Combined vaccine containing bordetella bronchiseptica attenuated strain HN60

Abstract

The invention relates to the technical field of biological pharmacy, and particularly provides a combined vaccine containing a bordetella bronchiseptica attenuated strain HN60. The antigen of the combined vaccine contains the bordetella bronchiseptica attenuated strain HN60 or a culture thereof, so that clinical symptoms occurring after bacterial infection can be relieved, the living quality of dogs is improved, and the occurrence rate of canine nest cough caused by mixed infection with other viruses is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and specifically provides a combined vaccine containing a Bordetella bronchiseptica attenuated strain HN60. Background Art

[0002] Canine infectious tracheobronchitis (kennel cough) is an important disease in dogs. It often occurs and spreads in groups of dogs raised in concentrated areas, such as kennels, stray dog ​​shelters, pet shops and pet hospitals. It is caused by infection with Bordetella bronchiseptica (Bb). It is also often mixed with pathogens such as canine parainfluenza virus (CPIV) and canine adenovirus type 2 (CAV-2). Dogs of different breeds, genders and ages can be infected with this disease, but young dogs are the most sensitive and the harm is more serious. Clinically, paroxysmal cough, tracheobronchitis and rhinitis are the main symptoms.

[0003] Bordetella bronchiseptica was first isolated from the respiratory tract of dogs with canine distemper. It is a Gram-negative coccobacillus belonging to the genus Bordetella. It can infect a variety of mammals, causing atrophic rhinitis and pneumonia in pigs, bronchial pneumonia in rabbits and guinea pigs, and respiratory infections in cats, mice and horses. It is highly contagious to dogs and can be easily transmitted through direct contact between dogs.

[0004] There are four phenotypes of Bordetella bronchiseptica growing on the Bauer blood plate, namely, phase I, phase II, phase III and rough type. The colony morphology of phase I bacteria on the Bauer blood plate is small pearl-like colonies, some strains are hemolytic, and express a series of virulence factors. The colony morphology of phase III bacteria on the Bauer blood plate is large gray-white flat colonies, which are not hemolytic and do not express corresponding virulence factors. Studies have shown that phase I strains are highly virulent strains, and other variant strains are weak strains.

[0005] Vaccination plays an important role in preventing infectious canine tracheobronchitis. The present invention selects a wild strain of Bordetella bronchiseptica isolated in China for cultivation to obtain a weakened strain, which is used to prepare an attenuated vaccine composition, which has an active prevention and efficient control effect on canine infectious tracheobronchitis.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first object of the present invention is to provide the use of Bordetella bronchiseptica attenuated strain HN60 or its culture in a combined vaccine.

[0008] The second object of the present invention is to provide a combined vaccine.

[0009] In order to achieve the above object, the present invention particularly adopts the following technical scheme:

[0010] The use of the attenuated strain HN60 of Bordetella bronchiseptica or its culture in a combination vaccine. The preservation number of the attenuated strain HN60 of Bordetella bronchiseptica is CCTCCNO:M20221719, and it is preserved in the China Center for Type Culture Collection, the preservation address is Wuhan University, Wuhan, China, and the preservation date is November 3, 2022.

[0011] A combined vaccine comprises an antigen and a pharmaceutically acceptable adjuvant.

[0012] The antigen includes an immune effective amount of the attenuated strain HN60 of Bordetella bronchiseptica or its culture. The attenuated strain HN60 of Bordetella bronchiseptica is deposited with CCTCCNO: M20221719 in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China, and the deposit date is November 3, 2022.

[0013] Furthermore, the antigen includes an immunologically effective amount of live whole virus antigens of the attenuated strain HN60 of Bordetella bronchiseptica or its culture.

[0014] Preferably, the content of the Bordetella bronchiseptica attenuated strain HN60 is ≥1×10 7 CFU / head, preferably 1×10 7 ~1×10 9 CFU / head.

[0015] Preferably, the Bordetella bronchiseptica attenuated strain HN60 culture is selected from 1 to 60 generations.

[0016] Furthermore, the antigen also includes an immunologically effective amount of canine parainfluenza virus antigen and / or an immunologically effective amount of canine adenovirus antigen.

[0017] Furthermore, the canine parainfluenza virus antigen is a live whole virus antigen of the canine parainfluenza virus HeN20 strain or its culture, preferably with a content of ≥10 3.0 TCID 50 / head, more preferably 10 3.0 ~10 6.0 TCID 50 / First portion.

[0018] Preferably, the canine parainfluenza virus HeN20 strain culture is selected from 1 to 20 generations.

[0019] Furthermore, the canine adenovirus antigen is a live whole virus antigen of canine adenovirus type 2 HL50 strain or its culture, preferably with a content of ≥10 4.0 TCID 50 / head, more preferably 10 4.0 ~10 6.0 TCID 50 / First portion.

[0020] Preferably, the canine adenovirus type 2 HL50 strain is selected from passages 1 to 70.

[0021] Furthermore, the pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA (a lyoprotectant commonly used in the art, composed of sucrose, phosphate, glutamic acid and albumin), sugars, gelatin, proteins, substances containing proteins or / and buffer;

[0022] Preferably, the sugar is selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; the protein is selected from albumin or casein; the substance containing protein is bovine serum or skim milk; the buffer is phosphate buffer;

[0023] Preferably, the lyoprotectant is 20w / v% sucrose and 2w / v% gelatin;

[0024] Preferably, the volume ratio of the antigen to the pharmaceutically acceptable excipient is 1:1.

