CN114767847B - Novel crown recombinant protein vaccine adjuvant and application thereof - Google Patents

Novel crown recombinant protein vaccine adjuvant and application thereof Download PDF

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CN114767847B
CN114767847B CN202210710925.XA CN202210710925A CN114767847B CN 114767847 B CN114767847 B CN 114767847B CN 202210710925 A CN202210710925 A CN 202210710925A CN 114767847 B CN114767847 B CN 114767847B
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recombinant protein
vaccine
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new crown
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CN114767847A (en
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叶亮
胡灏
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Shenzhen University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
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    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Abstract

The application relates to the technical field of biomedicine, in particular to a new crown recombinant protein vaccine adjuvant and application thereof, and provides application of long-chain thymic stromal lymphopoietin serving as the new crown recombinant protein vaccine adjuvant in preparation of new crown recombinant protein vaccines. Long-chain thymic stromal lymphopoietin is used as a new crown recombinant protein vaccine adjuvant, which can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of new crown virus, can effectively inhibit the combination of S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; in addition, the long-chain thymic stromal lymphopoietin is used as a vaccine adjuvant, has small side effect, is easy to store and can be widely used, and a new strategy is provided for the development of new crown recombinant protein vaccines.

Description

Novel crown recombinant protein vaccine adjuvant and application thereof
Technical Field
The application belongs to the technical field of biological medicines, and particularly relates to a novel crown recombinant protein vaccine adjuvant and application thereof.
Background
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, new coronavirus for short) is a single-stranded RNA virus of the family of beta-coronaviridae. The research finds that the new coronavirus envelope surface Spike protein (Spike protein, S) is combined with the host cell surface receptor Angiotensin converting enzyme-2 (ACE 2), so that the virus invades the host cell. The S protein mainly comprises an N-terminal S1 subunit and a C-terminal S2 subunit, S1 is mainly responsible for binding of viruses with host receptors, and S2 mainly plays a role in membrane fusion, wherein a Receptor Binding Domain (RBD) of the S1 subunit is a key region for directly binding ACE 2.
As SARS-CoV-2 virus has very high mutation property, the prevention and control of epidemic caused by new coronavirus mutant strain is still the first problem of world public health at present. Vaccination is the most economical and effective means of public health intervention for the prevention and control of infectious diseases. Therefore, the development of safe and effective new coronary vaccines is the most effective measure for preventing and stopping new coronary pandemics.
At present, vaccines produced in China are inactivated vaccines, adenovirus vector vaccines and recombinant protein vaccines, wherein the recombinant protein vaccines have no live viruses, do not need to worry about the advantages of virus leakage and the like, and are easy to store, transport and the like. The recombinant protein vaccine generally consists of an antigen and an adjuvant, wherein the antigen generally selects S protein or RBD protein, so the adjuvant is a key link for successfully developing the SARS-CoV-2 recombinant protein vaccine.
Recent studies have found that aluminum adjuvant and MF59 can promote the production of specific and neutralizing antibodies of the new corona vaccine, but at the same time, obvious limitations are exposed and the mechanism of action is not completely clear. Such as: firstly, the aluminum salt and MF59 adjuvant mainly enhance systemic IgG antibodies and neutralizing antibodies aiming at new coronavirus S protein and RBD protein through muscle or subcutaneous immunization, but do not promote the generation of respiratory tract mucosa antibody IgA, which suggests that the adjuvant vaccine is not beneficial to inhibiting the spread infection of the new coronavirus; ② aluminum salts and MF59 adjuvant lack or induce weaker CD4 + T and CD8 + T cell response, which impairs the ability of cellular immunity against new coronaviruses; ③ the aluminum salt and the adjuvant MF59 have obvious side effects, and usually cause symptoms such as erythra, granuloma, headache, nerve diseases, etc. MF59 fusion inactivated vaccine immunization does not induce CD8 + T cell immunity, often leads to symptoms such as red swelling, fever, pain, stomach discomfort at the injection site. Fourthly, the aluminum adjuvant vaccine can not be frozen and is not easy to store. Since the new coronavirus is mainly infected and spread through the respiratory tract, the mucous membrane of the respiratory tract becomes the first line of defense against the new coronavirus. The design of stable and efficient mucosal immune recombinant protein vaccines is one of the directions for research of new corona vaccines. And the selection of a proper mucosal immune adjuvant is the key for the successful development of a mucosal immune vaccine.
