurological disease was sometimes observed in live-born piglets soon after birth. Such clinical manifestations seen in the farmed pigs infected with the GIV strain of JEV were consistent with those seen elsewhere in the world where other JEV genotypes circulate 15–19 . Additionally, there were the anticipated impacts on reproduction due to testicular infection in boars, which resulted in poor semen quality or no viable sperm 20 . Apart from the impact on boars, JEV infection does not frequently result in disease in healthy, non-pregnant adult or growing pigs. However, they are a potential reservoir for virus amplification which has public health implications 2 . While veterinary vaccines for JEV are commercially available in some countries for the vaccination of pigs and/or horses (e.g., Nisseiken inactivated and live attenuated, Japan; Anyang300 live-attenuated, South Korea) 21 , none are available in Australia. Currently, all commercially available vaccines are based on GIII JEV strains, while the Australian outbreak involved a strain of GIV – a much rarer JEV genotype with antigenic differences 22,23 . While cross-protection is expected between vaccines produced to different genotypes, this protection might not be optimal 21 . In addition to orthoflaviviruses that can infect both vertebrate and arthropod hosts, there are insect-specific orthoflaviviruses, which can infect arthropod hosts but are not replication competent in vertebrate cells. As such, these viruses offer opportunities to develop candidate vaccines that will negate problems with live attenuated vaccines, such as reversion to virulence, and provide additional production safety. A vaccine technology based on modifying an insect-specific orthoflavivirus (Binjari virus, BinJV) to produce chimeric viruses that display the surface proteins of pathogenic orthoflaviviruses (such as JEV, West Nile virus (WNV), dengue virus and Zika virus) has recently been developed 24 . These chimeric viruses grow efficiently in mosquito cell cultures but have been shown not to replicate in vertebrate cells, providing a high level of safety for vaccine production and administration. In response to the 2022 Australian JEV outbreak, a chimeric virus containing the prM and E surface glycoprotein genes of the outbreak strain (JEV NSW/22 ) in the BinJV genome backbone (BinJ/JEV NSW22 -prME, herein abbreviated to BinJ/JEV NSW/22 ) was rapidly constructed and produced to high titre in C6/36 cells 22 . Using a sensitive immunodeficient mouse model of JEV NSW/22 disease 25 , vaccination with BinJV/JEV NSW/22 was shown to generate a protective anti-JEV immune response against lethal virus challenge 22 . In this study we assessed the application of BinJ/JEV NSW/22 as a vaccine for pigs, using an established pig model of JEV GIV infection 15 and investigated the protection provided to young pigs against challenge with JEV NSW/22 . Immunogenicity and safety of the vaccine was also assessed in older multiparous sows. Materials & Methods Ethics approvals All animal work was conducted in accordance with the “ Australian code for the care and use of animals for scientific purposes ” as defined by the National Health and Medical Research Council of Australia. Animal experiments in mice and multiparous sows, performed at the University of Queensland were approved by the University of Queensland Animal Ethics Committee (permit numbers 2020/AE000118, 2022/AE000862). The multiparous sow experiment was performed under the Australian Department of Agriculture, Fisheries and Forestry (DAFF) permit 2023/010. Dealing with JEV was approved under the Queensland Biosecurity Act (PRID000998). Vaccination and challenge studies in weaner pigs were conducted with approval of the Elizabeth Macarthur Agriculture Institute Animal Ethics Committee (Project ID 341), Biosafety Committee (Approval M22/07) and DAFF permit (2022/047). Cell lines and Culture C6/36 cells ( Aedes albopictus , ATCC CRL-1660) were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640) medium, supplemented with 5% Australian sourced fetal bovine serum (FBS) at 28 o C. Detailed handling of C6/36 cells for vaccine production are provided in a later section. Vero76 cells (ATCC-CRL1587; Cercopithecus aethiops , African green monkey kidney) and Vero cells (ATCC CCL-81) were maintained in Dulbecco’s modified Eagle medium (DMEM) and supplemented with 5% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin and 2 mM L-glutamine. Virus Isolates and Culture The 2022 Australian outbreak strain of JEV (JEV NSW/22 ) was previously isolated from an infected porcine fetus (Genbank: OP904182) (O883/NSW/22). A stock of this isolate (O909) was generated by passage in cell culture using Vero76 cells for vaccine efficacy studies. The chimeric virus BinJ/JEV NSW/22− prME (herein described as BinJ/JEV NSW/22 ) has been previously reported 22 . This virus was generated by replacing the prM and E genes of BinJV with the corresponding sequences amplified from JEV NSW/22 and the genome of the rescued virus was deep sequenced to confirm its authenticity 22 . Stocks of JEV NSW/22 and BinJ/JEV NSW/22 were generated by infecting Vero (JEV NSW/22 only) or C6/36 cell monolayers with virus at a multiplicity of infection (MOI) of 0.1 and incubating at 28 o C (C6/36 cells) or 37 o C with 5% CO 2 (Vero76 cells) for 1 hr. Following this, inoculum was removed and replaced with fresh maintenance medium containing 2% FBS and incubated at the appropriate temperature for 5–7 days post-infection (dpi), before harvesting the medium and centrifuging at 3,000 rpm, 4 o C for 10 min. The clarified supernatant was then supplemented with additional FBS to increase the total concentration to 10%, aliquoted and stored at -80 o C until required. The titre of virus stocks was calculated using the method of Reed and Muench 26 to determine the 50% tissue culture infectious dose (TCID 50 ) in Vero or C6/36 cells for JEV NSW/22 and C6/36 cells for BinJ/JEV NSW/22 , after immunolabelling the cells with the pan-orthoflavivirus NS1 monoclonal antibody 4G4 27 or anti-JEV mAb 989 28 described previously 15,22 . In some instances, TCID 50 values were converted to infectious units per mL (IU/mL) using the Poisson distribution, under the assumption of a single-hit infection model where 1 TCID 50 corresponds to 0.69 IU, and assuming random distribution of particles Production of Master Seed Banks Master Seed Cell Bank . A Master Seed Cell Bank (MSC) of C6/36 cells ( Aedes albopictus , ATCC CRL-1660) was set down for vaccine antigen production. C6/36 cells at 6 passages following their receipt from ATCC were passaged 19 times in serum free medium (sf900-III, Gibco) and a further 5 times in RPMI/5% Australian sourced FBS. As adherent cultures, the cells were dislodged using a cell scraper to ablate the requirement for use of animal derived materials such as trypsin. Characterisation of the MSC was performed on cell supernatant or cell lysates, as stipulated by the company. Karyotyping of the cells was performed at Monash Health Pathology (Clayton, Vic, Australia), while species identification was performed at Cellbank Australia (Westmead, NSW, Australia). Extraneous agent testing for viral, bacterial and fungal contaminants of the cells was performed at three NATA-accredited (National Association of Testing Authorities, Australia) laboratories: Australian Centre for Disease Preparedness (Geelong, Vic, Australia), Eurofins Laboratories Pty Ltd (Girraween, NSW, Australia) and the Elizabeth Macarthur Agricultural Institute Laboratory Services (Menangle, NSW, Australia). To confirm the absence of exogenous viral agents, deep sequencing was performed on RNA extracted from conditioned C6/36 media. The RNA was extracted using the Macherey-Nagal Nucleospin RNA virus kit as per the manufacturer’s instructions, with the following modifications: carrier RNA was omitted from the lysis buffer; 20 uL of Proteinase K (20 mg/ml) was added to the initial lysis mixture and incubated at 70 o C for 5 min; and an in-column DNAse I digestion step was performed after the first wash step by adding 5 uL DNAse I (NEB). The purified RNA was sequenced at the Australian Genome Research Facility (AGRF, Melbourne, Australia) using the NovaSeq Illumina platform, generating 2 x 150-base paired reads. The paired reads were then mapped using the reference genomes of exogenous viral agents as scaffolds and Geneious Prime software (version 2023.1.1). Master Seed Virus Bank : A Master Seed Virus Bank (MSV) was prepared by inoculating a culture of cells from the MCS with BinJ/JEV NSW/22 at an MOI of 0.1. The virus, as culture supernatant was harvested and stored as indicated earlier as the MSV. The BinJ/JEV NSW/22 MSV underwent identical extraneous agent testing as for the C6/36 MCS. Vaccine Preparation Purified BinJ/JEV NSW/22 vaccine preparations : A gradient purified preparation of BinJ/JEV NSW/22 was used as a reference vaccine 22 . Briefly, virions were precipitated from BinJ/JEV NSW/22 -infected culture supernatant by polyethylene glycol precipitation, before ultracentrifugation through a sucrose cushion and potassium tartrate gradient. Purified virus was collected, and buffer exchanged with sterile phosphate buffered saline (PBS) using a 30 kDa molecular weight cut off Amicon filter and stored at 4 o C. The protein concentration of purified virus was quantified by determining the amount of E protein against BSA standards following SDS-PAGE, total protein staining (SYPRO Ruby, Invitrogen) and analysis using ImageJ software 22 . Clarified culture supernatant vaccine preparation: C6/36 cells in RPMI/2% FBS were inoculated with BinJ/JEV NSW/22 and the supernatant harvested after 5–7 days. The culture supernatant was clarified by centrifugation. For the mouse study, a portion of the clarified supernatant was used non-inactivated, while the remaining supernatant was inactivated with formalin (as below). For the pig vaccine studies, the clarified supernatant was formalin inactivated. Prior to inactivation, the vaccine virus titre (as a measure of antigen concentration) was determined via TCID 50 using the methods described above. Deep sequencing was performed as for the MSC above. Formalin inactivation of vaccine A range of formalin concentrations (0%, 0.05%, 0.08%, 0.1%) were assessed for inactivation of BinJ/JEV NSW/22 in clarified culture supernatant from infected C6/36 cells (Table S3). Virus inactivation was monitored by TCID 50 and immunofluorescence assay (IFA) following three serial passages on C6/36 cells. Briefly, 2.5% formalin (diluted in PBS) was added drop-wise to BinJ/JEV NSW/22 viral (or mock-infected) culture supernatant, to the final desired concentration, or the supernatants were left untreated. The supernatants were incubated at 20°C for 1 hr followed by transfer to 4°C for the remaining period. Inactivation was monitored by passage of 0.3 ml of the virus/formalin preparations onto monolayers of C6/36 cells. After 7 days, the inoculated cells were seeded onto coverslips, fixed and tested by IFA with mAb 4G4 and using previously described methods 24,29 , while 0.3 ml of the culture medium was transferred to fresh C6/36 cell monolayers. A total of three passages were performed in this manner to assess viral inactivation. IFA results were recorded as the proportion of antigen-positive ([-] no positive cells, [+] weak positive, very few cells positive, [++] 90% of cells positive). Mouse immunogenicity studies Six to 8 week-old female CD1 mice (Animal Resources Centre, Murdoch, Western Australia) were immunised twice via the subcutaneous route at the base of the tail three weeks apart with 2 µg purified non-inactivated BinJ/JEV NSW/22 diluted in PBS or 100 µL of BinJV/JEV NSW22 in clarified C6/36 culture supernatant, either untreated or formalin inactivated (0.08% for 28 days at 4°C), with a pre-inactivation titre of 1.98 x 10 7 infectious units (IU)/mL. All doses were supplemented with the adjuvant Advax2™ (Vaxine Pty Ltd, Adelaide, Australia, 1 mg/mouse). On the day of the second injection (bleed 1), a serum sample was taken from each mouse via tail-bleed and assessed for neutralising antibodies to JEV NSW/22 using a virus microneutralisation assay as previously described using Vero cells (CCL-81) and 60 infectious units of JEV NSW/22 22 , as determined by back titration. Serum samples were also taken two (bleed 2) or 13 (bleed 3) weeks after dose two of the vaccine. Vaccination studies in weaner pigs This study was conducted at the Elizabeth Macarthur Agricultural Institute. Weaned, 4-week-old male piglets with an average body weight of 7.27 kg (range 3.49–10.60 kg) were obtained from a commercial pig farm. Forty-five piglets, born to gilts with negative serology (blocking ELISA) for JEV and related orthoflaviviruses (Murray Valley encephalitis virus (MVEV) and WNV), and with individual negative serology results for JEV and related orthoflaviviruses were chosen for the study. Piglets were randomly assigned to one of five vaccine treatment groups (n = 9) and received the appropriate vaccine as a 2 mL injection with a 28-day interval between prime and booster doses (Tables 1 , S4). The treatments were: 1) Sham vaccine - cell culture medium (Minimal Essential Medium, MEM); 2) Purified BinJ/JEV NSW/22 − 5 µg/mL in PBS (gradient purified as in 22 ) with 10 mg/mL Advax2™; 3) Formalin-inactivated BinJ/JEV NSW/22 (higher concentration) Emulsigen and gel – Undiluted clarified culture supernatant with a pre-inactivation titre of 1.98 x 10 7 IU/mL prepared with 12% v/v Emulsigen (MVP Adjuvants Phibro Animal Health Corp., Omaha, NE, USA) and 4% v/v aluminium hydroxide gel (SPI Pharma, Wilmington, DE, USA). 4) Formalin-inactivated BinJ/JEV NSW/22 (lower concentration) Emulsigen and gel – 1:5 dilution of the undiluted clarified culture supernatant used for Group 3 in PBS with the same final concentration of Emulsigen and aluminium hydroxide gel. 5) Formalin-inactivated BinJ/JEV NSW/22 (higher concentration) and gel – Undiluted clarified culture supernatant used for Group 3, formulated without Emulsigen but with 16% v/v aluminium hydroxide gel. All formalin-inactivated preparations were produced by adding formalin to a final concentration of 0.08% and incubation for 28 days at 4°C. Piglets were housed in indoor biological containment level 2 animal housing with additional JEV-specific biosecurity measures for containment and personal protection. Water and a commercial pellet diet appropriate to age was supplied ad libitum (Grolean weaner pellets and pig grower pellets, Vella Stock Feeds, Glendenning, NSW). Air temperature was controlled by central air-handling set initially at 30°C for 4-week-old pigs and reduced by 2°C per week until 20°C, where it was maintained for the remainder of the trial. After challenge with JEV, each room contained pigs from a mix of treatment groups except for the no-vaccine control group. The priming dose of vaccine was administered on day 0 (after 7 days of acclimation) when piglets were approximately 5 weeks of age. A booster of the same vaccine was administered on day 28. All animals were given a 2 mL dose of the relevant preparation by intramuscular injection in the neck using a 21-guage needle. The injection site was examined, and rectal temperature was taken each day for 5 days around the time of the priming dose of vaccine. Blood samples were collected from the jugular vein on days − 7, 0, 7, 14, 21, 28, 35 and 42. The serological response (determined by blocking ELISA) to vaccination was used to select two groups of vaccinated pigs for challenge with infectious JEV along with the unvaccinated control group. Challenge of weaner pigs with JEV Pigs were challenged with JEV by intradermal injection on day 42 (two weeks after administration of the booster vaccine). A dose of 1 x 10 5.0 TCID 50 of JEV NSW/22 diluted to 1 ml in serum free cell culture medium was administered by intradermal injection using a 25-gauge needle with equal volumes in four locations (two injection sites on the left and right lateral flank, respectively). The pigs were observed at least twice daily with once daily measurement of rectal temperature, clinical