Respiratory mucosa vaccine platform with novel coronavirus spike protein as carrier

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a vaccine preparation strategy taking new coronavirus spike protein as a respiratory mucosa vaccine antigen protein carrier, which is used for preventing respiratory pathogen infection. The novel coronavirus spike protein has good potential as a respiratory mucosa vaccine antigen vector, and can be used for enhancing mucosa and system immune response aiming at a target pathogen.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of the SARS-CoV-2 spike protein in respiratory mucosal vaccine antigen vectors. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is the pathogen that causes coronavirus disease 2019 (COVID-19) and has a strong ability to spread from person to person. Some infected individuals develop severe clinical manifestations such as viral pneumonia. As the virus continues to spread globally, its genome accumulates mutations, giving rise to multiple variants with significantly altered transmissibility, pathogenicity, and immune evasion capabilities. These variants, particularly the Omicron variant, are able to partially overcome the neutralizing antibody immune barrier established by previous natural infection or vaccination, and have rapidly become the dominant circulating strain globally.

[0003] Vaccination is the most effective means of controlling the spread of infectious diseases and reducing their severity. To address the challenge of immune evasion posed by the mutation of the novel coronavirus, developing novel vaccines that can be administered via the upper respiratory tract mucosa has become a crucial strategy and urgent need in current epidemic prevention and control. Existing mainstream vaccines (including inactivated vaccines, adenovirus vector vaccines, recombinant protein subunit vaccines, and mRNA vaccines) are mostly administered via intramuscular injection. They primarily induce humoral and cellular immunity, producing high titers of neutralizing antibodies (mainly IgG) in the systemic circulation. However, they struggle to effectively induce local mucosal immune responses (especially secretory IgA) at the gateway to viral invasion—the respiratory mucosa. Therefore, their effectiveness in preventing initial infection is limited. Furthermore, the levels of neutralizing antibodies induced by existing vaccines decay over time, and intramuscular injection relies on specialized medical personnel and equipment, making large-scale implementation costly.

[0004] In contrast, vaccines administered via nasal drops or sprays through the respiratory mucosa are not only simpler to administer but also mimic the natural infection route. They directly activate secretory IgA-mediated mucosal immunity and tissue-resident memory T-cell responses in the respiratory mucosa, establishing an immune defense at the initial site of viral invasion and more effectively blocking infection. Although adenovirus vector vaccines have been designed for mucosal administration, the vector itself can induce an immune response against the adenovirus coat protein. This can lead to rapid clearance of the vaccine by the body during subsequent homologous booster immunizations due to pre-existing immunity, thus affecting vaccine efficacy.

[0005] Recombinant protein-based mucosal vaccines offer several unique advantages: they do not contain viral genetic material and are not dependent on viral vectors, thus ensuring higher safety and avoiding the influence of pre-existing immunity, making them suitable for repeated booster immunizations. Whether through natural infection or prior vaccination, the IgA memory response established in the respiratory mucosa can assist vaccine antigens in crossing the mucosal epithelium during booster immunization, allowing them to be taken up and processed by submucosal immune cells, thereby enhancing the strength and duration of the subsequent immune response. However, similar to intramuscular injection, antigen proteins delivered via the mucosal route still require effective vaccine adjuvants to elicit an immune response. Due to the unique structure and function of the respiratory tract, the safety requirements for adjuvants are more stringent than for intramuscular injection; currently, no respiratory mucosal adjuvant has been approved for clinical use.

[0006] The spike protein of SARS-CoV-2 is a key protein mediating viral binding to the host cell surface receptor (ACE2) and membrane fusion. It is the main target of neutralizing antibodies and the core antigen in the design of most current COVID-19 vaccines (including mRNA vaccines, adenovirus vector vaccines, and recombinant subunit vaccines). This invention demonstrates that, without the use of any adjuvants, administering the SARS-CoV-2 spike protein to mice via nasal instillation can induce the production of respiratory mucosal IgA antibodies and serum IgG antibodies. Furthermore, mixing the influenza A virus hemagglutinin (HA) protein with the SARS-CoV-2 spike protein and administering it via nasal instillation can also successfully induce respiratory mucosal IgA and serum IgG antibodies against HA. Moreover, by inserting the receptor binding site (RBS) of HA into the SARS-CoV-2 spike protein to construct a fusion protein, and administering it via nasal instillation to mice, respiratory mucosal IgA antibodies and serum IgG antibodies against the HA receptor binding site can be induced. Summary of the Invention

[0007] The present invention aims to provide a vaccine preparation strategy that uses the spike protein of the novel coronavirus as a respiratory mucosal vaccine antigen protein carrier for the prevention of respiratory pathogen infection.

