INACTIVATED PISCINE ORTHOREOVIRUS VACCINE

Abstract

The invention derives from the discovery that the immunogenicity of piscine orthoreovirus (PRV) can be significantly increased by inactivating the virus in a way different from the prior art: by incubation with an aziridine the vaccine potency against challenge infection by cohabitation is much improved over prior art approaches. This allowed the development of an effective inactivated vaccine against infection and disease caused by PRV.

Claims

1. Composition comprising inactivated orthoreovirus, characterised in that the orthoreovirus had been inactivated by incubation with an aziridine.

2. The composition according to claim 1, characterised in that the orthoreovirus is piscine orthoreovirus (PRV).

3. The composition according to claim 1 or 2, characterised in that the aziridine is ethylenimine.

4. Method for the production of a composition comprising aziridine-inactivated PRV, the method comprising the steps of: a. obtaining a composition comprising PRV, and b. inactivating the PRV in said composition by incubation with an aziridine.

5. The method according to claim 4, characterised in that the aziridine is ethylenimine or binary ethylenimine.

6. The composition according to any one of claims 1-3, or the composition as obtainable by the method of claim 4 or 5, for use in a vaccine for the protection of fish against infection or disease caused by PRV.

7. Vaccine for fish against infection or disease caused by PRV, said vaccine comprising the composition according to any one of claims 1-3, or the composition as obtainable by the method of claim 4 or 5, and a pharmaceutically acceptable carrier.

8. The vaccine according to claim 7, characterised in that the vaccine comprises an adjuvant.

9. Method for protecting a fish against infection or disease caused by PRV, the method comprising the administration to said fish of the vaccine according to claim 7 or 8.

10. Use of the composition according to any one of claims 1-3, or of the composition as obtainable by the method of claim 4 or 5, for the manufacture of a vaccine for the protection of fish against infection or disease caused by PRV.

11. Method for the manufacture of a vaccine for the protection of fish against infection or disease caused by PRV, the method comprising the step of formulating the composition according to any one of claims 1-3, or the composition as obtainable by the method of claim 4 or 5, into a vaccine.

Description

EXAMPLES

Example 1: Materials and Methods

1.1. Amplification of PRV

[0162] 150 naive Atlantic salmon pre-smolt fish were inoculated to amplify virus in vivo, for antigen preparation and for challenge trials. The inoculum was a Norwegian isolate of PRV, strain V3279, which in turn had been obtained as a sonicate from whole blood of infected Atlantic salmon. The inoculum was 0.1 ml/fish given i.p. The inoculum was prepared on the day of infection, diluted 1:10 in EMEM and kept on ice prior to inoculation. The pre-smolts were then kept in fresh water for 2 weeks at 12° C.

[0163] Next the blood from the 150 PRV inoculated fish was collected with disposable syringes, and pooled in heparin coated 2 ml Vacutainers™ (2-4 fish per tube). After heparinisation, the blood was pooled in 50 ml centrifuge tubes and centrifuged at 2000×g for 10 minutes at 4° C. The blood plasma (about 26 ml) was discarded, and the red blood cell pellet was resuspended in 5× the volume (125 ml) of L 15 medium (Sigma) and 50 μg/mL gentamicin.

1.2. Isolation and Purification of PRV Antigen

[0164] The pelleted infected erythrocytes were subsequently sonicated on ice to lyse the cells and release the PRV. The equipment used was a digital sonifier, model SPLe (Branson), using 50 ml centrifugation tubes, and approximately 25 ml of sample volume per sonication. The samples were given 8 pulses of 10 seconds at 25 kHz, with 30 second pauses in between. Next the sonicates were centrifuged at 2000×g for 5 minutes at 4° C. to pellet the cell debris and the PRV containing supernatant was collected and stored at 4° C. until use.

[0165] The sonicate was then purified as described by James et al., 2016 (supra), by removing impurities through extraction with a resin. In short: Capto™ Core 700 resin (GE healthcare) was washed three times with virus dilution buffer (150 mM NaCl, 15 mM MgCl.sub.2, 10 mM Tris, at pH 8.2). After the final wash, the beads were topped with an equal volume of virus dilution buffer to create a Capto Core slurry. For isolating the PRV from the sonicated Atlantic salmon red blood cells, a mixture of 20% Capto Core slurry and 80% lysed Atlantic salmon red blood cells was prepared and incubated by turning the tubes end-over-end for 45 minutes at 4° C. The resin beads were then pelleted by centrifugation at 800×g for 10 minutes, at 4° C., and the supernatant was transferred to a fresh tube. After this first Capto Core extraction, 4 more were performed on the same material and in the same way. The selection of 5 rounds of purification in total was a balance between loss of virus and loss of impurities such as cytokines and haemoglobin which may interfere with vaccination.

