WO2018223668A1 - 一种预防和/或治疗猪圆环病毒感染的二联疫苗组合物及其制备方法和应用 - Google Patents

一种预防和/或治疗猪圆环病毒感染的二联疫苗组合物及其制备方法和应用 Download PDF

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WO2018223668A1
WO2018223668A1 PCT/CN2017/118343 CN2017118343W WO2018223668A1 WO 2018223668 A1 WO2018223668 A1 WO 2018223668A1 CN 2017118343 W CN2017118343 W CN 2017118343W WO 2018223668 A1 WO2018223668 A1 WO 2018223668A1
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porcine circovirus
strain
circovirus type
culture
vaccine composition
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PCT/CN2017/118343
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English (en)
French (fr)
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田克恭
李向东
肖燕
习向锋
孙进忠
张许科
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普莱柯生物工程股份有限公司
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Priority to US16/620,685 priority Critical patent/US11116836B2/en
Priority to JP2019567613A priority patent/JP6914370B2/ja
Publication of WO2018223668A1 publication Critical patent/WO2018223668A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/295—Polyvalent viral antigens; Mixtures of viral and bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2750/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to a double vaccine composition for preventing and/or treating porcine circovirus infection, a preparation method and application thereof, and belongs to the field of animal virology.
  • PCV Porcine circoviruses
  • PCV1 porcine circovirus type 1
  • PCV2 porcine circovirus type 2
  • PCV1 was first identified in PK cell culture as a contaminant in 1974 and was not pathogenic to pigs.
  • PCV2 was first reported in 1998, which can cause Porcine circovirus associated diseases (PCVAD) in pigs under clinical conditions, mainly causing multiple systemic exhaustion syndrome, pneumonia, porcine dermatitis and nephrotic syndrome in weaned piglets.
  • reproductive disorders mainly manifested by respiratory, urinary, intestinal, lymphatic, cardiovascular, neurological, reproductive systems, and dysfunction of the skin, causing significant economic losses to pig farming worldwide.
  • PCV2 Clinically, with the widespread use of PCV2 vaccine, the variability of PCV2 is accelerated under immunological stress, and a new type of strain between PCV2b and PCV2d is becoming popular.
  • the virus is characterized by the ORF2 gene. There are mutations or recombination of different gene subtypes. Due to the prevalence of the new PCV2 subtype strain, there is a difference between the antigen and the existing gene subtypes, and the existing commercial vaccines are prepared by using PCV2a or PCV2b as a vaccine strain, which cannot be the latest epidemic. The PCV2 strain is fully protected.
  • the present invention provides a vaccine composition for preventing and/or treating mixed infection of porcine circovirus, which can provide effective protection against mixed infection of different types of porcine circovirus, showing Significant immunological properties.
  • Another object of the present invention is to provide a vaccine composition for the prevention and/or treatment of a mixed infection of porcine circovirus type 2 of different porcine circoviruses and different gene subtypes.
  • Another object of the present invention is to provide a vaccine composition for preventing and/or treating porcine circovirus infection of different geographical origins.
  • Another object of the present invention is to provide a method for preparing a vaccine composition for preventing and/or treating porcine circovirus infection, comprising: step (1) respectively proliferating porcine circovirus type 3 virus, porcine circovirus type 2 Virus; step (2) inactivates the step (1) proliferation of porcine circovirus type 3 virus, proliferating porcine circovirus type 2 virus; step (3) mixing the steps (2) inactivated The porcine circovirus type 3 virus antigen, the inactivated porcine circovirus type 2 virus antigen, is added with an adjuvant and emulsified.
  • Another object of the present invention is to provide a use of the above vaccine composition for the preparation of a medicament for preventing and/or treating a disease associated with porcine circovirus infection.
  • the vaccine composition of the invention has good immunogenicity, and once immunized, it can stimulate the body to rapidly produce immunity, effectively protect the attack of the epidemic strain, has a good protective effect; can achieve with a lower antigen content. Good immune protection, further reducing production costs;
  • the vaccine composition prepared by the present invention for the first time using the porcine circovirus type 3 and the porcine circovirus type 2 strain can not only carry out infection or mixed infection of porcine circovirus type 2 and porcine circovirus type 3 Immune protection, can also protect against infection or mixed infection of different genetic subtypes of porcine circovirus type 2 strain;
  • the vaccine of the present invention can provide complete protection against mixed infection of porcine circovirus type 3 strain and porcine circovirus type 2 strain of different geographical origins, and has broad-spectrum protection ability.
  • Porcine circovirus type 3 is a genomic 2.0 kb circovirus, which is less than 50% homologous to the known circovirus, whether nucleotide or amino acid sequence. It is a new porcine circovirus. It can be mixed with a variety of pathogens to cause porcine dermatitis and nephrotic syndrome, proliferative necrotizing pneumonia, reproductive disorders and heart and multi-system inflammatory response.
  • “porcine circovirus type 2 new gene subtype” refers to a novel subtype of PCV2 gene, which has a mutation in the ORF2 gene or a recombination of different gene subtypes, with a tag sequence of PCV2b, but in genetic evolution analysis Forming an independent branch, the clinical manifestations of pigs infected with the subtype of the gene include: persistent high temperature, loss of appetite, depression, rough hair, weight loss and slowing of growth, and different degrees of lungs in the necropsy Consolidation, lymphadenopathy, and necrosis of the kidney.
  • the present invention relates to a vaccine composition for preventing and/or treating porcine circovirus infection, wherein the vaccine composition comprises an immunological amount of porcine circovirus type 3 antigen, an immunological amount of porcine circovirus type 2 antigen, and A pharmaceutically acceptable carrier.
  • a vaccine composition prepared by mixed infection of porcine circovirus may comprise an immunological amount of an inactivated antigen of porcine circovirus type 3 or a culture thereof, an attenuated whole virus antigen, an immunological amount of porcine circovirus type 2 or The inactivated antigen of the culture, the attenuated whole viral antigen, and the pharmaceutically acceptable carrier.
  • the porcine circovirus type 3 antigen and the porcine circovirus type 2 antigen contained in the vaccine composition of the invention have good immunogenicity, can rapidly activate the immune reaction at a low content, and protect the pig from the pig. Attack of the strain.
  • Vaccine composition refers to a pharmaceutical composition comprising porcine circovirus type 3 and type 2 immunogenicity.
  • the pharmaceutical composition can induce, stimulate or enhance the immune response of pigs to porcine circovirus types 3 and 2.
  • Inactivated antigen also known as an inactivated vaccine, refers to a suspension of inactivated virus that is used as an antigen to produce immunity.
  • inactivated vaccines include whole virus vaccines and lytic vaccines.
  • Inactivated vaccines can be readily produced using known methods. For example, a whole virus inactivated vaccine can be obtained by treating the virus with a formaldehyde solution. The lytic vaccine can be prepared from the viral envelope after treatment with ether.
  • Attenuated whole virus antigen refers to a virus that has been virulence weakened but still replicates in the host or on the cell.
  • the term "attenuated” as used in the present invention is used to artificially reduce pathogen toxicity by mutating a gene in a manner that causes the pathogen to lose pathogenicity but remains immunogenic. Typically, attenuation is achieved by UV radiation, chemical treatment, or continuous high-order subculture in vitro. Alternatively, virulence is attenuated by artificial genetic alterations, such as deletion of specific nucleotides in known sequences.
  • the porcine circovirus type 3 antigen is a porcine circovirus type 3 SG strain antigen, and the SG strain is deposited as CCTCC NO.V201712;
  • the porcine circovirus type 2 antigen is a porcine circovirus type 2 HH3 strain antigen, and the HH3 strain has a deposit number of CCTCC NO. V201726.
  • Porcine Circovirus type 3 (strain SG), preserved in the China Center for Type Culture Collection, the deposit number is CCTCC NO.V201712, and the deposit date is March 23, 2017. The deposit address is: Wuhan, China. ⁇ Wuhan University.
  • the novel porcine circovirus type 2 genome provided by the present invention has a full length of 1768 nucleotides and has a tag sequence of PCV2b, but forms an independent branch in the genetic evolution analysis, and a mutation or a different gene is present in the ORF2 gene. Recombination is a novel subtype of the PCV2 gene.
  • Porcine Circovirus type 2 (strain HH3), preserved in the China Center for Type Culture Collection, the deposit number is CCTCC NO.V201726, and the deposit date is June 4, 2017. The deposit address is: Wuhan, China. ⁇ Wuhan University.
  • the strain used in the vaccine composition of the invention has good immunogenicity, and the prepared vaccine composition can effectively stimulate the immune system with low content, and can directly produce immune immunity after one immunization, and completely protect the body, and the protection rate. Up to 100%.
