CN115715801A - Vaccine and preparation method and application thereof - Google Patents
Vaccine and preparation method and application thereof Download PDFInfo
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- CN115715801A CN115715801A CN202110976364.3A CN202110976364A CN115715801A CN 115715801 A CN115715801 A CN 115715801A CN 202110976364 A CN202110976364 A CN 202110976364A CN 115715801 A CN115715801 A CN 115715801A
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Abstract
The invention relates to an integrated vaccine, a preparation method and application thereof. The integrated vaccine comprises nanoparticles with a core-shell structure, wherein the nanoparticles are composed of an aluminum hydroxide adjuvant and a protein antigen, a single protein antigen is used as a shell of each nanoparticle, and the aluminum hydroxide adjuvant is used as an inner core. The vaccine of the invention can effectively induce an organism to generate specific cellular immune response and humoral immune response.
Description
Technical Field
The invention relates to the field of medicines, in particular to a vaccine and a preparation method and application thereof.
Background
Vaccines are immunogenic substances that elicit body-specific immune responses, are the most cost-effective treatment for the prevention and treatment of human diseases, and by administering vaccines, antigens can be recognized by their own antigen-presenting cells (e.g., dendritic cells), and specific immune responses can be rapidly activated, initiating defense mechanisms against foreign bodies carrying antigenic determinants. Conventional inactivated pathogen vaccines and attenuated live vaccines use the pathogen as a carrier, which is prone to cause safety problems. The vaccine medicine prepared by directly using the pathogen protein antigen can avoid using virus as a carrier, and has better safety. Because the vaccine preparation contains various components and has a complex action mechanism, the inactivated or attenuated pathogen vaccine has the risk of generating virulence recovery, causes serious adverse reaction and has higher requirement on the safety of the production environment. Just because of concerns about safety issues that may be caused by pathogens, conventional vaccines are not suitable for virulent pathogens with high pathogenicity. The pathogen protein antigen is directly used for subunit vaccine drug development, and the composition of the vaccine preparation can be well controlled. The definite composition of the components of the subunit vaccine ensures the safety of the subunit vaccine in the using and preparing processes. However, because of its low immunogenicity, the immune response triggered by it is weak, so it needs to be supplemented with immune adjuvant (such as aluminum adjuvant or CpG oligonucleotide, etc.) to play a better role.
Aluminum hydroxide adjuvant is used as the most widely used immunoadjuvant, and is used for the treatment of various diseases including rabies, avian influenza, and pediatric diphtheria because of its excellent safety. Currently, the aluminum hydroxide adjuvants in clinical use are often in the form of gels containing aluminum hydroxide clusters of 1-10 μm in size. It adsorbs antigen with anion in the form of electrostatic adsorption, and activates antigen presenting cells by intramuscular injection or subcutaneous injection to trigger immune response of the body. The currently commonly used aluminum hydroxide adjuvant has weak in-vivo binding capacity with antigen and is difficult to deliver to lymph node antigen presenting cells, so that the problems of poor immune activation efficiency, incapability of effectively activating cellular immunity of organisms and the like exist. First, traditional aluminum hydroxide adjuvants are difficult to maintain in an in vivo environment andbinding between adsorbed antigens. Journal (Nature Medicine,2020,26 (3), 430-440) reports that in vivo environment, aluminum hydroxide adjuvant can be rapidly dissociated from its adsorbed antigen, and only three days after subcutaneous inoculation, the inoculated vaccine can be basically eliminated by the body, and the function of effectively activating immune response can not be achieved. Second, traditional aluminum hydroxide gels are large in size (1-10 μm) and difficult to access to lymph nodes, and release antigen at the site of inoculation in the form of a "sponge", resulting in inefficient access of antigen to lymph nodes for contacting antigen presenting cells. Furthermore, conventional aluminum hydroxide adjuvants are not believed to have the ability to activate cellular immunity. Previous studies on aluminum adjuvants found that although aluminum adjuvants have some activating effect on Th 2-type humoral immunity, it is difficult to control CD8 + T cell-mediated cellular immunity produces effects. In conclusion, various drawbacks of conventional aluminum hydroxide adjuvants in antigen delivery limit their further application in immunotherapy. Therefore, it is of great interest to explore ways of delivering aluminum adjuvants and protein antigens that are not easily dissociated in vivo and that can effectively activate immune responses (especially cellular immune responses).
Disclosure of Invention
Based on this, the object of the present invention is to provide a vaccine which can induce the body to generate specific cellular immune response and humoral immune response strongly and effectively.
The specific technical scheme is as follows:
a vaccine comprises nanoparticles of a core-shell structure, wherein the nanoparticles are composed of an aluminum hydroxide adjuvant and a protein antigen, a single protein antigen is used as an outer shell of each nanoparticle, and the aluminum hydroxide adjuvant is used as an inner core of each nanoparticle.
It is another object of the present invention to provide a method for preparing a vaccine comprising nanoparticles having a core-shell structure; the preparation method comprises the following steps:
dissolving a protein antigen in a buffer system to be used as a liquid A;
dissolving aluminum salt in a buffer system to obtain solution B;
dissolving hydroxide in a buffer system to obtain a third solution;
mixing the solution A and the solution B, adding the solution C, mixing until the pH value in a mixed system is 10-14, and then aging the obtained mixed system to obtain the nano-particles with a protein antigen as a shell and an aluminum hydroxide adjuvant as an inner core;
the molar ratio of the protein antigen to the aluminum ions in the aluminum salt in the raw material is 1: (1-50).
The invention further aims to provide application of the vaccine in preparing a medicament for preventing and/or treating viral pneumonia related diseases. Further, the viral pneumonia is related diseases caused by 2019-nCov infection.
