Influenza virus nano vaccine as well as preparation method and application thereof

Abstract

The invention discloses an influenza virus nano vaccine as well as a preparation method and application thereof. The invention relates to the field of biomedicine, and provides an influenza virus nano vaccine, which comprises nano particles and an STING agonist adsorbed on the surfaces of the nano particles, the nanoparticle comprises a polysaccharide antigen conjugate and a dendritic macromolecule, wherein the polysaccharide antigen conjugate is formed by coupling beta-glucan and influenza virus antigen protein, and the dendritic macromolecule and the polysaccharide antigen conjugate are adsorbed together. The immune response induced by the vaccine is higher than that of monomer immunogen protein without the adjuvant, body fluid, cell and mucosa immune response can be remarkably activated, lethal attack of an organism to H1N1 and H5N1 viruses can be effectively protected through nasal spray inoculation, and the nano vaccine can stimulate cross immune response and immune protection effect to different subtype influenza viruses.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an influenza virus nanovaccine, its preparation method, and its application. Background Technology

[0002] Influenza virus is the main pathogen causing influenza (flu) in humans. It belongs to the Orthomyxoviridae family and is mainly divided into types A, B, and C. Influenza caused by types A (H1N1 and H3N2) and B (Yamagata, BY, and Victoria, BV) viruses poses a significant threat to human health, imposing a huge public health and economic burden. Vaccination is the most effective measure to prevent influenza. Currently, the global research and production of influenza vaccines mainly uses chicken embryo technology, but this still has many limitations, such as a long production cycle (approximately 6 months), difficulty in quickly responding to antigenic drift / transformation, viral adaptive mutations in chicken embryos, and limited use by people with egg allergies. Studies have shown that the protection rate of chicken embryo vaccines is 40%-60%. Globally available influenza vaccines include inactivated influenza vaccines (IIV), live attenuated influenza vaccines (LAIV), and recombinant influenza vaccines (RIV). However, these vaccines have significant shortcomings. For example, IIV and LAIV require manipulation and purification of live viruses, and their processes are complex and cannot quickly address the challenges posed by influenza virus mutations. Some RIV vaccines require adjuvants to function. To effectively overcome the challenges posed by influenza virus mutations and prolong the protective effect of vaccines, it is necessary to establish new influenza vaccine research and development platforms, such as broad-spectrum vaccines with novel adjuvants combined with conserved influenza antigens, mRNA vaccines, and viral vector vaccines. Recombinant protein vaccines are vaccines prepared by expressing immunogens in vitro using expression systems such as bacteria, yeast, plants, insects, or mammalian cells, followed by separation and purification processes to obtain the corresponding proteins, and then combining them with appropriate adjuvants. In addition to direct combination with adjuvants, protein subunit vaccines can also be prepared as nanoparticle vaccines or virus-like particle vaccines, thereby enhancing the display and delivery efficiency of immunogens.

[0003] Nanoparticle vaccines are vaccine platforms designed based on nanotechnology. By loading antigens, adjuvants, or immunomodulatory molecules into nanocarriers, they can significantly improve the immunogenicity and safety of vaccines. By mimicking pathogen activation of antigen-presenting cells, they enhance the effective delivery and presentation of antigen molecules, thereby inducing a highly efficient immune response. Nanoparticles can serve as alternative mucosal adjuvants, providing sustained protection for antigen delivery to the nasal cavity, thus enhancing the immunogenicity of the antigen. In recent years, nanoparticle-based formulations have been widely used in mucosal immunotherapies.

[0004] β-glucan is widely found in the cell walls of fungi, yeast, and some bacteria. It can serve as a pathogen-associated molecular pattern (PAMP), binding to pattern recognition receptors (PRRs) such as Dectin-1, TLR2 / 6, and CR3. This activates the NF-κB and MAPK pathways, enhancing antigen presentation and dendritic cell maturation, promoting T cell activation, activating memory B cells and T cells, prolonging vaccine efficacy, and stimulating Th1, Th17, and cytotoxic T cell responses, thus improving antibody neutralization capacity. Dendritic macromolecules (PAMAMs) are highly branched nanomaterials with numerous amino functional groups on their surface. They can efficiently bind antigens through electrostatic interactions or covalent bonds, forming stable nanocomplexes and protecting antigens from degradation. Furthermore, PAMAM's "proton sponge effect" buffers the acidic environment of lysosomes, promoting antigen escape into the cytoplasm, enhancing MHC-I antigen presentation, and activating CD8+. + T cells simultaneously carry antigens and immunostimulatory molecules, synergistically activating multiple signaling pathways. 2′,3′-cGAMP, as a STING agonist, can initiate the expression of type I interferon and pro-inflammatory cytokines, enhance the activation and cross-presentation capacity of antigen-presenting cells, and act as a mucosal adjuvant. Summary of the Invention

[0005] The purpose of this invention is to provide an influenza virus nanovaccine based on β-glucan, dendritic macromolecules and 2′,3′-cGAMP and its preparation method.

[0006] In one aspect, the present invention claims protection for an influenza virus nanovaccine.

[0007] The influenza virus nanovaccine claimed in this invention may include nanoparticles and a STING agonist adsorbed on the surface of the nanoparticles. The nanoparticles may include a polysaccharide antigen conjugate formed by coupling β-glucan and influenza virus antigen protein, as well as dendritic macromolecules adsorbed together with the polysaccharide antigen conjugate. Furthermore, the dendritic macromolecule may be PAMAM. Even further, the PAMAM may be an ethylenediamine core, generation 0 (GO).

[0008] Furthermore, the STING agonist is 2′,3′-cGAMP.

