Application of neutralizing antibodies in combination with tiaconazole and / or verteporfen in the preparation of drugs against bat coronavirus WIV1

Abstract

This invention discloses the application of neutralizing antibodies in combination with tiaconazole and / or verteporfen in the preparation of drugs against bat coronavirus WIV1, belonging to the field of biomedicine. This invention demonstrates that the combination of neutralizing antibody ADG20 with tiaconazole or verteporfen can effectively block intercellular transmission of bat coronaviruses, broadening the application of tiaconazole and verteporfen in clinical practice. This invention solves the problem of insufficient efficacy caused by the inability of neutralizing antibodies to completely eradicate viral infections in clinical applications, providing a new therapeutic drug for combating bat coronavirus WIV1 infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of neutralizing antibodies in combination with tiaconazole or verteporfen in the preparation of drugs against bat coronavirus WIV1. Background Technology

[0002] Emerging and re-emerging coronavirus epidemics have become a major threat to global public health security. Coronaviruses are positive-sense single-stranded RNA viruses, and their infection can cause symptoms such as fever, cough, and chest discomfort. Severe cases are often accompanied by typical signs of viral pneumonia, including progressive dyspnea and diffuse infiltration of both lungs. The source of coronavirus transmission can be traced back to its natural host—bats. Among them, WIV1, as a typical representative of bat coronaviruses, has a high degree of phylogenetic relationship with severe acute respiratory syndrome coronavirus. Studies have confirmed that WIV1 can use the angiotensin-converting enzyme 2 (ACE2) receptor of different hosts to invade non-natural host cells, and its affinity for human ACE2 is particularly significant. Therefore, this cross-species infection characteristic suggests that WIV1 poses a risk of transmission from wild animals to humans, and it is urgent to pay attention to its potential threat of causing emerging infectious diseases and to take adequate preventive measures.

[0003] In clinical practice, neutralizing antibodies offer advantages such as rapid onset of action and precise targeting. Neutralizing antibodies directly prevent viral invasion of host cells by targeting the receptor-binding domain of the viral spike protein (S protein), blocking the binding of the virus to the host cell surface receptor ACE2, and efficiently clearing viral particles through an immune response mediated by the antibody's Fc fragment. Currently available neutralizing antibodies for preventing coronavirus infection include sotopemumab and betalovimab; however, due to continuous viral mutations, these antibodies are gradually becoming ineffective. Furthermore, our previous research found that neutralizing antibodies cannot completely block the transmission of bat coronavirus WIV1 because the virus can achieve large-scale infection of neighboring cells without releasing progeny viral particles outside the cell, further limiting the clinical application of neutralizing antibodies. Therefore, it is necessary to find drug combinations that strongly antagonize the transmission of bat coronaviruses.

[0004] Tiaconazole is an antifungal drug primarily used to treat fungal skin infections such as tinea corporis, tinea cruris, tinea manuum, tinea pedis, and tinea versicolor, and can also be used for vaginal candidiasis. It exerts its antifungal effect by inhibiting the synthesis of fungal cell membranes. Some studies have found that tiaconazole inhibits the autophagy-associated cysteine ​​protease ATG4B. Verteporfen is a drug mainly used in photodynamic therapy to treat age-related macular degeneration and pathological myopia-related choroidal neovascularization. It is also an autophagy inhibitor that inhibits autophagosome formation by directly targeting and modifying p62. No research on the antiviral effects of tiaconazole and verteporfen against bat coronavirus WIV1 has been reported in the current technical literature. Summary of the Invention

[0005] Currently, there are no drugs available for the treatment of bat coronavirus infection in clinical practice, and existing neutralizing antibodies cannot completely block the intercellular transmission of bat coronavirus. The purpose of this invention is to solve this problem by providing the application of neutralizing antibodies in combination with tiaconazole and / or verteporfen in the preparation of drugs against bat coronavirus WIV1.

