CN111358942A - Vaccine and preparation method thereof - Google Patents
Vaccine and preparation method thereof Download PDFInfo
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- CN111358942A CN111358942A CN201811594091.0A CN201811594091A CN111358942A CN 111358942 A CN111358942 A CN 111358942A CN 201811594091 A CN201811594091 A CN 201811594091A CN 111358942 A CN111358942 A CN 111358942A
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- A—HUMAN NECESSITIES
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- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P35/00—Antineoplastic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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Abstract
The invention provides a vaccine and a preparation method thereof, wherein the vaccine comprises a bacterial outer membrane vesicle and an antigen; wherein the antigen and the bacterial outer membrane vesicle are associated with each other to form a nanoparticle; the anti-tumor vaccine prepared by combining the bacterial outer membrane vesicle and the tumor specific antigen simultaneously causes congenital and specific immune response, achieves the aim of treating tumors by utilizing an organism autoimmune system, and has the advantages of strong specificity, small side effect, wide application prospect and huge market value.
Description
Technical Field
The invention relates to the technical field of biology, and relates to a vaccine and a preparation method thereof.
Background
Tumor vaccines utilize tumor-specific antigens to activate Antigen Presenting Cells (APCs), thereby initiating Antigen-specific effector T cell responses and killing cancer cells. Immunotherapy in this manner may be beneficial for patients who do not respond to other immunotherapy or lack the target. The tumor antigen is mainly protein or polypeptide, so the tumor antigen is easy to degrade and has weaker immunogenicity, and is difficult to induce a sufficiently strong anti-tumor immune effect, so an adjuvant is needed to assist the antigen to activate specific immune response when the tumor vaccine is prepared.
There are many known adjuvant mechanisms of action, such as sustained release of antigen at the site of injection to promote APC maturation, upregulation of cytokines and chemokines, recruitment of immune cells to the site of antigen injection, activation of inflammasome, and the like. In 1973, Ralph Steinman and Zanvil Cohn described the critical role of DCs in mediating innate and inducing adaptive immune responses, and since then, DCs were considered by immunologists to be the most effective APCs capable of activating both initial and memory immune responses. The antigen presented by the mature DC with high expression and co-stimulation molecules can be activated to generate specific immune response by the initial T cell, while the antigen presented by the immature DC can generate immune tolerance by regulating or inhibiting the differentiation of the T cell, so that in the application of tumor vaccines, a proper adjuvant is selected to stimulate the body to generate innate immune response, thereby stimulating the maturation of the DC cell and further causing the specific immune response aiming at the specific antigen, and the application becomes a research hotspot.
Therefore, a novel vaccine adjuvant is developed and used for stimulating an organism to generate innate immune response and promoting DC cells to mature, so that the purpose of treating tumors by utilizing an organism autoimmune system is achieved, and the novel vaccine adjuvant has a wide application prospect and a huge market value.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides a vaccine and a preparation method thereof, the anti-tumor vaccine prepared by combining the bacterial outer membrane vesicle and the tumor specific antigen can simultaneously cause congenital and specific immune response, so as to achieve the purpose of treating tumors by utilizing the body autoimmune system, and the vaccine has wide application prospect and great market value.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a vaccine comprising a bacterial outer membrane vesicle and an antigen;
wherein the antigen is associated with a bacterial outer membrane vesicle to form a nanoparticle.
The invention provides an anti-tumor vaccine prepared by combining bacterial Outer Membrane Vesicles (OMVs) and tumor specific antigens, wherein the particle size of the anti-tumor vaccine is 20-250 nm; in the vaccine, OMV is used as an adjuvant in a tumor vaccine to perform the functions of stimulating DC cell maturation and causing the innate immune response of an organism, so that the antigen-specific effector T cell reaction is started and cancer cells are killed, the adjuvant (namely the bacterial outer membrane vesicle) is combined with the antigen, so that the adjuvant can be simultaneously transported to the same DC cell, the innate immunity and the specific immunity are simultaneously activated, the treatment effect of the vaccine is enhanced, and the purpose of treating tumors by utilizing the autoimmune system of the organism is achieved.