[0009] Further, the influenza virus antigen protein is an influenza virus HA protein. In some embodiments, the influenza virus HA protein is an HA protein derived from type A influenza virus or type B influenza virus. The type A influenza virus may be an H1-H16 subtype influenza virus; the type B influenza virus may be a Victoria lineage Bv or a Yamagata lineage By influenza virus. In one embodiment of the present invention, the influenza virus HA protein is an HA protein derived from H1N1 influenza virus. Furthermore, the amino acid sequence of the HA protein derived from H1N1 influenza virus may be as shown in SEQ ID NO:1.

[0010] Furthermore, the influenza virus antigen protein may also be other conserved protective antigens besides the influenza virus HA protein, such as NA, M2e, NP, etc.

[0011] Furthermore, the polysaccharide antigen conjugate can be obtained by first covalently coupling β-glucan with octaglutamic acid to obtain a covalent conjugate, and then covalently binding the covalent conjugate to the influenza virus HA protein.

[0012] Furthermore, the dendritic macromolecules electrostatically adsorb the polysaccharide antigen conjugate.

[0013] Furthermore, the STING agonist is electrostatically adsorbed onto the surface of the nanoparticles.

[0014] Furthermore, the influenza virus nanovaccine can be prepared by a method including the following steps: (A1) β-glucan was mixed with sodium periodate and dissolved in a buffer solution for reaction. After dialysis, β-glucan with aldehyde groups was obtained.

[0015] (A2) The aldehyde-containing β-glucan obtained in (A1), N -(2-aminoethyl)maleimide and sodium cyanoborohydride were mixed and dissolved in a buffer solution and reacted. After dialysis, β-glucan with maleimide groups was obtained. (A3) The maleimide-containing β-glucan and octaglutamic acid obtained in (A2) are mixed and dissolved in a buffer solution to prepare a negatively charged β-glucan and octaglutamic acid conjugate, denoted as β-glucan-8E conjugate; the amino acid sequence of octaglutamic acid is EEEEEEEEC.

[0016] (A4) The influenza virus HA protein and 2-iminothion were mixed and dissolved in a buffer solution for reaction, and the HA protein with thiol groups was obtained after dialysis.

[0017] (A5) The β-glucan-8E conjugate obtained in (A3) and the thiol-containing HA protein obtained in (A4) are mixed and reacted to prepare a polysaccharide antigen conjugate.

[0018] (A6) The polysaccharide antigen conjugate obtained in (A5) is mixed and reacted with the dendritic macromolecule to obtain the nanoparticles.

[0019] (A7) The nanoparticles obtained in (A6) are mixed and reacted with the STING agonist to obtain the nanovaccine.

[0020] In step (A1), the concentration of β-glucan in the reaction system can be 4 mg / mL, and the concentration of sodium periodate in the reaction system can be 20 mM. The buffer solution can be sodium acetate buffer (e.g., 20 mM sodium acetate buffer) with a pH of 5.6. The reaction conditions can be 20-30°C (e.g., 25°C) in the dark for 20-30 minutes (e.g., 20 minutes). The dialysate used for the dialysis can be phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The molecular weight cutoff of the dialysis bag used for the dialysis can be 2 kDa.

[0021] In (A2), the aldehyde-containing β-glucan, N The mass ratio of -(2-aminoethyl)maleimide to sodium cyanoborohydride can be 1:(1-2):(1-5) (e.g., 1:1.5:2). The buffer solution can be a phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The reaction temperature can be 1-5°C (e.g., 4°C), and the time can be 8-16 h (e.g., 16 h). The dialysate used for the dialysis can be a phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The molecular weight cutoff of the dialysis bag used for the dialysis can be 2 kDa.

[0022] In (A3), the mass ratio of the maleimide-containing β-glucan to the octameric glutamic acid can be 10:(1-2) (e.g., 10:1). The buffer solution can be a phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The reaction temperature can be 1-5°C (e.g., 4°C), and the reaction time can be 1-3 h (e.g., 2 h).

[0023] In step (A4), the molar ratio of the influenza virus HA protein to 2-iminothione can be 1:(20-60) (e.g., 1:40). The buffer solution can be a phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The reaction temperature can be 1-5°C (e.g., 4°C), and the time can be 8-16 h (e.g., 16 h). The dialysate used for the dialysis can be a phosphate buffer (e.g., 20 mM phosphate buffer) with a pH of 7.4. The molecular weight cutoff of the dialysis bag used for the dialysis can be 7 kDa.

[0024] In step (A5), the mass ratio of the β-glucan-8E conjugate to the thiol-containing HA protein can be (1-1.2):1 (e.g., 1:1). The pH of the reaction can be 7.4 (e.g., the reaction can be carried out in phosphate buffer (pH 7.4), the temperature can be 1-5°C (e.g., 4°C), and the time can be 8-16 h (e.g., 16 h).

[0025] In step (A6), the molar ratio of the polysaccharide antigen conjugate to the dendritic macromolecule can be (50-10):1 (e.g., 15:1). The pH of the reaction can be 7.4 (e.g., the reaction can be carried out in phosphate buffer (pH 7.4), the temperature can be 1-5°C (e.g., 4°C), and the time can be 8-24 h (e.g., 16 h).

[0026] In (A7), the mass ratio of the nanoparticles to the STING agonist can be 10:(1-2) (e.g., 10:1). The pH of the reaction can be 7.4 (e.g., the reaction can be carried out in phosphate buffer (pH 7.4), the temperature can be 1-5°C (e.g., 4°C), and the time can be 8-16 h (e.g., 8 h).

[0027] In one embodiment of the present invention, the β-glucan is β-D-glucan derived from barley.

[0028] Secondly, the present invention claims protection for a method for preparing an influenza virus nanovaccine.

[0029] The method for preparing an influenza virus nanovaccine claimed in this invention may include (A1) to (A7) described in the first aspect above.

[0030] Thirdly, the present invention claims protection for the use of the influenza virus nanovaccine described in the first aspect above in the preparation of products for the prevention of influenza.

