Coronavirus

Abstract

The present invention provides a live attenuated coronavirus comprising a variant replicase gene encoding a polyprotein comprising a mutation in one or more of the non-structural proteins (nsp)-10, nsp-14, nsp-15, or nsp-16. The coronavirus may be used as a vaccine for the treatment and / or prevention of a disease (e.g., infectious bronchitis) in a subject.

Description

[0001] This application is a divisional application based on a patent application with an application date of July 23, 2015, a priority date of July 23, 2014, an application number of 201580037402.1, and an invention name of “Coronavirus”. Field of the Invention

[0002] The present invention relates to an attenuated coronavirus comprising a variant replicase gene, which results in the virus having reduced pathogenicity. The present invention also relates to the use of such coronavirus in vaccines to prevent and / or treat diseases. Background of the Invention

[0004] Avian infectious bronchitis virus (IBV), the causative agent of infectious bronchitis (IB), is a highly infectious and contagious pathogen of poultry that replicates primarily in the respiratory tract, but also in epithelial cells of the intestine, kidney, and oviduct. IBV is a member of the order Nidovirales, family Coronaviridae, subfamily Coronavirinae, and genus Gammacoronavirus; genetically very similar coronaviruses cause disease in turkeys, guinea fowl, and pheasants.

[0005] Clinical symptoms of IB include sneezing, tracheospasm, nasal discharge, and asthma. Meat birds have reduced weight gain, while egg-laying birds lay fewer eggs and produce eggs of poor quality. Respiratory tract infections predispose chickens to secondary bacterial infections, which can be fatal in chicks. The virus can also cause permanent damage to the oviducts, especially in chicks, leading to reduced egg production and quality; and permanent damage to the kidneys, sometimes leading to kidney disease, which can be fatal.

[0006] IBV has been reported to cause more economic losses to the poultry industry than any other infectious disease. Although live attenuated and inactivated vaccines are commonly used for the control of IBV, the protection obtained by using the vaccine may be lost due to vaccine breakdown or the introduction of new IBV serotypes unrelated to the vaccine used, which poses a risk to the poultry industry.

[0007] In addition, in order to improve the efficiency and cost-effectiveness of the vaccination program, it is industrially necessary to develop a vaccine suitable for use in the egg. The major challenge associated with inoculation in the egg is that the virus must be able to replicate in the presence of maternally derived antibodies against the virus, without causing disease to the embryo. Current IBV vaccines are derived after multiple passages in embryonated eggs, which results in the pathogenicity of chickens having a reduction, so that they can be used as live attenuated vaccines. However, this type of virus almost always shows increased virulence to the embryo, and therefore cannot be used for inoculation in the egg, because they cause the hatchability of reduction. Observe the reduction of hatchability 70% in some cases.

[0008] Attenuation after multiple passages in embryonated eggs also has other shortcomings.It is a kind of empirical method, because the attenuation of virus is random, and all different when going down to the next generation virus at each time, therefore for the purpose of attenuation, the same virus will cause the set of different mutations causing attenuation by the going down of the egg of different series.Also there is the efficacy problem relevant to this process: some sudden changes will affect the duplication of virus, and some sudden changes may make virus too attenuated.Sudden change also can occur in S gene, and it also can affect immunogenicity, so that required immune response is affected, and potential vaccine may not be protected for required serotype.In addition, there is the problem relevant to the reversal of toxicity and vaccine stability.

[0009] It is important to develop new and safer vaccines for controlling IBV. Therefore, there is a need for IBV vaccines that are not associated with these problems, particularly vaccines that can be used for in ovo administration. SUMMARY OF THE INVENTION

[0011] To rationally attenuate IBV, the inventors have used a reverse genetics approach. This approach is more controllable than random attenuation after multiple passages in embryonated eggs, because the position of each mutation is known and its effect on the virus, i.e. the cause of attenuation, can be deduced.

[0012] Using their reverse genetics approach, the inventors have identified various mutations that result in viruses with reduced pathogenicity levels. The pathogenicity level can be reduced so that when the virus is administered to embryonated eggs, it is able to replicate without being pathogenic to the embryo. Such viruses are suitable for in ovo vaccination, which is a significant advantage and an improvement over attenuated IBV vaccines produced after multiple passages in embryonated eggs.

[0013] Thus, in a first aspect, the present invention provides a live attenuated coronavirus comprising a variant replicase gene encoding a polyprotein comprising a mutation in one or more of nonstructural protein (nsp)-10, nsp-14, nsp-15 or nsp-16.

[0014] The variant replicase gene may encode a protein comprising one or more amino acid mutations selected from the group consisting of:

[0015] Pro to Leu at position 85 of SEQ ID NO:6,

[0016] Val to Leu at position 393 of SEQ ID NO:7;

[0017] Leu to Ile at position 183 of SEQ ID NO:8;

[0018] Val to Ile at position 209 in SEQ ID NO:9.

[0019] The replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6.

[0020] The replicase gene may encode a protein comprising the following amino acid mutations: Val to Leu at position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8; and Val to Ile at position 209 of SEQ ID NO:9.

[0021] The replicase gene can encode a protein comprising the following amino acid mutations: Pro to Leu at position 85 of SEQ ID NO:6; Val to Leu at position 393 of SEQ ID NO:7; Leu to Ile at position 183 of SEQ ID NO:8; and Val to Ile at position 209 of SEQ ID NO:9.

[0022] Compared with the sequence shown in SEQ ID NO: 1, the replicase gene may contain one or more nucleotide substitutions selected from the following:

[0023] C to T at nucleotide position 12137;

[0024] G to C at nucleotide position 18114;

[0025] T to A at nucleotide position 19047; and

[0026] G to A at nucleotide position 20139.

[0027] The coronavirus may be infectious bronchitis virus (IBV).

[0028] The coronavirus may be IBV M41.

[0029] The coronavirus may comprise at least a portion of the S protein from an IBV serotype other than M41.

[0030] For example, the S1 subunit or the entire S protein may be from an IBV serotype other than M41.

[0031] The coronavirus according to the first aspect of the invention has reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type replicase, such that when the virus is administered to embryonated eggs, it is able to replicate without being pathogenic to the embryo.

[0032] In a second aspect, the present invention provides a variant replicase gene as defined in relation to the first aspect of the invention.

[0033] In a third aspect, the present invention provides a protein encoded by the variant coronavirus replicase gene according to the second aspect of the present invention.

[0034] In a fourth aspect, the present invention provides a plasmid comprising the replicase gene according to the second aspect of the present invention.

[0035] In a fifth aspect, the present invention provides a method for producing a coronavirus according to the first aspect of the present invention, comprising the following steps:

[0036] (i) transfecting a plasmid according to the fourth aspect of the present invention into a host cell;

[0037] (ii) infecting a host cell with a recombinant virus comprising a coronavirus strain genome having a replicase gene;

[0038] (iii) allowing homologous recombination to occur between the replicase gene sequence in the plasmid and the corresponding sequence in the recombinant viral genome to produce a modified replicase gene; and

[0039] (iv) selecting a recombinant virus comprising the modified replicase gene.

[0040] The recombinant virus can be a vaccinia virus.

[0041] The method may further comprise the following steps:

[0042] (v) recovering a recombinant coronavirus comprising the modified replicase gene in the DNA of the recombinant virus from step (iv).

[0043] In a sixth aspect, the present invention provides a cell capable of producing the coronavirus according to the first aspect of the present invention.

[0044] In a seventh aspect, the present invention provides a vaccine comprising the coronavirus according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0045] In an eighth aspect, the present invention provides a method for treating and / or preventing a disease in a subject, comprising the step of administering to the subject a vaccine according to the seventh aspect of the present invention.

[0046] Further aspects of the present invention provide:

[0047] - A vaccine according to the seventh aspect of the present invention for use in treating and / or treating a disease in a subject.

[0048] Use of the coronavirus according to the first aspect of the present invention in the preparation of a vaccine for treating and / or preventing a disease in a subject.

[0049] The disease may be infectious bronchitis (IB).

[0050] The administration method of the vaccine may be selected from the group consisting of eye drop administration, intranasal administration, drinking water administration, post-hatching injection and in ovo injection.

[0051] The vaccination may be by in ovo vaccination.

