CN117860879A - Nanometer vaccine delivery system and preparation method and application thereof - Google Patents
Nanometer vaccine delivery system and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a nano vaccine, which is prepared from the following raw materials: a slow release carrier, an antigen, an adjuvant, and a cationic component; the mass ratio of the slow release carrier, the antigen, the adjuvant and the cation component is (1-30): 1: (0.1-1): (0.05 to 0.5); the slow release carrier comprises sodium alginate; the antigen comprises at least one of a protein, a recombinant subunit, and a polypeptide; the cation component comprises at least one of calcium ion, manganese ion, aluminum ion, polyamine, basic amino acid and polyamino acid composed of 2-30 basic amino acids. The nanometer vaccine comprises sodium alginate serving as a slow-release carrier, an antigen, an adjuvant and a cation component in a proper proportion, wherein the sodium alginate is used as the slow-release carrier, and is promoted to form a net structure through the action of the cation component, and the antigen and the adjuvant are wrapped to form stable antigen-carrier-adjuvant nanoparticle gel. The nanometer vaccine can induce more powerful immune response, and has good stability and biocompatibility.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a nano vaccine delivery system, a preparation method and application thereof.
Background
Malignant tumors, i.e. cancers, have a high incidence worldwide. Malignant tumor incidence will also increase rapidly worldwide as the population ages and the population increases. Thus, malignancy poses a considerable threat to human health.
There are three traditional treatments for cancer, namely surgery, chemotherapy and radiation, all of which have their advantages and disadvantages. Surgery can directly ablate macroscopic tumor tissue, however recent studies have shown that the post-operative wound healing response may lead to the growth of metastatic tumors. The radiotherapy and chemotherapy can kill tumor cells in a large area, but the tumor cells are easy to be tolerated and recurred, and meanwhile, the radiotherapy and chemotherapy can kill normal cells, so that the prognosis of a patient is easy to be poor. In recent years, immunotherapy has rapidly progressed, and has become a mature cancer treatment strategy in addition to surgery, chemotherapy, and radiotherapy. Immunotherapy refers to the use of immunological principles and methods to artificially interfere with or regulate the immune function of the body in a hypoimmunity or hyperimmunity state, and to enhance or attenuate the immune response, so as to achieve the purpose of treating diseases. The hot spots of current tumor immunotherapy are focused on the fields of immune checkpoint blocker therapy, chimeric antigen receptor-T cell therapy (ChimericAntigen Receptor T-Cell Immunotherapy, CAR-T) and therapeutic tumor vaccines. These treatments have their advantages and disadvantages. For example, the treatment of the immune checkpoint blocker PD-1 (Programmed Cell Death Protein-1) has the advantages of durable curative effect, low recurrence rate, wide treatment spectrum and the like, but also has the defects of poor effect on cold tumor, toxic and side effects and the like; the CAR-T therapy has the advantages of strong specificity, overcoming the restriction of MHC, reducing the risk of immune escape caused by the down regulation of MHC molecules by tumor cells, good effect on acute leukemia, non-Hodgkin lymphoma and other blood tumors, and the like, but also has the problems of killing normal cells, inducing cytokine storm, difficult homing of CAR-T cells, poor treatment effect on solid tumors, high cost and the like.
Therapeutic vaccines are a representative strategy for cancer immunotherapy, aimed at inducing in vivo specific immune responses against a specific antigen or group of antigens by boosting or reactivating the patient's own immune system. Compared with other treatment methods, the therapeutic cancer vaccine is used as a treatment method of active immune intervention, has stronger operability and does not cause strong side effects, thereby becoming a research hot spot of immunotherapy. Tumor vaccines can be divided into three major classes, namely cell vaccines, protein/polypeptide vaccines and nucleic acid vaccines, depending on the mechanism of action. Wherein the cell vaccine can be classified into tumor cell vaccine and DC vaccine; protein/polypeptide vaccines can be divided into protein vaccines, short peptide vaccines and long peptide vaccines; nucleic acid vaccines can be classified as DNA vaccines, RNA vaccines and viral vector vaccines. The U.S. Food and drug administration (Food andDrugAdministration, FDA) approved a first Dendritic Cell (DC) tumor vaccine, profnge, for the treatment of metastatic castration-resistant prostate cancer (CRPC) 4, after which research and results in the tumor vaccine field grew as a spring bamboo shoot after rain.
