Broad-spectrum vaccine against avian reovirus

Abstract

A broad-spectrum vaccine against avian Reovirus is disclosed, which is effective in reducing the infection of avian Reovirus in an avian target. The vaccine comprises antigenic material derived from avian Reovirus of two genotype groups: 1 and 4, as defined herein. This vaccine is effective against all avian Reoviruses, homologous or heterologous to the vaccine, including recent virulent break-through strains.

Claims

1. A vaccine for reducing infection by avian Reovirus, the vaccine comprising an avian Reovirus antigenic material that is derived from avian Reoviruses from more than a single genotype group, and a pharmaceutically acceptable carrier, wherein the avian Reovirus antigenic material consists of antigenic material derived from a replicative form of an avian Reovirus from genotype group 1 and from a replicative form of an avian Reovirus from genotype group 4; wherein the avian Reovirus antigenic material comprises inactivated avian Reovirus; wherein the avian Reovirus from genotype group 1 comprises genetic information encoding a sigmaC protein comprising an amino acid sequence that has at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1; and wherein the avian Reovirus from genotype group 4 comprises genetic information encoding a sigmaC protein comprising an amino acid sequence that has at least 85% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 4.

2. The vaccine of claim 1, wherein the antigenic material derived from avian Reovirus from genotype group 1, is derived from avian Reovirus from genotype subgroup 1B; wherein the avian Reovirus from genotype group 1B comprises genetic information encoding a sigmaC protein comprising an amino acid sequence that has at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1.

3. The vaccine of claim 1, wherein the vaccine comprises an adjuvant.

4. The vaccine of claim 1, wherein the vaccine comprises additional antigenic material that is derived from a micro-organism pathogenic to an avian, but not from an avian Reovirus.

5. The vaccine of claim 1, wherein the step of inactivating both the avian Reovirus from genotype group 1 and the avian Reovirus from genotype group 4 is performed by an inactivation method selected from the group consisting of heat, radiation, formalin, beta-propiolactone, binary ethyleneimine, and beta-ethanolamine.

6. The vaccine of claim 5, wherein the vaccine comprises additional antigenic material that is derived from a micro-organism pathogenic to an avian, but not from an avian Reovirus.

7. A method for the preparation of the vaccine of claim 1, comprising the step of inactivating both the avian Reovirus from genotype group 1 and the avian Reovirus from genotype group 4 by a method selected from the group consisting of heat, radiation, formalin, beta-propiolactone, binary ethyleneimine, and beta-ethanolamine.

8. A method for the preparation of the vaccine of claim 1, comprising the step of admixing the avian Reovirus antigenic material with an adjuvant.

9. A method for reducing infection by avian Reovirus in an avian, comprising administering the vaccine of claim 1 to the avian.

Description

EXAMPLES

(1) 1. Avian Reovirus Isolation and Sample Preparation

(2) Avian Reovirus was isolated from chicken organs by culturing tissue homogenates on chicken embryo liver cells (CEL). In short: tissues such as tendon, liver, jejunum, and blood were obtained from (potentially) infected chickens. Blood was centrifuged and serum collected. Samples of tissues were cut out. These were placed in 6 ml tubes containing about 0.5 cc of glass beads of about 1 mm diameter, and 1 ml of phosphate buffered saline (PBS) which contained a cocktail of antibiotic- and antifungal compounds. Samples were homogenised by shaking for 20 minutes in a Mixer Mill ball mill (Retsch), followed by clarification by centrifugation. Supernatant was collected and could be stored frozen at 70 C. for later use.

(3) Primary CEL cells were prepared fresh before use, according to a standard protocol, in short: 14-16 day old SPF chicken embryos were used to obtain livers. The livers were washed twice in PBS to remove blood, and were then incubated while stirring in a trypsin/PBS solution at 37 C. for 15 minutes. the trypsin was neutralised with FCS, and the trypsinised mixture was centrifuged for 10 min. at 600g. The pellet was resuspended in standard growth medium (comprising a cocktail of antibiotics and 5% fetal calf serum (FCS)). This was filtered once to remove clumps, and then the CEL cells were counted and used directly.

(4) CEL were seeded at 1.510{circumflex over ()}6 cells/ml in 5 ml growth medium (5% FCS) into T25 culture flasks, and incubated overnight in a moisturised incubator at 37 C. and at 5% CO.sub.2, to allow establishment of a monolayer. Next day the culture medium was replaced by medium comprising 2% FCS, and the flasks were inoculated with a virus sample as 50 l serum or tissue supernatant, next the flasks were incubated for 4-7 days

(5) After the first round of incubation, the CEL cell-layer often appeared damaged from debris, so that no distinct cytopathic effect (cpe) was visible, so a second passage was usually given: fresh CEL were prepared, seeded, and incubated as monolayer, then 50 l of the culture supernatant from the first passage was inoculated. This was incubated for 4 days, after which avian Reovirus specific cpe (CEL cells seem to turn dark and granular, with translucent blast-like vesicles) could be clearly observed in a the flask. For cpe negative flasks a third passage could be given. Finally the culture supernatant of the 2nd or 3rd passage was harvested and stored frozen (70 C.) for later use.

(6) The isolates were named and numbered, whereby SL stands for service laboratory, followed by two digits for the year of isolation, a chronological isolate number, and the 2 letter code of the country of the donor material used for the isolation.

