Attenuated IBV with extended cell culture and tissue tropism

Abstract

The present invention relates to the IBV (Infectious Bronchitis Virus) deposited at BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof and any IBV having all the identifying characteristics of the deposited IBV. Further, the present invention relates to an immunogenic composition comprising said deposited IBV.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing pursuant to 37 CFR 1.821-1.825. The Sequence Listing accompanying this application is hereby incorporated by reference in its entirety. Background Art

[0003] Avian coronavirus infectious bronchitis virus (IBV) is a prototype gamma coronavirus of the Nidovirales order, Coronaviridae. Infectious bronchitis virus infects the upper respiratory epithelium of chickens, causing respiratory diseases, usually complicated by secondary bacterial pathogens (Cook et al. 2012. Avian Pathol. 41: 239-250). Some IBV strains additionally affect the renal tubules, oviducts and part of the gastrointestinal tract, causing pathological damage and clinical signs in these organ systems. The virus has a worldwide presence in both commercial chickens and free-range chickens. Due to its high genome variability, IBV is divided into widely diverse genotypes, serotypes and protectotypes. IBV is currently regarded as one of the most economically relevant viral pathogens in the poultry industry.

[0004] Infectious bronchitis virus is an enveloped virus with a 27.6kb positive single-stranded RNA genome (Cavanagh 2007. Vet. Res. 38: 281-297). The first two-thirds of the viral genome contains a large coding region (also designated as gene 1), divided into two open reading frames 1a and 1b, which encode at least 15 non-structural proteins involved in RNA replication, editing and transcription. The last third of the viral genome encodes structural proteins: spike protein (S, encoded by gene 2), envelope protein (E, encoded by gene 3c), membrane protein (M, encoded by gene 4), and nucleocapsid protein (N, encoded by gene 6). Proteins S, E and M are part of the viral envelope, while protein N forms the ribonucleoprotein core together with the viral RNA. The coronavirus spike protein determines the tropism of the host species (Kuo et al. 2000. J. Virol. 74: 1393-1406). It is a dimeric or trimeric transmembrane protein that is proteolytically cleaved into two subunits, S1 and S2. The glycosylated S1 domain forms the 'head' of the spike protein and contains a receptor binding domain that interacts with 2,3-linked sialic acids on the surface of host cells (Promkuntod et al. 2014. Virology. 448: 26-32). The S2 domain is the remainder of the extracellular domain ('stem'), the transmembrane domain, and the intracellular domain located in the cytoplasm.

[0005] The attenuated live IBV vaccine strains H52 and H120 widely used so far were developed in the Netherlands in the 1960s by serial passage of Massachusetts serotype IBV strains in embryonated chicken eggs (Bijlenga et al. 2004; Avian Pathol. 33:550-557). The vaccine strains must also be propagated in embryonated chicken eggs for vaccine production. Today, IBV vaccines (both inactivated and live) are still propagated in embryonated chicken eggs, which is cumbersome and expensive.

[0006] The only cell line-adapted IBV described so far is the IBV strain Beaudette, which replicates efficiently in Vero and BHK cells. Casais et al. 2003 (J. Virol. 77; 9084-9089) showed that the S protein of Beaudette is a determinant of cell line tropism by using the extracellular domain sequence of the Beaudette spike to generate recombinant IBV, which is able to transfer this extended cell line tropism to another IBV (M41). Fang et al. 2005 (Biochemical and Biophysical Research Communication 336; pp. 417-423) disclosed that the adaptation of Beaudette for propagation in Vero cells resulted in 49 amino acid modifications, 26 of which were located in the spike protein. However, recombinant IBV with the Beaudette spike is not suitable as a vaccine. Ellis et al. 2018 (J. Virol. 92 (23)) describe that recombinant Beaudette constructs with chimeric spikes having heterologous S1 subunits from M41 or QX combined with Beaudette spike S2 ​​subunits do not confer adequate protection against S1 homologous challenge. In addition, Beaudette wild type does not provide protection against homologous challenge like other licensed vaccines belonging to the Massachusetts serotype (Hodgson et al. 2004: J Virol 78: 13804–13811 or Geilhausen et al. 1973: Archiv für die gesamte Virusforschung 40: 285-290).

[0007] In summary, providing an IBV vaccine with extended cell tropism or tissue tropism by exchanging the spike protein for a heterologous Beaudette spike protein (recombinant IBV) does not result in an IBV vaccine providing sufficient efficacy, and the use of the Beaudette spike sequence will be limited to protection against challenge with Massachusetts serotype strains and lack cross-protection against further genotypes. Further, in contrast to the recombinant IBV disclosed in the prior art, the IBV of the present invention is not a GMO (genetically modified organism).

[0008] Thus, there is a need for effective IBV vaccines with extended cellular or tissue tropism for efficient production.Preferably, such new IBV vaccines with extended cellular or tissue tropism are non-recombinant vaccines. DETAILED DESCRIPTION

[0009] Before describing aspects of the present invention, it must be noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antigen" includes multiple antigens, reference to "a virus" is a reference to one or more viruses and equivalents thereof known to those skilled in the art, and so forth. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods, apparatus, and materials are now described, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing cell lines, vectors, and methods as reported in the publications that may be used in conjunction with the present invention. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention.

[0010] Composition of matter

[0011] The present invention solves the problems inherent in the prior art and provides a significant advancement in the state of the art.

[0012] In general, the present invention provides an IBV (Infectious Bronchitis Virus) deposited at BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof, or any IBV having all the identifying characteristics of the IBV deposited under DPS RE RSCIC 16.

[0013] Further, the present invention provides an IBV (infectious bronchitis virus) of BVR deposited at IZSLER under accession number DPS RE RSCIC 16, any attenuated progeny IBV thereof having extended cell tropism or tissue tropism and being protective against challenge with virulent M41, or any attenuated IBV having extended cell tropism or tissue tropism and being protective against challenge with virulent M41.

[0014] Advantageously, experimental data show that the deposited strains and their progeny have extended cell tropism or tissue tropism, and they can infect and / or replicate in different cell lines and tissue cells. Surprisingly, the deposited strains and their progeny still maintain vaccine efficacy before at least several passages, but have improved safety.

[0015] The deposited IBV (IB66HP) strain was deposited at BVR (Biobank of Veterinary Resources of the Istituto ZooprofilatticoSperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini" Biobank of Veterinary Resource) of IZSLER under the Budapest Treaty under the accession number DPS RE RSCIC 16. The date of deposit or transfer was March 28, 2019. The deposited microorganisms tested were alive.

[0016] The term "IBV" refers to the infectious bronchitis virus well known to those skilled in the art. The term "IBV" includes all strains, genotypes, protective types and serotypes of infectious bronchitis virus.

[0017] The term "protective against virulent M41 challenge" means that the IBV provides protection against virulent M41 challenge or infection to a subject vaccinated with the IBV.

[0018] In a specific aspect of the IBV according to the invention, the IBV is attenuated.

[0019] The term "attenuated" refers to a pathogen with reduced virulence compared to a wild-type isolate. In the present invention, an attenuated IBV is an IBV in which the virulence has been reduced so that it does not cause clinical signs of IBV infection, but is able to induce an immune response in a target animal, but may also mean that clinical signs are reduced in incidence or severity in animals infected with the attenuated IBV compared to a "control group" of animals infected with non-attenuated IBV and not receiving the attenuated virus. In this context, the term "reduced / reduced" means a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, still more preferably 80%, still more preferably 90%, even more preferably 95%, and most preferably 100% compared to a control group infected with non-attenuated IBV as defined above. Thus, an attenuated IBV strain is a strain suitable for incorporation into an immunogenic composition comprising a modified live IBV.

[0020] In another specific aspect of the IBV according to the present invention, the IBV is attenuated in one-day-old chickens.

[0021] In another specific aspect of the IBV according to the present invention, the IBV is inactivated.

[0022] Any conventional deactivation method can be used for the purpose of the present invention. Therefore, deactivation can be carried out by chemical treatment and / or physical treatment known to those skilled in the art. Preferred deactivation methods include adding cyclized diethyleneimine (BEI), including adding a solution of 2-bromoethylamine hydrobromide (BEA), and the 2-bromoethylamine hydrobromide has been cyclized to diethyleneimine (BEI). Preferred further chemical deactivators include but are not limited to Triton X-100, sodium deoxycholate, hexadecyltrimethylammonium bromide, β-propiolactone, thimerosal, phenol and formaldehyde (formalin). However, deactivation can also include a neutralization step. Preferred neutralizing agents include but are not limited to sodium thiosulfate, sodium bisulfite, etc.

[0023] Preferred formalin inactivation conditions include about 0.02% (v / v)-2.0% (v / v), more preferably about 0.1% (v / v)-1.0% (v / v), still more preferably about 0.15% (v / v)-0.8% (v / v), even more preferably about 0.16% (v / v)-0.6% (v / v), and most preferably about 0.2% (v / v)-0.4% (v / v) formalin concentration. The incubation time depends on the resistance of the IBV. Generally speaking, the inactivation process is carried out until the growth of the IBV cannot be detected in a suitable culture system.

[0024] Preferably, the inactivated IBV of the present invention is formalin inactivated, preferably using the concentrations as described above.

[0025] The inactivated IBV of the present invention can be incorporated into liposomes using known techniques, such as those described in Nature, 1974, 252, 252-254 or Journal of Immunology, 1978, 120, 1109-13. In another embodiment of the present invention, the inactivated IBV of the present invention can be conjugated to a suitable biological compound such as a polysaccharide, peptide, protein, etc. or a combination thereof.

[0026] In another specific aspect of the IBV according to the invention, the IBV is non-recombinant.

[0027] As used herein, the term "non-recombinant" refers to an RNA genome (or RNA sequence, cDNA sequence or protein) that has not been recombinantly introduced by human intervention (e.g., insertions, deletions, inversions, relocations or point mutations). The term "non-recombinant" as used in relation to a virus means a virus that is not produced by recombinant artificial manipulation of the viral genome. The term "non-recombinant virus" excludes recombinant genetically modified viruses. The term "genetically engineered" refers to an IBV that has been mutated using a "reverse genetics" approach, in which a viral cDNA or chemically synthesized RNA is involved.

[0028] The terms "protein", "amino acid" and "polypeptide" are used interchangeably. The term "protein" refers to an amino acid sequence consisting of naturally occurring amino acids and their derivatives. Naturally occurring amino acids are well known in the art and are described in standard textbooks of biochemistry. Within the amino acid sequence, the amino acids are linked by peptide bonds. Further, the two ends of the amino acid sequence are referred to as the carboxyl terminus (C-terminus) and the amino terminus (N-terminus). The term "protein" includes substantially purified proteins or protein preparations that additionally contain other proteins. Further, the term also refers to protein fragments. In addition, it includes chemically modified proteins. Such modifications can be artificial modifications or naturally occurring modifications, such as phosphorylation, glycosylation, myristylation, etc.

