Nanoparticle vaccine for mucosal immunity as well as preparation method and application of nanoparticle vaccine

Abstract

The invention relates to a nanoparticle vaccine for mucosal immunity and a preparation method and application thereof. The nanoparticle is of a core-shell structure, the core is a compound with a hydrophobic group, the shell is maltodextrin, and the surface of the shell is loaded with a conjugate of an antigen protein coupled cationic polymer and an adjuvant. The prepared nano-particle vaccine can be used for mucosal immunity, co-delivery of a vaccine adjuvant and high-molecular-weight antigen protein is achieved, mucosal immune response of a body can be remarkably enhanced, meanwhile, strong systemic humoral immunity and cellular immune response can be induced, and the nano-particle vaccine is uniform in particle size and good in dispersity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a nanoparticle vaccine for mucosal immunization and a preparation method and application thereof. Background Art

[0002] The natural route of infection of many antigenic proteins begins at the mucosal surface of the upper respiratory tract. Among them, the nasal cavity is a reservoir for early viral infection and a source of toxins for later lung infection, and is also the first line of defense against viruses. Intranasal vaccination can simulate the normal viral infection route, stimulate the respiratory mucosa to produce local specific immune responses and effective cross-protective immune responses. Currently, about twenty mucosal vaccines have entered clinical trials worldwide, but most of them are nasal vaccines with adenovirus as the carrier. Since most people have neutralizing antibodies to adenovirus, they will have an inhibitory effect after vaccination and affect their effectiveness.

[0003] In recent years, the development of nano-vaccine platforms for co-delivery of antigens and adjuvants has made rapid progress, such as liposomes, amphiphilic block copolymers and other nano-carriers used for delivery platforms. Nanoparticle vaccines based on nanoparticle delivery systems have similar molecular sizes and large specific surface areas to pathogens, can simulate pathogen invasion, are easily recognized by receptors, and enhance antigen presentation; they can display multiple antigen epitopes and can also be loaded with adjuvants to induce stronger immune responses, and are one of the most promising vaccine development platforms. At present, more than 20 candidate nanoparticle vaccines have entered the clinical trial stage, and about 60 are in the preclinical development stage, but most of them cannot effectively stimulate the body to produce mucosal immune responses.

[0004] Maltodextrin is a plant polysaccharide with mucosal adjuvant activity and immunomodulatory effects. It can also stimulate cellular and humoral immune responses, promote antigen presentation, and induce the secretion of cytokines such as IFN-γ, IL-5, and IL-12. 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (2',3'-cGAMP) is an agonist of the cGAS-STING pathway, which can promote the production of proinflammatory cytokines and type I interferons, induce the maturation of antigen-presenting cells, upregulate co-stimulatory signals, and enhance their ability to present or cross-present antigens. However, 2',3'-cGAMP directly mixed with antigens has disadvantages such as poor bioavailability and easy diffusion, resulting in toxic side effects.

[0005] In summary, almost all vaccines currently administered by intramuscular injection cannot induce an effective mucosal immune response to prevent the virus from invading the upper respiratory tract. Therefore, how to obtain a nanoparticle vaccine for mucosal immunity has become an urgent problem to be solved. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a nanoparticle vaccine for mucosal immunization and a preparation method and application thereof. The nanoparticle vaccine prepared by the present invention can be used for mucosal immunization, realizes the co-delivery of vaccine adjuvants and large molecular weight antigen proteins, can significantly enhance the body's mucosal immune response, and can also induce strong systemic humoral immunity and cellular immune response, and has uniform particle size and good dispersibility.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a nanoparticle vaccine for mucosal immunization, wherein the nanoparticle has a core-shell structure, the core is a compound having a hydrophobic group, the shell is maltodextrin, and the surface of the shell is loaded with a conjugate of an antigen protein coupled to a cationic polymer and an adjuvant.

[0009] In the present invention, a compound with a hydrophobic group is connected to maltodextrin to form a nanoparticle with a core-shell structure, and an antigen protein is loaded on the shell. Since the nanoparticle has a similar molecular size and a large specific surface area to the pathogen, it can simulate the invasion of the pathogen, be easily recognized by the receptor, and enhance the antigen presentation effect. In addition, maltodextrin and adjuvants have a strong synergistic effect and can significantly enhance the immune effect.

