CN114767847B - New crown recombinant protein vaccine adjuvant and its application - Google Patents

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

New crown recombinant protein vaccine adjuvant and its application

technical field

The application belongs to the technical field of biomedicine, and in particular relates to a new crown recombinant protein vaccine adjuvant and its application.

Background technique

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, SARS-CoV-2 for short) is a single-stranded RNA virus of the β-coronaviridae family. The study found that the Spike protein (S) on the surface of the new coronavirus envelope binds to the receptor angiotensin-converting enzyme 2 (ACE2) on the surface of the host cell, thereby allowing the virus to invade the host cell. The S protein is mainly composed of the N-terminal S1 subunit and the C-terminal S2 subunit. S1 is mainly responsible for the binding of the virus to the host receptor, and S2 mainly plays the role of membrane fusion. The receptor binding domain (RBD) of the S1 subunit is the key region for direct binding to ACE2.

Due to the very high mutation characteristics of the SARS-CoV-2 virus, the prevention and control of the epidemic caused by the mutant strain of the new coronavirus is still the number one problem in the world's public health. Vaccination is the most cost-effective and effective public health intervention for the prevention and control of infectious diseases. Therefore, developing a safe and effective new crown vaccine is the most effective measure to prevent and stop the new crown pandemic.

At present, the domestically produced vaccines are inactivated vaccines, adenovirus vector vaccines and recombinant protein vaccines. Among them, recombinant protein vaccines have no live virus, so there is no need to worry about virus leakage, and they are easy to store and transport. Recombinant protein vaccines are generally composed of antigens and adjuvants. The antigens are generally selected from S protein or RBD protein. Therefore, adjuvants are the key to the successful development of SARS-CoV-2 recombinant protein vaccines.

Recent studies have found that aluminum adjuvant and MF59 can promote the production of COVID-19 vaccine-specific antibodies and neutralizing antibodies, but at the same time, they have exposed obvious limitations and the mechanism of action is not fully understood. For example: ① Aluminium salt and MF59 adjuvant mainly enhance systemic IgG antibodies and neutralizing antibodies against SARS-CoV-2 S protein and RBD protein through intramuscular or subcutaneous immunity, but do not promote the production of respiratory mucosal antibody IgA, suggesting that such adjuvant vaccines It may be unfavorable to curb the spread of new coronavirus infection; ② Aluminum salt and MF59 adjuvant lack or induce weak CD4 ⁺ T and CD8 ⁺ T cell responses, weakening the ability of cellular immunity against new coronavirus; ③ Aluminum salt and MF59 adjuvant have obvious side effects, usually leading to symptoms such as rashes, granulomas, headaches, and neurological disorders. Immunization with MF59 fusion inactivated vaccine does not induce CD8 ⁺ T cell immunity, which usually results in symptoms such as redness, swelling, fever, pain, and stomach discomfort at the injection site. ④ Aluminum-adjuvanted vaccines cannot be frozen and are 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. Designing a stable and efficient mucosal immune recombinant protein vaccine is one of the directions of new crown vaccine research. The selection of appropriate mucosal immune adjuvants is the key to the success of mucosal immune vaccine development.

SUMMARY OF THE INVENTION

The purpose of this application is to provide a new crown recombinant protein vaccine adjuvant and its application, which aims to solve the problem that the vaccine adjuvant commonly used in the new crown vaccine in the prior art weakens the ability of cellular immunity to resist the new crown virus and has relatively large side effects.

In order to realize the above-mentioned application purpose, the technical scheme adopted in this application is as follows:

In the first aspect, this application provides the application of long-chain thymic stromal lymphopoietin as a new crown recombinant protein vaccine adjuvant in the preparation of a new crown recombinant protein vaccine.

In a second aspect, the application provides a new crown recombinant protein vaccine adjuvant, and the new crown recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin.

In a third aspect, the present application provides a new crown vaccine, and the new crown vaccine includes a new crown recombinant protein vaccine adjuvant.