[0052] The present invention also provides a method for producing a vaccine according to the seventh aspect of the present invention, comprising the step of infecting cells according to the sixth aspect of the present invention with the coronavirus according to the first aspect of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 - Growth kinetics of M41-R-6 and M41-R-12 compared to M41-CK (M41EP4) on CK cells.

[0054] Figure 2 - Clinical symptoms, snicking and wheezing associated with M41-R-6 and M41-R-12 compared to M41-CK (M41EP4) and Beau-R (bars show from left to right mock, Beau-R, M41-R 6, M41-R 12, M41-CK EP4 at each time point).

[0055] Figure 3 - Cialis activity of the virus in tracheal rings isolated from trachea taken from infected chicks. 100% ciliary activity indicates no viral influence; no pathogenicity, 0% activity indicates a complete loss of ciliary activity, complete ciliary arrest, indicating that the virus is pathogenic (bars from left to right show mock, Beau-R, M41-R 6, M41-R12, M41-CK EP4 at each time point)

[0056] Figure 4- Clinical symptoms associated with M41R-nsp10rep and M41R-nsp14,15,16rep compared to M41-R-12 and M41-CK (M41 EP5), snicking (bars show from left to right the mimic, M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 at each time point).

[0057] Figure 5 - Ciliary activity of M41R-nsp10rep and M41R-nsp14,15,16rep in tracheal rings isolated from tracheas taken from infected chicks compared to M41-R-12 and M41-CK (bars show mock; M41-R12; M41R-nsp10rep; M41R-nsp14,15,16rep and M41-CK EP5 at each time point from left to right).

[0058] Figure 6 - Clinical symptoms associated with M41R-nsp10,15rep, M41R-nsp10,14,15rep, M41R-nsp10,14,16rep, M41R-nsp10,15,16rep and M41-K compared to M41-CK, snicking (bars from left to right show mimic, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 at each time point).

[0059] Figure 7 - Clinical symptoms, wheezing, associated with M41R-nsp10,15rep, M41R-nsp10,14,15rep, M41R-nsp10,14,16rep, M41R-nsp10,15,16rep and M41-K compared to M41-CK (bars from left to right show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 at each time point).

[0060] Figure 8-Ciliary activity of M41R-nsp10,15rep, M41R-nsp10,14,15rep, M41R-nsp10,14,16rep, M41R-nsp10,15,16rep and M41-K in tracheal rings isolated from tracheas taken from infected chicks compared to M41-CK (bars show mock, M41R-nsp10,15rep1; M41R-nsp10,14,16rep4; M41R-nsp10,15,16rep8; M41R-nsp10,14,15rep10; M41-K6 and M41-CK EP4 at each time point from left to right).

[0061] Figure 9 - Growth kinetics of rIBVs on CK cells compared to M41-CK. Fig.9A Results for M41-R and M41-K are shown. Fig. 9B Results are shown for M41-nsp10 rep; M41R-nsp14,15,16rep; M41R-nsp10,15rep; M41R-nsp10,15,16rep; M41R-nsp10,14,15rep; and M41R-nsp10,14,16.

[0062] Fig.10 - Positions of amino acid mutations in the mutated nsp10, nsp14, nsp15 and nsp16 sequences.

[0063] Figure 11 - A) Snicking; B) respiratory symptoms (combined wheezing and rales) and C) ciliary activity of rIBV M41R-nsp 10,14rep and rIBV M41R-nsp 10,16rep compared to M41-CK. DETAILED DESCRIPTION OF THE INVENTION

[0065] The present invention provides a coronavirus comprising a variant replicase gene, which, when expressed in a coronavirus, causes the virus to have reduced pathogenicity compared to a corresponding coronavirus comprising a wild-type replicase gene.

[0066] Coronavirus

[0067] Gammacoronavirus is a genus of animal viruses belonging to the Coronaviridae family. Coronaviruses are enveloped viruses with a positive-sense single-stranded RNA genome and helical symmetry.

[0068] The genome size of coronaviruses ranges from approximately 27 to 32 kilobases, which is the longest of any known RNA virus.

[0069] Coronaviruses primarily infect the upper respiratory tract or gastrointestinal tract of mammals and birds. Five to six currently known coronavirus strains infect humans. The most widely known human coronavirus, SARS-CoV, which causes severe acute respiratory syndrome (SARS), has a unique pathogenesis because it causes infections of both the upper and lower respiratory tracts and can also lead to gastroenteritis. Middle East respiratory syndrome coronavirus (MERS-CoV) also causes lower respiratory tract infections in humans. Coronaviruses are believed to cause a significant percentage of all common colds in human adults.

[0070] Coronaviruses also cause a range of diseases in livestock animals and domestic pets, some of which can be severe and pose a threat to the agricultural industry. Economically significant livestock animal coronaviruses include infectious bronchitis virus (IBV), which causes respiratory disease primarily in chickens and severely affects the poultry industry worldwide; porcine coronavirus (transmissible gastroenteritis, TGE) and bovine coronavirus, both of which cause diarrhea in young animals. There are two forms of feline coronavirus, feline enteric coronavirus, which is a pathogen of minor clinical significance, but spontaneous mutations of this virus can cause feline infectious peritonitis (FIP), a disease associated with high mortality.

[0071] There are also two types of canine coronavirus (CCoV), one of which causes mild gastrointestinal disease and the other has been found to cause respiratory disease. Mouse hepatitis virus (MHV) is a coronavirus that causes an epidemic rodent disease with high mortality, particularly in colonies of laboratory mice.

[0072] Coronaviruses can be divided into four groups as follows:

[0073] Alpha

[0074] Canine coronavirus (CCoV)

[0075] Feline coronavirus (FeCoV)

[0076] Human coronavirus 229E (HCoV-229E)

[0077] Porcine epidemic diarrhea virus (PEDV)

[0078] Transmissible gastroenteritis virus (TGEV)

[0079] Human coronavirus NL63 (NL or New Haven)

[0080] Beta

[0081] Bovine coronavirus (BCoV)

[0082] Canine respiratory coronavirus (CRCoV) – common in Southeast Asia and Micronesia

[0083] Human coronavirus OC43 (HCoV-OC43)

[0084] Mouse hepatitis virus (MHV)

[0085] Porcine hemagglutinating encephalomyelitis virus (HEV)

[0086] · Rat coronavirus (RCV). Rat coronavirus is fairly prevalent in eastern Australia, where it was detected in native and wild rodent populations in March / April 2008.

[0087] (No common name yet) (HCoV-HKU1)

[0088] Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)

[0089] Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

[0090] Gamma

[0091] Infectious bronchitis virus (IBV)

[0092] Turkey coronavirus (Bluecomb disease virus)

[0093] Pheasant coronavirus

[0094] Guinea fowl coronavirus

[0095] Delta

[0096] Bulbul coronavirus (BuCoV)

[0097] Thrush coronavirus (ThCoV)

[0098] Munia coronavirus (MuCoV)

[0099] Porcine coronavirus (PorCov) HKU15

[0100] The variant replicase gene of the coronavirus of the present invention can be derived from an alphacoronavirus such as TGEV; a betacoronavirus such as MHV; or a gammacoronavirus such as IBV.

[0101] As used herein, the term "derived from" means a replicase gene comprising a nucleotide sequence substantially identical to a wild-type replicase gene of a related coronavirus. For example, a variant replicase gene of the present invention may have up to 80%, 85%, 90%, 95%, 98% or 99% identity to a wild-type replicase sequence. The variant coronavirus replicase gene encodes a protein that, when compared to the wild-type sequence of a nonstructural protein, comprises a mutation in one or more of nonstructural protein (nsp)-10, nsp-14, nsp-15 or nsp-16.

[0102] IBV

[0103] Avian infectious bronchitis (IB) is an acute and highly contagious respiratory disease of chickens that causes significant economic losses. The disease is characterized by respiratory symptoms, including gasping, coughing, sneezing, tracheal rales, and nasal discharge. In young chickens, severe respiratory distress may occur. In layers, respiratory distress, nephritis, decreased egg production, and loss of internal egg mass and eggshell quality are common.

[0104] In broilers, coughing and quacking are common clinical signs that spread rapidly among all birds in the house. Morbidity in unvaccinated flocks is 100%. Mortality varies according to age, virus strain, and secondary infection, but can be as high as 60% in unvaccinated flocks.