observation by a veterinarian and collection of an EDTA-treated blood sample for 10 days after challenge. Serum samples were also collected at weekly intervals on days 49, 56 and 63. The pigs were humanely euthanized at the completion of the experiment 21–23 days after JEV challenge by intravenous administration of pentobarbitone sodium (Lethobarb, Virbac) after intramuscular injection of the sedative azaperone (Stresnil, Elanco) 15 . Additional blood samples and fresh palatine tonsil were collected at this time to assess final JEV infection status and serological profile. Serum samples collected from pigs were assessed for antibodies to JEV and other orthoflaviviruses using blocking ELISA and neutralisation assay protocols. EDTA-treated blood and tonsil tissues were assessed for JEV by RT-qPCR and by virus isolation (tonsil), according to previously described methods 15 . Sow Vaccination Studies This study was conducted at the Queensland Animal Science Precinct (QASP), University of Queensland, Gatton Campus. Ten multiparous sows (1.5–3 yrs of age), that had not been infected with JEV and with no recent clinical history of systemic disease, were sourced from a commercial piggery in the Darling Downs region (Qld). Upon arrival, the sows were weighed, (average weight of 263 kg (range 240–290)) and housed at QASP in individual pens in a Department of Agriculture, Fisheries & Forestry (DAFF)-approved PC2 facility, and allowed to acclimate for 8 days prior to the first blood sampling and prime vaccination. Each animal was confirmed to be orthoflavivirus seronegative by blocking ELISA (orthoflavivirus group common mAb 6B6C-1 30 ). Animals received three vaccinations. The priming dose of the vaccine was administered on day 0 and a booster of the same batch of vaccine was administered on day 28. A third dose of the vaccine (new batch) was administered on day 70 (i.e. 6 weeks after the second dose). For each vaccine dose, each animal was given 4 mL of the inactivated vaccine formulation (higher concentration), double the dose used in the experimental efficacy study in weaner pigs (i.e., ~ 6.6 x 10 7 IU). The vaccine was administered by intramuscular injection in the shoulder muscles using an 18-gauge needle. Blood samples were collected from the auricular veins immediately prior to the first vaccination and subsequently every 14 days until termination of the trial. Animal weights were recorded every 10–15 days. After each vaccination, the injection site was marked with an indelible marker for easy site examination and monitoring. Animals were observed for about 30–60 minutes for localized and generalized hypersensitivity reactions. After the third vaccination, animals were observed for about 2 hours before they were humanely euthanised. Serum Neutralisation Assays The porcine sera were assessed for a JEV-specific antibody response using either a modified immunoplaque, 50% plaque reduction neutralisation (PRNT 50 ) assay (sow sera) 31 , or a 50% microneutralisation assay (Microneut 50 , weaner sera). The PRNT 50 was performed using Vero76 cells, using the following modifications: incubation with Medium 199, 2% carboxymethyl-cellulose (Sigma-Aldrich) and 3% heat-inactivated FBS was for 30 hours at 37°C and 5% CO 2 ; the primary antibody was rabbitinised anti-orthoflavivirus 4G2 purified antibody and the secondary antibody was IRDye800 donkey anti-rabbit (LiCor Biosciences, NE, USA). The virus used in the assays (JEV NSW/22 ) was diluted to give between 60–65 plaques per well, and the PRNT 50 titre was defined as the highest dilution that resulted in a 50% reduction in the number of plaques when compared to virus only controls. The geometric mean titres (GMTs) were calculated using Microsoft Excel. One-way analysis of variance (ANOVA) was performed for multiple comparison analysis with the α-level set at 0.05 with a Tukey’s post test (GraphPad Prism, V9). The microneut 50 , was performed essentially as previously described 32 , using Vero76 cells and an incubation period of 3 days and 158 infectious particles, as determined by back titration. The 50% cutoff was determined to be the average of the ODs of the virus only control. Blocking ELISA (bELISA) assays Antibodies to JEV in the pig sera (weaner trial) were detected by the mAb 989 blocking ELISA, as previously described 15 . Results were expressed as a percentage inhibition (PI%) relative to the reactivity of a negative control sample. A positive result was indicated by a PI > 60%, a negative result by a PI < 40% with other results considered equivocal. To provide end-point titres for the sera from experimentally infected pigs, samples were titrated in a 2-fold dilution series then tested in the same assay. The endpoint titre was defined as the highest dilution that gave a PI > 40%. The mouse sera were also assessed in a mAb 989 blocking ELISA. For this assay, BinJ/JEV NSW/22 virions, as a serum-free culture supernatant, were coated onto Greiner Bio-One high binding microplates at a pre-determined optimal concentration and the mAb 989 was applied at a non-saturating concentration as a HRP conjugate, with conjugation performed as per the manufacturer’s conditions (Lightning-Link HRP, Abcam). The orthoflavivirus-group reactive mAb 6B6C‐1 bELISA 30 , was performed as previously described 33,34 , with the use of a lysate of WNV infected cells. In the 6B6C‐1 orthoflavivirus assay, a positive result was indicated by a PI > 30%. Results Considerations for the development of a livestock-targeted JEV vaccine We have previously shown that purified BinJ/JEV NSW/22 formulated with a clinical grade adjuvant (Advax) induces a virus-neutralising antibody response in mice that protects against challenge with the JEV NSW/22 strain 22 . However, to develop a cost-effective veterinary vaccine, a vaccine derived of unpurified clarified culture supernatant harvested from BinJ/JEV NSW/22 -infected C6/36 cells was preferred. In anticipation of testing requirements for vaccine registration for use in livestock, Master cell (C6/36) and vaccine virus (BinJ/JEV NSW/22 ) stocks were produced and tested. Upon receipt of the C6/36 Aedes albopictus line from ATCC, the cells were passaged 25 times, with the majority (n = 19) of the passages under animal-derived component-free conditions. A Master Seed Cell Bank (MSC) was set down and characterised in terms of identity and the presence of extraneous agents. DNA barcoding using sequencing of the cytochrome c oxidase subunit I (COI) confirmed the cell derivation to be Aedes albopictus . Chromosomal enumeration of 20 cells of the C6/36 MSC revealed the presence of six chromosomes (Fig. S1). Extraneous agent testing confirmed the absence of persistent infection with bacteria, including mycoplasma, fungi and a panel of pathogenic viruses of health and biosecurity concern. The absence of detection of viral pathogens was also supported by deep sequencing analysis of extracted cellular RNA, which similarly did not reveal the presence of adventitious agents. The BinJ/JEV NSW/22 Master Seed Virus (MSV) also underwent extraneous agent testing, with no evidence of the same range of agents. To remove the Genetically Modified Organism (GMO) status of the vaccine, and the associated regulatory restrictions, various formalin inactivation parameters were assessed, in line with standard vaccine inactivation methodologies 35–37 . Unpurified BinJ/JEV NSW/22 vaccine consisting of clarified supernatant from infected C6/36 cell cultures was inactivated with 0.08% formalin following incubation at 20°C for 1 hour and then at 4°C for at least 28 days. This inactivation condition was selected based on the results of preliminary studies testing various formalin concentrations and incubation times; inactivation was confirmed by an absence of virus replication in cell culture following three serial passages (see Fig. S2 and Table S3) Safety and immunogenicity of unpurified, inactivated BinJ/JEV NSW/22 vaccine formulations in mice To determine if the vaccine using inactivated-unpurified material was capable of inducing a JEV-specific antibody response, CD1 mice were immunised via the subcutaneous route with 100 µL of either the inactivated formulation or a mock-inactivated (non-inactivated) preparation of the same antigen (5.75 x 10 5 IU/dose pre-inactivation titre), both supplemented with 1 mg/mouse of the adjuvant Advax2. These vaccines were given twice at 3-week intervals. After one dose, both vaccine preparations induced JEV-specific antibody responses as determined by microneutralisation assay, with 3/5 in the non-inactivated vaccine group and 4/5 in the inactivated vaccine group producing detectable neutralising antibodies (Fig. 1A). For comparative reference, a third group that received two doses of 2 µg purified vaccine (~ 10 7 IU/dose) without inactivation produced a strong neutralising response in all five mice (Fig. 1A). Seroconversion was also monitored by blocking ELISA two weeks after the second dose (second bleed) and again 11 weeks later (third bleed, Fig. 1B). Three of the five mice that received the non-inactivated vaccine returned a positive bELISA result when the serum was assessed at a minimum 1 in 100 dilution and 4/5 in the inactivated vaccine group produced a positive result. However, when serum was taken at a later time point, the antibodies levels were higher, with 4/5 mice receiving the non-inactivated unpurified and 5/5 mice receiving the inactivated formulation positive at or above a 1:100 serum dilution in blocking ELISA (Fig. 1B), although the increase in antibody levels was not significant (one-way ANOVA with Tukey’s multiple comparison test). Importantly, the mice displayed no adverse effects after two doses of either vaccine. These data indicated that a crude inactivated BinJ/JEV NSW/22 vaccine was safe and immunogenic in mice and induced an antibody response against JEV. Safety and immunogenicity of unpurified, inactivated vaccines in weaner pigs To determine if the BinJ/JEV NSW/22 vaccine was safe and efficacious in pigs, 5-week old male pigs, born to orthoflavivirus antibody-naïve gilts were vaccinated twice, 28 days apart, with one of five different BinJ/JEV NSW/22 vaccine formulations (Table S4, Fig. 2A). No adverse reactions such as swelling, erythema or tenderness were observed at the site of injection in any of the young pigs in the 5 days subsequent to the prime and booster doses for any of the vaccine preparations. There was no significant difference in the rectal temperature between groups (including the no-vaccine control) on any of the three days following the priming dose of vaccine (Fig. S3; p < 0.01). A JEV serological response was detected in all vaccinated weaner pigs by bELISA within seven days of the priming dose for those in vaccine groups 2–4 (Fig. 2B, Table S4) and robust immune responses following the boosting dose of vaccine for vaccine groups 3 and 4 (Fig. 2C, Table S5), with bELISA end point titres of 80–10,240. The immune response developed by pigs injected with the formalin-inactivated clarified supernatant preparation of BinJ/JEV NSW/22 with the adjuvant aluminum hydroxide gel only (Group 5) was inferior, with 100% positive in the bELISA only after a booster dose of vaccine (Table S4). As expected, the vaccine formulated with purified virus, without inactivation (group 2), produced the highest titres in virus neutralisations assays (Fig. 2D, Tables 1, S6). The majority of bELISA-positive samples from the weaner pigs vaccinated with the inactivated, clarified supernatant vaccine preparations (groups 3–5) also had neutralising activity against wild-type JEV, with the GMTs ranging between 17.1–31 (Tables 1, S6; Fig. 2D). Two neutralising assay formats were employed to assess the sera. Both assays indicated modest but superior responses of animals in Group 3 (higher dose with Emulsigen and gel) compared to other formulations of vaccines using unpurified, inactivated virus (Groups 4, 5; Fig. 2D, Tables 1, S6), although the increase in response was not statistically significant (One way ANOVA with Tukey’s multiple comparison test, p = 0.1228 for group 3 vs group 4 microneutralisation 50 assay). In the PRNT 50 (Table S6), some animals did not return a positive result, however, there was a clear reduction in plaque numbers for these samples that was not present in the unvaccinated animals indicating that a neutralising response had been generated, albeit below the limit of detection of the assay. This was confirmed for animals 90024, 90052 and 90023 in the lower dose clarified supernatant inactivated vaccine group (group 4) whereby a positive neutralisation response was detected by a microneutralisation 50 assay. One animal from group 3 (denoted 90042), did not produce a detectable neutralising antibody response in either neutralisation assay (Fig. 2D, Tables 1, S6) and had a much lower bELISA end point titre at days 35 and 42 post priming vaccine dose (1–2 weeks post booster vaccine dose) of 80 and 320 vs 640 - >5120 and 640 − 10,240 for other animals in that vaccine group (Fig. 2C, Table S5). Table 1 JEV neutralising antibody titres in sera of young pigs 2 weeks after the booster dose of each vaccine formulation as tested by microneutralisation 50 assay. Group Vaccine Treatment Range of micro-neut 50 Median Geometric Mean Titre* 1 No vaccine < 10 < 10 < 10 2 Purified BinJ/JEV NSW/22 with Advax2. Non-inactivated 20–160 80 63.5 3 Formalin-inactivated BinJ/JEV NSW/22 , higher dose + Emulsigen and aluminium hydroxide gel < 10–80 40 31 4 Formalin-inactivated BinJ/JEV NSW/22 , lower dose + Emulsigen and aluminium hydroxide gel 10–40 10 17.1 *For calculation purposes, where the microneutralisation 50 titre is below the minimum dilution factor tested, the value was set to 1. A . Timeline of BinJ/JEV NSW/22 vaccinations, WT JEV NSW22 challenge, and sample/data collections. B. serum percentage inhibition (PI) values against JEV antigen as measured by JEV-specific bELISA. Sera were assessed at a 1/10 dilution. Samples are considered positive if the PI is ≥ 60%. Samples were collected weekly post-the priming vaccination, up until day 28 (day of booster vaccination). C. Serum end-point JEV-specific bELISA titres for serum collected post-vaccine boost and post-JEV challenge (day 42), for the groups kept on for the challenge study. A positive bELISA result was considered ≥ 40%. Limit of quantification 1 in 10 dilution. D . Day 42 post-prime serum end-point neutralising antibody titres against JEV using microneutralisation 50 assay. Limit of quantification is 1 in 10. BinJ/JEV NSW/22 vaccine formulations: 1) no vaccine, black; 2) purified virions, non-inactivated with Advax adjuvant, red; 3) Formalin-inactivated BinJ/JEV NSW/22 as clarified cell culture supernatant (SN), higher concentration with Emulsigen/aluminium hydroxide gel adjuvant, blue; 4) Formalin-inactivated BinJ/JEV NSW/22 as clarified cell culture SN, lower concentration with Emulsigen/aluminium hydroxide gel adjuvant, purple; 5) Formalin-inactivated BinJ/JEV NSW/22 as clarified cell culture supernatant, higher concentration with aluminium hydroxide gel adjuvant only, orange. Protection of vaccinated weaner pigs from JEV challenge Although pigs in group 2 (purified, non-inactivated vaccine) had superior neutralising responses (Tables 1, S6; Fig. 2D), the objective was to proceed with a vaccine that could be produced cost-effectively (without extensive downstream processing and purification) and did not have the regulatory constraints associated with a GMO. The groups receiving the formalin-inactivated BinJ/JEV NSW/22 as clarified culture supernatant and adjuvanted with Emulsigen and aluminium hydroxide gel (Groups 3 and 4) were selected along with the negative control group (sham vaccine, Group 1) for challenge two weeks after the administration of the booster vaccine. After challenge, all of the unvaccinated weaner pigs were confirmed to have been infected with JEV, based on positive RT-qPCR tests of EDTA-treated blood with peak viraemia usually identified between 3 to 5 days after challenge (Table 2). Further, JEV RNA was detected and virus was isolated in cell culture from the tonsils of each of