(7) The isolated virus samples could be used directly for RNA isolation, RT-PCR and sequence analysis. The isolates that were used in animal experiments were given further treatment: samples used for challenge inoculations were amplified directly from the third round amplified isolate, to obtain higher titres and larger volumes by proliferation on CEL of CEF cells in successively larger culture vessels, such as in T75 (15 ml culture) or T175 (25 ml culture) in Cellstar flatbottom cell-culture flasks (Greiner Bio-One), or in 490 cm.sup.2 roller bottle flasks (150 ml culture) (Corning, Fischer Scientific), all in standard culture medium and under standard culturing conditions. In this way, challenge inocula were prepared of: SL11A823-2 BE (genotype group 1): 7.95 Log 10 TCID50/ml SL11A294-12 FR (genotype group 2): 6.32 Log 10 TCID50/ml SL10A1581-32 ES (genotype group 3): 5.95 Log 10 TCID50/ml SL11A823-1 BE (genotype group 4): 7.70 Log 10 TCID50/ml

(8) Samples of avian Reovirus breakthrough isolates that were used for the preparation of vaccines were first plaque purified 3 times as described below, and then amplified in similar ways.

(9) 2. Confirmation of Virulence of Viral Isolates in Target Animals

(10) In line with Koch's postulates, the pathogenicity and reisolation of the avian Reoviral isolates was demonstrated in experimental infections of chickens. One day old chicks were used, either from specified pathogen free (SPF) mothers, or from Reovirus vaccinated mothers. The test in these last chicks, which were positive for maternally-derived antibodies (MDA+) against classical Reovirus strains, served to confirm the vaccination-breakthrough capability of these viral isolates. Viruses used for the challenge inoculations were isolates: SL11A823-1BE, SL11A823-2BE, SL11A0294-12 FR, and SL10A1581-32 ES, that were isolated and amplified as described above. Inoculations were applied orally to mimic the natural infection route, or alternatively by intramuscular route, which had been observed to be an even more effective route of challenge-inoculation.

(11) 2.1. Experimental Design

(12) 200 Day old SPF chicks, of mixed sex, were marked using wingbands, and placed in separate negative pressure isolators, at 20 chicks per group. Chicks were White Leghorn layers, feed and water were available ad libitum, and evidently weak or small chicks were not included. In addition 10 hatchmates were bled to provide serum samples to test antibody status.

(13) A challenge inoculation was administered the same day of placement, and in total 10 isolators were used to house the different groups: 2 isolators for each of the inoculums with avian Reovirus isolates, with one group receiving an oral and the other an intramuscular inoculation. As positive control, one isolator was added in which the chicks received a control inoculation with the live attenuated Reovirus vaccine strain 1133 by intramuscular route. Also one isolator housed a negative control group that received only a mock inoculation.

(14) All inoculations were given in a 0.1 ml dose per chick, and at 4.5 Log 10 TCID50/chick, for both the oral and the intramuscular route. The virus-inoculum dilutions were prepared fresh and within 1 hour before administration, and were kept on ice until use. Left-over inocula were used for back-titration, to confirm the inoculum dose that had been applied.

(15) All chicks were observed daily during the course of the experiment by qualified personnel for the occurrence of clinical signs of disease or other abnormalities. Animals showing pain or discomfort were euthanized and subjected to post-mortem examination.

(16) Selected chicks were taken for sampling at 3 and at 10 days post inoculation, whereby blood and tissue samples were collected. From the blood, serum was obtained, half of which was used for virus re-isolation, and half for antibody determination. The various tissues (tendon, liver and jejunum) were used for homogenisation and virus reisolation.

(17) 2.2. Results of the Virulence Confirmation Experiments

(18) The negative control chicks, or any of their samples, as well as the sera from the day zero hatchmates, did not show any signs of disease or infection that would be relevant to the experiment. Also the positive control group inoculated with avian Reovirus 1133 vaccine strain, displayed virus positive tissue(tendon, liver) and serum samples (at 10 days p.i.) as expected, so that the experiment was valid.

(19) Back-titration of the inoculation samples showed all inoculations were within 0.2 Log 10 TCID50 of the intended 4.5 Log 10 TCID50 dose/chick.

(20) 2.2.1. General Observations: Viral reisolations from blood were only positive at 3 days p.i., not at 10 days p.i. Detection of seroresponse proved negative at 3 or 10 days p.i. for the field isolates, only isolate 1133 inoculates did become Reo antibody positive at 10 days p.i. Apparently antibody titres were not yet high enough at this time to allow detection by the commercial test for avian Reovirus antibodies used (IDEXX REO antibody Elisa). Intramuscular inoculation caused much more samples to be positive for virus re-isolation than oral inoculation, for all of the virus isolates tested, and both at 3 and at 10 days p.i. All field isolates were virulent both by oral and by i.m. route. Clinical signs observed were depression and growth retardation, lameness in the inoculated leg, or complete immobility. Also intestinal inflammation and liver necrosis was frequently observed. Occasionally even mortality was observed in the test animals: for the SL11A823-2 BE (genotype group 1) isolate by oral route, and for the SL10A1581-32 ES (genotype group 3) isolate by i.m. route. While strain 1133 demonstrated a preference for tendon, the field isolates were reisolated in highest amounts from liver or jejunum; the highest reisolation overall was obtained for each of the field isolates from jejunum at 7 days p.i.
3. Nucleic Acid Isolation, Amplification and DNA-sequencing

(21) Culture supernatant from a 2nd or 3rd isolation passage, was used to isolate total viral avian Reovirus RNA. This was used to prepare and amplify cDNA, which could then be used for DNA sequencing. Standard methods and conditions were applied for all procedures, employing commercial kits and automated laboratory equipment.

