Application of carvedilol in preparation of novel coronavirus respiratory mucosa vaccine

Abstract

The invention relates to the technical field of medicines, and relates to application of carvedilol in preparation of a novel coronavirus respiratory mucosa vaccine. The novel coronavirus respiratory mucosa vaccine is a vaccine which takes carvedilol as a unique immunologic adjuvant or a composite adjuvant containing capsaicin and is inoculated through a nasal drop or nasal spray way to prevent novel coronavirus infection. The recombinant new coronavirus spike protein is used as an antigen and is mixed with carvedilol, and the mixture is inoculated to a narla golden hamster in a nasal drop manner. Experimental results show that carvedilol can effectively promote a body to generate a specific antibody aiming at spike protein, so that the IgG level in serum is remarkably improved, and IgA response in alveolar lavage fluid is also enhanced. Therefore, a basis is provided for development of carvedilol as a respiratory mucosa vaccine adjuvant.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of carvedilol in the preparation of a novel coronavirus respiratory mucosal vaccine. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) is the pathogen causing 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. Since the outbreak in early 2020, the virus has continued to spread globally, and its genome has accumulated mutations, giving rise to several variants with significantly altered transmissibility, pathogenicity, and immune evasion capabilities. These variants, especially the Omicron variant first detected in South Africa in November 2021, can partially overcome the neutralizing antibody immune barrier established by previous natural infection or vaccination, and quickly became the dominant circulating strain globally, replacing previously prevalent variants such as Delta. The current consensus is that the novel coronavirus will coexist with humans for a long time.

[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] Therefore, mucosal vaccines based on recombinant protein technology exhibit unique advantages. These vaccines do not contain viral genetic material, have high safety profiles, and because they do not rely on viral vectors, they avoid interference from pre-existing immunity, making them suitable for repeated booster immunizations. More importantly, the IgA memory response already formed in the respiratory mucosa by previous immunizations (whether natural infection or vaccination) helps promote the passage of vaccine antigens across the mucosal epithelium during booster immunization, allowing them to be captured and processed by submucosal immune cells, thereby significantly enhancing the strength and durability of subsequent immune responses.

[0006] The spike protein of the novel coronavirus is a key protein mediating viral binding to the host cell surface receptor (ACE2) and membrane fusion. Therefore, it is the primary target of neutralizing antibodies and the core antigen in the design of the vast majority of current COVID-19 vaccines (including mRNA vaccines, adenovirus vector vaccines, and recombinant subunit vaccines). Vaccines using these various technological approaches have been approved and are used on a large scale globally.

[0007] Catecholamines, primarily including adrenaline, noradrenaline, and dopamine, are important neurotransmitters and hormones in the human body. They exert their effects by activating adrenergic receptors on the cell surface. These receptors are widely distributed on various immune cells and can exert immunosuppressive effects under acute stress. These effects include: inhibiting the production of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and interleukin-12 by macrophages through activation of β2-adrenergic receptors, while promoting the production of the anti-inflammatory cytokine interleukin-10; inhibiting the differentiation and function of Th1 cells (which dominate cellular immunity) and Th17 cells (which dominate inflammatory responses), while promoting the generation of Th2 cells and regulatory T cells; and inhibiting antibody production by B cells. Therefore, inhibiting catecholamines can relieve immunosuppression and activate the immune response by activating downstream signals triggered by adrenergic receptors. Catecholamine inhibitors mainly include β-adrenergic receptor blockers and α-adrenergic receptor blockers. In particular, blocking β2-receptors has an activating effect on macrophages, T cells, and B cells.

[0008] Given the immune-enhancing activity of catecholamine inhibitors, exploring their feasibility as adjuvants for respiratory mucosal vaccines is a promising research direction. Summary of the Invention

[0009] The present invention aims to provide a recombinant subunit vaccine against SARS-CoV-2, which uses carvedilol as an adjuvant and is administered via the respiratory mucosa for the prevention of SARS-CoV-2 infection.

[0010] This invention utilizes the spike protein of the SARS-CoV-2 envelope as an antigen. The antigen protein solution is mixed with carvedilol solution and inoculated into Syrian golden hamsters via nasal drops. Subsequently, spike protein IgG antibodies in hamster serum and spike protein IgA antibodies in bronchoalveolar lavage fluid are detected. The results show that carvedilol can significantly enhance the production of spike protein IgG antibodies in serum and IgA antibodies in bronchoalveolar lavage fluid, demonstrating its potential as an adjuvant for respiratory mucosal vaccines. Attached Figure Description

[0011] Figure 1 Anti-SARS-CoV-2 spike protein receptor-binding domain (RBD) IgG antibody in hamster serum;

[0012] Among them, enzyme-linked immunosorbent assay (ELISA) was used to detect anti-RBD specific IgG antibodies in hamster serum at days 21 and 35 after the first and second immunizations, as shown in the figure.

[0013] Figure 2 RBD IgA antibodies in hamster bronchoalveolar lavage fluid;

[0014] Among them, enzyme-linked immunosorbent assay (ELISA) was used to detect RBD IgA antibodies in hamster bronchoalveolar lavage fluid on day 35 after the second immunization. Detailed Implementation

[0015] 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.