these pigs at the completion of the trial (day 21 or 23 after challenge). JEV detection (summarised in Table 2) confirmed an infection rate for the unvaccinated pigs of 100% (70.1–100%, 95% CI), under these experimental conditions. Antibodies to JEV were detected in the unvaccinated piglets at day 7 after challenge (Fig. 2C) and the titre of antibodies determined by bELISA ranged from 160–640 (Fig. 2, Table S5). The rectal temperature of unvaccinated pigs was higher than vaccinated pigs on Days 9 and 12 (Fig. S4). There was no evidence of JEV infection by RT-qPCR of blood and tonsils, nor JEV viral isolation from the tonsils in 17/18 pigs from the two vaccinated groups that were challenged with JEV whilst one pig in group 3 was positive for JEV, indicating an infection rate of 5.6% (1.0–25.8%, 95% CI, Table 2). It is worth noting that the sole vaccinated animal (90042) that became infected (based on detection of virus in blood and tonsils) had a lower bELISA titre (80) at day 35 and the absence of detectable neutralising activity at the time of challenge compared to other members of the group (Tables 1, S5, S6; Fig. 2). Overall, this indicated a vaccine efficacy of 94.4%. Table2 . Detection of JEV RNA by RT-qPCR in blood and tonsil samples collected after intradermal JEV infection challenge Treatment Group Animal ID JEV RT-PCR (Ct) # Days Post-challenge for EDTA blood samples Tonsil at end of trial (Day 21 or 23*) l 2 3 4 5 6 7 9 12 1. No vaccine 90003 neg. neg. 33.73 35.05 neg. neg. neg. neg. neg. 33.10* 90016 neg. neg. 30.27 31.2 neg. neg. neg. neg. neg. 31.34* 90017 neg. 34.78 30.26 33.16 neg. 36.62 neg. neg. neg. 26.84* 90019 neg. neg. 31.02 32.9 34.56 neg. 36.62 neg. neg. 34.60* 90025 neg. neg. 35.16 33.13 36.67 neg. neg. neg. neg. 26.30* 90027 neg. neg. 33.84 33.96 35.5 neg. neg. neg. neg. 28.79* 90063 neg. neg. 36 32.92 37 neg. neg. neg. neg. 29.29* 90064 35.48 36.46 35.34 35.96 neg. neg. neg. neg. neg. 29.27* 90066 neg. neg. neg. 36.81 33.2 neg. neg. neg. neg. 24.82* 3. Higher dose BinJ/JEV as clarified supernatant, inactivated 90000 neg. neg. neg. neg. no sample neg. neg. neg. neg. neg. 90001 neg. neg. neg. neg. neg. neg. neg. no sample neg. neg. 90004 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90014 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90030 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90042 neg. neg. 36.98 32.3 33.26 neg. neg. neg. neg. 25.34 90051 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90062 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90071 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 4. Lower dose BinJ/JEV as clarified supernatant, inactivated 90018 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90023 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90024 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90039 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90046 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90049 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90052 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90058 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. 90068 neg. neg. neg. neg. neg. neg. neg. neg. neg. neg. # Data are the Ct values for positive samples. Negative tests (no evidence of amplification at 45 cycles) are indicated (neg). Safety and immunogenicity of BinJ/JEV NSW/22 vaccination in multiparous sows A cohort of 10 multiparous sows, confirmed to be orthoflavivirus seronegative in pan-orthoflavivirus bELISA (Table S7), were vaccinated with the same batch of vaccine used in the group 3 weaner pigs – i.e. formalin-inactivated BinJ/JEV NSW/22 (higher concentration) with Emulsigen and aluminium hydroxide gel, undiluted clarified culture supernatant. The sows received three doses of 4 mL of the vaccine (twice the dose given to the weaner pigs) four weeks apart, with the third dose give six weeks after the first booster vaccination (dose 2, Fig. 3A). The animals were monitored for clinical signs post vaccination in addition to the immune response elicited. Neither following the prime or booster vaccinations, were there any local tissue reactions to the vaccine and none of the sows showed clinical signs indicative of systemic reactions, including hypersensitivity. The vaccinations had no effect on the animals’ appetite and overall demeanour. One animal was culled two weeks after receiving the first booster vaccine dose, due to conditions unrelated to the vaccinations (laminitis which was not responding to antibiotics and anti-inflammatory drugs). At the termination of the study, all nine remaining animals had gained an average weight of 17 kg (range 7–36 kg; Fig. S5). The immune response to the vaccine was monitored by a JEV bELISA and JEV neutralisation assay. While all animals gave negative results in the pan-orthoflavivirus assay upon arrival, the serum from one sow (582) returned a low-range positive result (PI > 60) in the JEV-specific bELISA (Table S8). However, it was negative in a JEV PRNT 50 assay and by an in-house developed anti-JEV total IgG lateral flow assay (data not shown), collectively indicating that this animal was unlikely to have been exposed to JEV prior to vaccination, and the bELISA result was more likely due to exposure to another orthoflavivirus. A JEV-specific serological response in all vaccinated animals was detected by bELISA in samples collected as early as 14 days post-prime vaccination (Fig. 3, Tables S8, S9), with the immune response boosted after the second dose, as indicated by the increase in titres at day 42 (Fig. 3). PRNT 50 assays confirmed that all sows produced a neutralising immune response against JEV as early as 14 days post-priming vaccine dose (PRNT 50 20–80, Table S10). When the sera were assessed in the JEV bELISA, end-point titres rose from a range of 20–160 following the priming vaccine, to 640–2560 two weeks post the booster vaccination (Fig. 3, Table S9). The results confirmed the safety of the inactivated BinJ/JEV NSW/22 vaccine formulation in older pigs and demonstrated its effectiveness in inducing a robust antibody response to JEV. Figure 3. Vaccination of sows with BinJ/JEV NSW/22. A) Timeline of vaccinations and blood sample collections; B) End-point titres of anti-JEV antibodies in sows at different times after vaccination determined by titration in the JEV bELISA. C) Anti-JEV neutralising titres as determined by PRNT 50 14- and 28- days post-priming vaccine dose of BinJ/JEV NSW/22 . Statistics by one-way ANOVA with Tukey’s post-test, whereby p = 0.0277 (*), 0.0021 (**) or < 0.0001 (***). ND = not detected. Discussion There is an urgent need for effective control of JEV infection in piggeries in the likelihood that the virus has become endemic in Australia, having already contributed to outbreaks of disease in piggeries over two summer periods, 2021/2022 and 2024/2025. A chimeric virus based on the genome backbone of the insect-specific orthoflavivirus BinJV, with the prM and E gene sequences from the Australian 2022 JEV (GIV) strain (BinJ/JEV NSW/22 ) was previously constructed as a candidate vaccine against JEV and shown to be safe and protective in mouse models of this disease 22 . To further assess the use of this vaccine in a veterinary context we prepared a range of formulations of the vaccine, using varying purity levels of virus, different adjuvants and with and without formalin inactivation to undertake vaccine trials in pigs. Our study confirmed that a relatively crude, inactivated formulation of BinJ/JEV NSW/22 was safe and immunogenic in both weaner pigs and multiparous sows and induced an immune response that could prevent viraemia and infection of target tissues in 94% of vaccinated young pigs. These studies corroborate and extend our previous findings that a live purified version of the vaccine protected immunodeficient mice from JEV challenge 22 . While the vaccine challenge study in weaner pigs was conducted after two immunisations with the inactivated, unpurified vaccine, our data provide evidence of neutralising responses after a single dose of the inactivated crude vaccine in weaner pigs and sows, similar to what has been reported for non-inactivated purified versions of BinJ/JEV NSW/22 in mice 22 , suggesting pigs also develop a rapid immune response to this type of vaccine. As imported vaccines are based on viruses of a different genotype (currently GIII), a locally produced vaccine based on the genotype of JEV responsible for the outbreak in Australia (GIV) maximises the potential for protection and also averts possible biosecurity risks arising from imported vaccines. Vaccine genotype matching may be important to provide maximum protection given prior research showing that the GIII-based live-attenuated at222-derived JEV vaccine conferred sterilising immunity against natural infection with GI in only a small proportion of vaccinated piglets 38 . Although experimental challenge would be needed for confirmation, field-based estimates indicated that a GIII live-attenuated JEV vaccine provided reduced protection against JEV-associated stillbirth and abortion in gilts when GI viruses were circulating, compared with its higher estimated efficacy against the homologous GIII strains 39 . Our data demonstrates that our BinJ/JEV NSW/22 vaccine protects against JEV infection, with a vaccine efficacy that is comparable to reported efficacies of other JEV vaccines in pigs 40 . Vaccination of pigs for JEV is practiced in Asian countries where the virus has been endemic with a range of live attenuated and cell culture derived inactivated virus vaccines 41 . There is limited efficacy data for these products 42 . Small scale experimental and field trial evaluations indicated similar efficacy for the attenuated S − strain vaccine 43 , formalin inactivated JEV 44 , recombinant virus vaccines 45 and a combination of formalin inactivated JEV with a DNA vaccine 46 . In our study, one vaccinated pig with lower bELISA titre than other vaccinates and an absence of a neutralising titre, developed viraemia. This may be the result of insufficient B-cell priming for this particular animal. Insufficient B-cell priming against JEV infection has been reported in the mouse model previously 47 . As this vaccine shows considerable promise, it is important to initiate studies to confirm safety by vaccinating animals across the range of ages and classes (breed, age, sex and reproductive status) that will be considered for administration of vaccine in a JEV control program in Australian piggeries. In particular, further efficacy data is required to confirm that the immune response and prevention of viraemia and tonsillar infection in weaner/growing pigs translates to prevention of reproductive disease in gilts/sows and infertility of boars. Our small-scale trial on multiparous sows provides preliminary confidence that the inactivated vaccine also induced JEV-specific neutralising antibody responses, which has been indicated as a correlate of protection for JEV in pigs and humans 46,48,49 . Thus, it is possible that vaccination will also protect breeder pigs (sows and boars) from JEV-induced reproductive impacts. Furthermore, the vaccine was well tolerated in these older animals, even after receiving three “double doses”, demonstrating that the vaccine is also safe in older pigs kept under conventional farming conditions with likely exposure to mosquitoes and other insects. However, safety and efficacy data will be best obtained through a large-scale field trial, as it is essential to vaccinate a large number of animals to exclude the potential for a low level of adverse outcomes and to assess the longevity of the protective immune response. In addition to the disease control benefits for pig producers, there are also possible public health implications by reducing the number of pigs that become viraemic and in turn amplify this virus to increase the proportion of infected mosquitoes. Although waterbirds are the natural reservoir for JEV, in particular, feral pigs likely play an epidemiological role in the transmission and maintenance of JEV on the Australian mainland 50 . A major advantage of our chimeric virus technology for production of a new vaccine is the ability to rapidly pivot to target new genotypes of JEV. Indeed, the BinJ/JEV NSW/22 vaccine virus took less than a month to generate once the sequence of the outbreak strain was available 22 . Furthermore, we have also successfully prepared BinJ/JEV chimeras for each of the 5 JEV genotypes and have shown in mice that the antibodies induced by the BinJ/JEV NSW/22 vaccine also neutralise other JEV genotypes 22 . The flexibility of the platform was recently shown for another veterinary vaccine candidate, whereby a BinJV chimera displaying the prM/E structural protein genes of a subtype of WNV, Kunjin strain, elicited protective immune responses in Australian saltwater crocodiles against skin lesions when applied as either an inactivated or non-inactivated antigen formulation 51 . The design of the vaccine also allows the development of serological tests that can differentiate vaccinated pigs from those that have had a natural infection (DIVA). Since the NS1 protein of JEV is immunodominant during natural infection, but is not included in the vaccine, this antigen is the ideal candidate for DIVA. The use of an unpurified vaccine formulated as clarified culture supernatant greatly reduces the downstream processing of the vaccine and significantly lowers the cost of production, an essential property of an economically feasible vaccine for production animals. In this context, the ability of the BinJ/JEV NSW/22 vaccine virus to grow efficiently in mosquito cell culture while being unable to replicate in vertebrate cells also enhances ease of production under low biosecurity requirements. While the crude vaccine preparations used to immunise pigs in this study were produced in adherent cultures of C6/36 cells in RPMI media supplemented with 2% FBS, we have recently shown that this vaccine virus can also be grown to high titre in serum-free-medium in suspension cultures of C6/36 cells 52 . As part of the present study, through extensive innocuity tests, we have also confirmed the absence of adventitious agents in the C6/36 cells line, corroborating the deep sequencing data of Miller and colleagues 53 . Further, formalin inactivation removes any GMO-associated regulatory restrictions of the vaccine while not compromising its protective capability. In summary, we have provided evidence that the BinJ/JEV NSW/22 vaccine candidate delivered as an inactivated, clarified cell culture supernatant provides protection in pigs against JEV viraemia and does not cause adverse reactions in the vaccinated animals. This vaccine is currently being assessed by Australian regulatory authorities for commercial manufacture and registration for use in pigs. Declarations Competing Interests Data included in this publication are based on a patent (WO/2018/176075) on which J.J.H., H.B-O., R.A.H., and J.H-P are inventors. J.N. and M.W. are paid employees of Tréidlia BioVet Pty Ltd, Australia. The remaining authors declare no conflicts of interest. Author Contribution Project oversight: J.H-P, P.D.K. H.B-O, R.A.H., M.W. Experimental design: P.M.H, H.D., J.J.H., G.H., J.N., R.A.H., H.B-O., P.D.K., J.H-P. Experiments: P.M.H, H.D., J.J.H., G.H., J.R.P., J.N., I.E.M., J.N., H.B-O., A.H.Y.L., N.K.Y.Y., M.S.M. Writing, original draft preparation: P.M.H, H.D., J.J.H., G.H., R.A.H., H.B-O., P.D.K., J.H-P. Writing, review and editing: all authors. Acknowledgement We are indebted to the following groups for assistance during this study: Virology Laboratory, EMAI: Veterinary professional staff for management and sampling of pigs in animal house trials. Technical and support staff for the care and management of pigs and provision of laboratory support associated with sample management, processing and testing; JBS Pork Australia for generously providing the weaned pigs and Greg Tuckett and Chrissie Kracht for providing training and expert advice on their care, management and sample collection; Sow trial at the Queensland Animal Science Precinct (QASP), UQ: We are grateful for the expert knowledge and skills of key personnel at QASP for assistance with facility and AEC approvals. We are indebted to the expertise of Milou Dekkers, Stacey Groves, and Chelsey Baker (all QASP) for oversight of the trial and animal welfare. We also thank other QASP staff who at various times assisted with husbandry; We are thankful for staff at Tréidlia BioVet who contributed to the supply of the formulated vaccines. We also thank Vaxine Pty Ltd for generous supply of the Advax adjuvant. 