CN112402603A - Temperature-sensitive adjuvant for aerosol immunization and preparation method thereof - Google Patents
Temperature-sensitive adjuvant for aerosol immunization and preparation method thereof Download PDFInfo
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Abstract
The invention provides a temperature-sensitive adjuvant for aerosol immunization, which is used for preparing poultry vaccines and enables the vaccines to generate phase transformation along with the change of external temperature, wherein the temperature-sensitive adjuvant at least comprises 1-20 wt% of a nutritional agent, 0.1-3.0 wt% of a surfactant, 0.5-8.0 wt% of an adjuvant and the balance of deionized water; in actual application, 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 mixed solution (Adg: Vat); the phase transition temperature of the vaccine mixed solution is 36-40 ℃, and the time for changing the liquid phase into the colloid is within 90 seconds.
Description
Technical Field
The invention relates to an adjuvant for a vaccine and a preparation method thereof, in particular to a temperature-sensitive adjuvant which is suitable for aerosol immunization and can change phase along with temperature change and a preparation method thereof.
Background
Among the various means used to control pandemics, including newcastle disease, for example, in birds, vaccines often play an essential and important role. In particular, when a large number of specific viral strains of the aforementioned epidemic diseases cause infection and a serious epidemic situation develops in a short time, it is generally impossible to control the infection by physical means such as environmental sanitation or by using chemical agents such as antibiotics or antiviral agents, and in such cases, epidemic prevention is usually performed only depending on the extent and range of infection control by vaccination.
Second, avian newcastle disease is an acute infectious disease caused by a newcastle disease virus strain (NDV) of animal paramyxoviruses. However, until now there has been no reliable and reliable effective therapeutic agent. Therefore, conventionally, the preventive measures adopted to prevent such infectious diseases are usually a method of performing planned immunization on chickens of different ages in days to vaccinate newcastle in time to rapidly increase the antibody level in the body.
Although there are many ways of vaccinating newcastle disease, such as injection, nasal drop, eye drop, drinking water, or aerosol immunization, the effect of different immunization methods is different, wherein the effect of aerosol immunization is stronger than that of eye drop, nasal drop, and drinking water, the level of antibody generated is higher, and the antibody is generated faster than that of injection immunization. In addition, aerial fog immunization does not need to grab chickens, is convenient to operate, saves time and labor, does not frighten chicken flocks, and is a preferred immunization mode for large-scale chicken farms.
The aerosol immunization method is to use a sprayer or an atomizer to enable vaccine liquid to form atomized particles with a specific particle size range, the atomized particles can be uniformly dispersed in the air and can enter an animal body from a nasal cavity along with the respiration of the animal or can be attached to the surface of the skin of the animal and absorbed into the body through the skin, and good and consistent immunization effects can be generated for animal groups. However, aerosol immunization also has the disadvantages of high technical requirements and easy immune failure due to improper operation, and particularly, atomized particles after vaccine atomization are easily reduced or evaporated due to the influence of ambient temperature and humidity, and rather, adverse reactions of respiratory tracts are caused. Since the vaccine solution is mainly composed of antigen solution and adjuvant used in combination with antigen solution, development of an adjuvant suitable for aerosol immunization is strongly desired in various circles, so as to solve the problem that the conventional aerosol immunization is easily affected by operation technology.
Disclosure of Invention
In view of the above, the present inventors have made extensive studies to solve the problems of the conventional art, and as a result, have developed a temperature-sensitive adjuvant that is superior in coating rate and water retention, and that can be rapidly converted from a liquid phase to a gel to be attached to the respiratory tract or skin surface of an animal when it is mixed with a viral antigen liquid in a specific ratio, thereby increasing the content of the viral antigen in the body of the animal, increasing the time required for stimulating the immune system, and further increasing the memory immune effect of the animal.
