Vaccine

Abstract

An isolated infectious bronchitis (IB) virus (IBV) QX1285 strain under accession number DPS RE RSCIC 41 in the IZSLER biobank. A vaccine comprising the isolated infectious bronchitis virus and a carrier. The virus may be inactivated. The virus may alternatively be live and attenuated. The vaccine may be formulated for an avian. The avian may be a chicken. The vaccine may be administrated via drinking water. Also claimed is an isolated infectious bronchitis virus having an S1 protein encoded by SEQ ID NO: 1 or a sequence having at least 96% sequence identity thereto, for use as a vaccine against infectious bronchitis. Also claimed is a method of vaccinating an avian against infectious bronchitis, comprising administering the vaccine. Also claimed is a process for preparing a live infectious bronchitis vaccine comprising passaging the virus to reduce pathogenicity, harvesting the attenuated virus, and processing the virus to produce a vaccine. The virus may be passaged at least 50 times. The virus may be passaged by culturing in allantoic fluid of chicken embryos.

Description

Technical Field The present invention relates to a novel infectious bronchitis virus (IBV) serotype, to isolated attenuated IBV strains derived therefrom, and to live or inactivated vaccines against infectious bronchitis made using the isolated attenuated IBV strain. This invention also relates to a method for protecting avians, such as poultry, against infectious bronchitis using these vaccines. Background to the Invention Infectious bronchitis virus is a coronavirus that causes respiratory disease in avian such as poultry (e.g., chickens). IBV causes an acute, highly contagious disease in poultry of all ages, affecting the respiratory, reproductive and renal systems. Young birds up to 4 weeks of age are most susceptible. Disease symptoms include, e.g., respiratory distress, reduced weight, reduced egg production (e.g., up to 70%), increased frequency of abnormal eggs, and increased rates of mortality. In addition, coinfection with Mycoplasma gallisepticum, M synoviae, Escherichia coli, and / or Avibacterium paragallinarum can exacerbate disease. Infection with IBV can therefore have a significant adverse impact on the production of poultry, and is generally controlled through vaccination of flocks. Several different genotypes and serotypes of IB viruses have been identified. The S1 region of the spike glycoprotein can be sequenced to determine the genetic type of the virus, which correlates with the virus serotype. The determined amino acid sequence can then be compared to published sequences to determine the degree of correlation to known strains Exemplary genotypes (or “strains”) of IB virus include 793B, Massachusetts, ltaly02, D274, Arkansas, B1648 and D1466. (See, e.g., Worthington et al. (June 2008), Avian Pathology 37:247-257) and “QX” or “QXIBV” (See Liu et al. (2006), Archives of Virology 151:1133-1148). EP0639640 describes the identification of a novel IBV strain belonging to a new IB serotype, and to a vaccine for infectious bronchitis derived from an attenuated form of that viral strain. US4751079 describes novel vaccines which are characterized in that they are derived from infectious bronchitis viruses which spontaneously hemagglutinate chicken erythrocytes. US9296991 describes vaccine compositions comprising live, attenuated or killed strains of IB-QX-like viruses There remains a need in the art for new vaccine compositions and methods of vaccination that provide specific protection against infectious bronchitis virus (IBV), and in particular against IB-QX and IB-QX-like IB viruses. Summary of the Invention The present invention satisfies the aforementioned need in the art by providing IB viruses that are useful, inter alia, as antigenic components in vaccine compositions that protect against infection by IB-QX and IB-QX-like viruses. The invention includes live, attenuated versions of IB-QX-like viruses. Suitable live, attenuated strains can be produced, e.g., by serially passaging IB-QX-like viruses until adequate attenuation is obtained. The present invention also includes inactivated versions of IB-QX-like viruses. IB-QX-like viruses for use in the context of the present invention can be obtained, e.g., from deposited strains of IB-QX-like viruses, field cases of IB-QX-like virus infection, or by construction of recombinant IB viruses expressing defined, predetermined gene segments such as a particular S1 gene sequence. The present invention also provides vaccine compositions comprising live, attenuated or killed strains of IB-QX-like viruses, as well as methods for making live, attenuated and / or killed strains of IB-QX-like viruses. The present invention also provides methods for vaccinating a bird against infectious bronchitis by administering to the bird a vaccine composition comprising the live, attenuated or killed strains of IB-QX-like viruses according to the invention. There is also provided a method of vaccinating an avian against infectious bronchitis (IB), said method comprising administering the vaccine composition disclosed herein to said avian. According to a further aspect of the invention, there is provided the isolated attenuated infectious bronchitis (IB) virus disclosed herein for use in vaccinating an avian. In particular, the compositions, methods and uses disclosed herein are suitable and effective in the vaccination of an avian, in particular a chicken, against infectious bronchitis (IB). There is also provided a process for the preparation of live infectious bronchitis vaccine which comprises passaging the infectious bronchitis virus in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity whilst retaining its immunogenicity, harvesting the attenuated virus and processing the harvested material to produce a vaccine. According to a further aspect of the present invention, there is provided a kit of parts to vaccinate an avian against IB, including the isolated attenuated infectious bronchitis (IB) virus disclosed herein or the composition as disclosed herein and instructions for use. The kit of parts generally includes a plurality of unit dosages of the composition. Brief description of the Figures Figure 1 shows a comparison of the results obtained using ELISA kits from different manufacturers as described in Example 9. Detailed