[0010] Preferably, the hydrophobic group comprises C 10 -C 20 Any one or a combination of at least two of a hydrocarbon group, an ester group, an aromatic group, an amine group or an amide group. 10 -C 20 , for example, it could be C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 or C 10 wait.

[0011] Preferably, the compound having a hydrophobic group includes any one of L-phenylalanine methyl ester, hexadecylamine, octadecyl mercaptan or octadecylamine, or a combination of at least two thereof.

[0012] Preferably, the compound having a hydrophobic group is connected to maltodextrin via chemical bonds to form nanoparticles.

[0013] Preferably, the chemical bond comprises any one of an amide bond, an ester bond or a thioether bond, or a combination of at least two of them.

[0014] Preferably, the glucose equivalent of the maltodextrin is 4-20, for example, it can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

[0015] Preferably, the maltodextrin is carboxylated maltodextrin.

[0016] Preferably, the antigen protein includes any one or a combination of at least two of the novel coronavirus antigen protein, novel coronavirus antigen protein RBD, novel coronavirus antigen protein RBD dimer, novel coronavirus antigen protein RBD trimer, S protein antigen, respiratory syncytial virus antigen F protein, G protein, and influenza virus antigen HA protein or NA protein.

[0017] Preferably, the antigen protein includes a new coronavirus antigen protein.

[0018] In one embodiment of the present invention, a novel coronavirus antigen protein is selected. This protein mainly plays the role of an antigen in the structure of the present invention. When connected to nanoparticles, it can significantly enhance the immune response. In theory, different antigen proteins can have good effects when connected to the structure of the present invention.

[0019] Preferably, the structure of the novel coronavirus antigen protein is an RBD trimer.

[0020] Preferably, the amino acid sequence of the new coronavirus antigen protein is SEQ ID NO: 1.

[0021] SEQ ID NO: 1:

[0022] MFVFLVLLPLVSSQENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLLVPRGSGGSGGSGGLEVLFQGPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVL YNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPEQSYGFOPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKHHHHHH.

[0023] Preferably, the coupling agent and the nanoparticles are connected by electrostatic adsorption.

[0024] Preferably, the adjuvant comprises one or a combination of at least two of 2',3'-cGAMP, CpG or PolyI:C.

[0025] Preferably, the adjuvant and the conjugate are connected by electrostatic adsorption.

[0026] Preferably, the cationic polymer comprises any one of polyethyleneimine, dendrimer or chitosan, or a combination of at least two thereof.

[0027] Preferably, the cationic polymer has a molecular weight of 0.5-30 kDa, for example, 0.5 kDa, 1.5 kDa, 1.8 kDa, 25 kDa, 3.0 kDa, 5.0 kDa, 6.0 kDa, 10.0 kDa, 15.0 kDa, 20.0 kDa, 25.0 kDa, 28.0 kDa or 30.0 kDa.

[0028] Preferably, the mass ratio of the nanoparticles, the conjugate and the adjuvant is 1:(5-20):(0.5-2). The (5-20) may be, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, etc.

[0029] Preferably, the particle size of the nanoparticle vaccine is 20-200 nm, for example, it can be 20 nm, 40 nm, 60 nm, 80 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm or 200 nm, etc.

[0030] In the present invention, the size of the nanoparticles is within the above range and has a similar molecular size to that of pathogens, and can be more easily recognized by receptors, thereby presenting antigens and significantly enhancing the immune effect.

[0031] In a second aspect, the present invention provides a method for preparing a nanoparticle vaccine for mucosal immunization according to the first aspect, the preparation method comprising the following steps: carboxylating maltodextrin and grafting a compound having a hydrophobic group to form nanoparticles, coupling the antigen protein with a cationic polymer to form a conjugate, mixing the nanoparticles and the conjugate for a first time to form a nanoparticle-conjugate, and then adding an adjuvant for a second mixing.

[0032] In the present invention, hydrophobic groups are grafted onto carboxyl maltodextrin to self-assemble to form negatively charged nanoparticles. A cationic polymer with a positive charge is coupled to an antigen protein by a chemical modification method to form a conjugate of an antigen protein coupled to a cationic polymer. The conjugate of negatively charged nanoparticles and a cationic polymer coupled to a novel coronavirus antigen protein is electrostatically adsorbed to form nanoparticles loaded with antigen proteins. An adjuvant is further added to the mixture to obtain a nanoparticle vaccine. The present invention ingeniously utilizes electrostatic adsorption to obtain a nanoparticle vaccine, and the method is simple and easy to operate.

