H52 IBV vaccines with heterologous spike proteins

Abstract

The present invention relates to a H52IBV (Infectious Bronchitis Virus) encoding a heterologous S (spike) protein or fragment thereof. Further, the present invention relates to an immunogenic composition comprising said H52IBV encoding a heterologous S (spike) protein or fragment thereof. Furthermore, the invention relates to a method for immunizing a subject comprising administering to the subject an immunogenic composition of the invention. Furthermore, the invention relates to a method of treating or preventing clinical signs caused by IBV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to the invention.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201980071601.2, filed on October 28, 2019, with the invention title of "H52 IBV Vaccine with Heterologous Spike Protein".

[0002] Sequence Listing

[0003] This application contains a sequence listing in accordance with 37 C.F.R. 1.821–1.825. The sequence listing attached to this application is incorporated herein by reference in its entirety. Background of the Invention

[0005] Avian coronavirus infectious bronchitis virus (IBV) is the prototype gamma coronavirus of the family Coronaviridae in the order Nidovirales. Infectious bronchitis virus mainly infects the upper respiratory epithelium of chickens, causing respiratory diseases, usually complicated by secondary bacterial pathogen infections (Cook et al. 2012. Avian Pathol. 41:239-250). Some IBV strains also affect the renal tubules, oviducts, and parts of the gastrointestinal tract, resulting in pathological lesions and clinical signs in these organ systems. The virus is widespread in commercial and backyard poultry. Due to its high genomic variability, IBV is divided into multiple genotypes, serotypes, and protective types. IBV is currently considered one of the most economically relevant viral pathogens in the poultry industry.

[0006] Infectious bronchitis virus is an enveloped virus with a positive-sense single-stranded RNA genome of 27.6 kb (Cavanagh 2007. Vet. Res. 38:281-297). The first two-thirds of the viral genome contains a large coding region (also known as gene 1), which is divided into two open reading frames 1a and 1b, encoding 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 viral RNA. The coronavirus spike protein determines host species tropism (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 highly glycosylated S1 domain forms the "head" of the spike protein and contains the receptor-binding domain that interacts with 2,3-linked sialic acid on the surface of host cells (Promkuntod et al. 2014. Virology. 448:26-32). The S2 domain contains the rest of the ectodomain ("stalk"), the transmembrane domain, and the intracellular domain located in the cytoplasm.

[0007] To date, the most widely used live attenuated IBV vaccine strains were developed in the 1960s in the Netherlands by serial passage of Massachusetts-like IBV strains (Bijlenga et al. 2004; Avian Pathol. 33:550-557). However, since the 1970s, new IBV serotypes have emerged, and traditional Massachusetts-like vaccines do not provide sufficient protection against them (Cook et al. 2012. Avian Pathol. 41:239-250). Therefore, there is still a need for new and efficient IBV vaccines against other IBV serotypes.

[0008] IBV Beaudette (Geilhausen et al. 1973. Arch Gesamte Virusforsch.: 40(3)(1973), pp. 285-290) and H120 (G. Bijlenga et al. 2004. Avian Pathol.: 33(6); pp. 550-557) are attenuated IBVs. However, attenuation may lead to loss of immunogenicity.

[0009] In addition, recombinant IBV has been generated. Zhou et al. 2016 (Arch Virol.;161:3179–3187) disclosed H120 (Massachusetts genotype) IBV with the spike protein of Beaudette (Massachusetts genotype). Hodgson et al. 2004 (J Virol 78:13804–13811) disclosed Beaudette (Massachusetts genotype) IBV with the spike protein of M41 (Massachusetts genotype). In addition, Armesto et al. 2011 (PLoS One:6(8):e24352) disclosed Beaudette (Massachusetts genotype) IBV with a heterologous spike protein from 4 / 91 (4 / 91 genotype).

[0010] However, neither of the recombinant IBVs disclosed by Zhou et al. 2016 and Hodgson et al. 2004 can be regarded as IBV with a heterologous spike protein, because both the IBV and the inserted spike protein are from the same genotype / serotype (Massachusetts). Moreover, all the vaccines mentioned are based on the backbone of Beaudette or have the spike protein from Beaudette.

[0011] In addition, although Beaudette was described decades ago and the recombinant methods using Beaudette have been known for more than a decade, there is no Beaudette-based vaccine and such a recombinant vaccine (with a heterologous spike protein) available for commercial purchase. The Beaudette-based recombinant IBV is not suitable as a vaccine. Wei et al 2014 (Apl Microbiol Biotechnol 98) disclosed Beaudette IBV with the S1 subunit of H120.

[0012] Ellis et al 2018 (J. Virol. 92(23)), Hodgson et al (J. Virol. 78(24)), and Armesto et al. 2011 (PLoS One: 6(8): e24352) all disclose Beaudette IBV with M41 or 4 / 91 spike proteins. However, Ellis et al 2018 (J. Virol. 92(23)) describes recombinant Beaudette having a chimeric spike protein with a heterologous S1 subunit from M41 or QX combined with the Beaudette spike protein S2 subunit, which does not provide sufficient protection against S1 homologous challenge ("Single inoculation of specific pathogen-free chickens with rIBV expressing the S1 of the virulent strains M41 or QX, namely BeauR-M41(S1) and BeauR-QX(S1), provides incomplete protection against homologous challenge based on ciliary motility and clinical signs"; abstract). Further, Ellis et al 2018 (J. Virol. 92(23)) describes that the full-length S gene (S1 and S2 from M41) only provides partial protection against homologous serotype IBV challenge (page 12), suggesting that the IBV Beaudette strain is not suitable as a backbone for recombinant IBV vaccines. Hodgson et al (J. Virol. 78(24)) further discloses that the Baudette strain "is also considered to have poor immunogenicity" and thus, "it has never been used as a vaccine strain" (page 13802, left column, second paragraph). Therefore, there is a need to separately generate novel and highly effective IBV vaccines and recombinant IBV vaccines. In addition, there is a need for highly effective IBV vaccine vectors. DETAILED DESCRIPTION OF THE INVENTION

[0014] 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" refers to one or more viruses known to those of ordinary skill in the art and their equivalents, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. For the purposes of describing and disclosing cell lines, vectors, and methodologies reported in publications that can be used with the present invention, all publications mentioned herein are incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0015] Composition of substances

[0016] The present invention solves the problems inherent in the prior art and provides a significant improvement over the prior art.

[0017] Generally, the present invention provides an H52 infectious bronchitis virus (IBV) encoding a heterologous IBV spike (S) protein or a fragment thereof.

[0018] The term "H52 IBV" is well known to those skilled in the art. The term "IBV" refers to infectious bronchitis virus. The term "H52" defines a specific IBV strain. The H52 strain is well known to those skilled in the art and belongs to the Massachusetts genotype. IBV strains are typically distinguished by the coding sequence of the spike protein S1 subunit (Valastro et al. 2016. Infect Genet Evol. 39:349-364), but can also be distinguished by their complete nucleotide sequence or the sequence of specific proteins such as the spike protein, nucleocapsid protein, envelope (E) protein, or membrane (M) glycoprotein. Since the spike protein determines the host tropism and antigenicity of IBV, IBV genotypes are classified by the coding sequence of spike protein subunit 1. Alternatively, IBV strains can be distinguished by their serotype. Serotype classification involves treating the virus with neutralizing antibodies.

[0019] In addition, H52 and H120 can be distinguished by higher pathogenicity when administered to young chickens.

[0020] How to obtain H52 IBV is common knowledge to those skilled in the art. The H52 IBV strain can be commercially available, such as the exemplified Nobilis IB H52 (MSD Animal Health), AviPro IB H52 (Lohmann Animal Health GmbH & Co. KG), Bronchovac (Ceva), etc. In addition, McDonald et al. 1980 (Avain Pathology 9: 245-259) disclosed that H52 IBV can be obtained from the Central Veterinary Laboratory Rotterdam, Kusters (J. gen Virol 68: 343-352) disclosed that H52 IBV can be obtained from the Poultry Health Institute Dorn in the Netherlands (now GD Animal Health), and then Chen et al. 2007 (Avian Pathology 36(4): 269-274) disclosed that H52 IBV can be obtained from the China Institute of Veterinary Drug Control. In addition, H52 IBV has been used as a vaccine strain for decades (Bijlenga et al. 2004, Avian Pathology 33(6): 550-557), so it can be isolated in the wild. The methods for isolating and identifying the H52 IBV strain are well known to those skilled in the art. For example, the identification of the H52 IBV strain can be carried out as described by 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). Zwaagstra et al. 1992 and Handberg et al. 1999, for example, disclosed Massachusetts-specific primers (targeting the S and N proteins respectively) for RT-PCR and sequencing and reference sequences for comparison. In addition, H52 IBV has been sequenced and the genomic sequence can be obtained, such as EU817497. Therefore, the viral genome can be generated by synthesizing its sequence and can be generated by applying a reverse genetics system.

[0021] The term "spike protein" refers to a specific protein of IBV well-known to those skilled in the art. The spike protein is the main inducer of antibodies and protective immune responses. In addition, the spike (S) protein promotes the entry of IBV into cells by binding to the cellular receptors of host cells and also by mediating virus-cell membrane fusion with host cells. In addition, it determines the tissue and cell tropism of the virus strain.

[0022] The term "heterologous S (spike protein)" refers to a spike protein or a fragment thereof that has been introduced into H52 IBV and is from a different genotype or serotype than H52 IBV. Since H52 is of the Massachusetts genotype and serotype, the heterologous spike protein is non-Massachusetts genotype or serotype.

[0023] The terms "protein", "amino acid" and "polypeptide" are used interchangeably. The term "protein" refers to an amino acid sequence composed 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. In addition, 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. In addition, the term also relates to protein fragments. Moreover, it includes chemically modified proteins. Such modifications can be artificial or naturally occurring modifications, such as phosphorylation, glycosylation, myristoylation, etc.

[0024] Furthermore, the present invention also provides an immunogenic composition comprising H52 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.

[0025] In addition, the present invention also provides an immunogenic composition comprising the IBV (infectious bronchitis virus) described herein. Furthermore, the present invention also provides an immunogenic composition comprising H52 IBV (infectious bronchitis virus) encoding a heterologous S (spike) protein or a fragment thereof.

[0026] 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 can be a cell- and / or antibody-mediated immune response to the immunogenic composition of the present invention. Preferably, the immunogenic composition induces an immune response, and more preferably confers protective immunity against one or more clinical signs of IBV infection. A host is also referred to as an "object". Preferably, any host or object described or mentioned herein is avian or poultry.

[0027] Generally, 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 gamma-delta T cells that specifically target one or more antigens contained in the immunogenic composition of the present invention. Preferably, the host will exhibit a protective immune response or a therapeutic response.

[0028] A "protective immune response" or "protective immunity" is demonstrated by a decrease or disappearance of the clinical signs normally exhibited by an infected host, a shorter recovery time and / or a shorter duration of infection in the infected host, or a decrease in the pathogen titer in tissues or body fluids or excretions.

[0029] If a host exhibits a protective immune response, resulting in an increased resistance to new infections and / or a reduced clinical severity of the disease, the immunogenic composition is described as a "vaccine".

[0030] H52-IBV – defined by protein coding sequence

[0031] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV has the nucleotide sequence shown in EU817497 (SEQ ID NO:78) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0032] The term "nucleic acid" or "nucleic acid sequence" or "nucleotide sequence" refers to a polynucleotide comprising a DNA molecule, an RNA molecule, a cDNA molecule or a derivative. This term encompasses single-stranded and 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, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or artificially modified polynucleotides, such as biotinylated polynucleotides. Furthermore, the terms "nucleic acid" and "polynucleotide" are used interchangeably and refer to any nucleic acid. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine and uracil).

[0033] The term "RNA" refers to any ribonucleic acid. This term encompasses single-stranded and double-stranded RNA. The RNA of the present invention includes isolated RNA (i.e., isolated from its natural environment) and genetically modified forms. In addition, chemically modified RNA is also included, including naturally occurring modified RNA such as methylated RNA, or artificially modified RNA such as biotinylated RNA. The term "RNA" also specifically includes RNA composed of bases other than the four biologically occurring nucleotides / bases (adenine, guanine, cytosine, and uracil).

[0034] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a spike (S) protein, and the spike protein has the amino acid sequence shown in AF352315 (SEQ ID NO:79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0035] It must be understood that the spike protein or nucleic acid sequence can be used to determine whether any IBV strain is derived from H52. However, since the H52 IBV is used as a backbone and the H52 spike protein or nucleic acid sequence is replaced by a heterologous spike protein or a fragment thereof, the final IBV having a heterologous spike protein does not contain any H52 spike protein or only a remaining part of the H52 spike protein.