In a fourth aspect, the present application provides a method for preparing a new crown vaccine, comprising the following steps:

Dissolving the new crown recombinant protein vaccine adjuvant and immunogen in the buffer respectively to obtain the new crown recombinant protein vaccine adjuvant buffer and immunogen buffer;

The new crown recombinant protein vaccine adjuvant buffer and the immunogen buffer are mixed to prepare the new crown vaccine.

The application of the long-chain thymic stromal lymphopoietin provided in the first aspect of the application as an adjuvant for a new crown recombinant protein vaccine in the preparation of a new crown recombinant protein vaccine. Since long-chain thymic stromal lymphopoietin is an epithelial cell-derived interleukin (IL)-7-like cytokine, it mainly binds to the TSLP receptor (TSLPR) and the IL-7 receptor alpha chain (IL-7Rα). The heterodimeric receptor complex activates downstream signal transduction. As an adjuvant for the new coronavirus recombinant protein vaccine, it can promote the production of antibodies, has a strong affinity for the S1 protein and RBD protein of the new coronavirus, and can effectively inhibit the The S1 protein binds to ACE2 and can effectively neutralize the binding of the SARS-CoV-2 pseudovirus to ACE2, thereby preventing the virus from entering and infecting cells; it can also enhance cellular immune responses. In addition, long-chain thymic stromal lymphopoietin acts as a The use of vaccine adjuvants has less side effects, is easy to store, and can be widely used, providing a new strategy for the development of new coronavirus recombinant protein vaccines.

The new crown recombinant protein vaccine adjuvant provided in the second aspect of this application, the new crown recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin. This application uses long-chain thymic stromal lymphopoietin as an adjuvant for the new coronavirus recombinant protein vaccine, and the obtained vaccine can promote the production of antibodies, has strong affinity for the S1 protein and RBD protein of the new coronavirus, and can effectively inhibit the S1 protein and the RBD protein. ACE2 binds, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; at the same time, it can also enhance cellular immune response, with better immune effect, and also has high biological compatibility.

The third aspect of this application provides a new crown vaccine. The new crown vaccine includes a new crown recombinant protein vaccine adjuvant. Since the provided new crown recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin, the prepared new crown vaccine can effectively promote antibodies It can also prevent the virus from entering and infecting cells; at the same time, it can also enhance the cellular immune response, has a better immune effect, and the obtained vaccine has good stability and high safety, which is conducive to wide use.

A fourth aspect of the present application provides a preparation method for a new crown vaccine, which includes separately providing a new crown recombinant protein vaccine adjuvant buffer and an immunogen buffer, and then mixing to obtain a vaccine; the preparation method is simple and fast, and does not need to provide large-scale equipment.

Description of drawings

In order to illustrate the technical solutions in the embodiments of the present application more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only for the present application. In some embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the timeline of intranasal immunization of mice provided in the examples of the present application.

FIG. 2 is an analysis diagram of lfTSLP as an adjuvant to promote the production of S1 protein-specific antibodies provided in the examples of the present application.

Figure 3 is an analysis diagram of the affinity and inhibitory effect of lfTSLP as an adjuvant on S1/RBD provided in the examples of the present application.

FIG. 4 is a graph showing the response analysis of lfTSLP promoting vaccine-specific germinal centers provided in the examples of the present application.

Detailed ways

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clear, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

In this application, the term "and/or", which describes the relationship between related objects, means that there can be three relationships, for example, A and/or B, which can mean that A exists alone, A and B exist at the same time, and B exists alone Happening. where A and B can be singular or plural. The character "/" generally indicates that the associated objects are an "or" relationship.

In this application, "at least one" means one or more, and "plurality" means two or more. "At least one item(s) below" or similar expressions refer to any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (one) of a, b, or c", or "at least one (one) of a, b, and c", can mean: a, b, c, a-b ( That is, a and b), a-c, b-c, or a-b-c, where a, b, and c can be single or multiple respectively.