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The anamnestic neutralizing antibody response is critical for protection of mice from challenge following vaccination with a plasmid encoding the Japanese encephalitis virus premembrane and envelope genes. J Virol 73 , 5527–5534 (1999). https://doi.org/10.1128/JVI.73.7.5527-5534.1999 Vannice, K. S. et al. The future of Japanese encephalitis vaccination: expert recommendations for achieving and maintaining optimal JE control. NPJ Vaccines 6 , 82 (2021). https://doi.org/10.1038/s41541-021-00338-z Hombach, J., Solomon, T., Kurane, I., Jacobson, J. & Wood, D. Report on a WHO consultation on immunological endpoints for evaluation of new Japanese encephalitis vaccines, WHO, Geneva, 2–3 September, 2004. Vaccine 23 , 5205–5211 (2005). https://doi.org/10.1016/j.vaccine.2005.07.002 Furlong, M. et al. Japanese Encephalitis Enzootic and Epidemic Risks across Australia. Viruses 15 (2023). https://doi.org/10.3390/v15020450 Habarugira, G. et al. A chimeric vaccine protects farmed saltwater crocodiles from West Nile virus-induced skin lesions. NPJ Vaccines 8 , 93 (2023). https://doi.org/10.1038/s41541-023-00688-w Dawurung, J. S. et al. Serum-Free Suspension Culture of the Aedes albopictus C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production. Viruses 17 (2025). https://doi.org/10.3390/v17020250 Miller, J. R. et al. Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation. Gigascience 7 , 1–13 (2018). https://doi.org/10.1093/gigascience/gix135 Additional Declarations Competing interest reported. Data included in this publication are based on a patent (WO/2018/176075) on which J.J.H., H.B-O., R.A.H., and J.H-P are inventors. J.N. and M.W. are paid employees of Tréidlia BioVet Pty Ltd, Australia. The remaining authors declare no conflicts of interest. 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Queensland","correspondingAuthor":false,"prefix":"","firstName":"Henry","middleName":"","lastName":"de Malmanche","suffix":""},{"id":592223050,"identity":"80b52b12-11e9-4d82-8cb5-3b16b596047f","order_by":2,"name":"Jessica J Harrison","email":"","orcid":"","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"J","lastName":"Harrison","suffix":""},{"id":592223051,"identity":"989c7dff-a28a-4d40-95f5-57fbdd39ffb5","order_by":3,"name":"Gervais Habarugira","email":"","orcid":"","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Gervais","middleName":"","lastName":"Habarugira","suffix":""},{"id":592223052,"identity":"44cd31bb-391f-41d0-af71-ff596ac412dc","order_by":4,"name":"James R Potter","email":"","orcid":"","institution":"University of 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Y","lastName":"Lau","suffix":""},{"id":592223056,"identity":"0267c45a-028a-4a2f-b235-a79cda4376d4","order_by":8,"name":"Nicholas K Y Yuen","email":"","orcid":"","institution":"University of Queensland","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"K Y","lastName":"Yuen","suffix":""},{"id":592223057,"identity":"88581e9f-49e0-4cde-b562-6024980030d5","order_by":9,"name":"Jawad Nazir","email":"","orcid":"","institution":"Tréidlia Biovet","correspondingAuthor":false,"prefix":"","firstName":"Jawad","middleName":"","lastName":"Nazir","suffix":""},{"id":592223058,"identity":"caf8a7ab-b217-4b0d-9f2e-b47928f7c2dd","order_by":10,"name":"Mark White","email":"","orcid":"","institution":"Tréidlia Biovet","correspondingAuthor":false,"prefix":"","firstName":"Mark","middleName":"","lastName":"White","suffix":""},{"id":592223059,"identity":"c21abc08-69e0-4d30-bd8e-089d1e540632","order_by":11,"name":"Roy A Hall","email":"","orcid":"","institution":"University of 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Hobson-Peters","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYBACxgYog58dzGImUssBIJbsOUCkFjAAKTa4kUCkFuYG3oOPP1Tcs2e4+cbwBkOFdWID+xkDAg7jSzY4cKY4sXF2jrEFw5n0xAaeHEJaeMwkDrYlJDBL55hJMLYdTmxgIKzF/MfBfwn2bJJngFr+AbXwvyFsC8PBhgTGHgmgdYwNQC0ShGxp5kuWOHMsIXEGT1qxRcKxdOM2iWcFeLUYtvce/FBRk2Bvf/zwxhsfaqxl+/mTN+DX0syD4EgkAAk2vOqBQJ4BWQsh1aNgFIyCUTAyAQCWPEO2KaoAZAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Queensland","correspondingAuthor":true,"prefix":"","firstName":"Jody","middleName":"","lastName":"Hobson-Peters","suffix":""}],"badges":[],"createdAt":"2026-02-03 11:53:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8775364/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8775364/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103777190,"identity":"4bba82e0-b047-4fb6-9a3b-44d70ee20b68","added_by":"auto","created_at":"2026-03-02 19:10:26","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntibody responses in mice to BinJ/JEV\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eNSW/22 \u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003ewhen administered as different formulations.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Anti-JEV\u003csub\u003eNSW/22\u003c/sub\u003e\u003csup\u003e \u003c/sup\u003eneutralising antibody responses expressed as serum end point titres. Limit of quantification is 1 in 20. The serum was taken two weeks post dose 1 for the formulations of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003ein clarified C6/36 culture supernatant, either non-inactivated or formalin inactivated (inactivated), or 13 weeks post dose 2 for the formulation of 2 µg non-inactivated purified BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e\u003csub\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/sub\u003evirions.\u003cstrong\u003e (B) \u003c/strong\u003eAntibody responses in the mice were measured two (bleed 2) or 13 (bleed 3) weeks after dose 2 of the vaccine and analysed in JEV-specific blocking ELISA. Antibody responses are expressed as an end point titre with a limit of quantification of 1:100.\u0026nbsp; ND = not detected.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8775364/v1/e66697c0fb14568b18e41567.jpg"},{"id":103777192,"identity":"33e84b27-aba5-4c61-9f0c-990ace60466c","added_by":"auto","created_at":"2026-03-02 19:10:26","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":198998,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmune response of weaner pigs after vaccination with BinJ/JEV\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eNSW/22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Timeline of BinJ/JEV\u003csub\u003eNSW/22 \u003c/sub\u003evaccinations, WT JEV\u003csub\u003eNSW22 \u003c/sub\u003echallenge, and sample/data collections. \u003cstrong\u003eB.\u003c/strong\u003e serum percentage inhibition (PI) values against JEV antigen as measured by JEV-specific bELISA. Sera were assessed at a 1/10 dilution. Samples are considered positive if the PI is ≥60%. Samples were collected weekly post-the priming vaccination, up until day 28 (day of booster vaccination). \u003cstrong\u003eC.\u003c/strong\u003e Serum end-point JEV-specific bELISA titres for serum collected post-vaccine boost and post-JEV challenge (day 42), for the groups kept on for the challenge study. A positive bELISA result was considered ≥40%. Limit of quantification 1 in 10 dilution. \u003cstrong\u003eD\u003c/strong\u003e. Day 42 post-prime serum end-point neutralising antibody titres against JEV using microneutralisation\u003csub\u003e50\u003c/sub\u003e assay. Limit of quantification is 1 in 10. BinJ/JEV\u003csub\u003eNSW/22 \u003c/sub\u003evaccine formulations: 1) no vaccine, black; 2)\u0026nbsp; purified virions, non-inactivated with Advax adjuvant, red; 3) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22 \u003c/sub\u003eas clarified cell culture supernatant (SN), higher concentration with Emulsigen/aluminium hydroxide gel adjuvant, blue; 4) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22 \u003c/sub\u003eas clarified cell culture SN, lower concentration with Emulsigen/aluminium hydroxide gel adjuvant, purple; 5) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22 \u003c/sub\u003eas clarified cell culture supernatant, higher concentration with aluminium hydroxide gel adjuvant only, orange.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8775364/v1/916ab530aa671fc077870a56.jpg"},{"id":104400331,"identity":"e6905898-5548-4e4f-82c7-911e17acebd3","added_by":"auto","created_at":"2026-03-11 12:09:39","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69642,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVaccination of sows with BinJ/JEV\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eNSW/22. \u003c/strong\u003e\u003c/sub\u003eA) Timeline of vaccinations and blood sample collections;\u003cstrong\u003e \u003c/strong\u003eB) End-point titres of anti-JEV antibodies in sows at different times after vaccination determined by titration in the JEV bELISA. C) Anti-JEV neutralising titres as determined by PRNT\u003csub\u003e50\u003c/sub\u003e 14- and 28- days post-priming vaccine dose of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e. Statistics by one-way ANOVA with Tukey’s post-test, whereby p = 0.0277 (*), 0.0021 (**) or \u0026lt;0.0001 (***). ND = not detected.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8775364/v1/8487bef48d8f97ceaee4fb71.jpg"},{"id":104407920,"identity":"e95d0d54-6a25-4731-8424-034f7f2244a1","added_by":"auto","created_at":"2026-03-11 12:40:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1903514,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8775364/v1/9b3360e5-0eee-4c2f-88c1-c0b6d0a369b6.pdf"},{"id":103777193,"identity":"9a57c00c-750d-45dd-922c-fe48440b55b2","added_by":"auto","created_at":"2026-03-02 19:10:26","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4233637,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYINFORMATIONfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-8775364/v1/384964d6a22a0f4038515f30.docx"}],"financialInterests":"Competing interest reported. Data included in this publication are based on a patent (WO/2018/176075) on which J.J.H., H.B-O., R.A.H., and J.H-P are inventors. J.N. and M.W. are paid employees of Tréidlia BioVet Pty Ltd, Australia. The remaining authors declare no conflicts of interest.","formattedTitle":"A novel chimeric vaccine protects pigs from infection with Japanese encephalitis virus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eJapanese encephalitis virus (JEV, \u003cem\u003eOrthoflavivirus japonicum\u003c/em\u003e) is a mosquito-borne orthoflavivirus that sometimes causes fatal encephalitis in humans, reproductive disease in pigs and occasionally neurological disease in horses\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e. There are five genotypes (GI to GV) of JEV with GI and GIII the most prevalent\u003csup\u003e4,5\u003c/sup\u003e. Prior to the southern hemisphere summer of 2021/2022, JEV was considered exotic to Australia, with isolated incursions limited to the islands of the Torres Strait\u003csup\u003e6\u0026ndash;8\u003c/sup\u003e and Cape York Peninsula\u003csup\u003e9\u0026ndash;11\u003c/sup\u003e, none of which led to the establishment of transmission on the Australian mainland. However, in early 2022, a large outbreak of the rarer GIV of JEV was documented across Queensland (Qld), New South Wales (NSW), Victoria (Vic) and South Australia (SA), involving greater than 80 piggeries and causing 42 cases of human disease, resulting in seven fatalities as reported by the Australian Department of Health and Aged Care\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e. The virus now appears to have a strong foothold on the Australian mainland, with detections in mosquito populations (\u003cem\u003eCulex\u003c/em\u003e species) during the 2024/2025 Australian summer, evidence for active infections in feral and farmed pigs, in addition to human infections with several deaths (8 cases, 3 fatal, Tables S1, S2). Thus, JEV is now likely endemic in Australia.\u003c/p\u003e \u003cp\u003eThe 2022 outbreak of JEV infection caused large scale economic and welfare impacts through reproductive losses for the pig industry in eastern Australia. In NSW, impacted piggeries saw up to 60% of porcine fetuses and neonates affected on farms during the summer of 2021/22 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.agriculture.gov.au/biosecurity-trade/pests-diseases-weeds/animal/japanese-encephalitis\u003c/span\u003e\u003cspan address=\"https://www.agriculture.gov.au/biosecurity-trade/pests-diseases-weeds/animal/japanese-encephalitis\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swinehealth.org/wp-content/uploads/2024/03/2024-JEV-Economic-Assessment-SHIC-White-Paper-Final.pdf\u003c/span\u003e\u003cspan address=\"https://www.swinehealth.org/wp-content/uploads/2024/03/2024-JEV-Economic-Assessment-SHIC-White-Paper-Final.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) Most of the losses in pigs were due to abortions and stillbirths often associated with severe congenital defects. Neurological disease was sometimes observed in live-born piglets soon after birth. Such clinical manifestations seen in the farmed pigs infected with the GIV strain of JEV were consistent with those seen elsewhere in the world where other JEV genotypes circulate\u003csup\u003e15\u0026ndash;19\u003c/sup\u003e. Additionally, there were the anticipated impacts on reproduction due to testicular infection in boars, which resulted in poor semen quality or no viable sperm\u003csup\u003e20\u003c/sup\u003e. Apart from the impact on boars, JEV infection does not frequently result in disease in healthy, non-pregnant adult or growing pigs. However, they are a potential reservoir for virus amplification which has public health implications\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile veterinary vaccines for JEV are commercially available in some countries for the vaccination of pigs and/or horses (e.g., Nisseiken inactivated and live attenuated, Japan; Anyang300 live-attenuated, South Korea)\u003csup\u003e21\u003c/sup\u003e, none are available in Australia. Currently, all commercially available vaccines are based on GIII JEV strains, while the Australian outbreak involved a strain of GIV \u0026ndash; a much rarer JEV genotype with antigenic differences\u003csup\u003e22,23\u003c/sup\u003e. While cross-protection is expected between vaccines produced to different genotypes, this protection might not be optimal\u003csup\u003e21\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn addition to orthoflaviviruses that can infect both vertebrate and arthropod hosts, there are insect-specific orthoflaviviruses, which can infect arthropod hosts but are not replication competent in vertebrate cells. As such, these viruses offer opportunities to develop candidate vaccines that will negate problems with live attenuated vaccines, such as reversion to virulence, and provide additional production safety. A vaccine technology based on modifying an insect-specific orthoflavivirus (Binjari virus, BinJV) to produce chimeric viruses that display the surface proteins of pathogenic orthoflaviviruses (such as JEV, West Nile virus (WNV), dengue virus and Zika virus) has recently been developed\u003csup\u003e24\u003c/sup\u003e. These chimeric viruses grow efficiently in mosquito cell cultures but have been shown not to replicate in vertebrate cells, providing a high level of safety for vaccine production and administration. In response to the 2022 Australian JEV outbreak, a chimeric virus containing the prM and E surface glycoprotein genes of the outbreak strain (JEV\u003csub\u003eNSW/22\u003c/sub\u003e) in the BinJV genome backbone (BinJ/JEV\u003csub\u003eNSW22\u003c/sub\u003e-prME, herein abbreviated to BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e) was rapidly constructed and produced to high titre in C6/36 cells\u003csup\u003e22\u003c/sup\u003e. Using a sensitive immunodeficient mouse model of JEV\u003csub\u003eNSW/22\u003c/sub\u003e disease\u003csup\u003e25\u003c/sup\u003e, vaccination with BinJV/JEV\u003csub\u003eNSW/22\u003c/sub\u003e was shown to generate a protective anti-JEV immune response against lethal virus challenge\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study we assessed the application of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e as a vaccine for pigs, using an established pig model of JEV GIV infection\u003csup\u003e15\u003c/sup\u003e and investigated the protection provided to young pigs against challenge with JEV\u003csub\u003eNSW/22\u003c/sub\u003e. Immunogenicity and safety of the vaccine was also assessed in older multiparous sows.