Preparation method and application of atomized protective agent for pseudorabies aerosol live vaccine of swine

Abstract

The present invention provides an aerosol protective agent for a porcine pseudorabies live vaccine, and relates to the field of animal vaccines. The aerosol protective agent is an aqueous solution containing the following ingredients: 3-24 g / L trehalose, 3-24 g / L L-arginine, 3-24 g / L proline and 1-10% glycerol, with a pH value of 7.0-7.8. The present invention also provides a method for preparing a porcine pseudorabies live vaccine aerosol, comprising: preparing a solution of a commercially available porcine pseudorabies live vaccine freeze-dried product with an aerosol protective agent, and preparing the aerosol live vaccine by ultrasonic atomization; the aerodynamic mass median particle size of the aerosol live vaccine is 1-10 μm, and after being retained in the air for 30 minutes, it still has a high titer, a high immune protection rate, simple preparation, and low cost.

Description

Technical Field

[0001] The present invention relates to the field of animal vaccines, and in particular to a preparation method and application of an aerosol protective agent for a porcine pseudorabies aerosol live vaccine. Background Art

[0002] Pseudorabies (PR), also known as Aujeszky's disease, is an acute infectious disease caused by the pseudorabies virus (PRV) in a variety of domestic and wild animals, with fever, severe itching (except in pigs), and encephalomyelitis as the main symptoms. PRV is primarily transmitted through the mouth and nose, proliferating in the host's respiratory epithelial cells. It then infects the tonsils and lungs, spreading throughout the body by free diffusion or bloodstream transmission. Furthermore, PRV can enter the trigeminal and olfactory nerve endings to invade the host's central nervous system, ultimately causing central nervous system disorders characterized by non-purulent meningitis. Currently, pseudorabies has been reported in over 50 countries and regions worldwide. Vaccination is the primary method of preventing the disease. Commercially available pseudorabies vaccines for pigs are mainly divided into live and inactivated vaccines. Live vaccines are administered by intramuscular injection or intranasal drops, while inactivated vaccines are administered by intramuscular injection. The inactivated pseudorabies vaccine for porcine swine is favored by some farms due to its excellent biosafety and effectiveness in the PR decontamination process. However, compared to live vaccines, it has the disadvantage of being less effective. Furthermore, nasal administration of live vaccines can simulate natural infection to a certain extent. Therefore, farms still prefer the classic live attenuated vaccine for pseudorabies prevention and control.

[0003] With the continuous expansion of pig farming, the traditional manual, head-to-head vaccination method has become increasingly problematic. These include high labor costs, stress and disease transmission risks associated with frequent pig handling and the use of needles, which hinder effective disease prevention and control and the healthy development of the pig industry. Aerosol vaccination has emerged as a promising approach for improving vaccination methods, offering advantages such as significantly reduced labor costs, resolving the conflict between vaccination and cross-infection, significantly reducing animal stress, and significantly stimulating mucosal immunity. The formulation of a live pseudorabies vaccine for porcine administration not only improves vaccination convenience but also significantly reduces labor and time. Aerosol vaccine particle size is the primary factor influencing the success of aerosol vaccination. Successful aerosol vaccination requires the delivery of vaccine antigen particles to the appropriate target tissue. The location and efficiency of aerosolized particles in the respiratory tract are influenced by factors such as particle size (aerodynamic parameters), size distribution, particle shape, and density. Due to the unique anatomy of the pig's nasal concha and trachea, only particles with a mass median aerodynamic diameter (MMAD) of 1-10 μm can enter the respiratory tract. Therefore, the particle size of aerosolized live pseudorabies vaccines must be controlled within 1-10 μm. Furthermore, aerosol immunization must maintain the activity of the vaccine antigen to ensure an effective immune response. Aerosol protectants significantly affect the size of aerosol live vaccine particles and the activity of the antigens contained therein. However, no aerosol protectants suitable for aerosolized live pseudorabies vaccines are currently available. Summary of the Invention

[0004] The purpose of the present invention is to provide an aerosol protective agent for pseudorabies aerosol live vaccine of pigs. The aerosol protective agent is mixed with the existing commercially available pseudorabies live vaccine of pigs for aerosol immunization. The aerodynamic mass median particle size of the aerosol live vaccine is 1-10 μm. After being retained in the air for 30 minutes, it still has a high titer and a high immune protection rate.

