Preparation of mucous membrane adjuvant for recruiting pre-DC and application of mucous membrane adjuvant in influenza vaccine

Abstract

The invention relates to the field of immunology, in particular to preparation of a mucous membrane adjuvant for recruiting pre-DC and application of the mucous membrane adjuvant in influenza vaccines. Tridecafluoroheptyl ethylene oxide and mannose are grafted on polyethyleneimine, the polyethyleneimine and structural protein hemagglutinin of influenza viruses are adsorbed to form a nano vaccine MF25PEI / HA, secretion of CCL2 is promoted by activating a TLR4 pathway of nasal cavity cDC, pre-DC is recruited and differentiated into cDC, and the network steady state of the nasal cavity cDC is maintained. HA uptake is increased by targeting cDC, cDC maturation is stimulated, and high-level immune response is induced. After intranasal immunization of MF25PEI / HA, secretion of high-level IgA and IgG is caused, activation of B cells, T cells and Tfh cells is promoted, and differentiation of memory B cells and memory T cells is induced. Effective protection can be provided for the influenza H1N1 virus, and a new thought is provided for research and development of respiratory mucosa vaccines.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular to the preparation of a mucosal adjuvant for recruiting pre-DCs and its application in influenza vaccine. BACKGROUND

[0002] Influenza virus is a zoonotic pathogen with a wide host range, causing not only economic losses in the livestock industry but also serious threats to public health. Influenza A virus, due to its high infectivity, rapid mutation ability, and wide host adaptability, has become the most prevalent subtype, and its variation and spread have been associated with multiple historical pandemics. Influenza virus is mainly transmitted through respiratory droplets or indirectly through contact with contaminated surfaces, leading to infection. Enhancing mucosal immunity is an effective strategy to block early viral invasion and transmission. Traditional vaccines stimulate systemic immune responses through intramuscular or subcutaneous injection, but have limited protection for the respiratory mucosa. In contrast, respiratory mucosal vaccines have unique advantages. For influenza A virus, blocking the initial infection in the body can reduce the risk of cross-species transmission. However, the nasal mucosa vaccination for influenza vaccine is hindered by the mucosal barrier and the insufficient number of conventional dendritic cells (cDCs) residing in the submucosal layer, which cannot induce a high-efficiency immune response.

[0003] Fluorine-modified polymers can stably encapsulate proteins due to their low surface tension, hydrophobicity, and self-assembly ability. Their hydrophobic and oleophobic properties mean they have good biocompatibility and lower cytotoxicity, are easy to penetrate the mucosal barrier, and are easily endocytosed by cells but not easily fused with cell membranes. As a protein delivery and vaccine adjuvant platform, it has broad prospects, and combining with mannose can further enhance antigen presentation by dendritic cells.

[0004] cDCs are derived from pre-DCs, originating from bone marrow, and after entering the blood circulation, they migrate to peripheral tissues to differentiate and mature. cDCs can form a semi-continuous network on the surface of the nasal mucosa, acting as an immune sentinel, detecting and capturing antigens, and further migrating to draining lymph nodes, thereby initiating innate and adaptive immune responses against viral infection. Studies have found that during viral infection, the number of cDCs increases as the inflammatory response intensifies. During influenza virus infection, the increase in cDCs in the lungs is due to increased migration of pre-DCs from bone marrow to blood and increased recruitment from blood to lung tissue, mediated by upregulation of CCR2 expression and increased secretion of CCL2 in pre-DCs in bone marrow. Studies have shown that activation of the TLR4 pathway can promote CCL2 secretion, and lipopolysaccharide (LPS) as a TLR4 pathway agonist can significantly increase CCL2 expression levels after inhalation, recruiting pre-DCs to the site of lung inflammation. SUMMARY

[0005] Therefore, the present application aims to provide a mucosal adjuvant preparation for recruiting pre-DC and its application in influenza vaccine, which provides effective protection against influenza H1N1 virus infection.

[0006] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a polymer, as shown in formula (I):

[0008] (I);

[0009] Wherein, n is the degree of polymerization.

[0010] In some specific embodiments of the present application, n of the above-mentioned polymer is any integer from 35 to 58;

[0011] x is any integer from 10 to 250;

[0012] m is any integer from 1 to 6;

[0013] R is selected from:

[0014] (1) a mannose residue, a glucose residue, a galactose residue, a fucose residue, a xylose residue, a rhamnose residue, or a D-, L-, DL-configuration thereof;

[0015] (2) a derivative in which the C1-C6 hydroxyl group of the above-mentioned residue is substituted with -H, -CH3, -COCH3, -SO3H, -PO3H, -CH2COOH, -CH2CH2NH2;

[0016] (3) a group formed by connecting the aforementioned residue or derivative to a connecting arm through an O-glycoside, S-glycoside, N-glycoside or carboxylic acid ester bond;

[0017] (4) a pharmaceutically acceptable salt, stereoisomer or solvate thereof.

[0018] The present application also provides a preparation method of the above-mentioned polymer, comprising:

[0019] tridecafluoroheptyl oxirane is grafted with polyethyleneimine to obtain a graft product, and the graft product is grafted with a mannose derivative to obtain the mucosal delivery adjuvant;

[0020] a reactant molar ratio of the tridecafluoroheptyl oxirane to the polyethyleneimine is 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.

[0021] In some embodiments of the present application, the reactant molar ratio of the mannose derivative to the polyethyleneimine in the above preparation method is 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0022] In some embodiments of the present application, the polyethyleneimine in the above preparation method is PEI 1.8K , and the mannose derivative is Mannose-PEG-COOH.

[0023] In some embodiments of the present application, the above preparation method comprises:

[0024] a methanol solution of tridecafluoroheptyl oxirane is added dropwise into a methanol solution of PEI 1.8K , stirred, gradient dialyzed to obtain a grafted product;

[0025] Mannose-PEG-COOH, EDC, NHS, DMSO are mixed, stirred, and then mixed with the grafted product, reacted, dialyzed to obtain the mucosal delivery adjuvant;

[0026] In some embodiments of the present application, the reactant molar ratio of the tridecafluoroheptyl oxirane to the PEI 1.8K is 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43:1, 45:1, 46:1, 47:1, 48:1, 49:1, or 50:1.

[0027] The reactant molar ratio of the Mannose-PEG-COOH to the graft product is 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, or 1:2.

[0028] In some embodiments of the present application, the above preparation method comprises:

[0029] The reactant molar ratio of the Mannose-PEG-COOH, the EDC, and the NHS is 1:1.2:1.2. 1.8K The reactant molar ratio of the Mannose-PEG-COOH to the graft product is 1:1.

[0030] The reactant molar ratio of the Mannose-PEG-COOH, the EDC, and the NHS is 1:1.2:1.2.

[0031] The reactant molar ratio of the Mannose-PEG-COOH to the graft product is 1:1.

[0032] In some embodiments of the present application, the above preparation method comprises: 1.8K As shown in formula (II):

[0033] (II);

[0034] wherein n is any integer from 35 to 58.

[0035] In some embodiments of the present application, the Mannose-PEG-COOH in the above preparation method is as shown in formula (III):

[0036] (III);

[0037] wherein x is any integer from 10 to 250.

[0038] m is any integer from 1 to 6.

[0039] R is selected from:

[0040] (1) a mannose residue, a glucose residue, a galactose residue, a fucose residue, a xylose residue, a rhamnose residue, or a D-, L-, DL-configuration thereof;

[0041] (2) a derivative in which a C1-C6 hydroxyl group of the above residue is substituted with –H, –CH3, –COCH3, –SO3H, –PO3H, –CH2COOH, –CH2CH2NH2;

[0042] (3) a group formed by linking the aforementioned residue or derivative to a linking arm through an O-glycoside, S-glycoside, N-glycoside, or carboxylate bond;

[0043] (4) a pharmaceutically acceptable salt, stereoisomer, or solvate thereof.

