A rabies inactivated vaccine diluent and its preparation method and application

Abstract

The present invention discloses a rabies inactivated vaccine diluent and its preparation method and application, belonging to the technical field of vaccine diluents. The invention provides an inactivated vaccine diluent to solve the technical problems of low antibody titer, long production time and short antibody maintenance time after animal inactivated vaccine immunization. The invention provides a rabies inactivated vaccine diluent, which is composed of the following components: phosphate buffer, MONTANIDE TM GEL 02 PR adjuvant, canine interferon, and ricin B chain protein. This diluent can significantly increase the rabies neutralizing antibody titer after rabies inactivated vaccine administration.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccine diluents, and particularly relates to a rabies inactivated vaccine diluent and a preparation method and application thereof. Background Art

[0002] Due to the complex properties and functions of adjuvants, preservatives and other additives involved in inactivated rabies vaccines, the vast majority of veterinary rabies vaccines on the market are aqueous solutions. Moreover, due to the complexity of the vaccine ingredients, the transportation and storage costs have increased significantly.

[0003] Vaccine immunization is the best means of preventing and controlling infectious diseases. Vaccine strains currently used on the market are mainly classified into two categories: one is attenuated live vaccines, and the other is inactivated vaccines or subunit vaccines. Attenuated live vaccines enter the animal body after immunization and can infect host cells and continue to replicate. In clinical practice, piglets can obtain passive immunity through colostrum after birth. In the presence of passive immunity, the maternal antibodies obtained by the piglets will interfere with the replication of the attenuated live vaccine in the body, resulting in immunization failure. In a group, it is often difficult to find a balance due to individual differences, so multiple immunizations are required to achieve a high immunization coverage rate, thereby reducing individual cases of immunization failure.

[0004] In addition, live vaccines usually use phosphate buffered saline or physiological saline as diluents, which will result in a long immune blank period, and this immune blank period may be the peak period of viral infection and disease. Summary of the Invention

[0005] The purpose of the present invention is to provide a live vaccine diluent to solve the technical problems of low antibody titer, long production time and short antibody maintenance time after animal live vaccine immunization.

[0006] The present invention provides a diluent for inactivated rabies vaccine, which is composed of the following components: phosphate buffer, MONTANIDE TM GEL 02 PR adjuvant, canine interferon, and ricin B chain protein.

[0007] It is further defined that the canine interferon is canine alpha interferon.

[0008] Further defined, the diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 14% to 15% by mass of MONTANIDE TM GEL 02 PR adjuvant, canine interferon 5×10 6 IU and ricin B chain protein 3×10 4 IU.

[0009] It is further defined that the diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 14% by mass of MONTANIDE TM GEL 02 PR adjuvant, canine interferon 5×10 6 IU and ricin B chain protein 3×10 4 IU.

[0010] It is further defined that the diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 15% by mass of MONTANIDE TM GEL 02 PR adjuvant, canine interferon 5×10 6 IU and ricin B chain protein 3×10 4 IU.

[0011] The present invention provides a method for preparing the above-mentioned diluent, characterized in that the method comprises the following specific steps:

[0012] 1) Montanide TM GEL 02 PR adjuvant and phosphate buffer were mixed in proportion for 30 minutes, sterilized and filtered to obtain a mixed solution;

[0013] 2) Add canine interferon-alpha and ricin B chain protein to the mixed solution obtained in step 1) and mix well to obtain a rabies inactivated vaccine dilution solution.

[0014] Further defined, the method for preparing phosphate buffer in step 1) is:

[0015] (1) Solution A: 0.15 mol / L sodium hydrogen phosphate solution: 21.30 g of sodium hydrogen phosphate and purified water to 1000 ml;

[0016] (2) Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml;

[0017] (3) Take 87.0 ml of solution A and 13.0 ml of solution B, pH 7.6, and sterilize at 121°C for 30 minutes.

[0018] The present invention provides a method for preparing an inactivated rabies vaccine, which utilizes the above-mentioned diluent in the dilution process.

[0019] The present invention provides the use of the above-mentioned diluent in diluting vaccines.

[0020] The present invention provides application of the above-mentioned diluent in improving the immune function of vaccines.

