Feline calicivirus 60 strain and application thereof

Abstract

The invention relates to the field of veterinary biological products, in particular to a feline calicivirus 60 strain and application thereof. The preservation number of the feline calicivirus 60 strain is CCTCC No.V202319, and the feline calicivirus 60 strain belongs to an epidemic feline calicivirus wild strain, has good immunogenicity, and can provide effective immune protection for epidemic strains in different regions.

Description

Technical Field

[0001] The present application relates to the field of veterinary biological products, and specifically to feline calicivirus 60 strain and its application. Background Art

[0002] Feline calicivirus disease is an upper respiratory tract disease caused by feline calicivirus (FCV). FCV mainly infects cats and wild cats such as tigers, cheetahs, and lions. Cats of different breeds and ages can be infected with FCV, and kittens are more susceptible. The main symptoms of sick cats are fever, eye and nasal discharge, oral ulcers, and swollen or ulcerated foot pads. In 1957, Fastier et al. first isolated FCV, and subsequent studies found that FCV has been widely prevalent worldwide. In 1999, Fan Quanshui et al. first isolated FCV in China.

[0003] At present, vaccination is the main means of preventing the disease, but FCV has genetic diversity, and the prevalent strains in different regions still have significant variations, which leads to the phenomenon of occasional disease after vaccination. Therefore, it is urgent to screen out strains that have immune protection against prevalent strains in different regions, which is of great significance for the prevention and control of feline calicivirus disease.

[0004] In view of this, this application is hereby filed. Summary of the invention

[0005] The purpose of the present application is to provide a feline calicivirus strain, which belongs to the existing epidemic strain and has good immunogenicity and can provide effective immune protection against epidemic strains in different regions.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] Feline calicivirus strain 60, the deposit number is CCTCC No.V202319.

[0008] Application of feline calicivirus 60 strain in the preparation of medicines for preventing diseases related to feline calicivirus infection.

[0009] Furthermore, the drug is an inactivated feline calicivirus vaccine.

[0010] A vaccine composition comprises an immunizing amount of antigens of feline calicivirus strain 60 and a pharmaceutically acceptable carrier.

[0011] Furthermore, the antigen is 1 to 15 generations of feline calicivirus 60 strains or their cultures.

[0012] Furthermore, the antigen is an inactivated 1 to 15 generation feline calicivirus 60 strain or a culture thereof, preferably, the antigen content is 10 8.0~10 10.0 TCID 50 / ml, preferably 10 9.0 TCID 50 / ml.

[0013] Furthermore, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes one or more of aluminum gel adjuvant, saponin, avridine, DDA, oil-in-water emulsion, water-in-oil emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, and Gel adjuvant.

[0014] Furthermore, the concentration of the adjuvant ranges from 5% V / V to 50% V / V, preferably 10% V / V.

[0015] A method for preparing a vaccine composition comprises culturing and proliferating the feline calicivirus 60 strain, inactivating the proliferated feline calicivirus 60 strain or its culture, and adding a pharmaceutically acceptable carrier to the inactivated feline calicivirus 60 strain or its culture.

[0016] Furthermore, the inactivation is inactivation with an inactivating agent.

[0017] Compared with the prior art, the technical effects of this application are:

[0018] The feline calicivirus strain isolated in the present application is genetically a prevalent wild strain of feline calicivirus. Immunizing animals with a vaccine composition containing the strain can enable the animal body to quickly produce high titer specific antibodies, which can effectively prevent epidemics caused by feline calicivirus and provide effective immune protection against prevalent strains in different regions. DETAILED DESCRIPTION

[0019] Hereinafter, embodiments of the present application will be described.

[0020] The term "Feline calicivirus" (FCV) refers to a member of the Caliciviridae family and is characterized by flu-like symptoms such as upper respiratory tract infection. Calicivirus usually affects the throat, eyes, nasal cavity and mouth of cats, but sometimes also affects the lungs, musculoskeletal system and intestines.

[0021] The term "feline calicivirus strain (FCV strain)" in this application, unless otherwise specified, refers to the wild-type feline calicivirus strain (FCV wild-type strain), the virulent feline calicivirus strain (FCV virulent strain), and the wild-type FCV strain, and can be used interchangeably.

[0022] The present application relates to Feline Calicivirus 60 strain, with a deposit number of CCTCC No. V202319. Feline Calicivirus 60 strain (Feline calicivirus, Strain 60) is deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: V202319, and the deposit address is Wuhan University, Wuhan, China, and the deposit date is March 21, 2023.

