Exosome-like body co-loaded with antigen and trichosanthin, preparation method of exosome-like body, antigen-adjuvant integrated vaccine preparation and application of antigen-adjuvant integrated vaccine preparation

Abstract

The invention relates to an antigen and trichosanthin (TCS) co-loaded exosome, a preparation method thereof, an antigen adjuvant integrated vaccine preparation and application. The exosome comprises a cell membrane vesicle, an antigen unit combined on the surface of the cell membrane vesicle, and a trichosanthin unit combined on the surface of the cell membrane vesicle. The exosome can effectively induce generation of antigen-specific antibodies and is beneficial to reduction of off-target effects. The exosome can be used for preparing an antigen adjuvant integrated vaccine preparation, and can be further applied to preparing a medicine for inducing the neutralization of a specific antibody. According to the present invention, the antigen unit of the antibody can be SARS-CoV-2RBD, and the corresponding exosome-like body can effectively induce the maturation of the dendritic cells in vitro, and can effectively induce the generation of the SARS-CoV-2RBD specific antibody in vivo, such that the SARS-CoV-2 infection can be effectively resisted;

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, and in particular to an exosome-like vesicle co-loaded with an antigen and trichosanthin (TCS) and a preparation method thereof, and an antigen-adjuvant integrated vaccine preparation and application thereof. Background Art

[0002] The main symptoms of COVID-19 (Corona Virus Disease 2019) caused by SARS-CoV-2 include high fever, myalgia, dyspnea, and lung inflammation. Patients with severe lung damage may develop Acute Respiratory Distress Syndrome (ARDS), which has a high mortality rate. Vaccines are still the most effective means of prevention for diseases caused by the new coronavirus. However, the continuous mutation of the virus and the prevalence of mutant strains have made breakthrough infections a common phenomenon. Traditional subunit vaccines are safe, but they are time-consuming and costly to produce, require strict quality control, and are difficult to achieve large-scale immunization when new mutant strains break out. Therefore, it is currently an urgent need to develop a new vaccine and immunization technology that is efficient, safe, more economical, and more productive. Summary of the invention

[0003] Based on this, the purpose of the present invention includes providing an exosome-like substance co-loaded with antigen and trichosanthin (TCS) and a preparation method thereof. The exosome-like substance co-loaded with antigen and TCS can effectively induce the production of antigen-specific antibodies and is conducive to reducing off-target effects. The exosome-like substance co-loaded with antigen and TCS can be used to prepare an antigen-adjuvant integrated vaccine preparation, and can further be used to prepare a drug capable of inducing specific antibody neutralization.

[0004] In a first aspect of the present invention, there is provided an exosome-like substance co-carrying an antigen and trichosanthin, which comprises a cell membrane vesicle, an antigen unit bound to the surface of the cell membrane vesicle, and a trichosanthin unit bound to the surface of the cell membrane vesicle.

[0005] In some embodiments, the exosome-like particles satisfy one or more of the following characteristics:

[0006] The antigenic unit is the SARS-CoV-2 receptor binding domain;

[0007] The cell source of the cell membrane vesicles is selected from human embryonic kidney cells HEK 293T;

[0008] The average particle size of the cell membrane vesicles is 140-150 nm (optionally 144 nm);

[0009] In the exosome-like vesicles co-loaded with antigen and trichosanthin, the content ratio of the trichosanthin unit to the total protein in the cell membrane vesicles is (0.01-0.1) μmol / mg.

[0010] In some embodiments, the antigen unit is bound to the surface of the cell membrane vesicle through the transmembrane domain of the viral glycoprotein, wherein the antigen unit is bound to one side of the transmembrane domain of the viral glycoprotein, and the transmembrane domain passes through the membrane structure of the cell membrane vesicle.

[0011] In some embodiments, the exosome-like particles satisfy one or more of the following characteristics:

[0012] A linker unit may or may not be provided between the antigen unit and the transmembrane domain of the viral glycoprotein, wherein the linker unit has a length of 8 to 12 (optionally 10) amino acids;

[0013] The transmembrane domain of the viral glycoprotein is also connected to the intracellular domain of the viral glycoprotein, and the intracellular domain of the viral glycoprotein is located inside the cell membrane vesicle;

[0014] The viral glycoprotein is ZEBOV viral glycoprotein.

[0015] In some embodiments, the trichosanthin unit is bound to the surface of the cell membrane vesicle via a lipid unit, and the lipid unit is inserted into the membrane structure of the cell membrane vesicle.

[0016] In some embodiments, the exosome-like particles satisfy one or more of the following characteristics:

[0017] The lipid unit is selected from one or more of distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine;

[0018] The trichosanthin unit is connected to the lipid unit via a polyethylene glycol unit, the weight average molecular weight of the polyethylene glycol unit is 1800Da to 2200Da (optionally 2000Da), and the polyethylene glycol unit is connected to the sulfhydryl site in the trichosanthin unit;

[0019] The trichosanthin unit is connected to the lipid unit via a thioether bond formed by a maleimide group and a thiol group.

[0020] In a second aspect of the present invention, the present invention provides a method for preparing exosome-like vesicles co-loaded with antigen and trichosanthin, comprising the following steps:

[0021] A viral vector comprising a coding sequence of the antigen unit is obtained by using a gene sequence including a signal peptide, an antigen unit, a transmembrane domain and an intracellular domain of a viral glycoprotein in sequence, and then transfected into a host cell to obtain an engineered cell expressing the antigen unit on the cell membrane surface; wherein a connecting unit is provided or not provided between the antigen unit and the transmembrane domain of the viral glycoprotein, and the length of the connecting unit is 8 to 12 (optionally 10) amino acids;

[0022] Cultivating the engineered cells, and subjecting the culture to cell disruption, differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface;

[0023] The lipid-modified trichosanthin is co-incubated with the cell membrane vesicles with the antigen unit bound to the surface to obtain exosome-like vesicles carrying the antigen unit and the trichosanthin unit.

[0024] In some embodiments, the preparation method meets one or more of the following characteristics:

[0025] The viral vector is selected from a lentiviral vector;

[0026] The host cell is selected from HEK 293T cells;

[0027] The lipid-modified trichosanthin has the following structure: lipid unit-polyethylene glycol unit-trichosanthin unit;

[0028] In the step of culturing the engineered cells, culture conditions suitable for the growth of the engineered cells are adopted;

[0029] In the step of culturing the engineered cells, the culturing is terminated when the cells grow to 70%-90%;

[0030] The cell disruption includes firstly hypotonic cell disruption and then ultrasonic cell disruption; the hypotonic cell disruption includes: placing the cells in a hypotonic lysis solution and stirring at 4°C for 2 hours, wherein the hypotonic lysis solution is a 0.1×PBS buffer solution containing a protease inhibitor; the ultrasonic cell disruption method is: 200W ultrasonic for 3 minutes, interval 3s, and ultrasonic for 3s;

[0031] The differential centrifugation comprises: first centrifuging at 10,000×g for 10 min at 4°C, and then centrifuging the supernatant at 100,000×g for 60 min at 4°C;

[0032] The extrusion comprises: sequentially extruding using liposome extrusion membranes with pore sizes of 1 μm, 0.4 μm and 0.2 μm, and the number of extrusions of each membrane with a pore size is 10 to 12 times; the liposome extrusion membrane is a polycarbonate membrane;

[0033] In the step of co-incubating the lipid-modified trichosanthin with the cell membrane vesicles with the antigen unit bound to the surface, in the culture system after the co-incubation, the final concentration of the trichosanthin component and the final concentration of the total protein in the cell membrane vesicles is (0.01-0.1) μmol:1 mg; or, in the exosome-like vesicles co-loaded with antigen and trichosanthin, the content ratio of the trichosanthin unit to the total protein in the cell membrane vesicles is (0.01-0.1) μmol / mg;

[0034] The step of culturing the engineered cells, disrupting the culture, performing differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface comprises the following steps: culturing the engineered cells, disrupting the culture, performing differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface comprises the following steps: culturing the engineered cells, digesting after culturing, centrifuging, washing with PBS, discarding the supernatant, resuspending the cells with a hypotonic lysis solution and stirring, disrupting the cells with ultrasound, performing differential centrifugation, collecting the precipitate obtained by the last centrifugation, resuspending the collected precipitate with PBS, and extruding in sequence from large pore size to small pore size with a liposome extrusion membrane with a pore size of 1 to 0.2 μm to obtain cell membrane vesicles with the antigen unit bound to the surface;

[0035] After the step of co-incubating the lipid-modified trichosanthin with the cell membrane vesicles with the antigen unit bound to the surface, the method further comprises the step of ultrafiltration centrifugation of the co-incubation system using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and collecting the material on the upper layer of the ultrafiltration membrane to obtain the exosome-like vesicles co-loaded with the antigen and trichosanthin;

[0036] The exosome-like vesicles co-loaded with antigen and trichosanthin are as defined in the first aspect of the present invention.

[0037] In the third aspect of the present invention, the present invention provides an antigen-adjuvant integrated vaccine preparation, which includes at least one of the exosome-like vesicles co-loaded with antigen and trichosanthin protein described in the first aspect of the present invention and the exosome-like vesicles co-loaded with antigen and trichosanthin protein prepared by the preparation method described in the second aspect of the present invention.

[0038] In the fourth aspect of the present invention, the present invention provides the use of the exosome-like substances co-loaded with antigen and trichosanthin protein as described in the first aspect of the present invention, or the exosome-like substances co-loaded with antigen and trichosanthin protein prepared by the preparation method described in the second aspect of the present invention, or the antigen-adjuvant integrated vaccine preparation described in the third aspect of the present invention in the preparation of drugs capable of inducing specific antibody neutralization.

[0039] In some embodiments, the drug capable of inducing specific antibody neutralization is used to combat the invasion and / or infection of SARS-CoV-2, wherein the antigenic unit is a SARS-CoV-2 receptor binding domain.

[0040] The exosome-like vesicles co-loaded with antigen and TCS provided by the present invention integrate the antigen and the adjuvant trichosanthin (TCS) on the surface of cell membrane vesicles, which can effectively induce the production of antigen-specific antibodies and can be used to prepare drugs that can induce specific antibody neutralization. Moreover, the integrated delivery of antigen and TCS can inhibit the rapid degradation of free adjuvant and promote the entry of adjuvant into cells, which is beneficial to reduce off-target effects, can make vaccines safer, and has great potential for clinical application.

[0041] Exosomes that co-load the SARS-CoV-2 receptor binding domain (which can be recorded as SARS-CoV-2RBD) and TCS and the vaccines prepared therefrom can effectively induce the maturation of dendritic cells (DCs) in vitro and effectively induce the production of SARS-CoV-2RBD-specific antibodies in vivo, thereby effectively combating SARS-CoV-2 infection.

[0042] The method for preparing exosome-like vesicles co-loaded with antigen and TCS provided by the present invention can omit or simplify the purification step of the antigen protein, and can greatly improve the yield and production efficiency in the large-scale production link. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present invention. The various sizes of each component shown in the drawings are arbitrarily shown and may be accurate or may not be drawn according to the actual scale. For example, in order to make the illustration clearer, the size of the components is appropriately exaggerated in some places in the drawings. Unless otherwise specified, the components in the figure are not drawn to scale. The present invention does not limit each size of each component.

[0044] In the following description, the same reference numerals denote the same parts.