Although tumor vaccines have good clinical results in some patients, there are still problems in how efficient the vaccine works. The method comprises the following specific aspects: (1) The polypeptide antigen has smaller molecules, can be rapidly cleared by blood injection into a human body, is easily hydrolyzed and metabolized by relevant protease in the human body to reduce the antigen concentration, is difficult to accumulate in immune tissue organs such as lymph nodes, spleens and the like, so that the probability of taking up the antigen by DC cells is greatly reduced, and the tumor vaccine injected subcutaneously also faces similar rapid clearing and degradation problems; (2) The antigen is difficult to practice and escape after being taken into lysosomes by DC cells, and can not be released into cytoplasm after being treated and combined with MHC I molecules to be presented on the surface of DC cells, so that the anti-tumor immune response level caused by vaccine is very low; (3) The injection of antigen alone can easily cause immune tolerance, tumor vaccine is usually combined with adjuvant to activate effective immune response, but at present, many adjuvants have the problems of poor stability, short half-life, low cell uptake rate and the like, and in addition, the combination effect of the antigen and the adjuvant at the same time and at the same position is difficult to realize by simply injecting the antigen and the adjuvant, namely, the two lack of space-time co-localization capability; (4) Because the antigen and the adjuvant have short in vivo maintenance time, the vaccine effect is not durable enough, and the dosage and the inoculation frequency of the antigen and the adjuvant need to be increased to maintain the vaccine effect, which brings stronger side effects and the risk of immune tolerance to patients. Therefore, in order to induce a more durable, more potent immune response in the body, there is a need to develop vaccine delivery systems that can simultaneously load antigen and adjuvant, protecting them from clearance.
Disclosure of Invention
Based on the above, the invention aims to provide a nano vaccine which can induce more effective immune response, and has good stability and good biocompatibility.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
In a first aspect of the invention, there is provided a nanovaccine prepared from the following raw materials: a slow release carrier, an antigen, an adjuvant, and a cationic component; the mass ratio of the slow release carrier, the antigen, the adjuvant and the cation component is (1-30): 1: (0.1-1): (0.05 to 0.5);
the slow release carrier comprises sodium alginate; the antigen comprises at least one of a protein, a recombinant subunit, and a polypeptide; the cation component comprises at least one of calcium ion, manganese ion, aluminum ion, polyamine, basic amino acid and polyamino acid composed of 2-30 basic amino acids.
In some embodiments, the basic amino acid comprises at least one of arginine, lysine.
In some preferred embodiments, the cationic component is polyarginine consisting of 1-25 arginine residues.
In some embodiments, the antigen comprises at least one of a CD8 epitope short peptide, a CD8 epitope long peptide, a CD4 epitope short peptide, a CD4 epitope long peptide, a recombinant novel coronavirus S protein, a DNA capable of expressing a recombinant protein, an RNA capable of expressing a recombinant protein.
In some embodiments, the calcium ions are derived from at least one of the following compounds: caCl (CaCl) 2 、CaCO 3 、Ca(OH) 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or the manganese ions are derived from at least one of the following compounds: mnCl 2 、MnCO 3 、Mn(OH) 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or, the aluminum ion is derived from at least one of the following compounds: alCl 3 、Al(OH) 3 。
In some embodiments, the polyamine comprises at least one of a polymer comprising greater than 2 amino groups; preferably, the polyamine includes at least one of an amino acid polymer having an amino group, spermidine, and Polyetherimide (PEI for short).
In some embodiments, the adjuvant comprises at least one of CpG, LPS, poly (I: C), MPL, manganese adjuvant, oil adjuvant.
In some embodiments, the particle size of the nanovaccine is 10nm to 1000nm; preferably, the particle size of the nano vaccine is 10 nm-500 nm.