  • the vaccine composition of the present invention is capable of fully protecting against different genetic subtypes of PCV2 and individual infections and mixed infections of PCV3.
  • the vaccine composition of the invention can provide effective protection against strains of different geographical origins, and expands the application range of the vaccine.
  • the porcine circovirus type 3 SG strain antigen is an inactivated whole virus antigen of a porcine circovirus type 3 SG strain or a culture thereof.
  • the culture of the porcine circovirus type 3 SG strain is a culture of ⁇ 1 generation;
  • the porcine circovirus type 2 HH3 strain antigen is a porcine circovirus type 2 HH3 strain or a culture thereof is inactivated
  • the whole virus antigen, the culture of the porcine circovirus type 2 HH3 strain is a culture of ⁇ 1 generation.
  • the culture of the porcine circovirus type 3 SG strain is a culture of ⁇ 5 passages
  • the porcine circovirus type 2 The HH3 strain culture was a culture of ⁇ 5 passages.
  • the culture of the porcine circovirus type 3 SG strain is a culture of 5 to 55 passages, and the porcine circovirus
  • the culture of type 2 HH3 strain was a culture of 5 to 48 passages.
  • the amount of the component or component of the composition of the invention is preferably a therapeutically effective amount.
  • the therapeutically effective amount refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing excessive side effects.
  • the precise amount of ingredients used and compositions to be administered will vary depending on various factors such as the type of disease being treated, the type and age of the animal to be treated, the mode of administration, and other ingredients in the composition.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof is ⁇ 10 5.0 TCID 50 /ml before inactivation
  • the porcine circovirus type 2 HH3 strain or the culture inactivated whole virus antigen content is ⁇ 10 5.0 TCID 50 /ml before inactivation.
  • the inactivated whole virus antigen of the porcine circovirus type 3 SG strain or the culture thereof in the vaccine composition of the invention has good immunogenicity, and once immunized, it can stimulate the body to rapidly produce immunity, before using inactivation 10 5.0 TCID 50 / ml content, can also achieve good immune protection, achieving 100% protection.
  • the porcine circovirus type 2 HH3 strain inactivated whole virus antigen in the vaccine composition of the invention has good immunogenicity, and one immunization can stimulate the body to rapidly produce immunity when using 10 5.0 TCID 50 / before inactivation.
  • the content of ml is also good, it can achieve a good immune protection and achieve a 100% protection rate.
  • the porcine circovirus type 3 SG strain or the culture thereof inactivates the whole virus antigen content before inactivation 10 5.0 to 10 7.0 TCID 50 / Ml
  • the porcine circovirus type 2 HH3 strain or its culture inactivated whole virus antigen content is 10 5.0 to 10 7.0 TCID 50 /ml before inactivation.
  • the porcine circovirus type 3 SG strain or the culture thereof inactivates the whole virus antigen content to be 10 6.0 TCID 50 /ml before inactivation,
  • the inactivated whole virus antigen content of the porcine circovirus type 2 HH3 strain or its culture was 10 6.0 TCID 50 /ml before inactivation.
  • the inactivated whole virus antigen content of the porcine circovirus type 3 SG strain or the culture thereof may also be selected from the range of 10 5.0 to 10 6.0 TCID 50 /ml, or 10 6.0 to 10 7.0 TCID. 50 / ml.
  • the inactivated whole virus antigen content of the porcine circovirus type 2 HH3 strain or a culture thereof may also be selected from the range of 10 5.0 to 10 6.0 TCID 50 /ml, or 10 6.0 to 10 7.0 TCID 50 /ml.
  • the pharmaceutically acceptable carrier is an adjuvant
  • the adjuvant includes white oil, drake oil, and animal oil, vegetable oil or mineral oil; Or aluminum hydroxide, aluminum phosphate and metal salts; or Montanide TM Gel, carbomer, squalane or squalene, ISA206 adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil emulsion .
  • the vaccine composition of the present invention is Montanide TM Gel.
  • the amount of adjuvant suitable for use in the compositions of the present invention is preferably an effective amount.
  • the "effective amount” refers to the amount necessary for the adjuvant to be necessary or sufficient to exert its immunological effects in the host when administered in combination with the antigen of the present invention without causing excessive side effects.
  • the precise amount of adjuvant to be administered will vary depending on various factors such as the ingredients employed and the type of disease being treated, the type and age of the animal to be treated, the mode of administration, and other ingredients in the composition.
  • the adjuvant is contained in an amount of 5 to 20 V/V%.
  • the adjuvant content is 10 V/V%.
  • the vaccine compositions of the invention may be formulated using conventional techniques, preferably together with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example, oil can help stabilize the formulation and additionally act as a vaccine adjuvant.
  • Oil adjuvants can be either natural or synthetically obtained.
  • adjuvant refers to a substance that is added to the compositions of the present invention to increase the immunogenicity of the composition.
  • Known adjuvants include, but are not limited to, (1) aluminum hydroxide, saponine (eg QuilA), affine, DDA, (2) polymer of acrylic acid or methacrylic acid, maleic anhydride and alkenyl derivative polymers, (3) the vaccine may be an oil in water, water-oil or water-in-oil emulsion made in the form, or (4) Montanide TM Gel.
  • the emulsion may be based on light liquid paraffinic oils, isoprenoid oils such as squalane or squalene; olefins, especially those produced by oligomerization of isobutylene or decene, linear alkyl acid or Esters formed by alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di(octanoate/caprate), triglyceride (caprylate/caprate), propylene glycol dioleate; branched fatty acid esters Or an ester of an alcohol, especially an isostearate.
  • the oil is used with an emulsifier to form an emulsion.
  • the emulsifier is preferably a nonionic surfactant, in particular a polyoxyethylated fatty acid (for example oleic acid), sorbitan, mannitol (for example dehydrated mannitol oleate), glycerol, polyglycerol, propylene glycol and optionally B.
  • the acrylic or methacrylic polymer is crosslinked by a polyalkenyl ether of a sugar or a polyol. These compounds are called carbomers.
  • the present invention is selected adjuvant Montanide TM Gel.
  • the invention also relates to a method for preparing the vaccine composition, wherein the method comprises: step (1) proliferating and culturing the porcine circovirus type 3 SG strain or a culture thereof, and proliferating and cultivating the pig ring a virus type 2 HH3 strain or a culture thereof; and a step (2) inactivating the step (1) proliferation cultured porcine circovirus type 3 SG strain or a culture thereof, inactivating the step (1) proliferation culture Porcine circovirus type 2 HH3 strain or culture thereof; step (3) mixing the inactivated porcine circovirus type 3 SG strain or culture thereof, and the inactivated porcine circovirus 2 The HH3 strain or the culture thereof is added with an adjuvant and emulsified.
  • compositions of the present invention may further incorporate additional agents into the compositions of the present invention.
  • the composition of the present invention may further comprise the following agents, such as: drugs, immunostimulants (eg, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony-stimulating factor (GM-CSF) ), macrophage colony-stimulating factor (M-CSF) and interleukin 2 (IL2), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellants, and preservatives.
  • agents such as: drugs, immunostimulants (eg, ⁇ -interferon, ⁇ -interferon, ⁇ -interferon, granulocyte macrophage colony-stimulating factor (GM-CSF) ), macrophage colony-stimulating factor (M-CSF) and interleukin 2 (IL2), antioxidants, surfactants, colorants, volatile oils, buffers, dispersants, propellant
  • the vaccine composition according to the invention may be prepared as an oral dosage form or a parenteral dosage form.
  • Non-oral dosage forms are preferably administered by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or epidural routes.
  • the present invention also relates to the use of the above vaccine composition for the preparation of a medicament for preventing and/or treating a disease associated with porcine circovirus infection, wherein the related disease caused by the porcine circovirus infection is a different subtype of PCV2 And porcine circovirus type 3 infection alone or a mixed disease caused by infection.
  • the term "related disease caused by mixed infection of porcine circovirus” as used in the present invention is used to mean a disease caused by a mixed infection of porcine circovirus types 3 and 2.
  • Non-exhaustive includes, but not limited to, weaned piglet multisystemic wasting syndrome, porcine dermatitis and nephrotic syndrome, reproductive disorders, and cardiac and multi-system inflammatory responses.
  • porcine circovirus type 3 and type 2 mixed infections means inhibition of porcine circovirus type 3 and type 2 replication, inhibition of porcine circovirus type 3 and type 2 transmission or Prevents porcine circovirus types 3 and 2 from colonizing in their hosts, as well as alleviating the symptoms of diseases or conditions of porcine circovirus type 3 and type 2 infection. If the viral load is reduced, the condition is alleviated, and/or the food intake and/or growth is increased, then the prevention and/or treatment can be considered to have achieved an effect.