[0045] Figure 1Schematic diagram of the preparation of exosome-like vesicles (RBD-vesicle-TCS) carrying both RBD and TCS on the surface of cell membrane vesicles in one embodiment of the present invention, which can be obtained by co-incubating cell membrane vesicles (RBD-vesicles) carrying RBD on the surface with lipid-modified TCS (lipid-TCS);

[0046] Figure 2 The immunofluorescence characterization of RBD expression on the surface of 293T cells and 293T-RBD cells in one embodiment of the present invention; wherein, 293T cells refer to 293T cells that have not been modified to express RBD on the surface, and 293T-RBD cells refer to 293T cells that express RBD on the cell surface, and each scale bar is 50 μm; the DAPI column performs nuclear fluorescence staining, the RBD column performs immunofluorescence staining of RBD, and the overlay column corresponds to the superposition of DAPI fluorescence staining and RBD fluorescence staining;

[0047] Figure 3 The Western blot characterization results of the RBD expression of 293T cells and 293T-RBD cells in one embodiment of the present invention; wherein, "RBD" corresponds to the expression of SARS-CoV-2RBD in the cells, and "GAPDH" corresponds to the expression of the internal reference glyceraldehyde-3-phosphate dehydrogenase;

[0048] Figure 4 In one embodiment of the present invention, the particle size distribution diagram (A) of the exosome-like particles derived from 293T-RBD cells, the transmission electron micrograph (B), and the summary results of the average particle size (average diameter), Zeta potential (Zeta potential) and polydispersity index (PDI) (C) of the exosome-like particles derived from 293T-RBD cells; Figure 4 The scale bar in B is 100 nm;

[0049] Figure 5 This is a diagram showing the expression and content analysis of RBD on the cell membrane in one embodiment of the present invention; Figure 5 Middle A shows the expression of RBD on the cell membrane from 293T cells and 293T-RBD cells; Figure 5 Middle B is the protein immunoblot of RBD standard and 293T-RBD cell membrane; Figure 5 Middle C is the standard curve obtained by gray value analysis of RBD standard using Image-J software, the horizontal axis is protein amount (protein amount, unit is ng), and the vertical axis is OD value (OD value);

[0050] Figure 6 Evaluation of the immune effect of exosomes co-loaded with RBD and TCS in one embodiment of the present invention; Figure 6A in the figure is the mouse immunization scheme, "immunization" means immunizing mice, and "serum collection" means collecting serum. The time is measured in weeks. The mice were immunized for the first, second, and third time at weeks 0, 1, and 2, respectively. Serum was collected for testing one week after the second and third immunizations (i.e., weeks 2 and 3). Figure 6 Middle B is an analysis of the RBD-specific IgG antibody level in mouse serum; Figure 6 Center C is a schematic diagram of a pseudovirus neutralization experiment; Figure 6 D in the middle is the effect of pseudovirus neutralization of antibodies in mouse serum, and the ordinate is the luminescence value;

[0051] Figure 7 The preparation and characterization results of lipid-modified TCS (DSPE-PEG-TCS) in one embodiment of the present invention; wherein A is a polyacrylamide gel electrophoresis diagram of TCS; wherein B is a rapid protein purification liquid chromatogram of TCS; wherein C is a comparison of polyacrylamide gel electrophoresis diagrams of TCS and DSPE-PEG-TCS; wherein D is a rapid protein purification liquid chromatogram of the reaction solution of DSPE-PEG-MAL (MAL represents a maleimide group) and TCS; wherein E is a mass spectrum of TCS, and the abscissa is the mass-to-charge ratio (mass, m / z); wherein F is a mass spectrum of DSPE-PEG-TCS;

[0052] Figure 8 Schematic diagram of the preparation and characterization results of exosome-like vesicles co-loaded with RBD and TCS (denoted as RBD / TCS-CMVs) in one embodiment of the present invention; A is a schematic diagram of the preparation of RBD / TCS-CMVs, which is obtained by co-incubating cell membrane vesicles derived from RBD-293T cells and loaded with RBD on the surface (denoted as RBD-293T CMVs or RBD-CMVs) with DSPE-PEG-TCS; B is a particle size distribution diagram of RBD-CMVs and RBD / TCS-CMVs, with the abscissa axis being particle size (size) and the ordinate axis being intensity (intensity); C is a comparison diagram of the Zeta potential (Zeta potential) of RBD-CMVs and RBD / TCS-CMVs;

[0053] Fig. 9The in vitro and in vivo immunostimulatory effects of RBD / TCS-CMVs in one embodiment of the present invention; wherein A is an analysis diagram of the effect of RBD / TCS-CMVs in promoting high expression of the maturation marker CD86 by bone marrow-derived dendritic cells (BMDC); "control" is a PBS negative control group; "RBD-CMVs" are cell membrane vesicles with RBD on the surface but not bound to TCS; "RBD-CMVs+freeTCS" is a physical mixture group of RBD-CMVs and free TCS; "RBD / TCS-CMVs" is an antigen-adjuvant integrated exosome group with RBD and TCS co-loaded on the surface; and "LPS" is an LPS (lipopolysaccharide) positive control group (final concentration 500 ng / mL); wherein B is an analysis diagram of the effect of RBD / TCS-CMVs in promoting high expression of the maturation marker MHC-II by BMDC; wherein C is an analysis diagram of the effect of RBD / TCS-CMVs inducing mice to produce RBD-specific IgG antibodies.

[0054] Table 1.

[0055]

[0056] DETAILED DESCRIPTION

[0057] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. For example, the features described or described as part of an embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0059] the term

[0060] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0061] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, which include any combination of any two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions: A, B, and "a combination of A and B".

[0062] In the present invention, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0063] In the present invention, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other expressions that mean "one or more" are also understood in the same way unless otherwise specified.

[0064] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0065] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0066] Herein, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute a limitation on the scope of protection of the present invention. If multiple "preferred" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "preferred" item is independent.

[0067] In the present invention, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the contents covered, but should not be understood as limiting the previous technical solution, nor can they be understood as limiting the scope of protection of the present invention. In the present invention, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0068] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "optional" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. For example, the descriptions such as "optionally contain", "optionally include" in this application mean "contain or do not contain". "Optional component X" means that component X exists or does not exist.

[0069] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0070] The terms "contain", "include" and "include" used in the present invention are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions, timing, states, etc. for the occurrence of actions.

[0071] In the present invention, the technical features or technical solutions described in an open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0072] In the present invention, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers in the numerical interval, including the two endpoint integers of the numerical range, and each integer between the two endpoints, is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0073] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0074] In the present invention, the term "room temperature" or "normal temperature" generally refers to 4°C to 35°C, for example, 20°C ± 5°C. In some embodiments of the present invention, "room temperature" or "normal temperature" refers to 10°C to 30°C. In some embodiments of the present invention, "room temperature" or "normal temperature" refers to 20°C to 30°C.

[0075] In the present invention, when it comes to the unit of the data range, if there is a unit only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same. For example, 3-5h means that the units of the left endpoint "3" and the right endpoint "5" are both h (hours).

[0076] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0077] The mass or weight of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the proportional relationship of the mass or weight between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass or weight described in the description of the embodiments of the present invention may be units known in the chemical industry such as μg, mg, g, and kg.

[0078] In the present invention, if there are multiple steps involved in the method flow, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0079] In the present invention, the terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to an epitope on a predetermined antigen.

[0080] In the present invention, the term "antibody" is a protein that binds to a specific antigen, and it generally refers to all proteins and protein fragments containing a complementarity-determining region (CDR), especially full-length antibodies or antibody functional fragments. The term "full-length antibody" includes polyclonal antibodies and monoclonal antibodies, and the term "antibody functional fragment" is a substance that contains part or all of the antibody CDR, which lacks at least one amino acid present in the full-length chain but is still able to specifically bind to the antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules (including complete antibodies) for binding to a given epitope. In some embodiments, the antibody or antibody functional fragment can bind to the receptor binding domain (RBD) or a functional fragment containing RBD, and this binding is usually specific. In some embodiments, the antibody functional fragment comprises a single heavy chain (or a portion of a heavy chain) and a single light chain (or a portion of a light chain). In other embodiments, the antibody functional fragment comprises at least one heavy chain (or a portion of a heavy chain) and an equal number of light chains (or a portion of a light chain). In other embodiments, the antibody functional fragment comprises at least one heavy chain variable region and at least one light chain variable region, and further, the number of heavy chain variable regions and the number of light chain variable regions are equal. Antibody functional fragments can be produced by recombinant nucleic acid technology, or can be produced by enzymatic cleavage or chemical cleavage of antigen binding molecules (including complete antibodies).

[0081] In the present invention, the term "complementarity determining region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed "US Department of Health and Human Services, NIH, 1991, and later versions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the circumstances, the term "CDR" is used to refer to a region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to its recognized antigen or epitope. In another specific embodiment, CDR refers to the hypervariable regions of the heavy and light chains of immunoglobulins as defined by IMGT.

[0082] In a first aspect of the present invention, an exosome-like substance co-loaded with antigen and trichosanthin (TCS) is provided, which comprises a cell membrane vesicle, an antigen unit bound to the surface of the cell membrane vesicle, and a trichosanthin unit bound to the surface of the cell membrane vesicle.

[0083] In the present invention, “exosome-like cells co-carrying antigen and trichosanthin” and “exosome-like cells co-carrying antigen and TCS” have the same meaning and can be used interchangeably.

[0084] In the exosome-like vesicles co-loaded with antigens and TCS provided in the first aspect, cell membrane vesicles are used as the delivery system. "Cell membrane vesicles" are nanoscale vesicles prepared by extrusion or ultrasound of cell membranes, and are composed of lipids (phospholipids, cholesterol), proteins and carbohydrates (glycolipids, glycoproteins). The cell membrane vesicles obtained by mechanical extrusion and other methods have no significant differences in size and some protein markers from exosomes, so they are also called "exosome-like". Please refer to the literature [4] "Wen, Y., et al., Cell-derived nanovesicles prepared by membrane extrusion are good substitutes for natural extracellular vesicles. Extracellular Vesicle, 2022.1: p.100004.".

[0085] Unless otherwise specified, the "exosome-like" referred to in the present invention has cell membrane vesicles similar to exosomes, has the inherent functions or signaling molecules of its source cells, and can promote the interaction between the delivery system and the target cells. Please refer to the literature [5] "Huang, LL, et al., Cell-Membrane-Based Biomimetic Systems with Bioorthogonal Functionalities. Acc Chem Res, 2020. 53(1): p. 276-287.".

[0086] Trichosanthin (TCS) is a type I ribosome inactivating protein extracted from the traditional Chinese medicinal plant Trichosanthes kirilowii, which has the effect of stimulating immunity. Trichosanthin has a tumor immune activation effect: Trichosanthin has been found to stimulate the maturation of bone marrow-derived dendritic cells (BMDCs) at the cellular level, and can increase the infiltration of immune cells in the tumor site in animals, thereby activating anti-tumor immunity (see reference [7] "Tang, Y., et al., Recombinant cancer nanovaccine for targeting tumor-associated macrophage and remodeling tumor microenvironment. Nano Today, 2021.40: p.101244."). The crystal structure and full sequence of trichosanthin have been resolved, and the use of genetic engineering technology to prepare recombinant trichosanthin has been widely studied (reference [6] "Shaw, PC, et al., Cloning of trichosanthin cDNA and its expression in Escherichia coli. Gene, 1991. 97 (2): p. 267-72."). Compared with traditional methods, trichosanthin prepared by genetic engineering technology has good stability and is conducive to quality control.

[0087] In the present invention, the immune activation property of trichosanthin (TCS) is utilized, and TCS is used as an adjuvant in exosome-like vaccines, and exosomes that co-load antigens and TCS are used to achieve integrated delivery of antigens and adjuvants.