In a second aspect of the present invention, there is provided a method of preparing a nanovaccine as described above, comprising the steps of: (1) Uniformly mixing the antigen and the cation component to obtain an antigen-cation component mixture; (2) Dropwise adding the adjuvant into the antigen-cation component mixture, and uniformly mixing to obtain an adjuvant-antigen-cation mixture; (3) And (3) dropwise adding sodium alginate into the adjuvant-antigen-cation mixture, and uniformly mixing to obtain the nano vaccine.
In a third aspect, the invention provides an application of the nano vaccine or the nano vaccine prepared by the preparation method in preparation of a medicament for improving T cell immune response of an organism.
In a fourth aspect, the invention provides an application of the nano vaccine or the nano vaccine prepared by the preparation method in preparation of medicines for preventing and treating solid tumors or infectious diseases.
In some embodiments, the solid tumor comprises colorectal cancer, melanoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, head and neck tumor, prostate cancer, liver cancer, nasopharyngeal cancer, epithelial ovarian cancer, esophageal cancer, cervical cancer.
In some embodiments, the infectious disease comprises a novel coronavirus infection, influenza, hepatitis b.
In some embodiments, the medicament has at least one of the following efficacy: (1) Enhancement of anti-tumor immune response, and enhancement of IFN-gamma secretion level of T cells aiming at tumor antigens; (2) tumor volume reduction or tumor elimination; (3) Fever, weight loss and pain symptoms caused by tumors are reduced, alleviated or eliminated.
According to the invention, the nano vaccine is obtained through optimization, and comprises a slow release carrier sodium alginate, an antigen, an adjuvant and a cation component in a proper proportion, wherein the sodium alginate is used as a slow release carrier, and ionic bonds are formed between the cation component and the negatively charged sodium alginate to promote the sodium alginate to form a network structure, wrap the antigen and the adjuvant to form a stable antigen-carrier-adjuvant nano particle gel, inhibit release of internal substances and improve the slow release effect of the antigen substances. After the nano vaccine is injected into a body, the antigen and cations in body fluid are subjected to displacement reaction and slowly released, so that the antigen is prevented from being immediately released into the body to be degraded, and sodium alginate can be safely degraded in the body. And the network structure can avoid the rapid metabolism of the encapsulated adjuvant in vivo, thus realizing the co-localization delivery of the antigen and the adjuvant.
In the nano vaccine system, the interaction of positive and negative charges can promote the polymerization of vaccine particles to a certain extent, so that the uptake and presentation effect of DC cells are improved, and the immune response of T cells is improved. Firstly, the cationic component with polymerization promoting effect is optimized and obtained, and then the T cell immune response induced by the prepared nano vaccine is further researched. The cation component selected by the invention, especially polyarginine and polyamine polymer, can be well cooperated with sodium alginate slow release carrier to form more stable and uniform vaccine particles, so that the vaccine particles are easier to be absorbed by DC cells or macrophages, and better immune effect is obtained.
Drawings
FIG. 1 shows the particle size measurement results of nano-vaccines prepared with different cationic components.
Figure 2 shows the results of T cell immune response in human body by nanometer vaccine prepared with different cation components.
FIG. 3 shows the scanning electron microscope results of the nanovaccine prepared with the cationic component poly-Arg (15).
Figure 4 shows the results of T cell immune response in the body with nanovaccines prepared with different adjuvants.
FIG. 5 shows the effect of nanovaccine composition on T cell immune response in an organism.
FIG. 6 shows the results of cellular immunization of nanovaccines with novel coronavirus protein S protein as antigen.
FIG. 7 shows the results of humoral immunity of the nanovaccine against novel coronavirus protein S protein.
Figure 8 is the therapeutic effect of a nanovaccine binding to CD4 epitope and CD8 epitope on tumors.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. The various chemicals commonly 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 a non-exclusive inclusion. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to the elements or modules listed but may alternatively include additional steps not listed or inherent to such process, method, article, or device.
