Thermosensitive adjuvant for aerosol immunization and preparation method thereof

Abstract

The invention provides a temperature-sensitive adjuvant for aerosol immunization, which is used for preparing poultry vaccines and enables the vaccines to undergo phase transition as the external temperature changes. The temperature-sensitive adjuvant at least comprises 1wt% to 20wt% of a nutrient, 0.1wt% to 3.0wt% of a surfactant, 0.5wt% to 8.0wt% of an auxiliary agent, and the rest is deionized water. When actually used, the temperature-sensitive adjuvant (Adj) and a virus antigen solution (Vat) are mixed in a ratio of 1:1 (v / v) to 1:5 (v / v) to prepare a vaccine mixture (Adg:Vat). The phase transition temperature of the vaccine mixture is between 36 and 40°C, and the time for the liquid phase to transition to a colloid is within 90 seconds.

Description

Technical Field

[0001] The present invention relates to a vaccine adjuvant and a preparation method thereof, in particular to a temperature-sensitive adjuvant suitable for aerosol immunization and capable of undergoing phase changes with temperature changes and a preparation method thereof. Background Art

[0002] Vaccines usually play an indispensable role in various means of controlling pandemics, such as Newcastle disease in poultry. In particular, when a particular virus strain of such an epidemic causes a large number of infections and a major outbreak occurs in a short period of time, it is generally impossible to combat it with physical means such as environmental hygiene control or chemical agents such as antibiotics or antiviral agents. In this case, vaccination is usually the only means to suppress the degree and scope of infection for prevention.

[0003] Secondly, avian Newcastle disease is an acute infectious disease caused by the Newcastle disease virus strain (NDV) of the animal paramyxovirus. However, there is no reliable and effective treatment drug so far. Therefore, in the conventional technology, the preventive measures used to prevent the occurrence of such infectious diseases are usually a method of implementing planned immunization of chickens of different ages, timely vaccinating Newcastle vaccines, and quickly improving the body's antibody level.

[0004] Although there are many ways to vaccinate against Newcastle disease, such as injection, nasal drops, eye drops, drinking water, or aerosol immunization, the effects of different immunization methods are different. Among them, the effect of aerosol immunization is stronger than eye drops, nasal drops, and drinking water, and the antibody level it can produce is higher, and it produces antibodies faster than injection immunization. In addition, aerosol immunization does not require catching chickens, is easy to operate, saves time and effort, and does not disturb the chickens. It is the preferred immunization method for large chicken farms.

[0005] Aerosol immunization uses a sprayer or atomizer to form atomized particles of a specific particle size range from the vaccine liquid. The atomized particles will be evenly dispersed in the air, and can enter the animal's body through the nasal cavity as the animal breathes, or adhere to the surface of the animal's skin and be absorbed into the body through the skin, which can produce a good and consistent immune effect in the animal group. However, aerosol immunization also has its disadvantages. It has high technical requirements, and improper operation can easily lead to immunization failure. In particular, the atomized particles of the vaccine after aerosolization are easily affected by the ambient temperature and humidity and shrink or evaporate, which can cause adverse reactions in the respiratory tract. Since the vaccine liquid is mainly composed of an antigen liquid and an adjuvant used in combination with the antigen liquid, all walks of life are eagerly looking forward to the development of an adjuvant suitable for aerosol immunization to solve the problem that the conventional aerosol immunization is easily affected by the operating technology. Summary of the invention

[0006] In view of this, the inventors have devoted themselves to studying various possible solutions to the problems of the conventional technology, and have developed a temperature-sensitive adjuvant that can not only improve the problems of the conventional technology, but also address the deficiencies of the prior art. The temperature-sensitive adjuvant of the present invention has excellent coverage and water retention, and after being mixed with the viral antigen liquid in a specific ratio, it can quickly transform from a liquid phase to a colloid when it contacts the animal body and adhere to the respiratory tract or skin surface of the animal, which can increase the content of the viral antigen in the organism, increase the time of stimulating the immune system, and thus increase the memory immune effect of the animal.