[0036] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a spike (S) protein, and the spike protein has the amino acid sequence shown in SEQ ID NO:1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0037] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a nucleocapsid (N) protein, and the nucleocapsid protein has the amino acid sequence shown in AY044185 (SEQ ID NO:80) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to at least one of the above sequences.

[0038] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a nucleocapsid (N) protein, and the nucleocapsid protein has an amino acid sequence as shown in AF352310 (SEQ ID NO: 81) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0039] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a nucleocapsid (N) protein, and the nucleocapsid protein has an amino acid sequence as shown in SEQ ID NO: 2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0040] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has an envelope (E) protein, and the envelope protein has an amino acid sequence as shown in AF317210 (SEQ ID NO: 82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0041] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has an envelope (E) protein, and the envelope protein has an amino acid sequence as shown in SEQ ID NO: 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0042] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a membrane glycoprotein (M), and the membrane glycoprotein has an amino acid sequence as shown in AF286185 (SEQ ID NO: 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0043] In another specific aspect of the IBV or immunogenic composition of the present invention, the H52 IBV strain has a membrane glycoprotein (M), and the membrane glycoprotein has an amino acid sequence as shown in SEQ ID NO: 4 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0044] The terms "identity" or "sequence identity" are known in the art and refer to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, i.e., the relationship between a reference sequence and a designated sequence to be compared with the reference sequence. After the designated sequence is optimally aligned with the reference sequence to yield the highest degree of sequence similarity, sequence identity is determined by sequence comparison and is determined by the matching between sequence strings. Based on such alignment, sequence identity can be determined on a position-by-position basis. For example, if the nucleotide or amino acid residue at a certain position is the same, the sequences are "identical" at that particular position. The total number of such position identities is divided by the total number of nucleotides or residues in the reference sequence to yield the percentage of sequence identity. Sequence identity can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A.N., ed., Oxford University Press, New York (1988), Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinge, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988), the teachings of which are incorporated herein by reference. Preferred methods for determining sequence identity are designed to give the maximum match between the tested sequences. Methods for determining sequence identity are compiled in publicly available computer programs for determining sequence identity between designated sequences.Examples of such programs include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research, 12(1):387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S.F. et al., J. Molec. Biol., 215:403-410 (1990)). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIH Bethesda, MD 20894, Altschul, S.F. et al., J. Molec. Biol., 215:403-410 (1990), the teachings of which are incorporated herein by reference). These programs use default gap weights to optimally align sequences to produce the highest level of sequence identity between a specified sequence and a reference sequence. For example, a polynucleotide having a nucleotide sequence that has at least, for example, 85%, preferably 90%, even more preferably 95% "sequence identity" with a reference nucleotide sequence means that the nucleotide sequence of the specified polynucleotide is identical to the reference sequence, except that the specified polynucleotide sequence may include up to 15, preferably up to 10, even more preferably up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in a polynucleotide having a nucleotide sequence that has at least 85%, preferably 90%, even more preferably 95% identity relative to a reference nucleotide sequence, up to 15%, preferably 10%, even more preferably 5% of the nucleotides in the reference nucleotide sequence may be deleted or replaced with another nucleotide, or up to 15%, preferably 10%, even more preferably 5% of the nucleotides of the entire reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or at any position between those terminal positions, be scattered among the individual nucleotides of the reference sequence, or be in one or more contiguous groups within the reference sequence. Similarly, a polypeptide having an amino acid sequence that has at least, for example, 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence means that the amino acid sequence of the specified polypeptide is identical to the reference sequence, except that the specified polypeptide sequence may include up to 15, preferably up to 10, even more preferably up to 5 amino acid changes per 100 amino acids of the reference amino acid sequence.In other words, in order to obtain a specified polypeptide sequence having at least 85%, preferably 90%, even more preferably 95% sequence identity with a reference amino acid sequence, up to 15%, preferably up to 10%, even more preferably up to 5% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or up to 15%, preferably up to 10%, even more preferably up to 5% of the total number of amino acids in the reference sequence may be inserted into the reference sequence. These alterations to the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence or at any position between those terminal positions, be scattered among the individual residues of the reference sequence, or in one or more contiguous groups within the reference sequence. Preferably, conservative amino acid substitutions at non-identical residue positions vary. However, conservative substitutions are not included as matches when determining sequence identity.

[0045] The terms "identity", "sequence identity", and "percent identity" are used interchangeably herein. For the purposes of the present invention, the percent identity of two amino acid sequences or two nucleic acid sequences is defined herein as the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of positions shared by the sequences (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences have the same length.

[0046] Sequence comparisons can be performed over the full length of the two sequences being compared or over a fragment of the two sequences. Typically, the comparison is made over the full length of the two sequences being compared. However, sequence identity can be compared in regions of, for example, 20, 50, 100 or more contiguous amino acid residues.

[0047] Those skilled in the art will recognize that different computer programs can be utilized to determine the homology between two sequences. For example, mathematical algorithms can be used to accomplish sequence comparison and determination of the percentage identity between two sequences. In a preferred embodiment, the percentage identity between two amino acid or nucleic acid sequences is determined using the following algorithm: the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm, which has been incorporated into the GAP program in the Accelrys GCG software package (available from http: / / www.accelrys.com / products / gcg / ), using the Blosum 62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Those skilled in the art will understand that when different algorithms are used, all of these different parameters will produce slightly different results, but the overall percentage identity of the two sequences will not change significantly.

[0048] The protein or nucleic acid sequences of the present invention can further be used as a "query sequence" to search public databases, for example to identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0), as described in Altschul, et al. (1990) J. Mol. Biol. 215:403-10. The BLAST protein search can be performed using the BLASTP program with a score = 50 and word length = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain a gapped alignment for comparison purposes, the Gapped BLAST program can be used, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the National Center for Biotechnology Information homepage http: / / www.ncbi.nlm.nih.gov / .

[0049] As used herein, the term "identical to the sequence of SEQ ID NO:X" is to be specifically understood as equivalent to the term "identical in length to the sequence of SEQ ID NO:X" or to the term "identical to the sequence of SEQ ID NO:X over the full length of the sequence of SEQ ID NO:X". In this text, "X" is any integer selected from 1 to 85, whereby "SEQ ID NO:X" represents any SEQ ID NO mentioned herein.

[0050] Heterologous S protein

[0051] IBV strain

[0052] 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. The first identified IBV serotype was the Massachusetts type, but in addition to the originally identified Massachusetts type, several other serotypes have been identified in the United States, including Arkansas and Delaware.

[0053] The IBV strain Beaudette is of the Massachusetts type and was produced by at least 150 passages in chicken embryos. The 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 in addition to Beaudette are H120, H52, and M41. The H120 strain was passaged 120 times.

[0054] IBV QX has been described as a virulent field isolate of IBV and was originally isolated in China. However, the virus has spread to Europe and has been found in parts of Western Europe, mainly in the Netherlands, but also in Germany, France, Belgium, Denmark, and the United Kingdom. Additionally, the QX genotype or serotype has been described in some countries in Asia and Africa.

[0055] IBV 4 / 91, commonly known as 793B, was first reported in the United Kingdom in the early 1990s and has now spread to many parts of the world. CR88 is an attenuated strain belonging to this serotype and is commercially available as a vaccine.

[0056] In the late 1990s, strains named "Italien-02" or "Italy-02" were isolated in Italy. Sequence analysis of one of these isolates was published in 2002 (NCBI-BLAST, accession number AJ457137). However, studies have shown that this Italian-02 strain is widespread in Europe and, except for the IBV variant 4 / 91, it has become one of the most predominant genotypes in the UK, Spain, France, and the Netherlands.

[0057] Since 1996, a new genotype of infectious bronchitis virus (IBV) called Q1 has been prevalent in China and was first reported in Italy in 2011. Q1 is associated with increased mortality, kidney lesions, and glandular gastritis.

[0058] In addition, strains D274, B1648 / D8880, D1466, V1397, and Arkansas have also been identified in Europe.

[0059] How to obtain any IBV strain is common knowledge to those skilled in the art. IBV strains can be commercially purchased, obtained from research institutions, or the genes can be synthesized into complementary DNA because the IBV strains have been sequenced and the sequences have been published and are thus available for use. In addition, IBV strains can be isolated from the wild. Methods for isolating and identifying 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 undzum Vorkommen sowie zur des Genotyps IBV QX in spezifischpathogenfreien(SPF)Broilern,Freie Berlin), Worthington et al. 2009 (Avian Pathology 37(3), 247-257), Liu et al. 2009 (Virus Genes 38:56-65), Dolz et al. 2006 (Avian Pathology 35(2):77-85), Farsang et al. 2002 (Avian Pathology 31:229-236) and Feng et al. 2014 (Virus Genes 49:292-303) describe how to isolate and differentiate different IBV strains.

[0060] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous spike protein is a non-Massachusetts genotype or serotype.

[0061] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or its fragment is derived from an IBV having a genotype or serotype selected from the following group: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California1734 / 04, California99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, LDT3, Maine (e.g., Maine 209), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88).

[0062] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or fragment thereof is derived from an IBV selected from the following genotypes or serotypes: 4 / 91, QX, Q1, Italy 02, Arkansas, Connecticut, Georgia, LDT3, PL84084, Variant 2, and Brazil.

[0063] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or fragment thereof is derived from an IBV selected from the following genotypes or serotypes: 4 / 91, QX, Q1, Arkansas, Variant 2, and Brazil.

[0064] In another specific aspect of the IBV or immunogenic composition according to the present invention, the 4 / 91 strain is selected from the following group: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, 4 / 91 - pathogenicity, 4 / 91 attenuated, IB4-91, and CR88.

[0065] In another specific aspect of the IBV or immunogenic composition according to the present invention, the QX strain is selected from the following group: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03, and GB341 / 96.

[0066] In another specific aspect of the IBV or immunogenic composition according to the present invention, the Q1 strain is selected from the following group: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21, and Chile-295-10.

[0067] In another specific aspect of the IBV or immunogenic composition according to the present invention, the Arkansas strain is selected from the following group: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01, and AL / 4614 / 98.

[0068] In another specific aspect of the IBV or immunogenic composition according to the present invention, the variant 2 strains are selected from the following group: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8, and IB VAR2-06.

[0069] In another specific aspect of the IBV or immunogenic composition according to the present invention, the Brazil strains are selected from the following group: BR-1, BR-2, 23 / 2013, and IBV / Brasil / 351 / 1984.

[0070] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or a fragment thereof is from genotype or serotype 4 / 91.

[0071] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or a fragment thereof is from genotype or serotype 4 / 91, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with SEQ ID NO:5 or 6.

[0072] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or a fragment thereof is from genotype or serotype QX, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with SEQ ID NO:7 or 8.

[0073] In another specific aspect of the IBV or immunogenic composition according to the present invention, the heterologous S protein or a fragment thereof is from genotype or serotype Q1, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity with SEQ ID NO:9 or 10.

[0074] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from the Arkansas genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO: 11 or 12.

[0075] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from the variant 2 genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO: 13 or 14.

[0076] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is from the Brazil genotype or serotype, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity with SEQ ID NO: 15 or 16.

[0077] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein or fragment thereof is selected from SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0078] In another specific aspect of the IBV or immunogenic composition of the present invention, the heterologous S protein is the full-length spike protein.

[0079] The experimental data of the present invention show that fragments of the spike protein sequence such as the extracellular domain of the spike protein can be used. However, the full-length spike protein sequence can also be used.

[0080] In another specific aspect of the IBV or immunogenic composition of the present invention, the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.

[0081] In another specific aspect of the IBV or immunogenic composition of the present invention, the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length from the N-terminus.

[0082] The term "N-terminus" is well known to those skilled in the art. The N-terminus is also referred to as the amino terminus, NH2-terminus, N-terminus, or amine terminus. When translating a protein from messenger RNA, it is produced from the N-terminus to the C-terminus. Thus, the N-terminus is the starting point of the amino acid chain (protein or polypeptide) that contains the amine group (-NH2).

[0083] In another specific aspect of the IBV or immunogenic composition of the present invention, the length of the fragment of the heterologous S (spike) protein is at least 1000 amino acids.

[0084] In another specific aspect of the IBV or immunogenic composition of the present invention, the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.

[0085] The term "extracellular domain" is well known to those skilled in the art. The spike protein contains different functional parts, namely a signal sequence, an extracellular domain, a transmembrane domain, and an intracellular domain (from the N-terminus to the C-terminus). Thus, after cleavage of the signal sequence, the N-terminus of the spike protein begins with the extracellular domain. The length of the extracellular domain of the IBV spike protein is approximately 1077 amino acids, varying by a few amino acids in length depending on the IBV strain.

[0086] In another specific aspect of the IBV or immunogenic composition of the present invention, wherein the heterologous S (spike) protein or its fragment replaces the homologous S protein or its fragment.