[0031] The influenza virus may be type A or type B. Further, the influenza virus may be H1N1 or H5N1. The type A influenza virus may be an H1-H16 subtype; the type B influenza virus may be a Victoria lineage Bv or a Yamagata lineage By influenza virus.

[0032] In one embodiment of the present invention, the influenza virus is specifically the H1N1 influenza virus strain A / Beijing / 501 / 2009(H1N1)(BJ501) and the H5N1 influenza virus strain A / Ostrich / SuZhou / 097 / 03.

[0033] Experiments have shown that the influenza virus HA protein is selected as the antigen in this invention, and β-glucan is used as an adjuvant to couple with negatively charged octaglutamate. β-glucan and HA protein are coupled to form a polysaccharide antigen conjugate, which adsorbs dendritic macromolecules to form nanoparticles. The STING agonist 2′,3′-cGAMP is adsorbed onto the surface of the nanoparticles to form a nanovaccine, which induces a high level of protective immune response in the body and has mucosal protective efficacy.

[0034] Specifically, the technical advantages of this invention are reflected in the following aspects: (1) This invention uses dendritic macromolecule PAMAM as a delivery platform, electrostatically adsorbs β-glucan-HA protein conjugate and mucosal adjuvant 2′,3′-cGAMP, and constructs antigen-adjuvant nanoparticles, achieving simultaneous delivery of antigen and adjuvant. The immune response induced by the nanovaccine obtained by this method is higher than that of monomeric immunogen proteins. Nasal spray inoculation can effectively protect mice from lethal challenges to H1N1 and H5N1 viruses, and the nanovaccine can stimulate cross-immune responses.

[0035] (2) The influenza virus nanovaccine of the present invention has HA protein coupled with polysaccharide adjuvant β-glucan, and adsorbs mucosal adjuvant 2′,3′-cGAMP through dendritic macromolecule PAMAM. The two adjuvants work together to provide comprehensive immune protection of body fluids, cells and mucosa. At the same time, the positive charge on the surface of PAMAM can enhance endocytosis, thereby evading lysosomes or directly penetrating the membrane to deliver antigens to the cytoplasm, further activating the cellular response and enhancing the depth and breadth of the immune response.

[0036] (3) The influenza virus nanovaccine of the present invention can significantly activate humoral, cellular and mucosal immune responses in mice and can exert cross-immune protection. The influenza virus nanovaccine can exert immune protection after being challenged by H1N1 and H5N1 viruses respectively. Attached Figure Description

[0037] Figure 1This is a schematic diagram of the reaction for vaccine preparation. 8E-COOH represents the octameric glutamate polypeptide EEEEEEEEC.

[0038] Figure 2 SDS-PAGE electrophoresis was used to analyze polysaccharide antigen conjugates. Lane 1 was for the standard protein marker; lane 2 for HA protein; and lane 3 for β-glucan-HA conjugate.

[0039] Figure 3 The average particle size, polydispersity index (PDI), and zeta potential of the nanovaccine were analyzed using dynamic light scattering. In the diagram, A represents the particle size distribution of the nanovaccine; B represents the zeta potential of the nanovaccine. Nanoparticles labeled "HA-GP" are β-glucan-HA protein nanoparticles with dendritic macromolecule PAMAM as a carrier, and nanovaccines labeled "HA-GP-cG" are nanovaccines after HA-GP adsorbs 2′,3′-cGAMP.

[0040] Figure 4 To analyze the morphology of nanovaccines using transmission electron microscopy.

[0041] Figure 5 Fourier transform infrared spectroscopy analysis of nano-vaccines. Among them, those labeled "HA-GP-cG" represent nano-vaccines after HA-GP adsorbs 2′,3′-cGAMP (i.e., the influenza virus nano-vaccines finally obtained in step one); those labeled "HA-G8E" represent β-glucan-HA conjugates; those labeled "G8E" represent β-glucan-8E conjugates; and those labeled "HA" represent influenza virus serum HA protein.

[0042] Figure 6 This invention relates to the influenza virus nanovaccine that induces humoral immunity in mice. In the figures, A represents the nanovaccine immunization process; B represents the serum IgG titer of mice after the first and second immunizations; C represents the serum IgA titer of mice after the second immunization; D represents the titer of H1N1-specific hemagglutination inhibition antibody in the serum after the second immunization; and E represents the titer of H5N1-specific hemagglutination inhibition antibody in the serum after the second immunization.

[0043] Figure 7 This invention demonstrates how the influenza virus nanovaccine induces cellular immunity in mice. Wherein, A represents the CD4+ concentration in the spleen of mice in each group after secondary immunization. + T cell ratio; B represents the IFNγ concentration in the spleen of mice in each group after secondary immunization. + CD4 + T cell ratio; C represents IL-4 levels in the spleen of mice in each group after secondary immunization. + CD4 + T cell ratio; D represents IL17A levels in the spleen of mice in each group after secondary immunization. + CD4 + T cell ratio; E represents the CD8+ concentration in the spleen of mice in each group after secondary immunization.+ T cell ratio; F represents the IFNγ concentration in the spleen of mice in each group after secondary immunization. + CD8 + T cell ratio; G represents IL-4 levels in the spleen of mice in each group after secondary immunization. + CD8 + T cell ratio.

[0044] Figure 8 This invention relates to the influenza virus nanovaccine that induces mucosal immunity in mice. In this context, A represents the IgG titer in the bronchoalveolar lavage fluid of mice in each group after a second immunization; B represents the IgG titer in the nasal lavage fluid of mice in each group after a second immunization; and C represents the IgA titer in the nasal lavage fluid of mice in each group after a second immunization.

[0045] Figure 9 This invention evaluates the immunoprotective effect of the influenza virus nanovaccine. In the figures, A represents the change in body weight of mice in each group 14 days after H1N1 challenge; B represents the survival rate of mice in each group after H1N1 challenge; C represents the pathological changes in lung tissue of mice in each group on day 5 after H1N1 challenge; D represents the change in body weight of mice in each group within 14 days after H5N1 challenge; E represents the survival rate of mice in each group after H5N1 challenge; and F represents the pathological changes in lung tissue of mice in each group on day 5 after H5N1 challenge.