[0005] The purpose of the present invention is achieved by adopting the following technical solutions:

[0006] The invention discloses an atomized protective agent for aerosol live vaccine of pseudorabies in pigs, which is an aqueous solution containing the following ingredients: 3-24 g / L trehalose, 3-24 g / L L-arginine, 3-24 g / L proline, 1-10% glycerol, and a pH value of 7.0-7.8.

[0007] In the present invention, the atomized protective agent is an aqueous solution containing the following ingredients: 5-20 g / L trehalose, 5-20 g / L L-arginine, 5-20 g / L proline, 2-8% glycerol, and a pH value of 7.0-7.8.

[0008] In the present invention, the solvent of the aerosol protective agent is a phosphate buffer solution with a pH value of 7.0-7.8, a 10-500 mM Tris buffer solution or a physiological saline solution.

[0009] In the present invention, the concentration of the phosphate buffer is 0.005-0.5M.

[0010] The present invention also provides a method for preparing a live aerosol vaccine of pseudorabies by using the atomized protective agent.

[0011] In the present invention, the method comprises dissolving the pseudorabies live vaccine with the above-mentioned aerosol protective agent and then aerosolizing.

[0012] Beneficial Effects: This invention, for the first time, has identified an aerosol protective agent for pseudorabies live vaccine aerosol administration. This protective agent, when mixed with an existing commercially available pseudorabies live vaccine, is then administered via aerosol. The aerodynamic mass median particle size of the aerosol live vaccine is 1-10 μm, and after 30 minutes of airborne retention, it maintains a high titer and high immune protection rate. Immunizing pigs with pseudorabies live vaccine via aerosol administration not only provides convenient vaccination but also significantly saves labor and time, facilitating large-scale promotion on farms and enabling automation. DETAILED DESCRIPTION

[0013] The present invention will be further described below with reference to specific embodiments, but the present invention is not limited thereto.

[0014] Sources of biological materials in the present invention:

[0015] 1. The "porcine pseudorabies live vaccine (SA215 strain) (Pseudorabies)" involved in the present invention was purchased from Xuke Bioengineering Co., Ltd., with approval number: Veterinary Drug 221271061.

[0016] 2. The pseudorabies live vaccine (Bartha-K61 strain) (pseudo-anqing) involved in the present invention was purchased from Huapai Bioengineering Group Co., Ltd., with approval number: Veterinary Drug 221017018.

[0017] 3. The "porcine pseudorabies live vaccine (HB-98 strain) (Keweining)" involved in the present invention was purchased from Wuhan Keqian Biological Co., Ltd., with approval number: Veterinary Drug No. 170041056.

[0018] 4. The pseudorabies virus ZJ01 strain (abbreviated as PRVZJ01) involved in the present invention is disclosed in Gu Z, Dong J, Wang J, Hou C, Sun H, Yang W, Bai J, Jiang P. A novel inactivated gE / gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerging variant PRV challenge. Virus Res. 2015, 195: 57-63.

[0019] Example 1 Preparation and testing of porcine pseudorabies aerosol live vaccine

[0020] 1. Preparation of atomized protective agent

[0021] Atomized protective agent A is an aqueous solution containing the following substances: 10 g / L trehalose, 10 g / L L-arginine, 10 g / L proline, 5% (volume percentage concentration) glycerol, 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4.

[0022] 2. Preparation of aerosol live vaccine

[0023] Take 1 bottle of porcine pseudorabies live vaccine (SA215 strain, 10 6 TCID 50 / bottle), pseudorabies live vaccine (Bartha-K61 strain, 10 6 TCID 50 / bottle) and pseudorabies live vaccine (HB-98 strain, 10 6 TCID 50 / bottle) freeze-dried vaccine, add 2 mL of aerosol protectant A to each bottle for reconstitution, and then add 18 mL of aerosol protectant A and mix evenly to obtain the vaccine solution.

