Dendritic cell-targeted allergen nanovaccine and uses thereof

Abstract

The present application relates to a dendritic cell-targeted allergen nanovaccine. The present application discloses a nanovaccine which is internally wrapped with an allergen and externally coupled with a dendritic cell targeting molecule. The present application also provides a preparation method and use of the nanovaccine. The nanovaccine can be specifically taken up by dendritic cells, and induce cell tolerance, and is used for specific immunotherapy of allergic diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine. Specifically, the present application relates to a dendritic cell-targeted allergen nanovaccine and uses thereof. BACKGROUND

[0002] Allergic diseases have significantly increased in incidence in the past few decades and have become a global public health concern. It is urgent to develop effective prevention and treatment measures. Allergen-specific immunotherapy (AIT) is the only long-term effective treatment targeting the cause. Currently, allergen crude extract is still used as a vaccine in clinical practice. However, due to its complex composition and difficulty in achieving standardization in production, the dose cannot be accurately controlled, which seriously affects the safety and effectiveness of treatment and limits its clinical application. Therefore, it is urgent to develop a safer, more effective and economical vaccine.

[0003] Dendritic cells (DCs) are professional antigen-presenting cells (APCs) and are currently the strongest APCs known to present antigens in vivo. They are also the only APCs that can activate naive T cells and play an important role in innate and adaptive immune responses. DCs are widely distributed in the dermis, mucosal lamina propria and blood, and express pattern recognition receptors (such as Toll-like receptors (TLRs), C-type lectin receptors (CLRs), costimulatory molecules (CD80, CD86, CD40, etc.) and adhesion molecules) on the surface, which are involved in DC recognition and antigen presentation, migration and T cell activation. Currently, a variety of DC subtypes have been identified, including plasmacytoid DCs (pDCs), conventional DCs (cDCs) and inflammatory DCs (iDCs). Most DCs in the human body are in a non-mature state, and antigen stimulation can induce DC maturation, upregulate the expression of costimulatory molecules and chemokine receptors on the surface, and increase the secretion of inflammatory cytokines IL-1β, IL-12, IL-6 and TNF. These mature DCs enter the lymph nodes and activate T cells are effector T cells.

[0004] Moreover, DCs play an important role in maintaining central and peripheral tolerance, and they induce Treg cells to achieve immune tolerance. Immature DCs (imDCs) ingest antigens to form tolerogenic DCs (tolDCs), which express low levels of costimulatory molecules on their surface and can induce T cells to become Tregs, thereby participating in immune tolerance. Studies have also shown that increasing the uptake of allergen vaccines by APCs can promote the formation of immune tolerance.

[0005] However, there is no allergen vaccine in the art that can target DC cells to induce immune tolerance.

[0006] Therefore, there is a need in the art to develop an allergen vaccine that targets DC cells. SUMMARY

[0007] The purpose of the present application is to provide a dendritic cell-targeted allergen nanovaccine, as well as a preparation method and use thereof.

[0008] In a first aspect of the present application, a dendritic cell (DC cell)-targeted vaccine preparation is provided, which comprises:

[0009] (1) a vaccine antigen;

[0010] (2) a nanocarrier; and

[0011] (3) a DC cell-targeting molecule;

[0012] wherein the vaccine antigen is encapsulated inside the nanocarrier, and the DC cell-targeting molecule is modified on the outer surface of the nanocarrier.

[0013] In another preferred embodiment, the vaccine antigen is an allergen.

[0014] In another preferred embodiment, the allergen is selected from the group consisting of a crude allergen extract, a naturally purified allergen, a single allergen extract, a recombinant allergen, or a combination thereof.

[0015] In another preferred embodiment, the allergen is a natural extract of mites, German cockroaches, mugwort, Aspergillus fumigatus, dog dander, cat dander, shrimp, wheat, peanuts, peaches, fish, crabs, shrimp, chickens, eggs, milk, sesame, soybeans, common ragweed, mugwort, goosefoot, and salsify, or a single allergen extract thereof, or a recombinant allergen component thereof, or a combination thereof.

[0016] In another preferred embodiment, the nanocarrier is a polymeric nanoparticle, a liposome, a mesoporous silica nanoparticle, a PLGA nanoparticle, or a combination thereof.

