Nanovaccine delivery system, preparation method therefor, and use thereof

Abstract

Provided is a nanovaccine. The nanovaccine is prepared from the following starting materials: a sustained-release carrier, an antigen, an adjuvant, and a cationic component. The mass ratio of the sustained-release carrier to the antigen to the adjuvant to the cationic component is (1-30):1:(0.1-1):(0.05-0.5). The sustained-release carrier comprises sodium alginate. The antigen comprises at least one of a protein, a recombinant subunit, and a polypeptide. The cationic component comprises at least one of a calcium ion, a manganese ion, an aluminum ion, a polyamine, a basic amino acid, and a polyamino acid consisting of 2-30 basic amino acids. The nanovaccine comprises the sustained-release carrier sodium alginate, the antigen, the adjuvant, and the cationic component in a proper ratio. Sodium alginate is used as a sustained-release carrier and is promoted to form a network structure under the action of the cationic component, so as to encapsulate the antigen and the adjuvant, thereby forming a stable antigen-carrier-adjuvant nanoparticle gel. The nanovaccine can induce a stronger immune response and has good stability and good biocompatibility.

Description

A nano vaccine delivery system and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202410037434.2, filed with the Patent Office of China on January 10, 2024, entitled "A Nanovaccine Delivery System, Its Preparation Method and Application", the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention belongs to the field of biomedicine technology, and specifically relates to a nano vaccine delivery system and a preparation method and application thereof. Background Art

[0004] Malignant tumors, also known as cancer, have a high incidence worldwide. With the aging of the population and the increase in population size, the incidence of malignant tumors is expected to increase rapidly worldwide. Therefore, malignant tumors pose a considerable threat to human health.

[0005] There are three traditional treatments for cancer: surgery, chemotherapy, and radiotherapy. Each has its advantages and disadvantages. Surgery can directly remove visible tumor tissue, but recent studies have shown that the postoperative wound healing response may lead to the growth of metastatic tumors. Chemoradiotherapy and radiotherapy can kill tumor cells extensively, but they can easily lead to tumor cell tolerance and recurrence. They also kill normal cells, which can lead to poor patient prognosis. In recent years, immunotherapy has developed rapidly 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 intervene in or modulate the body's immune function in immune-compromised or immune-hyperactive states, enhancing or weakening the immune response to achieve the goal of treating the disease. Current hot topics in tumor immunotherapy are immune checkpoint blockade therapy, chimeric antigen receptor T-cell immunotherapy (CAR-T), and therapeutic tumor vaccines. Each of these treatments has its advantages and disadvantages. For example, treatment with the immune checkpoint blocker PD-1 (Programmed Cell Death Protein-1) has advantages such as long-lasting efficacy, low recurrence rate, and a broad therapeutic spectrum, but it also has disadvantages such as poor effect on "cold tumors" and toxic side effects; CAR-T therapy has the advantages of strong specificity, overcoming the restriction of MHC, reducing the risk of immune escape caused by tumor cells downregulating the expression of MHC molecules, and good effect on blood tumors such as acute leukemia and non-Hodgkin's lymphoma, but it also brings problems such as killing normal cells, inducing cytokine storms, difficulty in homing of CAR-T cells, poor treatment effect on solid tumors, and high cost.

[0006] Therapeutic vaccines are a representative strategy in cancer immunotherapy, aiming to induce specific immune responses against a specific antigen or group of antigens by strengthening or reactivating the patient's own immune system. Compared to other treatments, therapeutic cancer vaccines, as an active immune intervention, are more feasible and less prone to severe side effects, making them a research hotspot in immunotherapy. Tumor vaccines can be divided into three main categories based on their mechanism of action: cell-based vaccines, protein / peptide vaccines, and nucleic acid vaccines. Cell-based vaccines are categorized into tumor cell vaccines and DC vaccines; protein / peptide vaccines are categorized into protein vaccines, short peptide vaccines, and long peptide vaccines; and nucleic acid vaccines are categorized into DNA vaccines, RNA vaccines, and viral vector vaccines. In April 2010, the US Food and Drug Administration (FDA) approved the first dendritic cell (DC) tumor vaccine, Provenge, for the treatment of metastatic castrate-resistant prostate cancer (CRPC). Since then, research and achievements in the field of tumor vaccines have mushroomed.