It should be understood that, in various embodiments of the present application, the size of the sequence numbers of the above-mentioned processes does not imply the sequence of execution, some or all of the steps may be executed in parallel or sequentially, and the execution sequence of each process should be based on its functions and It is determined by the internal logic and should not constitute any limitation on the implementation process of the embodiments of the present application.

The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. As used in the embodiments of this application and the appended claims, the singular forms "a" and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

The weight of the relevant components mentioned in the description of the examples of this application can not only refer to the specific content of each component, but also can represent the proportional relationship between the weights of the components. It is within the scope disclosed in the description of the embodiments of the present application that the content of the ingredients is scaled up or down. Specifically, the mass in the description of the embodiments of the present application may be a mass unit known in the chemical field such as μg, mg, g, kg, etc.

The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implying the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, a feature defined as "first" or "second" may expressly or implicitly include one or more of that feature.

The first aspect of the embodiments of this application provides the application of a long-chain thymic stromal lymphopoietin as an adjuvant for a new crown recombinant protein vaccine in the preparation of a new crown recombinant protein vaccine.

The application of the long-chain thymic stromal lymphopoietin provided in the first aspect of the examples of this application as an adjuvant for a new crown recombinant protein vaccine in the preparation of a new crown recombinant protein vaccine. Since long-chain thymic stromal lymphopoietin is an epithelial cell-derived interleukin (IL)-7-like cytokine, it mainly binds to the TSLP receptor (TSLPR) and the IL-7 receptor alpha chain (IL-7Rα). The heterodimeric receptor complex activates downstream signal transduction. As an adjuvant for the new coronavirus recombinant protein vaccine, it can promote the production of antibodies, has a strong affinity for the S1 protein and RBD protein of the new coronavirus, and can effectively inhibit the The S1 protein binds to ACE2 and can effectively neutralize the binding of the SARS-CoV-2 pseudovirus to ACE2, thereby preventing the virus from entering and infecting cells; it can also enhance cellular immune responses. In addition, long-chain thymic stromal lymphopoietin acts as a The use of vaccine adjuvants has less side effects, is easy to store, and can be widely used, providing a new strategy for the development of new coronavirus recombinant protein vaccines.

In some embodiments, the provided long-chain thymic stromal lymphopoietin (ifTSLP for short) is a subtype of thymic stromal lymphopoietin, and the long-chain thymic stromal lymphopoietin contains 159 amino acids and is a Epithelial cell-derived interleukin (IL)-7-like cytokine that binds to the heterodimeric receptor complex composed of TSLP receptor (TSLPR) and IL-7 receptor alpha chain (IL-7Rα) to activate downstream signaling divert.

In some embodiments, long-chain thymic stromal lymphopoietin induces mucosal immunity and inhibits viral infection by promoting the production of respiratory mucosal antibody IgA.

In some embodiments, the long-chain thymic stromal lymphopoietin enhances the ability of cellular immunity against SARS-CoV-2 by inducing responses from CD4 ⁺ T cells, CD8 ⁺ T cells and germinal centers.

In some embodiments, long-chain thymic stromal lymphopoietin induces vaccine-specific IgGl antibody production to promote vaccine-specific germinal center responses.

A second aspect of the embodiments of the present application provides a novel coronavirus recombinant protein vaccine adjuvant, and the novel coronavirus recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin.

The new crown recombinant protein vaccine adjuvant provided in the second aspect of the embodiment of the present application, the new crown recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin. This application uses long-chain thymic stromal lymphopoietin as an adjuvant for the new coronavirus recombinant protein vaccine, and the obtained vaccine can promote the production of antibodies, has strong affinity for the S1 protein and RBD protein of the new coronavirus, and can effectively inhibit the S1 protein and the RBD protein. ACE2 binds, and can effectively neutralize the combination of SARS-CoV-2 pseudovirus and ACE2, thereby preventing the virus from entering and infecting cells; at the same time, it can also enhance cellular immune response, with better immune effect, and also has high biological compatibility.

