CN108324940B - Use of glycerol-3-phosphate, adjuvant and vaccine agent containing glycerol-3-phosphate - Google Patents
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Abstract
The invention discloses application of glycerol-3-phosphate, an adjuvant containing the glycerol-3-phosphate and a vaccine agent, wherein the dosage of the glycerol-3-phosphate as the adjuvant in the vaccine is as follows: each single vaccine liquid contains 1-8 mg of glycerol-3-phosphoric acid. An adjuvant containing glycerol-3-phosphate comprises 54 μ L of physiological saline and 1-8 mg of glycerol-3-phosphate. The adjuvant has the advantages of easily available raw materials, simple preparation process, low cost, stable performance, small toxic and side effects, capability of quickly stimulating immune response, safety and reliability in use within the range of immune dose, capability of effectively inducing antigen-specific humoral immune response and remarkably enhancing antigen-specific humoral immune response, and the effect is superior to that of a non-adjuvant group and is equivalent to that of an aluminum adjuvant group.
Description
Technical Field
The invention relates to application of glycerol-3-phosphate, an adjuvant and a vaccine agent containing the glycerol-3-phosphate, belonging to the field of immunology.
Background
The vaccine is the most effective means for preventing and controlling infectious diseases of human beings, more than 250 ten thousand deaths can be avoided by vaccination every year, the vaccine put into use in the market generally consists of an antigen and an adjuvant, and the immune adjuvant plays a crucial role in playing the role of the vaccine. The adjuvant can quickly activate the immune system, which is more important for children and the old with low immunity. The development of technologies such as bioinformatics, structural biology, reverse genetics and the like provides a new direction and thought for the development of novel vaccines in the future, and the rapid development of the novel vaccines enables adjuvant safety and vaccine immunogenicity enhancing effects to be concerned. Adjuvants currently approved for human use, including aluminum salt adjuvants, MF59, AS03, AS04, and liposomes, have not been overlooked for some local skin inflammatory reactions, IgE antibody reactions, and safety issues. Therefore, the development of a novel high-efficiency, economical and side-effect-free immunologic adjuvant is a core task of future adjuvant development.
Glycerol-3-phosphate (G3P) is a very important substance in the growth process of prokaryotes, plants, animals and humans, and is an important metabolite associated with diseases, and is an important component of the human glycolysis and fat metabolism pathways. Medium chain fatty acid triglycerides (MCT) are hydrolyzed to free fatty acids and glycerol by esterases, and hepatocytes take up glycerol and react to glycerol-3-phosphate by glycerol kinase. There is evidence that medium chain fatty acid triglycerides (MCT) are able to enhance immune responses and alter the type of immune response. It is not reported whether glycerol-3-phosphate has an effect of enhancing immune response in animal experiments, but G3P is an important form of broad-spectrum antigen inducer in plants, called Systemic Acquired Resistance (SAR). The primary infection causes SAR and protects distal tissues from secondary infection, and genetic mutants deficient in G3P biosynthesis cannot induce SAR and can re-induce SAR only when supplied exogenously with G3P, thus G3P serves to enhance the immune response. Evidence suggests that G3P levels in plants are associated with the defence action of the fungal pathogen, Bacillus thuringiensis (Colletotrichum higginsianum), and that an increase in G3P enhances the defence action of Arabidopsis thaliana against attack by Bacillus thuringiensis. At present, no research report of using glycerol-3-phosphate as a vaccine adjuvant has appeared.
Disclosure of Invention
In order to solve the problems of high price, insignificant effect of promoting immune response and the like of the existing vaccine adjuvant, the invention provides the application of the glycerol-3-phosphate in preparing the vaccine adjuvant, and provides an adjuvant and a vaccine agent which are economic, effective, safe and stable and contain the glycerol-3-phosphate and a preparation method thereof.
The invention is realized by the following technical scheme: the application of glycerol-3-phosphate in preparing vaccine adjuvant.
The dosage of the glycerol-3-phosphate as an adjuvant in the vaccine is as follows: each single vaccine liquid contains 1-8 mg of glycerol-3-phosphoric acid.