[0023] Feline calicivirus strain 60 is genetically a prevalent wild strain of feline calicivirus. Immunizing animals with a vaccine composition containing the strain can rapidly produce high titer specific antibodies in the animal body, effectively preventing epidemics caused by feline calicivirus, and providing effective immune protection against prevalent strains in different regions. Therefore, the present application provides the use of feline calicivirus strain 60 in the preparation of a drug for preventing diseases related to feline calicivirus infection.

[0024] In some embodiments, the medicament is an inactivated feline calicivirus vaccine.

[0025] The present application relates to a vaccine composition, wherein the vaccine composition comprises an immunizing amount of an antigen of the feline calicivirus strain 60 and a pharmaceutically acceptable carrier.

[0026] In some embodiments, the antigen may be 1-15 passages of feline calicivirus 60 strains, or 1-15 passages of feline calicivirus 60 strain cultures. Preferably, 1-15 passages of feline calicivirus 60 strains or cultures thereof are inactivated or attenuated.

[0027] The term "vaccine composition", also known as an immunogenic composition, refers to a preparation containing an immunogen, including, for example, whole cells, inactivated or attenuated, live viruses or bacteria, or polysaccharides, or a combination thereof, which is administered to stimulate a humoral and cellular immune response in a recipient to one or more antigens present in the immunogenic composition. Immunization is the process of administering a vaccine composition and stimulating an immune or immunogenic response to the antigen in a host, preferably an animal such as a cat.

[0028] The term "immunizing amount" should be understood as an "immunologically effective amount", also known as an immunoprotective amount or an effective amount for producing an immune response, which is an amount of antigen that can effectively induce an immune response in the recipient, and this amount is sufficient to prevent or improve the signs or symptoms of the disease, including adverse health effects or its complications. The immune response may be sufficient for diagnostic purposes or other tests, or may be suitable for preventing signs or symptoms of the disease, including adverse health consequences or complications caused by infections caused by pathogens. Humoral immunity or cell-mediated immunity or both can be induced. The immune response of animals to the immunogenic composition can be indirectly assessed by, for example, measuring antibody titers, lymphocyte proliferation analysis, or directly assessed by monitoring signs or symptoms after attacking with wild-type strains, and the protective immunity provided by the vaccine can be assessed by measuring, for example, clinical signs of the subject such as mortality, reduction in morbidity, temperature values, overall physiological condition of the subject, and overall health and performance. The immune response may include, but is not limited to, inducing cellular and / or humoral immunity.

[0029] The term "antigen of feline calicivirus" refers to any composition containing at least one feline calicivirus antigen form, the antigen of the feline calicivirus can induce, stimulate or resist the immune response of feline calicivirus infection, and the antigen form includes but is not limited to inactivated, attenuated or subunit antigens. Wherein, the inactivated antigen can be inactivated by various methods including chemical treatment, physical treatment (such as sonication, irradiation, heat) or any other common method sufficient to prevent organism replication or growth to maintain its immunogenicity, preferably the pathogen is inactivated after collection and optionally clarified and purified, by chemical treatment using, for example, formalin or formaldehyde, β-propiolactone, ethyleneimine, diethyleneimine BEI, thimerosal, etc.; The method of inactivation is well known to those skilled in the art, such as by β-propiolactone (Plana-Duran et al., Vet.Microbiol., 1997, 55: 361-370) or by BEI treatment (US5587164).

[0030] As an embodiment of the present application, in the vaccine composition described in the present application, the antigen is an antigen of the inactivated 1 to 15 generation feline calicivirus 60 strain or its culture, and the antigen content is 10 8.0 ~10 10.0 TCID 50 / ml, can be but not limited to 10 8.0 TCID 50 / ml, 10 8.1 TCID 50 / ml, 10 8.2 TCID 50 / ml, 10 8.3 TCID 50 / ml, 108.4 TCID 50 / ml, 10 8.5 TCID 50 / ml, 10 8.6 TCID 50 / ml, 10 8.7 TCID 50 / ml, 10 8.8 TCID 50 / ml, 10 8.9 TCID 50 / ml, 10 9.0 TCID 50 / ml, 10 9.1 TCID 50 / ml, 10 9.2 TCID 50 / ml, 10 9.3 TCID 50 / ml, 10 9.4 TCID 50 / ml, 10 9.5 TCID 50 / ml, 10 9.6 TCID 50 / ml, 10 9.7 TCID 50 / ml, 10 9.0 TCID 50 / ml, 10 9.9 TCID 50 / ml or 10 10.0 TCID 50 / ml.