[0105] The first IBV serotype identified was Massachusetts, but in the United States, several serotypes other than the originally identified Massachusetts are currently circulating, including Arkansas and Delaware.

[0106] IBV strain Beaudette was derived after at least 150 passages in chicken embryos. IBV Beaudette is no longer pathogenic to hatching chickens but rapidly kills embryos.

[0107] H120 is a commercially available live attenuated IBV Massachusetts serotype vaccine strain attenuated by approximately 120 passages in embryonated chicken eggs. H52 is another Massachusetts-type vaccine and represents an earlier and slightly more pathogenic passage virus (passage 52) during the development of H120. Vaccines based on H120 are commonly used.

[0108] IB QX is a virulent field isolate of IBV. From 2004 onwards, severe egg production problems were identified with a very similar virus in parts of Western Europe, mainly in the Netherlands, but also reported from Germany, France, Belgium, Denmark and the UK.

[0109] The virus isolated from the Dutch case was identified by the Dutch Institute in Deventer as a new strain, which they called D388. The link to IB QX came from further testing, which showed that the virus had 99% similarity to the IB QX virus. A live, attenuated QX-like IBV vaccine strain has now been developed.

[0110] IBV is an enveloped virus that replicates in the cytoplasm and contains a non-segmented single-stranded positive-sense RNA genome. IBV has a 27.6 kb RNA genome and, like all coronaviruses, contains four structural proteins that interact with the genomic RNA; spike glycoprotein (S), small membrane protein (E), membrane integral protein (M) and nucleocapsid protein (N).

[0111] The genome is organized in the following manner: 5'UTR - polymerase (replicase) gene - structural protein gene (SEMN) - UTR 3'; where UTRs are untranslated regions (~500 nucleotides each in IBV).

[0112] The lipid envelope contains three membrane proteins: S, M and E. The IBV S protein is a type I glycoprotein that oligomerizes in the endoplasmic reticulum and assembles into homotrimers inserted into the viral membrane via a transmembrane domain and associates with the M protein through non-covalent interactions. After incorporation into coronavirus particles, the S protein is responsible for binding to target cell receptors and fusion of the viral and cell membranes. The S glycoprotein consists of four domains: a signal sequence that is cleaved during synthesis; an extracellular domain present on the outside of the virion particle; a transmembrane region responsible for anchoring the S protein to the lipid bilayer of the virion particle; and a cytoplasmic tail.

[0113] All coronaviruses also encode a set of accessory protein genes of unknown function that are not essential for in vitro replication but may play a role in pathogenesis. IBV encodes two accessory genes, gene 3 and gene 5, which express two accessory proteins, 3a, 3b and 5a, 5b, respectively.

[0114] The variant replicase gene of the coronavirus of the present invention can be derived from IBV. For example, the IBV can be IBV Beaudette, H120, H52, IB QX, D388 or M41.

[0115] The IBV may be IBV M41. M41 is the prototype Massachusetts serotype isolated in the United States in 1941. It is an isolate used as a pathogenic laboratory virus strain in many laboratories around the world and can be obtained from ATCC (VR-21 TM). The attenuated variant is also used by several vaccine producers as an IBV vaccine against the Massachusetts serotype, which has caused problems in the art. The inventors chose to use this strain because they have been working with the virus for many years and because the sequence of the complete viral genome is available. The M41 isolate used by the inventors, M41-CK, is adapted to grow in primary chicken kidney (CK) cells and is therefore considered suitable for recovery from the cDNA of the complete genome as an infectious virus. It is a representative of pathogenic IBV and can therefore be analyzed for mutations that result in loss or reduction of pathogenicity.

[0116] The genomic sequence of IBV M41-CK is provided as SEQ ID NO:1.

[0117] SEQ ID NO:1 IBV M41-CK sequence

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127] Replicase

[0128] In addition to structural and accessory genes, two-thirds of the coronavirus genome contains the replicase gene (at the 5' end of the genome), which is expressed as two polyproteins, pp1a and pp1ab, where pp1ab is an extension product of pp1a as a result of a -1 ribosomal shift mechanism. These two polyproteins are cleaved by two types of virus-encoded proteases, typically producing 16 nonstructural proteins (Nsp1-16); IBV lacks Nsp1 and thus encodes Nsp2-16.

[0129] Thus, gene 1 in IBV encodes 15 (compared to 16 in other coronaviruses) nonstructural proteins (nsp2-16) associated with RNA replication and transcription.

[0130] The term "replicase protein" is used herein to refer to the pp1a and pp1ab polyproteins or to the individual nsp subunits.

[0131] The term "replicase gene" is used herein to refer to a nucleic acid sequence encoding a replicase protein.

[0132] An overview of the functions of coronavirus nsp proteins is provided in Table 1.

[0133] Table 1

[0134]

[0135]

[0136] The variant replicase gene encoded by the coronavirus of the present invention contains mutations in one or more segments of the sequence encoding nsp-10, nsp-14, nsp-15 or nsp-16.

[0137] Nsp10 has RNA binding activity and appears to participate in homotypic and / or heterotypic interactions with other NSPs from the PP1A / PP1AB regions. 10 -helix and an α / β fold of three β strands. Two zinc-binding sites formed by conserved cysteine ​​residues and one histidine residue (Cys-74 / Cys-77 / His-83Cys-90; Cys-117 / Cys-120 / Cys-128 / Cys-130) have been identified. The protein has been shown to bind single-stranded and double-stranded RNA and DNA without obvious specificity. Nsp-10 can cross-link with nsp-9, indicating the presence of a complex network of protein-protein interactions involving nsp-7, nsp-8, nsp-9 and -nsp10. In addition, nsp-10 is known to interact with nsp-14 and nsp-16.

[0138] Nsp-14 contains a 3' to 5' exoribonuclease (ExoN) activity domain in the amino-terminal region. SARS-CoV ExoN has been shown to have a metal ion-dependent 3' to 5' exoribonuclease activity that acts on both single-stranded and double-stranded RNA, but not on DNA. Nsp-14 has been shown to have proof-reading activity. This nsp has also been shown to have N7-methyltransferase (MT) activity in the carboxyl-terminal region.

[0139] Nsp-15-related NendoU (nidoviral endoribonuclease, specific for U) RNase activity has been reported in many coronaviruses, including SARS-CoV, MHV, and IBV. The activity has been consistently reported to be inhibited by Mn 2+ ions are significantly enhanced, and in Mg 2+ and Ca 2+ There is little activity in the presence of . NendoU cleaves on the 3' side of uridylic acid residues in both single-stranded and double-stranded RNA. Biologically relevant substrates of coronavirus NendoUs remain to be identified.

[0140] Nsp-16 has been predicted to mediate ribose-2'-O-methyltransferase (2'-O-MTase) activity, and reverse genetics experiments have shown that the 2'-O-MTase domain is essential for viral RNA synthesis in HCoV-229E and SARS-CoV. The enzyme can participate in the generation of the cap 1 structure of coronavirus RNA, and it can also cooperate with NendoU and ExoN in other RNA processing pathways. 2'-O-MTase can also methylate specific RNAs to protect them from NendoU-mediated cleavage.

[0141] The genomic and protein sequences of nsp-10, -14, -15 and -16 are provided as SEQ ID NOs: 2-5 and 6-9, respectively.

[0142] SEQ ID NO: 2 (nsp-10 nucleotide sequence -nucleotides 11884-12318 of SEQ ID NO: 1)

[0143]

[0144] SEQ ID NO: 3 (nsp-14 nucleotide sequence -nucleotides 16938-18500 of SEQ ID NO: 1)

[0145]

[0146] SEQ ID NO: 4 (nsp-15 nucleotide sequence -nucleotides 18501-19514 of SEQ ID NO: 1)

[0147]

[0148]

[0149] SEQ ID NO: 5 (nsp-16 nucleotide sequence -nucleotides 19515-20423 of SEQ ID NO: 1)

[0150]

[0151] SEQ ID NO: 6 (nsp-10 amino acid sequence)

[0152]

[0153] SEQ ID NO: 7 (nsp-14 amino acid sequence)

[0154]

[0155] SEQ ID NO: 8 (nsp-15 amino acid sequence)

[0156]

[0157] SEQ ID NO: 9 (nsp-16 amino acid sequence)

[0158]

[0159] Reduced pathogenicity

[0160] The live attenuated coronavirus of the present invention comprises a variant replicase gene, which results in the virus having reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type gene.

