CN111658617A - Aluminum adjuvant-containing vaccine freeze-dried preparation and preparation method and application thereof - Google Patents

Aluminum adjuvant-containing vaccine freeze-dried preparation and preparation method and application thereof Download PDF

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CN111658617A
CN111658617A CN201910970876.1A CN201910970876A CN111658617A CN 111658617 A CN111658617 A CN 111658617A CN 201910970876 A CN201910970876 A CN 201910970876A CN 111658617 A CN111658617 A CN 111658617A
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孙逊
张志荣
龚涛
白淑婷
侯盈盈
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Abstract

The invention discloses a freeze-dried preparation of a vaccine containing an aluminum adjuvant, and a preparation method and application thereof. The aluminum salt vaccine lyophilized preparation contains: the freeze-drying method provided by the invention avoids the massive aggregation of aluminum hydroxide in the vaccine and reduces the damage of the antigen activity. The invention can improve the stability of the vaccine in the freeze-drying process and the storage process, can enable the vaccine to get rid of cold chain transportation, reduce the cost, save energy and particularly solve the technical problem that the low-temperature cold chain in remote areas is difficult to guarantee.

Description

Aluminum adjuvant-containing vaccine freeze-dried preparation and preparation method and application thereof
Technical Field
The invention belongs to the technical field of vaccine production processes, and particularly relates to a freeze-dried preparation containing an aluminum adjuvant vaccine, a preparation method and application thereof.
Background
The application of the vaccine greatly reduces the morbidity and mortality of common epidemic diseases, so that a plurality of epidemic diseases are effectively controlled. In recent years, with the deeper understanding of the mechanisms related to the immune system, the development of vaccines has advanced greatly, which not only performs more excellently in the field of traditional prevention and treatment, but also starts to show up in the head and horn in the diseases such as HIV and cancer. Vaccines have become an indispensable drug in human health care today.
As the application of vaccines is more and more extensive, the stability problem during the production, transportation and storage of the vaccines is more and more concerned. Lyophilization techniques provide more favorable conditions for vaccine stability. The dried protein medicine is in a loose cake shape, has low water content and more stable physical and chemical properties, can prolong the storage life, reduce the weight, reduce the transportation cost and the like. However, lyophilization is a complex phase transition process, and various stresses, including low temperature stress, freezing stress, drying stress, etc., are present in the lyophilized product throughout the process, which are often factors that lead to denaturation of the proteins in the product. Most of the freeze-drying processes used at present are complex, because the quality of freeze-dried products is obviously affected due to the setting and the change of various parameters in the freeze-drying technology, and in the freeze-drying process, the quality of vaccines is adversely affected due to improper temperature and time, so that the vaccines lose activity. Various freeze-drying protective agents and excipients can be added to prevent the protein drug from being denatured, but the complex components of the freeze-drying protective agents can easily cause adverse reactions, and the complex freeze-drying process also causes the cost of the freeze-dried preparation to be high. Thus, although some lyophilized vaccine formulations are already on the market, the production of a vaccine lyophilized product with high efficiency, safety and low cost is still a problem to be solved.
The aluminum adjuvant is proposed from the beginning of the 20 th century to the use history of up to eighty years, which fully proves the safety and the effectiveness of the aluminum adjuvant, and most of the vaccines such as hepatitis B vaccine, HPV vaccine, tetanus vaccine and the like which are commercially available at present contain the aluminum adjuvant. The aluminum adjuvant adsorbs the antigen by mixing with the antigen to form an antigen reservoir in the body so as to prolong the retention time of the antigen in the body; and locally stimulate related cells to release inflammatory factors, promote antigen presentation of antigen presenting cells, induce generation of various immune cells, induce Th2 type immune response, and generate more effective immune response than free antigen ("Munks, M.W. Toward san understating of the administration of aluminum", Marrack et al, Natureevews.immunology, Vol. 9 of 2009, No. 4, pp 287-93, No. 4 of 2009). Most vaccines now use aluminium adjuvants. However, all aluminum-adjuvant-containing vaccines currently on the market cannot be frozen or lyophilized (vaccine (6 th edition), Stanley Hem et al, people health press, first chapter of the book, fifth chapter, page 85, 2017), and a non-scattering coagulum is formed after freezing, so that the structure and immunogenicity of the vaccine are destroyed. Therefore, the product must be preserved at 2-8 ℃, which brings inconvenience to the storage and transportation of the vaccine.
The breadth of China is broad, the climate difference between the south and the north is large, the cold chain long-distance transportation brings many challenges, in addition, the influence of factors such as the vehicle condition of a refrigerated truck, the external environment temperature, the road condition, the flight arrangement and the like in the cold chain transportation process can cause the problems of cold chain 'chain breakage' and the like in the transportation link, and the stability and the effectiveness of the vaccine are directly influenced. Moreover, the high cost and high risk transportation mode also severely limits the use of vaccines in remote areas such as rural areas.
Therefore, how to provide a vaccine preparation containing aluminum adjuvant which can be lyophilized, avoid cold chain transportation, improve the stability and safety of the vaccine, and reduce the cost and risk is a problem which needs to be solved clinically.
Disclosure of Invention
One of the objectives of the present invention is to provide a lyophilized preparation of aluminum-adjuvant-containing vaccine, a method for preparing the same, and uses thereof, wherein the lyophilized preparation has the same immunogenicity and higher thermal stability compared with the non-lyophilized preparation, and can reduce the production cost and solve the problem of cold chain transportation of the vaccine.
One of the objects of the present invention is to provide a lyophilized vaccine preparation containing an aluminum adjuvant, preferably an aluminum salt adjuvant, and more preferably an aluminum hydroxide adjuvant.
One of the objectives of the present invention is to provide a method for freeze-drying aluminum adjuvant-containing vaccine, which is characterized in that the vaccine preparation is nanoparticle or microparticle, preferably nanoparticle, preferably with a particle size of 500nm to 50nm, more preferably 300nm to 50nm, and even more preferably 150nm to 80 nm.
The invention aims to provide a method for freeze-drying a vaccine containing an aluminum adjuvant, which is characterized in that a freeze-drying protective agent is added into a vaccine preparation.
The freeze-drying protective agent comprises polysaccharides, polyols, polymers, amino acids, surfactants and anhydrous solvents, wherein the polysaccharides comprise one or more of trehalose, sucrose, glucose, maltose, glucan and dextrin, the polyols comprise mannitol and glycerol, the polymers comprise one or more of PVP, PEG and PVA, the surfactants comprise one or more of Tween 80, poloxamer 188 and Arabic gum, the amino acids comprise one or more of L-serine, arginine, tryptophan, lysine hydrochloride, sodium glutamate, alanine, glycine, sarcosine and phenylalanine, and the anhydrous solvents comprise DMSO, preferably one or more of trehalose, sucrose, glucose, sorbitol and mannitol.
The mass concentration of the freeze-drying protective agent is 2-15% (w/v), more preferably 2-10%, and even more preferably 5%.
The aluminum adjuvant nanoparticles are aluminum salt nanoparticles formed by the interaction of aluminum salt and an anionic polymer or a derivative thereof. Preferably, the aluminum hydroxide is generated by using aluminum sulfate under an alkaline condition, the aluminum hydroxide has positive charges, the anionic polymer or the derivative thereof has negative charges, and the anionic polymer can modify the aluminum hydroxide through electrostatic interaction, so that the precipitation of the aluminum hydroxide is effectively limited and the aluminum hydroxide is excessively aggregated and grown, and a nano-scale aluminum hydroxide semi-finished product with better stability and easy dispersion is obtained.
The anionic polymer or the derivative thereof used in the vaccine freeze-dried preparation is characterized in that the natural or endogenous anionic polymer material comprises: one or more of gamma-polyglutamic acid, mucopolysaccharide, polymannuronic acid, polyguluronic acid, hyaluronic acid, chondroitin, heparin sodium, fucoidan, fucogalactan, alginate, agar, gellan, ghatti gum, karaya gum, tragacanth, keratan, alginic acid, dextran, xylomannan sulfate, gellan gum, xanthan gum, carrageenan; the semisynthetic derivative anionic polymeric material comprises: one or more of heparin sulfate, chondroitin sulfate, keratan sulfate, dextran sulfate, carboxymethyl cellulose, cross-linked caramel, carboxymethyl starch, carboxymethyl dextran, carboxymethyl chitosan, hyaluronic acid derivatives, rhamsan sulfate, cellulose sulfate, curdlan sulfate, and chitosan phosphate; the fully synthetic anionic polymeric material comprises: polyanionic polypeptide, polyacrylic acid, polymethacrylic acid, polyglutamic acid, polyaspartic acid grafted polyethylene glycol, polyglutamic acid grafted polyethylene glycol, polycarbophil, carboxyvinyl polymer, maleic anhydride copolymer, and thiolated polyacrylate.