[0025] Furthermore, the combined vaccine is a freeze-dried powder and is administered by subcutaneous injection.

[0026] Compared with the prior art, the technical effects of the present invention are:

[0027] The Bordetella bronchiseptica attenuated strain HN60 or its culture in the present invention can be used for the preparation of combined vaccines. For example, after immunizing dogs with a combined vaccine prepared with a canine parainfluenza virus antigen and / or an immunologically effective amount of a canine adenovirus antigen, the clinical symptoms after bacterial infection can be alleviated, the quality of life of the dogs can be improved, and the incidence of "kennel cough" caused by mixed infection with other viruses can be reduced. DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The term "immunologically effective amount" refers to the amount necessary to exert their immunological effect in the subject to which the vaccine is administered without causing undue side effects. The exact amount of the ingredients used and the vaccine to be administered will vary depending on factors such as the type of disease being treated, the type and age of the animal being treated, the mode of administration, and the other ingredients in the vaccine.

[0030] The term "pharmaceutically acceptable excipient" refers to an excipient that does not irritate the body and does not interfere with the biological activity and properties of the compound being used.

[0031] The term "combination vaccine" refers to a mixed preparation containing two or more different antigens, including multivalent vaccines (used to prevent diseases caused by different microorganisms) and multivalent vaccines (used to prevent the same disease caused by different subtypes or serotypes). The advantage of combination vaccines over single vaccines is that one shot of vaccine can prevent several diseases at the same time. Its development purpose is to prevent more types of diseases while reducing the number of vaccine injections.

[0032] The Bordetella bronchiseptica attenuated strain HN60 (Bordetellabronchiseptica HN60) involved in the present invention has a deposit number of CCTCCNO: M20221719, which is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the deposit date is November 3, 2022. The Bordetella bronchiseptica attenuated strain HN60 is obtained by cultivating the Bordetella bronchiseptica virulent strain HN03, and the two are homologous. The Bordetella bronchiseptica virulent strain HN03 (Bordetellabronchiseptica HN), with a deposit number of CCTCCNO: M20221718, is deposited in the China Center for Type Culture Collection, the deposit address is Wuhan University, Wuhan, China, and the deposit date is November 3, 2022.

[0033] The attenuated strain HN60 of Bordetella bronchiseptica has stable biological characteristics, good immunogenicity, safety and reliability. The prepared attenuated live vaccine can produce a high level of antibodies and has a good protective effect against the strong toxin attack of Bordetella bronchiseptica.

[0034] The pathogenicity test showed that the pathogenicity of Bordetella bronchiseptica HN60 to dogs decreased significantly between the 1st and 60th generations. The dogs did not show clinical symptoms after 14 days of observation after the 60th generation of inoculation. Therefore, compared with its parent, the highly virulent Bordetella bronchiseptica HN03, the pathogenicity of this strain is significantly reduced, and it is a weak strain.

[0035] Immunogenicity tests showed that Bordetella bronchiseptica HN60 cultured to the 60th generation still had good immunogenicity. Dogs could resist the attack of the highly virulent Bordetella bronchiseptica HN03 28 days after vaccination. At the same time, dogs that were not vaccinated with Bordetella bronchiseptica HN60 culture could not resist the attack of Bordetella bronchiseptica HN03 and all became ill.

[0036] The virulence reversion test showed that Bordetella bronchiseptica HN60 cultured between the 1st and 60th generations did not revert to virulence after continuous contact and passage in the dog group after inoculation. Therefore, after Bordetella bronchiseptica HN60 was inoculated in the dog group, it would not re-evolve into a virulent virus and cause disease, and its safety was guaranteed.

[0037] Compared with its parent, the highly virulent Bordetella bronchiseptica HN03, the cultures of Bordetella bronchiseptica HN60 from the 1st to the 60th generations commonly showed a nucleotide base G inserted into the BvgS gene at the 2004 position, resulting in a frameshift mutation of the BvgS gene after the insertion site. The toxicity is lower than that of the parental highly virulent strain, and it has genetic stability.

[0038] The common characteristic changes in viral genes of different generations of Bordetella bronchiseptica HN60 cultures are most likely the reason for the reduction in virulence of its parental virulent strain, and these changes did not lead to changes in its immunogenicity.

[0039] Therefore, the Bordetella bronchiseptica attenuated strain HN60 or its culture can be used to prepare a drug for preventing Bordetella bronchiseptica infection, and the drug can be a single vaccine or a combined vaccine.

[0040] The present invention provides a combined vaccine, which comprises an antigen and a pharmaceutically acceptable adjuvant. The antigen is the above-mentioned immune effective amount of the Bordetella bronchiseptica attenuated strain HN60 or its culture.

[0041] In some embodiments, the antigen is an immunologically effective amount of a live whole virus antigen of the attenuated strain HN60 of Bordetella bronchiseptica or its culture. Specifically, the content of the attenuated strain HN60 of Bordetella bronchiseptica is ≥1×10 7 CFU / head, preferably 1×10 7 ~1×10 9 CFU / head portion. The culture of Bordetella bronchiseptica attenuated strain HN60 is selected from 1 to 60 generations, for example, it can be selected from the culture of 1 generation, 2 generations, 3 generations, 4 generations, 5 generations, 6 generations, 7 generations, 8 generations, 9 generations, 10 generations, 11 generations, 12 generations, 13 generations, 14 generations, 15 generations, 16 generations, 17 generations, 18 generations, 19 generations, 20 generations, 21 generations, 22 generations, 23 generations, 24 generations, 25 generations, 26 generations, 27 generations, 28 generations, 29 generations, 30 generations, 35 generations, 40 generations, 45 generations, 50 generations, 55 generations, and 60 generations.