Reference in the present disclosure to "at least one" means one or more than one. "and/or", describes an association relationship of an association object, and indicates that there may be three relationships, for example, a and/or B, and may indicate: a exists alone, A and B exist together, and B exists alone.
The following description is made with reference to specific embodiments.
Sodium alginate was purchased from Shanghai Ala Biochemical technologies Co. Lana 4, GGFNFRTL, mLana 4, VGFNFRTL, mITGB1: TYSVNGYNEAIVHVVE are all available from Jiangsu Style technologies Inc. CpG2395 (TAKARA) (hereinafter, referred to as CpG) was purchased from Takara doctor materials, inc., and CpG1826 (Invivogen) and CpG2395 (Invivogen) were purchased from Invivogen, inc. The antibodies PerCP-Cy5.5-TCRβ, BV510-CD8, PE-Cy7-CD44, APC-IFN- γ, FITC-CD4 were all purchased from Biolegend, eFlour 780-availability from eBioscience. Cell culture 1640 medium and foetal calf serum were purchased from gibico company.
Arginine (Arg) in the present invention refers to L-arginine (L-Arg).
The polyamino acid consisting of 2-30 basic amino acids refers to a compound (peptide) formed by connecting 2-30 basic amino acids together through peptide bonds, and is named as poly-AA (n), wherein n is more than or equal to 2 and less than or equal to 30. For example, polyarginine consisting of 15 Args refers to a peptide formed by joining 15 Args together with peptide bonds, designated poly-Arg (15).
The invention is supported by national natural science foundation (foundation codes: 82130076 and 81972668).
EXAMPLE 1 results of particle size of nanoparticle prepared from different cationic Components
This example investigated the polymerization of different cationic components on the nano-vaccine particles. Wherein CD8 epitope peptide mLama4 is used as an antigen, and CpG is used as an adjuvant.
The nano vaccine is prepared and obtained according to the following method:
(1) Mu.l of antigen water (ddH 2 O) solution or dimethyl sulfoxide (DMSO) solution (10 mg/ml) is respectively mixed with 10 μl of different cation component water solution (2 μmol/ml) uniformly, and then water is added to dilute to 200 μl to obtain antigen-cation component mixture; the cationic component comprises PEI20000 and Mn 2+ (derived from MnCl) 2 )、Al 3+ (from Al (OH) 3 )、Ca 2+ (derived from CaCl) 2 ) Lys, poly-Lys (3), poly-Lys (15), poly-Lys (20), arg, poly-Arg (3), poly-Arg (15), poly-Arg (20); wherein the Control group (Control) is ddH 2 O replaces the cationic component.
(2) Slowly dripping 50 μl of adjuvant CpG water solution (2 mg/ml) into the antigen-cation component mixture, and shaking and mixing at 500rpm to obtain adjuvant-antigen-cation mixture;
(3) Mu.l of sodium alginate (Alg) aqueous solution (40 mg/ml) was slowly added dropwise to the adjuvant-antigen-cation mixture, and the nanovaccine was obtained by shaking and mixing uniformly at 500 rpm.
ddH for nanometer vaccine containing different cation components obtained by the preparation method 2 O is diluted 10000 times, and the particle size of the nano vaccine particles prepared by different cation components is measured by a Malvern Panalytical Zetasizer Ultra dynamic light scattering system. The results are shown in FIG. 1, mn 2+ Arg, poly-Arg (15), poly-Lys (20) all promote the polymerization of the nano-vaccine particles.
Example 2 noNanometer vaccine prepared from same cation component induces CD8 + Results of T cell immune response levels
This example investigated the effect of different cationic components on the immune effect of a nanovaccine.
A nanovaccine of different cationic composition was obtained according to the preparation method of example 1. Wherein, CD8 epitope peptide mLama4 is taken as an antigen, cpG is taken as an adjuvant, PEI and Mn are taken as the adjuvant 2+ Arg, poly-Arg (15) and poly-Lys (20) are grouped together into 5 groups as cationic components.