Description of the Invention The present invention provides isolated attenuated IB virus herein designated as QX1285. It has been found that this specific isolated attenuated virus is particularly effective when administered as a vaccine to protect against avian infectious bronchitis, in particular when caused by IBV of the QX serotype. The attenuated IB virus QX1285 of the present invention is able to protect against infectious bronchitis caused by a IB-QX-like virus. As used herein, the term “isolated” means that the viruses are not contained within a tissue of a live animal. As used herein the term “poultry” refers to any domesticated fowl, for example chicken, turkey, duck, pheasant etc. According to one aspect of the invention there is provided a novel infectious bronchitis virus serotype which has been isolated and deposited on 16 April 2024 with the Biobank of Veterinary Resouces (BVR) of the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna (IZSLER Biobank), Italy, under the Budapest Treaty, and designated accession no. DPS RE RSCIC 41 and / or an infectious bronchitis strain which is cross-neutralised significantly by antisera raised in chickens against said deposited strain. According to a further aspect of the present invention, there is provided an isolated infectious bronchitis (IB) virus having an S1 protein which is encoded by the nucleotide sequence of SEQ ID NO:1 or a sequence having at least 96% sequence identity thereto for use as a vaccine against infectious bronchitis. QX1285 is an IB-QX-like virus. As used herein, the term “IB-QX-like virus” means an IB virus with an S1 protein encoded by a nucleotide sequence that is at least 95% identical to the nucleotide sequence that encodes the S1 protein of the original IB-QX strain (AF193423), for example is at least 96% identical, for example 97% identical, 98% identical or 99% identical to the original IB-QX strain (AF193423). The nucleotide sequence that encodes the S1 protein of the original IB-QX strain is available under NCBI Genbank Accession No. AF193423. In addition, the invention features an IB virus with an S1 protein encoded by a nucleotide sequence having at least 75% identity with the nucleotide sequence that encodes the S1 protein of the original IB-QX strain, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the nucleotide sequence has at least 96% sequence identity, generally at least 97% or 98% sequence identity, with the nucleotide sequence that encodes the S1 protein of the original IB-QX strain. To attenuate the novel IBV serotype of the present invention the virus may be passaged in embryonated eggs, for example of domesticated poultry, such as chicken. Inoculation of the eggs can be via the allantoic cavity, chorioallantoic membrane, yolk sac, amniotic cavity or even direct into the embryo. For example, embryonated chicken eggs are inoculated with a quantity of the IB-QX-like virus via the allantoic cavity. The inoculated eggs are incubated at suitable incubation conditions, times and temperatures, for example 37°C for 24 to 72 hours (depending on virulence). The allantoic fluid is then harvested from the eggs. The virus is then said to have been passaged “1 x.” The harvested allantoic fluid from the first passage, at appropriate dilution, is then inoculated into new embryonated eggs, which are incubated at suitable conditions, humidity, times and temperatures, for example 37° C for 24-48 hours, and the allantoic fluid is harvested from this second set of eggs. The virus is then said to have been passaged “2x” and this can be denoted as “p2”. Continued passaging in this manner can continue indefinitely. Alternative environments conducive to virus replication that can be used to passage IB viruses include, e.g., cell cultures such as chicken kidney cell cultures or chicken embryonic fibroblast cultures. Additionally, a viral isolate can be obtained by plating out the virus as is known in the art. The virus can be passaged at regular intervals of from 7 hours up to 4 days. More usually passaging takes place between 16 to 36 hours, for example every 24 hours. One skilled in the art would be able to determine a suitable period of incubation, and the incubation period may be adjusted as passaging continues. Likewise, other incubation temperatures may be used. For example, embryonated eggs may be incubated at temperatures ranging from 20° C. to 42° C. Alternatively, attenuation may be achieved by passaging the virus in avian cell culture, such as chicken embryo kidney (CEK) cells. Samples of virus can be tested after each passage (or at regular intervals of passage) for degree of virulence. The degree of virulence can be determined by, for example, administering the passaged virus to chicks and assessing various parameters indicative of infectious bronchitis. Exemplary parameters include: (i) ciliary activity of tracheal explants; (ii) clinical signs such as, e.g., watery exudates from eye or nose, gasping, or diarrhoea; (iii) gross pathological examination of, e.g., upper airways, kidneys, spleen and / or intestine; and (iv) histology of the trachea, lung and kidney. The degree of virulence can also be estimated by observing deformation of the chicken embryos used to culture the virus. The novel IBV serotype of the present invention can also be produced by using recombinant, or “reverse genetics” methods, as is well-known in the art. For example, Casais et al. (2003) J. Virol. 77:9084-9089, describe the construction of a recombinant IB virus expressing a heterologous spike gene (See also Hodgson et al. (2004) J. Virol. 78:13804-13811). This system involves the use of an IB virus infectious clone, i.e., a full length IB virus cDNA cloned into a vector such as, e.g., a vaccinia viral vector. (See, e.g., Casais et al. (2001) J. Virol. 