Microneedle patch vaccine for inducing mucosal immune response and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses a microneedle patch vaccine for inducing mucosal immune response and a preparation method and application thereof, specifically discloses a novel vaccine for inducing mucosal immune response, and the vaccine is delivered by a microneedle patch.The DNA nanoparticles wrapped by chitosan oligosaccharide are creatively combined with the microneedle technology, and it is proved that the strategy can induce a strong antigen-specific immune response in mucosal tissues such as lungs.The technology can be universally applied to the development of a novel mucosal vaccine for preventing mucosal route infectious pathogens (such as new coronavirus).

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a microneedle patch vaccine for inducing mucosal immune response and a preparation method and application thereof. BACKGROUND

[0002] The frequent emergence and re-emergence of highly pathogenic microorganisms have become a serious threat to human public health, including severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, avian influenza, Ebola virus (EBOV), human immunodeficiency virus (HIV) and Mycobacterium tuberculosis (TB). Most of these pathogens mainly infect the host through the mucosal route, such as skin epithelium, respiratory tract, ocular mucosa, gastrointestinal tract and reproductive tract. Therefore, the mucosal surface, as the first defense system against the invasion of these pathogens, plays a key role in this system, which is composed of physical, chemical, biological and immune barriers. The physical barrier is mainly composed of epithelial cell tight junctions, ciliary oscillation and mucus produced by goblet cells in the mucosal tissue; the chemical barrier can include antibacterial proteases, various digestive enzymes, lysozyme, special pH environment and mucopolysaccharides; the microbiome (gut flora and reproductive tract flora) and its metabolites constitute the biological barrier; the mucosal immune barrier is mainly formed by secretory IgA (sIgA), cytokines, mucosa-associated lymphoid tissue (MALT) and diffuse innate and adaptive immune cells. Through the inoculation of mucosal vaccines, the proliferation and functional regulation of mucosa-associated immune cells such as mucosal epithelial cells, mucosa-associated T and B lymphocytes, and the formation of mucosal immune tissues such as regulatory T cells and mucosal immune cell populations can be induced. Therefore, the immune protection of mucosal vaccines can reduce the infection rate, reduce the severity and duration of the disease, and play a role in controlling and preventing the disease in epidemiology. It is worth noting that mucosal tissue accounts for about 60% of the total immune cells in adults. In addition, although the physical, chemical and biological barriers of the mucosal surface can effectively defend against the invasion of pathogens, these barriers also hinder the entry of traditional vaccines into the human body. Therefore, there are many challenges in the development of safe and effective mucosal vaccines.

[0003] In the past few decades, great achievements have been made in vaccine technology. Vaccines based on different technology platforms, including inactivated virus, subunit protein, viral vector vaccine, and mRNA-based vaccine, have been widely used in clinics. However, currently clinically available vaccines are mainly for inoculation by systemic vaccination (i.e. intramuscular injection or subcutaneous injection). Although the traditional strategy of systemic vaccination can effectively induce pathogen-specific antibody and / or cellular immune responses in the peripheral blood system, it is generally less effective in inducing mucosal immunity. In addition, vaccination based on intramuscular injection has the following disadvantages: it usually requires cold chain distribution and strict storage conditions, as well as professional operation of medical staff. Due to the trauma and pain caused by intramuscular injection, the acceptance of vaccinated patients is greatly reduced, and the vaccination coverage of vaccines is reduced. In view of this, there is an urgent need for a platform to quickly develop new vaccine technologies that can induce mucosal immune responses in the mucosa, and it is of great significance to explore new vaccination methods to improve the effectiveness and coverage of vaccination in different populations.

[0004] Recently, transdermal drug delivery with biocompatible microneedles (MN) is a rapidly growing field. The MN matrix consists of an array of micrometer-sized needles that provide a painless way to deliver active substances through the skin. Notably, needles with lengths from 50 μm to 1100 μm can effectively deliver antigens to the epidermal and dermal regions rich in Langerhans cells and dendritic cells, thereby inducing a strong immune response. At the same time, MN-based vaccines can be easily self-administered at home with minimal training, reducing the risk of cross-infection and accidents during mass vaccination during a pandemic. Therefore, MN-based delivery systems as a new vaccine modality are becoming a promising strategy. However, it is not yet clear whether MN-mediated intradermal immunity can induce an immune response in mucosal tissues.

[0005] In addition, the manufacturing process of MN-based vaccines can require more stringent conditions than MN-based chemical drugs, as the main components of vaccines are unstable biological macromolecules, including protein subunits, DNA, mRNA, or recombinant virus particles. Therefore, it is crucial to use suitable materials to effectively protect the loaded bioactive compounds. Our previous studies have shown that chitosan oligosaccharide (COS) is a positively charged compound that can encapsulate and protect negatively charged DNA-based vaccines or recombinant virus-based vaccines on nanoparticles, thereby inducing strong systemic and mucosal immunity. Most importantly, its mucosal adhesion and adjuvant properties endow it with the potential to induce strong mucosal immunity. SUMMARY

[0006] It is an object of the first aspect of the present invention to provide a novel vaccine that can induce mucosal immunity.

[0007] The second aspect of the present application aims to provide a method for preparing the vaccine of the first aspect of the present application.

[0008] The third aspect of the present application aims to provide the use of the vaccine of the first aspect of the present application in the preparation of a product.

[0009] To achieve the above-mentioned objects, the technical solutions adopted by the present application are as follows:

[0010] The first aspect of the present application aims to provide a novel vaccine capable of inducing mucosal immune response, wherein the vaccine is delivered by a microneedle patch.

[0011] Preferably, the vaccine induces mucosal immune response by being delivered by the microneedle patch to be delivered subcutaneously or intradermally.

