CN116790511A - Canine adenovirus type 2 attenuated strain, vaccine composition prepared from same and application thereof - Google Patents
Canine adenovirus type 2 attenuated strain, vaccine composition prepared from same and application thereof Download PDFInfo
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- CN116790511A CN116790511A CN202210274441.5A CN202210274441A CN116790511A CN 116790511 A CN116790511 A CN 116790511A CN 202210274441 A CN202210274441 A CN 202210274441A CN 116790511 A CN116790511 A CN 116790511A
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- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
-
- 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
- A61P31/14—Antivirals for RNA 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
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
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Abstract
The invention provides a canine adenovirus type 2 HL50 vaccine strain which has good immunogenicity. The invention also provides a vaccine composition comprising an immunizing amount of live whole virus antigen of canine adenovirus type 2 HL50 strain or a culture thereof, and a pharmaceutically acceptable carrier. The vaccine composition can prevent and treat canine adenovirus type 1 and canine adenovirus type 2 infection; can also prevent and treat infection of foxes, and can prevent and treat infection of wild strains from different regions, and can block continuous infection of canine adenovirus type 1 and/or canine adenovirus type 2.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a canine adenovirus type 2 natural attenuated strain, a vaccine composition prepared from the canine adenovirus type 2 natural attenuated strain and application of the vaccine composition.
Background
Canine adenovirus is divided into two types, wherein canine adenovirus type 1 (CAV-1) mainly causes canine acute sepsis hepatitis and bear, fox encephalitis; the pathogenicity is strong, and the range of infection hosts is wide; canine adenovirus type 2 (CAV-2) mainly causes infectious laryngotracheitis, puppy pneumonia and the like of dogs, and brings great harm to the economic animal breeding industry for dogs and fur.
In the prior art, CAV-1 attenuated vaccine can cause ocular and renal lesions and cause transient blue eye, so CAV-2 attenuated vaccine is adopted in the world at present. However, at present, symptoms such as cough and the like appear in some beagle farms in China, and as a result, CAV-2 infection is detected to be positive, but the canines are vaccinated according to a normal immunization program, which indicates that the vaccine on the market at present cannot resist the infection of CAV-2 wild toxin.
Therefore, it is highly desirable to provide a vaccine against the existing epidemic canine adenovirus, which can prevent diseases caused by canine adenovirus infection, and has a preventive effect on clinical application, such as different regions of infected dogs, and has a very practical significance.
Disclosure of Invention
The invention relates to a canine adenovirus type 2 natural attenuated strain with good immunogenicity, which is canine adenovirus type 2 HL50 strain, the preservation number is CCTCC NO: V202215, the canine adenovirus type 2 natural attenuated strain is preserved in China center for type culture Collection, the preservation address is the university of Chinese martial arts, and the preservation date is 2022, 3 months and 1 days.
The invention also relates to a vaccine composition, wherein the vaccine composition comprises an immunizing amount of live whole virus antigen of canine adenovirus type 2 HL50 strain or a culture thereof, and a pharmaceutically acceptable carrier. It has good immunogenicity, and can prevent and treat canine adenovirus type 2 wild strain infection.
The invention also relates to a vaccine composition which has good immunogenicity and can prevent and treat the infection of canine adenovirus type 1 wild strain.
The invention also relates to a vaccine composition which has good immunogenicity and can prevent and treat canine adenovirus type 2 wild strain infection of dogs.
The invention also relates to a vaccine composition which has good immunogenicity and can prevent and treat the infection of canine adenovirus type 1 wild strain of dogs.
The invention also relates to a vaccine composition which has good immunogenicity and can prevent the infection of the canine adenovirus type 1 wild strain of foxes.
The invention also relates to a vaccine composition which can prevent and treat the infection of canine adenovirus type 1 wild strains from different regions to dogs.
The invention also relates to a vaccine composition which can prevent and treat the infection of canine adenovirus type 2 wild strains from different regions to dogs.
The invention also relates to a vaccine composition which can block continuous infection of canine adenovirus type 1 and/or canine adenovirus type 2.
The invention also relates to application of the vaccine composition in preparing medicines for preventing and/or treating canine adenovirus type 1 and/or canine adenovirus type 2 infection.
Detailed Description
Hereinafter, embodiments of the present invention will be described.
Definition of the definition
The term "immunologically effective amount" refers to the amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The exact amounts of ingredients used and the compositions to be administered will vary depending upon factors such as the type of disease being treated, the type and age of the animal to be treated, the manner of administration, and other ingredients in the composition.
The term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate the body and does not impede the use of the biological activity and properties of the compound.
The term "preventing" as used herein refers to reducing all the actions of detoxification after infection by inhibiting canine adenovirus and/or canine distemper virus and/or canine parvovirus infection or delaying onset of disease by administering a vaccine composition according to the present invention. The term "treatment" refers to all actions that result in alleviation or amelioration of symptoms caused by canine adenovirus and/or canine distemper virus and/or canine parvovirus infection by administration of the vaccine composition according to the present invention.
Detailed Description
In order to solve the defects in the prior art, the invention provides a canine adenovirus type 2 vaccine composition, wherein the vaccine composition comprises an immunity amount of canine adenovirus type 2 HL50 strain antigen and a freeze-drying protective agent; the canine adenovirus type 2 HL50 strain antigen is a live whole virus antigen of canine adenovirus type 2 HL50 strain or a culture thereof, the canine adenovirus type 2 HL50 strain (Canine adenovirus, strain HL50) is preserved with the CCTCC NO: V202215, and is preserved in China center for type culture collection, the preservation address is the university of Wuhan and Wuhan in China, and the preservation time is 2022 years, 3 months and 1 day.
The canine adenovirus type 2 attenuated strain has stable biological characteristics, good immunogenicity, safety and reliability, and the prepared attenuated live vaccine can generate higher-level antibodies and has good protection effect on canine adenovirus type 1 and canine adenovirus type 2 virulent attacks.
The vaccine composition can protect the existing epidemic strains, has a preventive effect, can treat infected dogs, and can further block continuous infection of canine adenovirus type 1 and canine adenovirus type 2.