[0024] The vaccine solutions of the three strains were poured into an ultrasonic nebulizer (ZL201521097267.3) for atomization with an oscillation frequency of 1.7 MHz to obtain aerosol live vaccines.

[0025] 3. Testing of the properties of aerosol live vaccines

[0026] (1) Particle size detection

[0027] An ultrasonic nebulizer (ZL201521097267.3) was connected to a nebulization box (648 L, 1.8 m × 0.6 m × 0.6 m, temperature controlled at 20-30°C) to introduce the aerosol vaccine of each strain into the nebulization box. The bottom opening of the nebulization box was connected to the detection port of the aerosol particle size spectrometer. Aerosol live vaccines of pseudorabies virus SA215 strain, Bartha-K61 strain, and HB-98 strain were prepared using the method described in Title 2 of this Example. After 5 minutes of aerosol live vaccine spraying, the aerosol particle size spectrometer was turned on and the aerosol live vaccine particles were detected. The results are as follows: the mean aerodynamic mass median diameter (MMAD) of the aerosol vaccine particles of the live pseudorabies vaccine (SA215 strain) of porcine was 5.91 μm, and the geometric standard deviation (GSD) was 1.57; the MMAD of the aerosol vaccine particles of the live pseudorabies vaccine (Bartha-K61 strain) of porcine was 5.97 μm, and the GSD was 1.57; the MMAD of the aerosol vaccine particles of the live pseudorabies vaccine (HB-98 strain) of porcine was 5.84 μm, and the GSD was 1.50.

[0028] (2) Activity detection

[0029] An ultrasonic nebulizer (ZL201521097267.3) was connected to a nebulization chamber (648 L, 1.8 m × 0.6 m × 0.6 m, temperature controlled at 20-30°C) to introduce aerosol vaccines of various strains. The bottom opening of the nebulization chamber was connected to a BioSampler air microbial sampler (SKC, USA). Aerosol live vaccines of pseudorabies virus strains SA215, Bartha-K61, and HB-98 were prepared using the method described in Title 2 of this Example. The ultrasonic nebulizer was turned off after 5 minutes of spraying. Thirty minutes after the nebulizer was turned off, the air microbial sampler was turned on, and vaccine aerosols were collected from the nebulization chamber for 5 minutes. The nebulization chamber temperature was maintained at 20-30°C and the humidity was ≥40% throughout the entire process.

[0030] By TCID 50 The active titer of the vaccine virus strain in the sampler was determined by the method, and the copy number of the vaccine virus strain in the vaccine solution before aerosolization and in the sampler after aerosolization was detected by quantitative PCR. According to the copy number ratio of the two, the titer of the aerosol vaccine was corrected and calculated to be 0.44 (SA215 strain), 0.4 (Bartha-K61 strain), and 0.5 (HB-98 strain) lower than that before aerosolization. The specific method is as follows:

[0031] 1) Before atomization, TCID 50The antigen titer A1 in each vaccine solution was detected by the method (Nie J, Sun Y, Peng F, Li X, Yang Y, Liu X, Li Y, Liu C, Bai Z. Production Process Development of Pseudorabies Virus Vaccine by Using a Novel Scale-Down Model of a Fixed-Bed Bioreactor. J Pharm Sci. 2020, 109(2): 959-965.), and the pseudorabies virus fluorescence quantitative PCR detection method (Ma W, Lager KM, Richt JA, Stoffregen WC, Zhou F, Yoon KJ. Development of real-time polymerase chain reaction assays for rapid detection and differentiation of wild-type pseudorabies and gene-deleted vaccine viruses. J Vet Diagn Invest. 2008, 20(4): 440-7.) was used to detect the gB gene copy number B1 of the antigen in the vaccine solution.