In other words, the invention can provide a temperature-sensitive adjuvant for aerosol immunization, which is used for preparing poultry vaccines and enables the vaccines to generate phase transformation along with the change of external temperature, wherein the temperature-sensitive adjuvant at least comprises 1 wt% -20 wt% of a nutritional agent, 0.1 wt% -3.0 wt% of a surfactant, 0.5 wt% -8.0 wt% of an adjuvant and the balance of deionized water; wherein the nutritional agent comprises chitosan or chitosan derivative, and lactose; the surfactant is at least one selected from tween 20, tween 40, tween 60 and tween 80; 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 when actually administered, 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 mixed solution (Adg: Vat); the phase transition temperature of the vaccine mixed solution is 36-40 ℃, and the time for changing the liquid phase into the colloid is within 90 seconds.
According to an embodiment of the present invention, the coating rate of the temperature-sensitive adjuvant on the virus antigen solution is 50% or more.
According to an embodiment of the present invention, the viscosity of the temperature-sensitive adjuvant is between 5 Pa · s and 100Pa · s.
According to an embodiment of the invention, wherein the evaporation rate of the colloid is at 1.5 μ g/cm2S is less than or equal to.
According to an embodiment of the present invention, the surface tension of the colloid is between 20 to 150N/m
According to an embodiment of the present invention, the osmotic pressure of the colloid is 280-300 mOsm/kg.
According to an embodiment of the present invention, wherein the chitosan derivative is selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate, chitosan quaternary ammonium salt, chitosan oligosaccharide, or chitin.
In an embodiment of the present invention, the temperature-sensitive adjuvant further comprises a pH adjuster, so that the pH of the temperature-sensitive adjuvant is between 4.0 and 8.0.
In addition, the invention also provides a preparation method of the temperature-sensitive adjuvant for aerosol immunization, which comprises the following steps: (a) adding lactose into deionized water, stirring to dissolve completely, adding chitosan or chitosan derivative, and mixing to obtain a first component; (b) adding the auxiliary agent into deionized water, and stirring until the auxiliary agent 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; adding the second component into the first component, continuously stirring for 30-100 minutes, adding the third component, and continuously stirring for 30-100 minutes to obtain the temperature-sensitive adjuvant; wherein in the first component, the weight percentage concentration of the lactose is 1-10 wt%, and the weight percentage concentration of the chitosan or the chitosan derivative is 2-15 wt%; in the second component, the weight percentage concentration of the auxiliary agent is 10 wt% to 50 wt%, 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 5 wt% to 30 wt%, and the surfactant is at least one selected from tween 20, tween 40, tween 60 and tween 80; and in 100 parts by weight of the temperature-sensitive adjuvant, the weight part of the first component is between 70 and 85 parts by weight; the weight part of the second component is between 10 and 20; the weight portion of the third component is between 5 and 10.
Drawings
FIG. 1 is a standard manufacturing flow chart showing the temperature-sensitive adjuvant of the present invention.
Detailed Description
The following detailed description and specific examples are given for illustrative purposes only, and are not intended to limit the scope of the present disclosure; however, it should be understood by those skilled in the art that the present invention is not limited to these examples, and other equivalent functions and steps can be used to achieve the same purpose.
Further, the following examples further illustrate the practical range of the present invention, but are not intended to limit the scope of the present invention in any manner.
First, a description will be given, illustratively, to specific terms or terms used in the present specification; however, the following description is only exemplary and should not be construed as limiting the present disclosure and claims. Unless defined otherwise herein, the scientific and technical terms used herein have the same meaning as commonly understood and used by one of ordinary skill in the art to which this invention belongs.
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, the vaccine can prevent, delay or reduce the severity of disease progression in a subject exposed to the same or a related antigen (relative to a non-vaccinated subject). The protective immunity provided by the vaccine may be humoral (antibody-mediated) immunity or cellular immunity, or both. For example, vaccination may eliminate or reduce the load of pathogens or infected cells, or produce any other measurable reduction in infection. Vaccination may also reduce the tumor burden in an immunized (vaccinated) subject.
As used herein, the term "adjuvant" refers to a compound that, when administered in conjunction with an antigen, enhances the subject's immune response to that antigen.