[0012] Preferably, the vaccine is delivered by the microneedle patch in a process of delivering nanoparticles.

[0013] Preferably, the vaccine comprises a backing and a microneedle array containing nanoparticles attached to the side of the backing.

[0014] Preferably, the nanoparticles comprise a nucleic acid molecule and chitosan oligosaccharide (COS); or the nanoparticles comprise a biomaterial associated with the nucleic acid molecule and chitosan oligosaccharide.

[0015] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID ON:1 or SEQ ID ON:2.

[0016] Preferably, the biomaterial comprises at least one of a1) to a3):

[0017] a1) an expression cassette containing the nucleic acid molecule;

[0018] a2) a recombinant vector containing the nucleic acid molecule or the expression cassette in (1);

[0019] a3) a recombinant cell containing the nucleic acid molecule, the expression cassette in (1) or the recombinant vector in (2).

[0020] Preferably, the recombinant vector is a plasmid vector, a viral vector or a cellular vector.

[0021] Preferably, the plasmid vector can be an optional plasmid, the viral vector can be an optional virus, and the cellular vector does not include propagation material.

[0022] Preferably, the microneedle array comprises a plurality of microneedles, wherein each microneedle comprises a substrate and nanoparticles loaded in the substrate.

[0023] Preferably, the base body comprises at least one of sodium hyaluronate, polyvinylpyrrolidone, chondroitin sulfate, sodium hydroxyethyl cellulose, collagen, sucrose, trehalose, maltose, chitosan and dextran.

[0024] Preferably, the backing is formed by cross-linking and / or drying curing an aqueous solution containing one or more of polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethyl cellulose, trehalose, hyaluronic acid, polylactic acid-glycolic acid copolymer, polylactic acid, galactose, polyvinylpyrrolidone, one or more of polyvinyl alcohol.

[0025] Preferably, the microneedle has a tip and a base end, the tip is away from the backing, the microneedle is attached to the backing via the base end, the distance from the tip to the base end is 500 μm-1 mm, the size of the base end diameter is 400 μm-700 μm, and the microneedle contains 72 microneedle tips.

[0026] Preferably, the nanoparticle vaccine is concentrated on the microneedle tip with a length of 200-400 μm.

[0027] Preferably, the nanoparticle is approximately spherical.

[0028] Preferably, the nanoparticle has a particle size of 130-160 nm.

[0029] Preferably, the preparation method of the nanoparticle vaccine comprises the following steps: slowly adding a solution of COS to a solution containing nucleic acid molecules or biological materials under the condition of vortex, continuing to vortex for 20-40 seconds, placing at 4-5℃ for 10-20 minutes, concentrating and purifying the formed DNA nanoparticle vaccine solution using ultracentrifugation until no free DNA is detected in the centrifugate, and confirming that the nanoparticle purification is complete, i.e., obtaining the nanoparticle.

[0030] The nanoparticle has a regular shape, is spherical, has good dispersibility, and has no obvious adhesion, damage, collapse and other phenomena, and the nanoparticle vaccine has high transfection efficiency and antigen-presenting cell uptake efficiency.

[0031] Most of the vaccines currently used in clinical practice are based on intramuscular injection delivery, which induces a lower mucosal immune response, and the present application proposes to deliver vaccines through microneedle transdermal immunization to induce mucosal immune indicators such as serum IgA antibodies and lung T lymphocyte responses, which are stronger than intramuscular injection, so this vaccine delivery method can improve the problem of insufficient mucosal immunity induced by current vaccines.

[0032] The vaccine provided by the present application fully utilizes the advantages of the microneedle itself and the advantages of the nanoparticle vaccine, and through the nanoparticle vaccine loaded in the microneedle, rapid humoral immunity can be achieved after the microneedle product is applied to the skin, the produced antibody has the effects of neutralization and clearance of pathogenic microorganisms and toxins produced by the pathogenic microorganisms, and has a good protective effect on extracellularly infected pathogenic microorganisms. Moreover, the vaccine existing in the microneedle patch not only can long-acting the vaccine, but also reduces the discomfort of the vaccine muscle injection to the vaccinated person and the use of medical resources. The microneedle generally includes a plurality of microneedles with a length of generally not more than 1 mm, the microneedle can form a micro channel in the stratum corneum of the skin, break through the barrier of the stratum corneum of the skin, and promote the penetration of the drug, thereby reducing the accumulation amount of the drug in the stratum corneum and increasing the dose of the drug reaching the epidermis, dermis and subcutaneous tissue.

[0033] In a second aspect, the present application provides a method for preparing the vaccine of the first aspect, comprising the following steps:

[0034] (1) mixing the nanoparticles and the matrix to form a mixed solution, placing the mixed solution in the forming holes of the microneedle mold and filling at least a part of the volume of the forming holes, centrifuging to form microneedles, and a plurality of microneedles constitute a microneedle array;

[0035] (2) applying a solution containing a backing material on the bottom end surface of the microneedle and the upper surface of the microneedle mold which is not covered by the microneedle to form a backing solution layer, centrifuging, and solidifying to obtain a microneedle patch.

[0036] Preferably, the centrifuging condition in step (1) is 4-5℃, 4000-5000rpm, centrifuging for 3-5 minutes.

[0037] Preferably, the centrifuging condition in step (2) is 4-5℃, 4000-5000rpm, centrifuging for 7-12 minutes.

[0038] Preferably, the solidifying includes dry solidification or thermal solidification.

[0039] In a third aspect, the present application provides the use of the vaccine of the first aspect in the preparation of a product.

[0040] Preferably, the product includes a drug, a medical device, a reagent and a kit, and the drug includes a vaccine.

