Canine distemper-canine parvorabies-rabies triple inactivated vaccine as well as preparation method and application thereof
By preparing triple inactivated vaccines including canine distemper, canine parvo and rabies viruses, the problem of the existing vaccines requiring separate vaccination and unstable immunity effects is solved, and simultaneous immune protection in the canine body is achieved.
Patent Information
- Application Number
- CN202510392583.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-31
- Publication Date
- 2025-08-12
AI Technical Summary
Existing canine distemper, canine parvovirus and rabies vaccines are usually designed separately and require separate vaccination, which increases cost and complexity. The immunization effect of the existing vaccines is unstable and cannot effectively prevent epidemic strains, which poses safety and risk of virus shedding.
A triple inactivated vaccine containing canine distemper virus CDV GS20-11, canine parvovirus CPV-S5 and rabies virus HEP-Flury-dG was developed, and the triple inactivated vaccine was mixed with immune adjuvants such as MONTANIDE GEL 01.
It achieves simultaneous immunity of canine distemper, canine tiny and rabies in the canine body, provides continuous immune protection, excellent immune effect, and avoids the need for multiple vaccinations.
Smart Images
Figure CN120459286A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a triple inactivated canine distemper-canine parvovirus-rabies vaccine, a preparation method thereof, and an application thereof. Background Art
[0002] Canine distemper, canine parvovirus and rabies are common and important infectious diseases in dogs, characterized by high mortality and wide spread.
[0003] Canine distemper virus (CDV) belongs to the genus Morbillivirus of the family Paramyxoviridae. It is a single-stranded negative-sense RNA virus that is highly contagious and capable of cross-species transmission. It can infect dogs, cats and a variety of wild animals, causing damage to the respiratory, digestive and nervous systems, with a mortality rate of over 50%; Canine parvovirus (CPV) is a member of the family Parvoviridae, a single-stranded DNA virus that mainly causes hemorrhagic enteritis and myocarditis in puppies, with a mortality rate of up to 90%; Rabies virus (RABV) belongs to the family Rhabdoviridae, a single-stranded negative-sense RNA virus that causes fatal encephalitis through neuroinvasion, with a mortality rate of 100% after infection.
[0004] At present, vaccines for canine distemper virus include inactivated vaccines and live attenuated vaccines (such as the Onderstepoort strain). In the early stage, the pathogenicity of the virus was reduced by inactivation or attenuation technology, but there are certain safety issues (such as reversion to virulence) and unstable immune effects (affected by immunization dose, route and host differences); the immunogenicity of inactivated vaccines for canine parvovirus is weak and multiple booster immunizations are required. Although the attenuated vaccines (such as CPV-2b and CPV-2c) have significant immune effects, there is a risk of virus shedding and the risk of interfering with diagnosis; the existing vaccines for canine distemper virus, canine parvovirus and rabies virus are mostly inactivated vaccines and live attenuated vaccines, and the immunogenicity of inactivated vaccines is weak and multiple booster immunizations are required; at the same time, although the existing vaccines can prevent these diseases, they are usually designed for a single disease; and the existing canine distemper vaccine and canine parvovirus vaccine are mostly based on old strains, and their antigenicity is different from the prevalent strains, resulting in the immune effect failing to achieve the ideal level of protection.
[0005] Moreover, vaccines against the three viruses mentioned above often need to be administered separately, which increases the cost and complexity of vaccination. Therefore, the development of a vaccine that can simultaneously prevent canine distemper, canine parvovirus and rabies has important clinical significance and market value. Summary of the Invention
[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a triple inactivated canine distemper-canine parvovirus-rabies vaccine and a preparation method and application thereof.
[0007] The first object of the present invention is to provide a triple inactivated vaccine of canine distemper-canine parvovirus-rabies.
[0008] The second object of the present invention is to provide the use of the triple inactivated vaccine in the preparation of a medicament for immunizing canine distemper virus, canine parvovirus and / or rabies virus.
[0009] The third object of the present invention is to provide a method for preparing a triple inactivated vaccine of canine distemper-canine parvovirus-rabies.
[0010] The fourth object of the present invention is to provide a triple inactivated vaccine prepared by the above preparation method.