In this context, the values and parameters defining the scope of the invention are inherently related to the standard deviation found in its respective testing method, and are therefore usually expressed in approximate numerical values, although the numerical values are expressed as precisely as possible in the specific examples. In this context, "about" generally refers to a value that represents an actual value within an acceptable standard error of the mean, e.g., within 10%, 5%, 1%, or 0.5% of a particular value or range, as determined by a consideration of persons of ordinary skill in the art to which the invention pertains.
Referring to fig. 1, a flow chart of a standard manufacturing process of the temperature-sensitive adjuvant of the present invention is shown, which comprises the following steps:
step T1: lactose is added into deionized water and stirred until completely dissolved, and then chitosan or chitosan derivatives are added and fully mixed to obtain the first component.
Step T2: the adjuvant is added into deionized water and stirred until completely dissolved to obtain a second component.
Step T3: and adding the surfactant into deionized water, and stirring until the surfactant is completely dissolved to obtain a third component.
Step T4: and firstly, adding the second component into the first component, continuously stirring for 30-100 minutes, then adding the third component, and continuously stirring for 30-100 minutes to obtain the temperature-sensitive adjuvant.
The chitosan or chitosan derivative and the lactose in the first component are nutritional ingredients serving as adjuvants, and the reaction ratio of the chitosan or chitosan derivative and the lactose is adjusted to have the characteristic of phase transition along with the change of temperature; the weight ratio of chitosan or chitosan derivative to lactose is between 10: 1-1: 1, preferably between 8: 1-1: 1, more preferably between 5: 1-1: 1, preferably between 3: 1-1: 1. Since lactose is solid at room temperature, it is necessary to dissolve lactose in deionized water and then mix it with chitosan or a chitosan derivative in step T1. The lactose is present in the first component in a weight percentage of 1 to 10 wt%, and the chitosan or chitosan derivative is present in the first component in a weight percentage of 2 to 15 wt%.
The degree of deacetylation 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%; most preferably in the range of 80% to 100%. In addition, the molecular weight of chitosan is generally in the range of 5 to 200 ten thousand; preferably in the range of 10 to 200 ten thousand; more preferably in the range of 50 to 200 ten thousand; the optimum range is 100-200 ten thousand.
In addition, according to an aspect of the present invention, the chitosan derivative that can be used in the temperature-sensitive adjuvant of the present invention is not particularly limited; for example, the chitosan sugar derivative is selected from carboxymethyl chitosan sugar, chitosan sugar hydrochloride, chitosan sugar lactate, chitosan sugar acetate, chitosan sugar sulfate, chitosan sugar quaternary ammonium salt, chitosan oligosaccharide sugar, or chitin; preferably selected from carboxymethyl chitosans, chitosans hydrochloride, chitosans lactate, chitosans acetate, chitosans sulfate, or chitosans quaternary ammonium salts; more preferably selected from carboxymethyl chitosans, chitosans hydrochloride, chitosans lactate, chitosans acetate; most preferably selected from carboxymethyl chitosans or chitosans lactate.
According to the technical idea of the invention, the weight percentage concentration of the adjuvant in the second component is between 10 wt% and 50 wt%. Furthermore, the adjuvant is at least one selected from the group consisting of glycerin, propylene glycol, ethylene glycol, diethylene glycol monoethyl ether, glycerol ester, and polyethylene glycol fatty acid ester; preferably at least one selected from the group consisting of glycerol, propylene glycol, ethylene glycol, and diethylene glycol monoethyl ether; more preferably at least one selected from the group consisting of glycerin, propylene glycol, and ethylene glycol; at least one of glycerin and propylene glycol is preferable.
In addition, in the third component, the weight percentage concentration of the surfactant is 5 wt% to 30 wt%, and the surfactant is at least one selected from tween 20, tween 40, tween 60 and tween 80.