[0044] In some embodiments of the present application, the Mannose-PEG-COOH of the above preparation method is shown as formula (IV):

[0045] (IV).

[0046] In some embodiments of the present application, the tridecafluoroheptyl oxirane of the above preparation method is shown as formula (V):

[0047] (V).

[0048] The present application also provides the use of the above polymer in the preparation of biological products for preventing and treating respiratory infections.

[0049] In some embodiments of the present application, the respiratory infection of the above use includes influenza.

[0050] In some embodiments of the present application, the prevention and treatment of the above use is achieved by any one of the following:

[0051] (I) promoting the secretion of CCL2 by activating the TLR4 pathway of conventional dendritic cells in the nasal cavity site, recruiting precursor dendritic cells to the nasal cavity site, and maintaining the network homeostasis of conventional dendritic cells in the nasal cavity site;

[0052] (II) increasing the uptake of antigens by targeting conventional dendritic cells, stimulating the maturation of conventional dendritic cells, and inducing a high level of immune response;

[0053] The high level of immune response includes at least one of the following:

[0054] (i) an increased level of IgA secretion;

[0055] (ii) an increased level of IgG secretion;

[0056] (iii) enhanced B cell activation;

[0057] (iv) enhanced T cell activation;

[0058] (v) enhanced Tfh cell activation;

[0059] (vi) enhanced differentiation of memory B cells;

[0060] (vii) memory T cell differentiation enhancement.

[0061] The present application also provides a biological product comprising the mucosal delivery adjuvant described above.

[0062] In some embodiments of the present application, the biological product described above further comprises an acceptable excipient or adjuvant.

[0063] The present application also provides a method for preparing a biological product, comprising preparing based on the mucosal delivery adjuvant described above.

[0064] The present application also provides a vaccine comprising the mucosal delivery adjuvant described above.

[0065] The present application also provides an influenza vaccine comprising the mucosal delivery adjuvant described above and a hemagglutinin protein.

[0066] In some embodiments of the present application, the method for preparing the influenza vaccine described above comprises mixing the mucosal delivery adjuvant described above and the hemagglutinin protein of the influenza virus at a mass ratio of 10:1, and then combining with an acceptable excipient or adjuvant to obtain the influenza vaccine.

[0067] In some embodiments of the present application, the dosage form of the influenza vaccine described above is a nasal spray.

[0068] In some embodiments of the present application, the excipient or adjuvant of the biological product or the influenza vaccine described above comprises at least one of a binding agent, a filler, a disintegrating agent, a lubricant, an isotonicity adjusting agent, a solubilizing agent, a co-solvent, a preservative, a coloring agent, a suspending agent, a wetting agent, an emulsifying agent, and a surfactant.

[0069] The present application also provides a method for preventing or treating a disease, comprising administering the biological product described above.

[0070] In some embodiments of the present application, the disease of the method for preventing or treating a disease described above is influenza, and the biological product is the influenza vaccine described above.

[0071] In some embodiments of the present application, the administration mode of the method for preventing or treating a disease described above is nasal mucosal administration.

[0072] The present application has the following beneficial effects:

[0073] Nano-vaccine MF 25 PEI / HA promotes the secretion of CCL2 by activating the TLR4 pathway of DCs in the nasal cavity site, recruits pre-DCs to the nasal cavity site, and further increases the uptake of HA by targeting DCs, stimulates DCs maturation, and induces high-level immune response. MF 25High levels of IgA and IgG secretion are induced after intranasal immunization with PEI / HA, which promotes the activation of B cells, T cells and Tfh cells, and induces the differentiation of memory B cells and memory T cells. The present application can provide effective protection against influenza H1N1 virus, and provides a new idea for the development of respiratory mucosal vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0074] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0075] Figure 1 Protein image results of Western Blot before and after purification are shown;

[0076] Figure 2 Chemical synthesis flowchart of MFPEI is shown;

[0077] Figure 3 Particle size distribution and zeta potential diagram of the nano vaccine are shown;

[0078] Figure 4 MF 25 Transmission electron microscope image of PEI / HA;

[0079] Figure 5 MF 25 Particle size monitoring results of PEI / HA in PBS and FBS for 7 consecutive days are shown;

[0080] Figure 6 F 25 NMR spectrum of PEI and F 50 NMR spectrum of PEI and F

[0081] Figure 7 Fourier transform infrared spectrum images of FPEI and MFPEI are shown;

[0082] Figure 8 MF 25 In vitro DC uptake of PEI / HA, wherein the left and right groups are HA or MF 25 CLSM image of PEI / HA after 4 h incubation with DC2.4 cells, and the middle group is DC2.4 cells pretreated with mannose for 4 h to block the mannose receptor;

[0083] Figure 9 Flow cytometry analysis of DC2.4 cells on HA, F 25 PEI / HA, MF 25 Uptake level of PEI / HA;

[0084] Figure 10Flow cytometry analysis of CD80 and CD86 expression of BMDCs after incubation with different components for 24 h;

[0085] Figure 11 Flow cytometry analysis of TNF-α, IFN-γ, IL-12, IL-6 secretion levels of BMDCs after incubation with different components;

[0086] Figure 12 Flow cytometry analysis of cDCs maturation in cervical lymph nodes three days after immunization with different components;

[0087] Figure 13 Flow cytometry analysis of cDCs maturation in spleen three days after immunization with different components;

[0088] Figure 14 Flow cytometry analysis of TNF-α, IFN-γ, IL-12, IL-6 secretion levels in cervical lymph nodes (A) and spleen (B) three days after immunization with different components;

[0089] Figure 15 Flow cytometry analysis of mucin aggregation after incubation of different components with different concentrations of artificial mucus;

[0090] Figure 16 Flow cytometry analysis of mucin aggregation after incubation of different components with different concentrations of artificial mucus;

[0091] Figure 17 In vivo fluorescence images and fluorescence intensity of nasal cavity after intranasal immunization of mice;

[0092] Figure 18 Fluorescence imaging of nasal cavity tissue sections 3 h after intranasal immunization;

[0093] Figure 19 Flow cytometry analysis of TLR4 + DC and macrophage percentages;

[0094] Figure 20 Western Blot analysis of MF 25 Expression of NF-κB and p-NF-κB in DC2.4 after incubation with PEI / HA;

[0095] Figure 21 qRT-PCR analysis of the relative mRNA level of CCL2 in nasal cavity tissue 3 h after intranasal immunization;

[0096] Figure 22 Immunofluorescence images of CCL2 expression in nasal cavity tissue 3 h after intranasal immunization;

[0097] Figure 23Flow cytometry analysis of the percentage of pre-DCs in the nasopharynx-associated lymphoid tissue 24 h after intranasal immunization;

[0098] Figure 24 Flow cytometry analysis of the dynamic changes of cDC numbers in the nasal cavity after intranasal immunization;

[0099] Figure 25 Flow chart of animal experiment immunization and sample collection. Mice in each group were intranasally immunized (10 µg HA per mouse, n = 6) on days 0, 14 and 28, lavage fluid, lung alveolar lavage fluid and cervical lymph nodes and spleen were collected on day 42, cervical lymph nodes and spleen were collected on day 60, and serum was continuously collected until day 120. The legend in the figure is applicable to Figures 26-28 , Figures 30-34 ;

[0100] Figure 26 Figure showing HA-specific IgA antibody titers in the nasal lavage fluid, bronchoalveolar lavage fluid (BALF) and serum on day 42 after immunization. The legend is shown in Figure 25 ;

[0101] Figure 27 Figure showing flow cytometry analysis of the percentage of CD3 + CD4 + T cells (A), CD3 + CD8 + T cells (B) in the cervical lymph nodes on day 42. The legend is shown in Figure 25 ;

[0102] Figure 28 Figure showing flow cytometry analysis of the percentage of Tfh cells (A) and CD19 + IgD + B cells (B) in the cervical lymph nodes on day 42. The legend is shown in Figure 25 ;