[0021] Beneficial effects: Most of the existing veterinary rabies inactivated vaccines are liquid dosage forms. After diluting the inactivated rabies vaccine with the vaccine diluent, not only can the rabies neutralizing antibody titer be significantly enhanced, but also the dosage form of the existing vaccine can be changed, and the storage and transportation costs of the vaccine can be reduced. The present invention provides an inactivated rabies vaccine diluent, which is composed of 0.15 mol / L phosphate buffer, MONTANIDE TM GEL 02 PR adjuvant, canine interferon, and ricin B chain protein are composed of ricin B chain protein, which can significantly increase the rabies neutralizing antibody titer after inactivated rabies vaccination. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a picture of the vaccine dilution of the present invention. DETAILED DESCRIPTION

[0023] Inactivated rabies vaccine (dG strain), batch number 1902011, was produced by Guangzhou South China Agricultural University Biopharmaceutical Co., Ltd.

[0024] Inactivated rabies vaccine (CTN-1 strain), batch number 201910008, is produced by Tangshan Yian Bioengineering Co., Ltd.

[0025] Inactivated rabies vaccine (G52 strain), batch number L467700, is produced by Merial’s French production plant.

[0026] The ricin B chain protein was obtained by gene cloning. See Xing Lijie, Luo Xiaoling, Ma Qingyuan, et al. Cloning, expression and identification of ricin B chain[J]. Journal of Food and Biotechnology, 2009, 28(1):4.

[0027] Canine interferon α recombinant canine α-interferon (lyophilized), batch number 20200301, produced by Tianjin Ruipu Biotechnology Co., Ltd.

[0028] The mixing ratio of the vaccine of the present invention and the diluent is 1:1, that is, 1 ml of the vaccine is mixed with 1 ml of the diluent.

[0029] MONTANIDE TM GEL 02 PR adjuvant is SEPPIC's MONTANIDE TM GEL 02.

[0030] Example 1. Rabies inactivated vaccine diluent and preparation method thereof

[0031] (1) Components and contents of the composition

[0032] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TMGEL 02 PR adjuvant 14%, canine interferon 5×10 6 IU, ricin B chain protein 3×10 4 IU.

[0033] (2) Preparation and treatment methods of the composition:

[0034] 1) Preparation of 0.15 mol / L phosphate buffer

[0035] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0036] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0037] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0038] 2) Montanide TM GEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0039] 3) Add canine interferon and ricin B chain protein to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0040] Quantitatively pack 1.0ml (1 head) per bottle, cap, and sample Figure 1 shown.

[0041] Example 2. Rabies inactivated vaccine diluent and preparation method thereof

[0042] (1) Components and contents of the composition:

[0043] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TM GEL 02 PR adjuvant 15%, canine interferon 5×10 6 IU, ricin B chain protein 3×10 4 IU.

[0044] (2) Preparation and treatment methods of the composition:

[0045] 1) Preparation of 0.15 mol / L phosphate buffer

[0046] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0047] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0048] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0049] 2) Montanide TM GEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0050] 3) Add canine interferon and ricin B chain protein to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0051] Dispense the solution in 1.0 ml (1 portion) per bottle and cap.

[0052] The following experiments verify the experimental results:

[0053] Example 3.

[0054] 1. Using the inactivated rabies vaccine diluent (the vaccine diluent of Example 1) and the inactivated rabies vaccine (dG strain)

[0055] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0056] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (dG strain).

[0057] Subcutaneous injection, injection volume is 2.0ml.

[0058] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0059] Example 4.

[0060] It involves inactivated rabies vaccine (dG strain).

[0061] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0062] Subcutaneous injection, injection volume is 1.0ml.

[0063] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0064] Example 5.

[0065] The inactivated rabies vaccine diluent (the vaccine diluent of Example 1) and the inactivated rabies vaccine (CTN-1 strain) were used.

[0066] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0067] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0068] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0069] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0070] Example 6.

[0071] Inactivated rabies vaccine (CTN-1 strain).

[0072] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0073] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0074] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0075] Example 7.