The invention further aims to provide application of the vaccine in preparing a medicament for preventing and/or treating tumors. Further, the tumor is a malignant tumor.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a vaccine composed of nanoparticles with a core-shell structure, wherein the nanoparticles take a protein antigen as a shell and an aluminum hydroxide adjuvant wrapped in the protein antigen as an inner core, the protein antigen and the aluminum hydroxide adjuvant are tightly combined through covalent interaction and are not easy to dissociate in vivo, and the synchronous delivery of the protein antigen and the aluminum hydroxide adjuvant is realized; meanwhile, the nano particles in the vaccine have small particle size and can easily enter lymph nodes to contact antigen presenting cells so as to activate immune response; finally, the vaccine of the invention can induce strong cellular immune response and humoral immune response in vivo, and can reduce the potential aluminum toxicity of the aluminum hydroxide adjuvant.
The method directly chelates aluminum ions in the protein antigen by using the protein antigen as a template through specific reaction conditions of the method, so that aluminum hydroxide clusters are generated in situ in the protein antigen to form the nanoparticle with a shell-core structure by using an aluminum hydroxide cluster adjuvant as an inner core and using the protein antigen as an outer shell. In addition, the method is simple and controllable, and can be universally applied to preparation of different protein vaccines.
Drawings
FIG. 1 is a map of the putamen structure of nanoparticles in the mode antigen OVA integrated vaccine of example 2, wherein the left panel a is a transmission electron micrograph of the mode antigen OVA integrated vaccine, and the right panel b is a partial enlarged view of the nanoparticles;
FIG. 2 is a transmission electron micrograph of the CT26 whole antigen integrated vaccine of the mouse colon cancer cell of example 3;
FIG. 3 is a graph showing the particle size characterization of nanoparticles of the antigen-adjuvant integrated vaccine; wherein, the graph a is a particle size characterization graph of nanoparticles of the antigen-adjuvant integrated vaccine of the mouse colon cancer cell CT26 holoantigen of the example 3, the graph b is a particle size characterization graph of nanoparticles of the antigen-adjuvant integrated vaccine of the model antigen OVA of the example 2, and the graph c is a particle size characterization graph of nanoparticles of the antigen-adjuvant integrated vaccine of the 2019 novel coronavirus Receptor Binding Domain (RBD) of the example 1;
FIG. 4 is a representation of co-localization of an aluminum hydroxide adjuvant component with a protein antigen in nanoparticles of the antigen-adjuvant integrated vaccine;
FIG. 5 is X-ray photoelectron spectroscopy analysis data of an aluminum hydroxide adjuvant component of the antigen-adjuvant integrated vaccine;
FIG. 6 is data of the uptake analysis of components in the antigen-adjuvant integrated vaccine by dendritic cells;
figure 7 is serum neutralizing antibody levels after 2019 immunization of mice with an antigen-adjuvant integrated vaccine of the novel coronavirus Receptor Binding Domain (RBD);
FIG. 8 shows the result of analysis of cell-killing T cells after 2019 mice immunized with the antigen-adjuvant integrated vaccine of the novel coronavirus Receptor Binding Domain (RBD); wherein, the graph a is the analysis of the proportion of the killer T cells, the graph b is the analysis of the proportion of the killer T cells of the degranulation enzyme positive cells, and the graph c is the analysis of the proportion of the killer T cells of the perforin positive cells;
FIG. 9 shows the anti-tumor effect of the antigen-adjuvant integrated tumor vaccine immunized mice;
FIG. 10 is the data of the dendritic cell activation analysis after the mice are immunized with the antigen-adjuvant integrated tumor vaccine;
FIG. 11 shows the data of the analysis of memory T cells after immunization of mice with the antigen-adjuvant-integrated tumor vaccine.
Detailed Description
Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or apparatus that comprises a list of steps is not limited to only those steps or modules recited, but may alternatively include other steps not recited, or may alternatively include other steps inherent to such process, method, article, or apparatus.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
Some of the terms to which the invention relates:
protein antigens in the context of the present invention are defined as protein molecules that contain epitopes that bind to T cells or B cell receptors and are capable of stimulating an immune response, particularly a B cell response and/or a T cell response, in a subject. "epitope" refers to the region of a protein antigen that interacts with B and/or T cell proteins, i.e., the B cell receptor and the T cell receptor.
A tumor or cancer in the present invention refers to an abnormal growth of cells, which may be benign or malignant.
Some embodiments of the present disclosure provide a vaccine comprising core-shell structured nanoparticles composed of an aluminum hydroxide adjuvant and a protein antigen, wherein each of the nanoparticles has a single protein antigen as an outer shell and the aluminum hydroxide adjuvant as an inner core. The vaccines in the embodiments of the present invention are also referred to as "integrated vaccines" or "antigen-adjuvant integrated vaccines" in the embodiments.
It is understood that the protein antigen of the present invention is not limited to the type thereof, as long as it can be chelated with aluminum ions and achieve in situ generation of aluminum hydroxide clusters in the protein antigen. In some embodiments, the structure of the protein antigens of the invention enables the aluminum core to be restricted within the protein.
It is understood that the protein antigen of the present invention may be natural or synthetic.
Protein antigens used in embodiments of the present disclosure may be derived from pathogens, cancer cells, autoantigens, or allergens. In certain embodiments, the protein antigen is a protein associated with autoimmunity. In yet other embodiments, the protein antigen is a protein associated with an allergy.
In certain embodiments, the protein antigen is a pathogen protein antigen or a tumor-associated protein antigen.
In certain embodiments, the protein antigen is a tumor associated protein antigen. It is understood that any protein antigen capable of inducing tumor immune effect can be the tumor-associated protein antigen of the present invention. It is understood that the tumor associated protein antigen of the present invention may be natural tumor self protein antigen, or tumor recombinant protein antigen artificially prepared by genetic engineering technology, or tumor synthetic protein antigen artificially synthesized by in vitro methods.