[0006] This invention confirms that the combination of neutralizing antibodies with tiaconazole or verteporfen can effectively block the intercellular transmission of bat coronaviruses, thus broadening the application of tiaconazole and verteporfen in clinical practice.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides the use of neutralizing antibodies in combination with tiaconazole and / or verteporfen in the preparation of drugs against bat coronavirus WIV1, comprising one or more of the following applications:

[0009] Application of neutralizing antibodies and tiaconazole in the preparation of drugs against bat coronavirus WIV1;

[0010] The use of a composition containing neutralizing antibodies and tiaconazole in the preparation of an anti-bat coronavirus WIV1 drug;

[0011] Application of neutralizing antibodies and verteporfen in the preparation of drugs against bat coronavirus WIV1;

[0012] The use of a composition containing neutralizing antibodies and verteporfen in the preparation of an anti-bat coronavirus WIV1 drug;

[0013] Application of neutralizing antibodies, tiaconazole, and verteporfen in the preparation of drugs against bat coronavirus WIV1;

[0014] The use of a composition containing neutralizing antibodies, tiaconazole, and verteporfen in the preparation of an anti-bat coronavirus WIV1 drug.

[0015] The neutralizing antibody is an antibody that targets the receptor-binding domain of the coronavirus S protein, preferably including one or more of ADG200, 10-40 (PDB number: 7TTY, https: / / www.rcsb.org / structure / 7TTY), and ADG20 (PDB number: 7U2D, https: / / www.rcsb.org / structure / 7U2D). More preferably, the neutralizing antibody is ADG20.

[0016] The structural formula of the thiaconazole is as follows: .

[0017] The structural formula of the verteporfen is as follows: .

[0018] The aforementioned anti-bat coronavirus WIV1 drug includes any one or more of the following drugs: drugs that inhibit the replication of bat coronavirus WIV1, drugs that block the transmission of bat coronavirus WIV1, and drugs that prevent and / or treat diseases caused by bat coronavirus WIV1 infection.

[0019] In some embodiments, the effective dose of neutralizing antibody in the anti-bat coronavirus WIV1 drug is 2-20 μg / mL. Preferably, the effective dose of neutralizing antibody is 5-10 μg / mL; more preferably, the effective dose of neutralizing antibody is 6.66 μg / mL.

[0020] In some embodiments, the effective dose of tiaconazole in the anti-bat coronavirus WIV1 drug is 5-15 μM. Preferably, the effective dose of tiaconazole is 8-12 μM; more preferably, the effective dose of tiaconazole is 10 μM.

[0021] In some embodiments, the effective dose of verteporfen in the anti-bat coronavirus WIV1 drug is 2-10 μM. Preferably, the effective dose of verteporfen is 3-6 μM; more preferably, the effective dose of verteporfen is 3.3 μM.

[0022] This invention also provides the use of tiaconazole in the preparation of drugs against bat coronavirus WIV1.

[0023] The present invention also provides a pharmaceutical composition comprising the above-mentioned neutralizing antibody, and one or both of tiaconazole and verteporfen. Further, the pharmaceutical composition may also comprise other anti-coronavirus agents.

[0024] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of an anti-bat coronavirus WIV1 drug.

[0025] The present invention also provides an anti-bat coronavirus WIV1 drug comprising the above-described pharmaceutical composition as the active ingredient. Further, the anti-bat coronavirus WIV1 drug also comprises pharmaceutically acceptable excipients.

[0026] In some embodiments, the excipients include any one or more combinations of fillers, diluents, solvents, solubilizers, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, wetting agents, emulsifiers, absorption delay agents, salts, drug stabilizers, binders, excipients, disintegrants, flow agents, gliding agents, lubricants, wetting agents, sweeteners, colorants, flavoring agents, seasonings, buffers, masking agents, and antioxidants.

[0027] In some implementations, the dosage forms of the anti-bat coronavirus WIV1 drug include: tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder inhalers.

[0028] In some implementations, the administration methods of the anti-bat coronavirus WIV1 drug include: oral administration, oral or nasal inhalation, or intravascular, intravenous, intraperitoneal, intramuscular, subcutaneous, or epidural injection.