[0088] In the present invention, "trichosanthin unit" is also referred to as TCS unit, which refers to trichosanthin or a trichosanthin derivative that can play a similar or better adjuvant role. In some embodiments, the amino acid sequence of the trichosanthin unit is shown in SEQ ID No.: 1:

[0089] MDVSFRLSGATSSSYGVFISNLRKALPNERKLYDIPLLRSSLPGSQRYALIHLTNYADETISVAIDVTNVYIMGYRAGDTSYFFNEASATEAAKYVFKDAMRKVTLPYSGNYERLQTAAGKIRENI PLGLPALDSAITTLFYYNANSAASALMVLIQSTSEAARYKFIEQQIGKRVDKTFLPSLAIISLENSWSALSKQIQIASTNNGQFESPVVLINAQNQRVTITNVDAGVVTSNIALLLNRNNMALEG.

[0090] In the present invention, "antigenic unit" refers to a functional fragment containing a receptor binding domain (RBD). The "antigenic unit" involved in the present invention may be RBD, but may not be limited thereto.

[0091] In the exosome-like vesicles co-loaded with antigen and TCS of the present invention, the antigen unit and the TCS unit are respectively bound to different sites on the surface of the cell membrane vesicle.

[0092] The exosome-like vesicles co-loaded with antigen and TCS provided by the present invention integrate the antigen and the adjuvant trichosanthin (TCS) on the surface of cell membrane vesicles, which can effectively induce the production of antigen-specific antibodies and can be used to prepare drugs that can induce specific antibody neutralization. Moreover, the integrated delivery of antigen and TCS can inhibit the rapid degradation of free adjuvant and promote the entry of adjuvant into cells, which is beneficial to reduce off-target effects, can make vaccines safer, and has great potential for clinical application.

[0093] In some embodiments, the antigen unit is a SARS-CoV-2 receptor binding domain. In this case, an exosome-like substance having both antigen RBD and adjuvant trichosanthin bound to the surface is provided, which can induce humoral immunity and cellular immunity, thereby resisting the invasion and / or infection of SARS-CoV-2 virus.

[0094] In some embodiments, the amino acid sequence of the antigenic unit is shown in SEQ ID No.: 2:

[0095] RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT GCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF.

[0096] In the SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) virus, the receptor binding domain (RBD) is located on the spike protein of SARS-CoV-2, which is directly involved in SARS-CoV-2 recognizing and binding to host cell receptors. This RBD (denoted as SARS-CoV-2RBD) can be used as an antigen to induce neutralizing antibodies that can effectively neutralize the virus and prevent the virus from infecting host cells.

[0097] SARS-CoV-2 is a type of single-stranded positive-sense RNA virus with an envelope structure (reference [1] "Hu, B., et al., Characteristics of SARS-CoV-2 and COVID-19. Nat Rev Microbiol, 2021.19(3): p.141-154."). It has the appearance of a 60-140 nm sphere, and the surface of the virus particle has a 9-12 nm long spike protein. Functionally, it is responsible for recognizing receptors on the surface of host cells and mediating the fusion of the viral envelope with the cell membrane, thereby causing the virus to enter the host cell. Structurally, Spike consists of two subunits, S1 and S2, and the receptor binding domain (RBD) is located in the S1 subunit. RBD is directly involved in the virus's recognition of the angiotensin converting enzyme 2 (ACE2) receptor on host cells (reference [2] "Jackson, CB, et al., Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol, 2022. 23 (1): p. 3-20."). Most SARS-CoV-2 neutralizing antibodies can bind to RBD and block the RBD-ACE2 interaction, thereby inhibiting the virus from attaching to target cells. RBD is also a relatively important vaccine target. It can not only induce high-quality neutralizing antibody responses, but also has multiple T cell epitopes, can induce potent cellular immune responses, and has a low risk of antibody-dependent enhancement (ADE).

[0098] Exosomes that co-load the SARS-CoV-2 receptor binding domain (which can be recorded as SARS-CoV-2RBD) and TCS and the vaccines prepared therefrom can effectively induce the maturation of dendritic cells (DCs) in vitro and effectively induce the production of SARS-CoV-2RBD-specific antibodies in vivo, thereby effectively combating SARS-CoV-2 infection.

[0099] In some embodiments, the cell source of the cell membrane vesicles is selected from human embryonic kidney cells HEK 293T.

[0100] In some embodiments, cell membrane vesicles are recorded as CMVs, and exosome-like vesicles co-loaded with antigen and TCS are recorded as exosome-like vesicles co-loaded with RBD and TCS are recorded as TCS / RBD-CMVs.

[0101] In some embodiments, the average particle size of the cell membrane vesicles is 140-150 nm, and may further be 144 nm, which can be obtained by testing and analyzing with a particle size analyzer.

[0102] In some embodiments, in the exosome-like vesicles co-loaded with antigen and trichosanthin, the content ratio of trichosanthin unit (TCS unit) to the total protein in the cell membrane vesicle is (0.01-0.1) μmol:1 mg, that is, (0.01-0.1) μmol / mg, at this time, each milligram of total protein in the cell membrane vesicle contains about 0.01-0.1 micromole trichosanthin unit. The content ratio of TCS unit to the total protein in the cell membrane vesicle can also be any one of the following content ratios or any two content ratios: 0.01 μmol / mg, 0.02 μmol / mg, 0.03 μmol / mg, 0.04 μmol / mg, 0.05 μmol / mg, 0.06 μmol / mg, 0.07 μmol / mg, 0.08 μmol / mg, 0.09 μmol / mg, 0.01 μmol / mg, etc.

[0103] In some embodiments, in the exosome-like vesicles co-loaded with antigen and trichosanthin, the ratio of the content of TCS unit to the total protein in the cell membrane vesicles is 0.2 micromoles:5 mg, that is, 0.04 μmol / mg.

[0104] In some embodiments, the antigen unit is bound to the surface of the cell membrane vesicle through the transmembrane domain of the viral glycoprotein, wherein the antigen unit is bound to one side of the transmembrane domain of the viral glycoprotein, and the transmembrane domain passes through the membrane structure of the cell membrane vesicle. The antigen unit can be expressed on the cell membrane surface by utilizing the property of the transmembrane domain that can pass through the cell membrane.

[0105] In the present invention, unless otherwise specified, "transmembrane domain" and "transmembrane structural domain" have the same meaning and can be used interchangeably.

[0106] In the present invention, unless otherwise specified, "intracellular domain" and "intracellular structural domain" have the same meaning and can be used interchangeably.

[0107] In some embodiments, a linker unit may or may not be provided between the antigenic unit and the transmembrane domain of the viral glycoprotein.

[0108] In some embodiments, a linker is provided between the antigen unit and the transmembrane domain of the viral glycoprotein. The length of the linker may be 8 to 12 amino acids, and may further be 10 amino acids. In some embodiments, the sequence of the linker is GGGGSGGGGS (SEQ ID No.: 3).

[0109] In some embodiments, the transmembrane domain of the viral glycoprotein is also connected to the intracellular domain of the viral glycoprotein, and the intracellular domain of the viral glycoprotein is located inside the cell membrane vesicle. At this time, the antigen unit is bound to the surface of the vesicle through the transmembrane domain and the intracellular domain of the viral glycoprotein, wherein the antigen unit is bound to one side of the transmembrane domain of the viral glycoprotein, the transmembrane domain passes through the membrane structure of the cell membrane vesicle, and the intracellular domain is located inside the cell membrane vesicle.

[0110] In some embodiments, the viral glycoprotein is ZEBOV virus (Zaire ebolavirus) glycoprotein.

[0111] In some embodiments, the trichosanthin unit (TCS unit) is bound to the surface of the cell membrane vesicle through a lipid unit, and the lipid unit is inserted into the membrane structure of the cell membrane vesicle. At this time, the hydrophobic force between the lipid unit and the membrane structure of the cell membrane vesicle is used to insert the lipid unit into the membrane structure of the cell membrane vesicle, thereby binding the TCS unit to the surface of the cell membrane vesicle.

[0112] In some embodiments, the lipid unit is a phospholipid unit, and further can be a neutral phospholipid unit. Further, the lipid unit can be selected from one or more of distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylethanolamine (DPPE), etc.

[0113] In some embodiments, the trichosanthin unit is connected to the lipid unit via a polyethylene glycol unit. The weight average molecular weight of the polyethylene glycol unit may be, but is not limited to, 800Da to 5000Da, further in the following ranges: 1800Da to 2200Da, 2000Da to 5000Da. The polyethylene glycol unit may be referred to as a PEG unit. In some embodiments, the weight average molecular weight of the polyethylene glycol unit is 2000Da.

[0114] Without limitation, the polyethylene glycol unit may be linked to a sulfhydryl site in the trichosanthin unit.

[0115] In some embodiments, the trichosanthin unit is linked to the lipid unit via a thioether bond (eg, a thioether bond formed by a maleimide group and a sulfhydryl group).

[0116] For example, when the lipid unit is linked to the trichosanthin unit through the maleimide group (MAL) in lipid-PEG-MAL, it can be linked to the sulfhydryl group in the trichosanthin unit to form a thioether bond through reaction. Trichosanthin is rich in sulfhydryl groups and can react with maleimide-containing compounds at pH 6.5-7.5 to form a thioether bond.

[0117] In the second aspect of the present invention, the present invention provides a method for preparing exosome-like vesicles co-loaded with antigen and TCS, which can be used to prepare the exosome-like vesicles co-loaded with antigen and TCS according to the first aspect of the present invention.

[0118] In some embodiments, a method for preparing exosome-like vesicles co-loaded with antigen and TCS is provided, comprising the following steps:

[0119] S100: using a gene sequence including a signal peptide, an antigen unit, a transmembrane domain and an intracellular domain of a viral glycoprotein in sequence, obtaining a viral vector containing a coding sequence of the antigen unit, and then transfecting it into a host cell to obtain an engineered cell expressing the antigen unit on the cell membrane surface; wherein a linker unit is provided or not provided between the antigen unit and the transmembrane domain of the viral glycoprotein, and optionally, the linker unit is 8 to 12 (e.g., 10) amino acids in length;

[0120] S200: culturing the engineered cells, and subjecting the culture to cell disruption, differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface; wherein the cell disruption may include one or both of hypotonic cell disruption and ultrasonic cell disruption;

[0121] S300: incubate the lipid-modified trichosanthin with the cell membrane vesicles with the antigen unit bound to the surface to obtain exosome-like vesicles co-loading the antigen unit and the trichosanthin unit (which is the aforementioned exosome-like vesicles co-loading the antigen and TCS).

[0122] The method for preparing exosome-like vesicles co-loaded with antigen and TCS provided by the present invention can omit or simplify the purification step of the antigen protein, which is beneficial to reducing costs and can greatly improve the yield and production efficiency in the large-scale production link.

[0123] S100: Prepare engineered cells that express target antigen units on the cell membrane surface.

[0124] The "signal peptide" involved in the aforementioned preparation method is used to promote the localization of the target protein on the cell membrane. A suitable signal peptide can be selected according to the characteristics of the target protein and the host cell. In some embodiments, the sequence of the signal peptide is MMKSIQLCILLWCLRAVC (SEQ ID No.: 4).

[0125] In some embodiments, the sequence of the transmembrane domain (TMD) of the viral glycoprotein is QWIPAGIGVTGVIIAVIALFCIC (SEQ ID No.: 5). By utilizing the property of the transmembrane domain that can pass through the cell membrane, the antigen unit can be expressed on the cell membrane surface.

[0126] In some embodiments, the sequence of the intracellular domain of the viral glycoprotein is KFVF (SEQ ID No.: 6).