The nano vaccine is injected into mice by soles on the 0 th day, the 3 rd day and the 6 th day respectively by adopting about 20g of 6-7 week-old C57BL/6 mice, and each mouse is injected with 50 mu l each time. Mice were sacrificed on day 10 for cervical dislocation and treated as follows: (1) the spleens of mice were surgically isolated and placed in PBS buffer and gently ground with syringe needles; (2) blowing off with a pipetting gun, and filtering with a 100 μm filter screen to obtain spleen cell suspension; (3) centrifuging the spleen cell suspension at 300g for 3 minutes, discarding the supernatant, adding 1ml of erythrocyte lysate, and uniformly mixing for 3 minutes; (4) the lysis reaction was stopped by adding 5ml PBS buffer, centrifuging at 300g for 3 min and discarding the supernatant, and adding 1640 medium (10% FBS) to prepare 60X 10 6 Spleen cell suspension of cells/ml; (5) spleen cells were stimulated with mLama4 peptide in U-bottom 96 well plates in a total volume of 200. Mu.l with a final concentration of mLama4 of 2ug/ml, and the number of spleen cells per well of 1.8X10 6 The method comprises the steps of carrying out a first treatment on the surface of the (6) Mu.l of Golgi apparatus transport blocker BrefeldinA (BFA) (500 ug/ml) was added to each well 30 minutes after the initiation of stimulation; (7) after the total stimulation time reached 5.5 hours, the spleen cell suspension was transferred to a flow tube and washed with 1ml PBS buffer, centrifuged at 300g for 3 minutes, and the supernatant was discarded; (8) the following fluorescent antibodies were added to each tube of spleen cells for staining for 30 minutes: perCP-Cy5.5-TCRβ, BV510-CD8, PE-Cy7-CD44, eFlour 780-virability, after staining, adding 1ml PBS buffer solution for washing, centrifuging for 3 minutes at 300g, and discarding the supernatant; (9) sequentially adding a cell fixing solution and a membrane rupture buffer solution for fixing and rupture, dyeing with APC-IFN-gamma for 30 minutes, adding 1ml PBS buffer solution for cleaning after dyeing, centrifuging 300g for 3 minutes, and discarding the supernatant; is (1)CD44 detection by flow cytometry, resuspended in 300. Mu.l PBS buffer + CD8 + IFN-gamma secretion levels from T cells.
The results are shown in FIG. 2, and each group of nano-vaccines can generate T cell immune response. In particular, nanovaccines made with poly-Arg (15) as cationic component are better at inducing CD8 + T cell immune response. About 2-fold increase compared to Arg and poly-Lys (20) groups compared to PEI and Mn groups 2+ The group was increased by about 4-fold, demonstrating that poly-Arg (15) as a cationic component was effective in increasing CD8 + T cell immune response.
EXAMPLE 3 nanovaccine scanning electron microscope results prepared with cationic component poly-Arg (15)
This example shows the variation of the nano vaccine prepared from the cationic component poly-Arg (15) under a scanning electron microscope.
A nanovaccine containing the cationic component poly-Arg (15) was obtained according to the preparation method of example 1. Wherein CD8 epitope peptide mLama4 is used as an antigen, cpG is used as an adjuvant, and a control group is ddH 2 O replaces the nano vaccine made of cationic component, and the experimental group is the nano vaccine made of poly-Arg (15) as cationic component.
As shown in FIG. 3, compared with the control group (FIG. 3A), poly-Arg (15) (FIG. 3B) can promote the formation of crosslinked gel by sodium alginate to form a stable network structure, thereby inhibiting the release of the antigen and the adjuvant coated by the crosslinked gel and enabling the nano vaccine to have a slow release effect. Further magnification observation, poly-Arg (15) (FIG. 3D) formed particles more uniformly and smaller in particle size than the control group (FIG. 3C). This is consistent with the results of examples 1 and 2, where the use of poly-Arg (15) as the cationic component to prepare the nanovaccine is more advantageous for improving the uptake and presentation functions of DC cells and for improving the vaccine-induced T cell immune response.
