CN111053892B

CN111053892B - Broad-spectrum enterovirus-resistant protein medicine and application thereof

Info

Publication number: CN111053892B
Application number: CN201911343541.3A
Authority: CN
Inventors: 杨霞; 姜世勃; 陆路; 刘泽众
Original assignee: Shanxi Jinbo Bio Pharmaceutical Co ltd; Fudan University
Current assignee: Shanxi Jinbo Bio Pharmaceutical Co ltd; Fudan University
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2023-11-17
Anticipated expiration: 2039-12-24
Also published as: CN111053892A

Abstract

The invention relates to a broad-spectrum anti-enterovirus protein medicine and application thereof, and discloses a method for inhibiting enterovirus growth in vitro and preventing or controlling enterovirus infected cells by using an anhydride modified protein or a composition containing the anhydride modified protein. The invention also discloses a biological agent for preventing and treating various enterovirus infections. The biological agent is protein treated by acid anhydride. Specific anhydridized proteins are anhydridized human serum albumin and bovine beta lactoglobulin. The protein can effectively inhibit recently large-scale outbreaks of enterovirus 68 type, enterovirus 71 type and coxsackievirus 16 type infected cells. The inhibitor has the advantages of broad-spectrum inhibition of enterovirus invasion to cells, prevention of virus diffusion, stability, low cost and the like.

Description

Broad-spectrum enterovirus-resistant protein medicine and application thereof

Technical Field

The invention relates to the field of biological medicine, in particular to a broad-spectrum enterovirus-resistant protein inhibitor and application thereof.

Background

Enteroviruses (EV) belong to the Picornaviridae family (Picornaviridae), enteroviruses. Consists of more than 100 subtypes of virus. The virus is free of capsids and can be divided into 4 subtypes (A, B, C, D). Enteroviruses are distributed worldwide and can cause a wide range of diseases in humans. Diseases caused include mild cold, respiratory disease, severe hand-foot-and-mouth disease, myocarditis, pancreatitis, meningoepithymitis, poliomyelitis, and the like. In 2014, enterovirus type 68 (EV-D68) has exploded on a large scale in the United states and has been shown to spread globally. In recent years, outbreaks of large-scale hand-foot-and-mouth disease have been caused annually in both enterovirus type 71 in the asia-pacific region (EV-a 71) and Coxsackievirus type 16 (cv-a 16).

EV-D68 has historically rarely caused large-scale infection in humans. EV-D68 caused 1153 cases of severe respiratory distress and 107 cases of acute myelitis in the United states until 2014, and resulted in 19 deaths. Then, canada diagnosed approximately 700 cases of EV-D68 infection. EP reports 408 cases of EV-D68 infection. A total of 25 cases of EV-D68 infection were reported in China and Thailand. People begin to pay attention to the hazard of EV-D68. EV-D68, while belonging to enteroviruses, is similar to rhinoviruses and is transmitted mainly through the respiratory tract. Infection with EV-D68 causes severe lower respiratory symptoms as well as neurological complications including muscle weakness, acute flaccid paralysis, and cranial neuropathy.

The hand-foot-mouth disease can be caused by more than 20 enteroviruses, and is a common childhood infectious disease in China. The infant mainly shows rash on hands, feet, buttocks and the like, and herpes on two sides of the oral cavity. Severe cases can lead to neurological symptoms leading to aseptic meningitis, brainstem encephalitis, and acute flaccid paralysis. But also can cause respiratory symptoms to cause neurogenic edema, hemorrhage and the like. In recent years, about 200 ten thousand people in China are infected with hand-foot-and-mouth disease each year, and the number of people suffering from the disease and the number of people dying each year are in the first place in the class-C infectious diseases in China for a plurality of years. EV-A71 and CV-A16 are two main pathogens causing hand-foot-and-mouth disease in China. In severe cases of hand-foot-and-mouth disease and in cases of death, EV-A71 infection occupies the vast majority.

So far, only two inactivated vaccines of EV-A71 are approved in China, but the vaccines cannot cross-protect other intestinal infections such as CVA16, EV-D68 and the like. There is no approved medicine for treating enterovirus infection at home and abroad. In view of the severe situation of enterovirus infection in China, development of medicines capable of effectively treating enterovirus infection is urgently needed.