[0041] Preferably, the product further comprises a pharmaceutically acceptable excipient.

[0042] Preferably, the excipient includes a carrier, a diluent, an excipient, a preservative, an antibacterial agent and / or an immunoadjuvant.

[0043] Preferably, the product has any one of the following functions: c1)-c3).

[0044] c1) preventing or treating respiratory viral infection related diseases;

[0045] c2) eliciting or enhancing systemic and mucosal T cell immune responses;

[0046] c3) inducing antibodies specific to respiratory viruses.

[0047] The beneficial effects of the present application are:

[0048] The present application creatively combines chitosan oligosaccharide-coated DNA nanoparticles with microneedle technology and demonstrates that this strategy can induce a strong antigen-specific immune response in mucosal tissues such as the lungs. This technology can be widely used in the development of new mucosal vaccines to prevent mucosal route infection pathogens (such as the new coronavirus).

[0049] The vaccine provided by the present application has sufficient mechanical strength to pierce the skin, thereby delivering the nanoparticle vaccine into the subcutaneous tissue and cells of the body, and has high dissolution efficiency and good stability. Specifically, by acting on the skin of mice, the results show that the microneedle patch can effectively penetrate the skin of mice, and the tip containing the nanoparticle vaccine is completely dissolved within 5 minutes, leaving a micron-sized hole on the skin; the microneedle tip is dissolved in phosphate buffered saline within 20 seconds, while the support layer remains relatively intact; there is no decrease in the level of in vitro transfection and the level of induced antibodies in vivo after being placed at room temperature for more than 30 days.

[0050] In one embodiment of the present application, the SARS-CoV-2 structural S protein and N protein sequences obtained from NCBI are analyzed, and the gene sequences are optimized based on the codon usage bias of mammalian cells and the inventors' experience in codon optimization, to finally screen and optimize the nucleic acid molecules shown as SEQ ID ON:1 and SEQ ID ON:2. The nucleic acid molecules are cloned into an expression vector and used to prepare a nanoparticle vaccine or a microneedle patch, which produces a good mucosal T cell immune response and a systemic T cell immune response.

[0051] Compared with traditional muscle injection delivery of vaccines, the microneedle patch technology can achieve painless self-administration through simple training, does not require professional medical personnel and equipment, can achieve self-inoculation, thereby greatly improving the compliance of users, and does not produce sharp medical waste after use, increasing the safety of vaccine use. Secondly, microneedles do not require cold chain transportation and storage, which has great economic effect, especially for underdeveloped areas, which can effectively improve their vaccine coverage. More importantly, the microneedle patch used in the present application induces a stronger mucosal T cell immune response and a systemic T cell immune response than muscle injection.

[0052] The vaccine is prepared by micro-needle template reverse molding, and the method is simple, convenient to operate, low in price, reusable, does not need high technical requirements, easy to control the basic morphology of the micro-needle array, high in safety, and suitable for promotion. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 Figure 1 is a transmission electron microscope image, size distribution and Zeta potential of pVAX-S+N@COS, pVAX-S@COS and pVAX-N@COS nanoparticles.

[0054] Figure 2 Figure 2 is an in vitro transfection effect of pVAX-GFP@COS and pVAX-luci@COS nanoparticles; wherein A is a fluorescence signal imaging result graph; B is a flow cytometry result graph; C is a GFP expression efficiency statistical result graph; and D is a bioluminescence signal imaging statistical result graph; in the graph, ns represents no significant difference, ** represents P<0.01, and **** represents P<0.001.

[0055] Figure 3 Figure 3 is the uptake efficiency of Cy5-pVAX-S@COS in HEK-293T cells at different times; in the graph, *** represents P<0.005, and **** represents P<0.001.

[0056] Figure 4 Figure 4 is a co-localization observation result graph of Cy5-labeled pVAX-S (red) and LysoTracker (green) in HEK-293T cells by confocal microscopy.

[0057] Figure 5 Figure 5 is a physicochemical property of a micro-needle vaccine (pVAX-S+N@Rho-COS / MN); wherein A is a perspective image of pVAX-S+N@Rho-COS-MN; B is a perforation effect of pVAX-S+N@Rho-COS-MN and an influence on mouse skin, before (left) and after (right) perforation; and C is a dissolution condition of the micro-needle at different time points in phosphate buffered saline.

[0058] Figure 6 Figure 6 is an in vivo fluorescence imaging result of luciferase expression in mice 24 hours and 48 hours after receiving pVAX-luci@COS nanoparticle vaccine by different administration routes.

[0059] Figure 7Humoral immune responses to S and N proteins in BALB / c after vaccination with DNA nanoparticle vaccines via intramuscular or intradermal microneedle-mediated intradermal administration; where A-B are the serum anti-S protein IgG endpoint titers on day 21 or 42 post-immunization, endpoint titers are defined by the lowest dilution where the OD value is twice the standard deviation of the mean of the blank wells; C is the serum anti-N protein IgG antibody titer on day 42 post-immunization; D is the serum anti-S protein IgG1 and IgG2c antibody responses (1:20 dilution) on day 42; where ns indicates no significant difference, * represents P<0.05, and ** represents P<0.01.

[0060] Figure 8 Neutralization efficiency of 1:20 diluted mouse serum on day 42 against SARS-CoV-2 wild type and Beta mutant strains; where ns indicates no significant difference, * represents P<0.05, and ** represents P<0.01.

[0061] Figure 9 Characteristics of DNA nanoparticle vaccines in microneedles stored at room temperature for 30 days; where A is the in vitro transfection efficiency of DNA nanoparticle vaccines stored in microneedles; B-C are the serum anti-S protein or anti-N protein IgG endpoint titers on day 21 post-immunization using vaccines stored in microneedles at room temperature for 30 days; where ns indicates no significant difference, ** represents P<0.01, *** represents P<0.005, and **** represents P<0.001.