According to the technical idea of the present invention, in 100 parts by weight of the temperature-sensitive adjuvant, the first component is generally between 70 parts by weight and 85 parts by weight, preferably between 75 parts by weight and 85 parts by weight, and most preferably between 80 parts by weight and 85 parts by weight; the second component is generally between 10 parts by weight and 20 parts by weight, preferably between 10 parts by weight and 18 parts by weight, and most preferably between 10 parts by weight and 15 parts by weight; the third component is generally between 5 parts by weight and 10 parts by weight, preferably between 5 parts by weight and 8 parts by weight, and most preferably between 5 parts by weight and 7 parts by weight.
The temperature-sensitive adjuvant prepared by the steps comprises 1-20 wt% of a nutrient, 0.1-3.0 wt% of a surfactant, 0.5-8.0 wt% of an adjuvant and the balance of deionized water. In actual application, 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 mixed solution (Adg: Vat); the phase transition temperature of the vaccine mixed solution is 36-40 ℃, and the time for changing the liquid phase into the colloid is within 90 seconds. The coating rate of the temperature-sensitive adjuvant to the virus antigen solution is 50% or more, preferably 60% or more, more preferably 70% or more, and most preferably 80% or more.
Next, the present invention will be further described with reference to the following examples.
Preparation example 1
First, 10g of lactose was put into 840g of deionized water, stirred at room temperature until completely dissolved, and 30g of carboxymethyl chitosan (degree of deacetylation 80%, molecular weight about 80 ten thousand) was added and stirred at 15rpm until completely mixed to obtain the first component.
Then, 20g of glycerol is put into 100g of deionized water and stirred until the glycerol is completely dissolved to obtain a second component; 5g of tween80 was further put into 60g of deionized water and stirred until completely dissolved to obtain a third component.
After 880g of the first component, 120g of the second component and 65g of the third component are obtained through the steps, the second component is added into the first component, stirring is continued for 100 minutes, then the third component is added, stirring is continued for 30-100 minutes, the pH value is adjusted to be 4-8 by using a pH value adjusting agent, and then the temperature-sensitive adjuvant S1 is obtained. Next, the viscosity of temperature sensitive adjuvant S1 was measured using a viscosity agent and recorded in table 1.
Preparation example 2
First, 30g of lactose was put into 840g of deionized water, stirred at room temperature until completely dissolved, and 50g of carboxymethyl chitosan (degree of deacetylation 80%, molecular weight about 80 ten thousand) was added and stirred at 15rpm continuously until completely mixed to obtain the first component.
Then, 40g of glycerol is put into 100g of deionized water and stirred until the glycerol is completely dissolved to obtain a second component; then 10g of tween80 was put into 60g of deionized water and stirred until completely dissolved to obtain a third component.
After 920g of the first component, 140g of the second component and 70g of the third component are obtained through the steps, the second component is firstly put into the first component, stirring is continuously carried out for 100 minutes, then the third component is put into the first component, stirring is continuously carried out for 30-100 minutes, the pH value is adjusted to be 4-8 by a pH value adjusting agent, and then the temperature-sensitive adjuvant S2 is obtained. Next, the viscosity of temperature sensitive adjuvant S2 was measured using a viscosity agent and recorded in table 1.
Preparation example 3
First, 50g of lactose was put into 840g of deionized water, stirred at room temperature until completely dissolved, 70g of carboxymethyl chitosan (degree of deacetylation 80%, molecular weight about 80 ten thousand) was added, and stirring was continued at 15rpm until completely mixed to obtain a first component.
Then, 50g of glycerol is put into 100g of deionized water and stirred until the glycerol is completely dissolved to obtain a second component; 15g of tween80 was further put into 60g of deionized water and stirred until completely dissolved to obtain a third component.
After 960g of the first component, 150g of the second component and 75g of the third component are obtained through the steps, the second component is added into the first component, stirring is continuously carried out for 100 minutes, then the third component is added, stirring is continuously carried out for 30-100 minutes, the pH value is adjusted to be 4-8 through a pH value adjusting agent, and then the temperature-sensitive adjuvant S3 is obtained. Next, the viscosity of temperature sensitive adjuvant S3 was measured using a viscosity agent and recorded in table 1.