  • the PCV2 gene subtype is a PCV2a, PCV2b, PCV2d, PCV2new gene subtype.
  • the vaccine composition of the invention can provide effective protection against different PCV2 gene subtypes and PCV3, expand the application range of the vaccine, and can prevent and/or treat individual infection and mixed infection of PCV2 gene subtypes and PCV3.
  • the diseases caused by the porcine circovirus infection include weaning piglet multisystemic wasting syndrome, porcine dermatitis nephrotic syndrome, reproductive disorders, and heart and many Systemic inflammatory response.
  • the chemical reagents used in the examples of the present invention are of analytical grade and are purchased from Sinopharm Group.
  • the disease material was added to the DMEM culture solution at a ratio of 1:10 (volume ratio) and ground to prepare a tissue suspension. After repeated three cycles of freezing and thawing, the tissue suspension was centrifuged at 12000 r/min for 15 min, and the supernatant was collected; After filtration through a 0.22 ⁇ m filter, the filtrate was passaged on PK15 cells, cultured at 37 ° C for 1 h, replaced with DMEM medium containing 2% yak serum, and cultured at 37 ° C for 5 days. The virus-containing culture solution was harvested, and after the culture solution was freeze-thawed twice, the virus was harvested.
  • the virus culture obtained by the above steps was taken, and the nucleic acid of the virus sample was extracted by the nucleic acid extraction kit, and PCR amplification was performed using the circovirus specific primers. The result showed that the 2000 bp target band was amplified by PCR.
  • the PCR product was sent to a sequencing company for nucleotide sequence determination, and the sequence determination results were subjected to genetic evolution analysis.
  • the whole genome sequence and amino acid sequence of the virus strain were less than 50% homologous to other circoviruses already reported, and according to the standards of the International Committee of Virology, the same virus in the genus Cyclone should be With >75% homology of the genomic nucleotide sequence, the Cap protein has >70% amino acid sequence homology, which is affirmative, it is a new porcine circovirus, also on the current pig body The third ring of viruses found.
  • porcine circovirus type 3 design-specific primers 235 suspected PCV3 positive samples collected from all over the country were analyzed by quantitative PCR, and 121 PCV3 viruses were isolated and screened.
  • the genomic nucleotide sequence homology is as high as 98.9 ⁇ 99.6%, and the amino acid sequence homology of Cap protein is as high as 97.7 ⁇ 99.5%.
  • the porcine circovirus type 3 virus was named porcine circovirus type 3 SG strain and submitted for preservation.
  • the cultures of the porcine circovirus type 3 SG strain screened in Example 2 were inserted into the monolayer-derived PK15 passage cells at 1% (V/V) of the amount of the virus culture solution, and placed at 37 ° C for adsorption. After 30 minutes, the cell maintenance solution was added and cultured at 37 °C. The cells were observed 1 to 2 times a day, and the cells grew well. After 4 to 7 days of culture at 36 to 37 ° C, the cell culture was harvested, and after the harvested cell culture was frozen and thawed 2-3 times, the virus solution was harvested, and the virus virulence was measured.
  • the virus solution was filtered through a hollow fiber (0.5 ⁇ m - 2 ⁇ m) filter column to remove cell debris, and then inactivated by adding 0.1% to 0.2% formaldehyde solution at 37 ° C for 24 hours to inactivate the complete virus antigen to prepare a vaccine.
  • the disease material was added to the DMEM culture solution at a ratio of 1:10 (volume ratio) and ground to prepare a tissue suspension. After repeated three cycles of freezing and thawing, the tissue suspension was centrifuged at 12000 r/min for 15 min, and the supernatant was collected, and the supernatant was collected. The filtrate was filtered through a 0.22 ⁇ m filter; the filtrate was passaged on PK15 cells, cultured at 37 ° C for 1 h, replaced with DMEM medium containing 2% yak serum, and cultured at 37 ° C for 5 days. The virus-containing culture solution was harvested, and after the culture solution was freeze-thawed twice, the virus was harvested.
  • the virus culture harvested in the above procedure was taken, and the nucleic acid of the virus sample was extracted with a nucleic acid extraction kit, and PCR amplification was performed using a PCV2-specific primer, and the result showed that a 1.7 kb target band was amplified by PCR.
  • the PCR product was sent to a sequencing company for nucleotide sequence determination, and the sequence determination results were subjected to genetic evolution analysis. The results showed that the whole genome sequence of the virus strain was less than 96% homologous to other PCV2s already reported, and the amino acid sequence was less than 94%. Further analysis of the strain was between PCV2b and PCV2d, and there was a mutation in the ORF2 gene. Or the recombination of different gene subtypes, belonging to a new PCV2 gene subtype in genetic evolution analysis.
  • porcine circovirus type 2 design specific primers 42 suspected PCV2 positive samples collected from sporadic cases in the post-immunization PCV2 vaccine from all over the country were analyzed by quantitative PCR, and 16 PCV2 viruses were screened and isolated. Among these 16 strains, the genomic nucleotide sequence homology is as high as 99.6-100%, and the Cap protein amino acid sequence homology is as high as 99.5-100%. Further analysis belongs to a gene subtype. After the animal pathogenicity test and immunogenicity test, a strain of PCV2 strain with strong pathogenicity, good immunogenicity and broad spectrum was screened out. The porcine circovirus type 2 virus was named as porcine circovirus type 2 HH3 strain and submitted for preservation.
  • the cultures of the porcine circovirus type 2 HH3 strain screened in Example 6 were inserted into the monolayer-derived PK15 passage cells at 1% (V/V) of the amount of the virus culture solution, and placed at 37 ° C for adsorption. After 30 minutes, the cell maintenance solution was added and cultured at 37 °C. Observe 1 or 2 times a day, and the cells grow well. After 4 to 7 days of culture at 36-37 ° C, the cell culture is harvested. After harvesting the cell culture for 2-3 times, the virus solution is harvested and the virus is detected in the virus solution. price.
  • the virus solution was filtered through a hollow fiber (0.5 ⁇ m to 2 ⁇ m) filter column to remove cell debris, and then inactivated by adding 0.1% to 0.2% formaldehyde solution at 37 ° C for 24 hours to inactivate the complete virus antigen to prepare a vaccine.
  • the porcine circovirus type 3 SG strain inactivated antigen prepared in Example 4, and the porcine circovirus type 2 HH3 strain inactivated antigen prepared in Example 8 were mixed in proportion and slowly added to the water-soluble adjuvant Gel adjuvant ( In the French company, the process of adding is continuously stirred for 12 minutes with an emulsifier of 800 rpm and mixed.
  • the specific formulation of the vaccine is shown in Table 3.
  • Group 5 Group 7, Group 9, Group 11, and Group 13 challenged porcine circovirus type 3 SG strain 10 5.0 TCID 50 / head, group 6, 8 Group, group 10, group 12, group 14 challenged porcine circovirus type 2 HH3 strain 10 5.0 TCID 50 / head, continuous observation of each piglet after challenge, according to the clinical symptoms, pathological changes and virus test results of each piglet The determination is made, and the specific results are shown in Table 4.
  • porcine circovirus type 3 and type 2 inactivated vaccines could provide 100% (5/5) protection for piglets after immunization of one piglet, while all the piglets in the challenge control group were infected. It is indicated that the porcine circovirus type 3 and type 2 inactivated vaccine provided by the invention has good protective effect.
  • Example 11 Broad-spectrum protection test of porcine circovirus type 3 and type 2 inactivated vaccine
  • 100 healthy piglets with PCV2, PCV3 antigen and antibody negative by ELISA at 28 to 30 days of age were randomly divided into 20 groups, 5 groups/group, and vaccines prepared in Example 15 to Group 24 to 24, Groups 25 to 34 Not immunized as a challenge control group.
  • Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium.
  • the challenge was carried out on the 28th, and the 15th and 25th groups were challenged with the virulent strain of the porcine circovirus 2a subtype HN06 strain newly isolated from Henan province of China; the 16th and 26th groups were newly used from China.
  • the virulent strain of the porcine circovirus 2b subtype JS04 strain isolated from Jiangsu province was challenged; the 17th and 27th groups were challenged with the virulent strain of the porcine circovirus 2d subtype JL13 strain newly isolated from Jilin province, China. Groups 18 and 28 were challenged with the newly bred circovirus 2new subtype CQ14 strain isolated from Chongqing, China; the 19th and 29th groups were newly isolated from Guangdong province, China.