[0062] Figure 10 Elispot detection of S and N protein-specific T cell responses in spleen and lung induced by DNA nanoparticle vaccines post-immunization.

[0063] Figure 11 ICS detection of S1, S2, and N polypeptide library-specific CD8+ T cell cytokine levels induced by DNA nanoparticle vaccines post-immunization against SARS-CoV-2; where A, C, and E are the S1, S2, or N-specific CD8 + IFN-γ + T cell cytokine levels in pVAX-S+N@COS-MN and pVAX-S+N@COS-IN groups, respectively; B, D, and F are the S1, S2, or N-specific CD8 + IL2 + , CD8 + TNF-α + , and CD8 + IL2 + TNF-α +T cell cytokine levels; in the figure, n* represents P<0.05, ** represents P<0.01, and **** represents P<0.001. DETAILED DESCRIPTION

[0064] The present application will be described in detail with specific embodiments, but the scope of the present application is not limited thereto.

[0065] The materials, reagents, etc. used in the present embodiment are materials and reagents obtained from commercial channels, unless otherwise specified.

[0066] As used herein, the term "particle" refers to a geometric body having a specific shape within a size range, a state of matter characterized by the presence of discrete particles, pellets, beads, or granules, regardless of their size, shape, or morphology.

[0067] As used herein, the term "particle size" or "equivalent particle size" refers to the diameter of a homogeneous sphere (or combination) that most closely approximates the physical property or behavior of the measured particle when the physical property or behavior of the measured particle is compared to that of the sphere (or combination).

[0068] As used herein, the term "average particle size" refers to the diameter of a hypothetical spherical particle that is composed of uniform spherical particles, compared to an actual particle group composed of particles of different sizes and shapes, and if the total length of the particle size of the two is the same, the diameter of the spherical particle is referred to as the average particle size of the actual particle group. Methods for measuring average particle size are known to those skilled in the art, such as light scattering; average particle size measuring instruments include, but are not limited to, a Malvern particle size analyzer.

[0069] As used herein, the term "room temperature" refers to 25±5°C.

[0070] As used herein, the term "immunoadjuvant" refers to a substance that is administered to the body together with or prior to an antigen, which enhances immunogenicity or changes the type of immune response. The immunoadjuvant can be immunogenic (e.g., BCG) or non-immunogenic (e.g., aluminum hydroxide adjuvant).

[0071] As used herein, the term "antigen" or "immunogen" refers to a substance capable of inducing a specific immune response in a host. The antigen can include an entire organism (e.g., an inactivated, attenuated, or live organism); a subunit or portion of an organism; a recombinant vector containing an insert having immunogenicity; a DNA portion or fragment capable of inducing an immune response upon presentation to a host; a protein, glycoprotein, lipoprotein, polypeptide, peptide, antigenic epitope, hapten, toxin, antitoxin, or any combination thereof.

[0072] Example 1 Design, optimization, and synthesis of SARS-CoV-2 S protein gene and N gene

[0073] The inventors obtained SARS-CoV-2 structural S protein and N protein sequences, including S protein sequence (YP_009724390.1) and N protein sequence (YP_009724397.2) from the National Center for Biotechnology Information (NCBI). The gene sequences were optimized according to the codon usage bias of mammalian cells, while based on the experience of codon optimization in the laboratory, the above-mentioned genes were obtained by gene synthesis method, and the sequences of the optimized S gene and N gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Then the codon-optimized S gene and N gene were cloned into the pVAX-1 expression vector to obtain pVAX-S and pVAX-N.

[0074] Example 2 Preparation of DNA@COS nanoparticles

[0075] The preparation method of DNA@COS nanoparticles is as follows: 2 mg of COS was dissolved in 2 mg / mL PBS. Under vortex conditions, the same volume of COS (purchased from Aladdin, CAS: 148 411-57-8) solution was slowly added to the DNA solution (pVAX-S, pVAX-N and pVAX-GFP or pVAX-luciferase prepared in Example 1, pVAX-GFP and pVAX-luciferase as control group, stored in the laboratory) (final concentration 500 μg / mL), continue to vortex for 30 seconds, and place at 4°C for 15 minutes, to obtain DNA nanoparticles (pVAX-S@COS), according to the same method, pVAX-N@COS was prepared by replacing pVAX-S solution with pVAX-N solution, and pVAX-S+N@COS was prepared by mixing pVAX-S solution and pVAX-N solution at a concentration ratio of 1:1 using the above method. The formed DNA nanoparticle vaccine solution was concentrated and purified by ultracentrifugation until no free DNA was detected in the centrifugate, confirming that the nanoparticle purification was completed. Respectively named pVAX-S@COS, pVAX-N@COS, pVAX-S+N@COS, pVAX-GFP@COS and pVAX-luci@COS.

[0076] The prepared DNA nanoparticles were further characterized by transmission electron microscopy (TEM), and the results are as follows: Figure 1As shown, due to ionic interactions, positively charged COS and negatively charged plasmid DNA solutions can self-assemble to form nanoparticles (DNA@COS). The zeta potentials of pVAX-S and pVAX-N plasmid DNA are -20.87±3.50 (mV) and -16.15±0.90 (mV), respectively. However, after the addition of positively charged COS (55.54±2.22 (mV)), the charges of pVAX-S@COS and pVAX-N@COS are opposite, with zeta potential values ​​of 45.11±0.75 (mV) and 44.18±0.04 (mV), respectively. These results confirm that COS and DNA can form nanoparticles through positive and negative charge interactions. Transmission electron microscopy (TEM) observations showed that pVAX-S@COS, pVAX-N@COS and pVAX-S+N@COS were spherical with particle sizes of approximately 130.53±0.26nm, 137.27±1.04nm and 159.87±1.52nm, respectively.