[0029] In another specific aspect of the IBV according to the present invention, the IBV is a Massachusetts genotype or serotype.

[0030] IBV strains can be classified by serotype and genotype. Serotype classification involves treating the virus with neutralizing antibodies, while genotype classification generally involves examining the sequence of the S1 (spike) protein. However, different IBV strains are well known to those skilled in the art. Infectious bronchitis virus was first discovered in the United States in the 1930s.

[0031] The first IBV serotype identified was Massachusetts, and remained the only serotype until the discovery of a different IBV serotype in 1956. Today, IBV Mass (Massachusetts) viruses can be identified in many countries throughout the world.

[0032] IBV strain Beaudette is of the Massachusetts type and was derived after at least 150 passages in chicken embryos. IBV strain Beaudette was originally isolated by Beaudette and Hudson (J. Am. Vet. Med. A. 90, 51-60, 1937) and passaged in chicken embryos. Other Massachusetts type IBV strains besides Beaudette are H120, H52, and M41. The H120 strain was passaged 120 times in embryonated chicken eggs.

[0033] Where to obtain any IBV strain is within the general knowledge of those skilled in the art. IBV strains may be purchased commercially, obtained from scientific institutions, or the genome may be synthesized as complementary DNA, as IBV strains have been sequenced and the sequences have been published and are therefore available. In addition, IBV strains may be isolated from samples obtained from infected chickens. Methods for isolating and characterizing IBV strains are well known to those skilled in the art. Valter Leonardo de Quadros 2011 (Dissertation, Das Bronchitis Virus(IBV):Molekularbiologische Untersuchungen zur Diagnostik und zum Vorkommen sowiezur des Genotyps IBV QX in spezifisch pathogenfreien(SPF)Broilern, Freie Berlin et al. and Farsang et al. 2002 (Avian Pathology 31: 229-236) describe how to isolate and differentiate different IBV strains.

[0034] In another specific aspect of the IBV according to the present invention, the IBV is of the M41, H52 or H120 strain.

[0035] In another specific aspect of the IBV according to the present invention, the IBV is the M41 strain.

[0036] In another specific aspect of the IBV according to the present invention, the IBV is a H52 or H120 strain.

[0037] Where to obtain IBV M41, H52 or H120 is within the general knowledge of those skilled in the art. Exemplarily, the IBVH52 strain can be purchased commercially, such as the exemplary Nobilis IB H52 (MSD Animal Health), AviProIB H52 (Lohmann Animal Health GmbH & Co. KG), Bronchovac (Ceva), etc. The IBV H120 strain can be purchased commercially, such as the exemplary BIORAL H120 (Boehringer Ingelheim), HatchPak IB H120 (Boehringer Ingelheim), AviPro IB H120 (Lohmann Animal Health GmbH & Co. KG), Poulvac IB H120 (Zoetis), etc. The IBV M41 strain can be purchased commercially, such as the exemplary Volvac IBFit (Boehringer Ingelheim). Further, McDonald et al. 1980 (Avain Pathology 9:245-259) disclosed that IBV H52 can be obtained through the Central Veterinary Laboratory Rotterdam, Kusters (J. gen Virol 68:343-352) disclosed that IBV H52 can be obtained through the Poultry Health Institute Dorn (GD Animal Health) in the Netherlands, and Chen et al. 2007 (Avian Pathology 36 (4): 269-274) disclosed that IBV H52 can be obtained through the China Institute of Veterinary Drug Control. In addition, IBV M41, H52 or H120 have been used as vaccine strains for decades and can therefore be found and isolated from the wild. Methods for isolating IBV M41, H52 or H120 strains and characterizing IBV H52 strains are well known to those skilled in the art. Illustratively, the IBV H52 strain can be characterized as described in Zwaagstra et al. 1992 (J. Clin. Microbiol. 30(1):79-84), Handberg et al. 1999 (Avian Pathology 28:327-335) or Callison et al. 2006 (Journal of Virological Methods 138:60-65).For example, Zwaagstra et al. 1992 and Handberg et al. 1999 disclose Massachusetts specific primers (for S and N proteins, respectively) for RT-PCR and sequencing and reference sequences for comparison. Further, H52 IBV has been sequenced and the genome sequence is available, for example EU817497. Thus, the viral genome can be generated by synthesizing its sequence and after applying the reverse genetics system.

[0038] Extended cell tropism or tissue tropism

[0039] In another specific aspect of the IBV according to the present invention, the IBV has extended cell tropism or tissue tropism.

[0040] The term "cell or tissue" is known to those skilled in the art. The term cell includes cell lines, such as cell lines listed elsewhere herein, and primary cells. The term tissue includes cells from tissues such as those listed elsewhere herein, illustratively such as primary chicken embryo cells or primary chicken fibroblasts from the lung or liver. Included is the propagation of cells or tissues (cells) in culture outside an organism. The term "culture" relates to the propagation of cells (e.g., cell line cells or primary cells or tissue cells) outside an organism under defined culture conditions known to those skilled in the art.

[0041] The term "extended tropism" means that the IBV of the present invention can be propagated in cells (e.g., cell lines) or tissue cells (except primary chicken embryo cells from the kidney). In contrast, the IBV vaccines or non-cell-adapted wild-type IBV described in the prior art (cell-adapted IBV Beaudette strain is described) can only be propagated in embryonated chicken eggs or primary chicken embryo cells from the kidney (after adaptation). Accordingly, the IBV of the present invention with extended cell tropism or tissue tropism has the ability to infect and / or replicate in one or more cell lines or tissue cells other than primary chicken embryo cells from the kidney. Preferably, the IBV of the present invention with extended cell tropism or tissue tropism has the ability to infect and / or replicate in one or more cell lines as listed herein. Accordingly, for example, an IBV with extended cell tropism or tissue tropism may have the ability to infect and / or replicate in PBS-12SF or HEK 293T cells.

[0042] Advantageously, experimental data show that the deposited strains and their progeny have extended cell or tissue tropism and are able to infect and replicate in cells of different cell lines and tissues.

[0043] In another specific aspect of the IBV according to the present invention, the IBV infects and / or replicates in at least one cell line or cell selected from the following list: primary chicken embryo cells or primary chicken fibroblasts from the lung or liver, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryonic kidney cell lines, baby hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, pig kidney cell lines and insect cell lines.

[0044] In another specific aspect of the IBV according to the present invention, the IBV infects and / or replicates in at least one cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver carcinoma), MDCK (Madin-Darby canine kidney), MDBK (Madin-Darby bovine kidney), PK15 (porcine kidney), PK2A (porcine kidney), SF9, SF21 and SF+ (Spodoptera frugiperda).

[0045] In another specific aspect of the IBV according to the present invention, the IBV infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104, MDCK, SF9 and RK13.

[0046] All mentioned cell lines are well known to those skilled in the art, and are commercially available and / or publicly available. MDCK cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC CCL-34 or ATCC CRL-2285. DF-1 cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC CRL-12203). PBS-12SF cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC PTA-8565, or are deposited in RRID under CVCL-1K17. BHK-21 cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC CCL-10. HEK 293T cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC CRL-3216. Vero cells are exemplarily deposited in the American Tissue Culture Collection under accession number ATCC CCL-81. MA104 cells are exemplarily deposited in the American Tissue Culture Collection under the accession number ATCC CRL-2378. RK13 cells are exemplarily deposited in the American Tissue Culture Collection under the accession number ATCC CCL-37. SF9 cells are exemplarily deposited in the American Tissue Culture Collection under the accession number ATCC CRL-3357 or ATCC PTA-3099.

[0047] Preferably, IBV infects and / or replicates in the EB66, BHK, Vero, MA104, MDCK, SF9 and RK13 cell lines.

[0048] In another specific aspect of the IBV according to the present invention, the primary chicken embryonic cells are fibroblasts or cells derived from liver or lung tissue.

[0049] Function Definition - Protection

[0050] In another specific aspect of the IBV according to the present invention, the IBV is protective against virulent M41 challenge or infection.

[0051] The terms "protective" or "protective immune response" or "protective immunity" are defined elsewhere herein.

[0052] In another specific aspect of the IBV according to the invention, protection against virulent M41 challenge or infection is determined by ciliary arrest scores, reduced clinical signs of the respiratory system, reduced viral RNA load in kidney tissue, or reduced viral shedding.

[0053] In another specific aspect of the IBV according to the invention, the attenuation of the IBV is increased compared to an IBV without extended cell tropism or tissue tropism.

[0054] The term "increased attenuation" means that the efficacy parameters (particularly ciliary arrest, as well as rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load or viral shedding) are reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% compared to subjects of the same species immunized with IBV without extended cell tropism or tissue tropism. How to measure the improvement of efficacy parameters is within the general knowledge of those skilled in the art.

[0055] In another specific aspect of the IBV according to the invention, the attenuation of the IBV is increased compared to IBV M41 without extended cell tropism or tissue tropism.

[0056] In another specific aspect of the IBV according to the invention, the attenuation of the IBV is increased after application in 1 day old chickens compared to an IBV without extended cell tropism or tissue tropism.

[0057] In another specific aspect of the IBV according to the invention, the attenuation of the IBV is increased after application in 1 day old chickens compared to IBV M41 without extended cell tropism or tissue tropism.

[0058] All identification characteristics

[0059] In another specific aspect of the IBV according to the invention, all identifying characteristics of the deposited IBV mean that the IBV is attenuated, has extended cell tropism or tissue tropism, and is protective against challenge or infection with virulent M41.

[0060] In another specific aspect of the IBV according to the invention, all identifying characteristics of the deposited IBV mean that the IBV is attenuated, has extended cell tropism or tissue tropism, and a protection profile that is the same or similar to that of the deposited IBV.

[0061] In another specific aspect of the IBV according to the present invention, all the identifying characteristics of the deposited IBV mean that the IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and has the same or similar protection profile as the deposited IBV.

[0062] In another specific aspect of the IBV according to the present invention, all the identifying characteristics of the deposited IBV mean that the IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and is protective against challenge or infection with the virulent M41.

[0063] Descendants

[0064] In another specific aspect of the IBV according to the invention, the progeny comprises at most 15 passages in cell culture of the IBV deposited at BVR at IZSLER under the accession number DPS RE RSCIC 16.

[0065] In another specific aspect of the IBV according to the invention, the progeny comprises at most 10 passages in cell culture of the IBV deposited at BVR at IZSLER under the accession number DPS RE RSCIC 16.