75:12359-12369). Starting with an IB virus infectious clone, recombinant IB viruses expressing the S1 protein of any other IB virus can be constructed. Thus, using the system of Casais et al. or variations thereof, recombinant IB viruses can be easily made that express the S1 protein from any IB-QX-like virus, thereby producing recombinant IB-QX-like viruses. Recombinant IB-QX-like viruses thus produced can be used in the context of the present invention in the same manner that naturally obtained IB-QX-like viruses (e.g., field isolates) are used, as described in detail herein. The recombinant or attenuated IBV prepared as described above, or the deposited attenuated strain, may be used in the preparation of a live vaccine. According to a further aspect of the present invention there is provided a live infectious bronchitis vaccine for use in immunizing poultry, said vaccine derived from the IBV described above. According to another aspect of the invention there is provided a process for the preparation of a live infectious bronchitis vaccine which comprises passaging the novel IBV serotype, or strain as hereinbefore described, in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity whilst retaining its immunogenicity and processing the material harvested to produce a vaccine. Preferably the virus is passaged at least 30 times. The present invention also relates to the use of an attenuated infectious bronchitis virus strain, as hereinbefore described, for the preparation of a vaccine for use in vaccinating poultry against IBV. Such live vaccines may be administered by eye drop, nose drop, in drinking water, or by spraying the birds, at any age from one day old up to point of lay (about 18 weeks). The dosage used is preferably in the range of 103 0 to 1070 EIDso per bird, for example 037 to 1053, preferably between 1040 and 1050 EIDso per bird. In one embodiment, the vaccine is administered via drinking water. Alternative forms of administration include spray administration or administration by droplet. Such an attenuated IB vaccine may be administered in combination with other live avian vaccines, for example Newcastle Disease Virus (NDV), Marek’s Disease Virus (MDV), Infectious Bursal Disease (IBD), reovirus, Avian Encephalomyelitis, Chicken Anaemia Agent (CAA) and other IBV serotypes. Alternatively, the novel IBV serotype according to the invention and / or the novel attenuated IBV strain described above may be presented as an inactivated vaccine. For both live and inactivated vaccine production the IBV is usually grown in embryonated specific pathogen free (SPF) chicken eggs. After harvesting, the virus may be inactivated, for use in a killed vaccine, using for example formaldehyde, p-propiolactone or binary ethyleneimine (BEI). After inactivation and, if necessary, adjusting of the pH and neutralising of the inactivating agent, the inactivated virus may be mixed with an adjuvant. The adjuvant can be aluminium hydroxide or a composition consisting of mineral oil (e.g., Marcol 82) or a plant oil and one or more emulsifiers (for example Tween 80 and / or Span 80). For example, live attenuated virus can be harvested, mixed with a stabilizer, and freeze dried. Suitable stabilizers include a first stabilizer comprising polyvinyl pyrrolidone, dextran 40, peptone, monosodium glutamate, potassium hydroxide, potassium dihydrogen phosphate in water and / or a second stabilizer comprising gelatine, peptone, arginine, and disodium phosphate dihydrate in water. Other suitable stabilizers can be used. If the vaccine of the invention is to be coadministered as an inactivated vaccine (for example with other inactivated vaccines), then it may be administered subcutaneously, in ovo or intramuscularly. The IBV vaccine of the present invention may be administered in combination with other inactivated avian vaccines, for example NDV, CAA, Egg Drop Syndrome 1976 and / or with vaccines specific to other IBV serotypes. According to yet a further aspect of the invention there is provided a method for protecting poultry against IBV comprising administering a vaccine as hereinbefore described to susceptible birds. According to a further aspect of the present invention, there is provided a kit of parts to vaccinate an avian against IB, including the isolated attenuated infectious bronchitis (IB) virus disclosed herein or the composition as disclosed herein and instructions for use. The kit of parts generally includes the composition as disclosed herein. The kit of parts may include a plurality of unit dosages of the composition. Kits can further include instructions for performing the methods described herein and / or interpreting the results. In addition, software can be included in the kit for assessing the efficacy of the methods of vaccination. Preferably, the kits are packaged in a container suitable for commercial distribution, sale, and / or use, containing the appropriate labels, for example, labels including the identification of the pharmaceutical / biological compounds included. Throughout the Application, where a composition is described as having, including, or comprising specific components, or where methods are described as having, including, or comprising specific process steps, it is contemplated that composition of the present teachings also consists essentially of, or consists of, the recited components, and that the methods of the present teachings also consist essentially of, or consist of, the recited process steps. In the Application, where an element or component is said to be included in and / or selected from a list of recited elements or components, or where a method is said to include certain steps, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and / or features of a composition, or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein. It should be understood that the expression “at least one of” includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The use of the singular herein, for example, “a,” “an,” and “the,” includes the plural (and vice versa) unless specifically stated otherwise. Where the use of the term “about” is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously. Features, integers, characteristics, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. At various places in the present specification, values are disclosed in groups or in ranges. It is specifically intended that the description include each and every individual sub combination of the members of such groups and ranges and any combination of the various endpoints of such groups or ranges. For example, an integer in the range of 0 to 40 is specifically intended to individually disclose 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40, and an integer in the range of 1 to 20 is specifically intended to individually disclose 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. The use of any and all examples, or exemplary