[0087] In another specific aspect of the IBV or immunogenic composition of the present invention, wherein the heterologous S (spike) protein or its fragment replaces the naturally occurring S protein or its fragment.

[0088] In another specific aspect of the IBV or immunogenic composition of the present invention, wherein the heterologous S (spike) protein or its fragment replaces the S protein or its fragment in H52 IBV.

[0089] In another specific aspect of the IBV or immunogenic composition of the present invention, the IBV is attenuated.

[0090] The term "attenuated" refers to a pathogen having reduced virulence compared to a wild-type isolate. In the present invention, an attenuated IBV is a strain in which the virulence has been reduced such that it does not cause the clinical signs of IBV infection but is capable of inducing an immune response in the target animal, but may also refer to a reduction in the occurrence or severity of clinical signs in animals infected with the attenuated IBV compared to "control" animals infected with non-attenuated IBV or not receiving the attenuated virus. In the present text, the term "reduce" refers to a reduction of at least 10%, preferably 25%, even more preferably 50%, still more preferably 60%, even more preferably 70%, still more preferably 80%, even more preferably 90%, even more preferably 95%, and most preferably 100% compared to the 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.

[0091] In another specific aspect of the IBV or immunogenic composition of the present invention, the IBV is inactivated.

[0092] Any conventional inactivation method can be used for the purposes of the present invention. Thus, inactivation can be carried out by chemical and / or physical treatment methods known to those skilled in the art. Preferred inactivation methods include the addition of bicyclic ethyleneimine (BEI), including the addition of a solution of 2-bromoethylamine hydrobromide (BEA) that has been cyclized to bicyclic ethyleneimine (BEI). Other preferred chemical inactivators include, but are not limited to, Triton X-100, sodium deoxycholate, cetyltrimethylammonium bromide, beta-propiolactone, thimerosal, phenol, and formaldehyde (formalin). However, inactivation may also include a neutralization step. Preferred neutralizing agents include, but are not limited to, sodium thiosulfate, sodium bisulfite, etc.

[0093] Preferred formalin inactivation conditions include a formalin concentration of 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). The incubation time depends on the resistance of the IBV. Generally, the inactivation procedure is carried out until no IBV growth is detected in a suitable culture system.

[0094] Preferably, the inactivated IBV of the present invention is formalin inactivated, preferably using the concentrations described above herein.

[0095] 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 with a suitable biocompound such as a polysaccharide, peptide, protein, etc. or a combination thereof.

[0096] In another specific aspect of the IBV or immunogenic composition of the present invention, the IBV is genetically engineered.

[0097] The term "genetically engineered" refers to an IBV that has been mutated by using "reverse genetics" methods. Preferably, the IBV of the present invention has been genetically engineered. Reverse genetics techniques involve the preparation of synthetic recombinant viral RNA. However, "reverse genetics" techniques are well known to those skilled in the art.

[0098] In another specific aspect of the IBV or immunogenic composition of the present invention, the IBV is a recombinant IBV.

[0099] As used herein, the term "recombinant" relates to an RNA genome (or RNA sequence, cDNA sequence or protein) that has any modification that is not naturally present in the corresponding RNA genome (or RNA sequence, cDNA sequence or protein). For example, if an RNA genome (or RNA sequence, cDNA sequence or protein) contains insertions, deletions, inversions, relocations or point mutations that are artificially introduced, for example, by human intervention, it is considered "recombinant". Thus, the RNA genome sequence (or RNA sequence, cDNA sequence or protein) is not related to all or part of the sequence (or RNA sequence, cDNA sequence or protein) that is naturally associated with it. The term "recombinant" as used with respect to a virus refers to a virus produced by artificial manipulation of the viral genome. The term "recombinant virus" includes genetically modified viruses.

[0100] In another specific aspect of the IBV or immunogenic composition of the present invention, the IBV is chimeric.

[0101] The term "chimeric" refers to an IBV that contains one or more nucleotide sequences from another coronavirus, preferably from another IBV strain. For example, the IBV H52 that encodes a heterologous S (spike) protein or a fragment thereof is a chimeric IBV.

[0102] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition is a vaccine. The term "vaccine" has been described elsewhere herein. However, an immunogenic composition is described as a "vaccine" if the host exhibits a protective immune response, whereby resistance to new infection is enhanced and / or the clinical severity of the disease is reduced.

[0103] In another specific aspect of the immunogenic composition according to the present invention, the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0104] The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating agents, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0105] "Diluents" may include water, saline, glucose, ethanol, glycerol, etc. Isotonic agents may include sodium chloride, glucose, mannitol, sorbitol, and lactose, etc. Stabilizers include albumin and alkali metal salts of ethylenediaminetetraacetic acid, etc.

[0106] In another specific aspect of the immunogenic composition of the present invention, the pharmaceutically acceptable carrier is phosphate buffered saline.

[0107] Preferably, the immunogenic composition further comprises a sucrose gelatin stabilizer.

[0108] Preferably, the pharmaceutically acceptable carrier is chitosan.

[0109] Chitosan is a natural deacetylated polysaccharide derived from chitin in crustaceans (such as shrimps, crabs), insects, and other invertebrates. Recently, Rauw et al. in 2009 (Vet Immunol Immunop 134:249 - 258) demonstrated that chitosan enhanced the cellular immune response of live Newcastle disease vaccine and promoted its protective effect. In addition, Wang et al., in 2012 (Arch Virol (2012) 157:1451 - 1461) showed the results of the potential of chitosan as an adjuvant for live attenuated influenza vaccine.

[0110] Preferably, the immunogenic composition may further comprise one or more other immunomodulators, such as interleukins, interferons, or other cytokines. The amounts and concentrations of adjuvants and additives useful within the scope of the present invention can be readily determined by a person skilled in the art.

[0111] In certain aspects, the immunogenic compositions of the invention contain an adjuvant. As used herein, "adjuvant" can include aluminum hydroxide and aluminum phosphate, saponins such as Quil A, QS-21 (Cambridge Biotech, Cambridge, Massachusetts), GPI-0100 (Pharmaceutical Formulations, Inc., Birmingham, Alabama), water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions. The emulsion can be based in particular on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils such as squalane or squalene; oils resulting from the oligomerization of olefins, especially isobutene or decene. Esters of acids or alcohols containing straight-chain alkyls, especially vegetable oils, ethyl oleate, propylene glycol di(caprylate / caprate), glyceryl tri(caprylate / caprate) or propylene glycol dioleate. Esters of branched-chain fatty acids or alcohols, especially isostearates. The oil is used in combination with an emulsifier to form an emulsion. The emulsifier is preferably a nonionic surfactant, especially esters of sorbitol, mannitol (such as dehydrated mannitol oleate), ethylene glycol, polyglycerol, propylene glycol and oleic, isostearic, ricinoleic or hydroxystearic acid, which may be optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, especially Pluronic products, especially L121. See Hunter et al., "The Theory and Practical Application of Adjuvants" (Ed. Stewart-Tull, D.E.S.), John Wiley & Sons, Agricultural Industry Standards, pp. 51-94 (1995) and Todd et al., "Vaccine 15:564-570" (1997). Typical 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 MF59 emulsion described on page 183 of the same book.

[0112] Another example of an adjuvant is a compound selected from polymers of acrylic acid or methacrylic acid and copolymers of maleic anhydride and vinyl derivatives. Preferred adjuvant compounds are crosslinked polymers of acrylic acid or methacrylic acid, especially polymers crosslinked with polyalkenyl ethers of sugars or polyols. These compounds are known as carbomers (Phameuropa, Volume 8, Number 2, June 1996). Those skilled in the art may also refer to U.S. Patent No. 2,909,462, which describes the crosslinking of such acrylic polymers with polyhydroxy compounds having at least 3 hydroxyl groups, preferably not more than 8 hydroxyl groups, and in which the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic groups having at least 2 carbon atoms. Preferred groups are those containing 2 to 4 carbon atoms, such as vinyl, allyl, and other vinyl unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. Products sold under the name Carbopol (BFGoodrich, Ohio, USA) are particularly suitable. They are crosslinked with allyl sucrose or allyl pentaerythritol. Among them, Carbopol 974P, 934P, and 971P may be mentioned. The most commonly used is Carbopol 971P. Among the copolymers of maleic anhydride and vinyl derivatives, there is the copolymer EMA (Monsanto), which is a copolymer of maleic anhydride and ethylene. These polymers dissolve in water to produce an acidic solution, which will be neutralized, preferably to physiological pH, in order to prepare an adjuvant solution to be incorporated into an immunogenic, immunological, or vaccine composition.

[0113] Further suitable adjuvants include, but are not limited to, the RIBI adjuvant system (RIBI Inc.), Block copolymer (CytRx, Atlanta, Georgia), SAF-M (Chiron, Emeryville, California), monophosphoryl lipid A, Avridine lipid amine adjuvant, heat-labile endotoxin from Escherichia coli (recombinant or otherwise), cholera toxin, IMS1314 or muramyl dipeptide, or naturally occurring or recombinant cytokines or their analogs, or stimulants for the release of endogenous cytokines, and the like.

[0114] The amount of adjuvant added is expected to be from about 100 micrograms to about 10 milligrams per dose, preferably from about 100 micrograms to about 10 milligrams per dose, more preferably from about 500 micrograms to about 5 milligrams per dose, even more preferably from about 750 micrograms to about 2.5 milligrams per dose, and most preferably about 1 milligram per dose. Additionally, the concentration of the adjuvant, calculated by the volume of the final product, may be from about 0.01 to 50%, preferably from about 2% to 30%, more preferably from about 5% to 25%, more preferably from about 7% to 22%, and most preferably a concentration of 10% to 20%.

[0115] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof. The terms "treating and / or preventing", "clinical symptoms" and "in need" have been defined elsewhere.

[0116] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge with IBV strains of a heterologous spike protein genotype or serotype.

[0117] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge with strains of genotype 4 / 91, QX, Q1, Arkansas, variant 2 or Brazil.

[0118] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition protects against challenge with strains of genotype 4 / 91.

[0119] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is formulated for single-dose administration.

[0120] The volume of a single dose has been defined elsewhere herein.

[0121] Furthermore, it has been shown that one dose of the immunogenic composition of the invention is effective after administration of such a single dose of the immunogenic composition of the invention.

[0122] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spray, via drinking water or by eye drops.

[0123] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises from 1 to 10 log 10 EID 50 of said IBV per dose.

[0124] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises from 2 to 5 log 10 EID 50 of said IBV per dose.

[0125] In another specific aspect of the immunogenic composition according to the invention, the immunogenic composition comprises from 2 to 4 log 10 EID 50 of said IBV per dose.

[0126] Kit

[0127] If desired, the composition may be present in a package or dispensing device that contains one or more unit dosage forms containing the active ingredient. For example, the package may comprise a metal or plastic foil, such as a blister pack. The package or dispenser device may be accompanied by instructions for administration, preferably for administration to a subject, especially a poultry. Associated with such a container may be a notice that is written in a form prescribed by a government agency that regulates the manufacture, use, or sale of drugs or biological products, and that reflects the agency's approval of the preparation, use, or sale of the medicament for human use.

[0128] Accordingly, the present invention provides a kit comprising the IBV or immunogenic composition described herein.

[0129] In a specific aspect of the kit according to the present invention, the kit further comprises instructions for the treatment and / or prevention of avian diseases.

[0130] In a specific aspect of the kit according to the present invention, the kit further includes instructions for the treatment and / or prevention of poultry diseases.

[0131] In a specific aspect of the kit according to the present invention, the kit further includes instructions for the treatment and / or prevention of IB (Infectious Bronchitis).

[0132] Therapeutic method

[0133] In addition, the present invention provides a method for immunizing a subject, comprising administering to the subject an immunogenic composition as described herein.

[0134] The term "immunize" refers to active immunization by administering an immunogenic composition to a subject to be immunized, thereby eliciting an immune response against the antigen contained in such immunogenic composition.

[0135] Preferably, immunization results in a decrease in the incidence of a specific IBV infection in a population or a decrease in the severity of clinical signs caused by or associated with a specific IBV infection.

[0136] In addition, immunizing a subject in need with the immunogenic composition provided herein results in preventing the subject from being infected with IBV. Even more desirably, the result of immunization is an effective and long-lasting immune response against IBV infection. It is understood that the period 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, more preferably more than 6 months. It should be understood that immunization may not be effective for all immunized subjects. However, the term requires that a large proportion of the population of subjects be effectively immunized.

[0137] Preferably, in this case, a group of subjects is envisioned which will typically develop clinical signs normally caused by or associated with IBV infection, i.e., unimmunized. Whether the subjects of the group are effectively immunized can be further determined by those skilled in the art. Preferably, compared to subjects that are unimmunized or immunized with an immunogenic composition available prior to the present invention but subsequently infected with a specific IBV, if at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, more preferably at least 95%, and most preferably 100% of the subjects in a group have a reduction in the incidence or severity of clinical signs of at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%, then the immunization should be effective..

