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WO2001096377A2 - Vaccin contre les tremblements congenitaux du porc - Google Patents

Vaccin contre les tremblements congenitaux du porc Download PDF

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Publication number
WO2001096377A2
WO2001096377A2 PCT/US2001/019220 US0119220W WO0196377A2 WO 2001096377 A2 WO2001096377 A2 WO 2001096377A2 US 0119220 W US0119220 W US 0119220W WO 0196377 A2 WO0196377 A2 WO 0196377A2
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WIPO (PCT)
Prior art keywords
pcv
seq
sequences
pig
nucleic acid
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PCT/US2001/019220
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English (en)
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WO2001096377A3 (fr
Inventor
Suresh K. Mittal
Gregory W. Stevenson
Jiwon Choi
Matti Kiupel
Charles L. Kanitz
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Purdue Research Foundation
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Priority to CA002410229A priority Critical patent/CA2410229A1/fr
Priority to AU2001266940A priority patent/AU2001266940A1/en
Priority to EP01944540A priority patent/EP1290016A2/fr
Priority to MXPA02012352A priority patent/MXPA02012352A/es
Priority to JP2002510517A priority patent/JP2004503234A/ja
Priority to US10/311,423 priority patent/US20040071728A1/en
Publication of WO2001096377A2 publication Critical patent/WO2001096377A2/fr
Publication of WO2001096377A3 publication Critical patent/WO2001096377A3/fr

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    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA 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
    • 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
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/10011—Circoviridae
    • C12N2750/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the identification of an association between porcine circovirus (PCV) and congenital tremors in pigs and to related diagnostic and therapeutic compositions and methods.
  • PCV porcine circovirus
  • the invention more particularly provides specific congenital tremors associated PCN nucleic acids and polypeptides.
  • Porcine circovirus was initially discovered as a noncytopathic contaminant of PK-15, a porcine kidney cell line (Tischer et al., Medizinische Mikrobiologie und Parasitologie 226: 153-167, 1974). This virus was characterized in 1982 (Tischer et al., Nature, 295:64-66, 1982) and classified among the circoviridae with the chicken anemia virus (Yuasa et al., Avian Diseases 23: 366, 1979), the psittacine beak and feather disease virus
  • Circoviral genome consists of a single copy of circular single-stranded ambisense DNA genome (Lukert et al., Sixth Report of the International Committee on Taxonomy of Viruses, 166-168, 1995). The size of the genome varies between 1.7 and 2.3 kb. Circoviruses are non-enveloped and have icosahedral symmetry. The PCV, derived from the PK- 15 cells has first been considered not to be pathogenic.
  • PK-15-PCV Phylogenetic analysis of PK-15-PCV, chicken and psittacine animal circoviruses, plant geminiviruses and nanoviruses (previously known as plant circoviruses) classified PK-15-PCV as most closely related to psittacine beak and feather disease virus; both PK-15-PCV and psittacine circovirus shared features with and were intermediate between the 2 plant viral groups (Niagro et al., Archives of Virology 143:1723-1744, 1998).
  • PK-15-PCV and/or psatticine circovirus originated from a plant nanovirus that infected a vertebrate host and recombined with a vertebrate-infecting RNA virus, most likely a calicivirus (Gibbs & Weiller, Proceedings of the National Academy of Sciences, USA, 96:8022-8027, 1999).
  • PMWS postweaning multisystemic wasting syndrome
  • PMWS-PCV Isolates of PMWS-PCV differ from PK-15-PCV antigenically and genetically (Allan et al., Journal of Veterniary Diagnostic Investigations 10:3-10, 1998; Hamel el al, Journal of Virology 72:5262-5267, 1998). These PMWS-associated PCV were thus referred to asPCV2 as opposed to the original PK-15 cell culture isolate referred to as PCV 1 (Meehan et al, Journal of General Virology 79:2171-2179, 1998).
  • CT Congenital tremors
  • pigs are associated with myelin deficiency and may be caused by genetic abnormalities (Harding et al, Vet Rec 92:527-529, 1973; Patterson et al., J Neurochem 26:481-485, 1976), in-utero trichlorfon toxicity (Knox et al, Nord Veterinaermed 30:538-545, 1978) and in-utero infection with classical swine fever virus (Harding et al, Vet Rec 79:388-390, 1966) or Aujeszky's virus (Mare et al, J Am Vet Med Assoc 164:309-310, 1974).
  • CT Congenital tremors
  • CT type A2 The most common form of CT in North America is transmissible and classified as type A2 (Done et al., Veterinary Annual 16:98-102, 1976).
  • the epidemiology of CT type A2 has been reviewed (Bolin et al, eds. Leman AD, Straw BE, Mengeling WL, D'Allaire S, Taylor DJ, 7th ed., pp.247-249, 1992).
  • PCV was re-isolated from intestinal tissues but not nervous tissues of pigs with CT and not from tissues of normal control pigs derived from sham-inoculated dams.
  • PCV DNA was also found in samples from swine herds with CT, using PCR primers sets designed to amplify a PMWS PCV isolate (G.W. Stevenson et al, IX International Symposium, College Station, Texas, June 1999).
  • genetic analysis of PCV isolates associated with CT has not been reported yet. Thus there is a need to confirm the association of PCV with congenital tremors and to precisely identify what types of PCV are involved in the disease. Indeed only such an analysis may allow to produce diagnostic and therapeutic tools against congenital tremors in pigs, as currently no effective diagnostic tests nor vaccines for congenital tremors are available.
  • the invention is based on the cloning of porcine circovirus (PCV) strains from pigs with congenital tremors.
  • PCV porcine circovirus
  • the present invention provides a method of diagnosis a pathological cause of congenital tremors in a pig, which method comprises determining whether the pig has been infected by a porcine circovirus strain of type 1 or type 2.
  • the invention further provides a method for the prevention or treatment of congenital tremors in a pig, which method comprises administering to the pig an effective amount of an immunogenic PCV 1 or PCV 2 polypeptide or of a nucleic acid encoding this polypeptide.
  • Another subject of the invention is the new PCV nucleic acid sequences identified by the inventors and the polypeptides encoded by these sequences, as well as the new PCV isolates and immunogenic preparations thereof.
  • the invention relates to an isolated porcine circovirus (PCV), which nucleic acid has a sequence that is identical to a sequence selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 7.
  • PCV porcine circovirus
  • the invention further relates to an isolated nucleic acid from porcine circovirus (PCV), which nucleic acid comprises a sequence coding for a circovirus polypeptide having a sequence selected from the group consisting of sequences coded by any of ORF1 to ORF11 of any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 7.
  • PCV porcine circovirus
  • Another subject of the invention is an expression vector comprising this nucleic acid operatively associated with an expression control sequence.
  • This expression vector may be associated to a pharmaceutically acceptable excipient to form a vaccine, that also is part of the invention.
  • the invention is further directed to a host cell comprising this expression vector.
  • the invention also provides a method for producing a PCV protein, which method comprises culturing this host cell under conditions that result in expression of the nucleic acid coding for a circovirus.
  • polypeptides comprising the amino acid sequences encoded by any of ORF1 to ORF11 of SEQ ID NO: 1 to NO: 7 are also described herein, as well as the antibodies directed against these polypeptides.
  • a further subj ect of the invention is a method for culturing a porcine circovirus strain which method comprises introducing a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:l TO SEQ ID NO: 6 into a suitable host cell under conditions that result in the production of porcine circovirus particles having a genome that comprises a sequence selected from the group consisting of SEQ ID NO: 1 TO SEQ ID NO: 6.
  • the invention also encompasses The isolated PCV strains which have a genome comprising a sequence selected from SEQ ID NO: 1 to SEQ ID NO:6.
  • Figure 1 is anucleotide sequence comparison of PMWS-PCV-P1, P2, P3, P4, and CT-PCV-P5, -P6, -P7.
  • the nucleotide sequence of PMWS-PCV and PK-15-PCV were taken from Hamel et al, 1998 and Meehan et al., 1997, respectively.
  • Figure 2 is a schematic representation of 11 ORFs of PMWS-PCV-P 1 ( Figure 1
  • FIG. 2A shows a distance matrix analysis of the full genome of porcine and bovine circovirus isolates. Branch length is proportional to the phylogenetic distance of the isolates. The bar represents 10% difference between two sequences. Unrooted tree; the type 1 strains (CT-P7, PMWS-AF012107, and the PK-15 cell line derived strains) were used as outgroup.
  • PK-15 cell line derivedstrains PK-ISA(U49186), PK-15B (Y09921), PK-15C (AF071879); PMWS strains:
  • PMWS-AF012107 P/48121 (Imp.1011 48121, AF055393), P/ISU-31 (AJ223185), P/48285 (Imp.1011 48285, AF055394), P/AF027217, P/ISUVDL (ISUVDL 98-15237, AF147751), (Imp.999 (AF055391), P/Im ⁇ .1010 (Imp.lOlO-Stoon, AF055392), strains without described disease: Tainan (AF166528), MLTW98 (AF154679), B9 (AF086834), 9741 (AF086835), 412 450 (AF085695), M226 (AF086836), strains with type names referring to restriction enzyme fragmentation pattern: PCV 2-B (AF112862), PCV 2-C (AF109398), PCV 2-D (AF117753), PCV 2-E (AF109399), bovine circovirus: Bovine CV (AF109397).
  • Figure 4 is a protein homology comparison of open reading frame 1 (ORF1) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK- 15-PCV.
  • ORF1 open reading frame 1
  • Figure 5 is a protein homology comparison of open reading frame 2 (ORF2) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK-15-PCV.
  • ORF2 open reading frame 2
  • Figure 6 is a protein homology comparison of open reading frame 3 (ORF3) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS-
  • Figure 7 is a protein homology comparison of open reading frame 4 (ORF4) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK-15-PCV.
  • Figure 8 is a protein homology comparison of open reading frame 5 (ORF5) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK-15-PCV.
  • Figure 9 is a protein homology comparison of open reading frame 6 (ORF6) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCN and the PK-15-PCN.
  • Figure 10 is a protein homology comparison of open reading frame 7 (ORF7) of PMWS- and CT-PCN isolates of the invention with the published sequences of a PWMS-
  • Figure 11 is a protein homology comparison of open reading frame 8 (ORF8) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK-15-PCV.