[0031] As a preferred embodiment of the present application, the inactivated whole virus antigen content of the feline calicivirus 60 strain or its culture is 10 9.0 TCID 50 / ml.

[0032] The term "1st to 15th generation feline calicivirus 60 strain culture" refers to a culture obtained by continuously and naturally subculturing the feline calicivirus 60 strain on F81 cells until it is adapted to growth and replication on F81 cells (i.e., cell lesions such as rounding, aggregation, and shedding appear on the cells), and then continuing to naturally subculture on F81 cells to the 1st to 15th generations.

[0033] The term "pharmaceutically acceptable carrier" refers to all other ingredients in the vaccine composition of the present application except the feline calicivirus antigen, a carrier or diluent that does not stimulate the body and does not hinder the biological activity and characteristics of the compound used, preferably an adjuvant. The term "adjuvant" may include aluminum gel adjuvants; saponins (saponin), such as Quil A, QS-21 (Cambridge Biotech Incorporation, Cambridge MA), GPI-0100 (Galenica Pharmaceuticals Incorporation, Birmingham AL); oil-in-water emulsions; water-in-oil emulsions such as the SPT emulsion described on page 147 of "Vaccine design, the Subunit and adiuvant approach" (Plenum Press, 1995) written by Powell M and Newman M and the MF59 emulsion described on page 183; water-in-oil-in-water emulsions; polymers of acrylic acid or methacrylic acid; one or more copolymers of maleic anhydride and alkenyl derivatives. The term "emulsion" may be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oils resulting from olefin oligomerization, such as squalane or squalene oil, especially isobutylene or decene; linear alkyl-containing esters of acids or alcohols, more particularly vegetable oils, ethyl oleate, propylene glycol di-(caprylate / decene), glycerol tri-(caprylate / decene) or propylene glycol dioleate; esters of branched fatty acids or alcohols, especially isostearates. The oil is used in combination with an emulsifier to form an emulsion. Emulsifiers are preferably nonionic surfactants, especially esters of sorbitan, esters of mannide (such as anhydrous mannitol oleate), esters of fatty glycols, esters of polyglycerols, esters of propylene glycol and esters of oleic acid, isostearic acid, ricinoleic acid or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene block copolymers, especially Pluronic products, in particular L121. See Hunter et al., The theory and practical application of adjuvants (Ed. by DES Stewart-Tull, John Wiley and Sons, New York, 1995: 51-94) and Todd et al., Vaccine (1997, 15: 564-570).The term "polymer of acrylic acid or methacrylic acid" is preferably a cross-linked acrylic acid or methacrylic acid polymer, especially cross-linked with a polyalkenyl ether of sugar or a polyol, such compounds being known as carbomers (Carbopol, trade name) (Phameuropa, 1996, 8 (2)). Those skilled in the art may also refer to U.S. Pat. No. 2,909,462, which describes such acrylic acid polymers cross-linked with polyhydroxylated compounds having at least 3 hydroxyl groups, preferably not more than 8, wherein the hydrogen atoms of at least 3 hydroxyl groups are replaced by unsaturated aliphatic radicals having at least 2 carbon atoms. Preferred groups are those containing 2-4 carbon atoms, such as vinyl, allyl and other ethylenically unsaturated groups. The unsaturated groups themselves may contain other substituents, such as methyl. The products sold under the name of EMA (Monsanto) by BF Goodrich, Ohio, USA are particularly suitable. They are crosslinked with allyl sucrose or with allyl pentaerythritol. Among these, mention may be made of carbomers 974P, 934P and 971P, most preferably carbomer 971P is used. The term "copolymers of maleic anhydride and alkenyl derivatives" also considers copolymers of maleic anhydride and ethylene EMA (Monsanto), these polymers dissolving in water to give an acidic solution, which is neutralized, preferably to physiological pH, in order to give an adjuvant solution into which the immunogenic, immunogenic or vaccine composition itself can be incorporated. The term "adjuvant" also includes, but is not limited to, RIBI adjuvant system (Ribi Incorporation), Block co-polymer (CytRx, Atlanta GA), SAF-M (Chiron, Emeryville CA), monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin (recombinant or other), cholera toxin, IMS1314, muramyl dipeptide, Gel adjuvant, etc. Preferably, the adjuvant includes one or more of aluminum gel adjuvant, saponin, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide or Gel adjuvant.