[0007] In other words, the present invention can provide a temperature-sensitive adjuvant for aerosol immunization, which is used to prepare poultry vaccines and enables the vaccine to undergo phase transition as the external temperature changes. The temperature-sensitive adjuvant comprises at least 1wt% to 20wt% of a nutrient, 0.1 to 3.0wt% of a surfactant, 0.5 to 8.0wt% of an auxiliary agent, and the remainder is deionized water; wherein the nutrient comprises chitosan or a chitosan derivative, and lactose; the surfactant is selected from tween 20, tween 40, tween 50, tween 60, tween 70, tween 80, tween 90, tween 100, tween 110, tween 120, tween 130, tween 140, tween 150, tween 160, tween 170, tween 180, tween 190, tween 210, tween 230, tween 240, tween 250, tween 260, tween 270, tween 280, tween 290, tween 300, tween 310, tween 320, tween 330, tween 340, tween 350, tween 360, tween 60, and tween80; the adjuvant is at least one selected from glycerol, propylene glycol, ethylene glycol, diethylene glycol monoethyl ether, glyceride, and polyethylene glycol fatty acid ester; and in actual administration, the temperature-sensitive adjuvant (Adj) and the virus antigen liquid (Vat) are mixed in a ratio of 1:1 (v / v) to 1:5 (v / v) to prepare a vaccine mixture (Adg:Vat); the phase transition temperature of the vaccine mixture is between 36 and 40°C, and the time for transition from liquid phase to colloid is within 90 seconds.

[0008] According to one embodiment of the present invention, the coverage rate of the temperature-sensitive adjuvant on the viral antigen solution is above 50%.

[0009] According to one embodiment of the present invention, the viscosity of the temperature-sensitive adjuvant is between 5 and 100 Pa·s.

[0010] According to one embodiment of the present invention, the evaporation rate of the colloid is 1.5 μg / cm 2 ·s or less.

[0011] According to one embodiment of the present invention, the surface tension of the colloid is between 20 and 150 N / m.

[0012] According to an embodiment of the present invention, the osmotic pressure of the colloid is between 280 and 300 mOsm / kg.

[0013] According to one embodiment of the present invention, the chitosan derivative is selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate, chitosan quaternary ammonium salt, chito oligosaccharide, or chitin.

[0014] According to one embodiment of the present invention, a pH adjuster is further included to make the pH value of the temperature-sensitive adjuvant between 4.0 and 8.0.

[0015] In addition, the present invention can also provide a method for preparing a thermosensitive adjuvant for aerosol immunization, which comprises the following steps: (a) adding lactose into deionized water and stirring until completely dissolved, then adding chitosan or a chitosan derivative and mixing them thoroughly to obtain a first component; (b) adding an adjuvant into deionized water and stirring until completely dissolved to obtain a second component; (c) adding a surfactant into deionized water and stirring until completely dissolved to obtain a third component; wherein (d) firstly adding the second component into the first component, continuously stirring for 30 to 100 minutes, then adding the third component, and continuing stirring for 30 to 100 minutes to obtain The temperature-sensitive adjuvant is obtained; wherein in the first component, the weight percentage concentration of the lactose is 1-10wt%, and the weight percentage concentration of the chitosan or chitosan derivative is 2-15wt%; in the second component, the weight percentage concentration of the auxiliary agent is 10wt%-50wt%, and the auxiliary agent is at least one selected from glycerol, propylene glycol, ethylene glycol, diethylene glycol monoethyl ether, glyceride, and polyethylene glycol fatty acid ester; in the third component, the weight percentage concentration of the surfactant is 5wt%-30wt%, and the surfactant is at least one selected from tween20, tween 40, tween 60, and tween 80; and in 100 parts by weight of the temperature-sensitive adjuvant, the weight of the first component is between 70 parts by weight and 85 parts by weight; the weight of the second component is between 10 parts by weight and 20 parts by weight; and the weight of the third component is between 5 parts by weight and 10 parts by weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The following is a flow chart showing the standard preparation of the temperature-sensitive adjuvant of the present invention. DETAILED DESCRIPTION

[0017] Below, different specific embodiments are listed for the implementation of the present invention and described and explained in more detail so as to make the spirit and content of the present invention more complete and easy to understand; however, those with ordinary knowledge in this art should understand that the present invention is certainly not limited to these examples, and other same or equivalent functions and step sequences can also be used to achieve the present invention.

[0018] In addition, the following specific examples can further demonstrate the practical application scope of the present invention, but are not intended to limit the scope of the present invention in any form.