Disclosure of Invention
The application aims to provide a new crown recombinant protein vaccine adjuvant and application thereof, and aims to solve the problems that the common vaccine adjuvant of the new crown vaccine in the prior art weakens the capability of cellular immunity to resist new crown viruses and has larger side effect.
In order to achieve the purpose of the application, the technical scheme adopted by the application is as follows:
in a first aspect, the present application provides the use of long-chain thymic stromal lymphopoietin as an adjuvant in a new crown recombinant protein vaccine for the preparation of a new crown recombinant protein vaccine.
In a second aspect, the present application provides a novel crown recombinant protein vaccine adjuvant comprising long-chain thymic stromal lymphopoietin.
In a third aspect, the present application provides a new corona vaccine comprising a new corona recombinant protein vaccine adjuvant.
In a fourth aspect, the present application provides a method for preparing a novel corona vaccine, comprising the steps of:
respectively dissolving the new crown recombinant protein vaccine adjuvant and the immunogen in a buffer solution to obtain a new crown recombinant protein vaccine adjuvant buffer solution and an immunogen buffer solution;
and mixing the new crown recombinant protein vaccine adjuvant buffer solution and the immunogen buffer solution to prepare the new crown vaccine.
The application of the long-chain thymic stromal lymphopoietin provided by the first aspect of the application as an adjuvant of a new crown recombinant protein vaccine in preparing the new crown recombinant protein vaccine. Because the long-chain thymic stromal lymphopoietin is an Interleukin (IL) -7-like cytokine derived from epithelial cells, the long-chain thymic stromal lymphopoietin mainly combines with a heterodimer receptor complex consisting of a TSLP receptor (TSLPR) and an IL-7 receptor alpha chain (IL-7R alpha) to activate downstream signal transduction, and the long-chain thymic stromal lymphopoietin is used as a new crown recombinant protein vaccine adjuvant, can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of new crown virus, can effectively inhibit the combination of the S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; in addition, the long-chain thymic stromal lymphopoietin is used as a vaccine adjuvant, has small side effect, is easy to store and can be widely used, and a new strategy is provided for the development of new crown recombinant protein vaccines.
In the new crown recombinant protein vaccine adjuvant provided by the second aspect of the application, the new crown recombinant protein vaccine adjuvant comprises long-chain thymic stromal lymphopoietin. The long-chain thymic stromal lymphopoietin is used as a new crown recombinant protein vaccine adjuvant, the obtained vaccine can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of new crown virus, can effectively inhibit the combination of S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; meanwhile, the immune response of cells can be enhanced, and the immune response has a better immune effect and higher biocompatibility.
The third aspect of the application provides a new corona vaccine, which comprises a new corona recombinant protein vaccine adjuvant, and the provided new corona recombinant protein vaccine adjuvant comprises long-chain thymic stromal lymphopoietin, so the prepared new corona vaccine can effectively promote the generation of antibodies and prevent viruses from entering and infecting cells; meanwhile, the vaccine can enhance cellular immune response, has a good immune effect, is good in stability and high in safety, and is beneficial to wide application.
The fourth aspect of the application provides a method for preparing a new corona vaccine, which comprises the steps of respectively providing a new corona recombinant protein vaccine adjuvant buffer solution and an immunogen buffer solution, and mixing to obtain the vaccine; the preparation method is simple and quick, and large-scale instruments and equipment do not need to be provided.
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In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments or the prior art descriptions will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a timeline of nasal drop immunization of mice provided in the examples of the present application.
FIG. 2 is a graph of an assay of lfTSLP as an adjuvant to facilitate the production of antibodies specific to S1 protein, as provided in the examples herein.
FIG. 3 is a graph showing the affinity and inhibition of S1/RBD by lfTSLP as an adjuvant provided in the examples of the present application.
FIG. 4 is a graph of a response analysis of the lfTSLP promotion vaccine specific development center provided in the examples of the present application.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present application more clearly apparent, the present application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In this application, the term "and/or" describes an association relationship of associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a is present alone, A and B are present simultaneously, and B is present alone. Wherein A and B can be singular or plural. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
In the present application, "at least one" means one or more, "a plurality" means two or more. "at least one of the following" or similar expressions refer to any combination of these items, including any combination of the singular or plural items. For example, "at least one (a), b, or c", or "at least one (a), b, and c", may each represent: a, b, c, a-b (i.e. a and b), a-c, b-c, or a-b-c, wherein a, b, and c can be single or multiple respectively.
