# US 7,829,101 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 7,829,101 B2 - Document ID: US7829101B2 - Status: Granted - Kind: B2 - Application number: 12/137,960 - Filed: 2008-06-12 - Published: 2009-01-15 - Granted: 2010-11-09 - Earliest claimed: 2004-12-30 - Assignees: Boehringer Ingelheim Vetmedica GmbH - Inventors: Marc Eichmeyer; Greg Nitzel; Merrill Schaeffer - Web page: https://patentgrep.com/patent/US7829101B2 ## CPC - A61K 39/12: Viral antigens - A61K 33/00: Medicinal preparations containing inorganic active ingredients - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics - A61P 31/12: Antivirals - A61P 31/20: for DNA viruses - A61P 37/00: Drugs for immunological or allergic disorders - A61P 37/04: Immunostimulants - C07K 14/005: from viruses - A61K 2039/5258: {Virus-like particles} - A61K 2039/545: {characterised by the dose, timing or administration schedule} - A61K 2039/552: {Veterinary vaccine} - A61K 2039/55505: {Inorganic adjuvants} - A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers} - 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) ## 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 for an immunogenic composition administered as a single dose immunogenic composition, said kit comprising: i) a container containing a single dose immunogenic composition, wherein said single dose immunogenic composition has 4 μg to 400 μg of recombinant PCV2 ORF2 protein per dose and a polymer of acrylic or methacrylic acid therein, wherein administration of said single dose immunogenic composition provides protective immunity, and ii) a container containing an immunogenic composition comprising PRRS antigen. ### Claim 2 (depends on 1) The kit of claim 1, wherein said recombinant PCV2 ORF2 protein 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, 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; and iv) any polypeptide that is encoded by a polynucleotide that is at least 90% homologous to the polynucleotide of iii). ### Claim 3 (depends on 1) The kit of claim 1, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 4 (depends on 1) The kit of claim 1, wherein each said dose of said single dose immunogenic composition further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salt, and combinations thereof. ### Claim 5 (depends on 4) The kit of claim 4, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 6 (depends on 1) The kit of claim 1, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 7 (depends on 1) The kit of claim 1, wherein said polymer of acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 8 (depends on 6) The kit of claim 6, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 9 (depends on 1) The kit of claim 1, wherein said kit further comprises written instructions or an instruction manual, including the information to administer both immunogenic compositions to a pig. ### Claim 10 (depends on 1) The kit of claim 1, wherein said immunogenic composition containing recombinant PCV2 ORF2 is administered temporally prior to said immunogenic composition comprising PRRS antigen. ### Claim 11 (depends on 1) The kit of claim 1, wherein each dose of said single dose immunogenic composition of recombinant PCV2 ORF2 has a volume of at least 1 ml. ### Claim 12 (depends on 1) The kit of claim 1, wherein each dose of said single dose immunogenic composition of recombinant PCV2 ORF2 has a volume of at least 2 ml. ### Claim 13 (depends on 1) The kit of claim 1, wherein said recombinant PCV2 ORF2 protein is present in each dose of said single dose immunogenic composition in an amount from 4 μg to 100 μg. ### Claim 14 (depends on 1) The kit of claim 1, wherein said recombinant PCV2 ORF2 protein is present in each dose of said single dose immunogenic composition in an amount from 4 μg to 50 μg. ### Claim 15 A kit for an immunogenic composition administered as a single dose immunogenic composition, said kit comprising i) a container containing a single dose immunogenic composition, wherein said single dose immunogenic composition has 4 μg to 16 μg of recombinant PCV2 ORF2 protein per dose and a polymer of acrylic or methacrylic acid therein, and ii) a container containing an immunogenic composition comprising PRRS antigen. ### Claim 16 (depends on 15) The kit of claim 15, wherein said recombinant PCV2 ORF2 protein 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, 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; and iv) any polypeptide that is encoded by a polynucleotide that is at least 90% homologous to the polynucleotide of iii). ### Claim 17 (depends on 15) The kit of claim 15, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 18 (depends on 15) The kit of claim 15, wherein each said dose of said single dose immunogenic composition further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salts, and combinations thereof. ### Claim 19 (depends on 18) The kit of claim 18, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 20 (depends on 15) The kit of claim 15, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 21 (depends on 15) The kit of claim 15, wherein said polymer of acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 22 (depends on 20) The kit of claim 20, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 23 (depends on 15) The kit of claim 15, wherein said kit further comprises written instructions or an instruction manual, including the information to administer both immunogenic compositions to a pig. ### Claim 24 (depends on 15) The kit of claim 15, wherein said immunogenic composition containing recombinant PCV2 ORF2 is administered temporally prior to said immunogenic composition comprising PRRS antigen. ### Claim 25 (depends on 15) The kit of claim 15, wherein each dose of said single dose immunogenic composition of recombinant PCV2 ORF2 has a volume of at least 1 ml. ### Claim 26 (depends on 15) The kit of claim 15, wherein each dose of said single dose immunogenic composition of recombinant PCV2 ORF2 has a volume of at least 2 ml. ### Claim 27 A method for lessening the severity of clinical symptoms associated with PCV2 infection, said method comprising the step of administering a single dose of an immunogenic composition into pigs, wherein said immunogenic composition comprises 4 μg to 400 μg of recombinant PCV2 ORF2 protein per dose, a polymer of acrylic or methacrylic acid, and an immunogenic composition that comprises PRRS antigen, wherein the severity of clinical symptoms are lessened in pigs receiving said immunogenic composition in comparison to pigs not receiving said immunogenic composition and wherein said clinical symptoms are selected from the group consisting of wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea and combinations thereof. ### Claim 28 (depends on 27) The method of claim 27, wherein said recombinant PCV2 ORF2 protein is i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, 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 29 (depends on 27) The method of claim 27, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 