阅读说明：本技术 一种疫苗载体、其制备方法及应用 (Vaccine vector, preparation method and application thereof ) 是由 宋万通 徐玉笛 赵佳雨 刘丽萍 汤朝晖 陈学思 于 2021-07-13 设计创作，主要内容包括：本发明涉及生物医药技术领域,尤其涉及一种疫苗载体、其制备方法和应用。本发明疫苗载体由氧化甘露聚糖和阳离子聚合物组成；所述阳离子聚合物包括聚乙烯亚胺、聚酰胺、聚β氨酯中的至少一种,或这些阳离子聚合物的衍生物,或这些阳离子聚合物及其衍生物与聚乳酸或与聚乳酸-羟基乙酸的嵌段共聚物或接枝共聚物。本发明疫苗载体的抗原负载量大、疫苗制备条件简单温和、稳定性好,并且具有出色的淋巴结靶向和树突细胞靶向能力,能够引起强烈的抗原特异性免疫响应。(The invention relates to the technical field of biological medicines, in particular to a vaccine vector, and a preparation method and application thereof. The vaccine carrier consists of oxidized mannan and cationic polymer; the cationic polymer comprises at least one of polyethyleneimine, polyamide and poly beta-urethane, or derivatives of the cationic polymers, or block copolymers or graft copolymers of the cationic polymers and derivatives thereof and polylactic acid or polylactic acid-glycolic acid. The vaccine vector has the advantages of large antigen load, simple and mild vaccine preparation conditions, good stability, excellent lymph node targeting and dendritic cell targeting capabilities and capability of causing strong antigen specific immune response.)

1. A vaccine carrier, which is characterized by consisting of oxidized mannan and cationic polymer;

the cationic polymer comprises at least one of I) to III):

I) at least one of polyethyleneimine, polyamide and poly-beta-urethane or at least one of derivatives thereof;

II) a block copolymer or a graft copolymer of the polymer shown in I) and polylactic acid;

III), I) and a block copolymer or a graft copolymer of the polymer shown in the formula (I) and polylactic acid-glycolic acid.

2. The vaccine carrier of claim 1, wherein the cationic polymer is at least one of a branched polyethyleneimine, a linear polyethyleneimine, and a dendrimer, or a derivative thereof.

3. The vaccine vector according to claim 1, wherein the oxidized mannan has an oxidation degree of 30 to 70%.

4. The vaccine vector according to claim 1, wherein the weight average molecular weight of the oxidized mannan is 20kDa to 80 kDa.

5. Use of a vaccine vector according to any one of claims 1 to 4 in the preparation of a protein vaccine, an mRNA vaccine or a DNA vaccine.

6. A vaccine comprising the vaccine vector of any one of claims 1 to 4 and an antigen.

7. A method of producing the vaccine of claim 6, comprising:

preparing an antigen solution, a cationic polymer solution and a mannitol oxide solution respectively;

mixing an antigen solution with a cationic polymer solution under a vortex condition to obtain a mixed solution A;

and (3) vortexing the mixed solution A for 30s, adding the oxidized mannan solution, and vortexing for 30s to obtain the vaccine.

8. The method of claim 7, further comprising the steps of adding an immunostimulant, vortexing for 30s, prior to said adding the oxidized mannan solution.

9. The method of claim 7, wherein the antigen is mRNA or DNA encoding a tumor-specific or tumor-associated antigen, or mRNA or DNA encoding a viral or other microbial, pathogen target protein.

10. The method of claim 7, wherein the immunostimulatory agent is at least one of granulocyte-macrophage colony stimulating factor, ranibimod (R848), oligonucleotide (CpG. ODN), polyinosinic acid-polycytidylic acid Poly (I: C), LPS, MPLA.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a vaccine vector, and a preparation method and application thereof.