[0161] As used herein, the term "attenuated" refers to a virus that exhibits such reduced pathogenicity and can be classified as non-virulent. A live attenuated virus is a weakened replicating virus that is still able to stimulate an immune response and produce immunity but does not cause actual disease.

[0162] The term "pathogenicity" is used herein according to its normal meaning, referring to the potential of a virus to cause disease in a subject. Typically, the pathogenicity of a coronavirus is determined by measuring disease-related symptoms (e.g., sneezing, snicking, and decreased tracheal ciliary activity).

[0163] The term "reduced pathogenicity" is used to describe that the level of pathogenicity of a coronavirus is reduced, lessened or diminised compared to a corresponding wild-type coronavirus.

[0164] In one embodiment, the coronavirus of the present invention has reduced pathogenicity compared to the parental M41-CK virus or a control coronavirus from which the virus is derived. The control coronavirus can be a coronavirus with known pathogenicity, such as a coronavirus expressing a wild-type replicase protein.

[0165] The pathogenicity of coronaviruses can be assessed using methods well known in the art. Typically, pathogenicity is assessed by measuring clinical symptoms in subjects (e.g., chickens) challenged with the virus.

[0166] As an illustration, chickens can be challenged at 8-24 days of age by nasal or ocular inoculation. Clinical symptoms associated with IBV infection can be assessed 3-10 days post infection. Clinical symptoms commonly assessed to determine the pathogenicity of coronaviruses (e.g., IBV) include panting, coughing, sneezing, snicking, depression, ruffled feathers, and loss of tracheal ciliary activity.

[0167] The variant replicase of the present invention, when expressed in a coronavirus, can result in reduced levels of clinical symptoms compared to a coronavirus expressing a wild-type replicase.

[0168] For example, a coronavirus expressing a variant replicase can cause less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the number of snicks per bird per minute compared to the number of snicks caused by a virus expressing a wild-type replicase.

[0169] A coronavirus expressing a variant replicase according to the invention may cause asthma at less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20% or less than 10% of the times in a flock of birds infected with a virus expressing a wild-type replicase.

[0170] A coronavirus expressing a variant replicase according to the invention may cause ciliary activity in the trachea that is at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the level of ciliary activity in the trachea of ​​an uninfected bird.

[0171] Coronavirus expressing a variant replicase according to the present invention may cause clinical symptoms as defined in Table 2 at a lower level than coronavirus expressing a wild-type replicase.

[0172] Table 2. IBV severity limits based on clinical symptoms :

[0173]

[0174] When expressed in a coronavirus, the variant replicases of the invention can cause the virus to replicate at non-pathogenic levels in eggs.

[0175] When developing vaccines for in ovo administration to chicken embryos, two points must be considered: the effect of maternal antibodies on the vaccine and the effect of the vaccine on the embryo. Maternal antibodies are known to interfere with active immunity. For example, vaccines with mild virus strains do not induce protective antibody levels when administered to broiler chickens with maternal antibodies because these strains are neutralized by the maternal antibody pool.

[0176] Therefore, the virus particles must be efficient enough in replication and reproduction to ensure that they are not neutralized by maternally derived antibodies against the virus. Maternally derived antibodies are a limited set of effective antibodies that decrease with age of the chicken, and neutralization of the virus in this manner is not equivalent to the long-term immunity established by the embryo / chicken. In order to develop long-term immunity against the virus, the embryo and hatching chicken must develop a suitable protective immune response that is different from the effect of maternally derived antibodies.

[0177] To be useful for in ovo vaccination, the virus must also be unable to replicate and multiply at levels that render it pathogenic to the embryo.

[0178] With respect to embryos, reduced pathogenicity may mean that the coronavirus causes less reduction in hatchability compared to the corresponding wild-type control coronavirus. Thus, the term "non-pathogenic to embryos" in the context of the present invention may mean "does not cause reduced hatchability" when compared to a control coronavirus.

[0179] Suitable variant replicases may be identified using methods known in the art.For example, comparative challenge experiments may be conducted following in ovo vaccination of embryos with or without maternally derived antibodies (ie, wherein the egg-laying individuals have or have not been vaccinated against IBV).

[0180] If the variant replicase allows the virus to multiply at too high a level, the embryo will not hatch or will not survive after hatching (ie, the virus is pathogenic to the embryo). A virus that is pathogenic to the embryo can kill the embryo.

[0181] If the variant replicase results in a significant reduction in viral replication and reproduction, the virus will be neutralized by maternally derived antibodies. Subsequent challenge of chicks with IBV will result in the development of clinical signs (e.g., asthma, snicking, loss of ciliary activity) and disease onset in the challenged chicks; since it will not be able to develop effective immunity against the virus.

[0182] Variants

[0183] As used herein, the term "variant" is synonymous with "mutant" and refers to a nucleic acid or amino acid sequence that differs from the corresponding wild-type sequence.

[0184] Variant / mutant sequence can be naturally occurring, or can be artificially created (e.g., by site-directed mutagenesis). Mutants can have at least 70, 80, 90, 95, 98, or 99% sequence identity with the corresponding portion of the wild-type sequence. Mutants can have less than 20, 10, 5, 4, 3, 2, or 1 mutations on the corresponding portion of the wild-type sequence.

[0185] The term "wild type" is used to refer to a gene or protein having the same nucleotide or amino acid sequence, respectively, as a native gene or protein (ie, a viral gene or protein).

[0186] The identity comparison can be carried out by the naked eye, or more generally by means of an easily available sequence alignment program. These commercially available computer programs can calculate the % identity between two or more sequences. The suitable computer program for carrying out such comparison is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). The example of other software that can perform sequence alignment includes but is not limited to BLAST software package (see Ausubel et al., 1999ibid–Chapter 18), FASTA (Atschul et al., 1990, J.Mol.Biol., 403-410) and GENEWORKS alignment tool suite, ClustalX (see Larkin et al. (2007) Clustal W and Clustal X version 2.0.Bioinformatics, 23: 2947-2948). Both BLAST and FASTA can be used for offline and online searching (see Ausubel et al., 1999 ibid, pp. 7-58 to 7-60). However, for some applications, the GCG Bestfit program is preferred. A new tool called BLAST 2Sequences can also be used to align protein and nucleotide sequences (see FEMS Microbiol Lett 1999 174(2):247-50; FEMS Microbiol Lett 1999 177(1):187-8 and tatiana@ncbi.nlm.nih.gov).

[0187] The sequences may have one or more deletions, insertions or substitutions of amino acid residues which produce silent changes and result in functionally equivalent molecules. Amino acid substitutions may be made intentionally based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues, as long as activity is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; amino acids with similar hydrophilicity values ​​that do not carry an electric polar head group include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.

[0188] Conservative substitutions can be made, for example, according to the following table. Amino acids in the same block in the second column, and preferably in the same row in the third column, can be substituted for each other:

[0189]

[0190] The coronavirus of the present invention may comprise a variant replicase gene encoding a protein comprising a mutation compared to any one of SEQ ID NO: 6, 7, 8 or 9, which, when expressed in a coronavirus, results in a virus having reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type replicase.

[0191] The variant replicase gene may encode a protein comprising at least one or more amino acid mutations in any combination of nsp-10, nsp-14, nsp-15 and nsp-16.

[0192] The variant replicase gene of the coronavirus of the present invention can encode a protein comprising Fig.10 Mutations defined in the M41 mod sequence presented in .

[0193] The variant replicase gene of the coronavirus of the present invention may encode a protein comprising one or more amino acid mutations selected from the following:

[0194] Pro to Leu at position 85 of SEQ ID NO:6,

[0195] Val to Leu at position 393 of SEQ ID NO:7;

[0196] Leu to Ile at position 183 of SEQ ID NO:8;

[0197] Val to Ile at position 209 in SEQ ID NO:9.

[0198] The variant replicase gene of the coronavirus of the present invention can encode a protein that does not contain mutations in nsp-2, nsp-3, nsp-6 or nsp-13.

[0199] The variant replicase gene of the coronavirus of the present invention can encode a protein that does not contain a mutation in nsp10, which corresponds to the threonine to isoleucine mutation caused by the mutation at nucleotide position 12,008 in the nsp10 gene reported by Ammayappan et al. (Arch Virol (2009) 154: 495-499).