In certain embodiments, the native tumor self-protein antigen is any protein antigen produced by a tumor cell that, upon targeting by a T cell response, ideally causes a meaningful reduction in tumor growth or prevention of tumorigenesis, and has a limited impact on healthy, non-cancerous cells. In some embodiments, the native tumor self-protein antigen can be a whole protein antigen produced by a tumor cell or a part of protein antigen produced by a tumor cell. In certain embodiments, the native tumor self-protein antigen may comprise a region of protein from a tumor tissue (e.g., a cancer cell). In other embodiments, the native tumor self-protein antigen may be a protein or fragment of a protein expressed by tumor tissue (but not healthy tissue).
In certain embodiments, the tumor-associated protein antigen can be from a hematological tumor, non-limiting examples of which include leukemias, including acute leukemias (e.g., acute lymphocytic leukemia, promyelocytic, acute myelocytic leukemia, myelomonocytic cells, acute myelogenous leukemia and myeloblasts, monocytes and erythroleukemia), chronic leukemias (e.g., chronic myelogenous leukemia and chronic lymphocytic leukemia), lymphomas, hodgkin's lymphoma, non-hodgkin's lymphoma, heavy chain disease, myelodysplastic syndrome, multiple myeloma, fahrenheit macroglobulinemia, polycythemia vera, hairy cell leukemia and myelodysplasia. In certain embodiments, the tumor-associated protein antigen can be from a solid tumor, non-limiting examples of which are sarcomas and epithelial cancers, including myxosarcoma, fibrosarcoma, chondrosarcoma, liposarcoma, osteogenic sarcoma and other sarcomas, synovioma, leiomyosarcoma, rhabdomyosarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ewing's tumor, colon cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, basal cell carcinoma, adenocarcinoma, hepatocellular carcinoma, squamous cell carcinoma, sweat gland carcinoma, medullary thyroid carcinoma, bronchial cancer, renal cell carcinoma, papillary thyroid carcinoma, pheochromocytoma, wilms' tumor, papillary carcinoma, papillary adenocarcinoma, cervical cancer, sebaceous gland carcinoma, medullary carcinoma, testicular cancer, hepatoma, bile duct carcinoma, seminoma, bladder carcinoma, and CNS tumors (e.g., craniopharyngioma, ependymoma, medulloblastoma, glioma, astrocytoma, pinealoma, acoustic neuroma, angioma, meningioma, glioma, oligodendroglioma, and retinoblastoma). In several examples, the tumor is lymphoma, melanoma, breast cancer, lung cancer, or colon cancer. In certain embodiments, the tumor-associated protein antigen is derived from a protein antigen of a breast cancer, such as ductal carcinoma or lobular carcinoma. In certain embodiments, the tumor associated protein antigen is a protein antigen from prostate cancer. In certain embodiments, the tumor-associated protein antigen is a protein antigen from a skin cancer such as basal cell carcinoma, kaposi's sarcoma, squamous cell carcinoma, or melanoma. In certain embodiments, the tumor-associated protein antigen is a protein antigen from a lung cancer, such as an adenocarcinoma, a large cell carcinoma, a bronchoalveolar carcinoma, or a small cell carcinoma. In certain embodiments, the tumor-associated protein antigen is a protein antigen from a brain cancer, such as a glioblastoma or a meningioma. In certain embodiments, the tumor-associated protein antigen is a protein antigen from colon cancer. In certain embodiments, the tumor-associated protein antigen is a protein antigen from liver cancer, e.g., hepatocellular carcinoma. In certain embodiments, the tumor-associated protein antigen is a protein antigen from pancreatic cancer. In certain embodiments, the tumor-associated protein antigen is a protein antigen from a renal cancer, such as a renal cell carcinoma. In certain embodiments, the tumor-associated protein antigen is a protein antigen from testicular cancer. In certain embodiments, the tumor-associated protein antigen is a protein antigen derived from a precancerous lesion, such as a variant of carcinoma in situ or a vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.
In certain embodiments, the protein antigen is a pathogen protein antigen. It is understood that the pathogen protein antigen of the present invention may be a natural pathogen protein antigen, or a pathogen protein antigen artificially prepared by genetic engineering techniques, or a pathogen protein antigen artificially synthesized by an in vitro method. In certain embodiments, the native pathogen protein antigen is any protein antigen produced by an antigenic body (e.g., a virus, bacterium, or fungus). In certain embodiments, the native pathogen protein antigen may be a region of a protein from a pathogen. In other embodiments, the native pathogen protein antigen may be an intact protein or a protein fragment derived from a pathogen.
In additional embodiments, the pathogen protein antigen may be from an infectious microorganism that is a virus, a bacterium, a fungal parasite.
In further embodiments, the pathogen protein antigen is derived from a bacterium, non-limiting examples of which include Escherichia coli (Escherichia coli), chlamydia trachomatis (Chlamydia trachomatis), burkholderia cepacia (Burkholderia cepacia), klebsiella oxytoca (Klebsiella oxytoca), helicobacter pylori (Helicobacter pylori), bacteroides fragilis (Bacterioides fragilis), acinetobacter baumannii (Acinetobacter baumannii), enterobacter aerogenes (Enterobacter aerogenes), citrobacter freundii (Citrobacter freundii), campylobacter jejuni (Campylobacter jejuni), enterobacter cloacae (Enterobacter clobacter clavulane), pseudomonas aeruginosa (Pseudomonas aeruginosa), neisseria pneumoniae (k. Pneuentialba), clostridium difficini (Clostridium difficile), clostridium difficile (Clostridium typ), clostridium typhimurium), clostridium typhii (Clostridium typhii), clostridium typhimurium (Clostridium typhii), mycobacterium tuberculosis (Clostridium typhus (Clostridium typhi), clostridium typhi, clostridium sp Enterococcus faecalis (Enterococcus faecium), enterococcus faecium (Enterococcus faecium), vancomycin-resistant Enterococcus (VRE), listeria monocytogenes (listeriomonas), staphylococcus aureus (Staphylococcus aureus), nocardia necatrix (Nocardia farcina), propionibacterium acnes (p.acnes), methicillin-sensitive Staphylococcus aureus (MSSA), streptococcus pyogenes (Streptococcus pyogenes), leprosy bacillus (m.leprae), methicillin-resistant Staphylococcus aureus (MRSA), staphylococcus epidermidis (Staphylococcus epidermidis), group a Streptococcus (Streptococcus), group B Streptococcus (Streptococcus agalactiae), or group C Streptococcus.