[0127] In some embodiments, a linker unit is provided between the antigenic unit and the transmembrane domain of the viral glycoprotein. Further, the linker unit sequence may be as defined in the first aspect, for example, GGGGSGGGGS (SEQ ID No.: 3).

[0128] In some embodiments, a gene sequence comprising a signal peptide, RBD, a transmembrane domain and an intracellular domain of the ZEBOV virus is constructed, and further, the encoded amino acid sequence is MMKSIQLCILLWCLRAVCRVQPTESIVRFPNITNLCP FGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGGGGSGGGGSQWIPAGIGVTGVIIAVIALFCICKFVF (SEQ ID No.: 7).

[0129] In some embodiments, the viral vector is selected from a lentiviral vector

[0130] In some embodiments, the host cell is selected from 293T cells (HEK 293T cells).

[0131] In some embodiments, the antigen unit is RBD, and the resulting engineered cells can be referred to as RBD-cells.

[0132] In some embodiments, the antigen unit is RBD (further such as SARS-CoV-2RBD), the host cell is 293T cell (HEK 293T cell), and the obtained engineered cell can be recorded as RBD-293T cell or 293T-RBD cell.

[0133] Step S200: preparing cell membrane vesicles with target antigen units bound to their surfaces.

[0134] It is understood that the culture conditions suitable for the growth of the engineered cells are used, which may be the same or similar culture conditions as those for unengineered host cells.

[0135] In some embodiments, in step S200, "culturing the engineered cells" can end the culturing when the cells grow to 70%-90%.

[0136] Taking HEK 293T cells as host cells as an example, the engineered cells can be cultured in the following manner: DMEM containing 10% (v / v) fetal bovine serum (FBS) is used for culture at 37° C. and 5% (v / v) CO 2 , and the cells are cultured until they grow to 70%-90%.

[0137] In some embodiments, in step S200, a method combining hypotonic cell rupture and differential centrifugation can be used to remove organelles and extract cell membranes, and then cell membrane vesicles are obtained by extrusion. At this time, it is beneficial to increase the yield, shorten the operation time, and be more economical in cost. In some embodiments, the culture is subjected to hypotonic cell rupture, differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface.

[0138] In some embodiments, after culturing the engineered cells, the cell membrane vesicles with antigen units bound to their surfaces are collected by cell disruption, differential centrifugation, and extrusion.

[0139] In some embodiments, cell disruption may include one or both of hypotonic disruption of cells, ultrasonic disruption of cells.

[0140] In some embodiments, cell disruption includes hypotonic cell disruption and ultrasonic cell disruption. Further, cell disruption may include first hypotonic cell disruption and then ultrasonic cell disruption. Further, cell disruption may include first hypotonic cell disruption and then ultrasonic cell disruption. In some embodiments of the present invention, "hypotonic cell disruption" is performed as follows: cells are placed in a hypotonic lysis solution and stirred at 4°C for 2 hours. In some preferred examples, the hypotonic lysis solution is a 0.1×PBS buffer containing a protease inhibitor.

[0141] In some embodiments of the present invention, "ultrasonic cell disruption" is performed in the following manner: 200W ultrasonic wave for 3 minutes, interval of 3 seconds, ultrasonic wave for 3 seconds. Ultrasonic cell disruptor can be used to ultrasonically disrupt cells.

[0142] In some embodiments, in step S200, the differential centrifugation method is: first centrifugation at 10,000×g, and then centrifugation of the supernatant at 100,000×g. In some embodiments, first centrifugation at 10,000×g at 4°C for 10 min, and then centrifugation of the supernatant at 100,000×g at 4°C for 60 min.

[0143] In some embodiments, the extrusion step is performed using a liposome extruder.

[0144] In some embodiments, extrusion is performed in the following manner: a liposome extrusion membrane with a pore size of 1 to 0.2 μm is used to extrude in sequence from large pore size to small pore size (for example, 1 μm, 0.4 μm, and 0.2 μm pore sizes are used to extrude in sequence), and the number of extrusions of each pore size membrane can be 1 time or more, such as greater than or equal to 10 times, such as 10 to 15 times, such as 10 to 12 times, such as 11 times.

[0145] In the present invention, the liposome extrusion membrane refers to a membrane suitable for collecting vesicles by extrusion, such as a polycarbonate membrane.

[0146] In some embodiments, the extrusion comprises: sequentially extruding using liposome extrusion membranes with pore sizes of 1 μm, 0.4 μm and 0.2 μm, and the number of extrusions of each pore size membrane is 10 to 12. Further, the liposome extrusion membrane is a polycarbonate membrane.

[0147] In some embodiments, step S200 includes: culturing the engineered cells, digesting after the culture, centrifuging, breaking the cells, collecting the precipitate by differential centrifugation, and extruding after resuspending to obtain cell membrane vesicles with the antigen unit bound to the surface. A washing step may also be included after centrifugation and before breaking the cells, for example, washing with PBS, which may be washed once or more times, such as washing with PBS twice.

[0148] In some embodiments, step S200 includes: culturing the engineered cells, digesting after the culture, centrifuging, hypotonic cell rupture, ultrasonic cell disruption, collecting the precipitate by differential centrifugation, resuspending and then extruding to obtain cell membrane vesicles with the antigen unit bound to the surface.

[0149] In some embodiments, step S200 includes: culturing engineered cells (such as RBD-293T cells, and further, the RBD is SARS-CoV-2RBD), terminating the culture when the cells grow to 70%-90%, digesting, centrifuging, and sequentially performing hypotonic cell rupture, ultrasonic cell disruption, differential centrifugation and extrusion treatment to obtain cell membrane vesicles with antigen units bound to the surface.

[0150] In some embodiments, step S200 includes: culturing the engineered cells, performing cell disruption, differential centrifugation and extrusion on the culture, and obtaining cell membrane vesicles with the antigen unit bound to the surface includes the following steps: culturing the engineered cells, digesting after the culture, centrifuging, washing with PBS, discarding the supernatant, resuspending the cells with a hypotonic lysis solution and stirring, then ultrasonically disrupting the cells, and then performing differential centrifugation, collecting the precipitate obtained by the last centrifugation, resuspending the collected precipitate with PBS, and extruding from the large pore size to the small pore size using a liposome extrusion membrane with a pore size of 1 to 0.2 μm to obtain cell membrane vesicles with the antigen unit bound to the surface.

[0151] In some embodiments, step S200 includes: culturing engineered cells (as used in any embodiment of the context, such as RBD-293T cells, further, RBD is SARS-CoV-2RBD, it can be understood that the engineered cells are cultured under conditions suitable for cell growth), culturing until the cells grow to 70%-90%, digesting, centrifuging at 1500 rpm for 3 minutes, washing with PBS, discarding the supernatant, resuspending the cells with a hypotonic lysis solution (a non-limiting example of a hypotonic lysis solution is 0.1×PBS containing a protease inhibitor), stirring at 4° C. (e.g., stirring for 2 hours), and then ultrasonically disrupting the cells (e.g., For example, 200W ultrasound for 3 minutes, interval of 3 seconds, ultrasound for 3 seconds), then differential centrifugation (centrifuge the cell solution at 10000×g for 10 minutes at 4°C, take the supernatant and centrifuge it at 100,000×g for 60 minutes at 4°C), collect the precipitate, resuspend the collected precipitate with PBS, and extrude it from large pore size to small pore size using a liposome extrusion membrane with a pore size of 1 to 0.2 μm (such as using 1 μm, 0.4 μm, and 0.2 μm pore size for extrusion in sequence, the number of extrusions for each pore size is as described above, for example, it can be greater than or equal to 10 times, such as 11 times), to obtain cell membrane vesicles with the antigen unit bound to the surface.

[0152] In some embodiments, step S200 includes: culturing engineered cells (such as used in any embodiment of the context) under conditions suitable for cell growth, discarding the supernatant when the cells grow to 70%-90%, washing with PBS (such as washing twice), digesting with trypsin until the cells fall off, terminating the digestion with a serum-containing culture medium, centrifuging at 1500 rpm for 3 minutes, and then washing with PBS (such as washing twice), discarding the supernatant, resuspending the cells with a hypotonic lysis solution (such as 0.1×PBS containing protease inhibitors) and stirring at 4°C for 2 hours (the cells are ruptured under hypotonic conditions by stirring the hypotonic lysis solution), and then ultrasonically disrupting the cells. The ultrasonic disruption conditions are 200W, 3min, 3s interval, and ultrasonication for 3s. The resulting cell solution is centrifuged at 10000×g for 10min at 4°C, the supernatant is taken and centrifuged at 100,000×g for 60min at 4°C, and the precipitate is collected (the collected precipitate is the cell membrane). The collected precipitate (i.e., the cell membrane obtained by centrifugation) is resuspended in PBS and extruded in sequence using liposome extrusion membranes (such as polycarbonate membranes) with pore sizes of 1 μm, 0.4 μm, and 0.2 μm. The membranes with each pore size are extruded 10 to 15 times (such as 10 to 12 times, further such as 11 times) to obtain cell membrane vesicles with the antigen unit bound to the surface.

[0153] Step S300: preparing exosome-like vesicles co-carrying target antigen units and trichosanthin units.

[0154] In step S300, as described above, the main driving force for the co-incubation to obtain the exosome-like vesicles carrying the antigen unit and the trichosanthin unit is the hydrophobic interaction between the lipid unit and the membrane structure of the cell membrane vesicle.

[0155] In some embodiments, the lipid-modified trichosanthin has the following structure: lipid unit-polyethylene glycol unit-trichosanthin unit. The definitions of lipid unit, polyethylene glycol unit and trichosanthin unit can refer to the first aspect of the present invention.

[0156] In some embodiments, the lipid-modified trichosanthin is phospholipid-modified trichosanthin, and further, the phospholipid-modified trichosanthin may have the following structure: phospholipid unit-polyethylene glycol unit-trichosanthin unit, wherein "-" may be a chemical bond or a linker. The definition of the phospholipid unit may refer to the first aspect of the present invention.

[0157] In some embodiments, the exosome-like vesicles co-loaded with antigen and TCS are as defined in the first aspect of the present invention.

[0158] In some embodiments of the present invention, cell membrane vesicles carrying RBD on the surface (also called RBD engineered exosomes) can be prepared by the following method: a cell line stably expressing RBD (such as SARS-CoV-2 RBD) on the surface is successfully constructed by infecting 293T cells with lentivirus, and then cell membrane vesicles expressing RBD are isolated from the cell line by cell disruption, differential centrifugation and extrusion, which can induce humoral immunity in mice with the help of aluminum adjuvant. In some embodiments, the prepared RBD engineered exosomes are mixed with aluminum adjuvants and then immunized with mice, which can induce the production of RBD-specific IgG and inhibit pseudoviruses from entering cells expressing ACE2 in vitro.

[0159] In some embodiments of the present invention, Trichosanthes eggs are DSPE-PEGylated so that TCS can be inserted into the membrane structure of cell membrane vesicles, such as the aforementioned cell membrane vesicles with RBD on the surface. At this time, the constructed biomimetic vaccine integrating antigen and adjuvant can effectively induce the maturation of dendritic cells (DCs) in vitro and the production of RBD-specific antibodies in vivo. This engineered exosome-like vaccine platform can reduce or avoid the purification steps of antigen proteins, and can improve the yield and production efficiency in large-scale production; in addition, integrated delivery can inhibit the rapid degradation of free adjuvants, promote the entry of adjuvants into cells, reduce off-target effects, make vaccines safer, and have greater clinical application potential.