[0007] Although tumor vaccines have good clinical effects on some patients, they still face many challenges in terms of how to effectively function. Specifically, there are the following aspects: (1) The molecules of polypeptide antigens are small and can be quickly cleared after being injected into the body. At the same time, they are easily hydrolyzed and metabolized by related proteases in the body, which reduces the antigen concentration and makes it difficult to accumulate in immune tissues and organs such as lymph nodes and spleen, resulting in a significant decrease in the probability of DC cells taking up antigens. Tumor vaccines injected subcutaneously also face similar problems of rapid clearance and degradation; (2) After the antigens are taken up by DC cells and enter the lysosomes, it is difficult for them to escape from the lysosomes and cannot be released into the cytoplasm after processing and interact with MHC. Class I molecules are combined and presented together on the surface of DC cells, so the level of anti-tumor immune response induced by the vaccine is very low; (3) Injecting only antigens can easily lead to immune tolerance. Tumor vaccines are usually used in combination with adjuvants to activate effective immune responses. However, many adjuvants currently have problems such as poor stability, short half-life, and low cellular uptake. In addition, simply injecting antigens and adjuvants makes it difficult to achieve the combined effect of the two at the same time and in the same part, that is, the two lack the ability to co-localize in time and space; (4) Because the antigens and adjuvants maintain their effects in the body for a short time, the vaccine effect is not long-lasting. It is necessary to increase the dosage of antigens and adjuvants and the number of vaccinations to maintain the vaccine effect, which brings stronger side effects and the risk of immune tolerance to patients. Therefore, in order to induce the body to produce a more lasting and effective immune response, it is urgent to develop a vaccine delivery system that can simultaneously load antigens and adjuvants and protect them from being cleared. Summary of the Invention

[0008] Based on this, the object of the present invention is to provide a nanovaccine that can induce a more potent immune response, has good stability and good biocompatibility.

[0009] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions.

[0010] The first aspect of the present invention provides a nano vaccine, which is prepared from the following raw materials: a sustained-release carrier, an antigen, an adjuvant, and a cationic component; the mass ratio of the sustained-release carrier, antigen, adjuvant, and cationic component is (1-30):1:(0.1-1):(0.05-0.5);

[0011] The sustained-release carrier comprises sodium alginate; the antigen comprises at least one of a protein, a recombinant subunit and a polypeptide; and the cationic component comprises at least one of calcium ions, manganese ions, aluminum ions, polyamines, basic amino acids and polyamino acids composed of 2-30 basic amino acids.

[0012] In some embodiments, the basic amino acid includes at least one of arginine and lysine.

[0013] In some preferred embodiments, the cationic component is polyarginine composed of 1-25 arginine amino acids.

[0014] In some embodiments, the antigen includes 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, and an RNA capable of expressing a recombinant protein.

[0015] In some embodiments, the calcium ions are derived from at least one of the following compounds: CaCl2, CaCO3, Ca(OH)3; and / or, the manganese ions are derived from at least one of the following compounds: MnCl2, MnCO3, Mn(OH)3; and / or, the aluminum ions are derived from at least one of the following compounds: AlCl3, Al(OH)3.

[0016] In some embodiments, the polyamine includes at least one of polymers containing more than two amino groups; preferably, the polyamine includes at least one of amino acid polymers having amino groups, spermidine, and polyetherimide (PEI).

[0017] In some embodiments, the adjuvant comprises at least one of CpG, LPS, Poly(I:C), MPL, manganese adjuvant, and oil adjuvant.

[0018] In some embodiments, the particle size of the nanovaccine is 10 nm to 1000 nm; preferably, the particle size of the nanovaccine is 10 nm to 500 nm.