[0033] Preferably, the method for preparing the nanoparticles comprises oxidizing carboxylated maltodextrin and then adding a compound having a hydrophobic group and sodium cyanoborohydride.

[0034] Preferably, the mass ratio of the carboxylated maltodextrin, the compound having a hydrophobic group and sodium cyanoborohydride is 1:(0.1-1):(0.4-4). The (0.1-1) may be, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1. The (0.4-4) may be, for example, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3. or 4.

[0035] Preferably, the preparation of the conjugate comprises adding a blocking agent to the antigen protein and then adding a cross-linking agent.

[0036] Preferably, the cross-linking agent includes any one or a combination of at least two of N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS), N-ε-maleimidocaproyloxysulfosuccinimide ester (Sulfo-EMCS), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (Sulfo-SMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS).

[0037] Preferably, the molar ratio of the cross-linking agent to the antigen protein is (10-30): 1. The (10-30) may be, for example, 10, 12, 15, 18, 20, 24, 25, 28 or 30.

[0038] Preferably, in the first mixing, the mass ratio of the nanoparticles to the conjugate is 1:(5-20); wherein (5-20) may be, for example, 5, 6, 8, 10, 12, 15, 16, 18 or 20.

[0039] Preferably, in the second mixing, the mass ratio of the nanoparticle-conjugate to the adjuvant is 1:(0.5-2). The (0.5-2) may be, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.5, 1.6, 1.8 or 2.

[0040] In a third aspect, the present invention provides a use of the nanoparticle vaccine for mucosal immunization according to the first aspect in the preparation of a medicament for a disease associated with antigen protein infection.

[0041] Preferably, the pharmaceutical composition further contains pharmaceutically acceptable excipients.

[0042] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of a carrier, a diluent, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0043] Compared with the prior art, the present invention has at least the following beneficial effects:

[0044] 1. The nano vaccine system involved in the present invention realizes the co-delivery of multiple vaccine adjuvants and large molecular weight antigen proteins, and the prepared nanoparticles have uniform particle size and good dispersibility.

[0045] 2. The mucosal adjuvants maltodextrin and 2',3'-cGAMP involved in the present invention have a synergistic effect, and the immune response induced by them is significantly higher than the immune response produced by no adjuvant or a single adjuvant.

[0046] 3. The novel coronavirus nanoparticle vaccine involved in the present invention can stimulate mice to produce a strong mucosal immune response after nasal inoculation, shows a high level of neutralizing antibody titer in mouse serum, can induce a significant T cell immune response, has great market application value and broad application prospects; and its preparation method is simple and efficient, providing a new approach and option for the effective prevention of novel coronavirus infection and the preparation of protein nano vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 Schematic diagram of the nanovaccine preparation process.

[0048] Figure 2 These are the results of dynamic light scattering detection of sample particle size, where Figure a is the result of maltodextrin-L-phenylalanine methyl ester nanoparticles, Figure b is the result of nanoparticle-conjugate, and Figure c is the result of nanoparticle vaccine.

[0049] Figure 3 This is a transmission electron microscopy image of the nanovaccine.

[0050] Figure 4 The following are the results of infrared spectrum analysis of each group of samples.

[0051] Figure 5 It is a bar graph of antigen-specific IgA antibody titers in bronchoalveolar lavage fluid.

[0052] Figure 6 It is a bar graph of antigen-specific IgA antibody titers in nasal wash fluid.

[0053] Figure 7 It is a bar graph of antigen-specific IgA antibody titers in mouse sera.

[0054] Figure 8 It is a bar graph of the titer of antigen-specific IgG and its subtype antibodies in mouse serum, wherein Figure a is a bar graph of IgG antibody titer, Figure b is a bar graph of IgG1 antibody titer, and Figure c is a bar graph of IgG2a antibody titer.