In additional embodiments, the pathogen protein antigen is from a virus, non-limiting examples of which include coronavirus, vaccinia virus, dengue virus (degue virus), HSV2, human Papilloma Virus (HPV), ebola virus (Ebola virus), EBV, hepatitis a virus (hepis a virus), HIV, marburg virus (Marburgvirus), hepatitis b virus (hepis bvvirus), hepatitis C virus (hepis C virus), hepatitis D virus (hepis D virus), cytomegalovirus (CMV), HSV1, influenza a virus (Influenza a virus), west nile virus (wennlevirus), human Rhinovirus (HRV), human respiratory virus (RSV), or zika virus. In some embodiments, non-limiting examples of the coronavirus include SARS coronavirus (SARS-CoV) or middle east respiratory syndrome coronavirus (MERS-CoV) or SARS-Cov-2. In other embodiments, the pathogen protein antigen is a protein antigen of SARS-Cov-2. For example, the S protein, S1 protein or RBD protein of SARS-Cov-2.
In additional embodiments, the pathogen protein antigen is from a parasite, non-limiting examples of which include Trichomonas (Trichomonas), leishmania (Leishmania), plasmodium (Malaria), cryptosporidium (Cryptosporidium), trypanosoma (Trypanosoma), and Schistosoma (Schistosoma).
In some embodiments, the nanoparticles have a particle size of 100nm or less, further, 50nm or less, or 30nm or less, or from 2nm to 50nm, from 2nm to 30nm, or from 2nm to 15nm. Generally, the particle size of the nanoparticles is small, so that the nanoparticles can enter lymph nodes to contact antigen presenting cells, and the cellular immunity of the organism can be activated better.
In some of these embodiments, the diameter of the core of the nanoparticle is ≦ 10nm, and further 2nm to 10nm.
In some of these embodiments, the number of aluminum atoms in the aluminum hydroxide adjuvant encapsulated by each protein antigen is 1 to 50. It will be appreciated that the number of aluminium atoms in the aluminium hydroxide adjuvant encapsulated by each of the protein antigens may be 1 to 50, or 1 to 40, or 1 to 35, or 1 to 30, or 5 to 50, or 10 to 35, or 10 to 30, or 10 to 25.
In some of these embodiments, the aluminum hydroxide adjuvant is bound to the protein antigen by covalent interaction.
In some embodiments, the main component of the aluminum hydroxide adjuvant is aluminum metahydroxide, and the other components besides the aluminum metahydroxide comprise aluminum hydroxide, aluminum oxide and aluminum potassium sulfate.
It is understood that the vaccine of the present invention is preferably in the form of injection, nasal drop, spray or powder for injection. In certain embodiments, the injectable formulation is an intramuscular injection or an intravenous injection.
In certain embodiments, the vaccines of the present invention are administered by mucosal site vaccination or injection.
Some embodiments of the present invention also provide a method of preparing a vaccine comprising nanoparticles having a core-shell structure; the preparation method comprises the following steps:
dissolving a protein antigen in a buffer system to be used as a liquid A;
dissolving aluminum salt in a buffer system to obtain solution B;
dissolving hydroxide in a buffer system to obtain a third solution;
mixing the solution A and the solution B, adding the solution C, mixing until the pH value in a mixed system is 10-14, and then aging the mixed system to obtain the nano-particles with a protein antigen as a shell and an aluminum hydroxide adjuvant as an inner core;
the molar ratio of the protein antigen to the aluminum ions in the aluminum salt is 1 (1-50).
In some of these embodiments, the molar ratio of the protein antigen to aluminum ion in the aluminum salt is 1: (10 to 50).
In some embodiments, the molar ratio of the protein antigen to the aluminum ion in the aluminum salt is 1 (15-25) or 1 (25-35) or 1 (35-50).
The invention adopts a biomineralization method, takes a single protein antigen as a growth template of an aluminum hydroxide adjuvant, chelates a certain equivalent amount of aluminum ions in the protein antigen in a buffer system, slowly adds hydroxide solution, the aluminum ions in the protein antigen react with hydroxide ions to primarily generate hydrated alumina, and the aluminum hydroxide adjuvant cluster kernel which takes aluminum metahydroxide (the main component of the traditional aluminum hydroxide gel adjuvant) as the main component can be obtained through slow aging growth. The dosage of the protein antigen and the aluminum is controlled to be the ratio, aluminum ions in the aluminum salt are chelated in the protein antigen and are not dissociated to the outside of the protein antigen to form an aluminum hydroxide cluster, so that a protein antigen shell structure can be well maintained, and the protein antigen shell structure is very key for ensuring the immune activation effect of the vaccine.
In some embodiments, the concentration of the protein antigen in the nail fluid is between 0.01 and 0.5. Mu. Mol/mL, further between 0.01 and 0.1. Mu. Mol/mL, and further (0.033. + -. 0.01). Mu. Mol/mL.
In some embodiments, the concentration of aluminum ions in the solution B is 0.5 to 5. Mu. Mol/mL, further 0.5 to 3. Mu. Mol/mL, and further 0.5 to 2. Mu. Mol/mL.