[0032] 2) After atomization, the aerosolized live vaccine is collected using a BioSampler air microbial sampler to obtain a sample solution;

[0033] 3) Detecting the antigen titer A2 and gB gene copy number B2 in the sample liquid, the specific method is the same as step 1); calculating the ideal titer A3 of the sample liquid by the following formula: A3 = B2 / B1 × A1;

[0034] 4) The drop in aerosol vaccine titer compared to before atomization = A3-A2.

[0035] The titer A2 of the aerosol vaccine of the porcine pseudorabies live vaccine (SA215 strain) was calculated to be 3.5lgTCID 50 The drop in titer before atomization was 0.44 titers (i.e., a drop of 0.44lgTCID 50 ); Pseudorabies live vaccine (Bartha-K61 strain) aerosol vaccine titer A2 is 3.75lgTCID 50 The drop in titer before atomization was 0.40 titer (i.e., a drop of 0.40 lgTCID 50 ); the titer A2 of the porcine pseudorabies live vaccine (HB-98 strain) was 3.5 lgTCID 50The drop in titer before atomization was 0.50 titer (i.e., a drop of 0.50 lgTCID 50 ).

[0036] 4. Immunity and effects of aerosol vaccines

[0037] (1) Grouping and scheme: 25 28-day-old weaned piglets with negative PRV antigen and antibody tests were selected and randomly divided into 5 groups, with 5 pigs in each group. Groups 1-3 were the immune challenge group, group 4 was the challenge control group (not immune but challenged), and group 5 was the healthy control group (not immune and not challenged). The 5 pigs in groups 1-3 were placed in 3 piglet atomization boxes respectively. Each atomization box (648L, 1.8m×0.6m×0.6m, temperature controlled at 20-30℃) was connected to an ultrasonic atomizer (ZL201521097267.3). One bottle of porcine pseudorabies live vaccine (SA215 strain), one bottle of porcine pseudorabies live vaccine (Bartha-K61 strain), and one bottle of porcine pseudorabies live vaccine (HB-98 strain) freeze-dried vaccine were taken respectively. Aerosol live vaccine was prepared in the piglet atomization box according to the method in Title 2 of this Example. The piglets in the atomization box were immunized. After the atomization was completed (the atomization time was 10 minutes), the piglets were allowed to stay in the atomization box for another 30 minutes. The immunization dose was 1.28×10 5 TCID 50 / head (The dose of aerosol vaccine for piglets was calculated according to the formula in the following reference: HaoF, Bai Y, Xie X, Yuan T, WeiY, Xiong Q, GanY, Zhang L, Zhang Z, Shao G, FengZ. Phenotypic characteristics and protective efficacy of an attenuated Mycoplasma hyopneumoniae vaccine by aerosol administration. Vaccine. 2022, 40(42): 6074-6083.). Two weeks after aerosol immunization, the pigs in the immunization and challenge groups and the challenge control group were challenged with 1 mL of 10 6 TCID 50A novel inactivated gE / gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge (Gu Z, Dong J, Wang J, Hou C, Sun H, Yang W, Bai J, Jiang PA). A healthy control group of pigs received the same dose of BHK-21 cell culture supernatant intranasally. Following challenge, the five groups of piglets were housed separately and observed for 14 days. Rectal temperatures were measured daily, and clinical symptoms were observed and recorded.

[0038] Criteria for determining if pigs are infected with pseudorabies:

[0039] ① Body temperature ≥40.5℃ for at least 3 days.

[0040] ② Clinical symptoms such as depression, loss of appetite or difficulty breathing appear.

[0041] ③ Central nervous system symptoms or death, manifested as muscle tremors, increased salivation, ataxia and nystagmus; or opisthotonos, epilepsy, circling; or side-lying and paddling exercises and other neurological symptoms, and in severe cases, death.

[0042] If the patient meets ③ or both ① and ②, the disease can be diagnosed.

[0043] (2) Results: Two days after the challenge, the challenge control group showed clinical manifestations such as fever (≥40.5℃ and maintained for more than 3 days), depression, vomiting, coughing, dyspnea and neurological symptoms. All pigs in the challenge control group died within 8 days. Only a few pigs in each immune challenge group showed mild body temperature reaction (less than 40.5℃) or mild respiratory symptoms. The pigs in the healthy control group all showed normal performance. This shows that the live aerosol vaccine for pseudorabies in pigs can effectively prevent pseudorabies virus infection.