[0042] In some embodiments, the antigen further comprises an immunologically effective amount of a canine parainfluenza virus antigen and / or an immunologically effective amount of a canine adenovirus antigen.

[0043] For example, the canine parainfluenza virus antigen is a live whole virus antigen of the canine parainfluenza virus HeN20 strain or its culture, the canine parainfluenza virus HeN20 strain (Canine Parainfluenza Virus, Strain HeN20), which is deposited in the China Center for Type Culture Collection, with the accession number CCTCCNO: V202214, the deposit address is Wuhan University, Wuhan, China, and the deposit date is March 1, 2022.

[0044] In some embodiments, the content of canine parainfluenza virus HeN20 strain is ≥10 3.0 TCID 50 / head, more preferably 10 3.0 ~10 6.0 TCID 50 The canine parainfluenza virus HeN20 strain culture is selected from passages 1 to 20, and can be selected from passages 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

[0045] For example, the canine adenovirus antigen is a live whole virus antigen of canine adenovirus type 2 HL50 strain or its culture. Canine adenovirus type 2 HL50 strain (Canineadenovirus2, Strain HL50) is deposited in the China Center for Type Culture Collection, with the accession number CCTCCNO: V202215, the deposit address is Wuhan University, Wuhan, China, and the deposit date is March 1, 2022.

[0046] In some embodiments, the content of canine adenovirus type 2 HL50 strain is ≥10 4.0 TCID 50 / head, more preferably 10 4.0 ~10 6.0 TCID 50 The canine adenovirus type 2 HL50 strain is selected from passages 1 to 70, and can be selected from a culture of passage 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 or 70.

[0047] In some embodiments, the pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA, sugars, gelatin, proteins, substances containing proteins or / and buffers; sugars are preferably selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; proteins are preferably selected from albumin or casein; substances containing proteins are preferably bovine serum or skim milk; buffers are preferably phosphate buffers; lyoprotectants are preferably 20w / v% sucrose and 2w / v% gelatin. In particular, when such lyoprotectants are added to vaccines, the vaccines are very suitable for freeze drying. Therefore, in a more preferred form of this embodiment, the combination vaccine is in a freeze-dried form, and the route of administration can be subcutaneous injection.

[0048] In some embodiments, the volume ratio of the antigen to the pharmaceutically acceptable excipient is 1:1.

[0049] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0050] Example 1 Isolation and Identification of Bordetella bronchiseptica in Dogs

[0051] 1.1 Isolation and identification of Bordetella bronchiseptica in dogs

[0052] Wild strains of Bordetella bronchiseptica were isolated from dogs clinically infected with Bordetella bronchiseptica. The clinical symptoms of infected animals included coughing, sneezing, runny nose, etc., which were used as the criteria for selecting animals.

[0053] Nasal or throat swabs were collected aseptically from dogs with clinical symptoms, and the collected swabs were inoculated on Bjornsberg blood agar plates and MacConkey agar plates, respectively, and cultured at 37°C for 40 to 48 hours. The colony morphology was observed, and typical colonies were picked for subculture, purification, and Gram staining microscopy. The results showed that 13 suspected Bordetella bronchiseptica colonies were isolated from 123 canine nasopharyngeal swabs collected, that is, small pearly, milky white colonies grown on Bjornsberg blood agar plates for 40 to 48 hours, and some strains had β-hemolysis; when cultured on MacConkey plates at 37°C for 40 to 48 hours, the colony morphology was colorless or tea-yellow with neat edges, smooth and moist, and smaller colonies. Gram staining microscopy of the 13 suspected isolated bacteria were all negative for cocci.

[0054] The above-mentioned isolated strains were identified by the PCR identification method of Bordetella bronchiseptica reported by Hozbor, D. (Hozbor, D., F. Fouque, and N. Guiso, Detection of Bordetella bronchiseptica by the polymerase chain reaction. Research in Microbiology, 1999. 150 (5): 333-341.), and the results showed that all 13 suspected strains amplified a specific band of 237 bp in size, and PCR identification showed that they were all Bordetella bronchiseptica. According to the report of Bai Xue et al. (Bai Xue, et al., Isolation and Identification of Bordetella bronchiseptica in Dogs. Animal Husbandry and Veterinary Medicine, 2010. 42 (2): 55-58.), the isolated strains were biochemically identified, and the results showed that the biochemical test results of the 13 isolated strains were consistent with the biochemical characteristics of Bordetella bronchiseptica.

[0055] The above results confirmed that the isolated pathogenic strain was a wild strain of Bordetella bronchiseptica canine.

[0056] 1.2 Screening of highly virulent strains of Bordetella bronchiseptica in dogs

[0057] 1.2.1 Virulence to mice The isolates were cultured on TSA containing 10% newborn calf serum at 37°C for 24 h, and then the culture was suspended in PBS (0.01 mol / L, pH 7.2) to prepare a plate with a viable bacterial content of 5 × 10 8 CFU / mL of bacterial suspension. The bacterial suspension was injected intraperitoneally into BALB / c mice, with the injection volume of 0.2 mL (1×10 8 CFU / each), and the challenge was observed for 10 days. The results showed that the challenge dose of BJ01, HN01, HN03 and HN04 was 1×10 8 CFU / mouse, it can cause the death of all mice, while the other strains cannot cause all the death.