Further, as a preferable example, the glycerol-3-phosphoric acid is used in an amount of 2 mg.
Another objective of the invention is to provide an adjuvant containing glycerol-3-phosphate, which comprises 54 mu L of physiological saline and 1-8 mg of glycerol-3-phosphate.
A vaccine formulation comprising glycerol-3-phosphate, said vaccine formulation being 300 μ L and consisting of:
a single vaccine liquid,
1-8 mg of glycerol-3-phosphoric acid,
The balance being normal saline.
The preparation method of the vaccine agent containing the glycerol-3-phosphate comprises the following steps:
(1) adding 1-8 mg of glycerol-3-phosphoric acid into 54 mu L of physiological saline, and obtaining an adjuvant mixed solution after the glycerol-3-phosphoric acid is completely dissolved;
(2) adding a single part of vaccine liquid into the adjuvant mixed liquid obtained in the step (1) to obtain the vaccine liquid containing the adjuvant;
(3) and (3) adding physiological saline into the adjuvant-containing vaccine liquid obtained in the step (2) to 300 mu L, and uniformly mixing to obtain the vaccine containing the glycerol-3-phosphoric acid.
Compared with the prior art, the invention has the following advantages and effects: (1) the adjuvant has the advantages of easily available raw materials, simple preparation process, low cost, stable performance and small toxic and side effects, can quickly stimulate an immune response reaction, promote the immune response of organisms and effectively improve the immune response level, and the glycerol-3-phosphate immune response reactivity is faster than that of an aluminum adjuvant, so that the adjuvant is very important for quickly inducing the immune response in case of sudden infectious diseases. (2) Glycerol-3-phosphate is an intermediate of animal and plant carbohydrate and lipid metabolism, avoids stimulation of exogenous substances to mice, has better safety theoretically when being used as an adjuvant compared with exogenous substances, has good safety when being observed by pathological tissue slices, and very accords with the conditions of high efficiency, economy and safety of the novel adjuvant.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to be illustrative only and are not intended to limit the scope of the invention, and the following examples are intended to be exemplary only, with no specific conditions being indicated, and the preferred conditions described herein being performed either under conventional conditions or under conditions recommended by the manufacturer.
Example 1
The hepatitis A vaccine provided in this example with the glycerol-3-phosphate adjuvant is: dissolving 2 mul of glycerol-3-phosphate in 54 mul of normal saline to obtain 1mg of glycerol-3-phosphate adjuvant solution, adding HAV antigen to adjuvant according to the conventional method (namely, single injection dosage used in animal experiment), finally adding normal saline (PH 7.4) to 300 mul, and mixing uniformly to obtain the hepatitis A vaccine containing glycerol-3-phosphate.
Glycerol-3-phosphate, also known as glycerophosphate, of the formula: c3H9O6P, molecular weight: 172.07 available from J&K, CASNO.57-03-4; the attenuated live vaccine for hepatitis A has antigen titer of 18EU/ml and is purchased from the institute of medical biology of Beijing cooperative medical college of Chinese medical science.
The immunity test and effect of the hepatitis A vaccine containing glycerol-3-phosphate adjuvant are as follows:
A. immunization
Randomly dividing 6-8 weeks old and 18-22g clean grade ICR mice into a glycerol-3-phosphate adjuvant group, an aluminum hydroxide adjuvant group, an adjuvant-free group and a blank group, wherein the total number of the groups is four, and each group comprises 7 mice; al (OH)3The adjuvant was provided by storage in the laboratory at a concentration of 12.5 mg/ml.
For the glycerol-3-phosphate adjuvant group: the hepatitis A vaccine of example 1 containing the glycerol-3-phosphate adjuvant group was injected into mice at a dose of 300. mu.L per mouse, wherein 300. mu.L of the vaccine contained 1mg of the glycerol-3-phosphate adjuvant and 18EU as hepatitis A antigen.
For the aluminum hydroxide adjuvant group: 300 mu g of aluminum hydroxide and 18EU of hepatitis A antigen are mixed, then physiological saline is added to 300 mu L, and the mixture is injected into mice by multiple points through skin, wherein the injection dose is 300 mu L per mouse.