[0034] As one embodiment of the present application, in the vaccine composition described in the present application, the pharmaceutically acceptable carrier includes an adjuvant, and the adjuvant includes: (1) aluminum gel adjuvant, saponin, avridine, DDA; (2) water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion; or (3) polymers of acrylic acid or methacrylic acid, copolymers of maleic anhydride and alkenyl derivatives; and one or more of RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, and Gel adjuvant.

[0035] Preferably, the saponin is Quil A, QS-21, GPI-0100.

[0036] Preferably, the emulsion is an SPT emulsion, an MF59 emulsion, or an emulsion formed by combining an oil with an emulsifier, and the emulsion can be based on light liquid paraffin oil, isoprenoid oils produced by olefin oligomerization (such as squalane or squalene oil, olefins, especially oils produced by oligomerization of isobutylene or decene), linear alkyl-containing esters of acids or alcohols (more especially vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprylate), glycerol tri-(caprylate / caprylate) or propylene glycol dioleate), esters of branched fatty acids or alcohols ( emulsifiers are nonionic surfactants (especially esters of polyoxyethylated fatty acids (e.g. oleic acid), esters of sorbitan, esters of mannide (e.g. anhydrous mannitol oleate), esters of aliphatic diols, esters of glycerol, esters of polyglycerol, esters of propylene glycol, and esters of oleic acid, esters of isostearic acid, esters of ricinoleic acid or esters of hydroxystearic acid, which esters may be ethoxylated, ethers of fatty alcohols and polyols (e.g. oleyl alcohol), polyoxypropylene-polyoxyethylene block copolymers (especially Especially L121)).

[0037] Preferably, the polymer of acrylic acid or methacrylic acid is a cross-linked acrylic acid or methacrylic acid polymer, especially a compound carbomer cross-linked with a polyalkenyl ether of a sugar or a polyalcohol, preferably carbomer 974P, 934P and 971P.

[0038] Preferably, the copolymer of maleic anhydride and an alkenyl derivative is a copolymer of maleic anhydride and ethylene EMA.

[0039] Preferably, the adjuvant is a high molecular weight polymer water-soluble adjuvant.

[0040] The concentration of the adjuvant ranges from 5% to 50% V / V, preferably 10% V / V.

[0041] The present application also relates to a method for preparing the vaccine composition, which comprises: culturing and proliferating the feline calicivirus 60 strain, inactivating the proliferated feline calicivirus 60 strain or its culture, and adding a pharmaceutically acceptable carrier to the inactivated feline calicivirus 60 strain or its culture.

[0042] As an embodiment of the present application, the inactivated whole virus antigen is obtained by obtaining the strain cell whole virus culture fluid by continuously and naturally passage the feline calicivirus strain on cells, and then inactivating it with an inactivating agent.

[0043] As an embodiment of the present application, the inactivator includes but is not limited to β-propiolactone BPL, diethyleneimine BEI, formalin, formaldehyde, N-acetylenimine AEI, and polyhexamethyleneguanidine hydrochloride PHMG; preferably, the inactivator is β-propiolactone BPL, and its final concentration in the vaccine composition is 0.05% V / V.

[0044] The term "prevent" when referring to feline calicivirus infection means inhibiting the replication of feline calicivirus, alleviating the symptoms of a disease or condition caused by feline calicivirus infection, or shortening the duration of disease symptoms.

[0045] The present application also relates to the use of the vaccine composition in the preparation of a medicament for preventing diseases related to feline calicivirus infection.

[0046] The present application is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0047] Example 1 Isolation and Identification of Feline Calicivirus

[0048] Eye and nose swabs of cats suspected of being infected with feline calicivirus were collected from multiple pet hospitals in Tianjin, Henan, Shanghai, Sichuan and other places, and 153 samples were found to be infected with feline calicivirus by PCR. The collected eye and nose swabs were diluted 10 times with PBS, centrifuged at 5000 rpm for 10 minutes, and the supernatant was filtered with a 0.45μm filter and inoculated into cat kidney cells (F81 cells). The cytopathic effect was observed daily, and the culture was repeatedly frozen and thawed at -70℃ and inoculated into F81 cells. The virus was collected by blind transmission in the same way until cytopathic effect was produced. The results showed that 153 samples could produce obvious cytopathic effect after inoculation, and further sequence comparison was performed by molecular biological methods, and it was determined that they were all feline caliciviruses. Five strains of viruses from different regions were further screened out, numbered 32, 33, 35, 60, and 72 respectively.