[0011] In order to achieve the above object, the present invention is implemented through the following scheme:
[0012] A triple inactivated canine distemper-canine parvovirus-rabies vaccine, comprising an inactivated canine distemper virus antigen, an inactivated canine parvovirus antigen, an inactivated rabies virus antigen, and an immune adjuvant;
[0013] The inactivated canine distemper virus antigen is an inactivated antigen of the canine distemper virus CDV GS20-11 strain, the inactivated canine parvovirus antigen is an inactivated antigen of the canine parvovirus CPV-S5 strain, and the inactivated rabies virus antigen is an inactivated antigen of the rabies HEP-Flury-dG strain.
[0014] The CDV GS20-11 strain is recorded at https: / / www.ncbi.nlm.nih.gov / nuccore / MW600730.1, GenBank: MW600730.1.
[0015] Preferably, the immune adjuvant is MF59, aluminum hydroxide gel, mineral oil and / or MONTANIDE GEL 01.
[0016] More preferably, the immune adjuvant is MONTANIDE GEL01.
[0017] Further preferably, the volume concentration of the MONTANIDE GEL 01 in the triple inactivated vaccine is 10-20%.
[0018] More preferably, the volume concentration of MONTANIDE GEL 01 in the triple inactivated vaccine is 10%.
[0019] The present invention also seeks to protect the use of any of the aforementioned triple inactivated vaccines in the preparation of a medicament for immunizing against canine distemper virus, canine parvovirus and / or rabies virus.
[0020] The present invention also claims protection for a method for preparing a triple inactivated canine distemper-canine parvovirus-rabies vaccine, comprising the following steps:
[0021] S1. Cultivate canine distemper virus, canine parvovirus, and rabies virus, respectively, and collect canine distemper virus fluid, canine parvovirus fluid, and rabies virus fluid;
[0022] S2. The canine distemper virus solution, canine parvovirus solution and rabies virus solution obtained in step S1 were filtered through 0.22μm~0.45μm filters, respectively, and then concentrated, and then inactivated to obtain inactivated canine distemper virus solution, inactivated canine parvovirus solution and inactivated rabies virus solution;
[0023] S3. The inactivated canine distemper virus solution, inactivated canine parvovirus solution and inactivated rabies virus solution obtained in step S2 are mixed in a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and then emulsified with an immune adjuvant to obtain a triple inactivated vaccine.
[0024] Preferably, in step S1, canine distemper virus is cultured using Vero-DogSLAM cells, canine parvovirus is cultured using F81 cells, and rabies virus is cultured using BHK-21 cells.
[0025] Preferably, β-propiolactone is used for inactivation treatment in step S2.
[0026] Preferably, in step S2, the solution is filtered through a 0.45 μm filter.
[0027] Preferably, in step S2, a 100KD ultrafiltration tube is used for concentration.
[0028] Preferably, in step S3, the inactivated canine distemper virus solution, the inactivated canine parvovirus solution and the inactivated rabies virus solution are mixed in a volume ratio of 1:1:1.
[0029] Preferably, the immune adjuvant in step S3 is MF59, aluminum hydroxide gel, mineral oil and / or MONTANIDE GEL01.
[0030] More preferably, the immune adjuvant is MONTANIDE GEL01.
[0031] The present invention also seeks to protect the triple inactivated vaccine prepared by any of the above-mentioned preparation methods.
[0032] Compared with the prior art, the present invention has the following beneficial effects:
[0033] The present invention provides a triple inactivated canine distemper, canine parvovirus, and rabies vaccine, comprising an inactivated canine distemper virus antigen, an inactivated canine parvovirus antigen, an inactivated rabies virus antigen, and an immune adjuvant. The inactivated canine distemper virus antigen is an inactivated antigen of the canine distemper virus (CDV) GS20-11 strain, the inactivated canine parvovirus antigen is an inactivated antigen of the canine parvovirus (CPV)-S5 strain, and the inactivated rabies virus antigen is an inactivated antigen of the rabies HEP-Flury-dG strain. The present invention also provides a method for preparing the triple inactivated vaccine. The resulting triple inactivated vaccine can effectively induce neutralizing antibodies against the three viruses in dogs and provide sustained immune protection. Therefore, vaccination with the triple inactivated canine distemper-canine parvovirus-rabies vaccine of the present invention not only achieves the simultaneous immunization of dogs against canine distemper virus, canine parvovirus and rabies virus with a single shot of the vaccine, but also achieves excellent immune effects. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the phylogenetic tree of canine distemper virus (CDV GS20-11 strain, GenBank: MW600730.1) in Example 1;
[0035] Figure 2 Figures 2A and 2B are graphs showing the protein content of each virus solution before and after concentration; Figure A shows the G protein content of the rabies virus solution before and after concentration; Figure B shows the N protein content of the canine distemper virus solution before and after concentration; and Figure C shows the VP2 protein content of the canine parvovirus solution before and after concentration.