[0077] Example 3 Transfection efficiency of DNA@COS nanoparticles in cells

[0078] In this example, cells were transfected with pVAX-GFP / pVAX-luci plasmids encoding luciferase to determine whether the nanoparticles can be normally expressed in cells. The details are as follows:

[0079] HEK-293T cells were incubated with different concentrations of pVAX-GFP@COS (0.5 μg / mL, 1 μg / mL, 2 μg / mL, and 4 μg / mL). At 48 hours, the treated cells were imaged for fluorescence signals using an inverted fluorescence microscope. GFP expression efficiency was then quantified by flow cytometry and analyzed using FlowJo software. Similarly, HEK-293T cells were treated with pVAX-luci@COS, and the bioluminescent signals of the treated cells were imaged using a luciferase kit using a microplate luminescence detector.

[0080] like Figure 2 As shown, the transfection efficiency of DNA@COS nanoparticles increased in a dose-dependent manner. Consistent with the above observations, the fluorescence intensity of pVAX-luci@COS nanoparticles, which encode luciferase and catalyze the oxidation of its substrate luciferin to produce bioluminescence, increased in a dose-dependent manner when transfected into HEK-293T cells.

[0081] Example 4 Uptake efficiency of DNA@COS nanoparticles in cells

[0082] To evaluate DNA@COS nanoparticles in HEK-293T cells, the inventors used the Label DNA was labeled with fluorescent (Cy5) Label IT® Nucleic Acid Labeling Kit (purchased from Mirus, CAS: MIR 3300). Specifically, 293T cells were incubated with 2 pg / mL DNA@COS nanoparticles (pVAX-S@COS, pVAX-N@COS, pVAX-GFP@COS or pVAX-luci@COS) for 2 hours, 4 hours and 8 hours, and the uptake of nanoparticles was observed using confocal laser scanning microscope and quantified by flow cytometry.

[0083] As shown in FIG. 6, the uptake of nanoparticles was significantly increased after pVAX-S@COS was incubated with HEK-293T cells for 4 hours, and the uptake efficiency remained until 8 hours; pVAX-N@COS, pVAX-GFP@COS and pVAX-luci@COS also had the same effect. Figure 3

[0084] Example 5 DNA@COS nanoparticle-mediated lysosome escape

[0085] DNA@COS nanoparticles were labeled with Label IT® Nucleic Acid Labeling Kit with fluorescent (Cy5), and incubated with HEK-293T cells for 1 hour, 2 hours, 4 hours and 8 hours, respectively. The lysosomes of HEK-293T cells were stained with lysotracker dye kit (purchased from Beyotime, CAS: C1047S). The localization of lysosomes and nanoparticles was observed using confocal laser scanning microscope. As shown in FIG. 7, there was a large amount of red fluorescence overlapping with green fluorescence after HEK-293T cells were incubated with Cy5-pVAX-S@COS nanoparticles for 1 hour and 2 hours, indicating that DNA@COS nanoparticles were effectively transported to endo / lysosomes. However, the co-localization phenomenon was significantly reduced at 4 hours and 8 hours, which indicated that pVAX-S@COS could effectively escape from endo / lysosomes. pVAX-N@COS, pVAX-GFP@COS and pVAX-luci@COS also had the same effect.

[0086] Figure 4 Example 6 Preparation of microneedle patch vaccine

[0087] As shown in FIG. 8, the microneedle patch vaccine was prepared by coating the microneedle with DNA@COS nanoparticles (pVAX-S@COS, pVAX-N@COS, pVAX-GFP@COS or pVAX-luci@COS). The microneedle patch vaccine was then used to vaccinate mice.

[0088] ​​The microneedle patch was prepared using centrifugal infusion polydimethylsiloxane (PDMS) mold method as follows: DNA@COS nanoparticles were suspended in an aqueous solution containing 20% sucrose (the final concentration of pVAX-S@COS and pVAX-N@COS was 1.5 mg / mL) for a short time, then 200 μL of the solution was added to the PDMS mold with a pipette and centrifuged for 3 minutes (4500 rpm, 4°C) to fill the cavities. The residual suspension was removed with a pipette. Subsequently, the mold was centrifuged at 25°C for 30 minutes to dry. Then, 18% polyvinyl alcohol (PVA) aqueous solution was added to the mold, centrifuged for 10 minutes (4500 rpm, 4°C), and stored overnight in a desiccator to obtain the microneedle patch, denoted as pVAX-S+N@COS-MN.

[0089] The pVAX-S+N@COS-MN prepared by the above method had consistent geometric dimensions, with a base radius of 600 μm and a height of 1000 μm.

[0090] To investigate the distribution of DNA@COS nanoparticles in the microneedle, COS was labeled with a red fluorescent dye (rhodamine) to obtain pVAX-S+N@Rho-COS nanoparticles, and a microneedle patch containing red fluorescent dye-labeled microneedles (denoted as S+N@Rho-COS / MN) was prepared according to the above method. By observation, as shown in FIG. 8A, the pVAX-S+N@Rho-COS nanoparticles were concentrated in the tip of the microneedle with a length of about 300 μm. Figure 5