[0055] Fig. 9 CD4+ cells that produce IFN-γ, IL-4, and IL-17A in spleen culture + 、CD8 + The result of T cell percentage is shown in Figure a, where Figure a is IFN-γ + CD4 + The result of T cell percentage is shown in Figure B, and Figure B is IL-4 + CD4 + The result graph of T cell percentage, graph c is IL-17A + CD4 + The result graph of T cell percentage, and graph d is IFN-γ + CD8 + The result graph of T cell percentage, graph e is IL-4 + CD8 + The result graph of T cell percentage, graph f is IL-17A + CD8 + Results graph of T cell percentage.

[0056] Fig.10 A bar graph showing the titers of neutralizing antibodies induced by the vaccine. DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0058] Example 1

[0059] This example is used to prepare a novel coronavirus nanoparticle vaccine

[0060] (1) Preparation of maltodextrin-L-phenylalanine methyl ester

[0061] 5mg / mL of maltodextrin (MD), 1M chloroacetic acid and 3M NaOH were incubated at room temperature for 1 hour. Then, the pH was adjusted to 7.0 by adding 3M hydrochloric acid. The reaction mixture was fully dialyzed with 20mM pH 5.6 sodium acetate buffer for 4 hours. 0.4mL of the above-mentioned carboxylated maltodextrin was oxidized with 100μL of 0.1M sodium periodate at room temperature for 1 hour. After dialysis with 20mM pH 7.4 phosphate buffer, maltodextrin was incubated at room temperature for 36 hours with L-phenylalanine methyl ester and sodium cyanoborohydride at a mass ratio of 1:0.5:1. The reaction mixture was fully dialyzed with deionized water to obtain nanoparticles MD-Phe.

[0062] (2) Preparation of conjugates

[0063] The antigen protein used in this embodiment is a new coronavirus antigen protein, the structure of the new coronavirus antigen protein is an RBD trimer, and the amino acid sequence is SEQ ID NO: 1.

[0064] The above-mentioned novel coronavirus antigen protein was incubated with N-ethylmaleimide (NEM) in a 20mM pH 7.4 phosphate buffer at a molar ratio of 1:5 at 4°C for 2 hours to block the free thiol groups on the antigen protein. The thiol-alkylated RBD trimer was reacted with N-ε-maleimidocaproyl-oxysuccinimide ester (EMCS) at a molar ratio of 1:20 at 4°C for 4 hours, and then fully dialyzed in a 20mM pH 7.4 phosphate buffer. The cationic polymer polyethyleneimine (PEI) was dissolved in 20mM pH 7.4 phosphate buffer, and then 20 times the molar amount of 2-iminothiolane was added and activated at room temperature for 3 hours. 2-Iminothiolane converts the amino part of polyethyleneimine into thiol. After activation, the unreacted 2-iminothiolane was dialyzed with a dialysis bag. The activated antigen protein RBD trimer obtained above was mixed with the activated polyethyleneimine and reacted overnight at 4°C. After sufficient dialysis, the conjugate RBD-PEI (RP) was obtained.

[0065] (3) Preparation of Nanovaccines

[0066] MD-Phe and RP were mixed at a mass ratio of 1:10, and the nanoparticle-conjugate (RP-MP) was obtained by electrostatic adsorption at 4°C. RP-MP and adjuvant 2',3'-cGAMP were mixed at a mass ratio of 1:0.5 at 4°C to obtain the nanovaccine. The specific preparation process of the novel coronavirus nanoparticle vaccine is as follows: Figure 1 shown.

[0067] Example 2

[0068] This example characterizes the structure of the novel coronavirus nanoparticle vaccine

[0069] (1) Dynamic light scattering

[0070] The size and distribution of nanoparticles were measured by dynamic light scattering (DLS). Figure 2 As shown in Figure a, the size of MD-Phe is 122 nm and the PDI is 0.18. Figure 2 As shown in (b), the size of RP-MP is 59 nm and the PDI is 0.17. Figure 2 As shown in c, the size of the nanovaccine is 51 nm and the PDI is 0.12. This indicates that the electrostatic interaction between MD-Phe and RBD-PEI and 2',3'-cGAMP can compress the conformation of MD-Phe, thereby significantly reducing its molecular size. The Zeta potential values ​​of MD-Phe and RP-MP are -13.0±1.2mV and +46.3±2.7mV, respectively. After adsorbing RP-MP with 2',3'-cGAMP, the Zeta potential of the vaccine is +18.1±2.0mV.