[0036] Figure 3 Figure 2 is the result of PCR detection in Example 2; A is the PCR identification result of CDV fire extinguishing virus solution; B is the PCR identification result of CPV inactivated virus solution; C is the PCR identification result of RABV inactivated virus solution;
[0037] Figure 4 1 is a graph showing the observation results of pathological changes in cells inoculated with canine distemper virus after fire extinguishing in Example 2; a is a graph showing the observation results of cells inoculated with canine distemper virus; b is a graph showing the observation results of cells inoculated with F3 generation CDV virus solution;
[0038] Figure 5 Graphs showing the observation results of pathological changes in cells inoculated with inactivated canine parvovirus in Example 2; a graph showing the observation results of cells inoculated with canine parvovirus; and b graph showing the observation results of cells inoculated with F3 generation CPV virus solution.
[0039] Figure 6Graphs showing the IFA test results of cells inoculated with inactivated rabies virus in Example 2; a graph showing the IFA test results of cells inoculated with rabies virus; b graph showing the IFA test results of cells inoculated with F3 generation RABV virus solution;
[0040] Figure 7 Figures 1 and 2 are photographic observation results of the triple inactivated vaccine before and after emulsification in Example 3; Figure A is a photographic observation result of the triple inactivated vaccine before emulsification; Figure B is a photographic observation result of the triple inactivated vaccine after emulsification;
[0041] Figure 8 A is a graph showing the sterility test results in Example 3; A is a graph showing the TSB inoculation results; B is a graph showing the TG inoculation results; and C is a graph showing the GA agar inoculation results. DETAILED DESCRIPTION
[0042] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.
[0043] Example 1 Virus culture and selection of cultured cells
[0044] 1. Experimental Methods
[0045] Canine distemper virus (CDV GS20-11 strain, GenBank: MW600730.1) was inoculated into Vero-DogSLAM cells at an MOI of 1, canine parvovirus (CPV-S5 strain) was inoculated into F81 cells at an MOI of 1, and rabies virus (HEP-Flury (dG) strain) was inoculated into BHK-21 cells at an MOI of 1. The phylogenetic tree of canine distemper virus (CDV GS20-11 strain, GenBank: MW600730.1) is shown in FIG. Figure 1 shown.
[0046] The virus-inoculated cells were then cultured in a cell culture incubator at 37°C and 5% (v / v) CO2. After culturing for 2 hours, the complete culture medium containing 2% (v / v) FBS was replaced and culture was continued for 5 days.
[0047] After the culture was completed, the culture fluid of each virus-inoculated cell was collected and centrifuged at 10,000 rpm for 15 min, and the supernatant was collected to obtain canine distemper virus liquid, canine parvovirus liquid and rabies virus liquid, respectively.
[0048] Then, the virus titers of the canine distemper virus solution, canine parvovirus solution and rabies virus solution were determined respectively according to the TCID50 method.
[0049] 2. Experimental Results
[0050] The virus titer of canine distemper virus solution was 10 4.5 TCID 50 / mL, the virus titer of canine parvovirus solution is 10 6 TCID 50 / mL, the virus titer of rabies virus liquid is 10 5.2 TCID 50 / mL, indicating that the cells used are suitable for the culture of canine distemper virus, canine parvovirus and rabies virus.
[0051] Example 2 Concentration of Virus Liquid and Verification of Inactivated Viruses
[0052] 1. Experimental Methods
[0053] According to the method shown in Example 1, canine distemper virus liquid, canine parvovirus liquid and rabies virus liquid were collected respectively.