[0138] In addition, the present invention provides a method of 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 as described herein.

[0139] The term "treating or preventing" refers to reducing the incidence of a specific IBV infection in a population, or reducing the severity of clinical signs caused by or associated with a specific IBV infection. Thus, the term "treating or preventing" also refers to a group of subjects receiving an effective amount of the immunogenic composition provided herein, which, compared to a group of subjects not receiving such an immunogenic composition, reduces the number of subjects in the population infected with a specific IBV (= reduces the incidence of a specific IBV infection), or alleviates the severity of clinical signs normally associated with or caused by an IBV infection, or reduces viral shedding after a specific IBV infection, or prevents or reduces egg production decline in laying hens after infection with a specific IBV.

[0140] "Treating or preventing" generally involves administering an effective amount of the immunogenic composition of the present invention to a subject or group of subjects in need of or likely to benefit from such treatment / prevention. The term "treating" refers to administering an effective amount of the immunogenic composition once a subject or at least some of the subjects in the group have been infected with such an IBV and wherein these subjects have shown some clinical signs caused by or associated with such an IBV infection. The term "preventing" refers to administering to a subject prior to the subject being infected with IBV, or at least in the situation where none of the subjects in the subject or group of subjects have shown any clinical signs caused by or associated with such an IBV infection. The terms "preventing" and "prevention" are used interchangeably in this application.

[0141] As used herein, the term "effective amount" refers to, but is not limited to, an amount of 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 particular IBV infection in a population or reducing the severity of the clinical signs of a particular IBV infection.

[0142] Preferably, compared to a subject that is untreated or treated with an immunogenic composition available prior to the present invention and subsequently infected with a particular IBV, the incidence or severity of the clinical symptoms is reduced by at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%.

[0143] The term "clinical signs" as used herein refers to the signs of a subject infected with IBV. The clinical signs of infection depend on the pathogen selected. Examples of such clinical signs include, but are not limited to, respiratory distress, nephritis, salpingitis, abnormal egg production, ruffled feathers, depression, decreased growth rate, and decreased appetite. Signs of respiratory distress include respiratory signs, including wheezing, coughing, sneezing, tracheal rales, nasal and ocular secretions, tracheal lesions, and ciliostasis within the trachea. Signs of nephritis include kidney lesions and watery diarrhea. Signs of abnormal egg production include reduced egg production, smaller egg size, poorer eggshell quality, decreased internal egg quality, thinner egg white, and ciliostasis within the oviduct. However, clinical signs also include, but are not limited to, clinical signs that can be directly observed in a live animal. Examples of clinical signs that can be directly observed in a live animal include nasal and ocular secretions, coughing, wheezing, sneezing, tracheal rales, ruffled feathers, conjunctivitis, weight loss, decreased growth rate, decreased appetite, dehydration, watery diarrhea, lameness, lethargy, emaciation, and lack of movement, among others.

[0144] Preferably, compared to a subject that is untreated or treated with an immunogenic composition available prior to the present invention and subsequently infected with a particular IBV, a reduction in the incidence or severity of the clinical symptoms in a treated subject refers to a reduction in ciliostasis, a reduction in rales, a reduction in egg drop, a reduction in kidney lesions, a reduction in watery diarrhea, a reduction in weight loss, a reduction in viral load, a reduction in viral shedding, or a combination thereof.

[0145] The term "in need of" or "in need" as used herein refers to administering / treating in relation to enhancing or improving health or clinical signs or having any other positive medical effect on the health of a subject receiving the immunogenic composition of the present invention.

[0146] The terms "reduce" or "reduction" or "mitigate" or "decrease" are used interchangeably in this application. The term "reduce" means that, compared to an untreated (unimmunized) subject that is subsequently infected with a specific IBV, the clinical signs are reduced by at least 10%, more preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95%, and most preferably 100%.

[0147] In addition, compared to subjects in an unimmunized control group of the same species, the present invention provides a method for reducing ciliary stasis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition as described herein.

[0148] As shown in the examples, the immunogenic compositions provided herein have been shown to be effective in reducing ciliary stasis.

[0149] The term "ciliary stasis" refers to a reduction in the movement of cilia in the trachea. Thus, ciliary stasis can be determined by examining the inner layer of the tracheal ring to determine the movement of cilia. How to determine the movement of cilia in the trachea is common knowledge to those skilled in the art.

[0150] Preferably, compared to subjects in an unimmunized control group of the same species, starting from the 10th day after challenge or infection, more preferably starting from the 5th day after challenge or infection, more preferably starting from the 4th day after challenge or infection, more preferably starting from the 3rd day after challenge or infection, and most preferably starting from the 1st or 2nd day after challenge or infection with IBV, the movement of cilia is not reduced.

[0151] The term "reduction of ciliary stasis" means that, compared to subjects in an unimmunized control group of the same species, ciliary stasis 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%. How to measure the reduction of ciliary stasis is common knowledge to those skilled in the art.

[0152] In one aspect of the present invention, the subject is avian.

[0153] The term "avian" is well known to those skilled in the art. The term "avian" includes all birds, including domestic fowl.

[0154] In one aspect of the present invention, the subject is poultry.

[0155] The term "poultry" is well known to those skilled in the art. The term "poultry" includes chicken, turkey, quail, pheasant, guineafowl, goose and duck. In addition, the term "chicken" includes broilers, laying hens, and breeding chickens of both, also known as parent stock chickens.

[0156] In one aspect of the invention, the subject is selected from chicken, turkey, quail or pheasant.

[0157] In one aspect of the invention, the subject is a chicken.

[0158] In one aspect of the invention, the immunogenic composition is administered once.

[0159] Understand that a single dose means administered only once. As shown in the examples, the immunogenic compositions provided herein have been shown to be efficacious after administering a single dose to a subject in need.

[0160] The dose volume per poultry depends on the route of inoculation and the age of the poultry.

[0161] Generally, at any age, the eye-drop vaccine is administered in a volume of 1 to 100 μl per dose. Preferably, the total volume of a single dose of the eye-drop vaccine is between about 5 μl and 70 μl, more preferably between about 20 μl and 50 μl, preferably 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl or 50 μl per single dose. Preferably, the total volume of a single dose of the eye-drop vaccine is between about 5 μl and 70 μl, more preferably between about 20 μl and 50 μl, preferably 20 μl, 25 μl, 30 μl, 35 μl, 40 μl, 45 μl or 50 μl per single dose.

[0162] For day-old poultry, the spray vaccine may contain a dose volume of 25 to 1000 μl. Preferably, the total volume of a single dose of the spray vaccine is between about 50 μl and 5000 μl, more preferably between about 75 μl and 2000 μl, more preferably between about 100 μl and 1000 μl, even more preferably between about 200 μl and 900 μl, more preferably between about 300 μl and 800 μl, even more preferably between about 400 μl and 700 μl, preferably 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 per single dose. Most preferably, the total volume of a single dose is 400 μl, 450 μl, 500 μl, 550 μl, 600 μl, 650 μl or 700 μl.

[0163] Vaccines for in ovo inoculation can contain a dose volume of 50 to 100 μl, preferably 50 μl. Preferably, the total volume of a single dose of in ovo vaccine is from about 10 μl to 250 μl, more preferably from about 15 μl to 200 μl, even more preferably from about 20 μl to 150 μl, even more preferably from about 30 μl to 100 μl, even more preferably between about 30 μl and 75 μl, and preferably a single dose of 30 μl, 35 μl, 40 μl, 45 μl, 50 μl, 55 μl, 60 μl, 65 μl, 70 μl or 75 μl. Most preferably, the total volume of a single dose is 40 μl, 45 μl, 50 μl, 55 μl or 60 μl.

[0164] Vaccines for intramuscular or subcutaneous inoculation or a single dose of drinking water vaccine can contain a dose having a volume of 30 μl to 1000 μl. Preferably, the total volume of a single dose is between about 30 μl and 1000 μl, more preferably between about 50 μl and 500 μl, more preferably between about 75 μl and 250 μl, even more preferably between about 100 μl and 200 μl, and most preferably a single dose of 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.

[0165] In one aspect of the invention, the immunogenic composition is administered in two or more doses.

[0166] However, the immunogenic composition can be administered in two or more doses, with a first dose being administered before a second dose (booster).

[0167] In a preferred aspect of the two-dose regimen, the first and second doses of the immunogenic composition are administered in the same amount. Preferably, each dose is the preferred amount as specified above. In addition to the first and second dose regimens, alternative embodiments include additional subsequent doses. For example, a third, fourth or fifth dose can be administered in these aspects. Preferably, the amounts administered in the subsequent third, fourth and fifth dose regimens are the same as the first dose, and the time intervals between the doses are consistent with the time between the first and second doses as described above.

[0168] Preferably, the first administration of the vaccine is carried out within three weeks of age, more preferably within one week of age, and most preferably on the first day of age by the following method. The second administration can be carried out within 20 weeks of age, preferably within 16 - 18 weeks of age, and more preferably within 6 - 12 weeks of age. Exemplarily, the initial (first) vaccination is carried out at 1 - 10 days of age, and the second vaccination (boost) is carried out at 6 - 12 or 16 - 18 weeks of age using a live vaccine or an inactivated vaccine. More preferably, the initial (first) vaccination is carried out on the first day of age, and the second vaccination (boost) is carried out at 6 - 12 weeks of age or 16 - 18 weeks of age using a live vaccine or an inactivated vaccine.

[0169] In the case of using an in - ovo vaccine, it is preferred that the first administration is carried out when the embryo is between 15 and 19 days old, preferably on the 17th, 18th or 19th day, and most preferably on the 18th day. The second administration can be carried out within the first three weeks after birth, preferably within the first 10 days after birth.

[0170] In one aspect of the present invention, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, via spraying, via drinking water or via eye drops.

[0171] The immunogenic composition is preferably administered locally or systemically. Suitable routes of administration for routine use are oral or parenteral administration, such as intranasal, intravenous, intradermal, transdermal, intramuscular, intraperitoneal, subcutaneous, and inhalation, in ovo, by spraying, by drinking water or by eye drops. However, depending on the nature and mode of action of the compound, the immunogenic composition can also be administered by other routes. For example, such other routes include transdermal, intravenous, intravascular, intra - arterial, intranasal, intrathoracic, intratracheal, intracardiac, intraperitoneal, intraperitoneal, intramedullary, extraperitoneal, rectal and vaginal. However, most preferably, the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spraying, by drinking water or by eye drops.

[0172] The live IBV vaccine is preferably administered alone by eye drops, intranasally, intramuscularly or subcutaneously.

[0173] More preferably, a mass administration method is used, including vaccination by drinking water and aerosol. Also preferred is the use of the vaccine as an embryo vaccine (so - called in - ovo vaccine), as further described below.

[0174] For example, broiler chickens can be vaccinated at one day of age or 1 - 3 weeks of age, especially for broiler chickens with a high MDA level. Laying hens or breeding hens can initially be vaccinated at 1 - 10 days of age and boosted with the vaccine at 7 - 12 or 16 - 18 weeks of age.

[0175] In - ovo administration

[0176] As described above, the present invention also provides an IBV vaccine that can be safely administered via the in-ovo route while being able to induce a protective immune response. In-ovo administration is well known to those skilled in the art and can be carried out by those skilled in the art without much effort. In-ovo administration of the vaccine involves administering the vaccine to an avian embryo contained within an egg (for a review of in-ovo vaccination, see: Ricks et al., Advances in Veterinary Medicine. 495 - 515, 1999). The vaccine can be injected into any suitable compartment of the egg (such as the allantoic fluid, yolk sac, amnion, air cell or into the embryo), as described in the art (Sharma; Am. J. Vet. Res. 45 1619 - 1623, 1984). Preferably, the vaccine is administered beneath the shell (air cell) membrane and the chorioallantoic membrane.

[0177] Preferably, the vaccine is injected into the embryonated egg at a late stage of embryonic development, typically in the last quarter of the incubation period, preferably 3 - 4 days before hatching. It is preferably carried out when the embryo is 15 to 19 days old, preferably on the 17th, 18th or 19th day, most preferably on the 18th day. Subsequently, the inoculated embryonated eggs are transferred to an incubator for hatching. The process of in-ovo administration can be automated using robotic injection techniques described in the prior art.

[0178] Conventional vaccines commonly used for post-hatch vaccination of poultry cannot be used for in-ovo vaccination because late embryos are highly sensitive to most of the vaccine viruses tested. However, International Patent Application WO 01 / 64244 discloses that an IBV vaccine can be used for in-ovo administration but must be applied at a very low dose. In addition, Wakenell et al., 1986 (Am. J. Vet. Res., 47 933 - 938) disclosed that passage of an IB vaccine virus in tissue culture renders the virus non-pathogenic to embryos.