Background

The tumor immunotherapy makes great progress, and the tumor vaccine is increasingly regarded as an immunotherapy mode with great potential. Tumor vaccines have the advantages of being highly specific, safe and effective for long periods of time. Nano-tumor vaccines have gained increasing attention in recent years because nano-tumor vaccines can: 1) the tumor antigen and the adjuvant are loaded, and the degradation and the premature dispersion of the antigen and the adjuvant are prevented; 2) the immunostimulation effect is improved by the co-delivery of the antigen and the adjuvant; 3) the lymph nodes are efficiently refluxed and captured by antigen presenting cells by utilizing the unique nano-size advantage. Nevertheless, the development of the current nano vaccine is not satisfactory, and the obtained immune stimulation effect and anti-tumor effect are limited, thereby limiting the clinical popularization.

The primary site of action of the vaccine is in secondary lymphoid organs such as lymph nodes. Thus, effective lymph node reflux is critical for the vaccine to elicit an effective immune response. It is known that the particle size has an important influence on the backflow of the nano-vaccine to the lymph nodes, and the nano-particles with the size between 20 and 200 nanometers can efficiently flow back to the lymph nodes. However, stimulation of an effective immune response often requires a multi-step process including antigen capture by Dendritic Cells (DCs), DC cell activation, antigen cross-presentation, and the like. The existing vaccine vectors are often single in function and cannot efficiently stimulate to generate immune response. For example, Zhiping Zhang et al uses simple PLGA as a vaccine carrier to carry antigens and adjuvants, but has limited anti-tumor effects. Simple stacking of multiple functions has led to increased complexity of vaccine vectors (Biomaterials 32(2011) 3666. 3678). As reported by rodneya, rosalia et al, the components of the nanocarrier include CD40 antibody, antigenic protein, Pam3Csk4 and Poly (I: C) adjuvant, and the preparation process is complicated (Biomaterials 40(2015) 88-97). Randalltoy et al reported a vaccine whose components included PLGA, R848, protein antigens, PEI and negatively charged PUCC et al (Journal of controlled Release330(2021) 866-877). The complex preparation process of the vaccine greatly limits the clinical application of the vaccine.

Researches prove that the lymph node reflux effect can be improved by modifying the surface of the nanoparticle, for example, the surface modification of PEG can reduce the protein adsorption of the surface of the nanoparticle and enhance the reflux to the lymph node. Mannan is a polysaccharide of bacterial origin, consisting of a plurality of repeating sugar units, which are recognized by pattern recognition receptors on the surface of macrophages, B cells and DC cells. In particular, mannose receptors expressed on the surface of DC cells and DC cell-specific intercellular adhesion molecules (DC-SIGN) are able to recognize mannans and mediate the occurrence of phagocytosis. In addition, mannan, as a TLR4 agonist, is able to induce DC cell activation. Therefore, the use of mannan as a component of vaccine vectors has significant potential. In the prior art, the research on the application of mannan to vaccines mainly adopts a mixture of mannan and antigen directly. As in the immunostimulating composition and vaccine composition patent (CN201180033660.4) disclosed by the australian essard biopharmaceutical company, a composition comprising mannan is used, but it puts high demands on the molecular weight distribution and oxidation degree of mannan, at least 75% of mannan has a molecular weight of more than 1000kDa, and antigen cross-presentation cannot be efficiently achieved, and the immune effect is limited. Moon et al reported that a class of mannan-coated nanocapsules was used for mRNA antigen delivery (Nano Letter (20) 1499-1509). Although the nanocapsule has certain flexibility, the preparation process of the nanocapsule needs to firstly use the silicon ball as a template and remove the silicon oxide by a corrosion method at the later stage, so that the process is complicated and complex, and the large-scale production is difficult.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel vaccine vector, a preparation method and applications thereof. The vaccine vector provided by the invention has high immunocompetence and simple structure, and can be widely used for preparing protein vaccines, mRNA vaccines or DNA vaccines.