[0103] Figure 29 Figure showing ELISA analysis of the secretion levels of IL-12, IL-6, TNF-α and IFN-γ in the cervical lymph nodes on day 42 after immunization;

[0104] Figure 30 Figure showing flow cytometry analysis of the percentage of B cells in the spleen (A), HA-specific IgG antibody titers in the serum (B), HAI titers (C) and the proliferation rate of splenocytes after in vitro culture and stimulation with the same antigen (D) on day 42 after immunization. The legend is shown in Figure 25 ;

[0105] Figure 31 Figure showing flow cytometry analysis of the percentage of CD3 + CD4+ T cells (A) and CD3 + CD8 + Percentage of T (B) cells, legend see Figure 25 ;

[0106] Figure 32 Percentage of memory B cells in cervical lymph nodes (A) and in spleen (B) 60 days post immunization analyzed by flow cytometry, legend see Figure 25 ;

[0107] Figure 33 Antibody titers of HA-specific IgG in serum at day 60 (A), day 90 (B), day 120 (C) analyzed by ELISA, legend see Figure 25 ;

[0108] Figure 34 Percentage of T cells in spleen 60 days post immunization analyzed by flow cytometry, legend see Figure 25 ;

[0109] Figure 35 Timeline of immunization and virus challenge, the legend therein applies to Figures 36-39 ;

[0110] Figure 36 Body weight change (A) and survival rate (B) of mice after PR8 virus challenge; clinical score at day 5 after PR8 virus challenge (C), legend see Figure 35 ;

[0111] Figure 37 Image of H&E staining of lung tissue at day 5 after PR8 virus challenge, legend see Figure 35 ;

[0112] Figure 38 Image of lung tissue at day 5 after PR8 virus challenge, legend see Figure 35 ;

[0113] Figure 39 Lung index and lung virus titer at day 5 after PR8 virus challenge, legend see Figure 35 ;

[0114] Figure 40 Cell viability of DC2.4 cells incubated with different components for 24 h;

[0115] Figure 41 Body weight change (A) of mice after intranasal immunization with different components; H&E images of heart, liver, spleen, lung, kidney tissue sections of mice at day 14 post immunization (B); hematological evaluation of mice at day 14 post immunization (C). DETAILED DESCRIPTION

[0116] The mucosal adjuvant preparation for recruiting pre-DC and its application in influenza vaccine are disclosed, and those skilled in the art can refer to the content herein to improve the process parameters appropriately. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are all regarded as included in the present application. The method and application of the present application have been described by preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0117] It should be understood that the expression "one or more of" includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0118] The terms "comprising", "having", or "including", including their grammatical synonyms, should be generally understood as open-ended and non-limiting, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0119] It should be understood that the order of steps or the order of performing certain actions is not important as long as the present application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0120] The use of any and all examples, or exemplary language herein, for example, "for instance" or "such as", is intended merely to better illustrate the present application and does not indicate that any non-recited element is essential to the practice of the present application. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present application.

[0121] In addition, the numerical ranges and parameters regarding the present application are approximate values, and the related values in the specific embodiments have been presented as accurately as possible. However, any numerical value inevitably contains a standard deviation due to the individual test method. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, numbers, values and percentages used in the present disclosure are modified by "about". Here, "about" generally means within plus or minus 10%, 5%, 1% or 0.5% of a particular value or range.

[0122] In one implementation, after grafting tridecafluoroheptyl oxirane and mannose on polyethyleneimine (PEI), and adsorbing the structural protein hemagglutinin (HA) of influenza virus, a nano vaccine (MF 25 PEI / HA) is formed.

[0123] In a specific implementation, the expression and purification method of the HA protein is as follows:

[0124] The sequence encoding the PR8 full-length HA protein is cloned into the PCMV vector, expressed in HEK293F cells and purified. The cell density in the logarithmic growth phase is adjusted to 2 x 10 6 ~3 x 10 6 The cells are transfected using PEI (MW 40000), and the cells are harvested after 72 h, centrifuged (200 g, 5 min, 4 °C) to collect the supernatant, and the His-tagged target protein is purified using a Ni-NTA affinity column. The equilibration buffer (20 mM Tris, 300 mM NaCl, 5% glycerol, pH 7.0) and the eluent (20 mM Tris, 300 mM NaCl, 5% glycerol, 500 mM imidazole, pH 7.0) are prepared. The supernatant is added to the Ni-NTA affinity column, and the binding is performed overnight at 4 °C, and the elution is performed using different concentrations of imidazole equilibration buffer, wherein the HA protein is eluted in 250 mM imidazole equilibration buffer. The HA protein is dialyzed in PBS (molecular weight cut-off 3500 Da) and concentrated using a 10 kDa ultrafiltration tube, and the protein concentration is determined using a BCA kit.

[0125] The synthesis method of the MFPEI / HA nanovaccine is as follows:

[0126] A methanol solution (20 mg / mL) of tridecafluoroheptyl oxirane is slowly added dropwise to a methanol solution (20 mg / mL) of dendritic PEI 1.8K The molar ratio of tridecafluoroheptyl oxirane to PEI 1.8K is 25:1. The reaction is stirred at room temperature for 48 h. The reactants are dialyzed (molecular weight cut-off 500 Da) in 5%~1% methanol solution and deionized water for 48 h and then freeze-dried to obtain F 25 PEI. Mannose-PEG-COOH, EDC and NHS are added to DMSO at a molar ratio of 1:1.2:1.2, stirred at room temperature for 30 min in the dark, and F 25 PEI is added dropwise to DMSO at a molar ratio of 1:1 with Mannose-PEG-COOH, and the reaction is performed at room temperature for 24 h. The reactants are dialyzed (molecular weight cut-off 1500 Da) in deionized water for 48 h and then freeze-dried to obtain MF 25 PEI, and the fluorine content and mannose grafting are characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy (FT-IR) and EDS. MF 50 PEI is synthesized in the same way as above, and the molar ratio of tridecafluoroheptyl oxirane to PEI 1.8K is 50:1.

[0127] MF 25 PEI and HA were incubated in deionized water at a series of different mass ratios for 30 min at 4 ℃ to obtain MF 25 PEI / HA. MF 50 The preparation of PEI / HA was the same as above.

[0128] The reagents involved in the present application: Ni-NTA affinity column was purchased from Shanghai Yisheng Company; BCA protein quantification kit, DAPI containing mounting medium were purchased from Shanghai Biyun Tian Company; PEI 1.8K was purchased from Shanghai Maikelin Company, CAS: 25987-06-8, average polymerization degree 35-58; Tridecafluoroheptyl oxirane was purchased from Shanghai Maier Company, CAS: 38565-52-5; Mannose-PEG-COOH was purchased from Xi'an Ruishi Biological Company, CAT: R-66673, Lot: R3J0230316; FITC and Cy5.5-NHS were purchased from Aladdin Company (Shanghai, China); Trizol reagent was purchased from Japan Takara Company; NF-KB p65 Rabbit mAb, Phospho-NF-KB p65 Rabbit mAb were purchased from the United States Cell Signaling Technology (CST) Company; Anti-TNF-α, anti-IL-12, anti-IL-6 and anti-IFN-γ ELISA kits were purchased from Thermo Fisher Scientific Company (USA); Anti-CD80-FITC, anti-CD86-FITC, anti-TLR4 (CD284) / MD2-PE, anti-CD3-APC, anti-CD4-FITC, anti-CD8-FITC, anti-CD19-APC, anti-IgD-FITC, anti-CD138-PerCP / Cy5.5, anti-CD62L-PerCP, anti-CD279 (PD-1)-PE / Cy7, anti-CXCR5-PerCP / Cy5.5, anti-CD135-PE were purchased from Biolegend (USA); HRP labeled goat anti-mouse IgA, anti-B220-FITC, anti-CD43-FITC, anti-CD11b-PE / Cy5, anti-CD11c-APC, anti-CD135-PE, anti-CD44-PE, anti-CD38-PE / CF594 were purchased from Proteintech (China).