[0019] The second aspect of the present invention provides a method for preparing the nanovaccine as described above, comprising the following steps: (1) uniformly mixing the antigen and the cationic component to obtain an antigen-cationic component mixture; (2) dropwise adding the adjuvant to the antigen-cationic component mixture, mixing uniformly to obtain an adjuvant-antigen-cationic mixture; and (3) dropwise adding sodium alginate to the adjuvant-antigen-cationic mixture, mixing uniformly to obtain the nanovaccine.

[0020] The third aspect of the present invention provides the use of the nanovaccine as described above or the nanovaccine prepared by the preparation method as described above in the preparation of a drug for enhancing the T cell immune response of an organism.

[0021] In a fourth aspect, the present invention provides the use of the nanovaccine as described above or the nanovaccine prepared by the preparation method as described above in the preparation of drugs for preventing and treating solid tumors or infectious diseases.

[0022] In some embodiments, the solid tumor comprises colorectal cancer, melanoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, head and neck cancer, prostate cancer, liver cancer, nasopharyngeal cancer, epithelial ovarian cancer, esophageal cancer, or cervical cancer.

[0023] In some embodiments, the infectious diseases include novel coronavirus infection, influenza, and hepatitis B.

[0024] In some embodiments, the drug has at least one of the following effects: (1) enhanced anti-tumor immune response, with increased levels of IFN-γ secreted by T cells against tumor antigens; (2) reduced tumor volume or tumor elimination; (3) reduced, alleviated, or eliminated fever, weight loss, and pain symptoms caused by tumors.

[0025] The present invention obtains a nano vaccine through optimization. The nano vaccine comprises a slow-release carrier sodium alginate, an antigen, an adjuvant and a cationic component in an appropriate ratio. Sodium alginate is used as a slow-release carrier. The cationic component forms an ionic bond with the negatively charged sodium alginate, promoting the formation of a network structure of the sodium alginate, encapsulating the antigen and adjuvant, forming a stable antigen-carrier-adjuvant nanoparticle gel, inhibiting the release of internal substances, and improving the slow-release effect of the antigen substance. When the nano vaccine is injected into the body, the antigen undergoes a replacement reaction with the cations in the body fluid and is slowly released to prevent the antigen from being immediately released into the body and being degraded, while the sodium alginate can be safely degraded in the body. In addition, the network structure can prevent the encapsulated adjuvant from being rapidly metabolized in the body, achieving co-localized delivery of the antigen and adjuvant.

[0026] In the nanovaccine system described in the present invention, the interaction between positive and negative charges promotes the polymerization of vaccine particles to a certain extent, thereby improving the uptake and presentation of DC cells and enhancing the immune response of T cells. First, the cationic components with polymerization-promoting effects were optimized, and then the T cell immune response induced by the nanovaccine prepared therefrom was further studied. The cationic components selected in the present invention, especially polyarginine and polyamine polymers, can cooperate well with the sodium alginate sustained-release carrier to form more stable and uniform vaccine particles, making them more easily taken up by DC cells or macrophages, thereby achieving better immune effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the particle size test results of nanovaccines prepared with different cationic components.

[0028] Figure 2 shows the results of nanovaccines prepared with different cationic components causing the body to produce T cell immune responses.

[0029] Figure 3 shows the scanning electron microscopy results of the nanovaccine prepared with the cationic component poly-Arg (15).

[0030] Figure 4 shows the results of nanovaccines prepared with different adjuvants inducing T cell immune responses in the body.

[0031] FIG5 shows the effect of the composition of the nanovaccine on the body's T cell immune response.

[0032] Figure 6 shows the cellular immunity results of the nanovaccine using the novel coronavirus protein S protein as the antigen.

[0033] Figure 7 shows the humoral immunity results of the nanovaccine using the novel coronavirus protein S protein as the antigen.

[0034] FIG8 shows the therapeutic effect of nanovaccines combining CD4 epitopes and CD8 epitopes on tumors. DETAILED DESCRIPTION

[0035] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0037] The terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0038] In this disclosure, "at least one" refers to one or more than one. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone.

