CN116115748A - Al-Poly (I: C) composite adjuvant formed based on covalent interaction - Google Patents
Al-Poly (I: C) composite adjuvant formed based on covalent interaction Download PDFInfo
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Abstract
The invention discloses an Al-Poly (I: C) composite adjuvant formed based on covalent interaction. The main components of the composition comprise: the covalent bonding of the hydroxyl-containing aluminum adjuvant and the phosphate-containing nucleotide chain Poly (I: C) of the composite adjuvant is the covalent bonding of the hydroxyl group on the aluminum adjuvant and the phosphate group on the Poly (I: C) chain, and the chemical bonding mode ensures that the adjuvant exerts stronger immune effect. The immunogenicity is superior to that of aluminum adjuvant and Poly (I: C) adjuvant which are used independently, can induce high-level antigen-specific humoral immunity and cellular immune response at the same time, and has the effect of saving antigen.
Description
Technical Field
The invention belongs to the fields of biochemical engineering and biological medicine, and in particular relates to a compound adjuvant which is used for promoting vaccine preparations to induce disease-specific cellular immune responses in a host.
Background
Adjuvants can enhance their immunogenicity or enhance the host's immune response to antigens, playing an irreplaceable role in the play of vaccine immunization.
In the vaccine industry, aluminum adjuvants are the most traditional, most important, and most widely used vaccine adjuvants. There are 25 aluminum-containing adjuvants in FDA approved human vaccines. There are several explanations of the mechanism of action of aluminum salt-based adjuvants, one of which is the depot effect. The antigen is electrostatically adsorbed onto the adjuvant, which can be released continuously at the site of injection, enhancing the uptake and presentation of the antigen by Antigen Presenting Cells (APCs). After undergoing lysosomal disruption, the aluminum salt adjuvant induces NLRP3 inflammatory corpuscle activation under the stimulation of active oxygen, stimulates DCs to generate IL-1 beta and IL-18, promotes effector T cell CD4+ T cells to differentiate into effector B cells in Th 2 type immune response, and generates antibody IgG 1 IgE. In addition, aluminum salts as adjuvants can disrupt dendritic cell membranes, promote complement activation, and enhance immune responses by B cells and dendritic cells.
Polyinosinic acid-polycytidylic acid (abbreviated as Poly (I: C)) is a double-stranded product formed by pairing artificially synthesized Poly I and Poly C, and is a high-efficiency interferon inducer. Poly (I: C) recognizes activation of TLR 3 by TLR 3.Poly (I: C) can induce stable maturation of DCs, thereby inducing Th 1 type responses.
While a single adjuvant cannot meet all requirements of various disease prevention and treatment on the immune system, for example, aluminum adjuvant cannot induce Th 1 type reaction or has extremely low induction level; for the introduction of Poly (I: C) into the cytoplasm of non-immune cells (e.g., fibroblasts) may induce aberrant expression of class I and class II MHC molecules, there is a potential risk of autoreactive T cells and development of autoimmune diseases; in addition, poly (I: C) is readily hydrolyzed by nucleases in human and primate serum after injection and is difficult to exert in vivo. Therefore, the compound adjuvant is developed by compounding different adjuvants by utilizing physical or chemical action, and is an important means for solving the function deficiency of a single adjuvant.
Disclosure of Invention
The invention provides a method for forming a composite adjuvant based on covalent bonding, which makes clear the interaction between the adjuvants, maximally exerts the performance of the composite adjuvant, improves the compounding effect of a vaccine preparation, and lays a foundation for researching the structure-activity relationship of each component of the vaccine preparation.