[0200] Ammayappan et al. (as above) reported the identification of sequence changes responsible for the attenuation of the IBV strain Arkansas DPI. The study identified 17 amino acid changes in multiple IBV proteins after multiple passages of the virus in embryonated eggs (about 100 generations). It was not studied whether the attenuated virus (Ark DPI 101) could replicate in the presence of maternally derived antibodies to the virus in the eggs without being pathogenic to the embryo. Given that the virus is produced by multiple passages in SPF embryonated eggs (similar methodology to that used for classical IBV vaccines), it is possible that the virus is pathogenic to the embryo. If hens are inoculated with similar serotypes, the virus may also be sensitive to maternally derived antibodies.

[0201] The variant replicase gene of the coronavirus of the present invention may encode a protein comprising any combination of one or more amino acid mutations listed above.

[0202] The variant replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6.

[0203] The variant replicase gene may encode a protein comprising an amino acid mutation from Val to Leu at position 393 of SEQ ID NO:7.

[0204] The variant replicase gene may encode a protein comprising an amino acid mutation from Leu to Ile at position 183 of SEQ ID NO:8.

[0205] The variant replicase gene may encode a protein comprising an amino acid mutation from Val to Ile at position 209 of SEQ ID NO:9.

[0206] The variant replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6 and from Val to Leu at position 393 of SEQ ID NO:7.

[0207] The variant replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6 and from Leu to Ile at position 183 of SEQ ID NO:8.

[0208] The variant replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6 and from Val to Ile at position 209 of SEQ ID NO:9.

[0209] The variant replicase gene may encode a protein comprising an amino acid mutation from Val to Leu at position 393 of SEQ ID NO:7 and from Leu to Ile at position 183 of SEQ ID NO:8.

[0210] The variant replicase gene may encode a protein comprising an amino acid mutation from Val to Leu at position 393 of SEQ ID NO:7 and from Val to Ile at position 209 of SEQ ID NO:9.

[0211] The variant replicase gene may encode a protein comprising an amino acid mutation from Leu to Ile at position 183 of SEQ ID NO:8 and from Val to Ile at position 209 of SEQ ID NO:9.

[0212] The variant replicase gene may encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6, from Val to Leu at position 393 of SEQ ID NO:7, and from Leu to Ile at position 183 of SEQ ID NO:8.

[0213] The variant replicase gene can encode a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:6, from Leu to Ile at position 183 of SEQ ID NO:8, and from Val to Ile at position 209 of SEQ ID NO:9.

[0214] The variant replicase gene can encode a protein comprising amino acid mutations from Pro to Leu at position 85 of SEQ ID NO:6, from Val to Leu at position 393 of SEQ ID NO:7, and from Val to Ile at position 209 of SEQ ID NO:9.

[0215] The variant replicase gene can encode a protein comprising an amino acid mutation from Val to Leu at position 393 of SEQ ID NO:7, from Leu to Ile at position 183 of SEQ ID NO:8, and from Val to Ile at position 209 of SEQ ID NO:9.

[0216] The variant replicase gene can encode a protein comprising amino acid mutations from Pro to Leu at position 85 of SEQ ID NO:6, from Val to Leu at position 393 of SEQ ID NO:7, from Leu to Ile at position 183 of SEQ ID NO:8, and from Val to Ile at position 209 of SEQ ID NO:9.

[0217] Variant replicase genes can also be defined at the nucleotide level.

[0218] For example, the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may contain one or more nucleotide substitutions selected from the following regions: 11884-12318, 16938-18500, 18501-19514 and 19515-20423 of SEQ ID NO:1.

[0219] For example, the nucleotide sequence of the variant replicase gene of the coronavirus of the present invention may comprise one or more nucleotide substitutions selected from the following compared to the sequence shown in SEQ ID NO: 1:

[0220] C to T at nucleotide position 12137;

[0221] G to C at nucleotide position 18114;

[0222] T to A at nucleotide position 19047; and

[0223] G to A at nucleotide position 20139.

[0224] As used herein, the term "substitution" is synonymous with the term mutation, and means that the nucleotide at a specified position is different from that of the wild-type nucleotide sequence.

[0225] Compared to the sequence shown in SEQ ID NO: 1, the nucleotide sequence may include any combination of nucleotide substitutions selected from the group consisting of:

[0226] C to T at nucleotide position 12137;

[0227] G to C at nucleotide position 18114;

[0228] T to A at nucleotide position 19047; and

[0229] G to A at nucleotide position 20139.

[0230] The nucleotide sequence may comprise the substitution C12137T.

[0231] The nucleotide sequence may comprise the substitution G18114C.

[0232] The nucleotide sequence may comprise the substitution T19047A.

[0233] The nucleotide sequence may comprise the substitution G20139A.

[0234] The nucleotide sequence may comprise the substitutions C12137T and G18114C.

[0235] The nucleotide sequence may comprise the substitutions C12137T and T19047A.

[0236] The nucleotide sequence may comprise the substitutions C12137T and G20139A.

[0237] The nucleotide sequence may comprise the substitutions G18114C and T19047A.

[0238] The nucleotide sequence may comprise the substitutions G18114C and G20139A.

[0239] The nucleotide sequence may comprise the substitutions T19047A and G20139A.

[0240] The nucleotide sequence may comprise the substitutions C12137T, G18114C and T19047A.

[0241] The nucleotide sequence may comprise the substitutions C12137T, T19047A and G20139A.

[0242] The nucleotide sequence may comprise the substitutions C12137T, G18114C and G20139A.

[0243] The nucleotide sequence may comprise the substitutions G18114C, T19047A and G20139A.

[0244] The nucleotide sequence may comprise the substitutions C12137T, G18114C, T19047A and G20139A.

[0245] The nucleotide sequence may not contain the substitution corresponding to the C12008T substitution reported by Ammayappan et al. (supra).

[0246] The nucleotide sequence can be natural, synthetic or recombinant. It can be double-stranded or single-stranded, it can be DNA or RNA or a combination thereof. It can be, for example, cDNA, PCR product, genomic sequence or mRNA.

[0247] The nucleotide sequence may be codon-optimized for production in the host / host cell of choice.

[0248] It may be isolated, or as part of a plasmid, virus or host cell.

[0249] Plasmids

[0250] Plasmids are extrachromosomal DNA molecules separate from chromosomal DNA that are capable of replicating independently of the chromosomal DNA. They are usually circular and double-stranded.

[0251] Plasmids or vectors (as they are sometimes called) can be used to express proteins in host cells. For example, bacterial host cells can be transfected with a plasmid that encodes a particular protein in order to express the protein. The term also includes yeast artificial chromosomes and bacterial artificial chromosomes that can accommodate longer portions of DNA.

[0252] Plasmid of the present invention comprises the nucleotide sequence of the determined region that can encode replicase protein.It can also comprise one or more other coronavirus nucleotide sequences or can encode the nucleotide sequence of one or more other coronavirus proteins such as S gene and / or gene 3.

[0253] The plasmid may also contain a resistance marker, such as the guanine xanthine phosphoribosyltransferase gene (gpt) from Escherichia coli, which confers resistance to mycophenolic acid (MPA) in the presence of xanthine and hypoxanthine and is controlled by the vaccinia virus P7.5 early / late promoter.

[0254] Recombinant vaccine virus

[0255] The present invention also relates to a recombinant vaccinia virus (rVV) comprising a variant replicase gene as defined herein.

[0256] Recombinant vaccinia viruses (rVV) can be prepared using a vaccinia-virus based reverse genetics system.

[0257] In this regard, the present invention also provides a method for preparing a virus particle by:

[0258] (i) transfecting the plasmid described in the previous section into a host cell;

[0259] (ii) Infecting the virus with a recombinant virus comprising the genome of a coronavirus strain having a replicase gene

[0260] Infect host cells;

[0261] (iii) allowing the replicase gene sequence in the plasmid and the corresponding gene in the recombinant viral genome to

[0262] Homologous recombination occurs between the sequences to produce a modified replicase gene;

[0263] (iv) selecting a recombinant virus comprising the modified replicase gene.