[0039] The following describes the details in conjunction with specific embodiments.

[0040] Sodium alginate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Lama4:GGFNFRTL, mLama4:VGFNFRTL, and mITGB1:TYSVNGYNEAIVHVVE were all purchased from Jiangsu GenScript Technology Co., Ltd. CpG2395 (TAKARA) (hereinafter referred to as CpG) was purchased from Bio-Rad Biotechnology Co., Ltd. CpG1826 (invivogen) and CpG2395 (invivogen) were both purchased from Invivogen. Antibodies used for flow cytometry staining, including PerCP-Cy5.5-TCRβ, BV510-CD8, PE-Cy7-CD44, APC-IFN-γ, and FITC-CD4, were all purchased from Biolegend, and eFlour780-viability was purchased from eBioscience. Medium 1640 and fetal bovine serum for cell culture were purchased from Gibico.

[0041] The arginine (Arg) mentioned in the present invention refers to L-arginine (L-Arg).

[0042] The polyamino acid composed of 2-30 basic amino acids described in the present invention refers to a compound (peptide) formed by 2-30 basic amino acids linked together by peptide bonds, denoted as poly-AA(n), where 2≤n≤30. For example, polyarginine composed of 15 Arg refers to a peptide formed by 15 Arg linked together by peptide bonds, denoted as poly-Arg(15).

[0043] Example 1 Results of the particle size of nanovaccine particles prepared with different cationic components

[0044] This example studies the polymerization effect of different cationic components on nanovaccine particles, wherein the CD8 epitope peptide mLama4 is used as the antigen and CpG is used as the adjuvant.

[0045] Nano vaccines were prepared as follows:

[0046] (1) 50 μl of antigen water (ddH2O) solution or dimethyl sulfoxide (DMSO) solution (10 mg / ml) was mixed with 10 μl of different cationic component aqueous solutions (2 μmol / ml) and diluted with water to 200 μl to obtain an antigen-cationic component mixture; the cationic component included PEI20000, Mn 2+ (derived from MnCl2), Al 3+ (derived from Al(OH)3), Ca 2+ (derived from CaCl2), Lys, poly-Lys(3), poly-Lys(15), poly-Lys(20), Arg, poly-Arg(3), poly-Arg(15), poly-Arg(20); among them, the control group (Control) used ddH2O instead of cationic components.

[0047] (2) Slowly add 50 μl of adjuvant CpG aqueous solution (2 mg / ml) dropwise to the antigen-cation component mixture and shake at 500 rpm to mix uniformly to obtain an adjuvant-antigen-cation mixture;

[0048] (3) 250 μl of sodium alginate (Alg) aqueous solution (40 mg / ml) was slowly added dropwise to the adjuvant-antigen-cation mixture, and the mixture was shaken and mixed at 500 rpm to obtain the nanovaccine.

[0049] The nanovaccines containing different cationic components obtained by the above preparation method were diluted 10,000 times with ddH2O, and the particle size of the nanovaccine particles prepared with different cationic components was measured using a Malvern Panalytical Zetasizer Ultra dynamic light scattering system. The results are shown in Figure 1. 2+ , Arg, poly-Arg(15), and poly-Lys(20) can all promote the aggregation of nanovaccine particles.

[0050] Example 2 Nanovaccines prepared with different cationic components induce CD8 + T cell immune response level results

[0051] This example studies the effects of different cationic components on the immune effect of nanovaccines.

[0052] Nano vaccines with different cationic components were obtained according to the preparation method of Example 1. Among them, CD8 epitope peptide mLama4 was used as antigen, CpG was used as adjuvant, PEI, Mn 2+, Arg, poly-Arg(15), and poly-Lys(20) as cationic components are divided into 5 groups.