The anionic polymer can be modified by derivatization, but is not limited to modification of PEG, carboxyl, sulfate radical, sulfite radical, phosphate radical and phosphite radical.
The anionic polymer may be a linear polymer, a crosslinked polymer, or a branched copolymer.
An object of the present invention is to provide a lyophilized formulation of an aluminum-adjuvanted vaccine, characterized in that the antigen is selected from the group consisting of: the protein antigens comprise hepatitis B surface antigen HBsAg, recombinant PreS1, PreS2, recombinant core protein, hepatitis C virus antigen, hepatitis E virus antigen, anthrax toxin binding protein, streptococcus pneumoniae protein, group A M protein, streptococcus C5a peptidase, zonulin binding protein, serine carboxylesterase, meningococcal outer membrane protein type B OMP, chicken ovalbumin, pale dense helical body surface lipoprotein, bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, soybean albumin, wheat albumin, myoglobin, collagen and fibrillin; or a toxoid such as diphtheria toxoid, tetanus toxoid, cholera toxin, or a polysaccharide such as group a polysaccharide, capsular polysaccharide, pneumococcal polysaccharide, typhoid polysaccharide, meningococcal polysaccharide, or a virus-like particle such as human papilloma virus, rotavirus, or an inactivated or attenuated vaccine antigen such as pertussis toxin, hepatitis a virus, encephalitis b virus, rabies virus, polio virus antigen, influenza virus, cytomegalovirus, rhino influenza antigen, varicella-zoster virus, mumps antigen, rubella virus, smallpox virus, measles, chicken pox, yellow fever, poliomyelitis virus strain, respiratory syncytial virus; or attenuated and killed bacteria such as live Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordetella pertussis, group A Streptococcus, Neisseria meningitidis, Legionella pneumophila, Vibrio cholerae, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Treponema pallidum; or polysaccharide-protein complexes such as pneumococcal polysaccharide conjugates, meningococcal polysaccharide-protein conjugates; or nucleic acids, single and double stranded nucleic acids, supercoiled plasmid DNA, linear plasmid DNA, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, mammalian artificial chromosomes, RNA; or synthetic polypeptides such as: group A streptococcus M peptide, TRP2, HGP100, p15E, pseudomonas aeruginosa synthetic peptide, rubella virus synthetic peptide; or one or more of tumor cell lysate antigen, bacterial lysate antigen and tumor whole cell antigen.
The invention provides a freeze-dried vaccine preparation containing an aluminum adjuvant, which is characterized by also containing other adjuvants except the aluminum adjuvant, wherein the other adjuvants comprise: cytokines such as GM-CSF, interferon, interleukins IL-2, IL-6, IL-1, IL-12, Toll-like receptor agonists such as CpG-ODN, double stranded RNA (PolyI: C), MPLA, polysaccharide adjuvants such as inulin, chitosan, dextran, cholera toxin B subunits, E.coli heat labile enterotoxin LT, lipopolysaccharide LPS, heat shock protein HSP, saponin adjuvants, Limquid.
As one of the preferred embodiments of the present invention, a lyophilized formulation containing aluminum hydroxide nanoparticles is characterized in that the nanoparticles are formed by mainly compounding an anionic polymer or its derivative with aluminum hydroxide while adsorbing antigen and/or other adjuvants, and then adding a lyoprotectant, wherein the ratio of aluminum hydroxide: anionic polymer or derivative thereof: antigen: other adjuvants: the proportion of the freeze-drying protective agent is 0.152-1.52: 0.12-3: 0.01-0.1: 0-0.1: 10 to 150. Further preferred are aluminum hydroxide: anionic polymer or derivative thereof: antigen: other adjuvants: the proportion of the freeze-drying protective agent is 0.152-1.52: 0.12-2.4: 0.01-0.1: 0.0001 to 0.1: 20 to 150.
The preferred anionic polymer or its derivative in the invention is one or more of PEG-poly (AGE-Suc-Eti), chondroitin sulfate, polyglutamic acid grafted polyethylene glycol and gamma-polyglutamic acid.
As a preferred embodiment of the invention, the anionic polymer derivative used in the lyophilized vaccine preparation of the invention is PEG-poly (AGE-Suc-Eti), herein referred to as PEG-Eti, wherein the length of PEG unit is between 20 and 120, and the length of poly (AGE-Suc-Eti) unit is between 1 and 50. The structural formula is as follows:
Figure RE-GDA0002261843690000041
as one of the preferable embodiments of the invention, the anionic polymer used in the lyophilized vaccine preparation of the invention is chondroitin sulfate, and the structural formula is as follows:
Figure RE-GDA0002261843690000051
as one of the preferable embodiments of the invention, the anionic polymer used in the lyophilized vaccine preparation of the invention is polyglutamic acid grafted polyethylene glycol, the length of the polyglutamic acid unit is 50-220; the grafted polyethylene glycol unit length is 500-1200, and the molecular weight of the polymer is 30000-70000 daltons.
The structural formula is as follows:
Figure RE-GDA0002261843690000052
as one of the preferable embodiments of the invention, the anionic polymer used in the lyophilized vaccine preparation of the invention is gamma-polyglutamic acid which is obtained by biological fermentation and has a molecular weight of 1000-
The structural formula is as follows:
Figure RE-GDA0002261843690000053
one of the purposes of the invention is to provide a preparation method of a freeze-dried preparation containing an aluminum adjuvant vaccine, which is characterized by comprising the following steps:
(1) mixing Hepes buffer solution with anionic polymer material or its derivative, antigen and/or adjuvant uniformly to obtain phase A;
(2) taking an aluminum sulfate aqueous solution as a B phase;
(3) mixing the A phase and the B phase to obtain a semi-finished product;
(4) adding a freeze-drying protective agent into the semi-finished product, and uniformly mixing;
(5) pre-freezing the semi-finished product at-40 deg.C to-80 deg.C for 4-24 h;
(6) when the temperature of the freeze dryer is reduced to-40 ℃, the vacuum pressure of a freeze dryer system is reduced to 0.1-0.01 mbar, and the freeze drying time is more than or equal to 24 hours.
As a preferred embodiment of the invention, the following are calculated at a final volume of 1 ml:
wherein the Hepes buffer solution in the step (1) is 30-200 mM.
Wherein the amount of the anion composition material or the derivative thereof in the step (1) is 0.1mg to 3 mg.
Wherein the antigen amount in the step (1) is 0.01-0.1 mg.
Wherein the amount of the aluminum sulfate in the step (2) is 0.975-9.75 mu mol, and the aluminum content in the aluminum sulfate is converted into Al (OH)3The amount of the compound is 0.152-1.52 mg.
Wherein, the mixing mode in the step (3) is mechanical mixing, including but not limited to stirring, vortex, ultrasonic and constant speed injection pump mixing.
Wherein, the amount of the freeze-drying protective agent in the step (4) is 20-150 mg, and preferably 50 mg.
The particle size of the nanoparticle semi-finished product prepared by the steps is 80-300 nm before freeze-drying, the potential is + 10-minus 30mV, the freeze-dried product after freeze-drying is loose and quick to redissolve, the product is completely dissolved within 20s, the particle size after redissolution is 80-300 nm, and the potential is + 10-minus 30 mV.
The preparation method of the invention is simple and rapid, has low finished product and mild conditions, and can not cause the denaturation and inactivation of the antigen.
Compared with the existing commercial aluminum-adjuvant-containing vaccine (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001), the aluminum hydroxide freeze-dried preparation containing the aluminum adjuvant does not have the phenomena of obvious agglomeration and inactivation of antigen after being frozen.
The invention selects OVA, HBsAg and tetanus toxoid as model antigens, and in vivo and in vitro experiments prove that the vaccine freeze-dried preparation containing the aluminum adjuvant and the preparation which is not freeze-dried have the same immune effect, thereby effectively ensuring the activity of the vaccine while realizing freeze-drying of the vaccine.
As one of the preferable embodiments of the invention, when the aluminum content of the prepared vaccine freeze-dried preparation is 0.166mg/ml, the antibody IgG, IgG1 and IgG2a subtype levels generated by the recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001) are equivalent to those of the commercial hepatitis B vaccine with the aluminum content of 0.35mg/ml, and therefore, the application obtains the equivalent immune effect with the existing commercial product under the lower adjuvant dosage, and therefore, the application greatly reduces the dosage of the aluminum adjuvant and has higher safety.