In some embodiments, the concentration of hydroxide ions in the solution is 1 μmol/mL to 10 μmol/mL, further 3 μmol/mL to 8 μmol/mL, and further (6.6. + -. 0.2) μmol/mL.
In some of these embodiments, the addition of the liquid C is carried out until the pH in the mixed system is between 11 and 13, preferably at a pH of 12. + -. 0.5.
In some of these embodiments, the temperature of the aging is from 0 ℃ to 6 ℃, and further from 0 ℃ to 4 ℃.
In some of these embodiments, the aging time is from 2 hours to 18 hours, further from 6 hours to 12 hours, further from 6 hours to 10 hours, or from 6 hours to 8 hours.
In certain embodiments, the aluminum salt refers to a salt comprising a trivalent positive aluminum ion and an acid anion, for example, the aluminum salt is potassium aluminum sulfate or aluminum sulfate.
In certain embodiments, the hydroxide is at least one of sodium hydroxide and potassium hydroxide, such as sodium hydroxide.
In some of these embodiments, the mixing is performed by a microfluidic system or a micro-syringe pump.
In some of these embodiments, the microfluidic system or the micro syringe pump has a bolus rate of (0.1 ± 0.05) mL/min.
In certain of these embodiments, the mixing is performed with ice bath stirring.
In some of these embodiments, the aging is followed by a purification step, which is a purification method conventionally used in the art, for example, a solution dialysis method.
In some of these embodiments, the solution dialysis method employs ultrafiltration tubes with a molecular weight cut-off (3500 ± 500) for dialysis.
The buffer system of the present invention is a buffer system conventionally used in the art. In some embodiments, the buffer system is a phosphate buffer system.
The present invention will be described in further detail with reference to specific examples.
RBD protein (2019 novel coronavirus receptor binding domain protein): purchased from yinqiao shenzhou company.
OVA protein antigen: purchased from Sigma-Aldrich.
Mouse colon cancer cells CT26 tumor cells: purchased from ATCC.
BSA protein: purchased from biofrox.
Commercial aluminum hydroxide gel tumor vaccine: purchased from InvivoGen.
Example 1.2019 preparation of antigen-adjuvant Integrated vaccine of novel coronavirus Receptor Binding Domain (RBD)
(1) Weighing RBD protein (with the molecular weight of 30 kDa) (0.033 mu mol,1 eq.v.) and dissolving in sterile 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution A;
(2) Weighing aluminum potassium sulfate (1.65 mu mol,50 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Weighing sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain a third solution;
(4) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(5) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, pushing for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when the pH value is monitored by a pH meter until the pH value reaches 12.
(6) And transferring the mixed system into a refrigerator at 4 ℃, incubating and aging overnight, and purifying through an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated novel coronavirus recombinant protein vaccine with the RBD protein antigen as the shell and the aluminum metahydroxide cluster as the core and having the nucleocapsid structure.
Example 2 preparation of antigen-adjuvant Integrated vaccines for OVA protein antigens
(1) Weighing OVA protein (with the molecular weight of 45 kDa) (0.033 mu mol,1 eq.) and dissolving in sterile 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution A;
(2) Weighing aluminum potassium sulfate (1.32 mu mol,40eq v.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Weighing sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain a third solution;
(4) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the sterile syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, enabling the push injection time to be 2min, and slowly injecting the solution B into the solution A;
(5) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, carrying out push injection for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when a pH meter monitors that the pH value reaches 12.
(6) And transferring the mixed system into a refrigerator at 4 ℃ for incubation and aging overnight, and purifying through an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated vaccine with the OVA protein antigen as the shell and the aluminum metahydroxide cluster as the core and the core-shell structure.
Example 3 preparation of an antigen-adjuvant Integrated vaccine for tumor holoprotein antigens
(1) After counting mouse colon cancer cell CT26 tumor cells using a blood cell technique plate, about 2X 10 cells were taken 7 Repeatedly freezing and thawing the cells for three times, centrifuging, taking supernate, filtering the supernate with a 0.22 mu m filter membrane, and quantifying the extracted tumor whole protein antigen by using a BCA method on the supernate;
(2) According to the protein quantification result in the step (1), dissolving the tumor holoprotein antigen (with the average molecular weight of 65 kDa) (0.033 mu mol,1 eq.v.) in sterile 1mL phosphate buffer solution, and stirring uniformly in an ice bath to obtain liquid A;
(3) Weighing aluminum potassium sulfate (0.825 mu mol,25 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(4) Weighing 1.6mg of sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL of phosphate buffer solution, and uniformly stirring in an ice bath to obtain a third solution;
(5) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(6) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, pushing for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when the pH value is monitored by a pH meter until the pH value reaches 12.
(7) And transferring the mixed system into a refrigerator at 4 ℃, incubating and aging overnight, and purifying through an ultrafiltration tube (MWCO = 3500) to obtain the tumor vaccine with a core-shell structure, wherein the tumor vaccine takes a tumor whole protein antigen as a shell and an aluminum metahydroxide cluster as a core.
Example 4 preparation of antigen-adjuvant-Integrated vaccines for BSA protein
(1) Weighing BSA (bovine serum albumin) with the molecular weight of 65KDa (0.033 mu mol,1 eq.v.) and dissolving in sterile 1mL phosphate buffer solution, and uniformly stirring in an ice bath to obtain a solution A;
(2) Weighing aluminum potassium sulfate (0.99 mu mol,30 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Weighing sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain a third solution;
(4) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(5) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, pushing for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when the pH value is monitored by a pH meter until the pH value reaches 12.
(6) And transferring the mixed system into a refrigerator at 4 ℃, incubating and aging overnight, and purifying through an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated vaccine with a core-shell structure, wherein the BSA protein antigen is taken as a shell and the aluminum metahydroxide cluster is taken as an inner core.