  • Figure 12 is a protein homology comparison of open reading frame 9 (ORF9) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS- PCV and the PK-15-PCV.
  • Figure 13 is a protein homology comparison of open reading frame 10 (ORFIO) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS-PCV and the PK-15-PCV.
  • ORFIO open reading frame 10
  • Figure 14 is a protein homology comparison of open reading frame 11 (ORFl 1) of PMWS- and CT-PCV isolates of the invention with the published sequences of a PWMS-PCV and the PK-15-PCV.
  • the present invention solves the problem of identifying with certainty the strains of etiological agent for congenital tremors.
  • Cloning and sequencing porcine circovirus (PCV) strains from pigs with congenital tremors and comparing them with PCV strains associated with PMWS have resulted in the development of many useful materials available, such as primers, probes or viral strains.
  • the invention permits cloning strains and culturing virus of known CT pathogenicity.
  • the invention is based, in part, on the discovery that PCV genomic DNA present in neurons and, to a lesser degree, glial cells in the brain and spinal chord of pigs shared very close sequence similarity, greater than about 95%, to the genomic DNA of a strain associated with postweaning multisystemic wasting syndrome (PMWS).
  • PMWS postweaning multisystemic wasting syndrome
  • PCV type I is composed of PK-15-PCVs and our old isolate of CT-PCV
  • PCV type 2 contains PMWS and our new CT isolates.
  • Additional work established the tissue distribution and genetic type of PCV in 1-2 day-old pigs with naturally occurring CT type A2 using in-situ hybridization, polymerase chain reaction (PCR) and frozen-tissue-section indirect fluorescent antibody tests.
  • PCR polymerase chain reaction
  • CT affected and clinically normal pigs originating from four Midwestern U. S. farms undergoing outbreaks of CT type A2 were selected. All CT and most normal pigs were infected with PCV.
  • PCV2 can be transmitted from an infected sow to its litter in utero.
  • PCV2 alone or in combination of a co-factor therefore can be congenitally transmitted.
  • the invention provides diagnostic and therapeutic methods and materials based on these reagents, and identifies PCV, particularly PCV type 2, as a target for diagnostic evaluation and therapeutic, particularly immunological, intervention.
  • PCV refers specifically to a pig circovirus, e.g. , as shown in the distance matrix analysis of the full genome of porcine and bovine circovirus isolates ( Figure 3).
  • PCV means PMWS-PCV-P1 (SEQ ID NO:l),
  • PCV includes CT-PCV-P7 (SEQ ID NO:7), PK-15 PCV (Meehan et al, 1997), and PMWS-PCV (Hamel et al, 1998).
  • a "PCV polypeptide” refers to a polypeptide gene product encoded by a PCV open reading frame (ORF).
  • Each PCV has 11 ORFs, and thus there is an ORFl, ORF2, ORF3, ORF4, ORF5, ORF6, ORF7, ORF8, ORF9, ORFIO, and ORF11 for each strain, as set forth above.
  • ORFs and polypeptides they encode are set forth in the Tables in Example 1, infra, and in Figures 4-14.
  • the term "vaccine” refers to a composition (protein or vector; the latter may also be loosely termed a "DNA vaccine", although RNA vectors can be used as well) that can be used to elicit protective immunity in a recipient.
  • a vaccine of the invention can elicit immunity in a portion of the population, as some individuals may fail to mount a robust or protective immune response, or, in some cases, any immune response. This inability may stem from the individual's genetic background or because of an immunodeficiency condition (either acquired or congenital) or immunosuppression (e.g., treatment with immunosuppressive drugs to prevent organ rej ection or suppress an autoimmune condition).
  • an immunodeficiency condition either acquired or congenital
  • immunosuppression e.g., treatment with immunosuppressive drugs to prevent organ rej ection or suppress an autoimmune condition.
  • immunotherapy refers to a treatment regimen based on activation of a pathogen-specific immune response.
  • a vaccine can be one form of immunotherapy.
  • Charging dendritic cells with PCV polypeptide (a "PCV antigen"), optionally with a stimulatory cytokine such as GM-CSF or Flt3 ligand ex vivo (followed by transplantation into the subject) or in vivo is also a form of immunotherapy.
  • prophylactic administration of the vaccine can protect the recipient subject from PCV infection, e.g., to prevent infectious mononucleosis or lymphoproliferative diseases .
  • Therapeutic administration of the vaccine or immunotherapy can protect the recipient from PCV-infection-mediated pathogenesis, e.g., to treat a disease or disorder such as PMWS or CT.
  • the term "subject” as used herein refers to an animal that supports PCV.
  • the term refers to a pig.
  • the term "vector for expression in pigs" or "porcine expression vector” as used herein means that the vector at least includes a promoter that is effective in porcine cells, and preferably that the vector is safe and effective in pigs. Such a vector will, for example, omit extraneous genes not involved in developing immunity. If it is a viral vector, it will omit regions that permit replication and development of a robust infection, and will be engineered to avoid development of replication competence in vivo.
  • Such vectors are preferably safe for use in pigs on a farm; in a more preferred embodiment, the vector is approved by a government regulatory agency (such as the United States Department of Agriculture (USD A)) for clinical testing or use in pigs. Specific vectors are described in greater detail below.
  • USD A United States Department of Agriculture
  • immunogenic polypeptide means that the polypeptide is capable of eliciting a humoral or cellular immune response, and preferably both.
  • An immunogenic polypeptide is also antigenic.
  • a molecule is "antigenic” when it is capable of specifically interacting with an antigen recognition molecule of the immune system, such as an immunoglobulin (antibody) or T cell antigen receptor.
  • An antigenic polypeptide contains an epitope of at least about 5, and preferably at least about 10, amino acids.
  • An antigenic portion of a polypeptide can be that portion that is immunodominant for antibody or T cell receptor recognition, or it can be a portion used to generate an antibody to the molecule by conjugating the antigenic portion to a carrier polypeptide for immunization.
  • a molecule that is antigenic need not be itself immunogenic, i.e., capable of eliciting an immune response without a carrier.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • An adjuvant can serve as a tissue depot that slowly releases the antigen and also as a lymphoid system activator that non-specifically enhances the immune response (Hood et al., Immunology, Second Ed, 1984, Benjamin/Cummings: Menlo Park, California, p. 384).
  • a primary challenge with an antigen alone, in the absence of an adjuvant will fail to elicit a humoral or cellular immune response.
  • Adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, and potentially useful human adjuvants such as BCG (bacille Calmette-Gueri ) and Corynehacterium parvum.
  • BCG Bacille Calmette-Gueri
  • Corynehacterium parvum Alternatively, or in addition, immunostimulatory proteins, as described below, can be provided as an adjuvant or to increase the immune response to a vaccine.
  • the adjuvant is pharmaceutically acceptable.
  • phrases "pharmaceutically acceptable” or " veterinary acceptable” refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to an animal.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Sterile water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical
  • an isolated biological material can be free of some or all cellular components, i.e., components of the cells in which the native material occurs naturally (e.g., cytoplasmic or membrane component).
  • a material shall be deemed isolated if it is present in a cell extract or if it is present in a heterologous cell or cell extract.
  • nucleic acid molecules an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
  • Isolated nucleic acid molecules include sequences inserted into plasmids, cosmids, artificial chromosomes, and the like, i.e., when it forms part of a chimeric recombinant nucleic aid construct.
  • a recombinant nucleic acid is an isolated nucleic acid.
  • An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein.
  • An isolated organelle, cell, or tissue is removed from the anatomical site in which it is found in an organism.
  • An isolated material may be, but need not be, purified.
  • purified refers to material that has been isolated under conditions that reduce or eliminate the presence of unrelated materials, i.e., contaminants, including native materials from which the material is obtained.
  • a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell.
  • substantially free is used operationally, in the context of analytical testing of the material.
  • purified material substantially free of contaminants is at least 50%) pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art.
  • An "open-reading frame", "coding sequence” or a sequence “encoding” an expression product, such as a RNA, polypeptide, protein, or enzyme is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme.
  • a coding sequence for a protein may include a start codon (usually ATG) and a stop codon.
  • a coding sequence is "under the control of or “operatively associated with” transcriptional and translational control sequences in a cell when RNA pofymerase transcribes the coding sequence into RNA, particularly mRNA, which is then trans-RNA spliced (if it contains introns) and translated into the protein encoded by the coding sequence.
  • the "expression control sequences” are transcriptional or translational control sequences including enhancer, repressor or promoter sequences.
  • a “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • vector means the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.
  • vectors include plasmids, phages, viruses, etc.; they are discussed in greater detail below.
  • Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted.
  • a common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites.
  • a "cassette” refers to a DNA coding sequence or segment of DNA that codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame.
  • foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA.
  • a segment or sequence of DNA having inserted or added DNA, such as an expression vector can also be called a "DNA construct.”
  • a common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell.
  • a plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA.
  • Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme.
  • Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA.
  • Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms.
  • a large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts.
  • Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art.
  • Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.
  • the terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become manifest, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence.
  • a DNA sequence is expressed in or by a cell to form an "expression product" such as a protein.
  • the expression product itself, e.g. the resulting protein, may also be said to be “expressed” by the cell.
  • An expression product can be characterized as intracellular, extracellular or secreted.
  • intracellular means something that is inside a cell.
  • extracellular means something that is outside a cell.
  • a substance is "secreted” by a cell if it appears in significant measure outside the cell, from somewhere on or inside the cell.
  • transfection means the introduction of a foreign nucleic acid into a cell.
  • transformation means the introduction of a "foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence.
  • the introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, may include regulatory or control sequences, such as start, stop, promoter, signal, secretion, or other sequences used by a cell's genetic machinery.
  • the gene or sequence may include nonfunctional sequences or sequences with no known function.
  • a host cell that receives and expresses introduced DNA or RNA has been "transformed” and is a "transformant” or a “clone.”
  • the DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or cells of a different genus or species.
  • the term "host cell” means any cell of any organism that is selected, modified, transformed, grown, or used or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme.
  • Suitable host cells include primary macrophages, particularly porcine macrophages, or such a macrophage cell line, porcine kidney cells, or other mammalian cells in which PCV can produce virus, or that support a viral infection, or both.
  • expression system means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.
  • Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors.