For the non-adjuvant group: the hepatitis A antigen 18EU was mixed with physiological saline to 300uL, and then injected into mice at multiple subcutaneous injection doses of 300. mu.L per mouse.
For the blank group: saline was injected alone at a dose of 300uL per mouse.
Immunization protocol: the injection is subcutaneously injected into mice at multiple points for one time at week 0
B. ELISA for detection of serum anti-HAV IgG levels
At weeks 4, 8, 12, 16, and 20 after immunization, mouse tail vein blood was collected, serum was isolated, and serum anti-HAV IgG level was measured by ELISA, according to the instructions of mouse IgG ELISA kit manufactured by KPL corporation.
C. Data analysis
The obtained experimental data were t-tested with Graphpad prism5 statistical software, with P <0.05 as the statistical significance of the differences.
The antibody level trends of mouse serum anti-HAV IgG antibody levels in groups of 4, 8, 12, 16 and 20 weeks after immunization are increased and then decreased, and are highest in 8 weeks after immunization, the antibody levels in the groups of 4, 8 and 12 weeks after immunization are higher than those in a single antigen group, and the results of the glycerol-3-phosphate experimental groups in 4 weeks and 8 weeks have statistical significance compared with the single antigen group (P is less than 0.05); the results of the week 4 glycerol-3-phosphate experimental group were higher than those of the aluminum adjuvant group and statistically significant (P <0.05), see table 1.
TABLE 1
Example 2
The hepatitis A vaccine provided in this example with the glycerol-3-phosphate adjuvant is: dissolving 4. mu.l of glycerol-3-phosphate in 54. mu.l of physiological saline to obtain a glycerol-3-phosphate adjuvant solution with the content of 2mg, adding HAV antigen to the adjuvant according to the conventional method (namely, the single injection dosage used in animal experiments), finally adding physiological saline (PH 7.4) to 300. mu.L, and uniformly mixing to obtain the hepatitis A vaccine containing the glycerol-3-phosphate. The aluminium hydroxide adjuvant and HAV antigen were obtained in the same manner as in example 1.
The immunization test for the hepatitis A vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as in example 1, and the results are shown in Table 2. Table 2 shows the serum anti-HAV IgG antibody levels in each experimental group of mice within 20 weeks after the adjuvant provided in example 2 was used.
TABLE 2
Data analysis shows that the antibody level trends of mouse serum anti-HAV IgG antibody levels in groups of 4, 8, 12, 16 and 20 weeks after immunization are increased and then decreased, and are highest in 8 weeks after immunization, the antibody levels in the groups of 4, 8, 12, 16 and 20 weeks after immunization are higher than those in a single antigen group, the results of the glycerol-3-phosphate experimental groups in 4 weeks and 8 weeks have statistical significance (P is less than 0.05) compared with the single antigen group, and the glycerol-3-phosphate antibody water level in 4 weeks is larger than that in an aluminum adjuvant group on average and has statistical significance; the antibody level of the aluminum adjuvant group was greater than that of the antigen group alone at 4, 8, 12, 16, and 20 weeks.
Example 3
The hepatitis A vaccine provided in this example with the glycerol-3-phosphate adjuvant is: after dissolving 8. mu.l of glycerol-3-phosphate in 54. mu.l of physiological saline, 4mg of glycerol-3-phosphate adjuvant solution was obtained, each single HAV antigen (i.e., single injection dose used in animal experiments) was added to the adjuvant as usual, and finally physiological saline (pH 7.4) was added to 300. mu.l, and mixed well to obtain a hepatitis A vaccine containing glycerol-3-phosphate. The aluminium hydroxide adjuvant and HAV antigen were obtained in the same manner as in example 1.
The immunization test for the hepatitis A vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as in example 1, and the results are shown in Table 3. Table 3 shows the serum anti-HAV IgG antibody levels in each experimental group of mice within 20 weeks after the adjuvant provided in example 3 was used.