[0066] In another specific aspect of the IBV according to the invention, the progeny comprises at most 5 passages in cell culture of the IBV deposited at BVR at IZSLER under the accession number DPS RE RSCIC 16.

[0067] Advantageously, experimental data show that the strain of preservation and its progeny have extended cell tropism or tissue tropism, and they can infect and / or replicate in different cell lines and tissue cells. Surprisingly, the strain of preservation and its progeny still maintain vaccine efficacy before at least several passages, and have improved safety.

[0068] In another specific aspect of the IBV according to the present invention, the progeny IBV is attenuated, has extended cell tropism or tissue tropism, and is protective against virulent M41 challenge or infection.

[0069] In another specific aspect of the IBV according to the present invention, the progeny IBV is attenuated, has extended cell tropism or tissue tropism, and a protection profile that is the same or similar to that of the deposited IBV.

[0070] In another specific aspect of the IBV according to the present invention, the progeny IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and has the same or similar protection profile as the deposited IBV.

[0071] In another specific aspect of the IBV according to the present invention, the progeny IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and is protective against virulent M41 challenge or infection.

[0072] Nucleotide sequences and plasmids

[0073] Further, the present invention provides a nucleotide sequence encoding an IBV as described herein. Thus, the present invention provides a nucleotide sequence encoding the IBV (Infectious Bronchitis Virus) of BVR deposited at IZSLER under accession number DPS RE RSCIC 16, any attenuated progeny IBV thereof having extended cell tropism or tissue tropism and being protective against virulent M41 challenge, or any attenuated IBV having extended cell tropism or tissue tropism and being protective against virulent M41 challenge.

[0074] Further, the present invention provides a plasmid comprising a nucleotide sequence encoding an IBV as described herein. Thus, the present invention provides a plasmid comprising a nucleotide sequence encoding the IBV (Infectious Bronchitis Virus) of BVR deposited at IZSLER under accession number DPS RE RSCIC 16, any attenuated progeny IBV thereof having extended cell tropism or tissue tropism and being protective against virulent M41 challenge, or any attenuated IBV having extended cell tropism or tissue tropism and being protective against virulent M41 challenge.

[0075] The term "nucleic acid" or "nucleic acid sequence" or "nucleotide sequence" refers to a polynucleotide, including a DNA molecule, an RNA molecule, a cDNA molecule or a derivative. The term includes single-stranded polynucleotides as well as double-stranded polynucleotides. The nucleic acids of the present invention include isolated polynucleotides (i.e., isolated from their natural environment) and genetically modified forms. In addition, also included are chemically modified polynucleotides, including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides such as biotinylated polynucleotides. Further, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to any nucleic acid. The terms "nucleic acid" and "polynucleotide" specifically also include nucleic acids composed of nucleotides having nucleobases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0076] The term "plasmid" refers to cytoplasmic DNA that replicates within a bacterial host cell independently of the bacterial chromosome.

[0077] cell

[0078] Further, the present invention provides a cell comprising an IBV or a plasmid as described herein.The cell may be a eukaryotic cell or a prokaryotic cell.

[0079] In another specific aspect of the cell according to the invention, the cell is a cell line or cell selected from the following list: primary chicken embryo cells, chicken embryo fibroblast cell line, duck embryo stem cell line, human embryonic kidney cell line, baby hamster kidney cell line, African green monkey kidney cell line, rabbit kidney cell line, canine kidney cell line, chicken liver cell line, bovine kidney cell line, porcine kidney cell line and insect cell line.

[0080] In another specific aspect of the cell according to the invention, the cell is a cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby dog ​​kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A, SF9, SF21 and SF+ (Spodoptera frugiperda).

[0081] In another specific aspect of the cell according to the invention, the cell is a cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0082] In another specific aspect of the cell according to the invention, the primary chicken embryonic cell is a fibroblast or a cell derived from liver or lung tissue.

[0083] Viral particles, immunogenic compositions and vaccines

[0084] Further, the present invention provides a viral particle comprising the IBV as described herein.

[0085] Further, the present invention provides an immunogenic composition comprising the IBV as described herein.

[0086] Further, the present invention provides a vaccine comprising the IBV as described herein.

[0087] Further, the present invention provides an improved live vaccine comprising an IBV as described herein, having extended cell tropism or tissue tropism.

[0088] The term "immunogenic composition" refers to a composition comprising at least one antigen that elicits an immune response in a host to which the immunogenic composition is administered. Such an immune response may be a cellular and / or antibody-mediated immune response to the immunogenic composition of the invention. Preferably, the immunogenic composition induces an immune response, and more preferably, confers protective immunity against one or more clinical signs of IBV infection. The host is also described as a "subject". Preferably, any host or subject described or referred to herein is avian or poultry.

[0089] Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or γ-δ T cells specific for one or more antigens included in the immunogenic composition of the invention. Preferably, the host will display a protective immune response or a therapeutic response.

[0090] A "protective immune response" or "protective immunity" will be demonstrated by a reduction or absence of clinical signs normally exhibited by an infected host, a more rapid recovery time, and / or a reduced duration of infectivity or reduced pathogen titers in the tissues or body fluids or excretions of the infected host.

[0091] In situations where the host exhibits a protective immune response such that resistance to new infection will be enhanced and / or the clinical severity of disease will be reduced, the immunogenic composition is described as a "vaccine."

[0092] The terms "modified live" and "attenuated" are used interchangeably herein.

[0093] In another specific aspect of the immunogenic composition or vaccine according to the present invention, the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier.

[0094] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0095] "Diluents" may include water, saline, dextrose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid, among others.

[0096] In another specific aspect of the immunogenic composition or vaccine according to the invention, the pharmaceutically acceptable carrier is phosphate buffered saline.

[0097] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0098] Preferably, the pharmaceutically acceptable carrier is chitosan.

[0099] Chitosan is a natural deacetylated polysaccharide from chitin in crustaceans (e.g. shrimp, crab), insects and other invertebrates. Recently, Rauw et al. 2009 (Vet Immunol Immunop 134: 249–258) demonstrated that chitosan enhanced the cellular immune response of a live Newcastle disease vaccine and promoted its protective effect. Further, Wang et al. 2012 (Arch Virol (2012) 157: 1451–1461) have shown results revealing the potential of chitosan as an adjuvant for use in live attenuated influenza vaccines.

[0100] Preferably, the immunogenic composition may further include one or more other immunomodulators, such as interleukins, interferons or other cytokines.The amounts and concentrations of adjuvants and additives that can be used in the context of the present invention can be readily determined by the skilled person.

[0101] In some aspects, the immunogenic composition of the present invention contains an adjuvant. As used herein, "adjuvant" may include aluminum hydroxide and aluminum phosphate, saponins such as QuilA, QS-21 (Cambridge Biotech Inc., Cambridge MA), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. Emulsions may be based in particular on light liquid paraffin oil (European drug type); isoprenoid oils such as squalane or squalene; oils resulting from oligomerization of olefins, particularly isobutylene or decene; esters of acids or alcohols containing linear alkyl groups, more particularly vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), tri(caprylate / caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, particularly isostearates. Oils are used in combination with emulsifiers to form emulsions. Emulsifiers are preferably nonionic surfactants, in particular sorbitan, mannide (e.g. anhydromannide oleate), ethylene glycol, polyglycerol, propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular Pluronic products, especially L121. See Hunter et al., The Theory and Practical Application of Adjuvants (ed. Stewart-Tull, DES), John Wiley and Sons, NY, pp. 51-94 (1995), and Todd et al., Vaccine 15:564-570 (1997). Exemplary adjuvants are the SPT emulsion described on page 147 of "Vaccine Design, The Subunit and Adjuvant Approach", edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same book.

[0102] A further example of adjuvant is a compound selected from a polymer of acrylic acid or methacrylic acid and a copolymer of maleic anhydride and an alkenyl derivative. Advantageous adjuvant compounds are polymers of acrylic acid or methacrylic acid, which are especially cross-linked with polyalkenyl ethers of sugars or polyols. These compounds are known by the term carbomer (Phameuropa Vol. 8, No. 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes such acrylic polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyls, preferably no more than 8 hydroxyls, wherein the hydrogen atoms of the at least three hydroxyls are replaced with unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred radicals are those containing 2 to 4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated radical itself may contain other substituents, such as methyl. Products sold under the name carbomer; (BF Goodrich, Ohio, USA) are particularly suitable. They are cross-linked with allyl sucrose or allyl pentaerythritol. Among them, Carbomer 974P, 934P and 971P can be mentioned. Most preferred is the use of Carbomer 971P. Among the copolymers of maleic anhydride and alkenyl derivatives there is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. The dissolution of these polymers in water results in an acid solution which will preferably be neutralized to physiological pH in order to give an adjuvant solution into which the immunogenic composition, the immunological composition or the vaccine composition itself will be incorporated.

[0103] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (Ribi Inc.), block copolymers (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, avridine lipid-amine adjuvant, heat-labile enterotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS 1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or analogs thereof, or stimulators of endogenous cytokine release, and the like.

[0104] It is expected that the adjuvant can be added in an amount of about 100 μg to about 10 mg / dose, preferably in an amount of about 100 μg to about 10 mg / dose, more preferably in an amount of about 500 μg to about 5 mg / dose, even more preferably in an amount of about 750 μg to about 2.5 mg / dose, and most preferably in an amount of about 1 mg / dose. Alternatively, the adjuvant can be in a concentration of about 0.01% to 50% by volume of the final product, preferably in a concentration of about 2% to 30%, more preferably in a concentration of about 5% to 25%, still more preferably in a concentration of about 7% to 22%, and most preferably in a concentration of 10% to 20%.

[0105] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine is effective for treating and / or preventing clinical signs caused by IBV in a subject in need thereof. The terms "treating and / or preventing", "clinical signs" and "in need thereof" have been defined elsewhere.

[0106] In another specific aspect of the immunogenic composition or vaccine according to the present invention, said immunogenic composition or vaccine is formulated for single dose administration.

[0107] Volumes for single doses are defined elsewhere herein.

[0108] Furthermore, it has been shown that one dose of the immunogenic composition of the invention is effective following such single dose administration of such immunogenic composition or vaccine.

[0109] In another specific aspect of the immunogenic composition or vaccine according to the invention, the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, in ovo, via spray, via the drinking water or by eye drops.

[0110] In another specific aspect of the immunogenic composition or vaccine according to the present invention, the immunogenic composition or vaccine comprises 1 to 10 log 10 EID50 / IBV dose.

[0111] In another specific aspect of the immunogenic composition or vaccine according to the present invention, the immunogenic composition or vaccine comprises 2 to 5 log 10 EID50 / IBV dose.

[0112] In another specific aspect of the immunogenic composition or vaccine according to the present invention, the immunogenic composition or vaccine comprises 2 to 4 log 10 EID50 / IBV dose.