[0058] 1.2.2 LD in mice 50 The LD of four strains of Bordetella bronchiseptica, BJ01, HN01, HN03 and HN04, against mice were determined. 50 Specifically, when the bacterial count is 10 9 CFU / mL, and make 10-fold serial dilutions to 10 7 CFU / mL, the bacterial solution of different dilutions was intraperitoneally injected into 8 16-18g BALB / c mice, each group was kept in isolation, and the mice in each group were observed for 10 days after inoculation. The mortality of mice in each group was recorded, and the LD to mice was calculated by Karber method. 50The mice were challenged with BJ01, HN01, HN03 and HN04 strains respectively, and the LD 50 The results showed that the LD of BJ01, HN01, HN03 and HN04 strains in mice 50 They are 1.51×10 7 , 3.87×10 7 , 1.43×10 7 and 2.37×10 7 CFU / mouse, the results showed that HN03 strain was the most virulent to mice.

[0059] 1.3 Toxicity test on dogs

[0060] The HN03 strain, which was the most virulent to mice, was tested for virulence to dogs. The HN03 strain was cultured on TSA containing 10% newborn calf serum at 37°C for 24 h, and then the culture was suspended in PBS (0.01 mol / L, pH 7.2) to prepare a viable bacterial cell culture solution with a viable bacterial content of 1×10 9 CFU / mL of bacterial suspension. Healthy susceptible dogs with negative Bb antigen and antibody were inoculated with bacterial suspension by nasal drops, with an inoculation volume of 2 mL for each dog. The dogs were observed for 14 consecutive days after the challenge, and the clinical symptoms of the test dogs after inoculation were observed. The results showed that 3 / 3 of the dogs inoculated with HN03 strain had sneezing, 2 / 3 had coughing, and 3 / 3 had eye secretions and runny nose, which could cause typical clinical symptoms in the inoculated dogs. The canine bronchiseptica Bordetella HN03 strain was submitted for preservation as a weakened original strain.

[0061] Example 2 Cultivation of a canine bronchiseptica Bordetella attenuated strain

[0062] 2.1 Cultivation of a low-virulence strain of Bordetella bronchiseptica in dogs

[0063] 2.1.1 Cultivate canine bronchiseptica Bordetella HN03 strain by streaking on Baojiang's blood agar plates and culturing at 42°C for 5 to 7 days per generation. Pick out large gray, flat colonies and continue to subculture according to the above method. Continue to subculture according to the above method until the 60th generation. The colony morphology is large gray, flat colonies, which agglutinate with O factor serum but not with K factor serum, and are phase III bacteria.

[0064] 2.1.2 Safety of strains The bacterial solution prepared by 60 generations of high temperature culture was inoculated into 5 healthy susceptible dogs aged 6 to 8 weeks, 2 ml / dog (containing 2×10 10CFU live bacteria), and another 5 were taken as the control group, each of which was inoculated with 2ml of BHI medium (Brain-Heart Infusion Broth) and observed for 14 days. The inoculated dogs' spirit, feeding, and clinical symptoms such as coughing, sneezing, vomiting, eye secretions, and nasal secretions were observed. The body temperature of the experimental dogs was measured before and after inoculation. The results showed that after the 60th generation of bacteria were inoculated in dogs, 5 / 5 were normal. The results showed that the F60 generation of Bordetella bronchiseptica HN03 strain was safe. We renamed the Bordetella bronchiseptica HN03 strain from the 60th generation as HN60 strain and submitted it for deposit.

[0065] 2.2 Genetic characteristics of bacterial strains

[0066] The whole genome sequencing of the original strain HN03 before attenuation and the attenuated strain HN60 was performed respectively. The sequencing comparison results showed that a nucleotide base G was inserted into the BvgS gene of the attenuated strain HN60 at the 2004 position, resulting in a frameshift mutation of the BvgS gene after the insertion site.

[0067] 2.3 Genetic stability

[0068] The Bordetella bronchiseptica HN60 strain prepared by the present invention is streaked and cultured on a Baojiang blood plate, cultured in a 37°C incubator for 40-48h, and then a single colony is picked on the cultured plate and placed on the Baojiang blood plate, and passage is performed continuously for 60 times to observe the colony morphology. The results show that the colonies of each generation of 60 passages are large gray, flat colonies, and no variation occurs. The O factor and K factor serum agglutination tests are performed on the 1st, 10th, 20th, 30th, 40th, 50th and 60th generation cultures, respectively. The colonies of different generations all agglutinate with the O factor serum, and do not agglutinate with the K factor serum, and are all phase III bacteria. At the same time, the primers BvgS-F / BvgS-R (see Table 1) are used to extend and sequence the 1st, 10th, 20th, 30th, 40th, 50th and 60th generation templates of the HN60 strain, and the sequencing results show that the site of the strains of different generations has not changed, and can be stably inherited.