The technical scheme of the invention is as follows:
an Al-Poly (I: C) composite adjuvant formed based on covalent interaction comprises an aluminum adjuvant containing hydroxyl groups and ribonucleic acid chain polyinosinic acid-polycytidylic acid (abbreviated as Poly (I: C)) containing phosphate groups, wherein the covalent bonding of the composite adjuvant is the covalent bonding of the hydroxyl groups on the aluminum adjuvant and the phosphate groups on the ribonucleic acid chain Poly (I: C)), the combination of the hydroxyl groups on the aluminum adjuvant and the ribonucleic acid chain Poly (I: C) is stable, and the hydroxyl groups on the excessive aluminum adjuvant can continuously adsorb antigens, so that the stable combination of the aluminum adjuvant, the Poly (I: C) and the antigens is realized. The chemical bonding mode of the invention enables the adjuvant to exert stronger immune effect, the immunogenicity of the adjuvant is superior to that of an aluminum adjuvant and a ribonucleic acid chain Poly (I: C) adjuvant which are independently used, the adjuvant can induce high-level antigen-specific humoral immunity and cellular immune response at the same time, and the adjuvant has the effect of saving antigens.
For the above technical scheme, preferably, the mass ratio of the hydroxyl-containing adjuvant to the ribonucleic acid chain containing phosphate group in the vaccine preparation is 1-100: 1, preferably 5 to 50:1. the hydroxyl groups on the excessive aluminum adjuvant can continuously adsorb the antigen, so that stable combination of the aluminum adjuvant, the Poly (I: C) and the antigen is realized.
For the above technical solution, preferably, the aluminum adjuvant containing hydroxyl group includes aluminum hydroxide adjuvant, aluminum phosphate adjuvant, and hydroxyapatite adjuvant.
The preparation method of the composite adjuvant based on covalent interaction comprises the following steps:
1) Preparing an aluminum adjuvant solution from an aluminum adjuvant containing hydroxyl groups, and uniformly mixing; wherein the concentration of the aluminum adjuvant solution containing hydroxyl is 1 mg/mL-50 mg/mL, preferably 5 mg/mL-20 mg/mL;
2) Adding ribonucleic acid chain Poly (I: C) solution containing phosphate groups into the aluminum adjuvant solution to obtain a mixed preparation; wherein the concentration of the solution of ribonucleic acid chain Poly (I: C) containing phosphate groups is 0.01 mg/mL-5 mg/mL, preferably 0.1 mg/mL-4 mg/mL; the volume ratio of the aluminum adjuvant solution containing hydroxyl to the ribonucleic acid chain Poly (I: C) solution containing phosphate groups is 10:1-1:3, preferably 1:2;
3) And vibrating the mixed preparation for a certain time to ensure that the mixed preparation and the mixed preparation are fully combined, thus obtaining the composite adjuvant.
For the above technical solution, in the step 3), the time of oscillation is preferably 10 to 60 minutes, and the rotation speed is 800 to 2000rpm.
In another aspect, the present invention provides a vaccine formulation comprising a complex adjuvant formed on the basis of covalent interactions as described above and an antigen, said antigen being one or more antigens, such as hepatitis b surface antigen; such as hepatitis b vaccine.
The preparation method of the vaccine preparation comprises the steps of mixing the composite adjuvant with 0.08 mg/mL-0.64 mg/mL antigen solution, wherein the composite adjuvant and the antigen solution are 10:1 to 1:1 (preferably 3:1-1:1) and uniformly shaking to obtain the vaccine preparation.
The vaccine preparation of the invention comprises the following main components in the content range:
the mass ratio of the aluminum adjuvant containing hydroxyl and ribonucleic acid chain Poly (I: C) containing phosphate groups to the antigen is 50 mug-2 mg:0.5-500 mug and 0.1-30 mug;
the preferred ranges are as follows:
the mass ratio of the hydroxyl-containing aluminum adjuvant, the ribonucleic acid chain Poly (I: C) containing phosphate groups and the antigen is 0.1-1.0mg, 1-250 mug and 0.5-15 mug;
the most preferred ranges are as follows:
the mass ratio of hydroxyl-containing aluminum adjuvant, phosphate-containing ribonucleic acid chain Poly (I: C) to antigen is 0.2-0.5mg, 2-100 μg and HBsAg 1-10 μg.
In still another aspect, the present invention also provides the use of the vaccine formulation described above for the preparation of an induced IgG antibody product, said antibody being IgG 1 And IgG 2c In (a) and (b)At least one kind.
