WO2024032805A1 - Coronavirus porcin recombinant - Google Patents

Coronavirus porcin recombinant Download PDF

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Publication number
WO2024032805A1
WO2024032805A1 PCT/CN2023/112866 CN2023112866W WO2024032805A1 WO 2024032805 A1 WO2024032805 A1 WO 2024032805A1 CN 2023112866 W CN2023112866 W CN 2023112866W WO 2024032805 A1 WO2024032805 A1 WO 2024032805A1
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Prior art keywords
coronavirus
pedv
porcine
porcine coronavirus
infection
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PCT/CN2023/112866
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English (en)
Inventor
Yaowei Huang
Yajuan JIAO
Bin Wang
Jiaqi Yu
Ning Chen
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Zhejiang University
Boehringer Ingelheim Vetmedica (China) Co., Ltd.
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Publication of WO2024032805A1 publication Critical patent/WO2024032805A1/fr

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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20061—Methods of inactivation or attenuation
    • C12N2770/20062—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention relates the field of animal health.
  • the present invention relates to a recombinant porcine coronavirus, in particular a recombinant porcine epidemic diarrhea virus (PEDV) , comprising a deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.
  • PDV porcine epidemic diarrhea virus
  • NSP2 complete nonstructural protein 2
  • the present invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention and the uses thereof.
  • the porcine epidemic diarrhea virus is an enveloped, positive-sense single-stranded RNA virus that causes acute diarrhea, vomiting, and dehydration in pigs.
  • clinical signs including acute watery, diarrhea, vomiting, and dehydration
  • the gross and histological changes in the gut of animals infected with PEDV can cause gross pathological lesions in the small intestine.
  • PEDV was first identified in Europe but has become increasingly problematic in many Asian countries, including Korea, China, Japan, the Philippines, and Thailand. Since 2013, PEDV emerged in the U.S. and the economic impact of PEDV infection has already been substantial.
  • the present invention provides a recombinant porcine coronavirus, comprising a deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.
  • NSP2 nonstructural protein 2
  • the present invention provides a recombinant porcine coronavirus, which is
  • a recombinant PEDV with a genomic sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, sequence identity with the recombinant PEDV deposited at CCTCC under the accession number CCTCC V202263 on August 10, 2022.
  • the present invention provides an isolated nucleic acid coding for the recombinant porcine coronavirus of the present invention.
  • the present invention provides a vector comprising the nucleic acid of the present invention.
  • the present invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention.
  • the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention in the manufacture of immunogenic composition for
  • the present invention provides a use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention in the manufacture of immunogenic composition for
  • the present invention provides a method for
  • said method comprises administering to the pig an immunogenic composition of the present invention.
  • the present invention provides a method for i) reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet, and/or ii) reducing the mortality caused by an infection with a PEDV in a piglet, wherein the piglet is to be suckled by a sow to which the immunogenic composition of the present invention has been administered.
  • the present invention provides a method of attenuating a porcine coronavirus, comprising deleting the complete nonstructural protein 2 (NSP2) coding sequence from its genome.
  • NSP2 nonstructural protein 2
  • the present invention provides a method for producing the recombinant porcine coronavirus of the present invention, comprising
  • the present invention provides a method of differentiating animals infected with a porcine coronavirus from animals vaccinated with the immunogenic composition of the present invention, comprising
  • Figure 1 The strategy for rescuing PEDV ZJU/G2/2013 strain with NSP2 coding sequence completely deleted.
  • Figure 2 The method for rescue of infectious virus.
  • Figure 6 The growth curve of rPEDV-delNsp2 virus.
  • FIG. 15 Construction and rescue of rSADS-CoV- ⁇ nsp2.
  • A Organization of the SADS-CoV "SeACoV-p10" genome (upper) . Detail showing the nsp2 gene before and after deletion (lower) .
  • C Confirmation of nsp2 deletion in rSADS-CoV- ⁇ nsp2.
  • Sequencing results show the fragment containing the nsp2 deletion region of the plasmid harboring the infectious cDNA clone of rSADS-CoV- ⁇ nsp2 (upper) , or the same region by RT-PCR amplification of viral RNA extracted from rSADS-CoV- ⁇ nsp2-infected Vero cells (lower) .
  • the corresponding amino acids are indicated below.
  • A, B and/or C encompasses “A” , “B” , “C” , “A and B” , “A and C” , “B and C” , and “A and B and C” .
  • all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the technical field to which this invention belongs. Although any methods and materials similar 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. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the virus strains, the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. None herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
  • the present invention is based on the surprising finding that a deletion of the NSP2 coding sequence of a porcine coronavirus such as a PEDV results in attenuation of the virus, and the attenuated live virus may be suitable for providing an effective protection against porcine coronavirus such as PEDV infection or challenge in pigs.
  • the invention provides a recombinant porcine coronavirus, comprising a deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.
  • NSP2 nonstructural protein 2
  • recombinant refers to a porcine coronavirus that has been altered, rearranged, or modified by genetic engineering. However, the term does not refer to alterations in polynucleotide or amino acid sequence that result from naturally occurring events, such as spontaneous mutations.
  • the deletion of the complete nonstructural protein 2 (NSP2) coding sequence is a complete deletion of the NSP2 gene.
  • said virus is capable of infecting a host cell.
  • a host cell may be a cell or a cell line from pigs.
  • “Infecting a host cell” in particular includes attachment of the virus to a host cell, entry of the virus into the cell, disassembly of the virion, replication and transcription of the viral genome, expression of viral proteins and assembly and release of new infectious viral particles.
  • the recombinant porcine coronavirus is attenuated.
  • the recombinant porcine coronavirus has lower virulence than a corresponding wildtype porcine coronavirus which does not comprise the deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.
  • NSP2 complete nonstructural protein 2
  • the porcine coronavirus may be an alphacoronavirus, a betacoronavirus, or a deltacoronavirus.
  • porcine coronavirus belongs to alphacoronavirus
  • porcine hemagglutinating encephalomyelitis virus belongs to betacoronavirus
  • porcine deltacoronavirus belongs to delta-coronavirus.
  • the porcine coronavirus may be selected from the group consisting of porcine epidemic diarrhea virus (PEDV) , swine acute diarrhea syndrome-coronavirus (SADS-CoV) , transmissible gastroenteritis virus (TGEV) , porcine respiratory coronavirus (PRCV) , porcine hemagglutinating encephalomyelitis virus (PHEV) , and porcine deltacoronavirus (PDCoV) .
  • porcine epidemic diarrhea virus PEDV
  • SADS-CoV swine acute diarrhea syndrome-coronavirus
  • TGEV transmissible gastroenteritis virus
  • PRCV porcine respiratory coronavirus
  • PHEV porcine hemagglutinating encephalomyelitis virus
  • PDCoV porcine deltacoronavirus
  • the porcine coronavirus is a PEDV. In some embodiments, the porcine coronavirus is a genotype 2 PEDV.