[0264] The term "modified replicase gene" refers to a replicase gene that includes a variant replicase gene as described herein in relation to the first aspect of the invention. Specifically, the term refers to a gene derived from a wild-type replicase gene but comprising a nucleotide sequence that causes it to encode a variant replicase protein as defined herein.

[0265] The recombination may involve all or part of the replicase gene. For example, the recombination may involve a nucleotide sequence encoding any combination of nsp-10, nsp-14, nsp-15 and / or nsp-16. The recombination may involve a nucleotide sequence encoding an amino acid mutation as defined above or comprising a nucleotide substitution as defined above.

[0266] The genome of the coronavirus strain may lack the portion of the replicase protein that corresponds to the portion provided by the plasmid, thereby forming a modified protein by insertion of the nucleotide sequence provided by the plasmid.

[0267] A recombinant virus is a virus that is adapted to allow homologous recombination between its genome and a plasmid. Vaccinia virus is particularly suitable because homologous recombination is routinely used for sequence insertion and deletion in the vaccinia virus genome.

[0268] The above method optionally comprises the following steps:

[0269] (v) recovering a recombinant coronavirus comprising the modified replicase gene in the DNA of the recombinant virus from step (iv).

[0270] Methods for recovering recombinant coronaviruses (eg, recombinant IBV) are known in the art (see Britton et al. (2005) at page 24; and PCT / GB2010 / 001293).

[0271] For example, the DNA of the recombinant virus from step (iv) can be inserted into a plasmid and used to transfect cells expressing cytoplasmic T7 RNA polymerase. The cells can be pre-infected, for example, with a fowlpox virus expressing T7 RNA polymerase. The recombinant coronavirus can then be isolated, for example, from the growth medium.

[0272] When the plasmid is inserted into the vaccinia virus genome, an unstable intermediate is formed. Recombinants containing the plasmid can be selected, for example, using a resistance marker on the plasmid.

[0273] Positive recombinants can then be verified, for example, by PCR and sequencing, to contain the modified replicase gene.

[0274] Large quantities of recombinant virus, such as recombinant vaccinia virus (rVV), comprising a modified replicase gene may be grown up and the DNA extracted to perform step (V).

[0275] Suitable reverse genetics systems are known in the art (Casais et al. (2001) J. Virol 75: 12359-12369; Casais et al. (2003) J. Virol. 77: 9084-9089; Britton et al. (2005) J. Virological Methods 123: 203-211; Armesto et al. (2008) Methods in Molecular Biology 454: 255-273).

[0276] cell

[0277] Coronaviruses can be used to infect cells.

[0278] For example, coronavirus particles can be harvested from the supernatant by methods known in the art and optionally purified.

[0279] The cells can be used to produce coronavirus particles.

[0280] Therefore, the present invention also provides a method for producing a coronavirus, comprising the following steps:

[0281] (i) infecting cells with the coronavirus according to the present invention;

[0282] (ii) allowing the virus to replicate in the cell; and

[0283] (iii) Harvesting the progeny viruses.

[0284] The present invention also provides the cell that can produce the coronavirus according to the present invention using the reverse genetics system.For example, the cell can include the recombinant viral genome, which includes the nucleotide sequence that can encode the replicase gene of the present invention.

[0285] The cells may be capable of producing a recombinant virus (eg, vaccinia virus) containing a replicase gene.

[0286] Alternatively, the cells may be capable of producing recombinant coronaviruses by a reverse genetics system. The cells may express or induce expression of T7 polymerase to rescue recombinant virus particles.

[0287] vaccine

[0288] Coronavirus can be used to produce vaccines. The vaccine can be a live attenuated form of the coronavirus of the present invention, and can also contain a pharmaceutically acceptable carrier. As defined herein, "pharmaceutically acceptable carriers" suitable for the present invention are well known to those skilled in the art. Such carriers include, but are not limited to, water, saline, buffered saline, phosphate buffer, alcohol / water solution, emulsion or suspension. Other conventionally used diluents and excipients can be added according to conventional techniques. Such carriers can include ethanol, polyols and suitable mixtures thereof, vegetable oils and injectable organic esters. Buffers and pH regulators can also be used. Buffers include, but are not limited to, salts prepared from organic acids or bases. Representative buffers include, but are not limited to, organic acid salts, such as salts of citric acid, such as citrate, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, Tris, trimethylammonium hydrochloride, or phosphate buffer. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose, trehalose, sucrose, and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous carriers may include fluid and nutrient supplements, electrolyte supplements, such as those based on Ringer's dextrose, etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents (e.g., EDTA), inert gases, etc., may also be provided in pharmaceutical carriers. The present invention is not limited by the choice of carrier. It is within the technical scope of the art to prepare these pharmaceutically acceptable compositions from the above components with suitable pH isotonicity, stability, and other conventional features. For example, see articles such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, pub! ., 2000; and The Handbook of Pharmaceutical Excipients, 4.sup.th edit., eds. RC Rowe et al., APhAPublications, 2003.

[0289] The vaccines of the present invention will be administered in a "therapeutically effective amount", which refers to the amount of active ingredient (e.g., an agent according to the present invention) sufficient to achieve a beneficial or desired result when administered to a subject or patient. The effective amount can be administered in one or more applications, applications, or dosages. The therapeutically effective amount of the composition according to the present invention can be easily determined by one of ordinary skill in the art. In the context of the present invention, a "therapeutically effective amount" is an amount that produces an objectively measured change in one or more parameters associated with infectious bronchitis, which is sufficient to produce a beneficial or desired result. The effective amount can be administered in one or more applications. For the purposes of the present invention, the effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to reduce the incidence of infectious bronchitis. As used herein, the term "therapeutic" encompasses the full spectrum of treatments for a disease, illness or condition. The "therapeutic agents" of the invention may act in a prophylactic or preventive manner (including integration into programs designed to target animals that may be identified as at risk (pharmacogenetics)); or in an ameliorative or curative manner; or may act to slow the rate or extent of progression of at least one symptom of the disease or condition being treated.

[0290] The invention also relates to a method of producing such a vaccine comprising the step of infecting cells (eg Vero cells) with viral particles comprising a replicase protein as defined in relation to the first aspect of the invention.

[0291] Vaccination method

[0292] The coronavirus of the present invention can be used to treat and / or prevent diseases.

[0293] "Treatment" means administering a vaccine to a subject with an existing disease to alleviate, reduce or ameliorate at least one symptom associated with the disease, and / or to slow, reduce or block the progression of the disease.

[0294] "Prevention" means administering a vaccine to a subject who has not yet contracted a disease, and / or does not show any symptoms of a disease, to prevent or attenuate the cause of the disease (e.g., infection), or to reduce or prevent the development of at least one symptom associated with the disease.

[0295] The illness can be any disease caused by a coronavirus, such as respiratory illness and / or gastroenteritis in humans, and hepatitis, gastroenteritis, encephalitis, or respiratory disease in other animals.

[0296] The disease may be infectious bronchitis (IB); porcine epidemic diarrhea; transmissible gastroenteritis; mouse hepatitis virus; porcine hemagglutinating encephalomyelitis; severe acute respiratory syndrome (SARS); or blue crown disease.

[0297] The disease may be infectious bronchitis.

[0298] Vaccines can be administered to hatching chicks or chickens, for example, by eye drops or intranasal administration. Although accurate, these methods can be expensive, for example, for large broiler flocks. Alternatives include spray inoculation applied to drinking water, but it is difficult to ensure uniform vaccine application using this method.

[0299] The vaccine may be provided in a form suitable for its administration, such as eye drops for intraocular use.

[0300] The vaccine can be administered by in ovo vaccination, for example by injection of embryonated eggs. In ovo vaccination has the advantage of providing early stage resistance to the disease. Unlike spray vaccination and administration through drinking water, it also facilitates the administration of a uniform dose to each subject.

[0301] The vaccine may be administered to any suitable compartment of the egg, including the allantoic fluid, yolk sac, amnion, air sac or embryo. It may be administered under the shell (air cell) membrane and the chorioallantoic membrane.

[0302] The vaccine is usually injected into embryonated eggs during late embryonic development, usually during the last quarter of the incubation period (e.g., 3-4 days before hatching). In chickens, the vaccine can be administered between days 15-19 (e.g., on day 17 or 18) of the 21-day incubation period.