[0044] This example demonstrates that aerosolized protective agent A effectively protects against different pseudorabies virus vaccine strains. The aerosolized vaccine exhibits minimal loss of activity after 30 minutes of airborne retention. Furthermore, the vaccine effectively prevents pseudorabies virus infection in immunized pigs, making aerosolized immunization with live pseudorabies vaccines a reality. Compared to conventional immunization methods involving intranasal or intramuscular injections, this method not only improves the convenience of vaccination but also significantly reduces labor and time.

[0045] 5. Replace the solvent in atomizer protective agent A

[0046] In Title 1 of this Example, atomizer protectant A is an aqueous solution containing the following substances: 10 g / L trehalose, 10 g / L L-arginine, 10 g / L proline, 5% (volume percentage) glycerol, 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4. Atomizer protectant A is prepared by dissolving trehalose, L-arginine, proline, and glycerol in phosphate buffer (an aqueous solution containing 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4). The phosphate buffer in atomizer protectant A is replaced with 10-500 mM Tris buffer or physiological saline, respectively, to obtain the corresponding atomizer protectants. The buffer was replaced with a 10-500 mM Tris buffer and a nebulizer containing normal saline. Aerosolized live vaccines of various strains were prepared according to Examples 1-4. The aerodynamic mass median diameter, titer after 30 minutes of airborne residence, and immune efficacy were measured. Results: The mean aerodynamic mass median diameter (MMAD) of the various pseudorabies aerosol vaccines (SA215, Bartha-K61, and HB-98 strains) ranged from 5.82 to 5.95 microns, with a geometric standard deviation (GSD) of 1.51 to 1.62. After 30 minutes of airborne residence, the aerosol vaccine showed a titer decrease of 0.42 to 0.53 compared to the pre-nebulization titer. Only a few of the immunized pigs developed mild temperature reactions or mild respiratory symptoms. Pigs in the challenge control group developed typical symptoms of pseudorabies, while pigs in the healthy control group appeared normal.

[0047] Example 2 Effects of different aerosol protective agents on the particle size distribution and activity of pseudorabies aerosol live vaccine

[0048] 1. Preparation of atomized protective agent

[0049] Atomized protective agent A is an aqueous solution containing the following substances: 10 g / L trehalose, 10 g / L L-arginine, 10 g / L proline, 5% (volume percentage concentration) glycerol, 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4.

[0050] Atomized protective agent B is an aqueous solution containing the following substances: 5 g / L trehalose, 5 g / L L-arginine, 5 g / L proline, 2% (volume percentage concentration) glycerol, 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4.

[0051] Atomization protective agent C is an aqueous solution containing the following substances: 20 g / L trehalose, 20 g / L L-arginine, 20 g / L proline, 8% (volume percentage concentration) glycerol, 8 g / L NaCl, 0.2 g / L KCl, 1.44 g / L Na2HPO4, 0.24 g / L KH2PO4, pH 7.4.

[0052] Control atomized protective agent 1: deionized water solution containing 5% glycerol and 0.1% polyvinyl pyrrolidone, pH 7.5.

[0053] Control aerosol protectant 2: deionized water solution containing 5% glycerol and 100 mM Tris-HCl, pH 7.4.

[0054] The control atomized protective agent 3 is an aqueous solution containing the following substances: 0.1 g / L chitosan (molecular weight 150 KDa), 20 g / L PEG6000 (polyethylene glycol 6000), 2 g / L cyclodextrin, 10 g / L sodium thiosulfate, 10 g / L L-histidine, pH 7.4.

[0055] 2. Preparation of aerosol vaccine

[0056] 1 bottle of porcine pseudorabies live vaccine (SA215 strain, 10 6 TCID 50 2 mL of aerosol protectant was added to each bottle for reconstitution, and then 18 mL of the same aerosol protectant was added and mixed to obtain the vaccine solution corresponding to each aerosol protectant.

