# US 9,919,041 B2 · PCV2 immunogenic compositions and methods of producing such compositions > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,919,041 B2 - Document ID: US9919041B2 - Status: Granted - Kind: B2 - Application number: 14/661,689 - Filed: 2015-03-18 - Published: 2015-07-09 - Granted: 2018-03-20 - Earliest claimed: 2004-12-30 - Assignees: Boehringer Ingelheim Vetmedica GmbH - Applicants: Boehringer Ingelheim Vetmedica, Inc. - Inventors: Marc Allan Eichmeyer; Gregory Paul Nitzel; Merrill Lynn Schaeffer - Web page: https://patentgrep.com/patent/US9919041B2 ## CPC - A61K 39/12: Viral antigens - A61P 31/12: Antivirals - C07K 14/005: from viruses - C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) - G01N 33/56983: {Viruses} - A61K 2039/545: {characterised by the dose, timing or administration schedule} - A61K 2039/55: {characterised by the host/recipient, e.g. newborn with maternal antibodies} - A61K 2039/552: {Veterinary vaccine} - A61K 2039/55511: {Organic adjuvants} - A61K 2039/55566: {Emulsions, e.g. Freund's adjuvant, MF59} - C12N 2710/14143: viral genome or elements thereof as genetic vector - C12N 2710/14163: by chemical treatment - C12N 2750/10022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2750/10034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - C12N 2750/10051: Methods of production or purification of viral material - C12N 2770/10034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract An improved method for recovering the protein expressed by open reading frame 2 from porcine circovirus type 2 is provided. The method generally involves the steps of transfecting recombinant virus containing open reading frame 2 coding sequences into cells contained in growth media, causing the virus to express open reading frame 2, and recovering the expressed protein in the supernate. This recovery should take place beginning approximately 5 days after infection of the cells in order to permit sufficient quantities of recombinant protein to be expressed and secreted from the cell into the growth media. Such methods avoid costly and time consuming extraction procedures required to separate and recover the recombinant protein from within the cells. ## Claims ### Claim 1 A kit comprising a container, wherein said container comprises: i) a recombinant PCV2 ORF2 protein administered as a single efficacious dose, wherein said efficacious dose has 4.0 μg to 400.0 μg of said recombinant PCV2 ORF2 protein; and ii) an additional component selected from the group consisting of antibiotics, virus inactivators, inactivated viral vector, virus inactivator neutralizers, and any combinations thereof. ### Claim 2 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for one or more clinical symptoms selected from the group consisting of PCV2 viral load in blood, PCV2 viral load in lymphoid tissues, mortality, weight loss, viral shedding, lymphoid infection, and any combinations thereof. ### Claim 3 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for PCV2 viral load in blood. ### Claim 4 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for PCV2 viral load in lymphoid tissues. ### Claim 5 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for mortality. ### Claim 6 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for weight loss. ### Claim 7 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for one or more clinical symptoms selected from the group consisting of wasting, paleness of skin, unthriftiness, respiratory distress, diarrhea, jaundice, cough, icterus, and any combinations thereof. ### Claim 8 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for lung infections. ### Claim 9 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for PCV2 viral shedding. ### Claim 10 (depends on 1) The kit of claim 1, wherein said single efficacious dose is efficacious for one or more clinical symptoms selected from the group consisting of cough, diarrhea, and any combinations thereof. ### Claim 11 (depends on 1) The kit of claim 1, wherein said single efficacious dose of PCV2 ORF2 protein comprises: i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; ii) any polypeptide that is at least 90% homologous to the polypeptide of i); iii) a polypeptide that is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 4; or iv) any polypeptide that is encoded by a polynucleotide that is at least 90% homologous to the polynucleotide of iii). ### Claim 12 (depends on 1) The kit of claim 1, wherein said additional component comprises an inactivated viral vector. ### Claim 13 (depends on 12) The kit of claim 12, wherein said inactivated viral vector is a recombinant baculovirus coding for the recombinant PCV2 ORF2 protein. ### Claim 14 (depends on 1) The kit of claim 1, wherein said single efficacious dose further comprises an additional component selected from the