Preparation example 4
First, 70g of lactose was put into 840g of deionized water, stirred at room temperature until completely dissolved, and 100g of carboxymethyl chitosan (degree of deacetylation 80%, molecular weight about 80 ten thousand) was added and stirred at 15rpm continuously until completely mixed to obtain the first component.
Then, 70g of glycerol is put into 100g of deionized water and stirred until the glycerol is completely dissolved to obtain a second component; then, 20g of tween80 was put into 60g of deionized water and stirred until completely dissolved to obtain a third component.
After 1010g of the first component, 170g of the second component and 80g of the third component are obtained through the steps, the second component is firstly put into the first component, the stirring is continuously carried out for 100 minutes, then the third component is put into the first component, the stirring is further continued for 30-100 minutes, the pH value is adjusted to be 4-8 by using a pH value adjusting agent, and then the temperature-sensitive adjuvant S4 is obtained. Next, the viscosity of temperature sensitive adjuvant S4 was measured using a viscosity agent and recorded in table 1.
TABLE 1
Analysis of phase inversion time and phase inversion temperature
Temperature-sensitive adjuvants S1 to S4 obtained in preparation examples 1 to 4 were mixed with Phosphate Buffered Saline (PBS) at volume ratios shown in table 2, respectively, and heated, and the process of converting the mixed solution from liquid to gel at different ratios was observed, and the phase transition temperature and the phase transition time were recorded in table 2.
TABLE 2
From the results in table 2, it can be seen that the phase transition temperature of the temperature-sensitive adjuvant S1 at different ratios is between 25 ℃ and 42 ℃, and the phase transition time is within 90 seconds; the phase transition temperature of the temperature-sensitive adjuvant S2 in different proportions is 29.5-45 ℃, and the phase transition time is within 120 seconds; the phase transition temperature of the temperature-sensitive adjuvant S3 in different proportions is 30-44 ℃, and the phase transition time is within 131 seconds; the phase transition temperature of the temperature-sensitive adjuvant S4 is 31.5-44.5 ℃ in different proportions, and the phase transition time is within 141 seconds. Because the body temperature of general poultry is about 36-40 ℃, the ratio of the mixing volume ratio of the temperature-sensitive adjuvant (Adj) and the virus antigen liquid (Vat) of the invention can be about 1: 5(v/v) -1: 1 (v/v); in certain embodiments, it is preferred that the ratio of about 1: 3(v/v) -2: a range of 3 (v/v); most preferably about 3: 7(v/v), wherein the phase transition time of the temperature-sensitive adjuvant S1 is within 90 seconds.
Analysis of surface tension and Evaporation Rate
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-P4, and then the osmotic pressure, surface tension and evaporation rate of the mixed solutions P1-P4 in different temperature environments were measured, and the results are shown in Table 3.
The osmotic pressure was measured using a micro-type osmotic pressure analyzer, and the surface tension was measured using a surface tensiometer. The evaporation rate was calculated by placing 15g of the sample in an evaporation pan (diameter: 7cm) and placing the evaporation pan in a fume hood under various temperature conditions (relative humidity: 50%), and measuring the weight of the remaining sample in the evaporation pan at regular intervals.
TABLE 3
The results in Table 3 show that the osmotic pressures of the mixed solutions P1 to P4 are 280 to 300mOsm/kg, the surface tensions decrease with the temperature increase, and the evaporation rates decrease with the temperature increase.
Effect test on coating percentage of adjuvant
80ml of temperature-sensitive adjuvants S1-S4 are respectively put into a beaker, stirred by a homogenizer at the rotating speed of 3000rpm, then 20ml of 1mg/ml bovine serum albumin solution (the weight of the bovine serum albumin is 20mg) is slowly added, and after all the adjuvants are added, the rotating speed is adjusted to 5000rpm and the stirring is continued for 10 minutes.