[0303] The process may be automated using a robotic injection process, such as those described in WO 2004 / 078203.

[0304] The vaccine can be administered together with one or more other vaccines, for example vaccines for other diseases, for example Newcastle disease virus (NDV). The present invention also provides vaccine compositions comprising a vaccine according to the present invention and one or more other vaccines. The present invention also provides test kits comprising a vaccine according to the present invention and one or more other vaccines for separate, sequential or simultaneous administration.

[0305] The vaccine or vaccine composition of the present invention can be used to treat a human, animal or avian subject. For example, the subject can be a chick, chicken or mouse (eg, a laboratory mouse, such as a transgenic mouse).

[0306] Typically, a physician or veterinarian will determine the actual dosage which will be most suitable for an individual subject or group of subjects and it will vary with the age, weight and response of the particular subject.

[0307] The composition may optionally include a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. The selection of a pharmaceutical carrier, excipient or diluent may be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include (or in addition to) a carrier, excipient or diluent, any suitable adhesive, lubricant, suspending agent, coating agent, solubilizing agent, and other carrier agents that may assist or increase viral delivery or immunogenicity.

[0308] The present invention will now be further described by way of examples, which are intended to help those of ordinary skill in the art to practice the present invention and are not intended to limit the scope of the present invention in any way. Example

[0309] Example 1: Generation of an IBV reverse genetics system based on M41-CK

[0310] The M41-CK full-length cDNA was generated by replacing the Beaudette cDNA in the vaccinia virus reverse genetics system previously described in PCT / GB2010 / 001293 (incorporated herein by reference) with a synthetic cDNA derived from the M41 consensus sequence.

[0311] The IBV cDNA in the rVV-BeauR-Rep-M41 structure of the recombinant vaccinia virus (rVV) is described in Armesto, Cavanagh and Britton (2009). PLoS ONE 4 (10): e7384. doi: 10.1371 / journal.pone.0007384, which consists of a replicase derived from the IBV Beaudette strain and structural and accessory genes and 3'UTR from IBV M41-CK, and further replaces the 5'UTR-Nsp2-Nsp3 sequence of Beaudette with the corresponding sequence from IBVM41-CK. The resulting IBV cDNA consists of 5'UTR-Nsp2-Nsp3 from M41, Nsp4-Nsp16 from Beaudette, and structural and accessory genes and 3'UTR from M41. The cDNA is further modified by the deletion of the Beaudette Nsp4-Nsp16 sequence. The resulting cDNA lacking Nsp4-16 was modified in four further steps, wherein the missing Nsps were replaced in sequence by the corresponding sequences from M41-CK, the replacement cDNAs being denoted as M41-CK Nsp4-8, Nsp9-12, Nsp12-14, and finally Nsp15-16. Each replacement cDNA contained approximately 500 nucleotides corresponding to the 3' most of the previously inserted M41 sequence at the 5' end, and approximately 500 nucleotides corresponding to the M41 S gene sequence at the 3' end. This allowed the insertion of the M41 cDNA sequence by homologous recombination and the sequential addition of the contiguous M41 replicase gene sequence. Synthetic cDNAs containing the Nsp sequences derived from M41 were added by homologous recombination using the transient dominant selection (TDS) system previously described by the inventors (see PCT / GB2010 / 001293). The M41-derived cDNAs containing sequences corresponding to M41 Nsps-10, -14, -15, and -16 contain modified amino acids at positions 85, 393, 183, and 209, respectively, such as Fig.10 shown.

[0312] A full-length cDNA representing the M41-CK genome was generated in a vaccinia virus representing the synthetic sequence. Two rIBVs, M41-R-6 and M41-R-12, were rescued and shown to grow in a similar manner to M41-CK ( Figure 1 ).

[0313] Example 2: Determination of the pathogenicity of the rescued M41 virus

[0314] The rescued viruses in Example 1 were used to infect 8-day-old specific pathogen-free (SPF) chicks by ocular and nasal inoculation to test their pathogenicity, as observed by clinical symptoms based on daily clinical signs from 3 to 7 days post-infection, and by ciliary activity on days 4 and 6 post-infection. The loss of ciliary activity is a recognized method for determining the pathogenicity of IBV. When compared to M41-CK, the two M41-R viruses were found to be non-pathogenic, although compared to uninfected control chicks ( Figure 2 ), they do show some clinical signs and some but inconsistent loss of ciliary activity.

[0315] Therefore, compared with the parental virus M41-CK, the M41-R molecular clone of M41-CK is not pathogenic.

[0316] The inventors identified several nucleotide differences in M41-R compared to the M41-CK sequence. Most of these are synonymous mutations, as the nucleotide changes do not affect the amino acid sequence of the protein associated with the sequence. However, four non-synonymous mutations were identified in the IBV replicase gene, which are specific to the Nsp-10, Nsp-14, Nsp-15and Nsp-16 components of the replicase gene, and these mutations result in changes in amino acids (Table 3).

[0317] Table 3. Nonsynonymous mutations identified in Nsps of the M41-R full-length genome

[0318] Replicase region Nucleotide position Nucleotide mutation Amino acid changes Nsp10 12137 C→T Pro→Leu Nsp14 18114 G→C Val → Leu Nsp15 19047 T→A Leu → Ile Nsp16 20139 G→A Val → Ile

[0319] Example 3: Repair of M41-R rIBVs

[0320] To determine whether the identified mutations were responsible for the loss of pathogenicity associated with M41-R, the Nsp10 mutation was repaired and the mutations in Nsp-14, -15 and -16 were repaired and showed a growth pattern similar to M41-CK (Figure 9). Therefore, the inventors generated rIBVs, M41R-nsp10rep and M41R-nsp14,15,16rep using the (TDS) system previously described by the inventors (see PCT / GB2010 / 001293) using synthetic cDNA containing the correct nucleotides.

[0321] The pathogenicity of rIBVs was assessed in chicks as described previously. Both rIBVs showed increased pathogenicity when compared to M41-R, but not to the levels observed with M41-CK ( Figure 4 and 5M41R-nsp14,15,16rep produced more clinical symptoms and more decreased ciliary activity than M41R-nsp10rep, and overall these results suggest that changes associated with the four Nsps appear to affect pathogenicity.

[0322] To determine the role of Nsps in pathogenicity, full-length cDNA corresponding to M41R-nsp10rep was used to repair mutations in Nsps14, 15, and 16 using synthetic cDNAs containing the correct nucleotides using the TDS system.

[0323] The following rIBVs were generated:

[0324] M41R-nsp10,15rep - M41-R with repaired mutations in Nsp-10 and Nsp-15

[0325] M41R-nsp10,14,15rep - M41-R with repaired mutations in Nsp-10, Nsp-14, and Nsp-15

[0326] M41R-nsp10,14,16rep - M41-R with repaired mutations in Nsp-10, Nsp-14, and Nsp-16

[0327] M41R-nsp10,15,16rep – M41-R with repaired mutations in Nsp-10, Nsp-15, and Nsp-16

[0328] All four mutations Nsp-10, Nsp-14, Nsp-15 and Nsp-16 were restored in M41-K–M41-R.

[0329] rIBV was shown to grow in a similar manner to M41-CK ( FIG. 9 ), and pathogenicity was assessed as previously described. M41-K (in which all four mutations had been repaired) resulted in clinical symptoms and 100% loss of ciliary activity (complete ciliary arrest) 4 days after infection ( Figure 6 , 7 and 8). With the exception of M41R-nsp10,15,16rep (which is essentially non-pathogenic), the other rIBVs exhibited varying degrees of pathogenicity. These results confirm that repair of all four Nsps restored the pathogenicity of M41-R; again supporting previous evidence that the mutations described in the four Nsps are involved in the attenuated M41-CK.

[0330] The inventors also generated rIBV M41R-nsp 10,14rep (nsp 10 and nsp 14 were repaired, nsp 15 and nsp 16 contained mutations) and rIBV M41R-nsp 10,16rep (nsp 10 and nsp 16 were repaired, nsp 14 and nsp 15 contained mutations) and evaluated the pathogenicity of these viruses.

[0331] rIBV M41R-nsp 10,14rep was less pathogenic than M41-K, but caused approximately 50% ciliary arrest at days 4-6 post infection. rIBV M41R-nsp 10,16rep was almost non-pathogenic and did not cause ciliary arrest (see Figures 11a-c).