As one of the preferred embodiments of the invention, when the aluminum content of the prepared vaccine freeze-dried preparation is 0.166mg/ml, compared with the commercial hepatitis B vaccine with the aluminum content of 0.35mg/ml (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001), the generated CD8 is generated+IFN-γ+The content of T cells is better, and therefore, the application obtains better cell immune effect under the condition of lower adjuvant dosage.
As one of the preferred embodiments of the present invention, when the prepared vaccine lyophilized preparation has an aluminum content of 0.105mg/ml, the produced antibody IgG and IgG1 levels are equivalent to those of a commercial tetanus vaccine with an aluminum content of 0.69mg/ml (Doctorou Biotechnology Ltd., product batch No. 20170813), the produced IgG2a subtype level is superior, the IgG1 subtype antibody level reflects the humoral immune response level, and the IgG2a subtype level reflects the cellular immune response level, so that the application can produce the humoral immune response equivalent to that of the commercial vaccine with a lower adjuvant dosage, and produce a better cellular immune response effect.
As one of the preferable embodiments of the invention, the prepared vaccine freeze-dried preparation can be used for freeze-dried powder injection, spray and the like; can be stored and transported at normal temperature, and avoids cold chain transportation.
Advantageous effects
The preparation method of the vaccine freeze-dried preparation is simple and quick, and has good repeatability, high stability and low cost.
The freeze-dried vaccine preparation has low aluminum content, and can induce equivalent or stronger immune response without the risk of local side reaction and metal ion accumulation under the condition that the aluminum content is far lower than that of the commercial aluminum gel adsorption vaccine.
The vaccine freeze-dried preparation has high thermal stability, can be stored and transported at normal temperature, can get rid of cold chain transportation, reduces the cost and improves the popularization rate of the vaccine in remote areas.
Compared with the prior art, the invention has the following advantages:
the freeze-drying protective agent used as the vaccine freeze-drying preparation containing the aluminum adjuvant has single component and is not easy to generate adverse reactions such as allergy and the like.
The invention can realize the freeze drying of the vaccine containing the aluminum adjuvant, greatly improve the stability of the vaccine containing the aluminum adjuvant, get rid of the cold chain transportation of the vaccine, save resources and reduce cost.
The freeze-dried vaccine preparation has low aluminum content, and can induce equivalent or stronger immune response without the risk of local side reaction and metal ion accumulation under the condition that the aluminum content is far lower than that of the commercial aluminum gel adsorption vaccine.
The preparation method is simple and rapid, the freeze-drying process is simple, the expanded production is easy, and the application prospect is wide.
Drawings
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings
FIG. 1a is a particle size diagram of a semi-finished product of aluminum hydroxide-PEG-Eti-HBsAg nanoparticles
FIG. 1b is a particle size diagram of a nanoparticle intermediate product of aluminum hydroxide-PEG-Eti-tetanus toxoid
FIG. 1c is a particle size diagram of a semi-finished product of aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles
FIG. 1d is a particle size diagram of a semi-finished product of aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles
FIG. 2a is a particle size diagram of aluminum hydroxide-PEG-Eti-HBsAg nanoparticle lyophilized preparation redissolved
FIG. 2b is a particle size diagram of the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle lyophilized preparation redissolved
FIG. 2c is a particle size diagram of aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle lyophilized preparation for reconstitution
FIG. 2d is a particle size diagram of the aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticle lyophilized preparation redissolved
FIG. 3 is a transmission electron microscope image of aluminum hydroxide-PEG-Eti-HBsAg nanoparticles before freeze-drying
FIG. 4 is a transmission electron microscope picture of aluminum hydroxide-PEG-Eti-HBsAg nano particle frozen preparation redissolution
FIG. 5 is an antibody of OVA nanoparticle lyophilized formulation of aluminum hydroxide immunized with mice at day 21
FIG. 6 shows the OVA nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IFN-gamma+CD8+Percentage of T cells
FIG. 7 shows the OVA nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IL-4+CD4+Percentage of T cells
FIG. 8 shows the antibody of HBsAg nanoparticle lyophilized preparation of aluminum hydroxide on day 21 of immunization of mice
FIG. 9 shows the antibody of HBsAg nanoparticle lyophilized preparation of aluminum hydroxide on day 35 of mouse immunization
FIG. 10 shows the HBsAg nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IFN-gamma+CD8+Percentage of T cells
FIG. 11 shows HBsAg nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IL-4+CD4+Percentage of T cells
FIG. 12 shows antibodies from mice immunized on day 21 with lyophilized formulation of tetanus toxoid nanoparticles of aluminum hydroxide
FIG. 13 shows antibodies from mice immunized on day 28 with lyophilized formulation of tetanus toxoid nanoparticles of aluminum hydroxide
FIG. 14 shows the antibody of HBsAg nanoparticle lyophilized preparation of aluminum hydroxide stored at 20 + -5 deg.C for one week on day 35 of immunized mice
FIG. 15 shows that HBsAg nanoparticle lyophilized preparation of aluminum hydroxide is stored at 20 + -5 deg.C for one week to immunize mouse splenocyte IFN-gamma+CD8+Percentage of cells
FIG. 16 shows that HBsAg nanoparticle lyophilized preparation of aluminum hydroxide is stored at 20 + -5 deg.C for one week to immunize mouse splenocyte IL-4+CD4+Percentage of T cells
Detailed description of the preferred embodiments
The following examples are further illustrative of the present invention and are in no way intended to limit the scope of the invention. The present invention is further illustrated in detail below with reference to examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation method used. Also, equivalent substitutions, combinations, improvements or modifications of the invention may be made by those skilled in the art based on the description of the invention, but these are included in the scope of the invention.
Example 1
Preparing a semi-finished product of aluminum hydroxide-chondroitin sulfate-egg white albumin (OVA) nanoparticles: add 60. mu.l of chondroitin sulfate 28mg/ml to 320. mu.l of Hepes buffer solution with 100mmol/LPH of 7.7, mix well as phase A; sucking 400 μ L of 10mmol/L aluminum sulfate solution, 40 μ L of 1000 μ g/ml OVA solution and 40 μ L of 100 μ g/ml oligodeoxynucleotide adjuvant CpG1826 solution, uniformly mixing to obtain phase B, dropwise adding the phase B into the phase A under the condition of vortex, and vortex for 30 s; 45mg of glucose was added to the nanoparticle solution and dissolved sufficiently.
Example 2
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: adding 60 μ L of 30mg/ml chondroitin sulfate into 400 μ L of 1000mmol/L Hepes buffer solution with pH of 7.6, and mixing well to obtain phase A; sucking 400 mu L of 10mmol/L aluminum sulfate solution and 400 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; 63mg trehalose was added to the nanoparticle solution and dissolved thoroughly.
Example 3
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: add 60. mu.l of chondroitin sulfate 28mg/ml to 320. mu.l of 90mmol/L Hepes buffer solution with pH 7.6, mix well as phase A; sucking 400 mu L of 10mmol/L aluminum sulfate solution and 350 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; 60mg of glucose was added to the nanoparticle solution and dissolved sufficiently.
Example 4
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: add 70. mu.l of 25mg/ml chondroitin sulfate to 330. mu.l of 100mmol/L Hepes buffer solution with pH 7.7, mix well as phase A; sucking 200 mu L of 20mmol/L aluminum sulfate solution and 400 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; add 50mg trehalose to the nanoparticle solution and dissolve it thoroughly.
Example 5
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: add 120. mu.l of 20mg/ml chondroitin sulfate to 350. mu.l of 80mmol/L Hepes buffer solution with pH 7.8, mix well as phase A; sucking 160 mu L of 20mmol/L aluminum sulfate solution and 200 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; 41.5mg trehalose was added to the nanoparticle solution and dissolved thoroughly.
Example 6
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: add 80. mu.l 25mg/ml chondroitin sulfate to 320. mu.l 100mmol/L Hepes buffer solution with pH 7.6, mix well as phase A; sucking 200 mu L L20 mmol/L aluminum sulfate solution and 350 mu L50 mu g/ml hepatitis B surface antigen solution, uniformly mixing to obtain phase B, dropwise adding the phase B into the phase A under the condition of vortex, and vortex for 30 s; add 40mg trehalose to the nanoparticle solution and dissolve thoroughly.