[0129] The English abbreviations involved in the present application and their meanings are: HA: hemagglutinin protein of influenza virus; PEI: polyethyleneimine; F 25PEI: polyethylenimine grafted with fluorocarbon linkers at a molar ratio of 1:25; F 50 PEI: polyethylenimine grafted with fluorocarbon linkers at a molar ratio of 1:50; MF 25 PEI: F grafted with mannose 25 PEI; MF 50 PEI: F grafted with mannose 50 PEI; MF 25 PEI / HA: MF 25 PEI / HA: MF 50 PEI / HA: MF 50 PEI / HA: MF

[0130] In Example 1 to Example 5 and Verification Example 1 of the present application, the raw materials and reagents used can be purchased from the market. Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary commercially available products, which can be purchased from the market.

[0131] The present application will be further described below in conjunction with examples.

[0132] Example 1: Preparation of a nano-vaccine

[0133] (I) Expression and purification of HA protein

[0134] The sequence encoding the full-length HA protein of PR8 was cloned into the PCMV vector, and the specific sequence was GenBank: J04572.1.

[0135] Expression and purification in HEK293F cells. Adjust the cell density in the logarithmic growth phase to 2 x 10 6 ~3 x 10 6 Use PEI (MW 40000) to transfect cells, harvest cells after 72 h, centrifuge (200 g, 5 min, 4 ℃) to collect the supernatant, and purify the His-tagged target protein with a Ni-NTA affinity column. Prepare an equilibration buffer (20 mM Tris, 300 mM NaCl, 5% glycerol, pH 7.0) and an eluent (20 mM Tris, 300 mM NaCl, 5% glycerol, 500 mM imidazole, pH 7.0). Add the supernatant to the Ni-NTA affinity column, bind overnight at 4 ℃, and elute with different concentrations of imidazole equilibration buffer, wherein the HA protein is eluted in 250 mM imidazole equilibration buffer. Dialyze the HA protein in PBS (molecular weight cut-off 3500 Da), and concentrate with a 10 kDa ultrafiltration tube, and determine the protein concentration with a BCA kit.

[0136] (II) Synthesis of MFPEI / HA Nanoparticle Vaccine

[0137] A methanol solution of tridecafluoroheptyl ethylene oxide (20 mg / mL) was slowly added dropwise to the dendritic PEI. 1.8K In a methanol solution of (20 mg / mL), tridecafluoroheptyl ethylene oxide and PEI 1.8K The molar ratio was 25:1. The mixture was stirred at room temperature for 48 h. The reactants were dialyzed against 5%–1% methanol and deionized water (molecular weight cutoff 500 Da) for 48 h, followed by freeze-drying to obtain F. 25 PEI. Add Mannose-PEG-COOH, EDC, and NHS to DMSO in a molar ratio of 1:1.2:1.2, stir at room temperature in the dark for 30 minutes, and then add F... 25 PEI was added dropwise to DMSO at a molar ratio of 1:1 to mannose-PEG-COOH, and the reaction was carried out at room temperature for 24 h. The reactants were dialyzed against deionized water (molecular weight cutoff 1500 Da) for 48 h and then freeze-dried to obtain MF. 25 PEI, and the grafting of tridecafluoroheptyl ethylene oxide and mannose were verified by NMR and Fourier transform infrared spectroscopy (FT-IR). MF 50 The synthesis of PEI is the same as described above, using tridecafluoroheptyl ethylene oxide and PEI. 1.8K The molar ratio is 50:1. See the reaction formula. Figure 2 .

[0138] MF 25 PEI and HA were incubated in deionized water at a series of different mass ratios at 4 °C for 30 min to obtain MF. 25 PEI / HA. MF 50 The preparation of PEI / HA is the same as described above.

[0139] Verification Example 1: Basic Characteristics of Nanoparticle Vaccines

[0140] (1) Identification of HA protein

[0141] The protein harvested in Example 1 was added to 5× protein loading buffer, heated in a metal bath at 95°C for 10 min, and then analyzed by Western blotting. The results are as follows: Figure 1 As shown.

[0142] (2) Particle size distribution

[0143] Using a ZS90 dynamic light scanning instrument (DLS, Malvern Zeta-sizer, UK), the inventors measured the adsorption of MF at a series of different ratios. 25 PEI / HA and MF 50The particle size and potential of PEI / HA (i.e., prepared in step (ii) in Example 1) were determined.

[0144] At the same time, scanning observation was performed using a transmission electron microscope (TEM, Tecnai G220, 10 times) and the images were observed.

[0145] The particle size distribution and potential were as shown in Table 1 and Table 2. Figure 3 The transmission electron microscope images were as shown in Figure 1 and Figure 2. Figure 4 It can be seen that: MF 25 PEI / HA and MF 50 PEI / HA showed the optimal ratio when the ratio of HA was 10:1, the particle size was the smallest, which was 179 nm and 322.5 nm, respectively, and the potential was 39 mV and 31.7 mV, respectively. Therefore, unless otherwise specified, all studies used the optimized weight ratio of 10:1 (Table 1, Table 2).

[0146] Table 1: Particle size and potential of MF 25 PEI / HA nanovaccine

[0147]

[0148] Table 2: Particle size and potential of MF 50 PEI / HA nanovaccine

[0149]

[0150] (3) Stability detection

[0151] The prepared MF 25 PEI / HA was placed in PBS and diluted 10 times, and then the particle size was detected using a ZS90 dynamic light scattering instrument for 7 consecutive days and recorded, and the results were as shown in Table 3. Figure 5 Figure 5 The table data corresponding to Table 3 in the above are as follows, and it can be seen from the analysis that the MF 25 PEI / HA had no significant change in particle size in 7 days, indicating that the MF 25 PEI / HA had good stability.

[0152] Table 3: Particle size monitoring results (nm) placed in PBS for 7 consecutive days

[0153]

[0154] Table 4: Particle size monitoring results (nm) placed in FBS for 7 consecutive days

[0155]

[0156] (4) MFPEI synthesis confirmation​

[0157] By nuclear magnetic resonance hydrogen spectrum ( 1 ¹H NMR and energy-dispersive spectroscopy (EDS) confirmed the grafting of tridecafluoroheptylethylene oxide and its fluorine content. Figure 6 As shown, a peak shift occurred in the NMR spectrum from 3.1 to 3.7 ppm, indicating that PEI... 1.8k The successful fluorination of F. EDS results further demonstrate the success of F. 25 PEI and F 50 The fluorine content in PEI was 10.79% and 19.73%, respectively.

[0158] The structure of MFPEI was characterized using Fourier transform infrared spectroscopy. Figure 7 As shown, in F 25 PEI, F 50 PEI, MF 25 PEI and MF 50 In the PEI spectrum, characteristic absorption peaks of the amide bond (6040 nm, 7840 nm) can be observed, indicating that the mannose grafting was successful. 25 FPEI and M 50 FPEI was successfully prepared.

[0159] (5) DC2.4 cell uptake

[0160] The inventor prepared MF 25 The PEI / FITC-HA nanovaccine was evaluated for its in vitro uptake by DC2.4. A brief description of the experimental procedure is as follows.

[0161] A DMSO solution of FITC (1 mg / mL, 2 mL) was added to a PBS solution of HA protein (2 mg / mL, 5 mL), and the mixture was stirred at 4°C for 12 h, followed by dialyzing in deionized water for 48 h (MW 3500 Da). FITC-labeled HA (FITC-HA) was obtained by freeze-drying. All the above processes were carried out under light-protected conditions. The preparation method of Cy5.5-labeled HA (Cy5.5-HA) was the same.