[0076] The inactivated rabies vaccine diluent (the vaccine diluent of Example 1) and the inactivated rabies vaccine (G52 strain) were used.

[0077] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0078] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0079] Subcutaneous injection, injection volume is 2.0ml.

[0080] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0081] Example 8.

[0082] It involves inactivated rabies vaccine (G52 strain).

[0083] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0084] Subcutaneous injection, injection volume is 1.0ml.

[0085] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0086] Example 9.

[0087] The inactivated rabies vaccine diluent (the vaccine diluent of Example 2) and the inactivated rabies vaccine (dG strain) were used.

[0088] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0089] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (dG strain).

[0090] Subcutaneous injection, injection volume is 2.0ml.

[0091] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0092] Example 10.

[0093] It involves inactivated rabies vaccine (dG strain).

[0094] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0095] Subcutaneous injection, injection volume is 1.0ml.

[0096] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0097] Example 11.

[0098] This example relates to an inactivated rabies vaccine diluent (the vaccine diluent of Example 2) and an inactivated rabies vaccine (CTN-1 strain).

[0099] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0100] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0101] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0102] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0103] Example 12.

[0104] This example relates to an inactivated rabies vaccine (CTN-1 strain).

[0105] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0106] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0107] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0108] Example 13.

[0109] The inactivated rabies vaccine diluent (the vaccine diluent of Example 2) and the inactivated rabies vaccine (G52 strain) were used.

[0110] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0111] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0112] Subcutaneous injection, injection volume is 2.0ml.

[0113] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0114] Example 14.

[0115] This example relates to an inactivated rabies vaccine (G52 strain).

[0116] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0117] Subcutaneous injection, injection volume is 1.0ml.

[0118] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0119] The results are shown in Table 1 and Table 2:

[0120] As shown in Table 1: The vaccine diluent mixed with the vaccine had no effect on the safety of the test dogs after vaccination, and passed the safety test.

[0121] As shown in Table 2: After the vaccine diluent is mixed with the vaccine, the positive rate can reach 100% one month after immunization, which enhances the immune effect of the vaccine; the positive rate can still be maintained at 100% 12 months after immunization, which extends the duration of vaccine immunity.

[0122] Table 3 shows that Examples 1-2 significantly enhance the immune response of inactivated rabies vaccines. All vaccines reached peak rabies neutralizing antibody levels 1-2 months after immunization. After using Examples 1-2, the peak increased nearly threefold. The average values ​​of rabies neutralizing antibody levels from 1 to 12 months after immunization were higher than those obtained with either vaccine alone.

[0123] Therefore, the vaccine diluent has good protection for the conversion rate to positivity in the initial stage of immunity, the peak value of immunity, and the positive rate at the end of immunity.

[0124] Table 1: Safety test results of inactivated rabies vaccine diluent

[0125]

[0126]

[0127] Table 2: Positive rate of inactivated rabies vaccine

[0128] batch batch number Before first exemption Before the second exemption 1 month 2 months 3 months 6 months 9 months 12 months 1 Example 3 0 - 100％ 100％ 100％ 100％ 100％ 100％ 2 Example 4 0 - 80％ 100％ 100％ 80％ 80％ 80％ 3 Example 5 0 80％ 100％ 100％ 100％ 100％ 100％ 100％ 4 Example 6 0 60％ 100％ 100％ 100％ 100％ 80％ 60％ 5 Example 7 0 - 100％ 100％ 100％ 100％ 100％ 100％ 6 Example 8 0 - 80％ 100％ 100％ 100％ 80％ 80％ 7 Example 9 0 - 100％ 100％ 100％ 100％ 100％ 100％ 8 Example 10 0 - 100％ 100％ 100％ 100％ 100％ 80％ 9 Example 11 0 100％ 100％ 100％ 100％ 100％ 100％ 100％ 10 Example 12 0 80％ 100％ 100％ 100％ 80％ 80％ 80％ 11 Example 13 0 - 100％ 100％ 100％ 100％ 100％ 100％ 12 Example 14 0 - 80％ 100％ 100％ 100％ 80％ 60％

[0129] Table 3: Results of the Immunity Duration Test of Inactivated Rabies Vaccine

[0130]

[0131]

[0132] 1) Mix one dose of canine live bivalent vaccine and one dose of vaccine diluent for inoculation

[0133] Canine distemper and parvovirus combined live vaccine, batch number A173B01, is produced by Intervet International Co., Ltd.