Disclosure of Invention

The anhydridized protein is one of the inventors, and internationally known AIDS specialist Jiang Shibo teaches that the research team can effectively prevent HIV virus from entering target cells by modifying beta-lactoglobulin (beta-LG) with 3-hydroxy-phthalic anhydride (3-hydroxyphthalic anhydride,3 HP) for the first time. The scientific research team then found that the anhydrated proteins have inhibitory effects on human papilloma virus, herpes simplex virus, respiratory syncytial virus, and ebola virus; but has no significant inhibitory effect on middle east respiratory syndrome virus and Zika virus. The anhydrified protein is described as a broad spectrum viral inhibitor, but not all viruses. Whether the protein modified by the anhydride can effectively inhibit the infection of enteroviruses or not has very important roles in developing the drug potential of the anhydride protein, exploring the mechanism of the anhydride protein and researching protein antiviral drugs. Furthermore, the effects of different protein classes are to be investigated.

The inventor discovers that serum albumin (HSA) and beta lactoglobulin modified by acid anhydride can effectively inhibit the growth of enteroviruses and prevent the enteroviruses from infecting cells. The inventors have also found that anhydride modified serum albumin (HSA) has a different effect on different viruses than beta lactoglobulin.

Enterovirus is a plus-sense single-stranded RNA virus. The virus encodes a large protein first, then is cleaved into the four structural proteins VP1-VP4 by the virus and host protease to form the viral capsid, and the seven nonstructural proteins 2A-2C and 3A-3D are involved in viral replication. Enterovirus invasion into target cells can be divided into multiple steps, mainly including recognition and binding of receptors, endocytosis of viruses, decolonization of viruses and release of viral RNAs. If the virus can be blocked from entering the target cells, the virus can be blocked outside the cells, so that the aim of inhibiting the primary infection and the secondary infection of the virus is fulfilled. Based on the thought, the protein modified by the acid anhydride can be effectively combined to the relevant position on the surface of the viral capsid through physical and other actions, so that the protein can be prevented from combining with a receptor or changing the conformation, and finally, the infection of viruses is blocked, and the antiviral action is exerted. Aiming at the lack of the existing enterovirus medicines, a candidate broad-spectrum protein medicine for effectively preventing and controlling enterovirus infection is provided.

In one aspect, the invention provides a method of inhibiting enterovirus growth, preventing or controlling enterovirus-infected cells in vitro with an anhydride modified protein or a composition comprising an anhydride modified protein, the method comprising the step of contacting the anhydride modified protein or the composition comprising an anhydride modified protein with an article or sample containing, suspected of containing or at risk of containing an enterovirus.

In one embodiment, the protein is selected from albumin and beta lactoglobulin. In one embodiment, the anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride or maleic anhydride.

In one embodiment, the protein is fully anhydrified as determined by the lysine and arginine modification ratio of the protein.

In one embodiment, the sample is selected from the group consisting of stool, saliva, blood, and serum. In another embodiment, the article is selected from the group consisting of sanitary napkins, pantiliners, wet wipes, and tissues; the paper towel comprises handkerchiefs, removable toilet paper, napkin or hand towel. The article or sample may further comprise, be suspected of comprising, or be at risk of comprising a virus selected from the group consisting of: human papilloma virus, herpes simplex virus, respiratory syncytial virus, and ebola virus.

In one embodiment, the enterovirus is selected from enterovirus type 68, enterovirus type 71 and said coxsackievirus, preferably coxsackievirus type 16.

In one embodiment, the albumin is selected from serum albumin or egg albumin, preferably human serum albumin, bovine serum albumin or chicken egg albumin. In one embodiment, the beta lactoglobulin is bovine beta lactoglobulin.

In one embodiment, the composition is in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol.

In one embodiment, the anhydride modified protein is prepared by mixing the anhydride with the protein.

In another aspect, the invention provides the use of an anhydride modified protein or a composition comprising an anhydride modified protein in the manufacture of a medicament for treating or preventing an enterovirus infection or a disease caused by an enterovirus infection in a subject.

In one embodiment, the protein is selected from albumin and beta lactoglobulin and the anhydride is selected from 3-hydroxyphthalic anhydride, succinic anhydride or maleic anhydride.

In one embodiment, the subject also has or is at risk of having a viral infection selected from the group consisting of: human papilloma virus, herpes simplex virus, respiratory syncytial virus, and ebola virus.

In one embodiment, the medicament is in the form of a tablet, capsule, powder, nasal drops or aerosol, or in the form of a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol.

In one embodiment, the disease is hand-foot-and-mouth disease.

In one embodiment, the subject is a mammal, such as a human.