Example 5 preparation of antigen-adjuvant Integrated vaccines
(1) Weighing OVA protein (with the molecular weight of 45 kDa) or BSA protein (with the molecular weight of 65 kDa) (0.033 mu mol,1 eq.v.) and dissolving in sterile 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution A;
(2) Weighing aluminum potassium sulfate (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Weighing sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain a third solution;
(4) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(5) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, pushing for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when the pH value is monitored by a pH meter until the pH value reaches 12.
(6) And transferring the mixed system into a refrigerator at 4 ℃, incubating and aging overnight, and purifying through an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated novel coronavirus recombinant protein vaccine with the RBD protein antigen as the shell and the aluminum metahydroxide cluster as the core and having the nucleocapsid structure.
Example 6 preparation of antigen-adjuvant Integrated vaccines
(1) Weighing and weighing OVA protein (molecular weight is 45 kDa) or BSA protein (molecular weight is 65 kDa) (0.033 mu mol,1 eq.v.) and dissolving in sterile 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution A;
(2) Weighing aluminum potassium sulfate (1.65 mu mol,50 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(4) And (3) transferring the mixed system in the step (3) to a refrigerator at 4 ℃, incubating and aging overnight, and then purifying through an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated novel coronavirus recombinant protein vaccine with the RBD protein antigen as the shell and the aluminum metahydroxide cluster as the core and having the core-shell structure.
Example 7 preparation of antigen-adjuvant Integrated vaccines
(1) Weighing OVA protein (with the molecular weight of 45 kDa) or BSA protein (with the molecular weight of 65 kDa) (0.033 mu mol,1 eq.v.) and dissolving in sterile 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution A;
(2) Weighing aluminum potassium sulfate (1.65 mu mol,50 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain solution B;
(3) Weighing sodium hydroxide (6.6 mu mol,200 eqv.) and dissolving in 1mL phosphate buffer solution, and stirring uniformly in ice bath to obtain a third solution;
(4) Stirring the solution A in ice bath, transferring the solution B into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting the parameters to be 0.1mL/min, and slowly injecting the solution B into the solution A after the push injection time is 2 min;
(5) And after fully stirring, transferring the solution A into a 1mL sterile syringe, placing the syringe in a mechanical push injection pump, setting parameters to be 0.1mL/min, carrying out push injection for 5min, slowly injecting the solution A into the mixed solution of the solution A and the solution B, and stopping adding the solution A when a pH meter monitors that the pH value reaches 12.
(6) And incubating and aging the mixed system at room temperature overnight, and purifying by an ultrafiltration tube (MWCO = 3500) to obtain the antigen-adjuvant integrated novel coronavirus recombinant protein vaccine with the RBD protein antigen as the shell and the aluminum metahydroxide cluster as the core and having the nucleocapsid structure.
Experiment I, transmission electron microscope detection shell-core structure of antigen-adjuvant integrated vaccine
The appearance of the antigen-adjuvant integrated vaccine in the embodiment 2-3 is observed by using a transmission electron microscope, 10 μ L of the antigen-adjuvant integrated vaccine prepared by the method in the embodiment 2-3 is absorbed and placed on a transmission electron microscope sample preparation copper net, the transmission electron microscope sample preparation copper net is placed in an ultra-clean workbench at room temperature for airing, and the sample appearance is observed by using a normal-temperature transmission electron microscope under the condition of 200 kV.
The results are shown in fig. 1-2, wherein fig. 1 is a transmission electron micrograph and an enlarged view of the model antigen OVA integrated vaccine of example 2, and fig. 2 is a transmission electron micrograph of the whole antigen integrated vaccine of mouse colon cancer cell CT26 of example 3. As can be seen from fig. 1 and 2, the black part with higher contrast is confined in the gray part with lower contrast in the TEM result, and the high-contrast inorganic aluminum adjuvant is judged to be in a single protein antigen by combining the TEM principle. The result shows that the antigen-adjuvant integrated vaccine with the nucleocapsid structure, which takes a single protein antigen as the shell and the aluminum metahydroxide cluster as the inner core, is successfully prepared, and the preparation method has universality.
Experiment II, particle size detection of antigen-adjuvant integrated vaccine
The mean particle size and distribution of the antigen-adjuvant integrated vaccine prepared by the method of examples 1-3 and 5-7 were measured by using a Malvern Zetasizer dynamic light scattering particle sizer, 0.1mL of the antigen-adjuvant integrated vaccine prepared by the method of examples 1-3 and 5-7 was added to a marvens micro sample test cell, the test material was set to protein, the test temperature was set to 25 ℃, the equilibration time was 120s, the test medium was water, and the test results of examples 1-3 are shown in fig. 3, where a in fig. 3 is an antigen-adjuvant integrated vaccine particle size characterization graph of the mouse colon cancer cell CT26 whole antigen of example 3, b in fig. 3 is an antigen-adjuvant integrated vaccine particle size characterization graph of the model antigen OVA of example 2, and c in fig. 3 is an antigen-adjuvant integrated vaccine particle size characterization graph of the 2019 novel coronavirus Receptor Binding Domain (RBD) of example 1. As can be seen from figure 3, the antigen-adjuvant integrated vaccine prepared by the invention has the average particle size of less than 30 nanometers for the first time, the diameter of the inner core of the nanoparticle is less than or equal to 10nm, and the distribution is uniform, so that the integrated vaccine can enter lymph nodes to contact antigen presenting cells to activate immune response. The particle size of the conventional aluminum hydroxide gel is generally 2-10 mu m, and the aluminum hydroxide gel can be nano-sized at most in the order of hundreds of nanometers even if a complex nano-sizing process is adopted. The average particle size of the antigen-adjuvant integrated vaccines of examples 5 to 7 is shown in table 1.