Example 4 nanovaccine induced CD8 prepared with different adjuvants + Results of T cell immune response levels
This example explores the CD8 pair with different nucleic acid sequences or different brands of CpG adjuvants + Influence of T cell immune response.
Nanovaccines comprising different adjuvants were obtained according to the preparation method of example 1, wherein CD8 epitope peptide mLama4 was used as antigen, poly-Arg (15) was used as cationic component, cpG (Xu) (CN 101979566 a), cpG1826 (invivogen), cpG2395 (TAKARA) were used as adjuvants and were grouped into 4 groups.
Research method the same as in example 2, CD44 was detected in a flow + CD8 + IFN-gamma secretion levels from T cells.
The results show (FIG. 4) that all four CpG adjuvants induced higher levels of CD8 + T cell immune response.
Example 5 composition of nanovaccine versus CD8 + Influence of T cell immune response
In order to study the effect of the composition of the nanovaccine on the immune effect, the present example uses the nanovaccine of different composition to immunize mice: (1) poly-Arg (15) group: adopts mlama4 as antigen, poly-Arg (15) as cation component, cpG as adjuvant, and sodium alginate (Alg) as slow release carrier. (2) Alg group: adopts mlama4 as antigen, cpG as adjuvant, sodium alginate (Alg) as slow release carrier, and does not add poly-Arg (15) as cation component. (3) group alg+poly-Arg (15): adopts mlama4 as antigen, poly-Arg (15) as cation component, cpG as adjuvant and sodium alginate (Alg) as slow-release carrier.
The nano vaccine preparation method is the same as in example 1.
The prepared nano vaccines of each group are respectively injected into mice on the 0 th day and the 3 rd day through soles, and the cervical dislocation of the mice is killed on the 7 th day and the experiment is carried out according to the step of the example 2. Streaming detection of CD8 + IFN-gamma secretion levels from T cells are shown.
As shown in FIG. 5, the result shows that the Alg+poly-Arg (15) group nano vaccine added with sodium alginate and poly-Arg (15) can effectively improve CD8 + The level of immune response of T cells was increased by about 1-fold compared to the Alg group and about 5-fold compared to the poly-Arg (15) group. The simultaneous addition of sodium alginate and polyArg15 can effectively increase the immune effect of the nano vaccine.
EXAMPLE 6 cellular immunization results of nanovaccines with novel coronavirus protein S protein as antigen
In the embodiment, whether the nano vaccine prepared by taking the novel coronavirus protein S protein (S protein) as an antigen can effectively induce cellular immunity is explored, and the nano vaccine is compared with a finished product S protein aluminum adjuvant vaccine.
Experimental grouping: (1) PBS group: mice were immunized with PBS sole. (2) CpG+S protein group: mice were immunized with protein S and adjuvant CpG sole. (3) CpG+S protein+poly-Arg (15) +Alg group: a nano vaccine is prepared by using S protein as an antigen, poly-Arg (15) as a cationic component, cpG as an adjuvant and sodium alginate (Alg) as a slow release carrier according to the method described in the example 1. (4) S protein vaccine group: the mice were immunized on sole with the finished S protein aluminum adjuvant vaccine.
Different vaccine groups immunized mice on day 0 and day 7, respectively. Mice were sacrificed after day 42 for cervical dislocation and experiments were performed as in example 2 (replacement with stimulation of spleen cells of mice with S protein at a final concentration of 50 μg/ml, with addition of FITC-CD4 fluorescent antibody staining), flow-through detection of CD8 + T and CD4 + IFN-gamma secretion levels from T cells.
As shown in fig. 6, the nanovaccine group was able to significantly stimulate T cell immune levels of the immune system in comparison to the PBS group, cpg+s protein group. Compared with S protein vaccine group S protein aluminum adjuvant vaccine, nanometer vaccine induced CD8 + T cell immune response levels were 8-fold, CD4 + T cell response levels were 12 times higher.