In still another aspect, the invention also provides an application of the vaccine preparation in preparing a product for inducing B cell activation and CTL killing cell medium, wherein the mark for inducing B cell activation is CD27+/B220+; the sign of induction of CTL killing was cd107 α+/cd8+.
In a further aspect, the invention also provides the use of the vaccine formulation described above for inducing antigen dose sparing products, with an increase in total IgG antibodies.
The invention has the beneficial effects that:
the composite adjuvant of the invention is different from the existing aluminum and Poly (I: C) composite adjuvant in that the important role of combining hydroxyl and phosphate groups on the aluminum adjuvant and Poly (I: C) in the covalent adsorption process is emphasized, so that the composite adjuvant can more effectively stimulate organisms to generate high-level humoral immunity and cellular immunity, and has the effect of saving antigen dose.
The invention can prepare the Al-Poly (I: C) composite adjuvant formed by ligand exchange into hepatitis B vaccine (HBsAg+Al+Poly (I: C) composite adjuvant group vaccine) containing Al and Poly (I: C). The vaccine more effectively activates the special IgG 1 And IgG 2c Antibodies, and significantly activate B cells and CTL killing media.
Animal experiments show that the hepatitis B vaccine prepared by the compound adjuvant is safe and has no visible toxicity to test animals. Can effectively stimulate animals to produce high-titer specific antibodies.
Drawings
FIG. 1 is a transmission electron micrograph of AlOOH (A), alOOH-Poly (I: C) -L (B), alOOH-Poly (I: C) -H (C) of example 1. Wherein the scale is 100nm. Transmission electron microscopy shows that different degrees of aggregation occur after covalent bonding of Poly (I: C) and AlOOH, and that groups AlOOH-Poly (I: C) -H aggregate to a higher degree.
FIG. 2 is a Fourier transform infrared absorption spectrum (1300-900 cm) of AlOOH, alOOH-Poly (I: C) -L, alOOH-Poly (I: C) -H in example 1 -1 ) A drawing. Results show 1068cm -1 The absorption peak of AlOOH hydroxyl is obviously weakened, which proves that OH is on AlOOH - The sites are modified by PO on Poly (I: C) 3- Occupying.
FIG. 3 is a graph of ELISA detection of HBsAg specific antibodies in mouse serum total IgG antibodies in example 2. The results demonstrate that AlOOH-Poly (I: C) -H complex adjuvant induced specific antibody levels more significantly than Alhydrogel. The AlOOH-Poly (I: C) -H composite adjuvant showed that increasing the dosage of Poly (I: C) in the composite significantly increased the IgG antibody level compared to the effect of AlOOH-Poly (I: C) -L composite adjuvant.
FIG. 4 is an ELISA for detecting HBsAg-specific antibody IgG in mouse serum in example 2 1 Antibody profile. Serum was isolated from mice immunized with various adjuvant combinations and IgG was measured in the serum 1 Titers. The results showed that AlOOH-Poly (I: C) -L, alOOH-Poly (I: C) -H complex adjuvant and Alhydrogel adjuvant group ratio induced IgG production 1 The effect is stronger, wherein the AlOOH-Poly (I: C) -H compound adjuvant has more remarkable effect.
FIG. 5 is an ELISA for detecting HBsAg-specific antibody IgG in mouse serum in example 2 2c Antibody profile. Serum was isolated from mice immunized with various adjuvant combinations and IgG was measured in the serum 2c Titers. The results show that AlOOH-Poly (I: C) -L, alOOH-Poly (I: C) -H, poly (I: C) -H all significantly improves IgG over Alhydrogel 2c Titers, wherein AlOOH-Poly (I: C) -H complex adjuvant effect is more pronounced.
