WO2023245415A1

WO2023245415A1 - Use of polygonatum cyrtonema hua. lectin in blocking invasion and infection of novel coronavirus

Info

Publication number: WO2023245415A1
Application number: PCT/CN2022/100123
Authority: WO
Inventors: 鲍锦库; 吴传芳; 郑茹潇; 聂川雄; 哈格·莱纳
Original assignee: 四川大学; 柏林自由大学
Priority date: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-12-28

Abstract

The present application provides a novel blocking method for blocking the invasion and infection of SARS-CoV-2 by using polygonatum cyrtonema Hua. lectin protein, use thereof, and a pharmaceutical composition and kit for blocking the invasion and infection of SARS-CoV-2.

Description

The use of Polygonatum agglutinin in blocking the invasion and infection of new coronavirus

Technical field

The invention belongs to the field of biomedicine, and specifically relates to a novel blocking method and use of using polygonatum lectin protein to block SARS-CoV-2 invading infection, as well as pharmaceutical compositions and medicines for blocking SARS-CoV-2 invading infection. box.

Background technique

Coronavirus disease 2019 (COVID-19), a respiratory disease caused by the novel coronavirus (SARS-CoV-2), is currently the most difficult public health problem in the world. It has caused large-scale outbreaks around the world and led to considerable The high morbidity and mortality rates seriously endanger human health and the world's economic and social development. Currently, scientists around the world are actively developing vaccines against the new coronavirus and new coronavirus drugs. There are currently a number of vaccines in use around the world, but the protective effect of the vaccines still needs to be tested.

The new coronavirus mainly has four structural proteins, namely the spike (or spike) glycoprotein (S protein), the small envelope glycoprotein (E, protein) and the membrane glycoprotein (M protein) that are highly exposed to the surface of the virus. Seed membrane protein, and nucleocapsid protein (N protein). The infectivity of SARS-CoV-2 virus is mainly determined by the S protein, which binds to the membrane receptor of the host cell, thereby mediating the fusion of the virus and the cell membrane, and is widely distributed on the epithelial cell membranes of the host's nasal cavity, lungs, small intestine and other organs and tissues. Angiotensin-converting enzyme 2 (ACE2) is the main receptor for S protein.

Research shows that the spike protein (S protein), which is highly exposed on the surface of the virus, is the main weapon for the virus to invade the body and is a key part of the virus to attach and infect host cells. By forming homotrimers, the S protein is formed on the surface of the virus. Distinctive spikes mediate membrane fusion and viral entry into cells. The receptor recognition binding domain (RBD) of S protein is crucial for its binding to ACE2. In view of the fact that the S protein plays an indispensable role in viral invasion and infection, the recognition and binding of the S protein to ACE2 triggers the virus to infect host cells. Therefore, any way to cut off or interfere with the interaction between the spike S protein and ACE2 is necessary. The role is crucial for controlling viral invasion, which is currently the main strategy for the design of drugs such as vaccines and neutralizing antibodies. Therefore, the design of drugs and vaccines targeting the S protein has become the most important direction in the research and development of preventive and therapeutic drugs for SARS-CoV-2. The peptide segments (skeleton) of specific parts of the S protein polypeptide chain, especially the key peptides targeting the RBD part The segment is currently the most important target for vaccine design, neutralizing antibodies and other drug development.

However, the S protein of SARS-CoV-2 is a highly glycosylated glycoprotein. Each S protein monomer contains at least 22 N-linked sugar chains, and the trimeric S protein has at least 66 sugar groups. ionization site, with a large number of oligomannose-type, high-mannose-type and mannose-containing complex sugar chains attached to the surface. This means that the new coronavirus is a veritable "sugar-coated virus", with so many glycosylation sites connected to a large number of polysaccharide chains, as if the surface is covered with a thick layer of "sugar chain barriers" or "sugar shield" ( Glycan shield). This dynamic, floating "sugar chain barrier" covers more than 90% of the virus surface, which may cause difficulties in the development of vaccines and targeted drugs. Antibodies generated by targeted drugs or injectable vaccines often have difficulty penetrating the thick sugar-coated surface layer and entering the interior to bind to the target peptide skeleton. At the same time, the "swimming and floating" sugar chains may cover or even hide the binding site of antibodies or drugs, making it difficult for antibodies and drugs to recognize the target site and thus have difficulty exerting their effect, thereby causing immune evasion and drug off-target. Studies have found that the sugar chains at N165 and N234 glycosylation sites of S protein can stabilize the open conformation of S protein, thereby promoting the binding of RBD and ACE2; not only that, the sugar chains at N165, 234, and 343 can also shield the S protein in its closed conformation. RBD, which in turn plays a protective role. Therefore, in addition to protecting and shielding the S protein peptide chain, the sugar chain can also promote the combination of the virus and the receptor protein, thereby promoting the occurrence of infection.