[0113] Cultivation methods

[0114] Further, the present invention provides a method for culturing an IBV in a cell line or tissue cell, comprising using an IBV as described herein. Thus, the present invention provides a method for culturing an IBV in a cell line or tissue cell, comprising using the following: an IBV of BVR deposited at IZSLER under accession number DPS RE RSCIC 16, any attenuated progeny IBV thereof having an expanded cell tropism or tissue tropism and being protective against a virulent M41 challenge, or any attenuated IBV having an expanded cell tropism or tissue tropism and being protective against a virulent M41 challenge.

[0115] In another specific aspect of the method for culturing IBV in a cell line or tissue cell according to the invention, IBV infects and / or replicates in a cell line or tissue cell as described herein.

[0116] In another specific aspect of the method for culturing IBV in a cell line or tissue cell according to the present invention, the cell line or tissue cell is selected from the following list: primary chicken embryo cells, chicken embryo fibroblast cell line, duck embryo stem cell line, human embryonic kidney cell line, baby hamster kidney cell line, African green monkey kidney cell line, rabbit kidney cell line, canine kidney cell line, chicken liver cell line, bovine kidney cell line, porcine kidney cell line and insect cell line.

[0117] In another specific aspect of the method for culturing IBV in cell lines or tissue cells according to the present invention, the cell line is selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby dog ​​kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A, SF9, SF21 and SF+ (Spodoptera frugiperda).

[0118] In another specific aspect of the method for culturing IBV in cell lines or tissue cells according to the present invention, the cell line is selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0119] Reagent test kit

[0120] When needed, the composition may be present in a package or dispenser device, which may contain one or more unit dosage forms containing active ingredients. The package may, for example, comprise metal or plastic foil, such as a blister pack. The package or dispenser device may be provided with instructions for use preferably for application to a subject, especially poultry. Associated with such containers may be notices in the form of regulations for the manufacture, use or sale of a drug or biological product, which reflect approval for use by manufacture, use or sale agencies.

[0121] The invention provides kits comprising an IBV or an immunogenic composition or vaccine as described herein.

[0122] In a particular aspect of the kit according to the invention, the kit further comprises instructions for use in the treatment and / or prevention of avian diseases.

[0123] In a particular aspect of the kit according to the invention, the kit further comprises instructions for use in the treatment and / or prevention of poultry diseases.

[0124] In a particular aspect of the kit according to the invention, the kit further comprises instructions for use in the treatment and / or prevention of IB.

[0125] In a particular aspect of the kit according to the invention, the kit further comprises a dispenser capable of administering the vaccine to said animal.

[0126] Treatment

[0127] Further, the present invention provides methods for immunizing a subject comprising administering to such subject an immunogenic composition as described herein.

[0128] The term "immunization" relates to active immunization by administering an immunogenic composition to a subject to be immunized, thereby eliciting an immune response against the antigens included in such immunogenic composition.

[0129] Preferably, the immunization results in a reduction in the incidence of, or a reduction in the severity of, clinical signs caused by or associated with a specific IBV infection in the flock of birds.

[0130] Further, immunization of a subject in need thereof with an immunogenic composition as provided herein results in preventing infection of the subject with an IBV infection. Even more preferably, the immunization results in an effective, durable immune response against an IBV infection. It should be understood that the period of time will last for more than 1 month, preferably more than 2 months, preferably more than 3 months, more preferably more than 4 months, more preferably more than 5 months, and more preferably more than 6 months. It should be understood that the immunization may not be effective in all subjects immunized. However, the term requires that a significant proportion of the subjects in the flock are effectively immunized.

[0131] Preferably, in this context a population of subjects is envisaged which would normally (i.e., if not immunized) develop clinical signs normally caused by an IBV infection or associated with an avian coronavirus or IBV infection. Whether the subjects of said population are effectively immunized can be determined without difficulty by a person skilled in the art. Preferably, the immunization should be effective if the clinical signs in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, even more preferably at least 95%, and most preferably 100% of the subjects of a given population are reduced in incidence or severity by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%, compared to subjects who were not immunized or immunized with an immunogenic composition available prior to the present invention, but subsequently infected with a specific IBV, are reduced in incidence or severity by at least 10%, more preferably at least 20%, even more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100%.

[0132] Further, the present invention provides a method for treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine as described herein.

[0133] As shown in the Examples, the immunogenic compositions or vaccines as provided herein have been demonstrated to be effective in treating or preventing clinical signs caused by IBV in a subject.

[0134] The term "treatment or prevention" refers to a reduction in the incidence of a specific IBV infection in a flock of birds, or a reduction in the severity of clinical signs caused by or associated with a specific IBV infection. Thus, the term "treatment or prevention" also refers to a reduction in the number of subjects in a flock of birds that become infected with a specific IBV (= a reduction in the incidence of a specific IBV infection), or a reduction in the severity of clinical signs typically associated with or caused by an IBV infection, or a reduction in viral shedding after infection with a specific IBV, or the prevention or reduction of a drop in egg production in laying hens after infection with a specific IBV, compared to a group of subjects whose subjects have not received such an immunogenic composition.

[0135] "Treatment or prevention" generally involves administering an effective amount of an immunogenic composition of the present invention to a subject or a group of subjects in need or who may benefit from such treatment / prevention. The term "treatment" refers to the administration of an effective amount of an immunogenic composition once a subject or at least some of the subjects in a group have been infected with such IBV, and wherein such subjects have shown some clinical signs caused by or associated with such IBV infection. The term "prevention" refers to administration to a subject prior to any infection of such subject by IBV, or at least when none of the subjects in such subject or a group of subjects show any clinical signs caused by or associated with such IBV infection. The terms "prophylaxis" and "preventing" are used interchangeably in this application.

[0136] As used herein, the term "effective amount" means but is not limited to an amount of an antigen that elicits or is capable of eliciting an immune response in a subject. Such an effective amount is capable of reducing the incidence of a specific IBV infection in a flock of birds, or reducing the severity of clinical signs of a specific IBV infection.

[0137] Preferably, clinical signs are reduced in incidence or severity by at least 10%, more preferably at least 20%, still more preferably at least 30%, even more preferably at least 40%, still more preferably at least 50%, even more preferably at least 60%, still more preferably at least 70%, even more preferably at least 80%, still more preferably at least 90%, still more preferably at least 95%, and most preferably 100%, compared to subjects who were not treated or who were treated with an immunogenic composition available prior to the present invention but who were subsequently infected with the particular IBV.

[0138] As used herein, the term "clinical signs" refers to signs of infection of a subject from IBV. Examples of such clinical signs include, but are not limited to, respiratory distress, nephritis, salpingitis, abnormal egg production, ruffled feathers, depression, reduced growth rate, and loss of appetite. Signs of respiratory distress include respiratory signs, including gasping, coughing, sneezing, tracheal rales, nasal and ocular discharge, tracheal damage, and ciliary arrest in the trachea. Signs of nephritis include kidney damage and watery diarrhea. Signs of abnormal egg production include decreased egg production, smaller sized eggs, inferior eggshells, reduced internal egg quality, eggs with thin albumen, and ciliary arrest in the oviduct. However, clinical signs also include, but are not limited to, clinical signs that can be observed directly from live animals. Examples of clinical signs that can be observed directly in live animals include nasal and ocular discharge, coughing, panting, sneezing, tracheal rales, ruffled feathers, conjunctivitis, weight loss, reduced growth rate, decreased appetite, dehydration, watery diarrhoea, lameness, lethargy, emaciation and weakness.

[0139] Preferably, the clinical signs of reduced incidence or severity in treated subjects are reduced ciliary arrest, reduced rales, reduced egg production, reduced kidney damage, reduced watery diarrhea, reduced weight loss, less viral load, reduced viral shedding, or a combination thereof, compared to subjects who were not treated or treated with an immunogenic composition available prior to the present invention but subsequently infected with a particular IBV.

[0140] As used herein, the term "in need of" or "in need of" means that the administration / treatment is associated with an enhancement or improvement of health or clinical signs, or any other positive medical effect on the health of the subject receiving the immunogenic composition according to the present invention.

[0141] Further, the present invention provides a method of reducing ciliary arrest in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine as described herein.

[0142] As shown in the Examples, the immunogenic compositions or vaccines as provided herein have been demonstrated to be effective in reducing ciliary arrest.

[0143] The term "ciliostasis" is well known to those skilled in the art. The surface of the trachea is covered with specialized epithelial cells, which are lined with numerous, motile, hair-like structures called cilia. The term "ciliostasis" includes a reduction or loss of cilia and / or a loss or partial loss of ciliary activity (movement). Ciliosis can be determined by examining the lining of the tracheal rings for movement of the cilia. How to determine ciliary movement in the trachea is within the general knowledge of those skilled in the art.

[0144] Preferably, ciliary movement is not reduced starting from day 10 after challenge or infection with IBV, more preferably starting from day 5 after challenge or infection, more preferably starting from day 4 after challenge or infection, more preferably starting from day 3 after challenge or infection, and most preferably starting from day 1 or day 2 after challenge or infection, compared to subjects in a non-immune control group of the same species.

[0145] The term "reduction of ciliary arrest" means that ciliary arrest is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% compared to a non-immunized control group of subjects of the same species. How to measure the reduction of ciliary arrest is within the general knowledge of a person skilled in the art.

[0146] Further, the present invention provides a method of reducing viral RNA load in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine as described herein.

[0147] The term "viral load" or "viral titer" is a measure of the severity of an active viral infection, and can be determined by methods known to those skilled in the art. The term "viral titer" is a measure of the viral preparation of infection units / volume. Viral titer is the endpoint in a biological procedure, and is defined as the dilution at which a certain proportion of tests performed in parallel show an effect (Reed and Muench, 1938). Determination can be based on, for example, detection of viral proteins by antibodies bound to viral proteins, and further detection or alternative detection by amplification methods such as RT-PCR detection of viral RNA. Monitoring viral RNA associated with virions in plasma by nucleic acid amplification methods is a widely used parameter to evaluate the state and progression of retroviral diseases, and to assess the effectiveness of preventive interventions and therapeutic interventions. Exemplarily, viral load or viral titer can be calculated by estimating the viral activity in the body fluids involved, such as the RNA copy number per milliliter of plasma.

[0148] The term "reduction in viral RNA load" means that the viral RNA load is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% compared to a non-immunized control group of the same species. How to measure the reduction in viral RNA load is within the general knowledge of a person skilled in the art.