FIG. 6 is an ELISA for detecting HBsAg-specific antibody IgG in mouse serum in example 2 1 /IgG 2c Scale bar graph. HBsAg, alhydrogel Induction with IgG 1 Mainly comprises; igG induced by Poly (I: C), alOOH+Poly (I: C) complex adjuvant group 2c Mainly comprises; and AlOOH+Poly (I: C) compound adjuvant group can induce more IgG at the same time 1 And IgG 2c 。
FIG. 7 is an ELISA for detecting IFN-. Gamma.secretion by mouse spleen cells in example 2. The results show that the AlOOH+Poly (I: C) -H complex adjuvant group, the Poly (I: C) adjuvant group and the Alhydrogel adjuvant group all increased IFN-gamma release levels in mouse spleen cells, with the AlOOH+Poly (I: C) -H complex adjuvant group having significant advantages.
FIG. 8 is a graph showing the response of flow cytometry in example 2 to detection of B cells specific for HBsAg following ex vivo stimulation of mouse spleen cells. The Al-Poly (I: C) -H composite adjuvant significantly activated B cells.
FIG. 9 is a graph showing the response of flow cytometry in example 2 to detection of HBsAg specific T cells after ex vivo stimulation of mouse spleen cells. The CTL killing medium CD107 alpha+ induced by the Al-Poly (I: C) -H composite adjuvant is obviously improved.
FIG. 10 is a graph of ELISA detection of HBsAg specific antibodies in mouse serum total IgG antibodies in example 3. The results showed that there was no significant decrease in AlOOH-Poly (I: C) -H complex adjuvant group with consistent total IgG antibody levels when the HBsAg antigen dose injected into each mouse was reduced from 8 μg to 2 μg.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
Example 1
A preparation method of hepatitis B vaccine injection.
Raw materials and sources: CHO cells expressed hepatitis B virus surface antigen and were prepared to 160. Mu.g/mL by injection of physiological saline. Homemade aluminum hydroxide adjuvants (prepared by the method of reference (Sun B, ji Z, liao Y-P, et al engineering an Effective Immune Adjuvant by Designed Control of Shape and Crystallinity of Aluminum Oxyhydroxide Nanoparticles [ J ]. ACS Nano,2013,7 (12): 10834-10849)), designated AlOOH, were prepared by preparing 20mg/mL with physiological saline for injection and mixing with ultrasound. Polyriboinosine-polyribocytidylic acid poly (I: C) was purchased from Invivo Gen and was prepared at 0.2mg/mL and 2mg/mL, respectively, by injecting physiological saline and frozen at-20 ℃.
The preparation method of the vaccine comprises the following steps: 500. Mu.L of poly (I: C) was placed in a sterile centrifuge tube, 250. Mu.L of self-made aluminum hydroxide adjuvant was added, and the mixture was adsorbed in a metal bath at 1500rpm for 1 hour. Adding 250 mu L of hepatitis B virus surface antigen into a sterile centrifuge tube after uniform mixing, and uniformly mixing to obtain 1mL of vaccine preparation, namely, the vaccine preparation prepared by using 0.2mg/mL of Poly (I: C) is marked as AlOOH-Poly (I: C) -L, and the vaccine preparation prepared by using 0.2mg/mL of Poly (I: C) is marked as AlOOH-Poly (I: C) -H.
Example 2
The effect of the vaccine formulation of example 1 of the present invention and commercial aluminium hydroxide adjuvants, poly (I: C) adjuvants in two-needle simple protein immunization was investigated in comparison.
The experiment is as follows:
CHO cells expressed hepatitis B virus surface antigen (HBsAg) and were prepared to 160. Mu.g/mL by injection of physiological saline. Commercial aluminum hydroxide adjuvant (Alhydrogel), available from InvivoGen, designated Alhydrogel, was dried at 60℃to a solid and then prepared to 20mg/mL with physiological saline for injection, and sonicated. Homemade aluminum hydroxide (prepared by the method of Sun B, ji Z, liao Y-P, et al engineering an Effective Immune Adjuvant by Designed Control of Shape and Crystallinity of Aluminum Oxyhydroxide Nanoparticles [ J ]. ACS Nano,2013,7 (12): 10834-10849), designated AlOOH, was prepared by preparing 20mg/mL of saline for injection, and mixing with ultrasound.
Polyriboinosine-polyribocytidylic acid poly (I: C) was purchased from InvivoGen corporation. The injection physiological saline is respectively prepared into 0.2mg/mL and 2mg/mL, and the injection physiological saline is frozen at the temperature of minus 20 ℃.