[0069] Table 1 Sequencing primer sequences Primer name Primer sequences BvgS-F 5'-CATTTCGAACGACGAACTCGCCT-3' BvgS-R 5'-CAATGTCCAGGATGTCGCCTATC-3'

[0070] 2.4 Virulence reversion test

[0071] 2.4.1 Preliminary test of toxicity reversion

[0072] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10 CFU / ml, 12 healthy susceptible dogs were inoculated with a dose of 2ml / dog, containing 2×10 10CFU / ml. The 12 vaccinated dogs were randomly divided into 6 groups, with 2 dogs in each group. Two dogs were dissected on the 3rd, 5th, 7th, 9th, 11th and 13th days after vaccination, and tonsils, submandibular lymph nodes, tracheal lymph nodes, lungs, liver, spleen, heart, kidneys, larynx, trachea and daily nasal swabs were taken. The content of Bordetella bronchiseptica in nasal swabs and organ tissues was detected by live bacteria counting method. At the same time, 2 blank dogs were set up without vaccination. Each tissue was dissected and the content of Bordetella bronchiseptica in each tissue was detected. The temperature was measured 3 days before vaccination and every day after vaccination. The results showed that there was no abnormality in body temperature after vaccination. See Table 2 for details. The clinical symptoms of the vaccinated dogs were observed every day after vaccination, and no abnormalities were found.

[0073] Table 2 Body temperature of test dogs before and after vaccination

[0074] Note: “ / ” means the experimental dogs were autopsied but body temperature was not measured.

[0075] Nasal swabs were collected daily before and after vaccination for live bacteria counts. The results are shown in Table 3. The results showed that the nasal swab content was the highest 2 days after vaccination, and then it showed a downward trend. By 10 days after vaccination, no Bordetella bronchiseptica could be detected in the nasal swabs. The vaccinated dogs were autopsied at different times after vaccination, and no abnormalities were found in any organs. The live bacteria counts of Bordetella bronchiseptica in each organ showed that the Bb content in the submandibular lymph nodes and organ lymph nodes was the highest among the organs, and its content was much higher than the Bordetella bronchiseptica content in the nasal swabs. Among them, the Bordetella bronchiseptica content in the submandibular lymph nodes and tracheal lymph nodes was the highest 7 days after vaccination, as shown in Table 4. Based on the above results, it was determined that the target tissues for virulence reversion were the submandibular lymph nodes and tracheal lymph nodes, and the collection time was 7 days after vaccination.

[0076] Table 3 Results of Bordetella bronchiseptica counts in nasal swabs before and after inoculation

[0077] Note: The unit is CFU / ml. “—” means no detection, and “ / ” means the test dog has been autopsied but not detected.

[0078] Table 4 Results of Bordetella bronchiseptica counts in different organs of dogs in each group

[0079] Note: The unit is CFU / g, “—” means not detected.

[0080] 2.4.2 Virulence reversion test

[0081] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10 CFU / ml, 2 dogs were inoculated intranasally, 2ml / dog, 7 days after inoculation, the 2 inoculated dogs were dissected, submandibular lymph nodes and tracheal lymph nodes were taken, weighed separately, added with PBS (0.01mol / L, pH7.2) at a ratio of 1:10 (w / v), ground with a grinder and filtered with an 80-mesh filter, and the filtrate of the ground submandibular lymph node of each dog was sampled for Bb live bacteria count, and the filtrate of the ground submandibular lymph node and organ lymph node of the 2 dogs was mixed and inoculated intranasally with another 2 blank dogs, 2ml / dog. According to the above method, the dog body was continuously passaged for 5 generations. If Bordetella bronchiseptica could not be re-isolated from the submandibular lymph node and tracheal lymph node during the passage process, the inoculation dose and the number of test animals should be doubled and the test should be repeated. The results showed that the canine bronchiseptica Bordetella HN60 strain was continuously propagated in dogs for two generations, and no abnormalities were found in the autopsy of the inoculated dogs. No bronchiseptica Bordetella was detected in the organs of the second-generation dogs. The results are shown in Table 5. The inoculation dose and the number of inoculated dogs were doubled and the test was repeated. The results showed that no bronchiseptica Bordetella was detected in the organs of the dogs in the second passage. The results are shown in Table 6. The above results show that the canine bronchiseptica Bordetella HN60 strain does not have a reversion to virulence.

[0082] Table 5 Results of Bb viable counts in different organs

[0083] Note: The unit is CFU / g, “—” means not detected.

[0084] Table 6 Results of Bb viable counts in different organs after doubling the inoculation dose and the number of inoculated dogs

[0085] Note: The unit is CFU / g, “—” means not detected.