[0046] Figure 10 This study evaluates the safety of the influenza virus nanovaccine of this invention in mice. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] The H1N1 influenza virus strain A / Beijing / 501 / 2009(H1N1)(BJ501) used in the following examples is described in the article “Wei Wang, et al. Monoclonal antibody against CXCL-10 / IP-10 ameliorates influenza A (H1N1) virus induced acute lung injury. Cell Res. 2013 Apr;23(4):577-80.” Under biosafety conditions, the public can obtain this virus strain from the applicant and it can only be used to repeat the experiments of this invention and may not be used for other purposes.

[0050] The H5N1 influenza virus strain A / Ostrich / SuZhou / 097 / 03 used in the following examples is described in the article “Yang P, et al. Characterization of a highly pathogenic avian influenza H5N1 virus isolated from an ostrich. Biochem Biophys Res Commun. 2010 Jun 11;396(4):973-7.”. Under biosafety conditions, the public can obtain this virus strain from the applicant and it can only be used to repeat the experiments of this invention and may not be used for other purposes.

[0051] The dendritic macromolecule PAMAM used in the following examples is a product of Sigma-Aldrich, catalog number 412368. Its linear molecular formula is [NH2(CH2)2NH2]:(G=O);dendri PAMAM(NH2)4. CAS number: 155773-72-1. Molecular weight: 516.68 g / mol. PubChem chemical code number: 24865805. Its structural formula is as follows:

[0052] The β-glucan used in the following examples is a product of Sigma, catalog number G6513 (β-D-glucan, derived from barley). CAS number: 9041-22-9. PubChem chemical code number: 329799952.

[0053] The 2′,3′-cGAMP used in the following examples is an InvivoGen product, catalog number tlrl-nacga23s.

[0054] The octaglutamic acid used in the following examples was synthesized by Jier Biochemical (Shanghai) Co., Ltd., with the amino acid sequence EEEEEEEEC (SEQ ID NO:2) and a molecular weight of 1154.07 g / mol.

[0055] Example 1: Preparation and Identification of the Influenza Virus Nanovaccine of the Present Invention I. Preparation of an influenza virus nanovaccine based on β-glucan, dendritic macromolecules, and 2′,3′-cGAMP. 1. Obtaining the influenza virus HA protein The antigen selected in this invention is the HA protein derived from the N1H1 influenza virus, whose amino acid sequence is shown in SEQ ID NO:1. Specifically, it is Influenza A H1N1 (A / California / 07 / 2009) Hemagglutinin / HAProtein (His Tag), a product of Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat: 11085-V08B. The SDS-PAGE results of this HA protein are as follows... Figure 2 As shown. All HA proteins used in the following text refer to this HA protein.

[0056] 2. Preparation of influenza virus nanovaccines based on β-glucan, dendritic macromolecules and 2′,3′-cGAMP A schematic diagram of the vaccine preparation reaction is shown below. Figure 1 As shown, the specific process includes the following steps: (1) Prepare 20mM sodium acetate buffer (pH5.6). Weigh β-glucan and sodium periodate separately, and dissolve and mix them in the above "20mM sodium acetate buffer (pH5.6)" at a final concentration of 4mg / mL and 20mM. React at 25°C in the dark for 20 minutes. Dialyze thoroughly to 20mM phosphate buffer (pH7.4) using a dialysis bag with a molecular weight cutoff of 2kDa to obtain β-glucan with aldehyde groups.

[0057] (2) Add β-glucan with aldehyde group, N β-(2-aminoethyl)maleimide and sodium cyanoborohydride were mixed in a mass ratio of 1:1.5:2 in phosphate buffer (pH 7.4) and reacted at 4°C for 16 hours. The mixture was then dialyzed thoroughly into 20 mM phosphate buffer (pH 7.4) using a dialysis bag with a molecular weight cutoff of 2 kDa to obtain β-glucan with maleimide groups. The β-glucan with maleimide groups and octaglutamic acid were then mixed in a mass ratio of 10:1 in phosphate buffer (pH 7.4) and reacted at 4°C for 2 hours to obtain a negatively charged β-glucan-8E conjugate. Note: 8E represents octaglutamic acid.

[0058] (3) Mix HA protein (prepared in the previous step) and 2-iminothione in a molar ratio of 1:40 in phosphate buffer (pH 7.4), react at 4°C for 16 hours, and dialyze thoroughly into 20 mM phosphate buffer (pH 7.4) using a dialysis bag with a molecular weight cutoff of 7 kDa to obtain HA protein with thiol groups.

[0059] (4) The negatively charged β-glucan-8E conjugate with maleimide groups obtained in step (2) and the thiol-containing HA protein obtained in step (3) were mixed at a mass ratio of 1:1 in phosphate buffer (pH 7.4) and reacted at 4°C for 16 hours to obtain a polysaccharide antigen conjugate (denoted as β-glucan-HA conjugate). The β-glucan-HA conjugate and dendritic macromolecules were mixed at a molar ratio of 15:1 in phosphate buffer (pH 7.4) and reacted at 4°C for 16 hours to obtain nanoparticles. Then, the nanoparticles and 2′,3′-cGAMP were mixed at a mass ratio of 10:1 in phosphate buffer (pH 7.4) and reacted at 4°C for 8 hours to obtain an influenza virus nanovaccine.