The experimental group vaccine was prepared as in example 1: 500. Mu.L Poly (I: C) was placed in a sterile centrifuge tube, 250. Mu.L of self-made aluminum hydroxide adjuvant was added, and the mixture was adsorbed in a metal bath at 1500rpm for 1 hour. Adding 250 mu L of hepatitis B virus surface antigen into a sterile centrifuge tube after uniform mixing, and uniformly mixing to obtain 1mL of vaccine preparation.
The experimental group and the control group vaccines were prepared as above, and the formulation was as follows (see table 1):
table 1 hepatitis b vaccine formulation for animal experiments
Note that: alOOH is a commercial aluminum hydroxide when preparing the Alhydrogel control group; when preparing AlOOH control group, alOOH is self-made aluminum hydroxide; alOOH is self-made aluminum hydroxide when preparing AlOOH+Poly (I: C) experimental group.
Method of comparative experiment:
animal immunization: female C57BL/6 mice, 6-8 weeks old, without specific pathogen, were given 7 per group, and were intramuscularly inoculated with the 8 groups of hepatitis B vaccine of Table 1 on days 0 and 21 at a dose of 50. Mu.L for the posterior leg quadriceps. Eyeball blood was collected at 42 days, 0.1mL of blood was collected via the orbit, serum was isolated, the mice were sacrificed after blood collection by freezing at-80℃before measurement, and spleen cells were isolated.
Immune response type and intensity evaluation:
1. HBsAg specific antibodies Total IgG, igG in mouse serum 1 、IgG 2c ELISA assay of (C): 4. Mu.g/mL of HBsAg solution was prepared with 50mM carbonate buffer (pH 9.6), added to ELISA plates at 50. Mu.L/well, and incubated at 4℃for 12h; washing five times, adding 10% FBS at 100 μl/well, blocking at 37deg.C for 2 hr, gradient diluting the above groups of serum samples to 150 μl, adding 50 μl of diluted serum sample to coated HBsAg plate, incubating at 37deg.C for 2 hr, washing 5 times, and detecting mouse total IgG, igG with specificity of 50 μl/Kong Jiaru 1 、IgG 2c Antibody, incubation at 37℃for 2 hours, 50. Mu.L/well TMB color development solution (V Liquid A ：V Liquid B =1: 1, from BD Biosciences) was developed for 2min, 25. Mu.L/well 1MH was added 2 SO 4 The a450 value was determined. The serum of normal mouse is used as negative control, the experimental group/negative control is more than or equal to 2.0 and positive, the highest dilution of the serum is the total IgG, igG 1 Or IgG 2c Titers. The results are shown in FIGS. 2-6.
2. ELISA assay of cytokine IFN-gamma secretion by mouse spleen cells: specific mouse IFN-gamma capture antibodies were formulated with 50mM carbonate buffer (pH 9.6) and added to the coated ELISA plates at 50. Mu.L/well and incubated overnight at 4 ℃. Washing, adding 10% FBS at 100 μl/well, and incubating at room temperature for 1 hr. The IFN-. Gamma.standard was prepared in a corresponding concentration gradient by washing, and 50. Mu.L/well of the standard and sample were added to ELISA plates and incubated at room temperature for 2 hours. Wash, formulate affinity streptavidin-HRP and add 50 μl/well to ELISA plate and incubate at room temperature for 1 hour. Washing, 50. Mu.L/well TMB color development solution (V) Liquid A ：V Liquid B =1: 1, from BD Biosciences) was developed for 2min, 25. Mu.L/well 1M H was added 2 SO 4 The A450 OD value was measured. Drawing standard A450 OD valueAnd calculating the IFN-gamma concentration of the sample according to the IFN-gamma concentration standard curve. The results are shown in FIG. 7.