The invention aims at how to evoke the specific immunity of an organism to the tumor, and materials with specific immunological functions (namely the bacterial outer membrane vesicle and the tumor specific antigen in the invention) are organically combined and assembled to prepare the anti-tumor vaccine so as to enhance the specific immune function of the organism, solve the defects of large side effect, poor specificity and the like in other cancer treatment schemes, and achieve better cancer treatment effect.
The bacterial outer membrane vesicle and the tumor specific antigen can be prepared by mixing in any proportion, which is determined according to the application purpose.
Preferably, the particle size of the vaccine is 20-250nm, which may be, for example, 20nm, 40nm, 60nm, 80nm, 100nm, 150nm, 200nm, 220nm or 250 nm.
Preferably, the binding means of the antigen to the bacterial outer membrane vesicles comprises electrostatic adsorption and/or chemical modification, preferably electrostatic adsorption.
Preferably, the source of the bacterial outer membrane vesicles includes gram-negative bacteria such as Salmonella typhimurium (Salmonella typhimurium), Escherichia coli (Escherichia coli), Acinetobacter baumannii (Acinetobacter baumannii), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Haemophilus influenzae (Haemophilus influenzae), Burkholderia pseudomallei (Burkholderia pseudomonilei), Vibrio cholerae (Vibrio cholerae), Neisseria meningitidis (Neisseria meningitidis), Bordetella pertussis (Bordetella pertussis perusas); the source of the bacterial outer membrane vesicle can be genetically modified strains or strains which are not genetically modified.
Preferably, the antigen comprises a tumor specific antigen.
Preferably, the tumor specific antigen comprises any one of or a combination of at least two of a melanoma specific antigen, a prostate cancer specific antigen, a breast cancer specific antigen, a blood cancer specific antigen, a bladder cancer specific antigen or a metastatic lung cancer specific antigen, preferably a melanoma specific antigen.
Preferably, the melanoma specific antigen comprises a TRP-2 antigen peptide and/or a gp100 antigen peptide.
In the present invention, the antigen is not particularly limited, and any antigen capable of combining with bacterial vesicles to form nanoparticles is within the scope of the present application.
Preferably, the N-terminus of the tumor specific antigen is modified.
Preferably, the modified molecule comprises a polypeptide, preferably a positively charged polypeptide, which does not affect the original function of the specific antigen.
Preferably, the positively charged polypeptide comprises any one of the polypeptide sequences Arg-Arg-Arg, Lys-Lys-Lys-Lys, or His-His-His-His, or a combination of at least two thereof.
Preferably, the vaccine is more readily taken up by DC cells and is functional due to the positively charged antigen on the surface.
During the course of a natural immune response, the host's immune cells recognize foreign microbial signals by a variety of mechanisms, including Toll-like receptors located on the surface of cell membranes, which recognize extracellular microbial components, such as LPS. LPS, if it enters the cytoplasm, can trigger activation of caspase-11 inflammasome, which in turn triggers an inflammatory response. However, many bacteria that have this phenomenon do not have the ability to invade cells themselves. The bacteria are capable of secreting a small vesicle, i.e. OMV, which is enriched with LPS. The bacteria achieve the purpose of transporting LPS by fusing OMV and host cells, a structure similar to 'OMV' appears in the host cells in the bacterial infection process, the purified OMV and macrophages are cultured together, and a large amount of LPS appears in cytoplasm of the cultured cells, so that the OMV can be determined to perform the main function of transporting LPS and can cause the innate immunity function. Certain components contained in OMVs, such as proteins, DNA, RNA, etc., also play a role in immune stimulation. Therefore, the invention selects the Outer Membrane Vesicle (OMV) as the adjuvant in the tumor vaccine to stimulate the maturation of DC cells and cause the function of the innate immune response of the organism; the adjuvant (namely the bacterial outer membrane vesicle) is combined with the antigen, so that the adjuvant can be transported to the same DC cell at the same time, and the innate immunity and the specific immunity are activated at the same time, so that the treatment effect of the vaccine is enhanced.