[0054] According to the TCID50 method, the virus titers of the canine distemper virus liquid, the canine parvovirus liquid and the rabies virus liquid were respectively determined, and then each virus liquid was filtered through a 0.45 μm microporous filter, and then each virus liquid was concentrated using a 100KD ultrafiltration tube to one tenth of the original volume to obtain concentrated canine distemper virus liquid, concentrated canine parvovirus liquid and concentrated rabies virus liquid, and the virus titer of each concentrated virus liquid was determined according to the TCID50 method, and the concentration efficiency of each virus liquid was calculated.
[0055] 10 μL of 5× protein loading buffer was added to 40 μL of the virus solution before and after concentration, and the mixture was evenly mixed. The mixture was placed in a 100°C metal bath for 10 minutes. After cooling to 25°C, the viral proteins were separated by centrifugation and transferred to nitrocellulose membranes. The concentration effect of the immunogen was confirmed by staining with the corresponding antibodies for CDV virus, CPV virus, and rabies virus. The antibody for CDV virus was purchased from VMRD, with the corresponding product number 1C42H11; the antibody for CPV virus was CPV-VP2, with the brand name Abnova and the product number MAB17265; and the antibody for rabies virus was FITC Anti-Rabies Monoclonal Globulin, with the brand name innogenetics and the product number 800-092.
[0056] Then, the concentrated canine distemper virus liquid and β-propiolactone were mixed in a volume ratio of 1:2000, stirred evenly, and placed at 4°C for 24 hours, and then placed in a 37°C water bath for 1 hour to obtain an inactivated canine distemper virus liquid; the concentrated canine parvovirus liquid and the concentrated rabies virus liquid were treated identically to obtain an inactivated canine parvovirus liquid and an inactivated rabies virus liquid.
[0057] The inactivated and inactivated CDV fluids were inoculated into 6-cm dishes of Vero-Dog SLAM cells at a confluency of 60-70% at a volume of 1 mL. The cells were incubated in a 37°C incubator for 2 hours. The maintenance medium was replaced with 2% fetal bovine serum (FBS). After 4 days of culture, the virus was harvested by repeated freeze-thaw cycles and blindly passaged for three generations (F1, F2, and F3). Vero-Dog SLAM cells were inoculated with the F1, F2, and F3 CDV fluids, respectively. Cytopathic effects were observed on Vero-Dog SLAM cells inoculated with the F3 CDV fluid to determine whether the inactivated CDV fluid was infective.
[0058] Then, PCR amplification was performed on Vero-DogSLAM cells inoculated with F1 generation canine distemper virus liquid, F2 generation canine distemper virus liquid, F3 generation canine distemper virus liquid, Vero-DogSLAM cells not inoculated with virus liquid, and Vero-DogSLAM cells inoculated with inactivated canine distemper virus liquid, respectively. The PCR amplification products were obtained and placed on 1% (w / w) agarose gel electrophoresis for identification.
[0059] PCR amplification used CDV-F (upstream primer, SEQ ID NO: 1) and CDV-R (downstream primer, SEQ ID NO: 2). The PCR reaction system consisted of 12.5 μL of 2× Taq PCR Mix, 1.0 μL each of upstream and downstream primers F and R (10 μM), 1.0 μL of template cDNA, and ddH2O to a total of 25 μL. The PCR program was as follows: 95°C initial denaturation for 5 min, 95°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 1 min, and 72°C final extension for 7 min, for 34 cycles. PCR products were identified by electrophoresis on 1% agarose gels.
[0060] The inactivated and inactivated canine distemper virus solutions were replaced with the inactivated and inactivated canine parvovirus solutions and the inactivated and inactivated rabies virus solutions, respectively. The Vero-DogSLAM cells were replaced with F81 cells and BHK-21 cells, respectively. The inactivated canine parvovirus solutions and the inactivated rabies virus solutions were tested according to the above method to observe whether they were infectious, and the response results were recorded.
[0061] For rabies viruses that do not cause pathological changes, immunofluorescence analysis (IFA) is used to detect whether the inactivated canine distemper virus solution is infectious. The specific steps of the IFA test are as follows: the infected cells after 4 days of culture are fixed with 80% (v / v) acetone for 30 minutes, washed three times with 0.01mM PBS (pH = 7.4), and then FITC-labeled RABV-G Mab is added. The cells are incubated in a 37°C incubator for 1 hour, washed three times with 0.01mM PBS, and observed under a fluorescence microscope.