Ctl primary killing media detection: the isolated spleen cells were restimulated with 2. Mu.g/mL HBsAg for 60h and collected. 500g was centrifuged for 6min, each set of cells was resuspended in DPBS, 1. Mu.L of CD16/CD32 (from BD Biosciences) was added and incubated at 4℃for 30min; the single cell suspension was added to the centrifuge tube as a sample stain and the blank and single stain were removed, the sample stain was added to 1. Mu.L of anti-mouse CD8 (APC) and CD 107. Alpha. -PE) fluorescent antibodies, and the single stain was added to 1. Mu.L of anti-mouse CD8 (APC) or CD 107. Alpha. -PE fluorescent antibodies (ex BioLgend). Incubating for 30min at 4 ℃ in dark; centrifuge 500g for 6min, resuspended with 300 μl DPBS; the cells were resuspended in a 1.5mL centrifuge tube using 300. Mu.L of DPBS with 1% FBS and detected using a flow cytometer for 6min after centrifugation at 500 g. The data was processed through flowjo_v10 and the results are shown in fig. 8.
B cell activation assay: the method was similar to CTL primary killing media detection, except that fluorescent staining antibodies were used as anti-mouse B220 (APC) and CD27 (FITC) fluorescent antibodies (purchased from BioLgend), the results are shown in fig. 9.
Example 3
The vaccine formulation of example 1 of the present invention was studied in comparison with the effect of simple protein immunization at two different doses.
The experiment is as follows:
CHO cells expressed hepatitis B virus surface antigen (HBsAg) and were prepared at 160. Mu.g/mL and 640. Mu.g/mL by injection of physiological saline. Commercial aluminum hydroxide adjuvant (Alhydrogel), available from InvivoGen, designated Alhydrogel, was dried at 60℃to a solid and then prepared to 20mg/mL with physiological saline for injection, and sonicated. Polyriboinosine-polyribocytidylic acid poly (I: C) was prepared to 2mg/mL from InvivoGen by injecting physiological saline and frozen at-20 ℃.
The experimental group vaccine was prepared as in example 1: 500. Mu.L Poly (I: C) was placed in a sterile centrifuge tube, 250. Mu.L of self-made aluminum hydroxide adjuvant was added, and the mixture was adsorbed in a metal bath at 1500rpm for 1 hour. Adding 250 mu L of hepatitis B virus surface antigen into a sterile centrifuge tube after uniform mixing, and uniformly mixing to obtain 1mL of vaccine preparation.
Table 2 hepatitis b vaccine formulation for animal experiments
Note that:
AlOOH is a commercial aluminum hydroxide when preparing the Alhydrogel control group; when preparing AlOOH control group, alOOH is self-made aluminum hydroxide; alOOH is self-made aluminum hydroxide when preparing AlOOH+Poly (I: C) experimental group.
Each mouse in the HBsAg-1 control group, the Poly (I: C) -H-1 control group, and the AlOOH+Poly (I: C) -H-1 experimental group was injected with 2. Mu.g of antigen; each mouse in the HBsAg-2 control group, the Poly (I: C) -H-2 control group, and the AlOOH+Poly (I: C) -H-2 experimental group was injected with 8. Mu.g antigen.
Method of comparative experiment:
animal immunization: female C57BL/6 mice, 6-8 weeks old, no specific pathogen, 7 per group, were inoculated intramuscularly with the 8 groups of hepatitis B vaccine of Table 2 on days 0 and 21, at a dose of 50. Mu.L for the posterior leg quadriceps. Eyeball blood was collected at 42 days, 0.1mL of blood was collected via the orbit, serum was isolated, the mice were sacrificed after blood collection by freezing at-80℃before measurement, and spleen cells were isolated.
Immune response type and intensity evaluation:
1. ELISA assay of HBsAg specific antibody Total IgG in mouse serum: 4. Mu.g/mL of HBsAg solution was prepared with 50mM carbonate buffer (pH 9.6), added to ELISA plates at 50. Mu.L/well, and incubated at 4℃for 12h; washing five times, adding 10% FBS at 100 μl/well, blocking at 37deg.C for 2 hr, gradient diluting the above groups of serum samples to 150 μl, adding 50 μl of diluted serum sample to coated HBsAg plate, incubating at 37deg.C for 2 hr, washing 5 times, and detecting mouse total IgG, igG with specificity of 50 μl/Kong Jiaru 1 、IgG 2c Antibody, incubation at 37℃for 2 hours, 50. Mu.L/well TMB color development solution (V Liquid A ：V Liquid B =1: 1, from BD Biosciences) developed for 2minAdd 25. Mu.L/well 1M H 2 SO 4 The a450 value was determined. The serum of normal mice is used as negative control, the experimental group/negative control is more than or equal to 2.0 and positive, and the highest dilution of the serum is the total IgG. The results are shown in FIG. 10.