After the vaccine composition provided by the invention is used for immunizing dogs in different areas, viral infection can be blocked (clinical symptoms appear), 100% protection can be provided for dogs, and the canine adenovirus type 2 HL50 attenuated live vaccine disclosed by the invention has good protective power on dogs infected with canine adenovirus type 1 and canine adenovirus type 2 in clinic, and shows good immune protection and safety against canine adenovirus type 1 and canine adenovirus type 2 infection in different areas.
After the vaccine composition provided by the invention is used for immunizing a diseased dog, the vaccine composition can still block virus infection (clinical symptoms) for dogs subjected to repeated infection, so that the vaccine composition has practical clinical treatment significance.
The amount of the ingredients or components of the compositions of the present invention is preferably an immunologically effective amount. By immunologically effective amount is meant that amount necessary to exert their immunological effects in the host to which the composition is administered without causing undue side effects. The exact amounts of ingredients used and the compositions to be administered will vary depending upon factors such as the type of disease being treated, the type and age of the animal to be treated, the manner of administration, and other ingredients in the composition.
As one embodiment of the present invention, the canine adenovirus type 2 HL50 strain antigen content in the vaccine composition of the present invention is not less than 10 3.0 TCID 50 Head.
As a preferred embodiment of the present invention, the canine adenovirus type 2 HL50 strain antigen content in the vaccine composition of the present invention is 10 3.0 ～10 6.0 TCID 50 Head.
The canine adenovirus type 2 HL50 strain antigen content can be selected from 10 3.0 TCID 50 Head, 10 3.1 TCID 50 Head, 10 3.2 TCID 50 Head, 10 3.3 TCID 50 Head, 10 3.4 TCID 50 Head, 10 3.5 TCID 50 Head, 10 3.6 TCID 50 Head, 10 3.7 TCID 50 Head, 10 3.8 TCID 50 Head, 10 3.9 TCID 50 Head, 10 4.0 TCID 50 Head, 10 4.1 TCID 50 Head, 10 4.2 TCID 50 Head, 10 4.3 TCID 50 Head, 10 4.4 TCID 50 Head, 10 4.5 TCID 50 Head, 10 4.6 TCID 50 Head, 10 4.7 TCID 50 Head, 10 4.8 TCID 50 Head, 10 4.9 TCID 50 Head, 10 5.0 TCID 50 Head, 10 5.1 TCID 50 Head, 10 5.2 TCID 50 Head, 10 5.3 TCID 50 Head, 10 5.4 TCID 50 Head, 10 5.5 TCID 50 Head, 10 5.6 TCID 50 Head, 10 5.7 TCID 50 Head, 10 5.8 TCID 50 Head, 10 5.9 TCID 50 Head, 10 6.0 TCID 50 Head.
As a more preferred embodiment of the invention, the vaccine combination of the inventionIn the composition, the antigen content of the canine adenovirus 2 type HL50 strain is 10 4.0 ～10 5.0 TCID 50 Head.
In one embodiment of the present invention, the canine adenovirus type 2 HL50 strain culture is a 1-70-generation culture in the vaccine composition of the present invention.
Canine adenovirus type 2 HL50 strain cultures may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70 cultures.
As an embodiment of the present invention, the vaccine composition of the present invention, the lyoprotectant comprises SPGA, saccharide, protein-containing substance or/and buffer; preferably, the saccharide is selected from sorbitol, mannitol, starch, sucrose, glucose, dextran, the protein is selected from albumin or casein, the protein-containing substance is bovine serum or skim milk, and the buffer is phosphate buffer; more preferably, the lyoprotectant is an aqueous solution of 40w/v% sucrose and 8w/v% gelatin, and the ratio of lyoprotectant to antigen is 1:1 by volume.
As one embodiment of the present invention, the vaccine composition of the present invention further comprises an immunizing amount of a canine distemper virus LT90 strain antigen, wherein the canine distemper virus LT90 strain antigen is a live whole virus antigen of the canine distemper virus LT90 strain or a culture thereof, and the canine distemper virus LT90 strain has a preservation number of cctccc NO: v202030. Canine distemper virus LT90 Strain (Canine Distemper Virus, strain LT 90) is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: v202030, the preservation address is Wuhan university of Chinese, the preservation date is 13 days of 5 months in 2020, and is disclosed in patent application CN 113827716A.
In the vaccine composition, the canine distemper virus LT90 strain is Asia-1, and can completely protect the main epidemic wild strain in China with the protection rate of 100% (5/5).
As one embodiment of the present invention, the vaccine composition of the present invention further comprises an immunizing amount of canine parvovirus JM35 strain antigen, wherein the canine parvovirus JM35 strain antigen is a live whole virus antigen of the canine parvovirus JM35 strain or a culture thereof, and the canine parvovirus JM35 strain has a accession number of cctccc NO: v201965. Canine parvovirus JM35 Strain (Canine Parvovirus, strain JM 35) was deposited at the chinese collection for typical cultures under the accession number cctccc NO: v201965, the preservation address is university of Wuhan and Wuhan in China, the preservation date is 10 months and 22 days in 2019, and is disclosed in patent application CN 113073083A.
In the vaccine composition, the canine parvovirus JM35 strain is new CPV-2a type, and can completely protect the main epidemic wild strain in China with the protection rate of 100% (5/5).
The triple vaccine composition can completely protect the canine adenovirus type 1 domestic main epidemic wild strain, the canine adenovirus type 2 domestic main epidemic wild strain, the canine distemper virus domestic main epidemic wild strain and the canine parvovirus domestic main epidemic wild strain, and shows better immune protection and safety than the existing commercial vaccine aiming at the existing epidemic strain.
As one embodiment of the present invention, in the vaccine composition of the present invention, the antigen content of the canine distemper virus LT90 strain is not less than 10 2.5 FAID 50 Head.
As a preferred embodiment of the present invention, the vaccine composition of the present invention has an antigen content of 10 in the LT90 strain of canine distemper virus 2.5 ～10 5.5 FAID 50 Head.