In some embodiments, the new crown recombinant protein vaccine adjuvant further includes at least one of aluminum adjuvant and MF59 adjuvant.

In some embodiments, the provided novel coronavirus recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin and aluminum adjuvant.

In some embodiments, the provided novel coronavirus recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin and MF59 adjuvant.

In some embodiments, the provided novel coronavirus recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin, aluminum adjuvant and MF59 adjuvant.

A third aspect of the embodiments of this application provides a new crown vaccine, and the new crown vaccine includes a new crown recombinant protein vaccine adjuvant.

The third aspect of the examples of this application provides a new crown vaccine, the new crown vaccine includes a new crown recombinant protein vaccine adjuvant, and since the provided new crown recombinant protein vaccine adjuvant includes long-chain thymic stromal lymphopoietin, the prepared new crown vaccine can effectively It can promote the production of antibodies, and prevent the virus from entering and infecting cells; at the same time, it can also enhance the cellular immune response, has a better immune effect, and the obtained vaccine has good stability and high safety, which is conducive to wide use.

In some embodiments, the novel coronavirus vaccine further includes an immunogen, wherein the immunogen includes a novel coronavirus immunogen.

In some embodiments, in the new crown vaccine, the molar ratio of the new crown recombinant protein vaccine adjuvant and the immunogen is 1~1.1:1~1.1.

In some specific embodiments, in the new crown vaccine, the molar ratio of the new crown recombinant protein vaccine adjuvant and the immunogen is 1:1.

A fourth aspect of the embodiments of the present application provides a method for preparing a new crown vaccine, comprising the following steps:

S01. Dissolve the new crown recombinant protein vaccine adjuvant and immunogen in the buffer respectively to obtain the new crown recombinant protein vaccine adjuvant buffer and immunogen buffer;

S02. Mix the new crown recombinant protein vaccine adjuvant buffer and immunogen buffer to prepare the new crown vaccine.

The fourth aspect of the embodiments of the present application provides a method for preparing a new crown vaccine, which includes separately providing a new crown recombinant protein vaccine adjuvant buffer and an immunogen buffer, and then performing a mixed treatment to obtain a vaccine; the preparation method is simple and fast, and does not require Provide large equipment.

In step S01, the provided buffer is selected from PBS buffer.

In some embodiments, the novel coronavirus 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 the buffer to obtain the new crown recombinant protein vaccine adjuvant buffer and the immunogen buffer.

In step S02, the new crown recombinant protein vaccine adjuvant buffer and the immunogen buffer are mixed, wherein the molar ratio of the provided new crown recombinant protein vaccine adjuvant buffer and the immunogen buffer is 1:1.

The following description will be given in conjunction with specific embodiments.

Example 1

A kind of new crown vaccine and preparation method thereof

The new crown vaccine provided includes the new crown recombinant protein vaccine adjuvant long-chain thymic stromal lymphopoietin and the immunogen new coronavirus S1 protein.

The preparation method of the new crown vaccine includes the following steps:

Dissolving the new crown recombinant protein vaccine adjuvant long-chain thymic stromal lymphopoietin and the immunogen new coronavirus S1 protein in PBS buffer respectively to obtain the new crown recombinant protein vaccine adjuvant buffer and immunogen buffer;

The new crown recombinant protein vaccine adjuvant buffer and the immunogen buffer are mixed in a molar ratio of 1:1 (that is, the new crown recombinant protein vaccine adjuvant is 2 μg, and the immunogen is 2 μg) to prepare the new crown vaccine.