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics approvals\u003c/h2\u003e \u003cp\u003eAll animal work was conducted in accordance with the \u0026ldquo;\u003cem\u003eAustralian code for the care and use of animals for scientific purposes\u003c/em\u003e\u0026rdquo; as defined by the National Health and Medical Research Council of Australia. Animal experiments in mice and multiparous sows, performed at the University of Queensland were approved by the University of Queensland Animal Ethics Committee (permit numbers 2020/AE000118, 2022/AE000862). The multiparous sow experiment was performed under the Australian Department of Agriculture, Fisheries and Forestry (DAFF) permit 2023/010. Dealing with JEV was approved under the Queensland Biosecurity Act (PRID000998). Vaccination and challenge studies in weaner pigs were conducted with approval of the Elizabeth Macarthur Agriculture Institute Animal Ethics Committee (Project ID 341), Biosafety Committee (Approval M22/07) and DAFF permit (2022/047).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell lines and Culture\u003c/h3\u003e\n\u003cp\u003eC6/36 cells (\u003cem\u003eAedes albopictus\u003c/em\u003e, ATCC CRL-1660) were maintained in Roswell Park Memorial Institute 1640 (RPMI 1640) medium, supplemented with 5% Australian sourced fetal bovine serum (FBS) at 28 \u003csup\u003eo\u003c/sup\u003eC. Detailed handling of C6/36 cells for vaccine production are provided in a later section. Vero76 cells (ATCC-CRL1587; \u003cem\u003eCercopithecus aethiops\u003c/em\u003e, African green monkey kidney) and Vero cells (ATCC CCL-81) were maintained in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM) and supplemented with 5% FBS, 50 U/ml penicillin, 50 \u0026micro;g/ml streptomycin and 2 mM L-glutamine.\u003c/p\u003e\n\u003ch3\u003eVirus Isolates and Culture\u003c/h3\u003e\n\u003cp\u003eThe 2022 Australian outbreak strain of JEV (JEV\u003csub\u003eNSW/22\u003c/sub\u003e) was previously isolated from an infected porcine fetus (Genbank: OP904182) (O883/NSW/22). A stock of this isolate (O909) was generated by passage in cell culture using Vero76 cells for vaccine efficacy studies. The chimeric virus BinJ/JEV\u003csub\u003eNSW/22\u0026minus;\u003c/sub\u003eprME (herein described as BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e) has been previously reported\u003csup\u003e22\u003c/sup\u003e. This virus was generated by replacing the prM and E genes of BinJV with the corresponding sequences amplified from JEV\u003csub\u003eNSW/22\u003c/sub\u003e and the genome of the rescued virus was deep sequenced to confirm its authenticity\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eStocks of JEV\u003csub\u003eNSW/22\u003c/sub\u003e and BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e were generated by infecting Vero (JEV\u003csub\u003eNSW/22\u003c/sub\u003e only) or C6/36 cell monolayers with virus at a multiplicity of infection (MOI) of 0.1 and incubating at 28 \u003csup\u003eo\u003c/sup\u003eC (C6/36 cells) or 37 \u003csup\u003eo\u003c/sup\u003eC with 5% CO\u003csub\u003e2\u003c/sub\u003e (Vero76 cells) for 1 hr. Following this, inoculum was removed and replaced with fresh maintenance medium containing 2% FBS and incubated at the appropriate temperature for 5\u0026ndash;7 days post-infection (dpi), before harvesting the medium and centrifuging at 3,000 rpm, 4 \u003csup\u003eo\u003c/sup\u003eC for 10 min. The clarified supernatant was then supplemented with additional FBS to increase the total concentration to 10%, aliquoted and stored at -80 \u003csup\u003eo\u003c/sup\u003eC until required. The titre of virus stocks was calculated using the method of Reed and Muench\u003csup\u003e26\u003c/sup\u003e to determine the 50% tissue culture infectious dose (TCID\u003csub\u003e50\u003c/sub\u003e) in Vero or C6/36 cells for JEV\u003csub\u003eNSW/22\u003c/sub\u003e and C6/36 cells for BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e, after immunolabelling the cells with the pan-orthoflavivirus NS1 monoclonal antibody 4G4\u003csup\u003e27\u003c/sup\u003e or anti-JEV mAb 989\u003csup\u003e28\u003c/sup\u003e described previously\u003csup\u003e15,22\u003c/sup\u003e. In some instances, TCID\u003csub\u003e50\u003c/sub\u003e values were converted to infectious units per mL (IU/mL) using the Poisson distribution, under the assumption of a single-hit infection model where 1 TCID\u003csub\u003e50\u003c/sub\u003e corresponds to 0.69 IU, and assuming random distribution of particles\u003c/p\u003e\n\u003ch3\u003eProduction of Master Seed Banks\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMaster Seed Cell Bank\u003c/span\u003e. A Master Seed Cell Bank (MSC) of C6/36 cells (\u003cem\u003eAedes albopictus\u003c/em\u003e, ATCC CRL-1660) was set down for vaccine antigen production. C6/36 cells at 6 passages following their receipt from ATCC were passaged 19 times in serum free medium (sf900-III, Gibco) and a further 5 times in RPMI/5% Australian sourced FBS. As adherent cultures, the cells were dislodged using a cell scraper to ablate the requirement for use of animal derived materials such as trypsin. Characterisation of the MSC was performed on cell supernatant or cell lysates, as stipulated by the company. Karyotyping of the cells was performed at Monash Health Pathology (Clayton, Vic, Australia), while species identification was performed at Cellbank Australia (Westmead, NSW, Australia). Extraneous agent testing for viral, bacterial and fungal contaminants of the cells was performed at three NATA-accredited (National Association of Testing Authorities, Australia) laboratories: Australian Centre for Disease Preparedness (Geelong, Vic, Australia), Eurofins Laboratories Pty Ltd (Girraween, NSW, Australia) and the Elizabeth Macarthur Agricultural Institute Laboratory Services (Menangle, NSW, Australia).\u003c/p\u003e \u003cp\u003eTo confirm the absence of exogenous viral agents, deep sequencing was performed on RNA extracted from conditioned C6/36 media. The RNA was extracted using the Macherey-Nagal Nucleospin RNA virus kit as per the manufacturer\u0026rsquo;s instructions, with the following modifications: carrier RNA was omitted from the lysis buffer; 20 uL of Proteinase K (20 mg/ml) was added to the initial lysis mixture and incubated at 70 \u003csup\u003eo\u003c/sup\u003eC for 5 min; and an in-column DNAse I digestion step was performed after the first wash step by adding 5 uL DNAse I (NEB). The purified RNA was sequenced at the Australian Genome Research Facility (AGRF, Melbourne, Australia) using the NovaSeq Illumina platform, generating 2 x 150-base paired reads. The paired reads were then mapped using the reference genomes of exogenous viral agents as scaffolds and Geneious Prime software (version 2023.1.1).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMaster Seed Virus Bank\u003c/span\u003e: A Master Seed Virus Bank (MSV) was prepared by inoculating a culture of cells from the MCS with BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e at an MOI of 0.1. The virus, as culture supernatant was harvested and stored as indicated earlier as the MSV. The BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e MSV underwent identical extraneous agent testing as for the C6/36 MCS.\u003c/p\u003e\n\u003ch3\u003eVaccine Preparation\u003c/h3\u003e\n\u003cp\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePurified BinJ/JEV\u003c/span\u003e \u003csub\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNSW/22\u003c/span\u003e \u003c/sub\u003e \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003evaccine preparations\u003c/span\u003e: A gradient purified preparation of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e was used as a reference vaccine\u003csup\u003e22\u003c/sup\u003e. Briefly, virions were precipitated from BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e-infected culture supernatant by polyethylene glycol precipitation, before ultracentrifugation through a sucrose cushion and potassium tartrate gradient. Purified virus was collected, and buffer exchanged with sterile phosphate buffered saline (PBS) using a 30 kDa molecular weight cut off Amicon filter and stored at 4 \u003csup\u003eo\u003c/sup\u003eC. The protein concentration of purified virus was quantified by determining the amount of E protein against BSA standards following SDS-PAGE, total protein staining (SYPRO Ruby, Invitrogen) and analysis using ImageJ software\u003csup\u003e22\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eClarified culture supernatant vaccine preparation:\u003c/h2\u003e \u003cp\u003eC6/36 cells in RPMI/2% FBS were inoculated with BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e and the supernatant harvested after 5\u0026ndash;7 days. The culture supernatant was clarified by centrifugation. For the mouse study, a portion of the clarified supernatant was used non-inactivated, while the remaining supernatant was inactivated with formalin (as below). For the pig vaccine studies, the clarified supernatant was formalin inactivated. Prior to inactivation, the vaccine virus titre (as a measure of antigen concentration) was determined via TCID\u003csub\u003e50\u003c/sub\u003e using the methods described above. Deep sequencing was performed as for the MSC above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFormalin inactivation of vaccine\u003c/h3\u003e\n\u003cp\u003eA range of formalin concentrations (0%, 0.05%, 0.08%, 0.1%) were assessed for inactivation of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e in clarified culture supernatant from infected C6/36 cells (Table S3). Virus inactivation was monitored by TCID\u003csub\u003e50\u003c/sub\u003e and immunofluorescence assay (IFA) following three serial passages on C6/36 cells. Briefly, 2.5% formalin (diluted in PBS) was added drop-wise to BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e viral (or mock-infected) culture supernatant, to the final desired concentration, or the supernatants were left untreated. The supernatants were incubated at 20\u0026deg;C for 1 hr followed by transfer to 4\u0026deg;C for the remaining period. Inactivation was monitored by passage of 0.3 ml of the virus/formalin preparations onto monolayers of C6/36 cells. After 7 days, the inoculated cells were seeded onto coverslips, fixed and tested by IFA with mAb 4G4 and using previously described methods\u003csup\u003e24,29\u003c/sup\u003e, while 0.3 ml of the culture medium was transferred to fresh C6/36 cell monolayers. A total of three passages were performed in this manner to assess viral inactivation. IFA results were recorded as the proportion of antigen-positive ([-] no positive cells, [+] weak positive, very few cells positive, [++]\u0026thinsp;\u0026lt;\u0026thinsp;30% of cells positive, [+++] strong positive, \u0026gt;\u0026thinsp;90% of cells positive).\u003c/p\u003e\n\u003ch3\u003eMouse immunogenicity studies\u003c/h3\u003e\n\u003cp\u003eSix to 8 week-old female CD1 mice (Animal Resources Centre, Murdoch, Western Australia) were immunised twice via the subcutaneous route at the base of the tail three weeks apart with 2 \u0026micro;g purified non-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e diluted in PBS or 100 \u0026micro;L of BinJV/JEV\u003csub\u003eNSW22\u003c/sub\u003e in clarified C6/36 culture supernatant, either untreated or formalin inactivated (0.08% for 28 days at 4\u0026deg;C), with a pre-inactivation titre of 1.98 x 10\u003csup\u003e7\u003c/sup\u003e infectious units (IU)/mL. All doses were supplemented with the adjuvant Advax2\u0026trade; (Vaxine Pty Ltd, Adelaide, Australia, 1 mg/mouse). On the day of the second injection (bleed 1), a serum sample was taken from each mouse via tail-bleed and assessed for neutralising antibodies to JEV\u003csub\u003eNSW/22\u003c/sub\u003e using a virus microneutralisation assay as previously described using Vero cells (CCL-81) and 60 infectious units of JEV\u003csub\u003eNSW/22\u003c/sub\u003e\u003csup\u003e22\u003c/sup\u003e, as determined by back titration. Serum samples were also taken two (bleed 2) or 13 (bleed 3) weeks after dose two of the vaccine.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVaccination studies in weaner pigs\u003c/h2\u003e \u003cp\u003eThis study was conducted at the Elizabeth Macarthur Agricultural Institute. Weaned, 4-week-old male piglets with an average body weight of 7.27 kg (range 3.49\u0026ndash;10.60 kg) were obtained from a commercial pig farm. Forty-five piglets, born to gilts with negative serology (blocking ELISA) for JEV and related orthoflaviviruses (Murray Valley encephalitis virus (MVEV) and WNV), and with individual negative serology results for JEV and related orthoflaviviruses were chosen for the study.\u003c/p\u003e \u003cp\u003ePiglets were randomly assigned to one of five vaccine treatment groups (n\u0026thinsp;=\u0026thinsp;9) and received the appropriate vaccine as a 2 mL injection with a 28-day interval between prime and booster doses (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, S4). The treatments were: \u003cb\u003e1)\u003c/b\u003e \u003cb\u003eSham vaccine\u003c/b\u003e - cell culture medium (Minimal Essential Medium, MEM); \u003cb\u003e2)\u003c/b\u003e \u003cb\u003ePurified BinJ/JEV\u003c/b\u003e\u003csub\u003e\u003cb\u003eNSW/22\u003c/b\u003e\u003c/sub\u003e \u0026minus;\u0026thinsp;5 \u0026micro;g/mL in PBS (gradient purified as in \u003csup\u003e22\u003c/sup\u003e) with 10 mg/mL Advax2\u0026trade;; \u003cb\u003e3)\u003c/b\u003e \u003cb\u003eFormalin-inactivated BinJ/JEV\u003c/b\u003e\u003csub\u003e\u003cb\u003eNSW/22\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(higher concentration) Emulsigen and gel\u003c/b\u003e \u0026ndash; Undiluted clarified culture supernatant with a pre-inactivation titre of 1.98 x 10\u003csup\u003e7\u003c/sup\u003e IU/mL prepared with 12% v/v Emulsigen (MVP Adjuvants Phibro Animal Health Corp., Omaha, NE, USA) and 4% v/v aluminium hydroxide gel (SPI Pharma, Wilmington, DE, USA). \u003cb\u003e4)\u003c/b\u003e \u003cb\u003eFormalin-inactivated BinJ/JEV\u003c/b\u003e\u003csub\u003e\u003cb\u003eNSW/22\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(lower concentration) Emulsigen and gel\u003c/b\u003e \u0026ndash; 1:5 dilution of the undiluted clarified culture supernatant used for Group 3 in PBS with the same final concentration of Emulsigen and aluminium hydroxide gel. \u003cb\u003e5)\u003c/b\u003e \u003cb\u003eFormalin-inactivated BinJ/JEV\u003c/b\u003e\u003csub\u003e\u003cb\u003eNSW/22\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(higher concentration) and gel \u0026ndash;\u003c/b\u003e Undiluted clarified culture supernatant used for Group 3, formulated without Emulsigen but with 16% v/v aluminium hydroxide gel. All formalin-inactivated preparations were produced by adding formalin to a final concentration of 0.08% and incubation for 28 days at 4\u0026deg;C.\u003c/p\u003e \u003cp\u003ePiglets were housed in indoor biological containment level 2 animal housing with additional JEV-specific biosecurity measures for containment and personal protection. Water and a commercial pellet diet appropriate to age was supplied \u003cem\u003ead libitum\u003c/em\u003e (Grolean weaner pellets and pig grower pellets, Vella Stock Feeds, Glendenning, NSW). Air temperature was controlled by central air-handling set initially at 30\u0026deg;C for 4-week-old pigs and reduced by 2\u0026deg;C per week until 20\u0026deg;C, where it was maintained for the remainder of the trial. After challenge with JEV, each room contained pigs from a mix of treatment groups except for the no-vaccine control group.