Example 7
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles: adding 160. mu.l of 10mg/ml chondroitin sulfate to 400. mu.l of 110mmol/L Hepes buffer solution with pH of 7.7, and mixing well to obtain phase A; sucking 200 mu L of 20mmol/L aluminum sulfate solution and 350 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; add 55mg of sucrose to the nanoparticle solution and dissolve it thoroughly.
Example 8
Preparing an aluminum hydroxide-PEG-Eti-egg white albumin (OVA) nanoparticle semi-finished product: add 60. mu.l 10mg/ml PEG-Eti to 380. mu.l 100mmol/L Hepes buffer solution with pH 8, mix well as phase A; sucking 200 μ L of 20mmol/L aluminum sulfate solution, uniformly mixing with 40 μ L of 1000 μ g/ml OVA solution and 40 μ L of 100 μ g/ml oligodeoxynucleotide adjuvant CpG1826 solution to obtain phase B, dropwise adding the phase B into the phase A under the condition of vortex, and vortex for 30 s; then 60mg of trehalose is added into the nanoparticle solution and fully dissolved.
Example 9
Preparing a semi-finished product of the aluminum hydroxide-PEG-Eti-HBsAg nano-particle: add 80. mu.l 10mg/ml PEG-Eti material to 320. mu.l 100mmol/L Hepes buffer at pH 8 and mix well as phase A; sucking 200 mu L of 20mmol/L aluminum sulfate solution and 400 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; then 50mg of trehalose is added into the nanoparticle solution and fully dissolved.
Example 10
Preparing a semi-finished product of the aluminum hydroxide-PEG-Eti-HBsAg nano-particle: add 90. mu.l 10mg/ml PEG-Eti material to 380. mu.l 100mmol/L Hepes buffer solution with pH 8.2, mix well as phase A; sucking 400 mu L of 10mmol/L aluminum sulfate solution and 350 mu L of 50 mu g/ml hepatitis B surface antigen solution, uniformly mixing the solution to be used as a phase B, dropwise adding the phase B into the phase A under the vortex condition, and vortexing for 30 s; and adding 70mg of sucrose into the nanoparticle solution, and fully dissolving.
Example 11
Preparing a semi-finished product of the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle: add 125. mu.l 10mg/ml PEG-Eti material and 150. mu.l 450Lf/ml toxoid solution to 300. mu.l 100mmol/L Hepes buffer solution with pH 8.3, mix well as phase A; sucking 375 mu L of 10mmol/L aluminum sulfate solution as a B phase, dropwise adding the B phase into the A phase under the vortex condition, and vortexing for 30 s; then 80mg of trehalose is added into the nanoparticle solution and fully dissolved.
Example 12
Preparing a semi-finished product of the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle: add 115. mu.l 10mg/ml PEG-Eti material and 160. mu.l 450Lf/ml toxoid solution to 350. mu.l 90mmol/L Hepes buffer solution with pH 8.2, mix well as phase A; sucking 300 mu L of 10mmol/L aluminum sulfate solution as a B phase, dropwise adding the B phase into the A phase under the vortex condition, and vortexing for 30 s; then 50mg of trehalose is added into the nanoparticle solution and fully dissolved.
Example 13
Preparing a semi-finished product of the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle: add 160. mu.l of 8mg/ml PEG-Eti material and 200. mu.l of 450Lf toxoid solution to 400. mu.l of 100mmol/L Hepes buffer solution with pH 8.2, mix well as phase A; sucking 400 mu L of 10mmol/L aluminum sulfate solution as a B phase, dropwise adding the B phase into the A phase under the vortex condition, and vortexing for 30 s; then 80mg of trehalose is added into the nanoparticle solution and fully dissolved.
Example 14
Preparing a semi-finished product of the aluminum hydroxide-chondroitin sulfate-tetanus toxoid nanoparticle: adding 160. mu.l of 10mg/ml chondrosulfate material and 200. mu.l of 450Lf toxoid solution to 400. mu.l of 100mmol/L Hepes buffer solution with pH 8.2, and mixing well to obtain phase A; sucking 400 mu L of 10mmol/L aluminum sulfate solution as a B phase, dropwise adding the B phase into the A phase under the vortex condition, and vortexing for 30 s; add 80mg of sucrose to the nanoparticle solution and dissolve it thoroughly.
Example 15
Preparing a semi-finished product of the aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles: adding 125 mul of 10mg/ml polyglutamic acid grafted polyethylene glycol material into 400 mul of 90mmol/L Hepes buffer solution with pH of 8, uniformly mixing, sucking 290 mul of 9mmol/L aluminum sulfate solution and 200 mul of 400ug/ml HBsAg solution, uniformly mixing, adding into the mixed solution, swirling for 30s, adding 60mg trehalose into the nanoparticle solution, and fully dissolving to obtain the chitosan nanoparticle.
Example 16
Preparing a semi-finished product of the aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticles: adding 120 mu L of 10mg/ml polyglutamic acid grafted polyethylene glycol material into 380 mu L of Hepes buffer solution with 100mmol/LPH of 8, uniformly mixing, sucking 280 mu L of 8mmol/L aluminum sulfate solution and 240 mu L of 10ug/ml HBsAg solution, uniformly mixing, adding into the mixed solution, swirling for 35s, adding 60mg sucrose into the nanoparticle solution, and fully dissolving to obtain the chitosan nanoparticle.
Example 17
Preparing a nano-particle semi-finished product of aluminum hydroxide-gamma-polyglutamic acid-egg white albumin (OVA): adding 110 mu L of 10mg/ml gamma-polyglutamic acid into 380 mu L of Hepes buffer solution with 80mmol/LPH of 8, uniformly mixing, sucking 500 mu L of 2 mmol/L aluminum sulfate solution, 100 mu L of 2mg/ml OVA solution and 10 mu L of 2mg/ml CpG1826 solution, uniformly mixing, adding the mixture into the solution, performing ultrasonic treatment for 5min at the power of 120w, adding 60mg of sucrose into the nanoparticle solution, and fully dissolving.
Example 18
Preparing a semi-finished product of aluminum hydroxide-chondroitin sulfate-egg white albumin (OVA) nanoparticles: adding 10ml of 10mg/ml chondroitin sulfate material and 3ml of 1mg/ml OVA solution into 20ml of Hepes buffer solution with the concentration of 100mmol/LPH of 7.9, uniformly mixing, adding a syringe No. 1, adding 33ml of 4.04mmol/L aluminum sulfate solution into a syringe No. 2, simultaneously passing through a special-shaped three-channel microfluidic device by a syringe pump at the speed of 50ml/min, and collecting mixed liquid; add 3.3g trehalose to the nanoparticle solution and dissolve thoroughly.
Example 19
Preparing a semi-finished product of aluminum hydroxide-chondroitin sulfate-egg white albumin (OVA) nanoparticles: adding 10ml of 10mg/ml chondroitin sulfate material and 3ml of 1mg/ml OVA solution into 20ml of Hepes buffer solution with the concentration of 100mmol/LPH of 7.8, uniformly mixing, adding a syringe No. 1, adding 33ml of 6.06mmol/L aluminum sulfate solution into a syringe No. 2, simultaneously passing through a special-shaped three-channel microfluidic device at the speed of 20ml/min by virtue of injection pumps, and collecting mixed liquid; then 3.5g of sucrose was added to the nanoparticle solution and dissolved sufficiently.
Example 20
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: packaging the aluminum hydroxide-chondroitin sulfate-chicken Ovalbumin (OVA) nanoparticle semi-finished product in the example 1 into a penicillin bottle, and pre-freezing for 6 hours at-40 ℃; vacuumizing to 0.1mbar, heating to-30 ℃, heating to-20 ℃ after 8-10 hours, and heating to-10 ℃ after 4-6 hours for 6-8 hours to obtain the composite material.
Example 21
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle semi-finished product in the example 2 is filled into a penicillin bottle and pre-frozen for 4 hours at the temperature of-40 ℃; vacuum pressure is controlled at 0.1mbar, temperature is constant at-50 deg.C, and freeze drying is carried out for 24 h.
Example 22
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle semi-finished product in the example 5 is filled into a penicillin bottle and pre-frozen for 5 hours at the temperature of-40 ℃; vacuum-pumping to 0.1mbar, constant temperature of-52 deg.C, and freeze-drying for 24 h.