  • Virus 2new subtype GD 15 virulent strains were challenged; Group 20 and Group 30 were challenged with the virulent strain of porcine circovirus type 3 HN12 strain isolated from Henan province, China; Group 21, Group 31 Toxic with the porcine circovirus type 3 JS08 strain, which was newly isolated from Jiangsu province, China; the 22nd and 32nd groups were challenged with the porcine circovirus type 3 JL11 strain, which was newly isolated from Jilin province, China. Group 23 and Group 33 were challenged with the virulent strain of porcine circovirus type 3 CQ04 strain newly isolated from Chongqing, China; the 24th and 34th groups were used.
  • porcine circovirus type 3 and type 2 inactivated vaccine provided by the invention can be effectively immunized against porcine circovirus type 2 of different geographical origins and different gene subtypes. Immune protection, and PCV2 cannot be detected from various organ tissues.
  • porcine circovirus type 3 and type 2 inactivated vaccine provided by the invention can be effectively immunized against porcine circovirus type 3 of different geographical sources, and can be effectively immunized from pigs.
  • the challenged PCV3 strain could not be detected in each organ tissue.
  • the vaccine composition provided by the present invention has a broad spectrum of immunogenicity and can provide complete protection for porcine circovirus types 3 and 2 strains of different geographical origins.
  • Example 12 Mixed infection protection test of porcine circovirus type 3 and type 2 inactivated vaccine
  • Group 35 was immunized with Vaccine 1 prepared in Example 9
  • Group 36 was immunized with Vaccine 4 prepared in Example 9
  • Group 37 was immunized with Vaccine 5 prepared in Example 9, and
  • Group 38 was not immunized as a challenge control group.
  • Each immunization group was injected with 2 ml/head of vaccine, and the challenge control group was inoculated with 2 ml/head of DMEM medium. After the immunization, the challenge was carried out on the 28th.
  • the immunized group was injected with 2 ml/head of vaccine, and the blank control group was inoculated with 2 ml/head of DMEM medium.
  • the results of the two groups of sows were counted. The results are shown in Table 8.
  • the piglets from the blank control group of group B were isolated and nested separately, and 15 litters were divided into two groups: group B1 (including B-1 to B-13), except for B-6 nest due to the whole nest of mummies. A total of 12 litters of piglets), B2 group (including B-14 to B-18 litters, except for B-16 litters and B-18 litters, due to the total nest of mummies, a total of 3 litters of piglets), B1 group before immunization
  • the vaccine 1 prepared in Example 9 and the B2 group were blank control groups.
  • the immunized group was injected with 2 ml/head of the vaccine, and the control group was inoculated with 2 ml/head of DMEM medium. Each piglet was continuously observed and judged according to the clinical symptoms, pathological changes and virus detection of each piglet. The specific results are shown in Table 9.
  • the PCR was performed by necropsy of the organs of the pigs. PCV3 and PCV2 were negative, while the piglets in the blank control group showed different degrees.
  • the body temperature increased by 40.5 °C or more for 5 days, and the clinical symptoms such as loss of appetite, depression, rough hair, weight loss and slowing of growth rate, some pigs died, and different degrees of lung consolidation and lymphadenopathy occurred in the necropsy.
  • the renal pathological changes of the necrotic point, PCR detection of each organ tissue, can be isolated again to porcine circovirus type 3 and porcine circovirus type 2.

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Abstract

本发明公开一种预防和/或治疗猪圆环病毒感染的疫苗组合物，其包含免疫量的猪圆环病毒3型抗原、免疫量的猪圆环病毒2型抗原以及药学上可以接受的载体。该疫苗组合物能预防和/或治疗猪圆环病毒不同基因亚型感染或混合感染导致的相关疾病，且能对不同地域来源的毒株均具有免疫保护效力。

Description

一种预防和/或治疗猪圆环病毒感染的二联疫苗组合物及其制备方法和应用 技术领域
本发明涉及一种预防和/或治疗猪圆环病毒感染的二联疫苗组合物、制备方法和应用，属于动物病毒学领域。
背景技术