  • the protein of interest is expressed in COS-1 or C 2 C 12 cells.
  • suitable cells include CHO cells, HeLa cells, 293T (human kidney cells), mouse primary myoblasts, and NIH 3T3 cells.
  • Sequence-conservative variants of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position.
  • “Function-conservative variants” are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like).
  • Amino acids with similar properties are well known in the art. For example, arginine, histidine and lysine are hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine.
  • Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm.
  • a “function-conservative variant” also includes a polypeptide or enzyme which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared.
  • homologous in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin,” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g. , myosin light chain, etc.)
  • sequence similarity in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra).
  • sequence similarity when modified with an adverb such as "highly,” may refer to sequence similarity and may or may not relate to a common evolutionary origin.
  • two DNA sequences are "substantially homologous" or “substantially similar” when at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc.
  • sequence comparison algorithms such as BLAST, FASTA, DNA Strider, etc.
  • An example of such a sequence is an allelic or species variant of the specific genes of the invention.
  • Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.
  • two amino acid sequences are "substantially homologous” or “substantially similar” when greater than 80% of the amino acids are identical, or greater than about 90% are similar (functionally identical).
  • the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison,
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al, supra). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m (melting temperature) of 55 °C
  • T m melting temperature
  • Moderate stringency hybridization conditions correspond to a higher T m , e.g., 40% formamide, with 5x or 6x SCC.
  • High stringency hybridization conditions correspond to the highest T m , e.g. , 50% formamide, 5x or 6x SCC.
  • SCC is a 0.15MNaC 1, 0.015M Na-citrate.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • the relative stability (corresponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • a minimum length for a hybridizable nucleic acid is at least about 10 nucleotides; preferably at least about 15 nucleotides; and more preferably the length is at least about 20 nucleotides.
  • standard hybridization conditions refers to a T m of 55 °C, and utilizes conditions as set forth above.
  • the T m is 60 °C; in a more preferred embodiment, the T m is 65 °C.
  • high stringency refers to hybridization and/or washing conditions at 68° C in 0.2XSSC, at 42 °C in 50% formamide, 4XSSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions.
  • oligonucleotide refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybridizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest. Oligonucleotides can be labeled, e.g., with 32 P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated.
  • a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid.
  • oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of the gene, or to detect the presence of nucleic acids encoding the protein.
  • an oligonucleotide of the invention can form a triple helix with a DNA molecule.
  • oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc.
  • PCV porcine circovirus
  • the PMWS-PCV isolates yielded an approximately 99%> nucleotide sequence identity with each other. Furthermore although the new CT-PCV isolates from the late 1990s and the old CT-PCV isolate from the late 1960's originated from neonatal pigs with CT type A2, they shared only 72% nucleotide sequence identity. The genomes of the 2 new CT-PCVs share high sequence homology with the type 2 PMWS-PCV isolates. On the other hand the CT-PCV-7 strain was found to be very close to type 1 PK-15-PCV variants.
  • a subject of the present invention is, thus, an isolated nucleic acid from PCV, which nucleic acid comprises a sequence selected from the group consisting of SEQ ID NO.
  • Another subject of the invention is an isolated- nucleic acid from PCV which has a sequence identical to a sequence selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 7.
  • the nucleic acid sequences of the invention may be useful to design probes or primers for detecting the presence of a PCV nucleic acid in a biological sample.
  • Such probes or primer may be more particularly in the form of oligonucleotides, that specifically hybridize to PCV nucleic acid sequences under conditions of high stringency.
  • Such oligonucleotides which preferably comprise at least about 20 bases that has a sequence found in 20 contiguous bases of SEQ ID NO. 1 to SEQ ID NO. 7 or a complement thereof.
  • the invention also provides a nucleic acid from PCV, which nucleic acid comprises a sequence coding for a circovirus polypeptide having a sequence selected from the group consisting of the aminoacid sequences coded by any of ORFl to ORF11 of any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 7. More particularly the nucleic acid of the invention comprises a sequence selected from any of ORFl to ORFl 1 of any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 7. Among these, ORFs 1, 2, 3 or 4, are particularly interesting.
  • the present invention encompasses conservative sequences, that is to say the sequences which do not change the functionality or the strain-specificity of the sequence described or of the polypeptides encoded by this sequence. These sequences are also called “function-conservative variants”. The sequences differing by degeneracy of the code, which are called “sequence-conservative variants”, also are encompassed.
  • the invention also covers the equivalent sequences in the sense that they are capable of hybridizing with the above sequence under high stringency conditions and/or have a very high homology with the strains of the invention.
  • Cloning vectors comprising any of these nucleic acid sequences are also part of the invention.
  • the preparation of such vectors is well-known by one skilled in the art and is described in the above definitions.
  • These nucleic acid sequences and their fragments can be advantageously used for in vitro or in vivo expression of a polypeptide with the aid of appropriate expression vectors.
  • vectors more particularly comprise a sequence selected from the group consisting of any of ORFl to ORF 11 of any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 7., operatively associated with an expression control sequence.
  • the vectors of the invention may be used to transfect host cells, which are also part of the present invention.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysome fusion), use of a gene gun, or a DNA vector transporter (see, e.g. , Wu et al, J. Biol. Chem.267:963-967, 1992; Wu and Wu, J. Biol. Chem.263:14621-14624, 1988; Hartmutet ⁇ /., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • the invention further provides a method for producing a PCV protein, which method comprises culturing a cell transfected with an expression vector as above-defined under conditions that result in expression of the nucleic acid coding for a circovirus protein.
  • E. coli or baculovirus are the expression systems that may be used (U.S. PatentNo.4,745,051) for that purpose.
  • the coding sequences may be integrated into the baculovirus genome (e.g. the baculovirus Autographa californica Nuclear Polyhedrosis Virus AcNPV) and the latter can be then propagated on insect cells, e.g. Spodoptera frugiperda Sf9 (deposit ATCC CRL 1711).
  • the invention is directed to the expression of PCV polypeptides or proteins in vitro, in vivo or ex vivo.
  • PCV polypeptides or proteins in vitro, in vivo or ex vivo.
  • one skilled in the art may select any suitable expression system, as detailed below.
  • host/expression vector combinations i.e., expression systems
  • useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e. g. , E.
  • yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof
  • vectors useful in eukaryotic cells such as vectors useful in insect or mammalian cells
  • vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • Expression of the protein or polypeptide may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control gene expression include, but are not limited to, cytomegalovirus (CMV) promoter, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, Cell 22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A.
  • CMV cytomegalovirus
  • promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and control regions that exhibit hematopoietic tissue specificity, in particular: beta-globin gene control region which is active in myeloid cells (Mogram et al, Nature 315:338-340, 1985; Kollias et al, Cell
  • hematopoietic stem cell differentiation factor promoters hematopoietic stem cell differentiation factor promoters, erythropoietin receptor promoter (Maouche et al, Blood, 15:2557, 1991), etc; and control regions that exhibit mucosal epithelial cell specificity.
  • Preferred vectors are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, and other recombinant viruses with desirable cellular tropism.
  • viral vectors such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, and other recombinant viruses with desirable cellular tropism.
  • a vector encoding an immunogenic polypeptide can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA.
  • Expression in targeted tissues can be effected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in International Patent
  • Viral vectors commonly used for in vivo or ex vivo targeting and vaccination procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques, 7:980-990, 1992).
  • the viral vectors are replication defective, that is, they are unable to replicate autonomously in the target cell.
  • the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome which are necessary for encapsidating the genome to produce viral particles.
  • DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), vaccinia virus, and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • vaccinia virus and the like.
  • particular vectors include, but are not limited to, a defective herpes virus 1 (HSVl) vector (Kaplitt etal, Molec. Cell. Neurosci. 2:320-330, 1991; International Patent Publication No. WO 94/21807, published September 29, 1994; International Patent Publication No.
  • WO 92/05263, published April 2, 1994 an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet etal. (J. Clin. Invest. 90:626-630, 1992; see also La Salle et al, Science 259:988-990, 1993); and a defective adeno-associated virus vector (Samulski etal, J. Virol.61 :3096-3101, 1987; Samulski etal, J. Virol.63:3822-3828, 1989; Lebkowski et al, Mol. Cell. Biol. 8:3988-3996, 1988).
  • Various companies produce viral vectors commercially, including but by no means limited to Avigen, Inc.
  • Adenovirus vectors are eukaryotic DNA viruses that can be modified to efficiently deliver a nucleic acid of the invention to a variety of cell types.
  • Various serotypes of adenovirus exist. Of these serotypes, preference is given, within the scope of the present invention, to using type 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (see WO94/26914).
  • Those adenoviruses of animal origin which can be used within the scope of the present invention include adenoviruses of canine, bovine, murine (example: Mavl, Beard et al.
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus (e.g. Manhattan or A26/61 strain (ATCC VR-800), for example).
  • CAV2 adenovirus e.g. Manhattan or A26/61 strain (ATCC VR-800), for example.
  • Various replication defective adenovirus and minimum adenovirus vectors have been described (WO94/26914, WO95/02697, WO94/28938, WO94/28152, WO94/12649, WO95/02697 WO96/22378).
  • The- replication defective recombinant adenoviruses according to the invention can be prepared by any technique known to the person skilled in the art (Levrero etal, Gene 101:195 1991; EP 185 573; Graham, EMBO J. 3:2917, 1984; Graham et al, J. Gen. Virol. 36:59, 1977). Recombinant adenoviruses are recovered and purified using standard molecular biological techniques, which are well known to one of ordinary skill in the art.
  • Adeno-associated viruses are DNA viruses of relatively small size which can integrate, in a stable and site-specific manner, into the genome of the cells which they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • the AAV genome has been cloned, sequenced and characterized. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (see WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488 528).
  • the replication defective recombinant AAVs according to the invention can be prepared by cotransfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line which is infected with a human helper virus (for example an adenovirus).
  • ITR inverted terminal repeat
  • Retrovirus vectors In another embodiment the gene can be introduced in a retroviral vector, e.g. , as described in Anderson et al. , U.S. Patent No.5,399,346; Mann et al. ,
  • the retroviruses are integrating viruses which infect dividing cells.
  • the retrovirus genome includes two LTRs, an encapsidation sequence and three coding regions (gag, pol and env).