language herein, for example, “such as,” “including,” or “for example,” is intended merely to illustrate better the present teachings and does not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present teachings. In any embodiment herein, the effective amount may be determined in relation to a subject. “Effective amount” refers to the amount of a compound or composition required to produce a desired effect. One non-limiting example of an effective amount includes amounts or dosages that yield acceptable pathogenicity and bioavailability levels for vaccine use. All documents referred to herein are incorporated by reference. Any modifications and / or variations to described embodiments that would be apparent to one of skill in art are hereby encompassed. Whilst the invention has been described herein with reference to certain specific embodiments and examples, it should be understood that the invention is not intended to be unduly limited to these specific embodiments or examples. Example 1 Description of forming the vaccine p63 The IB virus used to form the vaccine was isolated from a broiler farm. The virus was isolated from chicken respiratory tract and identified as a QX strain of IBV by PCR analysis. Passaging was performed by inoculating 9- to-12-day old SPF embryos with material from the previous virus passage. After inoculation, embryos were incubated at 37.5°C and >50% r.h. (relative humidity) for 48 to 72 hours. Inoculated eggs were candled daily for detection of embryo deaths. After incubation, embryos were chilled and allantoic fluid was harvested and used as an inoculum for a subsequent embryo passage. During passaging, morphological changes on embryos were observed that are characteristic for IB viruses: curling, stunting, dwarfing, clubbing of the down etc. Virus titrations were performed after each 5 to 10 passages. Virus titer was determined on SPF eggs and ranged from 106-8 EIDso / ml (passage 15) to 108-6 EIDso / ml (passage 59). After 15 passages, increased mortality rate was observed in inoculated embryos. Further passages were performed with a 10-fold dilution of the harvested allantoic fluid, and incubation time was reduced to 30 to 48 hours. Virus titre was determined on passages 18 and 33 and was shown to be 1075 EIDso / ml in both cases. Due to increased mortality of embryos, passaging from passage 42 onwards was performed with 100-fold dilution of the previous passage and with reduction of incubation time to 24-30 hours. Virus titre in passage 46 was shown to be 108 3 EIDso / ml. Further passaging was performed with a 1000-fold dilution of the previous passage. From passage 53 onwards, inoculation was performed with a 10,000-fold dilution of a previous passage and with 24 hours of incubation before harvest. Passaging was performed up to 63 times, generally 50 to 60 times. Harvested material of all passages was kept in multiple aliquots and stored at -80°C. Example 2 Materials and methods Two groups of one-day-old Specific Pathogen Free (SPF) chickens were included in the study. The chickens were 1 day old at vaccination. One group (G1) was vaccinated with the allantoic fluid from a passage p63 virus from Example 1 in a dose of 1042 EIDso by spray in 0.5ml. The other group (G2) was used as nonvaccinated challenge control group. Twenty-one days after the vaccination, chickens were challenged with virulent IB QX isolate. Birds were observed daily from day 0 to day 25 for general appearance, behaviour and clinical signs of IB during whole experiment. Four days after challenge the activity of cilia in tracheal explants were determined. Results and Conclusion: 100% of control chickens from group G2 showed extreme loss of ciliary activity and during the period between the vaccination and challenge, less than 10% of vaccinated or control chickens show abnormal clinical signs or die from causes not attributable to the vaccination. All the chickens in control group (G2) were affected by the challenge. 85% of chickens in the vaccinated group (G1) were protected against challenge. Example 3 Description of forming the vaccine p58 The isolated virus was prepared as described in Example 1 but was passaged 58 times. Harvested allantoic fluid was mixed with stabilizer and freeze dried. Example 4 Materials and methods Two groups of one-day-old commercial chickens with MDA (maternally derived antibodies) against IBV were included in the study. The chickens were 1 day old at vaccination. One group (G1) was vaccinated with a batch of the vaccine from Example 3 in a dose of 1037 EIDso by spray in 0.5ml. The other group (G2) was used as non-vaccinated challenge control group. Twenty-one days after the vaccination, chickens were challenged with virulent IB QX isolate. Birds were observed daily from day 0 to day 27 for general appearance, behaviour and clinical signs of IB during whole experiment. Six days after challenge the activity of cilia in tracheal explants were determined. Results and Conclusion: 100% of control chickens from group G2 showed extreme loss of ciliary activity and during the period between the vaccination and challenge, less than 10% of vaccinated or control chickens showed abnormal clinical signs or died from causes not attributable to the vaccination. All the chickens in control group (G2) were affected by the challenge 70% of chickens in the vaccinated group (G1) were protected against challenge. No mortality or clinical signs were observed during observation period in any of groups. Fisher’s exact test analysis showed the significant difference in protection levels between vaccinated chickens (G1) and respective non-vaccinated control chickens (G2), six days after challenge, with p = 0.0003 (the difference was considered significant with p <0.05). Efficacy of the Vaccine against Avian Infectious Bronchitis Virus, strain QX 1285 when used at a minimum dose 1037 EIDso and therefore onset of immunity 21 days after vaccination was demonstrated. Example 5 Materials and methods Two groups of one-day-old commercial chickens (commercial broilers) with MDA (maternally derived antibodies) against IBV were included in the study. The chickens were 1 day old at vaccination. One group (G1) was vaccinated with a batch of the vaccine from Example 3 in a dose of 103-7 EIDso in 0.02ml per chicken by oculo-nasal / eye-nose drop. The other group (G2) was