猪圆环病毒(Porcine circoviruses，PCV)是单股环状的DNA病毒，基因组长度约为1.7kb，是最小的动物DNA病毒之一。已经确定有两种类型的PCV，即猪圆环病毒1型(PCV1)和猪圆环病毒2型(PCV2)。PCV1于1974年首次在PK细胞培养物中作为一种污染物鉴定而发现，其对猪只没有致病性。PCV2于1998年首次报道，其在临床条件下能引起猪只的猪圆环病毒相关疾病(Porcine circovirus associated diseases，PCVAD)，主要引起断奶仔猪多系统衰竭综合征，肺炎，猪皮炎和肾病综合征和繁殖障碍，主要表现为呼吸、泌尿、肠道、淋巴、心血管、神经、繁殖系统以及皮肤的功能紊乱，对全世界的生猪养殖造成了重大的经济损失。
临床上，随着PCV2疫苗的普遍应用，在免疫压力下，PCV2的变异速度加快，一类介于PCV2b与PCV2d之间的新的基因亚型的毒株开始流行，这类病毒特征是ORF2基因中存在突变或存在不同基因亚型的重组。由于该新的PCV2基因亚型毒株的流行，其抗原与现有的基因亚型之间存在差异，而现有的商品化疫苗均以PCV2a或PCV2b为疫苗株来制备的，不能对最新流行的PCV2毒株进行完全的保护。
在一起猪的繁殖障碍病例中，分离到一株病毒基因组为2.0kb的圆环病毒，经后续试验证实，其与已知的圆环病毒无论在核苷酸还是氨基酸序列的同源性均小于50％，根据国际病毒学分类委员会的标准，圆环 病毒属中同一种的病毒应该具有＞75％的基因组核苷酸序列的同源性，Cap蛋白具有＞70％的氨基酸序列的同源性，据此可以肯定这是一种新的猪圆环病毒(PCV3)。
当初早在1985年由PCV2导致的系统疾病就已经以零星状态爆发，由于未能重视，导致九十年代末大规模爆发该疾病，而新的猪圆环病毒在猪皮炎肾病综合征(Porcine dermatitis and nephropathy syndrome，PDNS)和繁殖障碍方面具有与PCV2相似病原学特性，PCV2和PCV3之间蛋白同源性很低，使用PCV2疫苗不能对PCV3进行有效预防和交叉保护。
新的猪圆环病毒与PCV2现有的和新的基因亚型之间的混合感染进一步加重临床上PCV感染的复杂情况，因此针对该新临床疫情制备新的疫苗组合物，对猪场疾病控制十分重要。
发明内容
为解决现有技术的不足，本发明提供一种预防和/或治疗猪圆环病毒混合感染的疫苗组合物，该疫苗组合物能对不同类型猪圆环病毒混合感染提供有效的保护，显示出显著的免疫特性。
本发明的一个目的在于提供一种预防和/或治疗猪圆环病毒感染的疫苗组合物，所述疫苗组合物包含免疫量的猪圆环病毒3型抗原、免疫量的猪圆环病毒2型抗原以及药学上可以接受的载体。
本发明的另一个目的在于提供一种预防和/或治疗不同猪圆环病毒和不同基因亚型的猪圆环病毒2型混合感染的疫苗组合物。
本发明的另一个目的在于提供一种预防和/或治疗不同地域来源的猪圆环病毒感染的疫苗组合物。
本发明的另一个目的是提供一种预防和/或治疗猪圆环病毒感染的疫苗组合物的制备方法，包括：步骤(1)分别增殖猪圆环病毒3型病毒、猪圆环病毒2型病毒；步骤(2)分别灭活所述步骤(1)增殖的猪圆环病毒3型病毒、增殖的猪圆环病毒2型病毒；步骤(3)按比例混合所述步骤(2)灭活的猪圆环病毒3型病毒抗原、灭活的猪圆环病毒 2型病毒抗原，加入佐剂，乳化。
本发明的另一个目的在于提供上述疫苗组合物在制备预防和/或治疗猪圆环病毒感染的相关疾病的药物中的应用。
发明优点：
(1)本发明疫苗组合物具有良好的免疫原性，一次免疫，即能刺激机体快速地产生免疫力，有效保护流行株的攻击，具有很好的保护作用；能以较低的抗原含量达到良好的免疫保护作用，进一步降低生产成本；
(2)本发明首次采用猪圆环病毒3型和猪圆环病毒2型毒株制备的疫苗组合物，不仅能对猪圆环病毒2型和猪圆环病毒3型的感染或混合感染进行免疫保护，还能针对不同基因亚型猪圆环病毒2型毒株的感染或混合感染进行保护；
(3)本发明的疫苗，能针对不同地域来源的猪圆环病毒3型毒株、猪圆环病毒2型毒株混合感染提供完全保护，具有广谱的保护能力。
具体实施方式
以下，对本发明的实施方式进行说明。
猪圆环病毒3型为基因组2.0kb的圆环病毒，其与已知的圆环病毒无论是核苷酸还是氨基酸序列的同源性均小于50％，是一种新的猪圆环病毒，其能与多种病原混合感染引起猪的皮炎肾病综合征、增生性坏死性肺炎、繁殖障碍及心脏和多系统的炎症反应。
“猪圆环病毒2型new基因亚型”是指一种新的PCV2基因亚型，其ORF2基因中存在突变或存在不同基因亚型的重组，具有PCV2b的标签序列，但在遗传进化分析中组成一个独立分支，猪只感染该基因亚型的毒株后出现的临床表征有：持续性高温，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点。
本发明涉及一种预防和/或治疗猪圆环病毒感染的疫苗组合物，其中，所述疫苗组合物包含免疫量的猪圆环病毒3型抗原、免疫量的猪圆 环病毒2型抗原以及药学上可以接受的载体。
针对猪圆环病毒混合感染制备的疫苗组合物，可以包含免疫量的猪圆环病毒3型或其培养物的灭活抗原、减毒全病毒抗原，免疫量的猪圆环病毒2型或其培养物的灭活抗原、减毒全病毒抗原，以及药学上可以接受的载体。
本发明的疫苗组合物中包含的猪圆环病毒3型抗原、和猪圆环病毒2型抗原具有良好的免疫原性，能在低含量时就能快速的激活免疫反应，保护猪只免于毒株的攻击。
“疫苗组合物”指含有猪圆环病毒3型和2型免疫原性的药物组合物。该药物组合物可诱发、刺激或增强猪只针对猪圆环病毒3型和2型的免疫反应。
“灭活抗原”，也称作失活疫苗，指的是用作抗原以产生免疫力的灭活病毒的混悬液。灭活疫苗的例子包括全病毒疫苗和裂解型疫苗。使用已知方法可以很容易地产生灭活疫苗。例如，通过用甲醛溶液处理病毒可获得全病毒灭活疫苗。裂解型疫苗可在用醚处理后由病毒包膜制备得到。
“减毒全病毒抗原”指的是以毒力已经减弱但仍可在宿主体内或细胞上复制的病毒。本发明所用的术语“减毒”用于指以使病原丧失致病性、但保持免疫原性的方式对基因进行突变来人工降低病原体毒性。通常，通过UV辐射、化学处理或体外连续高阶继代培养实现减毒。或者通过人工的基因改变，例如将已知序列中的特定核苷酸缺失以使毒力减弱。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述的猪圆环病毒3型抗原为猪圆环病毒3型SG株抗原，所述SG株保藏号为CCTCC NO.V201712；所述的猪圆环病毒2型抗原为猪圆环病毒2型HH3株抗原，所述HH3株保藏号为CCTCC NO.V201726。
猪圆环病毒3型SG株(Porcine Circovirus type 3，strain SG)，保藏于中国典型培养物保藏中心，保藏号为CCTCC NO.V201712，保藏日期为2017年3月23日，保藏地址：中国武汉·武汉大学。
本发明提供的新的猪圆环病毒2型基因组全长为1768个核苷酸，具有PCV2b的标签序列，但在遗传进化分析中却组成一个独立分支，其ORF2基因中存在突变或不同基因亚型的重组，是一种新PCV2基因亚型。
猪圆环病毒2型HH3株(Porcine Circovirus type 2，strain HH3)，保藏于中国典型培养物保藏中心，保藏号为CCTCC NO.V201726，保藏日期为2017年6月4日，保藏地址：中国武汉·武汉大学。
本发明疫苗组合物中使用的毒株具有良好的免疫原性，其制备的疫苗组合物能以低含量有效刺激免疫系统，一次免疫，即能快速产生免疫效力，对机体产生完全保护，保护率可达100％。
本发明的疫苗组合物能针对PCV2不同的基因亚型和PCV3的单独感染和混合感染进行完全保护。
本发明的疫苗组合物能针对不同地域来源的毒株提供有效保护，扩大了疫苗的应用范围。
作为本发明的一种实施方式，本发明所述的疫苗组合物中，所述的猪圆环病毒3型SG株抗原为猪圆环病毒3型SG株或其培养物的灭活全病毒抗原，所述猪圆环病毒3型SG株的培养物为≥1代次的培养物；所述的猪圆环病毒2型HH3株抗原为猪圆环病毒2型HH3株或其培养物灭活全病毒抗原，所述猪圆环病毒2型HH3株的培养物为≥1代次的培养物。
“培养物”是病毒的不同代次传代培养物，本领域技术人员知晓不同代次之间其基因序列仅可能会发生微小的变异。
作为本发明的一种优选实施方式，本发明所述的疫苗组合物中，所述猪圆环病毒3型SG株的培养物为≥5代次的培养物，所述猪圆环病毒2型HH3株培养物为≥5代次的培养物。
作为本发明的一种更优选实施方式，本发明所述的疫苗组合物中，所述猪圆环病毒3型SG株的培养物为5～55代次的培养物，所述猪圆环病毒2型HH3株培养物为5～48代次的培养物。
本发明的组合物的成分或组分的量优选地是治疗有效量。所述治疗 有效量是指在组合物施用的宿主中发挥它们的免疫学作用而不导致过度副作用所必需量。所用的成分和待施用的组合物的精确的量将根据多种因素如治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前≥10 5.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前≥10 5.0TCID 50/ml。
本发明的疫苗组合物中猪圆环病毒3型SG株或其培养物的灭活全病毒抗原具有良好的免疫原性，一次免疫，即能刺激机体快速地产生免疫力，当使用灭活前10 5.0TCID 50/ml的含量时，也能达到良好的免疫保护作用，实现100％的保护率。
本发明的疫苗组合物中猪圆环病毒2型HH3株灭活全病毒抗原具有良好的免疫原性，一次免疫，即能刺激机体快速地产生免疫力，当使用灭活前10 5.0TCID 50/ml的含量时，也能达到良好的免疫保护作用，实现100％的保护率。