  • G2a genotype 2a
  • G2b genotype 2b
  • G2 field epidemic or pandemic
  • G2 comprises global field isolates, which are further clustered into 2a and 2b subgroups responsible for previous local epidemic outbreaks in Asia and recent pandemic outbreaks in North America and Asia, respectively.
  • the recombinant porcine coronavirus is derived from PEDV-ZJU/G2/2013 strain.
  • PEDV-ZJU/G2/2013 strain is a virulent strain isolated in Zhejiangzhou, China in 2013.
  • the genomic sequence of PEDV-ZJU/G2/2013 strain can be obtained from GenBank under the accession number KU558701.1.
  • An infectious clone of PEDV-ZJU/G2/2013 can be rescued according to the disclosure of Chinese Patent Application No: 202010162003.0 (published as CN111471709A) .
  • the porcine coronavirus is a SADS-CoV.
  • the recombinant porcine coronavirus is derived from SADS-CoV-CH/GD-01/2017 strain.
  • SADS-CoV-CH/GD-01/2017 strain is a virulent strain isolated in southern China in 2017. An infectious clone of SADS-CoV-CH/GD-01/2017 can be rescued according to the disclosure of Y. -L. Yang, et al. Virology 536 (2019) 110–118.
  • the genomic sequence of the SADS-CoV-CH/GD-01/2017 infectious clone can be obtained from GenBank under the accession number MK977618.
  • An exemplary NSP2 protein of PEDV has an amino acid sequence as shown in SEQ ID NO: 1, or a complete coding sequence as shown in SEQ ID NO: 2.
  • An exemplary NSP2 protein of SADS-CoV has an amino acid sequence as shown in SEQ ID NO: 3, or a complete coding sequence as shown in SEQ ID NO: 8.
  • An exemplary NSP2 protein of TGEV has an amino acid sequence as shown in SEQ ID NO: 4, or a complete coding sequence as shown in SEQ ID NO: 9.
  • An exemplary NSP2 protein of PRCV has an amino acid sequence as shown in SEQ ID NO: 5, or a complete coding sequence as shown in SEQ ID NO: 10.
  • An exemplary NSP2 protein of PHEV has an amino acid sequence as shown in SEQ ID NO: 6, or a complete coding sequence as shown in SEQ ID NO: 11.
  • An exemplary NSP2 protein of PDCoV has an amino acid sequence as shown in SEQ ID NO: 7, or a complete coding sequence as shown in SEQ ID NO: 12.
  • the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 1 and 3-7.
  • the complete NSP2 coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 2 and 8-12.
  • Sequence identity between two polypeptide sequences indicates the percentage of amino acids that are identical between the sequences. Methods for evaluating the level of sequence identity between amino acid or nucleotide sequences are known in the art. For example, sequence analysis softwares are often used to determine the identity of amino acid sequences. For example, identity can be determined by using the BLAST program at NCBI database.
  • sequence identity For determination of sequence identity, see e.g., Computational Molecular Biology, Lesk, A.M., 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 Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • sequence identity with the sequence of SEQ ID NO: X is equivalent to the term “sequence identity with the sequence of SEQ ID NO: X over the length of SEQ ID NO: X” or to the term “sequence identity with the sequence of SEQ ID NO: X over the whole length of SEQ ID NO: X” , respectively.
  • X is any integer selected from 1 to 32 so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.
  • the invention provides a recombinant porcine coronavirus, in particular the recombinant porcine coronavirus as mentioned above, which is
  • a recombinant PEDV with a genomic sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, sequence identity with the recombinant PEDV deposited at CCTCC under the accession number CCTCC V202263 on August 10, 2022.
  • the invention provides an isolated nucleic acid coding for the recombinant porcine coronavirus of the present invention.
  • isolated as used herein in the context of a nucleic acid, in particular means isolated from the environment of its production and preferably processed through one or more purifying steps that separate the nucleic acid from other molecules associated with it in the process of its production.
  • nucleic acid refers to polynucleotides including DNA molecules, RNA molecules, cDNA molecules or derivatives. The term encompasses single as well as double stranded polynucleotides.
  • the nucleic acid of the present invention encompasses isolated polynucleotides (i.e., isolated from its natural context) and genetically modified forms. Moreover, comprised are also chemically modified polynucleotides including naturally occurring modified polynucleotides such as glycosylated or methylated polynucleotides or artificial modified one such as biotinylated polynucleotides. Further, it is to be understood that the recombinant porcine coronavirus of the present invention may be encoded by a large number of polynucleotides due to the degenerated genetic code.
  • the invention provides a vector comprising the nucleic acid of the present invention.
  • vector encompasses phage, plasmid, viral or retroviral vectors as well artificial chromosomes, such as bacterial or yeast artificial chromosomes. Moreover, the term also relates to targeting constructs which allow for random or site-directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below.
  • the vector encompassing the nucleic acid of the present invention preferably, further comprises selectable markers for propagation and/or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art.
  • a plasmid vector can be introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerenes.
  • a plasmid vector may be introduced by heat shock or electroporation techniques.
  • the vector may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
  • the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
  • Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells are well known in the art. They, preferably, comprise regulatory sequences ensuring initiation of transcription and, optionally, poly-Asignals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E.
  • inducible expression control sequences may be used in an expression vector encompassed by the present invention.
  • Such inducible vectors may comprise tet or lac operator sequences or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. For example, the techniques are described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994) .
  • the invention provides an immunogenic composition comprising the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector of the present invention.
  • immunogenic composition refers to a composition that comprises at least one antigen, which elicits an immunological response in the host to which the immunogenic composition is administered.
  • immunological response may be a cellular and/or antibody-mediated immunological response to the immunogenic composition of the present invention.
  • the immunogenic composition induces an immunological response and, more preferably, confers protective immunity against one or more of the clinical signs of a porcine coronavirus infection.
  • the porcine coronavirus in the context of the present invention is most preferably PEDV.
  • the herein mentioned “porcine coronavirus infection” or “infection with a porcine coronavirus” , respectively is preferably understood to be a “PEDV infection” or “infection with a PEDV” , respectively.
  • an "antigen” as used herein refers to, but is not limited to, components which elicit an immunological response in a host to an immunogenic composition or vaccine of interest comprising such antigen or an immunologically active component thereof.
  • the antigen or immunologically active component may be a microorganism that is whole (in inactivated or modified live form) , or any fragment or fraction thereof, which, if administered to a host, can elicit an immunological response in the host.
  • the antigen may be or may comprise complete live organisms in either its original form or as attenuated organisms in a so called modified live vaccine (MLV) .
  • MMV modified live vaccine
  • the antigen may further comprise appropriate elements of said organisms (subunit vaccines) whereby these elements are generated either by destroying the whole organism or the growth cultures of such organisms and subsequent purification steps yielding in the desired structure (s) , or by synthetic processes induced by an appropriate manipulation of a suitable system like, but not restricted to bacteria, insects, mammalian or other species, and optionally by subsequent isolation and purification procedures, or by induction of said synthetic processes in the animal needing a vaccine by direct incorporation of genetic material using suitable pharmaceutical compositions (polynucleotide vaccination) .