Therefore, immune evasion and drug off-target problems caused by the high glycosylation of S protein are obstacles and challenges facing the development of SARS-CoV-2 vaccines and other drugs. In particular, the current challenge of the emergence of escape virus variants that reduce the effectiveness of existing vaccines highlights the need for broad-spectrum SARS-CoV-2 inhibitors and the need for new strategies to overcome the challenges of evolving new mutants.

Targeting the S protein of SARS-CoV-2, especially targeting some of the key peptides of its RBD, is currently the most important target for vaccine design, neutralizing antibodies and other drug development. However, since the S protein is a highly glycosylated glycoprotein, and its trimer has at least 66 N-glycosylation sites, the sugar shield covering the surface of the S protein can easily become a "golden bell" for the virus. ". It greatly affects the neutralizing effect of the antibodies and neutralizing antibodies produced by the vaccine on the virus and the protective effect on the host. At the same time, SARS-CoV-2 continues to evolve and mutate, producing a large number of virus mutant strains. Variations in the genomes of these mutant strains have caused changes in virus phenotypes: such as enhanced transmissibility or adverse changes in epidemic characteristics; enhanced virulence or increased clinical pathogenicity; changes in public health, social intervention measures, or existing diagnostics, Reduced effectiveness of vaccines and treatments, etc.

Five variant strains of concern (VOC) that have caused widespread epidemics around the world include: Delta, which is the most contagious of all mutant strains, Omicron, which spreads extremely fast, and highly virulent but Gamma, which is less prevalent, Beta, which has enhanced toxicity and transmissibility and evades vaccines, and Alpha, which attacks immunity and has enhanced transmissibility. Relevant mutations in the S protein of these mutant strains bring greater risks, causing immune evasion, failure of diagnosis and/or treatment, and reduction of vaccine efficacy. Especially Omicron, which has 34 S protein mutations: the neutralizing effect of vaccine antibodies on Omicron has dropped significantly, and the Omicron variant can escape the immune protection conferred by vaccines and natural infections, and may evolve rapidly; research shows that it is closely related to the new coronavirus Compared with the original strain, the neutralizing effect of Omicron stimulated by the vaccine dropped 22 times; Omicron can escape more than 85% of the antibodies; Omicron can completely resist or partially resist the neutralizing effect of all monoclonal antibodies in the experiment. .

The spike protein is highly glycosylated, but the glycans covering its surface are conserved across mutations. The global spread of mutant strains such as Delta and Omicron has broken through the protective barrier of vaccines, leading to the occurrence of "breakthrough infections." The present invention takes a unique approach to address issues such as vaccine off-targeting due to coronavirus glycosylation. It targets the conserved sugar chain of the S protein and uses the sugar-binding protein Polygonatum agglutinin to block and inhibit SARS-CoV-2 infection, and at the same time provides a Targeting the spike proteoglycan as a strategy to design broad-spectrum SARS-CoV-2 inhibitors may overcome the challenges of evolving new mutants.

In view of the possible bottlenecks in the development of new coronavirus drugs, and to avoid the sugar chain traps and obstacles that may be faced by the current vaccine design targeting specific parts of the peptide chain backbone of the S protein and the development of other targeted drugs, adopt a sugar chain based on the S protein. A new strategy that targets the S protein chain, using protein polypeptide compounds that specifically bind to its sugar chain, to block the S protein sugar chain, the "spike" of host cells infected by the new coronavirus, thereby blocking the recognition of the virus and the host cell receptor (ACE2). Combined to achieve the purpose of preventing and treating viral infections. Therefore, blocking the recognition and binding of viruses and host cells by binding and blocking the glycoprotein sugar chains on the surface of the virus may be an effective strategy to block and prevent viral infection. Therefore, infection blocking targeting sugar chains may become a A new breakthrough in drug research and development.

Contents of the invention

Lectins are a class of proteins or glycoproteins of non-immune origin that specifically recognize and bind to pairs of sugars and their complexes. Polygonatum cyrtonema Hua.Lectin (PCL) is a lectin isolated by the inventor from the traditional Chinese herbal medicine Polygonatum cyrtonema Hua. that specifically recognizes and binds mannose and sialic acid. In the process of extracting and separating Polygonatum agglutinin, two or three protein peaks usually appear, among which Peak II has a higher content. Therefore, the Polygonatum agglutinin is usually called Polygonatum agglutinin with Peak II, or Polygonatum agglutinin II. . Polygonatum agglutinin II is a 160 amino acid glycoprotein, a mannose/sialic acid-binding lectin. Its protein sequence is shown in GenBank Accession:AAM28644.1, and its encoding mRNA sequence is shown in GenBank Accession:AY099150.1 Show. The Polygonatum agglutinin described herein is Polygonatum agglutinin II.