作为本发明的一种优选实施方式，本发明的疫苗组合物中，所述猪圆环病毒3型SG株或其培养物灭活全病毒抗原含量为灭活前10 5.0～10 7.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前10 5.0～10 7.0TCID 50/ml。
作为本发明的一种更优选实施方式，本发明的疫苗组合物中，所述猪圆环病毒3型SG株或其培养物灭活全病毒抗原含量为灭活前10 6.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前10 6.0TCID 50/ml。
本发明疫苗组合物中，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量范围还可选自10 5.0～10 6.0TCID 50/ml，或10 6.0～10 7.0TCID 50/ml。所述猪圆环病毒2型HH3株或其培养物的灭活全病毒抗原含量范围还可选自10 5.0～10 6.0TCID 50/ml，或10 6.0～10 7.0TCID 50/ml。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述药学上 可以接受的载体为佐剂，所述佐剂包括白油、德雷克油，以及动物油、植物油或矿物油；或氢氧化铝、磷酸铝及金属盐；或Montanide TM Gel、卡波姆、角鲨烷或角鲨烯、ISA206佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂。
作为本发明的一种优选实施方式，本发明的疫苗组合物中，所述佐剂为Montanide TMGel。
适用于本发明的组合物的佐剂的量优选地是有效量。所述“有效量”是指佐剂在同本发明抗原联合施用时在宿主中发挥它们的免疫学作用而言必须或足够的而不导致过度副作用所必需量。待施用的佐剂的精确的量将根据多种因素如所用的成分和治疗的疾病的类型，待治疗的动物的类型和年龄，施用的方式，以及组合物中的其它成分而变化。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述佐剂含量为5～20V/V％。
作为本发明的一种优选实施方式，本发明的疫苗组合物中，所述佐剂含量为10V/V％。
本发明的疫苗组合物可使用常规技术来调配，优选为药学上可接受的载体一起调配。例如，油可有助于稳定调配物，且另外充当疫苗佐剂。油佐剂既可以是自然来源，也可以是经过人工合成获得的。
术语“佐剂”指加入到本发明的组合物中以增加组合物的免疫原性的物质。已知的佐剂包括，但不限于：(1)氢氧化铝、皂苷(Saponine)(例如QuilA)、阿夫立定、DDA，(2)丙烯酸或甲基丙烯酸的聚合物、顺丁烯二酸酐和链烯基衍生物的聚合物，(3)疫苗可以以水包油、油包水或水包油包水乳剂形式制成，或(4)Montanide TMGel。
尤其是，乳剂可以基于轻液体石蜡油、类异戊二烯油，例如角鲨烷或角鲨烯；烯烃，特别是异丁烯或癸烯低聚化产生的油，带直链烷基的酸或醇形成的酯，更特别是植物油，油酸乙基酯，丙二醇二(辛酸酯/癸酸酯)，甘油三(辛酸酯/癸酸酯)，丙二醇二油酸酯；分支脂肪酸酯或醇的酯，特别是异硬脂酸酯。油与乳化剂一起使用形成乳剂。乳化剂优选非离子表面活性剂，特别是聚氧乙烯化脂肪酸(例如油酸)，脱 水山梨糖醇、甘露醇(例如脱水甘露醇油酸酯)、甘油、聚甘油、丙二醇和可选地乙氧基化的油酸、异硬脂酸、蓖麻油酸、羟基硬脂酸形成的酯，脂肪醇和多元醇(例如油醇)的醚，聚氧丙稀-聚氧乙烯嵌段共聚物，特别是PluronicR，尤其是L121(参照Hunter等，1995，“The Theory and Practical Application ofAdjuvants”(Steward-Tull，D.E.S主编)John Wiley andSons，NY，51-94；Todd等，Vaccine，1997，15，564-570)。
特别地，丙烯酸或甲基丙烯酸聚合物通过糖或多元醇的聚链烯基醚交联。这些化合物被称作卡波姆。
优选地，本发明选用的佐剂为Montanide TMGel。
本发明还涉及一种制备所述疫苗组合物的方法，其中，所述方法包括：步骤(1)增殖培养所述猪圆环病毒3型SG株或其培养物、增殖培养所述猪圆环病毒2型HH3株或其培养物；以及步骤(2)灭活所述步骤(1)增殖培养的猪圆环病毒3型SG株或其培养物、灭活所述步骤(1)增殖培养的猪圆环病毒2型HH3株或其培养物；步骤(3)按比例混合所述灭活后的猪圆环病毒3型SG株或其培养物、所述灭活后的猪圆环病毒2型HH3株或其培养物，加入佐剂，乳化。
本发明疫苗组合物还可以进一步将其他的试剂加入到本发明的组合物中。例如，本发明的组合物还可以包含以下试剂，如：药物，免疫刺激剂(如：α-干扰素、β-干扰素、γ-干扰素、粒细胞巨噬细胞集落刺激因子(GM-CSF)、巨噬细胞集落刺激因子(M-CSF)和白介素2(IL2))、抗氧化剂、表面活性剂、着色剂、挥发性油、缓冲剂、分散剂、推进剂和防腐剂。为了制备这样的组合物，可以使用本领域公知的方法。
可以根据本发明的疫苗组合物制备成口服剂型或非口服剂型。
优选地可通过皮内、肌肉、腹膜内、静脉内、皮下、鼻内或硬脑膜外途径给予的非口服剂型。
本发明还涉及上述疫苗组合物在制备预防和/或治疗猪圆环病毒感染导致的相关疾病的药物中的应用，其中，所述猪圆环病毒感染导致的相关疾病为由PCV2不同基因亚型和猪圆环病毒3型单独感染或它们混合感染导致的相关疾病。
本发明所用术语“猪圆环病毒混合感染导致的相关疾病”用于指由猪圆环病毒3型和2型混合感染引起的疾病。非穷举性包括，断奶仔猪多系统衰竭综合征、猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应，但不限于此。
术语“预防和/或治疗”在涉及猪圆环病毒3型和2型混合感染时是指抑制猪圆环病毒3型和2型的复制、抑制猪圆环病毒3型和2型的传播或防止猪圆环病毒3型和2型在其宿主体内定居，以及减轻猪圆环病毒3型和2型感染的疾病或病症的症状。若病毒荷载量减少、病症减轻和/或摄食量和/或生长增加，那么就可以认为所述预防和/或治疗达到了效果。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述PCV2基因亚型为PCV2a、PCV2b、PCV2d、PCV2new基因亚型。
本发明的疫苗组合物能针对不同PCV2基因亚型以及PCV3提供有效保护，扩大了疫苗的应用范围，能对PCV2各基因亚型和PCV3的单独感染和混合感染进行预防和/或治疗。
作为本发明的一种实施方式，本发明的疫苗组合物中，所述猪圆环病毒感染导致的相关疾病包括断奶仔猪多系统衰竭综合征、猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应。
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
本发明实施例中所用到的化学试剂均为分析纯，购自国药集团。
本发明所述的实验方法，若无特殊说明，均为常规方法；所述的生物材料，若无特殊说明，均可从商业途径获得。
实施例1猪圆环病毒3型的分离鉴定
1、病料来源
在国内一商品化猪场，与历史平均值相比，出现母猪死亡率增加9.4％，受孕率降低了1.2％，木乃伊胎增加8.2％的现象。临床表现上，受影响的母猪表现出厌食，呈多灶性丘疹，斑点和表面皮炎的症状。在流产的窝中含有不同胎龄的木乃伊化胎儿，与PCV2相关流产的症状一致。虽然在母猪中观察到的总体临床表现以及流产症状与猪圆环病毒2型导致的繁殖障碍疾病一致，但所有母猪的不同组织，包括肾、淋巴结、肺、皮肤以及死胎，通过免疫组化和定量PCR对PCV2、PRRSV、PPV、CSFV、猪肺炎支原体检测均为阴性。为进一步查清原因，选取各组织病料进行病原分离。
2、病毒株的分离与培养
将病料以1∶10(体积比)加入DMEM培养液，研磨，制备组织悬液；组织悬液经反复3次冻融后，于12000r/min离心15min，收集上清液；上清液再经0.22μm滤膜滤器过滤，滤液在PK15细胞上传代，于37℃培养1h，替换加入含2％犊牛血清的DMEM培养液，于37℃培养5日。收获含病毒的培养液，培养液经2次冻融后，收获病毒。
3、PCR及测序分析鉴定病毒种属
取以上步骤收获的病毒培养物，用核酸提取试剂盒提取病毒样品的核酸，使用圆环病毒种属特异性引物进行PCR扩增鉴定，结果显示，PCR扩增出2000bp目的条带。PCR产物送测序公司进行核苷酸序列测定，序列测定结果进行遗传进化分析。结果显示该病毒株全基因组序列和氨基酸序列同已经报道过的其他圆环病毒的同源性都少于50％，而根据国际病毒学分类委员会的标准，圆环病毒属中同一种的病毒应该具有＞75％的基因组核苷酸序列的同源性，Cap蛋白具有＞70％的氨基酸序列的同源性，据此可以肯定，它是一种新的猪圆环病毒，也是目前猪体上发现的第三种圆环病毒。
实施例2猪圆环病毒3型疫苗株的筛选
根据上述分离到的猪圆环病毒3型设计特异性引物，通过定量PCR 分析从全国各地采集的235份疑似PCV3阳性样品，筛选分离出121株PCV3病毒，这121株中，各株之间其基因组核苷酸序列同源性高达98.9～99.6％，Cap蛋白氨基酸序列同源性高达97.7～99.5％。经本动物致病性试验及免疫原性测试，最终筛选出1株致病性强、免疫原性良好、广谱性强的PCV3病毒株。该猪圆环病毒3型病毒命名为猪圆环病毒3型SG株，提交保藏。
实施例3猪圆环病毒3型SG株致病性试验