[0053] C57BL / 6 mice weighing about 20g, aged 6 to 7 weeks, were used, with 3 mice in each group. The nanovaccine was injected into the mice through the paws on day 0, day 3, and day 6, with 50μl injected into each mouse each time. On day 10, the mice were killed by cervical dislocation and processed according to the following steps: ① The mouse spleen was surgically isolated and placed in PBS buffer, and the spleen was gently ground with a syringe needle; ② After being blown off with a pipette, it was filtered with a 100μm filter to prepare a spleen cell suspension; ③ The spleen cell suspension was centrifuged at 300g for 3 minutes and the supernatant was discarded, then 1ml of red blood cell lysis buffer was added and mixed for 3 minutes; ④ 5ml of PBS buffer was added to terminate the lysis reaction, centrifuged at 300g for 3 minutes and the supernatant was discarded, and then 1640 culture medium (10% FBS) was added to prepare 60×10 6 cells / ml of spleen cell suspension; ⑤ Stimulate spleen cells with mLama4 peptide in a U-bottom 96-well plate with a total volume of 200 μl, a final mLama4 concentration of 2 ug / ml, and 1.8×10 spleen cells per well. 6 ⑥ 30 minutes after the start of stimulation, 1 μl of Golgi transport blocker Brefeldin A (BFA) (500 ug / ml) was added to each well; ⑦ After the total stimulation time reached 5.5 hours, the spleen cell suspension was transferred to a flow cytometry tube, washed with 1 ml of PBS buffer, centrifuged at 300g for 3 minutes, and the supernatant was discarded; ⑧ Each tube of spleen cells was stained with the following fluorescent antibodies for 30 minutes: PerCP-Cy5.5-TCRβ, BV510-CD8, PE-Cy7-CD44, eFlour780-viability. After the staining was completed, 1 ml of PBS buffer was added for washing, centrifuged at 300g for 3 minutes, and the supernatant was discarded; ⑨ Cell fixative and permeabilization buffer were added in sequence for fixation and permeabilization, and APC-IFN-γ was stained for 30 minutes. After the staining was completed, 1 ml of PBS buffer was added for washing, centrifuged at 300g for 3 minutes, and the supernatant was discarded; ⑩ The cells were resuspended in 300 μl of PBS buffer and CD44 was detected by flow cytometry. + CD8 + IFN-γ secretion levels of T cells.

[0054] The results are shown in Figure 2. All groups of nanovaccines can induce T cell immune response. In particular, the nanovaccines made with poly-Arg (15) as the cationic component can better induce CD8 + T cell immune response. Compared with Arg group and poly-Lys(20) group, it increased by about 2 times, and compared with PEI group and Mn 2+The group increased by about 4 times, indicating that poly-Arg (15) as a cationic component can effectively increase CD8 + T cell immune response.

[0055] Example 3 Scanning electron microscopy results of nanovaccine prepared with cationic component poly-Arg (15)

[0056] This example observes the changes of the nanovaccine prepared with the cationic component poly-Arg (15) under a scanning electron microscope.

[0057] A nanovaccine containing the cationic component poly-Arg(15) was obtained according to the preparation method of Example 1. The CD8 epitope peptide mLama4 was used as the antigen, CpG was used as the adjuvant, the control group was a nanovaccine prepared with ddH2O instead of the cationic component, and the experimental group was a nanovaccine prepared with poly-Arg(15) as the cationic component.

[0058] As shown in Figure 3, compared with the control group (Figure 3A), poly-Arg (15) (Figure 3B) can promote the formation of cross-linked gels in sodium alginate to form a stable network structure, thereby inhibiting the release of the antigens and adjuvants encapsulated therein, so that the nanovaccine has a sustained release effect. Further magnification observation shows that compared with the control group (Figure 3C), the particles formed by poly-Arg (15) (Figure 3D) are more uniform and have a smaller particle size. This is consistent with the results of Examples 1 and 2. Nanovaccines made with poly-Arg (15) as the cationic component are more conducive to improving the uptake and presentation function of DC cells, and are conducive to improving the vaccine-induced T cell immune response.

[0059] Example 4 Nano-vaccines prepared with different adjuvants induce CD8 + T cell immune response level results

[0060] This example explores the effects of different nucleic acid sequences or different brands of CpG adjuvants on CD8 + Effects of T cell immune responses.