[0029] The present invention also provides a method for combating bat coronavirus WIV1, comprising: administering the above-mentioned anti-bat coronavirus WIV1 drug to a host that has been infected with bat coronavirus WIV1 or may be infected with bat coronavirus WIV1, thereby inhibiting the infection of bat coronavirus WIV1 or blocking the transmission of bat coronavirus WIV1.

[0030] This invention has the following advantages and beneficial effects:

[0031] This invention discovers that neutralizing antibodies can block bat coronavirus infection, but cannot block the intercellular transmission of bat coronaviruses. Combining neutralizing antibodies with tiaconazole or verteporfen can efficiently prevent the intercellular transmission of bat coronaviruses.

[0032] This invention provides the application of neutralizing antibodies in combination with tiaconazole or verteporfen in the preparation of drugs against bat coronaviruses. This invention not only enriches the development strategies for antiviral drugs but also demonstrates therapeutic potential against bat coronavirus infections in practical application.

[0033] This invention not only provides an innovative approach to address the problem of insufficient efficacy of neutralizing antibodies in clinical applications due to their inability to completely eradicate viral infections, but also opens up new avenues for reducing the treatment burden on patients and improving treatment safety.

[0034] Therefore, this invention not only has significant scientific research value, but also has broad prospects for clinical application. Attached Figure Description

[0035] Figure 1 The ability of neutralizing antibody ADG20 to block free WIV1 virus particles from bat coronavirus in supernatant.

[0036] Figure 2 The impact of neutralizing antibody ADG20 on the transmission of bat coronavirus WIV1

[0037] Figure 3 Effects of tiaconazole on Caco2 cell viability.

[0038] Figure 4 Effects of verteporfen on Caco2 cell viability.

[0039] Figure 5 The effect of tiaconazole combined with neutralizing antibodies on the transmission of bat coronavirus WIV1.

[0040] Figure 6 IC50 of tiaconazole combined with neutralizing antibodies inhibiting the transmission of bat coronavirus WIV1 50 .

[0041] Figure 7 Effects of tiaconazole on the replication of bat coronavirus WIV1.

[0042] Figure 8 Effects of tiaconazole combined with neutralizing antibodies on the RNA of bat coronavirus WIV1.

[0043] Figure 9 Effects of tiaconazole combined with neutralizing antibodies on the protein of bat coronavirus WIV1.

[0044] Figure 10 The effect of verteporfen combined with neutralizing antibodies on the transmission of bat coronavirus WIV1.

[0045] Figure 11 The IC50 of verteporfen combined with neutralizing antibodies inhibiting the transmission of bat coronavirus WIV1 50 .

[0046] Figure 12 The effect of verteporfen on the replication of bat coronavirus WIV1.

[0047] Figure 13 Effects of verteporfen combined with neutralizing antibodies on the genomic RNA of bat coronavirus WIV1.

[0048] Figure 14 Effects of verteporfen combined with neutralizing antibodies on the viral protein of bat coronavirus WIV1. Detailed Implementation

[0049] The following embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0050] Example 1: Detection of neutralizing antibody ADG20 blocking the transmission of bat coronavirus WIV1

[0051] (1) First, Caco2 cells were arranged at a ratio of 3×10 6 / wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 6-well cell culture plates. The virus solution was incubated with cells for 2 h, followed by medium replacement. Neutralizing antibody ADG20 was prepared at concentrations of 0, 0.22, 0.66, 2.22, 6.66, and 20 μg / mL. The corresponding antibody-containing medium was added to the infected cells, and the cells were incubated at 37 °C. Cell supernatants were collected at 24 and 48 h for virus titer determination. The six collected cell supernatant samples were serially diluted 10-fold, resulting in seven dilution gradients (-1, -2, -3, -4, -5, -6, -7). The diluted virus solution was then seeded into Caco2 cells. After 3 h of infection, the culture medium was discarded, and DMEM medium containing 1% methylcellulose and 2% FBS was added. Ninety-six hours after infection, when obvious fluorescent spots were observed, 4% PFA was added for fixation. The fluorescent spots in each well were counted under a fluorescence microscope. The amount of residual free infectious viral particles in the cell supernatant at different antibody concentrations was calculated to determine the concentration of neutralizing antibodies that could effectively block infection by free viral particles. Results are as follows: Figure 1 As shown, when the concentration of neutralizing antibody ADG20 reached 6.66 μg / mL, no infectious viral particles could be detected in the supernatant.