[0019] First, the specific terms or nouns used in this specification are described in a descriptive manner; however, the following description is only for illustrative purposes and is not intended to limit the scope of the present invention and the scope of the patent application. Unless otherwise defined in this specification, the meanings of scientific and technical terms used in this article are the same as those understood and used by those with ordinary knowledge in the technical field to which the present invention belongs.

[0020] As used herein, a "vaccine" or "vaccine fluid" is a composition that can be used to induce protective immunity in a recipient. Thus, after a subject has been vaccinated with an antigen, a vaccine can prevent, delay, or reduce the severity of disease development in a subject exposed to the same or a related antigen (relative to an unvaccinated subject). The protective immunity provided by a vaccine can be humoral (antibody-mediated) or cellular, or both. For example, vaccination can eliminate or reduce the load of pathogens or infected cells, or produce any other measurable reduction in infection. Vaccination can also reduce tumor burden in immunized (vaccinated) subjects.

[0021] As used herein, the term "adjuvant" refers to a compound that, when administered together with an antigen, enhances a subject's immune response to that antigen.

[0022] In this article, the numerical values ​​and parameters used to define the scope of the present invention are essentially inevitably containing standard deviations due to individual test methods, and are therefore mostly expressed as approximate numerical values, but in the specific embodiments, the relevant numerical values ​​are presented as accurately as possible. In this article, "about" is usually determined by the consideration of those with ordinary knowledge in the technical field to which the present invention belongs, and generally refers to the actual value falling within the acceptable standard error of the mean value, for example, the actual value is within ±10%, ±5%, ±1%, or ±0.5% of a specific value or range.

[0023] First, see Figure 1 , which is a standard production flow chart showing the temperature-sensitive adjuvant of the present invention, comprising the following steps:

[0024] Step T1: Add lactose into deionized water and stir until completely dissolved, then add chitosan or chitosan derivative and mix thoroughly to obtain the first component.

[0025] Step T2: Add the auxiliary agent into deionized water and stir until it is completely dissolved to obtain the second component.

[0026] Step T3: Add the surfactant into deionized water and stir until it is completely dissolved to obtain the third component.

[0027] Step T4: firstly add the second component into the first component, continue stirring for 30 to 100 minutes, then add the third component, continue stirring for 30 to 100 minutes to obtain the temperature-sensitive adjuvant.

[0028] Chitosan or chitosan derivatives and lactose in the first component are nutrient ingredients used as adjuvants, and the reaction ratio of chitosan or chitosan derivatives to lactose is adjusted to have the characteristic of being able to undergo phase transition with temperature changes; the weight ratio of chitosan or chitosan derivatives to lactose is between 10:1 and 1:1, preferably between 8:1 and 1:1, more preferably between 5:1 and 1:1, and most preferably between 3:1 and 1:1. Since lactose is solid at room temperature, lactose must be dissolved in deionized water in step T1 before being mixed with chitosan or chitosan derivatives. The weight percentage concentration of lactose in the first component is 1 to 10 wt%, and the weight percentage concentration of chitosan or chitosan derivatives in the first component is 2 to 15 wt%.

[0029] The deacetylation degree of chitosan is generally in the range of 50% to 100%, preferably in the range of 60% to 100%, more preferably in the range of 70% to 100%, and most preferably in the range of 80% to 100%. In addition, the molecular weight of chitosan is generally in the range of 50,000 to 2 million, preferably in the range of 100,000 to 2 million, more preferably in the range of 500,000 to 2 million, and most preferably in the range of 1 million to 2 million.

[0030] In addition, according to one aspect of the present invention, the chitosan derivative that can be used in the thermosensitive adjuvant of the present invention is not particularly limited; for example, the chitosan derivative is selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate, chitosan quaternary ammonium salt, chito-oligosaccharide, or chitin; preferably selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate, or chitosan quaternary ammonium salt; more preferably selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate; most preferably selected from carboxymethyl chitosan or chitosan lactate.

[0031] According to the technical concept of the present invention, in the second component, the weight percentage concentration of the auxiliary agent is 10wt% to 50wt%. Furthermore, the auxiliary agent is at least one selected from glycerol, propylene glycol, ethylene glycol, diethylene glycol monoethyl ether, glyceride, and polyethylene glycol fatty acid ester; preferably at least one selected from glycerol, propylene glycol, ethylene glycol, and diethylene glycol monoethyl ether; more preferably at least one selected from glycerol, propylene glycol, and ethylene glycol; and most preferably at least one selected from glycerol and propylene glycol.