Example 7 humoral immune results of nanovaccine against novel coronavirus protein S protein as antigen
In the embodiment, whether the nano vaccine prepared by taking the novel coronavirus protein S protein (S protein) as an antigen can effectively induce humoral immunity is explored, and the nano vaccine is compared with a finished product S protein aluminum adjuvant vaccine.
The experimental group is the same as in example 6.
The different vaccine groups were immunized on day 0 and day 7, respectively, and blood was collected from the posterior venous plexus of the eye of the mice on days 7, 14, 21, 28, 35, 42 in 1.5ml EP tubes, and the serum was isolated by centrifugation at 3000rpm for 5 minutes, and the relative titers of antibodies to total IgG and IgG1 in the serum were detected by ELISA, with endpoint titers defined as the reciprocal of the maximum serum dilution at which the absorbance was 2.5 times above background. The ELISA procedure was as follows: (1) coating the S protein as antigen on the bottom of a 96-well enzyme-labeled plate (BIOFIL), coating 100ng of the S protein on each well, and standing overnight at 4 ℃ in a total volume of 100 μl of PBS buffer; (2) antigen coated 96-well plates were washed six times with 0.15%200 μl PBST (PBS containing 0.05% tween 20) and 5% skim milk powder in PBS buffer was added to the plates, total volume was 200 μl, blocked for 2 hours at 37 ℃, and plates were washed six times with 0.15%200 μl PBST; (3) serial 10-fold dilution of serum samples of immunized mice, adding the diluted serum samples into 96-well plates, incubating the diluted serum samples at 37 ℃ for 1 hour at 100 mu l of each well, and washing the plates with 0.15%200 mu l of PBST for six times; (4) goat anti-mouse IgG-HRP (1:50000, cwbiotech) or goat anti-mouse IgG1-HRP (1:50000, proteintech) was diluted with 1% nonfat milk powder in PBS buffer, incubated at 37℃for 0.5 hours per well 100. Mu.l, and then plates were washed six times with 0.15% 200. Mu.l of PBST; (5) after 50. Mu.l of HRP substrate TMB was added to the 96-well plate and developed at 37℃for 10 minutes, the development was terminated by adding 50. Mu.l of 2mol/L sulfuric acid; (6) absorbance at 450nm was read with a microplate reader.
As shown in fig. 7, the nano vaccine of the present invention has the same capability of inducing humoral immunity as the commercial finished S protein aluminum adjuvant vaccine, the total IgG reflecting the total level of antibodies and the IgG1 titer reflecting the Th2 level are the same, and the duration of the antibody titer is also consistent, and the nano vaccine of the present invention is higher than the finished S protein aluminum adjuvant vaccine in the antibody titer level at 28 days, and it is considered that the nano vaccine of the present invention can induce humoral immunity earlier in the body than the finished S protein aluminum adjuvant vaccine.
Example 8 therapeutic Effect of nanovaccines binding to CD4 epitope and CD8 epitope on tumors
C57BL/6 female mice at 8 weeks of age were purchased from Guangdong far Biotechnology and inoculated 5X 10 by inoculating the right back of the mice 5 And constructing a tumor-bearing mouse model of the KP13 tumor by in vitro cultured KP13-mITGB1-mLAMA4 non-small cell lung cancer cells. All animal experiment operations are according to the practice issued by the scientific and technical division of the people's republic of ChinaThe regulations of animal administration and the guidelines on animals to be tested are followed.
Nanometer vaccine Vac mLama4+mITGB1 : CD4 epitope mITGB1 and CD8 epitope mLama4 are used as antigens, poly-Arg (15) is used as a cationic component, cpG is used as an adjuvant, and sodium alginate (Alg) is used as a slow release carrier. The nano vaccine preparation method is the same as in example 1.
Firstly, a tumor-bearing mouse model of a non-small cell lung cancer cell KP13 which simultaneously expresses a CD4 epitope mITGB1 and a CD8 epitope mLama4 antigen is constructed, tumor cells are inoculated on day 0, then sole injection treatment is carried out on 8 mice on days 7, 10 and 13 by using the nano vaccine respectively, and sole injection is carried out on 8 mice by using PBS as a control group. Then starting on day 7, tumors were measured every 3 days and the volume v= (length×width) was calculated 2 )/2。
The results are shown in fig. 8, compared with the PBS group, the nano vaccine treatment group has obviously slowed tumor growth, mice have obviously improved survival rate, and simultaneously, the tumors of half of the mice are completely eliminated after treatment, which proves that the nano vaccine with CD4 and CD8 epitopes as antigens has good tumor treatment effect.