The present invention measures the S protein binding activity and novel coronavirus (SARS-CoV-2, or novel coronavirus) antiviral activity of Polygonatum agglutinin (PCL), and finds that it can specifically and efficiently bind S protein sugar chains and spatially hinder Its binding to ACE2 and other cell receptors. PCL displays potent inhibitory effects on SARS-CoV-2 infection, including eliminating cytopathic effects, limiting viral spread, and reducing viral titers. When applied to persistent SARS-CoV-2 infection, it can also reduce virus replication by 4 orders of magnitude. PCL can bind to glycosylated spike protein trimers and spike protein RBD (S_Trimer, S_RBD) in a dose-dependent manner. PCL can also bind to delta mutant spike protein trimers with similar affinity levels. , can inhibit the infection of VeroE6 cells by Beta and Delta mutant strains at a level similar to its performance on wild-type SARS-CoV-2.

Antiviral activity experiments show that Polygonatum lectin protein can effectively block the infection of VERO cells by SARS-CoV-2. Adding mannose to block the mannose sugar binding site of Polygonatum lectin protein significantly reduces the antiviral ability of the lectin protein. Even lost. Therefore, Polygonatum agglutinin protein can be used as an active ingredient to prepare anti-novel coronavirus drugs. The present invention provides the possibility of using a polygonatum agglutinin protein to block the invasion and infection of SARS-CoV-2 and its mutants to achieve the effect of blocking and preventing viral infection.

Specifically, this application solves the technical problems existing in the field through the following technical solutions.

1. The use of Polygonatum agglutinin (PCL) in blocking and preventing the infection of host cells by the new coronavirus SARS-CoV-2 and its mutant strains such as Delta, Omicron, Gamma, Beta and Alpha mutant strains in vitro.

2. The use of Polygonatum agglutinin (PCL) in the preparation of drugs for preventing or treating infection by the new coronavirus SARS-CoV-2 and its mutant strains, such as Delta, Omicron, Gamma, Beta and Alpha mutant strains.

3. A pharmaceutical composition for preventing or treating infection by the new coronavirus SARS-CoV-2 and its mutant strains such as Delta, Omicron, Gamma, Beta and Alpha mutant strains, which contains Polygonatum agglutinin (PCL) and pharmaceutically acceptable carrier.

4. A pharmaceutical kit comprising the pharmaceutical composition of item 3 and instructions for use and optionally an administration tool.

5. A method of preventing or treating infection by the new coronavirus SARS-CoV-2 and its mutant strains such as Delta, Omicron, Gamma, Beta and Alpha mutant strains, wherein the method includes administering the pharmaceutical composition of item 3 to the subject Or provide a pill box of item 4.

6. The method of item 5, wherein the method further comprises administering to the subject another drug for treating infection by the new coronavirus SARS-CoV-2 and its mutant strains, such as Delta, Omicron, Gamma, Beta and Alpha mutant strains. .

Description of the drawings

Figure 1. (a) SARS-CoV-2 plaque image after treatment with PCL (500 μg/mL); (b) Inhibition curve of PCL against WT wild-type virus, beta and delta mutant strains in plaque reduction experiment. Values are expressed as mean ± standard deviation, n = 3.

Figure 2. (a) CLSM image of Dio-labeled WT SARS-CoV-2 virions binding to VeroE6 cells. Scale bar: 20 μm. Inactivated virus particles are marked in green and cell nuclei in blue. (b) Count of VeroE6 cells bound to viral particles. Values are expressed as mean ± standard deviation, n = 4. Unpaired t test P<0.001. (c) CLSM image of Delta mutant strain S protein RBD binding to VeroE6 cells in the presence of PCL. The RBD is labeled green and the nucleus is labeled blue. Scale bar: 20 μm.

Figure 3. (a) CPE observation of SARS-CoV-2 infected cells. Scale bar: 100 μm. (b) Inhibitory effect of PCL on viruses. Values are expressed as mean ± standard deviation, n = 3.

Figure 4. (a) Immunostaining image of infected cells. PCL concentration: 125μg/mL. Scale bar: 20 μm. (b) Plaque titration of supernatant. Values are expressed as mean ± standard deviation, n = 2.

Figure 5. (a) Binding analysis of PCL and trimeric spike protein, trimeric spike protein treated with PNGase F. (b) Binding analysis of PCL with eukaryotic expressed RBD and prokaryotic expressed RBD. (c) Binding analysis of PCL and the trimeric spike protein of the Delta mutant strain and the trimeric spike protein treated with PNGase F. (d) Dose-dependent binding of PCL to trimeric spike protein. (e) Dose-dependent binding of PCL to RBD. (f) Dose-dependent binding of PCL to the trimeric spike protein of the Delta mutant strain.