[0166] The presence of PRV was confirmed with RT-qPCR and the purity of the sample was confirmed with SDS-PAGE and a silver stain.

[0167] The supernatants from all purifications were pooled, resulting in a combined composition comprising PRV in which the Cq value was 23.23, and the amount of gene copies of Sigma1 was determined to be 1.13×10{circumflex over ( )}5/μl.

1.3. BEI Inactivation

[0168] The BEI was prepared from combining equal volumes of 1.09 M BEA and 1.91 M NaOH. This generated a stock-solution of 545 mM BEI.

[0169] The purified erythrocyte sonicate, containing the PRV in the virus dilution buffer (see above), was treated with 10 mM BEI at 20° C. for 28 hours while on a magnetic stirrer. After the incubation, any remaining BEI was neutralized with 15 mM Na-thiosulfate at 20° C. for 1 hour while on a magnetic stirrer. Finally, the pH was set to 7.6 using 4N hydrochloric acid.

1.4. UV-Inactivation

[0170] The UV inactivation was performed using standard equipment. In short: a total of 40.5 mL PRV in virus dilution buffer (the purified erythrocyte sonicate) was transferred into a reagent reservoir together with 4.5 mL of 500 μM riboflavin (vitamin B2) in 0.9% NaCl. Using a 50 mL syringe, the whole mixture was transferred into an R&D scale ELP bag provided by Terumo™ BCT for UV inactivation, and any trapped air was removed using the same syringe. The solution was inactivated using a Mirasol® PRT system (Terumo BCT) using the pre-programmed method no.: 150. After the run was completed, the ELP bag was pierced with a 21G needle to aspirate the UV-inactivated PRV sample. The sample was transferred into a 100 mL glass bottle and stored at 4° C. until use.

1.5. Quantitative PCR

[0171] Nucleic acid was isolated from the purified PRV samples as follows: 50 μL of the Capto Core purified erythrocyte lysate was added to 150 μL virus dilution buffer. Subsequently, 250 μL lysis buffer from the MagNA Pure LC Total Nucleic Acid Isolation Kit™ (Roche) was added. Total nucleic acids were isolated using a MagNA Pure™ 96 machine (Roche), using the ‘external lysis’ program, and using the MagNA Pure™ 96 DNA and Viral NA Small Volume™ kit (Roche).

[0172] Also, one sample with PBS as a negative control was included in the MagNA Pure isolation. A one-step RT-qPCR mastermix was prepared with 17 μL water for injection; 1 μL Superscript™ III RT/Platinum Taq mix (Invitrogen); 25 μL 2×SYBR™ Green reaction mix (Invitrogen); 2 μL primer mix; and 5 μL test sample. The primer mix consisted of 10 μM FWD 51 and 10 μM REV 51 primers:

TABLE-US-00001 FWD S1 (SEQ ID NO: 1): 5′-TGCGTCCTGCGTATGGCACC-3′, REV S1 (SEQ ID NO: 2):  5′-GGCTGGCATGCCCGAATAGCA-3′,

[0173] The RT-qPCR cycling and measurement was done using a CFX96 Real-Time System C1000™ thermal cycler (Bio-Rad). With each RT-qPCR run a standard consisting of a DNA template of the PRV 51 gene was included, in 10-fold fold dilutions between 4.37×10{circumflex over ( )} and 4.37×10{circumflex over ( )}1 copy numbers/μL.

[0174] The RT-qPCR protocol started with 55.0° C. for 3 minutes for the formation of cDNA; increased to 95.0° C. for 3 minutes for the RT enzyme inactivation. This was the start of the qPCR loop with the denaturation at 92.0° C. for 5 seconds, the annealing and elongation at 58.0° C. for 5 seconds, and the SYBR green measurement at 77° C. for 1 second. This loop was repeated for 39 more times. After the program had finished, a melt curve was performed starting at 73.0° C. and rising to 89.5° C. by steps of 0.3° C. every 5 seconds. During this procedure a SYBR green measurement was done every 5 seconds. The Cq values were determined and the copy numbers were calculated using CFX manager (BioRad)

1.6. Vaccine Formulation

[0175] The test vaccines were formulated as water-in-oil emulsions, with light liquid paraffin oil as adjuvant, and contained Polysorbate 80 and sorbitan mono-oleate as emulsifier, using standard procedures. Water:oil volume ratio of the vaccine was 40:60. The waterphases consisted for 95% of the inactivated PRV samples. The negative control vaccine was made using a saline sample.