Claims (10)
1. An Al-Poly (I: C) composite adjuvant formed based on covalent interactions, characterized in that the composite adjuvant comprises an aluminum adjuvant containing hydroxyl groups and ribonucleic acid chains containing phosphate groups, the ribonucleic acid chains containing phosphate groups being Poly (I: C), the covalent bonding of the composite adjuvant being covalent bonding of hydroxyl groups on the aluminum adjuvant and phosphate groups on the Poly (I: C).
2. The composite adjuvant according to claim 1, wherein the mass ratio of the hydroxyl-containing aluminum adjuvant to the ribonucleic acid chain Poly (I: C) containing a phosphate group is 1 to 100:1.
3. the composite adjuvant according to claim 1, wherein the hydroxyl-containing aluminum adjuvant is one or more of an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant and hydroxyapatite.
4. A method for preparing an Al-Poly (I: C) composite adjuvant based on covalent formation according to any one of claims 1-3, characterized in that it comprises the steps of:
1) Preparing an aluminum adjuvant solution from an aluminum adjuvant containing hydroxyl groups; wherein the concentration of the aluminum adjuvant solution is 1 mg/mL-50 mg/mL;
2) Adding ribonucleic acid chain Poly (I: C) containing phosphate group into the aluminum adjuvant solution to obtain a mixed preparation; wherein the concentration of the solution of ribonucleic acid chain Poly (I: C) containing phosphate groups is 0.01 mg/mL-5 mg/mL; the volume ratio of the aluminum adjuvant solution containing hydroxyl to the ribonucleic acid chain solution Poly (I: C) with phosphate groups is 10:1-1:3;
3) And vibrating the mixed preparation for a certain time to ensure that the mixed preparation and the mixed preparation are fully combined, thus obtaining the composite adjuvant.
5. The method according to claim 4, wherein in the step 3), the shaking time is 10 to 60 minutes and the rotation speed is 800 to 2000rpm.
6. A vaccine formulation comprising a covalently based complex adjuvant according to any one of claims 1 to 5, such as a hepatitis b vaccine.
7. Use of the vaccine formulation of claim 6 for the preparation of an induced IgG antibody product.
8. The use according to claim 7, characterized in that the antibody is IgG 1 And IgG 2c At least one of them.
9. Use of the vaccine formulation of claim 6 for the preparation of a product that induces B cell activation, CTL killing, cell mediators.
10. Use of the vaccine formulation of claim 6 for inducing antigen dose sparing products.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102949717A (en) * | 2012-07-06 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis B vaccine preparation containing poly I:C adjuvant |
| CN108853488A (en) * | 2018-06-29 | 2018-11-23 | 广东华南疫苗股份有限公司 | A kind of EV71 vaccine containing adjuvant combination |
| WO2019126371A1 (en) * | 2017-12-19 | 2019-06-27 | Massachusetts Institute Of Technology | Antigen-adjuvant coupling reagents and methods of use |
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| CN102949717A (en) * | 2012-07-06 | 2013-03-06 | 中国疾病预防控制中心病毒病预防控制所 | Novel hepatitis B vaccine preparation containing poly I:C adjuvant |
| WO2019126371A1 (en) * | 2017-12-19 | 2019-06-27 | Massachusetts Institute Of Technology | Antigen-adjuvant coupling reagents and methods of use |
| CN108853488A (en) * | 2018-06-29 | 2018-11-23 | 广东华南疫苗股份有限公司 | A kind of EV71 vaccine containing adjuvant combination |
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