Then, the mixed solution after stirring is heated to 38 ℃, so that the mixed solution is solidified into colloid, the colloid is put into 10ml PBS (pH 7.4,10mM,38 ℃) to be slightly shaken, so that the bovine serum albumin remained on the surface of the colloid and not coated is completely dissolved in the PBS, the clear solution is separated and put into a UV spectrophotometer, the absorption value under 280nm is measured, the concentration of the bovine serum albumin is converted by a standard calibration line of the bovine serum albumin solution, the total volume is 10ml, the total volume is the weight of the bovine serum albumin contained in the clear solution, and the calculation formula of the coating rate is as follows:
the calculated coating ratios are shown in table 4.
TABLE 4
| Example 1 | Example 2 | Example 3 | Example 4 | |
| Bovine serum albumin solution (ml) | 20 | 20 | 20 | 20 |
| Temperature sensitive adjuvant S1(ml) | 80 | 0 | 0 | 0 |
| Temperature sensitive adjuvant S2(ml) | 0 | 80 | 0 | 0 |
| Temperature sensitive adjuvant S3(ml) | 0 | 0 | 80 | 0 |
| Temperature sensitive adjuvant S4(ml) | 0 | 0 | 0 | 80 |
| Coating ratio (%) | 97.5 | 90.7 | 88.1 | 70.5 |
From the results of table 4, it is understood that the coating rates of the temperature-sensitive adjuvants S1, S2, and S3 used in examples 1 to 4 with respect to bovine serum albumin were 97.5%, 90.7%, 88.1%, and 70.5%, respectively.
Verification of Living toxin vaccine's potency
Firstly, Phosphate Buffer Solution (PBS) and temperature-sensitive adjuvants S1 to S4 obtained in preparation examples 1 to 4 were respectively put into a 2000 ml beaker according to the proportion shown in Table 5, a homogenizer was put into the beaker, the solution in the beaker was covered with a stirring rod of the homogenizer to 2/3 (about 15 cm), then the high-speed shearing force of the homogenizer (model: T.T-S.M-S, brand: Taiwan tripod machine, China) was turned to 3000rpm, and 400 ml of VII gene type Newcastle disease virus antigen liquid (10)6.5TCID50/ml) was added to the rotating solution; finally, when all antigen liquid is added, the rotating speed can be adjusted to 5000rpm, and the final stirring time is five to ten minutes, so that the live virus vaccines V1 to V4 are prepared.
Subsequently, the live vaccines V1 to V4 obtained above were each subjected to aerosol immunization with a nebulizer under the following conditions: the injection pressure is 2Kg/cm2～30Kg/cm2(ii) a The flow rate is 0.4-3L/min, the air flow temperature is 25-37 ℃, the air relative humidity is 50-80%, the vaccine solution is prepared into liquid aerosol which meets the requirements of 50-1000 microns of particle size distribution, 10-30% of fog particle concentration and microbiological activity on vaccine immunity, the chickens are immunized in the aerosol box in an atomizing mode, 10 immunized 1-day-old chickens exist in each aerosol box, and blood is collected in 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks and 6 weeks to separate serum to be tested.
Next, Hemagglutination (HA) tests were performed by adding 25. mu.l of 0.85% saline to each row of a 96-well U-disk, and then adding 25. mu.l of the test serum to row 1.
And then, after fully mixing by using a micropipette, adding 25 mu l of the serum to be detected in the row 2 into the second hole, and adding 25 mu l of the serum to be detected into the row 3, so that the sequence is diluted to the row 10, the antigen is diluted by 2 times to 1024 times, and 25 mu l of the mixed solution in the row 11 is not used. The test was repeated in each row and with 3 wells.
Then, the VII genotype Newcastle disease antigen solution was diluted with HI buffer to 25. mu.l of the VII genotype Newcastle disease antigen solution containing 8HAU per well, and 25. mu.l of the dilution was added to each well and left to stand for 15 minutes.