used as non-vaccinated challenge control group. Twenty-one days after the vaccination, chickens were challenged with virulent IB QX isolate. Birds were observed daily from day 0 to day 27 for general appearance, behaviour and clinical signs of IB during whole experiment, with special attention to symptoms of Infectious Bronchitis from day 0 (DO) until day 27 (D27). Six days after challenge chickens were euthanized and the activity of cilia in tracheal explants was examined and the efficacy of vaccination was determined. Results and Conclusion: 100% of control chickens showed cessation or extreme loss of ciliary activity. No more than 10% of vaccinated or control chickens showed abnormal clinical signs or died from causes not attributable to the vaccine during the period between the vaccination and challenge. Fisher’s exact test analysis showed the significant difference in protection levels between vaccinated chickens (G1) and respective non-vaccinated control chickens (G2), six days after challenge, with p <0,0001 (the difference was considered significant with p <0.05). As shown in the following table, 80% (16 out of 20) vaccinated birds (G1) were found to be protected. None of the control birds (G2) was found to be protected. The results demonstrate that the vaccine of Example 3 used at a minimum dose 103 7 EIDso and applied oculo-nasaly (eye-nose drop method) efficacy and therefore onset of immunity 21 days after vaccination was clearly demonstrated. Example 6 -- Determination of Vaccine Safety Materials and methods One group of one-day-old SPF chickens (White Leghorn / VALO BioMedia) were vaccinated against IBV using a vaccine prepared as described in Example 3, but passaged 54 times in a dose of 1065 EID50 in 0.5ml per chicken by oculo-nasal / eye-nose drop. The chickens were 1 day old at vaccination. Chickens were observed for any reaction to treatment, 1 and 4 hours after vaccination and then daily for general appearance, behaviour and signs of IB until the end of the study. On each of day five, seven and ten after inoculation, five chickens per group were euthanized and samples of trachea and kidney were collected (trachea for ciliary activity tests and kidney for histological examination). Results and Conclusion: No chicken died from causes not attributable to the vaccine virus. No immediate general or local reaction could be observed 1 and 4 hours after vaccination except in one chicken for two days, most probably as a consequence of vaccination. No mortality occurred during the observation period. The average ciliostasis scores (determined in accordance with Ph. Eur. 07 / 2020:0442) obtained on each sampling point are given in Table 1: Table 1 Day 5 Day 7 Day 10 12.4 5.6 3.6 Kidney histology (nephritis) was scored as: Scoring the level of nephritis. 1 (slight) - focal lymphocytic and histiocytic infiltration in the intertubular tissues: 2 (moderate) -- multifocal lymphocytic and histiocytic infiltration in the intertubular tissues; and 3 (marked) - diffuse lymphocytic and histiocytic infiltration in the intertubular 5 tissues, accompanied with atrophy of affected tubules and accumulation of urate crystals. The results of the kidney histological examination is summarised in Table 2 below: Table 2 Days post application Chicken number Score Mean 5 1. 2 1.0 2. 0 3. 0 4. 2 5. 1 7 6. 0 0.2 7. 0 8. 0 9. 1 10. 0 10 11. 0 0.0 12. 0 13. 0 14. 0 15. 0 Average score of group 0.4 In summary, the results show that the vaccine of Example 3 was safe when a 10 fold dose was applied by oculonasal (spray) route to SPF chickens. No chicken showed notable clinical signs or abnormal local or systemic reactions or signs of avian infectious bronchitis or died from causes attributable to the vaccine virus. Inflammatory lesions seen during the kidney histological examination were, at most, moderate. Example 7 Materials and methods Two groups of one-day-old SPF chickens were included in the study. One group (G1) was vaccinated with the vaccine prepared as described in Example 3, but passaged 54 times. Allantoic fluid of the p54 passaged virus was used in a dose of 1042 EIDso in 0.5ml per chicken by spray. The other group (G2) was vaccinated with a commercially available vaccine against IBV. strain QX in a dose of 1O3 OEIDso (minimum dose declared in the SPC) in 0.5ml by spray. Twenty-one days after the vaccination, chickens were challenged with the same virulent IB QX isolate. Birds were observed daily from day 0 to day 27 for general appearance, behaviour and clinical signs of IB during whole experiment, with special attention to symptoms of Infectious Bronchitis from day 0 (DO) until day 25 (D25). Four days after challenge chickens were euthanized and the activity of cilia in tracheal explants was examined and the efficacy of vaccination was determined. Results and Conclusion: G1 showed 90% protection after challenge. G2 showed 80% protection after challenge. Example 8 Materials and methods Three groups of one-day-old commercial broiler chickens were included in the study Groups G1 and G2 were vaccinated by spray with minimum dose of vaccines listed below. Group G3 was left unvaccinated. Twenty-one days after the vaccination, chickens in all 3 groups were challenged with the virulent IB QX isolate. Birds were observed daily (DO to D25) for general appearance, behaviour and clinical signs of IB during whole experiment. Four days after challenge the activity of cilia in tracheal explants were determined. Group G1 was vaccinated with a batch of the vaccine prepared as described in Exampie 3, but passaged 56 times, in a dose of 1O4-2EID5o in 0.5ml per chicken by spray. Group G2 was vaccinated with a commercially available vaccine, IB Primo QX lyophilizate for suspension for spray, Intervet International. The dose was 1O4OEIDso (minimum dose declared in the SPC) in 0.5ml by spray. Group G3 was unvaccinated. Results and Conclusion: G1 showed 95% protection after challenge. G2 showed 80% protection after challenge. G3 (unvaccinated) showed no protection after challenge. Example 9 Four groups of one-day-old SPF chickens were included in the study. Groups G1, G2 and G3 were vaccinated by spray at day old with minimum dose of vaccines listed below. Group G4 was left unvaccinated. At predefined time intervals after vaccination, blood and tear samples have been taken from all the chickens in each group and tested for presence of antibodies against IBV QX. Group G1 was vaccinated with the vaccine prepared as described in Example 3, but passaged 56 times, in a dose of 1042 EIDso in 0.5ml per chicken by spray. Group G2 was vaccinated with a commercially available vaccine, Poulvac IB QX lyophilizate for suspension for spray, Zoetis. The dose was 1O3 OEIDso (minimum dose declared in the SPC) in 0.5ml by spray. Group G3 was vaccinated with a commercially available vaccine, IB Primo QX, lyophilisate for oculo-nasal suspension for chickens, Intervet International. The dose was 1O4OEID5o (minimum dose declared in the SPC) in 0.5ml by spray. Group G4 was unvaccinated. 