[0057] 1 bottle of pseudorabies live vaccine (Bartha-K61 strain, 10 6 TCID 50 2 mL of aerosol protectant was added to each bottle for reconstitution, and then 18 mL of the same aerosol protectant was added and mixed to obtain vaccine solutions corresponding to each aerosol protectant.

[0058] 1 bottle of live porcine pseudorabies vaccine (HB-98 strain, 10 6 TCID 50 2 mL of aerosol protectant was added to each bottle for reconstitution, and then 18 mL of the same aerosol protectant was added and mixed to obtain vaccine solutions corresponding to each aerosol protectant.

[0059] 3. Testing of aerosol vaccine properties

[0060] The 18 vaccine solutions were each prepared using the method described in Example 1 to prepare aerosol live vaccines. The average aerodynamic mass median diameter of each aerosol vaccine particle was measured three times using the particle size measurement method described in Section 3 of Example 1. The titer of the aerosol live vaccine prepared with each aerosol protective agent was also measured using the activity measurement method described in Section 3 of Example 1 after 30 minutes of airborne suspension.

[0061] 4. Results

[0062] As shown in Table 1, the aerosol vaccine of live porcine pseudorabies vaccine (SA215 strain) formulated with aerosol protectant B had a mean aerodynamic mass median particle size of 5.79 μm and a geometric standard deviation of 1.57. After 30 minutes of airborne residence, the activity of the aerosol vaccine decreased by 0.53 titers. The aerosol vaccine of live porcine pseudorabies vaccine (SA215 strain) formulated with aerosol protectant C had a mean aerodynamic mass median particle size of 5.71 μm and a geometric standard deviation of 1.49. After 30 minutes of airborne residence, the activity of the aerosol vaccine of live porcine pseudorabies vaccine (SA215 strain) formulated with the control aerosol protectant 1 had a mean aerodynamic mass median particle size of 6.04 μm and a geometric standard deviation of 1.65. After 30 minutes of airborne residence, the activity of the aerosol vaccine decreased by 1.72 titers. The aerosol vaccine of live porcine pseudorabies vaccine (SA215 strain) formulated with control aerosol protectant 2 had a mean aerodynamic mass median particle size of 5.81 μm and a geometric standard deviation of 1.64. After 30 minutes of airborne residence, the activity decreased by 2.52 titers. The aerosol vaccine of live porcine pseudorabies vaccine (SA215 strain) formulated with control aerosol protectant 3 had a mean aerodynamic mass median particle size of 5.63 μm and a geometric standard deviation of 1.62. After 30 minutes of airborne residence, the activity decreased by 1.49 titers. Therefore, aerosol protectants B and C, like aerosol protectant A, effectively protect against the SA215 strain, with minimal loss of activity after 30 minutes of airborne residence. In contrast, control aerosol protectants 1, 2, and 3 showed weaker protection against the SA215 strain, with a greater loss of activity after 30 minutes of airborne residence.

[0063] Table 1 Average aerodynamic mass median particle size and activity test results of live pseudorabies aerosol vaccine (SA215 strain) formulated with different aerosol protective agents

[0064]