  • the gag, pol and env genes are generally deleted, in whole or in part, and replaced with a heterologous nucleic acid sequence of interest.
  • vectors can be constructed from different types of retrovirus, such as, HIV, MoMuLV ("murine Moloney leukaemia virus” MS V ("murine Moloney sarcoma virus"), HaS V ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
  • Suitable packaging cell lines have been described in the prior art, in particular the cell line P A317 (US 4,861,719); the PsiCRIP cell line (WO 90/02806) and the GP+envAm-12 cell line (WO 89/07150).
  • the recombinant retroviral vectors can contain modifications within the LTRs for suppressing transcriptional activity as well as extensive encapsidation sequences which may include a part of the gag gene (Bender et al, J. Virol. 61:1639, 1987).
  • Recombinant retroviral vectors are purified by standard techniques known to those having ordinary skill in the art.
  • Retrovirus vectors can also be introduced by DNA viruses, which permits one cycle of retroviral replication and amplifies tranfection efficiency (see WO 95/22617, WO 95/26411, WO 96/39036, WO 97/19182).
  • Lentivirus vectors can be used as agents for the direct delivery and sustained expression of a transgene in several tissue types, including brain, retina, muscle, liver and blood.
  • the vectors can efficiently transduce dividing and nondividing cells in these tissues, and maintain long-term expression of the gene of interest.
  • Lentiviral packaging cell lines are available and known generally in the art. They facilitate the production of high-titer lentivirus vectors for gene therapy.
  • An example is a tetracycline-inducible VSV-G pseudotyped lentivirus packaging cell line which can generate virusparticles at titers greater than 106 IU/ml for at least 3 to 4 days (Kafri, et al, J. Virol., 73: 576-584, 1999).
  • the vector produced by the inducible cell line can be concentrated as needed for efficiently transducing nondividing cells in vitro and in vivo.
  • Non-viral vectors can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.).
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Feigner and Ringold, Science 337:387-388, 1989; see Mackey, et al, Proc. Natl. Acad. Sci. U.S.A.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection), or use of a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263 : 14621 - 14624, 1988 ; Hartmut et al. , Canadian Patent Application No.2,012,311 , filed March 15, 1990; Williams et al, Proc. Natl. Acad. Sci. USA 88:2726-2730, 1991).
  • Receptor-mediated DNA delivery approaches can also be used (Curiel etal. , Hum. Gene Ther. 3:147-154, 1992; Wu and Wu, J. Biol. Chem. 262:4429-4432, 1987).
  • US Patent Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al, C.P. Acad. Sci., 321:893, 1998; WO 99/01157; WO 99/01158; WO 99/01175).
  • the polypeptide that is so produced may be recovered and preferably purified.
  • Methods for purification are well-known in the art.
  • the purification methods including, without limitation, preparative disc-gel electrophoresis and isoelectric focusing; affinity, HPLC, reversed-phase HPLC, gel filtration or size exclusion, ion exchange and partition chromatography; precipitation and salting-out chromatography; extraction; and countercurrent distribution.
  • the polypeptide can then be purified from a crude lysate of the host cell by chromatography on an appropriate solid-phase matrix.
  • antibodies produced against the protein or against peptides derived therefrom can be used as purification reagents.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • polyclonal antibodies to PCV polypeptides or derivative or analog thereof may be used for the production of polyclonal antibodies to PCV polypeptides or derivative or analog thereof.
  • various host animals can be immunized by injection with the antigenic polypeptide, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • the immunized animal is of the same species as the animal who will receive the antibodies in passive immunization, to avoid allergic reactions to the antibodies.
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497, 1975), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 4:72, 1983; Cote et al, Proc. Natl. Acad. Sci. U.S.A. 80:2026- 2030, 1983), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (International Patent Publication No. WO 89/12690, published 28 December, 1989).
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme- linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme- linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • the present invention further relates to a method of culturing a porcine circovirus strain which method comprises introducing a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO:l TO SEQ ID NO: 6 into a suitable host cell under conditions that result in the production of porcine circovirus particles having a genome that comprises a sequence selected from the group consisting of SEQ ID NO:l TO SEQ ID NO:6.
  • Sus ser of a or Tayassu tajacu more preferably neuronally derived pig cells (e.g, glial cells), pig kidney cells (e.g., PK-15 cells) or pig macrophage cells may be infected by introduction of PCV nucleic acids of the invention (e.g., single or preferably double stranded genomic DNA or one or more plasmids comprising PCV genomic DNA) into the cells.
  • PCV nucleic acids of the invention e.g., single or preferably double stranded genomic DNA or one or more plasmids comprising PCV genomic DNA
  • PCV nucleic acids of the invention e.g., single or preferably double stranded genomic DNA or one or more plasmids comprising PCV genomic DNA
  • PCV nucleic acids of the invention e.g., single or preferably double stranded genomic DNA or one or more plasmids comprising PCV genomic DNA
  • Example 1 includes a procedure for trans
  • one or more clones from the infected cell line may be selected and propagated.
  • the cells from the selected clones may be stored (e.g, frozen) and used as a master cell bank from which samples may be taken and used to generate multiple working cell lines.
  • Viral particles which are used for vaccine production and as a source of viral proteins may be obtained from the working cell lines.
  • a working cell line may be produced by thawing a sample of a frozen master cell line and expanding the cells in culture; the cells in the expanded cell culture are used as the working cell line.
  • the master cell line may be thawed and grown in a Nunc Cell Factory (Nalge Nunc International; Rochester, NY) for production of a large quantity of cells.
  • Viral particles may be obtained from the working cell lines by methods which are commonly known to those of ordinary skill in the art. For example, viral particles in the culture supernatant of the working cell lines may be harvested, filtered, purified (e.g, by gradient centrifugation) and used for vaccine generation. Diagnosis of Congenital Tremors
  • CT congenital tremors
  • diagnosis refers to the identification of the disease at any stage of its development, and also includes the determination of a predisposition of a foetus or new-born piglet to develop the disease or a predisposition of a sow to transmit the disease to the foetus.
  • the diagnostic method of the invention may involve the detection of any
  • the repartition of PCV strains between the two types is shown in Figure 3.
  • PCV strains of Type 1 more particularly include PK-15 PCV or the so- called "CT-PCV-7" strain that comprises nucleic acid sequence SEQ ID NO. 7.
  • PCV strains of Type 2 more particularly include the strains that comprise a nucleic acid sequence of any of SEQ ID NO. 1 to SEQ ID NO. 6.
  • PMWS (“PMWS-PCV”) are also included in the target PCV strains.
  • the diagnostic method of the invention may be performed by any standard technique well-known by one skilled in the art, as reviewed for example, in the International Application WO 99/18214.
  • the determination of the infection may encompass detecting the presence of a PCV nucleic acid in a biological sample of the test pig.
  • the determination of the infection may encompass detecting the presence of a PCV polypeptide in a biological sample of the test pig
  • the determination of the infection may be effected by detecting the presence of an antibody directed against a PCV polypeptide in a biological sample of the test pig.
  • the biological sample may be of any kind, including a fluid sample (blood, plasma, serum, cerebrospinal fluid, etc.) or an organ or tissue sample (ganglions, liver, etc.). Cells or cell extracts from the central nervous system may be used more particularly for post-mortem diagnosis.
  • a fluid sample blood, plasma, serum, cerebrospinal fluid, etc.
  • an organ or tissue sample ganglions, liver, etc.
  • Cells or cell extracts from the central nervous system may be used more particularly for post-mortem diagnosis.
  • test samples and methods are those that can be easily implemented by a veterinarian or the animal breeder on the farm. Accordingly, Western blot, immunofluorescence, ELISA or immunochromatography suit these applications very well.
  • a polypeptide of the invention or epitopic fragment thereof are immobilized onto a selected surface, for example, a surface capable of binding proteins such as the wells of a polystyrene microtiter plate.
  • a nonspecific protein such as a solution of bovine serum albumin (BSA) that is known to be antigenically neutral with regard to the test sample may be bound to the selected surface.
  • BSA bovine serum albumin
  • the immobilizing surface is then contacted with a sample, such as clinical or biological materials, to be tested in a manner conductive to immune complex (antigen/antibody) formation.
  • a sample such as clinical or biological materials
  • This may include diluting the sample with diluents, such as solutions of BSA, bovine gamma globulin (BGG) and/or phosphate buffered saline (PBS)/Tween.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the sample-contacted surface is washed to remove non-immunocomplexed material.
  • the washing procedure may include washing wiht a solution, such as PBS/Tween or borate buffer.
  • the second antibody may have an associated activity such as an enzymatic activity that will generate, for example, a color development upon incubating with an appropriate chromogenic substrate. Quantification may then be achieved by measuring the degree of color generation using, for example, a visible spectra spectrophotometer.
  • hybridization probes in solution hybridizations and in embodiments employing solid-phase procedures.
  • the test is adsorbed or otherwise affixed to a selected matrix or surface.
  • the fixed, single-stranded nucleic acid is then subjected to specific hybridization with selected probes.
  • one skilled in the art may use oligonucleotide primers in an amplification technique, such as PCR ("polymerase chain reaction"), to specifically amplify the target PCV nucleic acid potentially present in the biological sample.
  • PCR polymerase chain reaction
  • examples of such primers are given in Table 1 of Example 1.
  • the present invention contemplates vaccination or passive immunization to prevent or treat congenital tremors.
  • the antigenic or immunogenic compositions of the invention are broadly applicable to protect a pig or its progeny from infection by porcine circovirus.
  • the term "protect” is used herein to mean for the treatment or prevention of PCV infection, and congenital tremors.
  • any animal susceptible to this type of infection can be vaccinated.
  • Pigs may be treated at any age, and include new-bom piglets. Treatment of sows is particularly useful to protect foetus.
  • the present invention more particularly relates to antigenic or immunogenic compositions that comprise a circovirus antigen, a veterinary acceptable vehicle or excipient and generally a veterinary acceptable adjuvant.
  • immunogenic composition elicits an immunological response which can, but need not be, protective.
  • a vaccine composition elicits a protective response.
  • immunogenic composition includes a vaccine composition (as the former term can be protective composition).
  • the subject of the invention also is a method of immunization or of vaccination against congenital tremors, comprising the administration of an immunogenic composition or a vaccine against the porcine circovirus.