[0060] II. Structural Characterization of Influenza Virus Nanoparticle Vaccines 1. SDS-PAGE electrophoresis analysis of the coupling between polysaccharides and antigens. Prepare a 10% polyacrylamide gel for electrophoresis, and prepare HA protein (see step 1) and β-glucan-HA conjugate (see step 2, (4)). Add the sample and standard protein marker to the wells in sequence, set the electrophoresis parameters, remove the gel after electrophoresis and stain with Coomassie Brilliant Blue, then replace with destaining solution and destain repeatedly until clear electrophoretic bands appear. Figure 2 As shown, the HA protein is a single band with a molecular weight of approximately 60 kDa, while the molecular weight of the β-glucan-HA conjugate is above 180 kDa, which is significantly larger than the molecular weight of the single HA protein, indicating that the β-glucan and HA protein were successfully conjugated.

[0061] 2. Dynamic light scattering (DLS) analysis of vaccine hydration kinetics: diameter and zeta potential. The concentration of the influenza virus nanovaccine sample obtained in step one was adjusted to 0.2 mg / mL. 60 μL was added to a cuvette, and the nanovaccine particle size and polydispersity index (PDI) were measured using a Zetasizer Nano ZS at room temperature. The average particle size was calculated after three repeated scans. 800 μL of nanoparticles (HA-GP, i.e., β-glucan-HA protein nanoparticles with dendritic macromolecule PAMAM as a carrier prepared in the previous step) and vaccine (HA-GP-cG, i.e., the influenza virus nanovaccine sample formed after HA-GP adsorbs 2′,3′-cGAMP in the previous step) were added to the zeta potential sample cell, and the zeta potential was calculated after three scans at room temperature. Figure 3 As shown in Figure A, the nano-vaccine has a particle size of 113 nm and an average PDI of 0.28. Figure 3 As shown in Figure B, the zeta potential of β-glucan-HA protein nanoparticles (HA-GP) with dendritic macromolecule PAMAM as carrier is 9.37. After adsorbing 2′,3′-cGAMP, the zeta potential of (HA-GP-cG) drops to 6.07, indicating that the nanoparticles successfully adsorb 2′,3′-cGAMP, and the nanovaccine is successfully constructed.

[0062] 3. Transmission electron microscopy can characterize the morphology of nanovaccines. Prepare a 20 mg / mL uranyl acetate solution and filter it through a 0.22 μm filter membrane to obtain a negative staining dye. Prepare a 0.5 mg / mL vaccine sample (i.e., the influenza virus nano-vaccine finally obtained in step one). Pipette 5 μL of the vaccine sample onto a copper grid, let it stand for 1 minute, blot the sample dry with filter paper, wash it twice with an equal volume of deionized water, and stain it with uranyl acetate solution for 1 minute. After blotting off excess liquid with filter paper, dry it thoroughly to prepare the sample for later use. Figure 4 As shown, the vaccine exhibits a solid spherical morphology with a size of approximately 100 nm.

[0063] 4. Infrared spectroscopy analysis of changes in the chemical structure and functional groups of the vaccine before and after conjugation. The HA protein (see step 1), β-glucan-8E conjugate (see step 2, part (2)), β-glucan-HA conjugate (see step 2, part (4)), and nano-vaccine (i.e., the influenza virus nano-vaccine finally obtained in step 1) samples were dialyzed and replaced with deionized water, and then freeze-dried. After mixing a small amount of freeze-dried sample with KBr, the sample was thoroughly ground and pressed into transparent sheets, which were then placed in an FT-IR spectrometer at 4000-400 cm⁻¹. -1 The wavenumber range is scanned, repeated 30 times, and the average value is taken. For example... Figure 5 As shown, β-glucan-8E conjugate (8GE) at 1650 cm⁻¹ -1 (C=O stretching) and 1075cm-1 There is a characteristic transmission peak at (CO vibration), at 2900 cm⁻¹. -1 (OH stretching), 1430 cm -1 (OH vibration) and 900cm -1 The weak transmission peak at (C=O and OH vibrations) indicates that the β-glucan-8E conjugate contains a carboxyl group, and that 8E is successfully coupled with β-glucan. The β-glucan-HA conjugate (HA-8GE) shows a transmission peak at 2400 cm⁻¹. -1 The presence of a characteristic transmission peak at the (SH stretching) position indicates that the thiolized HA protein has been successfully coupled with β-glucan. Compared to the HA protein, both the β-glucan-HA conjugate (HA-G8E) and the nanovaccine (HA-GP-cG) contain the aforementioned transmission peak, indicating that the carboxyl-containing β-glucan has been successfully coupled with the antigen.

[0064] Example 2: Determination of antibody levels induced by the influenza nanovaccine of the present invention Thirty-two 6-8 week old female BALB / c mice were randomly divided into four groups: PBS group, adjuvant group, HA protein group, and vaccine group, with eight mice in each group. The mouse immunization procedure is as follows: Figure 6 As shown in Figure A.

[0065] PBS group: 60 μL of PBS solution was administered intranasally to each mouse.

[0066] HA group: Each mouse was immunized intranasally with 60 μL of HA protein solution, which contained 40 μg of HA protein.

[0067] Vaccine group: Each mouse was immunized intranasally with 60 μL of the influenza nanovaccine solution prepared in step one, which contained 40 μg of HA protein. Adjuvant group: Each mouse was immunized intranasally with 60 μL of adjuvant solution containing the same amounts of β-glucan, PAMAM and 2′,3′-cGAMP as the vaccine group.