[0065] As shown in Table 2, the aerosol vaccine of live pseudorabies vaccine (Bartha-K61 strain) formulated with aerosol protectant B had a mean aerodynamic mass median particle size of 5.88 μm and a geometric standard deviation of 1.51. After 30 minutes of airborne residence, the activity decreased by 0.45 titers. The aerosol vaccine of live pseudorabies vaccine (Bartha-K61 strain) formulated with aerosol protectant C had a mean aerodynamic mass median particle size of 5.93 μm and a geometric standard deviation of 1.51. After 30 minutes of airborne residence, the activity decreased by 0.51 titers. The aerosol vaccine of live pseudorabies vaccine (Bartha-K61 strain) formulated with control aerosol protectant 1 had a mean aerodynamic mass median particle size of 5.19 μm and a geometric standard deviation of 1.99. After 30 minutes of airborne residence, the activity decreased by 2.12 titers. The aerosol vaccine of live pseudorabies vaccine (Bartha K61 strain) formulated with control aerosol protectant 2 had a mean aerodynamic mass median particle size of 5.49 μm and a geometric standard deviation of 2.08; after 30 minutes of airborne residence, the activity decreased by 1.72 titers. The aerosol vaccine of live pseudorabies vaccine (Bartha K61 strain) formulated with control aerosol protectant 3 had a mean aerodynamic mass median particle size of 5.09 μm and a geometric standard deviation of 2.01; after 30 minutes of airborne residence, the activity decreased by 1.85 titers. Therefore, aerosol protectants B and C, like aerosol protectant A, effectively protect against the Bartha K61 strain, with minimal loss of activity after 30 minutes of airborne residence. In contrast, control aerosol protectants 1, 2, and 3 showed weaker protection against the Bartha K61 strain, with a greater loss of activity after 30 minutes of airborne residence.

[0066] Table 2 Average aerodynamic mass median particle size and activity test results of live pseudorabies aerosol vaccine (Bartha-K61 strain) formulated with different types of aerosol protective agents

[0067]

[0068] As shown in Table 3, the aerosol vaccine of live porcine pseudorabies vaccine (HB-98 strain) formulated with aerosol protectant B had a mean aerodynamic mass median particle size of 5.94 μm and a geometric standard deviation of 1.60. After 30 minutes of airborne residence, the activity decreased by 0.49 titers. The aerosol vaccine of live porcine pseudorabies vaccine (HB-98 strain) formulated with aerosol protectant C had a mean aerodynamic mass median particle size of 5.91 μm and a geometric standard deviation of 1.62. After 30 minutes of airborne residence, the activity decreased by 0.55 titers. The aerosol vaccine of live porcine pseudorabies vaccine (HB-98 strain) formulated with control aerosol protectant 1 had a mean aerodynamic mass median particle size of 5.80 μm and a geometric standard deviation of 1.65. After 30 minutes of airborne residence, the activity decreased by 1.66 titers. The aerosol vaccine of live pseudorabies vaccine (HB-98 strain) formulated with control aerosol protectant 2 had a mean aerodynamic mass median particle size of 5.64 μm and a geometric standard deviation of 1.52. After 30 minutes of airborne residence, the activity of the aerosol vaccine decreased by 1.83 titers. The aerosol vaccine of live pseudorabies vaccine (HB-98 strain) formulated with control aerosol protectant 3 had a mean aerodynamic mass median particle size of 5.67 μm and a geometric standard deviation of 1.51. After 30 minutes of airborne residence, the activity of the aerosol vaccine decreased by 1.58 titers. Therefore, aerosol protectants B and C, like aerosol protectant A, effectively protect against the HB-98 strain, with minimal loss of activity after 30 minutes of airborne residence. In contrast, control aerosol protectants 1, 2, and 3 showed weaker protection against the HB-98 strain, with significant loss of activity after 30 minutes of airborne residence.

[0069] Table 3 Average aerodynamic mass median particle size and activity test results of live pseudorabies aerosol vaccine (HB-98 strain) prepared with different types of aerosol protective agents

[0070]

[0071]

[0072] In summary, aerosol protectants A, B, and C effectively protected the activity of pseudorabies virus strains SA215, Bartha-K61, and HB-98, with minimal loss of activity after the aerosol vaccine remained in the air for 30 minutes. In contrast, the control aerosol protectants 1, 2, and 3 showed weaker protection against these strains, with significant loss of activity after the aerosol vaccine remained in the air for 30 minutes.

[0073] Example 3 Effects of different aerosol protective agents on the immune protection rate of pseudorabies aerosol live vaccine in pigs

[0074] 1. Preparation of atomized protective agent

[0075] Aerosol live vaccines of SA215, Bartha-K61 and HB-98 strains were prepared according to the methods in Titles 1, 2 and 3 of Example 2.