  • This method of immunization or vaccination uses in particular the vaccines as defined below.
  • a subject of the present invention is thus an antigenic preparation directed against congenital tremors (CT), comprising at least one porcine circovirus (PCV) antigen.
  • This antigen may consist of an attenuated live whole PCV, an inactivated whole PCV, a subunit antigen, a recombinant live vector, or a DNA vector.
  • a subject of the invention is an isolated porcine circovirus strain, which has a genome comprising a nucleic acid sequence selected from the group consisting of any of ORFl to ORFl l of any of the sequences of SEQ ID NO. 1 to SEQ ID NO. 7.
  • Purified preparations of virions may be obtained by one skilled in the art knowing these sequences.
  • These virus may be used in an antigenic composition for vaccinating pigs against congenital tremors.
  • the virus particles may be attenuated, inactivated, or killed, according to standard techniques well known by one skilled in the art, as described below.
  • the circoviruses may be obtained after passage in cells, in particular cell lines, e.g. PK/15 cells.
  • the culture supernatants or extracts, optionally purified by standard techniques, may be used as antigenic preparation.
  • the attenuation may be carried out according to the customary methods, e.g. by passage on cells, preferably by passage on pig cells, especially cell lines, such as PK 15 cells (for example from 50 to 150, especially of the - order of 100, passages).
  • These immunogenic compositions and vaccines comprise in general a veterinary acceptable vehicle or a diluent, with optionally in addition a veterinary acceptable adjuvant as well as optionally a freeze-drying stabilizer.
  • These antigenic preparations, immunogenic compositions and vaccines will preferably comprise from 10 3 to 10 7 TCID50 of the attenuated virus in question.
  • the inactivated immunogenic compositions and vaccines comprise, in addition, a veterinary acceptable vehicle or a diluent, with optionally in addition a veterinary acceptable adjuvant.
  • the circoviruses according to the invention are inactivated according to techniques known to persons skilled in the art.
  • the inactivation will be preferably carried out by the chemical route, e.g. by exposing the antigen to a chemical agent such as formaldehyde (formalin), paraformaldehyde, .beta.-propiolactone or ethyleneimine or its derivatives.
  • a chemical agent such as formaldehyde (formalin), paraformaldehyde, .beta.-propiolactone or ethyleneimine or its derivatives.
  • the preferred method of inactivation will be herein the exposure to a chemical agent and in particular to ethyleneimine or to .beta.-propiolactone.
  • the antigenic preparations, immunogenic compositions and vaccines will preferably comprise from 10 5 to 10 8 TCID50 of the inactivated whole virus in question.
  • the attenuated or inactivated antigenic preparations and the attenuated or inactivated immunogenic compositions and vaccines according to the invention will be supplemented with adjuvant, advantageously by being provided in the form of emulsions, for example water-in-oil or oil-in- water, according to techniques well known to persons skilled in the art.
  • adjuvant character will also come from the incorporation of a customary adjuvant compound into the active ingredient.
  • the adjuvants which may be used there may be mentioned by way of example aluminium hydroxide, the saponines (e.g. Quillaja saponin or Quil A; see
  • Vaccine Design The Subunit and Adjuvant Approach, 1995, edited by Michael F. Powel and Mark J. Newman, Plennum Press, New- York and London, p.210), Avridine.RTM. (Vaccine Design p. 148), DDA (Dimethyldioctadecyl-ammonium bromide, Vaccine Design p. 157), Polyphosphazene (Vaccine Design p. 204), or alternatively oil-in- water emulsions based on mineral oil, squalene (e.g. SPT emulsion, Vaccine Design p. 147), squalene (e.g. MF59, Vaccine Design p.
  • squalene e.g. SPT emulsion, Vaccine Design p. 147
  • squalene e.g. MF59, Vaccine Design p.
  • subunit antigen refers to an antigenic PCV polypeptide or an antigenic fragment thereof.
  • subunit or polypeptide vaccine refers to a vaccine comprising an immunogenic polypeptide and, generally, an adjuvant.
  • a “vector vaccine” comprise “recombinant live vectors” or "DNA vectors".
  • recombinant live vector refers to the vectors used to express an antigenic or immunogenic polypeptide for in vivo or ex vivo vaccination.
  • Preferred vectors are viral vectors, such as DNA-based vectors and retroviral vectors.
  • live vectors there may be used preferably live viruses, preferably capable of multiplying in pigs, nonpathogenic for pigs (naturally nonpathogenic or rendered as such), according to techniques well known to persons skilled in the art.
  • DNA vectors can also be used as vectors (WO 90/11092, WO 93/19813,WO 94/21797, WO 95/20660).
  • the vector is administered in vivo, but ex vivo transduction of appropriate antigen presenting cells, such as dendritic cells, with administration of the transduced cells in vivo, is also contemplated.
  • the vector may be in the form of a DNA molecule that can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.).
  • This embodiment is herein referred to as the "DNA vector” technology.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection (Feigner, et. al., Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417, 1987; Feigner and Ringold, Science 337:387-388, 1989; see Mackey, et al, Proc. Natl. Acad.
  • lipid compounds and compositions for transfer of nucleic acids are described in International Patent Publications WO95/18863 and WO96/17823, and in U.S. Patent No. 5,459,127.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al, supra).
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • a nucleic acid in vivo, is also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., International Patent Publication WO95/21931), peptides derived from DNA binding proteins (e.g., International Patent Publication WO96/25508), or a cationic polymer (e.g. , International Patent Publication
  • DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun (ballistic transfection), or use of a DNA vector transporter (see, e.g., Wu et al, J. Biol. Chem. 267:963-967, 1992; Wu and Wu, J. Biol. Chem. 263:14621-14624, 1988; Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990; Williams et al, Proc. Natl. Acad. Sci. USA 88:2726-
  • polypeptide vaccine formulations can be delivered by subcutaneous (s.c), intraperitoneal (i.p.), intramuscular (i.m.), subdermal (s.d.), intradermal (i.d.), or by administration to antigen presenting cells ex vivo followed by administration of the cells to the subject.
  • any of the gene delivery methods described above can be used to administer a vector vaccine to a subject, such as naked DNA and RNA delivery, e.g., by gene gun or direct injection.
  • Vaccination effectiveness may be enhanced by co-administration of an immunostimulatory molecule, such as an immunostimulatory or immunopotentiating, cytokine, lymphokine, or chemokine with the vaccine, particularly with a vector vaccine.
  • an immunostimulatory molecule such as an immunostimulatory or immunopotentiating, cytokine, lymphokine, or chemokine
  • cytokines or cytokine genes such as interleukin (IL)-l, IL-2, IL-3, IL-4, IL- 12, IL-13, granulocyte-macrophage (GM)-colony stimulating factor (CSF), macrophage inflammatory factor, as well as some key costimulatory molecules or their genes (e.g., B7.1, B7.2) can be used.
  • Mucosal vaccine strategies are particularly effective for many pathogenic bacteria, since infection often occurs via the mucosa.
  • mucosal vaccination strategies for both polypeptide and DNA vaccines are contemplated. While the mucosa can be targeted by local delivery of a vaccine, various strategies have been employed to deliver immunogenic proteins to the mucosa (these strategies include delivery of DNA vaccines as well, e.g., by using the specific mucosal targeting proteins as vector targeting proteins, or by delivering the vaccine vector in an admixture with the mucosal targeting protein).
  • the immunogenic polypeptide or vector vaccine can be administered in an admixture with, or as a conjugate or chimeric fustion protein with, cholera toxin, such as cholera toxin B or a cholera toxin A/B chimera (Hajishengallis et al, J Immunol., 154:4322-32, 1995; Jobling and Holmes, Infect Immun., 60:4915-24 , 1992).
  • Mucosal vaccines based on use of the cholera toxin B subunit have been described (Lebens and Holmgren, Dev Biol Stand 82:215-27, 1994).
  • an admixture with heat labile enterotoxin (LT) can be prepared for mucosal vaccination.
  • mucosal immunization strategies include encapsulating the immunogen in microcapsules (U.S. Patents No. 5,075,109, No. 5,820,883, and No. 5,853,763) and using an immunopotentiating membranous carrier (WO 98/0558).
  • Immunogenicity of orally administered immunogens can be enhanced by using red blood cells (rbc) or rbc ghosts (U.S. Patent No. 5,643,577), or by using blue tongue antigen (U.S. Patent No. 5,690,938).
  • Systemic administration of a targeted immunogen can also produce mucosal immunization (see, U.S. Patent No. 5,518,725).
  • Various strategies can be used to deliver genes for expression in mucosal tissues, such as using chimeric rhinoviruses (U.S. Patent No. 5,714,374), adenoviruses, or specific targeting of a nucleic acid (WO 97/05267).
  • the present invention further contemplates passive immunization with an antibody reactive with, and preferably generated against a PCV antigen. Passive immunization is particularly effective for an incipient or established infection, before the host's immune system can respond.
  • antibodies for use in passive immunization is from convalescent serum of affected animals of the same species as the infected host.
  • antibodies from pig sera can be isolated, preferably by affinity purification against a PCV polypeptide and used to passively immunize newly infected pigs.
  • antibodies can be generated against the immunogenic polypeptide, i.e., the vaccine strategy can also be used to generate antibodies for passive immunization.
  • the anti-PC V antibodies of the invention may be cross reactive, e.g., they may recognize various PCV strains. Polyclonal antibodies have greater likelihood of cross reactivity.
  • an immunogenic polypeptide of the invention can be used in an immunoassay to detect protective antibodies against Congenital Tremors in a pig. Based on the discoveries of the present invention, a high titer of antibody reactive with (specific for) a PCV antigen indicates that the individual may be protected from an infection by PCV, and consequently from congenital tremors. Low or no detectable antibodies reactive with a PCV antigen indicates that the individual may not be protected from infection.
  • the immunoassay of the invention can be used to detect antibody levels in subjects who have been exposed to a PCV infection, e.g., in convalescent serum. It can also be used to detect antibodies in subjects of unknown status. High level antibody titers in such subjects would indicate prior exposure, and possibly protective immunity, to PCV.
  • the immunoassay can be used to evaluate the effectiveness of a vaccine of the invention.
  • any of the immunoassay formats described above can be used in an immunoassay of the invention.