In yet another aspect, the present invention provides a composition for preventing or controlling an enterovirus infection comprising an anhydride modified protein and another agent for preventing or controlling an enterovirus infection,

In one embodiment, the protein is fully anhydrified as determined by the lysine and arginine modification ratio of the protein;

in one embodiment, the enterovirus is selected from enterovirus type 68, enterovirus type 71 and said coxsackievirus, preferably coxsackievirus type 16;

The invention also provides methods of treating enterovirus infection or a disease caused by enterovirus infection using the anhydride modified proteins.

The invention can effectively resist the infection of various enteroviruses, so that the enteroviruses are blocked in the invasion stage. And has inhibitory activity in vivo. Meanwhile, the preparation can be made into various dosage forms, is simple to operate, and can effectively become a candidate medicine for resisting enteroviruses.

Drawings

FIG. 1 shows lysine modification rates of HSA modified with 3HP at different concentrations;

FIG. 2 shows arginine modification rates of HSA modified with 3HP at different concentrations;

FIG. 3 is a SDS-PAGE running of HSA modified with 3HP at different concentrations;

FIG. 4 shows the inhibitory activity of 3HP-HSA and 3 HP-beta-LG on EV-D68 by CCK-8;

FIG. 5 shows the detection of EV-D68 inhibitory activity of 3HP-HSA using a plaque reduction assay;

FIG. 6 shows the activity of 3HP-HSA in inhibiting EV-A71 using a plaque reduction assay;

FIG. 7 is a graph showing the activity of 3HP-HSA in inhibiting CV-A16 using a plaque reduction assay;

FIG. 8 shows the detection of EV-D68 inhibitory activity of 3HP-HSA by q-PCR;

FIG. 9 shows the detection of EV-A71 inhibitory activity of 3HP-HSA by q-PCR;

FIG. 10 shows the activity of 3HP-HSA in inhibiting CV-A16 by q-PCR;

FIG. 11 is a fluorescence detection chart of the inhibition of EV-A71-GFP by 3 HP-HSA;

FIG. 12 shows cytotoxicity assays of 3HP-HSA on RD cells;

FIG. 13 shows the activity of 3 HP-beta-LG in inhibiting EV-A71 by CCK-8 assay;

FIG. 14 shows the activity of 3 HP-beta-LG in inhibiting CV-A16 by CCK-8 assay;

FIG. 15 is a graph showing the effect of addition of 3HP-HSA on inhibition of EV-D68 activity at various time points of viral addition;

FIG. 16 is a Time-of-remove experimental test;

FIG. 17 shows detection of the inhibition of EV-D68 activity by 3HP-HSA in mice.

Detailed Description

The details of one or more embodiments of the invention are set forth herein. Other features, objects, and advantages of the invention will be apparent from the description and examples, and from the claims. The following description is provided to aid in the understanding of the invention.

As used herein, "anhydride modified protein" refers to anhydride-derivatized/modified albumin and beta lactoglobulin. The degree of anhydride modification of a protein can be determined by the ratio of arginine and/or lysine modifications to the protein. The preparation of anhydride modified proteins and the degree of anhydride modification are known to those skilled in the art. See, for example, chinese patent applications CN201210066696.9 and CN201810691093.5. In this context, the degree of anhydride modification of a protein, as measured by the arginine and/or lysine modification ratio of the protein, may independently be 100%,99%,95%,93%,92%,90%,85%,80%, etc. The ratio of the components can be appropriately adjusted by those skilled in the art according to the anhydride modification/derivatization ratio. Preferably, the anhydride modification of the protein may be 100%. In this context, anhydride modified proteins, preferably 3-hydroxy-phthalic anhydride modified human serum albumin or 3-hydroxy-phthalic anhydride modified bovine beta lactoglobulin, can be prepared as follows: (1) Preparing 0.1M disodium hydrogen phosphate, adjusting the pH value to 8.5, and filtering and sterilizing by a filter membrane with the thickness of 0.2 mu M; (2) Dissolving Human Serum Albumin (HSA) or bovine beta-lactoglobulin (beta-LG) powder in a prepared disodium hydrogen phosphate solution to a concentration of 20mg/ml; (3) Anhydride (3-hydroxy-phthalic anhydride (3 HP), maleic anhydride (ML), succinic anhydride (SU)) was dissolved in DMSO to a concentration of 1M; (4) Adding an anhydride with a final concentration of 12mM to the human serum albumin and bovine beta-lactoglobulin solution; immediately and uniformly mixing; (5) Adjusting the pH to 8.5-9 by using 5M sodium hydroxide solution; (6) standing at room temperature for 20min; (7) Repeating the steps 4-6 four times to make the final addition concentration of the anhydride be 60mM; (8) After the last time of adding the anhydride, standing for 2 hours at room temperature to fully react the anhydride; (9) Loading the modified anhydrated protein into a dialysis bag (7.5 kDa), and placing the dialysis bag in PBS for dialysis for 48 hours, and replacing PBS buffer solution for three times; (10) The dialyzed proteins were concentrated by ultrafiltration using a 10kDa ultrafiltration tube.