[0071] (2) Negative staining electron microscopy

[0072] like Figure 3 As shown, the nano-vaccine showed a complete nano-particle morphology under transmission microscopy, with an average particle size of about 50nm. The vaccine was in a regular spherical shape without aggregation.

[0073] (3) FT-IR spectroscopy

[0074] The structure of the vaccine was characterized by infrared spectroscopy. Figure 4 As shown, maltodextrin (MD) is at 3384 cm -1 (OH expansion), 2930cm -1 (CH telescopic), 1650cm -1 (C=O stretch) and 930cm -1 Compared with MD, MD-Phe shows several peaks at 3384 cm -1 and 930cm -1 The peaks at 3420 cm -1 and 938cm -1 This is due to the oxidation of the hydroxyl moiety in MD. In addition, due to the introduction of carboxyl and Phe, 1650cm -1 The peak at 3403 cm becomes deeper and broader. -1 (NH telescopic), 2851cm -1(-CH2 symmetric stretching) and 981cm -1 Compared with PEI, RBD-PEI has a peak at 3403 cm due to the coupling of RBD trimer. -1 The peak at 3389 cm shifts rightward to 3389 cm -1 Compared with RBD-PEI, the vaccine was -1 Additional bands were observed at (-CH stretch), indicating the adsorption of 2',3'-cGAMP and MD-Phe.

[0075] Example 3

[0076] This example evaluates the mucosal immune response induced by the vaccine

[0077] Thirty 20g female BALB / C mice aged 6-8 weeks were randomly divided into five groups, namely PBS, RBD trimer, RBD-PEI, RP-MP and vaccine groups, with 6 mice in each group. The mice were immunized by intranasal administration on days 0, 14 and 28, respectively. Except for the PBS group, each sample contained 20μg of RBD trimer in PBS buffer at pH 7.4. The mice were euthanized on day 42, and specimens including blood, bronchoalveolar lavage (BAL) and nasal wash were collected.

[0078] (1) Detection of RBD-specific IgA antibody levels in bronchoalveolar lavage fluid

[0079] The ELISA method was used to detect the RBD-specific IgA antibody titer in the mouse bronchoalveolar lavage fluid. Figure 5 As shown, the specific IgA antibody titer induced in the alveolar lavage fluid of the vaccine group was around 100, while it was not detected in the other groups, indicating that the nanovaccine containing dual adjuvants can enhance the body's induction of the immune response of the lung mucosa.

[0080] (2) Detection of RBD-specific IgA antibody levels in nasal wash fluid

[0081] The ELISA method was used to detect the RBD-specific IgA antibody titer in the mouse nasal wash. Figure 6 As shown in Figure 2, no RBD-specific IgA antibody titers were detected in the nasal washes of the three samples of PBS, RBD trimer, and RBD-PEI, while the RBD-specific IgA antibody titers of the vaccine group were as high as 1.3×10 3 , which was much higher than that of the RP-MP group (84, P<0.01). This indicates that the nanovaccine containing dual adjuvants can effectively enhance the body's induction of the immune response of the nasal mucosa.

[0082] (3) Detection of RBD-specific IgA antibody levels in serum

[0083] The ELISA method was used to detect the RBD-specific IgA antibody titer in mouse serum. Figure 7 As shown, the RBD-specific IgA titer of the vaccine group (3.2×10 3 ) was significantly higher than that of the RBD-PEI group (65, P<0.01) and the RP-MP group (1.7×10 2 , P<0.01), while the IgA antibody titers were almost undetectable in the PBS group and the RBD trimer group.

[0084] Example 4

[0085] This example evaluates the humoral immune response induced by the vaccine

[0086] The RBD-specific antibody titer of serum on day 42 was measured by ELISA. Figure 8 As shown in a, the RBD-specific IgG antibody titers in the PBS and RBD trimer groups were almost undetectable. The RBD-specific IgG titers in the RBD-PEI and RP-MP groups were 1.8×10 3 and 6.4×10 3 This suggests that the combination of PEI adsorption and MD can additionally increase the RBD-specific IgG titer. The RBD-specific IgG titer of the vaccine group was as high as 9.9×10 4 , which is the highest among the five groups. This shows that the nanovaccine containing dual adjuvants induced a significant increase in the intensity of serum IgG antibody response.