(22) In short: viral RNA isolation was performed on 200 l samples of culture supernatant, using the MagNA Pure 96 (Roche). This equipment applies lysis of the sample, and isolates nucleic acid using magnetic beads. Next, the eluted nucleic acid was used to prepare first strand cDNA, by reverse transcription with the SuperScript III first strand synthesis kit (Invitrogen), according to the manufacturer's instructions.

(23) Used primers, specific for the avian Reovirus SigmaC gene were:

(24) TABLE-US-00001 REOFW1: (SEQIDNO:19) 5-AGTATTTGTGAGTACGATTG-3 REOREV5: (SEQIDNO:20) 5-GGCGCCACACCTTAGGT-3

(25) Whereby primer REO FW1 binds upstream of the SigmaC gene (approximate location on the avian Reovirus S1 genome segment: nucleotides 533-552), and primer REO REV5 binds at the 3end of the SigmaC gene (approximate S1 segment location: 1621-1605); this makes that the penultimate downstream nucleotides of the SigmaC gene were not determined. The primer REO FW1 was used for first strand synthesis, and both primers FW1 and REV5 were used for PCR amplification and sequencing reactions. These primers could be used for avian Reovirus samples from all genotype groups, however, occasionally the reverse primer was not effective enough, so that for some avian Reovirus samples additional primers were designed and used, to further amplify the viral nucleic acid.

(26) After cDNA synthesis, samples were amplified by PCR, using the FW1 and REV5 primers at 0.4 M final concentration in 50 l samples with 2 l cDNA sample, using the Phusion High-Fidelity DNA polymerase and master mix (Thermo Scientific). PCR conditions used were: 60 sec. 98 C.; 35 cycles of: 10 sec. 98 C., 30 sec. of 58 C., and 30 sec. 72 C.; followed by 10 min. at 72 C.

(27) cDNA preparation was verified by agarose gel-electrophoresis on a 1% agarose gel (Hispanagar), containing ethidium bromide, and by comparison to a Smartladder (Eurogentec) 200-10.000 bp marker lane. Gel bands of about 1.1 kbp were routinely obtained, illustrative of successful avian Reovirus SigmaC gene amplification.

(28) PCR samples positive for a 1088 bp band were purified using the QIAquick PCR purification kit (Qiagen). Next the DNA concentration was measured in a NanoDrop spectrophotometer (Thermo Scientific). Typically between 10 and 40 ng/l of viral cDNA was obtained.

(29) DNA sequence determination was done using automated cycle-sequencing equipment and sequence readings were assembled, aligned and analysed using computer software, all according to the manufacturer's instructions. In short: first cycle sequencing PCR was performed with 20 to 70 ng viral cDNA (typically 1 or 2 l of the purified PCR amplified viral cDNA sample), and 0.5 M of the FW1 or REV5 primer in 20 l per reaction, using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems). The sequencing PCR program was: 25 cycles of: 10 sec. 96 C., 5 sec. 50 C., and 2 min. 60 C. Next, samples were kept at 15 C. until analysis.

(30) Next the sequencing-PCR samples were purified using the Dye Ex Spin kit (Qiagen) according to the manufacturer's instructions, and samples were stored at 4 C. until the sequencing run commenced.

(31) DNA sequencing was performed by capillary electrophoresis using an ABI 3500 Genetic Analyzer and corresponding software (Applied Biosystems). Sequence data was then analysed using Sequencher v.54 software (Gene Codes Corp.).

(32) Some ambiguities in the assembled sequences could be resolved by use of additional primers, hybridising to internal regions of the SigmaC gene, to provide additional overlapping readings.

(33) 4. Sequence Alignments and Phylogeny

(34) Avian Reovirus amino acid sequences of SigmaC protein were analysed, both from sequenced field isolates as described above, and from public databases. The amino acid sequences were aligned using a combination of programs, such as: SIAS (http://imed.med.ucm.es/Tools/sias.html) for multiple pairwise sequence alignments, and MEGA6 (supra) for collection of results and phylogeny. Alignments were verified by one-on-one alignments using BlastP (supra), and the scores from this program were decisive in attributing a genotype group to a viral sequence.

(35) In several of the avian Reovirus outbreak isolates, the last 30 or so nucleic acids of the 3 region of the gene encoding the sigmaC protein could not be determined with confidence, as these bases directly followed the downstream PCR primer. Also, for several of the sequences from the public databases information on the C-terminal end of the sigmaC protein was missing. So to optimise the overlap in the alignments, the C-terminal end of the sigmaC protein's amino acid sequence was not used in these analyses. However this did not seem to affect the fidelity of the grouping, as Kant et al. (supra) had also noted: using less than a complete sigmaC protein's amino acid sequence still could provide reliable grouping. Similarly, an occasional sequence ambiguity did not hinder the analysis.

(36) Alignments were made using amino acids 1-277 of the SigmaC protein when possible; sequences that were much shorter were not included in the analyses. An slightly shorter sequence, such as ISR-5223 was still included as this provided a link to prior art type genotype grouping.