TABLE 3
As can be seen by data analysis, except for the blank group, anti-HAVA IgG was detected in each experimental group 4 weeks after immunization, and increased in time, peaked after 8 weeks, and gradually decreased thereafter. The glycerol-3-phosphate 4mg test group began to decline gradually at 12 weeks, but was greater than the non-adjuvant group, and was statistically significant (P <0.05) at 4 and 8 weeks, greater than the aluminum adjuvant group at 4 weeks, and statistically significant (P <0.05), but after 8 weeks, the antibody level was less than the aluminum adjuvant group. The glycerol-3-phosphate adjuvant is shown to enhance HAV antigen specific humoral immune response, and the effect is better than that of the adjuvant-free group, and the adjuvant effect is achieved.
Example 4
The hepatitis A vaccine provided in this example with the glycerol-3-phosphate adjuvant is: dissolving 16 mul of glycerol-3-phosphate in 54 mul of normal saline to obtain glycerol-3-phosphate adjuvant solution with the content of 8mg, adding HAV antigen into the adjuvant according to the conventional method (namely, the single injection dosage used in animal experiments), finally adding normal saline (PH is 7.4) to 300 mul, and uniformly mixing to obtain the hepatitis A vaccine containing the glycerol-3-phosphate. The aluminium hydroxide adjuvant and HAV antigen were obtained in the same manner as in example 1.
The immunization test for the hepatitis A vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as in example 1, and the results are shown in Table 4. Table 4 shows the serum anti-HAV IgG antibody levels in each experimental group of mice within 20 weeks after the adjuvant provided in example 4 was used.
TABLE 4
As can be seen from the data analysis, except for the blank group, anti-HAVA IgG was detected in each experimental group of mice after 4 weeks of immunization, and increased in the course of time, peaked after 8 weeks and gradually decreased at 12 weeks. The antibody level of the glycerol-3-phosphate 4mg experimental group is higher than that of the non-adjuvant group at 4 weeks and 8 weeks, which shows that the glycerol-3-phosphate adjuvant can enhance the HAV antigen specific humoral immune response, has better effect than that of the non-adjuvant group, and has adjuvant effect.
Example 5
The immunoassay for hepatitis B vaccine containing glycerol-3-phosphate adjuvant provided in this example: dissolving 4 mul of glycerol-3-phosphate in 54 mul of normal saline to obtain adjuvant solution of 2mg of glycerol-3-phosphate, adding each single part of hepatitis B surface antigen (namely single injection dosage used in animal experiments) into the adjuvant according to the conventional method, finally adding normal saline (PH 7.4) to 300 mul, and uniformly mixing to obtain the hepatitis B vaccine containing the glycerol-3-phosphate.
The hepatitis B surface antigen is a conventional commercial product containing 1 mu g of hepatitis B surface antigen and is purchased from institute of medical biology of Chinese academy of medical sciences.
The immunization test for the hepatitis B vaccine containing glycerol-3-phosphate adjuvant obtained in this example was performed as in example 1, and the effects are shown in Table 5. Table 5 shows the serum anti-HBsAg IgG antibody levels in each experimental group of mice within 20 weeks after the adjuvant provided in example 5 was used.
TABLE 5
The antibody level trends of mouse serum anti-HBV IgG antibody levels in groups of 4, 8, 12, 16 and 20 weeks after immunization are firstly increased and then decreased, and are the highest in 8 weeks after immunization, the antibody levels in the groups of 4, 8 and 12 weeks after immunization are higher than those in a single antigen group, the results of the glycerol-3-phosphate experimental groups in 4 weeks and 8 weeks are higher than those in a single antigen group, and the antibody level in 8 weeks is higher than that in an aluminum adjuvant group, but no statistical difference exists; the glycerol-3-phosphate adjuvant is shown to enhance the specific humoral immune response of the HBV antigen, the effect is better than that of the adjuvant-free group, the effect is equivalent to that of an aluminum adjuvant, and the adjuvant effect is achieved.