[0017] In another preferred embodiment, the DC cell-targeting molecule recognizes and binds to a DC cell-specific receptor.

[0018] In another preferred embodiment, the DC cell is an immature DC cell (imDC).

[0019] In another preferred embodiment, the DC cell specific receptor is dendritic cell specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), preferably human DC-SIGN.

[0020] In another preferred embodiment, the DC cell targeting molecule is selected from the group consisting of a protein, a peptide, a polysaccharide, or a combination thereof.

[0021] In another preferred embodiment, the DC cell targeting molecule is selected from the group consisting of a human antibody, a non-human antibody (e.g., a murine antibody), a chimeric antibody, an antibody Fab fragment, a single chain antibody, a nanobody, a ligand, and a combination thereof.

[0022] In another preferred embodiment, the DC cell targeting molecule is a natural ligand of DC-SIGN.

[0023] In another preferred embodiment, the DC cell targeting molecule is Lewis polysaccharide.

[0024] In another preferred embodiment, the DC cell targeting molecule is selected from the group consisting of Lewis X , Lewis Y , Lewis b , and Lewis a , and a combination thereof.

[0025] In another preferred embodiment, the DC cell targeting molecule is selected from the group consisting of an anti-human DC-SIGN antibody, an anti-human DC-SIGN antibody Fab fragment, a humanized anti-human DC-SIGN antibody, a single chain anti-human DC-SIGN antibody, an anti-human DC-SIGN nanobody, a ligand of DC-SIGN, preferably high mannose oligosaccharide-containing and fucose-containing Lewis polysaccharide (Lewis X , Lewis Y , Lewis b , and Lewis a .

[0026] In another preferred embodiment, the DC cell targeting vaccine formulation is a DC cell targeting molecule-nanocarrier-allergen triplex.

[0027] In another preferred embodiment, the DC cell targeting molecule is modified on the outer surface of the nanocarrier by covalent linkage or hydrophobic-hydrophilic interaction, preferably by covalent linkage.

[0028] In another preferred embodiment, the method of preparing the DC cell targeting vaccine formulation comprises the steps of:

[0029] (a) linking the DC cell targeting molecule to the nanocarrier to obtain a DC cell targeting molecule-nanocarrier;

[0030] (b) using the DC cell targeting molecule-nanocarrier obtained in step (a) to encapsulate a vaccine antigen to obtain a DC cell targeting vaccine formulation.

[0031] In a second aspect of the present application, there is provided a method for preparing a DC cell targeting vaccine formulation as described in the first aspect of the present application, the method comprising the steps of:

[0032] (a) linking the DC cell targeting molecule to the nanocarrier to obtain a DC cell targeting molecule-nanocarrier;

[0033] (b) using the DC cell targeting molecule-nanocarrier obtained in step (a) to encapsulate a vaccine antigen to obtain a DC cell targeting vaccine formulation.

[0034] In a third aspect of the present application, there is provided a vaccine composition comprising a DC cell targeting vaccine formulation as described in the first aspect of the present application.

[0035] In another preferred embodiment, the vaccine composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or a combination thereof.

[0036] In another preferred embodiment, the vaccine composition further comprises another drug, preferably a drug selected from the group consisting of a targeting monoclonal antibody (anti-IgE antibody, IL-4 antibody, etc.), an antihistamine drug, an immunosuppressant, a hormone, or a combination thereof.

[0037] In another preferred embodiment, the vaccine composition is monovalent or multivalent, i.e., the vaccine composition comprises a vaccine against one or more vaccine antigens.

[0038] In another preferred embodiment, the vaccine composition is in a dosage form of a liquid, a solid, or a gel.

[0039] In another preferred embodiment, the vaccine composition is administered by a method selected from the group consisting of subcutaneous injection, intradermal injection, intralymphatic injection, oral administration, epicutaneous immunotherapy, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, and oral and nasal cavity spray and aerosol inhalation.

[0040] In a fourth aspect of the present application, there is provided an immunotolerant DC cell, which is a DC cell immunologically activated by a DC cell targeting vaccine formulation as described in the first aspect of the present application.

[0041] In another preferred embodiment, the DC cell is an immature DC cell (imDC).

[0042] In another preferred embodiment, the activation is in vitro activation.