[0149] Further, the present invention provides an immunogenic composition or vaccine as described herein for use in a method of immunizing a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0150] Further, the present invention provides an immunogenic composition or vaccine as described herein for use in a method for treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0151] Further, the present invention provides an immunogenic composition or vaccine as described herein for use in a method of reducing ciliary arrest in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0152] Further, the present invention provides an immunogenic composition or vaccine as described herein, for use in a method for reducing the viral RNA load in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0153] In a particular aspect of the method or use according to the invention, the subject is a poultry.

[0154] The term "avian" is well known to those skilled in the art. The term "avian" includes all birds including poultry.

[0155] In a specific aspect of the method or use according to the invention, the subject is poultry.

[0156] The term "poultry" is well known to those skilled in the art. The term "poultry" includes chickens, turkeys, quails, pheasants, guinea fowl, geese and ducks. Further, the term "chicken" includes broilers, laying hens and breeding groups for both, also known as breeders.

[0157] In a particular aspect of the method or use according to the invention, the subject is selected from a chicken, a turkey, a quail or a pheasant.

[0158] In a specific aspect of the method or use according to the invention, the subject is a chicken.

[0159] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered once.

[0160] It is understood that a single dose is administered only once. As shown in the Examples, the immunogenic compositions as provided herein have been shown to be effective following administration of a single dose to a subject in need thereof.

[0161] The dose volume / bird depends on the route of vaccination and the age of the birds.

[0162] Typically, eye drop vaccines are administered at any age in a volume of 1 to 100 μl / dose. Preferably, a single dose for eye drop vaccines has a total volume of about 5 μl to 70 μl, and more preferably about 20 μl to 50 μl, wherein a single 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl or 50 μl dose is preferred. Most preferably, a single dose for eye drop vaccines has a total volume of about 30 μl to 50 μl, wherein a single 30 μl, 35 μl, 40 μl, 45 μl or 50 μl dose is preferred.

[0163] For day-old poultry, the spray vaccine may contain a dose in a volume of 25 to 1000 μl. Preferably, a single dose for the spray vaccine has a total volume of about 50 μl to 5000 μl, more preferably about 75 μl to 2000 μl, more preferably about 100 μl to 1000 μl, even more preferably about 200 μl to 900 μl, even more preferably about 300 μl to 800 μl, and even more preferably about 400 μl to 700 μl, wherein a single 400 μl, 425 μl, 450 μl, 475 μl, 500 μl, 525 μl, 550 μl, 575 μl, 600 μl, 625 μl, 650 μl, 675 μl or 700 μl dose is preferred. Most preferably, a single dose has a total volume of 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl or 700 μl.

[0164] The vaccine for in ovo vaccination may contain a dose in a volume of 50 to 100 μl, preferably 50 μl. Preferably, a single dose for in ovo vaccination has a total volume of about 10 μl to 250 μl, more preferably about 15 μl to 200 μl, even more preferably about 20 μl to 150 μl, even more preferably about 30 μl to 100 μl, even more preferably about 30 μl to 75 μl, and wherein a single 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl or 75 μl dose is preferred. Most preferably, a single dose has a total volume of 40 μl, 45 μl, 50 μl, 55 μl or 60 μl.

[0165] One dose of the vaccine for intramuscular or subcutaneous vaccination or the drinking water vaccine may contain a dose in a volume of 30 μl to 1000 μl. Preferably, a single dose has a total volume of about 30 μl to 1000 μl, more preferably about 50 μl to 500 μl, more preferably about 75 μl to 250 μl, and even more preferably about 100 μl to 200 μl, wherein a single 100 μl, 110 μl, 120 μl, 125 μl, 130 μl, 135 μl, 140 μl, 145 μl, 150 μl, 160 μl, 170 μl, 175 μl, 180 μl, 190 μl, 155 μl or 200 μl dose is most preferred.

[0166] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered in two or more doses.

[0167] However, the immunogenic composition may be administered in two or more doses, with a first dose being administered prior to a second (boosting) dose.

[0168] In a preferred aspect of the two administration schemes, the first dose and the second dose of the immunogenic composition are administered in the same amount. Preferably, each dose is the preferred amount specified above. In addition to the first dose and the second dose scheme, an alternative embodiment comprises further subsequent doses. For example, a third dose, a fourth dose or a fifth dose may be administered in these aspects. Preferably, the subsequent third dose, fourth dose and fifth dose scheme are administered in the same amount as the first dose, wherein the time period between the doses is consistent with the time arrangement between the first dose and the second dose mentioned above.

[0169] Preferably, the first administration of the vaccine is carried out in the first three weeks of age, more preferably in the first week of age, and most preferably in one day of age by the method described below. The second administration can be carried out in the first 20 weeks of age, preferably in 16-18 weeks of age, more preferably between 6-12 weeks of age. Exemplarily, the initial (first) vaccination is carried out at 1-10 days of age, and the second vaccination (reinforcement) is carried out with a live vaccine or an inactivated vaccine at 6-12 or 16-18 weeks of age. More preferably, the initial (first) vaccination is carried out at one day of age, and the second vaccination (reinforcement) is carried out with a live vaccine or an inactivated vaccine at 6-12 or 16-18 weeks of age.

[0170] Where in ovo vaccination is used, preferably the first administration is carried out when the embryo is 15 to 19 days old, preferably at 17, 18 or 19 days old, most preferably at 18 days old. The second administration may be carried out within the first three weeks of age, preferably within the first 10 days of age.

[0171] In a specific aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, in ovo, via spray, via the drinking water or by eye drops.

[0172] The immunogenic composition is preferably administered topically or systemically. Suitable routes of administration for conventional use are oral or parenteral administration, such as intranasal, intravenous, intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous, and inhalation, in eggs, via spray, via drinking water or by eye drops. However, depending on the properties and mode of action of the compound, the immunogenic composition may also be administered by other routes. For example, such other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intradermal, intracardiac, intralobal (intralobally), intralobal, intramedullary, intrapulmonary, rectal and intravaginal. However, most preferably, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in eggs, via spray, via drinking water or by eye drops.

[0173] The live IBV vaccine is preferably administered individually by eye drops, intranasally, intramuscularly or subcutaneously.

[0174] More preferably, a large number of application methods are used, including drinking water and aerosol spray vaccination. Also preferred is the use of the vaccine as an embryo vaccine (so called in ovo vaccine), as further described below.

[0175] For example, broilers can be vaccinated at one day old or 1-3 weeks old, especially for broilers with high levels of MDA. Egg-laying or breeding flocks can be initially vaccinated at 1-10 days of age and boosted with the vaccine at 7-12 or 16-18 weeks of age.

[0176] As outlined above, the present invention also provides an IBV vaccine that can be safely administered via the in ovo route and is simultaneously capable of inducing a protective immune response. In ovo administration is well known to those skilled in the art and can be easily performed by those skilled in the art. In ovo administration of the vaccine involves administering the vaccine to an avian embryo when contained in an egg (for a review of in ovo vaccination, see: Ricks et al., Advances in Vet. Med. 495-515, 1999). As described in the art (Sharma; Am. J. Vet. Res. 45 1619-1623, 1984), the vaccine can be administered to any suitable egg compartment (e.g., allantoic fluid, yolk sac, amnion, air cell, or embryo). Preferably, the vaccine is administered below the shell (air cell) membrane and the chorioallantoic membrane.

[0177] Preferably, the vaccine is injected into embryonated eggs during the later stages of embryonic development, generally during the last quarter of the incubation period, preferably 3-4 days before hatching. Preferably, administration is performed when the embryo is 15 to 19 days old, preferably 17, 18 or 19 days old, and most preferably 18 days old. Subsequently, the vaccinated embryonated eggs are transferred to an incubator for hatching. The process of intra-ovum administration can be automated using a robotic injection process as described in the prior art.

[0178] Conventional vaccines used for post-hatching vaccination of poultry cannot be used for in ovo vaccination because late embryos are highly susceptible to infection by most vaccine viruses examined. However, International Patent Application WO 01 / 64244 discloses that an IBV vaccine can be used for in ovo administration provided that it is administered at very low doses. Further, Wakenell et al. 1986 (Am. J. Vet. Res., 47 933-938) disclose that passage of an IB vaccine virus in tissue culture renders the virus non-pathogenic to the embryo.

[0179] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered via eye drops.

[0180] Typically, live vaccines for post-hatch administration contain 10 1 Up to 10 8 EID 50 (50% egg infection dose) / dose concentration of attenuated IBV, preferably 10 2 Up to 10 5 EID 50 / dose concentration, and more preferably 10 2 Up to 10 4 EID 50 / unit dose concentration, and even more preferably at 10 2 Up to 10 3 EID 50 / dose concentration.

[0181] Live vaccines for in ovo administration usually contain 10 2 Up to 10 7 EID 50 / embryo, preferably 10 2 Up to 10 3 EID 50 / embryo amount of attenuated IBV.

[0182] Preferably, the immunogenic composition of the present invention comprises an amount of about 1 to about 10 log 10 EID (Egg Infectious Dose) 50 / ml / dose of IBV of the present invention, preferably about 2 to about 8 log 10 EID 50 / dose, preferably about 2 to about 7 log 10 EID 50 / dose, more preferably from about 2 to about 6 log 10 EID 50 / dose, even more preferably from about 2 to about 5 log 10 EID 50 / dose, even more preferably from about 2 to about 4 log 10 EID 50 / dose, most preferably about 2 to about 3 log 10 EID 50 More preferably, the immunogenic composition of the invention comprises about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or log 10 EID 50 / dose of IBV of the present invention.

[0183] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine comprises 1 to 10 log10 EID 50 / dose of IBV.

[0184] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0185] In a particular aspect of the method or use according to the invention, the immunogenic composition or vaccine comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0186] In a specific aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered to a subject within the first week of age, within the first three days of age, within the first two days of age, or within the first day of age.

[0187] Preferably, the subject to be immunized is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days old. More preferably, the subject to be immunized is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days old. Most preferably, the subject to be immunized is 1, 2, 3, 4, 5, 6 or 7 days old.

[0188] However, it must be understood that after vaccination of subjects of a few days of age, the immune system of poultry does require several days to build up immunity against IBV infection. Therefore, preferably, subjects are immunized within the first 24 hours of age.

[0189] In a specific aspect of the method or use according to the invention, the immunogenic composition or vaccine is administered to a subject within the previous day of age. As shown in the Examples, the immunogenic composition as provided herein has been shown to be safe and effective when administered to 1 day old poultry.

[0190] In a specific aspect of the method or use according to the invention, the method results in an improvement in an efficacy parameter selected from the following: prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of decreased egg production, prevention or reduction of kidney damage, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, lower viral load, reduced viral shedding or a combination thereof, compared to subjects in an untreated control group of the same species.

[0191] The terms "treatment and / or prevention" have been defined elsewhere, wherein the terms "prophylaxis" and "preventing" or "prevention" are used interchangeably in this application. Further, the term "shedding" has also been defined elsewhere.