Example 6
The immunoassay for hepatitis B vaccine containing glycerol-3-phosphate adjuvant provided in this example: dissolving 8 mul of glycerol-3-phosphate in 54 mul of normal saline to obtain 4mg of glycerol-3-phosphate adjuvant solution, adding each single part of hepatitis B surface antigen (namely single injection dosage used in animal experiments) into the adjuvant according to the conventional method, finally adding normal saline (PH 7.4) to 300 mul, and uniformly mixing to obtain the hepatitis B vaccine containing the glycerol-3-phosphate.
The hepatitis B surface antigen is a conventional commercial product containing 1 mu g of hepatitis B surface antigen and is purchased from institute of medical biology of Chinese academy of medical sciences.
The immunization test for the hepatitis B vaccine containing glycerol-3-phosphate adjuvant obtained in this example was performed as in example 1, and the effects are shown in Table 6. Table 6 shows the serum anti-HBsAg IgG antibody levels in each experimental group of mice within 20 weeks after the adjuvant provided in example 6 was used.
TABLE 6
The antibody level trend of mouse serum anti-HBV IgG antibody levels in each group at 4, 8, 12, 16 and 20 weeks after immunization is firstly increased and then reduced, and is highest at 8 weeks after immunization, the antibody level in each group at 4, 8, 12, 16 and 20 weeks after immunization is higher than that in a single antigen group, the result of a 4-week glycerol-3-phosphate experiment group is higher than that of an aluminum adjuvant group, and the difference has statistical significance (P < 0.05).
Example 7
The vaccine containing the glycerol-3-phosphate adjuvant provided by the invention is as follows: after dissolving 2. mu.l of glycerol-3-phosphate in 54. mu.l of physiological saline, a glycerol-3-phosphate adjuvant solution with a content of 1mg was obtained, and each single rabies virus antigen (i.e., single injection dose used in animal experiments) was added to the adjuvant as usual, and finally physiological saline (pH 7.4) was added to 300. mu.l, and mixed well.
The glycerol-3-phosphate adjuvant vaccine was the same as that of example 1; the rabies virus antigen is a conventional commercial product containing 0.125IU of rabies virus antigen and is purchased from Dalian Hanxin biopharmaceutical Limited company.
The vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as example 1, and the effect is shown in Table 7. Table 7 shows the anti-rabies IgG antibody level in the serum of each experimental group of mice within 20 weeks after the adjuvant provided in example 7 was used.
TABLE 7
The antibody level trends of mouse serum anti-rabies virus IgG antibody levels in groups of 4, 8, 12, 16 and 20 weeks after immunization are firstly increased and then reduced, the antibody level trend is the highest in 8 weeks after immunization, and the antibody level of the adjuvant group is higher than that of the simple antigen group in 4, 8, 12, 16 and 20 weeks after immunization; the results of the week 4 glycerol-3-phosphate experimental group were higher than those of the aluminum adjuvant group with no statistical difference.
Example 8
The vaccine containing the glycerol-3-phosphate adjuvant provided by the invention is as follows: after 4. mu.l of glycerol-3-phosphate was dissolved in 54. mu.l of physiological saline, a glycerol-3-phosphate adjuvant solution with a content of 2mg was obtained, and each single rabies virus antigen (i.e., single injection dose used in animal experiments) was added to the adjuvant as usual, and finally physiological saline (pH 7.4) was added to 300. mu.l, and mixed well.
The glycerol-3-phosphate adjuvant vaccine was the same as that of example 1; the rabies virus antigen is a conventional commercial product containing 0.125IU of rabies virus antigen and is purchased from Dalian Hanxin biopharmaceutical Limited company.
The vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as example 1, and the effect is shown in Table 8.
Table 8 shows the anti-rabies IgG antibody level in the serum of each experimental group of mice within 20 weeks after the adjuvant provided in example 8 was used.
TABLE 8
The antibody level trends of mouse serum anti-rabies virus IgG antibody in groups of 4, 8, 12, 16 and 20 weeks after immunization are firstly increased and then reduced, the antibody level trend is the highest in 8 week after immunization, and the antibody level of the adjuvant group is higher than that of the simple antigen group in 4, 8, 12 and 16 weeks after immunization; the results of the week 4 glycerol-3-phosphate experimental group were higher than those of the aluminum adjuvant group with no statistical difference.