[0179] In one aspect of the present invention, the immunogenic composition is administered by eye drops.

[0180] Typically, live vaccines used for post-hatch administration include attenuated IBV at a concentration of 10 1 to 10 8 EID 50 (50% egg infective dose), preferably 10 2 to 10 5 EID 50 , more preferably 10 2 to 10 4 EID 50 , even more preferably 10 2 to 10 3 EID 50 .

[0181] Live vaccines for in ovo administration generally contain from 50 to 100 μl, preferably 50 μl, of 10 2 -10 7 EID 50 / embryo, preferably 10 2 -10 3 EID 50 / embryo of attenuated IBV.

[0182] Preferably, the immunogenic composition of the present invention comprises the IBV of the present invention in an amount of from about 1 to about 10 log 10 EID (egg infective dose) 50 , preferably from about 2 to about 8 log 10 EID 50 , preferably from about 2 to about 7 log 10 EID 50 , more preferably from about 2 to about 6 log 10 EID 50 of the amount, even more preferably from about 2 to about 5 log 10 EID 50 of the amount, even more preferably from about 2 to about 4 log 10 EID 50 of the amount, most preferably from about 2 to about 3 log 10 EID 50 of the amount. More preferably, the immunogenic composition of the present invention comprises the IBV of the present invention in an amount of 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 .

[0183] In one aspect of the present invention, the immunogenic composition comprises from 1 to 10 log 10 EID50 of IBV per dose.

[0184] In one aspect of the present invention, the immunogenic composition comprises from 2 to 5 log 10 EID50 of IBV per dose.

[0185] In one aspect of the present invention, the immunogenic composition comprises from 2 - 4 log 10 EID50 of IBV per dose.

[0186] In one aspect of the present invention, the immunogenic composition is administered within one week, within three days, within two days or within one day of age of the subject.

[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 inoculating the subject, it does take a few days for the immune system of the poultry to build immunity against IBV infection. Therefore, it is preferably to perform the immunization within the first 24 hours after the subject is born.

[0189] In one aspect of the invention, the immunogenic composition is administered within the first day after the subject is born. As shown in the examples, the immunization compositions provided herein have been proven to be safe and effective when administered to 1-day-old poultry.

[0190] In one aspect of the invention, the method results in an improvement in efficacy parameters selected from the following group compared to subjects in an untreated control group of the same species: prevention or reduction of ciliary stasis, prevention or reduction of rales, prevention or reduction of egg production decline, prevention or reduction of kidney lesions, prevention or reduction of watery diarrhea, reduction of weight loss, reduction of viral load, reduction of virus shedding or a combination thereof.

[0191] The terms "treatment and / or prevention" have been defined elsewhere, where the terms "prevention" and "prevention" or "avoidance" are used interchangeably in this application. In addition, the term "shedding" has also been defined elsewhere.

[0192] The terms "reduce", "decrease", "alleviate" or "become lower" mean that the efficacy parameters (ciliary stasis, rales, egg production decline, kidney lesions, watery diarrhea, weight loss, viral load, virus 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 in a non-immunized control group of the same species. How to measure the improvement of efficacy parameters is common knowledge to those skilled in the art.

[0193] The term "viral load" is well known to those skilled in the art. The term viral load may be used interchangeably with viral titer herein. 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 determination can be based on the detection of viral proteins, such as by binding of an antibody to a viral protein and further detection, or, by amplification methods such as RT-PCR to detect viral RNA. Monitoring of virus-associated viral RNA in plasma by nucleic acid amplification methods is a widely used parameter for assessing the status and progression of retroviral diseases and for evaluating the effectiveness of preventive and therapeutic interventions. For example, viral load or viral titer can be calculated by estimating the number of viable viruses in the body fluid involved, such as the number of RNA copies per milliliter of plasma.

[0194] The term "ciliary stasis" is well known to those skilled in the art. The surface of the trachea is covered with special epithelial cells lined with numerous, active, hair-like structures called cilia. The term "ciliary stasis" includes a decrease or loss of cilia and / or a loss or partial loss of ciliary activity. Those skilled in the art can determine ciliary stasis without undue experimentation.

[0195] The term "rales" is well known to those skilled in the art. However, the term "rales" includes tracheal rales and refers to the sounds emanating from the bronchi. Those skilled in the art can determine rales without undue experimentation.

[0196] The term "egg drop" is well known to those skilled in the art. The term "egg drop" includes a decrease in egg production.

[0197] In one aspect of the invention, treatment or prophylaxis results in a prevention or reduction of ciliary stasis compared to an untreated control subject of the same species.

[0198] In one aspect of the invention, treatment or prophylaxis results in a prevention or reduction of renal lesions compared to an untreated control subject of the same species.

[0199] In one aspect of the invention, treatment or prophylaxis results in a prevention or reduction of egg drop compared to an untreated control subject of the same species.

[0200] The invention further provides an IBV or immunogenic composition as described herein for therapeutic use.

[0201] The invention further provides an IBV or immunogenic composition as described herein for use as an immunogen or vaccine.

[0202] The invention also provides an IBV or immunogenic composition as described herein for use as a medicament.

[0203] The present invention further provides the use of the IBV or immunogenic composition described herein in the preparation of a medicament.

[0204] The present invention further provides the use of the IBV or immunogenic composition described herein in the treatment and / or prevention of IBV infection in a subject.

[0205] The present invention further provides an immunogenic composition comprising an H52 infectious bronchitis virus (IBV) encoding a heterologous spike (S) protein or a fragment thereof, wherein the H52 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having an amino acid sequence as shown in SEQ ID NO.2, SEQ ID NO:3, SEQ ID NO:4, AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82) or AF286185 (SEQ ID NO:83), or a sequence having at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith, and wherein the heterologous S protein or fragment thereof is selected from the genotypes or serotypes of 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil, or an amino acid sequence as shown in SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0206] In another specific aspect of the immunogenic composition according to the present invention, the heterologous S protein is a full-length spike protein.

[0207] In another specific aspect of the immunogenic composition according to the present invention, the fragment of the heterologous spike (S) protein has a length of at least 500, 750, 1000 or 1077 amino acids starting from the N-terminus.

[0208] In another specific aspect of the immunogenic composition according to the present invention, the fragment of the heterologous spike (S) protein is the extracellular domain of the spike protein.

[0209] In another specific aspect of the immunogenic composition according to the present invention, the IBV is attenuated.

[0210] The present invention further provides a method for preparing an immunogenic composition for treating and / or preventing IBV infection in a subject, the method comprising:

[0211] a.) providing a H52 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein, or a membrane glycoprotein (M), having an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AF352315 (SEQ ID NO79), AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82), or AF286185 (SEQ ID NO:83), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity therewith; and

[0212] b.) providing a heterologous S protein or a fragment thereof, selected from the list of genotypes or serotypes of 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil, or an amino acid sequence as shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity therewith; and

[0213] c.) replacing the spike protein or a fragment thereof of the H52 IBV of a) with the heterologous S (spike) protein or a fragment thereof of b) to obtain a H52 IBV having the heterologous S protein or a fragment thereof; and

[0214] d.) obtaining the H52 IBV having the heterologous S protein or a fragment thereof; and

[0215] e.) adding a pharmaceutically acceptable carrier.

[0216] The term "obtaining" includes harvesting, isolating, purifying, and / or formulating (e.g., completing, inactivating, and / or mixing) the IBV H52 having the heterologous S protein or a fragment thereof.

[0217] The term "harvesting" refers to the collection or recovery of said IBV H52 having a heterologous S protein or a fragment thereof from transfected or infected cells or cell lines. Any conventional method known in the art can be used, such as any isolation method. Methods well known in the art include centrifugation or filtration, such as using a semi-permeable membrane with a certain pore size.

[0218] The term "isolation" includes the step of isolating said IBV H52 having a heterologous S protein or a fragment thereof. Methods of isolating from transfected or infected cells or cell lines are known to those skilled in the art. These methods include physical and / or chemical methods, including but not limited to freeze-thaw cycles, sonication, etc.

[0219] Methods for "purifying" said IBV H52 having a heterologous S protein or a fragment thereof from an isolate are known to those skilled in the art, such as in "Protein Purification Methods - A Practical Approach" (E.L.V. Harris and S. Angel, eds., IRL Press at Oxford University Press). These methods include but are not limited to separation by centrifugation and / or filtration, precipitation, size exclusion (gel filtration) chromatography, affinity chromatography, metal chelate chromatography, ion exchange chromatography, covalent chromatography, hydrophobic interaction chromatography, etc. The carrier can be obtained in a pure form, or free or substantially free of other cell materials or culture media, etc. After said isolation and / or purification, the antigen exhibits a purity of at least 80%, preferably 80%-90%, more preferably 90%-97%, most preferably more than 97%, up to an absolutely pure form without any contamination.

[0220] According to another aspect, "obtaining" as used herein can also include further finishing steps as part of the final formulation process, such as adding buffers, inactivation, neutralization steps, etc.

[0221] In another specific aspect of the method for preparing an immunogenic composition according to the present invention, the fragment of the heterologous S (spike) protein is the extracellular domain of the spike protein.

[0222] In another specific aspect of the immunogenic composition according to the present invention, the pharmaceutically acceptable carrier is selected from solvents, dispersion media, coatings, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption retardants, adjuvants, immunostimulants, and combinations thereof.

[0223] In another specific aspect of the method for preparing an immunogenic composition according to the present invention, the heterologous S protein is the full-length spike protein.

[0224] The present invention further relates to a plasmid comprising a nucleic acid encoding a portion of the H52 IBV (Infectious Bronchitis Virus) genome, said genome comprising a heterologous IBV S (spike) protein or a fragment thereof, said plasmid being, for example, the pUC57-s H52 rIBVCR88 S Ecto donor plasmid (SEQ ID NO:21).

[0225] Clause

[0226] The following clauses are also described herein:

[0227] 1. H52 IBV (Infectious Bronchitis Virus) encoding a heterologous IBV S (spike) protein or a fragment thereof.

[0228] 2. An immunogenic composition comprising H52 IBV (Infectious Bronchitis Virus) encoding a heterologous S (spike) protein or a fragment thereof.

[0229] 3. An immunogenic composition comprising the IBV (Infectious Bronchitis Virus) of clause 1.

[0230] IBV H52 – defined by protein coding sequence

[0231] 4. The IBV or immunogenic composition of any one of clauses 1 to 3, wherein the H52 IBV has or consists of or comprises a nucleotide sequence as shown in EU817497 (SEQ ID NO 78) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0232] 5. The IBV or immunogenic composition of any one of clauses 1 to 4, wherein the H52 IBV has or consists of or comprises a spike (S1) protein having an amino acid sequence as shown in AF352315 (SEQ ID NO 79) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0233] 6. An immunogenic composition of IBV or any one of Articles 1 to 5, wherein the H52 IBV strain has or consists of or contains a spike (S) protein, and the spike protein has an amino acid sequence as shown in SEQ ID NO 1 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0234] 7. An IBV or immunogenic composition according to any one of Articles 1 to 6, wherein the H52 IBV has or consists of or contains a nucleocapsid (N) protein, and the nucleocapsid (N) protein has an amino acid sequence shown in AY044185 (SEQ ID NO 80) or AF352310 (SEQ ID NO 81) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0235] 8. An IBV or immunogenic composition according to any one of Articles 1 to 7, wherein the H52 IBV has or consists of or contains a nucleocapsid (N) protein, and the nucleocapsid (N) protein has an amino acid sequence as shown in SEQ ID NO: 2 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0236] 9. An IBV or immunogenic composition according to any one of Articles 1 to 8, wherein the H52 IBV has or consists of or contains an envelope (E) protein, and the envelope (E) protein has an amino acid sequence shown in AF317210 (SEQ ID NO 82) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity therewith.

[0237] 10. An IBV or immunogenic composition according to any one of clauses 1 to 9, wherein the H52 IBV has or consists of or comprises an envelope (E) protein having the amino acid sequence shown in SEQ ID NO: 3 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0238] 11. An IBV or immunogenic composition according to any one of clauses 1 to 10, wherein the H52 IBV has or consists of or comprises a membrane glycoprotein (M) having the amino acid sequence shown in AF286185 (SEQ ID NO 83) or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0239] 12. An IBV or immunogenic composition according to any one of clauses 1 - 11, wherein the H52 IBV has or consists of or comprises a membrane glycoprotein (M) protein having the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0240] Heterologous S protein

[0241] 13. An IBV or immunogenic composition according to any one of clauses 1 - 12, wherein the heterologous spike is of a non-Massachusetts genotype or serotype.

