An artificially designed heat-resistant multimeric protein, scaffolds and applications and methods in the preparation of vaccines

Abstract

The application belongs to the technical field of nano-vaccine, and relates to an artificially designed heat-resistant multimeric protein, a scaffold and application and a method in preparation of a vaccine. The application provides an artificially designed heat-resistant multimeric protein. The artificially designed heat-resistant multimeric protein can be used for preparing a vaccine, and significantly improves the heat stability and immunogenicity of the vaccine antigen.

Description

Technical Field

[0001] This invention belongs to the field of nanovaccine technology, specifically relating to an artificially designed heat-resistant polymer protein, a scaffold, and its application and method in vaccine preparation. Background Technology

[0002] Vaccines are active immunizing agents used to prevent infectious diseases, made from pathogenic microorganisms (such as bacteria, rickettsiae, viruses, etc.) and their metabolites through artificial attenuation, inactivation, or genetic modification. Their main components are proteins or live microorganisms. Vaccination is recognized worldwide as the most economical and effective means of preventing, controlling, or eliminating vaccine-targeted diseases. An effective vaccine needs to elicit a protective immune response against a specific type of pathogen without causing disease itself, and maintain high thermal stability to avoid dependence on the cold chain. Traditional vaccines are mainly based on attenuated or inactivated live pathogens. Although they can elicit a strong immune response, they often have safety concerns regarding the regaining of pathogenicity. Subunit vaccines have higher safety but weaker immunogenicity, requiring adjuvants and multiple administrations. Furthermore, due to the limitations of their components, accidental exposure to environmental stressors (e.g., light or temperature) can lead to loss of immunogenicity in traditional vaccines, necessitating strict cold chain storage and transportation. Therefore, there is an urgent need to develop novel vaccines with high immunogenicity, high safety, and high stability.

[0003] Multimeric protein scaffolds have been widely used in the packaging and transportation of biological systems, and reusing these scaffolds for targeted delivery of multivalent immunogens is a major emerging trend in the vaccine field. Multimeric proteins possess a well-defined parallel multimeric structure with high stability, allowing the introduction of target-binding antigens and hinge regions, and achieving the desired multivalentity through self-assembly without disrupting target antigen binding. Compared to monomeric vaccines, multimeric proteins exhibit multiple antigenic epitopes on their surface, significantly enhancing the binding strength between antigens and immune cell receptors, thus eliciting a stronger immune signaling response. Simultaneously, multimeric protein vaccines generally have a larger particle size than monomeric vaccines, making them easily taken up by antigen-presenting cells and less easily excreted by the body, prolonging the antigen's retention time in the body and thus maintaining a durable immune response.

[0004] Multimeric proteins offer numerous advantages as scaffolds for multivalent antigen delivery, but selecting scaffolds with ultra-high heat resistance to maintain antigen stability presents certain challenges. Developing scalable, low-cost, heat-stable antigen delivery platforms can overcome the stringent cold chain conditions required for vaccine development and transportation, which is crucial for vaccine stockpiling to prevent epidemics and for providing vaccines to resource-scarce regions. Although various protein scaffolds (such as ferritin, dihydrolipoic acid acetyltransferase (E2p), and non-structural protein 10 (nsp10)) are currently used for vaccine antigen delivery, these antigens are all natural proteins, and the scaffold proteins exhibit insufficient heat stability and adjuvant effects. Summary of the Invention

[0005] The purpose of this invention is to provide an artificially designed heat-resistant multimeric protein, a scaffold, and its application and method in vaccine preparation. The artificially designed heat-resistant multimeric protein of this invention can be used to prepare vaccines, significantly improving the thermal stability and immunogenicity of vaccine antigens.

[0006] This invention provides an artificially designed heat-resistant multimeric protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The present invention also provides the use of the protein in the preparation of heat-resistant multimeric protein scaffolds and / or vaccines.