Example 9
The vaccine containing the glycerol-3-phosphate adjuvant provided by the invention is as follows: after dissolving 8. mu.l of glycerol-3-phosphate in 54. mu.l of physiological saline, a 4mg glycerol-3-phosphate adjuvant solution was obtained, to which each single rabies virus antigen (i.e., a single injection dose used in an animal experiment) was added conventionally, and finally physiological saline (pH 7.4) was added to 300. mu.l, and mixed uniformly.
The glycerol-3-phosphate adjuvant vaccine was the same as that of example 1; the rabies virus antigen is a conventional commercial product containing 0.125IU of rabies virus antigen and is purchased from Dalian Hanxin biopharmaceutical Limited company.
The vaccine containing glycerol-3-phosphate adjuvant obtained in this example was the same as example 1, and the effect is shown in Table 9.
Table 9 shows the anti-rabies IgG antibody level in the serum of each experimental group of mice within 20 weeks after the adjuvant provided in example 9 was used.
TABLE 9
The antibody level trends of mouse serum anti-rabies virus IgG antibody in groups of 4, 8, 12, 16 and 20 weeks after immunization are firstly increased and then reduced, the antibody level trend is the highest in 8 week after immunization, and the antibody level of the adjuvant-containing group is higher than that of the simple antigen group in 4, 8, 12 and 16 weeks after immunization; the results of the week 4 glycerol-3-phosphate experimental group were higher than those of the aluminum adjuvant group with no statistical difference.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (3)
1. The application of glycerol-3-phosphate as an adjuvant in the preparation of vaccine agents is characterized in that: the dosage of the glycerol-3-phosphate as an adjuvant in the vaccine agent is as follows: the vaccine agent was 300 μ L, wherein: each single vaccine agent contains 1-8 mg of glycerol-3-phosphoric acid, vaccine liquid and the balance of normal saline;
the vaccine is hepatitis A vaccine, hepatitis B vaccine or rabies vaccine.
2. Use according to claim 1, characterized in that: the dosage of the glycerol-3-phosphate as an adjuvant in the vaccine agent is as follows: each single vaccine formulation contained 2mg of glycerol-3-phosphate.
3. A method for preparing the vaccine agent using glycerol-3-phosphate as adjuvant according to claim 1, comprising the steps of:
(1) adding 1-8 mg of glycerol-3-phosphoric acid into 54 mu L of physiological saline, and obtaining an adjuvant mixed solution after the glycerol-3-phosphoric acid is completely dissolved;
(2) adding a single part of vaccine liquid into the adjuvant mixed liquid obtained in the step (1) to obtain the vaccine liquid containing the adjuvant;
(3) and (3) adding physiological saline into the adjuvant-containing vaccine solution obtained in the step (2) to 300 mu L, and uniformly mixing to obtain the vaccine agent containing the glycerol-3-phosphoric acid, wherein the vaccine is a hepatitis A vaccine, a hepatitis B vaccine or a rabies vaccine.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1184431A (en) * | 1995-04-20 | 1998-06-10 | 化工产品开发公司 | Therapeutic composition comprising an antigen or an in vivo generator for generating a compound comprising an amino acid sequence |
| CN104208664A (en) * | 2013-05-30 | 2014-12-17 | 上海医药工业研究院 | Vaccine composition liquid preparation used for nose and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1184431A (en) * | 1995-04-20 | 1998-06-10 | 化工产品开发公司 | Therapeutic composition comprising an antigen or an in vivo generator for generating a compound comprising an amino acid sequence |
| CN104208664A (en) * | 2013-05-30 | 2014-12-17 | 上海医药工业研究院 | Vaccine composition liquid preparation used for nose and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| Glycerol-3-phosphate is a critical mobile inducer of systemic immunity in plants;Bidisha Chanda et al.;《nature genetics》;20110327;第43卷(第5期);421-427 * |
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