[0049] Example 2 Pathogenicity Study of Feline Calicivirus

[0050] Thirty healthy susceptible antigen antibody-negative cats aged 2 months were randomly divided into 6 groups, with 5 cats in each group. The grouping and infection situation are shown in Table 1.

[0051] Table 1 Pathogenicity study groups Group Inoculation strain Vaccination dose 1 32 strains <![CDATA[Instill 1 ml (10 7.0 TCID 50 / ml)]]> 2 33 strains <![CDATA[Instill 1 ml (10 7.0 TCID 50 / ml)]]> 3 35 strains <![CDATA[Instill 1 ml (10 7.0 TCID 50 / ml)]]> 4 60 plants <![CDATA[Instill 1 ml (10 7.0 TCID 50 / ml)]]> 5 72 strains <![CDATA[Instill 1 ml (10 7.0 TCID 50 / ml)]]> 6 RPMI 1640 medium 1ml per nose drop

[0052] The cats were observed for 14 days after virus inoculation, and their body temperature, appetite, eye and nasal secretions, oral cavity, paw pads and other clinical manifestations were observed and recorded daily. The specific results are shown in Table 2.

[0053] Table 2 Pathogenicity study results

[0054] The results showed that all five strains of feline calicivirus could cause 100% disease in cats (5 / 5), with strain 60 of feline calicivirus being the most pathogenic.

[0055] At the same time, in order to verify the stability of the pathogenicity of different generations of feline calicivirus 60 strains, a group of cats (5) with negative feline calicivirus antigen and antibody were inoculated with cultures of feline calicivirus 60 strains of the 5th, 7th, 10th, and 15th generations, and 1 ml (10 7.0 TCID 50 / ml), and 5 cats were used as the control group. The clinical changes of the cats were observed and recorded daily until 14 days after inoculation.

[0056] The results showed that 100% of the 5th, 7th, 10th and 15th generations of feline calicivirus 60 strains developed disease (5 / 5) after 14 days of observation after inoculation, indicating that the pathogenicity of feline calicivirus 60 strains of different generations is stable.

[0057] Example 3 Preparation of Feline Calicivirus 60 Strain Antigen

[0058] Cell recovery: F81 cells were taken out of the liquid nitrogen tank, shaken in a 37°C water bath to melt, and then transferred to a 50ml centrifuge tube. 15ml of culture medium was added dropwise, and the cells were centrifuged at 100g for 5min in a horizontal rotor centrifuge. The supernatant was discarded, and the cells were resuspended in 20ml of culture medium. The cells were transferred to a 125ml shake flask and cultured in a shaker containing 5% carbon dioxide.

[0059] Cell passaging: 1.5×10 6 cells / ml initial density, culture volume 30ml, inoculated into 125ml shake flask. Placed in a shaker at 37℃, 5% carbon dioxide concentration, orbit radius 25mm, speed 120rpm, humidity 80%. Cultured until the third day, according to 1.5×10 6 cells / ml and gradually increase the cell seed amount to prepare seed cells for the 3L reactor.

[0060] Virus proliferation in 3L reactor: Adjust the initial cell density to 3.0×10 in 3L reactor. 6 cells / m, culture volume is 1L, MOI=0.01-0.05, inoculate 60 strains of feline calicivirus, parameter settings: temperature 37°C, pH 7.0±0.2, dissolved oxygen 40%, associated stirring speed at 100-150rpm, oxygen 0-0.1vvm. Air 0.01-0.2vvm, surface ventilation (ventilation and oxygen supply from the surface) 0.02-0.2vvm. Harvest when the cell viability drops below 90%. The harvested virus liquid is filtered by centrifugation or hollow fiber (0.5μm-2μm) filter column to remove cell debris, and then an inactivator is added for inactivation, and the virus antigen after complete inactivation is used for preparing a vaccine.