It should be understood that, in various embodiments of the present application, the sequence numbers of the above-mentioned processes do not mean the execution sequence, some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its function and inherent logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in the examples of this application and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weight of the related components mentioned in the description of the embodiments of the present application may not only refer to the specific content of each component, but also represent the proportional relationship of the weight among the components, and therefore, the content of the related components is scaled up or down within the scope disclosed in the description of the embodiments of the present application as long as it is scaled up or down according to the description of the embodiments of the present application. Specifically, the mass in the description of the embodiments of the present application may be a mass unit known in the chemical industry field such as μ g, mg, g, kg, and the like.
The terms "first" and "second" are used for descriptive purposes only and are used for distinguishing purposes such as substances from one another, and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the present application. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature.
The first aspect of the embodiment of the application provides an application of long-chain thymic stromal lymphopoietin as an adjuvant of a new crown recombinant protein vaccine in preparation of the new crown recombinant protein vaccine.
The application of the long-chain thymic stromal lymphopoietin provided by the first aspect of the embodiment of the application as an adjuvant of a new crown recombinant protein vaccine in the preparation of the new crown recombinant protein vaccine. Because the long-chain thymic stromal lymphopoietin is an Interleukin (IL) -7-like cytokine derived from epithelial cells, the long-chain thymic stromal lymphopoietin mainly combines with a heterodimer receptor complex consisting of a TSLP receptor (TSLPR) and an IL-7 receptor alpha chain (IL-7R alpha) to activate downstream signal transduction, and the long-chain thymic stromal lymphopoietin is used as a new crown recombinant protein vaccine adjuvant, can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of new crown virus, can effectively inhibit the combination of the S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; in addition, the long-chain thymic stromal lymphopoietin is used as a vaccine adjuvant, has small side effect, is easy to store and can be widely used, and a new strategy is provided for the development of new crown recombinant protein vaccines.
In some embodiments, long-chain thymic stromal lymphopoietin (lfTSLP) is provided as a subtype of thymic stromal lymphopoietin, and long-chain thymic stromal lymphopoietin comprises 159 amino acids, is an epithelial cell-derived Interleukin (IL) -7-like cytokine, and is capable of binding to a heterodimeric receptor complex consisting of TSLP receptor (TSLPR) and IL-7 receptor alpha chain (IL-7 ra) to activate downstream signaling.
In some embodiments, long-chain thymic stromal lymphopoietin inhibits viral infection by promoting respiratory mucosal antibody IgA production, inducing mucosal immunity.
In some embodiments, long-chain thymic stromal lymphopoietin is produced by induction of CD4 + T cell, CD8 + T cells and germinal centers react to enhance the cellular immunity against the new coronavirus.
In some embodiments, long-chain thymic stromal lymphopoietin induces vaccine-specific IgG1 antibody production to promote vaccine-specific germinal center responses.
In a second aspect of the embodiments of the present application, there is provided a novel crown recombinant protein vaccine adjuvant, which comprises long-chain thymic stromal lymphopoietin.
In the new crown recombinant protein vaccine adjuvant provided by the second aspect of the embodiment of the application, the new crown recombinant protein vaccine adjuvant comprises long-chain thymic stromal lymphopoietin. The vaccine obtained by using the long-chain thymic stromal lymphopoietin as a new crown recombinant protein vaccine adjuvant can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of a new crown virus, can effectively inhibit the combination of the S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; meanwhile, the immune response of cells can be enhanced, and the immune response has a better immune effect and higher biocompatibility.
In some embodiments, the novel crown recombinant protein vaccine adjuvant further comprises at least one of an aluminum adjuvant, MF59 adjuvant.
In some embodiments, provided novel crown recombinant protein vaccine adjuvants include long-chain thymic stromal lymphopoietin and aluminum adjuvants.
In some embodiments, provided novel crown recombinant protein vaccine adjuvants include long-chain thymic stromal lymphopoietin and MF59 adjuvant.