[0160] In some embodiments of the present invention, TCS is lipidated and then connected to the surface of RBD-cell membrane vesicles (RBD-CMVs) through the hydrophobic force between lipids to prepare exosome-like RBD / TCS-CMVs carrying both RBD and TCS. Such exosomes can effectively stimulate the maturation of bone marrow-derived dendritic cells in vitro and effectively induce the production of RBD-specific neutralizing antibodies in vivo.

[0161] In some embodiments of the present invention, in step S300: the lipid-modified trichosanthin is co-incubated with the cell membrane vesicles with the antigen unit bound to the surface (for example, co-incubated at 4°C, and further incubated at 4°C for 2h) to obtain exosome-like vesicles carrying the antigen unit and the trichosanthin unit. In some embodiments, in the culture system after the incubation, the final concentration of the trichosanthin component and the final concentration of the total protein in the cell membrane vesicles is (0.01-0.1) μmol:1 mg, that is, (0.01-0.1) μmol / mg. At this time, the amount of TCS units corresponding to each mg of total protein in the cell membrane vesicles is about 0.01-0.1 micromoles. The concentration ratio of the final concentration of trichosanthes protein component to the final concentration of total cell membrane vesicle protein can also be any one of the following ratios or an interval consisting of any two ratios: 0.01 μmol / mg, 0.02 μmol / mg, 0.03 μmol / mg, 0.04 μmol / mg, 0.05 μmol / mg, 0.06 μmol / mg, 0.07 μmol / mg, 0.08 μmol / mg, 0.09 μmol / mg, 0.01 μmol / mg, etc. In some embodiments, in the culture system after the incubation, the final concentration of the trichosanthes protein component is 200 μmol / L, and the final concentration of total cell membrane vesicle protein is 5 mg / mL (i.e., 5 g / L).

[0162] In some embodiments of the present invention, in step S300, after the step of "co-incubating the lipid-modified trichosanthin with the cell membrane vesicles having the antigen unit bound to the surface", the step also includes separating and purifying the formed co-loaded antigen and TCS-like exosomes from the co-incubation system. Furthermore, the co-loaded antigen and TCS-like exosomes can be collected by ultrafiltration centrifugation. Furthermore, the separation and purification can be achieved by ultrafiltration centrifugation using an ultrafiltration tube with a molecular weight cutoff of 100 kDa. Furthermore, ultrafiltration centrifugation can be performed using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa (in the form of an ultrafiltration tube) at 4°C and 3000 rpm (it can be understood that the ultrafiltration centrifugation is performed for an appropriate time), and the material on the upper layer of the ultrafiltration membrane can be collected to obtain the aforementioned co-loaded antigen and TCS-like exosomes.

[0163] In some embodiments, after the step of co-incubating the lipid-modified trichosanthin protein with the cell membrane vesicles with the antigen unit bound to the surface, the step also includes ultrafiltration and centrifugation of the co-incubation system using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and collecting the material on the upper layer of the ultrafiltration membrane to obtain the exosome-like vesicles co-loaded with the antigen and trichosanthin protein.

[0164] In the present invention, the protein concentration or protein content test may be performed using the BCA method, for example, a BCA protein concentration quantification kit (manufacturer such as Shanghai Bio-Tech Biotechnology Co., Ltd.) may be used.

[0165] In the third aspect of the present invention, the present invention provides an antigen-adjuvant integrated vaccine preparation, which includes at least one of the exosome-like vesicles co-loaded with antigen and TCS described in the first aspect of the present invention and the exosome-like vesicles co-loaded with antigen and TCS prepared by the preparation method described in the second aspect of the present invention.

[0166] For the "antigen-adjuvant integrated vaccine preparation" provided by the present invention, in the exosome-like vesicles that co-load antigen and TCS, the antigen unit and the adjuvant unit (TCS unit) are both bound to the surface of the cell membrane vesicles and can be delivered as an integrated whole. Therefore, the corresponding vaccine preparation is called the "antigen-adjuvant integrated vaccine preparation."

[0167] This "antigen-adjuvant integrated vaccine preparation" can effectively induce the production of antigen-specific antibodies and can be used to prepare drugs that can induce specific antibody neutralization. Moreover, the integrated delivery of antigen and TCS can inhibit the rapid degradation of free adjuvant and promote the entry of adjuvant into cells, which is beneficial to reduce off-target effects, making the vaccine safer and having great potential for clinical application.

[0168] Without limitation, the vaccine preparation may be in the form of an injection preparation. In addition to the exosome-like vesicles carrying antigens and TCS provided by the present invention, a solvent may also be included. The solvent may be water.

[0169] In the fourth aspect of the present invention, the present invention provides the use of the exosome-like particles co-loaded with antigen and TCS as described in the first aspect of the present invention, or the exosome-like particles co-loaded with antigen and TCS prepared by the preparation method described in the second aspect of the present invention, or the antigen-adjuvant integrated vaccine preparation described in the third aspect of the present invention in the preparation of a drug capable of inducing specific antibody neutralization.

[0170] In the present invention, unless otherwise specified, "drug" includes any agent, compound, composition or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by the "drug" in vivo is not particularly limited, and may be systemic effects or may only produce effects locally. The activity of the "drug" is not particularly limited, and may be an active substance that can interact with other substances, or may be an inert substance that does not interact.

[0171] The specific antibodies in “capable of inducing specific antibody neutralization” can specifically bind to the antigen units contained in the aforementioned exosome-like or the aforementioned antigen-adjuvant integrated vaccine preparation.

[0172] In some embodiments, the drug capable of inducing specific antibody neutralization is used to combat the invasion and / or infection of SARS-CoV-2. Furthermore, the antigen unit may be a SARS-CoV-2 receptor binding domain.

[0173] Without limitation, drugs that can induce specific antibody neutralization can be used to immunize the human body through different immune pathways, such as respiratory immunization, muscle immunization, subcutaneous immunization, etc.

[0174] In the fifth aspect of the present invention, a method for combating viral invasion and / or infection is also provided, comprising administering to a subject a therapeutically effective amount of the co-loaded antigen and TCS-like exosomes described in the first aspect of the present invention, or administering to a subject a therapeutically effective amount of the co-loaded antigen and TCS-like exosomes prepared by the preparation method described in the second aspect of the present invention, or administering to a subject a therapeutically effective amount of the antigen-adjuvant integrated vaccine preparation described in the third aspect of the present invention. The aforementioned "combat viral invasion and / or infection" can be used for prevention. Specific antibodies that can specifically bind to the aforementioned exosome-like exosomes or the aforementioned antigen-adjuvant integrated vaccine preparation can be produced in the subject's body, and the purpose of combating viral invasion and / or infection can be achieved by inducing the specific antibodies to neutralize the antigens carried by the virus.

[0175] In the present invention, the "subject" refers to an animal, preferably a mammal, and more preferably a human.

[0176] In the present invention, "therapeutically effective amount" refers to the amount of active pharmaceutical ingredient that will cause a biological or medical response in an individual, for example, the amount of active pharmaceutical ingredient that brings physiological and / or pharmacological positive effects to an individual, wherein the physiological and / or pharmacological positive effects include but are not limited to reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing down or delaying disease progression or preventing disease, etc.

[0177] The administration route in this method can be an injection method, such as intramuscular injection or intraperitoneal injection.

[0178] Some specific embodiments are provided below.

[0179] Embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manuals of this area (such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)" etc.) or normal conditions, and can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0180] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0181] In the following examples, unless otherwise specified, "room temperature" refers to 20°C to 30°C.

[0182] Example 1. Construction of RBD engineered cell lines

[0183] 1.1 Materials and Instruments

[0184] (1) Materials

[0185] DMEM culture medium (Dalian Meilun Biotechnology Co., Ltd.); Polybrene (Jiman Biotechnology); Lipofectamine3000 (Invitrogen); LB medium (Oxoid, UK); RIPA (strong) lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.); Cocktail protease inhibitor (Dalian Meilun Biotechnology Co., Ltd.); BCA protein concentration quantification kit (Shanghai Biyuntian Biotechnology Co., Ltd., which can be recorded as BCA kit); Puromycin sterile solution (Dalian Meilun Biotechnology Co., Ltd.); Plasmid extraction kit (Qiagen, Germany). Double antibody refers to penicillin and streptomycin in a molar ratio of 1:1. 70% (w / v) ethanol is a mixed solvent obtained by mixing ethanol and water in a volume ratio of 7:3.

[0186] (2) Instruments

[0187] Constant temperature water bath (Shanghai Titan Technology Co., Ltd., Shanghai, China); constant temperature shaker (Taicang Experimental Equipment Factory, Taicang, China); clean bench (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory, Shanghai, China); gel imaging system (ChemiDoc MPTM Imaging System, BIO-RAD, California, USA); manual pipette (Eppendorf, Hamburg, Germany); inverted fluorescence microscope (CARL, ZEISS, Germany).

[0188] (3) Cells

[0189] HEK 293T cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and are also referred to as 293T cells in this case.

[0190] 1.2 Experimental methods

[0191] Cell culture

[0192] Take out the frozen HEK 293T cells from the liquid nitrogen tank and quickly put them into a 37°C constant temperature water bath. After the freezing solution is thawed, centrifuge (1500rpm, 3min), discard the supernatant, resuspend with DMEM culture medium containing 10% (v / v) fetal bovine serum and 1% (v / v) double antibody, and transfer to a cell culture bottle. Culture at 37°C, 5% (v / v) CO2. When the cell growth density reaches 80% of the flat bottom area, the cells are passaged: discard the cell culture medium, rinse the cells once with 2mL of PBS, add an appropriate amount of trypsin cell digestion solution, aspirate and discard the trypsin digestion solution after digestion for a suitable time, add 1mL of DMEM culture solution to terminate digestion, and transfer the cells to a suitable culture bottle or culture dish according to the number for culture.

[0193] 1.2.2. Construction of lentiviral plasmid and lentiviral transfection

[0194] 1.2.2.1. Construction of RBD recombinant plasmid

[0195] The target gene consists of five parts: signal peptide, SARS-CoV-2 RBD, linker (connector peptide), ZEBOV glycoprotein transmembrane domain and ZEBOV glycoprotein intracellular domain. Using pLVX-Puro plasmid as a vector, the target gene sequence was subcloned into the lentiviral vector pLVX-Puro, the cloning site was Xhol / BamHI, the gene optimization host was human, and the plasmid containing the target gene was synthesized and constructed by Shanghai Jierui Bioengineering Co., Ltd. The amino acid sequence encoded by the target gene is MMKSIQLCILLWCLRAVCRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFGGGGSGGGGSQWIPAGIGVTGVIIAVIALFCICKFVF (SEQ ID No.:7), wherein the signal peptide, SARS-CoV-2 RBD, linker, ZEBOV glycoprotein transmembrane domain and ZEBOV glycoprotein intracellular domain correspond to positions 1-18, 19-241, 242-251, 252-274 and 275-278, respectively.

[0196] 1.2.2.2. Large-scale extraction of RBD recombinant plasmid

[0197] Take 200 μL of punctured bacteria containing recombinant plasmids in 5 mL LB culture medium, place in a 37°C constant temperature shaker to shake the culture medium at 200 rpm, culture for 4-5 hours until the culture medium becomes turbid, transfer the culture medium containing the bacteria to 500 mL LB culture medium with a final concentration of 100 μg / mL ampicillin, continue shaking and culture for 12 hours, centrifuge the culture medium at 10,000 × g for 3 minutes, discard the supernatant, and collect the bacteria. Use the instructions of the Qiagen plasmid extraction kit to extract a large amount of plasmids containing the target gene from the bacteria.