[0068] Mice were immunized on days 0 and 28. Blood samples of 200 μL were collected from the orbital sinus on days 0, 28, and 42. Serum was obtained and stored at -80°C for later use. On day 42, mice were challenged with 50 LD of the virus via intranasal instillation. 50 H1N1 (A / Beijing / 501 / 2009(H1N1)(BJ501)) and 10LD 50H5N1 (A / Ostrich / SuZhou / 097 / 03). Anti-HA specific IgG in mouse serum was detected by ELISA. The titer of HA specific IgA antibody in the serum of mice 14 days after the second immunization (i.e., serum taken on day 42) was detected by ELISA. The specific procedures are as follows: Dilute HA protein to 2 μg / ml using ELISA antigen coating buffer (Solepro, C1055), add 100 μl / well to a 96-well plate, and incubate overnight at 4°C; wash the plate 3 times with PBST; add 300 μL of 1% BSA to each well and block at 37°C for 1 h; dilute serum samples with 1% BSA (initial dilution 1:400 or 1:800), perform serial 2-fold dilutions, and add to subsequent wells, incubating at 37°C for 1 h; wash the plate 3 times with PBST; add diluted antibodies: goat anti-mouse IgG (Abcam, ab205719, 1:8000), IgA (Abcam, ab97235, 1:8000), and incubate at 37°C for 45 min; wash the plate 5 times with PBST; add 50 μL of TMB (Aladdin) to each well and incubate at 37°C for 5 min; add 50 μL of TMB to each well. ELISA stop solution (Solepro, C1058); OD readings were obtained using a microplate reader. 450 Values, with the PBS group as the negative counterweight, OD 450 >2.1 times OD 450 Negative wells are positive wells. The hemagglutination inhibition (HI) assay was used to detect H1N1 strain A / Beijing / 501 / 2009(H1N1)(BJ501) and H5N1 strain A / Ostrich / SuZhou / 097 / 03 in the serum of mice 14 days after the second immunization (i.e., serum taken on day 42). The specific operation is as follows: Mix the receptor-destroying enzyme (Japan Biotech, 340016) and the serum to be tested at a ratio of 1:4 and incubate at 37°C for 18 hours; inactivate the treated serum at 56°C for 30 minutes; add 50 μL of the treated serum to be tested to V-type plates A1-A10, add 25 μL of PBS to A11, and add 50 μL of PBS to A12 as a control; use a multichannel pipette to take 25 μL of serum from row A, dilute the serum 2-fold from row A to row H, and discard 25 μL from row H; add 25 μL of antigen-virus solution containing 4 agglutination units to columns A1-H9 and A11, add 25 μL of PBS to column A12, mix well and incubate at room temperature for 20-30 minutes; add 50 μL of 1% chicken erythrocyte suspension (Sbjbio, SBJ-RBC-C001) to each well, mix well, incubate at room temperature for 30 minutes, and observe the results of the erythrocyte agglutination inhibition experiment.

[0069] The results are as follows Figure 6 As shown in Figure B, HA-specific IgG was detectable in the vaccine-treated mice after the first immunization, and the serum HA-specific IgG titer increased significantly after the second immunization. HA-specific IgG was not detected in other groups. Figure 6 As shown in Figure C, compared with the PBS group, the serum HA-specific IgA titer in the vaccine group mice was significantly increased after the second immunization. HA-specific IgA was not detected in the HA group or the adjuvant group. Hemagglutination inhibition assay results showed that, compared with the PBS group, the vaccine group had a significantly increased H1N1-specific hemagglutination inhibition antibody level. There was no significant difference between the HA group and the PBS group. Figure 6 In addition, the H5N1-specific hemagglutination inhibition antibody level was significantly increased in the vaccine group (D). Figure 6 (E). The above results indicate that the influenza virus nanovaccine prepared in this invention can significantly activate the humoral immune response in mice through nasal drop immunization.

[0070] Example 3: Determination of cellular immune response induced by the influenza virus nanovaccine of the present invention Thirty-two 6-8 week old female BALB / c mice were randomly divided into four groups: PBS group, adjuvant group, HA protein group, and vaccine group, with eight mice in each group.

[0071] PBS group: 60 μL of PBS solution was administered intranasally to each mouse.

[0072] HA group: Each mouse was immunized intranasally with 60 μL of HA protein solution, which contained 40 μg of HA protein.

[0073] Vaccine group: Each mouse was immunized intranasally with 60 μL of the influenza nanovaccine solution prepared in step one, which contained 40 μg of HA protein. Adjuvant group: Each mouse was immunized intranasally with 60 μL of adjuvant solution containing the same amounts of β-glucan, PAMAM and 2′,3′-cGAMP as the vaccine group.

[0074] Immunization was performed on days 0 and 28. On day 14 after the second immunization, mouse spleens were collected, and spleen cells were isolated for flow cytometry analysis. Surface staining antibodies (CD3, CD45, CD4, and CD8a) were added, and the cells were incubated at 4°C in the dark for 30 minutes. Fixative solution was added, and the cells were incubated at room temperature in the dark for 20 minutes. Cells were fixed and permeabilized using BD Cytoperm fixation / permeabilization solution (BD Biosciences): 250 μL of fixation / permeabilization solution was added to each tube, and the cells were incubated at 4°C for 20 minutes, avoiding vortexing. The cells were washed twice with 1×BD Perm / Wash™ buffer. Intracellular staining antibodies (IFNγ, IL4, and IL17A) were added, and the cells were incubated at room temperature in the dark for 30 minutes. The stained cell samples were then analyzed by flow cytometry.

[0075] like Figure 7 As shown, compared with the PBS group, CD4+ cells in the spleen cells of the vaccine group mice... + T cells and CD8+ The proportion of T cells was significantly increased in all groups. Compared with the PBS group, the IFNγ level in the spleen of the vaccine-treated mice was significantly higher. + CD4 + T cells, IL4 + CD4 + T cells, IL17A + CD4 + T cells, IL4 + CD8 + T cells and IFN-γ + CD8 + The proportion of T cells was significantly increased, and IFNγ + CD4 + T cells and IFN-γ + CD8 + The proportion of T cells was higher than that of IL4. + CD4 + T cells and IL4 + CD8 + T cells. The above results demonstrate that the influenza virus nanovaccine prepared in this invention can significantly activate cellular immunity in mice via nasal drops.

[0076] Example 4: Determination of mucosal immune response induced by the influenza virus nanovaccine of the present invention Thirty-two 6-8 week old female BALB / c mice were randomly divided into four groups: PBS group, adjuvant group, HA protein group, and vaccine group, with eight mice in each group.