The vaccine carrier provided by the invention consists of oxidized mannan and cationic polymer;

the cationic polymer comprises at least one of I) to III):

I) at least one of polyethyleneimine, polyamide and poly-beta-urethane or at least one of derivatives thereof;

II) a block copolymer or a graft copolymer of the polymer shown in I) and polylactic acid;

III), I) and a block copolymer or a graft copolymer of the polymer shown in the formula (I) and polylactic acid-glycolic acid.

Mannan is a polysaccharide of bacterial origin, consisting of a plurality of repeating sugar units, which are recognized by pattern recognition receptors on the surface of macrophages, B cells and DC cells. In particular, mannose receptors expressed on the surface of DC cells and DC cell-specific intercellular adhesion molecules (DC-SIGN) are able to recognize mannans and mediate the occurrence of phagocytosis. Additionally, mannan, as a TLR4 agonist, is able to stimulate activation of DC cells.

The cationic polymer can be compounded with protein antigen, mRNA antigen or DNA antigen to form nano particles through electrostatic interaction, meanwhile, immunostimulant such as CpG, Poly (I: C), LPS, MPLA and the like can be loaded, and the membrane-breaking effect of cation can be utilized to promote the generation of antigen cross presentation.

In the present invention, the cationic polymer is a polymer represented by I): the polyurethane comprises at least one of polyethyleneimine, polyamide and poly-beta-urethane or derivatives thereof, namely at least one of polyethyleneimine, polyamide and poly-beta-urethane, and at least one of polyethyleneimine derivatives, polyamide derivatives and poly-beta-urethane derivatives.

In some embodiments, the cationic polymer is at least one of a branched polyethyleneimine, a linear polyethyleneimine, and a dendritic polyamide.

In some embodiments, the oxidized mannans have a degree of oxidation of 30 to 70%.

In some embodiments, the oxidized mannan has a weight average molecular weight of 20kDa to 80 kDa.

The invention also provides application of the vaccine vector in preparation of protein vaccines, mRNA vaccines or DNA vaccines.

The invention also provides a vaccine comprising the vaccine vector and the antigen.

The invention also provides a preparation method of the vaccine, which comprises the following steps:

preparing an antigen solution, a cationic polymer solution and an oxidized mannan solution respectively;

mixing an antigen solution with a cationic polymer solution under a vortex condition to obtain a mixed solution A;

and (3) vortexing the mixed solution A for 30s, adding the oxidized mannan solution, and vortexing for 30s to obtain the vaccine.

In the invention, the step of adding an immunostimulant and vortexing for 30s can be further included before the step of adding the oxidized mannan solution.

Further, the antigen is mRNA or DNA encoding a tumor specific or tumor associated antigen, or mRNA or DNA encoding a virus or other microorganism, pathogen.

Specifically, the immunostimulant is at least one of granulocyte-macrophage colony stimulating factor, Rasimote (R848), oligonucleotide (CpG. ODN), polyinosinic acid-polycytidylic acid Poly (I: C), LPS and MPLA, and the types of the immunostimulant include but are not limited to the above, and biological adjuvants, inorganic adjuvants and artificial adjuvants which are commonly used in the field are all within the protection scope of the invention.

The vaccine provided by the invention has a core-shell structure, a cationic polymer and an antigen are compounded to form an inner core, and oxidized mannan is modified on the surface of the inner core to serve as a shell.

The vaccine carrier provided by the invention consists of oxidized mannan and cationic polymer;

the cationic polymer comprises at least one of I) to III):

I) at least one of polyethyleneimine, polyamide and poly-beta-urethane or at least one of derivatives thereof;

II) a block copolymer or a graft copolymer of the polymer shown in I) and polylactic acid;

III), I) and a block copolymer or a graft copolymer of the polymer shown in the formula (I) and polylactic acid-glycolic acid.

The vaccine carrier provided by the invention has the advantages of large antigen load, simple and mild vaccine preparation conditions, good stability, stronger lymph node targeting and dendritic cell targeting capabilities, and capability of causing strong antigen specific immune response, so that the cancer vaccine prepared by the vaccine carrier has a remarkable inhibition effect on tumor growth.