In a second aspect, the invention provides the use of a bacterial outer membrane vesicle as a vaccine adjuvant.
In a third aspect, the present invention provides a method for preparing a vaccine, comprising the steps of:
(1) connecting a modifying molecule at the N end of the antigen to prepare a solution;
(2) dispersing the solution of the step (1) in the bacterial outer membrane vesicle solution to form nanoparticles, namely the vaccine.
Preferably, the antigen of step (1) comprises a tumor specific antigen, preferably a melanoma specific antigen.
Preferably, the melanoma specific antigen comprises a TRP-2 antigen peptide and/or a gp100 antigen peptide.
Preferably, the modified molecule of step (1) comprises a polypeptide, preferably a positively charged polypeptide.
Preferably, the solvent of the solution of step (1) comprises dimethyl sulfoxide.
Preferably, the temperature of the formulation of step (1) is room temperature.
The temperature of the room temperature includes 20-30 deg.C, such as 20 deg.C, 21 deg.C, 22 deg.C, 23 deg.C, 24 deg.C, 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C or 30 deg.C.
Preferably, the source of the bacterial outer membrane vesicles of step (2) comprises gram-negative bacteria.
Preferably, the dispersing condition of the step (2) is ice bath ultrasound.
Preferably, the solvent of the bacterial outer membrane vesicle solution of step (2) is PBS.
Preferably, the particle size of the nanoparticles in the step (2) is 20-250 nm.
As a preferred technical scheme, the preparation method of the vaccine specifically comprises the following steps:
(1) connecting a modified molecule with positive charges at the N end of the tumor specific antigen, and dissolving the modified molecule in dimethyl sulfoxide to obtain a solution;
(2) and (2) dispersing the solution obtained in the step (1) in a bacterial outer membrane vesicle solution secreted by gram-negative bacteria under the ice bath ultrasonic condition to form nanoparticles with the diameter of 20-250nm, namely the vaccine.
The technical scheme is summarized as follows:
extracting OMV secreted by gram-negative bacteria by using an ultracentrifugation method, dissolving the OMV in a PBS solution, and quantifying by using a BCA method; connecting a modifying molecule at the N end of the tumor specific antigen to ensure that the modifying molecule has positive charges and is dissolved in a dimethyl sulfoxide solvent at room temperature; mixing OMV and antigen peptide according to a certain proportion, and performing ultrasonic treatment for 30s under the ice bath condition to disperse the antigen peptide in a certain volume of PBS solution containing bacterial outer membrane vesicles, wherein the system forms nanoparticles through electrostatic adsorption to obtain an anti-tumor vaccine; observing the particle size and the morphology of the anti-tumor vaccine by using a transmission electron microscope; observing the vaccine intake condition of mouse bone marrow-derived dendritic cells (BMDCs) by using a single photon laser confocal imaging system; evaluating the ability of the vaccine to stimulate BMDC cell maturation using flow assays; the treatment effect of the invention is verified by animal experiments.
Compared with the prior art, the invention has the following beneficial effects:
(1) the vaccine provided by the invention has the characteristics of high safety and convenience in preparation, is quick and simple in preparation, high in efficiency and time-saving, is favorable for popularization and application, has great potential in the aspect of tumor treatment due to strong specificity and small side effect, is expected to be applied to clinic as a novel nano-drug, and has good drug development potential;
(2) the dendritic cells for communicating innate immunity and acquired immunity are used as main research objects, the OMV is used as a tumor vaccine adjuvant for the first time, and the OMV and tumor antigen peptide are used as immunostimulants to combine, so that the defects that tumor specific antigens are easy to degrade, have weak immunogenicity, and are difficult to induce strong anti-tumor immune effect are overcome, and the treatment effect and specificity of the vaccine are improved;
(3) the invention has wide application range, can select different strains, or carry out genetic engineering transformation on certain strains, and can be applied to different purposes; meanwhile, different tumor specific antigens are selected, and the method can be applied to different tumor types.