5 Sampling: • Tears at 14, 21 and 28 days after vaccination * Blood at 21 and 28 days after vaccination 10 Results and Conclusion: BioCheck indirect ELISA Positive samples / total Average S / P ratio of the group Tears* Sera** Tears* Days after vaccination 14 21 28 28 28 Group G4 (control) 0 / 10 0 / 10 0 / 10 0 / 10 0.06 G1 3 / 10 10 / 10 10 / 10 1 / 10 1.06 G2 2 / 10 8 / 10 10 / 10 1 / 10 0.65 G3 1 / 10 - 5 / 10 0 / 10 0.68 *Tears were diluted 1:50 *sera samples were diluted 1:500 15 Samples with S / P ratio >0.200 were considered positive, according to manufacturers' instructions IDEXX indirect ELISA, 28 days after vaccination Group Positive samples / total Average S / P ratio of the group Tears* Sera** Tears* G4 (control) 0 / 10 0 / 10 0.01 G1 7 / 10 1 / 10 0.49 G2 5 / 10 0 / 10 0.21 G3 4 / 10 0 / 10 0.32 *Tears were diluted 1:50 *sera samples were diluted 1:500 Samples with S / P ratio >0.200 were considered positive, according to manufacturers' instructions IDvet indirect ELISA, 28 days after vaccination Group Positive samples / total Average S / P ratio of the group Tears* Sera** Tears* G4 (control) 0 / 10 0 / 10 0.03 G1 10 / 10 1 / 10 0.75 G2 8 / 10 0 / 10 0.53 G3 5 / 10 0 / 10 0.44 *Tears were diluted 1:50 *sera samples were diluted 1:500 10 Samples with S / P ratio >0.200 were considered positive, according to manufacturers' instructions Idvet competitive ELISA, 28 days after vaccination Group Positive samples / total Tears* Sera* G4 (control) 0 / 10 0 / 10 G1 7 / 10 3 / 10 G2 2 / 10 4 / 10 G3 4 / 10 3 / 10 15 Tears and sera samples were diluted 1:10 according to manufacturer's instructions Samples with PI value over 40% were considered positive according to manufacturer's instructions. Figure 1 shows a comparison of the results obtained using ELISA kits from different manufacturers. Virus neutralisation (VN) assay VN assay was performed to quantify the amount of neutralising antibodies after vaccination. The test has been performed on chicken embryo kidney cells using QX antigen at concentration of 100 TCIDso. VN titer has been determined as a last dilution of sample that still causes virus neutralisation. Results VN titer in sera 28 days p.v. and tears 21 days p.v. Group G1 G2 G3 Tears Sera Tears Sera Tears Sera Average VN titer (logz) 3.1 2.9 2.2 3.1 1.6 1.3 Positive / total samples 7 / 10 9 / 10 6 / 10 9 / 10 5 / 10 5 / 10 Example 10 Production of a Master Seed Virus (MSV) 12-day old SPF embryos were inoculated with 102 0 log EIDso of virus inoculum prepared from passage 54 (see Examplel). After inoculation, eggs were incubated at 37.5°C and 70% r.h. (relative humidity) for 24 hours. After cooling, the eggs overnight, the harvest was performed by aspiration of allantoic fluid from the eggs. Harvested allantoic fluid was first centrifuged and then the supernatant was filtered through 0.45pm and 0.22pm sterile filter cartridges. No antibiotic was added in any step of the production. Harvested material was sampled for absence of microbial contamination and virus titer and then frozen at <-70aC. Master seed virus was prepared by freeze drying the harvested antigen with the addition of stabilizer comprising polyvinyl pyrrolidone, dextran 40, peptone, monosodium glutamate, potassium hydroxide, potassium dihydrogen phosphate, and sucrose in water.. After freeze drying, vials of the MSV were sampled for quality control and stored at <-40°C. Example 11 Materials and methods Three groups of one-day-old SPF chickens were included in the study. Group G3 was a control group, and was divided into 2 subgroups (G3A and G3B) on day 21. Twenty-one days after the vaccination, chickens in all 3 groups were challenged with the virulent IB QX isolate. Birds were observed daily for general appearance, behaviour and clinical signs of IB during whole experiment. Six days after challenge the activity of cilia in tracheal explants were determined. Group G1 was used for oculo-nasal (eye-nose drop method) vaccination with a batch of vaccine which had been passaged 58 times (p58), in a dose of 103-7 EIDso in 0.02ml per chicken at 1-day of age. Group G2 was used for oral vaccination with a batch of vaccine which had been passaged 58 times (p58), in a dose of 1037 EIDso in 0.02ml per chicken. Group G2 were vaccinated at 7 days of age. Group G3 was unvaccinated. On day 21, 20 chickens from G1 and 5 chickens from G3A were challenged with virulent IB QX (i.e., 21 days after vaccination of Group G1). On day 28, 20 chickens from G2 and 5 chickens from G3B were challenged with virulent IB QX (i.e., 21 days after vaccination of Group G2). Challenge was performed for all groups using the eye-drop method and a dose of 1034 EIDso in a volume of 0.1 ml / chicken. Results and Conclusion: G1 showed 85% protection after challenge. G2 showed 80% protection after challenge. G3A (unvaccinated) showed no protection after challenge. G3B (unvaccinated) showed no protection after challenge. Example 12 Materials and methods Two groups of one-day-old MDA positive chickens were included in the study. Group G2 was a control group. There were 20 chickens in Group G1 and 10 chickens in Group G2. Twenty-one days after the vaccination, chickens in both groups were challenged with the virulent IB QX isolate in a dose of 103 4 EIDso per chicken. Birds were observed daily (DO to D27) for general appearance, behaviour and clinical signs of IB during whole experiment. Six days after challenge the activity of cilia in tracheal explants were determined. Group G1 was used for oculo-nasal (eye-nose drop method) vaccination at one day of age with a batch of the vaccine passaged 58 times (p58), in a dose of 103 9 EIDso in 0.5ml per chicken by spray. Group G2 was