[0086] 2.5 Horizontal transmission test of Bordetella bronchiseptica in dogs

[0087] The viable bacterial content of Bordetella bronchiseptica HN60 strain culture was adjusted to 1×10 10CFU / ml, 2 healthy susceptible dogs were inoculated with 2ml / dog by nasal drops, and 2 blank dogs were transferred after inoculation and placed in the same cage with the inoculated dogs for co-rearing. Nasal swabs were collected from the dogs before co-rearing, 3 days, 7 days, 10 days, 14 days, and 21 days of co-rearing to detect the content of live Bordetella bronchiseptica in the nasal swabs; blood was collected before inoculation or cohabitation, 7 days, 14 days, and 21 days after inoculation or cohabitation to detect Bb microagglutination antibodies; on the 21st day after inoculation, all dogs were dissected and submandibular lymph nodes and tracheal lymph nodes were taken, and grinded separately for Bordetella bronchiseptica count. The results showed that Bordetella bronchiseptica could be detected in the inoculated dogs on the 3rd and 7th days, but Bordetella bronchiseptica was not detected on the 10th day and later after inoculation. Bordetella bronchiseptica was not detected in the nasal swabs of the cohabiting dogs from the 3rd to the 21st day of co-rearing. The specific results are shown in Table 7. The microagglutination antibodies of Bordetella bronchiseptica in the serum of dogs at different time points after vaccination or cohabitation were measured. The results showed that the Bordetella bronchiseptica antibodies of the vaccinated dogs were all lower than 1:20 before vaccination. 7-21 days after vaccination, a higher level of Bordetella bronchiseptica antibodies was produced, and the antibodies showed an upward trend. The Bordetella bronchiseptica antibodies of the cohabiting dogs were all lower than 1:20 before cohabitation and 7-14 days after cohabitation. The specific results are shown in Table 8. On the 21st day of vaccination or cohabitation, all dogs were autopsied. No abnormalities were found in the autopsies of all dogs. The submandibular lymph nodes and tracheal lymph nodes of each dog were taken for Bordetella bronchiseptica count. The results showed that Bordetella bronchiseptica were detected in both the submandibular lymph nodes and tracheal lymph nodes of the vaccinated dogs, while Bordetella bronchiseptica were not detected in the submandibular lymph nodes and tracheal lymph nodes of the two cohabiting dogs. The specific results are shown in Table 9. Based on the above results, there is no horizontal transmission after dogs are inoculated with Bordetella bronchiseptica HN60 strain.

[0088] Table 7 Results of Bordetella bronchiseptica counts in nasal swabs of dogs at different time points of inoculation or cohabitation (CFU / ml)

[0089] Note: “—” means not detected.

[0090] Table 8 Results of Bordetella bronchiseptica microagglutination antibodies in serum of dogs at different time points of inoculation or cohabitation

[0091] Table 9 Results of Bordetella bronchiseptica detection in different lymph nodes of dogs vaccinated or cohabiting for 21 days

[0092] Note: The unit is CFU / g, “—” means not detected

[0093] Example 3Bb, Preparation of CPIV dual live vaccine

[0094] 3.1 Pathogen proliferation

[0095] The attenuated strain HN60 of Bordetella bronchiseptica was cultured on a solid medium of Bo Jiang's blood plate, and a single colony was selected and cultured in a liquid medium containing BHI for 16 h. The colony was then transferred and expanded at a ratio of 1% until the viable bacterial concentration was not less than 5.0×10 9 CFU / mL.

[0096] The canine parainfluenza virus HeN20 strain was inoculated into Vero cell suspension, and DMEM culture medium containing 2% newborn calf serum was added, and cultured at 37°C and 5% CO2. After 80% of the cells were cytopathic, the virus was harvested, the virus titer was determined, and the cells were stored at low temperature.

[0097] 3.2 Preparation of protective agent

[0098] Add 20 g of sucrose and 2 g of gelatin to every 100 ml of deionized water, and after fully melting, sterilize with high-pressure steam (121°C, 30 min).

[0099] 3.3 Vaccine preparation

[0100] The prepared and stored Bordetella bronchiseptica HN60 bacterial liquid and canine parainfluenza virus HeN20 strain virus liquid were mixed in proportion, mixed with the protective agent in a ratio of 1:1 (volume ratio), and freeze-dried. The specific ratio of the vaccine content is shown in Table 10.

[0101] Table 10 Content ratio of Bb and CPIV dual live vaccine

[0102] Example 4Bb, Immunogenicity of CPIV dual live vaccine

[0103] 40 healthy susceptible dogs of about 60 days of age were randomly divided into 8 groups, 5 in each group, and immunized with the Bb and CPIV dual live vaccine prepared in Example 3. Group 1 and Group 2 were immunized with vaccine 1, Group 3 and Group 4 were immunized with vaccine 2, Group 5 and Group 6 were immunized with vaccine 3, and Group 7 and Group 8 were the control group. Group 1, Group 3, and Group 5 were immunized with a single dose; Group 2, Group 4, and Group 6 were immunized with a single dose repeatedly; at an interval of 21 days, the dogs were injected subcutaneously. 28 days after immunization, the immunized dogs in Group 1, Group 3, and Group 5 and the control dogs in Group 7 were challenged with HN03 strain bacterial solution by nasal drops, 2 ml / dog (live bacteria content was 1.0×10 10CFU / mL), observe for 14 days after the virus attack, record the clinical symptoms of each dog respectively, score the clinical symptoms according to the scoring criteria in Table 11, and perform statistical analysis on the average clinical symptom score of each group of dogs 14 days after the virus attack. The average clinical symptom score of the immunized dogs should be significantly lower than that of the control dogs.

[0104] Table 11 Symptoms and scoring table of dogs after Bordetella infection

[0105] After the challenge, the dogs were observed for 14 days, and the number and frequency of coughing and sneezing were recorded and scored according to the scoring table. The results showed that 5 / 5 dogs in the control group had sneezing and coughing symptoms, and the clinical symptom scores of the Bb and CPIV two-in-one live vaccine groups were significantly lower than those of the challenge control group, which met the efficacy test standards. The specific results are shown in Table 12.