(37) The alignment scores were rounded-off to whole integers, in line with the score results as provided by the BLAST program. However this means these numbers are accurate up to 0.49%, which over a length of 277 amino acids corresponds to 2 amino acids.

(38) Tables describing the various sequences analysed, and their grouping are given in the Tables section below, and a graphical representations of the phylogeny of the five genotype groups are presented in the Figures. From the many field sample tested, only a selected number of relevant and representative samples are reproduced here: Table 1 describes the most relevant of these field samples, and a further selection was made for those for which the full amino acid sequence is presented in the attached sequence listing, see Table 2. In Table 3 are listed the most relevant of the publicly available sequences, with their corresponding Database accession numbers, and their prior genotype grouping.

(39) Table 4 presentsin several sectionsthe results of pairwise multiple alignments of avian Reovirus SigmaC amino acid sequences for the different genotype groups; the amino acid sequence of the genotype group's reference strain was aligned to the listed sequences. The results are in percent amino acid identity, and are presented for representative members of the genotype (sub)group, as well as for the reference strains of the other genotype groups. From these alignments, the cut-off values were deducted that serve to characterise the different genotype groups as defined herein.

(40) 5. Plaque Purification of Field-isolates

(41) For two of the avian Reovirus breakthrough strains, plaque isolation was performed to obtain a clonally pure viral isolate for vaccination studies. These were isolates: SL11A823-1 BE, and: SL11A823-2BE, respectively belonging to genotype groups: 4 and 1. This was done using standard isolation of viral plaques on CEL cells in tissue culture dishes under agar. In short: dilutions were prepared of the two virus isolates, up to 110{circumflex over ()}8. Next 6 cm diameter cell-culture dishes were inoculated with 1 ml of CEL cells at 510{circumflex over ()}6 cells/ml, 1 ml viral dilution, and 3 ml standard growth medium with FCS and antibiotics. The dishes were incubated O/N. The next day supernatant medium was discarded, and the monolayer was washed twice with PBS. Next the dishes were given a 5 ml agarose overlay of a 1:1 mixture of: bacto agar dissolved at 2.25% in distilled water at 49 C., and 2 culture medium at 37 C. Dishes were incubated for 4 days, and stained with neutral red: each dish was given 2 ml of a solution of 0.04% v/v neutral red in PBS. This was incubated for 6 hours, after which viral plaques were visible.

(42) For each viral isolate, several individual plaques were picked from a dish with clearly separated plaques from high dilutions. The plaques were taken up into 0.5 ml of PBS. Next each plaque isolate was given one round of amplification on CEL cells in a T25 culture flask.

(43) The plaque isolations were repeated for two more rounds using 0.1 ml of the plaque amplificate from the previous round, to a total of three rounds of plaque purification; negative control dishes and flasks were always included.

(44) The 3rd round plaque purified avian Reovirus isolates were then amplified on primary chicken embryo fibroblast cells (CEF) in larger tissue culture flasks; CEF cells were prepared essentially in the same way as CEL cells, except that whole embryos were used for the trypsinisation. The amplifications yielded large volumes of high titred virus stocks for further use; titres of 7-8 Log 10 TCID50/ml could routinely be obtained.

(45) 6. Vaccine Preparation

(46) The plaque purified avian Reovirus isolates were further amplified for the production of experimental vaccines. To this purpose the viruses were propagated on Vero cells, the culture supernatants were harvested, inactivated with formaldehyde, and the inactivated viral antigen was used for emulsification with a mineral oil adjuvant into a water-in-oil emulsion vaccine. In short: a suspension of Vero cells at 110{circumflex over ()}5 cells/ml was seeded into 1750 cm.sup.2 rollerbottles with 500 ml of standard growth medium with 5% FCS and antibiotics. The rollerbottles were inoculated with 1 ml of the amplified plaque purified avian Reovirus isolates SL11A823-1BE or SL11A823-2BE, and incubated at 37 C. while rolling.

(47) After 5-6 days 100% cpe was observed, and the culture-supernatant was harvested. Diluted formalin was added to the culture-supernatant to a final concentration of 0.2% v/v. This mixture was left to inactivate for 48 hours at 37 C., while stirring at 200 rpm.

(48) After the inactivation, the avian Reovirus antigens were concentrated about 20 fold by ultra-filtration. At the laboratory scale this was performed using a Centriprep centrifugal filter device (Millipore) with a 10 kDa cut-off membrane: 15 ml volume samples were centrifuge for 40 minutes at 3000g at 20 C. The concentrated viral antigen was stored refrigerated at 2-8 C. until further use.

(49) An adjuvated vaccine was prepared by combining the avian Reovirus antigenic materials with an oily phase. The watery phase had been prepared by stirring aseptically water-for-injection with the concentrated viral antigen. The oily phase contained liquid paraffin as mineral oil and emulsifiers which (at the laboratory scale) were homogenised with the oil using an Ultra Turrax (IKA), next the oily phase was filter-sterilised through a 0.2 m Ultipor nylon filter (Pall). The oily and the watery phases were then emulsified using an Ultra Turrax, in runs of a few minutes to avoid heating up the mixture over 40 C. Homogeneity was then checked by light-microscopy. This was repeated until all water vesicles were smaller than 3 m.

(50) Next, the ready vaccine was dispensed into labelled sterile bottles under aseptic conditions, the bottles were closed with nitryl rubber stoppers, and sealed with a coded aluminium cap. The final vaccine product was stored refrigerated until use.