Then, 50. mu.L of 0.96% erythrocyte suspension was added to each well, and after standing at room temperature for 60 minutes, the hemagglutination was judged to be present or not, and the antibody titer was determined as the highest dilution factor, and then the average of the antibody titers of 10 chicken test results in each example was recorded in Table 5.
TABLE 5
From the results of table 5, it is understood that the in vivo potency of the live vaccines V1 to V4 administered to the chickens in examples 5 to 8 to which the temperature-sensitive adjuvant of the present invention was administered rapidly increased to 60 or more in week 1, reached the maximum value to 100 or more in week 2, and declined to 70 or more in week 3, but the in vivo potency was maintained at 70 or more.
In summary, the present invention has been described with reference to the above embodiments, but the present invention is not limited to these embodiments. Those skilled in the art to which the invention pertains will readily appreciate that various modifications and adaptations can be made without departing from the spirit and scope of the invention; for example, the technical contents exemplified in the above embodiments are combined or changed to new embodiments, and such embodiments are also regarded as the contents of the present invention. Therefore, the protection sought herein also includes the claims and their equivalents.
Claims (10)
1. The temperature-sensitive adjuvant for aerosol immunization is characterized by being used for preparing poultry vaccines and enabling the vaccines to be subjected to phase transition along with the change of external temperature, wherein the temperature-sensitive adjuvant at least comprises 1-20 wt% of a nutritional agent, 0.1-3.0 wt% of a surfactant, 0.5-8.0 wt% of an adjuvant and the balance of deionized water; wherein
The nutrient comprises chitosan or chitosan derivatives and lactose;
the surfactant is at least one selected from tween 20, tween 40, tween 60 and tween 80;
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 application, 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 mixed solution (Adg: Vat); the phase transition temperature of the vaccine mixed solution is 36-40 ℃, and the time for changing the liquid phase into the colloid is within 90 seconds.
2. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the coating rate of the temperature-sensitive adjuvant on a virus antigen solution is 50% or more.
3. The temperature-sensitive adjuvant for mist immunization according to claim 1, wherein the viscosity of the temperature-sensitive adjuvant is 5 to 100 Pa-s.
4. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the evaporation rate of the colloid is 1.5 μ g/cm2S is less than or equal to.
5. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the surface tension of the colloid is 20 to 150N/m.
6. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the colloid has an osmotic pressure of 280 to 300 mOsm/kg.
7. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the chitosan has a deacetylation degree in the range of 55 to 80% and a molecular weight in the range of 50 to 100 ten thousand.
8. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, wherein the chitosan derivative is selected from carboxymethyl chitosan, chitosan hydrochloride, chitosan lactate, chitosan acetate, chitosan sulfate, chitosan quaternary ammonium salt, chitosan oligosaccharide, or chitin.
9. The temperature-sensitive adjuvant for aerosol immunization according to claim 1, further comprising a pH adjuster such that the pH of the temperature-sensitive adjuvant is 4.0 to 8.0.
10. A preparation method of a temperature-sensitive adjuvant for aerosol immunization is characterized by comprising the following steps:
(a) adding lactose into deionized water, stirring to dissolve completely, adding chitosan or chitosan derivative, and mixing to obtain a first component;
(b) adding the auxiliary agent into deionized water, and stirring until the auxiliary agent 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; wherein
(d) Firstly, the second component is added into the first component, stirring is continuously carried out for 30-100 minutes, then the third component is added, and stirring is continuously carried out for 30-100 minutes, so that the temperature-sensitive adjuvant is obtained; wherein
In the first component, the weight percentage concentration of the lactose is 1-10 wt%, and the weight percentage concentration of the chitosan or the chitosan derivative is 2-15 wt%;
in the second component, the weight percentage concentration of the auxiliary agent is 10 wt% to 50 wt%, 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 5 wt% to 30 wt%, and the surfactant is at least one selected from tween 20, tween 40, tween 60 and tween 80; and
in 100 parts by weight of the temperature-sensitive adjuvant,
the weight part of the first component is between 70 and 85;
the weight part of the second component is between 10 and 20;
the weight portion of the third component is between 5 and 10.
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