Example 23
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: packaging the aluminum hydroxide-PEG-Eti-chicken Ovalbumin (OVA) nanoparticle semi-finished product obtained in the example 8 into a penicillin bottle, and pre-freezing for 4 hours at-40 ℃; vacuumizing to 0.1mbar, heating to-30 ℃, heating to-20 ℃ after 8-10 hours, heating to-10 ℃ after 4-6 hours, and maintaining for 6-8 hours.
Example 24
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: the aluminum hydroxide-PEG-Eti-HBsAg nanoparticle semi-finished product in the example 9 is filled into a penicillin bottle and pre-frozen for 6h at-80 ℃; vacuum pressure is controlled at 0.01mbar, temperature is constant at-40 deg.C, and freeze drying is carried out for 24 h.
Example 25
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle semi-finished product obtained in the example 11 is filled into a penicillin bottle and pre-frozen for 6 hours at the temperature of-40 ℃; vacuum pressure is controlled at 0.02mbar, temperature is constant at-50 deg.C, and freeze drying is carried out for 24 h.
Example 26
The freeze-drying method of the aluminum hydroxide nanoparticles comprises the following steps: the aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticle semi-finished product in the example 15 is filled into a penicillin bottle and pre-frozen for 5 hours at the temperature of-40 ℃; vacuum pressure is controlled at 0.02mbar, temperature is constant at-52 deg.C, and freeze drying is carried out for 24 h.
Example 27
Appearance and redissolution of the aluminum hydroxide nanoparticle lyophilized preparation: as shown in fig. 1, the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle lyophilized preparation prepared in example 21 has a uniform color, and is loose and full; as shown in FIG. 2, the aluminum hydroxide-chondroitin sulfate-HBsAg nano-grade semi-finished product prepared in example 21 is clear and transparent, has no precipitate, and has no visible particle foreign matter; as shown in FIG. 3, the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle lyophilized preparation prepared in example 21 can be rapidly reconstituted within 10s by adding 1.26mL of water for injection, and the appearance is clear and transparent after reconstitution.
Example 28
Particle size determination of nanoparticles: the particle size distribution of the nanoparticles was determined using a Zetasizer Nano ZS90 laser particle size analyzer. Respectively taking 200 mul of the nanoparticle semi-finished product solution in examples 2, 9, 11 and 15, or respectively adding 860 mul, 1260 mul, 830 mul and 1025 mul of injection water into the freeze-dried powder in examples 20, 24, 25 and 26 for redissolving, then taking 200 mul of the nanoparticle solution, respectively placing the samples into a sample pool for detection, and setting the measurement temperature to be 25 ℃, and the results are shown in figures 1a-d and 2a-d, wherein figure 1a is a particle size diagram of the aluminum hydroxide-PEG-Eti-HBsAg nanoparticle semi-finished product in example 9, figure 1b is a particle size diagram of the aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle semi-finished product in example 11, figure 1c is a particle size diagram of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle semi-finished product in example 2, figure 1d is a particle size diagram of the aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticle semi-finished product in example 15, figure 1d, Fig. 2a is a particle size diagram of aluminum hydroxide-PEG-Eti-HBsAg nanoparticle lyophilized formulation redissolved in example 24, fig. 2b is a particle size diagram of aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle lyophilized formulation redissolved in example 25, fig. 2c is a particle size diagram of aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle lyophilized formulation redissolved in example 21, and fig. 2d is a particle size diagram of aluminum hydroxide-polyglutamic acid grafted polyethylene glycol-HBsAg nanoparticle lyophilized formulation redissolved in example 26. It can be seen that the particle size of the nanoparticle semi-finished product and the lyophilized redissolved preparation is not greatly changed, the particle size of the nanoparticles is between 80-200nm, the PDI is less than 0.3, the distribution is uniform, and the specific particle size results are shown in Table 1
TABLE 1 detection of particle size before and after nanoparticle lyophilization
Sample (I) Size/nm PDI
Example 8 110.2±8.93 0.160±0.023
Example 23 redissolved particle size 124.6±9.13 0.176±0.031
Example 1 105.6±5.23 0.208±0.043
Example 20 redissolved particle size 104.2±7.34 0.234±0.039
Example 9 90.02±7.33 0.197±0.015
Example 24 redissolved particle size 108.07±13.07 0.204±0.027
Example 2 133.03±16.09 0.211±0.022
Example 21 redissolved particle size 140.6±14.28 0.181±0.069
Example 11 85.43±12.87 0.182±0.029
Example 25 redissolved particle size 90.23±11.34 0.198±0.013
Example 15 113.37±5.23 0.198±0.043
Example 26 redissolved particle size 125.21±12.56 0.212±0.032
Example 29
Nano particle transmission electron microscope: dropwise adding the nanoparticle solution on a copper mesh, standing for 3min, dyeing for 2min by using 2% phosphotungstic acid, sucking away redundant dye liquor on the copper mesh by using filter paper, airing the sample at room temperature, observing the sample under the condition of 200kv, and observing by using a transmission electron microscope. The results are shown in fig. 3 and fig. 4, wherein fig. 3 is a transmission electron microscope image of the aluminum hydroxide-PEG-Eti-HBsAg nanoparticle semifinished product (before freeze-drying) of example 9, and fig. 4 is a transmission electron microscope image of the aluminum hydroxide-PEG-Eti-HBsAg nanoparticle frozen preparation of example 24 after reconstitution with water for injection. The figure shows that the nanoparticles are round particles before and after freeze-drying, the particle size is between 80 and 200nm, and the freeze-drying process does not influence the morphology of the nanoparticles.
Example 30
Screening the dosage of the freeze-drying protective agent: the preparation method of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles comprises the following steps: add 200. mu.l 10mg/ml chondroitin sulfate to 300. mu.l 100mmol/L Hepes buffer solution with pH 7.6, mix well as phase A; 300 μ L of 10mmol/L aluminum sulfate solution and 200 μ L of 50 μ g/ml hepatitis B surface antigen solution are sucked and mixed uniformly as phase B, phase B is added dropwise to phase A under the condition of vortex, and vortex for 30 s. 10mg, 20mg, 30mg, 50mg, and 100mg of trehalose were added, and after sufficient dissolution, the nanoparticle lyophilized preparation was prepared according to the method in example 21. The change in the measured particle diameter is shown in Table 2
TABLE 2 influence of trehalose in different proportions on the particle size of the lyophilized preparation of Alumoxychloride-chondroitin sulfate-HBsAg sodium
Figure RE-GDA0002261843690000151
As a result, the trehalose content is less than 2%, and the particle size of the aqueous solution of the lyophilized preparation is more than 300nm and up to 500nm, so that the concentration of the lyoprotectant is preferably 2% or more. When the trehalose proportion reaches 10%, the particle size of the aqueous solution nanoparticles of the freeze-dried preparation is less than 200 nm. Thus the trehalose of the invention may be used in an amount of 2% to 10%, preferably 5%.
Example 31
Screening the types of the freeze-drying protective agents: the preparation method of the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticles comprises the following steps: add 160. mu.l 10mg/ml chondroitin sulfate to 320. mu.l 100mmol/L Hepes buffer solution with pH 7.6, mix well as phase A; 400 μ L of 10mmol/L aluminum sulfate solution and 400 μ L of 50 μ g/ml hepatitis B surface antigen solution are sucked and mixed uniformly as phase B, phase B is added dropwise to phase A under the condition of vortex, and vortex for 30 s. Glucose, sucrose, trehalose, sorbitol and mannitol were added in different amounts, and after sufficient dissolution, the nanoparticle lyophilized preparation was prepared according to the method in example 20. The change in the measured particle diameter is shown in Table 3
TABLE 3 influence of different lyoprotectants on the particle size of the nanoparticle lyophilized formulation
Figure RE-GDA0002261843690000152
From the results, various freeze-drying protective agents can be used for preparing the aluminum hydroxide-chondroitin sulfate-HBsAg nanoparticle freeze-drying preparation.
Example 32
The particle size change of the nanoparticle lyophilized preparation at 20 + -5 deg.C storage is shown in Table 4
TABLE 4 nanoparticle lyophilized formulations of aluminum ammoxid with particle size variation at 20 + -5 deg.C storage
Figure RE-GDA0002261843690000161
Example 33
The particle size change of the nanoparticle lyophilized preparation at 37 + -5 deg.C is shown in the following table
TABLE 5 nanoparticle lyophilized formulations of aluminum ammonium oxide with variation in particle size at 37 + -5 deg.C storage
Figure RE-GDA0002261843690000162
Example 34
Stability accelerated test of nanoparticle lyophilized preparation, the nanoparticle lyophilized preparations of examples 5 and 22 were placed at 43 ℃ and RH 75% at different time points and tested for particle size change, and the results are shown in Table 6
TABLE 6 variation of particle size at different time points for the nanoparticle semi-finished product and lyophilized formulation
Figure RE-GDA0002261843690000171
The experimental result shows that the nanoparticle freeze-dried preparation has better stability than the nanoparticle without freeze-drying, and can be kept stable for at least 40 days under the conditions of 43 ℃ and RH 75%.