  • an ELISA assay is used in which a PCV polypeptide is adsorbed to the solid phase, sera (preferably in serial dilution) is contacted with the solid phase, and antibody binding is detected, e.g., with a labeled antibody specific for antibodies in the serum.
  • a competitive ELISA format could be used, in which anti-PCV antibodies in the serum sample compete for binding to the solid phase polypeptide against labeled antibodies specific for a PCV polypeptide, e.g., prepared as described above.
  • the polypeptide is labeled, and antibody specific for the polypeptide adsorbed to the solid phase support. The presence of antibodies in the biological sample (e.g., serum) will result in competition for the polypeptide, preventing binding of the label to the adsorbed antibodies.
  • convenient chromatographic immunoassay formats as described below, can be used.
  • immunoassays described here refer to testing for the presence of anti-PCV antibodies in serum
  • any biological sample that provides antibodies can be tested, including without limitation, blood, serum, plasma, tissue samples, lymph, mucosal secretions, sputum, synovial fluid and other inflammatory fluids, and the like.
  • kits of the invention provides a PCV polypeptide and an antibody detector, such as a labeled antibody specific for antibodies from the subject to be tested. The amounts of each can be pre-measured to provide a specified number of assays.
  • the kit will include an assay container, such as a plate, preferably of plastic or a material treated to avoid non-specific binding of protein.
  • an assay container such as a plate, preferably of plastic or a material treated to avoid non-specific binding of protein.
  • the term container has its broadest meaning, i.e., any receptacle for holding material or reagents. It can be fabricated from glass, plastic, ceramic, metal, etc.
  • the kit includes an immunochromatographic membrane or support, to which one reagent, either a PCV polypeptide or an antibody specific for the PCV polypeptide has been irreversibly coupled.
  • immunochromatographic assays are particularly useful under field conditions, where laboratory equipment is not available. Examples of such assays are provided in U.S. Patents No. 5,248,619, No. 5,451,504, No. 5,500,375, No. 5,624,809, and No. 5,658,801.
  • a kit of the invention preferably includes packaging and instructions for its use, e.g., on the packaging or package insert.
  • the immunogenic composition or vaccine according to the invention may therefore comprise another valency corresponding to another pig pathogen chosen from parvovirus (US 6,217,883), from PRRS (Porcine Reproductory and Respiratory Syndrome) and/or Mycoplasma hyopneumoniae, and/or E.
  • the programme of immunization or vaccination and the vaccines according to the invention will combine immunizations or vaccinations against the circovirus, and the PRRS (WO 93/07898, WO 94/18311, FR-A-2 709 966 ; C. Charreyre et al., Proceedings of the 15.sup.th IPVS Congress, Birmingham, England, Jul.
  • the subject of the present invention is therefore also multivalent immunogenic compositions and vaccines, multivaccine kits, and combined immunization or vaccination methods which makes it possible to use such combined immunization or vaccination programs.
  • Example I Sequence Analysis of Porcine Circovirus Associated With Congenital Tremors In Pigs.
  • PMWS or CT followed by microscopic examination of tissue sections. Presence of PCV in tissue was further confirmed by in situ hybridization using PCV-specific oligonucleotide probe, indirect fluorescent assay (IF A) using antiserum against PCV and PCR using primers specific to PCV.
  • IF A indirect fluorescent assay
  • PCV isolates collected from pigs showing signs of PMWS were named as PMWS-PCV-P1, PMWS-PCV-P2, PMWS-PCV-P3, and PMWS-PCV-P4.
  • CT-PCV-P5 Two PCV isolates collected from pigs showing signs of CT were named as CT-PCV-P5, and CT-PCV P6.
  • PCNS PCV contaminated cell line
  • EMEM Eagle's minimum essential medium
  • PCV-specific primers This PCV isolate was named as CT-PCV-P7.
  • PCNS cells grown in EMEM were harvested when cells started floating in the medium.
  • the cell pellet was lysed by SDS-pronase (500 ⁇ g/ml pronase in 10 mM Tris, pH7.4, 10 mM EDTA, and 0.5 % SDS) and incubated at 37 °C overnight.
  • SDS-pronase 500 ⁇ g/ml pronase in 10 mM Tris, pH7.4, 10 mM EDTA, and 0.5 % SDS
  • Lymph nodes for PMWS-PCV-P I, -P2, -P3, -P4, and CT-PCV-P6 and liver for CT-PCV-P5 were homogenized in EMEM using a tissumizer followed by sonication using a sonicator. Tissue homogenates were incubated with the equal volume of SDS-pronase (1 mg/ml pronase in 20 mM Tris, pH7.4, 20 mM EDTA, and 1 % SDS) at
  • PCN2-2 1 679-660 5'CCAACAAAATCTCTATACCC3'
  • PCN4-1 14 611-629 5 ⁇ GCAGGGCCAGAATTCAAC3'
  • PCN4-2 15 1100-1079 5'CGTCTTCGGAAGGATTATTCAG3 '
  • primers PCV2-1. 2-2, 2-3, 2-4, 4-1,4-2 and primers PCV I- 1, 1-2, 7-1, 7-2 are based upon the sequence of PMWS-PCV (Hamel et al.; 1999) and PK-1 5-PCV (Meehan et al. 1997). respectively.
  • PCR products were cloned into the Smal site of pUC18 by blunt-end ligation using T4 DNA ligase (New England Bio Lab).
  • T4 DNA ligase New England Bio Lab
  • pUC18 containing PCR products from nt 1076-679 amplified with PCV2-1 & 2 primers and PCR products from nt 7-1657 amplified with PCV2-3 & 4 primers were digested with Stul and Kpnl.
  • the 4 kb Stul - Kpnl fragment from pUC 18 containing PCV2-1 & 2 amplified PCR product was used to insert a 1.3 kb Stwl - Kpnl fragment from pUC18 containing PCV2-3 & 4 amplified PCR product to result in pUC 18 containing PCV genome from nucleotide 1076-1768 and 1- 1657.
  • the resultant plasmids containing the genome of either PMWS-PCV-P I, -P2 or -P3 were named pPCV-PI, pPCV-P2 and pPCV-P3, respectively. These plasmids when digested with S ⁇ cII 135 produced the linearized-form of complete PCV genomes.
  • the S ⁇ cII-digested-religated or unligated pPCV-P 1, -P2 and -P3 were used to transfect PCV -free PK-1 5 cells.
  • Cells were harvested after third passage and analyzed for the presence of PCV antigen by IFA.
  • a number of cells transfected with S ⁇ cII-digested religated PCV D ⁇ A were positive for PCV antigen, whereas cells transfected with S ⁇ cII-digested unligated PCV D ⁇ A were negative for PCV antigen by IFA.
  • PK-15 cells transfected with PCV D ⁇ A were analyzed by EM. Small spherical viruses, approximately 17nm in diameter, were observed. The detection of PCV antigen and the observation of virus particles in cells transfected with PCV D ⁇ A indicated that the cloned full-length circular PCV D ⁇ A results in viral replication and production of virus particles. Since the full-length PCV genomes amplified by PCR were infectious, the inventors used PCR technique to amplify genomes of other PCV field strains.
  • the inventors sequenced the entire genomes of 4 PCV isolates associated with PMWS (PMWS-PCV P 1, -P2, -P3 and -P4), 2 PCV isolates associated with CT in the late 1990s (CT-PCV-P5 and -P6)), and one PCV isolate associated with CT in the late 1960s (CT-PCV-P7). Sequences of these isolates were compared with that of the previously described PMWS-PCV isolate (Hamel et al, 1998) and PK-15-PCV isolate (Meehan et al, Journal of General Virology 78:221-227, 1997).
  • Genomes of PMWS-PCV-PI, -P2, and -P4 were 1768 nucleotides (nt) long, whereas PMWS-PCV-P3 was 6 nucleotides shorter than the rest of PMWS PCV isolates due to a 6-nt deletion between 820 to 825 nt (Fig. 1). All the PMWS-PCV isolates of the invention had an overall 99% nt sequence identity with each other. The orientation and the relative length of each ORF of PMWS-PCV-PI are shown (Fig. 2A). The coding strand, number of amino acids, and the location of each ORF in the genomes of PMWS-PCVs are listed (Table 2).
  • V indicate viral strand that is encapsidated into virus particles and C indicates the complementary strand to viral strand.
  • ORFl The amino acid sequence of ORFl was highly homologous (approximately 99%) homology at amino acid level) among all these PMWS-PCV isolates. Observed changes in amino acid residues in various ORFs of these PMWS-PCV isolates are listed (Table 3). The ORF2 had more amino acid changes than ORFl among PMWS-PCVS, but still had an approximately 97% homology. Open reading frames 3, 4, 7, and 8 were identical among PMWS-PCV isolates and there were only few changes in the rest of ORFs (Table 3).
  • Stop Indicates the stop codon resulting in the end of ORF.
  • CT-PCV-P5 and -P6 Two new CT-PCV isolates (CT-PCV-P5 and -P6), which were isolated in the late 1990s, were 1768 nt long (Fig. 2). These CT-PCVs had approximately 99% nt sequence identity. Interestingly, new CT-PCV isolates also demonstrated an approximately 99%) nt sequence identity with the new PMWS-PCV isolates.
  • the genomes of PMWS-PCV-PI and CT-PCV-P5 were identical. Both PMWS-PCV and new CT-PCV genomes encode 11 potential ORFs. The amino acid changes in the various ORFs of new CT-PCV compared to those of PMWS-PCV-PI are listed (Table 3). The genome of the old CT-PCV (CT-PCV-P7) was 1759 nucleotides long
  • FIG. 1 The CT PCV-P7 genome also encoded 11 potential ORFs. The orientation and relative length of each ORF are shown in Fig. 2B.
  • V indicates viral strand that is encapsidated into virus particles and C indicates the complementary strand to viral strand.
  • CT-PCV-P7 had only approximately 72% nt sequence identity with PMWS-PCVs and both new CT-PCVS, but shared a surprising approximately 98% nt sequence identity with PK-15-PCV. Amino acid sequences of all ORFs of CT-PCV-P7 were also highly homologous to those of PK-15-PCV. Amino -acid changes in various ORFs of CT-PCV-P7 compared to their counterpart in PK- 15 -PCV are listed (Table 3).