[0061] Nanovaccines containing different adjuvants were obtained according to the preparation method of Example 1, in which CD8 epitope peptide mLama4 was used as the antigen, poly-Arg (15) was used as the cationic component, and CpG (Xu) (CN 101979566A), CpG1826 (invivogen), CpG2395 (invivogen), and CpG2395 (TAKARA) were used as adjuvants and were divided into 4 groups.

[0062] The research method was the same as in Example 2, and CD44 was detected by flow cytometry. + CD8 + IFN-γ secretion levels of T cells.

[0063] The results showed (Figure 4) that all four CpG adjuvants could induce high levels of CD8 + T cell immune response.

[0064] Example 5 Effect of Nano-Vaccine Composition on CD8 + Effects of T cell immune response

[0065] In order to study the effect of nanovaccine composition on immune effect, this example uses nanovaccines with different compositions to immunize mice: (1) Poly-Arg (15) group: mlama4 is used as antigen, poly-Arg (15) as cationic component, CpG as adjuvant, and sodium alginate (Alg) is not added as sustained-release carrier. (2) Alg group: mlama4 is used as antigen, CpG as adjuvant, sodium alginate (Alg) is used as sustained-release carrier, and poly-Arg (15) is not added as cationic component. (3) Alg + poly-Arg (15) group: mlama4 is used as antigen, poly-Arg (15) as cationic component, CpG as adjuvant, and sodium alginate (Alg) is used as sustained-release carrier.

[0066] The method for preparing the nano vaccine is the same as that in Example 1.

[0067] The nanovaccines prepared in each group were injected into mice through the soles of their feet on day 0 and day 3, and the mice were killed by cervical dislocation on day 7 and the experiment was carried out according to the steps of Example 2. + The levels of IFN-γ secretion by T cells are shown in the figure.

[0068] As shown in Figure 5, the results showed that the Alg+poly-Arg(15) nanovaccine group with the addition of sodium alginate and poly-Arg(15) could effectively increase the CD8 + The immune response level of T cells increased by about 1 times compared with the Alg group and about 5 times compared with the poly-Arg(15) group, indicating that the simultaneous addition of sodium alginate and polyArg15 can effectively enhance the immune effect of the nanovaccine.

[0069] Example 6 Cellular Immunity Results of Nanovaccines Using Novel Coronavirus Protein S Protein as Antigen

[0070] This example explores whether a nanovaccine prepared with the novel coronavirus protein S protein (S protein) as an antigen can effectively induce cellular immunity, and compares it with a finished S protein aluminum adjuvant vaccine.

[0071] Experimental groups: (1) PBS group: mice were immunized with PBS on their paws. (2) CpG+S protein group: mice were immunized with S protein and adjuvant CpG on their paws. (3) CpG+S protein+poly-Arg(15)+Alg group: S protein was used as the antigen, poly-Arg(15) as the cationic component, CpG as the adjuvant, and sodium alginate (Alg) as the sustained-release carrier. Nano-vaccines were prepared according to the method described in Example 1 and mice were immunized with their paws. (4) S protein vaccine group: mice were immunized with the finished S protein aluminum adjuvant vaccine on their paws.

[0072] The mice were immunized with different vaccine groups on days 0 and 7, respectively. After day 42, the mice were sacrificed by cervical dislocation and the experiment was carried out according to the steps of Example 2 (splenic cells of the mice were stimulated with S protein at a final concentration of 50 μg / ml and FITC-CD4 fluorescent antibody staining was added). CD8 + T and CD4 + IFN-γ secretion levels of T cells.

[0073] As shown in Figure 6, compared with the PBS group and the CpG+S protein group, the nano vaccine group was able to significantly stimulate the immune system's T cell immunity level. Compared with the S protein vaccine group, i.e., the finished S protein aluminum adjuvant vaccine, the nano vaccine induced CD8 + The T cell immune response level is 8 times that of CD4 + The T cell response level was 12-fold higher.