Drawings
FIG. 1 is a schematic diagram showing the morphology of an antitumor vaccine in example 1 of the present invention;
FIG. 2 is a diagram illustrating the detection results of a single-photon confocal laser imaging system in embodiment 2 of the present invention;
FIGS. 3(A) and 3(B) are graphs showing the results of analysis by the flow cytometer in example 3 of the present invention;
FIGS. 4(A) and 4(B) are results of animal experiments in example 4 of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1
Inoculating DH5- α strain into corresponding culture medium, culturing at 37 deg.C and 220rpm overnight, centrifuging at 4 deg.C for 20min with 6000 × g of centrifuge, filtering the supernatant with 0.45 μm filter membrane, concentrating the filtrate to 100mL with 100kDa ultrafiltration tube, filtering the concentrated product with 0.22 μm filter membrane to remove other cells and debris, ultracentrifuging the obtained filtrate at 4 deg.C and 150000 × g for 3h, resuspending the obtained OMVs in PBS, and detecting the protein concentration with BCA;
connecting 4 arginines to the N end of a tumor specific antigen TRP2 antigen peptide to ensure that the arginines have positive charges and are dissolved in a dimethyl sulfoxide solvent at room temperature; OMV and antigen peptide were mixed at a ratio of 1:1, sonicated in ice bath for 30s, diluted to appropriate concentration with PBS, observed for morphology and particle size using a biological transmission electron microscope, and photographed by a field emission transmission electron microscope (F-20), see FIG. 1.
Example 2
Adding excessive cy 5.5-N-hydroxysuccinimide ester into the OMV solution, and reacting overnight at 4 ℃ to prepare OMV-cy 5.5; modifying FITC fluorescent molecules at the N end of an antigen peptide (modified TRP2 antigen peptide is used in the example and is positively charged), preparing an anti-tumor vaccine by using OMV-Cy5.5 and TRP2-FITC, culturing BMDC cells, connecting 0.4milli BMDC to each dish by using a confocal glass-bottom dish, adding 10 mu g of OMV anti-tumor vaccine, and incubating for 24 h;
the nuclei were stained with Hoechst33342 diluted 1000 times for 5min, washed three times with PBS, fixed with tissue fixative for 15min, washed three times with PBS, and finally detected with a single photon laser confocal imaging system, and the results are shown in FIG. 2, and the uptake of the anti-tumor vaccine by the BMDC cells can be seen in FIG. 2.
Example 3
Culturing BMDC cells, dividing into 5 groups, namely a Control group, an MPLA positive Control group, a tumor specific antigen group (TRP 2 is used in the example), an OMV group and an anti-tumor vaccine group (OMV-TRP 2 is used in the example), wherein each group contains about 4 ten thousand cells, and the added materials are all the amount of 10 mu g of OMV;
after incubating the material and the cells for 24 hours, centrifuging at 1000rpm for 5 minutes by using a centrifuge to collect the cells, diluting the PE anti-mouse CD11c antibody and the FITC anti-mouse CD80 antibody by using a FACSBuffer100 times, preparing an antibody staining solution, adding 50 mu L of each group of cells, staining the cells in a dark place for at least 20 minutes at 4 ℃, supplementing 1mL of FACS Buffer, centrifuging at 2000 rpm for 5 minutes, discarding the supernatant, shaking for resuspension, analyzing by using a flow cytometer, and detecting CD86 in the same way to evaluate the BMDC maturation, wherein the results are shown in FIG. 3(A) and FIG. 3(B) and reflect the maturation of the BMDC cells after being stimulated by the anti-tumor vaccine.
Example 4
The C57BL/6 black mice were divided into two groups: control group and experimental group (in this example, the anti-tumor antigen peptide used was a modified TRP2 antigen peptide), 3 of each group; all the old rat tails were intravenously injected with 40 ten thousand B16 cells and were administered at the later stage on days 3, 6, and 11; wherein, the Control group is injected with 200 mu L PBS per mouse, and the experimental group is injected with 100 mu g OMV anti-tumor vaccine per mouse; the preparation method of the anti-tumor vaccine is based on the steps, the experiment on the 17 th day is finished, the mice are killed by using a carbon dioxide method, the lungs are dissected and taken out, the lung metastasis condition is observed, and as shown in fig. 4(a) and 4(B), the anti-tumor vaccine group has a remarkable inhibiting effect on the lung metastasis condition of the B16 cells as can be seen from fig. 4(a) and 4 (B).