[0242] 14. An IBV or immunogenic composition of any one of clauses 1-13, wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from: Arkansas (e.g., Arkansas 99), Brazil (e.g., BR-1, BR-2, 23 / 2013, IBV / Brasil / 351 / 1984), California (e.g., California1734 / 04, California99), Connecticut, Delaware (e.g., Delaware 98), Dutch (e.g., D207, D212, D274, D3128, D3896, D8880, D1466), Florida, Georgia (e.g., Georgia GA-07, GA-08, GA-12, GA-13), Gray, Holte, Iowa (e.g., Iowa 97 and Iowa 69), Italy (e.g., Italy 02), JMK, LDT3, Maine (e.g., Maine 209), Pennsylvania (e.g., Pennsylvania 1220 / 98, Pennsylvania Wolg / 98), PL84084, Qu (e.g., Qu-mv), QX (e.g., GB341 / 96), Q1, SE 17, variant 2 (e.g., IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016) and 4 / 91 (793B, CR88).

[0243] 15. An IBV or immunogenic composition of any one of clauses 1-14, wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from: 4 / 91, QX, Q1, Italy 02, Arkansas, Conneticut, Georgia, LDT3, PL84084, variant 2 or Brazil.

[0244] 16. An IBV or immunogenic composition of any one of clauses 1-15, wherein the heterologous S protein or fragment thereof is from an IBV having a genotype or serotype selected from: 4 / 91, QX, Q1, Arkansas, variant 2 and Brazil.

[0245] 17. The IBV or immunogenic composition of clause 16, wherein the 4 / 91 strain is selected from: Spain / 98 / 328, Spain / 92 / 35, IR-3654-VM, FR-CR88061-88, FR-85131-85, UK-1233-95, UK / 3 / 91, Spain / 00 / 336, UK / 7 / 91, 4 / 91-pathogenicity, 4 / 91-attenuated, IB4-91 and CR88.

[0246] 18. The IBV or immunogenic composition of clause 16, wherein the QX strain is selected from: FR-L1450T-05, FR-L1450L-05, NL-L1449T-04, NL-L1449K-04, IBV / Ck / SP / 170 / 09, IBV / Ck / SP / 79 / 08, IBV / Ck / SP / 248 / 09, HBN, IBVQX, LX4, BJQ, CK / CH / LGD / 03 and GB341 / 96.

[0247] 19. The IBV or immunogenic composition of clause 16, wherein the Q1 strain is selected from: CK / CH / LDL / 98I, CK / CH / LSD / 08-10, J2, Q1, AR08ER22, AR08BA21 and Chile-295-10.

[0248] 20. The IBV or immunogenic composition of clause 16, wherein the Arkansas strain is selected from: Ark99, ArkGA, ArkDPI, AL / 5364 / 00, ARKDPI11, AL / 0803 / 01, AL / 7149 / 00, ArkDPI101, AL / 1221 / 01, AL / 1793 / 01 and AL / 4614 / 98.

[0249] 21. The IBV or immunogenic composition of clause 16, wherein the variant 2 strain is selected from: IS / 1494 / 06, IBV / Ck / EG / CU / 4 / 2014, gammaCoV / Ck / Poland / G052 / 2016, Eg / CLEVB-2 / IBV / 012, D1344 / 2 / 4 / 10_EG, TR8 and IB VAR2-06.

[0250] 22. The IBV or immunogenic composition of clause 16, wherein the Brazil strain is selected from BR-1, BR-2, 23 / 2013 and IBV / Brasil / 351 / 1984.

[0251] 23. The IBV or immunogenic composition of any one of clauses 1-22, wherein the heterologous S protein or its fragment is from the 4 / 91 genotype or serotype.

[0252] 24. An IBV or immunogenic composition according to any one of clauses 1 - 23, wherein the heterologous S protein or fragment thereof is from a 4 / 91 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:5 or 6, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO:5 or 6 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0253] 25. An IBV or immunogenic composition according to any one of clauses 1 - 24, wherein the heterologous S protein or fragment thereof is from a QX genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:7 or 8, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO:7 or 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0254] 26. An IBV or immunogenic composition of any one of clauses 1-25, wherein the heterologous S protein or fragment thereof is from a Q1 genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:9 or 10, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO:9 or 10 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0255] 27. An IBV or immunogenic composition of any one of clauses 1-26, wherein the heterologous S protein or fragment thereof is from an Arkansas genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO:11 or 12, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO:11 or 12 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0256] 28. An IBV or immunogenic composition according to any one of clauses 1-27, wherein the heterologous S protein or fragment thereof is from a variant genotype 2 or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 13 or 14, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO: 13 or 14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0257] 29. An IBV or immunogenic composition according to any one of clauses 1-28, wherein the heterologous S protein or fragment thereof is from a Brazil genotype or serotype having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity to SEQ ID NO: 15 or 16, or the heterologous S protein or fragment thereof comprises or consists of the amino acid sequence shown in SEQ ID NO: 15 or 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0258] 30. An IBV or immunogenic composition according to any one of clauses 1-29, wherein the heterologous S protein or fragment thereof is selected from SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0259] 31. An IBV or immunogenic composition according to any one of clauses 1-30, wherein the heterologous S protein is a full-length spike protein.

[0260] 32. An IBV or immunogenic composition according to any one of clauses 1-31, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length.

[0261] 33. An IBV or immunogenic composition according to any one of clauses 1-32, wherein the fragment of the heterologous S (spike) protein is at least 500, 750, 1000 or 1077 amino acids in length starting from the N-terminus.

[0262] 34. An IBV or immunogenic composition according to any one of clauses 1-33, wherein the fragment of the heterologous S (spike) protein is at least 1000 amino acids in length.

[0263] 35. An IBV or immunogenic composition according to any one of clauses 1-34, wherein the heterologous S (spike) protein is the extracellular domain of the spike protein.

[0264] 36. An IBV or immunogenic composition according to any one of clauses 1-35, wherein the heterologous S (spike) protein or a fragment thereof replaces the homologous S protein or a fragment thereof.

[0265] 37. An IBV or immunogenic composition according to any one of clauses 1-36, wherein the heterologous S (spike) protein or a fragment thereof replaces the naturally occurring S protein or a fragment thereof.

[0266] 38. An IBV or immunogenic composition according to any one of clauses 1-37, wherein the heterologous S (spike) protein or a fragment thereof replaces the S protein or a fragment thereof of H52.

[0267] 39. An IBV or immunogenic composition according to any one of clauses 1-38, wherein the IBV is attenuated.

[0268] 40. An IBV or immunogenic composition according to any one of clauses 1-39, wherein the IBV is inactivated.

[0269] 41. An IBV or immunogenic composition according to any one of clauses 1-40, wherein the IBV is genetically engineered.

[0270] 42. An IBV or immunogenic composition according to any one of clauses 1-41, wherein the IBV is a recombinant IBV.

[0271] 43. An immunogenic composition according to any one of clauses 2-42, wherein the immunogenic composition is a vaccine.

[0272] 44. An immunogenic composition according to any one of clauses 2-43, wherein the immunogenic composition comprises a pharmaceutically acceptable carrier.

[0273] 45. The immunogenic composition of clause 44, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.

[0274] 46. An immunogenic composition according to any one of clauses 2-45, wherein the immunogenic composition is effective in treating and / or preventing clinical signs caused by IBV in a subject in need thereof.

[0275] 47. An immunogenic composition according to any one of clauses 2-46, wherein the immunogenic composition protects against challenge with an IBV strain of a heterologous spike protein serotype.

[0276] 48. An immunogenic composition according to any one of clauses 2-47, wherein the immunogenic composition protects against challenge with a 4 / 91, QX, Q1, Arkansas, variant 2, or Brazil genotype strain.

[0277] 49. An immunogenic composition according to any one of clauses 2-48, wherein the immunogenic composition protects against challenge with a 4 / 91 genotype strain.

[0278] 50. An immunogenic composition according to any one of clauses 2-49, wherein the immunogenic composition is formulated for administration in a single dose form.

[0279] 51. An immunogenic composition according to any one of clauses 2-50, wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, in ovo, by spray, by drinking water, or by eye drops.

[0280] 52. An immunogenic composition according to any one of clauses 2-51, wherein the immunogenic composition comprises 1-10 log 10 EID 50 / dose of IBV.

[0281] 53. An immunogenic composition according to any one of clauses 2-52, wherein the immunogenic composition comprises 2-5 log 10 EID 50 / dose of IBV.

[0282] 54. An immunogenic composition according to any one of clauses 2-53, wherein the immunogenic composition comprises 2-4 log 10 EID 50 / dose of IBV.

[0283] 55. A kit comprising an IBV or an immunogenic composition according to any one of clauses 1-54.

[0284] 56. The kit according to clause 55, wherein the kit further comprises instructions for treating and / or preventing avian diseases.

[0285] 57. The kit according to clause 55, wherein the kit further includes instructions for treating and / or preventing poultry diseases.

[0286] 58. The kit according to clause 55, wherein the kit further comprises instructions for treating and / or preventing IB.

[0287] 59. A method of immunizing a subject, comprising administering to the subject an immunogenic composition according to any one of clauses 2 - 54.

[0288] 60. A method of treating or preventing clinical signs caused by IBV in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of clauses 2 - 54.

[0289] 61. A method of reducing ciliary stasis in a subject in need thereof compared to an unimmunized control group of the same species, the method comprising administering to the subject a therapeutically effective amount of an immunogenic composition according to any one of clauses 2 - 54.

[0290] 62. An immunogenic composition according to any one of clauses 2 - 54, for use in a method of immunizing a subject, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0291] 63. An immunogenic composition according to any one of clauses 2 - 54, for use in a method of 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.

[0292] 64. An immunogenic composition according to any one of clauses 2 - 54, for use in a method of reducing ciliary stasis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the immunogenic composition.

[0293] 65. The method or use according to any one of clauses 59 - 64, wherein the subject is a bird.

[0294] 66. The method or use according to any one of clauses 59 - 65, wherein the subject is a poultry.

[0295] 67. The method or use according to any one of clauses 59 - 66, wherein the subject is selected from a chicken, a turkey, a quail or a pheasant.

[0296] 68. The method or use according to any one of clauses 59 - 67, wherein the subject is a chicken.

[0297] 69. The method or use according to any one of clauses 59 - 68, wherein the immunogenic composition is administered once.

[0298] 70. The method or use according to any one of clauses 59 - 69, wherein the immunogenic composition is administered in two or more doses.

[0299] 71. A method or use according to any one of clauses 59 - 70, wherein the immunogenic composition is administered subcutaneously, intramuscularly, orally, by in ovo injection, by spraying, via drinking water or by eye drops.

[0300] 72. A method or use according to any one of clauses 59 - 71, wherein the immunogenic composition is administered by eye drops.

[0301] 73. A method or use according to any one of clauses 59 - 72, wherein the immunogenic composition comprises 1 - 10 log 10 EID 50 / dose of IBV.

[0302] 74. A method or use according to any one of clauses 59 - 73, wherein the immunogenic composition comprises 2 - 5 log 10 EID 50 / dose of IBV.

[0303] 75. A method or use according to any one of clauses 59 - 74, wherein the immunogenic composition comprises 2 - 4 log 10 EID 50 / dose of IBV.

[0304] 76. A method or use according to any one of clauses 59 - 75, wherein the immunogenic composition is administered to the subject within the first week, within the first three days, within the first two days or on the first day of the subject's age.

[0305] 77. A method or use according to any one of clauses 59 - 76, wherein the immunogenic composition is administered to the subject on the first day of the subject's age.

[0306] 78. A method or use according to any one of clauses 59 - 77, wherein the method results in an improvement in efficacy parameters selected from the following compared to untreated control subjects of the same species: prevention or reduction of ciliary stasis, prevention or reduction of rales, prevention or reduction of egg production decline, prevention or reduction of kidney lesions, prevention or reduction of watery diarrhea, prevention or alleviation of weight loss, reduction of viral load, reduction of virus shedding or a combination thereof.

[0307] 79. A method or use according to any one of clauses 59 - 78, wherein the treatment or prevention results in the prevention or reduction of ciliary stasis compared to untreated control subjects of the same species.

[0308] 80. A method or use according to any one of clauses 59 - 79, wherein the treatment or prevention results in the prevention or reduction of kidney lesions compared to untreated control subjects of the same species.

[0309] A method or use according to any one of clauses 59 - 80, wherein said treatment or prevention results in a prevention or reduction in egg drop compared to an untreated control subject of the same species.

[0310] An IBV or immunogenic composition according to any one of clauses 1 - 54, for use in therapy.

[0311] An IBV or immunogenic composition according to any one of clauses 1 - 54, used as an immunogen or a vaccine.

[0312] An IBV or immunogenic composition according to any one of clauses 1 - 54, used as a medicament.

[0313] Use of an IBV or immunogenic composition according to any one of clauses 1 - 54 in the manufacture of a medicament.