[0052] (2) To detect the effect of neutralizing antibody ADG20 on the overall transmission level of coronavirus, a mixed culture mode of donor and recipient cells was adopted, in which both donor and recipient cells were Caco 2. Donor cells were infected with bat coronavirus WIV1 at MOI=3 for 2 h, and then digested and counted for later use. Recipient cells were simultaneously labeled with cytoplasmic blue fluorescent dye (50 μM), incubated for 2 h, and then digested and counted for later use. Donor and recipient cells were mixed at a ratio of 1:6 to prepare cell suspensions containing different concentrations of neutralizing antibody, and then cultured at 3.6 × 10⁻⁶. 5 Cells / well were seeded into 24-well plates and co-cultured for 48 h. Cells were then harvested for flow cytometry analysis to detect recipient cell infection status, representing the intercellular transmission of bat coronavirus WIV1. Results are as follows: Figure 2As shown, the virus was still able to spread even with the addition of excessive amounts of the neutralizing antibody ADG20. This highlights the inadequacy of neutralizing antibodies in blocking the transmission of bat coronaviruses and the necessity of using drugs to block intercellular transmission of the virus.

[0053] Example 2: Detection of cytotoxicity of drugs (tiaconazole, verteporfen)

[0054] Cell viability was assessed using the CCK8 assay to evaluate drug (tiaconazole, verteporfen) toxicity. Caco2 cells were seeded into 96-well plates (2.5 × 10⁻⁶ cells / well) one day in advance. 4 The next day, when cell confluence reached over 85%, cells were treated with different concentrations of the drug (30, 10, 3.33, 1.11, 0.33, 0.11 μM). First, the original culture medium was discarded and the cells were washed with PBS buffer. Then, culture medium containing the corresponding drug concentration was added to the wells, and after standing for 48 h, CCK8 assay was performed. An enhanced CCK8 kit was used, with a 10% CCK8 test solution prepared. 100 μL of the solution was added to each well of a 96-well plate, and the absorbance at 450 nm was measured after 30 min using a microplate reader. A blank control group without cells was included to remove background interference. Finally, cell viability was calculated using the following formula: Cell viability = [OD(drug-treated) - OD(blank)] / [OD(0-drug-treated) - OD(blank)] × 100%. The specific cell viability values ​​at each concentration were calculated and displayed as a bar chart. The results showed that the drug toxicity of tiaconazole was within the acceptable range of 10-0.11 μM. At the highest concentration, treatment for 48 h showed no significant change in cell viability, while treatment at concentrations exceeding 10 μM resulted in significant cytotoxicity. Figure 3 Vertepofen's toxicity is within the acceptable range of 10-0.11 μM, with no significant change in cell viability after treatment at the highest concentration for 48 h. However, significant cytotoxicity occurs above 10 μM. Figure 4 ).

[0055] Example 3: Detection of the blocking effect of tiaconazole combined with neutralizing antibody ADG20 on the transmission of bat coronavirus WIV1

[0056] 1. Detection of the effect of tiaconazole combined with neutralizing antibody ADG20 on blocking the transmission of bat coronavirus WIV1

[0057] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates. The virus solution was incubated with the cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of tiaconazole (30, 10, 3.3, 1.1, 0.33 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to the wells. A blank group (PBS), a positive drug control group (E64D 40 μM and 6.66 μg / mL ADG20), and a negative control group (0.1% DMSO and 6.66 μg / mL ADG20) were set up and incubated for 48 h.