[0032] In addition, in the third component, the weight percentage concentration of the surfactant is 5wt% to 30wt%, and the surfactant is at least one selected from tween 20, tween 40, tween 60, and tween 80.

[0033] According to the technical idea of ​​the present invention, in 100 parts by weight of the thermosensitive adjuvant, the first component is generally between 70 and 85 parts by weight, preferably between 75 and 85 parts by weight, and optimally between 80 and 85 parts by weight; the second component is generally between 10 and 20 parts by weight, preferably between 10 and 18 parts by weight, and optimally between 10 and 15 parts by weight; the third component is generally between 5 and 10 parts by weight, preferably between 5 and 8 parts by weight, and optimally between 5 and 7 parts by weight.

[0034] As above, the temperature-sensitive adjuvant prepared by the above steps includes 1wt% to 20wt% of a nutrient, 0.1 to 3.0wt% of a surfactant, 0.5 to 8.0wt% of an auxiliary agent, and the rest is deionized water. In actual administration, the temperature-sensitive adjuvant (Adj) and the virus antigen solution (Vat) are mixed in a ratio of 1:1 (v / v) to 1:5 (v / v) to prepare a vaccine mixture (Adg:Vat); the phase transition temperature of the vaccine mixture is between 36 and 40°C, and the time for the phase transition from liquid to colloid is within 90 seconds. Furthermore, the coverage rate of the temperature-sensitive adjuvant for the virus antigen solution is above 50%, preferably above 60%, more preferably above 70%, and most preferably above 80%.

[0035] Next, the present invention is further described below in conjunction with specific embodiments.

[0036] 《Preparation Example 1》

[0037] First, 10 g of lactose was added to 840 g of deionized water and stirred at room temperature until completely dissolved, and then 30 g of carboxymethyl chitosan (with a deacetylation degree of 80% and a molecular weight of about 800,000) was added and stirred at 15 rpm until completely mixed to obtain the first component.

[0038] Next, 20 g of glycerol was added into 100 g of deionized water and stirred until completely dissolved to obtain a second component; and 5 g of tween 80 was added into 60 g of deionized water and stirred until completely dissolved to obtain a third component.

[0039] After obtaining 880 g of the first component, 120 g of the second component, and 65 g of the third component through the above steps, the second component was first added to the first component, and the mixture was stirred for 100 minutes, and then the third component was added, and the mixture was stirred for 30 to 100 minutes and the pH value was adjusted to between 4 and 8 with a pH adjuster, thereby obtaining a thermosensitive adjuvant S1. Next, the viscosity of the thermosensitive adjuvant S1 was measured using a viscosity meter and recorded in Table 1.

[0040] Preparation Example 2

[0041] First, 30 g of lactose was added to 840 g of deionized water and stirred at room temperature until completely dissolved, and then 50 g of carboxymethyl chitosan (with a deacetylation degree of 80% and a molecular weight of about 800,000) was added and stirred at 15 rpm until completely mixed to obtain the first component.

[0042] Next, 40 g of glycerol was added into 100 g of deionized water and stirred until completely dissolved to obtain a second component; and 10 g of tween 80 was added into 60 g of deionized water and stirred until completely dissolved to obtain a third component.

[0043] After obtaining 920 g of the first component, 140 g of the second component, and 70 g of the third component through the above steps, the second component was first added to the first component, and the mixture was stirred for 100 minutes, and then the third component was added, and the mixture was stirred for 30 to 100 minutes and the pH value was adjusted to between 4 and 8 with a pH adjuster, thereby obtaining a thermosensitive adjuvant S2. Next, the viscosity of the thermosensitive adjuvant S2 was measured using a viscosity meter and recorded in Table 1.

[0044] Preparation Example 3

[0045] First, 50 g of lactose was added to 840 g of deionized water and stirred at room temperature until completely dissolved, and then 70 g of carboxymethyl chitosan (with a deacetylation degree of 80% and a molecular weight of about 800,000) was added and stirred at 15 rpm until completely mixed to obtain the first component.