[0162] DC2.4 cells were seeded into 24-well plates containing crawling slides (2 × 10⁻⁶ cells per well). 5 (cells / well), FITC-HA and MF were added respectively. 25 PEI / FITC-HA (0.02 mg HA / mL) was incubated for 4 h. To evaluate the targeting effect of mannose, DC2.4 cells were blocked with mannose (10 mg / mL) for 4 h beforehand, washed with PBS, and then incubated with MF. 25PEI / FITC-HA. After 4 h incubation, the cells were fixed with 200 μL of 4% paraformaldehyde for 15 min after washing with PBS for three times. DAPI was added to the mounting medium on the glass slide in advance, and then the coverslips were inverted on the glass slide with mounting medium. The whole process was performed in the dark. The uptake of DC2.4 cells was observed by laser confocal microscopy (CLSM). In addition, after 4 h incubation according to the above experimental procedures, the medium was discarded, and the cells were collected after washing with PBS. The uptake of FITC-HA was detected by flow cytometry.

[0163] The results of the relevant experiments are shown in Figure 8 MF 25 PEI / FITC-HA group showed obvious green fluorescence in the cells, while the fluorescence of the FITC-HA group was negligible, indicating that MF 25 PEI significantly enhanced the uptake of HA by DC2.4 cells. In addition, the intracellular fluorescence decreased after pre-incubation with Mannose, indicating that MF 25 PEI / HA can increase the uptake of DC2.4 cells by Mannose-mediated targeting. The results of flow cytometry are consistent with the above, as shown in Figure 9 MF 25 PEI / HA-treated cells showed significantly enhanced uptake.

[0164] (6) MF 25 Detection of PEI / HA stimulating DC maturation in vitro

[0165] The maturation of DCs is crucial for promoting antigen cross-presentation and cytokine secretion, both of which are essential for adaptive immune responses. The inventors evaluated the effect of MF 25 PEI / HA nanovaccine on DC maturation, as follows.

[0166] Immature BMDCs were seeded in a 24-well plate (1 × 10 5 cells / well) and treated with HA, PEI / HA, MF 25 PEI / HA and MF 50 PEI / HA (0.02 mg HA / mL) for 24 h. After centrifugation, the supernatant was retained (4 °C, 200 g, 5 min). The harvested BMDCs from the precipitate were combined with anti-CD86-FITC, anti-CD80-FITC and anti-DC11c-APC at 4 °C for 30 min, and the BMDCs were washed with PBS for flow cytometry analysis to detect the expression levels of CD80 and CD86. The levels of TNF-α, IFN-γ, IL-12 and IL-6 cytokines in the supernatant were detected using an ELISA kit.

[0167] Experimental results are as follows Figure 10 As shown, CD11c in the HA group and the PEI / HA group + CD80 + and CD11c + CD86 + The proportion of DCs was significantly lower. MF 25 CD11c in PEI / HA group + CD80 + and CD11c + CD86 + The proportion of DCs was the highest, increasing by 4.4–5.3 times compared to the HA group and by 10.2–20.1 times compared to the PBS group. ELISA analysis of cytokine levels in the BMDCs supernatant showed that, compared to the HA group, MF… 25 The levels of TNF-α, IFN-γ, IL-6, and IL-12 in the PEI / HA group increased by 2.3 to 2.7 times. Figure 11 These results highlight MF 25 PEI / HA effectively stimulates the maturation of BMDCs in vitro.

[0168] (7) MF 25 Detection of PEI / HA in vivo stimulation of cDC maturation

[0169] The inventors evaluated MF 25 The effects of PEI / HA nanovaccines on cDC maturation in vivo are detailed below.

[0170] Mice were intranasally inoculated with PBS, HA, MF59+HA, and MF 25 PEI / HA, MF 50 PEI / HA (10 μg HA / animal, n = 3). Neck lymph nodes and spleen were collected 3 days post-immunization to prepare single-cell suspensions. These suspensions were bound to anti-CD64-FITC, anti-CD11c-APC, anti-MHC-II-PE, anti-CD86-PerCP, and anti-CD80-PerCP at 4 ℃ for 30 min, and then analyzed by flow cytometry after passing through PBS to detect the expression levels of CD80 and CD86. The levels of TNF-α, IFN-γ, IL-12, and IL-6 cytokines in the supernatant were detected using an ELISA kit.

[0171] Experimental results are as follows Figure 12 As shown, compared with the HA group, MF 25 CD64 in PEI / HA group - MHC-II +CD11c + CD80 + and CD64 - MHC-II + CD11c + CD86 + The ratio of cDCs was significantly increased by 2.7-fold and 4-fold, respectively, indicating that MF 25 PEI / HA could promote the maturation of cDCs in cervical lymph nodes. Consistent with the above results, in the spleen Figure 13 , MF 25 PEI / HA group, CD64 - MHC-II + CD11c + CD80 + and CD64 - MHC-II + CD11c + CD86 + The ratio of cDCs was 4.5-fold and 4-fold higher than that in the HA group. In addition, the inventors further performed ELISA analysis to determine the levels of secreted cytokines in the supernatant of cervical lymph nodes and spleen. As shown in Figure 14 , the secretion levels of IL-6, IL-12, TNF-α and IFN-γ were the highest in the MF 25 PEI / HA group. Notably, compared with the common influenza vaccine adjuvant MF59, the cytokine content in MF 25 PEI / HA was increased by 1.3-2-fold. In summary, these findings indicated that MF 25 PEI had stronger ability to induce the maturation of cDCs in vivo, thus providing further support for subsequent immune activation.

[0172] Example 2: Evaluation of mucosal penetration effect of MF 25 PEI / HA

[0173] (I) In vitro mucosal penetration level

[0174] (1) Detection of anti-mucin aggregation of MF 25 PEI in vitro

[0175] The inventors prepared MF 25 PEI / FITC-HA nanovaccine to evaluate the ability to resist mucin adhesion, as follows.

[0176] PEI / FITC-HA, F 25 PEI / FITC-HA, F 50 PEI / FITC-HA, MF 25PEI / FITC-HA, MF 50 PEI / FITC-HA was mixed with different concentrations of artificial mucus (0.1%, 0.3%, 0.5% porcine gastric mucin, PBS) and incubated in a 37 ℃ shaker for 30 min. After centrifugation (room temperature, 1500 rpm, 2 min), the precipitate was washed twice with PBS, then treated with NaOH for 10 min. The amount of FITC-HA was detected by fluorescence spectroscopy (λex = 488 nm, λem = 525 nm).

[0177] The experimental results are shown in Figure 15 Compared with the PEI / FITC-HA group, F 25 PEI / FITC-HA and F 50 The mucin aggregation level of PEI / FITC-HA was significantly reduced, and MF 25 PEI / FITC-HA and MF 50 The aggregation level of PEI / FITC-HA was further reduced by 2.1~2.6 times. The reason for the reduction of mucin aggregation is that the surface energy of the fluorine-containing material is low, thereby minimizing its interaction with mucin. In addition, PEG in the grafted Mannose-PEG-COOH has high hydrophilicity and electrical neutrality, which further avoids being adhered by mucin and enhances its ability to penetrate into the submucosa.

[0178] (2) MF 25 Detection of PEI in vitro nasal mucosa penetration

[0179] RPMI2650 cells are a kind of human nasal epithelial cells, which are often used in vitro to simulate the nasal epithelial barrier.

[0180] The inventors evaluated the ability of MF 25 PEI / FITC-HA nanovaccine to penetrate the nasal epithelial barrier in vitro, as follows.