Example 2: Virus-Inactivation Test

[0176] The efficiency of the inactivation by the two methods applied (UV-light exposure and aziridine incubation) was tested in vivo, next to a sample from the same PRV batch that had not been inactivated, as well as a PRV positive control sample.

[0177] The samples were each tested in a group of 10 Atlantic salmon pre-smolts, strain Stofnfiskur. The fish were acclimatized for 1 week in 12° C. freshwater. The water was kept at between 65 and 100% oxygen. Next fish were anaesthetized and inoculated i.p. with 0.1 ml from one of the three samples. All fish were marked per group by fin clipping. All samples were kept on ice until inoculation.

[0178] From all groups 5 animals were taken at 3 days post inoculation; plasma samples were obtained, and RT-qPCR of these samples served as baseline for the RT-qPCR from plasma samples taken at 14 days post inoculation.

2.1. Results

[0179] No amplification of PRV was detected in any of the fish samples inoculated with BEI-inactivated or with UV-inactivated virus, neither at 3 nor at 14 dpi. All samples from the untreated PRV, and most samples from the positive control group tested positive for PRV at 14 dpi.

[0180] It was concluded that both methods of inactivation resulted in complete inactivation of the PRV.

Example 3: Vaccination-Challenge Study

[0181] Atlantic salmon parr, strain Stofnfiskur, of 25-35 grams each were used. The population was tested for most of the relevant salmon pathogens, viral and bacterial, including PRV, by RT-qPCR. Only fish that scored negative for prior contamination, and that were apparently intact and healthy were used. The fish were kept in fresh water during acclimatisation and immunisation, and were kept in seawater (salinity 32 promille, 70% oxygen) during challenge. All water was at 12° C. Fish mean weight at start was determined at start of acclimatisation.

[0182] Test groups had 26 fish, and the unvaccinated control group had 32 fish. Also 10 fish were used as day zero controls. At challenge, 6 weeks after vaccination, infected shedder fish were added in a ratio of 1:5 to the total number of fish in the experiment.

[0183] Fish were marked by transponder tag 2 weeks before the experiment, and were acclimatised in the test tank 5 days before the experiment. Before vaccination, the fish were starved for 48 hrs, and sedated and anesthetized with benzocaine and iso-eugenol. Vaccination was with 0.1 ml, by i.p. injection. Challenge by co-habitation started at 6 weeks p.v., and continued for another 14 weeks.

[0184] Blood samples were taken at 4, 6, and 8 weeks post challenge. Tissue samples were taken at 6 and 8 weeks p.c.: heart samples were kept in formalin 4%. Blood was heparinized and centrifuged at 3000×g, for 10 min. at 4° C. Plasma and cells were stored separately at −80° C. until use.

3.1. Histopathological Scoring

[0185] Sections of heart muscle for histopathology were processed and stained with haematoxylin and eosin following standard procedures. Individual samples were examined for heart lesions in consistence with HSMI. The grade of changes was scored from 0 to 4 using criteria as described in Table 1.

TABLE-US-00002 TABLE 1 Heart lesions scoring of epicard HSMI-related heart lesion scores - Epicardium Score Description 0 Normal appearance. 1 Focal/multifocal (2-4 foci) of inflammatory cells lifting the epicardial layer from the surface of the heart, typically 2-3 cell layers thick 2 Diffuse infiltration of inflammatory cells (mononuclear) >5 cell layers thick in most of the epicard present. The infiltration of cells is multifocal to diffuse and can involve parts or the entire epicardium available for assessment 3 Diffuse infiltration of inflammatory cells (mononuclear) >10 cell layers thick in most of the epicard present. Moderate pathological changes consisting of moderate number of inflammatory cells in the epicardium 4 Diffuse infiltration of inflammatory cells (mononuclear) >15 cell layers thick in most of the epicard present. Severe pathological changes characterised by intense infiltration of inflammatory cells in the epicardium

3.2. Statistical Analyses

[0186] The PRV RT-qPCR results and the histopathology scores were analysed statistically using the Mann Whitney compare ranks test. All statistical analysis described were performed with GraphPad™ Prism (GraphPad Software Inc., USA) and p-values of p≤0.05 were considered as significant.

3.3. Results:

[0187] Average weight of the fish at marking was 30.8 grams, and at vaccination was 32.9 grams.