[0314] Use of an IBV or immunogenic composition according to any one of clauses 1 - 54 in the treatment and / or prevention of IBV infection in a subject.

[0315] 87. An immunogenic composition comprising an H52 infectious bronchitis virus (IBV) encoding a heterologous spike (S) protein or a fragment thereof, wherein the H52 IBV comprises a nucleocapsid (N) protein, an envelope (E) protein or a membrane glycoprotein (M) having or comprising or consisting of the following sequences: the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82) or AF286185 (SEQ ID NO:83) or sequences having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, and wherein the heterologous S protein or a fragment thereof is selected from the following genotypes or serotypes: 4, 91, QX, Q1, Arkansas, variant 2 and Brazil, or selected from the following amino acid sequences: SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto, or the heterologous S protein or a fragment thereof comprises or consists of the following amino acid sequences: SEQ ID NO:5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16, or sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98% or 99.99% sequence identity thereto.

[0316] 88. The immunogenic composition of clause 87, wherein the heterologous S protein is a full-length spike protein.

[0317] 89. The immunogenic composition of clause 87, wherein the fragment of the heterologous spike (S) protein is at least 500, 750, 1000 or 1077 amino acids in length starting from the N-terminus.

[0318] 90. The immunogenic composition of clause 87 or 89, wherein the fragment of the heterologous spike (S) protein is the extracellular domain of the spike protein.

[0319] 91. An immunogenic composition according to any one of clauses 92 - 95, wherein the IBV is attenuated.

[0320] 92. A method for preparing an immunogenic composition for treating and / or preventing IBV infection in a subject, comprising:

[0321] a.) providing an H52 IBV comprising a spike (S) protein, a nucleocapsid (N) protein, an envelope (E) protein, or a membrane glycoprotein (M), having or comprising or consisting of the following amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, AF352315 (SEQ ID NO 79), AY044185 (SEQ ID NO 80), AF352310 (SEQ ID NO 81), AF317210 (SEQ ID NO 82), or AF286185 (SEQ ID NO: 83), or a sequence having at least 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto; and

[0322] b.) providing a heterologous S protein or a fragment thereof selected from the genotypes or serotypes of 4 / 91, QX, Q1, Arkansas, variant 2, and Brazil, or selected from the amino acid sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto, or providing a heterologous S protein or a fragment thereof comprising or consisting of the amino acid sequences shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity thereto; and

[0323] c.) replacing the spike protein of the H52 IBV of a) with the heterologous S (spike) protein or a fragment thereof of b) such that the H52 IBV has the heterologous S protein or a fragment thereof; and

[0324] d.) Obtaining said H52 IBV with a heterologous S protein or a fragment thereof; and

[0325] e.) Adding a pharmaceutically acceptable carrier.

[0326] 93. The method of clause 92, wherein the fragment of said heterologous S (spike) protein is the extracellular domain of said spike protein.

[0327] 94. The method of clause 92 or 93, wherein the pharmaceutically acceptable carrier is selected from solvents, dispersion media, coating agents, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immunostimulants, and combinations thereof.

[0328] 95. The method of any one of clauses 92 or 94, wherein the heterologous S protein is the full-length spike protein. Brief Description of the Drawings

[0330] Figure 1 Intra-ovum replication kinetics of H52 rIBV CR88 S ecto compared to recombinant wild-type viruses CR88 and H52. Data points represent the mean of 5 samples at each time point. Error bars represent the standard deviation.

[0331] Figure 2 Ciliary stasis score overview Calculation of the sum of 10 individual scores for 10 loops of one animal, represented by a dot in the figure. The maximum ciliary stasis corresponds to a score of 40, while no ciliary stasis corresponds to a score of 0. Means and significance (p < 0.0007) were calculated using GraphPad Prism and a conventional one-way ANOVA test.

[0332] Figure 3 Overview of kidney tissue RT-qPCR results. Each avian animal is represented by a data point.

[0333] Figure 4 Overview of RT-PCR results of nasal swab eluates. Each individual bird is represented as a data point.

[0334] Figure 5 Ciliary stasis score overview Calculation of the sum of 10 individual scores for 10 loops of one animal, represented by a dot in the figure. The maximum ciliary stasis corresponds to a score of 40, while no ciliary stasis corresponds to a score of 0. Means and significance (p < 0.0001) were calculated using GraphPad Prism and a conventional one-way ANOVA test.

[0335] Sequence Overview

[0336] SEQ ID NO:1: H52 IBV spike (S) protein.

[0337] SEQ ID NO:2: H52 IBV nucleocapsid (N) protein.

[0338] SEQ ID NO:3: H52 IBV envelope (E) protein.

[0339] SEQ ID NO:4: H52 IBV membrane glycoprotein (M).

[0340] SEQ ID NO:5 and 6: Heterologous S protein or fragment thereof from genotype or serotype 4 / 91.

[0341] SEQ ID NO:7 and 8: Heterologous S protein or fragment thereof from genotype or serotype QX.

[0342] SEQ ID NO:9 and 10: Heterologous S protein or fragment thereof from genotype or serotype Q1.

[0343] SEQ ID NO:11 and 12: Heterologous S protein or fragment thereof from genotype or serotype Arkansas.

[0344] SEQ ID NO:13 and 14: Heterologous S protein or fragment thereof from genotype or serotype Variant 2.

[0345] SEQ ID NO:15 and 16: Heterologous S protein or fragment thereof from genotype or serotype Brazil.

[0346] SEQ ID NO:17: Nucleic acid coding sequence of IBV CR88 spike protein.

[0347] SEQ ID NO:18: Nucleic acid coding sequence of extracellular domain of IBV H52 spike protein.

[0348] SEQ ID NO:19: pUC57-s H52 rIBV donor plasmid.

[0349] SEQ ID NO:20: Nucleic acid coding sequence of extracellular domain of IBV CR88 spike protein.

[0350] SEQ ID NO:21: pUC57-s H52 rIBV CR88 S Ecto donor plasmid.

[0351] SEQ ID NO:22 to SEQ ID NO:77: Primers.

[0352] SEQ ID NO:78: EU817497 (H52 IBV nucleotide sequence)

[0353] SEQ ID NO:79: AF352315 (Amino acid sequence of H52 IBV S protein)

[0354] SEQ ID NO:80: AY044185 (Amino acid sequence of H52 IBV N protein)

[0355] SEQ ID NO:81: AF352310 (Amino acid sequence of H52 IBV N protein)

[0356] SEQ ID NO:82: AF317210 (Amino acid sequence of H52 IBV E protein)

[0357] SEQ ID NO:83: AF286185 (Amino acid sequence of H52 IBV M protein)

[0358] SEQ ID NO:84 and 85: Primers Example

[0359] The examples set forth below illustrate specific embodiments of the invention. These examples are for illustration only and should not be construed as limiting the scope or basic principles of the invention.

[0360] Example 1: Generation of recombinant IBV H52 in which the coding sequence of the H52 spike protein or the extracellular domain of the spike protein is replaced by the coding sequence of a heterologous spike protein or the extracellular domain of the spike protein

[0361] Construction of the donor plasmid

[0362] A detailed example of replacing the extracellular domain of the H52 spike protein with the extracellular domain of CR88 is described: The nucleic acid coding sequence of the IBV CR88 spike protein (SEQ ID NO: 17) was synthesized by a commercial supplier. It was used as a template to replace the nucleic acid coding sequence of the extracellular domain of the IBV H52 spike protein (SEQ ID NO: 18) in the pUC57-s IBV-5-1b-S-SIR-3T donor plasmid described by van Beurden et al. (Virol J. 2017;14(1):109), hereinafter referred to as the pUC57-s H52 rIBV donor plasmid (SEQ ID NO: 19). Bases 1717 to 4941 of SEQ ID NO: 19 were replaced with the corresponding nucleic acid coding sequence of the extracellular domain of the IBV CR88 spike protein (SEQ ID NO: 20), which corresponds to bases 55 to 3285 of SEQ ID NO 15. This generated the pUC57-s H52 rIBV CR88 S Ecto donor plasmid (SEQ ID NO: 21), in which the extracellular domain of the IBV CR88 spike protein is encoded by bases 1717 to 4947. For this purpose, the pUC57-s H52 rIBV donor plasmid (SEQ ID NO: 19) was digested with unique restriction enzyme sites 5' (EcoRV) and 3' (PmlI) close to the H52 spike protein coding sequence to linearize the plasmid and remove the H52 spike protein and flanking sequences. The QIAquick Gel Extraction Kit (Qiagen) was used to purify the band corresponding to the pUC57-sIBV H52 backbone without the H52 spike protein coding sequence. Using a high-fidelity DNA polymerase (NEB; primers are shown in Table 1), the nucleic acid coding sequence of the extracellular domain of the CR88 spike protein and the flanking 5' and 3' IBV H52 sequences were amplified in three separate PCR reactions. The PCR products were purified by QIAquick Gel Extraction (Qiagen) and used for Gibson assembly with the HiBuild DNA Assembly Cloning Kit (NEB) according to the kit protocol to generate the pUC57-s H52 rIBV CR88S Ecto donor plasmid.

[0363] Table 1 Gibson assembly primers designed by NEB using the NEBuilder online tool for generating PCR products to assemble the pUC57-s H52 rIBV CR88 S Ecto donor plasmid.

[0364]

[0365] Targeted RNA recombination and rescue of recombinant IBV

[0366] For the generation of recombinant IBV, a method of targeted RNA recombination as described by van Beurden et al. (Virol J. 2017;14(1):109) was adopted. Briefly, H52 murine (m) IBV was generated as described. To generate H52 rIBV CR88 S Ecto, LR7 cells were infected with H52 mIBV and electroporated with in vitro transcripts generated from the pUC57-s H52 rIBV CR88 S Ecto donor plasmid, which was then injected into 8-day-old SPF eggs containing embryos (VALOBioMedia). After incubation at 37.5 °C and 60% humidity for 9 days, RNA was isolated from the allantoic fluid of all eggs by the QIAamp Viral RNA mini kit (Qiagen) and using the SuperScript TM III One-Step RT-PCR System with Platinum TM Taq DNA Polymerase (ThermoFisher), and the rescued recombinant IBV was analyzed separately for each egg. Primers PO1323 and PO1729 (Table 2), which bind to the 1ab of H52 IBV and the extracellular domain of CR88 IBV S, are specific for recombinant IBV but not for mIBV. The allantoic fluid of the eggs inoculated with the highest dilution of LR7 cells was selected for two rounds of end-point dilution in 8-day-old SPF eggs. Nucleic acid isolation of the limited dilution samples was performed using the MagMAXTM Core Nucleic Acid Purification Kit (ThermoFisher) and the KingFisher TM Duo Prime Purification System (ThermoFisher), and the presence of rIBV was then analyzed by the above RT-PCR. After the second limited dilution, the experimentally positive allantoic fluid of the eggs inoculated at the highest dilution was used for propagation in 10-day-old SPF eggs containing embryos. The allantoic fluid was diluted 1:1000 in 1x PBS, and 100 μl was injected into each egg. The allantoic fluid was harvested 48 hours after inoculation, debris was removed, and it was stored at -80 °C. To confirm the sequence from the donor plasmid in the generated rIBV, viral nucleic acid was isolated using the QIAamp Viral RNA mini kit, and then SuperScript III One-Step RT-PCR was performed using the primers listed in Table 3. QIAquick PCR purification and subsequent Sanger sequencing were performed using the same primers by a commercial vendor.

[0367] Table 2 Primers for the identification of recombinant IBV after rescue

[0368]

[0369] Table 3 Sequencing primers for the confirmation of the donor region sequence in H52 rIBV CR88 S Ecto

[0370]

[0371] Generation and characterization of H52 recombinant IBV in which the coding sequence of the H52 spike protein or the extracellular domain of the spike protein is replaced by the coding sequence of the spike protein or the extracellular domain of the spike protein of another IBV genotype

[0372] The same method as described for generating and characterizing H52 rIBV CR88 S Ecto was applied to generate and characterize recombinant H52 IBV, in which the spike protein coding sequence (bases 1663 to 5151 of SEQ ID NO: 19) or the extracellular domain coding sequence of the H52 spike protein (bases 1717 to 4941 of SEQ ID NO: 19) was replaced by the coding sequence of the spike protein or the extracellular domain of the spike protein of the IBV serotypes and genotypes listed in Table 4.

[0373] Table 4 Primers for Gibson assembly of H52 rIBV donor plasmids with heterologous spike proteins or extracellular domains of spike proteins

[0374]

[0375]

[0376] The primers in Tables 2 and 3 were used to identify and sequence the different recombinant viruses and were modified for the corresponding spike protein sequences as necessary.