unvaccinated. Challenge was performed on day 21 for both groups using the eye-drop method and a dose of 103 4 EIDso in a volume of 0.1ml / chicken. Results and Conclusion: G1 showed 90% protection after challenge. G2 showed no protection after challenge. Example 13 Materials and methods Two groups of one-day-old SPF chickens were included in the study. Group 1 (45 chickens) were vaccinated oculo-nasally by spray at 1 day old using a batch of vaccine which had been passaged 58 times (p58), in a dose of 1037 EIDso in 0.5ml per chicken. Group 2 was a group of 15 chickens of the same age and origin. This group was the unvaccinated control group. On study day 14 (D14), vaccinated Group 1 was divided and 20 chickens were transferred to a separate isolator to form group GIA, with remainder as G1. Likewise, five chickens from Group 2 were removed to a separate isolator to form Group2A, with the remaining 10 chickens being retained as G2. On day 14, groups G1A (20 chickens) and G2A (5 chickens) were challenged with the virulent IB QX isolate in a dose of 103-4 EIDso per chicken in 0.1ml. Challenge was performed using the eye-drop method. On day 21, groups G1 (20 chickens) and G2 (5 chickens) were challenged with the virulent IB QX isolate in a dose of 1034 EIDso per chicken in 0.1ml. Challenge was performed using the eye-drop method. Birds were observed daily for general appearance, behaviour and clinical signs of IB during whole experiment. Six days after challenge (D20 for G1A and G2A; and D27 for G1 and G2), the activity of cilia in tracheal explants were determined. Results and Conclusion: Protection levels of 50% and 80% were observed 14 and 21 days after vaccination respectively, as shown in the following Table. Group Birds with ciliostasis score >1' (between brackets the scores of these birds) No affected versus total Percentage protection GIA 10(3; 2; 10; 3; 4; 3; 5; 3; 6; 7) 10 / 20 50% G2A 5 (10; 10; 10; 10; 10) 5 / 5 0% G1 4(3; 8; 5; 10) 4 / 20 80% G2 5(10; 10; 10; 10; 10) 5 / 5 0% Example 14 Materials and methods Two groups of one-day-old commercial broiler chickens were included in the study. Group 1 (25 chickens) were orally vaccinated at 7 days of age using a batch of vaccine which had been passaged 58 times (p58), in a dose of 1037 EIDso in 0.01ml per chicken. Group 2 was a group of 15 chickens of the same age and origin. This group was the unvaccinated control group. Blood was sampled on study days 6 (D6) and 27 (D27) from 25 chickens of G1 and 15 chickens of G2 in order to obtain information about anti-IBV antibody level in sera. Birds were observed daily for general appearance, behaviour and clinical signs of IB from day 0 (DO) until challenge day 28 (D28). On study day 28 (D28) 20 chickens from G1 and 10 chickens from G2 group were challenged with virulent IB QX isolate in a dose of 1034 EIDso per chicken. The surplus chickens were removed from the study. During the observation period after challenge (from D29 to D34) chickens were observed daily for general appearance, behaviour and presence of any clinical signs, with special attention to IB symptoms. Six days after challenge, chickens were euthanized and the activity of cilia in tracheal explants was examined (20 chickens from G1; all chickens from G2) and the efficacy of vaccination was determined. As shown in the following table, 85% (17 out of 20) of the vaccinated chickens (G1) were found to be protected whereas in the control group, all chickens were affected by challenge (protection level 0%). Group Birds with cihostasis score >11 (between brackets the scores of these birds) No. affected versus total Percentage protectioR^i|||||| G1 3(10; 10; 10) 3 / 20 85% G2 10(10; 10; 10; 10; 10; 10; 10; 10; 10; 10) 10 / 10 0% 1 more than 1 out of the 10 tracheal sections present less than 50% cilia movement. Example 15 Three groups of one day-old SPF chickens were included in the study. Two groups of 45, SPF chickens per group (G1 and G2) were used for vaccination with the Vaccine against Avian Infectious Bronchitis Virus, strain QX 1285, containing 103 7 EIDso. Vaccination was performed on one-day old SPF chickens by the spray method (G2) and on seven day-old chickens via the oral route (G1). A third group of 20 chickens of the same age and origin was kept as non-vaccinated control (G3). On study day 28 (D28) each vaccinated group (G1 and G2) was divided in two parts. Twenty chickens from G1 became group G1A and the remaining chickens continued the study as group G1. Twenty chickens from G2 became group G2A and the remaining chickens continued the study as group G2. Group G3 was a common control group for all vaccinated groups and was divided into three subgroups (G3, G3A and G3B) prior to challenge administrations. Each subgroup of group 3 (G3) consisted of 5 chickens. Sixty three days (9 weeks) after vaccination, 20 chickens from group 1A (G1 A) and 5 control chickens from group 3B (G3B) as well as 20 chickens from group 2A (G2A) and 5 control chickens from group 3A (G3A) were challenged. Seventy days (10 weeks) after vaccination 20 chickens from group 2 (G2), 20 chickens from group 1 (G1) and 5 control chickens from group 3 (G3) were challenged. Common control group for 20 chickens from G2 were 5 chickens from G3B. Challenge was performed for all seven groups with virulent IB QX isolate in a dose of 103 4 EIDso per chicken using the eye drop method. Birds were observed daily for general appearance, behaviour and the presence of any clinical signs, with special attention to symptoms of Infectious Bronchitis during the whole observation period until the end of the animal phase of the study. Six days after challenge, chickens were euthanized and the activity of cilia in tracheal explants was examined and the efficacy of vaccination was determined. Results and conclusion: Protection levels of 80% were observed 9 weeks after vaccination for both orally (group G1) and spray vaccinated (group G2) chickens. Protection level of 70% was observed 10 weeks after vaccination in orally vaccinated group, while spray vaccinated group was 60% protected. All the control groups challenged at different timepoints were not protected at all against challenge (protection level 0%). Duration of immunity was clearly demonstrated for up to 10 weeks after vaccination with Vaccine against Avian Infectious Bronchitis Virus, strain QX 1285.