Drawings

FIG. 1 is a drawing showing the preparation of PLA-PEI obtained in example 5¹H NMR spectrum;

FIG. 2 shows the particle size and potential of the cationic inner core of PLA-PEI, PLA-PEI-CpG-OVA and oxidized mannan/PLA-PEI-CpG-OVA prepared in example 6;

FIG. 3 shows the transmission electron microscope results of the oxidized mannan/PLA-PEI-CpG-OVA prepared in example 6;

FIG. 4 is a schematic diagram of the preparation of PAMAM-IMDQ derivatives of example 8;

FIG. 5 shows the result of BMDC activation by the oxidized mannan/PLA-PEI-CpG-OVA vaccine prepared in example 14;

FIG. 6 is the flow results of the oxidized mannan/PLA-PEI-CpG-OVA vaccine of example 15 promoting BMDC antigen cross presentation;

FIG. 7 is the therapeutic results of the oxidized mannan/PLA-PEI-CpG-OVA vaccine of example 16 for the B16-OVA tumor model;

FIG. 8 is an analysis of antigen specific responses after the oxidized mannan/PLA-PEI-CpG-OVA vaccine of example 17 was used in the B16-OVA model;

FIG. 9 shows the therapeutic results of the oxidized mannan/PLA-PEI-CpG-MC 38 antigen vaccine of example 18 in the MC38 tumor resection model.

Detailed Description

The invention provides a vaccine vector, a preparation method and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The invention relates to a vaccine vector, a preparation method and application thereof. The vaccine carrier consists of oxidized mannan and a cationic polymer, the cationic polymer composite protein antigen, mRNA antigen or DNA antigen are prepared into the inner core of the nano-particle when the vaccine is prepared, and then the oxidized mannan is modified to the surface through Schiff base reaction. The raw materials and equipment used in the embodiment of the present invention are known products and obtained by purchasing commercially available products.

Example 1

Preparation of oxidized mannan

Dissolving 1g of mannan in 10mL of sterile water, stirring the mixture under ice bath until the mannan is dissolved, dissolving 657mg of sodium periodate in 5mL of sterile water, and dropwise adding the mixture into the mannan solution. After 2h, the ice bath was removed and the reaction was carried out at room temperature in the dark for 12 h. The product is dialyzed, purified and freeze-dried to obtain the final product.

The oxidation degree of the final product is determined by the hydroxylamine hydrochloride method, which comprises the following specific steps: 0.1mg of oxidized mannan was dissolved in 25mL of an aqueous solution of hydroxylamine hydrochloride (0.25mol/L, containing 0.05% methyl orange), and allowed to stand at room temperature for 3 hours. Titration with a standard NaOH solution (0.1mol/L) gave a yellow change in red and the volume of NaOH consumed was recorded as V₁. Another portion of hydroxylamine hydrochloride solution (0.25mol/L, containing 0.05% methyl orange) was titrated with a standard NaOH solution (0.1mol/L) to turn red to yellow, and the volume of NaOH consumed was recorded as V₀. The degree of aldehyde substitution is calculated by the following formula:

N＝(V₁-V₀)×M×M_w/(1000W)×100％

in the formula, M is the molar concentration of the sodium hydroxide solution, M_wIs the relative molecular weight of the mannan repeat unit (164); w is the weight (g) of oxidized mannan used.

The degree of oxidation of the oxidized mannan was calculated to be 40%.

Example 2

Preparation of oxidized mannan/PEI-CpG-OVA vaccine

Dissolving OVA protein in sterile water for injection to obtain an antigen solution with the concentration of 1mg/mL, dissolving PEI in the sterile water for injection to obtain the concentration of 1mg/mL, dissolving CpG in the sterile water for injection to obtain the concentration of 1mg/mL, dissolving oxidized mannan in the sterile water for injection to obtain the concentration of 1mg/mL, adding 1 volume of OVA solution into 4 volumes of PEI solution under vortex, vortex for 30s, adding 1 volume of CpG into the mixture, vortex for 30s, adding 2 volumes of oxidized mannan solution, and vortex for 30s to obtain the oxidized mannan/PEI-CpG-OVA vaccine. The particle size was 211nm as determined by a Malvern particle sizer.