(51) In this way, avian Reovirus vaccines according to the invention were prepared. For a vaccine dose of 0.5 ml per chicken, the vaccine was made to contain 1% v/v per ml (over the final volume of the emulsified vaccine) of viral antigen of SL11A823-1BE (at 8.32 Log 10 TCID50/ml), and 2% v/v of SL11A823-2BE (at 6.45 Log 10 TCID50/ml). Control vaccines contained either no viral antigen, or only one of these two viral antigens.

(52) Similar vaccines were prepared to serve as comparative examples, containing other avian Reovirus antigens: either classical vaccine strains 1733 and 2408, as a multimeric single genotype group vaccine; or a combination of strains 1733, 2408 and ERS, containing antigens from genotype groups 1 (in two fold) and 5. The amounts of antigen used for these comparative vaccines were the same as in current commercial vaccines, and were measured in arbitrary Elisa units against a known standard reference.

(53) 7. Vaccination-challenge Experiments

(54) The water-in-oil vaccines prepared as described above were used in animal vaccination-challenge trials. In a series of experiments layer chickens were vaccinated with a vaccine according to the invention, based on avian Reovirus antigenic material from genotype groups 1 and 4, while their progeny was subjected by challenge inoculation to a severe infection with avian Reovirus from different genotype groups, to demonstrate the broad-spectrum protective- and cross-protective properties of such vaccines, to homologous- and heterologous challenge virus infection.

(55) 7.1. Experimental Design

(56) 7.1.1. Parental Vaccination

(57) Normal healthy SPF layer chickens were assigned to 8 groups of 12 chickens each. The chickens were White Leghorn layers, about 32 weeks of age, and to each group of 12 hens one rooster was added.

(58) The hens were vaccinated with one dose (0.5 ml) of the water-in-oil vaccine as described above, intramuscularly in the right breast muscle. The roosters were not vaccinated to provide negative control serum samples. From five weeks after vaccination eggs were collected daily for the subsequent challenge-inoculation experiments on the progeny. The eggs were stored at 4 C. until use.

(59) All chickens were housed in containment facilities, with Hepa filtered air in- and outlets. Standard chicken feed and tap water were available ad libitum.

(60) Chickens were assigned to the groups as they came to hand, although each group of hens was provided with one rooster. Chickens were marked individually using wing-bands or swift-tags.

(61) All chickens were placed one week prior to vaccination for acclimatisation, and were observed daily during the course of the experiment by qualified personnel for the occurrence of clinical signs of disease or other abnormalities.

(62) Parent Vaccination Schedule:

(63) Group 1: vaccine: SL11A823-1BE antigen Group 2: vaccine: SL11A823-2BE antigen Group 3: vaccine: SL11A823-1BE antigen and SL11A823-2BE antigen Group 4: mock vaccine (not containing avian Reovirus antigen) Group 5: classic avian Reovirus vaccine: strains 1733 and 2408 antigens (vaccine similar to the commercial vaccine Nobilis Reo Inac). Group 6: classic avian Reovirus vaccine (strains 1733 and 2408 antigens) with strain ERS antigen Roosters: no vaccine

(64) 7.1.2. Offspring Challenge Inoculation

(65) This experiment served to test the protection in progeny from vaccinated parents, for their ability to overcome a severe avian Reovirus infection, even when the challenge virus was from a different genotype group then the vaccine virus. Because of the scale and size, this was performed in two consecutive experiments, one testing challenge inoculation with isolates SL11A294-12 FR (genotype group 2) and SL10A1581-32 ES (genotype group 3), and two weeks later on new progeny from the same parents, a further series of challenge inoculations using isolates: SL11A823-1 BE (genotype group 4), and SL11A823-2 BE (genotype group 1).

(66) For both of these experiments, eggs collected from the parental vaccination experiment were incubateddivided by their parental treatment groupsin standard hatching incubators upto hatch at day 21. The day old chicks (of mixed sex) were gathered and were marked using wingbands. Evidently weak or small chicks were not included. From each parental treatment group 10 chicks were bled to provide serum samples to test the chicks' initial MDA status. Then chicks were divided by placement in separate negative pressure isolators, so that from each parental treatment group there was one isolator, containing 24-30 chicks. Feed and water were available ad libitum.

(67) The challenge inoculation was administered at the same day of placement: per isolator one type of challenge virus was used, whereby half of the chicks received the challenge by oral route and the other half by intramuscular route. Chicks were bled for collection of blood samples at 3 or at 10 days p.i.

(68) The challenge inoculations were given in a 0.1 ml dose with 4.5 Log 10 TCID50/chick, for each of the four challenge viruses tested, and was inoculated via the intramuscular (i.m.) route. The challenge virus dilutions were prepared fresh and within 1 hour before administration, and were kept on ice until use. Left-over inocula were used for back-titration, to confirm the inoculum dose that had been applied.

(69) All chicks were observed daily during the course of the experiment by qualified personnel for the occurrence of clinical signs of disease or other abnormalities. Animals showing pain or discomfort were euthanized and subjected to post-mortem examination.

(70) 7.2. Sample Analyses

(71) From the parents, blood samples were taken at 2 and 4 weeks after vaccination, as well as at 1 week before vaccination (day 0). From the offspring blood samples were taken at the start of the experiment and at 3 or 10 days p.i.

