Application of capsaicin in preparation of novel coronavirus respiratory mucosa vaccine

Abstract

The invention relates to the technical field of medicines, and relates to application of capsaicin in preparation of a novel coronavirus respiratory mucosa vaccine. The novel coronavirus respiratory mucosa vaccine is a vaccine which takes capsaicin 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 capsaicin, and then the mixture is inoculated to the narla golden hamster in a nasal drop manner. Experimental results show that capsaicin can effectively promote a body to generate a specific antibody aiming at spike protein, so that not only is the IgG level in serum remarkably improved, but also the IgA response in alveolar lavage fluid is enhanced. Therefore, a basis is provided for development of capsaicin 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 capsaicin 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 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] Nociceptors are far more than simple pain signal sensors; as key regulators of the immune system, they finely regulate the initiation, intensity, and resolution of the immune response at both local and systemic levels through neuropeptide release and direct cellular contact. These specialized sensory neurons (primarily unmyelinated C fibers and thinly myelinated Aδ fibers) detect tissue-damaging stimuli. When activated, in addition to transmitting pain signals to the central nervous system, they release neuropeptides locally via axonal reflexes. Among these, substance P and calcitonin gene-related peptide (CGRP) are core messengers regulating immunity. Substance P increases vascular permeability, causing plasma extravasation and edema, and directly acts on mast cells, macrophages, and neutrophils, prompting them to release inflammatory factors such as TNF-α, IL-1β, and histamine, thereby amplifying inflammation. CGRP, on the other hand, is a potent vasodilator, causing local redness and heat.

[0008] Most importantly, the study has revealed the presence of specific pain-sensing neurons innervating the spleen. These neurons primarily originate from the left T8–T13 dorsal root ganglion (DRG), with their fibers distributing along splenic vessels to the B-cell region. Upon antigen invasion, prostaglandin E2 (PGE2) accumulated in the spleen activates these pain-sensing neurons, prompting them to release CGRP. Subsequently, CGRP acts directly on B cells via its heterodimer receptor CALCRL-RAMP1, activating the cAMP signaling pathway, thereby significantly promoting germinal center responses, plasma cell differentiation, and the production of antigen-specific antibodies, enhancing humoral immunity.

[0009] Transient receptor potential vanillic acid isoform 1 (TRPV1), as an important class of pain receptors, can be activated or sensitized by heat, acid, capsaicin, and various inflammatory mediators (such as bradykinin, PGE2, and ATP), triggering Na+ receptor agonists.+ Ca 2+ Influx promotes the release of neuropeptides. Given the immunomodulatory function of pain receptors, exploring the feasibility of TRPV1 agonists as adjuvants for respiratory mucosal vaccines is a promising research direction. Summary of the Invention

[0010] The present invention aims to provide a recombinant subunit vaccine against COVID-19 administered via respiratory mucosa using the TRPV1 agonist capsaicin as an adjuvant for the prevention of COVID-19 infection.

[0011] This invention utilizes the spike protein of the SARS-CoV-2 envelope as an antigen. The antigen protein solution is mixed with capsaicin 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 capsaicin 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

[0012] Figure 1 Anti-SARS-CoV-2 spike protein receptor-binding domain (RBD) IgG antibody in hamster serum

[0013] 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 (Prime immunization) and the second (Boost immunization).

[0014] Figure 2 RBD IgA antibodies in hamster bronchoalveolar lavage fluid

[0015] 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

[0016] 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.

[0017] I. Reagents and Animals

[0018] 1. Syrian golden hamster, male, 8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0019] 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.

[0020] 3. TRPV1 small molecule agonist: capsaicin (hereinafter referred to as Cap), purchased from MCE (MedChemExpress), catalog number: HY-B1583R, dissolved in DMSO.

[0021] 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.

[0022] II. Experimental Methods:

[0023] (a) Immunization of Syrian golden hamsters

[0024] Twenty Syrian golden hamsters were randomly divided into four groups of five each. The animals were first anesthetized with isoflurane inhalation, followed by experimental vaccination.

[0025] Negative control group: PBS drops in both nostrils, 25 μL / nostril;

[0026] Single antigen (Str) nasal drops group: 25 μL / nostril for bilateral nasal drops, containing 10 μg of Str protein;

[0027] Str+Cap nasal drops: 25 microliters per nostril, containing 10 micrograms of Str protein and 10 micrograms of capsaicin;

[0028] Str+Cap adjuvant intramuscular injection group: 100 μL per side via bilateral intramuscular injection in the thigh, containing 10 μg of Str protein and 50 μg of capsaicin.

[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 fixed 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, and warm sterile saline was injected in divided doses, 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 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 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 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 using Graphpad Prism 5 software based on the absorbance values. 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 RBD IgG antibody titers in the sera of each group of hamsters are shown below. Figure 1 As shown: Nasal immunization with Str (a single SARS-CoV-2 spike protein extracellular trimer) induced extremely low levels of RBD IgG antibodies, while nasal immunization with Str combined with capsaicin induced high levels of RBD IgG antibodies. Intramuscular immunization with Str combined with capsaicin induced relatively low antibody levels. After booster immunization, the serum IgG antibody titers of the three groups of hamsters increased by 2.6, 9.8, and 3.7 times, respectively.

[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: nasal immunization with Str alone induces RBD IgA antibodies below the detection limit (i.e., the antibody is negative at 10-fold dilution). Nasal immunization with Str combined with capsaicin induces higher levels of RBD IgA antibodies. Intramuscular immunization with Str combined with capsaicin induces IgA antibodies below the detection limit (below 10, calculated as 10).

[0036] The results showed that intranasal administration of the SARS-CoV-2 envelope spike protein in combination with the TRPV1 agonist capsaicin could effectively induce serum IgG and respiratory IgA antibodies, and capsaicin can serve 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. Application of capsaicin in the preparation of a novel coronavirus respiratory mucosal vaccine.

2. The application according to claim 1, characterized in that: Capsaicin, as an effective upper respiratory tract mucosal immune adjuvant, is used as an immune adjuvant for the recombinant antigen or inactivated virus of the novel coronavirus, administered via nasal drops or nasal spray to prevent novel coronavirus infection.

3. The application according to any one of claims 1-2, characterized in that: The COVID-19 vaccine using capsaicin as an adjuvant can be administered via nasal drops or nasal spray.

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