[0008] Preferably, the vaccine includes a novel coronavirus vaccine.

[0009] The present invention also provides an artificially designed heat-resistant multimeric protein scaffold, wherein the heat-resistant multimeric protein scaffold is a fusion protein comprising a heat-resistant multimeric protein and SpyCatcher.

[0010] Preferably, the amino acid sequence of the heat-resistant multimeric protein scaffold is as shown in SEQ ID NO.2.

[0011] The present invention also provides a vaccine constructed based on the heat-resistant multimeric protein described in the above technical solution, the vaccine comprising a heat-resistant multimeric protein scaffold and an antigen; the heat-resistant multimeric protein scaffold and the antigen are covalently coupled; the heat-resistant multimeric protein scaffold is a fusion protein containing a heat-resistant multimeric protein and SpyCatcher; the antigen is linked with a SpyTag.

[0012] Preferably, the structure of the heat-resistant multimeric protein scaffold includes: a SpyCatcher attached to the N-terminus or C-terminus of the heat-resistant multimeric protein, and a His histidine tag introduced at the C-terminus or N-terminus;

[0013] The structure of the antigen includes: a signal peptide and a SpyTag linked to the N-terminus of the antigen, and a His histidine tag introduced at the C-terminus.

[0014] Preferably, the antigen includes the receptor-binding domain of the SARS-CoV-2S spike protein.

[0015] Preferably, the nucleotide sequence of the signal peptide is as shown in SEQ ID NO.4.

[0016] This invention also provides a method for constructing the vaccine described in the above technical solution, comprising the following steps:

[0017] 1) Construct a scaffold vector for expressing heat-resistant multimeric proteins, and express the heat-resistant multimeric protein scaffold to obtain the heat-resistant multimeric protein scaffold;

[0018] 2) Construct a vector for expressing the antigen, and express the antigen to obtain the antigen;

[0019] 3) The heat-resistant polymeric protein scaffold is mixed with the antigen and conjugated to obtain the vaccine;

[0020] The heat-resistant multimeric protein scaffold is coupled to the antigen via the SpyCatcher and SpyTag system.

[0021] There is no time limit for the order of steps 1) and 2).

[0022] This invention provides an artificially designed heat-resistant multimeric protein. The artificially designed heat-resistant multimeric protein of this invention can be used to prepare vaccines, significantly improving the thermal stability and immunogenicity of vaccine antigens. Specifically, the invention includes the design of a novel heat-resistant multimeric protein sequence and prediction of its spatial structure; the artificial synthesis of a novel heat-resistant multimeric protein scaffold and the construction and purification of a multivalent vaccine; the thermal stability assessment of the novel heat-resistant multimeric protein scaffold and the multivalent vaccine; and the immunogenicity assessment of the novel heat-resistant multimeric protein multivalent vaccine. Experimental results show that the artificially designed novel heat-resistant multimeric protein scaffold of this invention can be used for vaccine antigen delivery, significantly improving the thermal stability of vaccine antigens and reducing dependence on cold chains. The novel heat-resistant multimeric protein scaffold designed in this invention can self-assemble to construct multivalent vaccines without disrupting the binding of target antigens, significantly improving antigen presentation efficiency and affinity with immune cell receptors, thus enhancing the immunization effect. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The 3D spatial structure prediction diagram provided by this invention;

[0025] Figure 2 This is a schematic diagram of the construction of SpyCatcher-A301 and SpyTag-RBD provided by the present invention;

[0026] Figure 3 The image shows the purity identification results of SDS-PAGE / Coomassie Brilliant Blue staining provided by this invention;

[0027] Figure 4 The spatial structure diagrams of SC-A301 and RBD-A301 provided by this invention are for TEM observation.

[0028] Figure 5 The figure shows the SDS-PAGE results and changes in the proportion of soluble protein of SC-A301 under different temperature treatments provided by this invention.