[0134] 2) Inactivated rabies vaccine (CTN-1 strain), batch number 201910008, produced by Tangshan Yian Bioengineering Co., Ltd. and inactivated rabies vaccine (G52 strain), batch number L467700, produced by Merial S.A. in France, were mixed and administered.

[0135] 3) Mix 1 dose of inactivated rabies vaccine and 1 dose of vaccine diluent (ricin B chain protein) for inoculation.

[0136] The inactivated rabies vaccine (CTN-1 strain), batch number 201910008, was produced by Tangshan Yian Bioengineering Co., Ltd. The inactivated rabies vaccine (G52 strain), batch number L467700, was produced by Merial’s French production plant.

[0137] Comparative Example 1.

[0138] This comparative example relates to an inactivated rabies vaccine diluent.

[0139] (1) Components and contents of the composition

[0140] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TM GEL 02 PR adjuvant 14%, canine interferon 5×10 6 IU.

[0141] 1) Preparation of 0.15 mol / L phosphate buffer

[0142] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0143] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0144] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0145] 2) Montanide TM GEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0146] 3) Add canine interferon to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0147] Dispense the solution in 1.0 ml (1 portion) per bottle and cap.

[0148] Comparative Example 2.

[0149] This comparative example relates to an inactivated rabies vaccine diluent.

[0150] (1) Components and contents of the composition

[0151] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TM GEL 02 PR adjuvant 15%, canine interferon 5×10 6 IU.

[0152] 1) Preparation of 0.15 mol / L phosphate buffer

[0153] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0154] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0155] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0156] 2) Montanide TM GEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0157] 3) Add canine interferon to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0158] Dispense the solution in 1.0 ml (1 portion) per bottle and cap.

[0159] Comparative Example 3.

[0160] This comparative example relates to an inactivated rabies vaccine diluent.

[0161] (1) Components and contents of the composition

[0162] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TM GEL 02 PR adjuvant 14%, ricin B chain protein 3×10 4 IU.

[0163] 1) Preparation of 0.15 mol / L phosphate buffer

[0164] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0165] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0166] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0167] 2) Montanide TMGEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0168] 3) Add ricin B chain protein to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0169] Dispense the solution in 1.0 ml (1 portion) per bottle and cap.

[0170] Comparative Example 4.

[0171] This comparative example relates to an inactivated rabies vaccine diluent.

[0172] (1) Components and contents of the composition

[0173] 0.15 mol / L phosphate buffer 1 ml, MONTANIDE TM GEL 02 PR adjuvant 15%, ricin B chain protein 3×10 4 IU.

[0174] 1) Preparation of 0.15 mol / L phosphate buffer

[0175] Liquid A 0.15 mol / L disodium hydrogen phosphate solution: add 21.30 g disodium hydrogen phosphate and purified water to 1000 ml.

[0176] Solution B: 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml.

[0177] Take 87.0 ml of liquid A and 13.0 ml of liquid B, mix them, the pH value is 7.6, sterilize at 121℃ for 30 minutes, and set aside.

[0178] 2) Montanide TM GEL 02 PR adjuvant and 0.15 mol / L phosphate buffer were mixed in proportion for 30 minutes and sterilized by filtration.

[0179] 3) Add ricin B chain protein to the mixture in proportion and mix well to obtain the rabies inactivated vaccine dilution solution.

[0180] Dispense the solution in 1.0 ml (1 portion) per bottle and cap.

[0181] Comparative Example 5.

[0182] This comparative example relates to a rabies inactivated vaccine diluent (Example 1) and a canine distemper and parvovirus live dual vaccine.

[0183] Canine distemper and parvovirus combined live vaccine, batch number A173B01, is produced by Intervet International Co., Ltd.

[0184] Five healthy dogs with negative canine distemper neutralizing antibody were enrolled.