[0061] Example 4 Preparation of Feline Calicivirus 60 Strain Vaccine Composition

[0062] The inactivated antigen of feline calicivirus 60 strains prepared in Example 3 is slowly added to the adjuvant, and the mixture is stirred and mixed during the addition process. The adjuvant suitable for the present application can be an adjuvant known to those skilled in the art. In the present application, the adjuvant is a high molecular polymer water-soluble adjuvant, such as carbomer. The specific formula of the vaccine is shown in Table 3.

[0063] Table 3 Feline Calicivirus 60 strain vaccine ratio

[0064] Example 5 Immunogenicity Test of Feline Calicivirus 60 Strain Vaccine Composition

[0065] Twenty healthy susceptible antigen antibody negative cats aged 2 months were randomly divided into 4 groups, 5 in each group. Groups 7-9 were subcutaneously inoculated with 1 ml of inactivated vaccine 1, 2, and 3, respectively, and group 10 was subcutaneously injected with 1 ml of normal saline as a blank control. Two immunizations were performed with an interval of 21 days. 14 days after the second immunization, the test cats were challenged with feline calicivirus strain 60. Each cat was intranasally injected with 1 ml (10 7.0 TCID 50 / ml), and the body temperature, appetite, eye and nasal secretions, oral cavity, and foot pads of the cats were observed and recorded every day after the challenge. The clinical symptoms were scored daily according to the duration of clinical symptoms according to the scoring criteria in Table 4, and the sum of the clinical scores of each cat within 14 days after the challenge was calculated, and the differences in clinical scores between the immunization group and the control group were compared. The specific results are shown in Table 5.

[0066] Table 4 Scoring criteria for clinical symptoms after feline calicivirus challenge

[0067] Table 5 Results of immunogenicity test of feline calicivirus 60 strain vaccine composition

[0068] Note: Different superscript letters indicate significant differences (P<0.05).

[0069] The results showed that all the patients in the control group became ill, and the clinical scores of the group immunized with the feline calicivirus 60-strain vaccine composition after infection were significantly lower than those of the control group.

[0070] Immunogenicity tests showed that feline calicivirus 60 strain had good immunogenicity and could significantly alleviate clinical symptoms after challenge with virulent feline calicivirus.

[0071] At the same time, in order to verify the stability of the immunogenicity of different generations of feline calicivirus 60 strains, the 5th, 7th, 10th and 15th generations of feline calicivirus 60 strains were used respectively. On the 14th day after the second immunization, each immunization group and the control group were irradiated with feline calicivirus 60 strains at 1 ml (10 7.0 TCID 50 The cats were challenged with a dose of 100 mg / ml. After the challenge, the cats' body temperature, appetite, eye and nasal secretions, oral cavity and other clinical manifestations were observed and recorded every day.

[0072] The results showed that in the case of 5 / 5 cases in the control group, the clinical scores of the 5th, 7th, 10th and 15th generation immunization groups of feline calicivirus 60 strain after infection were significantly lower than those of the control group.

[0073] The immunogenicity tests of different generations showed that all 60 strains of feline calicivirus of different generations had good immunogenicity and could significantly alleviate clinical symptoms after attack by virulent feline calicivirus.

[0074] Example 6 Feline Calicivirus 60 strain vaccine composition immunity duration test

[0075] Twenty healthy susceptible antigen antibody negative cats aged 2 months were randomly divided into 4 groups, 5 in each group. Groups 11-13 were inoculated with inactivated vaccines 1, 2 and 3, 1 ml per cat subcutaneously. Group 14 was used as a control, and 1 ml of normal saline was inoculated per cat. Blood was collected regularly after immunization to determine the serum neutralizing antibody titer. The specific results are shown in Table 6.

[0076] Table 6 Results of the immune duration test of the feline calicivirus 60 strain vaccine composition

[0077] The results showed that after vaccination, the feline calicivirus 60-strain vaccine composition could quickly induce the production of FCV neutralizing antibodies, and the level was still maintained at a high level 12 months after immunization.

[0078] This indicates that the feline calicivirus 60 strain vaccine composition provided in the present application can enable the body to produce high titer specific antibodies in a short period of time after immunization, and the duration of immunity is at least 12 months, and can significantly alleviate clinical symptoms after a virulent attack of feline calicivirus.