Performance Testing

(1) Vaccine immunization experiment

Twelve purchased C57BL/6 mice with an average weight of 20 g were randomly divided into 2 groups (group A, group B). The new crown vaccine prepared in Example 1 (that is, the new crown recombinant protein vaccine adjuvant is 2 μg, the immunogen is 2 μg), immunized once every 10 days, immunized three times, and on the 10th day after the third immunization, blood was collected from the ocular marginal vein of all mice, and the serum was collected by centrifugation. The mice were euthanized, the spleen and lymph nodes were collected by dissection, and the lungs were lavaged with 500 μL of PBS for the detection of IgA in the respiratory mucosa.

(2) Detection of specific IgG and IgA in serum after immunization by ELISA

①Detection of specific IgG in serum after immunization by ELISA

1) Prepare antigen S1 or RBD (6 μg/mL) with a final concentration of 4 μg/mL in PBS, add 100 μL/well of antigen protein to a highly adsorbed eight-well enzyme labeling strip, coat overnight at 4°C and discard the liquid. Wash 5 times with 200 μL PBST;

2) Add 200 μL blocking solution (5 % BSA), block at room temperature for 2 h, discard the liquid, and wash 5 times with 200 μL PBST;

3) Add 100 μL/well of double-diluted serum, incubate at room temperature for 1 h, discard the liquid, and wash 5 times with 200 μL PBS;

4) Add 100 μL of Anti-IgG(H+L)-HRP diluted 1:2000 to each well, shake slightly at room temperature for 1 h, discard the antibody and wash 5 times with 200 μL PBST;

5) Add 100 μL of TMB substrate and shake slightly at room temperature until the sample clearly turns blue, then add 100 μL of 1M HCl to stop the reaction, and measure its OD value at OD 450 nm.

②Detection of serum specific IgA after immunization by ELISA

1) Prepare antigen S1 or RBD (6 μg/mL) with a final concentration of 4 μg/mL in PBS, add 100 μL/well of antigen protein to a high-adsorption eight-well enzyme labeling strip, coat overnight at 4°C and discard the liquid. Wash 5 times with 200 μL PBST;

2) Add 200 μL blocking solution (5 % BSA), block at room temperature for 2 h, discard the liquid, and wash 5 times with 200 μL PBST;

3) Add 100 μL/well of double-diluted serum, incubate at room temperature for 1 h, discard the liquid, and wash 5 times with 200 μL PBS;

4) Add 100 μL of Anti-IgA-HRP diluted 1:2000 to each well, shake slightly at room temperature for 1 h, discard the antibody and wash 5 times with 200 μL PBST;

Add 100 μL of TMB substrate and shake at room temperature until the sample turns blue, then add 100 μL of 1 M HCl to stop the reaction, and measure its OD value at OD 450 nm.

(3) Competitive ELISA to detect the inhibitory effect of serum on the binding of S1 to ACE2

1) Prepare hACE2-mFC with a final concentration of 6 μg/mL in PBS, add 100 μL/well to a highly adsorbed eight-well enzyme labeling strip, coat overnight at 4°C, discard the liquid, and wash 5 times with 200 μL PBST;

2) Add 200 μL of blocking solution (5% BSA), block at room temperature for 2 h, discard the liquid, and wash 5 times with 200 μL of PBST;

3) Double-diluted serum and S1 (final concentration of 6 μg/mL); the control group without serum was added to the sealed 96-well plate after mixing, and the liquid was discarded after incubating at 37°C for 1 h. , washed 5 times with 200 μL PBST;

4) Add 100 μL of Anti-His-HRP diluted 1:3000 to each well, shake slightly at room temperature for 1 h, discard the antibody and wash 5 times with 200 μL PBST;

5) Add 100 μL of TMB substrate and shake at room temperature slightly until the sample turns blue, then add 100 μL of 1M HCl to stop the reaction, and measure its OD value at OD450 nm.