[0029] Figure 6 The hydrodynamic particle size results of SC-A301 under different temperature treatments provided by this invention;

[0030] Figure 7 The reaction results of SC-A301 with ST-RBD after heat treatment at 75°C are provided by the present invention;

[0031] Figure 8 The figure shows the SDS-PAGE results and changes in the proportion of soluble protein of RBD-A301 under different temperature treatments provided by this invention.

[0032] Figure 9 The image shows the results of ELISA detection of the integrity of RBD-A301 vaccine antigen at different temperatures, as provided by this invention.

[0033] Figure 10 The results of the pseudovirus neutralization experiment provided by this invention show the antibody titers against RBD in the serum of each group.

[0034] Figure 11 The graph shows the time-varying antibody titers against RBD in each group of serum samples provided by this invention.

[0035] Figure 12The results of the plaque assay for detecting antibody titers against RBD in serum of each group provided by the present invention are shown in the figure.

[0036] Figure 13 The graph shows the percentage of different types of T cells in the spleen cells of mice in each group as determined by flow cytometry according to the present invention. Detailed Implementation

[0037] This invention provides an artificially designed heat-resistant multimeric protein, the amino acid sequence of which is shown in SEQ ID NO.1: MKLYEGHLKAEGFSFGIVASRFNHTLVERLVEGAIDCILRHGGSEDNIELARVP GSWEIPLIVKNMLLKEDVEGVIALGVLVRGQTPHFEYVASEVSEGLANLSLEL GKPVSFGVVTADTLEQAVERAGTKQGNKGWDAMLSTIEMANLFKKLG (abbreviated as A301). This invention artificially designs a novel amino-terminal sequence (A301), which, through structural simulation, can form a multimeric spatial structure. The formation of this multimeric structure has been experimentally confirmed. When used in the construction and delivery of multivalent vaccine antigens, it can significantly improve the thermal stability and immunogenicity of the vaccine antigen. This invention establishes a method for preparing a heat-stable vaccine scaffold based on a novel artificially designed heat-resistant multimeric protein, which can then be applied to vaccine preparation.

[0038] This invention also provides the application of the protein in the preparation of heat-resistant multimeric protein scaffolds and / or vaccines. Traditional vaccines are mainly inactivated, live attenuated, and subunit vaccines, whose main components are proteins or live microorganisms. These vaccines have certain deficiencies in safety, immunogenicity, and stability, including poor safety of live attenuated vaccines and low immunogenicity of subunit vaccines. Due to the limitations of traditional vaccine components, they are unstable and easily affected by changes in external environmental conditions (especially temperature), leading to loss of immunogenicity. They must rely on strict low-temperature storage and cold chain transportation. Currently, most scaffold proteins are derived from natural proteins, and the body may have already developed an immune response or tolerance to these proteins, resulting in the loss or weakening of the scaffold's adjuvant effect on the vaccine. This invention provides a non-natural heat-resistant multimeric protein based on artificial design for the construction and delivery of multivalent antigens and vaccines. This method can significantly improve the thermal stability, safety, and immunogenicity of vaccine antigens.

[0039] In this invention, the vaccine preferably includes a novel coronavirus (SARS-CoV-2) vaccine.

[0040] The present invention also provides an artificially designed heat-resistant multimeric protein scaffold, wherein the heat-resistant multimeric protein scaffold is a fusion protein comprising a heat-resistant multimeric protein and SpyCatcher.

[0041] In this invention, the amino acid sequence of the heat-resistant multimeric protein scaffold is shown in SEQ ID NO.2: The underlined part is spycatcher (SC); the double underlined part is the heat-resistant multimeric protein (nanoparticle) sequence (A301); the bold part is the flexible amino acid linker; the italicized part is the His histidine tag (HIS).