用28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪10头随机分成两组，5头/组，第1组用PCV3SG株(含10 5.0TCID 50/头)攻毒，肌肉注射，第2组空白对照组接种DMEM培养基，各组仔猪隔离饲养。攻毒后连续观察各组仔猪，根据其临床症状、病理变化和病毒检测进行判定，具体结果见表1。
表1猪圆环病毒3型SG株对仔猪致病性试验结果
Figure PCTCN2017118343-appb-000001
Figure PCTCN2017118343-appb-000002
结果显示，攻毒组所有猪只均出现3～5日持续性40.5℃以上高温，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型病毒，而空白对照组无异常，表明本发明猪圆环病毒3型SG株接种仔猪可导致仔猪发病，临床表征为典型的猪圆环病毒感染症状。
实施例4猪圆环病毒3型SG株抗原的制备
将实施例2筛选到的猪圆环病毒3型SG株不同代次的培养物按照病毒培养液量的1％(V/V)接入形成单层的PK15传代细胞中，置于37℃吸附30分钟，然后加入细胞维持液，置于37℃培养。每日观察1～2次，细胞生长良好，于36～37℃培养4～7日后收获细胞培养物，收获的细胞培养物冻融2-3次后，收获病毒液，并测定病毒毒价。将病毒 液用中空纤维(0.5μm～2μm)滤柱过滤，以除去细胞碎片，再加入0.1％～0.2％的甲醛溶液在37℃灭活24h，灭活完全后的病毒抗原用以制备疫苗。
实施例5猪圆环病毒2型的分离鉴定
1、病料来源
在国内一免疫商品化PCV2疫苗的猪场，零星出现母猪流产、木乃伊胎增加的现象。受影响的母猪表现出厌食症状，在流产的窝中含有不同胎龄的木乃伊化胎儿，与PCV2相关流产的症状一致。通过免疫组化和定量PCR检测，PCV2阳性。因其是在免疫PCV2商品化疫苗后依然从发病猪组织中检测到PCV2，原因值得深究，为进一步查清原因，选取各组织病料进行病原分离。
2、病毒株的分离与培养
将病料以1∶10(体积比)加入DMEM培养液，研磨，制备组织悬液；组织悬液经反复3次冻融后，于12000r/min离心15min，收集上清液，上清液再经0.22μm滤膜滤器过滤；滤液在PK15细胞上传代，于37℃培养1h，替换加入含2％犊牛血清的DMEM培养液，于37℃培养5日。收获含病毒的培养液，培养液经2次冻融后，收获病毒。
3、PCR及测序分析鉴定病毒
取以上步骤收获的病毒培养物，用核酸提取试剂盒提取病毒样品的核酸，使用PCV2特异性引物进行PCR扩增鉴定，结果显示，PCR扩增出1.7kb目的条带。PCR产物送测序公司进行核苷酸序列测定，序列测定结果进行遗传进化分析。结果显示该病毒株全基因组序列同已经报道过的其他PCV2的同源性都少于96％，氨基酸序列少于94％，进一步分析该毒株介于PCV2b与PCV2d之间，ORF2基因中存在突变或存在不同基因亚型的重组，在遗传进化分析中属于一个新的PCV2基因亚型。
实施例6猪圆环病毒2型疫苗株的筛选
根据上述分离到的猪圆环病毒2型设计特异性引物，通过定量PCR 分析从全国各地免疫商品化PCV2疫苗后零星发病的猪场采集的42份疑似PCV2阳性样品，筛选分离出16株PCV2病毒，在这16株之间，其基因组核苷酸序列同源性高达99.6～100％，Cap蛋白氨基酸序列同源性高达99.5～100％，进一步分析属于一个基因亚型。经本动物致病性试验及免疫原性测试，最终筛选出1株致病性强、免疫原性良好、广谱性强的PCV2病毒株。该猪圆环病毒2型病毒命名为猪圆环病毒2型HH3株，提交保藏。
实施例7猪圆环病毒2型HH3株致病性试验
用28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪10头随机分成两组，5头/组，第3组用PCV2HH3株(含10 5.0TCID 50/头)攻毒，肌肉注射，第4组空白对照组接种DMEM培养基，各组仔猪隔离饲养。攻毒后连续观察各组仔猪，根据其临床症状、病理变化和病毒检测进行判定，具体结果见表2。
表2猪圆环病毒2型HH3株对仔猪致病性试验结果
Figure PCTCN2017118343-appb-000003
Figure PCTCN2017118343-appb-000004
结果显示，攻毒组所有猪只均出现3～5日持续性40.5℃以上高温，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒2型病毒，而空白对照组无异常，表明本发明猪圆环病毒2型HH3株接种仔猪可导致仔猪发病，临床表征为典型的猪圆环病毒感染症状。
实施例8猪圆环病毒2型HH3株抗原的制备
将实施例6筛选到的猪圆环病毒2型HH3株不同代次的培养物按照病毒培养液量的1％(V/V)接入形成单层的PK15传代细胞中，置于37℃吸附30分钟，然后加入细胞维持液，置于37℃培养。每日观察1～2次，细胞生长良好，于36～37℃培养4～7日后收获细胞培养物，收 获的细胞培养物冻融2-3次后，收获病毒液，测定病毒液中病毒毒价。将病毒液用中空纤维(0.5μm～2μm)滤柱过滤，以除去细胞碎片，再加入0.1％～0.2％的甲醛溶液在37℃灭活24h，灭活完全后的病毒抗原用以制备疫苗。
实施例9猪圆环病毒3型、2型二联灭活疫苗的制备
用实施例4制备的猪圆环病毒3型SG株灭活抗原、实施例8制备的猪圆环病毒2型HH3株灭活抗原按比例混合后缓缓加入到水溶性佐剂Gel佐剂(法国赛比克公司)中，加的过程中不断用转速为800rpm乳化机搅拌12min，混匀。疫苗具体配方见表3。
表3猪圆环病毒3型、2型二联灭活疫苗配方及含量
组分 疫苗1 疫苗2 疫苗3 疫苗4 疫苗5
PCV3抗原(TCID 50/ml) 10 5.0 10 6.0 10 7.0 10 7.0 -
PCV2抗原(TCID 50/ml) 10 5.0 10 6.0 10 7.0 - 10 7.0
Gel佐剂(V/V) 10％ 10％ 10％ 10％ 10％
实施例10猪圆环病毒3型、2型二联灭活疫苗免疫原性试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪50头随机分成10组，5头/组，免疫实施例9制备的猪圆环病毒3型、2型二联灭活疫苗。第5～6组免疫疫苗1，第7～8组免疫疫苗2，第9～10组免疫疫苗3，第11～12组分别免疫疫苗4、疫苗5，第13～14组不免疫作为攻毒对照组。各免疫组注射疫苗2ml/头，攻毒对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，第5组、第7组、第9组、第11组、第13组攻毒猪圆环病毒3型SG株10 5.0TCID 50/头，第6组、第8组、第10组、第12组、第14组攻毒猪圆环病毒2型HH3株10 5.0TCID 50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测结果进行判定，具体结果见表4。
表4猪圆环病毒3型、2型二联灭活疫苗免疫原性试验结果
Figure PCTCN2017118343-appb-000005
Figure PCTCN2017118343-appb-000006
结果显示，猪圆环病毒3型、2型二联灭活疫苗免疫一次仔猪后，即能为仔猪提供100％(5/5)保护，而攻毒对照组仔猪攻毒后全部发病。表明本发明提供的猪圆环病毒3型、2型二联灭活疫苗具有很好的保护效力。
实施例11猪圆环病毒3型、2型二联灭活疫苗广谱性保护试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪100头随机分成20组，5头/组，第15～24组免疫实施例9制备的疫苗1，第25～34组不免疫作为攻毒对照组。各免疫组注射疫苗2ml/头，攻毒对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，第15组、第25组用新近从中国河南省分离的猪圆环病毒2a基因亚型HN06 株强毒株攻毒；第16组、第26组用新近从中国江苏省分离的猪圆环病毒2b基因亚型JS04株强毒株攻毒；第17组、第27组用新近从中国吉林省分离的猪圆环病毒2d基因亚型JL13株强毒株攻毒；第18组、第28组用新近从中国重庆市分离的猪圆环病毒2new基因亚型CQ14株强毒株攻毒；第19组、第29组用新近从中国广东省分离的猪圆环病毒2new基因亚型GD 15株强毒株攻毒；第20组、第30组用新近从中国河南省分离的猪圆环病毒3型HN12株强毒株攻毒；第21组、第31组用新近从中国江苏省分离的猪圆环病毒3型JS08株强毒株攻毒；第22组、第32组用新近从中国吉林省分离的猪圆环病毒3型JL11株强毒株攻毒；第23组、第33组用新近从中国重庆市分离的猪圆环病毒3型CQ04株强毒株攻毒；第24组、第34组用新近从中国广东省分离的猪圆环病毒3型GD05株强毒株攻毒；各组攻毒剂量均为10 5.0TCID 50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测进行判定，具体结果见表5～6。
表5猪圆环病毒3型、2型二联灭活疫苗针对PCV2感染广谱性保护试验结果
Figure PCTCN2017118343-appb-000007
Figure PCTCN2017118343-appb-000008