As used herein, "enteroviruses" belong to the genus picornaviridae, consisting of more than 100 subtypes of viruses. Pathogen enteroviruses include polioviruses, coxsackieviruses, and orphan viruses that cause cytopathic effects in the human gut (ECHO virus). The international committee for viral nomenclature in 1970 assigned these viruses to the enteroviruses genus of the picornaviridae family. Enteroviruses found after 67 types of the named 3 enteroviruses are named according to enterovirus ordinal numbers. 68, 69, 70, 71, 72 enteroviruses, etc.

Conventional pharmaceutical practices may be employed to provide suitable formulations or compositions for administration to subjects suffering from enterovirus infections. Any suitable route of administration may be employed, for example, parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, topical, or oral. The therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; for intranasal formulations, they may be in the form of powders, nasal drops or aerosols.

Methods for preparing formulations known in the art can be found, for example, "Remington's Pharmaceutical Sciences" (19 th edition), incorporated by reference, a.gennaro, 1995,Mack Publishing Company,Easton,Pa. Formulations for parenteral administration may, for example, contain excipients, sterile water or saline, poly (alkylene) glycols such as polyethylene glycol, vegetable oils or hydrogenated naphthalenes. Biocompatible, biodegradable glycolide polymers, glycolide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of compounds. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients such as lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or as a gel.

In this context, the anhydride modified protein may be prepared in the form of a composition, for example, a solution, tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol; such as solutions, liniments, sprays, toilet washes and dry powders.

The present invention provides compositions, preferably biological agents, for preventing or controlling enterovirus infections comprising anhydride modified proteins. Anhydride modified proteins are mixtures of anhydrides and proteins. Proteins used herein may include Human Serum Albumin (HSA), bovine beta-lactoglobulin beta-LG, bovine serum albumin, chicken egg albumin. Preferably, the proteins used herein are human serum albumin and bovine beta lactoglobulin. Biological agents can be prepared using 3-hydroxy-phthalic anhydride (3-hydroxyphthalic anhydride,3 HP), maleic anhydride (ML), and succinic anhydride (succinic anhydride, SU).

In this context, the anhydride solution is an anhydride powder dissolved in DMSO and may be present at a concentration of 0.5 to 10M, preferably 1M.

The protein solution may be prepared by dissolving the protein in a phosphate buffer. Phosphate buffers include, but are not limited to, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and the like. Disodium hydrogen phosphate is preferred. The phosphate buffer pH may be 7-10, preferably 8.5. The protein concentration may be 5mg/ml to 100mg/ml, preferably 20mg/ml. The pH of the anhydride-treated protein should be greater than 7, preferably 8.5, so that anhydride modification is sufficient.

The composition dosage form may be selected from: solutions, liniments, sprays, lotions and dry powders.

Herein, enteroviruses may include, but are not limited to enterovirus type 68, enterovirus type 71, coxsackievirus type 16.

Provided herein are methods of inhibiting infection by enteroviruses in vitro using an anhydrified protein, wherein the selected protein comprises: albumin, preferably human serum albumin and lactoglobulin, preferably bovine beta lactoglobulin.

Enteroviruses can be isolated from feces, saliva, blood, serum, surfaces of objects, and the like.

In this context, the acid anhydride may include 3-hydroxy-phthalic anhydride, maleic anhydride and succinic anhydride. The anhydride solution may be prepared by dissolving the anhydride powder in DMSO, and may be at a concentration of 0.5 to 10M, preferably 1M. Phosphate buffers may include, but are not limited to, disodium hydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, and the like. Disodium hydrogen phosphate is preferred. The phosphate buffer pH may be 7-10, preferably 8.5.

In the present invention, the anhydridized protein used is completely anhydridized by measurement of the modification rates of arginine and lysine.

Examples

Hereinafter, the present invention will be more specifically explained with reference to the following non-limiting examples.