[0058] Cell culture plates that had been co-cultured for 48 h were fixed and stained. Cells were fixed using high-concentration paraformaldehyde. 40 μL of 16% paraformaldehyde was added to each well and mixed with the existing culture medium to achieve a final paraformaldehyde concentration of 3-5%. The cells were then fixed at 4°C for at least 2 hours. After fixation, the culture medium in the cell plate was discarded, and 40 μL of 0.1% Triton was added to penetrate the cells. The cells were then incubated at 4°C for at least 15 minutes. After discarding the Triton, the cells were washed twice with PBS, and 40 μL of 5% BSA solution was added. The cells were then blocked at 4°C for at least 30 minutes. After discarding the 5% BSA solution, bat coronavirus S antibody (rabbit anti) was diluted 1:1000 to 5% BSA and added to the culture plate. The cells were incubated at 4°C for at least 16 hours. After discarding the primary antibody, the cells were washed twice with PBS buffer, and FITC-coupled fluorescent secondary antibody (goat anti-rabbit) was diluted 1:200 to 5% BSA and added to the culture plate. The cells were then incubated at 4°C in the dark for 2 hours. After 20 minutes, discard the II antibody, wash with PBS buffer, then stain the cell nuclei with DAPI staining solution. After staining in the dark for 20 minutes, discard the staining solution and wash again with PBS buffer. Finally, keep the cells moist with 20 μL PBS.

[0059] The prepared cell culture plates were imaged using a high-content imaging system. The specific imaging protocol for the high-content imaging system included: setting detailed parameters for the FITC and DAPI channels, adjusting the focal length according to the plate height, and setting the imaging range based on the plate bottom area to ensure complete recording. Each well was imaged, and the effective EGFP region was marked using EGFP fluorescence intensity as the readout threshold. The EGFP fluorescence area was then calculated to represent the extent of viral infection. Subsequently, the DAPI area was recorded using a similar method to represent the cell nucleus area, thus defining the statistical region for EGFP. The EGFP fluorescence area data was exported for analysis of drug inhibition effects.

[0060] The results showed that neutralizing antibody ADG20 (6.66 μg / mL) and tiaconazole (10 μM) had a good blocking effect on intercellular transmission of bat coronavirus WIV1 (EGFP fluorescence area change). Figure 5 Tiaconazole inhibits the intercellular transmission of WIV1 cells. 50 4.25 μM ( Figure 6 ).

[0061] 2. Detection of thiaconazole's inhibition of bat coronavirus WIV1 replication

[0062] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded into 96-well cell culture plates with bat coronavirus WIV1 at an initial infection dose of MOI = 3. The cells were incubated with the virus solution for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of tiaconazole (30, 10, 3.3, 1.1, 0.33 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration was added to each well. A blank control group (PBS), a positive control group (remdesivir 2 μM), and a negative control group (0.1% DMSO) were also established. The cells were incubated for 48 h. Fixation, staining, and imaging were performed according to the above steps. The results are shown below. Figure 7 As shown, tiaconazole alone, even at a high concentration of 30 μM, could not completely inhibit viral replication, with an EGFP fluorescence area of ​​approximately 20%.

[0063] 3. Effects of tiaconazole combined with neutralizing antibody ADG20 on the genomic RNA of bat coronavirus WIV1.

[0064] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates, and the virus solution was incubated with cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of tiaconazole (30, 10, 3.3, 1.1, 0.33 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to each well. A negative control group (0.1% DMSO and 6.66 μg / mL ADG20) was also set up. The cells were incubated for 48 h. Total RNA was extracted from the cells, and the inhibitory effect of the drug combination on virus transmission was detected by real-time quantitative polymerase chain reaction (RT-qPCR, primer sequences are shown in Table 1 below). The results are as follows. Figure 8 As shown, tiaconazole has antiviral effects in the concentration range of 0.33 μM to 10 μM, and at a concentration of 10 μM, tiaconazole has a very strong effect in blocking the intercellular transmission of WIV1.