The antigen content of canine distemper virus LT90 strain can be selected from 10 2.5 FAID 50 Head, 10 2.6 FAID 50 Head, 10 2.7 FAID 50 Head, 10 2.8 FAID 50 Head, 10 2.9 FAID 50 Head, 10 3.0 FAID 50 Head, 10 3.1 FAID 50 Head, 10 3.2 FAID 50 Head, 10 3.3 FAID 50 Head, 10 3.4 FAID 50 Head, 10 3.5 FAID 50 Head, 10 3.6 FAID 50/ Head part, 10 3.7 FAID 50 Head, 10 3.8 FAID 50 Head, 10 3.9 FAID 50 Head, 10 4.0 FAID 50 Head, 10 4.1 FAID 50 Head, 10 4.2 FAID 50 Head, 10 4.3 FAID 50 Head, 10 4.4 FAID 50 Head, 10 4.5 FAID 50 Head, 10 4.6 FAID 50 Head, 10 4.7 FAID 50 Head, 10 4.8 FAID 50 Head, 10 4.9 FAID 50 Head, 10 5.0 FAID 50 Head, 10 5.1 FAID 50 Head, 10 5.2 FAID 50 Head, 10 5.3 FAID 50 Head, 10 5.4 FAID 50 Head, 10 5.5 FAID 50 Head.
As a more preferred embodiment of the present invention, the vaccine composition of the present invention has an antigen content of 10 in the LT90 strain of canine distemper virus 3.5 ～10 4.5 FAID 50 Head.
In one embodiment of the present invention, in the vaccine composition of the present invention, the canine distemper virus LT90 strain culture is a culture of 1 to 100 generations.
The canine distemper virus LT90 strain culture may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 cultures.
As a means ofIn one embodiment of the present invention, the antigen content of the canine parvovirus JM35 strain in the vaccine composition of the present invention is not less than 10 4.0 FAID 50 Head.
As a preferred embodiment of the present invention, the vaccine composition of the present invention has an antigen content of 10 in the canine parvovirus JM35 strain 4.0 ～10 6.5 FAID 50 Head.
The antigen content of canine parvovirus JM35 strain is 10 4.0 FAID 50 Head, 10 4.1 FAID 50 Head, 10 4.2 FAID 50 Head, 10 4.3 FAID 50 Head, 10 4.4 FAID 50 Head, 10 4.5 FAID 50 Head, 10 4.6 FAID 50 Head, 10 4.7 FAID 50 Head, 10 4.8 FAID 50 Head, 10 4.9 FAID 50 Head, 10 5.0 FAID 50 Head, 10 5.1 FAID 50 Head, 10 5.2 FAID 50 Head, 10 5.3 FAID 50 Head, 10 5.4 FAID 50 Head, 10 5.5 FAID 50 Head, 10 5.6 FAID 50 Head, 10 5.7 FAID 50 Head, 10 5.8 FAID 50 Head, 10 5.9 FAID 50 Head, 10 6.0 FAID 50 Head, 10 6.1 FAID 50 Head, 10 6.2 FAID 50 Head, 10 6.3 FAID 50 Head, 10 6.4 FAID 50 Head, 10 6.5 FAID 50 Head.
As a more preferred embodiment of the present invention, the vaccine composition of the present invention has an antigen content of 10 in the canine parvovirus JM35 strain 5.0 ～10 6.0 FAID 50 Head.
In one embodiment of the present invention, the canine parvovirus JM35 strain culture is 1 to 100 generations of culture in the vaccine composition of the present invention.
The canine parvovirus JM35 strain culture may be selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 cultures.
The invention also provides a method for preparing the vaccine composition, wherein the method comprises the following steps: step (1) amplifying the canine adenovirus type 2 HL50 strain or a culture thereof; and (2) adding a freeze-drying protective agent into the canine adenovirus type 2 HL50 strain or the culture thereof amplified in the step (1), and uniformly mixing.
The invention also provides a method for preparing the vaccine composition, wherein the method comprises the following steps: step (1) amplifying the canine adenovirus 2 type HL50 strain or a culture thereof, the canine distemper virus LT90 strain or a culture thereof, and the canine parvovirus JM35 strain or a culture thereof respectively; and (2) adding a freeze-drying protective agent into the amplified canine adenovirus type 2 HL50 strain or a culture thereof, canine distemper virus LT90 strain or a culture thereof and the amplified parvovirus JM35 strain or a culture thereof in the step (1) and uniformly mixing.
The invention also provides application of the vaccine composition in preparing medicines for preventing and/or treating canine adenovirus infection.
The invention also provides a canine adenovirus type 2 attenuated strain HL50, wherein the canine adenovirus type 2 attenuated strain HL50 has a preservation number of CCTCC NO: V202215. Canine adenovirus 2 strain HL50 (canine adenovirus, strain HL 50) was deposited at the chinese collection of typical cultures, at the university of martial arts, at 2022, day 3 month 1.
The pathogenicity test shows that the canine adenovirus type 2 HL50 strain is cultured for 1 to 70 generations, and the pathogenicity of the canine is obviously reduced. Dogs were observed 21 days after 70 passages of inoculation, were free of clinical symptoms and were examined for tissue and organ changes. Therefore, compared with the parent virulent canine adenovirus type 2 HL006 strain, the virulence of the virus is obviously reduced, and the virus is an artificially attenuated virus strain.
The immunogenicity test shows that the canine adenovirus type 2 HL50 strain is cultured until the 70 th generation, and still has good immunogenicity. Dogs were resistant to challenge with virulent adenovirus type 2 HL006 strain 14 days after single dose repeat vaccination. Meanwhile, dogs which are not inoculated with adenovirus type 2 HL50 strain cultures cannot resist the attack of canine adenovirus type 2 HL006 strain, and all diseases occur.
Virulence reversion experiments show that viruses between 1 generation and 70 generation are cultured, and the viruses are continuously contacted and passaged for multiple times in the canine group after inoculation, so that reversion does not occur. Therefore, after the viruses are inoculated into the canine, the viruses cannot be evolved again to be virulent to cause diseases, and the safety is ensured.
Compared with the canine adenovirus type 2 parent virulent strain, cultures of canine adenovirus type 2 HL50 strain from generation 1 to generation 70 have the common occurrence of amino acid coded by each gene of the virus, so that the amino acid mutation of Fiber protein S492R is caused; the E3 gene (ORF 2) was inserted at position 1091 with nucleotide A, resulting in an amino acid mutation (M364N) and a stop codon back shift, 11 amino acids added (VACQINLPNFC).