[0079] Example 7 Broad-spectrum test of feline calicivirus 60 strain vaccine composition

[0080] 40 healthy susceptible antigen antibody negative cats aged 2 months were randomly divided into 8 groups, 5 in each group. Groups 15-18 were subcutaneously inoculated with 1 ml of inactivated vaccine 2, and groups 19-22 were subcutaneously injected with 1 ml of normal saline as blank controls. Two immunizations were performed with an interval of 21 days. 14 days after the second immunization, the test cats were challenged with feline calicivirus 32, feline calicivirus 33, feline calicivirus 35, and feline calicivirus 72 from Tianjin, Henan, Shanghai, and Sichuan, respectively. Each cat was nasally inoculated with 1 ml (10 7.0 TCID 50 / ml), and the body temperature, appetite, eye and nasal secretions, oral cavity, and foot pads of the cats were observed and recorded every day after the challenge. The clinical symptoms were scored daily according to the scoring criteria in Table 4 according to the duration of the clinical symptoms. The sum of the clinical scores of each cat within 14 days after the challenge was calculated, and the differences in clinical scores between the immunization group and the control group were compared. The specific results are shown in Tables 7, 8, 9, and 10.

[0081] Table 7 Broad-spectrum test results of feline calicivirus 60 strain vaccine composition (against feline calicivirus 32 strains)

[0082] Note: Different superscript letters indicate significant differences (P<0.05).

[0083] Table 8 Broad-spectrum test results of feline calicivirus 60 strain vaccine composition (against feline calicivirus 33 strains)

[0084] Note: Different superscript letters indicate significant differences (P<0.05).

[0085] Table 9 Broad-spectrum test results of feline calicivirus 60 strain vaccine composition (against feline calicivirus 35 strains)

[0086] Note: Different superscript letters indicate significant differences (P<0.05).

[0087] Table 10 Broad-spectrum test results of feline calicivirus 60 strain vaccine composition (against feline calicivirus 72 strains)

[0088] Note: Different superscript letters indicate significant differences (P<0.05).

[0089] The results showed that all the patients in the control group became ill, and the clinical scores of the group immunized with the feline calicivirus 60-strain vaccine composition after infection were significantly lower than those of the control group.

[0090] The results show that the feline calicivirus 60 strain vaccine composition provided by the present application can significantly alleviate clinical symptoms after being challenged with feline calicivirus from different geographical sources. The vaccine composition of the present application has a broad spectrum of immunogenicity and can significantly alleviate clinical symptoms after being challenged with feline calicivirus from different geographical sources.

[0091] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. Feline calicivirus strain 60, accession number is CCTCC No.V202319.

2. Application of the feline calicivirus 60 strains according to claim 1 in the preparation of medicines for preventing diseases related to feline calicivirus infection.

3. The use according to claim 2, characterized in that: The medicine is an inactivated feline calicivirus vaccine.

4. A vaccine composition, characterized in that The vaccine composition comprises an immunizing amount of the antigen of the feline calicivirus strain 60 according to claim 1 and a pharmaceutically acceptable carrier.

5. The vaccine composition according to claim 4, characterized in that The antigen is 1-15 generations of feline calicivirus 60 strains or their cultures.

6. The vaccine composition according to claim 5, characterized in that The antigen is the inactivated 1-15th generation feline calicivirus 60 strain or its culture, preferably, the antigen content is 10 8.0 ~10 10.0 TCID 50 / ml, preferably 10 9.0 TCID 50 / ml.

7. The vaccine composition according to any one of claims 4 to 6, characterized in that The pharmaceutically acceptable carrier includes an adjuvant, which includes one or more of aluminum gel adjuvant, saponin, avridine, DDA, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion, polymer of acrylic acid or methacrylic acid, copolymer of maleic anhydride and alkenyl derivative, RIBI adjuvant system, Block co-polymer, SAF-M, monophosphoryl lipid A, Avridine lipid-amine adjuvant, Escherichia coli heat-labile enterotoxin, cholera toxin, IMS1314, muramyl dipeptide, and Gel adjuvant.

8. The vaccine composition according to claim 7, characterized in that The concentration of the adjuvant ranges from 5% V / V to 50% V / V, preferably 10% V / V.

9. A method for preparing the vaccine composition according to any one of claims 4 to 8, characterized in that: The feline calicivirus 60 strain is cultured and proliferated, the proliferated feline calicivirus 60 strain or its culture is inactivated, and a pharmaceutically acceptable carrier is added to the inactivated feline calicivirus 60 strain or its culture.

10. The method according to claim 9, characterized in that The inactivation is inactivation with an inactivating agent.

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