In conclusion, the nano vaccine provided by the invention can induce more effective immune response, and has good stability and biocompatibility.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The nano vaccine is characterized by being prepared from the following raw materials: a slow release carrier, an antigen, an adjuvant, and a cationic component; the mass ratio of the slow release carrier, the antigen, the adjuvant and the cation component is (1-30): 1: (0.1-1): (0.05 to 0.5);
the slow release carrier comprises sodium alginate; the antigen comprises at least one of a protein, a recombinant subunit, and a polypeptide; the cation component comprises at least one of calcium ion, manganese ion, aluminum ion, polyamine, basic amino acid and polyamino acid composed of 2-30 basic amino acids.
2. The nanovaccine of claim 1, wherein the basic amino acid comprises at least one of arginine, lysine; preferably, the cationic component is polyarginine consisting of 10-25 arginines.
3. The nanovaccine of claim 1, wherein the antigen comprises at least one of a CD8 epitope short peptide, a CD8 epitope long peptide, a CD4 epitope short peptide, a CD4 epitope long peptide, a recombinant novel coronavirus S protein, a DNA capable of expressing a recombinant protein, an RNA capable of expressing a recombinant protein.
4. The nanovaccine of claim 1, wherein the calcium ion is derived from at least one of the following compounds: caCl (CaCl) 2 、CaCO 3 、Ca(OH) 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or the number of the groups of groups,
the manganese ions are derived from at least one of the following compounds: mnCl 2 、MnCO 3 、Mn(OH) 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or the number of the groups of groups,
the aluminum ions are derived from at least one of the following compounds: alCl 3 、Al(OH) 3 The method comprises the steps of carrying out a first treatment on the surface of the And/or the number of the groups of groups,
the polyamine includes at least one of polymers containing greater than 2 amino groups; preferably, the polyamine comprises at least one of amino acid polymer with amino group, spermidine, polyetherimide; and/or the number of the groups of groups,
the adjuvant comprises at least one of CpG, LPS, poly (I: C), MPL, manganese adjuvant and oil adjuvant.
5. The method for preparing the nano vaccine according to any one of claims 1 to 4, comprising the steps of: (1) Uniformly mixing the antigen and the cation component to obtain an antigen-cation component mixture; (2) Dropwise adding the adjuvant into the antigen-cation component mixture, and uniformly mixing to obtain an adjuvant-antigen-cation mixture; (3) And (3) dropwise adding sodium alginate into the adjuvant-antigen-cation mixture, and uniformly mixing to obtain the nano vaccine.
6. Use of a nanovaccine according to any one of claims 1 to 4 or a nanovaccine according to claim 5 for the preparation of a medicament for increasing the immune response of T cells in an organism.
7. Use of a nanovaccine according to any one of claims 1 to 4 or a nanovaccine obtained by a preparation method according to claim 5 for the preparation of a medicament for the prevention and treatment of solid tumors or infectious diseases.
8. The use of claim 7, wherein the solid tumor comprises colorectal cancer, melanoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, head and neck tumor, prostate cancer, liver cancer, nasopharyngeal cancer, epithelial ovarian cancer, esophageal cancer, cervical cancer.
9. The use of claim 7, wherein the infectious disease comprises a novel coronavirus infection, influenza, hepatitis b.
10. The use of claim 7, wherein the medicament has at least one of the following efficacy: (1) Enhancement of anti-tumor immune response, and enhancement of IFN-gamma secretion level of T cells aiming at tumor antigens; (2) tumor volume reduction or tumor elimination; (3) Fever, weight loss and pain symptoms caused by tumors are reduced, alleviated or eliminated.
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