  • the antigen may comprise whole organisms inactivated by appropriate methods in a so called killed vaccine (KV) .
  • an "immunological 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, directed specifically to an antigen or antigens included in the immunogenic composition of the present invention.
  • the host will display either a protective immunological response or a therapeutically response.
  • a “protective immunological response” or “protective immunity” will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and/or a lowered duration of infectivity or lowered pathogen titer in the tissues or body fluids or excretions of the infected host.
  • the immunogenic composition of the present invention further comprises a pharmaceutical acceptable carrier.
  • pharmaceutical-acceptable carrier includes any and all solvents, dispersion media, coatings, stabilizing agents, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, adjuvants, immune stimulants, and combinations thereof.
  • “Diluents” can include water, saline, dextrose, ethanol, glycerol, and the like.
  • Isotonic agents can include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others.
  • Stabilizers include albumin and alkali salts of ethylendiamintetracetic acid, among others.
  • the immunogenic composition is described as a “vaccine” .
  • the immunogenic composition of the present invention is a vaccine.
  • said vaccine further comprises an adjuvant.
  • Adjuvants can include aluminum hydroxide and aluminum phosphate, saponins e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge MA) , GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, AL) , water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
  • the emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type) ; isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di- (caprylate/caprate) , glyceryl tri- (caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters.
  • light liquid paraffin oil European Pharmacopea type
  • isoprenoid oil such as squalane or squalene
  • oil resulting from the oligomerization of alkenes in particular of isobutene or decene
  • the oil is used in combination with emulsifiers to form the emulsion.
  • the emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g., anhydromannitol oleate) , of glycol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic products.
  • mannide e.g., anhydromannitol oleate
  • glycol glycol
  • polyglycerol propylene glycol and of oleic
  • isostearic, ricinoleic or hydroxystearic acid which are optionally ethoxylated
  • polyoxypropylene-polyoxyethylene copolymer blocks in particular the Pluronic
  • the invention provides the immunogenic composition of the present invention for use in
  • the porcine coronavirus is PEDV
  • the infection with a porcine coronavirus is an infection with a PEDV
  • the porcine coronavirus is SADS-CoV
  • the infection with a porcine coronavirus is an infection with a SADS-CoV
  • the pig is a piglet or a sow.
  • the invention provides the immunogenic composition of the present invention for use in
  • the invention provides use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector according of the present invention in manufacture of an immunogenic composition for
  • the porcine coronavirus is PEDV
  • the infection with a porcine coronavirus is an infection with a PEDV
  • the porcine coronavirus is SADS-CoV
  • the infection with a porcine coronavirus is an infection with a SADS-CoV
  • the pig is a piglet or a sow.
  • the immunogenic composition further comprises a pharmaceutical acceptable carrier.
  • said immunogenic composition is a vaccine.
  • said vaccine further comprises an adjuvant.
  • the invention provides the use of the recombinant porcine coronavirus of the present invention, the isolated nucleic acid of the present invention, or the vector according of the present invention in the manufacture of an immunogenic composition for
  • the immunogenic composition further comprises a pharmaceutical acceptable carrier.
  • said immunogenic composition is a vaccine.
  • said vaccine further comprises an adjuvant.
  • the invention provides a method for
  • said method comprises administering to the pig an effective amount of the immunogenic composition of the present invention.
  • the porcine coronavirus is PEDV
  • the infection with a porcine coronavirus is an infection with a PEDV
  • the porcine coronavirus is SADS-CoV
  • the infection with a porcine coronavirus is an infection with a SADS-CoV
  • the pig is a piglet or a sow.
  • the invention provides a method for i) reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet, and/or ii) reducing the mortality caused by an infection with a PEDV in a piglet, wherein the piglet is to be suckled by a sow to which the immunogenic composition of the present invention has been administered.
  • the present invention provides a method of reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet and/or ii) reducing the mortality caused by an infection with a PEDV in a piglet, wherein said method comprises
  • Administering the immunogenic composition of the present invention results in the production of antibodies specific for PEDV in said sow.
  • the maternally derived antibodies from said sow are then passively transferred to the newborn piglets via colostrum and/or milk, and thus provide protection of the newborn piglets against PEDV infection.
  • immunizing relates to an active immunization by the administration of an immunogenic composition to a subject to be immunized, thereby causing an immunological response against the antigen included in such immunogenic composition.
  • immunization results in lessening of the incidence of the particular porcine coronavirus such as PEDV infection in a herd or in the reduction in the severity of clinical signs caused by or associated with the particular porcine coronavirus such as PEDV infection.
  • the immunization of a subject in need thereof with the immunogenic compositions as provided herewith results in preventing infection of a subject by porcine coronavirus such as PEDV infection. Even more preferably, immunization results in an effective, long-lasting, immunological-response against porcine coronavirus infection such as PEDV infection. It will be understood that the said period of time will last 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 is to be understood that immunization may not be effective in all subjects immunized. However, the term requires that a significant portion of subjects in a herd are effectively immunized.
  • a herd of subjects is envisaged in this context which normally, i.e., without immunization, would develop clinical signs normally caused by or associated with a porcine coronavirus infection such as PEDV infection. Whether the subjects of a herd are effectively immunized can be determined without further ado by the person skilled in the art.
  • the immunization shall be effective if clinical signs in at least 33%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, still more preferably in at least 95%and most preferably in 100%of the subjects of a given herd are lessened in incidence or severity by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, still more preferably by at least 95%and most preferably by 100%in comparison to subjects that are either not immunized or immunized with an immunogenic composition that was available prior to the present invention but subsequently infected by the particular porcine coronavirus such as PEDV.
  • PEDV porcine coronavirus
  • treating and/or preventing refers to the lessening of the incidence of the particular porcine coronavirus such as PEDV infection in a herd or the reduction in the severity of clinical signs caused by or associated with the particular porcine coronavirus such as PEDV infection.
  • the “treating and/or preventing” generally involves the administration of an effective amount of the immunogenic composition of the present invention to a subject or herd of subjects in need of or that could benefit from such a treatment/prophylaxis.
  • the term “treating” refers to the administration of the effective amount of the immunogenic composition once the subject or at least some subjects of the herd is/are already infected with such porcine coronavirus such as PEDV and wherein such subjects already show some clinical signs caused by or associated with such porcine coronavirus such as PEDV infection.
  • preventing refers to the administration of a subject prior to any infection of such subject with porcine coronavirus such as PEDV or at least where such subject or none of the subjects in a group of subjects do not show any clinical signs caused by or associated with the infection by such porcine coronavirus such as PEDV.
  • porcine coronavirus such as PEDV
  • prophylaxis and “preventing” are used interchangeable in this application.
  • an effective amount means, but is not limited to an amount of antigen, that elicits or is able to elicit an immune response in a subject. Such effective amount is able to lessen the incidence of the particular porcine coronavirus such as PEDV infection in a herd or to reduce the severity of clinical signs of the particular porcine coronavirus such as PEDV infection.