[0091] Further, to test whether the obtained microneedle patch (pVAX-S+N@Rho-COS-MN) has sufficient mechanical strength to pierce the skin, thereby delivering DNA@COS nanoparticles into the subcutaneous tissue and cells of the body, the inventors conducted a mouse experiment. Specifically, the mouse's back hair was cleaned with depilatory cream, then the mouse's back skin was pulled out and fixed on a hard and flat table top, then the microneedle patch (pVAX-S+N@Rho-COS-MN) was taken out and vertically forced into the mouse's fixed skin with the thumb, and the pressing lasted for one minute, then the hand was released, and the microneedle patch was removed after maintaining the effect on the skin for twenty minutes, and the effect of the microneedle on the skin was observed. The results showed that the microneedle patch could effectively penetrate the mouse skin, and the DNA@COS nanoparticles contained therein completely dissolved in 5 minutes, leaving micrometer-sized holes on the skin (FIG. 8B). Figure 5

[0092] ​​Further, to determine the dissolution efficiency of the obtained microneedle patch (pVAX-S+N@Rho-COS-MN), the inventors performed in vitro experiments. Specifically, a row of microneedles was carefully cut off with scissors, and then fixed on a glass slide with double-sided tape, and the glass slide was placed in a 10 cm culture dish; 10 mL of phosphate buffered saline (PBS, pH = 7.4) was added to the culture dish, and the dissolution of the microneedles was observed at different time points under a microscope. It was found that the tips of the microneedles dissolved in the phosphate buffered saline (PBS, pH = 7.4) within 20 seconds, while the support layer remained relatively intact. Figure 5 Example 7 Protein expression of microneedle vaccine in mice

[0093] Example 7 Protein expression of microneedle vaccine in mice

[0094] The pVAX-luci@COS (10 μg of plasmid per patch, prepared in Example 4) was loaded onto the microneedles (prepared according to the method of Example 6), and inserted into the exposed skin of 6-8 week old female BALB / c mice, and then the mice were imaged and quantified at different time points (24 h and 48 h) by intraperitoneal injection in the presence of 200 μL of D-luciferin potassium salt (150 μg / mL) substrate using an in vivo imaging system. As a control group, pVAX-luci@COS (10 μg of plasmid) was injected intramuscularly into the right quadriceps muscle of the mouse leg.

[0095] The results are shown in Figure 6 Figure 6. After 48 hours of intradermal administration mediated by microneedles, luciferase was expressed at a high level, which was significantly higher than that by intramuscular injection.

[0096] Example 8 Humoral immune response level of microneedle patch vaccine in mice

[0097] 1. Mouse immunization

[0098] Six to eight-week-old female BALB / c mice were randomly divided into four groups, namely G1 : pVAX-empty@COS-IM (the preparation method of pVAX-empty@COS is the same as that of Example 2) group, G2: pVAX-empty@COS-MN (prepared according to the method of Example 6) group, G3: pVAX-S+N@COS-IM group, and G4: pVAX-S+N@COS-MN group, 10 mice in each group, a total of three immunizations, 10 μg of plasmid each time, IM (intramuscular injection) is the method of intramuscular injection, the injection site is the quadriceps muscle of the right leg of the mouse, MN (microneedle patch) is the method of attaching the microneedle patch to the back of the mouse. The second immunization was performed on the 14th day after the first immunization, and the third immunization was performed on the 28th day. During the experiment, 5 mice were sacrificed on the 21st day and the 42nd day after the first immunization (orbital blood was taken, the corresponding tissues were taken, and the mice were euthanized) for detection of relevant immune indicators.

[0099] 2. Detection of the level of humoral immune response

[0100] Enzyme-Linked Immunosorbnent Assay (ELISA): The S and N proteins of SARS-CoV-2 were coated on the enzyme-labeled plate at 1 pg / mL, 100 pL / well, sealed and incubated overnight at 4°C. The next day, the solution in the wells was discarded, and the enzyme-labeled plate was washed with washing solution (PBST) 3 times, 300 pL / well, 3 minutes each time. Blocking solution (PBST containing 5% milk powder) was added, 200 pL / well, and incubated at 37°C for 1 hour. The blocking solution was discarded, and the enzyme-labeled plate was washed with washing solution (PBST) 3 times, 300 pL / well, 3 minutes each time. The serum sample of the mouse to be tested was diluted with PBS solution in turn by 2 times, and then 100 pL was added to the 96-well plate and incubated for 2 hours. After washing the plate, horseradish peroxidase-labeled mouse IgG, IgG1, IgG2c and IgA (1:5000 dilution) were added, and the plate was incubated in a 37°C incubator for 1 hour. The enzyme-labeled plate was washed as above, 50 pL / well of TMB substrate was added, and color development was performed at room temperature for 15 minutes. 50 pL / well of stop solution (2M H2SO4) was added to terminate color development. The absorbance (OD) value of each well was measured at 450 nm wavelength using an enzyme-labeled instrument.

[0101] Pseudovirus neutralization experiment: 293T-hACE2 cells were digested and plated, 30000 / well, 10% FBSDMEM was used for culture overnight; the next day, the mouse serum was inactivated in a 56°C water bath for 30 minutes, and the serum or antibody was diluted with 5% FBSDMEM medium, and an equal volume of pseudovirus was added and incubated at 37°C for 30 minutes (note to set up virus control and blank wells). The cells were washed with PBS to remove the serum, and 100 pL of the mixture was used to infect the cells. Incubate at 37°C. After overnight, the supernatant was discarded and replaced with 50 pL of 10% FBSDMEM culture. 48 hours after infection, luciferase expression was detected using a luciferase detection kit. Most of the supernatant was removed, leaving only 50 pL, 50 pL of detection solution was added to each well, incubated at room temperature for 2 minutes, mixed by gun, and 90 pL of detection solution was removed to a white 96-well plate for detection, and the fluorescence value was read.