The characteristic changes of the commonality of the occurrence of the amino acids encoded by the viral genes of different generations of cultures of canine adenovirus type 2 HL50 are probably the cause of the reduced virulence of its parent virulent strain, and at the same time, these changes do not lead to the change of the immunogenicity thereof.
The canine adenovirus type 2 attenuated strain Fiber amino acid sequence of the invention has the advantages of reduced toxicity and genetic stability compared with the parent attenuated strain due to the point mutation and the E3 gene insertion nucleotide A.
Other examples that may also contain pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteins such as albumin or casein, protein-containing substances such as bovine serum or skim milk, and buffers (e.g., phosphate buffers).
Particularly when such stabilizers are added to a vaccine, the vaccine is well suited for freeze-drying. Thus, in a more preferred form of this embodiment, the live attenuated vaccine is in a freeze-dried form.
Optionally, one or more compounds having adjuvant activity may be added to the vaccine. Such an adjuvant is not necessarily required for efficacy of a live attenuated canine adenovirus according to the invention, but would be worth adding an adjuvant, particularly for a combination vaccine comprising a live attenuated canine adenovirus according to the invention and an antigenic material from another pathogenic virus or microorganism. Adjuvants are non-specific stimulators of the immune system that enhance the host's immune response to a vaccine. Examples of adjuvants known in the art are Freund's complete and incomplete adjuvants, vitamin E, nonionic block polymers, muramyl dipeptide, ISCOMs (immunostimulatory complexes, see for example European patent EP 109942), saponins, mineral oils, vegetable oils, and carbomers (Carbopol).
Thus, in a preferred form of this embodiment, the live attenuated vaccine according to the invention comprises an adjuvant.
The advantages and features of the present invention will become more apparent from the following description of the embodiments. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
The chemical reagents used in the examples of the invention are all analytically pure and purchased from the national drug group. The experimental methods provided by the invention are conventional methods unless specified; the biological material, unless otherwise specified, is commercially available.
Example 1 obtaining canine adenovirus 2 type HL50 Strain
1. Taking well-grown MDCK cells, digesting the MDCK cells by pancreatin, inoculating the MDCK cells into a cell bottle, and continuously culturing the MDCK cells in a cell growth solution (pH is adjusted to be 6.8-7.2) containing 90-97% of DMEM culture solution and 3-10% of newborn calf serum by volume ratio at the temperature of 33-37 ℃ for inoculating viruses.
2. Synchronously inoculating canine adenovirus type 2 HL006 strain into MDCK cell suspension, continuously culturing with cell growth liquid (pH is regulated to 6.8-7.2) containing 95-99% of DMEM culture liquid and 1-5% of newborn calf serum at 33-37 ℃ for 36-48 h, and harvesting cell culture virus liquid when more than 80% of cells are diseased.
3. Repeating the steps to continuously subculture (50 generations) to obtain canine adenovirus type 2 attenuated strain, and sequencing the canine adenovirus type 2 attenuated strain, wherein the result shows that the Fiber protein S492R has stable variation of amino acid; the E3 gene (ORF 2) was inserted at position 1091 with nucleotide A, resulting in a stable mutation of amino acid M364N, and the stop codon was shifted back, resulting in an increase of 11 amino acids (VACQINLPNFC). The canine adenovirus type 2 attenuated strain is named canine adenovirus type 2 HL50 strain.
EXAMPLE 2 canine adenovirus 2 type HL50 strain biological property study
1. Pathogenicity test
15 healthy susceptible canine adenovirus type 1 and canine adenovirus type 2 antigen antibody negative dogs of 2-3 months of age are randomly divided into 3 groups of 5 dogs each, and the grouping and virus attack conditions are shown in table 1.
TABLE 1 grouping of pathogenicity test animals
| Group of | Strains for inoculation | Dose of inoculation |
| 1 | HL50 strain | Nose drops 2ml (10) 4.0 TCID 50 /ml) |
| 2 | HL006 strain | Nose drops 2ml (10) 4.0 TCID 50 /ml) |
| 3 | DMEM medium | Nose drops 2ml |
The dogs were observed for body temperature, spirit, appetite, faeces, ocular and nasal secretions, sneeze or not at 14 days after virus inoculation, and the specific results are shown in Table 2.
TABLE 2 pathogenicity of HL50 strains against canine
The results show that canine adenovirus type 2 HL006 strain can cause 100% of the disease of dogs (5/5), while canine adenovirus type 2 HL50 strain has normal body temperature, no clinical symptoms and no change of tissue and organ of the section examination.
The pathogenicity test shows that compared with the parent virulent strain HL006, the canine adenovirus type 2 HL50 strain has obviously reduced pathogenicity and is a attenuated virus strain.
Meanwhile, in order to verify the stability of different generations of the canine adenovirus type 2 HL50 strain, a group of canine adenovirus type 1 and canine adenovirus type 2 antigen antibody negative dogs (5 heads) are inoculated with canine adenovirus type 2 HL50 strain cultures of 1 generation, 10 generation, 30 generation, 50 generation and 70 generation, and 2ml (10) 4.0 TCID 50 Per ml)/head, 5 dogs served as control group. Clinical changes in dogs were observed and recorded daily until 14 days of inoculation.
The results show that the canine adenovirus type 2 HL50 strain cultures of the 1 st generation, the 10 th generation, the 30 th generation, the 50 th generation and the 70 th generation are observed for 14 days after inoculation, have normal body temperature, have no clinical symptoms and have no change in tissue organs of the section examination.
Different generations of pathogenicity tests show that the pathogenicity of different generations of cultures of the canine adenovirus type 2 HL50 strain is obviously reduced, and the canine adenovirus type 2 strain is a weakened virus strain.
2. Immunogenicity test
2.1 protective Effect on canine adenovirus 2 type HL006 Strain
10 healthy susceptible antigen antibody negative dogs of 2-3 months of age were randomly divided into 2 groups of 5 animals each, and the grouping and immunization conditions are shown in Table 3.