[0077] PBS group: 60 μL of PBS solution was administered intranasally to each mouse.

[0078] HA group: Each mouse was immunized intranasally with 60 μL of HA protein solution, which contained 40 μg of HA protein.

[0079] Vaccine group: Each mouse was immunized intranasally with 60 μL of the influenza nanovaccine solution prepared in step one, which contained 40 μg of HA protein. Adjuvant group: Each mouse was immunized intranasally with 60 μL of adjuvant solution containing the same amounts of β-glucan, PAMAM and 2′,3′-cGAMP as the vaccine group.

[0080] Mice were immunized on days 0 and 28. On day 14 after the second immunization, nasal and lung lavage fluids were collected and stored at -80°C for later use. The titers of HA-specific IgG and IgA antibodies in the nasal and lung lavage fluids were detected using ELISA. The specific procedures were as follows: A 2 μg / ml HA protein solution was prepared, and 100 μl / well was added to each well of a 96-well plate for coating. The plate was incubated overnight at 4°C or for 2 hours at 37°C, followed by washing three times with PBST. 250 μl / well of 1% BSA was used for blocking at 37°C for 1 hour. The initial dilution of the mouse nasal and lung lavage fluid samples was 1:40. These samples were then diluted twofold and added to subsequent wells. The plates were incubated at 37°C for 1 hour, followed by washing three times with PBST. Secondary antibodies (IgG, ab205719, 1:8000; IgA, aba97235, 1:8000) were added to each well, and the plates were incubated at 37°C for 45 minutes. The plates were then washed five times with PBST. Add 50 μl of TMB solution (Beyotime, P0209) to each well and incubate at 37°C for 5 min. Add 50 μl of ELISA stop solution (Beyotime, P0215) to each well. Read the OD value using a microplate reader. 450 OD of the hole to be tested 450 Reading > 2.1 times that of the negative control well (PBS group) OD 450 The positive hole is the one that is positive.

[0081] like Figure 8 As shown, compared with the PBS group, the levels of HA-specific IgG and IgA titers in the nasal lavage fluid and the level of HA-specific IgG titer in the bronchoalveolar lavage fluid of the vaccine group mice were significantly increased. These results indicate that the influenza virus nanovaccine prepared in this invention can stimulate a mucosal immune response through nasal drops.

[0082] Example 5: Protective effect of the influenza virus nanovaccine of the present invention on mice under lethal dose influenza virus challenge. Forty female BALB / c mice aged 6-8 weeks were randomly divided into four groups: PBS group, adjuvant group, HA protein group, and vaccine group, with 10 mice in each group.

[0083] PBS group: 60 μL of PBS solution was administered intranasally to each mouse.

[0084] HA group: Each mouse was immunized intranasally with 60 μL of HA protein solution, which contained 40 μg of HA protein.

[0085] Vaccine group: Each mouse was immunized intranasally with 60 μL of the influenza nanovaccine solution prepared in step one, which contained 40 μg of HA protein. Adjuvant group: Each mouse was immunized intranasally with 60 μL of adjuvant solution containing the same amounts of β-glucan, PAMAM and 2′,3′-cGAMP as the vaccine group.

[0086] Immunizations were administered on days 0 and 28, respectively. On day 14 following the second immunization, a lethal dose of A / Beijing / 501 / 2009(H1N1)(BJ501) (50LD) was administered nasally. 50 H1N1) and A / Ostrich / SuZhou / 097 / 03 (10LD) 50 H5N1 virus fluid. Mice weight was observed and recorded daily for 14 days post-infection, and survival rates were calculated. Five days post-infection, three mice from each group were sacrificed by cervical dislocation, and their lungs were harvested for pathological examination using hematoxylin and eosin (HE) assay.

[0087] The results showed that within 4-6 days after challenge, the body weight of mice in all groups decreased significantly. Seven days after challenge, the body weight of mice in the vaccine group gradually increased, and at 14 days, the survival rates were 60% (H1N1) and 40% (H5N1), respectively, while all mice in the PBS group died. Mice in the PBS group, HA protein group, and adjuvant group showed significant alveolar wall thickening and marked inflammatory responses, while mice in the vaccine group showed no obvious inflammatory response in their lungs. These results indicate that the influenza virus nanovaccine prepared in this invention provides effective immune protection for mice under lethal challenge from H1N1 and H5N1 viruses. See also Figure 9 .

[0088] Example 6: Safety Testing of Nano-Vaccines Thirty-two 6-8 week old female BALB / c mice were randomly divided into four groups: PBS group, adjuvant group, HA protein group, and vaccine group, with eight mice in each group.

[0089] PBS group: 60 μL of PBS solution was administered intranasally to each mouse.

[0090] HA group: Each mouse was immunized intranasally with 60 μL of HA protein solution, which contained 40 μg of HA protein.

[0091] Vaccine group: Each mouse was immunized intranasally with 60 μL of the influenza nanovaccine solution prepared in step one, which contained 40 μg of HA protein. Adjuvant group: Each mouse was immunized intranasally with 60 μL of adjuvant solution containing the same amounts of β-glucan, PAMAM and 2′,3′-cGAMP as the vaccine group.

[0092] The patients were immunized on days 0 and 28, respectively. Fourteen days after the second immunization, they were euthanized by cervical dislocation, and their hearts, livers, spleens, lungs, kidneys, brains, and uteruses were collected. Histopathological changes in these organs were examined using hematoxylin and eosin (HE) staining. The experimental results showed that the nasal influenza vaccine had no significant toxicity to the aforementioned organs. Figure 10The levels of creatine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), total protein (TP), albumin (ALB), and uric acid (UA) in mouse serum on day 42 were measured using a fully automated biochemical analyzer. CK and LDH are indicators of cardiac function, ALT, TP, and ALB are indicators of liver function, and UA represents renal excretion function. The potential toxicity of the vaccine to the heart, liver, and kidneys of mice was assessed by measuring the levels of these substances. As shown in Table 1, there were no significant differences between the adjuvant group, HA protein group, and vaccine group compared to the PBS group. Therefore, this vaccination did not have significant toxicity to the heart, liver, and kidneys of mice.