(72) The blood samples were transported to the laboratory at ambient temperature. After clotting at room temperature, serum was collected. For the progeny: half of the serum was stored at 70 C. for virus-isolation; remaining serum samples were heat inactivated for 30 minutes at 56 C., and subsequently, stored at 20 C. until use.

(73) The detection of avian Reovirus antibodies in the serum was done using the IDEXX REO antibody Elisa, according to the manufacturer's instructions.

(74) The experiments' validity was determined based on the absence of antibodies in the parents against avian Reovirus in the day 0 samples, and in the negative control samples.

(75) Offspring from the vaccinated hens was MDA+ against avian Reovirus, depending on their parents' treatment.

(76) Serum for virus reisolation had been obtained after clotting of the blood sample, and this was frozen at 70 C. without further treatment. When testing these samples, CEL cells had been seeded into 6 well tissue culture plates, at 2 ml per well with 110{circumflex over ()}6 cells/ml, in standard culture medium with 5% FCS and antibiotics. This was incubated overnight to form a monolayer. Next day the culture supernatant was removed and replaced with 4 ml standard medium without FCS. The wells were inoculated with 40 l of the test serum, and incubated for 5 days. Then 100 l of the well's supernatant was inoculated into wells of a new 6 well plate with CEL monolayer, and incubated again for 5 days. Next, avian Reovirus specific cpe was judged by light-microscopy. As negative samples remained negative even after a third passage, the two passages were used as standard.

(77) 7.3. Results of the Vaccination-challenge Experiments

(78) 7.3.1. Controls

(79) As all positive and negative control samples scored as was expected, therefore the experiment was considered valid.

(80) 7.3.2. Method of Assessing Results

(81) In assessing the results of the experiments on parent vaccination and offspring challenge, the many samples collected over the course of the experiment were analysed and compared. Serum was obtained and tested for antibodies and for virus reisolation; as for serology the 3-10 day measuring period was found not to be long enough to give clear positive results. Positive virus reisolation from serum was an indicator for an active avian Reovirus viraemia, and it was found that the virus reisolation from serum collected at 3 days after the intramuscular inoculation, gave a clear picture of the effect of the different treatments. This way it could be determined which vaccination of the parent could reduce the infection of avian Reovirus challenge virus in the progeny.

(82) For the treatment group receiving i.m. challenge, and serum collected at 3 days p.i., the serum from 5 chicks (one group only 4) per group were available for virus reisolation. As a cut-off, groups having 3 or more animals positive for virus reisolation in serum taken at 3 days p.i., were considered not to show reduction of challenge virus infection; 2 animals positive was considered doubtful; and zero or 1 positive was considered to show reduction of infection. Results are shown in Table 5, displaying if (YES), or if not (NO) reduction of infection by avian Reovirus challenge strain in the progeny was induced by the different vaccinations of the parents. Between brackets is indicated the basis for that conclusion by the number of chicks of the total tested, for which the serum sample taken at 3 d. p.i. was positive for avian Reovirus as determined by reading cpe after two passages on CEL cells.

(83) 7.3.3. Discussion of Results

(84) As is presented in Table 5, for all chicks derived from mock vaccinated parents (vaccination test group 4), avian Reovirus could be reisolated from serum taken at day 3 after inoculation, and this was the case for all of the challenge viruses applied. This indicated that the challenge virus had replicated unhindered in the chicks, and consequently that the chicks had not been protected by factors transferred from their (mock vaccinated) parents. Clinical signs were not overly apparent, as the chickens used here were of layer type, which are less sensitive than heavier type chickens such as broilers. Nevertheless clear differences in vaccination efficacy were observed.

(85) Parents in test group 5 were given a single vaccination with a vaccine containing antigenic material from two strains of classic avian Reovirus: strains 1733 and 2408, to mimic a broad genotype group 1 vaccine. This vaccine induced in offspring a significant reduction of infection by avian Reovirus challenge virus belonging to genotype groups 1 or 2 as defined herein. However no reduction of infection was induced against avian Reovirus challenge virus of genotype groups 3 or 4.

(86) Remarkably, parents in test group 6 did not provide a protection to their offspring that was any broader than that already provided by the genotype group 1 antigenic material as used for the vaccination of test group 5. This even though the vaccine used for group 6 had additional avian Reovirus antigenic material: from ERS strain (ERS strain belonging to genotype group 5 as defined herein). This shows that apparently there is no automatic broadening of protection obtained from the use of vaccines containing avian Reovirus antigenic material from more than a single genotype group.

(87) The vaccines used for test groups 1 or 2 contained antigenic material of avian Reovirus from either genotype group 1 or 4 (respectively). The parents from these test groups provided total protection in their offspring against the replication of avian Reovirus challenge virus that was of the same genotype as the vaccine strain. However no broad-spectrum or heterologous protection was induced, as these vaccines did not reduce infection by avian Reovirus from a genotype group that was different from their own genotype group.

(88) However, surprisingly it was found that a strong synergistic effect was obtained upon the combination of avian Reovirus antigenic material from genotype groups 1 and 4: parents receiving the vaccine of test group 3, which combined antigenic material from avian Reovirus genotype groups 1 and 4 (as defined herein) did provide broad-spectrum protection to their offspring: the chicks from these parents were able to reduce significantly the infection of all avian Reovirus challenge strains tested, both homologous and heterologous to the vaccine applied, and after a single vaccination.