[0042] This invention also provides a vaccine constructed based on the heat-resistant multimeric protein described above. The vaccine comprises a heat-resistant multimeric protein scaffold and an antigen; the heat-resistant multimeric protein scaffold and the antigen are covalently coupled; the heat-resistant multimeric protein scaffold is a fusion protein containing a heat-resistant multimeric protein and a SpyCatcher; the antigen is linked to a SpyTag. That is, in this invention, the heat-resistant multimeric protein scaffold and the antigen are coupled through a SpyCatcher and SpyTag system.

[0043] In this invention, the structure of the heat-resistant multimeric protein scaffold preferably includes: a SpyCatcher attached to the N-terminus or C-terminus of the heat-resistant multimeric protein, and a His-histidine tag introduced at the C-terminus or N-terminus; more preferably, a SpyCatcher attached to the N-terminus of the heat-resistant multimeric protein, and a His-histidine tag introduced at the C-terminus.

[0044] The antigen preferably comprises a signal peptide and a SpyTag linked to its N-terminus, and a His histidine tag introduced at its C-terminus. In this invention, the antigen preferably comprises the receptor-binding domain (RBD) of the SARS-CoV-2S spike protein. In this invention, the nucleotide sequence of the signal peptide is preferably as shown in SEQ ID NO. 4.

[0045] In this invention, the N-terminus is connected to a signal peptide and a SpyTag, and the C-terminus introduces a His histidine tag. The preferred amino acid sequence of the antigen is as shown in SEQ ID NO.3. The bolded part is the flexible amino acid linker; the double underlined part is the signal peptide (SP); the boxed part is the SpyTag (ST); the dashed part is the antigen (RBD); and the italic part is the His histidine tag (HIS).

[0046] In this invention, SpyCatcher (SC) and SpyTag (ST) are linked to the N-terminus of A301 and RBD respectively via flexible amino acids. The construction of the RBD antigen introduces a signal peptide (SP) at the front end of the sequence to ensure the effective synthesis and secretion of the antigen. A His histidine tag is introduced at the C-terminus for subsequent protein purification, ultimately yielding a heat-resistant multimeric protein scaffold SC-A301-HIS and antigen SP-ST-RBD-HIS.

[0047] This invention also provides a method for constructing a vaccine based on the heat-resistant multimeric protein described above, comprising the following steps:

[0048] 1) Construct a scaffold vector for expressing heat-resistant multimeric proteins, and express the heat-resistant multimeric protein scaffold to obtain the heat-resistant multimeric protein scaffold;

[0049] 2) Construct a vector for expressing the antigen, and express the antigen to obtain the antigen;

[0050] 3) The heat-resistant polymeric protein scaffold is mixed with the antigen and conjugated to obtain the vaccine;

[0051] The heat-resistant multimeric protein scaffold is coupled to the antigen via the SpyCatcher and SpyTag system.

[0052] This invention constructs a vector for expressing a thermostable multimeric protein scaffold and expresses the thermostable multimeric protein scaffold to obtain the scaffold. The construction preferably includes: synthesizing the amino acid sequence of the thermostable multimeric protein scaffold, and subcloning the synthesized cDNA sequence into a plasmid; in this invention, the plasmid is preferably pET28b. The constructed expression vector is preferably transformed into *E. coli* for amplification culture and induced to obtain the protein scaffold. In this invention, *E. coli* is preferably *E. coli* BL21. In this invention, the induction is preferably performed using 0.6 mM IPTG inducer. The protein scaffold of this invention is preferably purified using affinity chromatography and size exclusion chromatography.

[0053] This invention constructs a vector for expressing an antigen and expresses the antigen to obtain the antigen. Preferably, the antigen structure includes a signal peptide and a SpyTag linked to its N-terminus, and a His histidine tag introduced at its C-terminus. Preferably, the antigen includes the receptor-binding domain (RBD) of the SARS-CoV-2S spike protein. Preferably, the RBD antigen amino acid sequence is genetically synthesized, and the synthesized cDNA sequence is subcloned into the expression vector for antigen expression. Preferably, the expression vector includes pcDNA3.1. Preferably, the antigen is expressed in Expi293F cells using a transfection reagent. Preferably, affinity chromatography is used to purify the antigen.