Example 35
In vivo immunization experiment of mice: day 0, 7, 14 immunization, C57BL/6 mice were injected subcutaneously with 25. mu.l of free OVA solution, 25. mu.l of aluminum salt-OVA nanoparticle formulation (1.5. mu.g OVA per mouse). On day 21, blood was drawn from the orbit and the amount of OVA-specific antibodies in the serum was measured. The results are shown in FIG. 5, in which free represents the OVA antigen group, APN represents the aluminum hydroxide-PEG-Eti-OVA group (example 8), APN (L) represents the corresponding lyophilized formulation group (example 23), ASN represents the aluminum hydroxide-chondroitin sulfate-OVA group (example 1), and ASN (L) represents the corresponding lyophilized formulation group (example 20). According to experimental results, the aluminum-adjuvant-containing OVA vaccine lyophilized preparation of the present invention induces antibody levels comparable to those of the aluminum salt-OVA nanoparticle preparation without lyophilization, and has no significant difference (P is greater than 0.5), and is higher than free OVA, and has significant differences (P is 0.0028 between APN (L) and free in IgG antibody level, P is 0.0106 between APN (L) and free in IgG1 antibody level, and P is 0.0361 between APN (L) and free in IgG2a antibody (APN L) and free). Data analysis the mean values between groups were compared using a one-factor variance method. The value P less than 0.05 is statistically significant.
Example 36
Mice were immunized in vivo for 0, 7, 14 days, and C57BL/6 mice were injected subcutaneously with 25. mu.l of free OVA solution and 25. mu.l of aluminum salt-OVA nanoparticle formulation (1.5. mu.g OVA per mouse). On day 21, the spleen was removed from the sacrificed mice, red blood cells were lysed, stained, fixed, punched, re-stained, and finally the intracellular factor levels were flow-tested. The results are shown in FIGS. 6 and 7, in which free represents the OVA antigen group, APN represents the aluminum hydroxide-PEG-Eti-OVA group (example 8), APN (L) represents the corresponding lyophilized formulation group (example 23), ASN represents the aluminum hydroxide-chondroitin sulfate-OVA group (example 1), ASN (L) represents the corresponding lyophilized formulation group (example 20), and in which IFN-gamma of mouse splenocytes immunized with OVA nanoparticle lyophilized formulation of aluminum hydroxide in FIG. 6 is+CD8+T cell percentage, it can be seen from the results that the aluminum adjuvant-containing OVA vaccine lyophilized preparation of the present invention induces cytokine levels comparable to those of the aluminum salt-OVA nanoparticle preparation without lyophilization, with no significant difference (P of ASN and ASN (l) ═ 0.9907, P of APN and APN (l) ═ 0.9994), and higher than that of free OVA, with significant difference (P of ASN group and free group is 0.05); FIG. 7 shows the OVA nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IL-4+CD4+T cell percentage, it can be seen from the experimental results that the aluminum-adjuvant-containing OVA vaccine lyophilized preparation of the present invention induces cytokine levels comparable to those of the aluminum salt-OVA nanoparticle preparation without lyophilization, and has no significant difference (P of ASN and ASN (l) ═ 0.9199, and P of APN and APN (l) ═ 0.8946), and is higher than that of free OVA, and has significant difference (P of 0.0478 between APN group and free group). In summary, the lyophilization process does not affect the activity of the vaccine and adjuvant. Data analysis the mean values between groups were compared using a one-factor variance method. P<A value of 0.05 is statistically significant.
Example 37
In vivo immunization experiment of mice: day 0, 14, 28 immunization, BALB/c mice tail root injected subcutaneously with 100 μ l of Free hepatitis B antigen, commercial aluminum adjuvant adsorbed hepatitis B vaccine (2 μ g each HBsAg, 35 μ g aluminum, and aluminum salt-hepatitis B antigen nanoparticle formulation (2 μ g each HBsAg, 16.6ug), day 21, day 35, orbital bleeds, and the amount of HBsAg-specific antibody in serum was examined, the results are shown in FIGS. 8, 9, where Free HBsAg represents the Free hepatitis B surface antigen group, comercial represents the commercial vaccine group (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhentan, product lot number B201701001), APN represents the aluminum hydroxide-PEG-Eti-HBsAg group (example 9), APN (L) represents the corresponding lyophilized formulation group (example 24), ASN represents the aluminum hydroxide-chondroitin sulfate-HBsAg group (example 2), asn (l) represents the corresponding lyophilized formulation group (example 22), fig. 8 is the antibody level at day 21 of immunization of mice with the lyophilized formulation of HBsAg nanoparticles of aluminum hydroxide, and fig. 9 is the antibody level at day 35 of immunization of mice with the lyophilized formulation of HBsAg nanoparticles of aluminum hydroxide. The experimental results show that the lyophilized preparation of hepatitis B vaccine containing aluminum adjuvant of the present invention can induce mice to generate antigen-specific immune response, and induce mice to generate antibody level higher than free HBsAg, with significant difference (day 21 antibody level: P ═ 0.0026 between IgG antibody ASN (L) and free HBsAg, IgG1 antibody ASN (L) and free HBsAg P ═ 0.0003, IgG2a antibody ASN (L) and free HBsAg P ═ 0.0171, day 35 antibody level: IgG antibody ASN (L) and free HBsAg P ═ 0.0118, IgG1 antibody and free HBsAg P ═ 0.0478), and generate antibody level equivalent to that of non-lyophilized hepatitis B vaccine preparation, without significant difference (P >0.5), and the result proves that the activity of hepatitis antigen protein is not affected, and the adsorption of aluminum adjuvant is far lower than that of commercial aluminum vaccine ASN, and the application of aluminum gel vaccine is far lower than that of aluminum gel vaccine, the immune response induced is comparable or stronger without the risk of local side reactions and accumulation of metal ions. Data analysis the mean values between groups were compared using a one-factor variance method. P <0.05 values are statistically significant.
Example 38
The mice are immunized in vivo for 0, 14 and 28 days, 100 mu l of free hepatitis B antigen is injected subcutaneously at the tail root of the mice, the hepatitis B vaccine is adsorbed by a commercial aluminum adjuvant (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001, 2 mu g HBsAg and 35 mu g of aluminum for each), and the aluminum salt-hepatitis B antigen nanoparticle preparation (2 mu g HBsAg and 16.6 mu g of aluminum for each). On day 35, the spleen of the mice is sacrificed, red blood cells are lysed, and then the mice are stained, fixed, punched, re-stained, and finally the intracellular factor level is detected by a flow cytometer. The results are shown in FIGS. 10 and 11, in which Free HBsAg represents Free hepatitis B surface antigen group, commercial represents commercial vaccine group (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001), APN represents aluminum hydroxide-PEG-Eti-HBsAg group (example 9), APN (L) represents corresponding lyophilized preparation group (example 24), ASN represents aluminum hydroxide-chondroitin sulfate-HBsAg group (example 2), ASN (L) represents corresponding lyophilized preparation group (example 22), in which FIG. 10 is HBsAg nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocytes IFN-. gamma.+CD8+The percentage of cells, wherein the aluminum adjuvant-containing lyophilized formulation of hepatitis B vaccine described in the present invention induces mice to generate IFN γ + CD8+ T cells (asn (l) higher than those in free form by P ═ 0.0472 between free form and apn (l)) between free form and 0.0009 between free form, and is higher than that of commercial aluminum gel adsorption vaccine (recombinant hepatitis B vaccine (saccharomyces cerevisiae) shenzhen kangtai, product lot B201701001) (P ═ 0.0433). FIG. 11 shows HBsAg nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IL-4+CD4+T cell percentage, and results show that the aluminum adjuvant-containing hepatitis B vaccine lyophilized preparation induces mice to generate IL-4 higher than that of free antigen+CD4+T cells (APN (L) and P between free groups is 0.0016), and IL-4 produced by commercial aluminum gel adsorption vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001)+CD4+Comparable T cell levels (P)>0.05) and no significant difference between the lyophilized and the non-lyophilized formulations (ASN (l) and P between ASN)>0.9999). The results prove that the protein of the aluminum adjuvant-containing vaccine freeze-dried preparationThe activity is not damaged, the mouse can be induced to generate antigen specific immune response, and the invention induces the mouse to generate humoral immune response (IL-4) equivalent to that of the commercial aluminum gel adsorption vaccine (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001) under the condition that the aluminum content is far lower than that of the commercial aluminum gel adsorption vaccine+CD4+T), and produce higher levels of cellular immune responses (IFN γ)+CD8+T). Data analysis the mean values between groups were compared using a one-factor variance method. P<A value of 0.05 is statistically significant.