  • N-X-T or N-X-S, where 'X' is any amino acid were conserved in all our PCV isolates except ORF6 of CT-PCV-P7 where the amino acid residue TNT at number 6 was replaced with 'D'.
  • the smaller cluster contained the isolates from he different lineages of the PK-15 cell line, and 2 circovirus strains (PMWS accession number AF012107 and CT-PCV-P7) isolated from different pathological entities (PMWS and CT).
  • the other fairly large cluster contained the remaining 24 isolates, including
  • the goal of this study was to determine genetic variability in PCV associated with CT.
  • the PMWS-PCV isolates yielded an approximately 99%) nt sequence identity with each other and also 96% nt identity to PMWS-associated PCVs isolated in the U.K., Canada, France, and U.S. (Meehan et al, Journal of General Virology 79:2171-2179, 1998; Morozov et al, Journal of Clinical Microbiology 9:2535-2541, 1998; Hamel et al, Journal of Virology 72:5262-5267, 1998; Mankertz et al. Virus Research, 6665-77, 2000) indicating that various PMWS-PCV isolates are highly homologous regardless of their place of origin.
  • PK-15-PCV variants our old CT-PCV-P7 and a single uncharacterized PMWS isolate (AF012107) comprise PCV type I (PCV1) and the remaining 20 recent PMWS-PCVs and the 2 new CT-PCV field isolates (CT-PCV-P5 &
  • PCV 2 PCV type 2
  • PK-15-PCV (a PCV 1 isolate) was clinically nonpathogenic in inoculation studies in weaned pigs (Tischer et al., Archives of Virology 91 :271-276, 1986; Allan et al,
  • CT-PCV-P7 isolate also a PCV1
  • CT-PCV-P7 isolate also a PCV1
  • PK-15-PCV could also cause CT or whether CT-PCV-P7 is pathogenic in weaned pigs.
  • the age, route of infection and/or some other factors may determine the pathogenicity and clinical manifestations of PCV 1 and PCV2.
  • PCV2 is inoculated into germ-free pigs, lesions but not clinical disease typical of PMWS develop by 35 days postinoculation.
  • PCV2 is inoculated with porcine parvovirus or porcine reproductive and respiratory syndrome virus, replication of PCV is enhanced and PMWS is reproduced (Allan et al, Journal of Comparative Pathology 121, 1-11, 1999).
  • Other viruses might enhance PCV replication by directly or indirectly causing division of PCV-target cells.
  • the cluster of type 2 PCVs includes a single bovine-origin circovirus isolate. It is unknown how widespread circoviral infection is in bovids. Based on the high similarity between this single bovine isolate and PCV2's, the clue speculation that the two different PCV lineages previously and simultaneously evolved in porcine and bovine hosts as seen in the case of adenoviruses (Russell & Benk ⁇ , Encyclopedia of Virology, pp. 14-21, 1999) is contradicted. However, such PCV1 and PCV2 strain evolution may have occurred in 2 or more yet unidentified host species.
  • Example II Tissue distribution and Genetic Typing of Porcine Circoviruses In Pigs With Naturally Occurring Congenital Tremors.
  • Samples of cerebrum, cerebellum, pons, spinal cord segments Cl, C4, C7, T3, T6, T9, T12, L2, L5 and S2, lung, liver, kidney, spleen, tonsil, mesenteric and inguinal lymph nodes were collected in neutral buffered formalin or frozen at -20 °C for testing.
  • the detection system consisted of the antidigoxigenin antibody conjugated with alkaline phosphatase (dilution 1 :500) (Boehringer Mannheim Biochemica, Indianapolis, IN) applied at 37°C for 45 min and the substrates "NBT/X-Phos"(Nitro-blue tetrazoliun 5-Bromo-4-chloro-3-indolylphosphate) (Boehringer Mannheim Biochemica, Indianapolis, IN). Dye reduction to insoluble blue formazan was allowed for 45, 90 and 180 min on serial sections.
  • PCR testing Polymerase chain reaction (PCR) testing. PCR testing of cerebellum and liver samples from all pigs was accomplished to determine the genotype of PCV as type 1 ⁇ r type 2. Controls were the same as used for in situ hybridization testing. Tissues were homogenized in equal volume of TE (10 mM Tris-Hcl, 1 mM EDTA, pH 7.5) using a tissumizer. Total cellular DNA was extracted using a standard protocol (Sambrook et al, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, 1989). Primer sets were designed to be specific for PCV (Meehan et al, Journal of General Virology 78:221-227,
  • PCV2 (Hamel et al., Journal of Virology 72:5262-5267, 1998) and were used to amplify PCV sequences by PCR using Vent DNA polymerase (New England Biolab, Inc., Beverly, MA). PCR amplified DNA samples were analyzed on 1 % agarose gel by electrophoresis and the bands of the expected size were visualized with an UV transilluminator. Specificity of PCR results was confirmed by sequence analysis.
  • Frozen-section indirect immunofluorescent antibody testing Samples of liver and cerebellum that had previously tested positive for PCV by in-situ hybridization were selected from one pig from each herd. Indirect fluorescent antibody testing was completed to confirm the presence of PCV-specific antigen. Frozen tissue sections were prepared and indirect fluorescent antibody tests were performed by routine methods using a commercially available polyclonal antibody (Morozov and Paul, Iowa State University, Ames, IA) produced against purified PCV2 raised in a rabbit at a 1 :500 dilution and fluorescein-conjugated murine anti-rabbit IgG at 1 :250 dilution.
  • a commercially available polyclonal antibody (Morozov and Paul, Iowa State University, Ames, IA) produced against purified PCV2 raised in a rabbit at a 1 :500 dilution and fluorescein-conjugated murine anti-rabbit IgG at 1 :250 dilution.
  • Ames, IA swine influenza viruse
  • NYSL, Ames, IA porcine rotavirus
  • NYSL, Ames, IA porcine hemagglutinating encephalomyelitis virus
  • porcine parvovirus American Bioresearch, Sevierville, TN
  • transmissible, gastroenteritis virus American Bioresearch, Sevierville, TN
  • Samples of serum, spleen and lung were tested by virus isolation in swine primary alveolar macrophage cell cultures for porcine reproductive and respiratory syndrome (PRRS) virus.
  • Samples of brain, spleen and tonsil were tested by direct fluorescent antibody tests and virus isolation in swine turbinate cells for pseudorabies virus.
  • Samples of brain, spleen and tonsil were tested in swine turbinate and swine testicular cells for cytopathic viruses.
  • All 4 farms experiencing outbreaks of CT purchased all replacement breeding stock from outside sources. Sources of breeding stock were different for each farm and no farms shared common genetics. On the three farms, where pigs were retained to slaughter age, there was no recent history of PMWS. All pigs with CT that were selected for study were ⁇ 48 hours old and had moderate to severe tremors that were most severe when pigs attempted voluntary movements. Tremors partially abated when pigs rested. All pigs selected as age-matched, clinically normal, control pigs originated from lifters with no CT pigs. All pigs selected for testing were alert, active and otherwise clinically normal.
  • PCV nucleic acid was only in the cytoplasm of most positive macrophages and in the nuclei of few positive macrophages. There were more PCV-positive cells in the central nervous tissues of both CT and clinically normal pigs than in other tissues. Positive cells in the brain and spinal cord were predominantly large neurons with fewer positive small neurons and rare positive oligodendrocytes. Large neurons in cerebral and medullar nuclei were positive, Purkinje cells in the cerebellum were positive and large neurons in the spinal gray matter were positive, especially lower motor neurons. Like macrophages, positive neurons usually had PCVnucleic acid only in the cytoplasm and rarely in the nucleus.
  • PCV infected cells in the central nervous system were more numerous and more widely distributed (Table 5) in CT pigs than in clinically normal pigs.
  • CT pigs had large numbers of positive, large neurons diffusely distributed in the brain and spinal cord.
  • Clinically normal pigs had fewer PCV-positive, large neurons distributed multifocally in the brain and spinal cord.
  • PCV-specific antigens were demonstrated in approximately the same number and type of cells and in the same cellular locations as were PCV-specific nucleic acids with in situ hybridization.
  • PCR testing of cerebellum and liver from all pigs demonstrated amplification of PCV2 specific sequences but not PCV1 specific sequences in all positive pigs from all 4 farms.
  • PCV-infected cells there were many more PCV-infected cells in the brain and spinal cord of CT pigs when compared to clinically normal pigs due to more diffuse distribution and a larger proportion of infected cells.
  • the most commonly infected cells were large neurons in the brain and spinal cord and macrophages in non-neural tissues. Few oligodendrocytes were infected.
  • CT pigs are born with deficient myelin in the brain and spinal cord (Christensen, Nord Veterinaermed 8:921-943, 1956). Other studies demonstrated that CT pigs had abnormally immature myelin composed of disproportionately low cerebroside and high cholesterol esters relative to age-and-genetically matched normal control pigs (Patterson, J Neurochem 26:481-485, 1976). The hypothesis prior to this study was that oligodendrocytes would be the primary cell infected with PCV in the CNS accounting for reduced and abnormal myelin synthesis.
  • PCV2 can be transmitted from an infected sow to its litter.
  • PC V2 alone or in combination of a co-factor could be congenitally transmitted.
  • ORFs representing PMWS-PCV-P 1 ORF 1
  • Example V Development of ELISA to screen for PCV2 antibody
  • the inventors have more particularly developed an ELISA assay for screening pig sera to detect antibody against PCV2 by using bacterially expressed PMWS-PCV-PI ORF2 carboxy-portion purified protein to coat the plates.

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  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention repose sur le clonage de souches du circovirus porcin (CVP) provenant de porcs atteints de tremblements congénitaux. L'invention constitue une première étape dans la mise au point d'applications diagnostiques et thérapeutiques destinées à traiter les tremblements congénitaux chez les porcs. Le procédé diagnostique de l'invention consiste à déterminer si le porc a été infecté par une souche de circovirus porcin de type 1 ou de type 2. L'invention concerne également un procédé permettant de traiter ou de prévenir les tremblements congénitaux chez le porc, selon lequel on administre au porc une quantité efficace d'un polypeptide immunogène de CVP1 ou CVP2 ou d'un acide nucléique codant ledit polypeptide. L'invention concerne également de nouvelles séquences d'acide nucléique du CVP qui ont été identifiées par les inventeurs et les polypeptides codés par ces séquences, ainsi que de nouveaux isolats de CVP et des préparations immunogènes fabriquées à partir de ces derniers.