[0054] After obtaining the heat-resistant multimeric protein scaffold and the antigen, this invention mixes and conjugates the heat-resistant multimeric protein scaffold and the antigen to obtain a vaccine. In this invention, the conjugation is preferably performed in a neutral buffer solution. The neutral buffer solution is preferably neutral PBS phosphate buffer (containing Na₂HPO₄, KH₂PO₄, NaCl, and KCl). The heat-resistant multimeric protein scaffold and the antigen are conjugated using a SpyCatcher and SpyTag system. In this invention, the preferred conjugation conditions are 4°C for 16 hours.

[0055] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes an artificially designed heat-resistant multimeric protein, a scaffold, and its application and method in vaccine preparation, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0056] Example 1

[0057] Taking the novel coronavirus (SARS-CoV-2) as an example, this study verifies a method for preparing a heat-stable vaccine scaffold based on a novel artificially designed heat-resistant multimeric protein. Then, the prepared heat-stable vaccine scaffold is used to prepare a vaccine, thereby verifying the function of the vaccine scaffold.

[0058] 1. Novel heat-resistant multimeric protein sequence design and spatial structure prediction

[0059] This invention artificially modifies and designs the amino acid sequence of a novel heat-resistant multimeric protein (code name A301), and further uses relevant software to predict its 3D spatial structure. Figure 1 (Characterizing the 20-sided multimer morphology formed by 60-mers), and determining the spatial structure that can form multimers for subsequent construction and evaluation of multivalent antigens and vaccines.

[0060] 2. Construction and purification of novel heat-resistant multimeric protein scaffolds and multivalent vaccines

[0061] 2.1 Novel Heat-Resistant Polymeric Protein Scaffold and Construction of Multivalent Vaccines

[0062] The artificially designed multimeric protein A301 was used to construct a multivalent SARS-CoV-2 vaccine. The receptor-binding domain (RBD) of the SARS-CoV-2 S spike protein was selected as the multivalent vaccine antigen. This invention employs separate expression of the scaffold and antigen, coupled via covalent bonds. Specifically, the antigen is directionally loaded onto the surface of the multimeric protein using a spontaneously formed amide bond between the protein domain SpyCatcher and its peptide partner SpyTag to construct the multivalent vaccine. The specific construction method involves connecting SpyCatcher (SC) to the N-terminus of A301 via a flexible amino acid, and connecting SpyTag (ST) to the N-terminus of RBD via a flexible amino acid. For the construction of the RBD antigen, a signal peptide (SP) is introduced at the very beginning of the sequence to ensure effective antigen synthesis and secretion. His histidine tags are introduced at the C-terminus of A301 and RBD for subsequent protein purification, ultimately yielding SpyCatcher-A301 and SpyTag-RBD. (See below) Figure 2 . Figure 2 The diagram illustrates the construction of SpyCatcher-A301 and SpyTag-RBD, including the structural diagrams of SpyCatcher-A301 (abbreviated as SC-A301, specifically SC-A301-HIS) and SpyTag-RBD (abbreviated as ST-RBD, specifically SP-ST-RBD-HIS).

[0063] 2.2 Novel Heat-Resistant Polymeric Protein Scaffolds and Vaccine Expression and Purification

[0064] 2.2.1 Expression and purification of novel heat-resistant multimeric protein scaffolds

[0065] (1) Gene synthesis was performed on the amino acid sequence of the multimeric protein scaffold. The cDNA sequence of the synthesized multimeric protein scaffold was subcloned into the pET28b expression plasmid to construct pET28b-SpyCatcher-A301. pET28b-SpyCatcher-A301 was transformed into Escherichia coli BL21 for amplification culture. Protein expression was induced by adding 0.6 mM IPTG inducer.