[0008] This invention uses the SARS-CoV-2 envelope spike protein as an antigen. Inoculation of mice via nasal drop induced the production of respiratory mucosal IgA antibodies and serum IgG antibodies without the use of any immune adjuvants. In contrast, no respiratory mucosal IgA or serum IgG antibodies were detected when influenza A virus hemagglutinin (HA) was administered via the same route. Further experiments showed that mixing influenza A virus HA with the SARS-CoV-2 spike protein and administering it via nasal drop successfully induced respiratory mucosal IgA and serum IgG antibodies against HA. Further research revealed that inserting the receptor binding site (RBS) of HA into the SARS-CoV-2 spike protein to construct a fusion protein, and administering this fusion protein via nasal drop to mice, induced the production of respiratory mucosal IgA antibodies and serum IgG antibodies against the HA receptor binding site. These results demonstrate the potential of the SARS-CoV-2 envelope spike protein as an antigen carrier for respiratory mucosal vaccines. Attached Figure Description

[0009] Figure 1 .Titer of SARS-CoV-2 spike protein RBD IgG antibody in mouse serum;

[0010] Figure 2 The titer of influenza A virus HA IgG antibody in mouse serum;

[0011] Figure 3 The titer of SARS-CoV-2 spike protein RBD IgA antibody in mouse bronchoalveolar lavage fluid;

[0012] Figure 4 The titer of influenza A virus HA IgA antibody in mouse bronchoalveolar lavage fluid; Detailed Implementation

[0013] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0014] I. Reagents and Animals

[0015] 1. C57BL / 6 mice, female, 8 weeks old, Shanghai Lingchang Experimental Animal Technology Co., Ltd.

[0016] 2. Trimer of the full-length extracellular segment of the spike protein of the prototype SARS-CoV-2 strain (spike amino acid sequence can be found in GenBank: YP_009724390.1), in which the amino acid residues PRRARS at the furin protease site between the S1 / S2 subunits were replaced with GSAS, was expressed in CHO cells by the Department of Biomedical Protection, Naval Medical University and purified by nickel affinity chromatography.

[0017] 3. Hemagglutinin (HA) protein of influenza A virus was purchased from Sinocare Biotechnology Co., Ltd. Its amino acid sequence can be found in GenBank: ABD77675.1. It was expressed in human embryonic kidney 293 (HEK293) cells cultured in serum-free suspension, catalog number: 11684-V08H1.

[0018] 4. Fusion protein of SARS-CoV-2 spike protein and influenza A virus HA receptor binding site (RBS): A fusion protein was constructed by inserting the HA receptor binding site (RBS; amino acids 56-275 of HA) into the SARS-CoV-2 spike protein. Referring to the mutation site of the spike variant, the RBS encoding gene was inserted between amino acid residues 142 and 147 of the SARS-CoV-2 prototype spike protein (amino acid residues 163-146 were deleted, the fusion protein was named spike-142-HA-RBS), or between amino acid residues 248 and 249 (the fusion protein was named spike-248-HA-RBS). The two fusion proteins were named spike-142-HA-RBS and spike-248-HA-RBS, respectively, and both were expressed in serum-free suspension cultured HEK293 cells.

[0019] 4. Other reagents: The prototype strain of SARS-CoV-2 receptor-binding domain (RBD) protein expressed in HEK293 cells was purchased from Shanghai Nearshore Biotechnology Co., Ltd.; high-adsorption enzyme-labeled microplates were purchased from Nunc; horseradish peroxidase (HRP)-labeled anti-mouse IgG and IgA were purchased from Thermo Fisher; TMB chromogenic solution for enzyme-linked immunosorbent assay (ELISA) was purchased from Thermo Fisher.

[0020] II. Experimental Methods:

[0021] (a) Mouse Immunization

[0022] Thirty C57BL / 6 mice were randomly divided into six groups of five each. The animals were first anesthetized with isoflurane inhalation, followed by intranasal inoculation with the antigen protein.