TABLE 1
Experiment III, co-localization analysis of aluminum hydroxide adjuvant component and protein component of antigen-adjuvant integrated vaccine
After the antigen-adjuvant integrated vaccine of the model antigen OVA prepared by the method in example 2, 3mL of the antigen-adjuvant integrated vaccine solution was taken, proteins were fluorescently labeled by EDC/NHS reaction, and aluminum components of the antigen-adjuvant integrated vaccine were specifically labeled with fluorescent gallium (LumGa). And (3) performing polyacrylamide gel electrophoresis separation on the marked antigen-adjuvant integrated vaccine, and analyzing a fluorescence signal by using a small animal imager on the gel subjected to electrophoresis separation. The detection result is shown in fig. 4, the aluminum hydroxide adjuvant component and the protein antigen component in the antigen-adjuvant integrated vaccine still keep the co-localization combination state after electrophoresis, which shows that the protein antigen and the aluminum hydroxide adjuvant in the integrated vaccine of the invention are combined very tightly and are not easy to dissociate, thereby being beneficial to the adjuvant to realize the lasting effect. Figure 5 is an XPS analysis of the aluminium hydroxide component of the vaccine showing that the aluminium hydroxide component produced in the vaccine is predominantly aluminium metahydroxide, the composition of which is consistent with that of current commercial aluminium adjuvants.
Experiment four, the uptake of the aluminum hydroxide adjuvant of the antigen adjuvant integrated vaccine in the antigen presenting cells
After the antigen-adjuvant integrated vaccine prepared by the method in example 2, the aluminum hydroxide adjuvant part and the protein antigen part in the antigen-adjuvant integrated vaccine were fluorescently labeled by the method in experiment three, and DC2.4 cells were labeled by 1X 10 5 The cells were inoculated in a 24-well plate at a density of one well, cultured for 12 hours, replaced with a serum-free medium, and each of the labeled antigen-adjuvant integrated vaccine (RBD dose 5. Mu.g, adjuvant dose 0.5. Mu.g), free protein antigen (RBD dose 5. Mu.g, adjuvant dose 5. Mu.g) and vaccine protein incubated with commercial aluminum hydroxide gel (RBD dose 5. Mu.g) was added, incubated at 37 ℃ for 8 hours, washed 2 times with phosphate buffered saline, separated into single cell suspensions with 0.25 rpm TE, centrifuged at 4 ℃ for 5 minutes, and the supernatant was discarded. After the cells are resuspended, the uptake of the aluminum adjuvant part and the protein antigen part in the antigen-adjuvant integrated vaccine in the cells is detected by a flow cytometer. As shown in figure 6, after the antigen-adjuvant integrated vaccine of the invention is used, the uptake of the aluminum adjuvant and the protein antigen by the dendritic cells of the main antigen presenting cells can be remarkably increased.
Experiment five and 2019 detection of neutralizing antibody of mouse immune post-immunization of novel coronavirus Receptor Binding Domain (RBD) antigen-adjuvant integrated novel corona vaccine
Antigen adjuvant one was prepared as in example 1A novel coronavirus recombinant protein vaccine is formed and used for immunizing mice, and the immunization scheme is as follows: 24 female BALB/c mice (8 weeks old) of SPF grade were taken and randomized into 3 groups of 8 mice each. Mice were injected intramuscularly on the posterior medial thigh with phosphate buffered saline, RBD protein antigen (RBD dose 0.25 mgkg) on days 0 and 7 -1 ) New corona vaccine integrated with antigen adjuvant (RBD dosage 0.25 mgkg) -1 Adjuvant dose 0.25mgkg -1 ) The total injection is 2 times, and the dosage of each protein antigen is 5 mug/mouse. After 4 weeks mice were sacrificed, peripheral blood was taken from the orbit and serum RBD neutralizing antibody content was measured by ELISA. The experimental result is shown in fig. 7, the content of RBD neutralizing antibodies of mice can be significantly increased by the antigen-adjuvant integrated new corona vaccine inoculated through intramuscular injection, which is about 10 times of that of the control group and 2 times of that of the RBD protein antigen group. ( ns, P is more than or equal to 0.05; * P is less than 0.05; * P is less than 0.01; * P < 0.001; * P < 0.0001 )
Experiment six, 2019 detection of mouse immune activation effect of novel coronavirus Receptor Binding Domain (RBD) antigen-adjuvant integrated novel corona vaccine
SPF-grade female BALB/c mice (8 weeks old) are immunized according to the five experimental schemes, lungs, spleens and inguinal lymph nodes of the mice are picked up after the mice are sacrificed, after the mice are ground into single cell suspension, immune cells are marked by using a fluorescent flow antibody, the cells are washed by using phosphate buffer solution for 2 times, then 200 mu L of phosphate buffer solution is used for resuspension, and the detection is carried out by using a flow cytometer. The results of the experiment are shown in fig. 8, in which a of fig. 8 is a cell-killer T-cell fraction analysis, b of fig. 8 is a degranulation enzyme-positive cell-killer T-cell fraction analysis, and c of fig. 8 is a perforin-positive cell-killer T-cell fraction analysis. According to experimental results, the antigen-adjuvant integrated Xinguan vaccine can obviously increase the relative proportion and the activation degree of cell-killing T cells, and has obvious difference compared with a control group and an RBD protein antigen group. ( ns, P is more than or equal to 0.05; * P is less than 0.05; * P < 0.01; * P < 0.001; * P < 0.0001 )
Experiment seven, anti-tumor effect of antigen adjuvant integrated tumor vaccine immunized mouse