[0066] (2) The protein was purified by affinity chromatography (Ni-NTA) and size exclusion chromatography, and concentrated by ultrafiltration. The purity was identified by SDS-PAGE / Coomassie brilliant blue staining and SEC.

[0067] 2.2.2 RBD antigen expression and purification

[0068] (1) Gene synthesis of the amino acid sequence of RBD antigen was performed, and the cDNA sequence of the synthesized RBD was subcloned into the pcDNA3.1 expression plasmid to construct pcDNA3.1-ST-RBD. RBD antigen expression was performed in Expi293F cells using the transfection reagent (ExpiFectamine293 transfection reagent (Thermo Fisher)).

[0069] (2) The protein was purified by affinity chromatography (Ni-NTA) and concentrated by ultrafiltration. The purity was identified by SDS-PAGE / Coomassie brilliant blue staining and SEC.

[0070] 2.2.3 Coupling and purification of SC-A301 and ST-RBD

[0071] The purified SC-A301 and purified ST-RBD were coupled in neutral buffer (PBS) (4℃, 16h). The purity of the target protein was verified by SDS-PAGE analysis, and the concentration of the target protein was determined by BCA method for subsequent analysis.

[0072] 2.2.4 General Characterization of SC-A301 and RBD-A301

[0073] (1) Purity was determined by SDS-PAGE / Coomassie brilliant blue staining.

[0074] SDS-PAGE / Coomassie Brilliant Blue staining purity determination results are as follows: Figure 3 As shown, from left to right, they are: SC-A301, ST-RBD, and RBD-A301.

[0075] (2) Observation of the spatial structure of SC-A301 and RBD-A301 by negative staining transmission electron microscopy (TEM)

[0076] TEM observation of the spatial structure of SC-A301 and RBD-A301 (left image: SC-A301, right image: RBD-A301) results are shown below. Figure 4 As shown.

[0077] The results showed that ST-RBD, SC-A301, and RBD-A301 were all purified to high purity. Under the microscope, SC-A301 and RBD-A301 showed a uniform spherical polymer morphology, and the coupling of ST-RBD led to an increase in the particle size of A301, but did not affect the formation of its polymers.

[0078] 3. Evaluation of the thermal stability of novel heat-resistant multimeric protein scaffolds and multivalent vaccines

[0079] 3.2 Thermal stability analysis of SC-A301 and RBD-A301

[0080] 3.2.1 Thermal stability analysis of SC-A301

[0081] ① The purified SC-A301 was treated in PBS neutral buffer at 4–95℃ for 2 h. Centrifugation was used to remove any aggregates. SDS-PAGE was used to measure the change in the proportion of soluble protein at each treatment temperature. The SDS-PAGE results of SC-A301 at different temperatures (left image, the leftmost column of the left image represents the group not treated at 4–95℃ for 2 h) and the change in the proportion of soluble protein (right image, from left to right: solubility of the untreated group is 1, solubility of the 4℃ group is 0.95, solubility of the 25℃ group is 0.99, solubility of the 55℃ group is 0.93, solubility of the 75℃ group is 0.98, and solubility of the 95℃ group is 1.03) are shown in the figure below. Figure 5 As shown.

[0082] ② The purified SC-A301 was treated in PBS neutral buffer at a temperature range of 25-95℃ for 2h, and any aggregates were removed by centrifugation. The hydrodynamic particle size of the nanoparticles at each treatment temperature was measured by DLS.

[0083] The hydrodynamic particle size results of SC-A301 under different temperature treatments are shown in the figure below. Figure 6 .