Figure 6. Comparison of the results of the plaque reduction assay in Example 3 using ASL, ACL, LRL, ZCL and PCL.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

The laboratory of the inventor of the present application has previously prepared and detected the protein sample to be tested of Polygonatum cyrtonema (Hua. Lectin, PCL for short) protein (see An, J. et al. Anti-HIV I/II activity and molecular cloning of a novel mannose/sialic acid-binding lectin from rhizome of Polygonatum cyrtonema Hua.Acta biochimica et biophysica Sinica 2006,38,70-78). The inventor then used Fortbio Biolayer interferometry (BLI) to test the binding of polygonatum lectin in the previously prepared test protein sample to the S protein of the virus (wild type and Delta mutant strain) and its RBD, and used Confocal Laser Scanning Microscopy (CLSM) was used to study virus interactions on the aforementioned protein sample to be tested, Polygonatum agglutinin. Through the post-infection inhibition test, the inhibitory effect of the polygonatum agglutinin, the aforementioned protein sample to be tested, in multi-cycle viral infection was detected, and the virus TCID50 (half tissue infection dose) was measured to test the antiviral activity of the polygonatum agglutinin, the aforementioned protein sample to be tested. The inventor also tested the antiviral activity of the aforementioned protein sample to be tested, Polygonatum agglutinin (SARS-CoV-2 wild type, Beta and Delta mutant strains) through plaque reduction assay.

Example 1: Preparation of Polygonatum agglutinin protein

Polygonatum cyrtonema (Hua. Lectin, referred to as PCL) protein was prepared and tested according to the previous methods of the inventor's laboratory (An, J. et al. Anti-HIV I/II activity and molecular cloning of a novel mannose/sialic acid-binding lectin from rhizome of Polygonatum cyrtonema Hua.Acta biochimica et biophysica Sinica 2006,38,70-78), and some optimization has been carried out. The specific method is as follows:

1.Method

1.1 Coarse separation of PCL. Polygonatum cyrtonema Hua (PCL) was collected from Changning, Yibin, Sichuan; take the rhizome of fresh Polygonatum cyrtonema, grind it with a high-speed tissue masher, soak it in physiological saline overnight, filter it with gauze, and centrifuge (4°C, 45 00r/min , 30min), add solid ammonium sulfate to the supernatant to reach 30% saturation (g/L), overnight at 4°C, centrifuge at 6 000r/min for 30min, collect the supernatant, continue to add ammonium sulfate to reach 80% saturation (g/L) g/L), collect the precipitate by centrifugation as before, use pH 5.0, 20mM NaAc-HAc buffer for resuspension and dialysis, add PMSF to inhibit protease activity and reduce the degradation of the target protein. The operation process is all performed on ice.

1.2 Equilibrate the CM column with buffer. The CM-Sepharose cationic column uses pH 5, 20mM NaAc-HAc buffer. After 5-6 column volume equilibrium, the entire chromatography system reaches the target pH and ionic strength to prepare for the next step of loading the sample.

1.3 Ion exchange chromatography of PCL. After the CM column is equilibrated, the protein sample that has been previously dialyzed with a buffer (pH and ionic strength are the same as the buffer) is passed through the chromatography column. During this process, it is negatively charged at pH 9.0 (protein PI<9.0 ) proteins are bound to the anion exchange resin; proteins with positive charges (PI>5.0) at pH 5.0 are bound to the cation exchange resin. The impurity proteins flow out with the buffer, and after washing until the absorption value of the ultraviolet detector of the effluent is less than 0.02 at 280 nm, use 0.6 mol/L NaCl NaAc-HAc solution for ion linear gradient elution, pressurize with a peristaltic pump, and control The flow rate is 3ml/min. The effluent of the corresponding peak part is collected according to the display of UV detection, 5ml/tube. After UV and agglutination activity detection, the active part is collected and freeze-dried.

1.4 Molecular sieve chromatography of PCL. The above freeze-dried samples were dissolved in 0.14 mol/L NaCl, and the same solution was used to balance the Sephacryl S-100 column, and PCL molecular sieve chromatography was purified. The flow rate is 12ml/h, 3ml/tube, and tested by ultraviolet and coagulation activity. The second activity peak was collected, the collected liquid was dialyzed to remove salt, and then freeze-dried to obtain pure PCL.

1.5 PCL purity testing. The purity of PCL was identified using polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the separation gel is 15% (g/ml), stained with Coomassie Brilliant Blue R-250, destained with 10% (v/v) acetic acid destaining solution, and then the protein separation ribbon in the destained gel was scanned, photographed, and analyzed. image. It was detected as a single protein staining band by SDS-PAGE, and the purity reached 90%.

The detailed steps are as follows:

1.5.1 SDS-PAGE electrophoresis:

1) Reagent preparation

A. 30% (g/ml) Acr-Bis solution: 73.0g acrylamide (Acr), 2.0g methylene bisacryloyl (Bis), mix and add ddH ₂ O, dissolve and adjust to 250ml, brown The bottles were protected from light and stored at 4°C.