In some embodiments, provided novel crown recombinant protein vaccine adjuvants include long-chain thymic stromal lymphopoietin, aluminum adjuvant, and MF59 adjuvant.
In a third aspect of the embodiments of the present application, there is provided a novel corona vaccine comprising a novel corona recombinant protein vaccine adjuvant.
The third aspect of the embodiment of the application provides a new corona vaccine, which comprises a new corona recombinant protein vaccine adjuvant, and the provided new corona recombinant protein vaccine adjuvant comprises long-chain thymic stromal lymphopoietin, so that the prepared new corona vaccine can effectively promote the generation of antibodies and prevent viruses from entering and infecting cells; meanwhile, the vaccine can enhance cellular immune response, has a good immune effect, is good in stability and high in safety, and is beneficial to wide application.
In some embodiments, the neocorona vaccine further comprises an immunogen, wherein the immunogen comprises a neocorona virus immunogen.
In some embodiments, the molar ratio of the adjuvant to the immunogen in the new corona vaccine is 1-1.1: 1 to 1.1.
In some embodiments, the molar ratio of the new corona recombinant protein vaccine adjuvant to the immunogen is 1: 1.
in a fourth aspect, the embodiments of the present application provide a method for preparing a novel corona vaccine, comprising the following steps:
s01, respectively dissolving the new crown recombinant protein vaccine adjuvant and the immunogen in a buffer solution to obtain a new crown recombinant protein vaccine adjuvant buffer solution and an immunogen buffer solution;
s02, mixing the new crown recombinant protein vaccine adjuvant buffer solution and the immunogen buffer solution to prepare the new crown vaccine.
The fourth aspect of the embodiment of the application provides a method for preparing a new corona vaccine, which comprises the steps of respectively providing an adjuvant buffer solution and an immunogen buffer solution of the new corona recombinant protein vaccine, and mixing to obtain the vaccine; the preparation method is simple and quick, and large-scale instruments and equipment do not need to be provided.
In step S01, the buffer provided is selected from PBS buffer.
In some embodiments, the novel corona recombinant protein vaccine adjuvant is selected from long-chain thymic stromal lymphopoietin.
In some embodiments, the immunogen is selected from the novel coronavirus S1 protein.
Further, the new crown recombinant protein vaccine adjuvant and the immunogen are respectively dissolved in a buffer solution to obtain a new crown recombinant protein vaccine adjuvant buffer solution and an immunogen buffer solution.
In step S02, mixing the new crown recombinant protein vaccine adjuvant buffer solution and the immunogen buffer solution, wherein the molar ratio of the provided new crown recombinant protein vaccine adjuvant buffer solution to the immunogen buffer solution is 1: 1.
the following description will be given with reference to specific examples.
Example 1
New corona vaccine and preparation method thereof
The provided new corona vaccine comprises a new corona recombinant protein vaccine adjuvant long-chain thymic stromal lymphopoietin and immunogen novel coronavirus S1 protein.
The preparation method of the novel corona vaccine comprises the following steps:
respectively dissolving long-chain thymic stromal lymphopoietin serving as an adjuvant of the new crown recombinant protein vaccine and novel coronavirus S1 serving as an immunogen in a PBS buffer solution to obtain a new crown recombinant protein vaccine adjuvant buffer solution and an immunogen buffer solution;
and (2) mixing the new crown recombinant protein vaccine adjuvant buffer solution and the immunogen buffer solution according to the molar ratio of 1: 1 (namely 2 mug of new crown recombinant protein vaccine adjuvant and 2 mug of immunogen) to prepare the new crown vaccine.
Performance testing
Vaccine immunization experiment
12 purchased C57BL/6 mice each weighing 20g were randomly divided into 2 groups (group A, B). The mice in group A were subjected to nasal drip immunization for 30. mu.L of S1 protein (2. mu.g), the mice in group B were subjected to nasal drip immunization for 30. mu.L of the novel corona vaccine prepared in example 1 (i.e., the adjuvant of the novel corona recombinant protein vaccine is 2. mu.g, and the immunogen is 2. mu.g), immunization was performed once every 10 days for 3 times, and all mice were subjected to periocular vein bleeding on day 10 after the third immunization, and serum was collected by centrifugation. Mice were euthanized, spleens, lymph nodes were dissected and collected, and lungs were lavaged with 500 μ L PBS for detection of respiratory mucosal IgA.