PCT/US2001/019220 2000-06-15 2001-06-15 Vaccin contre les tremblements congenitaux du porc WO2001096377A2 (fr)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA002410229A CA2410229A1 (fr) 2000-06-15 2001-06-15 Vaccin contre les tremblements congenitaux du porc
AU2001266940A AU2001266940A1 (en) 2000-06-15 2001-06-15 Vaccine for congenital tremors in pigs
EP01944540A EP1290016A2 (fr) 2000-06-15 2001-06-15 Vaccin contre les tremblements congenitaux du porc
MXPA02012352A MXPA02012352A (es) 2000-06-15 2001-06-15 Vacuna para temblores congenitos en cerdos.
JP2002510517A JP2004503234A (ja) 2000-06-15 2001-06-15 ブタの先天性振せん用ワクチン
US10/311,423 US20040071728A1 (en) 2001-06-15 2001-06-15 Vaccine for congenital tremors in pigs

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US21171000P 2000-06-15 2000-06-15
US60/211,710 2000-06-15

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WO2001096377A2 true WO2001096377A2 (fr) 2001-12-20
WO2001096377A3 WO2001096377A3 (fr) 2002-06-13

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PCT/US2001/019220 WO2001096377A2 (fr) 2000-06-15 2001-06-15 Vaccin contre les tremblements congenitaux du porc

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JP (1) JP2004503234A (fr)
AU (1) AU2001266940A1 (fr)
CA (1) CA2410229A1 (fr)
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WO2003049703A2 (fr) 2001-12-12 2003-06-19 Virginia Tech Intellectual Properties, Inc. Clones d'adn infectieux chimeres, circovirus porcins chimeres et utilisation de ceux-ci
WO2005092069A2 (fr) 2004-03-25 2005-10-06 Virginia Tech Intellectual Properties, Inc. Clones d'adn infectieux chimeriques, circovirus porcins chimeriques et utilisations de ceux-ci
US7148015B2 (en) 1997-12-05 2006-12-12 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
WO2006132598A1 (fr) * 2005-06-07 2006-12-14 Temasek Life Sciences Laboratory Limited Vaccins contre le circovirus porcin de type 2
JP2007501207A (ja) * 2003-08-01 2007-01-25 クイーン　メアリー　アンド　ウェストフィールド　カレッジ ミクロソームワクチン
US7223407B2 (en) 1997-12-05 2007-05-29 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
WO2009093251A2 (fr) * 2008-01-24 2009-07-30 Gavish-Galilee Bio Applications Ltd Vaccin contenant un réovirus basé sur une séquence de protéine sigma c
WO2013030608A1 (fr) * 2011-08-30 2013-03-07 Szent István Egyetem Vaccin vétérinaire à base de nanoparticules
RU2520087C2 (ru) * 2006-12-15 2014-06-20 Бёрингер Ингельхайм Ветмедика, Инк. Лечение свиней с помощью антигена pcv2
CN104109704A (zh) * 2014-07-09 2014-10-22 福州大学 一种重组猪圆环病毒衣壳蛋白包涵体的分离纯化方法
EP2460821A3 (fr) * 2004-12-30 2014-12-10 Boehringer Ingelheim Vetmedica, Inc. Compositions immunogènes PCV2 et procédés de production de ces compositions
US9011872B2 (en) 2004-12-30 2015-04-21 Boehringer Ingelheim Vetmedica, Inc. PCV2 immunogenic compositions and methods of producing such compositions
US9011868B2 (en) 2005-12-29 2015-04-21 Boehringer Ingelheim Vetmedica, Inc. Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs
US9101561B2 (en) 2005-12-29 2015-08-11 Boehringer Ingelheim Vetmedica, Inc. Multivalent PCV2 immunogenic compositions and methods of producing such compositions
US9132186B2 (en) 2007-09-04 2015-09-15 Boehringer Ingelheim Vetmedica, Inc. Reduction of concomitant infections in pigs by the use of PCV2 antigen
US9132187B2 (en) 2007-02-13 2015-09-15 Boehringer Ingelheim Vetmedica, Inc. Prevention and treatment of sub-clinical PCVD
US9463231B2 (en) 2005-03-14 2016-10-11 Boehringer Ingelheim Vetmedica, Inc. Immunogenic compositions comprising Lawsonia intracellularis
US9505808B2 (en) 2013-10-02 2016-11-29 Boehringer Ingelheim Vetmedica, Inc. PCV2 ORF2 protein variant and virus like particles composed thereof
US9522182B2 (en) 2007-01-03 2016-12-20 Boehringer Ingelheim Vetmedica, Inc. Prophylaxis and treatment of PRDC
US9561270B2 (en) 2009-09-02 2017-02-07 Boehringer Ingelheim Vetmedica, Inc. Methods of reducing virucidal activity in PCV-2 compositions and PCV-2 compositions with an improved immunogenicity
US9636389B2 (en) 2006-12-11 2017-05-02 Boehringer Ingelheim Vetmedica, Inc. Effective method of treatment of porcine circovirus and Lawsonia intracellularis infections
US9669086B2 (en) 2008-01-23 2017-06-06 Boehringer Ingelheim Vetmedica, Inc. PCV2 Mycoplasma hyopneumoniae immunogenic compositions and methods of producing such compositions
US9919041B2 (en) 2004-12-30 2018-03-20 Boehringer Ingelheim Vetmedica, Inc. PCV2 immunogenic compositions and methods of producing such compositions
US9987348B2 (en) 2013-09-25 2018-06-05 Zoetis Services Llc PCV2B divergent vaccine composition and methods of use
CN111087452A (zh) * 2019-12-18 2020-05-01 中国农业科学院兰州兽医研究所 一种猪圆环病毒2b亚型Cap蛋白的原核可溶性表达方法
CN111154915A (zh) * 2020-01-06 2020-05-15 咸阳职业技术学院 一种猪圆环病毒4型与3型pcr鉴别诊断试剂盒及其检测方法
CN112014561A (zh) * 2020-09-03 2020-12-01 扬州大学 猪圆环病毒4型特异的抗原在制备检测猪圆环病毒4型抗体的试剂盒中的应用及试剂盒
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US7405075B2 (en) 1997-12-05 2008-07-29 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US10052375B2 (en) 1997-12-05 2018-08-21 Zoetics Services LLC Circovirus sequences associated with piglet weight loss disease (PWD)
US9717784B2 (en) 1997-12-05 2017-08-01 Zoetis Services Llc Circovirus sequences associated with piglet weight loss disease (PWD)
US7148015B2 (en) 1997-12-05 2006-12-12 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US9700613B2 (en) 1997-12-05 2017-07-11 Zoetis Services Llc Circovirus sequences associated with piglet weight loss disease (PWD)
US7740866B2 (en) 1997-12-05 2010-06-22 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7604808B2 (en) 1997-12-05 2009-10-20 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7179472B2 (en) 1997-12-05 2007-02-20 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7223407B2 (en) 1997-12-05 2007-05-29 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7223594B2 (en) 1997-12-05 2007-05-29 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7244433B2 (en) 1997-12-05 2007-07-17 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
US7258865B2 (en) 1997-12-05 2007-08-21 Wyeth Circovirus sequences associated with piglet weight loss disease (PWD)
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US9889187B2 (en) 2001-12-12 2018-02-13 Virginia Tech Intellectual Properties, Inc. Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof
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JP4903043B2 (ja) * 2003-08-01 2012-03-21 クイーン メアリー アンド ウェストフィールド カレッジ ミクロソームワクチン
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WO2005092069A3 (fr) * 2004-03-25 2006-06-08 Virginia Tech Intell Prop Clones d'adn infectieux chimeriques, circovirus porcins chimeriques et utilisations de ceux-ci
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US9919041B2 (en) 2004-12-30 2018-03-20 Boehringer Ingelheim Vetmedica, Inc. PCV2 immunogenic compositions and methods of producing such compositions
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US9463231B2 (en) 2005-03-14 2016-10-11 Boehringer Ingelheim Vetmedica, Inc. Immunogenic compositions comprising Lawsonia intracellularis
AU2006255818B2 (en) * 2005-06-07 2010-11-25 Temasek Life Sciences Laboratory Limited Porcine Circovirus Type 2 vaccines
WO2006132605A3 (fr) * 2005-06-07 2007-02-01 Temasek Life Sciences Lab Ltd Vaccins contre circovirus porcin de type 2
WO2006132598A1 (fr) * 2005-06-07 2006-12-14 Temasek Life Sciences Laboratory Limited Vaccins contre le circovirus porcin de type 2
US9315551B2 (en) 2005-06-07 2016-04-19 Temasek Life Sciences Laboratory Ltd. Porcine circovirus type 2 vaccines
US9011868B2 (en) 2005-12-29 2015-04-21 Boehringer Ingelheim Vetmedica, Inc. Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs
US9987349B2 (en) 2005-12-29 2018-06-05 Boehringer Ingelheim Vetmedica, Inc. Use of a PCV2 immunogenic composition for lessening clinical symptoms in pigs
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RU2520087C2 (ru) * 2006-12-15 2014-06-20 Бёрингер Ингельхайм Ветмедика, Инк. Лечение свиней с помощью антигена pcv2
US9522182B2 (en) 2007-01-03 2016-12-20 Boehringer Ingelheim Vetmedica, Inc. Prophylaxis and treatment of PRDC
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WO2009093251A3 (fr) * 2008-01-24 2009-10-22 Gavish-Galilee Bio Applications Ltd Vaccin contenant un réovirus basé sur une séquence de protéine sigma c
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TWI799365B (zh) * 2015-08-31 2023-04-21 德商百靈佳殷格翰維美迪加股份有限公司 用於先天性痙攣症之瘟疫病毒疫苗
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CN112014561B (zh) * 2020-09-03 2022-04-19 扬州大学 猪圆环病毒4型特异的抗原在制备检测猪圆环病毒4型抗体的试剂盒中的应用及试剂盒

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AU2001266940A1 (en) 2001-12-24
JP2004503234A (ja) 2004-02-05
WO2001096377A3 (fr) 2002-06-13
EP1290016A2 (fr) 2003-03-12
MXPA02012352A (es) 2004-09-06

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