[0106] Table 12 Results of immunogenicity test of Bb and CPIV combined live vaccine Bordetella

[0107] Note: “—” indicates no clinical symptoms, and different superscript letters indicate significant differences (P<0.05)

[0108] 21 days after the second vaccination, the immunized dogs in groups 2, 4, and 6 and the control dogs in group 8 were challenged with canine parainfluenza virus HeN0718 strain in the form of 2 ml (10 4.0 TCID 50 The dogs were challenged with 100 mg / ml of vaccinia virus per day. After the challenge, the dogs' body temperature, spirit, appetite, eye and nasal secretions, sneezing and coughing were observed and recorded every day. The scores were accumulated according to the scoring standard of "0.5 points for eye secretions and nasal secretions, 1 point for sneezing and coughing", and the total scores of each dog within 10 days after the challenge were calculated. SPSS software was used to perform statistical analysis using the T test method to determine whether the clinical symptom scores of the dogs in the immunization group were significantly lower than those of the control group (P<0.05). The specific results are shown in Table 13.

[0109] Table 13 Results of immunogenicity test of Bb and CPIV dual live vaccines for canine parainfluenza Group quantity Clinical symptoms Clinical symptom score 2 5 3 dogs had sneezing, 2 dogs had watery nasal discharge 3.200±0.942* 4 5 3 dogs had sneezing, 2 dogs had watery nasal discharge 2.600±1.025* 6 5 3 dogs had sneezing, 2 dogs had watery nasal discharge 2.000±1.046* 8 5 All dogs had eye and nasal discharge, and 4 dogs sneezed 11.20±1.348

[0110] The results showed that the clinical symptoms caused by the strong CPIV virus were significantly alleviated after dogs were immunized with the Bb and CPIV dual live vaccine prepared in Example 3, and all dogs in the control group became ill after the virus challenge.

[0111] It is proved that the Bb and CPIV dual live vaccine provided by the present invention has good protective power, shows good immune protection, effectively prevents infection by Bordetella bronchiseptica, and alleviates the clinical symptoms of canine parainfluenza.

[0112] Example 5 Preparation of Bb, CPIV, CAV-2 triple live vaccine

[0113] 5.1 Pathogen proliferation

[0114] The canine adenovirus type 2 HL50 strain virus seed was inoculated into MDCK cell suspension, and a cell growth medium containing 95% to 99% by volume of DMEM culture medium and 1% to 5% by volume of newborn calf serum (pH adjusted to 6.8 to 7.2) was added and cultured at 33° C. to 37° C. After 80% of the cells became cytopathic, the virus was harvested, the virus titer was determined, and the virus was stored at low temperature.

[0115] 5.2 Preparation of protective agent

[0116] Add 20 g of sucrose and 2 g of gelatin to every 100 ml of deionized water, and after fully melting, sterilize with high-pressure steam (121°C, 30 min).

[0117] 5.3 Vaccine preparation

[0118] The canine adenovirus type 2 HL50 strain virus liquid prepared and stored above, the Bordetella bronchiseptica HN60 strain bacterial liquid prepared and stored in Example 3, and the canine parainfluenza virus HeN20 strain virus liquid were mixed in proportion, mixed with the protective agent in a ratio of 1:1 (volume ratio), and freeze-dried. The specific ratio of the vaccine content is shown in Table 14.

[0119] Table 14 Content ratio of Bb, CPIV and CAV-2 triple live vaccine

[0120] Example 6 Immunogenicity of Bb, CPIV, and CAV-2 triple live vaccine

[0121] 60 healthy susceptible dogs of about 60 days of age were randomly divided into 12 groups, 5 in each group, and immunized with the Bb, CPIV, and CAV-2 triple live vaccine prepared in Example 5. Groups 9, 10, and 11 were immunized with 4 doses of vaccine, Groups 12, 13, and 14 were immunized with 5 doses of vaccine, Groups 15, 16, and 17 were immunized with 6 doses of vaccine, and Groups 18, 19, and 20 were the control groups. Groups 9, 12, and 15 were immunized with a single dose; Groups 10, 11, 13, 14, 16, and 17 were immunized with a single dose repeatedly; and the doses were subcutaneously injected at an interval of 21 days. 28 days after immunization, the immunized dogs in Groups 9, 12, and 15 and the control dogs in Group 18 were challenged with HN03 strain bacterial solution with 2 ml / dog (live bacteria content of 1.0×10 10 CFU / mL), observe for 14 days after the virus attack, record the clinical symptoms of each dog respectively, score the clinical symptoms according to the scoring criteria in Table 11, and perform statistical analysis on the average clinical symptom score of each group of dogs 14 days after the virus attack. The average clinical symptom score of the immunized dogs should be significantly lower than that of the control dogs.

[0122] After the challenge, the dogs were observed for 14 days, and the number and frequency of coughing and sneezing were recorded and scored according to the scoring table. The results showed that 5 / 5 dogs in the control group had sneezing and coughing symptoms, and the clinical symptom scores of the Bb, CPIV, and CAV-2 triple live vaccine immunization groups were significantly lower than those of the challenge control group, meeting the efficacy test standards. The specific results are shown in Table 15.

[0123] Table 15 Results of immunogenicity test of some Bb, CPIV and CAV-2 triple live vaccines against Bordetella

[0124] Note: “—” indicates no clinical symptoms, and different superscript letters indicate significant differences (P<0.05)

[0125] 21 days after the second vaccination, the immunized dogs in groups 10, 13, and 16 and the control dogs in group 19 were challenged with canine parainfluenza virus HeN0718 strain in the form of 2 ml (10 4.0 TCID 50 The dogs were challenged with 100 mg / ml of vaccinia virus per dog. After the challenge, the dogs' body temperature, spirit, appetite, eye and nasal secretions, sneezing and coughing were observed and recorded every day. The dogs were scored cumulatively according to the scoring standard of "0.5 points for eye secretions and nasal secretions, 1 point for sneezing and coughing", and the total score of each dog within 10 days after the challenge was calculated. SPSS software was used to perform statistical analysis using the T test method to determine whether the clinical symptom score of the dogs in the immunization group was significantly lower than that of the control group (P<0.05). The specific results are shown in Table 16.