\u003c/p\u003e \u003cp\u003eThe priming dose of vaccine was administered on day 0 (after 7 days of acclimation) when piglets were approximately 5 weeks of age. A booster of the same vaccine was administered on day 28. All animals were given a 2 mL dose of the relevant preparation by intramuscular injection in the neck using a 21-guage needle. The injection site was examined, and rectal temperature was taken each day for 5 days around the time of the priming dose of vaccine. Blood samples were collected from the jugular vein on days\u0026thinsp;\u0026minus;\u0026thinsp;7, 0, 7, 14, 21, 28, 35 and 42. The serological response (determined by blocking ELISA) to vaccination was used to select two groups of vaccinated pigs for challenge with infectious JEV along with the unvaccinated control group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eChallenge of weaner pigs with JEV\u003c/h2\u003e \u003cp\u003ePigs were challenged with JEV by intradermal injection on day 42 (two weeks after administration of the booster vaccine). A dose of 1 x 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JEV\u003csub\u003eNSW/22\u003c/sub\u003e diluted to 1 ml in serum free cell culture medium was administered by intradermal injection using a 25-gauge needle with equal volumes in four locations (two injection sites on the left and right lateral flank, respectively).\u003c/p\u003e \u003cp\u003eThe pigs were observed at least twice daily with once daily measurement of rectal temperature, clinical observation by a veterinarian and collection of an EDTA-treated blood sample for 10 days after challenge. Serum samples were also collected at weekly intervals on days 49, 56 and 63. The pigs were humanely euthanized at the completion of the experiment 21\u0026ndash;23 days after JEV challenge by intravenous administration of pentobarbitone sodium (Lethobarb, Virbac) after intramuscular injection of the sedative azaperone (Stresnil, Elanco)\u003csup\u003e15\u003c/sup\u003e. Additional blood samples and fresh palatine tonsil were collected at this time to assess final JEV infection status and serological profile.\u003c/p\u003e \u003cp\u003eSerum samples collected from pigs were assessed for antibodies to JEV and other orthoflaviviruses using blocking ELISA and neutralisation assay protocols. EDTA-treated blood and tonsil tissues were assessed for JEV by RT-qPCR and by virus isolation (tonsil), according to previously described methods\u003csup\u003e15\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eSow Vaccination Studies\u003c/h2\u003e \u003cp\u003eThis study was conducted at the Queensland Animal Science Precinct (QASP), University of Queensland, Gatton Campus. Ten multiparous sows (1.5\u0026ndash;3 yrs of age), that had not been infected with JEV and with no recent clinical history of systemic disease, were sourced from a commercial piggery in the Darling Downs region (Qld). Upon arrival, the sows were weighed, (average weight of 263 kg (range 240\u0026ndash;290)) and housed at QASP in individual pens in a Department of Agriculture, Fisheries \u0026amp; Forestry (DAFF)-approved PC2 facility, and allowed to acclimate for 8 days prior to the first blood sampling and prime vaccination.\u003c/p\u003e \u003cp\u003eEach animal was confirmed to be orthoflavivirus seronegative by blocking ELISA (orthoflavivirus group common mAb 6B6C-1\u003csup\u003e30\u003c/sup\u003e). Animals received three vaccinations. The priming dose of the vaccine was administered on day 0 and a booster of the same batch of vaccine was administered on day 28. A third dose of the vaccine (new batch) was administered on day 70 (i.e. 6 weeks after the second dose). For each vaccine dose, each animal was given 4 mL of the inactivated vaccine formulation (higher concentration), double the dose used in the experimental efficacy study in weaner pigs (i.e., ~\u0026thinsp;6.6 x 10\u003csup\u003e7\u003c/sup\u003e IU). The vaccine was administered by intramuscular injection in the shoulder muscles using an 18-gauge needle. Blood samples were collected from the auricular veins immediately prior to the first vaccination and subsequently every 14 days until termination of the trial. Animal weights were recorded every 10\u0026ndash;15 days. After each vaccination, the injection site was marked with an indelible marker for easy site examination and monitoring. Animals were observed for about 30\u0026ndash;60 minutes for localized and generalized hypersensitivity reactions. After the third vaccination, animals were observed for about 2 hours before they were humanely euthanised.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSerum Neutralisation Assays\u003c/h2\u003e \u003cp\u003eThe porcine sera were assessed for a JEV-specific antibody response using either a modified immunoplaque, 50% plaque reduction neutralisation (PRNT\u003csub\u003e50\u003c/sub\u003e) assay (sow sera)\u003csup\u003e31\u003c/sup\u003e, or a 50% microneutralisation assay (Microneut\u003csub\u003e50\u003c/sub\u003e, weaner sera). The PRNT\u003csub\u003e50\u003c/sub\u003e was performed using Vero76 cells, using the following modifications: incubation with Medium 199, 2% carboxymethyl-cellulose (Sigma-Aldrich) and 3% heat-inactivated FBS was for 30 hours at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e; the primary antibody was rabbitinised anti-orthoflavivirus 4G2 purified antibody and the secondary antibody was IRDye800 donkey anti-rabbit (LiCor Biosciences, NE, USA). The virus used in the assays (JEV\u003csub\u003eNSW/22\u003c/sub\u003e) was diluted to give between 60\u0026ndash;65 plaques per well, and the PRNT\u003csub\u003e50\u003c/sub\u003e titre was defined as the highest dilution that resulted in a 50% reduction in the number of plaques when compared to virus only controls. The geometric mean titres (GMTs) were calculated using Microsoft Excel. One-way analysis of variance (ANOVA) was performed for multiple comparison analysis with the α-level set at 0.05 with a Tukey\u0026rsquo;s post test (GraphPad Prism, V9).\u003c/p\u003e \u003cp\u003eThe microneut\u003csub\u003e50\u003c/sub\u003e, was performed essentially as previously described\u003csup\u003e32\u003c/sup\u003e, using Vero76 cells and an incubation period of 3 days and 158 infectious particles, as determined by back titration. The 50% cutoff was determined to be the average of the ODs of the virus only control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eBlocking ELISA (bELISA) assays\u003c/h2\u003e \u003cp\u003eAntibodies to JEV in the pig sera (weaner trial) were detected by the mAb 989 blocking ELISA, as previously described\u003csup\u003e15\u003c/sup\u003e. Results were expressed as a percentage inhibition (PI%) relative to the reactivity of a negative control sample. A positive result was indicated by a PI\u0026thinsp;\u0026gt;\u0026thinsp;60%, a negative result by a PI\u0026thinsp;\u0026lt;\u0026thinsp;40% with other results considered equivocal. To provide end-point titres for the sera from experimentally infected pigs, samples were titrated in a 2-fold dilution series then tested in the same assay. The endpoint titre was defined as the highest dilution that gave a PI\u0026thinsp;\u0026gt;\u0026thinsp;40%.\u003c/p\u003e \u003cp\u003eThe mouse sera were also assessed in a mAb 989 blocking ELISA. For this assay, BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e virions, as a serum-free culture supernatant, were coated onto Greiner Bio-One high binding microplates at a pre-determined optimal concentration and the mAb 989 was applied at a non-saturating concentration as a HRP conjugate, with conjugation performed as per the manufacturer\u0026rsquo;s conditions (Lightning-Link HRP, Abcam).\u003c/p\u003e \u003cp\u003eThe orthoflavivirus-group reactive mAb 6B6C‐1 bELISA\u003csup\u003e30\u003c/sup\u003e, was performed as previously described\u003csup\u003e33,34\u003c/sup\u003e, with the use of a lysate of WNV infected cells. In the 6B6C‐1 orthoflavivirus assay, a positive result was indicated by a PI\u0026thinsp;\u0026gt;\u0026thinsp;30%.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\"\u003e\n \u003ch2\u003eConsiderations for the development of a livestock-targeted JEV vaccine\u003c/h2\u003e\n \u003cp\u003eWe have previously shown that purified BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e formulated with a clinical grade adjuvant (Advax) induces a virus-neutralising antibody response in mice that protects against challenge with the JEV\u003csub\u003eNSW/22\u003c/sub\u003e strain\u003csup\u003e22\u003c/sup\u003e. However, to develop a cost-effective veterinary vaccine, a vaccine derived of unpurified clarified culture supernatant harvested from BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e-infected C6/36 cells was preferred. In anticipation of testing requirements for vaccine registration for use in livestock, Master cell (C6/36) and vaccine virus (BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e) stocks were produced and tested. Upon receipt of the C6/36 \u003cem\u003eAedes albopictus\u003c/em\u003e line from ATCC, the cells were passaged 25 times, with the majority (n\u0026thinsp;=\u0026thinsp;19) of the passages under animal-derived component-free conditions. A Master Seed Cell Bank (MSC) was set down and characterised in terms of identity and the presence of extraneous agents. DNA barcoding using sequencing of the cytochrome c oxidase subunit I (COI) confirmed the cell derivation to be \u003cem\u003eAedes albopictus\u003c/em\u003e. Chromosomal enumeration of 20 cells of the C6/36 MSC revealed the presence of six chromosomes (Fig. S1). Extraneous agent testing confirmed the absence of persistent infection with bacteria, including mycoplasma, fungi and a panel of pathogenic viruses of health and biosecurity concern. The absence of detection of viral pathogens was also supported by deep sequencing analysis of extracted cellular RNA, which similarly did not reveal the presence of adventitious agents. The BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e Master Seed Virus (MSV) also underwent extraneous agent testing, with no evidence of the same range of agents.\u003c/p\u003e\n \u003cp\u003eTo remove the Genetically Modified Organism (GMO) status of the vaccine, and the associated regulatory restrictions, various formalin inactivation parameters were assessed, in line with standard vaccine inactivation methodologies\u003csup\u003e35\u0026ndash;37\u003c/sup\u003e. Unpurified BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine consisting of clarified supernatant from infected C6/36 cell cultures was inactivated with 0.08% formalin following incubation at 20\u0026deg;C for 1 hour and then at 4\u0026deg;C for at least 28 days. This inactivation condition was selected based on the results of preliminary studies testing various formalin concentrations and incubation times; inactivation was confirmed by an absence of virus replication in cell culture following three serial passages (see Fig. S2 and Table S3)\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e\u003cstrong\u003eSafety and immunogenicity of unpurified, inactivated BinJ/JEV\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eNSW/22\u003c/strong\u003e\u003c/sub\u003e \u003cstrong\u003evaccine formulations in mice\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eTo determine if the vaccine using inactivated-unpurified material was capable of inducing a JEV-specific antibody response, CD1 mice were immunised via the subcutaneous route with 100 \u0026micro;L of either the inactivated formulation or a mock-inactivated (non-inactivated) preparation of the same antigen (5.75 x 10\u003csup\u003e5\u003c/sup\u003e IU/dose pre-inactivation titre), both supplemented with 1 mg/mouse of the adjuvant Advax2. These vaccines were given twice at 3-week intervals. After one dose, both vaccine preparations induced JEV-specific antibody responses as determined by microneutralisation assay, with 3/5 in the non-inactivated vaccine group and 4/5 in the inactivated vaccine group producing detectable neutralising antibodies (Fig. 1A). For comparative reference, a third group that received two doses of 2 \u0026micro;g purified vaccine (~\u0026thinsp;10\u003csup\u003e7\u003c/sup\u003e IU/dose) without inactivation produced a strong neutralising response in all five mice (Fig. 1A). Seroconversion was also monitored by blocking ELISA two weeks after the second dose (second bleed) and again 11 weeks later (third bleed, Fig. 1B). Three of the five mice that received the non-inactivated vaccine returned a positive bELISA result when the serum was assessed at a minimum 1 in 100 dilution and 4/5 in the inactivated vaccine group produced a positive result. However, when serum was taken at a later time point, the antibodies levels were higher, with 4/5 mice receiving the non-inactivated unpurified and 5/5 mice receiving the inactivated formulation positive at or above a 1:100 serum dilution in blocking ELISA (Fig. 1B), although the increase in antibody levels was not significant (one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test). Importantly, the mice displayed no adverse effects after two doses of either vaccine. These data indicated that a crude inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine was safe and immunogenic in mice and induced an antibody response against JEV.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003eSafety and immunogenicity of unpurified, inactivated vaccines in weaner pigs\u003c/h2\u003e\n \u003cp\u003eTo determine if the BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine was safe and efficacious in pigs, 5-week old male pigs, born to orthoflavivirus antibody-na\u0026iuml;ve gilts were vaccinated twice, 28 days apart, with one of five different BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine formulations (Table S4, Fig. 2A). No adverse reactions such as swelling, erythema or tenderness were observed at the site of injection in any of the young pigs in the 5 days subsequent to the prime and booster doses for any of the vaccine preparations. There was no significant difference in the rectal temperature between groups (including the no-vaccine control) on any of the three days following the priming dose of vaccine (Fig. S3; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\n \u003cp\u003eA JEV serological response was detected in all vaccinated weaner pigs by bELISA within seven days of the priming dose for those in vaccine groups 2\u0026ndash;4 (Fig.\u0026nbsp;2B, Table S4) and robust immune responses following the boosting dose of vaccine for vaccine groups 3 and 4 (Fig.\u0026nbsp;2C, Table S5), with bELISA end point titres of 80\u0026ndash;10,240. The immune response developed by pigs injected with the formalin-inactivated clarified supernatant preparation of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e with the adjuvant aluminum hydroxide gel only (Group 5) was inferior, with 100% positive in the bELISA only after a booster dose of vaccine (Table S4). As expected, the vaccine formulated with purified virus, without inactivation (group 2), produced the highest titres in virus neutralisations assays (Fig. 2D, Tables 1, S6). The majority of bELISA-positive samples from the weaner pigs vaccinated with the inactivated, clarified supernatant vaccine preparations (groups 3\u0026ndash;5) also had neutralising activity against wild-type JEV, with the GMTs ranging between 17.1\u0026ndash;31 (Tables 1, S6; Fig. 2D). Two neutralising assay formats were employed to assess the sera. Both assays indicated modest but superior responses of animals in Group 3 (higher dose with Emulsigen and gel) compared to other formulations of vaccines using unpurified, inactivated virus (Groups 4, 5; Fig. 2D, Tables 1, S6), although the increase in response was not statistically significant (One way ANOVA with Tukey\u0026rsquo;s multiple comparison test, p\u0026thinsp;=\u0026thinsp;0.1228 for group 3 vs group 4 microneutralisation\u003csub\u003e50\u003c/sub\u003e assay). In the PRNT\u003csub\u003e50\u003c/sub\u003e (Table S6), some animals did not return a positive result, however, there was a clear reduction in plaque numbers for these samples that was not present in the unvaccinated animals indicating that a neutralising response had been generated, albeit below the limit of detection of the assay. This was confirmed for animals 90024, 90052 and 90023 in the lower dose clarified supernatant inactivated vaccine group (group 4) whereby a positive neutralisation response was detected by a microneutralisation\u003csub\u003e50\u003c/sub\u003e assay. One animal from group 3 (denoted 90042), did not produce a detectable neutralising antibody response in either neutralisation assay (Fig. 2D, Tables 1, S6) and had a much lower bELISA end point titre at days 35 and 42 post priming vaccine dose (1\u0026ndash;2 weeks post booster vaccine dose) of 80 and 320 vs 640 - \u0026gt;5120 and 640\u0026thinsp;\u0026minus;\u0026thinsp;10,240 for other animals in that vaccine group (Fig. 2C, Table S5).