[0198] 1.2.2.3 Lentivirus packaging and cell transfection

[0199] 293T cells were seeded at an appropriate density in a 6 cm culture dish. 7.5 μg of psPAX2 plasmid, 2.5 μg of pMD2.G plasmid and 10 μg of pLVX-Puro plasmid containing the target gene were transfected into 293T cells using the Lipofectamine 3000 kit. The cell supernatant containing the lentivirus was collected 36 h and 72 h after transfection, filtered through a 0.45 μm filter and stored at 4°C for later use.

[0200] 293T cells were seeded at an appropriate density in a six-well plate. When the confluence reached 50%-70%, the culture medium was removed, and 1 mL of fresh culture medium, 1 mL of virus solution and 1.6 μL of 10 mg / μL polybrene were added for virus infection. Fresh culture medium was replaced after 12 hours, and culture medium containing 2 μg / mL puromycin was switched for screening after 48 hours. After all the cells in the control group died, 293T cells with stable expression of RBD were obtained, which can be recorded as "293T-RBD cells" or "RBD-293T cells". The cells were modified to express RBD on the surface.

[0201] 1.2.3. Cell Immunofluorescence

[0202] Place the cell slides in a 24-well plate in advance, inoculate 293T cells and 293T-RBD cells in a 24-well plate at an appropriate density, discard the culture medium after the cells are attached, wash the cells twice with PBS, add 4% (w / v) paraformaldehyde solution to fix at room temperature for 15 minutes, wash three times with PBS, 5 minutes each time, add PBS solution containing 1% (w / v) BSA to block for 15 minutes, dilute the primary antibody 100 times and incubate the primary antibody with the cells at 30 μL per slide, overnight at 4°C, wash three times with PBS solution containing 1% (w / v) BSA, dilute the secondary antibody 1000 times and incubate the secondary antibody with the cells at room temperature for 1 hour at 30 μL per slide, wash three times with PBS solution containing 1% (w / v) BSA, incubate the DAPI dye with the cells in the dark for 20 minutes, and then wash three times with PBS solution containing 1% (w / v) BSA. Add 3 μL of mounting medium onto the slide, place the slide containing cells on the mounting medium, and after 10 minutes, the mounting medium solidifies and the cells are observed using an inverted fluorescence microscope.

[0203] 1.2.4. Western Blot

[0204] An appropriate number of 293T cells and lentivirus-infected 293T cells were added to the protein lysis buffer and lysed on ice for 30 minutes. After centrifugation at 12000rpm for 30 minutes, the supernatant was taken and the protein concentration was determined using a BCA kit. After the protein concentration was adjusted to be consistent, a protein loading buffer was added, and the cells were heated at 100°C for 15 minutes for polyacrylamide gel electrophoresis. After the electrophoresis, the membrane was transferred using a semi-dry transfer apparatus to allow the protein to adhere to the polyvinylidene fluoride (PVDF) membrane. The membrane was placed in a serum-free rapid blocking solution for 15 minutes, and then the band containing the protein of specific molecular weight was cut according to the theoretical molecular weight, placed in a primary antibody solution diluted to an appropriate multiple, and incubated on a shaker overnight at 4°C. After washing 5 times with TBST, the band was placed in a secondary antibody solution, washed 5 times again, and exposed to color using an ECL luminescence kit and a gel imager.

[0205] Results and discussion

[0206] In order to express the antigen protein RBD on the cell surface, a target gene sequence containing a signal peptide (to promote protein secretion), RBD, the transmembrane domain and the intracellular domain of the ZEBOV virus was constructed, and a lentivirus containing the target gene was packaged and infected with 293T cells; after puromycin selection, cells without the target gene died, and the surviving cells were able to stably express RBD.

[0207] The RBD on the cell membrane was observed by cell immunofluorescence. The 293T-RBD cells infected with lentivirus could be bound by the anti-RBD antibody and thus stained with fluorescence (the original data was green fluorescence), while the fluorescence representing RBD in the control 293T cells was almost invisible, indicating that the 293T-RBD cells stably expressing RBD on the membrane were successfully constructed. Figure 2 (Colors are shown in grayscale). In addition, Western blotting was used to investigate whether lentivirus-infected cells expressed RBD. Figure 3 The results shown are as follows: compared with normal 293T cells that have not undergone the aforementioned surface expression RBD modification, 293T-RBD cells have a significant increase in RBD expression, indicating that a 293T cell line stably expressing RBD has been successfully constructed and can be recorded as a "stable cell line."

[0208] Example 2. Preparation, characterization and immune effect evaluation of exosome-like vaccines

[0209] Materials and instruments

[0210] 2.1.1. Materials

[0211] Prestained rainbow marker (Dalian Meilun Biotechnology Co., Ltd., China); RIPA (strong) lysis buffer (Shanghai Biotech Co., Ltd.); protease inhibitor cocktail (Dalian Meilun Biotechnology Co., Ltd.); imjectTMAlum Adjuvant (Thermo Scientific, USA); HRP-labeled goat anti-rabbit IgG (Shanghai Biotech Co., Ltd.); wild-type SARS-CoV-2 pseudovirus (Jiman Bio, China); firefly luciferase reporter gene assay kit (Dalian Meilun Biotechnology Co., Ltd.); TMB color development solution (Shanghai Biotech Co., Ltd.); TMB color stop solution (Shanghai Biotech Co., Ltd.).

[0212] 2.1.2. Instruments

[0213] Ultrasonic disruptor (Ningbo Xinzhi Biotechnology Co., Ltd., Ningbo, China); high-speed refrigerated centrifuge (MultifugeTM X1, Thermo Scientific, Massachusetts, USA); laser particle size analyzer (Malvern Panalytical, UK); water purifier (Ultra pure plus-12A, Shanghai Hetai Instrument Co., Ltd., China); liposome extruder (Avanti, USA); 120KV transmission electron microscope (Talos L120C, FEI, USA); microplate reader (Biotek Synergy4, USA).

[0214] 2.1.3. Experimental animals

[0215] BALB / c female mice (SPF grade) aged 6-8 weeks were purchased from Shanghai Slake Experimental Animal Co., Ltd. and kept at 25°C room temperature, 12 h day and night, regular addition of water and sterile food, and regular cage changes. All animal experimental operations followed the specifications and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0216] Experimental methods

[0217] 2.2.1. Preparation and characterization of cell membrane vesicles

[0218] The RBD-293T cells obtained in Example 1 were cultured at 37°C and 5% (v / v) CO2 using DMEM medium containing 10% (v / v) fetal bovine serum (FBS). When the cells grew to 70%-90%, the supernatant was discarded, the cells were washed twice with PBS, and trypsin was used to digest the cells until they fell off the bottom of the dish. The digestion was terminated with a serum-containing medium. The cells were centrifuged at 1500 rpm for 3 min and washed twice with PBS. The supernatant was then discarded. The cells were resuspended in a hypotonic lysis solution (0.1×PBS, containing protease inhibitors) and stirred at 4°C for 2 h to rupture the cells under hypotonic conditions. The cells were then ultrasonically disrupted using an ultrasonic cell disruptor under the conditions of 200 W, 3 min, 3 s interval, and 3 s ultrasonication. The resulting cell solution was centrifuged at 10,000×g at 4°C for 10 min, the supernatant was centrifuged at 100,000×g at 4°C for 60 min, and the precipitate was collected as the cell membrane.

[0219] The cell membrane obtained by centrifugation was resuspended in 800 μL PBS and extruded sequentially with polycarbonate membranes with pore sizes of 1 μm, 0.4 μm, and 0.2 μm. Each membrane with each pore size was extruded 11 times to obtain cell membrane vesicles. 100 μL of cell membrane vesicles was diluted with 900 μL PBS and the particle size and potential of the cell membrane vesicles were measured using a particle size analyzer.

[0220] Take a 200-mesh copper grid with intact morphology and no creases and place it in a glow discharge system for hydrophilization. Dilute the cell membrane vesicles with ultrapure water and gently blow several times with a pipette. Take 10 μL of the extracellular vesicle suspension and add it to the hydrophilized copper grid. Let it stand for 1 minute, carefully absorb the excess solution with absorbent paper, then add 10 μL of uranyl acetate to cover the copper grid for negative staining for 1 minute. After drying naturally, place it in a transmission electron microscope for observation.

[0221] 2.2.2. Semi-quantitative analysis of RBD expression on cell membrane by WB

[0222] The cell membranes extracted from 40 million cells were resuspended and mixed with 200 μL protein lysis buffer, then lysed on ice for 30 min, and then centrifuged at 12000×g for 20 min to collect the supernatant as the sample to be tested. SARS-CoV-2 RBD standard was diluted to 50 ng / μL, 25 ng / μL, 12.5 ng / μL, 6.25 ng / μL, and 3.125 ng / μL, and loaded after adding loading buffer, so that each sample had 500 ng, 250 ng, 125 ng, 62.5 ng, and 31.25 ng RBD protein, respectively. The experiment was carried out according to the protein immunoblotting steps in 1.2.4.

[0223] 2.2.3. Immunization of mice

[0224] Fifteen 6-8 week old Balb / c female mice were raised in an SPF animal breeding room for one week and then randomly divided into three groups, with 5 mice in each group. The mice were immunized according to the scheme shown in Table 2 below.

[0225] Table 2. Mouse immunization schedule

[0226] Immune pathway Antigen dose Aluminum adjuvant dosage CpG Dosage im 10 μg 100 μg 10 μg ip 10 μg 100 μg 10 μg blank - - -

[0227] In Table 2, im indicates intramuscular injection, ip indicates intraperitoneal injection, and blank indicates no treatment.

[0228] 2.2.4. ELISA determination of specific antibodies in sera of immunized mice

[0229] Two weeks after the first immunization, the mice were anesthetized with isoflurane, and blood was collected from the orbital venous plexus of the mice using a capillary tube. 300 μL of plasma was collected from each mouse, and the blood was allowed to stand at room temperature for 4 hours and then centrifuged twice at 8000 rpm to obtain the supernatant to obtain mouse serum.

[0230] 2 μg / mL RBD protein was added to ELISA special enzyme-labeled plate at 100 μL per well, placed in a 4°C refrigerator overnight to allow the antigen to attach to the bottom of the plate, the antigen was discarded, the plate was washed 3 times with PBST buffer, 100 μL / well of 1% (w / v) BSA was added as blocking solution, incubated at 37°C for 2 h, the blocking solution was discarded, the plate was washed 3 times with PBST buffer, 100 μL / well of 10-fold diluted mouse immune serum was added to each well, incubated at 37°C for 1 h, the serum was discarded, the plate was washed 3 times with PBST buffer, HRP-labeled goat anti-mouse IgG (1:1000 dilution) was added as secondary antibody, 100 μL per well, incubated at 37°C for 1 h, the secondary antibody was discarded, the plate was washed 3 times with PBST, 100 μL / well TMB color development solution was added, incubated at 37°C for 10 min, and 50 μL / well color stop solution was added. After shaking the plate, the absorbance value was measured at 450 nm.

[0231] 2.2.5. Pseudovirus neutralization experiments

[0232] See also Figure 6 Middle C.

[0233] 293T-hACE2 cells were inoculated in a 96-well plate at a density of 5000 / well, and the experiment could be started after the cells adhered to the wall. The mouse serum samples were placed in a 56°C water bath for 30 minutes for complement inactivation, and the serum samples were diluted a certain multiple with serum-free DMEM medium, and the pseudovirus concentrate was diluted a certain multiple. 50 μL of the diluted serum sample and 50 μL of the diluted pseudovirus were incubated at 37°C for 1.5 hours, and the old culture medium was replaced with this mixture, and polybrene was added to make the final concentration of 5 μg / mL. After 6 hours of culture in the incubator, the cells were replaced with fresh serum-containing DMEM culture medium, and the chemiluminescent signal of the pseudovirus in the 293ThACE2 cells was detected by an ELISA instrument after 48 hours of culture.