[0093] Table 1. Evaluation of vaccine toxicity to the heart, liver, and kidneys in mice.

[0094] Note: a Creatine kinase; b lactate dehydrogenase; c Alanine aminotransferase; d Total protein; e albumin; f Uric acid.

[0095] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. An influenza virus nanovaccine, characterized in that: The influenza virus nanovaccine comprises nanoparticles and a STING agonist adsorbed on the surface of the nanoparticles. The nanoparticles comprise a polysaccharide antigen conjugate formed by coupling β-glucan and influenza virus antigen protein, and dendritic macromolecules adsorbed together with the polysaccharide antigen conjugate.

2. The influenza virus nanovaccine according to claim 1, characterized in that: The dendritic macromolecule is PAMAM; and / or The STING agonist is 2′,3′-cGAMP; and / or The influenza virus antigen protein is the influenza virus HA protein.

3. The influenza virus nanovaccine according to claim 1, characterized in that: The influenza virus HA protein is derived from either type A influenza virus or type B influenza virus.

4. The influenza virus nanovaccine according to claim 3, characterized in that: The amino acid sequence of the HA protein derived from the H1N1 influenza virus is shown in SEQ ID NO:

1.

5. The influenza virus nanovaccine according to claim 1, characterized in that: The polysaccharide antigen conjugate is obtained by first covalently coupling β-glucan with octaglutamate to obtain a covalent conjugate, and then covalently binding the covalent conjugate to the influenza virus antigen protein; and / or, the dendritic macromolecule electrostatically adsorbs the polysaccharide antigen conjugate; and / or The STING agonist is electrostatically adsorbed onto the surface of the nanoparticles.

6. The influenza virus nanovaccine according to any one of claims 1-5, characterized in that: The influenza virus nanovaccine was prepared by a method comprising the following steps: (A1) β-glucan was mixed with sodium periodate and dissolved in a buffer solution for reaction. After dialysis, aldehyde-containing β-glucan was obtained. (A2) The aldehyde-containing β-glucan obtained in (A1), N -(2-aminoethyl)maleimide and sodium cyanoborohydride were mixed and dissolved in a buffer solution and reacted. After dialysis, β-glucan with maleimide groups was obtained. (A3) The maleimide-containing β-glucan and octaglutamic acid obtained in (A2) are mixed and dissolved in a buffer solution to prepare a negatively charged β-glucan and octaglutamic acid conjugate, denoted as β-glucan-8E conjugate. (A4) The influenza virus antigen protein and 2-iminothion were mixed and dissolved in a buffer solution for reaction, and the antigen protein with thiol groups was obtained after dialysis. (A5) The β-glucan-8E conjugate obtained in (A3) and the thiol-containing antigen protein obtained in (A4) are mixed and reacted to prepare a polysaccharide antigen conjugate; (A6) The polysaccharide antigen conjugate obtained in (A5) is mixed and reacted with the dendritic macromolecule to obtain the nanoparticles; (A7) The nanoparticles obtained in (A6) are mixed and reacted with the STING agonist to obtain the nanovaccine.

7. The influenza virus nanovaccine according to claim 6, characterized in that: In (A1), the concentration of β-glucan in the reaction system is 4 mg / mL, and the concentration of sodium periodate in the reaction system is 20 mM; and / or, the buffer solution is sodium acetate buffer with a pH of 5.6; and / or, the reaction conditions are 20-30°C in the dark for 20-30 minutes; and / or, the dialysate used for the dialysis is phosphate buffer with a pH of 7.4; and / or In (A2), the aldehyde-containing β-glucan, N The mass ratio of -(2-aminoethyl)maleimide to sodium cyanoborohydride is 1:(1-2):(1-5); and / or, the buffer solution is a phosphate buffer with a pH of 7.4; and / or, the reaction temperature is 1-5°C and the time is 8-16 h; and / or, the dialysate used for the dialysis is a phosphate buffer with a pH of 7.4; and / or In (A3), the mass ratio of the maleimide-containing β-glucan to octaglutamic acid is 10:(1-2); and / or, the buffer solution is a phosphate buffer with a pH of 7.4; and / or, the reaction temperature is 1-5°C and the time is 1-3 h; and / or In (A4), the molar ratio of the influenza virus antigen protein to 2-iminothione is 1:(20-60); and / or, the buffer solution is a phosphate buffer with a pH of 7.4; and / or, the reaction temperature is 1-5°C and the time is 8-16 h; and / or, the dialysate used for the dialysis is a phosphate buffer with a pH of 7.4; and / or In (A5), the mass ratio of the β-glucan-8E conjugate to the thiol-containing antigen protein is (1-1.2):1; and / or, the reaction is carried out at a pH of 7.4, a temperature of 1-5°C, and a time of 8-16 h; and / or In (A6), the molar ratio of the polysaccharide antigen conjugate to the dendritic macromolecule is (50-10):1; and / or, the reaction is carried out at a pH of 7.4, a temperature of 1-5°C, and a time of 8-24 h; and / or In (A7), the mass ratio of the nanoparticles to the STING agonist is 10:(1-2); and / or, the reaction is carried out at a pH of 7.4, a temperature of 1-5°C, and a time of 8-16 h.

8. A method for preparing an influenza virus nanovaccine, comprising (A1) to (A7) as described in claim 6 or 7.

9. The use of the influenza virus nanovaccine according to any one of claims 1-7 in the preparation of products for the prevention of influenza.

10. The application according to claim 9, characterized in that: The influenza virus in question is either type A or type B influenza virus.

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