(II) ELISA method for detecting serum specific IgG and IgA after immunization
ELISA method for detecting serum specific IgG after immunization
1) Preparing antigen S1 or RBD (6. mu.g/mL) with PBS (4. mu.g/mL of final concentration), adding 100. mu.L/well antigen protein into the high-adsorption eight-well enzyme label strip, coating overnight at 4 ℃, discarding liquid, and washing with 200. mu.L PBST for 5 times;
2) adding 200 μ L blocking solution (5% BSA), blocking at room temperature for 2 hr, discarding the solution, and washing with 200 μ L PBST for 5 times;
3) adding 100 μ L/well diluted serum, incubating at room temperature for 1h, discarding liquid, and washing with 200 μ L PBS for 5 times;
4) add 100. mu.L per well as 1: Anti-IgG (H + L) -HRP diluted at 2000, gently shaken at room temperature for 1H and then antibody discarded and washed 5 times with 200. mu.L PBST;
5) the addition of 100. mu.L of TMB substrate, gentle shaking at room temperature until the sample clearly developed a blue color, followed by the addition of 100. mu.L of 1M HCl to stop the reaction and determine its OD at OD450 nm.
② ELISA method for detecting serum specificity IgA after immunization
1) Preparing antigen S1 or RBD (6 mug/mL) with a final concentration of 4 mug/mL by PBS, adding 100 mug/well antigen protein into a high-adsorption eight-well enzyme label strip, discarding liquid after coating overnight at 4 ℃, and washing 5 times by 200 mug PBST;
2) adding 200 μ L blocking solution (5% BSA), blocking at room temperature for 2 hr, discarding the solution, and washing with 200 μ L PBST for 5 times;
3) adding 100 μ L/well diluted serum, incubating at room temperature for 1h, discarding liquid, and washing with 200 μ L PBS for 5 times;
4) add 100. mu.L per well as 1: 2000 diluted Anti-IgA-HRP, gently shaking for 1h at room temperature, discarding the antibody and washing 5 times with 200 μ L PBST;
the addition of 100. mu.L of TMB substrate, gentle shaking at room temperature until the sample clearly developed a blue color, followed by the addition of 100. mu.L of 1M HCl to stop the reaction and determine its OD at OD450 nm.
(III) competitive ELISA method for detecting inhibition effect of serum on binding of S1 and ACE2
1) Preparing hACE2-mFC with PBS to the final concentration of 6 mug/mL, adding 100 mug/hole in a high-adsorption eight-hole enzyme label strip, discarding liquid after coating overnight at 4 ℃, and washing 5 times with 200 mug PBST;
2) adding 200 μ L blocking solution (5% BSA), blocking at room temperature for 2 hr, discarding the solution, and washing with 200 μ L PBST for 5 times;
3) the diluted sera were mixed with S1 (final concentration 6. mu.g/mL); adding the control group without serum, mixing, adding into sealed 96-well plate, incubating at 37 deg.C for 1h, discarding liquid, and washing with 200 μ L PBST for 5 times;
4) add 100. mu.L per well as 1: 3000 diluted Anti-His-HRP, gently shaking for 1h at room temperature, discarding the antibody and washing 5 times with 200. mu.L PBST;
5) the addition of 100. mu.L of TMB substrate, gentle shaking at room temperature until the sample clearly developed a blue color, followed by the addition of 100. mu.L of 1M HCl to stop the reaction and determine its OD at OD450 nm.
(IV) detecting the binding effect of serum neutralization SARS-CoV-2 pseudovirus and ACE2
1) Cell plating: HEK293T-ACE2 cells to be infected were seeded into 96 well cell culture plates at approximately 2X 10 4 The incubator is used for overnight culture, so that the inoculation density of the cells is about 30 percent when virus infection is carried out the next day;
2) melting SARS-CoV-2-GFP pseudovirus and serum at 4 deg.C, mixing and incubating at room temperature for 1 h; (serum was diluted 2-fold, 4-fold, 8-fold with DMEM complete medium respectively; pseudovirus final concentration was 6.3X 10 4 TU/mL)；
3) Taking out and extracting the paved HEK293T-ACE2 cells, discarding the upper layer culture medium, sucking 100 mu L of the incubated pseudovirus-serum mixed solution into a 96-well plate paved with the HEK293T-ACE2 cells, repeating the three wells, infecting for 6 hours in a cell incubator, changing fresh DMEM complete culture medium to continuously culture for 48 hours, and observing the infection condition of the pseudovirus by using a fluorescence microscope.