[0377] Example 2: In ovo replication kinetics

[0378] Eight eight-day-old embryonated eggs were inoculated with 10 2 EID 50 of rIBV and respective controls. The eggs were incubated at 37.5 °C and 60% humidity and candled daily at 0, 8, 24, 34, 48, and 72 hours of incubation to record embryo mortality. Five preselected eggs were taken for each sample and time point and transferred to 4 °C for at least 2 hours. Subsequently, allantoic fluid was collected and stored at -80 °C. For analysis, samples were thawed and diluted 1:10 in 1xPBS without Ca and Mg, nucleic acids were extracted using the QIAamp DNA Blood Mini Kit (Qiagen), and carrier RNA was added using a Hamilton Starlet automated pipettor. The relative amount of IBV RNA in the extracted nucleic acids was analyzed by RT-qPCR, using a protocol adapted from Callison et al. (J Virol Methods. 2006;138(1-2):60-5). Briefly, the same primers and probes were used, and the temperature protocol was adjusted to be suitable for use Fast Virus 1-Step Master Mix (ThermoFisher) and ABI TM 7900HT Fast real-time PCR system (Thermo Fisher Scientific). Analyze all nucleic acid samples in triplicate, using 10-fold serial dilutions of IBV H52 as a reference.

[0379] Compared with the recombinant wild-type viruses H52 and CR88, slightly similar replication kinetics were observed for H52 rIBV CR88 S Ecto at early time points. However, after 32 hours, all viruses reached comparable ct values. All embryos survived 32 hours post-inoculation, while at the 48-hour time point post-infection, all remaining embryos in all samples were dead. Therefore, the replication of H52 rIBV CR88 Secto was considered equally efficient compared to the wild-type virus (see Figure 1 ).

[0380] Example 3: Preparation of vaccines and challenge viruses

[0381] To confirm the efficacy of H52 rIBV with heterologous spike proteins or spike protein ectodomains in chickens, aliquots of the virus stock were thawed, diluted 10-fold in 1xPBS, and the 50% embryo infectious dose (EID 50 ) was determined by inoculating 100 μl of each dilution into 5 eight-day-old embryonated eggs. The eggs were incubated at 37.5 °C and 60% humidity until 7 days post-inoculation. Eggs with dead embryos 24 hours after inoculation were excluded from the experiment. All other eggs with dead embryos 7 days post-inoculation were considered positive. On day 7 post-inoculation, all eggs with live embryos were candled from the bottom to identify stunted chicks that were considered positive. The EID 50 / ml was calculated using the Reed and Muench formula (Am JEpidemiol, 1938; 27(3):493–497). For immunization, the virus stock was diluted in 1xPBS to obtain a titer of 10 4.3 EID 50 / ml (10 3 EID 50 per chicken in 50 μl).

[0382] The challenge viruses of genotypes and serotypes 793B, QX, Q1, Ark, variant 2, and Brazil were propagated in 10-day-old embryonated SPF eggs. Twenty-four hours after inoculation, the eggs were transferred to 4 °C for at least 2 hours. The allantoic fluid was collected, aliquoted, and stored at -80 °C. The virus titer was determined as described above. The titer was set to 10 4.3from 0 to 10 5.3 EID 50 / ml (10 per chicken in 50 μl 3 from 0 to 10 4 EID 50 )

[0383] Example 4: Determination of Vaccine Efficacy

[0384] Fertilized SPF eggs were incubated in an egg incubator at 99.7°F and 50% humidity with rotation every hour for 18 days. On the 18th day of incubation, the eggs were candled, and the fertilized eggs were transferred to the incubator and incubated at 99°F and 70% humidity until hatching. Chicks without clinical signs or deformities were randomly assigned to the respective treatment groups and moved to separate isolators. At least two chicks were used as a strict negative control (SNC) group, five chicks were included in the challenge control (CC) group, and at least ten chicks in each group were inoculated with recombinant IBV having a heterologous spike protein or extracellular domain of the spike protein, followed by challenge. The animals were housed under husbandry conditions meeting local and national requirements for animal welfare recommendations. The lighting schedule was adjusted to 16 hours of light per day. Feed and water were provided ad libitum. After transfer to the isolator, chicks (1-day-old) were inoculated by eye drops (total volume 50 μl, 25 μl per eye) with 10 3 EID 50 / chick, while the SNC and CC groups remained untreated. On day 21 post-inoculation, the chicks in the CC group and the inoculated groups were challenged by eye drops (total volume 50 μl, 25 μl per eye) with 10 3 from 0 to 10 4 EID 50 / chick of their respective spike protein-homologous challenge strains (793B, QX, Q1, Ark, variant 2, or Brazil). On day 7 post-challenge, all chicks were euthanized, throat swabs were taken, the kidneys were removed, and stored in RNAlater stabilization solution (ThermoFisher) at 4°C for IBV-specific RT-qPCR analysis. In addition, the tracheas were removed and transferred to 50 ml tubes containing warm cell culture medium. Then, the tracheas were cleared of connective tissue and rinsed with cell culture medium. The tracheas were cut into tracheal rings using a McIlwain tissue chopper set to a section thickness of 0.6 - 0.8 mm. Three rings from the upper part, four rings from the middle part, and three rings from the lower part of each trachea were analyzed by light microscopy and scored for ciliary stasis (see Table 5). If more than 50% of the inner rings showed distinct ciliary movement (score 2 and lower), it was recorded as normal. If less than 50% of the ciliary movement (scores 3 and 4), the ring was recorded as positive for ciliary stasis.

[0385] For IBV-specific RT-qPCR analysis, kidney tissue sections were heated to room temperature and then transferred to separate 2 ml Precellys tubes filled with medium and PBS respectively. Using a tissue homogenizer (Bertin Instruments), the kidneys were homogenized at 6800 rpm for 1 x 20 seconds. Throat swabs were eluted with 2 ml of 1x PBS. Using MagMAX TM Core Nucleic Acid Purification Kit (ThermoFisher) and KingFisher TM Duo Prime Purification System (ThermoFisher), nucleic acids were isolated from 200 μl of eluate and tissue homogenates respectively. Except for performing the analysis in duplicate using a StepOnePlus TM Real-Time PCR System (ThermoFisher), RT-qPCR was performed as described above for in ovo kinetics.

[0386] Table 5: Ciliary Stasis Score in Tracheal Rings

[0387] Ciliary activity [%] Ciliary stasis score 100 0 <100-75 1 <75-50 2 <50-25 3 <25-0 4

[0388] Example 5: Efficacy of Recombinant IBV H52 Encoding Heterologous Spike Protein or Extracellular Domain of Spike Protein

[0389] The aim of this study was to demonstrate that vaccination with recombinant IBV H52 (Mass genotype) encoding heterologous spike protein or extracellular domain of spike protein could confer protection against challenge with a homologous challenge strain of spike protein.

[0390] The study analyzed whether recombinant IBV H52 (Mass genotype) encoding the extracellular domain of the spike protein of IBV CR88 (4 / 91 genotype) could confer protection against challenge with a highly virulent 793B strain (4 / 91 genotype), which was considered a homologous challenger of the encoded extracellular domain of the spike protein of IBV CR88 and a heterologous challenger of the IBV H52 backbone. Clinical signs of all chickens were observed daily. No clinical signs were recorded after vaccination or challenge. The titers of the 793B challenge virus applied on day 21 post-vaccination were determined by reverse titration of chickens vaccinated with H52 rIBV CR88 S Etco at 1 day of age to be 10 4.13 EID 50 / ml (target 10 4.3 EID 50 / ml), and 10 4.69 EID 50 / ml (target 10 4.3 EID 50 / ml). Ciliary motility stasis was scored as described above, and the results are shown in Figure 2as shown, and summarized in Table 6.

[0391] Table 6: Overview of ciliary stasis protection scores at 28 days post-inoculation and 7 days post-challenge. The average ciliary stasis score for each group was calculated by adding the total scores of each chicken in the group and then dividing the group total by the number of animals (highest score is 40, lowest score is 0). An animal was considered unaffected if at least 9 out of 10 rings showed normal ciliary motility.

[0392]

[0393] All animals in the strict negative control group showed normal ciliary movement, while 80% of the animals in the challenge control group had positive ciliary stasis. In contrast, 82% of the animals inoculated with H52 rIBV CR88 S Etco were protected. Additionally, the viral loads in the kidneys and nasal swabs of animals inoculated with H52 rIBV CR88 S Etco were reduced compared to those of animals in the challenge control group ( Figure 3 and Figure 4 ).

[0394] Furthermore, it was analyzed whether recombinant IBV H52 encoding the IBV QX spike protein could confer protection against challenge with the virulent D388 QX strain, which was considered a homologous challenge for the encoded IBV QX spike protein and a heterologous challenge for the IBV H52 backbone. Clinical signs of all chickens were observed daily. No clinical signs were recorded after inoculation or challenge. The titer determined by reverse titration with CH52 rIBV QX S at 1 day of age was 10 4 EID 50 / ml, while the QX vaccine inoculation exceeded 10 5 EID 50 / ml (target 10 4.3 EID 50 / ml). The titer determined by challenge with the D388 QX challenge virus at 21 days post-inoculation was 10 4.83 EID 50 / ml (target 10 4.3 EID 50 / ml). The ciliary stasis was scored as described above, and the results are as Figure 5 shown, and summarized in Table 7.

[0395] Table 7: Overview of ciliary stasis protection scores at 28 days post-inoculation and 7 days post-challenge. The average ciliary stasis score for each group was calculated by adding the total scores of each chicken in the group and then dividing the group total by the number of animals (highest score is 40, lowest score is 0). An animal was considered unaffected if at least 9 out of 10 rings showed normal ciliary motility.

[0396] Group Vaccine Challenge Average ciliary stasis score Unaffected [%] 1 - - 0 100 2 - D388 QX 38.4 0 3 QX vaccine D388 QX 3.5 100 3 H52 rIBV QX S D388 QX 5.9 100

[0397] All animals in the strict negative control group showed normal ciliary movement, while ciliary stasis was positive in all animals in the challenge control group. In contrast, 100% of the animals inoculated with H52 rIBV QX S or QX vaccine were protected.

[0398] Similar results were also obtained with other H52 rIBVs with heterologous spike proteins or extracellular domains of the spike protein.

[0399] This result highlights the suitability of IBV H52 as an effective backbone for generating recombinant IBVs with heterologous spike proteins, especially when showing excellent results compared to the data of the IBV Beaudette backbone in the prior art.

Claims

1. An infectious bronchitis virus H52 that encodes a heterologous infectious bronchitis virus S protein.

2. The infectious bronchitis virus H52 of claim 1, wherein the heterologous infectious bronchitis virus S protein is selected from the following genotypes or serotypes: 4 / 91, QX, Q1, Arkansas, Variant 2, and Brazil.

3. The infectious bronchitis virus H52 of any one of claims 1-2, wherein the heterologous infectious bronchitis virus S protein is a full-length S protein or its extracellular domain.

4. The infectious bronchitis virus H52 of any one of claims 1-3, wherein the heterologous infectious bronchitis virus S protein replaces the S protein of the infectious bronchitis virus H52.

5. The infectious bronchitis virus H52 of any one of claims 1-4, wherein the heterologous S protein is selected from the following genotypes or serotypes: 4 / 91, QX, Q1, Arkansas, Variant 2, and Brazil, or the heterologous S protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%, or 99.99% sequence identity therewith.

6. The infectious bronchitis virus H52 of any one of claims 1-5, wherein the infectious bronchitis virus H52 is attenuated or recombinant.

7. An immunogenic composition comprising the infectious bronchitis virus H52 of any one of claims 1-6.

8. The immunogenic composition of claim 7, wherein the immunogenic composition is a vaccine.

9. A kit comprising the infectious bronchitis virus H52 of any one of claims 1-6, or the immunogenic composition of any one of claims 7-8.

10. Use of the infectious bronchitis virus H52 of any one of claims 1-6 in the preparation of an immunogenic composition for immunizing a subject.

11. Use of the infectious bronchitis virus H52 of any one of claims 1-6 in the preparation of an immunogenic composition for treating or preventing clinical signs caused by infectious bronchitis virus in a subject in need thereof.

12. Use of the infectious bronchitis virus H52 of any one of claims 1-6 in the preparation of an immunogenic composition for reducing ciliary stasis in a subject in need thereof compared to an unimmunized control group of the same species.

13. The use of any one of claims 10-12, wherein the subject is a poultry.

14. Use according to any one of claims 10 - 12, wherein the immunogenic composition results in an improvement in a efficacy parameter selected from the following, compared to an untreated control group of the same species: prevention or reduction of ciliary stasis, prevention or reduction of rales, prevention or reduction of egg drop, prevention or reduction of kidney lesions, prevention or alleviation of watery diarrhea, prevention or reduction of weight loss, reduction of viral load, reduction of virus shedding or a combination thereof.

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