[0181] RPMI2650 cells were implanted in the upper chamber of the Transwell insert chamber (0.4 μm pore size, polycarbonate membrane) at a density of 2×10 5 / 1.13 cm 2 The outside medium needs to be changed every day, and the medium in the upper chamber is removed on the 4th day. Culture for 7~14 days, during which the intercellular resistance is detected every day, and when it reaches 700 Ω·cm 2 on the 14th day, it can be used for the next experiment. Wash three times with PBS, and add 500 μL of 1% BSA-containing HBSS balanced salt solution to the outside. In 200 μL of 1% BSA-containing HBSS balanced salt solution, MF 25 PEI / FITC-HA, MF50 PEI / FITC-HA, F 25 PEI / FITC-HA, F 50 PEI / FITC-HA, PEI / FITC-HA and FITC-HA were added to the upper chamber (2 μg HA / well, n = 3), and the fluorescence intensity of the outer medium was detected by fluorescence spectroscopy (λex= 488 nm, λem= 525 nm) after 6 h.

[0182] The calculation method is as follows:

[0183] The apparent permeability coefficient Papp= Q / Act;

[0184] Wherein, Q: the permeation amount of antigen (ng); A: the diffusion area of cell monolayer (cm 2 ); c: the initial concentration of antigen at the top end (ng / cm 3 ); t: transport time (s).

[0185] The model diagram is shown in Figure 16 , and the results are shown in Figure 16 B. Due to the reduction of surface energy after fluorination, MFPEI / HA shows significantly improved apparent permeability, and MF 25 PEI / FITC-HA and MF 50 PEI / FITC-HA is 2 times and 2.4 times higher than unfluorinated PEI. Both of them have superior mucosal penetration ability, but as an adjuvant MF 25 PEI has stronger ability to promote cell uptake and stimulate BMDCs maturation. 50 PEI has stronger ability to promote cell uptake and stimulate BMDCs maturation.

[0186] (II) In vivo mucosal penetration level

[0187] (1) MF 25 PEI prolongs the detection of HA nasal mucosa retention

[0188] The retention of antigen at the nasal site and the ability to penetrate the nasal mucosa after intranasal immunization is crucial for stimulating a strong immune response. The inventors prepared MF 25 PEI / Cy5.5-HA nanovaccine to evaluate its penetration at the nasal mucosa, which is described as follows.

[0189] Mice were intranasally inoculated with Cy5.5-HA, MF 25 PEI / Cy5.5-HA (5 μg HA / each, n = 3). The fluorescence intensity of the outer medium was detected by fluorescence spectroscopy (λex= 488 nm, λem= 525 nm) after 6 h. ®Fluorescence imaging of the nasal cavity in mice was performed at different time points (3 h, 6 h, 9 h, 12 h, 24 h, 48 h) by Lumina III, PerkinElmer, USA. The results are as follows: Figure 17 As shown, the fluorescence signal in the Cy5.5-HA group gradually weakened and almost disappeared by h9, which may be due to ciliary movement, mucus renewal and secretion, and the natural clearance mechanism of the nasal cavity. It is worth noting that MF... 25 The fluorescence signal of the PEI / Cy5.5-HA group remained strong at 9 h and persisted until 48 h, indicating that MF 25 PEI significantly improved the retention effect of HA in the nasal cavity.

[0190] (2) MF 25 Detection of PEI promoting HA penetration into the nasal mucosa

[0191] To further evaluate MF 25 The inventors prepared MF to enhance the nasal mucosa penetration ability of PEI. 25 For specific procedures regarding the PEI / FITC-HA nano-vaccine, please refer to the following.

[0192] After Cy5.5-HA, PEI / Cy5.5-HA, MF 25 Mice were sacrificed 3 hours after intranasal immunization with PEI / Cy5.5-HA. Nasal tissue was collected, embedded in OTC containers, frozen, sectioned, stained with DAPI, and observed under CLSM. Results are as follows: Figure 18 As shown, the fluorescence of the FITC-HA group almost disappeared on the mucosal surface, while the PEI / FITC-HA group showed weak fluorescence on the nasal mucosal surface. This may be because the positive charge of PEI promotes mucosal adhesion. 25 The PEI / FITC-HA group exhibited the strongest fluorescence signal and penetrated completely into the nasal submucosa. This indicates that the grafting of fluoroalkane and mannose-PEG-COOH enhanced its lipophilicity, effectively avoiding mucosal clearance and mucosal barrier obstruction, and promoting MF (fluid-mediated nasal mucosa). 25 Effective penetration of PEI / FITC-HA into the submucosa.

[0193] Example 3: Investigation into the immune response mechanism of nano-vaccines

[0194] Based on the aforementioned experimental tests, in order to accurately evaluate the immune response mechanism of the nanovaccine prepared in this application, the inventors conducted further tests, the specific process and results of which are briefly described below.

[0195] Put HA, MF 50 PEI / HA and MF 25Mice were immunized intranasally with PEI / HA (10 μg HA / mouse, n = 3). After 3 h, single-cell suspensions were collected from the nasal cavity, stained with anti-TLR4-PE, anti-CD11c-APC, anti-CD64-FITC and anti-CD11b-APC at 4 ℃ for 30 min, washed with PBS and analyzed by flow cytometry.

[0196] DC2.4 cells were seeded in 24-well plates (1×10⁻⁶ cells per well). 5 (per well / hole). Add HA, MF 50 PEI / HA, MF 25 Cells were incubated with PEI / HA for 12 h (2 μg HA / well). Anti-TLR4-PE staining was performed at 4 °C for 30 min, followed by flow cytometry analysis. Cells were lysed with RIPA and centrifuged at 12000 r for 20 min at 4 °C. The supernatant was then collected for Western blotting.

[0197] The results show MF 25 TLR4 in PEI / HA group + The proportion of DC cells was the highest, nearly 8.7 times higher than that of the HA group and higher than that of MF. 50 The PEI / HA group was 3.4 times higher ( Figure 19 In A), macrophages also showed the same trend ( Figure 19 (B) indicates MF 25 PEI / HA can effectively activate the TLR4 pathway in nasal cells. To further confirm MF 25 PEI / HA activates the TLR4 pathway, and in vitro, DC2.4 cells are inoculated with MF. 25 PEI / HA, MF 50 PEI / HA and HA were co-cultured for 12 h, followed by Western blot analysis. Results showed that MF... 25 PEI / HA group activates intracellular TLR4 / NF-κB pathway ( Figure 20 ).

[0198] In addition, HA and MF are used. 50 PEI / HA and MF 25 CCL2 secretion was detected in PEI / HA (10 μg / mouse, n = 3) mice immunized via the nose. One group was immunized with MF. 25PEI / HA 1 h before intraperitoneal injection of TLR4 pathway inhibitor, nasal tissues were collected 3 h after immunization for immunofluorescence staining and qRT-PCR analysis. The nasal cavity tissues were homogenized with Trizol reagent, and RNA was isolated by adding chloroform. RNA was precipitated by adding isopropanol, and then washed with 75% ethanol. Finally, the RNA was resuspended in DEPC water. RNA was reverse transcribed into cDNA using the HiScript II first strand cDNA synthesis kit. qRT-PCR was performed using AceQ Universal SYBR qPCR Master Mix and primers to analyze the expression level of CCL2 relative to the internal control gene GAPDH. QRT-PCR analysis showed that the expression level of CCL2 in the MF 25 PEI / HA group was significantly increased Figure 21 . In addition, nasal cavity tissues were collected for immunofluorescence staining for microscopic observation. The results, as shown in Figure 22 , MF 25 PEI / HA significantly enhanced CCL2 secretion, showing a more extensive distribution of green fluorescent signals in the nasal cavity tissues. In contrast, if the mice were pretreated with TLR4 inhibitor TAK-242 before immunization with MF 25 PEI / HA, the green fluorescent signal was significantly reduced.