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eJEV neutralising antibody titres in sera of young pigs 2 weeks after the booster dose of each vaccine formulation as tested by microneutralisation\u003csub\u003e50\u003c/sub\u003e assay.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVaccine Treatment\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRange of micro-neut\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMedian\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGeometric Mean Titre*\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo vaccine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePurified BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e with Advax2. Non-inactivated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u0026ndash;160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e63.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFormalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e, higher dose + Emulsigen and aluminium hydroxide gel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;10\u0026ndash;80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFormalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e, lower dose\u0026thinsp;+\u0026thinsp;Emulsigen and aluminium hydroxide gel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u0026ndash;40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003e*For calculation purposes, where the microneutralisation\u003csub\u003e50\u003c/sub\u003e titre is below the minimum dilution factor tested, the value was set to 1.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e. Timeline of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccinations, WT JEV\u003csub\u003eNSW22\u003c/sub\u003e challenge, and sample/data collections. \u003cstrong\u003eB.\u003c/strong\u003e serum percentage inhibition (PI) values against JEV antigen as measured by JEV-specific bELISA. Sera were assessed at a 1/10 dilution. Samples are considered positive if the PI is \u0026ge;\u0026thinsp;60%. Samples were collected weekly post-the priming vaccination, up until day 28 (day of booster vaccination). \u003cstrong\u003eC.\u003c/strong\u003e Serum end-point JEV-specific bELISA titres for serum collected post-vaccine boost and post-JEV challenge (day 42), for the groups kept on for the challenge study. A positive bELISA result was considered\u0026thinsp;\u0026ge;\u0026thinsp;40%. Limit of quantification 1 in 10 dilution. \u003cstrong\u003eD\u003c/strong\u003e. Day 42 post-prime serum end-point neutralising antibody titres against JEV using microneutralisation\u003csub\u003e50\u003c/sub\u003e assay. Limit of quantification is 1 in 10. BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine formulations: 1) no vaccine, black; 2) purified virions, non-inactivated with Advax adjuvant, red; 3) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e as clarified cell culture supernatant (SN), higher concentration with Emulsigen/aluminium hydroxide gel adjuvant, blue; 4) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e as clarified cell culture SN, lower concentration with Emulsigen/aluminium hydroxide gel adjuvant, purple; 5) Formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e as clarified cell culture supernatant, higher concentration with aluminium hydroxide gel adjuvant only, orange.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003eProtection of vaccinated weaner pigs from JEV challenge\u003c/h2\u003e\n \u003cp\u003eAlthough pigs in group 2 (purified, non-inactivated vaccine) had superior neutralising responses (Tables\u0026nbsp;1, S6; Fig.\u0026nbsp;2D), the objective was to proceed with a vaccine that could be produced cost-effectively (without extensive downstream processing and purification) and did not have the regulatory constraints associated with a GMO. The groups receiving the formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e as clarified culture supernatant and adjuvanted with Emulsigen and aluminium hydroxide gel (Groups 3 and 4) were selected along with the negative control group (sham vaccine, Group 1) for challenge two weeks after the administration of the booster vaccine.\u003c/p\u003e\n \u003cp\u003eAfter challenge, all of the unvaccinated weaner pigs were confirmed to have been infected with JEV, based on positive RT-qPCR tests of EDTA-treated blood with peak viraemia usually identified between 3 to 5 days after challenge (Table\u0026nbsp;2). Further, JEV RNA was detected and virus was isolated in cell culture from the tonsils of each of these pigs at the completion of the trial (day 21 or 23 after challenge). JEV detection (summarised in Table\u0026nbsp;2) confirmed an infection rate for the unvaccinated pigs of 100% (70.1\u0026ndash;100%, 95% CI), under these experimental conditions. Antibodies to JEV were detected in the unvaccinated piglets at day 7 after challenge (Fig.\u0026nbsp;2C) and the titre of antibodies determined by bELISA ranged from 160\u0026ndash;640 (Fig.\u0026nbsp;2, Table S5). The rectal temperature of unvaccinated pigs was higher than vaccinated pigs on Days 9 and 12 (Fig. S4).\u003c/p\u003e\n \u003cp\u003eThere was no evidence of JEV infection by RT-qPCR of blood and tonsils, nor JEV viral isolation from the tonsils in 17/18 pigs from the two vaccinated groups that were challenged with JEV whilst one pig in group 3 was positive for JEV, indicating an infection rate of 5.6% (1.0\u0026ndash;25.8%, 95% CI, Table\u0026nbsp;2). It is worth noting that the sole vaccinated animal (90042) that became infected (based on detection of virus in blood and tonsils) had a lower bELISA titre (80) at day 35 and the absence of detectable neutralising activity at the time of challenge compared to other members of the group (Tables\u0026nbsp;1, S5, S6; Fig.\u0026nbsp;2). Overall, this indicated a vaccine efficacy of 94.4%.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable2\u003c/strong\u003e. Detection of JEV RNA by RT-qPCR in blood and tonsil samples collected after intradermal JEV infection challenge\u0026nbsp;\u003c/p\u003e\n \u003ctable style=\"width:701.7pt;border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"3\" style=\"width:92.15pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003eTreatment Group\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" style=\"width:62.35pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003eAnimal ID\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"10\" style=\"width:547.2pt;border-top:solid windowtext 1.0pt;border-left:none;border-bottom:solid windowtext 1.0pt;border-right:none;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003eJEV RT-PCR (Ct)\u003csup\u003e#\u003c/sup\u003e\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\" style=\"width:442.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003e\u0026nbsp;Days Post-challenge for EDTA blood samples\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" style=\"width:105.2pt;border:none;border-bottom: solid windowtext 1.0pt;padding: 0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003eTonsil at end of trial\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003e(Day 21 or 23*)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003el\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:17.0pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003e2\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e36.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;background:#FCE4D6;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e35.34\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;background:#FCE4D6;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e35.96\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;background:#FBE4D5;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e29.27*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90066\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;background:#FCE4D6;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e36.81\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;border-bottom:solid windowtext 1.0pt;background:#FCE4D6;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e33.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;border-bottom:solid windowtext 1.0pt;background:#FBE4D5;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e24.82*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"9\" style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cstrong\u003e\u003cspan style='font-size:15px;font-family:\"Times New Roman\",serif;color:black;'\u003e3. Higher dose BinJ/JEV as clarified supernatant, inactivated\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eno sample\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan 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style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp 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style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n 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Lower dose BinJ/JEV as clarified supernatant, inactivated\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90018\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 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style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90049\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90052\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90058\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:62.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003e90068\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:53.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:48.0pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:105.2pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0cm 5.4pt 0cm 5.4pt;height:13.3pt;\"\u003e\n \u003cp style='margin:0cm;font-size:16px;font-family:\"Calibri\",sans-serif;text-align:center;'\u003e\u003cspan style='font-size:12px;font-family:\"Times New Roman\",serif;color:black;'\u003eneg.\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e# Data are the Ct values for positive samples. Negative tests (no evidence of amplification at 45 cycles) are indicated (neg).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003eSafety and immunogenicity of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccination in multiparous sows\u003c/h2\u003e\n \u003cp\u003eA cohort of 10 multiparous sows, confirmed to be orthoflavivirus seronegative in pan-orthoflavivirus bELISA (Table S7), were vaccinated with the same batch of vaccine used in the group 3 weaner pigs \u0026ndash; i.e. formalin-inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e (higher concentration) with Emulsigen and aluminium hydroxide gel, undiluted clarified culture supernatant. The sows received three doses of 4 mL of the vaccine (twice the dose given to the weaner pigs) four weeks apart, with the third dose give six weeks after the first booster vaccination (dose 2, Fig. 3A). The animals were monitored for clinical signs post vaccination in addition to the immune response elicited.\u003c/p\u003e\n \u003cp\u003eNeither following the prime or booster vaccinations, were there any local tissue reactions to the vaccine and none of the sows showed clinical signs indicative of systemic reactions, including hypersensitivity. The vaccinations had no effect on the animals\u0026rsquo; appetite and overall demeanour. One animal was culled two weeks after receiving the first booster vaccine dose, due to conditions unrelated to the vaccinations (laminitis which was not responding to antibiotics and anti-inflammatory drugs). At the termination of the study, all nine remaining animals had gained an average weight of 17 kg (range 7\u0026ndash;36 kg; Fig. S5).\u003c/p\u003e\n \u003cp\u003eThe immune response to the vaccine was monitored by a JEV bELISA and JEV neutralisation assay. While all animals gave negative results in the pan-orthoflavivirus assay upon arrival, the serum from one sow (582) returned a low-range positive result (PI\u0026thinsp;\u0026gt;\u0026thinsp;60) in the JEV-specific bELISA (Table S8). However, it was negative in a JEV PRNT\u003csub\u003e50\u003c/sub\u003e assay and by an in-house developed anti-JEV total IgG lateral flow assay (data not shown), collectively indicating that this animal was unlikely to have been exposed to JEV prior to vaccination, and the bELISA result was more likely due to exposure to another orthoflavivirus. A JEV-specific serological response in all vaccinated animals was detected by bELISA in samples collected as early as 14 days post-prime vaccination (Fig. 3, Tables S8, S9), with the immune response boosted after the second dose, as indicated by the increase in titres at day 42 (Fig. 3). PRNT\u003csub\u003e50\u003c/sub\u003e assays confirmed that all sows produced a neutralising immune response against JEV as early as 14 days post-priming vaccine dose (PRNT\u003csub\u003e50\u003c/sub\u003e 20\u0026ndash;80, Table S10). When the sera were assessed in the JEV bELISA, end-point titres rose from a range of 20\u0026ndash;160 following the priming vaccine, to 640\u0026ndash;2560 two weeks post the booster vaccination (Fig. 3, Table S9).\u003c/p\u003e\n \u003cp\u003eThe results confirmed the safety of the inactivated BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine formulation in older pigs and demonstrated its effectiveness in inducing a robust antibody response to JEV.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 3. Vaccination of sows with BinJ/JEV\u003c/strong\u003e \u003csub\u003e\u0026nbsp;\u003cstrong\u003eNSW/22.\u003c/strong\u003e\u0026nbsp;\u003c/sub\u003e A) Timeline of vaccinations and blood sample collections; B) End-point titres of anti-JEV antibodies in sows at different times after vaccination determined by titration in the JEV bELISA. C) Anti-JEV neutralising titres as determined by PRNT\u003csub\u003e50\u003c/sub\u003e 14- and 28- days post-priming vaccine dose of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e. Statistics by one-way ANOVA with Tukey\u0026rsquo;s post-test, whereby p\u0026thinsp;=\u0026thinsp;0.0277 (*), 0.0021 (**) or \u0026lt;\u0026thinsp;0.0001 (***). ND\u0026thinsp;=\u0026thinsp;not detected.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThere is an urgent need for effective control of JEV infection in piggeries in the likelihood that the virus has become endemic in Australia, having already contributed to outbreaks of disease in piggeries over two summer periods, 2021/2022 and 2024/2025. A chimeric virus based on the genome backbone of the insect-specific orthoflavivirus BinJV, with the prM and E gene sequences from the Australian 2022 JEV (GIV) strain (BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e) was previously constructed as a candidate vaccine against JEV and shown to be safe and protective in mouse models of this disease\u003csup\u003e22\u003c/sup\u003e. To further assess the use of this vaccine in a veterinary context we prepared a range of formulations of the vaccine, using varying purity levels of virus, different adjuvants and with and without formalin inactivation to undertake vaccine trials in pigs.