[0043] In another preferred embodiment, the in vitro activation comprises culturing the DC cell in the presence of the vaccine formulation for a period of time (e.g., 6-48 hours, preferably 20 hours) to obtain the immune activated DC cell.

[0044] In another preferred embodiment, the immune tolerant DC cell is capable of inducing Treg cell production.

[0045] In a fifth aspect of the present application, there is provided a cell preparation comprising the immune tolerant DC cell as described in the fourth aspect of the present application.

[0046] In another preferred embodiment, the cell preparation is a liquid preparation comprising viable cells.

[0047] In another preferred embodiment, the cell preparation is for intravenous administration.

[0048] In a sixth aspect of the present application, there is provided the use of the vaccine formulation targeting DC cells as described in the first aspect of the present application, or the vaccine composition as described in the third aspect of the present application, or the immune tolerant DC cell as described in the fourth aspect of the present application, or the cell preparation as described in the fifth aspect of the present application, for the preparation of a vaccine and / or a medicament for the prevention and / or treatment of an allergic disease.

[0049] In another preferred embodiment, the allergic disease comprises allergic asthma, allergic rhinitis, allergic dermatitis, or a combination thereof.

[0050] In another preferred embodiment, the allergic disease is an allergic disease caused by an allergen selected from the group consisting of mite, German cockroach, mugwort, Aspergillus fumigatus, dog dander, cat dander, shrimp, wheat, peanut, peach, fish, crab, shrimp, chicken, egg, milk, sesame, soybean, common ragweed, mugwort, goosefoot, common ragweed, and a combination thereof.

[0051] In a seventh aspect of the present application, there is provided a method for preparing an immune tolerant DC cell, the method comprising the steps of:

[0052] culturing a DC cell in the presence of the vaccine formulation as described in the first aspect of the present application to obtain the immune tolerant DC cell.

[0053] In an eighth aspect of the present application, there is provided a method for preventing and / or treating allergic diseases, comprising the step of administering to a subject in need thereof a DC cell targeted vaccine formulation as described in the first aspect of the present application, or a vaccine composition as described in the third aspect of the present application, or an immune-tolerant DC cell as described in claim 4, or a cell preparation as described in the fifth aspect of the present application, thereby preventing and / or treating allergic diseases.

[0054] In another preferred embodiment, the allergic diseases include allergic asthma, allergic rhinitis, allergic dermatitis, or a combination thereof.

[0055] In another preferred embodiment, the allergic diseases are allergic diseases caused by allergens selected from the group consisting of mites, German cockroaches, mugwort, Aspergillus fumigatus, dog dander, cat dander, shrimps, wheat, peanuts, peaches, fish, crabs, shrimps, chickens, eggs, milk, sesame, soybeans, common ragweed, mugwort, goosefoots, burrs, and combinations thereof.

[0056] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (e.g. in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0057] The following drawings are used to illustrate specific embodiments of the present application, and are not intended to limit the scope of the present application as defined by the claims.

[0058] Figure 1 The molecular structures of various Lewis are shown.

[0059] Figure 2 The intracellular fluorescence of Le X -lip-Der f2 targeting experiments, in which (A) is the fluorescence of different Der f2-FITC formulations incubated with THP-1 cells, and the vertical coordinate is the mean fluorescence intensity of FITC; (B) Der f2-FITC formulations were incubated with CD34 + DC cells, and the intracellular fluorescence was detected by laser confocal microscopy. The purple color is the DAPI-stained nucleus, the green color is the FITC fluorescence, and the Merge is the fusion of purple fluorescence and green fluorescence. *p<0.05.

[0060] Figure 3 The levels of cytokines in the supernatant of cells incubated with different Der f2 formulations and CD34+ DCs are shown.

[0061] Figure 4Different Der f2 preparations were shown to activate basophil cells sensitized with dust mite-specific IgE. Different Der f2 preparations were incubated with heparin-anticoagulated blood from 3 dust mite patients, and the percentage of CD63+ basophil cells out of basophil cells was determined. DETAILED DESCRIPTION

[0062] The inventors have developed, for the first time, a dendritic cell-targeted allergen nanovaccine. Specifically, the inventors have constructed a nanovaccine which encapsulates an allergen inside and couples a dendritic cell-targeting molecule outside. The nanovaccine can be specifically taken up by dendritic cells, inducing cell tolerance, and used for specific immunotherapy of allergic diseases. The inventors have also provided a preparation method and use of the nanovaccine. On this basis, the present application has been completed.