[0065] Table 1

[0066]

[0067] 4. Effects of tiaconazole combined with neutralizing antibody ADG20 on the viral protein of bat coronavirus WIV1

[0068] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates, and the virus solution was incubated with cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of tiaconazole (30, 10, 3.3, 1.1, 0.33 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to each well. A negative control group (0.1% DMSO and 6.66 μg / mL ADG20) was also set up. The cells were incubated for 48 h. After 48 h, cell proteins were extracted, and the inhibitory effect of the drug combination on virus transmission was detected by Western blotting (WB). The results are as follows. Figure 9 As shown, tiaconazole has antiviral effects in different concentration ranges, with a particularly strong effect in blocking intercellular transmission of WIV1 at a concentration of 10 μM.

[0069] Example 4: Detection of the blocking effect of verteporfen combined with neutralizing antibody ADG20 on the transmission of bat coronavirus WIV1

[0070] 1. Detection of the effect of verteporfen combined with neutralizing antibody ADG20 on blocking the transmission of bat coronavirus WIV1

[0071] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates. The virus solution was incubated with the cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of verteporfen (30, 10, 3.3, 1.1, 0.33 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to the wells. A blank group (PBS), a positive drug control group (E64D 40 μM and 6.66 μg / mL ADG20), and a negative control group (0.1% DMSO and 6.66 μg / mL ADG20) were set up and incubated for 48 h.

[0072] Cell culture plates that had been co-cultured for 48 h were fixed and stained. Cells were fixed using high-concentration paraformaldehyde. 40 μL of 16% paraformaldehyde was added to each well and mixed with the existing culture medium to achieve a final paraformaldehyde concentration of 3-5%. The cells were then fixed at 4°C for at least 2 hours. After fixation, the culture medium in the cell plate was discarded, and 40 μL of 0.1% Triton was added to penetrate the cells. The cells were then incubated at 4°C for at least 15 minutes. After discarding the Triton, the cells were washed twice with PBS, and 40 μL of 5% BSA solution was added. The cells were then blocked at 4°C for at least 30 minutes. After discarding the 5% BSA solution, bat coronavirus S antibody (rabbit anti) was diluted 1:1000 to 5% BSA and added to the culture plate. The cells were incubated at 4°C for at least 16 hours. After discarding the primary antibody, the cells were washed twice with PBS buffer, and FITC-coupled fluorescent secondary antibody (goat anti-rabbit) was diluted 1:200 to 5% BSA and added to the culture plate. The cells were then incubated at 4°C in the dark for 2 hours. After 20 minutes, discard the II antibody, wash with PBS buffer, then stain the cell nuclei with DAPI staining solution. After staining in the dark for 20 minutes, discard the staining solution and wash again with PBS buffer. Finally, keep the cells moist with 20 μL PBS.

[0073] The prepared cell culture plates were imaged using a high-content imaging system. The specific imaging protocol for the high-content imaging system included: setting detailed parameters for the FITC and DAPI channels, adjusting the focal length according to the plate height, and setting the imaging range based on the plate bottom area to ensure complete recording. Each well was imaged, and the effective EGFP region was marked using EGFP fluorescence intensity as the readout threshold. The EGFP fluorescence area was then calculated to represent the extent of viral infection. Subsequently, the DAPI area was recorded using a similar method to represent the cell nucleus area, thus defining the statistical region for EGFP. The EGFP fluorescence area data was exported for analysis of drug inhibition effects.

[0074] The results showed that the neutralizing antibody ADG20 (6.66 μg / mL) and verteporfen (3.33 μM) had a good blocking effect on intercellular transmission of bat coronavirus WIV1 (EGFP fluorescence area change). Figure 10 Vertepofen inhibited intercellular transmission of WIV1 at a concentration of 1.57 μM. Figure 11 ).

[0075] 2. Detection of verteporfen's inhibition of bat coronavirus WIV1 replication

[0076] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded into 96-well cell culture plates with bat coronavirus WIV1 at an initial infection dose of MOI = 3. The cells were incubated with the virus solution for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of verteporfen (10, 3.3, 1.1, 0.33, 0.11 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration was added to each well. A blank control group (PBS), a positive control group (remdesivir 2 μM), and a negative control group (0.1% DMSO) were also established. The cells were incubated for 48 h. Fixation, staining, and imaging were performed according to the above steps. The results are shown below. Figure 12 As shown, vertepofen alone, even at a high concentration of 10 μM, could not completely inhibit viral replication, with EGFP fluorescence covering 40%.