group consisting of cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, diluents, isotonic agents, immunomodulatory agents, and any combinations thereof. ### Claim 15 (depends on 14) The kit of claim 14, wherein said additional component is an adjuvant consisting of a polymer of acrylic or methacrylic acid cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 16 (depends on 15) The kit of claim 15, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 17 (depends on 16) The kit of claim 16, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 18 (depends on 1) The kit of claim 1, wherein said single efficacious dose further comprises a pharmaceutical acceptable salt. ### Claim 19 (depends on 1) The kit of claim 1, wherein each dose of said single efficacious dose has 4 μg to 50 μg of recombinant PCV2 ORF2 protein therein. ### Claim 20 (depends on 1) The kit of claim 1, wherein each dose of said single efficacious dose has 4 μg to 16 μg of recombinant PCV2 ORF2 protein therein. ### Claim 21 (depends on 1) The kit of claim 1, wherein said single efficacious dose is a vaccine. ### Claim 22 (depends on 1) The kit of claim 1, wherein one dose of said single efficacious dose has a volume of at least 1 ml. ### Claim 23 (depends on 1) The kit of claim 1, wherein one dose of said single efficacious dose has a volume of at least 2 ml. ### Claim 24 (depends on 1) The kit of claim 1, wherein said single efficacious dose is effective for lessening the severity of one or more clinical symptoms associated with a PCV2 infection. ### Claim 25 (depends on 1) A method of providing a protective effect against PCV2 infection in a pig after administration of a single efficacious dose of an immunogenic composition comprising the step of: administering the kit of claim 1 to said pig. ### Claim 26 (depends on 25) The method of claim 25, wherein said recombinant PCV2 ORF2 protein in said single efficacious dose is selected from the group consisting of: i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11; ii) any polypeptide that is at least 90% homologous to the polypeptide of i); iii) a polypeptide that is encoded by a DNA comprising the sequence of SEQ ID NO: 3 or SEQ ID NO: 4; and iv) any polypeptide that is encoded by a polynucleotide that is at least 90% homologous to the polynucleotide of iii). ### Claim 27 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for at least one clinical symptom selected from the group consisting wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea, and any combinations thereof. ### Claim 28 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for wasting. ### Claim 29 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for paleness of the skin. ### Claim 30 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for unthriftiness. ### Claim 31 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for respiratory distress. ### Claim 32 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for diarrhea. ### Claim 33 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for icterus. ### Claim 34 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for jaundice. ### Claim 35 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for lung lesions. ### Claim 36 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for PCV2 viral shedding. ### Claim 37 (depends on 25) The method of claim 25, wherein said single efficacious dose is efficacious for at least one clinical symptoms selected from the group consisting of cough, diarrhea, and any combinations thereof. ### Claim 38 (depends on 25) The method of claim 25, wherein said administration occurs when said pig is about 3 weeks of age. ### Claim 39 (depends on 25) The method of claim 25, wherein said single efficacious dose is administered intramuscularly, subcutaneously, intranasally, orally, or any combination thereof. ### Claim 40 (depends on 25) The method of claim 25, wherein said single efficacious dose in said kit comprises at least 4 μg recombinant PCV2 ORF2. ### Claim 41 A kit comprising a container, wherein said container comprises: i) a recombinant PCV2 ORF2 protein administered as a single efficacious dose, wherein said efficacious dose has 4.0 μg to 400.0 μg of said recombinant PCV2 ORF2 protein; and ii) an additional component selected from the group consisting of antibiotics, virus inactivators, inactivated viral vector, virus inactivator neutralizers, and any combinations thereof, wherein said recombinant PCV2 ORF2 protein in said single efficacious dose is selected from the group consisting of: i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10; and ii) any polypeptide that is at least 90% homologous to the polypeptide of i). ### Claim 42 (depends on 1) The kit of claim 1, wherein said antibiotic is Gentamicin. ### Claim 43 (depends on 41) The kit of claim 41, wherein said antibiotic is Gentamicin.