[0084] ③ After the purified SC-A301 was treated at 75℃ for 2 hours, it was coupled with ST-RBD, and SDS-PAGE was used to test whether the binding ability of SC-A301 and ST-RBD changed. Figure 7 The graph shows the reactivity of SC-A301 with ST-RBD after heat treatment at 75℃. Figure 7 In the diagram, SC-301 refers to SC-301 before coupling with ST-RBD, used to indicate the molecular weight position of the scaffold. Untreated refers to the bands of SC-301 after coupling with ST-RBD without undergoing 75℃, 2h treatment. 75℃ refers to the bands of SC-301 after coupling with ST-RBD with 75℃, 2h treatment. The results show that SC-A301 can react with ST-RBD before and after heat treatment, and SC-A301 retains its reactivity with ST-RBD after heat treatment at 75℃.

[0085] The results showed that with increasing processing temperature, the soluble protein ratio and hydrodynamic particle size of SC-A301 did not change significantly, and it maintained its reactivity with ST-RBD. These results all indicate that SC-A301 possesses high thermal stability.

[0086] 3.2.2 Thermal stability analysis of RBD-A301

[0087] ① RBD-A301 was treated in PBS neutral buffer at 4℃, 25℃, 37℃ and 65℃ for 2 h, then centrifuged to remove any aggregates, and the soluble protein and protein degradation were analyzed by SDS-PAGE / Coomassie staining.

[0088] The SDS-PAGE (left) and changes in the proportion of soluble protein of RBD-A301 under different temperature treatments (right, 4℃ (1), 25℃ (0.92), 37℃ (0.94), and 65℃ (0.9)) results are shown below. Figure 8 As shown.

[0089] ② RBD-A301 was incubated in neutral PBS buffer at 4°C, 25°C, 37°C, and 65°C for 2 hours. After centrifugation to remove any aggregates, the integrity of the vaccine antigen was further analyzed using an ELISA assay targeting RBD mAb. The results of ELISA detection of the integrity of the RBD-A301 vaccine antigen at different temperatures are shown in the figure below. Figure 9 As shown.

[0090] The results showed that the solubility of RBD-A301 did not change significantly with increasing temperature. Figure 8 The right image in the figure does not show protein degradation. Figure 8 As shown in the left figure, the immunogenicity of RBD-A301 was not significantly affected, indicating that it retained its complete antigenicity. All these results demonstrate that RBD-A301 has high thermal stability.

[0091] 4. Immunogenicity assessment of novel heat-resistant multimeric protein multivalent vaccine

[0092] 4.1 Humoral immunity assessment

[0093] BALB / c mice were divided into four groups: control group, SC-A301 group, free RBD group, and RBD-A301 group, with five mice in each group. The RBD-A301 group was subcutaneously immunized with a 6 μg dose of RBD-A301 vaccine. The RBD group was immunized with an equimolar amount of RBD as the RBD-A301 group. The SC-A301 group was immunized with an equimolar amount of A301 as the RBD-A301 group. The control group received no treatment. All mice were vaccinated using a primary-booster regimen, i.e., at week 0 and week 4. Serum was collected every two weeks, and the mice were euthanized at week 10.

[0094] ① A pseudovirus neutralization assay was conducted to detect the level of antibodies against RBD in serum and to analyze the immunogenicity of the RBD-A301 vaccine.

[0095] The results of the pseudovirus neutralization experiment, which determined the antibody titers against RBD in the serum of each group, are as follows: Figure 10As shown, the antibody titer in the blank group was 1, the antibody titer in the SC-A301 group was 1.5, the antibody titer in the free RBD group was 2.5, and the antibody titer in the RBD-A301 group was 5.6.

[0096] ② ELISA was used to detect the RBD antibody levels in the serum of each group over time to analyze the duration of immunity from the RBD-A301 vaccine.

[0097] The time-varying curves of antibody titers against RBD in serum of each group are shown in the figure below. Figure 11 As shown in the figure, the antibody titers for the blank group from weeks 0 to 10 were 1, 1.1, 1.3, 0.9, 0.9, and 1, respectively; for the SC-301 group, they were 1, 1.2, 1.2, 1.3, 1.4, and 1, respectively; for the free RBD group, they were 1, 1.4, 2.3, 4.3, 3.3, and 2.4, respectively; and for the RBD-A301 group, they were 1, 3.3, 5.2, 5.3, 5.4, and 5.32, respectively.