[0076] 2. Grouping and plan:

[0077] One hundred 28-day-old weaned piglets with negative PRV antigen and antibody tests were selected and randomly divided into 20 groups, with 5 pigs in each group. Groups S1-S6 were SA215 immune challenge groups, and were vaccinated with SA215 aerosol live vaccines prepared with 6 kinds of aerosol protective agents; groups B1-B6 were Bartha-K61 immune challenge groups, and were vaccinated with Bartha-K61 aerosol live vaccines prepared with 6 kinds of aerosol protective agents; groups H1-H6 were HB-98 immune challenge groups, and were vaccinated with HB-98 aerosol live vaccines prepared with 6 kinds of aerosol protective agents; a challenge control group (not immunized but challenged) and a healthy control group (not immunized and not challenged) were also set up. The pigs in the immune challenge group were placed in the piglet aerosol box corresponding to the aerosol vaccine, and the aerosol vaccine was prepared in the piglet aerosol box according to the method in Titles 2 and 3 of Example 2. The piglets in the aerosol box were immunized, and the immunization dose was 1.28×10 5 TCID 50 After the aerosolization is completed (the aerosolization time is about 10 minutes), the piglets are allowed to stay in the aerosol box for another 30 minutes. Two weeks after the aerosol immunization, the pigs in each immune challenge group and the challenge control group are challenged with 1 mL of 10 drops per head. 6 TCID 50 / mL PRVZJ01 strain cytotoxicity (Gu Z, Dong J, Wang J, Hou C, Sun H, Yang W, Bai J, Jiang P. A novel inactivated gE / gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerging variant PRV challenge. Virus Res. 2015, 195:57-63.). Healthy pigs in the control group were intranasally instilled with the same dose of BHK-21 cell culture supernatant. The pigs were observed for 14 days, with rectal temperatures measured daily and clinical symptoms recorded.

[0078] Criteria for determining if pigs are infected with pseudorabies:

[0079] (1) Body temperature ≥40.5℃ for at least 3 days.

[0080] (2) Clinical symptoms such as depression, loss of appetite, or difficulty breathing appear.

[0081] (3) Central nervous system symptoms or death, manifested as muscle tremors, increased salivation, ataxia and nystagmus; or opisthotonos, epilepsy, circling; or side-lying and paddling movements and other neurological symptoms, and in severe cases, death.

[0082] If (3) is met or both (1) and (2) are met at the same time, the disease can be diagnosed.

[0083] 3. Results:

[0084] Three days after challenge, the challenge control group developed fever, severe respiratory distress, and neurological symptoms, and all pigs in the challenge control group died within a week. Pigs immunized with the aerosol live vaccine prepared with aerosol protectants A, B, and C only occasionally developed mild body temperature reactions (less than 40.5°C) or mild respiratory symptoms after challenge, with a protection rate of 100%. However, among pigs immunized with the aerosol live vaccine prepared with control aerosol protectants 1, 2, and 3, some piglets developed typical pseudorabies symptoms (fever, depression, loss of appetite, or respiratory distress) three to seven days after challenge, with some pigs developing neurological symptoms such as ataxia six days after challenge. The protection rate did not exceed 60%. The specific results are shown in Table 4. The pigs in the healthy control group all appeared normal.

[0085] Table 4 Results of piglet groups and clinical symptoms and immune protection rates after challenge with aerosol live vaccines prepared with different aerosol protective agents

[0086]

Claims

1. An atomized protective agent for aerosol live vaccine of pseudorabies in pigs, characterized in that The atomized protective agent is an aqueous solution containing the following ingredients: 5-20 g / L trehalose, 5-20 g / L L-arginine, 5-20 g / L proline, 2-8% glycerol, and a pH value of 7.0-7.

8.

2. The atomized protective agent according to claim 1, characterized in that The solvent of the atomized protective agent is a phosphate buffer with a pH value of 7.0-7.8, a 10-500 mM Tris buffer or a physiological saline.

3. A method for preparing a live aerosol vaccine of pseudorabies using the aerosol protective agent according to claim 1.

4. The method according to claim 3, characterized in that The pseudorabies live vaccine is dissolved in the above-mentioned aerosol protective agent and then aerosolized.

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