Example 39
In vivo immunization experiment of mice: day 0, 7, 14 immunization, BALB/c mice were injected subcutaneously in the tail roots with 50 μ l free tetanus toxoid solution, 250 μ l commercial aluminum adjuvant adsorbed tetanus toxoid vaccine (Dow Europe Biotech Co., Ltd., product batch 20170813, 4.5Lf toxoid each, aluminum 173.075 μ g), and 50 μ l aluminum salt-tetanus toxoid nanoparticle formulation (4.5 Lf toxoid each, aluminum 10.125 μ g). The results of the orbital bleeds on days 21 and 28, and the amount of tetanus toxoid-specific antibodies in the serum were measured are shown in FIGS. 12 and 13, wherein commocial represents a commercially available adsorbed tetanus vaccine group (Duyulin Biotech Co., Ltd., product batch No. 20170813), Preparation represents a group of nanoparticle preparations without lyophilization, 5% sucrose represents a group of aluminum hydroxide-PEG-Eti-tetanus toxoid nanoparticle lyophilized preparations prepared by using 5% sucrose as a lyophilization protectant, 10% Trealose represents a result of the mouse immunization with 10% Trehalose as a lyophilization protectant for the 21-day antibody, and it is known that the lyophilized nanoparticles induce the IgG level of mice to produce IgG, which is higher than that of commercially available tetanus toxoid adsorbed by using an aluminum adjuvant (Duyulin Biotech Co., Ltd.), product lot No. 20170813), and there was no significant difference (P ═ 0.0306) between the levels induced by the non-lyophilized nanoparticles and the lyophilized nanoparticles to produce IgG in mice, both the levels induced by the non-lyophilized nanoparticles before and after lyophilization to produce IgG1 were comparable to those induced by commercial aluminum adjuvant adsorption of tetanus toxoid vaccine (medoxolin biotechnology, product lot No. 20170813), and there was no significant difference (P >0.05), and the level induced by the non-lyophilized nanoparticles to produce IgG2a was higher than that induced by commercial aluminum adjuvant adsorption of tetanus toxoid vaccine (medoxolin, product lot No. 20170813), both significant differences (P ═ 0.0161), and the level induced by the non-lyophilized nanoparticles and the lyophilized nanoparticles to produce IgG2a in mice was not significantly different (P > 0.05). Fig. 13 shows antibody results of the lyophilized formulation of tetanus toxoid nanoparticles with aluminum hydroxide on day 28 of mouse immunization, and from the experimental results, it can be seen that the levels of IgG and IgG1 produced by the mouse induced by the non-lyophilized nanoparticles are equivalent to those of IgG and IgG1 produced by the commercial aluminum adjuvant-adsorbed tetanus toxoid vaccine (guo european forest biotechnology limited, product lot No. 20170813), and there is no significant difference between the two (P >0.05) and between the non-lyophilized nanoparticles and the lyophilized nanoparticles (P > 0.05); the level of IgG2a production induced by the non-lyophilized nanoparticles was higher than that induced by commercial aluminum adjuvant-adsorbed tetanus toxoid vaccine (Dow Europe bioscience, Inc., product lot 20170813), the two were significantly different (P ═ 0.0284), and the level of IgG2a production induced by the non-lyophilized nanoparticles and the level of IgG2a production induced by the lyophilized nanoparticles (P >0.05) was not significantly different. The result proves that the tetanus vaccine freeze-dried preparation containing the aluminum adjuvant can induce mice to generate stronger antigen-specific immune response, the level of the generated antibody is the same as that of the tetanus vaccine preparation which is not freeze-dried, the antigen activity in the preparation is not destroyed in the freeze-drying process, and the humoral immune response (IgG, IgG1) of the tetanus vaccine (Chengdu European Lin biotechnology, Inc., product batch No. 20170813) with equivalent level adsorbed by the commercial aluminum adjuvant can be induced; and the induced cellular immune response (IgG2a) is stronger than that of the tetanus vaccine adsorbed by the commercial aluminum adjuvant (Chengdu European Lin Biotechnology corporation, product batch No. 20170813) under the condition that the content of the aluminum adjuvant is far lower than that of the commercial aluminum gel adsorption vaccine, and the invention has no local side reaction and toxic and side effect of metal ion accumulation. Data analysis the mean values between groups were compared using a one-factor variance method. P <0.05 values are statistically significant.
Example 40
Mouse immunization experiment of aluminum salt nanoparticle lyophilized preparation stored at room temperature: the aluminum salt nanoparticle lyophilized formulations of examples 22 and 24 were allowed to stand at 20. + -. 5 ℃ for 1 week, and then reconstituted and administered. Day 0, 14 and 28 immunization, injecting commercial aluminum adjuvant subcutaneously at tail root of BALB/c mouse to adsorb hepatitis B vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001, 2 ug HBsAg and 35 ug each, and aluminum salt-hepatitis B antigen nanoparticle preparation (2 ug HBsAg and 16.6ug each). Blood was collected from the orbit on day 35 and the amount of HBsAg-specific antibody in the serum was measured. The results are shown in FIG. 14, in which FreeHBsAg represents the free hepatitis B surface antigen group, comeseal represents the commercial vaccine group (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai, product batch No. B201701001), APN-L represents the aluminum hydroxide-PEG-Eti-HBsAg lyophilized preparation group (example 24), APN-R represents the aluminum hydroxide-PEG-Eti-HBsAg lyophilized preparation group (example 24) after being placed at 20 + -5 deg.C for 1 week, ASN-L represents the aluminum hydroxide-chondroitin sulfate-HBsAg lyophilized preparation group (example 22), and ASN-R represents the aluminum hydroxide-chondroitin sulfate-HBsAg lyophilized preparation group (example 22) after being placed at 20 + -5 deg.C for 1 week. The experimental result shows that the hepatitis B vaccine freeze-dried preparation containing the aluminum adjuvant can still induce a mouse to generate antigen specific immunoreaction after being placed for 1 week at room temperature, under the condition that the aluminum content is far lower than that of a commercial aluminum gel adsorption vaccine, the level of an antibody generated by the induced mouse is equivalent to that of a commercial hepatitis B vaccine preparation (a recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai and a product batch number B201701001), and no significant difference (P >0.5) exists, and the result proves that the freeze-drying mode can improve the stability of the aluminum-adjuvant-containing vaccine, and the freeze-drying preparation can be stored at room temperature, and further proves that under the condition that the aluminum content is far lower than that of the commercial aluminum gel adsorption vaccine, the induced mouse can generate the same immune effect as that of the commercial hepatitis B vaccine preparation (the recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhengkangtai and the product batch number B201701001). Data analysis the mean values between groups were compared using a one-factor variance method. P <0.05 values are statistically significant.