Preparation of antigen-adjuvant-Integrated tumors as in example 2Tumor vaccine, mice were immunized according to the following immunization protocol: 20 SPF-grade female C57/BL6 mice (8 weeks old) were taken and randomly divided into 4 groups of 5 mice each. Intramuscular injection of phosphate buffered saline, commercial aluminum hydroxide gel incubation of tumor protein antigen and antigen adjuvant integrated neocorona vaccine to the inner thigh of the mice on day 0, 4, 8 and 12, total injection for 4 times, and the dosage of tumor protein antigen is 5 mug/mouse. Mice were inoculated subcutaneously on the right back of the right side on day 15 at 5X 10 5 Mice were observed every two days for tumor growth on B16-F10 OVA cells. The experimental result is shown in fig. 9, the antigen-adjuvant integrated tumor vaccine can significantly inhibit the tumor growth of tumor-bearing mice, and has significant difference compared with a control group and an RBD protein antigen group. ( ns, P is more than or equal to 0.05; * P is less than 0.05; * P is less than 0.01; * P < 0.001; * P < 0.0001 )
Experiment eight, dendritic cell activation condition analysis after immunization of mice by antigen-adjuvant integrated tumor vaccine
SPF-grade female C57/BL6 mice (8 weeks old) were immunized according to the five experimental protocols (adding a group of "commercial aluminum hydroxide gel tumor vaccines": except for the different vaccines, other embodiments are the same as the group of "antigen-adjuvant integrated new crown vaccines" of the five experiments), inguinal lymph nodes and spleens of the mice were picked up after the mice were sacrificed, after grinding the mice into a single cell suspension, the immune cells were labeled with a fluorescent flow antibody, the cells were washed with a phosphate buffered saline solution for 2 times, then resuspended with 200. Mu.L of a phosphate buffered saline solution, and detected with a flow cytometer. As shown in fig. 10, the degree of activation of dendritic cells was significantly increased, and the level of activation-related marker molecules was significantly increased and significantly varied in mice immunized with the antigen-adjuvant-integrated tumor vaccine. ( ns, P is more than or equal to 0.05; * P is less than 0.05; * P < 0.01; * P < 0.001; * P < 0.0001 )
Experiment nine, memory T cell analysis data after immunization of mice by antigen-adjuvant integrated tumor vaccine
SPF-grade female C57/BL6 mice (8 weeks old) were immunized according to the five experimental protocols (adding a commercial aluminum hydroxide gel tumor vaccine group: except for the vaccine, other embodiments are the same as the antigen-adjuvant integrated new crown vaccine group of the five experimental protocols), the inguinal lymph node and spleen of the mice were picked up after the mice were sacrificed, after grinding the mice into a single cell suspension, the immune cells were labeled with a fluorescent flow antibody, the cells were washed with a phosphate buffered saline solution for 2 times, resuspended with 200. Mu.L of the phosphate buffered saline solution, and detected using a flow cytometer. The results are shown in fig. 11, the proportion of central memory T cells was significantly increased and significantly different in mice immunized with the antigen-adjuvant integrated tumor vaccine, and the immunized mice had better immunological memory effect. ( ns, P is more than or equal to 0.05; * P is less than 0.05; * P is less than 0.01; * P < 0.001; * P < 0.0001 )
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.
Claims (10)
1. The vaccine is characterized by comprising nanoparticles with a core-shell structure, wherein each nanoparticle is composed of an aluminum hydroxide adjuvant and a protein antigen, and the single protein antigen is taken as a shell of each nanoparticle and the aluminum hydroxide adjuvant is taken as an inner core of each nanoparticle.
2. The vaccine of claim 1, wherein the nanoparticles have a particle size of 100nm or less.
3. The vaccine of claim 2, wherein the nanoparticles have a particle size of 2nm to 50nm.
4. The vaccine of claim 1, wherein the aluminum hydroxide adjuvant is covalently bound to the protein antigen.
5. The vaccine of any one of claims 1 to 4, wherein the protein antigen is a pathogen protein antigen or a tumor-associated protein antigen.
6. The vaccine according to any one of claims 1 to 4, wherein the vaccine is in the form of injection, nasal drops, spray or powder injection.
7. A method for preparing a vaccine, wherein the vaccine comprises nanoparticles having a core-shell structure; the preparation method comprises the following steps:
dissolving a protein antigen in a buffer system to be used as a liquid A;
dissolving aluminum salt in a buffer system to obtain solution B;
dissolving hydroxide in a buffer system to obtain a third solution;
mixing the solution A and the solution B, adding the solution C, mixing until the pH value in a mixed system is 10-14, and then aging the obtained mixed system to obtain the nano-particles with a protein antigen as a shell and an aluminum hydroxide adjuvant as an inner core;
the molar ratio of the protein antigen to the aluminum ions in the aluminum salt in the raw material is 1: (1-50).
8. The method according to claim 7, wherein the molar ratio of the protein antigen to the aluminum ion in the aluminum salt is 1: (10 to 50).
9. The production method according to claim 7 or 8, wherein the pH in the mixed system is 11 to 13, preferably 12 ± 0.5;
and/or the aging temperature is 0-6 ℃, and the aging time is 2-18 hours;
and/or, the aluminum salt refers to a salt comprising a trivalent aluminum ion and an acid anion, further, the aluminum salt is aluminum potassium sulfate or aluminum sulfate;
and/or, the mixing is performed by a microfluidic system or a micro-syringe pump;
and/or, the aging process further comprises a purification step, and the purification method is a solution dialysis method.
10. Use of a vaccine according to any one of claims 1 to 6 or a vaccine prepared by a method according to any one of claims 7 to 9 for the manufacture of a medicament for the prevention and/or treatment of viral pneumonia-associated diseases; or the use of a vaccine according to any one of claims 1 to 6 or a vaccine prepared by the preparation method according to any one of claims 7 to 9 for the preparation of a medicament for the prevention and/or treatment of tumors.
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