TABLE-US-00003 TABLE 2 number of fish with heart lesions (in epicard) at 8 weeks post challenge of a certain score level. Number of fish with heart lesion score of: Vaccine group 0 1 2 3 4 1 Saline 2 0 11 8 3 2 UV-treated PRV 3 5 7 8 1 3 BEI-treated PRV 24 0 0 0 0

[0188] Thus, the percentage of fish with a HSMI specific hart lesions score of 2 or higher, at 8 weeks post challenge, was: [0189] Saline group: 92% [0190] UV-treated PRV: 67%. [0191] BEI-treated PRV: 0%

[0192] It was concluded that the challenge experiment was successful, as all non-vaccinated fish became very ill with signs of severe HSMI. The vaccine of UV-inactivated PRV gave hardly any protection. On the contrary, the vaccine of BEI-inactivated PRV gave excellent protection against signs of HSMI from the PRV challenge infection.

Example 4: qPCR Results of Vaccination-Challenge Study

[0193] 4.1. Quantification of Vaccine Dose

[0194] The purified PRV pool from which the vaccines had been prepared for the vaccination-challenge trial as described in Example 3 above, was tested for antigen content using the qPCR assay as described in Example 1.5 above, and showed the amount of gene copies of Sigma1 was 1.13×10{circumflex over ( )}5/μl.

[0195] When incorporating the dilution that occurred during vaccine preparation, this means the amount of PRV particles used for the vaccination was 4.6×10{circumflex over ( )}6 per animal dose.

[0196] A further qPCR protocol was employed to test samples of salmon heart tissue that were collected in the trial of Example 3.

[0197] 4.2. Further qPCR Test

[0198] Nucleic acid was isolated using RNAlater™ from conserved salmon heart tip samples as follows: 2×2×2 mm of the sample was added to 600 μL of RLT buffer and total nucleic acids were isolated using the RNeasy® 96 Kit (Qiagen), together with the TissueLyser™ II (Qiagen) for homogenisation of the tissue sample.

[0199] A one-step RT-qPCR mastermix (Thermo Scientific Verso™ 1-Step Q-RT-PCR Kit low ROX) was prepared with 1.85 μL RNAse free water; 0.25 μL Verso enzyme mix; 12.5 μL 2×1-step QPCR low ROX mix and 1.25 μL RT Enhancer. Next 15.85 μL of mastermix were added to 2.25 μL forward primer FWD L1 and 2.25 μL reverse primer REV L1; 0.65 μL TaqMan probe; and 4 μL test sample. The primers and probe (used at 10 μM each) are as follows:

TABLE-US-00004 FWD L1: (SEQ ID NO: 3) 5′-TGCTAACACTCCAGGAGTCATTG-3′ REV L1: (SEQ ID NO: 4) 5′-TGA ATCCGCTGCAGATGAGTA-3′: TaqMan probe L1: (SEQ ID NO: 5) 5′-6FAM-CGCCGGTAGCTCT-MGBNFQ-3′

[0200] The RT-qPCR cycling and measurement was done using an ABI PRISM® 7500 FAST Sequence Detection System (Applied Biosystems).

[0201] The RT-qPCR protocol started with 50.0° C. for 30 minutes for the formation of cDNA; increased to 95.0° C. for 15 minutes for the RT enzyme inactivation. This was the start of the qPCR loop with the denaturation at 95.0° C. for 15 seconds, and the annealing and elongation at 60.0° C. for 1 min. This loop was repeated for 39 more times. The Cq values were determined in the 7500 FAST™ System Software v1.5.1 (Applied Biosystem) with a threshold value of 0.1 and a baseline from cycle 3 to 15.

[0202] 4.3. Results of Heart Tip Sample qPCR

[0203] To evaluate vaccination-efficacy at 8 w. post challenge the reduction of virus load was tested by qPCR of samples from the tips of the heart ventricles from the salmon. In groups 1 and 2 (mock vaccine and UV-inactivated PRV vaccine, respectively) all samples were found to be positive for PRV. On the contrary, in group 3 (BEI-inactivated PRV vaccine) only 8 of 12 animals were found positive for PRV. In addition the amounts of virus detected in the group 3 samples that were PRV positive, was much reduced as compared to the amounts found in group 1 and 2 samples: the average Cq values in groups 1 and 2 were 22.1 and 22.0 respectively (n=12). Remarkably, for group 3, the average Cq value was 30.6 (n=8). The threshold for negative scores in this experiment was Cq=40.

[0204] 4.4. Conclusions:

[0205] The protective efficacy of the vaccine based on BEI-inactivated PRV was surprisingly good: the number of animals having PRV in their blood after challenge was reduced by ⅓ resulting from this vaccination. Also, the viral load in the BEI-PRV vaccinated animals was much reduced.

[0206] Such efficacy scores had not previously been observed or reported in literature. Both these results show a strong reduction of PRV replication, indicating significant viral clearance and/or resistance to infection.