In conclusion, the invention provides a vaccine and a preparation method thereof, the anti-tumor vaccine prepared by combining the bacterial outer membrane vesicle and the tumor specific antigen can simultaneously cause congenital and specific immune response, the purpose of treating the tumor by utilizing the body autoimmune system is achieved, the specificity is strong, the side effect is small, and the vaccine has wide application prospect and great market value.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. A vaccine comprising a bacterial outer membrane vesicle and an antigen;
wherein the antigen is associated with a bacterial outer membrane vesicle to form a nanoparticle.
2. The vaccine of claim 1, wherein the particle size of the vaccine is 20-250 nm;
preferably, the source of the bacterial outer membrane vesicles comprises gram-negative bacteria;
preferably, the binding means of the antigen to the bacterial outer membrane vesicles comprises electrostatic adsorption and/or chemical modification, preferably electrostatic adsorption.
3. The vaccine of claim 1 or 2, wherein the antigen comprises a tumor-specific antigen;
preferably, the tumor specific antigen comprises any one or a combination of at least two of melanoma specific antigen, prostate cancer specific antigen, breast cancer specific antigen, blood cancer specific antigen, bladder cancer specific antigen or metastatic lung cancer specific antigen, preferably melanoma specific antigen;
preferably, the melanoma specific antigen comprises a TRP-2 antigen peptide and/or a gp100 antigen peptide.
4. The vaccine of any one of claims 1-3, wherein the tumor specific antigen is modified at the N-terminus;
preferably, the modified modifying molecule comprises a polypeptide, preferably a positively charged polypeptide;
preferably, the positively charged polypeptide comprises any one of the polypeptide sequences Arg-Arg-Arg, Lys-Lys-Lys-Lys, or His-His-His-His, or a combination of at least two thereof.
5. Use of a bacterial outer membrane vesicle as a vaccine adjuvant.
6. A method for preparing a vaccine, comprising the steps of:
(1) connecting a modifying molecule at the N end of the antigen to prepare a solution;
(2) dispersing the solution of the step (1) in the bacterial outer membrane vesicle solution to form nanoparticles, namely the vaccine.
7. The method of claim 6, wherein the antigen of step (1) comprises a tumor specific antigen, preferably a melanoma specific antigen;
preferably, the melanoma specific antigen comprises a TRP-2 antigen peptide and/or a gp100 antigen peptide;
preferably, the modified molecule of step (1) comprises a polypeptide, preferably a positively charged polypeptide.
8. The method according to claim 6 or 7, wherein the solvent of the solution of step (1) comprises dimethyl sulfoxide;
preferably, the temperature of the formulation of step (1) is room temperature.
9. The method of any one of claims 6-8, wherein the source of the bacterial outer membrane vesicles of step (2) comprises gram-negative bacteria;
preferably, the dispersing condition of the step (2) is ice bath ultrasound;
preferably, the solvent of the bacterial outer membrane vesicle solution of step (2) is PBS;
preferably, the particle size of the nanoparticles in the step (2) is 20-250 nm.
10. The method according to any one of claims 6 to 9, comprising in particular the steps of:
(1) connecting a modified molecule with positive charges at the N end of the tumor specific antigen, and dissolving the modified molecule in dimethyl sulfoxide to obtain a solution;
(2) and (2) dispersing the solution obtained in the step (1) in a bacterial outer membrane vesicle solution secreted by gram-negative bacteria under the ice bath ultrasonic condition to form nanoparticles with the diameter of 20-250nm, namely the vaccine.
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| CN112773901A (en) * | 2021-01-29 | 2021-05-11 | 国家纳米科学中心 | Bacterial outer membrane vesicle carrier and preparation method and application thereof |
| CN112870340A (en) * | 2021-01-27 | 2021-06-01 | 四川大学 | Tumor vaccine based on breast cancer extracellular vesicles and preparation method thereof |
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