EXAMPLE 41
Mouse immunization experiment of aluminum salt nanoparticle lyophilized preparation stored at room temperature: the aluminum salt nanoparticle lyophilized formulations of examples 22 and 24 were allowed to stand at 20. + -. 5 ℃ for 1 week, and then reconstituted and administered. Day 0, 14 and 28 immunization, 100 μ l of free hepatitis B antigen was injected subcutaneously into tail root of BALB/c mice, and a commercial aluminum adjuvant adsorbed hepatitis B vaccine (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen kangtai, product batch No. B201701001, 2 μ g HBsAg and 35 μ g aluminum each) and an aluminum salt-hepatitis B antigen nanoparticle preparation (2 μ g HBsAg and 16.6 μ g aluminum each) were added. On day 35, the spleen was removed from the sacrificed mice, red blood cells were lysed, stained, fixed, punched, re-stained, and finally the intracellular factor levels were flow-tested. The results are shown in FIGS. 15 and 16, in which comercial represents the commercial vaccine group (recombinant hepatitis B vaccine (Saccharomyces cerevisiae) Shenzhen Kangtai, product lot B201701001), APN-L represents the aluminum hydroxide-PEG-Eti-HBsAg lyophilized preparation group (example 24), APN-R represents the aluminum hydroxide-PEG-Eti-HBsAg lyophilized preparation group (example 24) after being placed at 20 + -5 ℃ for 1 week, ASN-L represents the aluminum hydroxide-chondroitin sulfate-HBsAg lyophilized preparation group (example 22), and ASN-R represents the aluminum hydroxide-chondroitin sulfate-HBsAg lyophilized preparation group (example 22) after being placed at 20 + -5 ℃ for 1 week. FIG. 15 shows spleen CD8 of mice immunized with HBsAg nanoparticle lyophilized preparation of aluminum hydroxide+IFN-gamma in cells+Percentage, from the results, the formulation lyophilized and left for 1 week after lyophilization induced IFN-. gamma.production in mice+The induction amount of the CD8+ T cells is equivalent to that of a commercial vaccine group (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai, product batch number B201701001), and no significant difference (P201701001)>0.5); FIG. 16 shows HBsAg nanoparticle lyophilized preparation of aluminum hydroxide for immunizing mouse splenocyte IL-4+CD4+Percentage of T cells, as can be seen from the results, APN-R, i.e., the lyophilized preparation of APN after lyophilization, induced IL-4 production in mice when placed at room temperature for 1 week+CD4+Percentage of T cells versus commercial vaccine group (recombinant hepatitis B vaccine)The product batch number B201701001 of Shenzhen kangtai (Saccharomyces cerevisiae) is high, the two are significantly different (P ═ 0.0330) and the lyophilized preparation of ASN-R (ASN lyophilized) is placed at room temperature for 1 week to induce the mice to generate IL-4+CD4+The percentage of T cells is higher than that of the commercial vaccine group (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen kangtai, product batch number B201701001), and the two have significant difference (P ═ 0.0046). The result proves that the aluminum adjuvant-containing hepatitis B vaccine freeze-dried preparation can still induce a mouse to generate antigen-specific immune reaction after being placed for 1 week at room temperature, the level of the cytokine generated by the mouse under the condition that the aluminum content is far lower than that of a commercial aluminum gel adsorption vaccine is equivalent to or higher than that of a commercial hepatitis B vaccine preparation (recombinant hepatitis B vaccine (saccharomyces cerevisiae) Shenzhen Kangtai, product batch No. B201701001), and the freeze-drying mode can improve the stability of the aluminum adjuvant-containing vaccine, and the aluminum adjuvant-containing hepatitis B vaccine freeze-dried preparation can be transported and stored at room temperature. Data analysis the mean values between groups were compared using a one-factor variance method. P<A value of 0.05 is statistically significant.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A freeze-dried preparation of vaccine containing aluminium adjuvant is characterized by that the aluminium salt and anionic polymer or its derivative are interacted to form nano or micrometer particles, at the same time the antigen and/or vaccine adjuvant can be adsorbed, and the freeze-dried protective agent also is contained.
2. The aluminum adjuvanted vaccine-containing lyophilized formulation of claim 1 wherein the aluminum salt is aluminum sulfate, based on parts by weight, aluminum hydroxide: anionic polymer or derivative thereof: antigen: other adjuvants: the weight ratio of the freeze-drying protective agent is 0.152-1.52: 0.12-3: 0.01-0.1: 0-0.1: 20 to 150.
3. The aluminum-adjuvanted vaccine lyophilized formulation of any one of claims 1-2, wherein the anionic polymer comprises a natural or endogenous, semisynthetic derivative, or fully synthetic anionic polymeric material.
4. The aluminum-adjuvanted vaccine lyophilized formulation of any one of claims 1-2, wherein the anionic polymer or derivative thereof further comprises a derivatizing modification comprising: modification of PEG, carboxyl, carboxymethyl, sulfate, sulfite, phosphate, or phosphite.
5. The aluminum-adjuvanted vaccine lyophilisate according to any of claims 1 to 2, wherein the anionic polymer or derivative thereof comprises: one or more of a linear polymer, a crosslinked polymer, or a branched copolymer.
6. An aluminum-adjuvanted vaccine lyophilized formulation as claimed in any one of claims 1-2 wherein the lyoprotectant comprises one or more of polysaccharides including one or more of trehalose, sucrose, glucose, maltose, dextran, dextrin, polyols including mannitol, glycerol, polymers including one or more of PVP, PEG, PVA, surfactants including one or more of Tween 80, poloxamer 188, acacia, amino acids including one or more of L-serine, arginine, tryptophan, lysine hydrochloride, sodium glutamate, alanine, glycine, sarcosine, phenylalanine, anhydrous solvent including DMSO, and a polymer including one or more of amino acids including one or more of trehalose, sucrose, glucose, maltose, dextran, dextrin, and the like.
7. The aluminum-adjuvanted vaccine-containing lyophilized formulation of any one of claims 1-2, wherein the antigen is selected from the group consisting of: the protein antigen is selected from one or more of hepatitis B surface antigen HBsAg, recombinant PreS1, PreS2, recombinant core protein, hepatitis C virus antigen, hepatitis E virus antigen, anthrax toxin binding protein, streptococcus pneumoniae protein, group A M protein, streptococcus C5a peptidase, zonulin binding protein, serine carboxylesterase, meningococcal type B outer membrane protein OMP, chicken ovalbumin, pale dense helical body surface lipoprotein, bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, soybean albumin, wheat albumin, myoglobin, collagen and fibrillin; or the toxoid is selected from diphtheria toxoid, tetanus toxoid, cholera toxin, or polysaccharides such as one or more of group a polysaccharide, capsular polysaccharide, pneumococcal polysaccharide, typhoid polysaccharide, meningococcal polysaccharide, or virus-like particles such as human papilloma virus, rotavirus, or the inactivated or attenuated vaccine antigen is selected from pertussis toxin, hepatitis a virus, encephalitis b virus, rabies virus, polio-soxhlet strain virus antigen, influenza virus, cytomegalovirus, rhino influenza antigen, varicella-zoster virus, mumps antigen, rubella virus, smallpox virus, measles, varicella, yellow fever, poliomyelitis carbene strain, respiratory syncytial virus; or attenuated and killed bacteria such as live Mycobacterium tuberculosis, Corynebacterium diphtheriae, Bordetella pertussis, group A Streptococcus, Neisseria meningitidis, Legionella pneumophila, Vibrio cholerae, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Treponema pallidum; or a polysaccharide-protein complex such as one or more of a pneumococcal polysaccharide conjugate, a meningococcal polysaccharide-protein conjugate; or one or more of nucleic acids, single-and double-stranded nucleic acids, supercoiled plasmid DNA, linear plasmid DNA, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, mammalian artificial chromosomes, RNA; or synthetic polypeptide resisting one or more selected from group A streptococcus M peptide, TRP2, HGP100, p15E, pseudomonas aeruginosa synthetic peptide and rubella virus synthetic peptide; or one or more of tumor cell lysate antigen, bacterial lysate antigen and tumor whole cell antigen.
8. The aluminum-adjuvanted vaccine-containing lyophilized formulation of any one of claims 1-2, wherein the adjuvant comprises: the cell factor GM-CSF, interferon, interleukin IL-2, IL-6, IL-1, IL-12, Toll-like receptor agonist is selected from one or more of CpG-ODN, double-stranded RNA, monophosphoryl lipid A, polysaccharide adjuvant is selected from one or more of inulin, chitosan, glucan, cholera toxin B subunit, escherichia coli heat-labile enterotoxin, lipopolysaccharide, heat shock protein, saponin adjuvant, and Limquiod.
9. A process for the preparation of a lyophilised formulation of an aluminium-adjuvanted vaccine according to any of claims 1 to 9, characterised in that it comprises the following steps:
(1) mixing Hepes buffer solution with anionic polymer material or its derivative, antigen and/or adjuvant uniformly to obtain phase A;
(2) taking an aluminum sulfate aqueous solution as a B phase;
(3) mixing the A phase and the B phase to obtain a semi-finished product;
(4) adding a freeze-drying protective agent into the semi-finished product, and uniformly mixing;
(5) pre-freezing the semi-finished product at-40 deg.C to-80 deg.C for 4-24 h;
(6) when the temperature of the freeze dryer is reduced to below minus 40 ℃, the freeze dryer is vacuumized until the pressure of the freeze dryer system is 0.1 to 0.01mbar, and the freeze drying is carried out for more than or equal to 24 hours.
10. Use of a lyophilized formulation of an aluminium-adjuvanted vaccine as claimed in any one of claims 1 to 8 comprising: production, transportation and storage of preventive and therapeutic vaccines.
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