  • clinical signs are lessened in incidence or severity by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, still more preferably by at least 90%, still more preferably by at least 95%and most preferably by 100%in comparison to subjects that are either not treated or treated with an immunogenic composition that was available prior to the present invention but subsequently infected by the particular porcine coronavirus such as PEDV.
  • PEDV porcine coronavirus
  • clinical signs refers to signs of infection of a subject from porcine coronavirus such as PEDV.
  • examples for such clinical signs include but are not limited to virus load, diarrhea, shedding, increased body temperature, mortality, gross pathological lesions in the intestine, depression, weight loss, reduced growth rates and reduced appetite.
  • the clinical signs also include but are not limited to clinical signs that are directly observable from a live animal. Examples for clinical signs that are directly observable from a live animal include weight loss, reduced growth rates, reduced appetite, dehydration, watery diarrhea, vomiting, lameness, lethargy, wasting and unthriftiness and the like.
  • the clinical signs lessened in incidence or severity in a treated subject compared to subjects that are either not treated or treated with an immunogenic composition that was available prior to the present invention but subsequently infected by the particular porcine coronavirus such as PEDV refer to a reduction in weight loss, a lower virus load, a reduction of diarrhea, a reduced shedding, a reduced rectal temperature, mortality, reduced gross pathological lesions in the intestine, or combinations thereof.
  • reducing the mortality means that the mortality is reduced by at least 10%, more preferably by at least 20%, still more preferably by at least 30%, even more preferably by at least 40%, still more preferably by at least 50%, even more preferably by at least 60%, still more preferably by at least 70%, even more preferably by at least 80%, even more preferably by at least 90%, still more preferably by at least 95%most preferably by 100%in comparison to subjects that are not treated (not immunized) but subsequently infected by the particular porcine coronavirus such as PEDV.
  • the subject as mentioned is a pig, such as a piglet, or a sow.
  • the present invention provides a method for inducing the production of antibodies specific for porcine coronavirus such as PEDV in a subject, wherein said method comprises administering the immunogenic composition as described herein to said subject.
  • said subject is a pig, such as a piglet, or a sow.
  • antibodies specific for porcine coronavirus such as PEDV refers to detectable anti-porcine coronavirus such as anti-PEDV antibodies.
  • the anti-porcine coronavirus antibodies in the subject have been developed in response to the vaccination with the vaccine according to the present invention.
  • that antibody titer is detectable and quantifiable in a specific immune assay.
  • the immunogenic composition is administered to the pig within the first two months of age, more preferably, within the first month of age.
  • the immunogenic composition is administered to the pig within the first month of age.
  • the immunogenic composition can be administered to the pig exemplary within the first three weeks of age or within the first two weeks of age.
  • the immunogenic composition is administered to sows during pregnancy and lactation.
  • the immunogenic composition is administered to a pregnant sow at least two times before farrowing, preferably three times before farrowing, more preferably two times before farrowing ( "repeated doses" ) .
  • the pregnant sow is vaccinated with the immunogenic composition of the present invention twice with a single dose of said composition before farrowing.
  • the first administration should occur between 12 and 4 weeks before farrowing, more preferably between 9 and 5 weeks before farrowing.
  • the second administration should occur between 8 and 1 week before farrowing, more preferably between 6 and 1 week before farrowing.
  • the immunogenic composition is administered at two or more doses.
  • said immunogenic composition is administered to sows two times, the first administration between 9 and 5 weeks before farrowing and the second administration between 6 and 1 week before farrowing.
  • the immunogenic composition is, preferably, administered topically or systemically.
  • Suitable routes of administration conventionally used are oral or parenteral administration, such as intranasal, intravenous, intramuscular, intraperitoneal, subcutaneous, as well as inhalation.
  • the immunogenic composition may be administered by other routes as well.
  • said immunogenic composition is administered intranasal, mucosal, oral, intradermal or intramuscular.
  • the immunogenic composition comprises between 1x10 2 to 1x10 9 TCID 50 /ml, more preferably between 1x10 3 to 1x10 7 TCID 50 /ml and most preferably between 1x10 4 to 1x10 6 TCID 50 /ml of the recombinant porcine coronavirus of the present invention.
  • the immunogenic composition comprises between 1x10 3 to 1x10 7 TCID 50 /ml of the recombinant porcine coronavirus of the present invention.
  • TID 50 /ml refers to the measure of infectious virus titer. Specifically the tissue culture infectious dose fifty per milliliter (TCID 50 /ml) gives the dilution of a virus preparation at which 50%of a number of cell cultures inoculated in parallel with that dilution are infected.
  • the invention provides a method of attenuating a porcine coronavirus, comprising deleting the complete nonstructural protein 2 (NSP2) coding sequence from its genome.
  • NSP2 nonstructural protein 2
  • the method comprises deleting the complete NSP2 gene from its genome.
  • the method results in an attenuated virus capable of infecting a host cell.
  • the porcine coronavirus is an alphacoronavirus, a betacoronavirus, or a deltacoronavirus.
  • the porcine coronavirus is selected from the group consisting of porcine epidemic diarrhea virus (PEDV) , swine acute diarrhea syndrome-coronavirus (SADS-CoV) , transmissible gastroenteritis virus (TGEV) , porcine respiratory coronavirus (PRCV) , porcine hemagglutinating encephalomyelitis virus (PHEV) , and porcine deltacoronavirus (PDCoV) .
  • PDV porcine epidemic diarrhea virus
  • SADS-CoV swine acute diarrhea syndrome-coronavirus
  • TGEV transmissible gastroenteritis virus
  • PRCV porcine respiratory coronavirus
  • PHEV porcine hemagglutinating encephalomyelitis virus
  • PDCoV porcine deltacoronavirus
  • the porcine coronavirus is PEDV.
  • the porcine coronavirus is a genotype 2 PEDV.
  • the porcine coronavirus is a PEDV strain PEDV-ZJU/G2/2013.
  • the porcine coronavirus is SADS-CoV.
  • the porcine coronavirus is SADS-CoV strain SADS-CoV-CH/GD-01/2017.
  • the NSP2protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 1 and 3-7.
  • the complete NSP2 coding sequence has an nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 2 and 8-12.
  • the invention provides a method for producing the recombinant porcine coronavirus of the present invention, comprising
  • the host cell is an eukaryotic cell line. In some embodiments, the host cell is a Vero cell, a ST cell, a PK cell (e.g., a PK15 cell) or a BHK cell.
  • the invention provides a method of differentiating animals infected with a porcine coronavirus from animals vaccinated with the immunogenic composition of the present invention, comprising
  • the method comprises determining whether an NSP2 coding sequence, a NSP2 protein, or an antibody against the NSP2 protein is present in the sample.
  • sample refers to a sample of a body fluid, to a sample of separated cells or to a sample from a tissue or an organ.