(V) flow cytometry detection of the response of vaccine-specific Generation centers
1) Taking the spleen of the immunized mouse for three times, grinding lymph nodes into single cell suspension, transferring the single cell suspension into a 96-hole round bottom plate, and centrifuging (5min,1500 rpm);
2) and (3) sealing: 3% BSA was formulated in PBS and 50. mu.L of the BSA solution at 1: 1000 dilution CD16/32,4 ℃ blocking for 30min, centrifugation (5min,1500 rpm);
3) dyeing: using PE-labeled anti-murine CD19 antibody, PE/CY 7-labeled anti-murine CD4 antibody, FITC-labeled anti-murine PD1 antibody, APC-labeled anti-murine CXCR5 antibody, Pacific Blue-labeled anti-murine GL7 antibody, PerCP-CY 5.5-labeled anti-murine FAS antibody, BV 605-labeled anti-murine B220 antibody and AF 700-labeled anti-murine CD44 antibody (all antibodies were purchased from Biolegend), antibody staining of cells, staining at 4 ℃ for 30min, centrifugation (5min,1500 rpm);
4) cell fluorescence was detected on a flow cytometer.
(VI) flow cytometry detection of changes in DCs
1) Taking lymph nodes of the mice 5 days after the secondary immunization, grinding into single cell suspension, transferring into a 96-hole round bottom plate, and centrifuging (5min,1500 rpm);
2) and (3) sealing: 3% BSA was formulated in PBS and 50. mu.L of the BSA solution at 1: 1000 dilution CD16/32,4 ℃ blocking for 30min, centrifugation (5min,1500 rpm);
3) dyeing: using BV 605-labeled anti-murine CD11c antibody, AF 700-labeled anti-murine IA/IE antibody, FITC-labeled anti-murine CD103 antibody, PE-labeled anti-murine CD80 antibody, (all antibodies purchased from Biolegend), antibody staining of cells, staining at 4 ℃ for 30min, centrifugation (5min,1500 rpm);
4) cell fluorescence was detected on a flow cytometer.
Analysis of results
Vaccine immunization experiment
As shown in fig. 1, the time axis of nasal drop immunized mice shows that, at the cell layer level, lfTSLP can effectively neutralize the binding of the novel coronavirus and ACE2, and reduce virus-infected cells; it was further revealed by flow cytometry that lfTSLP can promote vaccine-specific germinal center responses. The results show that lfTSLP can be used as a novel mucosal adjuvant to play an important role in a new crown recombinant protein vaccine.
(II) ELISA method for detecting serum specific IgG and IgA after immunization
In order to detect the level of specific antibodies in serum, mice were subjected to nasal drip immunization and blood was collected according to the time axis of (fig. 1), and serum after three nasal drip immunization was detected by ELISA method, and the result is shown in fig. 2, in which a in fig. 2 is the level of S1 specific IgG antibodies in serum after three immunization; b in FIG. 2 is the level of RBD-specific IgG antibodies in serum after three immunizations; c of fig. 2 is S1-specific IgA antibody levels in serum after three immunizations; FIG. 2D is the level of RBD-specific IgA antibodies in serum after three immunizations; it can be seen that lfTSLP can promote the production of S1-specific antibody IgG (fig. 2A) IgA (fig. 2C), RBD-specific antibody IgG (fig. 2B) IgA (fig. 2D).
Affinity and inhibitory Effect of (tri) lfTSLP as an adjuvant on S1/RBD
In order to investigate the affinity of the adjuvant to S1/RBD and the inhibition effect of the adjuvant on the binding of S1 to hACE2, the affinity was analyzed by ELISA method, and the serum diluted by multiple times was added to the coated S1/RBD for reaction, as shown in FIG. 3, wherein A in FIG. 3 is the ELISA method for analyzing the affinity of the serum to S1; FIG. 3B shows the ELISA method for analyzing the affinity of serum to RBD; FIG. 3C shows the competitive ELISA assay for inhibition of binding of S1 to hACE2 in serum, and IC50 is the serum antibody inhibition rate; FIG. 3D is a graph showing the observation under a microscope of the fluorescence of GFP after infection of HEK293T-ACE2 cells for 48 h after incubation of SARS-CoV-2 pseudovirus with serum at different dilution times.