[0063] The present application uses a nanomaterial with good biocompatibility as a carrier, encapsulates an allergen, and couples a dendritic cell-targeting molecule outside, to construct a dendritic cell-targeting molecule-nano-allergen trimer. Experiments have shown that the allergen nanovaccine particles constructed by the present application can carry allergens to DCs expressing DC-SIGN, promote the uptake of allergens by DCs, and thus promote the induction of Treg cells and the formation of immune tolerance, and improve the therapeutic effect of the vaccine. The allergen nanovaccine particles can also avoid activating effector cells, and reduce the side effects of allergic reactions.

[0064] TERMS

[0065] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used have the meaning commonly understood by one of ordinary skill in the art to which the present application pertains. Before the present application is described, it is to be understood that this application is not limited to the particular methodology and experimental conditions described, as such can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present application will be limited only by the appended claims.

[0066] The term "about" can mean a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, varying depending on how the value or composition is measured or determined.

[0067] The term "optionally" or "optional" means that the subsequently described event or circumstance can or can not occur.

[0068] The terms "comprising" (or its derivatives) such as "comprise", "comprises" or "comprised" can be open-ended or closed-ended depending on the context in which it is used. In the context of describing a composition, the term "comprising" can mean that, with respect to a given compound, additional compound(s) can also be present. In the context of describing a process, the term "comprising" can mean that, with respect to a given process, additional process steps can also be performed.

[0069] It should be understood that, for any method described herein that includes more than one step, the order of the steps is not necessarily limited to the order described in these embodiments.

[0070] Allergic diseases and allergens

[0071] The etiology of allergic diseases is not fully understood. According to current research, it is generally divided into three stages: sensitization, challenge, and effect. In the sensitization stage, allergens enter the body and are taken up by dendritic cells (DCs), processed and presented to CD4+ T cells, which deliver signals to B cells, and activated B cells produce allergen-specific IgE antibodies. IgE antibodies can bind to the IgE high-affinity receptor on the surface of eosinophils / mast cells, sensitizing the cells; when the same allergen enters the body again, it can bind to the IgE that has been bound to the surface of eosinophils / mast cells, causing two or more IgE high-affinity receptors to bridge, activating eosinophils / mast cells, and releasing active mediators such as histamine, leukotrienes, platelet-activating factor, bradykinin, prostaglandins, interleukins, etc. Thus causing smooth muscle contraction, capillary dilation, increased permeability, and leukocyte chemotaxis to the local area. Ultimately causing bronchial spasm, mucosal congestion, edema, inflammation, and related reactions in the digestive tract.

[0072] The allergen of the present application refers to an antigen that can cause an allergic reaction in humans, also known as a sensitizing antigen or allergen.

[0073] The present application uses allergens as vaccine antigens. Allergens that can be used in the vaccine formulations of the present application include, but are not limited to, crude allergen extracts, naturally purified allergens, single allergen extracts, recombinant allergens, or combinations thereof.

[0074] In another preferred embodiment, the allergen is a natural extract of mites, German cockroaches, mugwort, Aspergillus fumigatus, dog dander, cat dander, shrimp, wheat, peanuts, peaches, fish, crabs, shrimp, chickens, eggs, milk, sesame, soybeans, common ragweed, mugwort, goosefoot, and sow thistle, or a single allergen extract thereof, or a recombinant allergen component thereof, or combinations thereof.

[0075] Dendritic cells

[0076] Dendritic cells (DCs) are professional antigen-presenting cells. DCs can induce immune tolerance by inducing Treg cells. Immature DCs (imDCs) take up antigens to form tolerogenic DCs, which have low expression of costimulatory molecules on their surface and can induce T cells to become Tregs, participating in immune tolerance.

[0077] DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin, CD209) is a CLR specifically expressed on the surface of DCs, which can bind to antigens (including invading pathogen antigens and body glycoproteins), promote the uptake and signal transmission of antigens, and participate in immune regulation, and is an ideal DC target. High-mannose oligosaccharides and Lewis X , Lewis Y , Lewis b and Lewis a polysaccharides containing fucose (Lewis X , Lewis Y , Lewis b and Lewis a ) are its natural ligands.