TABLE 3 grouping of immunogenicity test animals
| Group of | Strains for inoculation | Dose of inoculation |
| 4 | HL50 strain | Subcutaneous 1ml (10) 5.0 TCID 50 /ml) |
| 5 | DMEM medium | Subcutaneous 1ml |
The second immunization was performed 21 days after the first immunization. 14 days after the second immunization, 5 dogs inoculated with canine adenovirus type 2 HL50 strain and 5 dogs in the control group were each vaccinated with canine adenovirus type 2 HL006 strain in a nasal drop of 2ml (10 4.0 TCID 50 The composition can be used for treating canine body temperature, mental, appetite, feces, ocular and nasal secretion, sneeze, etc., by taking into account/ml)/head doseThe results are shown in Table 4.
TABLE 4 immunogenicity of HL50 strains against dogs
The results showed that canine adenovirus type 2 HL50 vaccinated dogs were fully protected, while control dogs were fully ill.
The immunogenicity test shows that the canine adenovirus type 2 HL50 strain has good immunogenicity and can produce good protection effect on canine adenovirus type 2 HL006 strain attack.
Meanwhile, in order to verify the stability of different generations of immunogenicity of the canine adenovirus type 2 HL50 strain, cultures of the canine adenovirus type 2 HL50 strain of the 1 st generation, the 10 th generation, the 30 th generation, the 50 th generation and the 70 th generation are respectively immunized, and secondary immunization is carried out 21 days after the primary immunization; 14 days after the second immunization, each immunization group together with the control group was treated with canine adenovirus strain 2 type HL006 in a nasal drop of 2ml (10 4.0 TCID 50 The composition can be used for treating canine body temperature, mental, appetite, fecal, ocular and nasal secretions, sneeze, etc., by taking into account the toxic materials of the head and head doses, and recording the results of the oral administration.
The results showed that dogs vaccinated with the canine adenovirus type 2 HL50 strain cultures of generation 1, 10, 30, 50, and 70 were fully protected and dogs in the control group were fully ill.
Different generations of immunogenicity experiments show that the canine adenovirus type 2 HL50 strain culture has good immunogenicity and can generate good protection effect on canine adenovirus type 2 HL006 strain attack.
3. Virus reversion safety test
Canine adenovirus 2 type HL50 strain 1 generation virus liquid (10) 6.5 TCID 50 Per ml) 3 healthy susceptible dogs were vaccinated by nasal drops of 2 ml. Clinical manifestations are observed by measuring temperature in the morning and evening every day after inoculation, splitting and killing are performed on the 5 th day after inoculation, lesions are observed, histology HE staining and immunohistochemical detection are performed on lung samples, canine adenovirus nucleic acid measurement is performed on lung grinding fluid, and canine lungs with high virus content are selected to be properly treated and used as inoculum for next generation virulence return, so that the canine adenovirus nucleic acid is transmitted in vivoGeneration 4.
Filtering lung grinding fluid obtained by taking the 4 th generation virulence return test, and inoculating 3 healthy susceptible dogs by 2ml nasal drops. Clinical manifestations were gently observed daily early and late, dissected and killed at day 21 post inoculation, lesions were observed, HE and immunohistochemical assays were performed on lung, intestinal, mesenteric lymph nodes and brain samples, and virus nucleic acid assays were performed on lung milling fluid.
If in the 1 st-4 th generation virulence return test, if the lung tissue cannot detect canine adenovirus nucleic acid in a certain generation test, the inoculation amount is properly increased, 6 dogs are inoculated in the same way, so that the detection rate of the viral nucleic acid is improved, and if the further test confirms that the canine adenovirus type 2 nucleic acid cannot be detected again in the lung after the secondary test, the result of the virulence return test can be judged to be true, and the virulence return test is not possible.
The results show that the body temperature and clinical manifestations of dogs inoculated with the 1 st-5 th generation virus liquid are not abnormal, the organs are not obviously diseased after sectioning, the lung alveoli are slightly thickened only in the 1 st generation of lung after HE staining, CAV-2 antigen positivity is visible in lung immunohistochemical detection, the lung HE staining is not abnormal in the 2 nd-5 th generation virulence return test, the immunohistochemical is negative, and the viral load in the lung is gradually reduced along with the increase of the generation. The experimental dogs which are bred together in the virulence return test have no abnormality in clinical observation, and serum CAV antibodies turn positive after 21 days of the literacy return test, and CAV-2 virus nucleic acid can be detected in a swab, so that canine adenovirus type 2 HL50 strain has no virulence return capability, but has certain horizontal transmission capability, and is a weak strain with good safety and immunogenicity.
The 10 th generation, 30 th generation, 50 th generation and 70 th generation virus solutions of the canine adenovirus 2 type HL50 strain are respectively verified by referring to the virus back-strengthening test steps, and the result shows that: the body temperature and clinical manifestation of dogs inoculated with the 1 st to 5 th generation virus liquid of each generation are not abnormal, the organs are not obviously diseased after sectioning, the lung of the 1 st generation only has slight pathological change after HE staining, CAV-2 antigen is positive in lung immunohistochemical detection, the lung HE staining is not abnormal in the 2 nd to 5 th generation virulence return test, the immunohistochemical is negative, and the viral load in the lung is gradually reduced along with the rise of the generation.
Therefore, the canine adenovirus type 2 HL50 strain cannot be evolved again into virulent strain to cause morbidity, and the safety is ensured.
4. Gene sequence analysis
Cultures of canine adenovirus type 2 HL50 strain 1 st to 70 th generation were subjected to genome amplification (each of the different generation cultures was amplified) by PCR. The obtained gene amplification product is recovered, purified, connected to a sequencing plasmid vector, and the nucleotide sequence of the virus gene is determined and converted into the amino acid sequence of the virus by computer software. And comparing the obtained amino acid sequence with the amino acid sequence of the parent virulent strain HL006 strain by using sequence analysis software, and describing the characteristics of the amino acid sequence of the virus.
The results show that the amino acid commonality of the codes of the genes of the canine adenovirus 2 type HL50 strain from the 1 st generation to the 70 th generation causes the amino acid variation of the Fiber protein S492R; the E3 gene (ORF 2) was inserted at position 1091 with nucleotide A, causing the amino acid M364N mutation and stop codon to be back shifted, adding 11 amino acids (VACQINLPNFC).
It is shown that the characteristic change of the commonality of the amino acid coded by the virus genes of different generations of subculture of canine adenovirus type 2 HL50 strain is probably the reason of the reduced virulence of the parent virulent strain.