B. 1.5M Tris-HCl (pH=8.8): Dissolve 45.4g Tris in ddH ₂ O, adjust the pH to 8.0 with concentrated hydrochloric acid, and adjust the volume to 250ml;

C. 1M Tris-HCl (pH=6.8): Dissolve 30.3g Tris in ddH ₂ O, adjust the pH to 6.8 with concentrated hydrochloric acid, and adjust the volume to 250ml;

D. 5× protein electrophoresis buffer: Tris 15.1g, glycine 94.0g, SDS 5.0g, add ddH ₂ O to dissolve, and adjust the volume to 1L;

E. 10% (g/ml) ammonium persulfate (Aps, freshly prepared): 1.0g Aps, add ddH ₂ O to 10ml;

F. 10% (g/ml) SDS: 10.0g SDS, add ddH ₂ O to dissolve, and adjust the volume to 100ml;

G. 5× protein loading buffer: 1M Tris-HCl (pH 6.8) 2.5ml, 50% (g/ml) SDS 2ml, glycerol 5mL, 0.1% (g/ml) bromophenol blue 50.0mg, ddH ₂ O 2.5mL, mix well, filter with a 0.45μm filter head, and divide into 1ml each tube. When using, add 50μl β-mercaptoethanol to each 1ml;

H. Coomassie Brilliant Blue staining solution: 1.0g Coomassie Brilliant Blue R250, 250ml isopropyl alcohol, 100ml glacial acetic acid, 650ml ddH ₂ O. After dissolving, filter with filter paper.

I. Decolorizing solution: 100ml of glacial acetic acid, 50ml of ethanol, 850ml of ddH ₂ O;

J. Separating gel (15%) (20ml): ddH ₂ O 6.6ml, 30% (g/ml) Acr-Bis 8ml, 1.5M Tris-HCl (pH 8.8) 5ml, 10% (g/ml) Aps 200μl (freshly prepared), 10% (g/ml) SDS 200μl, TEMED (N,N,N',N'-tetramethylethylenediamine) 8μl;

K. Stacking gel (5%) (10ml): ddH ₂ O 6.8ml, 30% (g/ml) Acr-Bis 1.7ml, 1.0M Tris-HCl (pH 6.8) 1.3ml, 10% (g/ml) Aps 100μl (freshly prepared), TEMED (N,N,N',N'-tetramethylethylenediamine) 10μl.

2) Preparation of rubber sheets

After installing the rubber plate model, add ultrapure water to check for leaks; then prepare the separation gel according to the formula table 1, use a pipette gun to add the separation gel mixture between the installed rubber plates, accounting for about 3/4 of the gap volume, and then Add distilled water to fill the remaining space, wait for the separation gel to solidify (about 30 minutes), pour out the upper layer of distilled water, add the configured stacking gel, insert the spotting comb, wait for the stacking gel to solidify, and then pull out the spotting comb;

Table 1

3) Sample processing

Add the purified polygonatum agglutinin sample to 5× loading buffer, and then heat it in a metal bath at 95°C-100°C for 10 minutes to denature the protein. The loading amount is about 50ng-100ng.

4)Electrophoresis

Place the prepared electrophoresis gel in the electrophoresis tank, and add the sample and Protein Marker into the sampling well. Dot 20ul of each sample; turn on the power and perform electrophoresis. First run the stacking gel at 80V (about 35min), and then change the voltage to 120V after it becomes a thinner strip. When the sample runs through the entire gel, turn off the power (about 1h20min), the total time is about 1.5h-2h; Peel and cut the stacking gel in preparation for staining.

5) Coomassie brilliant blue method

Place the peeled electrophoresis gel in Coomassie Brilliant Blue staining solution and place it on a shaker for 60 minutes to make it fully stained. Afterwards, pour away the dyeing solution, replace with the destaining solution and gently shake for 30 minutes to destain (the destaining time can be appropriately extended). Repeat this step 2 to 3 times until destaining is complete. Scan the destained electrophoresis gel into a picture and save it.

1.6 The purity is also tested using Pharmacia's fast protein chromatography (FPLC). Analysis conditions: TSK G3000SW column; mobile phase is 0.1mol/L PB containing 1% (g/ml) NaCl, pH=8; flow rate: 1ml/min, detection wavelength 280nm, detector 0.08AUFS. It was detected as a single protein peak by FPLC, and the purity reached 95%.

1.7 Take red blood cells from healthy rabbits and make a 2% (v/v) red blood cell suspension with physiological saline. The agglutination activity of PCL was carried out on a "V" type 96-well micro serum dilution plate. After two-fold serial dilution of 25 μl PCL (1 mg/ml) and an equal volume of 0.14 mol/L NaCl, add 25 μl rabbit red blood cells and mix on a micro shaker. Shake for 10 minutes, place at 25°C for 2 hours, and check the agglutination activity under a microscope. The results show that PCL can agglutinate rabbit red blood cells, and the minimum agglutination concentration is 0.25 μg/ml.

Example 2: Virus and cell culture

1. Minimum necessary culture of African green monkey kidney VeroE6 cells (ATCC CRL-1586) supplemented with 10% fetal calf serum (PAN Biotech), 100IU/mL penicillin G and 100μg/mL streptomycin (Carl Roth, Karlsruhe, Germany) medium (MEM; PAN Biotech, Aidenbach, Germany), cultured at 37°C, 5% _CO2 .