\u003c/p\u003e \u003cp\u003eOur study confirmed that a relatively crude, inactivated formulation of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e was safe and immunogenic in both weaner pigs and multiparous sows and induced an immune response that could prevent viraemia and infection of target tissues in 94% of vaccinated young pigs. These studies corroborate and extend our previous findings that a live purified version of the vaccine protected immunodeficient mice from JEV challenge\u003csup\u003e22\u003c/sup\u003e. While the vaccine challenge study in weaner pigs was conducted after two immunisations with the inactivated, unpurified vaccine, our data provide evidence of neutralising responses after a single dose of the inactivated crude vaccine in weaner pigs and sows, similar to what has been reported for non-inactivated purified versions of BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e in mice\u003csup\u003e22\u003c/sup\u003e, suggesting pigs also develop a rapid immune response to this type of vaccine.\u003c/p\u003e \u003cp\u003eAs imported vaccines are based on viruses of a different genotype (currently GIII), a locally produced vaccine based on the genotype of JEV responsible for the outbreak in Australia (GIV) maximises the potential for protection and also averts possible biosecurity risks arising from imported vaccines. Vaccine genotype matching may be important to provide maximum protection given prior research showing that the GIII-based live-attenuated at222-derived JEV vaccine conferred sterilising immunity against natural infection with GI in only a small proportion of vaccinated piglets\u003csup\u003e38\u003c/sup\u003e. Although experimental challenge would be needed for confirmation, field-based estimates indicated that a GIII live-attenuated JEV vaccine provided reduced protection against JEV-associated stillbirth and abortion in gilts when GI viruses were circulating, compared with its higher estimated efficacy against the homologous GIII strains\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur data demonstrates that our BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine protects against JEV infection, with a vaccine efficacy that is comparable to reported efficacies of other JEV vaccines in pigs\u003csup\u003e40\u003c/sup\u003e. Vaccination of pigs for JEV is practiced in Asian countries where the virus has been endemic with a range of live attenuated and cell culture derived inactivated virus vaccines\u003csup\u003e41\u003c/sup\u003e. There is limited efficacy data for these products\u003csup\u003e42\u003c/sup\u003e. Small scale experimental and field trial evaluations indicated similar efficacy for the attenuated S\u003csup\u003e\u0026minus;\u003c/sup\u003e strain vaccine\u003csup\u003e43\u003c/sup\u003e, formalin inactivated JEV\u003csup\u003e44\u003c/sup\u003e, recombinant virus vaccines\u003csup\u003e45\u003c/sup\u003e and a combination of formalin inactivated JEV with a DNA vaccine\u003csup\u003e46\u003c/sup\u003e. In our study, one vaccinated pig with lower bELISA titre than other vaccinates and an absence of a neutralising titre, developed viraemia. This may be the result of insufficient B-cell priming for this particular animal. Insufficient B-cell priming against JEV infection has been reported in the mouse model previously\u003csup\u003e47\u003c/sup\u003e. As this vaccine shows considerable promise, it is important to initiate studies to confirm safety by vaccinating animals across the range of ages and classes (breed, age, sex and reproductive status) that will be considered for administration of vaccine in a JEV control program in Australian piggeries.\u003c/p\u003e \u003cp\u003eIn particular, further efficacy data is required to confirm that the immune response and prevention of viraemia and tonsillar infection in weaner/growing pigs translates to prevention of reproductive disease in gilts/sows and infertility of boars. Our small-scale trial on multiparous sows provides preliminary confidence that the inactivated vaccine also induced JEV-specific neutralising antibody responses, which has been indicated as a correlate of protection for JEV in pigs and humans\u003csup\u003e46,48,49\u003c/sup\u003e. Thus, it is possible that vaccination will also protect breeder pigs (sows and boars) from JEV-induced reproductive impacts. Furthermore, the vaccine was well tolerated in these older animals, even after receiving three \u0026ldquo;double doses\u0026rdquo;, demonstrating that the vaccine is also safe in older pigs kept under conventional farming conditions with likely exposure to mosquitoes and other insects. However, safety and efficacy data will be best obtained through a large-scale field trial, as it is essential to vaccinate a large number of animals to exclude the potential for a low level of adverse outcomes and to assess the longevity of the protective immune response.\u003c/p\u003e \u003cp\u003eIn addition to the disease control benefits for pig producers, there are also possible public health implications by reducing the number of pigs that become viraemic and in turn amplify this virus to increase the proportion of infected mosquitoes. Although waterbirds are the natural reservoir for JEV, in particular, feral pigs likely play an epidemiological role in the transmission and maintenance of JEV on the Australian mainland\u003csup\u003e50\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eA major advantage of our chimeric virus technology for production of a new vaccine is the ability to rapidly pivot to target new genotypes of JEV. Indeed, the BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine virus took less than a month to generate once the sequence of the outbreak strain was available\u003csup\u003e22\u003c/sup\u003e. Furthermore, we have also successfully prepared BinJ/JEV chimeras for each of the 5 JEV genotypes and have shown in mice that the antibodies induced by the BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine also neutralise other JEV genotypes\u003csup\u003e22\u003c/sup\u003e. The flexibility of the platform was recently shown for another veterinary vaccine candidate, whereby a BinJV chimera displaying the prM/E structural protein genes of a subtype of WNV, Kunjin strain, elicited protective immune responses in Australian saltwater crocodiles against skin lesions when applied as either an inactivated or non-inactivated antigen formulation\u003csup\u003e51\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe design of the vaccine also allows the development of serological tests that can differentiate vaccinated pigs from those that have had a natural infection (DIVA). Since the NS1 protein of JEV is immunodominant during natural infection, but is not included in the vaccine, this antigen is the ideal candidate for DIVA.\u003c/p\u003e \u003cp\u003eThe use of an unpurified vaccine formulated as clarified culture supernatant greatly reduces the downstream processing of the vaccine and significantly lowers the cost of production, an essential property of an economically feasible vaccine for production animals. In this context, the ability of the BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine virus to grow efficiently in mosquito cell culture while being unable to replicate in vertebrate cells also enhances ease of production under low biosecurity requirements. While the crude vaccine preparations used to immunise pigs in this study were produced in adherent cultures of C6/36 cells in RPMI media supplemented with 2% FBS, we have recently shown that this vaccine virus can also be grown to high titre in serum-free-medium in suspension cultures of C6/36 cells\u003csup\u003e52\u003c/sup\u003e. As part of the present study, through extensive innocuity tests, we have also confirmed the absence of adventitious agents in the C6/36 cells line, corroborating the deep sequencing data of Miller and colleagues\u003csup\u003e53\u003c/sup\u003e. Further, formalin inactivation removes any GMO-associated regulatory restrictions of the vaccine while not compromising its protective capability.\u003c/p\u003e \u003cp\u003eIn summary, we have provided evidence that the BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e vaccine candidate delivered as an inactivated, clarified cell culture supernatant provides protection in pigs against JEV viraemia and does not cause adverse reactions in the vaccinated animals. This vaccine is currently being assessed by Australian regulatory authorities for commercial manufacture and registration for use in pigs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eData included in this publication are based on a patent (WO/2018/176075) on which J.J.H., H.B-O., R.A.H., and J.H-P are inventors. J.N. and M.W. are paid employees of Tr\u0026eacute;idlia BioVet Pty Ltd, Australia. The remaining authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eProject oversight: J.H-P, P.D.K. H.B-O, R.A.H., M.W. Experimental design: P.M.H, H.D., J.J.H., G.H., J.N., R.A.H., H.B-O., P.D.K., J.H-P. Experiments: P.M.H, H.D., J.J.H., G.H., J.R.P., J.N., I.E.M., J.N., H.B-O., A.H.Y.L., N.K.Y.Y., M.S.M. Writing, original draft preparation: P.M.H, H.D., J.J.H., G.H., R.A.H., H.B-O., P.D.K., J.H-P. Writing, review and editing: all authors.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe are indebted to the following groups for assistance during this study: Virology Laboratory, EMAI: Veterinary professional staff for management and sampling of pigs in animal house trials. Technical and support staff for the care and management of pigs and provision of laboratory support associated with sample management, processing and testing; JBS Pork Australia for generously providing the weaned pigs and Greg Tuckett and Chrissie Kracht for providing training and expert advice on their care, management and sample collection; Sow trial at the Queensland Animal Science Precinct (QASP), UQ: We are grateful for the expert knowledge and skills of key personnel at QASP for assistance with facility and AEC approvals. We are indebted to the expertise of Milou Dekkers, Stacey Groves, and Chelsey Baker (all QASP) for oversight of the trial and animal welfare. We also thank other QASP staff who at various times assisted with husbandry; We are thankful for staff at Tr\u0026eacute;idlia BioVet who contributed to the supply of the formulated vaccines. We also thank Vaxine Pty Ltd for generous supply of the Advax adjuvant.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eExtraneous agent testing data for the MSC and MSV are available from the corresponding author upon reasonable request, subject to legal and commercial restrictions. All other data are included in the manuscript and supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePark, S. L., Huang, Y. S. \u0026amp; Vanlandingham, D. L. Re-Examining the Importance of Pigs in the Transmission of Japanese Encephalitis Virus. \u003cem\u003ePathogens\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e (2022). https://doi.org/10.3390/pathogens11050575\u003c/li\u003e\n\u003cli\u003eMansfield, K. L., Hernandez-Triana, L. M., Banyard, A. C., Fooks, A. R. \u0026amp; Johnson, N. 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S. \u003cem\u003eet al.\u003c/em\u003e Serum-Free Suspension Culture of the Aedes albopictus C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production. \u003cem\u003eViruses\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e (2025). https://doi.org/10.3390/v17020250\u003c/li\u003e\n\u003cli\u003eMiller, J. R. \u003cem\u003eet al.\u003c/em\u003e Analysis of the Aedes albopictus C6/36 genome provides insight into cell line utility for viral propagation. \u003cem\u003eGigascience\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 1–13 (2018). https://doi.org/10.1093/gigascience/gix135\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Japanese encephalitis virus, Binjari, pig, vaccine, orthoflavivirus","lastPublishedDoi":"10.21203/rs.3.rs-8775364/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8775364/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWidespread infection with a rare genotype 4 (GIV) strain of Japanese encephalitis virus (JEV) throughout eastern Australia in 2022 created new threats to animal and public health. Extensive production losses were incurred by the reproductive impacts of JEV infection at piggeries. Further, as an amplifying host for JEV, there are broader One Health advantages in minimising JEV infection of pigs. A candidate vaccine for pigs was developed using a chimeric virus with the structural glycoprotein genes (prM and E) of the outbreak JEV strain (JEV\u003csub\u003eNSW/22\u003c/sub\u003e) inserted into an insect-specific orthoflavivirus (Binjari virus - BinJV) genome backbone (BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e). Vaccines prepared with adjuvants using purified chimeric virus or a non-purified, formalin-inactivated formulation, induced JEV-specific antibody responses in mice and pigs. No adverse effects of vaccine administration were observed in either species for either vaccine formulation when used with a prime and boost delivery 28 days apart. Ninety-four percent (17/18) of weaner pigs that received the inactivated vaccine were protected from challenge with 1 x 10\u003csup\u003e5.0\u003c/sup\u003e TCID\u003csub\u003e50\u003c/sub\u003e of JEV\u003csub\u003eNSW/22\u003c/sub\u003e, as determined by testing for JEV in blood and tonsils by RT-qPCR and virus isolation. In contrast, all control animals (n\u0026thinsp;=\u0026thinsp;9) became viraemic and harboured JEV in the tonsils. When adult sows (1.5 to 3 years of age) (n\u0026thinsp;=\u0026thinsp;9) were given a double-dose of the inactivated vaccine, there were no adverse effects, and each animal produced JEV-specific neutralising antibodies. Hence, BinJ/JEV\u003csub\u003eNSW/22\u003c/sub\u003e appears to be a promising candidate vaccine for prevention of JEV infection in pigs and can be used live or inactivated at varying levels of purity to provide manufacturers with logistical options for commercial production of the vaccine.\u003c/p\u003e","manuscriptTitle":"A novel chimeric vaccine protects pigs from infection with Japanese encephalitis virus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 19:10:21","doi":"10.21203/rs.3.rs-8775364/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-14T07:34:22+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-12T03:25:58+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T18:58:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165677703476800299497848634145859764785","date":"2026-03-27T14:52:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98309668287895091717620144542863121477","date":"2026-03-25T16:04:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-17T18:59:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"295644474467713290820946563617935222065","date":"2026-03-11T10:51:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-10T19:15:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-16T14:36:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-06T10:45:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Vaccines","date":"2026-02-03T11:03:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-vaccines","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"npjvaccines","sideBox":"Learn more about [npj Vaccines](http://www.nature.com/npjvaccines/)","snPcode":"41541","submissionUrl":"https://submission.springernature.com/new-submission/41541/3?","title":"npj Vaccines","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d87dddb8-b3ce-471b-9678-18ac00ed42e3","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63016255,"name":"Health sciences/Diseases"},{"id":63016256,"name":"Biological sciences/Immunology"},{"id":63016257,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-04-14T07:41:00+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 19:10:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8775364","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8775364","identity":"rs-8775364","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}