Example 3 preparation of canine adenovirus 2 type HL50 attenuated live vaccine
1. Proliferation of viruses
Synchronously inoculating the canine adenovirus 2 type HL50 strain prepared in the example 1 into MDCK cell suspension, adding DMEM culture medium containing 1% of neonatal bovine serum, placing at 37 ℃ and 5% of CO 2 Culturing. After 80% of cytopathy, the viruses are harvested, the virus titer is measured and the viruses are preserved at low temperature.
2. Preparation of protective agent
Every 100ml deionized water is added with 40g of sucrose and 8g of gelatin, and after full melting, the mixture is put into high-pressure steam for sterilization (30 min at 121 ℃).
3. Preparation of vaccine
Mixing the prepared and stored virus liquid with a protective agent according to a volume ratio of 1:1, and freeze-drying. The specific proportions of the vaccine contents are shown in Table 5.
Table 5 content ratio of canine adenovirus 2 type HL50 attenuated live vaccine
| Component (A) | Vaccine 1 | Vaccine 2 | Vaccine 3 |
| HL50 strain antigen (TCID) 50 ) Head part | 10 3.0 | 10 4.0 | 10 6.0 |
| Protective agent (V/V) | 50％ | 50％ | 50％ |
EXAMPLE 4 immunogenicity test of canine adenovirus 2 type HL50 attenuated live vaccine
The canine adenovirus antigen antibody negative dogs of 28 days old or more are randomly divided into 4 groups of 5 dogs each, and the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the immunization example 3 is immunized. Group 6 immunization vaccine 1, group 7 immunization vaccine 2, group 8 immunization vaccine 3, and group 9 immunization PBS were used as control groups. Subcutaneous injection, two immunizations, two days apart, and 14 days later to detoxify, canine adenovirus strain 2 HL006 was used to drip 2ml of nasal drops (10 4.0 TCID 50 The/ml)/head dose attacks the toxin, and the dose is daily in the morning and evening after the toxin is attackedThe dogs were observed and recorded for body temperature, spirit, appetite, abdomen, eyes, etc., and specific results are shown in table 6.
Table 6 results of immunogenicity test of canine adenovirus 2 type HL50 attenuated live vaccine
The results show that the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3 can block viral infection (clinical symptoms) after being immunized, and can provide 100% (5/5) protection for dogs, and the dogs in the control group are all ill after being challenged.
Proved by the experiment, the canine adenovirus type 2 HL50 attenuated live vaccine of the three test groups has good protective power and shows good immune protection and safety.
Example 5 broad-spectrum test of canine adenovirus 2 type HL50 attenuated live vaccine
The canine adenovirus antigen antibody negative dogs of 28 days old or more are randomly divided into 4 groups of 5 dogs each, and the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the immunization example 3 is immunized. Group 10 immunization vaccine 1, group 11 immunization vaccine 2, group 12 immunization vaccine 3, and group 13 immunization PBS were used as control groups. Subcutaneous injection, two immunizations, two days apart, and two days apart, 14 days later, of virus challenge, all with canine adenovirus type 1 YT-2 strain in 1ml vein (10 4.0 TCID 50 The body temperature, spirit, appetite, abdomen, eyes and the like of the dogs are observed and recorded every morning and evening after the toxin is attacked by the/ml)/head dose, and the specific results are shown in Table 7. The canine adenovirus type 1 YT-2 strain (Canine adenovirus, strain YT-2) has the accession number: CCTCC NO. V202216, which is preserved in China center for type culture Collection, with a preservation address of university of Wuhan and Wuhan in China, and a preservation date of 2022, 3 months and 1 day.
TABLE 7 broad-spectrum test results of canine adenovirus 2 type HL50 attenuated live vaccine
The results show that after dogs are immunized by using the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3, canine adenovirus type 1 infection (clinical symptoms appear) can be blocked, 100% (5/5) protection can be provided for dogs, and the dogs in the control group are all ill after the dogs are challenged.
Proved by the experiment, the canine adenovirus type 2 HL50 attenuated live vaccine of the three test groups has good broad spectrum.
Example 6 test of canine adenovirus 2 type HL50 attenuated live vaccine against fox
The canine adenovirus antigen antibody negative foxes more than 28 days old are randomly divided into 4 groups of 20, and 5 animals are used for immunizing the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3. Group 14 immunization vaccine 1, group 15 immunization vaccine 2, group 16 immunization vaccine 3, group 17 immunization PBS was used as a control group. Subcutaneous injection, two immunizations, two days apart, and two days apart, 14 days later, of virus challenge, all with canine adenovirus type 1 YT-2 strain in 1ml vein (10 4.0 TCID 50 The body temperature, spirit, appetite, abdomen, eyes and the like of the fox are observed and recorded in the morning and evening every day after the toxin is attacked by the/ml)/head dose, and the specific results are shown in Table 8.
Table 8 test results of canine adenovirus 2 type HL50 attenuated live vaccine on foxes
The results show that after the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3 is used for immunizing foxes, viral infection can be blocked (clinical symptoms appear), 100% (5/5) protection can be provided for foxes, and the foxes in the control group are all ill after toxicity attack.
Proved by the experiment, the canine adenovirus type 2 HL50 attenuated live vaccine of the three test groups has good protective power and shows good immune protection and safety.
Example 7 comparative immunogenicity test of canine adenovirus 2 type HL50 attenuated live vaccine
15 dogs negative for 45 day old canine adenovirus antigen antibodies were randomly divided into 3 groups, 5 dogs/group. Group 18 single dose repeated immunization example 3 prepared canine adenovirus 2 type HL50 strain attenuated live epidemic diseaseVaccine 1; group 19 single dose repeat immunization commercial canine distemper, parvovirus, infectious hepatitis, parainfluenza four-way live vaccine (Manhattan LPV 3); group 20 immunized PBS served as control. At intervals of 21 days, two immunizations were carried out, and after 14 days, virus was challenged with canine adenovirus strain 1 YT-2 in 1ml vein (10 4.0 TCID 50 The body temperature, spirit, appetite, abdomen, eyes and the like of the dogs are observed and recorded every morning and evening after the toxin is attacked by the/ml)/head dose, and the specific results are shown in Table 9.