[0192] The terms "reducing", "reduced", "reduction" or "lower" mean that the efficacy parameter (ciliostasis, rales, decreased egg production, kidney damage, watery diarrhea, weight loss, viral load, viral shedding) is reduced by at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, even more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% compared to a non-immunized control group of the same species. How to measure the improvement of an efficacy parameter is within the general knowledge of a person skilled in the art.

[0193] The term "viral load" is defined elsewhere herein.

[0194] The term "ciliar arrest" has been defined elsewhere herein.

[0195] The term "rales" is well known to those skilled in the art. However, the term "rales" includes tracheal rales and refers to sounds emitted from the bronchi. Rales can be easily determined by those skilled in the art.

[0196] The term "drop in egg production" is well known to the person skilled in the art. The term "drop in egg production" includes decreased egg production.

[0197] In a specific aspect of the method or use according to the invention, the treatment or prevention results in the prevention or reduction of ciliary arrest compared to an untreated control group of subjects of the same species.

[0198] In a specific aspect of the method or use according to the invention the treatment or prevention results in prevention or reduction of kidney damage compared to an untreated control group of subjects of the same species.

[0199] In a specific aspect of the method or use according to the invention the treatment or prevention results in the prevention or reduction of a decrease in egg production compared to an untreated control group of subjects of the same species.

[0200] The invention further provides an IBV, viral particle or immunogenic composition or vaccine as described herein for use in therapeutic use.

[0201] The invention further provides an IBV or a viral particle as described herein for use as an immunogen or vaccine.

[0202] The invention further provides an IBV, a viral particle or an immunogenic composition or vaccine as described herein for use as a medicament.

[0203] The invention further provides the use of an IBV, a viral particle or an immunogenic composition or vaccine as described herein for the manufacture of a medicament.

[0204] The present invention further provides use of an IBV, a viral particle or an immunogenic composition or vaccine as described herein for treating and / or preventing an IBV infection in a subject.

[0205] In another specific aspect of the IBV according to the invention, the IBV is phenotypically stable. Advantageously, experimental data show that the deposited strain and its progeny are phenotypically stable, as the extended cell culture tropism / tissue tropism and attenuation remain stable over a period of time (through passaging).

[0206] The term "phenotypically stable" means that the IBV retains its functional characteristics of extended cell culture tropism / tissue tropism and attenuation over a period of time (through passages). Preferably, the functional characteristics are still present after at least 3 passages of the IBV in cell culture or tissue culture, more preferably after at least 6 passages, even more preferably after at least 9 passages, even more preferably after at least 12 passages, and most preferably after 15 passages.

[0207] Technical Solution

[0208] This article also describes the following technical solutions:

[0209] 1. An IBV (Infectious Bronchitis Virus) deposited at BVR of IZSLER under accession number DPS RE RSCIC 16, any descendant IBV thereof or any IBV having all the identifying characteristics of the IBV deposited under DPS RE RSCIC 16.

[0210] 2. An IBV (Infectious Bronchitis Virus) of BVR deposited at IZSLER under accession number DPS RE RSCIC 16, any attenuated progeny IBV thereof having extended cell tropism or tissue tropism and being protective against challenge with virulent M41, or any attenuated IBV having extended cell tropism or tissue tropism and being protective against challenge with virulent M41.

[0211] 3. The IBV of technical solution 1 or 2, wherein the IBV is attenuated.

[0212] 4. The IBV of any one of technical solutions 1 to 3, wherein the IBV is attenuated in one-day-old chickens.

[0213] 5. The IBV of any one of technical solutions 1 to 4, wherein the IBV is non-recombinant.

[0214] 6. The IBV of any one of technical solutions 1 or 5, wherein the IBV is a Massachusetts genotype or serotype.

[0215] 7. The IBV of any one of technical solutions 1 or 6, wherein the IBV is a M41, H52 or H120 strain.

[0216] Extended cell tropism or tissue tropism

[0217] 8. The IBV of any one of technical solutions 1 or 7, wherein the IBV has extended cell tropism or tissue tropism.

[0218] 9. The IBV of any one of technical solutions 1 to 8, wherein the IBV infects and / or replicates in a cell line or cell selected from the following list: primary chicken embryo cells or primary chicken fibroblasts from the lung or liver, chicken embryo fibroblast cell line, duck embryo stem cell line, human embryonic kidney cell line, baby hamster kidney cell line, African green monkey kidney cell line, rabbit kidney cell line, canine kidney cell line, chicken liver cell line, bovine kidney cell line, pig kidney cell line and insect cell line.

[0219] 10. The IBV of any one of technical solutions 1 to 9, wherein the IBV infects and / or replicates in at least one cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby dog ​​kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A, SF9, SF21 and SF+ (spodoptera frugiperda).

[0220] 11. The IBV of any one of technical solutions 1 to 10, wherein the IBV infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104, MDCK, SF9 and RK13.

[0221] 12. The IBV of technical solution 9, wherein the primary chicken embryo cells are fibroblasts or cells derived from liver or lung tissue.

[0222] Function Definition - Protection

[0223] 13. The IBV of any one of technical solutions 1 to 12, wherein the IBV is protective against virulent M41 attack or infection.

[0224] 14. The IBV of technical solution 13, wherein the protection against virulent M41 attack or infection is determined by ciliary arrest score, reduced clinical signs of the respiratory system, reduced viral RNA load in kidney tissue, or reduced viral shedding.

[0225] 15. The IBV of any one of technical solutions 1 to 14, wherein the attenuation of the IBV is increased compared to an IBV without extended cell tropism or tissue tropism.

[0226] 16. The IBV of any one of technical solutions 1 to 15, wherein the attenuation of the IBV is increased compared to IBVM41 without extended cell tropism or tissue tropism.

[0227] 17. The IBV of any one of technical solutions 1 to 16, wherein the attenuation of the IBV is increased after application in one-day-old chickens compared to an IBV without extended cell tropism or tissue tropism.

[0228] 18. The IBV of any one of technical solutions 1 to 17, wherein the attenuation of the IBV is increased after application in one-day-old chickens compared to IBV M41 without extended cell tropism or tissue tropism.

[0229] All identification characteristics

[0230] 19. The IBV of any one of technical solutions 1 to 18, wherein all the identifying characteristics of the preserved IBV mean that the IBV is attenuated, has extended cell tropism or tissue tropism, and is protective against virulent M41 challenge or infection.

[0231] 20. The IBV of any one of technical solutions 1 to 19, wherein all the identifying characteristics of the deposited IBV mean that the IBV is attenuated, has extended cell tropism or tissue tropism, and has the same or similar protection profile as the deposited IBV.

[0232] 21. The IBV of any one of technical solutions 1 to 20, wherein all the identifying characteristics of the deposited IBV mean that the IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and has the same or similar protection profile as the deposited IBV.

[0233] 22. The IBV of any one of technical solutions 1 to 21, wherein all the identification characteristics of the preserved IBV mean that the IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and is protective against virulent M41 attack or infection.

[0234] Descendants

[0235] 23. The IBV of any one of technical solutions 1 to 22, wherein the progeny IBV is attenuated, has extended cell tropism or tissue tropism, and is protective against virulent M41 attack or infection.

[0236] 24. The IBV of any one of technical solutions 1 to 23, wherein the progeny IBV is attenuated, has extended cell tropism or tissue tropism, and a protection profile that is the same or similar to that of the preserved IBV.

[0237] 25. The IBV of any one of technical solutions 1 to 24, wherein the progeny IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and has the same or similar protection profile as the deposited IBV.

[0238] 26. The IBV of any one of technical solutions 1 to 25, wherein the progeny IBV is attenuated, infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13, and is protective against virulent M41 attack or infection.

[0239] 27. A plasmid comprising a nucleotide sequence encoding the IBV of any one of technical solutions 1 to 26.

[0240] 28. A cell comprising the IBV or plasmid of any one of technical solutions 1 to 27.

[0241] 29. A cell according to technical solution 28, wherein the cell is a cell line or cell selected from the following list: primary chicken embryo cells, chicken embryo fibroblast cell line, duck embryo stem cell line, human embryonic kidney cell line, baby hamster kidney cell line, African green monkey kidney cell line, rabbit kidney cell line, canine kidney cell line, chicken liver cell line, bovine kidney cell line, pig kidney cell line and insect cell line.

[0242] 30. A cell according to technical solution 28 or 29, wherein the cell is a cell line selected from the following list: DF-1 (Douglas Foster), EB66 (duck embryo stem cell line), PBS-12, PBS-12SF (serum-free PBS-12), BHK21 (baby hamster kidney), HEK 293T (human embryonic kidney), Vero (Verda Reno), MA104, RK13 (rabbit kidney), LMH (leghorn male liver cancer), MDCK (Martin Darby dog ​​kidney), MDBK (Martin Darby bovine kidney), PK15 (pig kidney), PK2A, SF9, SF21 and SF+ (Spodoptera frugiperda).

[0243] 31. The cell of any one of technical solutions 28 to 30, wherein the cell is a cell line selected from the following list: DF-1, EB66, PBS-12SF, BHK, HEK 293T, Vero, MA104 and RK13.

[0244] 32. The cells of technical solution 29, wherein the primary chicken embryo cells are fibroblasts or cells derived from liver or lung tissue.

[0245] 33. A virus particle comprising the IBV of any one of technical solutions 1 to 26.

[0246] 34. An immunogenic composition comprising the IBV of any one of technical solutions 1 to 26.

[0247] 35. A vaccine comprising the IBV of any one of technical solutions 1 to 26.

[0248] 36. An improved live vaccine with expanded cell tropism or tissue tropism, comprising the IBV of any one of technical solutions 1 to 26.

[0249] 37. The immunogenic composition or vaccine of any one of technical solutions 34 to 36, wherein the immunogenic composition or vaccine comprises a pharmaceutically acceptable carrier.

[0250] 38. The immunogenic composition or vaccine of technical solution 37, wherein the pharmaceutically acceptable carrier is phosphate-buffered saline.

[0251] 39. The immunogenic composition or vaccine of any one of technical solutions 34 to 38, wherein the immunogenic composition or vaccine is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof.

[0252] 40. The immunogenic composition or vaccine of any one of technical solutions 34 to 39, wherein the immunogenic composition or vaccine comprises 1 to 10 log 10 EID50 of IBV / dose.

[0253] 41. The immunogenic composition or vaccine of any one of technical solutions 34 to 40, wherein the immunogenic composition or vaccine comprises 2 to 5 log 10 EID 50 of IBV / dose.

[0254] 42. The immunogenic composition or vaccine of any one of technical solutions 34 to 41, wherein the immunogenic composition or vaccine comprises 2 to 4 log 10 EID 50 of IBV / dose.