[0102] The results showed that the microneedle-mediated pVAX-S+N@COS nanoparticles effectively induced S-specific IgG antibodies at day 21 and day 42 ( Figure 7 A and B), and induced N-specific IgG antibodies at day 42, with antibody levels comparable to the muscle injection group ( Figure 7 C). The results also showed that the microneedle group and the muscle injection group induced a T helper 1 (Th1) biased response with IgG2c subclass antibodies, but did not induce a Th2 biased response with IgG1 subclass antibodiesFigure 7 Figure 8D, which is consistent with previous studies that chitosan materials enhance antigen-specific Th1 responses. Importantly, the level of S-specific IgA antibody in the pVAX-S+N@COS-MN group was significantly higher than that in the pVAX-S+N@COS-IM group (Figure 8E), indicating that microneedle-mediated DNA@COS nanoparticles effectively induced a strong mucosal immunity, which plays a key role as the first line of defense against viral invasion. Figure 7 Figure 8D, which is consistent with previous studies that chitosan materials enhance antigen-specific Th1 responses. Importantly, the level of S-specific IgA antibody in the pVAX-S+N@COS-MN group was significantly higher than that in the pVAX-S+N@COS-IM group (Figure 8E), indicating that microneedle-mediated DNA@COS nanoparticles effectively induced a strong mucosal immunity, which plays a key role as the first line of defense against viral invasion.

[0103] Similar to the nanoparticles of traditional intramuscular injection, microneedle-mediated DNA@COS nanoparticles based on the nanoparticles of SARS-CoV-2 wild-type S protein can significantly inhibit the infection of SARS-CoV-2 wild-type and Beta mutant strains (Figure 8F). Figure 8 ).

[0104] Further, after storing the microneedles at room temperature for 30 days, the in vitro transfection effect of the microneedles was detected by flow cytometry. The specific process of detecting the in vitro transfection effect of the microneedles by flow cytometry is as follows: the microneedles were dissolved in double distilled water, and then HEK-293T cells were treated with pVAX-GFP loaded on COS at different concentrations (0.5 μg / mL, 1 μg / mL, 2 μg / mL and 4 μg / mL), 4 μg / mL pVAX-GFP and 16 μg / mL COS were used as negative controls; at 48 hours, the expression efficiency of GFP was quantified by flow cytometry. The results show that the DNA@COS nanoparticles stored at room temperature for 30 days still have a high transfection efficiency similar to that of freshly prepared nanoparticles (Figure 9A). Figure 9 Figure 9A). On day 21 after immunization with the microneedle vaccine, the serum anti-S protein or anti-N protein IgG of the mice was detected for endpoint antibody titers, and the results show that two doses of DNA immunization of the mice by the microneedle patch or intramuscular injection still induced S and N-specific antibodies (Figures 9B and 9C). Figure 9 Figure 9B and Figure 9C). Figure 9 Figure 9B and Figure 9C).

[0105] Example 9 Cellular immune response level of microneedle patch vaccine

[0106] 1. Isolation of mouse spleen lymphocytes

[0107] After blood collection, the mice were sacrificed by dislocation and the mice were soaked in 75% alcohol for a few minutes. The mice were placed on a clean bench and the spleen and lung were removed. 5-8 mL of mouse lymphocyte separation medium was added to a sterile petri dish. The mouse spleen was placed on a 200 mesh screen and the spleen was gently ground using a syringe plunger. The ground single cells were separated by the screen into the separation medium. The cells in the petri dish were mixed by blowing and transferred to a 15 mL centrifuge tube. Then 1 mL of 1640 medium was added to the top of the tube. The tube was centrifuged at 800g for 20-30 minutes at room temperature. The white layer of cells in the middle was removed and 1640 medium was added. The tube was centrifuged at 400g for 10 minutes. The supernatant was discarded and 1640 medium was added. The tube was centrifuged at 300g for 10 minutes. The supernatant was discarded and complete medium was added to resuspend the cells (usually 1 mL for one mouse). The cells were counted to obtain the mouse spleen lymphocytes.

[0108] Preparation of lung interstitial lymphocytes: The lung was first cut into small pieces of 0.5 cm and digested with collagenase I (1 mg / mL) and DNase I (10 μg / mL) for 1.5 hours. The remaining operations were similar to those for the spleen.

[0109] 2. Enzyme Linked Immuno SPOT Assay

[0110] 1) The ELISPOT plate was removed the day before and 30 μL (75% ethanol: PBS = 1:1) was added to each well for 30 seconds. The liquid was quickly shaken off and then washed with 200 μL of PBS (or sterile water) for 6 times. 2) The purified rat anti-mouse IFN-γ monoclonal antibody was diluted with sterile PBS to 5 ug / mL, and the stock solution was 0.5 mg / mL (i.e. diluted 100 times), 50 μL / well was added, and the plate was coated overnight at 4°C, and the plate was sealed with sealing film. 3) The coating antibody was shaken off and washed with sterile PBS for 6 times. 200 μL of R10 complete medium was added to each well, and the plate was sealed at 37°C for 2-4 hours. 4) During the sealing period, the spleen PBMC was separated. 5) The sealing liquid was discarded, 100 μL of spleen lymphocytes was added to each well, 5x10 5 cells / well (diluted with R10 complete medium to 5x10 6cell / mL cell suspension). Two replicates were performed for each sample. 6) Positive control (ConA, 10 pg / mL), peptides (4 pg / mL), mock (DMSO) were included in the experiment. Incubation was performed at 37 °C in a 5% CO2 incubator. 7) After 24 h, the cell suspension was discarded and the plate was washed 6 times with PBST. 8) The wash was flicked off and the plate was blotted dry on sterile paper. 9) The biotin-labeled rat anti-mouse IFN-g detection antibody was diluted 100-fold in 5% inactivated FBS / PBST, 100 pL / well was added and incubated overnight (16 h) at 4 °C. 10) The liquid was discarded and the plate was washed 6 times with 200 pL / well PBST and the wash was flicked off and the plate was blotted dry on sterile paper. 12) Streptavidin-conjugated alkaline phosphatase was prepared by diluting 1:2500 in 5% FBS / PBST, 100 pL / well was added and incubated for 2 h at 37 °C. 13) BCIP / NBT substrate was prepared and incubated for 30 min at 37 °C. 14) The plate was washed 5 times with 200 pL / well PBST and the wash was flicked off and the plate was blotted dry on sterile paper. 15) BCIP / NBT substrate was added, 100 pL / well, and the reaction was protected from light for 9 min. 16) The substrate was discarded and the plate was washed twice with water (tap water for 1 min) and the plate was air-dried before reading.