[0046] Next, 50 g of glycerol was added into 100 g of deionized water and stirred until completely dissolved to obtain a second component; and 15 g of tween 80 was added into 60 g of deionized water and stirred until completely dissolved to obtain a third component.

[0047] After obtaining 960 g of the first component, 150 g of the second component, and 75 g of the third component through the above steps, the second component was first added to the first component, and the mixture was stirred for 100 minutes, and then the third component was added, and the mixture was stirred for 30 to 100 minutes and the pH value was adjusted to between 4 and 8 with a pH adjuster, thereby obtaining a thermosensitive adjuvant S3. Next, the viscosity of the thermosensitive adjuvant S3 was measured using a viscosity meter and recorded in Table 1.

[0048] Preparation Example 4

[0049] First, 70 g of lactose was added to 840 g of deionized water and stirred at room temperature until completely dissolved. Then, 100 g of carboxymethyl chitosan (with a deacetylation degree of 80% and a molecular weight of about 800,000) was added and stirred at 15 rpm until completely mixed to obtain the first component.

[0050] Next, 70 g of glycerol was added into 100 g of deionized water and stirred until completely dissolved to obtain a second component; and 20 g of tween 80 was added into 60 g of deionized water and stirred until completely dissolved to obtain a third component.

[0051] After obtaining 1010 g of the first component, 170 g of the second component, and 80 g of the third component through the above steps, the second component was first added to the first component, and the mixture was stirred for 100 minutes, and then the third component was added, and the mixture was stirred for 30 to 100 minutes and the pH value was adjusted to between 4 and 8 with a pH adjuster, thereby obtaining a thermosensitive adjuvant S4. Next, the viscosity of the thermosensitive adjuvant S4 was measured using a viscosity meter and recorded in Table 1.

[0052] Table 1

[0053]

[0054] 《Analysis of phase transition time and phase transition temperature》

[0055] The temperature-sensitive adjuvants S1 to S4 obtained in the above Preparation Examples 1 to 4 were respectively mixed with phosphate buffered saline (PBS) in the volume ratios shown in Table 2 and heated. The process of the mixed solutions in different ratios transforming from liquid to gel was observed, and the phase transition temperature and phase transition time were recorded in Table 2.

[0056] Table 2

[0057]

[0058]

[0059] From the results in Table 2 above, it can be seen that the phase transition temperature of the thermosensitive adjuvant S1 at different ratios is between 25 and 42°C, and the phase transition time is within 90 seconds; the phase transition temperature of the thermosensitive adjuvant S2 at different ratios is between 29.5 and 45°C, and the phase transition time is within 120 seconds; the phase transition temperature of the thermosensitive adjuvant S3 at different ratios is between 30 and 44°C, and the phase transition time is within 131 seconds; the phase transition temperature of the thermosensitive adjuvant S4 at different ratios is between 31.5 and 44.5°C, and the phase transition time is within 141 seconds. Since the body temperature of general poultry is about 36-40°C, considering the storage method of the vaccine and the normal body temperature of poultry, the volume ratio of the temperature-sensitive adjuvant (Adj) and the virus antigen solution (Vat) of the present invention can be in the range of about 1:5 (v / v) to 1:1 (v / v); in some specific embodiments, it is preferably in the range of about 1:3 (v / v) to 2:3 (v / v); the best is about 3:7 (v / v), and the phase conversion time of the temperature-sensitive adjuvant S1 is within 90 seconds.

[0060] 《Surface tension and evaporation rate analysis》

[0061] The temperature-sensitive adjuvants S1 to S4 obtained in Preparation Examples 1 to 4 were mixed with PBS at a ratio of 3:7 (v / v) to obtain mixed solutions P1 to P4. The osmotic pressure, surface tension, and evaporation rate of the mixed solutions P1 to P4 in different temperature environments were then measured, and the results are recorded in Table 3.

[0062] The osmotic pressure is tested using a micro-osmotic pressure analyzer, and the surface tension is tested using a surface tensiometer. As for the evaporation rate, 15g of sample is placed in an evaporating dish (diameter: 7cm) in various temperature environments (relative humidity 50%), and the evaporating dish is placed in a fume hood. The weight of the remaining sample in the evaporating dish is measured at certain time intervals, and then the evaporation rate is calculated.