[0255] 43. A kit comprising the IBV of any one of technical solutions 1 to 26 or the immunogenic composition or vaccine of any one of technical solutions 34 to 42.

[0256] 44. A kit according to technical solution 43, wherein the kit further comprises instructions for treating and / or preventing poultry diseases.

[0257] 45. A kit according to technical solution 43, wherein the kit further comprises instructions for treating and / or preventing poultry diseases.

[0258] 46. ​​A kit according to technical solution 43, wherein the kit further comprises instructions for treating and / or preventing IB.

[0259] 47. A method for immunizing a subject, comprising administering to such subject an immunogenic composition or vaccine according to any one of technical solutions 34 to 42.

[0260] 48. A method for treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine according to any one of technical solutions 34 to 42.

[0261] 49. A method for reducing ciliary arrest in a subject in need thereof, compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine according to any one of technical solutions 34 to 42.

[0262] 50. A method for reducing viral RNA load in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition or vaccine according to any one of technical solutions 34 to 42.

[0263] 51. An immunogenic composition or vaccine according to any one of technical solutions 34 to 42, which is used in a method for immunizing a subject, the method comprising administering a therapeutically effective amount of the immunogenic composition or vaccine to the subject.

[0264] 52. An immunogenic composition or vaccine according to any one of technical solutions 34 to 42, for use in a method for treating or preventing clinical signs caused by IBV in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0265] 53. An immunogenic composition or vaccine according to any one of technical solutions 34 to 42, which is used in a method for reducing ciliary arrest in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0266] 54. An immunogenic composition or vaccine according to any one of technical solutions 34 to 42, which is used in a method for reducing the viral RNA load in a subject in need thereof compared to a non-immunized control group of subjects of the same species, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition or vaccine.

[0267] 55. The method or use of any one of technical solutions 47 to 54, wherein the subject is poultry.

[0268] 56. The method or use of any one of technical solutions 47 to 55, wherein the subject is poultry.

[0269] 57. The method or use of any one of technical solutions 47 to 56, wherein the subject is selected from chicken, turkey, quail or pheasant.

[0270] 58. The method or use of any one of technical solutions 47 to 57, wherein the subject is a chicken.

[0271] 59. The method or use of any one of technical solutions 47 to 58, wherein the immunogenic composition or vaccine is administered once.

[0272] 60. The method or use of any one of technical solutions 47 to 58, wherein the immunogenic composition or vaccine is administered in two or more doses.

[0273] 61. The method or use of any one of technical solutions 47 to 60, wherein the immunogenic composition or vaccine is administered subcutaneously, intramuscularly, orally, in ovo, via spray, via drinking water or by eye drops.

[0274] 62. The method or use of any one of technical solutions 47 to 61, wherein the immunogenic composition or vaccine is administered via eye drops.

[0275] 63. The method or use of any one of technical solutions 47 to 62, wherein the immunogenic composition or vaccine comprises 1 to 10 log 10 EID 50 / dose of IBV.

[0276] 64. The method or use of any one of technical solutions 47 to 63, wherein the immunogenic composition or vaccine comprises 2 to 5 log 10 EID 50 / dose of IBV.

[0277] 65. The method or use of any one of technical solutions 47 to 64, wherein the immunogenic composition or vaccine comprises 2 to 4 log 10 EID 50 / dose of IBV.

[0278] 66. The method or use of any one of technical solutions 47 to 65, wherein the immunogenic composition or vaccine is administered to a subject within the first week of age, within the first three days of age, within the first two days of age, or within the first day of age.

[0279] 67. The method or use of any one of technical solutions 47 to 66, wherein the immunogenic composition or vaccine is administered to a subject within the previous day of age.

[0280] 68. The method or use of any one of technical solutions 47 to 67, wherein the method results in an improvement in an efficacy parameter selected from: prevention or reduction of ciliary arrest, prevention or reduction of rales, prevention or reduction of decreased egg production, prevention or reduction of renal damage, prevention or reduction of watery diarrhea, prevention or reduction of weight loss, lower viral load, reduced viral shedding, or a combination thereof, compared to subjects in an untreated control group of the same species.

[0281] 69. The method or use of any one of technical solutions 47 to 68, wherein the treatment or prevention results in prevention or reduction of ciliary arrest compared to subjects in an untreated control group of the same species.

[0282] 70. The method or use of any one of technical solutions 47 to 69, wherein the treatment or prevention results in prevention or reduction of kidney damage compared to subjects in an untreated control group of the same species.

[0283] 71. The method or use of any one of technical solutions 47 to 70, wherein the treatment or prevention results in the prevention or reduction of decreased egg production compared to subjects in an untreated control group of the same species.

[0284] 72. The IBV of any one of technical solutions 1 to 26, the viral particle of technical solution 33, or the immunogenic composition or vaccine of any one of technical solutions 34 to 42, for therapeutic use.

[0285] 73. The IBV of any one of Technical Solutions 1 to 26 or the virus particle of Technical Solution 33, which is used as an immunogen or vaccine.

[0286] 74. The IBV of any one of technical solutions 1 to 26, the virus particle of technical solution 33, or the immunogenic composition or vaccine of any one of technical solutions 34 to 42, for use as a medicine.

[0287] 75. Use of the IBV of any one of Technical Solutions 1 to 26, the viral particles of Technical Solution 33, or the immunogenic composition or vaccine of any one of Technical Solutions 34 to 42 for the manufacture of a medicament.

[0288] 76. Use of the IBV of any one of technical solutions 1 to 26, the viral particles of technical solution 33, or the immunogenic composition or vaccine of any one of technical solutions 34 to 42 for treating and / or preventing IBV infection in a subject.

[0289] 77. The IBV of any one of technical solutions 1 to 26, wherein the progeny comprises up to 15 passages in cell culture of the IBV deposited at BVR of IZSLER under accession number DPS RE RSCIC16.

[0290] 78. The IBV of any one of technical solutions 1 to 26, wherein the progeny comprises up to 10 passages in cell culture of the IBV deposited at BVR of IZSLER under accession number DPS RE RSCIC16.

[0291] 79. The IBV of any one of technical solutions 1 to 26, wherein the progeny comprises up to 5 passages in cell culture of the IBV deposited at BVR of IZSLER under accession number DPS RE RSCIC16.

[0292] 80. The IBV of any one of technical solutions 1 to 26 or 77 to 79, wherein the IBV is phenotypically stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0293] Figure 1 .: About IB66HP infection Immunofluorescence staining of cells.

[0294] Figure 2 : Immunofluorescence staining for IB66HP 72 hours after infection of different cell lines. Negative controls were included but not shown.

[0295] Example

[0296] The following examples are set forth below to illustrate specific embodiments of the present invention. These examples are merely illustrative and should not be construed as limiting the scope or underlying principles of the present invention.

[0297] Example 1: Adaptation of IBV to cells and in vitro and in vivo characterization

[0298] Attenuated IBV M41 was used in The cells were serially passaged for 10 times. The material harvested after each passage was analyzed by EID. 50 and TCID 50 In fact, when inoculated with attenuated IBV Mass seed stock Infectious titers were already detected in the first passage after the cells were grown. EID for the first 10 passages 50 (for infectivity of embryonated eggs) and TCID 50 The results of the titer determinations (regarding the infectivity of cell cultures) are summarized in Table 1 and are for cells infected with IB66HP. Immunofluorescence signal detection of cells is summarized in Figure 1 The results confirmed that IBV is suitable for Cell culture in cells. Efficient adaptation after one passage in cells, while infectivity with respect to embryonated eggs was not affected. Passage 4 of IBV (IB66HP) was deposited under accession number DPS RE RSCIC 16 at BVR of IZSLER (Biobanking of Veterinary Resources of the Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna "Bruno Ubertini").

[0299] Table 1: IB66HP EID of cells throughout passage 50 and TCID 50 Summary of titers.

[0300] Passaging 1 2 3 4 5 6 7 8 9 10 <![CDATA[EID 50 / ml]]> 5.6 9 7.1 5.3 7 7 6.8 7.5 6.6 6.6 <![CDATA[TCID 50 / ml]]> 1 6.5 4.25 5.5 6.3 6.32 4.6 7 6 5.4

[0301] From Table 1 and other experiments further below, it is apparent that IB66HP is phenotypically stable in that the extended cell culture tropism / tissue tropism and attenuation are maintained over a period of time (through passaging).

[0302] exist Serial passaging of cells

[0303] Cells were 10 6 The cells were inoculated at a density of 10 cells / ml in a 250 ml shake flask with a total volume of 50 ml in GRO I medium (SIGMA, catalog: 141530C) supplemented with glutamine and 1 ml CHO feed (SIGMA, catalog: 1615). For the first passage, the cells were infected with a 1 / 10 dilution of the attenuated IBV Mass seed stock and the cells were incubated for 96 hours. For the next passages 2 to 10, freshly inoculated Inoculation with the harvested culture was performed intracellularly with a 1 / 10 dilution of the previous passage, which was then incubated for 48 to 96 hours under the same culture conditions. 4*10 5 Cells / ml were generated for storage at passage 4 (supernatant) and harvested 72 hours after infection, and the titer of the harvested material was determined by 50% embryonic infectious dose (EID 50 ) and immunofluorescence tissue culture infectious dose 50 (TCID 50 ) were determined, which were 10 5,75 / ml and 10 7.33 / ml.

[0304] 50% embryo infectious dose (EID 50 )

[0305] For EID 50 Titer determination, 10-day-old SPF embryonated chicken eggs were inoculated into the allantoic cavity with 100 μl of 10-fold serial dilutions / egg in 6 replicates / dilution. Infected eggs were incubated for 7 days and candled every 24 hours to determine mortality. Eggs with dead embryos before 24 hours of incubation were not included in the evaluation. EID was calculated as described by Reed & Muench 50 / mL.

[0306] 50% tissue culture infectious dose (TCID 50 )

[0307] For TCID 50 The determination of the viability of 2.5 to 3*10 cells with at least 90% viability was performed one day before infection with a 10-fold dilution series of passaged IBV Mass. 5Cells / well are inoculated into 96-well plates. Culture medium is removed from cells, and they are infected with 100 μl / well with 5 repetitions / dilutions. After static incubation for 72 hours at 37 ℃ and 7.5% CO2, culture medium is removed, and cells are fixed for 15 minutes at 4 ℃ with 50 μl 80% acetone / well. Washing steps with 1xPBS are carried out, and then incubated for 1 hour at 37 ℃ with 1:1000 dilution of the anti-IBV Mass antiserum (Charles River Laboratories, catalog: 10100454) of 50 μl / well. Plate is washed twice with 1xPBS, then 1:2000 dilution of the anti-chicken antibody (Invitrogen, catalog: A11039) of 50 μl Alexa488 conjugation is added, and incubated for 1 hour at 37 ℃. Subsequently, a final wash with 1x PBS was performed and 100 μl of 1x PBS was added to each well, followed by evaluation under an inverted fluorescence microscope. According to the formula of Reed & Muench, Infectious titers of IBV in cells.