Table 9 results of comparative immunogenicity test of canine adenovirus 2 type HL50 attenuated live vaccine
The result shows that after the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3 is used for immunizing dogs, the viral infection can be blocked (clinical symptoms are not generated), and 100% (5/5) protection can be provided for dogs; all the dogs in the control group developed after the challenge; however, the existing commercial vaccines cannot fully protect dogs.
The canine adenovirus type 2 HL50 attenuated live vaccine has good protective power, and shows better immune protection and safety than the existing commercial vaccine aiming at the existing epidemic strain.
Example 8 clinical trials of canine adenovirus 2 type attenuated live vaccine of HL50 strain
The eye, nose and anus swabs were collected from dogs in some pet hospitals or canine fields in the four places of Tianjin, hebei, henan and Hubei, respectively, and vaccine 1 was prepared for canine adenovirus type 1 or canine adenovirus type 2 positive canine immunization example 3, and the body temperature, spirit, appetite, abdomen and eyes of the dogs were observed and recorded daily, and specific results are shown in table 10.
Table 10 results of clinical trials of canine adenovirus 2 type HL50 attenuated live vaccine
The results show that the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the embodiment 3 can block viral infection (clinical symptoms) after being used for immunizing dogs in different areas, and can provide 100% protection for the dogs, so that the canine adenovirus type 2 HL50 attenuated live vaccine can clinically treat dogs infected with canine adenovirus type 1 or canine adenovirus type 2.
Proved by the demonstration, the canine adenovirus type 2 HL50 attenuated live vaccine has good protective power, and shows good immune protection and safety against canine adenovirus type 1 or canine adenovirus type 2 infection in different regions.
Further randomly selecting 5 dogs infected with canine adenovirus before immunization in Tianjin, hebei, henan and Hubei canine fields from each group after gradually returning to normal; in addition, 10 dogs negative for canine adenovirus antigen antibody are selected as a control, and randomly divided into two groups of 5 dogs. Wherein, the 21-1 group, the 22-1 group and the control 25 group are respectively prepared by using canine adenovirus type 1 YT-2 strain in 1ml (10 4.0 TCID 50 The composition is prepared by the steps of (1)/ml)/head dose detoxification, and observation and recording of the body temperature, spirit, appetite, abdomen, eyes and the like of a dog are carried out every day, morning and evening after detoxification; the canine adenovirus strain 2 HL006 was used in groups 23-1, 24-1 and control 26 to make nasal drops of 2ml (10 4.0 TCID 50 The results of the observation and recording of the body temperature, spirit, appetite, faeces, ocular and nasal secretions, sneeze and the like of dogs, after the detoxification, were shown in Table 11.
Table 11 clinical trial toxicity-counteracting results of canine adenovirus 2 type HL50 attenuated live vaccine
The results show that the dogs immunized with the canine adenovirus type 2 HL50 attenuated live vaccine prepared in the example 3 have no clinical symptoms after the challenge, and can provide 100% (5/5) protection for the dogs; the dogs in the control group all developed after challenge. This shows that the canine adenovirus 2 type HL50 attenuated live vaccine of the invention can have prolonged protection effect on dogs infected repeatedly after immunization, and the protection rate is 100%.
Further proves that the canine adenovirus type 2 HL50 attenuated live vaccine has good protective power and can block canine adenovirus type 1 or canine adenovirus type 2 continuous infection.
Example 9 preparation of triple live vaccine against canine distemper, parvovirus disease and infectious hepatitis
1. Proliferation of viruses
Synchronously inoculating the canine distemper virus LT90 strain into Vero cell suspension, adding a cell growth liquid (pH is adjusted to 6.8-7.2) containing 95-99% of DMEM culture liquid and 1-5% of newborn calf serum by volume ratio, continuously culturing at 33-37 ℃, after 72-96 h, freezing and thawing for toxin collection when more than 80% of cells are diseased, measuring the virus content, and storing at low temperature.
Synchronously inoculating canine parvovirus JM35 strain into F81 cell suspension, adding cell growth liquid (pH is regulated to 6.8-7.2) containing 90-98% of RPMI 1640 culture solution and 2-10% of newborn bovine serum by volume ratio, and culturing at 33-37 ℃. And (5) freeze thawing and toxin collection are carried out when cytopathy reaches about 80%, and virus content measurement is carried out, and the obtained product is stored at low temperature.
2. Preparation of protective agent
Every 100ml deionized water is added with 40g of sucrose and 8g of gelatin, and after full melting, the mixture is put into high-pressure steam for sterilization (121 ℃ for 30 min).
3. Preparation of vaccine
The prepared and stored canine distemper virus LT90 virus liquid, canine parvovirus JM35 virus liquid and canine adenovirus type 2 HL50 virus liquid prepared and stored in example 3 are mixed according to a proportion, mixed with a protective agent according to a volume ratio of 1:1, and freeze-dried. The specific proportions of the vaccine contents are shown in Table 12.
Table 12 content ratio of triple live vaccine against canine distemper, parvovirus disease and infectious hepatitis
EXAMPLE 10 immunogenicity test of triple live vaccine against canine distemper, parvovirus disease and infectious hepatitis
Over 28 days old, 60 dogs negative for canine distemper, canine parvovirus and canine adenovirus antigen antibody are randomly divided into 12 groups, 5 dogs in each group, and triple live vaccine for canine distemper, parvovirus disease and infectious hepatitis prepared in immunization example 9. Group 27, 28 single dose immunization vaccine 4, group 29, 30 single dose immunization vaccine 5, group 31, 32 single dose repeat immunization vaccine 4, group 33, 34 single dose repeat immunization vaccine 5, 21 days apart. The 35 th to 38 th groups of immune PBS served as a control group.
Group 27, 29 and 35 immunizations 21 days were followed to combat virulence by 2ml nasal drops and 4ml abdominal cavity drops with canine distemper virus strain C/HN/001 (10) 5.5 FAID 50 The body temperature, spirit, appetite, faeces, eye and nose secretions of dogs are observed and recorded daily, in the morning and evening after the challenge, and specific results are shown in Table 13.
TABLE 13 results of canine distemper antigen part immunogenicity test of canine triple live vaccine
The results show that after dogs are immunized by the triple live vaccine of canine distemper, parvovirus disease and infectious hepatitis prepared in the example 9, canine distemper virus infection (clinical symptoms appear) can be blocked, 100% (5/5) protection (prevention) can be provided for dogs, and the dogs in a control group are all ill after the dogs are attacked by the virus.