结果显示，第25～29组攻毒对照组攻毒后均不同程度出现体温升高40.5℃以上，持续3～5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒2型病毒；而第15～19组免疫组攻毒后无异常临床症状，剖检各组织器官也无异常，通过各脏器组织进行PCR检测，均显示PCV2阴性。表明本发明提供的猪圆环病毒3型、2型二联灭活疫苗一次免疫，即可对猪只针对不同地域来源、不同基因亚型的猪圆环病毒2型攻毒提供有效的、完全免疫保护，且从各脏器组织中不能检出攻毒的PCV2。
表6猪圆环病毒3型、2型二联灭活疫苗针对PCV3感染广谱性保护试验结果
Figure PCTCN2017118343-appb-000009
Figure PCTCN2017118343-appb-000010
结果显示，第30～34组攻毒对照组攻毒后均不同程度出现体温升高40.5℃以上，持续3～5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型病毒；而第20～24组免疫组攻毒后无异常临床症状，剖检各组织器官也无异常，通过各脏器组织进行PCR检测，均显示PCV3阴性。表明本发明提供的猪圆环病毒3型、2型二联灭活疫苗一次免疫，即可对猪只针对不同地域来源的猪圆环病毒3型攻毒提供有效的、完全免疫保护，且从各脏器组织中不能检出攻毒的PCV3株。
上述结果表明，本发明提供的疫苗组合物具有广谱的免疫原性，对于不同地域来源的猪圆环病毒3型、2型毒株均能提供完全保护。
实施例12猪圆环病毒3型、2型二联灭活疫苗混合感染保护试验
将28～30日龄经ELISA检测PCV2、PCV3抗原、抗体阴性的健康仔猪20头随机分成4组，5头/组。第35组免疫实施例9制备的疫苗1，第36组免疫实施例9制备的疫苗4，第37组免疫实施例9制备的疫苗5，第38组不免疫作为攻毒对照组。各免疫组注射疫苗2ml/头，攻毒对照组接种DMEM培养基2ml/头。免疫后28日进行攻毒，所有组攻毒猪圆环病毒3型SG株和猪圆环病毒2型HH3株混合病毒液，攻毒剂量为10 5.0TCID 50/头，攻毒后连续观察各仔猪，根据各仔猪临床症状、病理变化结果进行判定，具体结果见表7。
表7猪圆环病毒3型、2型二联灭活疫苗混合感染保护试验结果
Figure PCTCN2017118343-appb-000011
Figure PCTCN2017118343-appb-000012
结果显示，第38组攻毒对照组攻毒后均不同程度出现体温升高40.5℃以上，持续5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化；第35组免疫组攻毒后无异常临床症状，剖检各组织器官也无异常；而第36组、第37组免疫组不能有效阻止PCV2、PCV3的混合感染，依然呈发病状态。表明本发明提供的猪圆环病毒3型、2型二联灭活疫苗免疫一次，即可对猪只针对PCV2、PCV3联合攻毒提供有效的、完全免疫保护。
实施例13猪圆环病毒3型、2型二联灭活疫苗应用试验
在国内一商品化猪场，与历史平均值相比，出现母猪死亡率增加9.8％，受孕率降低了1.2％，木乃伊胎增加8.6％的现象。临床表现上，受影响的母猪表现厌食，呈多灶性丘疹，斑点和表面皮炎的症状。在流产的窝中含有不同胎龄的木乃伊化胎儿，将具有临床表现症状的母猪中选取36头怀孕母猪随机分成两组A组、B组，18头/组，A组为疫苗接种组，A组免疫实施例9制备的疫苗1，B组为空白对照组。免疫组注 射疫苗2ml/头，空白对照组接种DMEM培养基2ml/头。统计两组母猪产子情况，结果见表8。
表8免疫组和空白对照组母猪产子统计结果
Figure PCTCN2017118343-appb-000013
Figure PCTCN2017118343-appb-000014
结果显示，免疫组母猪产子无异常，生产健康仔猪，平均11.7头/窝，健康率高达99.5％，而空白对照组出现明显的木乃伊胎及弱仔猪情况，生产健康仔猪平均6.9头/窝，健康率58.8％，其中还有三头母猪出现流产情况，全窝木乃伊胎，免疫组和空白对照组差异显著。
表8的结果证明了本发明猪圆环病毒3型、2型二联灭活疫苗一次免疫，即对感染猪圆环病毒的母猪有着良好的免疫保护作用，且能针对已经感染了PCV病毒的母猪进行保护。
同时，对B组空白对照组所产仔猪分别隔离按窝饲养，15窝分成两组：B1组(包括B-1窝至B-13窝，除B-6窝因全窝木乃伊胎不计外，共12窝仔猪)、B2组(包括B-14至B-18窝，除B-16窝和B-18窝因全窝木乃伊胎不计外，共3窝仔猪)，B1组吃母乳之前免疫实施例9制备的疫苗1，B2组为空白对照组。免疫组注射疫苗2ml/头，对照组接种DMEM培养基2ml/头。连续观察各仔猪，根据各仔猪临床症状、病理变化和病毒检测进行判定，具体结果见表9。
表9猪圆环病毒3型、2型二联疫苗对仔猪的免疫保护试验
Figure PCTCN2017118343-appb-000015
Figure PCTCN2017118343-appb-000016
Figure PCTCN2017118343-appb-000017
结果显示，免疫组仔猪无异常临床症状，随机剖检各组织器官也无异常，通过剖检猪只各脏器组织进行PCR检测，均显示PCV3、PCV2阴性，而空白对照组仔猪均不同程度出现体温升高40.5℃以上，持续5天，食欲减退、精神沉郁、被毛粗乱、消瘦和生长速度减缓等临床症状，部分猪只死亡，剖检均出现不同程度肺实变、淋巴肿大、肾有坏死点的病理变化，通过各脏器组织进行PCR检测，可再次分离到猪圆环病毒3型和猪圆环病毒2型。
由于PCV在猪群中是可以通过垂直传播的，因此表9的结果证明了本发明猪圆环病毒3型、2型二联疫苗一次免疫，即对混合感染猪圆环病毒3型和2型的仔猪有着良好的免疫保护作用，且能针对已经混合感染了PCV3和PCV2病毒的仔猪进行治疗和保护，保护率为100％。
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已以优选实施例揭露如上，然而并非用以限定本发明，任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与修饰，均仍属于本发明技术方案的范围内。

Claims (13)

  1. 一种预防和/或治疗猪圆环病毒感染的疫苗组合物，其中，所述疫苗组合物包含免疫量的猪圆环病毒3型抗原、免疫量的猪圆环病毒2型抗原以及药学上可以接受的载体。
  2. 根据权利要求1所述的疫苗组合物，其中，所述的猪圆环病毒3型抗原为猪圆环病毒3型SG株抗原，所述SG株保藏号为CCTCC NO.V201712；所述的猪圆环病毒2型抗原为猪圆环病毒2型HH3株抗原，所述HH3株保藏号为CCTCC NO.V201726。
  3. 根据权利要求2所述的疫苗组合物，其中，所述的猪圆环病毒3型SG株抗原为猪圆环病毒3型SG株或其培养物的灭活全病毒抗原，所述猪圆环病毒3型SG株的培养物为≥1代次的培养物；
    所述的猪圆环病毒2型HH3株抗原为猪圆环病毒2型HH3株或其培养物灭活全病毒抗原，所述猪圆环病毒2型HH3株的培养物为≥1代次的培养物。
  4. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株的培养物为≥5代次的培养物，所述猪圆环病毒2型HH3株培养物为≥5代次的培养物。
  5. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株的培养物为5～55代次的培养物，所述猪圆环病毒2型HH3株培养物为5～48代次的培养物。
  6. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株或其培养物的灭活全病毒抗原含量为灭活前≥10 5.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前≥10 5.0TCID 50/ml。
  7. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株或其培养物灭活全病毒抗原含量为灭活前10 5.0～10 7.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前10 5.0～10 7.0TCID 50/ml。
  8. 根据权利要求3所述的疫苗组合物，其中，所述猪圆环病毒3型SG株或其培养物灭活全病毒抗原含量为灭活前10 6.0TCID 50/ml，所述猪圆环病毒2型HH3株或其培养物灭活全病毒抗原含量为灭活前10 6.0TCID 50/ml。
  9. 根据权利要求1所述的疫苗组合物，其中，所述药学上可以接受的载体为佐剂，所述佐剂包括白油、德雷克油，以及动物油、植物油或矿物油；或氢氧化铝、磷酸铝及金属盐；或Montanide TMGel、卡波姆、角鲨烷或角鲨烯、ISA206佐剂、皂苷、油包水乳剂、水包油乳剂、水包油包水乳剂；
    所述佐剂含量为5～20V/V％。
  10. 根据权利要求1所述的疫苗组合物，其中，所述佐剂为Montanide TMGel。
  11. 根据权利要求1所述的疫苗组合物，其中，所述佐剂含量为10V/V％。
  12. 权利要求1～11任一项所述的疫苗组合物在制备预防和/或治疗猪圆环病毒感染导致的相关疾病的药物中的应用，其中，所述猪圆环病毒感染导致的相关疾病为由猪圆环病毒2型不同基因亚型和猪圆环病毒3型单独感染或它们混合感染导致的相关疾病。
  13. 根据权利要求12所述的应用，其中，所述猪圆环病毒感染导致的相关疾病包括断奶仔猪多系统衰竭综合征、猪的皮炎肾病综合征、繁殖障碍及心脏和多系统的炎症反应。
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