[0078] Lewis antigens belong to the A, B, H, Lewis blood group family, and are named because of the expression of a group of glycoproteins called Lewis antigens on the surface of red blood cells. They are mainly synthesized in the epidermal layer cells. Lewis X (CAS No. 71208-06-5), Lewis Y (CAS No. 82993-43-9), Lewis b (CAS No. 80081-06-7), Lewis a (CAS No. 56570-03-7).

[0079] In the present application, by constructing an allergen nanovaccine coupled with a DC cell targeting molecule, the allergen vaccine is directly guided to DCs, which can promote the effective uptake of DCs to allergens, thereby promoting the induction of Treg cells and the formation of immune tolerance, and improving the therapeutic effect of the vaccine. Directly guiding the vaccine to DCs can also avoid the activation of effector cells by allergens and reduce the side effects of allergic reactions after the use of the vaccine.

[0080] In the nanovaccine of the present application, the DC cell targeting molecule that can be used includes but is not limited to: protein, peptide, polysaccharide, or a combination thereof.

[0081] In another preferred embodiment, the DC cell targeting molecule is selected from the group consisting of: an anti-human DC-SIGN antibody, an anti-human DC-SIGN antibody Fab fragment, a humanized anti-human DC-SIGN antibody, a single-chain anti-human DC-SIGN antibody, an anti-human DC-SIGN nanobody, a ligand of DC-SIGN, preferably Lewis X , Lewis Y , Lewis b and Lewis a polysaccharides containing fucose (Lewis X , Lewis Y , Lewis b and Lewis a ).

[0082] The dendritic cell-targeted allergen vaccine of the present application can be used for preventing and / or treating allergic diseases, and has a clinical application prospect. The dendritic cell-targeted allergen vaccine of the present application can be used for preventing and / or treating allergic diseases, and has a clinical application prospect.

[0083] Nanocarriers

[0084] The application of nanomaterials in the biological field provides a broad platform for the development of drugs. Nanocarriers have the advantages of good biocompatibility, improved protein stability, slow release, targeting, etc., and are widely used in the field of biological pharmaceuticals. Currently commonly used nanocarriers include liposomes, polymer particles, dendrimer polymers, nanocarbon tubes, and magnetic nanoparticles.

[0085] In the nanovaccine of the present application, the nanocarriers that can be used include, but are not limited to, polymer nanoparticles, liposomes, mesoporous silica nanoparticles, PLGA nanoparticles, or a combination thereof.

[0086] In a preferred embodiment of the present application, the nanocarrier used is a liposome.

[0087] Vaccine preparation targeting DC cells

[0088] As used herein, the terms "vaccine preparation targeting DC cells" and "vaccine preparation of the present application", "nanovaccine of the present application", "allergen nanovaccine" are used interchangeably and all refer to the vaccine preparation provided in the first aspect of the present application.

[0089] The present application provides a vaccine preparation targeting dendritic cells (DC cells), which contains:

[0090] (1) a vaccine antigen;

[0091] (2) a nanocarrier; and

[0092] (3) a DC cell targeting molecule;

[0093] wherein the vaccine antigen is encapsulated inside the nanocarrier, and the DC cell targeting molecule is modified on the outer surface of the nanocarrier.

[0094] The allergen loaded in the vaccine preparation of the present application can be an allergen crude extract, a naturally purified allergen, a single allergen extract, a recombinant allergen, or a combination thereof. Among them:

[0095] "allergen crude extract" refers to the allergen components extracted from the allergen raw material by chemical extraction method, which has complex components, including both sensitizing components and non-sensitizing components;

[0096] "naturally purified allergen" refers to the allergen sensitizing component obtained by separation and purification from the allergen raw material;

[0097] "single allergen extract" refers to an extract containing only one kind of allergen;

[0098] "Recombinant allergen" refers to an allergen component synthesized by molecular biology technology.