[0087] The antibody titer test results of IgG antibody subtype are as follows Figure 8 As shown in b and c, the RBD-specific IgG1 and IgG2a titers in the PBS, RBD trimer, and RBD-PEI groups were almost undetectable. The RBD-specific IgG1 titer in the vaccine group (3.2×10 4 ) and IgG2a titer (8.2×10 4 ) was higher than that of the RP-MP group (1.6×10 3 and 2.8×10 3 ). This indicates that the nanovaccine containing dual adjuvants can significantly increase RBD-specific IgG1 and IgG2a responses. In addition, the IgG2a / IgG1 ratio in the vaccine group was 2.6, which was higher than 1.8 in the RP-MP group. Therefore, the nanovaccine containing dual adjuvants can change the balance between Th1 and Th2.

[0088] Example 5

[0089] This example evaluates the cellular immune response induced by the vaccine

[0090] Thirty 20g female BALB / C mice aged 6-8 weeks were randomly divided into 5 groups, namely PBS, RBD trimer, RBD-PEI, RP-MP and vaccine groups, with 6 mice in each group. The mice were immunized by intranasal administration on days 0, 14 and 28, respectively. Except for the PBS group, each sample contained 20μg of RBD trimer in PBS buffer at pH 7.4. On day 42, the mice were euthanized, and the spleens of the mice were taken. Single cell suspensions were obtained by lysis, grinding and filtration, and RBD trimer was added for stimulation for 60h. The CD4 positive T cells in the cell suspension expressing IFN-γ, IL-4 and IL-17A (i.e. IFN-γ + CD4 + T cells, IL-4 + CD4 + T cells and IL-17A + CD4 + T cells) and the proportion of CD8 positive T cells expressing IFN-γ, IL-4 and IL-17A (i.e. IFN-γ + CD8 + T cells, IL-4 + CD8 + T cells and IL-17A + CD8 + T cells) ratio, the results are as follows Fig. 9 The results show that Fig. 9 As shown in (a), RBD-specific IFN-γ in the RBD trimer and RBD-PEI groups + CD4 + The percentage of T cells was comparable to that of the PBS group. + CD4 + The percentage of T cells was lower than that in the vaccine group (P < 0.05), but higher than that in the other three groups (P < 0.05). Fig. 9 As shown in b and c, IL-4 in the RBD-PEI group + CD4 + T cells and IL-17A + CD4 + The percentage of T cells was higher than that of the RBD and PBS groups, and lower than that of the RBD-PEI group. Importantly, the vaccine group showed the highest IL-4 + CD4 + T cells and IL-17A + CD4 + This suggests that maltodextrin and 2',3'-cGAMP can synergistically promote RBD-specific CD4 +T cells secrete Th1, Th2, and Th17 cytokines and induce a strong Th1 / Th2 balanced cellular immune response. Fig. 9 The vaccine groups shown in d, e and f showed the highest IFN-γ among the five groups. + CD8 + , IL-4 + CD8 + and IL-17A + CD8 + This suggests that maltodextrin and 2',3'-cGAMP can synergistically enhance the RBD-specific CD8 + T cell immune response.

[0091] Example 6

[0092] Detection of vaccine-induced neutralizing antibody titers

[0093] The serum of each group of mice was diluted in a 3-fold gradient ratio in a 96-well plate, and then mixed with pseudoviruses respectively, with phosphate buffer as a negative control, and incubated at 37°C for 2 hours. The serum / pseudovirus mixture was transferred to a 96-well plate filled with Vero cells, placed in an incubator at 37°C and 5% carbon dioxide for 96 hours, and the positive cell value was calculated by confocal cell imaging. After drawing the fitting curve, the reciprocal of the serum dilution corresponding to 50% neutralization was calculated, which was the 50% pseudovirus neutralizing antibody titer. The results are shown in Fig.10 As shown in the figure, the neutralizing antibody titers of the PBS, RBD trimer, and RBD-PEI groups were almost undetectable. The neutralizing antibody titer of the RP-MP group was low, about 90, while the neutralizing antibody titer of the vaccine group was much higher (1.8×10 3 , P<0.01). This indicates that adsorption with 2',3'-cGAMP can mainly increase the serum neutralizing antibody response.

[0094] In summary, the new coronavirus nanoparticle vaccine constructed by the present invention can stimulate mice to produce a strong mucosal immune response after nasal vaccination, shows a high level of neutralizing antibody titer in mouse serum, can induce a significant T cell immune response, has good market application value and broad application prospects; and its preparation method is simple and efficient, providing a new approach and option for the effective prevention of new coronavirus infection and the preparation of protein nano vaccines.