[0234] Results and discussion

[0235] In order to obtain exosome-like vesicles with RBD on the surface, the cell membrane of 293T-RBD cells was extracted by hypotonic cell rupture combined with differential centrifugation, and cell membrane vesicles were obtained by extrusion. Figure 4 The average particle size of this vesicle is about 144nm ( Figure 4 A), good dispersion, surface charge is slightly negative ( Figure 4 Middle B), transmission electron microscopy shows a bowl-shaped structure after the vesicles are air-dried and flattened ( Figure 4 Middle C).

[0236] In order to determine whether the cell membrane vesicles are loaded with RBD protein and the amount of RBD protein loaded, western blotting was used to characterize the RBD on the cell membrane, such as Figure 5 As shown in Figure 2, compared with the cell membrane extracted from 293T cells, there is a large amount of RBD expression on the cell membrane from 293T-RBD cells, such as Figure 5 As shown in A and B, after drawing and calculating the standard curve (such as Figure 5 C), every 1×10 7 The cell membrane obtained from the cells has about 3 μg of RBD. The cell membrane vesicles carrying RBD can be recorded as RBD-CMVs.

[0237] In order to explore whether cell membrane vesicles from 293T-RBD cells can induce specific humoral immunity in mice, cell membrane vesicles were used as antigens, mixed with aluminum adjuvant, and mice were immunized by intramuscular injection and intraperitoneal injection at an antigen dose of 10 μg RBD / mouse. The immunization scheme was as follows: Figure 6As shown in A, RBD-specific IgG can be detected in mouse serum one week after the second immunization ( Figure 6 B), indicating that cell membrane vesicles can effectively induce humoral immunity as an antigen delivery system, such as Figure 6 As shown in D, antibodies in mouse serum can effectively block the entry of SARS-CoV-2 pseudovirus into 293T-hACE2 cells, indicating that exosome-like vaccines expressing RBD have the potential to fight SARS-CoV-2.

[0238] Example 3. Preparation, characterization and immune effect evaluation of antigen-adjuvant integrated vaccine

[0239] Materials and instruments

[0240] Materials

[0241] The gene sequence of TCS (gift from Professor Pang-Chui Shaw of the Chinese University of Hong Kong); pTXB1 vector (New England Biolabs, USA); LB medium (Oxoid, UK); isopropyl β-D-1-thiogalactoside (IPTG), ampicillin, and L-cysteine ​​(Dalian Meilun Biotechnology Co., Ltd.); 4-Isothiocyanatophenyla-α-mannopyranoside (Beijing Kailaisen Technology Co., Ltd., Beijing, China); chitin resin (New England Biolabs, Massachusetts, USA); DSPE-PEG2000-MAL (Shanghai Aiweituo Pharmaceutical Technology Co., Ltd.); isoflurane (Shenzhen Ruiwode Life Science Co., Ltd.); CD11c-FITC, MHC-II-PE, and CD86-AF647 (BD Pharmingen, USA).

[0242] 3.1.20 Instruments

[0243] Laser particle size analyzer (Malvern Panalytical, UK); water purification instrument (Ultra pure plus-12A, Shanghai Hetai Instrument Co., Ltd., China); flow cytometer (ACEA NovoCyte 3000, USA); microplate reader (Biotek Synergy4, USA); MALDI-TOF mass spectrometer (MALDI-TOF / TOF 5800analyzer, AB Sciex, Massachusetts, USA); gel imaging system (ChemiDoc MPTM Imaging System, BIO-RAD, California, USA).

[0244] 3.1.3. Experimental animals

[0245] BALB / c female mice (SPF grade) aged 6-8 weeks were purchased from Shanghai Slake Experimental Animal Co., Ltd. and kept at 25°C room temperature, 12 h day and night, regular addition of water and sterile food, and regular cage changes. All animal experimental operations followed the specifications and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0246] Experimental methods

[0247] 3.2.1. Expression and purification of trichosanthin (TCS)

[0248] The recombinant plasmid (pTXB1-TCS) expressing trichosanthin (TCS) was constructed by subcloning by Shanghai Jierui Bioengineering Co., Ltd. Take 100 μL of glycerol bacteria containing the recombinant plasmid and add it to 5mL LB medium and shake it for 3-4h until the bacterial solution becomes turbid, then transfer it to 500mL LB medium and shake it until the bacteria grow to the logarithmic growth phase (37°C, 200rpm), that is, the OD value at 600nm (OD 600 absorbance value) reaches 0.5-0.6, then add IPTG with a final concentration of 0.3mM to induce protein expression, and continue to shake and culture in a shaker at 25°C, 150rpm for 12h.

[0249] The bacterial solution was centrifuged at 10,000 × g for 3 min at 4°C, the supernatant was discarded, the bacteria were collected, and the bacteria were resuspended in 15 mL of Binding buffer. The bacteria were placed in a probe ultrasonic disruptor and ultrasonically disrupted at 400 W in an ice bath (3 s interval, 3 s ultrasonication), and then centrifuged at 10,000 × g and 4°C for 30 min. The supernatant was collected, filtered with a 0.45 μm filter membrane, and the target protein in the filtrate was purified with a chitin column. The buffer containing the target protein was then concentrated to 1 mL by centrifugation at 4,500 rpm at 4°C using an ultrafiltration tube with a molecular weight cutoff of 10 kDa.

[0250] The gel exclusion chromatography column (desalting column, G25) was loaded into the fast protein purification liquid chromatograph (FPLC), and the column was balanced with ten column volumes of ultrapure water and 10 column volumes of PBS in turn until the detection baseline of UV and conductivity was stable. The concentrated TCS solution was injected, the flow rate was set to 1 mL / min, the detection wavelength was 280 nm, and the sample was collected when the UV detection baseline showed obvious fluctuations, and the small molecule salts that peaked later were discarded. The protein concentration was determined using the BCA protein quantification kit.

[0251] Use SDS-PAGE combined with Coomassie staining to analyze the purity of the product: take 40μL of the purified product and add 10μL of 5× loading buffer. Mix well and boil the sample in boiling water for 5 minutes. Perform protein gel electrophoresis. After the electrophoresis, place the gel in Coomassie stain and shake at room temperature for 4-5 hours. Then place the stained gel in 10% glacial acetic acid and shake for 3 hours to decolorize it, and then take pictures with a gel imager.

[0252] 3.2.2. Synthesis and purification of DSPE-PEG-TCS

[0253] Take 10mg DSPE-PEG-MAL and dissolve it in 2mL 10mM PBS (pH7.4), add 100μL 4.7mg / mL trichosanthes protein solution, react with 4℃ overnight, filter with 0.45μm filter membrane the next day and concentrate to 1mL with ultrafiltration tube with molecular weight cutoff of 10kDa, use FPLC to separate TCS from DSPE-PEG-TCS: load gel exclusion chromatography column (desalting column, G25) into fast protein liquid chromatography (FPLC), balance the column with ten column volumes of ultrapure water and 10 column volumes of PBS in turn, until the detection baseline of UV and conductivity is stable. Inject the concentrated reaction solution, set the flow rate to 1mL / min, the detection wavelength to 280nm, collect the sample when the UV detection baseline fluctuates significantly, and discard the unreacted TCS in the latter peak. Use BCA protein quantification kit to determine the protein concentration of the outflow sample of the first peak.

[0254] 3.2.3. SDS-PAGE characterization of DSPE-PEG-TCS

[0255] Adjust the concentration of TCS to the same as that of the sample obtained in 3.2.2, take 40 μL of the purified product and add 10 μL of 5× loading buffer, mix well and boil the sample in boiling water for 5 minutes, perform protein gel electrophoresis according to 1.2.4, and after the electrophoresis, place the gel in Coomassie stain and shake at room temperature for 4-5 hours, then place the stained gel in 10% (v / v) glacial acetic acid (the volume ratio of acetic acid and water is 10%:90%) and shake for 3 hours to decolorize, and then take pictures with a gel imager.

[0256] 3.2.4. Molecular weight characterization of TCS and DSPE-PEG-TCS by MALDI-TOF

[0257] TCS and the sample obtained in 3.2.2 were desalted by FPLC to dissolve the protein in ultrapure water. 10 mg / mL sinapinic acid was prepared with 30% acetonitrile solution as the matrix, mixed with the sample to be tested in a volume ratio of 2:1, and applied to the plate machine. The sample was detected in the "positive linear" mode, and the molecular weight range of the detection was 10 kDa to 60 kDa.

[0258] 3.2.5. Preparation and particle size determination of RBD / TCS-CMVS

[0259] The protein concentration of the synthesized and separated DSPE-PEG-TCS was determined by the BCA method, and incubated with the cell membrane vesicles expressing RBD at 4°C for 2h, so that the final concentration of TCS was 200μM and the final concentration of total protein in the cell membrane vesicles was 5mg / mL, and cell membrane vesicles co-loaded with TCS and RBD were prepared. After diluting it 10 times with ultrapure water, the particle size and Zeta potential of RBD / TCS-CMVS were measured with a laser particle size analyzer.

[0260] 3.2.6. Study on the effect of RBD-CMVs-TCS on stimulating the maturation of BMDCs (bone marrow-derived dendritic cells) in vitro

[0261] First, 4-5 week old BALB / c mice were euthanized, and the femur and tibia of the mice were taken. The serum-free DMEM was aspirated with a 1 mL syringe to flush out the bone marrow, which was evenly blown and filtered and collected by centrifugation. The red blood cells were lysed with red blood cell lysis buffer and then placed in DMEM culture medium containing GM-CSF at a final concentration of 20 ng / mL and IL-4 at 10 ng / mL and cultured for 3 days. On the third day, half of the culture medium was discarded and supplemented with fresh cytokine-containing culture medium, and cultured for another two days to obtain immature BMDCs.

[0262] Immature BMDCs were cultured at 4 × 10 4 / well were inoculated in a 12-well plate, and a PBS negative control group, an LPS positive control group (the final concentration of lipopolysaccharide LPS was 500 ng / mL), an RBD-CMVs group, an RBD-CMVs+free TCS group, and an RBD-CMVs-TCS group were set up. After 20 hours of in vitro stimulation, the cells were collected for flow cytometry detection, and the BMDCs maturation markers CD86 and MHC-II were used as evaluation indicators to study the in vitro stimulation effect of the vaccine on BMDCs.

[0263] 3.2.7. In vivo study of the effect of RBD-CMVs-TCS in inducing RBD-specific neutralizing antibodies

[0264] Twenty 6-8 week old Balb / c female mice were raised in an SPF animal breeding room for one week and randomly divided into 4 groups, 5 mice in each group, and mice were immunized according to the following scheme. Immunization was performed once a week. One week after the second immunization, blood was collected from the orbital venous plexus of mice using a capillary tube. 300 μL of plasma was collected from each mouse, and after standing at room temperature for 4 hours, it was centrifuged twice at 8000 rpm. The supernatant was obtained to obtain mouse serum, and specific antibodies in the mouse serum were detected according to the method of 2.2.4.

[0265] The in vivo study scheme of RBD-CMVs-TCS inducing RBD-specific neutralizing antibodies can be found in Table 3.

[0266] Table 3. Mouse immunization schedule

[0267] Grouping Immune pathway Antigen dose TCS dosage 1 im 10 μg 5μg 2 im 10 μg 10 μg 3 im 10 μg - 4 im - -

[0268] In Table 3, im means intramuscular injection.