  • Samples of body fluids can be obtained by well-known techniques and include, preferably, samples of blood, plasma, serum, or urine, more preferably, samples of blood, plasma or serum.
  • Tissue or organ samples may be obtained from any tissue or organ by, e.g., biopsy.
  • Separated cells may be obtained from the body fluids or the tissues or organs by separating techniques such as centrifugation or cell sorting.
  • a recombinant porcine coronavirus comprising a deletion of the complete nonstructural protein 2 (NSP2) coding sequence in its genome.
  • Clause 3 The recombinant porcine coronavirus of clause 1 or 2, wherein said virus is capable of infecting a host cell.
  • Clause 4 The recombinant porcine coronavirus of any one of clauses 1-3, wherein the recombinant porcine coronavirus is attenuated.
  • porcine coronavirus is selected from the group consisting of porcine epidemic diarrhea virus (PEDV) , swine acute diarrhea syndrome-coronavirus (SADS-CoV) , transmissible gastroenteritis virus (TGEV) , porcine respiratory coronavirus (PRCV) , porcine hemagglutinating encephalomyelitis virus (PHEV) , and porcine deltacoronavirus (PDCoV) .
  • porcine epidemic diarrhea virus PEDV
  • SADS-CoV swine acute diarrhea syndrome-coronavirus
  • TGEV transmissible gastroenteritis virus
  • PRCV porcine respiratory coronavirus
  • PHEV porcine hemagglutinating encephalomyelitis virus
  • PDCoV porcine deltacoronavirus
  • Clause 8 The recombinant porcine coronavirus of clause 7, wherein the porcine coronavirus is a genotype 2 PEDV.
  • Clause 11 The recombinant porcine coronavirus of clause 8, wherein the recombinant porcine coronavirus is derived from SADS-CoV-CH/GD-01/2017 strain.
  • Clause 12 The recombinant porcine coronavirus of any one of clauses 1-11, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 1 and 3-7.
  • Clause 13 The recombinant porcine coronavirus of any one of clauses 1-12, wherein the complete NSP2 coding sequence has an nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 2 and 8-12.
  • a recombinant PEDV with a genomic sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, sequence identity with the recombinant PEDV deposited at CCTCC under the accession number CCTCC V202263 on August 10, 2022.
  • Clause 15 An isolated nucleic acid coding for the recombinant porcine coronavirus according to any one of clauses 1 to 14.
  • Clause 16 A vector comprising the nucleic acid of clause 15.
  • Clause 17 An immunogenic composition comprising the recombinant porcine coronavirus according to any one of clauses 1 to 14, the isolated nucleic acid according to clause 15, or the vector according to clause 16.
  • Clause 23 The immunogenic composition for use according to clause 21, wherein the porcine coronavirus is SADS-CoV, and/or the infection with a porcine coronavirus is an infection with a SADS-CoV.
  • Clause 26 Use of the recombinant porcine coronavirus according to any one of clauses 1 to 14, the isolated nucleic acid according to clause 15, or the vector according to clause 16 in the manufacture of an immunogenic composition for
  • Clause 28 The use according to clause 26, wherein the porcine coronavirus is SADS-CoV, and/or the infection with a porcine coronavirus is an infection with a SADS-CoV.
  • Clause 29 The use according to any one of clauses 26-28, wherein the pig is a piglet or a sow.
  • Clause 30 Use of the recombinant porcine coronavirus according to any one of clauses 1 to 14, the isolated nucleic acid according to clause 15, or the vector according to clause 16 in the manufacture of an immunogenic composition for
  • said method comprises administering to the pig an immunogenic composition of any one of clauses 17-20.
  • Clause 35 The method according to clause 34, wherein the porcine coronavirus is PEDV, and the infection with a porcine coronavirus is an infection with a PEDV.
  • Clause 36 The method according to clause 34, wherein the porcine coronavirus is SADS-CoV, and/or the infection with a porcine coronavirus is an infection with a SADS-CoV.
  • Clause 37 The method according to any one of clauses 34-36, wherein the pig is a piglet or a sow.
  • Clause 38 A method for i) reducing or preventing the clinical signs or disease caused by an infection with a PEDV in a piglet, and/or ii) reducing the mortality caused by an infection with a PEDV in a piglet, wherein the piglet is to be suckled by a sow to which the immunogenic composition of any one of clauses 17-20 has been administered.
  • a method of attenuating a porcine coronavirus comprising deleting the complete nonstructural protein 2 (NSP2) coding sequence from its genome.
  • Clause 40 The method of clause 39, which comprises deleting the complete NSP2 gene from its genome.
  • Clause 41 The method of clause 39 or 40, which results in an attenuated virus capable of infecting a host cell.
  • Clause 42 The method of any one of clauses 39-41, wherein the porcine coronavirus is an alphacoronavirus, a betacoronavirus, or a deltacoronavirus.
  • porcine coronavirus is selected from the group consisting of porcine epidemic diarrhea virus (PEDV) , swine acute diarrhea syndrome-coronavirus (SADS-CoV) , transmissible gastroenteritis virus (TGEV) , porcine respiratory coronavirus (PRCV) , porcine hemagglutinating encephalomyelitis virus (PHEV) , and porcine deltacoronavirus (PDCoV) .
  • porcine epidemic diarrhea virus PEDV
  • SADS-CoV swine acute diarrhea syndrome-coronavirus
  • TGEV transmissible gastroenteritis virus
  • PRCV porcine respiratory coronavirus
  • PHEV porcine hemagglutinating encephalomyelitis virus
  • PDCoV porcine deltacoronavirus
  • Clause 45 The method of clause 44, wherein the porcine coronavirus is a genotype 2 PEDV.
  • Clause 46 The method of clause 45, wherein the porcine coronavirus is a PEDV strain PEDV-ZJU/G2/2013.
  • Clause 48 The method of clause 47, wherein the porcine coronavirus is a SADS-CoV strain SADS-CoV-CH/GD-01/2017.
  • Clause 49 The method of any one of clauses 39-48, wherein the NSP2 protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 1 and 3-7.
  • Clause 50 The method of any one of clauses 39-49, wherein the complete NSP2 coding sequence has an nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with any one of SEQ ID NOs: 2 and 8-12.
  • Clause 51 A method for producing the recombinant porcine coronavirus of any one of clauses 1-14, comprising
  • Clause 52 The method of clause 51, wherein the host cell is a Vero cell, a ST cell or a PK cell.
  • Clause 53 A method of differentiating animals infected with a porcine coronavirus from animals vaccinated with the immunogenic composition of any one of clauses 17 to 20, comprising
  • Clause 54 The method according to clause 53, comprising determining whether an NSP2 coding sequence, a NSP2 protein, or an antibody against the NSP2 protein is present in the sample.
  • the supernatant sample of the cells infected with PEDV ZJU/G2/2013 strain of G2 genotype was used to extract RNA by Trizol method, and reverse transcribed to obtain full-length cDNA as the template for amplification.