[0199] NALT located in the submucosa is the main site of immune cell aggregation. To evaluate whether MF 25 PEI / HA would recruit more pre-DCs to the induction site, intranasal immunization was performed 24 h in advance, with one group receiving intraperitoneal injection of CCL2 neutralizing antibody (10 mg / kg) for three consecutive days before immunization. Single cell suspension of NALT was collected, incubated with anti-CD11c-APC, anti-CD11b-PE / Cy5, anti-CD135-PE and anti-CD43-FITC at 4°C for 30 min, washed with PBS three times, and detected for pre-DCs levels by flow cytometry. The results, as shown in Figure 23 , compared with the HA group and the MF 50 PEI / HA group, the number of pre-DCs in the MF 25 PEI / HA group increased by 15.7 times and 2.4 times, respectively. When the CCL2 antibody was pre-neutralized, the level of pre-DCs decreased by 4.9 times, indicating that the recruitment of pre-DCs in the MF 25 PEI / HA group was significantly enhanced.

[0200] Mice were intranasally immunized to monitor the dynamic changes of cDC network at the nasal site. HA, MF 50 PEI / HA and MF 25PEI / HA (10 pg per mouse, n = 3) immunized mice intranasally, and collected the single-cell suspension of the nasal cavity tissue at 0 h, 24 h, 48 h, incubated with anti-CD64-FITC, anti-CD11c-APC, anti-MHC-II-PE at 4 °C for 30 min, washed with PBS for three times, and detected the level of cDC cells by flow cytometry. The results are shown in Figure 24 As shown in the figure, 24 h after immunization, the total number of cDCs showed a downward trend, which may be due to the migration of cDCs to lymph nodes after uptake of antigens. Among them, the MF 25 The PEI / HA group showed the smallest decrease and recovered to the steady state level first within 48 h. Although the MF 50 Both PEI / HA and HA groups showed different degrees of recovery, but the recovery rate was relatively slow, and reached 80.7% and 61.7% recovery rates at 48 h, respectively.

[0201] Example 4: Evaluation of the immune response effect of the nano vaccine

[0202] On the basis of the foregoing experimental detection, in order to accurately evaluate the immune response effect of the nano vaccine prepared in the present application, the inventors further carried out animal experiments, and the immune flow chart is as shown in Figure 25 The specific process and results are briefly introduced as follows.

[0203] (I) MF 25 PEI / HA enhances mucosal immune response

[0204] Female BALB / c mice (6-8 weeks old) were randomly divided into 5 groups, and were intranasally inoculated with PBS, HA, MF59+HA, MF 25 PEI / HA, MF 50 PEI / HA (10 pg HA per mouse, n = 6). The body weight of the mice was recorded every 4 days. Tail tip blood was collected on days 14, 28, 42, 60, 90, and 120, and centrifuged (4 °C, 2000 rpm, 5 min) to collect serum. On day 42, the nasal lavage fluid, BALF, and cervical lymph nodes and spleen were collected for further analysis. On day 60, the cervical lymph nodes and spleen were collected for further analysis.

[0205] The nasal lavage fluid, BALF, and serum were collected on day 42, and the IgA titer was detected by ELISA. The experimental results showed that the MF 25 The IgA titer in the nasal lavage fluid of the PEI / HA group was increased by 98 times compared with the HA group, and was increased by 5.4 times compared with the MF 50 The PEI / HA group was increased by 5.4 times compared with the MF59+HA group (P < 0.05) Figure 26A). Similar trends were also observed in BALF and serum, MF 25 The PEI / HA group had the highest antibody titers, with serum and BALF IgA antibody titers being 6.4 and 3.2 times higher than the MF59+HA group, respectively. Figure 26 B in Figure 26 (C in the middle).

[0206] T cells can coordinate adaptive immunity. CD8 + T cells play a crucial role in recognizing and eliminating infected cells, while CD4... + T cells recognize antigens and differentiate into various helper subsets to regulate immune responses. In particular, follicular helper T cells (Tfh) play a crucial role in humoral immunity, promoting the formation of germinal centers and the affinity maturation of B cells, thereby producing high-affinity antibodies.

[0207] To assess T cell differentiation and activation, cervical lymph nodes were collected on day 42 and processed into single-cell suspensions for flow cytometry analysis. Results are as follows: Figure 27 As shown, MF 25 CD4 in PEI / HA group + and CD8 + The proportion of T cells was the highest, 1.3–5.7 times and 1.2–3.5 times higher than other groups, respectively. PD-1 + CXCR5 + Tfh cells in MF 25 The proportions in the PEI / HA group were higher than those in the HA group, MF59+HA group, and MF group, respectively. 50 The PEI / HA group had 11.1 times, 4.7 times, and 5 times higher levels, respectively. Figure 28 A in the text indicates that MF 25 PEI / HA effectively induced Tfh cell activation, potentially promoting B cell maturation. Consistent with the above results, in MF... 25 IgD was found in PEI / HA + CD19 + The proportion of B cells increased by 1.5 to 5.9 times compared to other control groups. Figure 28 (B in the text), which indicates that MF 25 PEI / HA helps generate a wide range of antibodies to neutralize the virus. MF was detected by ELISA of cytokine (IL-6, IL-12, TNF-α, and IFN-γ) levels in cervical lymph nodes. 25 The PEI / HA group had the highest levels ( Figure 29 In summary, these results indicate that MF... 25 PEI / HA can effectively trigger a mucosal immune response, thereby providing a protective immune environment in the respiratory tract.

[0208] (2) MF 25 PEI / HA enhances the systemic immune response

[0209] To assess humoral response, spleen cells were collected from immunized mice on day 42 and analyzed by flow cytometry. Figure 30 As shown in A, MF 25 IgD in PEI / HA + CD19 + The proportion of B cells (30.2%) was significantly higher than that of HA (10.8%), MF59+HA (21.7%), and MF59+HA (21.7%). 50 PEI / HA (22.4%), indicating MF 25 PEI / HA showed a stronger ability to promote B cell activation. Serum ha-specific IgG titers were then further measured using ELISA. Compared to other control groups, MF... 25 The titer of HA-specific IgG in PEI / HA was significantly increased, reaching its peak 42 days after the initial immunization. Figure 30 (B in the text). Among them, MF 25 The HA-specific IgG titer of PEI / HA was 3.1–147 times higher than that of other controls, indicating that MF 25 PEI / HA can effectively promote IgG production by stimulating B cell activation. Furthermore, serum was collected on day 42 to measure hemagglutination inhibition titer (HAI), a key indicator of influenza vaccine efficacy; an HAI titer greater than 40 is considered effective. Results showed that, compared to other groups, MF... 25 The HAI titer was significantly higher in the PEI / HA group. Figure 30 The C values ​​(in the HA and MF59+HA groups) were 70 and 3.7 times higher, respectively, indicating that MF59+HA... 25 PEI / HA can induce protective humoral immunity.

[0210] To assess T-cell-mediated immune responses, collected spleen cells were restimulated with the same antigen, and a spleen cell proliferation assay was performed. Figure 30 As shown in D, MF 25 The proliferation rate of spleen cells in the PEI / HA group was 1.3–4.3 times that of other groups. Furthermore, ELISA results showed that MF… 25 The PEI / HA group showed significantly elevated levels of IL-12, IL-6, TNF-α, and IFN-γ, while other control groups did not show significant increases, indicating that MF... 25 PEI / HA exhibited a stronger ability to promote antigen-specific T cell proliferation. Consistent with the above findings, flow cytometry analysis also indicated that MF... 25 CD3 in PEI / HA + CD4 +and CD3 + CD8 + The proportion of T cells was 1.2 to 10.5 times higher than in other control groups. Figure 31 (A and B in the original text). In summary, these results indicate that MF... 25 PEI / HA can effectively trigger a strong humoral and cellular immune response, thereby achieving comprehensive systemic immunity.