[0269] 3.3. Results and discussion

[0270] First, this project used the E. coli expression system to express trichosanthin, and after chitin column purification and desalting by desalting column, trichosanthin with a molecular weight of about 27 kDa was obtained ( Figure 7 A and B). Since trichosanthin is rich in sulfhydryl groups, it can react with maleimide-containing compounds at pH 6.5-7.5 to form thioether bonds. Therefore, DSPE-PEG-MAL (weight average molecular weight of 2900 Da) was reacted with TCS to obtain DSPE-PEG-TCS. DSPE-PEG-TCS was separated from unreacted TCS using a protein purifier, as shown in FIG. Figure 7 As shown in C and D, DSPE-PEG-TCS has a large molecular weight, which preliminarily proves the successful synthesis of DSPE-PEG-TCS. Then we use matrix-assisted laser desorption time-of-flight mass spectrometry to characterize the molecular weight of the two samples, as shown in Figure 7 As shown in Figures E and F, the weight average molecular weight of TCS is about 28,000 Da, and the weight average molecular weight of DSPE-PEG-TCS is about 29,000 Da, indicating that DSPE-PEG-TCS was successfully synthesized.

[0271] The protein concentration of the synthesized DSPE-PEG-TCS was determined by the BCA (bicinchoninic acid) method, and then incubated with the RBD-CMVs prepared in Example 2 at 4°C for 2 h, so that the final concentration of TCS was 200 μM and the final concentration of total protein in the cell membrane vesicles was 5 mg / mL. This process utilizes the hydrophobic interaction between DSPE and the lipid membrane to insert DSPE-PEG-TCS into the exosome-like membrane ( Figure 8 In A), such as Figure 8 As shown in B and C, the particle size of the obtained RBD / TCS-CMVs is not much different from that of RBD-CMVs, and the negative charge is slightly weaker.

[0272] In order to explore whether RBD / TCS-CMVs have immunostimulatory function, RBD / TCS-CMVs were co-incubated with BMDCs to detect the expression of BMDC maturation markers, such as Fig. 9 As shown in A, compared with the RBD-CMVs and RBD-CMVs+free TCS groups, RBD / TCS-CMVs had the most significant effect in stimulating BMDC maturation, with significant differences compared with the control group ( Fig. 9 A and B).

[0273] In order to investigate whether RBD / TCS-CMVs can induce specific humoral immunity in mice, mice were immunized twice by intramuscular injection, such as Fig. 9 As shown in C, RBD-specific neutralizing antibodies were detected in the mouse serum one week after the second immunization, which was higher than that in the control group, and the neutralizing antibodies increased with the increase of TCS dose, indicating that RBD / TCS-CMVs has the potential to prevent SARS-CoV-2 infection.

[0274] The transmembrane domain and intracellular domain of the ZEBOV virus glycoprotein were fused with RBD to express RBD on the cell membrane, indicating that the transmembrane domain and intracellular domain of the ZEBOV virus glycoprotein can serve as membrane anchoring proteins. 293T cells constructed by lentiviral infection can stably express RBD, and RBD is also expressed on the cell membrane extracted by hypotonic rupture and differential centrifugation. The exosome-like cells obtained by extrusion can induce RBD-specific immune responses in mice as particulate vaccines. In addition, TCS can be inserted into the cell membrane in the form of DSPE-PEG-TCS through DSPE-PEG modification, thereby constructing exosome-like cells with antigens and adjuvants. The corresponding vaccine can stimulate the maturation of bone marrow-derived dendritic cells in vitro and induce the production of specific neutralizing antibodies in vivo, which has the potential to fight against SARS-CoV-2 invasion and / or infection.

[0275] At present, SARS-CoV-2 is still mutating, which is a major problem in vaccine development. The preparation method of the engineered exosomes provided by the present invention is simple. Once the corresponding stable strain (cell strain that stably expresses the target antigen protein) is constructed, the cell membrane with the antigen protein can be continuously extracted as a vaccine. Therefore, the rapid preparation and production of cell membrane vaccines for new mutant strains can be used as a method to solve the mutation problem, and then assisted by different vaccine adjuvants, the human body can be immunized in different immune pathways, such as respiratory immunity, muscle immunity, etc. In addition, the cell membrane vaccine can also be freeze-dried and stored, so that it does not need ultra-low temperature transportation and storage, and the stability of the vaccine can be maintained.

[0276] The technical features of the above-mentioned embodiments and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0277] The above embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of the present invention. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solution provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. An exosome-like substance co-loaded with antigen and trichosanthin, characterized in that: It comprises a cell membrane vesicle, an antigen unit bound to the surface of the cell membrane vesicle, and a trichosanthin unit bound to the surface of the cell membrane vesicle.

2. The exosome-like substance according to claim 1, characterized in that Meet one or more of the following characteristics: The antigenic unit is the SARS-CoV-2 receptor binding domain; The cell source of the cell membrane vesicles is selected from human embryonic kidney cells HEK 293T; The average particle size of the cell membrane vesicles is 140-150 nm; In the exosome-like vesicles co-loaded with antigen and trichosanthin, the content ratio of the trichosanthin unit to the total protein in the cell membrane vesicles is (0.01-0.1) μmol / mg.

3. The exosome-like substance according to claim 1, characterized in that The antigen unit is bound to the surface of the cell membrane vesicle through the transmembrane domain of the viral glycoprotein, wherein the antigen unit is bound to one side of the transmembrane domain of the viral glycoprotein, and the transmembrane domain passes through the membrane structure of the cell membrane vesicle.

4. The exosome-like substance according to claim 3, characterized in that: Meet one or more of the following characteristics: A linker unit may or may not be provided between the antigen unit and the transmembrane domain of the viral glycoprotein, and the linker unit has a length of 8 to 12 amino acids; The transmembrane domain of the viral glycoprotein is also connected to the intracellular domain of the viral glycoprotein, and the intracellular domain of the viral glycoprotein is located inside the cell membrane vesicle; The viral glycoprotein is ZEBOV viral glycoprotein.

5. The exosome-like substance according to any one of claims 1 to 4, characterized in that The trichosanthin unit is bound to the surface of the cell membrane vesicle via a lipid unit, and the lipid unit is inserted into the membrane structure of the cell membrane vesicle.

6. The exosome-like substance according to claim 5, characterized in that Meet one or more of the following characteristics: The lipid unit is selected from one or more of distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, and dipalmitoylphosphatidylethanolamine; The trichosanthin unit is connected to the lipid unit via a polyethylene glycol unit, the weight average molecular weight of the polyethylene glycol unit is 1800Da to 2200Da, and the polyethylene glycol unit is connected to the sulfhydryl site in the trichosanthin unit; The trichosanthin unit is connected to the lipid unit via a thioether bond formed by a maleimide group and a thiol group.

7. A method for preparing exosome-like vesicles co-loaded with antigen and trichosanthin, characterized in that: The steps include: A viral vector containing the coding sequence of the antigen unit is obtained by using a gene sequence including a signal peptide, an antigen unit, a transmembrane domain and an intracellular domain of a viral glycoprotein in sequence, and then transfected into a host cell to obtain an engineered cell expressing the antigen unit on the cell membrane surface; wherein a connecting unit is provided or not provided between the antigen unit and the transmembrane domain of the viral glycoprotein, and the length of the connecting unit is 8 to 12 amino acids; Cultivating the engineered cells, and subjecting the culture to cell disruption, differential centrifugation and extrusion to obtain cell membrane vesicles with the antigen unit bound to the surface; The lipid-modified trichosanthin is co-incubated with the cell membrane vesicles with the antigen unit bound to the surface to obtain exosome-like vesicles carrying the antigen unit and the trichosanthin unit.

8. The preparation method according to claim 7, characterized in that: Meet one or more of the following characteristics: The viral vector is selected from a lentiviral vector; The host cell is selected from HEK 293T cells; The lipid-modified trichosanthin has the following structure: lipid unit-polyethylene glycol unit-trichosanthin unit; In the step of culturing the engineered cells, culture conditions suitable for the growth of the engineered cells are adopted; In the step of culturing the engineered cells, the culturing is terminated when the cells grow to 70%-90%; The cell disruption comprises firstly hypotonic cell disruption and then ultrasonic cell disruption; The hypotonic cell rupture comprises: placing the cells in a hypotonic lysis solution and stirring at 4° C. for 2 hours, wherein the hypotonic lysis solution is a 0.1×PBS buffer solution containing a protease inhibitor; the ultrasonic cell rupture method comprises: 200W ultrasonic for 3 minutes, interval of 3 seconds, and ultrasonic for 3 seconds; The differential centrifugation comprises: first centrifuging at 10,000×g for 10 min at 4°C, and then centrifuging the supernatant at 100,000×g for 60 min at 4°C; The extrusion comprises: sequentially extruding using liposome extrusion membranes with pore sizes of 1 μm, 0.4 μm and 0.2 μm, and the number of extrusions of each membrane with a pore size is 10 to 12 times; the liposome extrusion membrane is a polycarbonate membrane; In the step of co-incubating the lipid-modified trichosanthin with the cell membrane vesicles with the antigen unit bound to the surface, in the culture system after the co-incubation, the final concentration of the trichosanthin component and the final concentration of the total protein in the cell membrane vesicles is (0.01-0.1) μmol:1 mg; or, in the exosome-like vesicles co-loaded with antigen and trichosanthin, the content ratio of the trichosanthin unit to the total protein in the cell membrane vesicles is (0.01-0.1) μmol / mg; The step of culturing the engineered cells, subjecting the culture to cell disruption, differential centrifugation and extrusion, and obtaining cell membrane vesicles with the antigen unit bound to the surface comprises the following steps: the step of culturing the engineered cells, subjecting the culture to cell disruption, differential centrifugation and extrusion, and obtaining cell membrane vesicles with the antigen unit bound to the surface comprises the following steps: culturing the engineered cells, digesting after culturing, centrifuging, washing with PBS, discarding the supernatant, resuspending the cells with a hypotonic lysis solution and stirring, ultrasonically disrupting the cells, and differentially centrifuging again, collecting the precipitate obtained from the last centrifugation, resuspending the collected precipitate with PBS, and sequentially extruding from a large pore size to a small pore size with a liposome extrusion membrane with a pore size of 1 to 0.2 μm to obtain cell membrane vesicles with the antigen unit bound to the surface; After the step of co-incubating the lipid-modified trichosanthin with the cell membrane vesicles with the antigen unit bound to the surface, the method further comprises the step of ultrafiltration centrifugation of the co-incubation system using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa, and collecting the material on the upper layer of the ultrafiltration membrane to obtain the exosome-like vesicles co-loaded with the antigen and trichosanthin; The exosome-like vesicles co-loaded with antigen and trichosanthin are as defined in any one of claims 1 to 7.

9. An antigen-adjuvant integrated vaccine preparation, characterized in that: It comprises at least one of the exosome-like vesicles co-loaded with antigen and trichosanthin as described in any one of claims 1 to 7 and the exosome-like vesicles co-loaded with antigen and trichosanthin prepared by the preparation method described in claim 7 or 8.

10. Use of the exosome-like substance co-loaded with antigen and trichosanthin as claimed in any one of claims 1 to 6, or the exosome-like substance co-loaded with antigen and trichosanthin prepared by the preparation method of claim 7 or 8, or the antigen-adjuvant integrated vaccine preparation as claimed in claim 9 in the preparation of a drug capable of inducing specific antibody neutralization; Preferably, the drug capable of inducing specific antibody neutralization is used to combat the invasion and / or infection of SARS-CoV-2, wherein: The antigenic unit is the SARS-CoV-2 receptor binding domain.

Images