(89) Tables

(90) TABLE-US-00002 TABLE 1 List of avian Reovirus field-outbreak isolates mentioned in the Examples Isolation Genotype Sample name year Country group SL06A0161-4 2006 US 4 SL06A0161-5 2006 US 4 SL09A0324-2 2009 DE 2 SL09A0324-3 2009 DE 2 SL09A0417-1 2009 BE 2 SL09A0459-11 2009 DK 4 SL09A0531-1 2009 PL 5 SL09A0877-4 2009 UK 3 SL09A0905-7 2009 DK 4 SL09A1218-3 2009 FR 4 SL10A0282-8 2010 LT 1 B SL10A0822-2 2010 UK 1 B SL10A1581-32 2010 ES 3 SL10A1597-4 2010 NL 1 B SL11A0268-12 2011 FR 2 SL11A0294-12 2011 FR 2 SL11A0712-12 2011 HU 3 SL11A0823-1 2011 BE 4 SL11A0823-2 2011 BE 1 B SL11A1174-2 2011 FR 4 SL11A1179-3 2011 FR 1 B SL11A1192-1 2011 LV 4 SL11A1414-13 2011 FR 5 SL11A1417-1 2011 BE 1 B SL11A1539-1 2011 NL 4 SL11A1646-41 2011 FR 5 SL11A1646-43 2011 FR 5 SL12A1142-1 2012 FR 1 B SL12A1627-4 2012 FR 1 B SL12A1628-1 2012 FR 1 B SL13A0226-1 2013 UK 1 B SL13A0226-2 2013 UK 4 SL13A0988-1 2013 BE 2 SL13A0988-2 2013 BE 1 B SL13A1000-5 2013 UK 1 B SL13A1000-6 2013 UK 1 B

(91) TABLE-US-00003 TABLE 2 Description of sequences presented in the sequence listing SEQ ID NO: name genotype group 1 SL11A0823-2_BE 1B 2 SL11A0294-12_FR 2 3 SL10A1581-32_ES 3 4 SL11A0823-1_BE 4 5 ERS 5 6 Reo-2177 1A 7 SL11A1417-1_BE 1B 8 SL10A0282-8_LT 1B 9 SL09A0324-2_DE 2 10 SL13A0988-1_BE 2 11 SL09A0417-1_BE 2 12 SL11A0712-12_HU 3 13 SL09A0877-4_UK 3 14 SL11A1539-1_NL 4 15 SL11A1192-1_LV 4 16 SL09A1218-3_FR 4 17 SL09A0531-1_PL 5 18 SL11A1646-43_FR 5 19 PCR primer: REO FW1 20 PCR primer: REO REV5

(92) TABLE-US-00004 TABLE 3 List of avian Reoviruses from prior art, mentioned in the Examples Year of GenBank acc. nr. or Genotype Reovirus name isolation Country SEQ ID NO group Prior classification 138 1992 US AF218359 1 A Kant, grp. 1 916 1992 TW AF297214 2 Kant, grp. 2 919 1992 TW AF204949 1 A Kant, grp. 1 1133 1973 US DQ868790 1 A 1733 1983 US AAB61607 1 A classic vaccine strains; 2177 1983 US SEQ ID NO: 6 1 A Kant, grp. 1; Lublin grp. 4 2408 1983 US AF204945 1 A 40973/2005 2005 US DQ872797 4 41560/2005 2005 US DQ872798 2 41565/2005 2005 US DQ872799 3 42563-1/2005 2005 US DQ872800 2 42563-4/2005 2005 US DQ872801 3 Kant, grp. 3 AVS-B 2006 US FR694197 4 Kant, grp. 4 ERS 1999 PL SEQ ID NO: 5 5 GEL01 96T 1996 DE AF354221 4 Kant, grp. 4 GEI10 97M 1997 DE AF354219 5 Kant, grp. 5; Lublin grp. 2 GEL05 96M 1996 DE AF354223 4 GEL06 97M 1997 DE AF354224 1 B Kant, grp. 1; Lublin, grp. 1 GEL12 98M 1998 DE AF354225 1 B GEL13A 98M 1998 DE AF354226 2 Kant, grp. 2; Lublin grp. 3 GEL13B 98M 1998 DE AF354227 3 Kant, grp. 3 ISR5215 2005/7 IL FJ793531 1 B Kant, grp. 1; Lublin, grp. 1 ISR5223 2005/7 IL FJ793544 5 Kant, grp. 5; Lublin, grp. 2 ISR59103 2005/7 IL AY332520 1 A Lublin, grp. 4 NLI12 96M 1996 NL AF354230 4 Kant, grp. 4 RAM1 1971 AU L38502 5 Kant, grp. 5 Som4 1971 AU L07069 5 Kant, grp. 5 VA-1 1984 IN EU681254 1 A