[0332] Therefore, genomes related to M41-R are potential backbone genomes for rationally attenuated IBVs.

[0333] Example 4: Vaccination / Challenge Studies with M41-R

[0334] The candidate vaccine virus was tested in studies in which fertilized chicken eggs were inoculated in ovo at 18 days of embryonic development and the hatchability of the inoculated eggs was determined. The clinical health of the chickens was studied and the chickens were challenged with virulent IB M41 virus at 21 days of age at a dose of 10 3.65 EID 50 Attack the chicken.

[0335] Clinical symptoms were studied after challenge protection by the vaccine and a ciliary arrest test was performed 5 days after challenge to investigate the effect of the challenge virus on ciliary motility and protection by the vaccine against ciliary arrest (inhibition of ciliary motility).

[0336] In ovo vaccination of commercial broiler eggs

[0337] The design of the experiment is given in Table 4 and the clinical results are given in Table 5. The hatchability of eggs inoculated with IB M41-R was good and the birds were healthy. After challenge in broiler chickens, IB M41-R protected them from clinical symptoms (placebo: 19 / 19 affected, IB M41-R: 3 / 18 affected and 1 dead). The results of the ciliary arrest test are given in Table 6. IB M41-R produced protection against ciliary arrest.

[0338] Table 4 - Design of hatchability, safety and efficacy studies of commercial eggs

[0339]

[0340] 1 Dose volume 0.1 ml, NA, not applicable.

[0341] 2 10 3.65 EID 50 / per dose.

[0342] Table 5 - Hatch percentage and clinical data before and after challenge in commercial chickens, see Table 1 for design.

[0343]

[0344] 1 Disturbed respiratory system

[0345] 2 Whizzing

[0346] 3 Changes in voice

[0347] 4 trouble breathing

[0348] 5 Swelling of the orbital sinus

[0349] 6 Uneven growth

[0350] 7 Weak

[0351] Table 6 - Results of the ciliary arrest test after challenge, see Table 1 for design.

[0352]

[0353]

[0354] In ovo vaccination of specific pathogen-free (SPF) eggs

[0355] The design of the study in SPF eggs is given in Table 7 and was similar to that of the study using commercial broiler chickens, but the inoculum dose of IB M41-R was higher (10 5 EID 50 per dose).

[0356] The results (Table 8) showed that the hatch percentage of IB M41-R was low, 19 out of 40 hatched, and the chicks were weak. Eight chicks died. The remaining 11 chickens were challenged and the chicks hatched from eggs that had been inoculated with saline were challenged.

[0357] In the ciliary arrest test after challenge, it appeared that all chickens vaccinated in ovo with IB M41-R were protected, whereas none of the controls were protected, see Table 9.

[0358] Table 7. Design of hatchability, safety and efficacy studies in SPF eggs

[0359]

[0360] 1 Dose volume 0.1 ml, NA, not applicable.

[0361] 2 Attack dose 10 3.3 EID 50 In 0.2 ml.

[0362] Table 8 - Hatching percentage and clinical data before and after challenge in SPF chickens, see Table 7 for design.

[0363]

[0364] Table 9. Results of the ciliary arrest test after challenge, see Table 7 for design.

[0365] deal with Protected / Total Protection percentage brine 0 / 11 0％ IB M41R 11 / 11 100％

[0366] In conclusion, IB M41-R was safe in commercial eggs and produced protection against clinical symptoms and to some extent against ciliary arrest.

[0367] A relatively small number of chicks hatched from SPF eggs inoculated with IB M41 R. This may be due to the use of 10 5 EID 50 of IB M41-R. This is 10 times higher than the dose used in previous studies with higher levels of hatchability. The lower hatch percentage could also be caused by the batch of SPF eggs having a particularly high susceptibility to the virus, as levels of embryonic mortality were also higher than previously observed in other studies.

[0368] After challenge, all chicks surviving hatching were fully protected against ciliary arrest. It was concluded that IB M41-R has great potential as a vaccine for in ovo administration.

[0369] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the described modes for implementing the present invention that are apparent to those skilled in the art of molecular biology, virology or related fields are intended to be within the scope of the following claims.

Claims

1. A live attenuated coronavirus comprising a variant replicase gene encoding a polyprotein comprising a mutation in one or more of nonstructural protein (nsp)-10, nsp-14, nsp-15 or nsp-16.

2. The coronavirus of claim 1, wherein the variant replicase gene encodes a protein comprising one or more amino acid mutations selected from the group consisting of: Pro to Leu at position 85 of SEQ ID NO: 6, Val to Leu at position 393 in SEQ ID NO: 7; Leu to Ile at position 183 in SEQ ID NO: 8; Val to Ile at position 209 in SEQ ID NO:

9.

3. The coronavirus according to claim 1 or 2, wherein the replicase gene encodes a protein comprising an amino acid mutation from Pro to Leu at position 85 of SEQ ID NO:

6.

4. A coronavirus according to any one of the preceding claims, wherein the replicase gene encodes a protein comprising the following amino acid mutations: Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO:

9.

5. A coronavirus according to any of the preceding claims, wherein the replicase gene encodes a protein comprising the following amino acid mutations: Pro to Leu at position 85 of SEQ ID NO: 6; Val to Leu at position 393 of SEQ ID NO: 7; Leu to Ile at position 183 of SEQ ID NO: 8; and Val to Ile at position 209 of SEQ ID NO:

9.

6. The coronavirus according to any one of the preceding claims, wherein the replicase gene comprises one or more nucleotide substitutions selected from the group consisting of: C to T at nucleotide position 12137; G to C at nucleotide position 18114; T to A at nucleotide position 19047; and G to A at nucleotide position 20139.

7. The coronavirus according to any one of the preceding claims, which is infectious bronchitis virus (IBV).

8. The coronavirus according to any one of the preceding claims, which is IBV M41.

9. The coronavirus according to claim 8, comprising an S protein, at least a portion of which is from an IBV serotype other than M41.

10. The coronavirus of claim 9, wherein the S1 subunit is from an IBV serotype other than M41.

11. The coronavirus of claim 9, wherein the S protein is from an IBV serotype other than M41.

12. A coronavirus according to any one of the preceding claims, having reduced pathogenicity compared to a coronavirus expressing the corresponding wild-type replicase, such that when the virus is administered to an embryonated egg, it is able to replicate without being pathogenic to the embryo.

13. A variant replicase gene as defined in any one of claims 1 to 6.

14. A protein encoded by the variant coronavirus replicase gene according to claim 13.

15. A plasmid comprising the replicase gene according to claim 13.

16. A method for producing a coronavirus according to any one of claims 1 to 12, comprising the following steps: (i) transfecting the plasmid according to claim 15 into a host cell; (ii) infecting the host cell with a recombinant virus, wherein the recombinant virus comprises a coronavirus strain genome having a replicase gene; (iii) allowing homologous recombination to occur between the replicase gene sequence in the plasmid and the corresponding sequence in the recombinant viral genome to produce a modified replicase gene; and (iv) selecting a recombinant virus comprising the modified replicase gene.

17. The method of claim 16, wherein the recombinant virus is a vaccinia virus.

18. The method according to claim 16 or 17, further comprising: The following steps are involved: (v) recovering a recombinant coronavirus comprising the modified replicase gene in the DNA of the recombinant virus from step (iv).

19. A cell capable of producing a coronavirus according to any one of claims 1 to 12.

20. A vaccine comprising a coronavirus according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier.

21. A method for treating and / or preventing a disease in a subject, comprising the step of administering a vaccine according to claim 20 to the subject.

22. The vaccine according to claim 20 for use in treating and / or preventing a disease in a subject.

23. Use of a coronavirus according to any one of claims 1 to 12 in the preparation of a vaccine for the treatment and / or prevention of a disease in a subject.

24. The method, vaccine or use according to claim 21, 22 or 23, wherein the disease is infectious bronchitis (IB).

25. The method of claim 21, wherein the administration method is selected from the group consisting of eye drop administration, intranasal administration, drinking water administration, post-hatch injection, and in ovo injection.

26. The method of claim 24, wherein the vaccination is in ovo.

27. A method for producing a vaccine according to claim 20, comprising the step of infecting cells according to claim 19 with a coronavirus according to any one of claims 1 to 12.

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