[0062] Then, when PCR amplification was performed according to the above method, when PCR amplification was performed on cells inoculated with canine parvovirus solution, the primers used were CPV-F (upstream primer, SEQ ID NO: 3) and CPV-R (downstream primer, SEQ ID NO: 4); when PCR amplification was performed on cells inoculated with rabies virus solution, the primers used were RABV-F (upstream primer, SEQ ID NO: 5) and RABV-R (downstream primer, SEQ ID NO: 6).
[0063] The primer information is shown in Table 1.
[0064] Table 1 Primer information
[0065]
[0066] 2. Experimental Results
[0067] The protein content identification results of each virus solution before and after concentration are as follows Figure 2 As shown, Figure 2 A in the figure is the result of identification of G protein content before and after concentration of rabies virus liquid. Figure 2 The 1 in A is the G protein content identification result of the rabies virus solution before concentration. Figure 2 2 in A is the identification result of G protein content in concentrated rabies virus solution; Figure 2 Figure B is the N protein content identification result of canine distemper virus liquid before and after concentration. Figure 2 The 1 in B is the identification result of N protein content of canine distemper virus solution before concentration. Figure 2 2 in B is the identification result of N protein content in concentrated canine distemper virus solution; Figure 2 Figure C shows the results of identifying the VP2 protein content of canine parvovirus solution before and after concentration. Figure 2 1 in C is the identification result of VP2 protein content of canine parvovirus solution before concentration. Figure 2 2 in C is the identification result of VP2 protein content in the concentrated canine parvovirus solution.
[0068] The results showed that after the virus liquid was concentrated, the protein content in the virus liquid was significantly increased compared with before concentration.
[0069] The virus titer of canine distemper virus solution before concentration is 10 4.125 TCID 50 / mL, the virus titer of the concentrated canine distemper virus solution is 10 5.25 TCID 50 / mL, the volume was concentrated about 13 times, and the concentration efficiency was 90%; the virus titer of canine parvovirus solution before concentration was 10 5.75 TCID 50 / mL, the virus titer of the concentrated canine parvovirus solution is 10 6.875 TCID 50 / mL, the volume was concentrated about 12 times, and the concentration efficiency was close to 90%; the virus titer of the rabies virus liquid before concentration was 10 4..625 TCID 50 / mL, the virus titer of the concentrated rabies virus solution is 10 5.50 TCID 50 / mL, the volume was concentrated about 10 times, and the concentration efficiency was close to 90%.
[0070] PCR identification results are shown in the figure Figure 3 As shown, Figure 3 A in the figure is the PCR identification result of CDV inactivated virus solution. Figure 3 Figure B is the PCR identification result of CPV inactivated virus solution. Figure 3 Figure C shows the PCR identification results of the RABV inactivated virus solution.
[0071] in Figure 3 1 is the PCR identification result of cells inoculated with F1 generation virus, 2 is the PCR identification result of cells inoculated with F2 generation virus, 3 is the PCR identification result of cells inoculated with F3 generation virus, 4 is the PCR identification result of cells not inoculated with virus, and 5 is the PCR identification result of cells inoculated with inactivated proviral solution.
[0072] The results showed that after PCR amplification, no target bands appeared in the first, second, and third generation cells harvested after inoculation of the inactivated virus solution, as well as in the cell control, while the corresponding target bands appeared in the positive control, indicating that the virus solution was effectively inactivated.
[0073] The pathological observation results of cells inoculated with inactivated canine distemper virus are shown in the figure below. Figure 4 As shown, Figure 4 Figure a is the observation result of cells inoculated with canine distemper virus. Figure 4 Panel b shows the observation results of cells inoculated with F3 generation CDV virus solution.
[0074] The pathological observation results of cells inoculated with inactivated canine parvovirus are shown in the figure below. Figure 5 As shown, Figure 5 Figure a shows the observation results of cells inoculated with canine parvovirus. Figure 5 Panel b shows the observation results of cells inoculated with F3 generation CPV virus solution.
[0075] The IFA test results of cells inoculated with inactivated rabies virus are shown in the figure below. Figure 6 As shown, Figure 6 Figure a is the IFA test result of cells inoculated with rabies virus. Figure 6 Figure b shows the IFA test results of cells inoculated with F3 generation RABV virus liquid.