Example 3

Preparation of PEI derivative (PEI-4BImi)

50mg of PEI (Mw ═ 10kDa) was dissolved in a DMSO solution, 25mg of benzimidazole-7-carboxylic acid, 60mg of EDC. HCl and 36mg of NHS were dissolved together in 10mL of DMSO, stirred at room temperature, activated for 30min, added to the DMSO solution of PEI, and reacted at room temperature for 72 hours. The product was purified by dialysis against sterile water for injection and lyophilized to give the final product PEI-4 BImi.

Example 4

Preparation of oxidized mannan/PEI-4 BImi-OVA vaccine

The PipE is dissolved in sterile water for injection, the concentration is 1mg/mL, OVA protein is dissolved in sterile water for injection, the concentration is 1mg/mL, 1 volume of OVA protein is added into 1 volume of PipE solution under the vortex, the vortex is carried out for 30s, and 2 volumes of oxidized mannan solution are added, thus preparing the PipE-OVA vaccine. The particle size was 225nm as determined by a Malvern particle sizer.

Example 5

Preparation of PLA-PEI

5g of PLA (molecular weight 15k) and 1.5 times the molar equivalent of N, N' -carbonyldiimidazole were dissolved in dry DMSO and stirred at room temperature for 24 hours. 1-fold molar equivalent of PEI (molecular weight 10k) was dissolved in dry DMSO and slowly added to the PLA solution and the reaction was stirred for an additional 24 h. And (4) dialyzing and purifying by using sterile water, and freeze-drying to obtain a final product. The cationic polymer of PLA-PEI obtained in example 5 was analyzed by hydrogen nuclear magnetic resonance spectroscopy. FIG. 1 shows the obtained PLA-PEI cationic polymer¹H NMR spectrum.

Example 6

Preparation of oxidized mannan/PLA-PEI-CpG-OVA vaccine

PLA-PEI nanoparticles were first prepared, 20mg PLA-PEI dissolved in 1mL DMSO, and another 15mL HEPES buffer was prepared at a concentration of 10 mM. The organic phase was slowly added dropwise to the aqueous phase under sonication (40% power, 10 min). The obtained solution is purified by sterile water dialysis to remove impurities such as DMSO and the like. And (4) fixing the volume to 20mL to obtain a cation inner core solution with the concentration of 1mg/mL, and storing at 4 ℃ for later use. Particle size and potential were measured using a malvern particle sizer, as shown in figure 2.

Dissolving OVA protein into sterile water for injection to obtain a concentration of 1mg/mL, dissolving CpG into sterile water for injection to obtain a concentration of 1mg/mL, dissolving oxidized mannan into sterile water for injection to obtain a concentration of 5mg/mL, adding 0.1 volume of CpG solution into 1 volume of PLA-PEI nanoparticle solution under vortex, vortex for 30s, adding 0.2 volume of OVA protein solution, vortex for 30s, adding the mixture into 1 volume of oxidized mannan solution, and vortex for 2min to obtain the oxidized mannan/PLA-PEI-CpG-OVA vaccine. Particle size and potential were measured using a malvern particle sizer, as described in figure 2. The vaccine structure was photographed using a transmission electron microscope, showing a more pronounced core-shell mechanism, as shown in fig. 3.

Example 7

Preparation of oxidized mannan and OVA complexes

Dissolving oxidized mannan in sterile water for injection at a concentration of 5mg/mL, dissolving OVA in sterile water for injection at a concentration of 1mg/mL, adding 0.2 volume of OVA to 1 volume of oxidized mannan solution, and vortexing for 30s to obtain oxidized mannan OVA complex. The particle size was measured using a malvern particle sizer.