[0063] Table 3

[0064]

[0065] The mixed solutions P1-P4 are all converted from liquid phase to colloid at 37°C. From the results in Table 3 above, it can be seen that the osmotic pressure of the mixed solutions P1-P4 is 280-300 mOsm / kg, the surface tension decreases with increasing temperature, and the evaporation rate also decreases with increasing temperature.

[0066] 《Adjuvant coating effect test》

[0067] 80 ml of the temperature-sensitive adjuvants S1 to S4 were placed in beakers respectively and stirred at 3000 rpm using a homogenizer. Then 20 ml of 1 mg / ml bovine serum albumin solution (bovine serum albumin weight is 20 mg) was slowly added. When all was added, the speed was adjusted to 5000 rpm and stirring was continued for 10 minutes.

[0068] Next, the stirred mixture was heated to 38°C to solidify it into a colloid, and the colloid was placed in 10 ml of PBS (pH 7.4, 10 mM, 38°C) and slightly shaken to completely dissolve the bovine serum albumin remaining on the surface of the colloid that was not coated in the PBS. The clarified liquid was separated and placed in a UV spectrophotometer to measure the absorbance at 280 nm and convert the concentration of bovine serum albumin to the standard calibration curve of the bovine serum albumin solution. Since the total volume of the clarified liquid is 10 ml, the total volume is converted to the concentration, which is the weight of the bovine serum albumin contained in the clarified liquid. The calculation formula for the coating rate is as follows:

[0069]

[0070] The calculated coverage is shown in Table 4.

[0071] Table 4

[0072] Example 1 Example 2 Example 3 Example 4 Bovine serum albumin solution (ml) 20 20 20 20 Thermosensitive adjuvant S1 (ml) 80 0 0 0 Thermosensitive adjuvant S2 (ml) 0 80 0 0 Thermosensitive adjuvant S3 (ml) 0 0 80 0 Thermosensitive adjuvant S4 (ml) 0 0 0 80 Coverage rate (%) 97.5 90.7 88.1 70.5

[0073] From the results in Table 4, it can be seen that the coverage rates of the temperature-sensitive adjuvants S1, S2, and S3 used in Examples 1 to 4 on bovine serum albumin were 97.5%, 90.7%, 88.1%, and 70.5%, respectively.

[0074] 《Potency Verification of Live Virus Vaccines》

[0075] First, phosphate buffered saline (PBS) and the temperature-sensitive adjuvants S1 to S4 obtained in Preparation Examples 1 to 4 were placed in a 2000 ml beaker at the ratios shown in Table 5, and a homogenizer was placed in the beaker. After the solution in the beaker covered the stirring rod of the homogenizer by 2 / 3 (about 15 cm), the high-speed shear force of the homogenizer (model: TT-SM-S, brand: Taiwan Da Ding Machinery) was turned to 3000 rpm, and then 400 ml of genotype VII Newcastle disease live virus antigen solution (10 6.5 TCID 50 / ml) is added to the rotating solution; finally, when all the antigen liquid is added, the speed can be adjusted to 5000 rpm, and the final stirring time is five to ten minutes to prepare live virus vaccines V1 to V4.

[0076] Next, the live virus vaccines V1 to V4 obtained above were respectively immunized by aerosol spraying, and the operating conditions were: spray pressure 2Kg / cm 2 ~30Kg / cm 2 The flow rate is 0.4-3L / min, the air flow temperature is 25-37°C, and the relative humidity is 50-80%. The vaccine solution is prepared into a liquid aerosol with a particle size distribution of 50-1000 microns, a mist particle concentration of 10-30%, and a microbiological activity that meets the requirements of vaccine immunization. Chickens are immunized by atomization in an aerosol box. There are 10 immunized one-day-old chickens in each aerosol box, and blood is collected at 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks to separate the test serum.

[0077] Next, a hemagglutination (HA) test was performed by adding 25 μl of 0.85% saline to each row of a 96-well U-shaped plate, and then adding 25 μl of the serum to be tested to the first row.

[0078] Next, after thorough mixing with a micropipette, take 25 μl of the serum to be tested in the second row and add it to the second well, and then take another 25 μl and add it to the third row, and so on until the 10th row, the antigen presents a 2-fold to 1024-fold dilution, and the 25 μl mixed solution in the 11th row is not used. Repeat the test with the same sample in 3 wells in each row.