[0076] The results showed that inactivated CDV and CPV did not cause cell pathology after passage 3. Furthermore, IFA testing of RABV showed that cells inoculated with F3 generation rabies virus fluid were also negative, indicating that the virus fluid was effectively inactivated.
[0077] Inoculation of inactivated virus liquid into cells showed that the inactivated canine distemper virus liquid, inactivated canine parvovirus liquid and inactivated rabies virus liquid did not cause damage to the unknown antigen protein structure. The cells were not damaged after inoculation of the inactivated virus liquid, indicating that the virus liquid inactivation process was effective.
[0078] Example 3 A triple inactivated vaccine of canine distemper, canine parvovirus and rabies
[0079] 1. Experimental Methods
[0080] 1. Preparation method
[0081] A method for preparing a triple inactivated canine distemper-canine parvovirus-rabies vaccine comprises the following steps:
[0082] S1. CDV GS20-11 strain, GenBank: MW600730.1) was inoculated into Vero-Dog SLSM cells at an MOI of 1, canine parvovirus (CPV-S5 strain) was inoculated into F81 cells at an MOI of 1, and rabies virus (HEP-Flury (dG) strain) was inoculated into BHK-21 cells at an MOI of 1. The virus-inoculated cells were then cultured in a cell culture incubator at 37°C, 5% (v / v) CO2 for 2 h, the culture medium was replaced, and the culture was continued for 5 days. The culture fluid of each virus-inoculated cell was then collected and centrifuged at 10,000 rpm for 10 min. The supernatant was collected to obtain canine distemper virus solution, canine parvovirus solution, and rabies virus solution, respectively;
[0083] S2. The canine distemper virus solution, canine parvovirus solution and rabies virus solution obtained in step S1 were filtered through a 0.45 μm microporous filter, and then each virus solution was concentrated using a 100KD ultrafiltration tube to one-tenth of the original volume to obtain a concentrated canine distemper virus solution, a concentrated canine parvovirus solution and a concentrated rabies virus solution;
[0084] The concentrated canine distemper virus solution and β-propiolactone were mixed in a volume ratio of 1:2000, stirred evenly, and then placed at 4°C for 24 hours, and then placed in a 37°C water bath for 1 hour to obtain an inactivated canine distemper virus solution; the concentrated canine parvovirus solution and the concentrated rabies virus solution were treated in the same manner to obtain an inactivated canine parvovirus solution and an inactivated rabies virus solution;
[0085] S3. The inactivated canine distemper virus solution obtained in step S2, the inactivated canine parvovirus solution and the inactivated rabies virus solution were mixed in a volume ratio of 1:1:1 to obtain a mixed virus solution;
[0086] The mixed virus solution and MONTANIDE GEL 01 (immune adjuvant) were mixed uniformly at a volume ratio of 9:1, and emulsified using a magnetic stirrer at 1000 rpm / min for 30 minutes to obtain a triple inactivated canine distemper-canine parvovirus-rabies vaccine;
[0087] At the same time, photos of the triple inactivated vaccine before and after emulsification were taken.
[0088] 2. Quality inspection
[0089] Sterility testing
[0090] Referring to the sterility test method in the 2020 edition of the "Chinese Veterinary Pharmacopoeia", the triple inactivated vaccine of canine distemper, canine parvovirus and rabies prepared in step 1 was tested for fungi, aerobic bacteria and anaerobic bacteria using TSB (trypticase soy peptone liquid medium), TG (thioglycollate fluid medium) and GA (casein agar medium), respectively. They were inoculated at a volume ratio of 1:100, and a negative control was set up. The results were observed and recorded after culturing at 37°C for 7 days.
[0091] 3. Vaccine safety testing
[0092] The triple inactivated canine distemper-parvovirus-rabies vaccine prepared in Example 1 was intraperitoneally injected into mice at a dose of 200 μL / mouse, and the mental status of the mice was observed within 21 days of immunization.
[0093] 2. Experimental Results
[0094] The photographic observation results of the triple inactivated vaccine before and after emulsification are shown in the figure below. Figure 7 As shown, Figure 7A in the figure is the photographic observation result of the triple inactivated vaccine before emulsification. Figure 7 B in the figure is the photographic observation result of the triple inactivated vaccine after emulsification.