(93) TABLE-US-00005 TABLE 4 Pairwise multiple alignments of avian Reovirus SigmaC amino acid sequences. Genotype group 1; Subgroup 1 A Reference sequence: Reo-1733 score Group Reo-138 83% members Reo-2177 95% VA-1 97% GenBank_KC963051.1_CHINA 98% ISR59103 99% GenBank_KC963052.1_CHINA 99% Reo-919 99% Reo-1133 100% Reo-2408 100% Reference strains, Genotype other groups Group 1B SL11A0823-2_BE 75% 2 SL11A0294-12_FR 55% 3 SL10A1581-32_ES 52% 4 SL11A0823-1_BE 49% 5 ERS 48% Genotype group 1; Subgroup 1 B Reference sequence: SL11A0823-2_BE score Group SL11A1417-1_BE 83% members GEL12_98M 85% GEL06_97M 87% ISR5215 88% SL13A0988-2_BE 90% SL13A0226-1_UK 92% SL10A0282-8_LT 92% SL13A1000-6_UK 92% SL10A0822-2_UK 93% SL11A1179-3_FR 94% SL10A1597-4_NL 94% SL12A1627-4_FR 96% GenBank_HE985297.1_FR 96% SL12A1142-1_FR 96% SL12A1628-1_FR 96% GenBank_HE985300.1_FR 97% SL13A1000-5_UK 97% Reference strains, Genotype other groups Group 1A Reo-1733 75% 2 SL11A0294-12_FR 55% 3 SL10A1581-32_ES 50% 4 SL11A0823-1_BE 51% 5 ERS 49% Genotype group 2 Reference sequence: SL11A0294-12_FR score Group Reo-916 59% members GenBank_JX983602.1_USA-GA 68% SL09A0324-2_DE 69% GenBank_JX983599.1_USA-GA 69% Reo-42563-1/2005 69% GEL13a_98M 70% SL13A0988-1_BE 70% Reo-41560/2005 71% SL09A0417-1_BE 81% SL09A0324-3_DE 91% SL11A0268-12_FR 96% Reference strains, Genotype other groups Group 1A Reo-1733 55% 1B SL11A0823-2_BE 55% 3 SL10A1581-32_ES 48% 4 SL11A0823-1_BE 52% 5 ERS 48% Genotype group 3 Reference sequence: SL10A1581-32_ES score Group GEL13B98M 61% members Reo-42563-4/2005 61% Reo-40963/2005 63% SL11A0712-12_HU 63% Reo-41565/2005 65% SL09A0877-4_UK 90% Reference strains, Genotype other groups Group 1A Reo-1733 52% 1B SL11A0823-2_BE 50% 2 SL11A0294-12_FR 48% 4 SL11A0823-1_BE 53% 5 ERS 50% Genotype group 4 Reference sequence: SL11A0823-1_BE score Group SL11A1539-1_NL 66% members SL13A0226-2_UK 66% GenBank_JX983600.1_USA-GA 67% SL11A1192-1_LV 67% SL06A0161-4_USA 67% SL11A1174-2_FR 67% SL06A0161-5_USA 68% AVS-B 68% SL09A1218-3_FR 68% Reo-40973/2005 68% GEL01_96T 92% GEL05_96M 97% NLI12_96M 97% SL09A0905-7_DK 97% SL09A0459-11_DK 98% Reference strains, Genotype other groups Group 1A Reo-1733 49% 1B SL11A0823-2_BE 51% 2 SL11A0294-12_FR 52% 3 SL10A1581-32_ES 53% 5 ERS 64% Genotype group 5 Reference sequence: ERS score Group ISR5223 74% members SL09A0531-1_PL 78% GEI10_97M 80% SL11A1646-43_FR 80% SL11A1414-13_FR 81% Som4 82% RAMI 82% SL11A1646-41_FR 83% Reference strains, Genotype other groups Group 1A Reo-1733 48% 1B SL11A0823-2_BE 49% 2 SL11A0294-12_FR 48% 3 SL10A1581-32_ES 50% 4 SL11A0823-1_BE 64%

(94) TABLE-US-00006 TABLE 5 Results of vaccination - challenge experiments Reduction of infection in progeny by different avian Reovirus challenge strains (No. of chicks per total positive for Reovirus (cpe) in serum at 3 d. p.i.) SL11A823-2 BE SL11A294-12 FR SL10A1581-32 ES SL11A823-1 BE Group Parental vaccine (genotype group 1) (genotype group 2) (genotype group 3) (genotype group 4) 1 SL11A823-1 BE (genotype group 4) NO (3/5) n.d. n.d. YES (0/5) 2 SL11A823-2 BE (genotype group 1) YES (0/5) n.d. n.d. NO (3/5) 3 823-1 + 823-2 (genotype groups 4 + 1) YES (1/5) YES (0/4) YES (1/5) YES (0/5) 4 mock vaccine NO (5/5) NO (5/5) NO (5/5) NO (5/5) 5 1733 + 2408 (genotype group 1) YES (1/5) YES (0/5) NO (3/5) NO (4/5) 6 1733 + 2408 + ERS (genotype groups 1 and 5) YES (0/5) YES (1/5) NO (5/5) NO (3/5) n.d. = not done

LEGEND TO THE FIGURES

(95) FIGS. 1-6:

(96) Phylogenetic trees of amino acid sequence alignments from sigmaC proteins of representative avian Reoviruses that were analysed and compared in the Examples, for all the genotype (sub)groups. The trees were drawn using MEGA6, by first calculating pairwise alignment scores, and then drawing unrooted trees using the neighbour-joining method (Saitou N. & Nei M., 1987, Mol. Biol. and Evol., vol. 4, p. 406-425). The scale bar indicates the relative genetic distance.

(97) FIG. 1: Genotype subgroup 1 A

(98) FIG. 2: Genotype subgroup 1 B

(99) FIG. 3: Genotype group 2

(100) FIG. 4: Genotype group 3

(101) FIG. 5: Genotype group 4

(102) FIG. 6: Genotype group 5