[0079] Then, the genotype VII Newcastle disease antigen solution was diluted with HI buffer to contain 8 HAU of the genotype VII Newcastle disease antigen solution in each 25 μl of the dilution solution, and 25 μl of the dilution solution was added to each well and allowed to stand for 15 minutes.

[0080] Next, 50 μL of 0.96% red blood cell suspension was added to each well, and after standing at room temperature for 60 minutes, the blood cells were judged to see whether there was agglutination. The blood cells that still had agglutination at the highest dilution multiple were the antibody titers. Then, the average antibody titers of the test results of 10 chickens in each group of each embodiment were recorded in Table 5.

[0081] Table 5

[0082]

[0083] From the results in Table 5, it can be seen that in Examples 5 to 8, the antibody titers of the chickens vaccinated with the live virus vaccines V1 to V4 added with the temperature-sensitive adjuvant of the present invention rapidly climbed to above 60 in the first week, reached a maximum of above 100 in the second week, and although it declined in the third week, the antibody titers in the chickens were still maintained above 70.

[0084] In summary, the content of the present invention has been illustrated by the above embodiments, but the present invention is not limited to these embodiments. Those with ordinary knowledge in the technical field to which the present invention belongs can make various changes and modifications without departing from the spirit and scope of the present invention; for example, the various technical contents illustrated in the above embodiments are combined or changed to form new embodiments, and these embodiments are of course regarded as the content of the present invention. Therefore, the scope of protection to be protected in this case also includes the claims and the scope defined therein.

Claims

1. A temperature-sensitive adjuvant for aerosol immunization, characterized in that: The invention is used for preparing poultry vaccines, and enables the vaccines to undergo phase transition as the external temperature changes. The temperature-sensitive adjuvant is composed of the following components: 1wt% to 20wt% of a nutrient, 0.1 to 3.0wt% of a surfactant, 0.5 to 8.0wt% of an auxiliary agent, and the rest is deionized water; The nutrient is composed of chitosan or a chitosan derivative and lactose; the chitosan or chitosan derivative is carboxymethyl chitosan with a deacetylation degree of 80% and a molecular weight of 800,000, and the weight ratio of the chitosan or chitosan derivative to the lactose is 10:1 to 1:1; The surfactant is tween 80; The auxiliary agent is glycerol; The pH value of the thermosensitive adjuvant is 4.0-8.0; as well as During actual administration, the temperature-sensitive adjuvant and the viral antigen liquid are mixed in a ratio of 1:1 (v / v) to 1:5 (v / v) to prepare a vaccine mixture; the phase transition temperature of the vaccine mixture is 36-40°C, and the time for transitioning from liquid phase to colloid is within 90 seconds.

2. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, characterized in that It further comprises a pH adjuster.

3. A method for preparing a temperature-sensitive adjuvant for aerosol immunization, characterized in that: The following steps are involved: (a) adding lactose into deionized water and stirring until completely dissolved, and then adding chitosan or a chitosan derivative and mixing them thoroughly to obtain a first component; (b) adding the auxiliary agent into deionized water and stirring until it is completely dissolved to obtain a second component; (c) adding the surfactant into deionized water and stirring until the surfactant is completely dissolved to obtain a third component; in (d) first adding the second component into the first component, continuously stirring for 30 to 100 minutes, then adding the third component, continuing stirring for 30 to 100 minutes and adjusting the pH value to obtain the temperature-sensitive adjuvant; in In the first component, the weight percentage concentration of the lactose is 1-10wt%, the weight percentage concentration of the chitosan or chitosan derivative is 2-15wt%, and the weight ratio of the chitosan or chitosan derivative to the lactose is 10:1-1:1; In the second component, the weight percentage concentration of the auxiliary agent is 10wt% to 50wt%, and the auxiliary agent is glycerol; In the third component, the weight percentage concentration of the surfactant is 5wt% to 30wt%, and the surfactant is tween 80; The chitosan or chitosan derivative is carboxymethyl chitosan with a deacetylation degree of 80% and a molecular weight of 800,000; The pH value of the thermosensitive adjuvant is 4.0-8.0; as well as In 100 parts by weight of the temperature-sensitive adjuvant, The weight portion of the first component is 70 parts by weight to 85 parts by weight; The weight portion of the second component is 10 to 20 parts by weight; The weight portion of the third component is 5 to 10 parts by weight.

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