Example 8

Preparation of PAMAM-IMDQ derivative bonded with IMDQ adjuvant

256mg of IMDQ (M. RTM. 359.21) was dissolved in dry DMSO with 2-fold molar equivalent of N, N' -carbonyldiimidazole and stirred at room temperature for 24 hours. 200mg of PAMAM (G4) was dissolved in dry DMSO, added to the above solution, and reacted at room temperature for 48 hours. After the reaction is finished, sterile water for injection is used for dialysis and purification, and freeze-drying is carried out, so as to obtain the final product PAMAM-IMDQ. The preparation process is shown in figure 4.

Example 9

Preparation of oxidized mannan/PAMAM-IMDQ-OVA vaccine

Dissolving OVA protein in sterile water for injection to obtain an OVA protein solution with the concentration of 1mg/mL, dissolving PAMAM-IMDQ in sterile water for injection to obtain the concentration of 1mg/mL, dissolving oxidized mannan in sterile water for injection to obtain the concentration of 1mg/mL, adding 1 volume of OVA protein solution into the PAMAM-IMDQ solution under the condition of vortex, vortex for 30s, adding the mixture into 2 times volume of oxidized mannan solution under the condition of vortex, and continuously vortex for 30s to obtain the oxidized mannan/PAMAM-IMDQ-OVA vaccine.

Example 10

Preparation of antigen protein derived from excised tumor tissue

Tumor tissue was obtained from tumor-bearing mice (e.g., MC38 tumor-bearing mice) and surgically excised. 2g of the excised tumor tissue was cut into small pieces, and 5mL of a 60. mu.M sodium hypochlorite solution was added thereto, followed by gentle grinding and then treated at 37 ℃ for 1 hour. After the incubation was completed, 10mL of PBS was added, centrifuged (8000rpm, 10min), the supernatant was discarded, 10mL of PBS was added, and the above centrifugation step was repeated. 5mL of PBS was added, sonicated (40% power, 20min), followed by repeated freeze-thaw cycles for 6 cycles. The mixture was centrifuged (8000rpm, 10min), the lower pellet was discarded, and the supernatant was retained. The supernatant was subjected to BCA protein quantification, and the supernatant was stored at-80 ℃ for further use.

Example 11

Preparation of oxidized mannan/PLA-PEI-CpG-MC 38 personalized antigen vaccine

CpG1826 was dissolved in sterile water to give a CpG solution at a concentration of 1 mg/mL. 1mL of the cationic inner core solution (1 mg/mL) was taken and 100. mu.L of the CpG solution was added with vortexing. After vortexing for 30s, the mixture was allowed to stand for 5 minutes. The MC38 antigen protein was diluted to give a protein solution with a concentration of 1 mg/mL. Add 200. mu.L of protein solution to the mixed solution of cationic core and CpG described above under vortexing, vortexing for 30s, and standing for 5 minutes. Obtaining the PLA-PEI-antigen protein complex.

Dissolving the oxidized mannan with sterile water to obtain oxidized mannan solution with concentration of 5 mg/mL. And (3) dripping the PLA-PEI-antigen protein complex into 1mL of oxidized mannan solution under the condition of vortex, and vortex for 30s to prepare the oxidized mannan/PLA-PEI-CpG-MC 38 antigen vaccine.

Example 12

Preparation of oxidized mannan/PEI-CpG-mRNA vaccine

Dissolving PEI with sterile water for injection to a concentration of 1mg/mL, dissolving mRNA encoding OVA antigen with sterile water for injection to a concentration of 1mg/mL, dissolving CpG1826 with sterile water for injection to a concentration of 1mg/mL, dissolving oxidized mannan with sterile water for injection to a concentration of 1mg/mL, adding 1 volume of mRNA into 5 volumes of PEI solution under vortex, continuing to vortex for 30s, adding 1 volume of CpG solution into the system, continuing to vortex for 30s, finally adding 2 volumes of oxidized mannan solution into the system, and vortex for 30s to prepare the oxidized mannan/PEI-CpG-mRNA vaccine.