[0099] The method for preparing the DC cell-targeted vaccine preparation of the present application comprises the steps of:

[0100] (a) connecting the DC cell-targeting molecule and the nanocarrier to obtain a DC cell-targeting molecule-nanocarrier targeting DC cells;

[0101] (b) using the DC cell-targeting molecule-nanocarrier obtained in step (a) to encapsulate a vaccine antigen to obtain a DC cell-targeted vaccine preparation.

[0102] Tolerogenic DC cells (tolDC)

[0103] Studies based on tolDC have been carried out in animal models of autoimmune diseases (type I diabetes, rheumatoid arthritis, multiple sclerosis, etc.) and organ transplantation, some of which are even in the clinical research stage, showing good therapeutic effect. The current treatment strategy is mainly to separate patient mononuclear cells in vitro, add tolerance inducers during the induction of DC and load antigens to make them into tolDC, and then reinfuse tolDC into the autologous body for treatment.

[0104] Based on this, the present application also provides a tolerogenic DC cell and a cell preparation containing the cell, wherein the DC cell is a DC cell immunologically activated by the DC cell-targeted vaccine preparation of the first aspect of the present application. The tolerogenic DC cell of the present application can induce the production of Treg cells, thereby achieving immunological tolerance.

[0105] Vaccine composition and cell preparation

[0106] The present application provides a vaccine composition containing the DC cell-targeted vaccine preparation of the first aspect of the present application.

[0107] The present application also provides a cell preparation containing the tolerogenic DC cell of the fourth aspect of the present application.

[0108] The vaccine composition of the present application comprises an immunologically effective amount of the vaccine preparation of the present application, and the vaccine can be monovalent or multivalent.

[0109] "Immunologically effective amount" refers to an amount administered to an individual in a single dose or part of a continuous dose that is effective for treatment or prevention. The amount can be determined according to the health and physiological conditions of the individual being treated, the category of the individual being treated (such as a human), the ability of the individual's immune system to synthesize antibodies, the degree of protection required, the formulation of the vaccine, the evaluation of the medical condition by the treating physician, and other relevant factors. It is expected that the amount will be within a relatively wide range, which can be determined by routine experiments.

[0110] The compositions or formulations of the present application can also contain a pharmaceutically acceptable carrier, diluent, or excipient. The term "pharmaceutically acceptable carrier" means a carrier for administration of a therapeutic agent, such as a vaccine formulation, cell preparation, or other therapeutic agent of the present application. This term refers to carriers that are nontoxic to the subject to be treated and that do not interfere with the effectiveness of the biological activity of the biological treatment being administered. The pharmaceutically acceptable carrier can include liquids such as water, saline, glycerol, and ethanol. Additionally, the carrier can contain auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like. Generally, the compositions can be presented for injection as a liquid solution or suspension, or as a solid form suitable for dissolution or suspension in liquid prior to injection. Such carriers are well known in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients is found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

[0111] Routes of administration

[0112] The vaccine compositions and / or cell preparations of the present application can be administered directly to a subject. The subject can be a human or a non-human mammal, preferably a human. The vaccine formulations of the present application can be administered directly to an individual using known methods. The vaccines are generally administered using the same routes of administration and / or mimicking the path of infection of the pathogen as conventional vaccines.

[0113] Routes of administration of the pharmaceutical compositions or vaccine compositions of the present application include, but are not limited to, intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, intravaginal, oral, or other parenteral routes of administration. Preferably, the route of administration includes intradermal injection, subcutaneous injection, intralymphatic injection, oral, nasal, epicutaneous immunotherapy. If desired, the route of administration can be combined or adjusted depending on the disease. The vaccine compositions can be administered in a single dose or multiple doses and can include administration of a booster dose to elicit and / or maintain immunity.

[0114] The vaccine compositions or cell preparations of the present application are administered to a human in a safe and effective amount. The appropriate vaccine dose varies for different allergens, but generally a safe and effective amount of the vaccine is at least about 5 μg of antigen per dose and, in most cases, no more than about 100 μg of antigen per dose, preferably the dose is about 5 μg to about 20 μg of antigen per dose. A safe and effective amount of the cell preparation is generally at least about 10 6 million cells per dose and, in most cases, no more than about 10 8 million cells per dose, preferably the dose is about 10 6 -10 7Cells / dose. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0115] The main advantages of this invention include:

[0116] 1) The allergen nanovaccine constructed in this invention can directionally carry allergens to DCs expressing DC-SIGN, promote the uptake of allergens by DCs, thereby promoting the induction of Treg cells and the formation of immune tolerance, and improving the therapeutic effect of the vaccine.