[0023] 1. Negative control group: PBS was administered via nasal drops, 25 μL per nostril, in both nostrils;

[0024] 2. COVID-19 Spike Nasal Drops: 25 μL / nostril, containing 10 μg spike protein;

[0025] 3. Influenza A virus HA nasal drops group: 25 μL / nostril for bilateral nasal drops, containing 10 μg of HA protein;

[0026] 4. COVID-19 spike + influenza A virus HA nasal drops group: The two protein solutions were mixed and then dripped into the nose, 25 μL / nostril, containing 10 μg each of spike and HA protein;

[0027] 5. Spike-142-HA-RBS nasal drops: 25 μL / nostril, containing 10 μg of spike-142-HA-RBS protein;

[0028] 6. Spike-248-HA-RBS fusion protein nasal drops: 25 μL / nostril, containing 10 μg of spike-248-HA-RBS protein;

[0029] Two nasal injections were administered, 21 days apart.

[0030] (II) Detection of serum antibodies and bronchoalveolar lavage fluid IgA antibodies in mice

[0031] On days 21 and 35, mice in each group were anesthetized with isoflurane inhalation. Blood was collected from the orbital region of the mice using capillary tubes, and the serum was separated by high-speed centrifugation and stored at -80°C.

[0032] On day 35, anesthetized mice were fixed in a supine position on the operating board with their necks extended. The glottis was exposed using a laryngoscope, and an endotracheal tube was gently inserted. A blunt-tipped catheter was then inserted through the endotracheal tube, and warm sterile saline was injected in fractions, approximately 0.1 mL each time. The lavage fluid was gently aspirated and collected. The collected lavage fluid was immediately placed on ice and centrifuged at 4°C for 10 minutes as soon as possible. The supernatant was collected and stored at -80°C.

[0033] IgG antibodies against SARS-CoV-2 spike protein and influenza A virus HA protein in mouse serum and IgA antibodies in bronchoalveolar lavage fluid were detected by ELISA. SARS-CoV-2 RBD protein (the receptor-binding region of the spike protein) and influenza A virus HA protein were coated onto high-adsorption ELISA microplates, 0.1 μg of protein per well, and placed in 4-well plates. oIncubate overnight at 4°C. The next day, aspirate the protein solution, wash the wells once with phosphate-buffered saline (PBS, pH 7.0), then block with PBS containing 3% bovine serum albumin (3% BSA-PBS) at room temperature for 2 hours. Aspirate the blocking solution and wash the wells three times with PBS. Add serially 2-fold diluted mouse serum (starting dilution 100) or bronchoalveolar lavage fluid (starting dilution 10) to each well, using 3% BSA-PBS, at a volume of 100 μL / well, and incubate at 4°C. o Incubate overnight at C. The next day, remove the serum diluent, wash the wells five times with PBS containing 0.05% Tween 20 (0.05% Tween 20-PBS), then add 1000-fold diluted HRP-labeled anti-mouse IgG or IgA (3% BSA-PBS, 100 μL / well), incubate at room temperature for 40 minutes. Remove the HRP antibody diluent, wash the wells five times with 0.05% Tween 20-PBS, add 100 μL of TMB chromogenic solution per well, incubate for 10 minutes, then add stop solution. Measure the absorbance at 450 nm and 630 nm using a microplate reader. Calculate the antibody titer for each serum sample based on the absorbance using Graphpad Prism 5 software. The antibody positivity standard is defined as an OD450-OD630 value in each experimental group being greater than 2.1 times the average OD450-OD630 value of the corresponding dilution in the negative control group.

[0034] RBD is the main target of neutralizing antibodies against SARS-CoV-2, and the level of RBD antibodies in serum represents the virus neutralizing capacity. The titer of RBD IgG antibodies in mouse serum is shown in the figure below. Figure 1 As shown, the results of enzyme-linked immunosorbent assay (ELISA) detection of SARS-CoV-2 spike protein RBD-specific IgG antibodies in mouse serum were obtained at two time points: day 21 (3 weeks after the initial immunization) and day 35 (2 weeks after the secondary immunization). The figure shows that on day 21, the RBD IgG antibody titers in the serum of mice in the spike nasal drop group, spike+HA nasal drop group, spike-142-HA-RBS nasal drop group, and spike-248-HA-RBS nasal drop group were similar, around 1.5 × 10³. On day 35, the antibody titers increased by 8.6, 8.0, 8.6, and 7.4 times, respectively.