[0016] I. Reagents and Animals

[0017] 1. Syrian golden hamster, male, 8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0018] 2. The full-length extracellular segment trimer of the spike envelope protein of the vaccine antigen SARS-CoV-2, in which the amino acid residues PRRARS at the furin protease site between the S1 and S2 subunits were replaced with GSAS, was expressed in CHO cells by the Department of Biomedical Protection, Naval Medical University, and purified using a nickel affinity chromatography column.

[0019] 2. Propranolol is a non-selective β-adrenergic receptor blocker with high affinity for both β1 and β2-adrenergic receptors; metoprolol tartrate is a selective β1-adrenergic receptor blocker; and carvedilol is a non-selective β / α-1 adrenergic receptor blocker. All three small molecule compounds were purchased from MCE (MedChemExpress) and dissolved in DMSO.

[0020] 4. Other reagents: The prototype strain of SARS-CoV-2 receptor-binding domain (RBD) protein expressed in 293 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.

[0021] II. Experimental Methods:

[0022] (a) Immunization of Syrian golden hamsters

[0023] Twenty-five Syrian golden hamsters were randomly divided into five groups of five each. The animals were first anesthetized with isoflurane inhalation, followed by experimental vaccination.

[0024] Negative control group: PBS drops in both nostrils, 25 μL / nostril;

[0025] Single antigen (Str) nasal drops group: 25 μL / nostril for bilateral nasal drops, containing 10 μg of Str protein;

[0026] Str+Propr nasal drops: 25 microliters per nostril, containing 10 micrograms of Str protein and 5 micrograms of propranolol;

[0027] Str+Metop nasal drops: 25 microliters per nostril, containing 10 micrograms of Str protein and 5 micrograms of metoprolol tartrate;

[0028] Str+Carve Nasal Drops: 25 μL / nostril for bilateral use, containing 10 μg of Str protein and 5 μg of carvedilol.

[0029] The patient was immunized twice, 21 days apart, with the second immunization administered on the same day after blood collection.

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

[0031] On days 21 and 35, hamsters were anesthetized with isoflurane inhalation, and blood was collected from the hamster's orbital cavity using a capillary tube. The serum was separated by high-speed centrifugation and stored at -80°C.

[0032] On day 35, the anesthetized hamster was placed in a supine position on the operating board with its neck extended. The glottis was exposed using a laryngoscope, and a tracheal tube was gently inserted. A blunt-tipped catheter was then inserted through the tracheal tube, usually into one main bronchus. Warm sterile saline was injected in fractions, approximately 0.2-0.3 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] Spike protein IgA antibodies in hamster serum and bronchoalveolar lavage fluid were detected by ELISA. RBD protein was coated onto highly absorbent enzyme-labeled microplates, 0.1 μg of protein per well, and incubated at 4°C. o Incubate overnight at room temperature (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 hamster 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 wells 5 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 wells 5 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 (OD) at 450 nm and 630 nm using a microplate reader. Calculate the antibody titer for each serum sample using Graphpad Prism 5 software based on the absorbance. The antibody positivity standard is defined as an OD450-OD630 value in each experimental group being greater than the average OD450-OD630 value of the corresponding dilution in the negative control group by 2.1 times.

[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 RBD IgG antibody titers in the sera of each group of hamsters are shown below. Figure 1As shown: Nasal immunization with Str (Spiral) alone, specifically the SARS-CoV-2 extracellular spike protein trimer, induced extremely low levels of RBD IgG antibodies. Nasal immunization with Str combined with propranolol or metoprolol tartrate did not significantly increase RBD IgG antibodies, while nasal immunization with Str combined with carvedilol significantly increased RBD antibody levels (statistical difference compared to Str alone, p < 0.05). After booster immunization, serum IgG antibody titers in the four groups of hamsters increased by 2.4, 2.6, 2.8, and 8.8 times, respectively (statistical difference between the Str combined with carvedilol immunization group and the Str alone immunization group, p < 0.0001).

[0035] After booster immunization, the RBD IgA antibody titers in the bronchoalveolar lavage fluid of each group of hamsters were as follows: Figure 2 As shown: Strole (Styrax) nasal immunization alone induced RBD IgA antibodies below the detection limit. While Strole combined with propranolol or metoprolol tartrate nasal immunization increased RBD IgA antibody titers, carvedilol increased RBD IgA antibody titers by 13.4 and 10.8 times, respectively, compared to propranolol or metoprolol tartrate. The statistical difference between the Strole combined with carvedilol nasal immunization group and the Strole alone nasal immunization group was statistically significant (p < 0.0001).

[0036] The results showed that among the three adrenergic receptor blockers, only carvedilol combined with the SARS-CoV-2 spike protein for nasal drops could effectively enhance the production of serum IgG and respiratory IgA antibodies, suggesting that carvedilol can be used as an effective adjuvant for upper respiratory tract mucosal vaccines.

[0037] 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. Carvedilol for use in the preparation of a new coronavirus respiratory mucosal vaccine.

2. Use according to claim 1, characterized in that: Carvedilol as an effective upper respiratory mucosal immune adjuvant is used as an immune adjuvant of a recombinant antigen or inactivated virus of a new coronavirus, inoculated by nose dropping or nasal spray, for preventing new coronavirus infection.

3. Use according to any one of claims 1-2, characterized in that: The new coronavirus vaccine with carvedilol as an adjuvant has an inoculation route including nose dropping and nasal spray.

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