The results show that lfTSLP has a strong affinity for S1 (FIG. 3A), RBD (FIG. 3B); the inhibition of binding of S1 to hACE2 was analyzed by competitive ELISA, and serum dilution was mixed with S1 at a final concentration of 6. mu.g/mL, and the mixture was added to hACE2 to react, indicating that lfTSLP inhibits binding of S1 to hACE2 (FIG. 3C); to test the neutralizing effect of lfTSLP on SARS-CoV-2 pseudovirus into ACE2 overexpressing cell line, serum with different dilution times was used to incubate SARS-CoV-2 pseudovirus with GFP and added to ACE2 overexpressing HEK293T cells for culture; GFP fluorescence was observed under the microscope after 48 h (FIG. 3D), and the results showed that the more serum, the less fluorescence, indicating that lfTSLP had a significant effect of inhibiting the binding of SARS-CoV-2 pseudovirus to ACE2 and into cells.
(IV) lfTSLP promotes vaccine-specific germinal center response
In order to explore the mechanism of lfTSLP in promoting antibody production, spleen and lymph nodes of mice after three immunizations were analyzed by flow cytometry, and the results are shown in fig. 4, in which a in fig. 4 is the ratio of Tfh cells in spleen and lymph nodes of mice after three immunizations; FIG. 4B is the ratio of GC B cells in the spleen and lymph nodes of mice after three immunizations; FIG. 4C is the proportion of DC cells in the lymph nodes of mice 5 days after the secondary immunization.
It can be seen that lfTSLP can increase the proportion of Tfh cells (fig. 4A), GC B cells (fig. 4B); meanwhile, the lymph nodes of mice 5 days after secondary immunization are analyzed, and lfTSLP is found to promote CD103 + Increase in the ratio (fig. 4C).
In conclusion, the long-chain thymic stromal lymphopoietin provided by the application is used as an adjuvant of a new crown recombinant protein vaccine in the preparation of the new crown recombinant protein vaccine. Because the long-chain thymic stromal lymphopoietin is an Interleukin (IL) -7-like cytokine derived from epithelial cells, the long-chain thymic stromal lymphopoietin mainly combines with a heterodimer receptor complex consisting of a TSLP receptor (TSLPR) and an IL-7 receptor alpha chain (IL-7R alpha) to activate downstream signal transduction, and the long-chain thymic stromal lymphopoietin is used as a new crown recombinant protein vaccine adjuvant, can promote the generation of antibodies, has stronger affinity to S1 protein and RBD protein of new crown virus, can effectively inhibit the combination of the S1 protein and ACE2, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; in addition, the long-chain thymic stromal lymphopoietin is used as a vaccine adjuvant, has small side effect, is easy to store and can be widely used, and a new strategy is provided for the development of new crown recombinant protein vaccines.
The above description is only exemplary of the present application and should not be taken as limiting the present application, as any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (3)

1. The application of long-chain thymic stromal lymphopoietin as an adjuvant of the new crown recombinant protein vaccine in the preparation of the new crown recombinant protein vaccine; during the use process, the new crown recombinant protein vaccine is acted in the form of nasal drop immunization, the dose of the nasal drop immunization is 30 mu L, the new crown recombinant protein vaccine comprises 2 mu g of new crown recombinant protein vaccine adjuvant, and 2 mu g of immunogen; in addition, in the form of nasal drip immunization, long-chain thymic stromal lymphopoietin enhances the proportion of Tfh cells and GC B cells in the germinal center by increasing the recruitment of migratory DC cells to the lymph node germinal center, promotes vaccine-specific germinal center responses, and induces high-affinity antibodies to enhance the ability to resist neocoronaviruses.
2. The use of claim 1, wherein said long-chain thymic stromal lymphopoietin inhibits viral infection by promoting respiratory mucosal antibody IgA production, inducing mucosal immunity.
3. The use of claim 1, wherein the long-chain thymic stromal lymphopoietin induces vaccine-specific IgG1 antibody production to promote vaccine-specific germinal center response.
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