  • Primer premier 5.0 software (see Table 1 for primer sequences) according to the genomic sequence of ZJU/G2/2013 strain in the GenBank (accession number: KU558701) to amplify nine truncated fragments covering the full length of the PEDV genome except for the NSP2 coding sequence. Primers were synthesized by Shangya Biotechnology Co., Ltd.
  • amino acid sequence of the NSP2 protein of PEDV ZJU/G2/2013 strain is set forth in SEQ ID NO: 1.
  • the NSP2 protein of PEDV ZJU/G2/2013 strain is encoded by the nucleotide sequence set forth in SEQ ID NO: 2.
  • PCR amplification was carried out with the full-length cDNA of PEDV as the template, and the polymerase used was Q5 high-fidelity DNA polymerase, which was purchased from NEB Company.
  • the products obtained by PCR were subjected to 1%agarose gel electrophoresis, and each fragment was consistent with the expected size.
  • the agarose gel containing the target DNA band was cut out, the target fragments were recovered by the kit, and the DNA concentration was determined. Gel recovery kits were purchased from AxyGen.
  • the fragments were assembled by seamless cloning according to the manufacturer's manual using the GeneArt TM High-Order Genetic Assembly System purchased from Thermo Scientific, Inc.
  • the 9 DNA fragments cloned above were ligated to the linearized pSB2 ⁇ vector (Yong-Le Yang, et al. Characterization of a novel bat-HKU2-like swine enteric alphacoronavirus (SeACoV) infection in cultured cells and development of a SeACoV infectious clone. Virology. 2019, 536: 110-118) to obtain PEDV-G2-ZJU- ⁇ Nsp2 infectious clone recombinant plasmid. The product was transformed into DH10B competent cells.
  • a single clone colony was picked and inoculated into 5 mL liquid LB medium containing chloramphenicol (40 ⁇ g/mL) resistance, incubated at 37°C for 16 hours, and then the plasmid was extracted according to the instructions of AxyPrep Plasmid DNA Mini Kit.
  • the PEDV-G2-ZJU- ⁇ Nsp2 infectious clone recombinant plasmid was obtained.
  • the full-length genome sequence was divided into 26 fragments, each of about 1400 bp, for sequencing.
  • the obtained recombinant plasmid was named pSB-PEDV-G2-ZJU- ⁇ Nsp2.
  • the glycerol bacteria containing the plasmid was inoculated into 1L 2 ⁇ YT liquid medium containing chloramphenicol (40 ⁇ g/mL) resistance according to the ratio of bacterial liquid to medium 1: 100, 37°C 200 rpm for 12 h.
  • the PEDV-G2-ZJU- ⁇ Nsp2 infectious cloning plasmid was extracted according to the instructions of the BAC/PAC DNA Isolation Maxi Kit, and stored at -20 °C for later use.
  • the fragment PEDV-N (the N gene sequence) was amplified and cloned into linearized pRK5 plasmid with EcoR I and Xba I double restriction reaction, to obtain the pRK5-PEDV-N plasmid.
  • the rescue of infectious virus was carried out according to Figure 2. Specifically, BHK-21 cells were plated in 12-well plates, placed in a 5%CO 2 , 37°C incubator, and transfected when the cell density reached about 75%. Following the instructions of 3000 transfection kit, the cell culture medium was discarded, the cells were washed twice with Opti-M EM, and 1.5 ⁇ g PEDV-G2-ZJU- ⁇ Nsp2 infectious cloning plasmid and 1 ⁇ g pRK5-PEDV-N plasmid was added to each well. The medium was changed 6 h after transfection and observed daily.
  • rPEDV-G2-ZJU- ⁇ Nsp2 the rescued virus supernatant was collected by repeated freezing and thawing, which was named rPEDV-G2-ZJU- ⁇ Nsp2, abbreviated as rPEDV- ⁇ Nsp2 or rPEDV-delNsp2.
  • Vero cells were plated in a 12-well plate, placed in a 5%CO 2 , 37°C incubator, and infected when the cell density reached about 90%. After 3 days of infection, anti-PEDV-Nsp2 rabbit polyclonal antibody was used to detect rescued viruses by indirect immunofluorescence (IFA) . The PEDV-Santibody was used as positive control. Results were shown in Figure 3.
  • RNA from cells infected with rPEDV-delNsp2 (48h) was extracted by Trizol method, and reverse transcribed into cDNA.
  • the cDNA was used as a template for PCR amplification and identified by agarose gel electrophoresis.
  • Figure 4 shows the RT-PCR identification of the rescue virus rPEDV-delNsp2.
  • the rescued Porcine Epidemic Diarrhea Virus rPEDV-G2-ZJU- ⁇ Nsp2 also named as Porcine Epidemic Diarrhea Virus rPEDV- ⁇ Nsp2 or Porcine Epidemic Diarrhea Virus rPEDV-delNsp2 was deposited at CCTCC (CHINA CENTER FOR TYPE CULTURE COLLECTION) , Wuhan University, Wuhan 430072, P. R. China) under the accession number CCTCC V202263 on August 10, 2022.
  • CCTCC CHINA CENTER FOR TYPE CULTURE COLLECTION
  • Vero cells were plated in a 6-well plate. After the cell density reached 95%, the cells were infected with rPEDV-G2-ZJU- ⁇ Nsp2. After 2 hours of infection, the supernatant was discarded, and pre-warmed 2%low-melting agarose was added to the 6-well plate. After solidification, it was transferred to a 37°C incubator and incubated upside down for 96h. The cells were fixed with 4%paraformaldehyde overnight at room temperature, then the agar was discarded and stained with crystal violet. After standing at room temperature for 30 min, the crystal violet solution was discarded, and the number of plaques were observed after washing with water.
  • Vero cells were plated in 8 35 mm cell culture dishes, placed in a 5%CO 2 , 37 °C incubator, and infected when the cell density reached about 90%.
  • rPEDV-G2-ZJU- ⁇ Nsp2 virus supernatant was collected from the 8 cell culture dishes respectively 2h, 6h, 12h, 18h, 24h, 36h, 48h, and 72h after infection by repeated freezing and thawing.
  • Vero cells were plated in 8 96-well cell culture plates, placed in a 5%CO 2 , 37 °C incubator, and when the cell density reached about 90%, infected with the previously collected 2h, 6h, 12h, 24h, 36h, 48h 72h virus after 10-fold gradient dilution.
  • Vero cells were plated in a 12-well plate, and the cells were infected with rPEDV-G2-ZJU- ⁇ Nsp2 after the cell density reached 95%. Supernatant and cells were collected 2h, 6h, 12h, 18h, 24h, 36h, 48h, 72h after infection. RNA from the samples was quantified by real-time PCR. The results showed that the RNA synthesis amount of rPEDV-G2-ZJU- ⁇ Nsp2 was significantly lower than that of the parental virus 24h after virus infection (Figure 7) .
  • Vero cells were plated in a 12-well cell plate, placed in a 5%CO 2 , 37°C incubator, and infected when the cell density reaches about 90%.