[0074] Example 7 Humoral Immunity Results of Nanovaccines Using Novel Coronavirus Protein S Protein as Antigen

[0075] This example explores whether a nanovaccine prepared with the novel coronavirus protein S protein (S protein) as an antigen can effectively induce humoral immunity, and compares it with a finished S protein aluminum adjuvant vaccine.

[0076] The experimental grouping is the same as that in Example 6.

[0077] Mice in different vaccine groups were immunized on days 0 and 7, and blood was collected from the retro-orbital venous plexus of mice on days 7, 14, 21, 28, 35, and 42 into 1.5 ml EP tubes. The serum was separated by centrifugation at 3000 rpm for 5 minutes, and the relative titers of total IgG and IgG1 antibodies in the serum were detected by ELISA. The endpoint titer was defined as the reciprocal of the maximum serum dilution at which the absorbance was 2.5 times higher than the background value. The ELISA experimental steps are as follows: ① The S protein was coated as an antigen on the bottom of a 96-well enzyme-labeled plate (BIOFIL), with 100 ng of S protein coated per well in a total volume of 100 μl PBS buffer, and the plate was placed at 4°C overnight; ② The antigen-coated 96-well plate was washed six times with 0.15% 200 μl PBST (PBS containing 0.05% Tween 20), and 5% skim milk powder prepared in PBS buffer was added in a total volume of 200 μl, blocked at 37°C for 2 hours, and washed six times with 0.15% 200 μl PBST; ③ The immunized mouse serum sample was serially diluted 10-fold and added to each well of the 96-well plate, 100 μl, incubated at 37°C for 1 hour, and then washed six times with 0.15% 200 μl PBST; ④ Goat anti-mouse IgG-HRP (1:50) was diluted with 1% skim milk powder dissolved in PBS buffer. ⑤ Add 100 μl of HRP (1:50,000, Cwbiotech) or goat anti-mouse IgG1-HRP (1:50,000, Proteintech) to each well, incubate at 37°C for 0.5 h, and then wash the plate six times with 200 μl of 0.15% PBST; ⑤ Add 50 μl of HRP substrate TMB to the 96-well plate, develop at 37°C for 10 min, and then add 50 μl of 2 mol / L sulfuric acid to stop the color development; ⑥ Read the absorbance at 450 nm using a microplate reader.

[0078] The results are shown in Figure 7. The nanovaccine of the present invention has the same ability to induce humoral immunity as the finished S protein aluminum adjuvant vaccine on the market. The total IgG and IgG1 titers reflecting the total antibody level and the Th2 reaction level are the same, and the duration of the antibody titer is also consistent. At 28 days, the antibody titer level of the nanovaccine of the present invention is higher than that of the finished S protein aluminum adjuvant vaccine. It can be considered that compared with the finished S protein aluminum adjuvant vaccine, the nanovaccine of the present invention can cause the body to induce humoral immunity earlier.

[0079] Example 8 Therapeutic Effects of Nanovaccines Combining CD4 and CD8 Epitopes on Tumors

[0080] 8-week-old C57BL / 6 female mice were purchased from Guangdong Zhiyuan Biotechnology Co., Ltd. and inoculated with 5×10 5A KP13 tumor-bearing mouse model was established using in vitro cultured KP13-mITGB1-mLAMA4 non-small cell lung cancer cells. All animal experiments were conducted in accordance with the "Regulations on the Administration of Laboratory Animals" and the "Guiding Opinions on the Ethical Treatment of Laboratory Animals" issued by the Ministry of Science and Technology of the People's Republic of China.

[0081] Nano vaccine VacmLama4+mITGB1: CD4 epitope mITGB1 and CD8 epitope mLama4 are used as antigens, poly-Arg (15) as the cationic component, CpG as the adjuvant, and sodium alginate (Alg) as the sustained-release carrier. The nano vaccine preparation method is the same as that in Example 1.