[0185] Mix the inactivated rabies vaccine diluent with 1 dose each of canine distemper and parvovirus live dual vaccine.

[0186] Subcutaneous injection, injection volume is 2.0ml.

[0187] Blood was collected before immunization and 1, 2, 3, and 6 months after immunization, and serum was separated and subjected to neutralization test to detect canine distemper neutralizing antibodies.

[0188] Comparative Example 6.

[0189] This comparative example relates to a rabies inactivated vaccine diluent (Example 2) and a canine distemper and parvovirus live dual vaccine.

[0190] Canine distemper and parvovirus combined live vaccine, batch number A173B01, is produced by Intervet International Co., Ltd.

[0191] Five healthy dogs with negative canine distemper neutralizing antibody were enrolled.

[0192] Mix the inactivated rabies vaccine diluent with 1 dose each of canine distemper and parvovirus live dual vaccine.

[0193] Subcutaneous injection, injection volume is 2.0ml.

[0194] Blood was collected before immunization and 1, 2, 3, and 6 months after immunization, and serum was separated and subjected to neutralization test to detect canine distemper neutralizing antibodies.

[0195] Comparative Example 7.

[0196] This comparative example relates to a canine distemper and parvovirus two-in-one live vaccine.

[0197] Canine distemper and parvovirus combined live vaccine, batch number A173B01, is produced by Intervet International Co., Ltd.

[0198] Five healthy dogs with negative canine distemper neutralizing antibody were enrolled.

[0199] Subcutaneous injection, injection volume is 2.0ml.

[0200] Blood was collected before immunization and 1, 2, 3, and 6 months after immunization, and serum was separated and subjected to neutralization test to detect canine distemper neutralizing antibodies.

[0201] Comparative Example 8.

[0202] This example relates to an inactivated rabies vaccine diluent (Comparative Example 1) and an inactivated rabies vaccine (CTN-1 strain).

[0203] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0204] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0205] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0206] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0207] Comparative Example 9.

[0208] This example relates to an inactivated rabies vaccine diluent (Comparative Example 2) and an inactivated rabies vaccine (CTN-1 strain).

[0209] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0210] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0211] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0212] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0213] Comparative Example 10.

[0214] This example relates to an inactivated rabies vaccine diluent (Comparative Example 3) and an inactivated rabies vaccine (CTN-1 strain).

[0215] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0216] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0217] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0218] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0219] Comparative Example 11.

[0220] This example relates to an inactivated rabies vaccine diluent (Comparative Example 4) and an inactivated rabies vaccine (CTN-1 strain).

[0221] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0222] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (CTN-1 strain).

[0223] Intramuscular injection, the injection volume is 2.0 ml, and the second vaccination is given 14 days after the first vaccination, the injection volume is 2.0 ml.

[0224] Blood was collected before the first vaccination, before the second vaccination, and 1, 2, 3, 6, 9, and 12 months after the second vaccination. The serum was separated and subjected to a fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0225] Comparative Example 12.

[0226] This example relates to an inactivated rabies vaccine diluent (Comparative Example 1) and an inactivated rabies vaccine (G52 strain).

[0227] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0228] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0229] Subcutaneous injection, injection volume is 2.0ml.

[0230] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0231] Comparative Example 13.

[0232] This example relates to an inactivated rabies vaccine diluent (Comparative Example 2) and an inactivated rabies vaccine (G52 strain).

[0233] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0234] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0235] Subcutaneous injection, injection volume is 2.0ml.

[0236] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0237] Comparative Example 14.

[0238] This embodiment relates to an inactivated rabies vaccine diluent (Comparative Example 3) and an inactivated rabies vaccine (G52 strain).

[0239] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0240] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0241] Subcutaneous injection, injection volume is 2.0ml.

[0242] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0243] Comparative Example 15.

[0244] This embodiment relates to an inactivated rabies vaccine diluent (Comparative Example 4) and an inactivated rabies vaccine (G52 strain).

[0245] Five healthy dogs with negative rabies neutralizing antibodies were enrolled.

[0246] Mix 1 portion of the inactivated rabies vaccine diluent and 1 portion of the inactivated rabies vaccine (G52 strain).