[0035] The titer of HA IgG antibody in mouse serum is as follows Figure 2As shown, the results of enzyme-linked immunosorbent assay (ELISA) detection of influenza A virus HA-specific IgG antibodies in mouse serum were obtained at two time points: day 21 (3 weeks after the primary immunization) and day 35 (2 weeks after the secondary immunization). The figure shows that, compared with the negative control group, on day 21, the HA IgG antibody titers in the serum of mice in the HA nasal drop group, spike+HA nasal drop group, spike-142-HA-RBS nasal drop group, and spike-248-HA-RBS nasal drop group were less than 100 (less than 100 is counted as 100), 122, 485, and 523, respectively; and on day 35 (2 weeks after the booster immunization), the antibody titers increased by 3.9, 7.4, and 8.0 times, respectively.

[0036] On day 35, the RBD IgA antibody titers in the bronchoalveolar lavage fluid of mice in each group were as follows: Figure 3 As shown, this is the result of enzyme-linked immunosorbent assay (ELISA) detection of SARS-CoV-2 spike protein RBD-specific IgA antibodies in mouse bronchoalveolar lavage fluid on day 35 after the primary immunization (i.e., 2 weeks after the secondary immunization). The figure shows that the RBD IgA antibody titers in the serum of mice in the spike nasal drop group, spike+HA nasal drop group, spike-142-HA-RBS nasal drop group, and spike-248-HA-RBS nasal drop group are similar, around 50.

[0037] The titers of HA IgA antibodies in the bronchoalveolar lavage fluid of each group of mice are as follows: Figure 4 The figure shows the results of enzyme-linked immunosorbent assay (ELISA) detection of influenza A virus HA-specific IgA antibodies in bronchoalveolar lavage fluid of mice on day 35 after the primary immunization (i.e., 2 weeks after the secondary immunization). The figure reveals that, compared with the negative control group, the serum HA IgA antibody titers in the HA nasal drop group, spike+HA nasal drop group, spike-142-HA-RBS nasal drop group, and spike-248-HA-RBS nasal drop group were less than 10 (less than 10 is counted as 10), 22, 44, and 54, respectively.

[0038] The results showed that intranasal administration of the SARS-CoV-2 envelope spike protein to mice without adjuvant administration induced respiratory mucosal IgA antibodies and serum IgG antibodies. Furthermore, it could assist in inducing respiratory mucosal IgA and serum IgG antibody responses with concurrent intranasal administration of influenza A virus (HA), and could serve as a protein carrier for the key antigen RBS in HA, inducing respiratory mucosal IgA and serum IgG antibody responses. These results indicate that the SARS-CoV-2 envelope spike protein possesses self-adjuvant activity and has potential application as an antigen carrier for respiratory mucosal vaccines.

[0039] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the present invention, shall still fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of SARS-CoV-2 spike protein as an antigen carrier in the preparation of respiratory mucosal vaccines.

2. The application according to claim 1, characterized in that, Without the addition of additional adjuvants, the spike protein of the novel coronavirus can induce respiratory mucosal IgA antibodies and serum IgG antibodies against the spike protein.

3. The application according to claim 1, characterized in that, The SARS-CoV-2 spike protein can act as a carrier protein, enhancing the immunogenicity of heterologous antigens applied in combination with it, and inducing respiratory mucosal IgA antibodies and serum IgG antibodies against the heterologous antigens.

4. The application according to claim 3, characterized in that, The heterologous antigen is a viral protein or its immunogenic fragment.

5. The application according to claim 4, characterized in that, The viral protein is selected from the hemagglutinin protein of influenza A virus or its key peptide containing a receptor binding site.

6. The application according to any one of claims 1-5, characterized in that, The vaccine is administered via mucosal route.

7. The application according to claim 6, characterized in that, The mucosal route is via nasal mucosal administration.

8. The application according to claim 7, characterized in that, The method of administration via the nasal mucosa is nasal drops or inhalation spray.

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