2. SARS-CoV-2 isolates, BetaCoV/Germany/BavPat1/2020(WT); B.1.351, hCoV-19/Netherlands/NoordHolland_20159/2021(beta) and B.1.617.2, SARS-CoV-2, Human, 2021, Germany ex India, 20A/452R (delta), propagated and cultured in VeroE6 cells for subsequent plaque determination.

Example 3: Plaque Reduction Assay

1. Prepare serial 10-fold dilutions of compound PCL in the cell culture medium described in Example 1 to obtain PCL preparation solutions with concentrations of 500 μg/ml, 50 μg/ml, 5 μg/ml, and 0.5 μg/ml respectively. Then incubate with 100 SARS-CoV-2 plaque forming units (PFU) for 1 hour at 37°C.

2. Incubate the incubated PCL-virus mixture with VeroE6 cells for 45 minutes at 37°C and 5% _CO2 . Cells were washed once with PBS and covered with sample-free overlay medium containing 1.5% (g/ml) carboxymethylcellulose for 72 hours.

3. Fix the cells with 2.5% (g/ml) formaldehyde for 24 hours, and stain the plaques with crystal violet. Inhibition rates were calculated by comparison with untreated controls.

4. Use GraphPad Prism 8.0.0 to calculate the half maximum inhibitory concentration (IC50) value. The results are shown in Figure 1a. The half maximum inhibitory concentration (IC50) of the SARS-CoV-2 virus is 36.0±3.4μg/mL (0.7μM) .

5. The determination of plaque reduction by PCL on virus mutant strains Beta and Delta is the same as above. The results are shown in Figure 1b. It can be seen that the half maximum inhibitory concentrations (IC50) of PCL against Beta and Delta strains are 51.4±3.4μg/mL (1.0μM) and 30.2±4.5μg/mL (0.6μM) respectively.

Comparative Example 1:

Allium sativum lectin (ASL) (purchased ASL from Eylabs, item number L-8007-1) and Allium chinense lectin (ACL) (prepared samples stored in this laboratory) were used respectively, and their specific separation For methods, see: [3] Smeets K, Van Damme EJ, Van Leuven F, Peumans WJ. Isolation and characterization of lectins and lectin-alliinase complexes from bulbs of garlic (Allium sativum) and ramsons (Allium ursinum). Glycoconj J.1997Apr; 14(3):331-43.doi:10.1023/a:1018570628180.PMID:9147057.), Lycoris radiata lectin (LRL) (prepared samples preserved in this laboratory, the specific separation method is shown in: [2] Rong Yanzhen. Study on the Purification and Properties of Lycoris Lycoris Lectin [D]. Sichuan University, 2005), Zephyranthes candida lectin (ZCL) (are the prepared samples preserved in this laboratory, and their specific For the separation method, please see: [1] Lu Hui, Wu Chuanfang, Long Hong, Wang Xinyan, Wang Ke, Gong Meng, Bao Jinku. Study on the isolation, purification and biological activity of Allium cepa Lectin [C]//. Chinese Society of Biochemistry and Molecular Biology The Eighth Member Congress and National Academic Conference Paper Abstracts. 2001: 332-333) replaced Polygonatum agglutinin and performed the same experiment as in Example 3. The test results are shown in Figure 6. The measured IC50 of ASL against SARS-CoV-2 is 73.7±12.8μg/mL (1.4μM), the IC50 of ZCL against SARS-CoV-2 is 44.6±3.9μg/mL (0.8μM), and the IC50 of PCL against SARS-CoV-2 The IC50 of 2 is 36.0±3.4μg/mL (0.7μM). ACL and LRL have almost no inhibitory effect on SARS-CoV-2. ZCL and PCL have similar inhibition rates, but the solubility of PCL in water is much higher than that of ZCL.

Example 4: Determination of binding of PCL to virus particles

1. Label purified virus particles with DiO (3,3′-dioctadecyloxacarbocyanine perchlorate, ThermoFisher Scientific, USA). Incubate 100 µL of virus solution with 5 µL of 20 µM DiO (dissolved in ethanol) for 45 minutes.

2. Remove free dye by spin column (Protein A HP SpinTrap ^™ , GE Healthcare, Germany).

3. Incubate 10 μL of labeled virions with 90 μL of PCL solution (1000 μg/mL) at 37°C for 45 minutes.

4. Incubate the mixture with Vero E6 cells on ice for 1 hour, then wash with PBS to remove unbound virus.

5. The cell nuclei were labeled with Hoechst 33342 (ThermoFisher Scientific, USA), and then the cells were observed with a confocal laser scanning microscope (SP8, Leica, Germany). The results are shown in Figure 2a. And the VeroE6 cells that bound to the virus particles were counted, and the results are shown in Figure 2b. It can be seen that PCL can effectively block the binding of SARS-CoV-2 virions to VeroE6 cells. Through image and counting analysis, PCL inhibited >95% of the binding of virions to cells.