[0117] 2) The allergen nanovaccine constructed in this invention can avoid allergen activation of effector cells and reduce the side effects of allergic reactions.

[0118] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0119] Example 1: Encapsulation of dust mite allergen coupled with Lewis X Preparation of nano-vaccines

[0120] Dioleoylphosphatidylcholine (DOPC) is coupled to avidin (SA) via EDC coupling to obtain SA-DOPC; then biotin-labeled Lewis... X (bio-Lex) reaction for 2 hours (37℃) yielded Le X -DOPC. Le X - DOPC reacts with recombinant house dust mite allergen class II component (Der f2), and impurities are removed by dialysis to obtain the coupled Lewis... X Lex-liposome-Der f2 (abbreviated as Le) is a liposomal vaccine containing Der f2. X -lip-Der f2).

[0121] Results: The particle size of the allergen nanoparticle vaccine of the present invention was approximately 100 nm. The allergen nanoparticles of the present invention were spherical as observed under a transmission electron microscope. The protein loading of the allergen nanoparticle vaccine was approximately 0.1 mg of Der f2 protein per milligram of liposomes.

[0122] Example 2: Targeting effect of allergen nanovaccines on dendritic cells

[0123] Der f2 was labeled with fluorescein isothiocyanate (FITC) to synthesize FITC-conjugated Der f2 (Der f2-FITC), liposomes encapsulating Der f2-FITC (Der f2-FITC-lip), and Lewis-conjugated Der f2. X Liposomes loaded with Der f2-FITC (Le X -lip-Der f2-FITC), using human monocytic leukemia cell lines THP-1 and CD34 expressing DC-SIGN, respectively. + Dendritic cells derived from stem cells (CD34) + Using DC as the research object, flow cytometry and laser confocal microscopy were used to detect the cells' ability to take up liposomes and Der f2.

[0124] Results: The results are as follows Figure 2 As shown. Flow cytometry results showed that, regardless of CD34 + In DC or THP-1, Le X The proportion of FITC-positive cells in the Lewis-lip-Der f2-FITC treatment group was significantly higher than that in the Der f2-FITC and Der f2-FITC-lip groups, indicating that Lewis X Coupled targeting of Der f2 to DC-SIGN promotes uptake of Der f2 by DCs. Results from laser confocal microscopy were consistent with those from flow cytometry.

[0125] Example 3: Allergen nanovaccine induces DC tolerance

[0126] Der f2, liposomes loaded with Der f2 (Der f2-lip), and Lewis were respectively conjugated. X Liposomes encapsulating Der f2 (Le X -lip-Der f2) was incubated with CD34+DC for 20 h, and the levels of cytokines were detected by flow cytometry multifactor detection microsphere immunoassay system (CBA).

[0127] Result: As Figure 3 As shown, compared with the Der f2 group and the Der f2-lip group, Le X The levels of IL-6, IL-4, and TNF-α in dendritic cells (DCs) treated with Lex-lip-Der f2 were significantly decreased. This result suggests that Lex-lip-Der f2 may reduce DC activation.

[0128] Example 4: DC-induced Treg cell formation under the action of allergen nanovaccines

[0129] Simultaneously, CD34+ stem cells and peripheral blood mononuclear cells (PBMCs) were isolated from the same donor (3-5 patients). The former induced CD34+ stem cells to be absorbed into the blood cells. + DC formation, the latter separating CD4 + Naive T cells.

[0130] CD34 + DC was coupled with Der f2, Der f2-encapsulated liposomes (Der f2-lip), and Lewis, respectively. X Liposomes carrying Derf2 (Le X -lip-Der f2) Incubate for 20 hours, CD4 + Naive T cells were labeled with CFSE, and then dendritic cells (DCs) stimulated with different Der f2 agents were mixed with CFSE-labeled CD4+. + Naive T cells were mixed at a 1:5 ratio and incubated for 6 days. Cells were collected and labeled with ECD-anti-human CD4 antibody, PE-anti-human CD127 antibody, PC5-anti-human CD45 antibody, and PC7-anti-human CD25 antibody. Flow cytometry was used to analyze the CD4+ antibody. + CD25 + CD127 - CD45 + SS low Treg cell population proliferation and Treg ratio.