[0111] 3. Intracellular cytokine staining (ICS)

[0112] Spleen lymphocytes and lung interstitial lymphocytes were isolated from mice according to the above method and adjusted to 2 x 10 6 / 200 μL, added to 96-well cell culture plates, 200 μL per well. Specific antigen peptides were added to the above cell suspension, and the negative control wells were added with DMSO of the corresponding concentration, and the positive control was added with PMA (40 ng / mL) + ionomycin (1000 ng / mL). 37°C, 5% CO2 culture for 1-2 hours. Add diluted BFA 10 μL (brefeldin, brefeldin), final concentration 10 μg / mL, mix well. 37°C, 5% CO2 culture for 5-6 hours. Blow the cells evenly and transfer them to a flow tube. Add 1 mL of PBS wash solution. 300g centrifuge at room temperature for 10 minutes. Discard the supernatant. Add surface antibodies such as anti-mouse CD3, CD4, CD8, etc. Shake for 2-3 seconds. Stain at room temperature for 20-30 minutes in the dark. Add 1 mL of PBS wash solution, shake well and centrifuge at 300g for 10 min at room temperature. Repeat the washing once. Shake the cells evenly, then add 200 μL of cell permeabilization solution cytofix / cytoperm (BD Biosciences), mix well. 4°C, avoid light permeabilization for 20 min. Add 1 mL of membrane breaking wash solution 1x perm / wash (BD Biosciences, original solution is 10x, before use dilute with sterile distilled water to 1x), 400g centrifuge for 10 minutes, discard the supernatant. Repeat once, discard the supernatant, add intracellular antibodies such as anti-IFN-γ, mix well. 4°C avoid light staining for 30-60 minutes. Add 1 mL of 1x perm / wash, 400g centrifuge for 10 minutes. Discard the supernatant, add 1 mL of PBS wash solution. Mix well and centrifuge at 400g for 10 minutes at room temperature. Add an appropriate amount of PBS to each tube after discarding the supernatant. Shake well. Detect on C6 or BD FACSFortessa flow cytometer, then analyze with FlowJo software.

[0113] The results show that at the systemic immune level, DNA@COS nanoparticles delivered by microneedles induce higher T cell immune responses than intramuscular injection, and after the third dose of immunization on day 42, S1-specific IFN-γ + The frequency of T cell response is further increased to 5.2 times the group of pVAX-S+N@COS-IM, S2-specific IFN-γ + The frequency of T cell response is 4.5 times higher. The N-specific IFN-γ + The trend of T cell response is higher than the IM group, although there is no statistical difference. At the mucosal immune level, it is found that in the lung mucosal tissue immunized with pVAX-S+N@COS-MN, SARS-CoV-2-specific IFN-γ + The frequency of T cell response is higher Figure 10) were significantly higher than that in pVAX-S+N@COS-IM group. In addition, the frequencies of CD8+IL2 + , CD8 + TNF-α + and CD8 + IL2 + TNF-α + T cells in pVAX-S+N@COS-MN group were significantly higher than that in pVAX-S+N@COS-IM group, especially when stimulated with S1 peptide pool Figure 11 ).

[0114] The embodiments of the present application are described in detail above with reference to the drawings, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A vaccine capable of inducing a mucosal immune response, wherein the vaccine is administered via a microneedle patch, the vaccine comprising a backing and a microneedle array containing nanoparticles attached to a side of the backing; The nanoparticles include nucleic acid molecules and chitosan oligosaccharides; The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID ON: 1 or SEQ ID ON: 2; The microneedle array comprises a plurality of microneedles, wherein each microneedle comprises a matrix and nanoparticles loaded in the matrix; The matrix comprises at least one of sodium hyaluronate, polyvinyl pyrrolidone, chondroitin sulfate, sodium hydroxyethyl cellulose, collagen, sucrose, trehalose, maltose, chitosan and dextran; The backing is formed by cross-linking and / or drying an aqueous solution containing one or more of the following substances: one or more of polyethylene glycol diacrylate, silk fibroin, methacrylate gelatin, carboxymethyl cellulose, trehalose, hyaluronic acid, polylactic acid-glycolic acid copolymer, polylactic acid, galactose, polyvinyl pyrrolidone, and polyvinyl alcohol.

2. The method for preparing the vaccine according to claim 1, comprising the following steps: (1) mixing the nanoparticles with the matrix to form a mixed solution, placing the mixed solution in the forming holes of the microneedle mold and filling at least a portion of the volume of the forming holes, centrifuging to form microneedles, wherein the plurality of microneedles constitute a microneedle array; (2) Applying a solution containing a backing material to the bottom surface of the microneedle and the upper surface of the microneedle mold not covered by the microneedle to form a backing solution layer, centrifuging, and solidifying to obtain the vaccine.

3. Use of the vaccine according to claim 1 in preparing a product; The function of the product is to prevent or treat diseases related to respiratory viral infections.

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