30 (depends on 27) The method of claim 27, wherein said immunogenic composition comprising recombinant PCV2 ORF2 protein further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salt, and combinations thereof. ### Claim 31 (depends on 30) The method of claim 30, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 32 (depends on 27) The method of claim 27, wherein said polymer of an acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 33 (depends on 27) The method of claim 27, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 34 (depends on 33) The method of claim 33, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 35 (depends on 27) The method of claim 27, wherein said single dose has a volume of at least 1 ml. ### Claim 36 (depends on 27) The method of claim 27, wherein said single dose has a volume of at least 2 ml. ### Claim 37 A method for lessening the severity of clinical symptoms associated with PCV2 infection, said method comprising the step of administering a single dose of an immunogenic composition into pigs, wherein said immunogenic composition comprises 4 μg to 100 μg of recombinant PCV2 ORF2 protein per dose, a polymer of acrylic or methacrylic acid, and an immunogenic composition that comprises PRRS antigen, wherein the severity of clinical symptoms are lessened in pigs receiving said immunogenic composition in comparison to pigs not receiving said immunogenic composition and wherein said clinical symptoms are selected from the group consisting of wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea and combinations thereof. ### Claim 38 (depends on 37) The method of claim 37, wherein said recombinant PCV2 ORF2 protein is i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, 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 39 (depends on 37) The method of claim 37, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 40 (depends on 37) The method of claim 37, wherein said immunogenic composition comprising recombinant PCV2 ORF2 protein further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salt, and combinations thereof. ### Claim 41 (depends on 40) The method of claim 40, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 42 (depends on 37) The method of claim 37, wherein said polymer of an acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 43 (depends on 37) The method of claim 37, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 44 (depends on 43) The method of claim 43, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 45 (depends on 37) The method of claim 37, wherein said single dose has a volume of at least 1 ml. ### Claim 46 (depends on 37) The method of claim 37, wherein said single dose has a volume of at least 2 ml. ### Claim 47 A method for lessening the severity of clinical symptoms associated with PCV2 infection, said method comprising the step of administering a single dose of an immunogenic composition into pigs, wherein said immunogenic composition comprises 4 μg to 50 μg of recombinant PCV2 ORF2 protein per dose, a polymer of acrylic or methacrylic acid, and an immunogenic composition that comprises PRRS antigen, wherein the severity of clinical symptoms are lessened in pigs receiving said immunogenic composition in comparison to pigs not receiving said immunogenic composition and wherein said clinical symptoms are selected from the group consisting of wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea and combinations thereof. ### Claim 48 (depends on 47) The method of claim 47, wherein said recombinant PCV2 ORF2 protein is i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, 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 49 (depends on 47) The method of claim 47, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 50 (depends on 47) The method of claim 47, wherein said immunogenic composition comprising recombinant PCV2 ORF2 protein further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salt, and combinations thereof. ### Claim 51 (depends on 50) The method of claim 50, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 52 (depends on 47) The method of claim 47, wherein said polymer of an acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 53 (depends on 47) The method of claim 47, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 54 (depends on 53) The method of claim 53, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 55 (depends on 47) The method of claim 47, wherein said single dose has a volume of at least 1 ml. ### Claim 56 (depends on 47) The method of claim 47, wherein said single dose has a volume of at least 2 ml. ### Claim 57 A method for lessening the severity of clinical symptoms associated with PCV2 infection, said method comprising the step of administering a single dose of an immunogenic composition into pigs, wherein said immunogenic composition comprises 4 μg to 16 μg of recombinant PCV2 ORF2 protein per dose, a polymer of acrylic or methacrylic acid, and a second immunogenic composition that comprises PRRS antigen, wherein the severity of one or more clinical symptoms are lessened in pigs receiving said single dose of said immunogenic composition in comparison to pigs not receiving said immunogenic composition and wherein said clinical symptoms are selected from the group consisting of wasting, paleness of the skin, unthriftiness, respiratory distress, diarrhea, icterus, jaundice, lung lesions, nasal shedding, cough, diarrhea and combinations thereof. ### Claim 58 (depends on 57) The method of claim 57, wherein said recombinant PCV2 ORF2 protein is i) a polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, 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 59 (depends on 57) The method of claim 57, wherein said PRRS antigen is Ingelvac PRRS MLV. ### Claim 60 (depends on 57) The method of claim 57, wherein said immunogenic composition comprising recombinant PCV2 ORF2 protein further comprises an additional component selected from the group consisting of an inactivated viral vector, cell culture supernate, BEI, sodium thiosulfate, carriers, adjuvants, media, viral inactivators, diluents, isotonic agents, immunomodulatory agents, antibiotics, pharmaceutical acceptable salt, and combinations thereof. ### Claim 61 (depends on 60) The method of claim 60, wherein said inactivated viral vector is a recombinant baculovirus coding for the PCV2 ORF2 protein. ### Claim 62 (depends on 57) The method of claim 57, wherein said polymer of an acrylic acid or methacrylic acid is cross-linked with polyalkenyl ethers of sugars or polyalcohols. ### Claim 63 (depends on 57) The method of claim 57, wherein said polymer of acrylic or methacrylic acid is a carbomer. ### Claim 64 (depends on 63) The method of claim 63, wherein said carbomer is included in an amount from about 500 μg to about 5 mg carbomer per dose. ### Claim 65 (depends on 57) The method of claim 57, wherein said single dose has a volume of at least 1 ml. ### Claim 66 (depends on 57) The method of claim 57, wherein said single dose has a volume of at least 2 ml.