Example 13

Preparation of oxidized mannan/PEI-CpG-DNA vaccine

Dissolving PEI with sterile water for injection to a concentration of 1mg/mL, dissolving DNA encoding OVA antigen with sterile water for injection to a concentration of 1mg/mL, dissolving CpG1826 with sterile water for injection to a concentration of 1mg/mL, dissolving oxidized mannan with sterile water for injection to a concentration of 1mg/mL, adding 1 volume of DNA into 5 volumes of PEI solution under vortex, continuing to vortex for 30s, adding 1 volume of CpG solution into the system, continuing to vortex for 30s, finally adding 2 volumes of oxidized mannan solution into the system, and vortex for 30s to prepare the oxidized mannan/PEI-CpG-DNA vaccine. The particle size was 241nm as determined by a Malvern particle sizer.

Example 14

Oxidized mannan/PLA-PEI-CpG-OVA vaccine activated BMDC

Bone marrow-derived DC cells (BMDCs) were seeded in 24-well plates at a density of 3 × 105 cells per well. Free OVA protein, free CpG, PLA-PEI-CpG-OVA, oxidized mannan/PLA-PEI-OVA and oxidized mannan/PLA-PEI-CpG-OVA vaccines are respectively added into different holes, and the amount of the OVA protein added into each hole is the same and is 20 mu g/mL. After 6h incubation, nonadherent or nonadherent cells were gently blown down and flow analysis was performed after staining with anti-murine PE-CD11C, APC/Cy7-MHC-II and APC-CD80 flow antibodies. All manipulations were done on ice, preventing unnecessary activation of BMDCs during the staining process. The BMDC activation profiles for the different groups are shown in fig. 5.

As can be seen from FIG. 5, both oxidized mannan and CpG can promote the activation of BMDC, but the oxidized mannan/PLA-PEI-CpG-OVA group has the strongest activation capability on BMDC, and is remarkably superior to other treatment groups, so that the oxidized mannan/PLA-PEI-CpG-OVA nano vaccine is proved to have remarkable immune activation capability.

Example 15

Oxidized mannan/PLA-PEI-CpG-OVA vaccines to promote antigen cross presentation

The vaccine used in this example was the oxidized mannan/PLA-PEI-CpG-OVA vaccine prepared in example 6. BMDCs were seeded in 24-well plates at a density of 3 × 105 cells per well. Free protein (free OVA), PLA-PEI-OVA and oxidized mannan/PLA-PEI-CpG-OVA vaccines were added to different wells, respectively, at a final concentration of 20. mu.g/mL of OVA protein per well. After 24H incubation, nonadherent or nonadherent cells were gently blown down and flow analysis was performed after staining with anti-murine FITC-CD11C and PE-H2Kb (conjugated SIINFEKL) antibodies. The results are shown in FIG. 6.

As can be seen from FIG. 6, the oxidized mannan/PLA-PEI-CpG-OVA vaccine can greatly promote the cross presentation capability of BMDC cells to antigens, the effect is obviously superior to that of a single OVA vaccine and oxidized mannan-OVA vaccine group, and the difference is very significant (p is less than 0.001). Compared with the oxidized mannan-OVA group alone, the content of the mannan-OVA is improved by 3 times.

Example 16

Oxidized mannan/PLA-PEI-CpG-OVA vaccine for B16-OVA model anti-tumor analysis

The vaccine used in this example was the oxidized mannan/PLA-PEI-CpG-OVA vaccine prepared in example 6And (5) seedling. In the B16-OVA model anti-tumor assay, 6 to 8 week old female C57 mice were injected subcutaneously with 3X 105B 16-OVA tumor cells, and the day of injection was scored as 0 day. Mice were randomly divided into 5 groups: 1) untreated group, 2) OVA proteome (free OVA), 3) aluminum adjuvant + OVA group (Al + OVA), 4) oxidized mannan-OVA, 5) oxidized mannan/PLA-PEI-CpG-OVA. On days 5,10 and 15, each group of mice received tail subcutaneous injection treatment. The single dose of OVA protein was 50. mu.g per mouse and the single dose of CpG was 25. mu.g per mouse. The tumor volume is measured every two days, and the formula of the tumor volume is V ═ a × b²X 0.5, where a is the length of the tumor and b is the width of the tumor. The results are shown in FIG. 7.