[0082] First, a tumor-bearing mouse model of non-small cell lung cancer cells KP13 expressing both CD4 epitope mITGB1 and CD8 epitope mLama4 antigens was established. Tumor cells were inoculated on day 0, and then the nanovaccine was injected into the soles of 8 mice on days 7, 10, and 13. PBS was injected into the soles of 8 mice as a control group. Then, starting from day 7, the tumor was measured every 3 days and the volume V = (length × width 2 ) / 2.

[0083] The results are shown in Figure 8. Compared with the PBS group, the tumor growth in the nanovaccine treatment group was significantly slowed down, and the survival rate of mice was also significantly improved. At the same time, the tumors of half of these mice were completely eliminated after treatment, which proves that the nanovaccine with CD4 and CD8 epitopes as antigens has a good tumor treatment effect.

[0084] In summary, the nanovaccine of the present invention can induce a more potent immune response, has good stability and good biocompatibility.

[0085] The technical features of the above-described embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0086] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A nano-vaccine, characterized in that, The nano-vaccine is prepared from the following raw materials: a sustained-release carrier, an antigen, an adjuvant, and a cationic component; the mass ratio of the sustained-release carrier, the antigen, the adjuvant, and the cationic component is (1-30):1:(0.1-1):(0.05-0.5); The sustained-release carrier includes sodium alginate; the antigen includes at least one of protein, recombinant subunit, and polypeptide; the cationic component includes 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 nano-vaccine according to claim 1, wherein The basic amino acid includes at least one of arginine and lysine; preferably, the cationic component is polyarginine composed of 10-25 arginines.

3. The nano-vaccine according to claim 1, characterized in that, The antigen includes at least one of CD8 epitope short peptide, CD8 epitope long peptide, CD4 epitope short peptide, CD4 epitope long peptide, recombinant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S protein, DNA capable of expressing recombinant protein, and RNA capable of expressing recombinant protein.

4. The nano-vaccine according to claim 1, characterized in that, The calcium ion is derived from at least one of the following compounds: CaCl2, CaCO3, Ca(OH)3; and / or, The manganese ion is derived from at least one of the following compounds: MnCl2, MnCO3, Mn(OH)3; and / or, The aluminum ion is derived from at least one of the following compounds: AlCl3, Al(OH)3; and / or, The polyamine includes at least one of polymers containing more than 2 amino groups; preferably, the polyamine includes at least one of amino acid polymers with amino groups, spermidine, and polyetherimide; and / or, The adjuvant includes at least one of CpG, LPS, Poly(I:C), MPL, manganese adjuvant, and oil adjuvant.

5. The preparation method of the nano-vaccine according to any one of claims 1 to 4, characterized in that, It includes the following steps: (1) Mix the antigen and the cationic component evenly to obtain an antigen-cationic component mixture; (2) Drop the adjuvant into the antigen-cationic component mixture and mix evenly to obtain an adjuvant-antigen-cationic mixture; (3) Drop sodium alginate into the adjuvant-antigen-cationic mixture and mix evenly to obtain the nano-vaccine.

6. Use of the nano-vaccine according to any one of claims 1 to 4 or the nano-vaccine prepared by the preparation method according to claim 5 in the preparation of a drug for enhancing the T cell immune response of the body.

7. Use of the nano-vaccine according to any one of claims 1 to 4 or the nano-vaccine prepared by the preparation method according to claim 5 in the preparation of a drug for preventing and treating solid tumors or infectious diseases.

8. The application according to claim 7, characterized in that, The solid tumors include colorectal cancer, melanoma, breast cancer, lung cancer, pancreatic cancer, gastric cancer, head and neck tumors, prostate cancer, liver cancer, nasopharyngeal cancer, epithelial ovarian cancer, esophageal cancer, and cervical cancer.

9. The application according to claim 7, wherein The infectious diseases include novel coronavirus infection, influenza, and hepatitis B.

10. The application according to claim 7, characterized in that The drug has at least one of the following effects: (1) Enhanced anti-tumor immune response and increased level of IFN-γ secreted by T cells against tumor antigens; (2) Reduced tumor volume or tumor elimination; (3) Alleviation, remission, or elimination of symptoms such as fever, weight loss, and pain caused by tumors.

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