[0098] ③ To evaluate the effectiveness of the neutralizing antibodies induced by the RBD-A301 multivalent vaccine in animals, the plaque reduction assay (FRNT) was used to detect whether the antibodies could inhibit true SARS-CoV-2 virus infection. Due to experimental limitations, this invention used pseudotyped SARS-CoV-2 virus for Vero cell infection.

[0099] The results of the plaque assay for detecting antibody titers against RBD in the serum of each group are as follows: Figure 12 As shown (each experiment used 6 mice).

[0100] The results showed that, compared with the free antigen group, the RBD-A301 multivalent vaccine group could induce higher antibody levels in mice and maintain higher immune persistence. The induced antibodies could further neutralize pseudoviruses, indicating that the antibodies are effective in neutralizing viral infections.

[0101] 4.2 Cellular immune assessment

[0102] To verify that RBD-A301 multivalent vaccination enhances T cell activation in animals, mice were sacrificed 10 days after vaccination, and their spleens were collected. Flow cytometry was used to assess the percentages of different lymphocyte types, including CD4+. + T and CD8 + An increase in the number of T cells, Th1 T cells, and CD8+ T cells indicates enhanced cellular immunity, while an increase in the number of Tfh T cells, which promote B cell maturation, indicates enhanced humoral immunity.

[0103] The results of flow cytometry analysis of the percentage of different types of T cells in the spleen cells of mice in each group are as follows: Figure 13 As shown.

[0104] Table 1. Percentage of different types of T cells

[0105] Blank group 0.02 0.01 0.05 SC-A301 group 0.08 0.03 0.07 Free RBD group 0.15 0.09 0.14 RBD-A301 group 0.43 0.24 0.56

[0106] The results showed that, compared with the free antigen group, the RBD-A301 multivalent vaccine group was able to induce higher levels of T cells in mice, including Th1, Tfh, and CD8. + T cells indicate that the RBD-A301 multivalent vaccine activates a stronger cellular immune response.

[0107] In summary, the novel thermostable multimeric protein A301, designed artificially, exhibits high thermostability. The RBD-A301 vaccine constructed using A301 as a scaffold also shows significantly improved thermostability and elicits a stronger immune response, including humoral and cellular immune responses, compared to the free RBD monomer group. This indicates that the novel thermostable multimeric protein can significantly improve the thermostability and immunogenicity of vaccine antigens.

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A man-made heat-resistant polymeric protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

1.

2. The use of the protein of claim 1 in the preparation of heat-resistant multimeric protein scaffolds and / or vaccines.

3. The application according to claim 2, characterized in that, The vaccines mentioned include those for the novel coronavirus.

4. A man-made heat-resistant multimeric protein scaffold, characterized in that, The amino acid sequence of the heat-resistant multimeric protein scaffold is shown in SEQ ID NO.

2.

5. A novel coronavirus vaccine constructed based on the heat-resistant multimeric protein of claim 1, the novel coronavirus vaccine comprising a heat-resistant multimeric protein scaffold and an antigen; the heat-resistant multimeric protein scaffold and the antigen being covalently coupled; the amino acid sequence of the heat-resistant multimeric protein scaffold is shown in SEQ ID NO. 2; the amino acid sequence of the antigen is shown in SEQ ID NO.

3.

6. A method for constructing the vaccine according to any one of claims 5, comprising the following steps: 1) Construct a vector for expressing a thermostable multimeric protein scaffold, and express the thermostable multimeric protein scaffold to obtain the thermostable multimeric protein scaffold; 2) Construct a vector for expressing the antigen, and express the antigen to obtain the antigen; 3) The heat-resistant polymeric protein scaffold is mixed with the antigen and conjugated to obtain the vaccine; There is no time limit for the order of steps 1) and 2).

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