As can be seen from FIG. 7, the oxidized mannan/PLA-PEI-CpG-OVA vaccine has the strongest anti-tumor capability, and the tumor inhibition rate reaches 94%, which is far higher than 24% of the tumor inhibition rate of the commercialized aluminum adjuvant + OVA antigen.

Example 17

Oxidized mannan/PLA-PEI-CpG-OVA vaccine for antigen specific response analysis after B16-OVA model

The vaccine used in this example was the oxidized mannan/PLA-PEI-CpG-OVA vaccine prepared in example 6. The specific response of splenocytes to antigen was determined using the elispot method after nucleocapsid protein vaccine was used in the B16-OVA model. The capture antibody was diluted to working concentration and added to ELISPOT well plates at 100 μ L per well, and the plates were incubated overnight at 4 degrees. The well plate was washed three times with PBS, and then 100. mu.L of blocking solution was added to each well, and left at room temperature for 2 hours. The blocking solution was discarded, and an antigen-containing RPMI1640 medium was prepared. Add 200. mu.L of RPMI1640 medium containing antigen per well. Spleen cells from treated mice were isolated and plated individually in well plates at a density of 2 × 105 cells per well. The well plates were incubated for 72 hours in a 37 degree incubator containing 5% carbon dioxide. Cells and medium were discarded and washed 2 times with deionized water. The detection antibody diluted to the working concentration was added, 100. mu.L per well, and incubated at room temperature for 2 h. The plates were washed 2 times with PBST, 100. mu.L of streptavidin-HRP was added to each well, and after incubation for 1h at room temperature, the plates were washed 3 times with PSB. 100 mul of color developing solution was added to each well until a plate appeared. The reaction was terminated by washing with deionized water. The results are shown in FIG. 8.

As can be seen from FIG. 8, after the oxidized mannan/PLA-PEI-CpG-OVA vaccine provided by the invention is administered, the specific immune response of an organism to an antigen can be obviously improved.

Example 18

Oxidized mannan/PLA-PEI-CpG-MC 38 vaccine for MC38 postoperative model anti-tumor analysis

The vaccine used in this example was the oxidized mannan/PLA-PEI-CpG-MC 38 antigen vaccine prepared in example 10. 6 to 8 week old female C57 mice were implanted with MC38 tumor cells subcutaneously at a density of 2X 10 cells per mouse⁶And (4) cells. When the tumor volume grows to about 200mm³In the present example, 90% of the tumor tissue was excised, and the day of excision was counted as 0 day, the MC38 antigen protein was prepared from the tumor tissue by the method of example 4, and the mice were randomly divided into five groups: 1) untreated group, 2) MC38 antigen, 3) aluminum adjuvant + MC38 antigen, 4) oxidized mannan-MC 38 antigen, 5) oxidized mannan/PLA-PEI-CpG-MC 38 antigen. Mice were given tail subcutaneous treatment with 50 μ g each of MC38 antigen protein single dose and 25 μ g each of CpG single dose on days 0,5 and 10, respectively. The results are shown in FIG. 9.

The results show that compared with the untreated group, the tumors of the MC38 antigen group, the aluminum adjuvant + MC38 antigen group, the oxidized mannan-MC 38 antigen group and the oxidized mannan/PLA-PEI-CpG-MC 38 antigen group are obviously reduced, wherein the tumor volume of the PLA-PEI-CpG-MC38 antigen group is not increased basically, and is far smaller than that of the other treated groups. The results show that the vaccine provided by the invention has a remarkable anti-tumor effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.