[0131] Result: Le X The -lip-Der f2 treatment group showed a trend of higher Treg cells than the Der f2 and Der f2-lip groups.

[0132] Example 5: Improved in vitro safety of allergen nano-vaccines

[0133] The safety of allergy vaccines was evaluated in vitro using a basophil activation assay. Different house dust mite allergen vaccines were incubated with heparin-anticoagulated blood from patients who were positive for house dust mite allergen-specific IgE. The activation level of basophils was analyzed by detecting the proportion of CD63.

[0134] Results: In three independent experiments, it was observed that under the same concentration of Der f2 stimulation, compared with Der f2 and Der f2-lip, Le... X -lip-Der f2 significantly reduced basophil activation levels. This indicates that Le X -lip-Der f2 offers higher security. Figure 4 ).

[0135] All documents referred to in the present application are incorporated herein by reference as if each were individually incorporated. In addition, it is to be understood that the application can be carried out by specifically different embodiments and that each disclosed embodiment can be implemented with or without the corresponding use of the other embodiments. Other embodiments will occur to those skilled in the art upon consideration of this disclosure or can be learned from practice of the application. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, and all changes coming within the meaning and equivalency range of the appended claims are intended to be embraced therein.

Claims

1. A vaccine formulation targeting dendritic cells, i.e., DC cells, characterized in that, The vaccine formulation targeting DC cells contains: (1) Vaccine antigen, wherein the vaccine antigen is an allergen, and the allergen is recombinant house dust mite allergen class II component Derf2; (2) Nanocarriers; and (3) DC cell targeting molecule, wherein the DC cell targeting molecule is Lewis X ; The vaccine antigen is encapsulated inside a nanocarrier, and the DC cell targeting molecule is modified on the outer surface of the nanocarrier. The vaccine formulation promotes the formation of immune tolerance; The nanocarrier is a liposome.

2. The vaccine formulation as described in claim 1, characterized in that, The DC cell-targeting molecules are modified on the outer surface of the nanocarrier through covalent linkage or hydrophobic-hydrophilic interactions.

3. The vaccine formulation as described in claim 1, characterized in that, The liposomes are dioleoylphosphatidylcholine SA-DOPC containing avidin.

4. The vaccine formulation as described in claim 1, characterized in that, The vaccine formulation is prepared using a method comprising the following steps: (S1) Dioleoylphosphatidylcholine (DOPC) was coupled with avidin (SA) via EDC coupling to obtain SA-DOPC. (S2) Add biotin-labeled Lewis X The reaction was carried out at 37°C for 2 hours to obtain Le. X -DOPC; (S3)Le X -DOPC reacts with recombinant house dust mite allergen class II component Der f2, and impurities are removed by dialysis to obtain the vaccine formulation.

5. The method for preparing a vaccine formulation targeting DC cells as described in claim 1, characterized in that, The method includes the following steps: (a) Connect the DC cell targeting molecule and the nanocarrier to obtain a DC cell targeting molecule-nanocarrier targeting DC cells; (b) The vaccine antigen is encapsulated using the DC cell targeting molecule-nanocarrier obtained in step (a) to obtain the DC cell-targeting vaccine formulation of claim 1.

6. A vaccine composition, characterized in that, The vaccine composition contains the vaccine formulation targeting DC cells as described in claim 1.

7. An immune-tolerant DC cell, characterized in that, The DC cells mentioned are DC cells that have been immune-activated by the vaccine formulation targeting DC cells as described in claim 1.

8. A cell preparation, characterized in that, The cell preparation contains immune-tolerant DC cells as described in claim 7.

9. The use of the vaccine formulation targeting DC cells as described in claim 1, or the vaccine composition as described in claim 6, or the use of the immune-tolerant DC cells as described in claim 7, characterized in that, Used in the preparation of vaccines and / or drugs for treating dust mite allergies.

10. A method for preparing immune-tolerant DC cells, the method comprising the steps of: DC cells are cultured in the presence of the vaccine formulation as described in claim 1 to obtain the immune-tolerant DC cells.

Images