[0095] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention are within the protection scope and disclosure scope of the present invention.

Claims

1. A nanoparticle vaccine for mucosal immunization, characterized in that: The nanoparticle vaccine is a core-shell structure, wherein the core is a compound having a hydrophobic group, the shell is maltodextrin, and the surface of the shell is loaded with a conjugate of an antigen protein coupled to a cationic polymer and an adjuvant.

2. The nanoparticle vaccine according to claim 1, characterized in that The hydrophobic group includes C 10 -C 20 Any one or a combination of at least two of a hydrocarbon group, an ester group, an aromatic group, an amine group or an amide group; Preferably, the compound having a hydrophobic group comprises any one or a combination of at least two of L-phenylalanine methyl ester, hexadecylamine, octadecyl mercaptan or octadecylamine; Preferably, the compound having a hydrophobic group is connected to maltodextrin via chemical bonds to form nanoparticles; Preferably, the chemical bond comprises any one of an amide bond, an ester bond or a thioether bond, or a combination of at least two of them.

3. The nanoparticle vaccine according to claim 1 or 2, characterized in that The maltodextrin is carboxylated maltodextrin; Preferably, the antigen protein includes any one or a combination of at least two of the novel coronavirus antigen protein RBD, the novel coronavirus antigen protein RBD dimer, the novel coronavirus antigen protein RBD trimer, S protein antigen, respiratory syncytial virus antigen F protein, G protein, and influenza virus antigen HA protein or NA protein; Preferably, the coupling agent and the nanoparticles are connected by electrostatic adsorption; Preferably, the adjuvant comprises one or a combination of at least two of 2',3'-cGAMP, CpG or PolyI:C; Preferably, the adjuvant and the conjugate are connected by electrostatic adsorption.

4. The nanoparticle vaccine according to any one of claims 1 to 3, characterized in that The cationic polymer includes any one of polyethyleneimine, dendrimer or chitosan or a combination of at least two thereof; Preferably, the cationic polymer has a molecular weight of 0.5-30 kDa.

5. The nanoparticle vaccine according to any one of claims 2 to 4, characterized in that The mass ratio of the nanoparticles, the conjugate and the adjuvant is 1:(5-20):(0.5-2); Preferably, the particle size of the nanoparticle vaccine is 20-200 nm.

6. A method for preparing a nanoparticle vaccine for mucosal immunization according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: carboxylating maltodextrin and then grafting a compound having a hydrophobic group to form nanoparticles, coupling an antigen protein with a cationic polymer to form a conjugate, mixing the nanoparticles and the conjugate for the first time to form a nanoparticle-conjugate, and then adding an adjuvant for a second mixing.

7. The preparation method according to claim 6, characterized in that: The preparation method of the nanoparticles comprises oxidizing carboxylated maltodextrin, and then adding a compound having a hydrophobic group and sodium cyanoborohydride; Preferably, the mass ratio of the carboxylated maltodextrin, the compound having a hydrophobic group and sodium cyanoborohydride is 1:(0.1-1):(0.4-4); Preferably, the preparation of the conjugate comprises adding a blocking agent to the antigen protein and then adding a cross-linking agent; Preferably, the cross-linking agent includes any one of N-ε-maleimidocaproyl-oxysuccinimide ester, N-ε-maleimidocaproyloxysulfosuccinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester sodium salt, m-maleimidobenzoyl-N-hydroxysuccinimide ester or m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester or a combination of at least two thereof; Preferably, the molar ratio of the cross-linking agent to the antigen protein is (10-30):

1.

8. The preparation method according to claim 6 or 7, characterized in that: In the first mixing, the mass ratio of the nanoparticles to the conjugate is 1:(5-20); Preferably, in the second mixing, the mass ratio of the nanoparticle-conjugate to the adjuvant is 1:(0.5-2).

9. Use of the nanoparticle vaccine for mucosal immunization according to any one of claims 1 to 5 in the preparation of a medicament for a disease associated with antigen protein infection.

10. The use according to claim 9, characterized in that: The drug also contains pharmaceutically acceptable excipients; Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of a carrier, a diluent, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

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