[0095] The results showed that the emulsified triple inactivated vaccine was clear, slightly viscous, and had no stratification. This indicated that the emulsification process was sufficient, with no obvious turbidity or particulate matter, and the vaccine had moderate fluidity.
[0096] Sterility test results are shown in the figure Figure 8 As shown, Figure 8 A in the figure is the TSB inoculation result diagram, Figure 8 B in the figure is the TG inoculation result diagram, Figure 8 Figure C shows the results of GA agar inoculation.
[0097] The results showed that the sterility test was negative, indicating that the vaccine maintained a good sterile state during the production and storage process; and after the prepared triple inactivated vaccine was intraperitoneally injected into mice at 200 μL / mouse, the mice were in good mental condition within 21 days, and no illness or death occurred.
[0098] Example 4 Verification of the immune efficacy of the triple inactivated vaccine
[0099] 1. Experimental Methods
[0100] A triple inactivated vaccine of canine distemper, canine parvovirus and rabies was prepared according to the method shown in Example 3.
[0101] Three beagle dogs were immunized with a triple inactivated canine distemper-parvovirus-rabies vaccine by subcutaneous injection in the neck at an immunization dose of 1 mL per dog (first immunization). Blood was collected from each beagle dog on the 0th, 7th, 14th and 21st day after the first immunization, and the antibody level was determined by serum neutralization test.
[0102] On the 21st day after the first immunization, blood was collected and the three beagle dogs were immunized for the second time with an immunization dose of 1 mL / dog (immunization with a triple inactivated vaccine of canine distemper, canine parvovirus and rabies). Blood was collected from each beagle dog on the 7th, 14th and 21st day after the second immunization, and the antibody level was determined by serum neutralization test.
[0103] The steps for determining antibody levels in serum neutralization tests are as follows:
[0104] The collected canine serum was placed at 37°C for 1 hour, then placed at 4°C for 12 hours, and then centrifuged at 1000 r / min for 5 minutes. The supernatant was aspirated to obtain the separated serum; the separated serum was placed in a 56°C water bath and complement was inactivated for 30 minutes. The inactivated serum was obtained and the neutralizing antibody levels of the three viruses were detected using the fixed virus-diluted serum method.
[0105] The inactivated serum was diluted with a starting dilution of 1:3 and then diluted in a 3-fold gradient for a total of 8 dilutions to obtain diluted serum. Then, each dilution was repeated in 4 wells and diluted serum was added at 50 μL / well. A standard serum group for RABV was established.
[0106] Take the virus with determined titer and dilute it to 100TCID based on the determined TCID50. 50 / 50μL; according to the corresponding wells, add 50μL of virus solution to each well, mix evenly, neutralize at 37℃ for 1h, and then add 100μL of the digested and blown-off Vero-dSLAM, BHK-21, and F81 cell suspensions to each well at a passage ratio of 1:3, and culture in a carbon dioxide incubator for 48h.
[0107] According to the experimental procedure of immunofluorescence analysis (IFA) shown in Example 2, the neutralizing antibody titer of the serum was calculated using the Reed-Muench method.
[0108] After the antibody levels were determined based on the serum neutralization test, the average of the antibody levels of the three beagle dogs was taken as the final antibody level result.
[0109] 2. Experimental Results
[0110] When beagles were immunized with the triple inactivated canine distemper-parvovirus-rabies vaccine prepared in Example 3, the antibody level test results after immunization were shown in Table 2.
[0111] Table 2 Antibody level test results after immunization
[0112]
[0113] The results showed that when beagle dogs were immunized with the triple inactivated canine distemper-parvovirus-rabies vaccine prepared in Example 3, the CPV antibody level in the beagle dogs was 1:1013, the RABV antibody level was 0.78 IU / mL, and the CDV antibody level was 1:17.5 on the 21st day after the first immunization.
[0114] On the 21st day after the second immunization, the CPV antibody level in the beagle dogs was 1:1307, the RABV antibody level was 4.6 IU / mL, and the CDV antibody level was 1:56.2.
[0115] The results show that the triple inactivated canine distemper-canine parvovirus-rabies vaccine prepared in Example 3 can achieve excellent immune effects on dogs. When injected into dogs for immunization, it can simultaneously achieve excellent immunity against canine distemper, canine parvovirus and rabies.