[0077] 3. Effects of verteporfen combined with neutralizing antibody ADG20 on the genomic RNA of bat coronavirus WIV1

[0078] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates, and the virus solution was incubated with cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of verteporfen (10, 3.3, 1.1, 0.33, 0.11 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to each well. A negative control group (0.1% DMSO and 6.66 μg / mL ADG20) was also set up. The cells were incubated for 48 h. The inhibitory effect of the drug combination on virus transmission was detected by real-time quantitative polymerase chain reaction (RT-qPCR, primer sequences are shown in Table 2 below). The results are as follows. Figure 13 As shown, verteporfen exhibits antiviral effects in the concentration range of 0.33 μM to 10 μM, with the strongest effect in blocking intercellular transmission of WIV1 at a concentration of 10 μM.

[0079] Table 2

[0080]

[0081] 4. Effects of verteporfen combined with neutralizing antibody ADG20 on the viral protein of bat coronavirus WIV1

[0082] First, CaCO2 cells were arranged at a ratio of 3 × 10 4 / Wells were seeded with bat coronavirus WIV1 at an initial infection dose of MOI = 0.3 in 96-well cell culture plates, and the virus solution was incubated with cells for 2 h. The original culture medium was discarded and the cells were washed with PBS buffer. Then, different concentrations of verteporfen (10, 3.3, 1.1, 0.33, 0.11 μM) were prepared, and 100 μL of culture medium containing the corresponding drug concentration and 6.66 μg / mL ADG20 was added to each well. A negative control group (0.1% DMSO and 6.66 μg / mL ADG20) was also set up. The cells were incubated for 48 h. After 48 h, cell proteins were extracted, and the inhibitory effect of the drug combination on virus transmission was detected by Western blotting (WB). The results are as follows. Figure 14 As shown, verteporfen exhibits antiviral effects across different concentration ranges, with the strongest effect in blocking intercellular transmission of WIV1 cells observed at a concentration of 10 μM.

[0083] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The application of neutralizing antibodies in combination with tiaconazole or verteporfen in the preparation of drugs against bat coronavirus WIV1, characterized in that: The application includes any one or more of the following applications: Application of neutralizing antibodies and tiaconazole in the preparation of drugs against bat coronavirus WIV1; The use of a composition containing neutralizing antibodies and tiaconazole in the preparation of an anti-bat coronavirus WIV1 drug; Application of neutralizing antibodies and verteporfen in the preparation of drugs against bat coronavirus WIV1; The use of a composition containing neutralizing antibodies and verteporfen in the preparation of an anti-bat coronavirus WIV1 drug; The neutralizing antibody mentioned is ADG20.

2. The application according to claim 1, characterized in that: The aforementioned anti-bat coronavirus WIV1 drug includes any one or more of the following drugs: drugs that inhibit the replication of bat coronavirus WIV1, drugs that block the transmission of bat coronavirus WIV1, and drugs that prevent and / or treat diseases caused by bat coronavirus WIV1 infection.

3. The application according to claim 1, characterized in that: In the aforementioned anti-bat coronavirus WIV1 drug, the effective dose of neutralizing antibody is 2-20 μg / mL; When the anti-bat coronavirus WIV1 drug contains tiaconazole, the effective dose of tiaconazole is 5-15 μM; when the anti-bat coronavirus WIV1 drug contains verteporfen, the effective dose of verteporfen is 2-10 μM.

4. A pharmaceutical composition, characterized in that: It includes the neutralizing antibody of claim 1, and one of tiaconazole and vertepofen.

5. The use of the pharmaceutical composition of claim 4 in the preparation of an anti-bat coronavirus WIV1 drug.

6. A drug against bat coronavirus WIV1, characterized in that: The pharmaceutical composition comprising claim 4.

7. The anti-bat coronavirus WIV1 drug according to claim 6, characterized in that: It also includes excipients that are acceptable in pharmaceutical manufacturing.

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