[0126] Table 13 Results of immunogenicity test of canine parainfluenza with Bb, CPIV and CAV-2 triple live vaccines

[0127] The results showed that the Bb, CPIV, CAV-2 triple live vaccine prepared in Example 5 could significantly alleviate the clinical symptoms caused by the strong CPIV virus after immunization of dogs, and all dogs in the control group became ill after the virus challenge.

[0128] Fourteen days after the second vaccination, the immunized dogs in groups 11, 14, and 17 and the control dogs in group 20 were challenged with canine adenovirus type 1 YT-2 strain (Canine adenovirus 1, Strain YT-2, China Center for Type Culture Collection, accession number CCTCCNO: V202216, deposit address: Wuhan University, Wuhan, China, deposit date: March 1, 2022) by intravenous injection of 1 ml (10 4.0 TCID 50 The dogs were challenged with 100 mg / ml of dapoxetine per day, and their body temperature, spirit, appetite, abdomen, eyes and other clinical manifestations were observed and recorded every day after the challenge. The specific results are shown in Table 14.

[0129] Table 14 Results of immunogenicity test of canine adenovirus of Bb, CPIV and CAV-2 triple live vaccine

[0130] The results showed that the Bb, CPIV, CAV-2 triple live vaccine prepared in Example 5 could block canine adenovirus infection (clinical symptoms) and provide 100% (5 / 5) protection (prevention) for dogs, while all dogs in the control group became ill after infection.

[0131] It is proved that the Bb, CPIV, CAV-2 triple live vaccine provided by the present invention has good protective power, shows good immune protection, effectively prevents infection by Bordetella bronchiseptica and canine adenovirus, and alleviates the clinical symptoms of canine parainfluenza.

[0132] Note that the above are only preferred embodiments of the present invention and the technical principles used. Those skilled in the art will understand that the present invention 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 scope of protection of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present invention, all of which belong to the protection scope of the present invention.

Claims

1. Use of the attenuated strain HN60 of Bordetella bronchiseptica or its culture in a combination vaccine. The deposit number of the attenuated strain HN60 of Bordetella bronchiseptica is CCTCCNO: M20221719, and it is deposited in the China Center for Type Culture Collection, with the deposit address at Wuhan University, Wuhan, China, and the deposit date is November 3, 2022.

2. A combined vaccine, characterized in that: The combined vaccine comprises an antigen and a pharmaceutically acceptable excipient; The antigen includes an immune effective amount of the attenuated strain HN60 of Bordetella bronchiseptica or its culture. The attenuated strain HN60 of Bordetella bronchiseptica is deposited with the China Center for Type Culture Collection, the address of which is Wuhan University, Wuhan, China, and the date of deposit is November 3, 2022.

3. The combined vaccine according to claim 2, characterized in that The antigen includes an immunologically effective amount of live whole virus antigens of the attenuated strain HN60 of Bordetella bronchiseptica or its culture; Preferably, the content of the Bordetella bronchiseptica attenuated strain HN60 is ≥1×10 7 CFU / head, preferably 1×10 7 ~1×10 9 CFU / head portion; Preferably, the Bordetella bronchiseptica attenuated strain HN60 culture is selected from 1 to 60 generations.

4. The combined vaccine according to claim 2, characterized in that The antigens also include an immunologically effective amount of a canine parainfluenza virus antigen and / or an immunologically effective amount of a canine adenovirus antigen.

5. The combined vaccine according to claim 4, characterized in that The canine parainfluenza virus antigen is a live whole virus antigen of the canine parainfluenza virus HeN20 strain or its culture, preferably with a content of ≥10 3.0 TCID 50 / head, more preferably 10 3.0 ~10 6.0 TCID 50 / first portion; Preferably, the canine parainfluenza virus HeN20 strain culture is selected from 1 to 20 generations.

6. The combined vaccine according to claim 4, characterized in that The canine adenovirus antigen is a live whole virus antigen of canine adenovirus type 2 HL50 strain or its culture, preferably with a content of ≥10 4.0 TCID 50 / head, more preferably 10 4.0 ~10 6.0 TCID 50 / first portion; Preferably, the canine adenovirus type 2 HL50 strain is selected from passages 1 to 70.

7. The combined vaccine according to any one of claims 2 to 6, characterized in that The pharmaceutically acceptable excipient is a lyoprotectant, which includes SPGA, sugars, gelatin, protein, a substance containing protein or / and a buffer; Preferably, the sugar is selected from sorbitol, mannitol, starch, sucrose, glucose or dextran; the protein is selected from albumin or casein; the substance containing protein is bovine serum or skim milk; the buffer is phosphate buffer; Preferably, the lyoprotectant is 20w / v% sucrose and 2w / v% gelatin; Preferably, the volume ratio of the antigen to the pharmaceutically acceptable excipient is 1:

1.

8. The combined vaccine according to claim 7, characterized in that The combined vaccine is freeze-dried powder and is administered by subcutaneous injection.

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