(4) Detection of the binding effect of serum neutralizing SARS-CoV-2 pseudovirus and ACE2

1) Cell plating: Inoculate the HEK293T-ACE2 cells to be infected in a 96-well cell culture plate with an inoculation volume of about 2×10 ⁴ . As for the overnight culture in the incubator, the cell inoculation density is about 30 when the virus is infected the next day. %;

2) After thawing SARS-CoV-2-GFP pseudovirus and serum at 4°C, mix and incubate at room temperature for 1 h; (serum is diluted 2 times, 4 times, and 8 times with DMEM complete medium respectively; the final concentration of pseudovirus is 6.3×10 ⁴ TU/mL);

3) Take out the extracted HEK293T-ACE2 cells, discard the upper medium, pipette 100 μL of the above incubated pseudovirus-serum mixture into the 96-well plate on which HEK293T-ACE2 cells have been plated, three replicate wells, After 6 h of infection in the cell incubator, the cells were replaced with fresh DMEM complete medium and continued to culture for 48 h, and the infection of pseudovirus was observed by fluorescence microscope.

(5) Detection of vaccine-specific germinal center responses by flow cytometry

1) Take the spleen of the mice after three immunizations, grind the lymph nodes into a single-cell suspension and transfer it to a 96-well round bottom plate, and centrifuge (5min, 1500rpm);

2) Blocking: prepare 3% BSA with PBS, add 50 μL of CD16/32 diluted 1:1000 in 3% BSA to each well, block at 4°C for 30 min, and centrifuge (5 min, 1500 rpm);

3) Staining: use PE-labeled anti-mouse CD19 antibody, PE/CY7-labeled anti-mouse CD4 antibody, FITC-labeled anti-mouse PD1 antibody, APC-labeled anti-mouse CXCR5 antibody, Pacific Blue-labeled anti-mouse GL7 antibody, PerCP- CY5.5-labeled anti-mouse FAS antibody, BV605-labeled anti-mouse B220 antibody and AF700-labeled anti-mouse CD44 antibody (all antibodies were purchased from Biolegend), stained cells with antibodies, stained at 4°C for 30 min, centrifuged (5 min, 1500rpm);

4) Detect cell fluorescence on a flow cytometer.

(6) Detection of DC changes by flow cytometry

1) Take the lymph nodes of mice 5 days after the secondary immunization, grind them into a single-cell suspension, transfer them to a 96-well round bottom plate, and centrifuge (5min, 1500rpm);

2) Blocking: prepare 3% BSA with PBS, add 50 μL of CD16/32 diluted 1:1000 in 3% BSA to each well, block at 4°C for 30 min, and centrifuge (5 min, 1500 rpm);

3) Staining: using BV605-labeled anti-mouse CD11c antibody, AF700-labeled anti-mouse IA/IE antibody, FITC-labeled anti-mouse CD103 antibody, PE-labeled anti-mouse CD80 antibody, (all antibodies were purchased from Biolegend Company), Cells were stained with antibodies, stained at 4°C for 30 min, and centrifuged (5 min, 1500 rpm);

4) Detect cell fluorescence on a flow cytometer.

Result analysis

(1) Vaccine immunization experiment

As shown in the timeline of intranasal immunization mice in Figure 1, the experimental results show that at the cell layer level, lfTSLP can effectively neutralize the binding of 2019-nCoV to ACE2 and reduce the virus-infected cells; flow cytometry further revealed that lfTSLP can Promotes vaccine-specific germinal center responses. The above results indicate that lfTSLP can play an important role as a new mucosal adjuvant in the new crown recombinant protein vaccine.

(2) Detection of specific IgG and IgA in serum after immunization by ELISA

In order to detect the level of specific antibodies in the serum, the mice were immunized by intranasal injection and blood was collected according to the time axis of (Fig. 1), and the serum after three intranasal immunizations was detected by ELISA. The results are shown in Fig. 2, and A in Fig. 2 is The level of S1-specific IgG antibody in the serum after three immunizations; Figure 2B is the RBD-specific IgG antibody level in the serum after three immunizations; Figure 2C is the S1-specific IgA antibody level in the serum after three immunizations; Figure 2D is the level of RBD-specific IgA antibody in serum after three immunizations; it can be seen that lfTSLP can promote S1-specific antibody IgG (as shown in Figure 2A) IgA (as shown in Figure 2C), RBD-specific antibody IgG (as shown in Figure 2B), and IgA (as shown in Figure 2B). Figure 2D) Generation.