[0116] Example 5 Immune effect test of CPV virus
[0117] 1. Experimental Methods
[0118] The canine parvovirus CPV-S5 strain is concentrated to one tenth of the original volume to obtain concentrated CPV-S5; the canine parvovirus CPV-1401 strain is concentrated to one tenth of the original volume to obtain concentrated CPV-1401.
[0119] Beagles were immunized with concentrated CPV-S5 and concentrated CPV-1401 at a hemagglutination titer of 8 log2 and a 1 mL immunization dose, and blood was collected on the 7th, 14th, 21st, and 28th days after immunization. The HI antibody titer was tested by hemagglutination (HA) and hemagglutination inhibition (HI) tests.
[0120] 2. Experimental Results
[0121] The results of the HI antibody titer test are shown in Table 3.
[0122] Table 3HI antibody titer test results
[0123]
[0124] The results showed that after beagle dogs were immunized with concentrated CPV-S5 strain, the HI antibody level in the beagle dogs was significantly higher than that in the beagle dogs immunized with concentrated CPV-1401 strain, indicating that the CPV strain combined in the triple inactivated canine distemper-canine parvovirus-rabies vaccine prepared in Example 3 of the present invention has better immunity.
[0125] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art will readily appreciate that other variations or modifications may be made based on the above descriptions and concepts. It is not necessary and impossible to provide an exhaustive list of all possible implementations. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.
Claims
1. A triple inactivated vaccine for canine distemper, canine parvovirus and rabies, characterized in that: The triple inactivated vaccine contains inactivated canine distemper virus antigen, inactivated canine parvovirus antigen, inactivated rabies virus antigen and immune adjuvant; The inactivated canine distemper virus antigen is an inactivated antigen of the canine distemper virus CDV GS20-11 strain, the inactivated canine parvovirus antigen is an inactivated antigen of the canine parvovirus CPV-S5 strain, and the inactivated rabies virus antigen is an inactivated antigen of the rabies HEP-Flury-dG strain.
2. The triple inactivated vaccine according to claim 1, characterized in that The immune adjuvant is MF59, aluminum hydroxide gel, mineral oil and / or MONTANIDE GEL 01.
3. The triple inactivated vaccine according to claim 2, characterized in that The volume concentration of the immune adjuvant in the triple inactivated vaccine is 10-20%.
4. Use of the triple inactivated vaccine according to any one of claims 1 to 3 in the preparation of a medicament for immunizing against canine distemper virus, canine parvovirus and / or rabies virus.
5. A method for preparing a triple inactivated vaccine of canine distemper, canine parvovirus and rabies, characterized in that: The following steps are involved: S1. Cultivate canine distemper virus, canine parvovirus, and rabies virus, respectively, and collect canine distemper virus fluid, canine parvovirus fluid, and rabies virus fluid; The canine distemper virus is the canine distemper virus CDV GS20-11 strain, the canine parvovirus is the canine parvovirus CPV-S5 strain, and the rabies virus is the rabies HEP-Flury-dG strain; S2. The canine distemper virus solution, canine parvovirus solution and rabies virus solution obtained in step S1 were filtered through 0.22μm~0.45μm filters, respectively, and then concentrated, and then inactivated to obtain inactivated canine distemper virus solution, inactivated canine parvovirus solution and inactivated rabies virus solution; S3. The inactivated canine distemper virus solution, inactivated canine parvovirus solution and inactivated rabies virus solution obtained in step S2 are mixed in a volume ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and then emulsified with an immune adjuvant to obtain a triple inactivated vaccine.
6. The preparation method according to claim 5, characterized in that In step S1, canine distemper virus is cultured using Vero-DogSLAM cells, canine parvovirus is cultured using F81 cells, and rabies virus is cultured using BHK-21 cells.
7. The preparation method according to claim 5, characterized in that In step S2, β-propiolactone is used for inactivation treatment.
8. The preparation method according to claim 5, characterized in that In step S3, the inactivated canine distemper virus solution, the inactivated canine parvovirus solution and the inactivated rabies virus solution are mixed in a volume ratio of 1:1:
1.
9. The preparation method according to claim 5, characterized in that The immune adjuvant in step S3 is MF59, aluminum hydroxide gel, mineral oil and / or MONTANIDE GEL 01.
10. The triple inactivated vaccine prepared by the preparation method according to any one of claims 5 to 9.