Example 5: Determination of the binding of PCL to the RBD of Delta mutant strain S protein

1. Seed VeroE6 cells on glass coverslips.

2. Dilute His-tag RBD with 2.5% (g/ml) BSA and incubate with PCL (1000μg/mL).

3. Label cells with Hoechst 33342, fix with 2.5% (g/ml) formaldehyde for 10 minutes, block with 2.5% (g/ml) BSA for 30 minutes, and then incubate with the sample for 1 hour.

4. Wash the cells twice with PBS and fix with 2.5% (g/ml) formaldehyde for 10 minutes. The cells were then incubated with 10 μg/mL 6-His antibody (FITC conjugated, ThermoFisher) for 1 hour.

5. Wash the cells again with PBS, and take Z-stack images on a confocal laser scanning microscope (SP8, Leica, Germany). The results are shown in Figure 2c. It can be seen that in the absence of PCL, Delta RBD strongly binds to the surface of VeroE6 cells. Adding PCL for pre-incubation can reduce the binding of Delta RBD to VeroE6 cells. The results are similar to those observed for the binding of PCL and WT SARS-CoV-2 virions, see Figure 2a. That is, PCL can block the binding of the virus to host cells and act as an entry inhibitor for SARS-CoV-2.

Example 6: Virus neutralization test

1. Cell preparation: One day before the experiment, take the Vero cells in good condition, digest them with trypsin, count them, dilute the cells with complete culture medium, prepare a cell suspension with a concentration of 1.5×10 ⁵ cells/mL, and add it to the cell suspension. Add 100 μL of cell suspension to each well of the 96-well cell culture plate and place it in a CO ₂ incubator to culture overnight. Before the infection experiment, observe the cell status. If the cell status is in good condition, wash it 2-3 times with PBS and add maintenance to each well. 100 μL of solution.

2. Sample processing: Plasma or serum is diluted 10 times with PBS, and the antibody is diluted with PBS to 0.1 μg/μL as the initial concentration.

3. Sample dilution: Dilute the sample at a two-fold ratio in a 96-well U-shaped plate. Set 10 replicates for each concentration to ensure that each well to be tested has a volume of 40 μL of sample to be tested after dilution.

4. Incubate the virus with the sample: Take the virus with a known TCID50 titer, dilute the virus with maintenance solution to a concentration of 100TCID50/35μL, then add 35μL of virus diluent to each sample well, place it in a safety cabinet and incubate at room temperature for 2 hours. , during this period, prepare the Vero cell plate and wash it twice with PBS. After the incubation is completed, take 35 μL/well of the incubation mixture of virus and sample, add it to the cells according to the corresponding holes, and place it in a CO ₂ incubator Incubate at 37°C for 2 hours.

5. Add 120 μL of virus maintenance solution to each cell culture well, place it in a cell culture incubator at 37°C for 3-5 days, and observe the cell CPE. The results are shown in Figure 3a. Addition of PCL completely eliminated CPE, and the cells performed comparable to uninfected controls. Interestingly, adding additional mannose to the PCL solution reduced the protective effect, leading to SARS-CoV-2-induced CPE in the cells. Based on the proportion of CPE appearing in 10 repetitions of each concentration, the virus inhibition rate was calculated, and finally the inhibition rate of 3 repetitions was calculated. The results are shown in Figure 3b. Its half-maximum inhibitory concentration (IC50) of SARS-CoV-2 virus is 1.443 μg/mL.

Example 7: Post-infection inhibition test

1. VeroE6 cells were seeded in 24-well plates for infection. After being infected with SARS-CoV-2 at MOI=0.01 for 1 hour, the cells were washed once with PBS to remove unbound virus particles.

2. Add PCL to the cell culture medium at different concentrations, then mix with the infected cells and culture for 24 hours.

3. Fix the cells with 2.5% (g/ml) formaldehyde, permeate the cells with 0.2% (g/ml) Triton X-100, and stain with a fluorescence microscope-specific antibody (primary antibody: Rabbit Anti-SARS-CoV-2 spike protein, Rockland; secondary antibody: Alexa 488-labeled goat anti-rabbit IgG, ThermoFisher), and DAPI was used to stain cell nuclei. The results are shown in Figure 4a.

4. The supernatant was titrated on VeroE6 cells using the plaque method according to the method of Example 2: plaque reduction assay. The results are shown in Figure 4b. Combined with the fluorescence image in Figure 4a, it can be seen that PCL above 125 μg/mL has a significant inhibitory effect on infection. The few infected cells were most likely cells infected during the first cycle before PCL was applied. It is suggested that PCL effectively inhibits the spread of SARS-CoV-2 between cells by inhibiting the entry and infection of progeny virus particles. When the PCL concentration was 1 mg/mL, the virus titer was reduced by 4 orders of magnitude, as shown in Figure 4b.