[0211] (III) MF 25 PEI / HA induces long-term immune responses

[0212] The formation of long-term immune memory is essential for vaccine-induced adaptive immunity because it provides durable protection against re-challenge by memory B cells and T cells through rapid recall responses. To assess MF... 25 The persistence of PEI / HA-induced humoral immune responses, according to Figure 25 The illustrated protocol was used to immunize mice. On day 60, memory B cells and T cells in the cervical lymph nodes and spleen tissue were analyzed by flow cytometry. Figure 32 As shown, compared with HA and MF59+HA, MF in cervical lymph nodes 25 PEI / HA group memory B cells (CD3) - B220 + CD38 + CD138 + The proportions of CD3 in the spleen increased significantly, by 13.3 times and 3.9 times, respectively. Furthermore, the proportions of CD3 in the spleen... - B220 + CD38 + CD138 + Memory B cells are still in MF 25 The highest proportion was found in the PEI / HA group, indicating that MF 25 PEI / HA assists in the activation of memory B cells. Additionally, serum antibody titers were continuously monitored at different time points. Excitingly, MF... 25 The PEI / HA-induced HA-specific IgG titer was significantly higher than that in other groups, decreased slightly over time, and maintained a high antibody titer for up to 120 days. Figure 33 (A through C in the original text). Furthermore, on day 120, MF... 25 The HA-specific IgG titer in the PEI / HA group was still 67 times higher than that in the HA group, indicating that MF 25 PEI / HA elicited durable and robust humoral immunity. Additionally, memory T cells, another key component of long-term adaptive immunity, were subsequently analyzed. Consistent with the above findings, MF... 25 CD3 in PEI / HA + CD8+ CD44 + CD62L + The proportion of memory T cells was 1.2-3.3 times higher than that of other control groups Figure 34 ). These results collectively indicate that MF 25 PEI / HA can effectively induce robust and durable humoral and cellular immune responses.

[0213] (IV) MF 25 PEI / HA induced immune protection against H1N1 influenza virus challenge

[0214] To evaluate the protective effect of MF 25 PEI / HA, mice were intranasally inoculated with MF 25 PEI / HA of different formulations, and then challenged with H1N1 PR8 virus (1 x 10 6 TCID 50 ) two weeks after the last immunization Figure 35 ). PBS-immunized mice showed significant body weight loss, reduced survival rate, and significantly increased clinical scores after virus challenge (A-C in Figure 36 ). In contrast, MF 25 PEI / HA provided complete protection against PR8 H1N1 virus infection, resulting in 100% survival, slight and reversible body weight loss, and only showing mild clinical scores. In addition, lung tissues of mice were collected 5 days after infection, and imaging and analysis were performed by lung index scoring and H&E staining. The results showed that PBS-immunized mice showed the highest lung index and severe lung pathology, including edema, hemorrhage, inflammatory cell infiltration, and alveolar wall thickening. In contrast, MF 25 PEI / HA immunization can significantly reduce these pathological changes, resulting in the lowest lung index and significantly reduced pathological scores in all groups Figures 37-39 ). Consistent with the above findings, lung virus titers of MF 25 PEI / HA were significantly reduced by 2-fold and 1.4-fold compared with HA and MF59+HA, respectively Figure 39 ). In summary, these results indicate that MF 25 PEI / HA intranasal immunization can provide effective immune protection against influenza virus infection.

[0215] Example 5: Safety evaluation of nanovaccine

[0216] On the basis of the foregoing experimental detection, the inventors evaluated the safety of the nanoadjuvant. The inventors detected the cytotoxicity of the nanoadjuvant, the body weight changes of mice during immunization, HE sections of major organs after immunization, and blood biochemical analysis after immunization, and the specific process and results are briefly introduced as follows.

[0217] DC2.4 cells were seeded in 96-well plates (1 x 10 4 cells / well) and incubated overnight. Different concentrations (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL) of MF 25 PEI, MF 50 PEI were added to each well and incubated for 24 h. Cell viability was determined by standard MTT assay.

[0218] DC2.4 cells were incubated with different concentrations of nano-vaccine for 24 h, and cell viability was determined by MTT assay. The results showed that MF 25 PEI / HA maintained more than 90% cell viability at a concentration of 20 μg / mL after 24 h of treatment, indicating that MF 25 PEI / HA had good biocompatibility. Figure 40

[0219] In addition, 6-8-week-old BALB / c mice were immunized intranasally with the aforementioned different components (10 μg HA per mouse, n = 3). On day 14 after immunization, the mice were sacrificed, and the heart, liver, spleen, lung, and kidney were collected. The collected organs were paraffin-embedded, sectioned, and stained with H&E for evaluation of toxic effects. Blood samples were collected for detection of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CR), and blood urea nitrogen (BUN) levels. The results showed that MF 25 PEI / HA immunized mice had stable weight gain during the entire immunization period Figure 41 (A), and there were no obvious pathological changes in the major organs Figure 41 (B). Hematological analysis further confirmed that MF 25 PEI / HA had no statistically significant difference from PBS in key biochemical indicators such as BUN, CR, AST, and ALT Figure 41 (C). These results collectively demonstrate that MF 25 PEI / HA has good in vivo safety.

[0220] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered within the scope of protection of the present application.​

Claims

1. A polymer, characterized in that, As shown in equation (I): （I）； Where n is the degree of aggregation.

2. The polymer according to claim 1, characterized in that, n is any integer from 35 to 58, x is any integer from 10 to 250, m is any integer from 1 to 6, and R is selected from: (1) Mannose residues, glucose residues, galactose residues, fucose residues, xylose residues, rhamnose residues, or their D-, L-, DL-configurations; (2) Derivatives in which the C1–C6 hydroxyl groups of the above residues are replaced by –H, –CH3, –COCH3, –SO3H, –PO3H, –CH2COOH, –CH2CH2NH2; (3) The aforementioned residues or derivatives are linked to the linker arm by an O-glycoside, S-glycoside, N-glycoside or carboxylic acid ester bond to form a group; (4) Its pharmaceutically acceptable salts, stereoisomers or solvates.

3. The method for preparing the polymer according to claim 1 or 2, characterized in that, include: Grafting tridecylfluoroheptyl ethylene oxide with polyethyleneimine, and then grafting the grafted product with a mannose derivative, yields the polymer. The molar ratio of the reactants of the tridecylfluoroheptyl ethylene oxide and the polyethyleneimine is (25~50):1; The molar ratio of the reactants of the mannose derivative and the polyethyleneimine is (1~2):(1~2).

4. The preparation method according to claim 3, characterized in that, The polyethyleneimine is PEI. 1.8K The mannose derivative is Mannose-PEG-COOH.

5. The use of the polymer as described in claim 1 or 2 in the preparation of mucosal delivery adjuvants.

6. A mucosal delivery adjuvant, characterized in that, It includes the polymer described in claim 1 or 2.

7. The use of the mucosal delivery adjuvant as described in claim 6 in the preparation of biological products for the prevention and treatment of respiratory tract infections.

8. The application as described in claim 7, characterized in that, include: The respiratory infections include influenza; and / or The prevention and control are achieved through any of the following: (I) By activating the TLR4 pathway of conventional dendritic cells in the nasal cavity, CCL2 secretion is promoted, precursor dendritic cells are recruited to the nasal cavity, and the network homeostasis of conventional dendritic cells in the nasal cavity is maintained. (II) By targeting conventional dendritic cells to increase antigen uptake, conventional dendritic cells are stimulated to mature, thus inducing a high level of immune response; The high level of immune response includes at least one of the following: (i) Increased IgA secretion levels; (ii) Increased IgG secretion levels; (iii) Enhanced B cell activation; (iv) Enhanced T cell activation; (v) Enhanced Tfh cell activation; (vi) Enhanced differentiation of memory B cells; (vii) Enhanced differentiation of memory T cells.

9. A biological product, characterized in that, Includes the mucosal delivery adjuvant and hemagglutinin protein as described in claim 6.

10. The biological product as described in claim 8, characterized in that, The preparation method includes: mixing the mucosal delivery adjuvant of claim 6 and hemagglutinin protein at a mass ratio of 10:1, and then combining them with acceptable excipients or adjuvants to obtain the biological product.

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