[0247] Subcutaneous injection, injection volume is 2.0ml.

[0248] Blood was collected before immunization and 1, 2, 3, 6, 9, and 12 months after immunization, and the serum was separated and subjected to fluorescent antibody neutralization test to detect rabies neutralizing antibodies.

[0249] The results are shown in Table 4-6:

[0250] As shown in Table 4, the mixed vaccination of comparative examples 1-4 with the vaccine had no effect on the safety of animals and passed the safety test.

[0251] As shown in Table 5, there was no significant difference in the antibody test results of Comparative Examples 5-7 each month, and the vaccine diluent could not enhance the immune effect of canine distemper in the canine bivalent vaccine.

[0252] As shown in Table 6: Compared with Example 1-2, the immune effect of Comparative Example 1-2 was not significant 1 to 2 months after immunization. After mixed vaccination with the inactivated rabies vaccine (CTN-1 strain), the rabies neutralizing antibody level of dogs vaccinated with Example 1-2 reached a maximum of 210.44 IU / ml one month after the second vaccination, while the maximum level of Comparative Example 1-2 was only 30.77 IU / ml. After mixed vaccination with the inactivated rabies vaccine (G52 strain), the rabies neutralizing antibody level of dogs vaccinated with Example 1-2 reached a maximum of 30.77 IU / ml one month after the second vaccination, while the maximum level of Comparative Example 1-2 was only 17.77 IU / ml.

[0253] Compared with Examples 1-2, although Comparative Examples 3-4 have similar immune effects 1 to 2 months after immunization, their immune peak duration is short and they return to normal levels 6 months after immunization, and cannot guarantee a positive rate close to 100% 12 months after immunization.

[0254] Therefore, the use of interferon or ricin B chain protein vaccine diluent alone cannot guarantee the immune effect of rabies inactivated vaccine and the positive rate 12 months after immunization.

[0255] Table 4: Safety test results of inactivated rabies vaccine diluent

[0256]

[0257]

[0258] Table 5: Results of the immunity duration test of canine distemper and parvovirus combined live vaccine

[0259]

[0260]

[0261] Table 6: Results of the Immunity Duration Test of Inactivated Rabies Vaccine

[0262]

[0263]

Claims

1. A diluent of an inactivated rabies vaccine, characterized in that: The diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 14% to 15% mass fraction of MONTANIDE GEL 02 PR adjuvant, 5×10 6 IU and ricin B chain protein 3×10 4 IU.

2. The diluent according to claim 1, characterized in that The diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 14% mass fraction of MONTANIDE GEL 02 PR adjuvant, 5×10 canine interferon 6 IU and ricin B chain protein 3×10 4 IU.

3. The diluent according to claim 1, characterized in that The diluent is composed of the following ingredients: 0.15 mol / L phosphate buffer, 15% by mass of MONTANIDE GEL 02 PR adjuvant, 5×10 6 IU and ricin B chain protein 3×10 4 IU.

4. The method for preparing the diluent according to any one of claims 1 to 3, characterized in that: The specific steps of the method are as follows: 1) Mix MONTANIDE GEL 02 PR adjuvant and phosphate buffer according to the appropriate proportion for 30 minutes, sterilize and filter to obtain a mixed solution; 2) Add canine interferon-alpha and ricin B chain protein to the mixture obtained in step 1) and mix well to obtain the inactivated rabies vaccine diluent.

5. The preparation method according to claim 4, characterized in that Method for preparing phosphate buffer in step 1): (1) Solution A 0.15 mol / L disodium hydrogen phosphate solution: 21.30 g disodium hydrogen phosphate and purified water to 1000 ml; (2) Solution B 0.15 mol / L potassium dihydrogen phosphate solution: 20.42 g potassium dihydrogen phosphate and purified water to 1000 ml; (3) Take 87.0 ml of liquid A and 13.0 ml of liquid B, with a pH value of 7.6, and sterilize at 121°C for 30 minutes.

6. A method for preparing an inactivated rabies vaccine, characterized in that: The diluent according to any one of claims 1 to 3 is used in the dilution process.

7. Use of the diluent according to any one of claims 1 to 3 in diluting a vaccine.

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