Example 8: BLI affinity determination

1. Prepare recombinant trimer spike protein S_Trimer and Delta mutant strain recombinant trimer spike protein S_Trimer_Delta (Sino Biological, China). Prokaryotic expression of spike protein RBD E.coli_RBD (Sangon Biotech, China), eukaryotic expression of spike protein RBD HEK293_RBD and Delta mutant spike protein RBD (OriGene Technologies, Inc., USA)

2. Using ForteBio Octet RED96e and K2 system (Pall ForteBio, USA), prewet the Ni-NTA biosensor (Pall ForteBio, USA) in dialysis buffer for 15 minutes before use.

3. Sugar-free trimeric spike proteins (S_Trimer' and S_Trimer_Delta') were composed of trimeric spike proteins (S_Trimer and S_Trimer_Delta) and PNGase F in PBS 7.4 buffer at a concentration of 20 μg/μL (Yeasen Biotechnology, China ) formed by incubation at 37°C. Use PBS buffer containing 0.02% (μl/ml) Tween20 to prepare 1μM and 10μM PCL solutions. Prepare gradient dilutions by serially diluting 10 μM PCL.

4. Fix the protein to be tested in step 1 on the surface of the biosensor tip (200 μl), and fix the signal value of each group at 3.0 nm (baseline equilibrium 100s; dissociation 300s). The following procedure was used to determine the binding of the protein to be tested to PCL: use PBS 7.4 containing 0.02% Tween20 to balance for 100s, use 1μM PCL containing 0.02% (μl/ml) Tween20 to bind for 180s, use PBS containing 0.02% (μl/ml) Tween20 Dissociate with PBS 7.4 for 300 s.

5. For the affinity determination of gradient dilution PCL and S_Trimer, S_Trimer_Delta, HEK293_RBD, use the following procedure: Use PBS 7.4 containing 0.02% (μl/ml) Tween20 to equilibrate for 180s, use PCL gradient dilution containing 0.02% (μl/ml) Tween20 Bind for 180 s and dissociate for 800 s using PBS 7.4 containing 0.02% (μl/ml) Tween20.

6. The entire experimental process was carried out at 30°C in a black 96-well microplate (Greiner Bio-One, Austria) with shaking at a rotation speed of 500 rpm, and the working volume of each well was 200 μL. The sensor immobilized with the corresponding protein to be tested was immersed in 200 μl PBS buffer as a control for correcting baseline drift. Sensing data were normalized and analyzed using Octet data analysis software (version 11.1.2.9). The results are shown in Figure 5. It can be seen that PCL can bind to the trimeric spike protein, but cannot bind to the sugar-free trimeric spike protein treated with PNGase F (WT SARS-CoV-2 and Delta mutant strains). PCL can bind to the RBD expressed by the eukaryotic expression of HEK239, but has almost no binding to the RBD expressed in E. coli (as a prokaryotic organism, E. coli cannot modify proteins with glycan chains). These results indicate that glycans on the spike protein are required for PCL binding. Further affinity analysis showed that PCL bound to S_Trimer in a dose-dependent manner with an affinity of 94.3nM. PCL binds to S_Trimer_Delta in a dose-dependent manner with an affinity of 172nM. PCL binds to HEK293_RBD in a dose-dependent manner with an affinity of 1.32 μM.

The specific embodiments described above further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Within the spirit and principles of the present invention, any modifications, equivalent substitutions, improvements, etc. shall be included in the protection scope of the present invention.

Claims

The use of Polygonatum agglutinin (PCL) in blocking and preventing the infection of host cells by the new coronavirus SARS-CoV-2 and its mutant strains such as Delta, Omicron, Gamma, Beta and Alpha mutant strains in vitro.
The use of Polygonatum lectin (PCL) in the preparation of drugs for preventing or treating infection by the new coronavirus SARS-CoV-2 and its mutant strains, such as Delta, Omicron, Gamma, Beta and Alpha mutant strains.
A pharmaceutical composition for preventing or treating infection by the new coronavirus SARS-CoV-2 and its mutant strains, such as Delta, Omicron, Gamma, Beta and Alpha mutant strains, which contains Polygonatum agglutinin (PCL) and a pharmaceutically acceptable carrier.
A pharmaceutical kit comprising the pharmaceutical composition of claim 3 and instructions for use and optionally administration means.
A method for preventing or treating infection by new coronavirus SARS-CoV-2 and its mutant strains such as Delta, Omicron, Gamma, Beta and Alpha mutant strains, wherein the method includes administering to a subject the pharmaceutical composition of claim 3 or A pill box according to claim 4 is provided.
The method of claim 5, wherein the method further comprises administering to the subject another drug for treating infection by the new coronavirus SARS-CoV-2 and its mutant strains, such as Delta, Omicron, Gamma, Beta and Alpha mutant strains.