  • PEDV-G2-ZJU- ⁇ Nsp2 cell samples were collected 2h, 6h, 12h, 18h, 24h, 36h, 48h, 72h after infection respectively for Western blotting experiments.
  • the experimental steps of the parental virus WT-PEDV-G2-ZJU are the same as above.
  • the Western blot results were consistent with the RNA quantification results, that is, the expression of rPEDV-G2-ZJU- ⁇ Nsp2 N protein was significantly lower than that of the WT-PEDV-G2-ZJU infection group 24 hours after virus infection (Figure. 8) , indicating that the virus replication ability of Nsp2-deletion strain is weaker than that of the wild strain.
  • the objective of this study is to evaluate the safety of different PEDV vaccine candidates administrated to 3-5-days-old PEDV IgA and IgG negative piglets.
  • PEDV IgA negative sows were randomly allocated to 4 groups, 4 sows per group for groups 1-4, 2 sows for group 5.
  • Piglets 3-5-days-old derived from the sows in different groups received different VPs (10 5.0 TCID 50 /ml, 2ml/piglet, oral (1ml) and intranasal administration (0.5ml/nostril) with each piglet) or MEM (2ml/piglet, oral (1ml) and intranasal administration (0.5ml/nostril) with each piglet) treatment.
  • the piglets included were healthy, had a weight > 0.9 kg on the farrowing day, PEDV genome negative in the fecal sample and PEDV antibody (IgA and IgG) negative in serum before treatment.
  • piglets in group 1, 2, 3 and 4 were inoculated according to Table 2, piglets in group 5 were inoculated with MEM as placebo and serve as negative control.
  • the primary parameters to be evaluated include morbidity and mortality after inoculation.
  • Mortality the dead animals associated with PEDV infection were analyzed as mortality.
  • Morbidity 1. Any Dead animals associated with PEDV infection were analyzed as morbidity; 2. Animals with a clinical score of two for two or more consecutive days were analyzed as morbidity.
  • the secondary parameters to be evaluated include:
  • CLIFA cell culture-immunofluorescence assay
  • RT-PCR Virus shedding
  • Figure 9 shows the morbidity and mortality after inoculation. Clinical score and survival rate are shown in Figure 10.
  • Figure 11 shows the virus shedding results detected by qRT-PCR.
  • Figure 12 shows the PEDV antibody levels at 14dpi in piglet serum detected by CCIFA.
  • Figure 13 shows the PEDV antibody in sow serum after exposure detected by CCIFA.
  • Figure 14 shows the PEDV antibody levels in piglet serum detected by ELISA.
  • rPEDV-D nsp2 deletion mutant
  • rPEDV-delNsp2 candidate in the above Example 2 and in the following this candidate is also termed “rPEDV-D” ) on lactogenic immunity to 3-5-day-old piglets against PEDV GII-WT challenge.
  • sows are assigned to four groups, 4 sows per group in group 1 to 3 to serve as vaccinate/challenge group or challenge control group, 2 sows in group 4 serve as negative control group.
  • Sows included in the study are negative for PEDV antibody and antigen, PPRSV (Ab) , CSFV (Ab) and PRV (gE Ab) .
  • SD means study day, also same as day post immunize in sows, named DPI.
  • Sows Serum sample of sows from SD 0, 14, 21, 28, 35, farrowing day and DPC 0 (challenge day) . Colostrum on farrowing day and milk on the piglets challenged day.
  • Piglets Serum sample of piglets from DPC 0, 7 and 14.
  • Sows Fecal sample of sows from SD 0, 1, 3, 7, 10, 22, 24, 28 and 31.
  • Piglets Fecal sample of piglets from DPC 0, 3, 7, 10 and 14.
  • Morbidity 1) To summarize severity of clinical signs, all animals with a clinical score of two for two clinical observation time point are counted, and the piglets are defined as morbidity. 2) dead animals associated with PEDV infection or PEDV-like lesion, characterized by thin and transparent intestinal walls are analyzed as morbidity.
  • Mortality The dead animals associated with PEDV infection are analyzed as mortality.
  • Piglets in Group 1 show at least a beneficial effect with respect to one or more of the following parameters: reduced clinical signs, virus shedding, morbidity and mortality, as compared to the piglets in the corresponding non-vaccinated challenge control group (Group 2) .
  • the supernatant sample of the cells infected with SADS-CoV CH/GD-01/2017 strain of G2 genotype was used to extract RNA by Trizol method, and reverse transcribed to obtain full-length cDNA as the template for amplification.
  • PCR amplification was carried out with the full-length cDNA of SADS-CoV as the template, and the polymerase used was Q5 high-fidelity DNA polymerase, which was purchased from NEB Company.
  • the products obtained by PCR were subjected to 1%agarose gel electrophoresis, and each fragment was consistent with the expected size.
  • the agarose gel containing the target DNA band was cut out, the target fragments were recovered by a kit, and the DNA concentration was determined. Gel recovery kits were purchased from AxyGen.
  • the fragments were assembled by seamless cloning according to the manufacturer's manual using the GeneArt TM High-Order Genetic Assembly System purchased from Thermo Scientific, Inc.
  • the 15 DNA fragments cloned above were ligated to the linearized pSB2 ⁇ vector to obtain SADS-CoV-CH/GD-01/2017- ⁇ Nsp2 infectious clone recombinant plasmid.
  • the product was transformed into DH10B competent cells.
  • a single clone colony was picked and inoculated into 5 mL liquid LB medium containing chloramphenicol (40 ⁇ g/mL) resistance, incubated at 37°C for 16 hours, and then the plasmid was extracted according to the instructions of AxyPrep Plasmid DNA Mini Kit.
  • the SADS-CoV-CH/GD-01/2017- ⁇ Nsp2 infectious clone recombinant plasmid was obtained.
  • the full-length genome sequence was divided into 26 fragments, each of about 1400 bp, for sequencing.
  • the obtained recombinant plasmid was named pSB-SADS-CoV-CH/GD-01/2017- ⁇ Nsp2.
  • the glycerol bacteria containing the plasmid was inoculated into 1L 2 ⁇ YT liquid medium containing chloramphenicol (40 ⁇ g/mL) resistance according to the ratio of bacterial liquid to medium 1: 100, 37°C 200 rpm for 12 h.
  • the SADS-CoV-CH/GD-01/2017- ⁇ Nsp2 infectious cloning plasmid was extracted according to the instructions of the BAC/PAC DNA Isolation Maxi Kit, and stored at -20 °C for later use.
  • the fragment SADS-CoV-N (the N gene sequence) was amplified and cloned into linearized pRK5 plasmid with EcoR I and Xba I double restriction reaction, to obtain the pRK5-SADS-CoV-N plasmid.
  • the rescue of infectious virus was carried out according to Figure 2. Specifically, BHK-21 cells were plated in 12-well plates, placed in a 5%CO 2 , 37°C incubator, and transfected when the cell density reached about 75%. Following the instructions of 3000 transfection kit, the cell culture medium was discarded, the cells were washed twice with Opti-MEM, and 1.5 ⁇ g SADS-CoV-CH/GD-01/2017- ⁇ Nsp2 infectious cloning plasmid and 1 ⁇ g pRK5-SADS-CoV-N plasmid was added to each well. The medium was changed 6 h after transfection and observed daily.
  • rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 the rescued virus supernatant was collected by repeated freezing and thawing, which was named rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2, abbreviated as rSADS-CoV- ⁇ Nsp2 or rSADS-CoV-delNsp2.
  • Vero cells were plated in a 12-well plate, placed in a 5%CO 2 , 37°C incubator, and infected when the cell density reached about 90%. After 3 days of infection, anti-SADS-CoV-Nsp2 rabbit polyclonal antibody was used to detect rescued viruses by indirect immunofluorescence (IFA) . IFA results are shown in Figure 15B.
  • RNA from cells infected with rSADS-CoV-delNsp2 48h was extracted by Trizol method, and reverse transcribed into cDNA.
  • the cDNA was used as a template for PCR amplification and identified by agarose gel electrophoresis and sequencing. The results are shown in Figure 15C, and the sequences in Figure 15C are designated as SEQ ID NOs: 65 and 66.
  • Vero cells were plated in 8 35 mm cell culture dishes, placed in a 5%CO 2 , 37 °C incubator, and infected when the cell density reached about 90%.
  • rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 virus supernatant was collected from the 8 cell culture dishes respectively 2h, 6h, 12h, 18h, 24h, 36h, 48h, and 72h after infection by repeated freezing and thawing.
  • Vero cells were plated in 8 96-well cell culture plates, placed in a 5%CO 2 , 37 °C incubator, and when the cell density reached about 90%, infected with the previously collected 2h, 6h, 12h, 24h, 36h, 48h 72h virus after 10-fold gradient dilution.
  • rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 virus The safety of rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 virus is evaluated by administering the virus to pigs, with wild-type SADS-CoV CH/GD-01/2017 strain as a control.
  • rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 virus The efficacy of rSADS-CoV-CH/GD-01/2017- ⁇ Nsp2 virus is evaluated by administering the virus to pigs which are then challenged with wild-type SADS-CoV.
  • SEQ ID NO: 1 amino acid sequence of PEDV NSP2
  • SEQ ID NO: 2 complete coding sequence of PEDV NSP2
  • SEQ ID NO: 3 amino acid sequence of SADS-CoV NSP2
  • SEQ ID NO: 4 amino acid sequence of TGEV NSP2
  • SEQ ID NO: 5 amino acid sequence of PRCV NSP2
  • SEQ ID NO: 6 amino acid sequence of PHEV NSP2
  • SEQ ID NO: 7 amino acid sequence of PDCoV NSP2
  • SEQ ID NO: 8 complete coding sequence of SADS-CoV NSP2
  • SEQ ID NO: 9 complete coding sequence of TGEV NSP2
  • SEQ ID NO: 10 complete coding sequence of PRCV NSP2
  • SEQ ID NO: 11 complete coding sequence of PHEV NSP2
  • SEQ ID NO: 12 complete coding sequence of PDCoV NSP2

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Abstract

La présente invention concerne le domaine de la santé animale. En particulier, la présente invention concerne un coronavirus porcin recombinant, en particulier un virus de la diarrhée épidémique porcine (PEDV) recombinante, comprenant une délétion de la séquence codante de la protéine 2 non structurale (NSP2) complète dans son génome. En outre, la présente invention concerne une composition immunogène comprenant le coronavirus porcin recombinant de la présente invention et ses utilisations.
PCT/CN2023/112866 2022-08-12 2023-08-14 Coronavirus porcin recombinant WO2024032805A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111471709A (zh) 2020-03-10 2020-07-31 浙江大学 一种基于dna质粒转染的拯救pedv zju/g2/2013株的反向遗传系统
US11351239B2 (en) * 2010-11-10 2022-06-07 Zoetis Services Llc Effective vaccination against porcine reproductive and respiratory syndrome (PRRS) virus prior to weaning

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11351239B2 (en) * 2010-11-10 2022-06-07 Zoetis Services Llc Effective vaccination against porcine reproductive and respiratory syndrome (PRRS) virus prior to weaning
CN111471709A (zh) 2020-03-10 2020-07-31 浙江大学 一种基于dna质粒转染的拯救pedv zju/g2/2013株的反向遗传系统

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS
"Computational Molecular Biology", 1988, OXFORD UNIVERSITY PRESS
"GenBank", Database accession no. KU558701.1
"Genbank", Database accession no. MK977618
"Sequence Analysis Primer", 1991, M STOCKTON PRESS
AUSUBEL: "Current Protocols in Molecular Biology", 1994, GREEN PUBLISHING ASSOCIATES AND WILEY INTERSCIENCE, N.Y.
GAO XIANG ET AL: "Characterization, pathogenicity and protective efficacy of a cell culture-derived porcine deltacoronavirus", VIRUS RESEARCH, AMSTERDAM, NL, vol. 282, 2 April 2020 (2020-04-02), XP086145643, ISSN: 0168-1702, [retrieved on 20200402], DOI: 10.1016/J.VIRUSRES.2020.197955 *
GRAHAM RACHEL L. ET AL: "The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication", vol. 79, no. 21, 1 November 2005 (2005-11-01), US, pages 13399 - 13411, XP093012794, ISSN: 0022-538X, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1262610/pdf/1282-05.pdf> DOI: 10.1128/JVI.79.21.13399-13411.2005 *
HUNTER ET AL.: "The Theory and Practical Application of Adjuvants", 1995, JOHNWILEY AND SONS, pages: 51 - 94
SAMBROOK: "Molecular Cloning A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
SUN WENCHAO ET AL: "Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China", VIRUS RESEARCH, AMSTERDAM, NL, vol. 278, 18 January 2020 (2020-01-18), XP086017443, ISSN: 0168-1702, [retrieved on 20200118], DOI: 10.1016/J.VIRUSRES.2020.197869 *
SUN Y ET AL: "Attenuation of a virulent swine acute diarrhea syndrome coronavirus strain via cell culture passage", VIROLOGY, ELSEVIER, AMSTERDAM, NL, vol. 538, 21 September 2019 (2019-09-21), pages 61 - 70, XP085895164, ISSN: 0042-6822, [retrieved on 20190921], DOI: 10.1016/J.VIROL.2019.09.009 *
TODD ET AL., VACCINE, vol. 15, 1997, pages 564 - 570
VON HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS
Y. PAN ET AL.: "Zhejiang virulent strain isolated by the inventor in 2017", VETERINARY MICROBIOLOGY, vol. 211, 2017, pages 15 - 21
YONG-LE YANG ET AL.: "Characterization of a novel bat-HKU2-like swine enteric alphacoronavirus (SeACoV) infection in cultured cells and development of a SeACoV infectious clone", VIROLOGY, vol. 536, 2019, pages 110 - 118, XP085801962, DOI: 10.1016/j.virol.2019.08.006

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