(3) The affinity and inhibitory effect of lfTSLP as an adjuvant on S1/RBD

In order to explore the affinity of the adjuvant for S1/RBD and its inhibitory effect on the binding of S1 to hACE2, ELISA was used to analyze the affinity, and the doubling diluted serum was added to the coated S1/RBD for reaction, as shown in Figure 3 As shown, Figure 3 A is the ELISA method to analyze the affinity of serum and S1; Figure 3 B is the ELISA method to analyze the serum and RBD affinity; Figure 3 C is the competitive ELISA method to analyze the inhibitory effect of serum and S1 and hACE2 binding , IC50 is the inhibition rate of serum antibody; D of Figure 3 shows the GFP fluorescence of HEK293T-ACE2 cells infected with SARS-CoV-2 pseudovirus after co-incubating with serum of different dilutions under a microscope for 48 h.

The results showed that lfTSLP had a strong affinity for S1 (as shown in Figure 3A) and RBD (as shown in Figure 3B); competitive ELISA was used to analyze the inhibitory effect on the binding of S1 to hACE2, and the serum was diluted to a final concentration of 6 μg/ After mixing evenly, mL of S1 was added to the coated hACE2 for reaction. The results showed that lfTSLP had an inhibitory effect on the binding of S1 to hACE2 (Fig. 3C). For the neutralization of cell lines, different dilutions of serum were used to co-incubate SARS-CoV-2 pseudovirus with GFP and then added to HEK293T cells overexpressing ACE2 for culture; GFP fluorescence was observed under a microscope after 48 h (Fig. 3D), the results showed that the more serum, the less fluorescence, indicating that lfTSLP has a significant effect on inhibiting the binding of SARS-CoV-2 pseudovirus to ACE2 and entering cells.

(IV) lfTSLP promotes vaccine-specific germinal center responses

In order to explore the mechanism by which lfTSLP promotes antibody production, the spleen and lymph nodes of mice after three immunizations were analyzed by flow cytometry. The results are shown in Figure 4. Figure 4 A shows the Tfh cells in the spleen and lymph nodes of mice after three immunization Figure 4 B is the proportion of GC B cells in the spleen and lymph nodes of mice after three immunizations; Figure 4 C is the proportion of DC cells in the lymph nodes of mice 5 days after the second 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 were analyzed, and it was found that lfTSLP could promote the increase of the proportion of CD103 ⁺ (Fig. 4C). ) .

In conclusion, the application of the long-chain thymic stromal lymphopoietin provided in this application as an adjuvant for a new crown recombinant protein vaccine in the preparation of a new crown recombinant protein vaccine. Since long-chain thymic stromal lymphopoietin is an epithelial cell-derived interleukin (IL)-7-like cytokine, it mainly binds to the TSLP receptor (TSLPR) and the IL-7 receptor alpha chain (IL-7Rα). The heterodimeric receptor complex activates downstream signal transduction. As an adjuvant for the new coronavirus recombinant protein vaccine, it can promote the production of antibodies, has a strong affinity for the S1 protein and RBD protein of the new coronavirus, and can effectively inhibit the The S1 protein binds to ACE2 and can effectively neutralize the binding of the SARS-CoV-2 pseudovirus to ACE2, thereby preventing the virus from entering and infecting cells; it can also enhance cellular immune responses. In addition, long-chain thymic stromal lymphopoietin acts as a The use of vaccine adjuvants has less side effects, is easy to store, and can be widely used, providing a new strategy for the development of new coronavirus recombinant protein vaccines.

The above descriptions are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection of the present application. within the range.

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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