Claims

1. An isolated infectious bronchitis (IB) virus deposited with the IZSLER under accession No. DPS RE RSCIC 41.

2. A vaccine comprising the isolated infectious bronchitis (IB) virus of claim 1 and a pharmaceutically acceptable carrier.

3. The vaccine as claimed in claim 2 wherein isolated IB virus is inactivated.

4. The vaccine as claimed in claim 2 wherein said isolated IB virus is live andattenuated.

5. The vaccine as claimed in any one of claims 2 to 4 formulated for administration to an avian.

6. The vaccine as claimed in claim 5 wherein the avian is a chicken.

7. The vaccine as claimed in any one of claims 2 to 6 for administration viadrinking water.

8. An isolated infectious bronchitis (IB) virus having an S1 protein which is encoded by the nucleotide sequence of SEQ ID NO:1 or a sequence having at least 96% sequence identity thereto for use as a vaccine against infectious bronchitis.

9. The vaccine as claimed in claim 8 wherein isolated IB virus is inactivated.

10. The vaccine as claimed in claim 8 wherein said isolated IB virus is live andattenuated.

11. The isolated infectious bronchitis (IB) virus of any one of claims 8 to 11 for use as an avian vaccine.

12. The isolated infectious bronchitis (IB) virus of claim 11 wherein said avian is a chicken.

13. A method of vaccinating an avian against infectious bronchitis (IB), said method comprising administering the vaccine composition of claims 2 to 12 to said avian.

14. The method as claimed in claim 13, wherein said avian is a chicken.

15. A process for the preparation of live infectious bronchitis vaccine which comprises passaging the infectious bronchitis virus in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity whilst retaining its immunogenicity, harvesting the attenuated virus and processing the harvested material to produce a vaccine.

16. The process according to claim 15 wherein the IBV is passaged at least 50 times.

17. The process according to claim 15 or claim 16 wherein the IBV is passaged by culturing in allantoic fluid of chicken embryos.25 07 24Amendments to the Claims have been filed as follows:CLAIMS1. An isolated infectious bronchitis (IB) virus deposited with the IZSLER under accession No. DPS RE RSCIC 41.

52. A vaccine comprising the isolated infectious bronchitis (IB) virus of claim 1 and a pharmaceutically acceptable carrier.

3. The vaccine as claimed in claim 2 wherein isolated IB virus is inactivated.

104. The vaccine as claimed in claim 2 wherein said isolated IB virus is live and attenuated.

5. The vaccine as claimed in any one of claims 2 to 4 formulated for15 administration to an avian.

6. The vaccine as claimed in claim 5 wherein the avian is a chicken.

7. The vaccine as claimed in any one of claims 2 to 6 formulated to be suitable20 for administration via drinking water.

8. An isolated infectious bronchitis (IB) virus having an S1 protein which is encoded by the nucleotide sequence of SEQ ID NO:1 or a sequence having at least 99% sequence identity thereto for use as a vaccine against25 infectious bronchitis.

9. The vaccine as claimed in claim 8 wherein isolated IB virus is inactivated.

10. The vaccine as claimed in claim 8 wherein said isolated IB virus is live and30 attenuated.

11. The isolated infectious bronchitis (IB) virus of any one of claims 8 to 11 for use as an avian vaccine.3525 07 2412. The isolated infectious bronchitis (IB) virus of claim 11 wherein said avian is a chicken.

13. A process for the preparation of live infectious bronchitis vaccine as claimed 5 in any one of claims 2 to 10 or virus as claimed in claims 11 and 12, whichcomprises passaging the infectious bronchitis virus in a culture on a suitable medium a sufficient number of times to reduce its pathogenicity whilst retaining its immunogenicity, harvesting the attenuated virus and processing the harvested material to produce a vaccine.1014. The process according to claim 12 wherein the IBV is passaged at least 50 times.

15. The process according to claim 12 or claim 13 wherein the IBV is passaged 15 by culturing in allantoic fluid of chicken embryos.

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