Group 28, group 30 and group 36 were challenged 14 days after immunization with canine parvovirus strain S0425 by subcutaneous injection in an amount of 4ml (10 6.5 FAID 50 Per ml)/dose, and the clinical manifestations of the dogs such as spirit, appetite, feces and the like are observed and recorded every day after the toxin is attacked. The specific results are shown in Table 14.
Table 14 results of canine parvoantigen partial immunogenicity test of canine triple live vaccine
The results show that after dogs are immunized by the triple live vaccine of canine distemper, parvovirus disease and infectious hepatitis prepared in the example 9, canine parvovirus infection can be blocked (clinical symptoms appear), 100% (5/5) protection (prevention) can be provided for dogs, and the dogs in a control group are all ill after the dogs are attacked by the virus.
Group 31, group 33 and group 37 were challenged 14 days later with canine adenovirus strain 1 YT-2 by intravenous injection of 1ml (10 4.0 TCID 50 Per ml)/dose, and after the toxin is removed, the clinical manifestations of the dogs such as body temperature, spirit, appetite, abdomen, eyes and the like are observed and recorded every day; group 32, 34 and 38 were challenged at 14 days with canine adenovirus strain 2 HL006 to drip 2ml of nasal drops (10 4.0 TCID 50 Per ml)/dose challenge; the dogs were observed and recorded for body temperature, spirit, appetite, eye and nose secretions, sneeze or the like in the morning and evening after toxin expelling. The specific results are shown in Table 15.
Table 15 results of canine adenovirus antigen partial immunogenicity test of canine triple live vaccine
The results show that after dogs are immunized by the triple live vaccine of canine distemper, parvovirus disease and infectious hepatitis prepared in the example 9, canine adenovirus type 1 and canine adenovirus type 2 infection (clinical symptoms appear) can be blocked, 100% (5/5) protection (prevention) can be provided for dogs, and the dogs in a control group are all ill after the dogs are challenged.
Proved that the triple live vaccine for canine distemper, parvovirus disease and infectious hepatitis provided by the invention has good protective power, shows good immune protection and safety, and effectively prevents the occurrence of canine distemper, canine parvovirus disease and canine infectious hepatitis.
The present invention is not limited to the above-mentioned embodiments, but is capable of modification and variation in all embodiments without departing from the spirit and scope of the present invention.
Claims (10)
1. The canine adenovirus 2 type HL50 strain has a preservation number of CCTCC NO: V202215, is preserved in China center for type culture Collection, has a preservation address of university of Wuhan and Wuhan in China, and has a preservation date of 2022, 3 months and 1 day.
2. A vaccine composition, wherein the vaccine composition comprises an immunizing amount of live whole virus antigen of canine adenovirus type 2 HL50 strain or a culture thereof, and a pharmaceutically acceptable carrier.
3. A vaccine composition according to claim 2, wherein the canine adenovirus type 2 HL50 strain has a culture of 1 to 70 passages.
4. The vaccine composition according to claim 2, wherein the canine adenovirus type 2 HL50 strain or a culture thereof has a live whole virus antigen content of 10 or more 3.0 TCID 50 Head part; preferably, the canine adenovirus type 2 HL50 strain or culture thereof has a live whole virus antigen content of 10 3.0 ～10 6.0 TCID 50 Head part; more preferably, the canine adenovirus type 2 HL50 strain or culture thereof has a live whole virus antigen content of 10 4.0 ～10 5.0 TCID 50 Head.
5. The vaccine composition of claim 2, wherein the pharmaceutically acceptable carrier is a lyoprotectant selected from the group consisting of SPGA, saccharides, proteins, protein-containing substances, and buffers; the saccharide comprises sorbitol, mannitol, starch, sucrose, glucose and dextran, the protein is albumin or casein, the substance containing the protein is bovine serum or skim milk, and the buffer solution is phosphate buffer solution; preferably, the lyoprotectant is an aqueous solution containing 40% w/v sucrose and 8% w/v gelatin.
6. A vaccine composition according to claim 2, wherein the volume ratio between the canine adenovirus type 2 HL50 strain, or a culture thereof, and the pharmaceutically acceptable carrier is 1:1.
7. The vaccine composition of claim 2, wherein the vaccine composition further comprises an immunizing amount of live whole virus antigen of canine distemper virus LT90 strain or a culture thereof, and/or the immunizing amount of live whole virus antigen of canine parvovirus JM35 strain or a culture thereof;
the collection number of the canine distemper virus LT90 strain is CCTCC NO. V202030, and the collection number of the canine parvovirus JM35 strain is CCTCC NO. V201965.
8. The vaccine composition according to claim 7, wherein the canine distemper virus LT90 strain is a culture of 1 to 100 generations and the canine parvovirus JM35 strain is a culture of 1 to 100 generations;
the content of the live whole virus antigen of the canine distemper virus LT90 strain or the culture thereof is more than or equal to 10 2.5 FAID 50/header; preferably, the live whole virus antigen content of the canine distemper virus LT90 strain or culture thereof is 10 2.5 ～10 5.5 FAID 50 Head part; more preferably, the live whole virus antigen content of the canine distemper virus LT90 strain or culture thereofIs 10 3.5 ～10 4.5 FAID 50 Head part;
the live whole virus antigen content of the canine parvovirus JM35 strain or the culture thereof is more than or equal to 10 4.0 FAID 50/header; preferably, the live whole virus antigen content of the canine parvovirus JM35 strain or culture thereof is 10 4.0 ～10 6.5 FAID 50 Head part; more preferably, the live whole virus antigen content of the canine parvovirus JM35 strain or culture thereof is 10 5.0 ～10 6.0 FAID 50 Head.
9. Use of a vaccine composition according to any one of claims 2 to 8 in the manufacture of a medicament for the prevention and/or treatment of canine adenovirus type 1 and/or canine adenovirus type 2 infections.
10. The use according to claim 9, wherein the use is in the manufacture of a medicament for the prevention and/or treatment of canine, fox canine adenovirus type 1 and/or canine adenovirus type 2 infections.
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