[0308] Infectivity of IB66HP on different cell lines

[0309] The next day, different cell lines were seeded into 96-well plates to reach 70-80% confluence. 5 Up to 10 5.75 TCID 50 The cells were infected with a 10-fold dilution series of IB66HP P8 at a titer of / ml, which was derived from Titering of cells. The medium was removed from the cells and they were infected with 100 μl / well in 4 replicates / dilution. After 72 hours, the TCID 50 In fact, the infectivity of IB66HP for different primary cells and cell lines was tested and the results are listed in Tables 2 and 3 and in Figure 2 Displayed in.

[0310] Table 2: IB66HP has extended tissue tropism. 5 TCID 50 TCID titer of IB66HP in various cell lines 50 Sure.

[0311] cell Species organize <![CDATA[Log TCID 50 / ml]]> EB66 duck Embryonic stem cells 5 BHK Hamster kidney 4 MA104 African green monkey kidney 2 Vero EU African green monkey kidney 2.67 RK13 rabbit kidney 3 CEK Chicken embryo Primary Kidney 5.33 CEH Chicken embryo Primary liver 3.67 CEL Chicken embryo Primary lung 6

[0312] Table 3: IB66HP has extended tissue tropism. 5.75 TCID 50 TCID titer of IB66HP in various cell lines50 Sure.

[0313] cell Species organize <![CDATA[Log TCID 50 / ml]]> SF9 Fall Armyworm Pupal ovary tissue 5.6 CEF Chicken embryo Primary fibroblasts 4.5 MDCK dog kidney 5.38

[0314] Conclusion of Example 1

[0315] Attenuated IBV M41 is suitable for cell culture. The deposited strains and their progeny show extended cell or tissue tropism, as they are able to infect primary cells and a wide range of different cell lines.

[0316] Example 2: Determination of the safety and efficacy of IB66HP in vivo

[0317] exist Attenuated IBV Mass allantoic fluid stock (P0) and passages 5 (P5) and 10 (P10) in cells were used to evaluate their safety and efficacy against virulent M41 challenge in 1-day-old SPF laying hens. Throughout the study, chickens were housed in isolation units and provided with water and commercial balanced formula feed ad libitum. Chickens were observed for clinical signs every day. Three groups of chickens were treated with 10 4 TCID 50 The dose of each chicken was vaccinated with P0, P5 and P10. One group was treated with a placebo and served as an attack control group. Seven days after vaccination, seven animals / groups vaccinated with P0, P5 or P10 were euthanized to evaluate safety via the score of tracheal ciliary arrest. For this reason, the trachea was taken out and cut into cross sections, wherein 3 lower, 4 middle and 3 upper sections were used for safety evaluation. All rings were evaluated for the beating of cilia by optical microscopy. Each ring was scored individually as described in Table 4.

[0318] Table 4: Ciliary arrest scoring scheme for safety evaluation

[0319] score Cilia activity [%] 0 100 1 75-99 2 50-74 3 25-49 4 0-24

[0320] 21 days after vaccination, all remaining chickens were vaccinated with 10 2.5 EID 50 / chicken were challenged with virulent IBVM41. As described in Table 4, efficacy was evaluated by ciliary arrest scoring 7 days after vaccination. If more than 50% of the inner rings show vigorous ciliary movement (score 2 and lower), the ring is recorded as normal. If less than 50% of the cilia are beating (scores 3 and 4), the ring is recorded as positive for ciliary arrest. If no less than 9 of the 10 rings show normal ciliary activity, the animal is considered protected. Throughout the study, all animals were observed daily for clinical signs, such as depression, respiratory, digestive or neurological signs, motor impairment, collapse or feathers standing up.

[0321] The objective of this study was to determine whether cell culture adaptability and cell passage of IB66HP had an effect on safety and efficacy when applied to 1-day-old chickens compared to the parental IBV M41att allantoic fluid stock. Safety evaluation by clinical signs and ciliary arrest scores in tracheal explants revealed that IB66HP was superior to IBV M41att in terms of efficacy and safety. P5 and P10 of IB66HP in cells have improved safety. Compared with IBV M41att, ciliostasis scores were significantly reduced for IB66HP P5 and P10 (Table 5). In addition, clinical signs in animals in the groups vaccinated with IB66HP P5 and P10 were reduced compared to animals vaccinated with M41att. Finally, IB66HP is more attenuated than M41att and has an improved safety profile for vaccination of 1-day-old chickens.

[0322] Table 5: IB66HP compared to M41att allantoic fluid stock at 5 days post vaccination Results of safety evaluations at cell passages 5 (P5) and 10 (P10). The mean ciliary arrest score per group was calculated by adding the total scores for the individual birds in each group and dividing the group total by the number of animals (highest possible score 40, lowest possible score 0). For unaffected animals, at least 9 out of 10 tracheal explants showed normal ciliary activity (score ≤ 2).

[0323] Vaccination Mean ciliary arrest score #animals / unaffected M41att P0 30.6 5 / 1 IB66HP P5 8.2 5 / 5 IB66HP P10 6.4 5 / 5 - 0.4 5 / 5

[0324] After challenge with virulent IBV M41, all animals in the unvaccinated control group showed clinical signs and high ciliary arrest scores. IB66HP cells at passages P5 and P10 were as effective as M41att in reducing clinical signs and tracheal damage (Table 6).

[0325] Table 6: IB66HP compared to M41att allantoic fluid stock solution at 7 days post-challenge Results of efficacy evaluation at cell passages 5 (P5) and 10 (P10). The mean ciliary arrest score per group was calculated by adding the total scores of the individual birds in each group and dividing the group total by the number of animals (highest possible score 40, lowest possible score 0). For unaffected animals, at least 9 out of 10 tracheal explants showed normal ciliary activity (score ≤ 2).

[0326]

[0327] In summary, IB66HP is an attenuated IBV strain that effectively infects and replicates in different cell lines and tissue cells. Surprisingly, it has improved safety for use in 1-day-old chickens compared to the parent egg-restricted IBV M41att strain. In addition, IB66HP maintains vaccine efficacy. In contrast, the only other known cell culture-adapted and highly attenuated IBV strain Beaudette does not confer sufficient protection after challenge infection. Therefore, IB66HP is the first described IBV that is attenuated, has extended cell culture tropism or tissue tropism, and demonstrates efficacy against virulent attack after use as a vaccine. Further, IB66HP has been shown to be phenotypically stable.

Claims

1. An infectious bronchitis virus deposited at BVR, IZSLER under accession number DPS RE RSCIC 16, or a progeny thereof, said progeny being up to 10 passages in cell culture of the infectious bronchitis virus deposited at BVR, IZSLER under accession number DPS RE RSCIC 16, wherein said infectious bronchitis virus or a progeny thereof has extended cell culture tropism or tissue tropism and is protective against virulent M41 challenge.

2. The infectious bronchitis virus or its progeny according to claim 1, wherein the infectious bronchitis virus or its progeny is attenuated.

3. The infectious bronchitis virus or its progeny according to claim 1, wherein the infectious bronchitis virus or its progeny is non-recombinant.

4. The infectious bronchitis virus or its progeny according to any one of claims 1 to 3, wherein the infectious bronchitis virus or its progeny is a Massachusetts genotype or serotype.

5. The infectious bronchitis virus or its progeny according to any one of claims 1 to 3, wherein the infectious bronchitis virus or its progeny infects and / or replicates in a cell line or cell selected from the following list: primary chicken embryo cells or primary chicken fibroblasts from the lung or liver, chicken embryo fibroblast cell lines, duck embryo stem cell lines, human embryonic kidney cell lines, baby hamster kidney cell lines, African green monkey kidney cell lines, rabbit kidney cell lines, canine kidney cell lines, chicken liver cell lines, bovine kidney cell lines, pig kidney cell lines and insect cell lines.

6. The infectious bronchitis virus or its progeny according to any one of claims 1 to 3, wherein the infectious bronchitis virus or its progeny infects and / or replicates in at least one cell line selected from the following list: DF-1, EB66, PBS-12, PBS-12SF, BHK21, HEK 293T, Vero, MA104, RK13, LMH, MDCK, MDBK, PK15, PK2A, SF9, SF21 and SF+.

7. A plasmid comprising a nucleotide sequence encoding the infectious bronchitis virus or its progeny according to any one of claims 1 to 6.

8. A cell comprising the infectious bronchitis virus or progeny thereof according to any one of claims 1 to 6 or the plasmid according to claim 7.

9. An immunogenic composition comprising the infectious bronchitis virus or a progeny thereof according to any one of claims 1 to 6.

10. A vaccine comprising the infectious bronchitis virus of any one of claims 1 to 6 or a progeny thereof.

11. Use of the infectious bronchitis virus or a progeny thereof according to any one of claims 1 to 6 in the preparation of an immunogenic composition for immunizing a subject.

12. Use of an infectious bronchitis virus or a progeny thereof according to any one of claims 1 to 6 for the preparation of an immunogenic composition for treating or preventing clinical signs caused by an infectious bronchitis virus in a subject in need thereof.

13. Use of an infectious bronchitis virus or a progeny thereof according to any one of claims 1 to 6 in the preparation of an immunogenic composition for reducing ciliary arrest in a subject in need thereof compared to a non-immunized control group of subjects of the same species.

14. The use according to any one of claims 11 to 13, wherein the immunogenic composition is a vaccine.

15. The use according to any one of claims 11 to 13, wherein the subject is a chicken.

16. The use according to any one of claims 11 to 13, wherein the dosage form of the immunogenic composition is a dosage form for one-time administration.

17. The use of claim 14, wherein the dosage form of the vaccine is a dosage form for one-time administration.

18. The use according to any one of claims 11 to 13, wherein the dosage form of the immunogenic composition is a dosage form for administration subcutaneously, intramuscularly, orally, in ovo, or by eye drops.

19. The use according to any one of claims 11 to 13, wherein the dosage form of the immunogenic composition is a dosage form for administration via spray.

20. The use according to any one of claims 11 to 13, wherein the dosage form of the immunogenic composition is a dosage form for administration via drinking water.

21. The use of claim 14, wherein the vaccine is in a dosage form for administration subcutaneously, intramuscularly, orally, in ovo, or via eye drops.

22. The use of claim 14, wherein the vaccine is in a dosage form for administration via spraying.

23. The use of claim 14, wherein the vaccine is in a dosage form for administration via drinking water.

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