Example 1: preparation of anhydride-modified proteins

Preparation of 3-hydroxy-phthalic anhydride modified human serum albumin (3-hydroxyphthalic anhydride-modified human serum album,3 HP-HSA) and 3-hydroxy-phthalic anhydride modified bovine beta lactoglobulin (3-hydroxyphthalic anhydride-modified bovine beta-lactoglobulin,3 HP-beta-LG):

(1) 0.1M disodium hydrogen phosphate was prepared, the pH was adjusted to 8.5, and the solution was filtered through a 0.2 μm filter to sterilize.

(2) Human Serum Albumin (HSA) and bovine beta-lactoglobulin (beta-LG) powders were dissolved in a prepared disodium hydrogen phosphate solution to a concentration of 20mg/ml.

(3) Anhydride (3-hydroxy-phthalic anhydride (3 HP), maleic anhydride (ML), succinic anhydride (SU)) was dissolved in DMSO to a concentration of 1M.

(4) An anhydride with a final concentration of 12mM was added to the human serum albumin and bovine beta lactoglobulin solution. Immediately mixing.

(5) The pH was adjusted to between 8.5 and 9 using 5M sodium hydroxide solution.

(6) Standing at room temperature for 20min.

(7) The steps 4-6 are repeated four times. The final addition concentration of anhydride was brought to 60mM.

(8) After the last addition of anhydride, the mixture was allowed to stand at room temperature for 2 hours to allow the anhydride to react sufficiently.

(9) The modified anhydrated protein was packed into dialysis bags (7.5 kDa) and dialyzed in PBS for 48h. During which the PBS buffer was replaced three times.

(10) The dialyzed proteins were concentrated by ultrafiltration using a 10kDa ultrafiltration tube.

(11) Proteins were quantified using the BCA kit from Takara. Specific operations are described with reference to the specification.

Example 2: preparation of proteins with different anhydride modification rates

(1) 20mg of human serum albumin was dissolved in 1ml of a 0.1M disodium hydrogen phosphate solution.

(2) 3-hydroxy-phthalic anhydride was dissolved in DMSO to a 1M mother liquor.

(3) To the human serum albumin solution was added 0mM, 3mM, 6mM, 12mM, 24mM, 48mM and 60mM of 3-hydroxy-phthalic anhydride, respectively.

(4) The pH was adjusted to 8.5-9.0 with 5M sodium hydroxide.

(5) The reaction was carried out at room temperature for 2 hours.

(6) The modified protein was quantified using the BCA kit from Takara. Specific operations are described with reference to the specification.

Example 3: detection of arginine and lysine modification rates of 3-hydroxy-phthalic anhydride modified human serum albumin with different concentrations

1. Lysine modification ratio:

(1) 3HP-HSA and HSA of different modified concentrations were diluted to 1mg/ml with phosphate buffer.

(2) 25 μl of each was added to the 96-well plate, and three replicates were set for each concentration.

(3) An equal volume of phosphate buffer was added as a blank.

(4) 0.1M sodium tetraborate solution, 25. Mu.L/well was added thereto, and the mixture was allowed to stand at room temperature for 5 minutes.

(5) 2,4, 6-trinitrobenzenesulfonic acid (2, 4,6-trinitrobenzene sulfonic acid, TNBS) was added, 10. Mu.L/well and the mixture was repeatedly sucked and mixed, and left to stand at room temperature for 5 minutes.

(6) The reaction was stopped by adding buffer (0.1M disodium hydrogen phosphate, 1.5mM sodium sulfite) at pH 8.5, 100. Mu.L/well.

(7) The absorbance of each well at 420nm was measured with a microplate reader.

(8) Lysine modification rate was calculated using the following formula. Lysine modification ratio (%) =100x [1- (OD 420nm experimental group-OD 420nm protein-free background control group mean)/(OD 420nm control group mean-OD 420nm protein-free background control group mean).

(9) The results are shown in Table 1 and FIG. 1

2. Arginine modification rate

(3) An equal volume of phosphate buffer was added as a blank.

(4) 0.5mM p-HPG, 60. Mu.L/well, was added and the pH was adjusted to 9.0. Incubate for 2h in the dark.

(5) Absorbance at 340nm was measured with a microplate reader.

(6) Arginine modification rate was calculated using the following formula. Arginine modification ratio (%) =100x [1- (OD 340nm experimental group-OD 340nm protein-free background control group mean)/(OD 340nm control group mean-OD 340nm protein-free background control group mean) ].

(7) The results are shown in Table 1 and FIG. 2.

3. Since the molecular weight of the protein gradually increases with the degree of modification of the acid anhydride. Thus, different concentrations of 3-hydroxy-phthalic anhydride modified human serum albumin were run by SDS-PAGE. The results are shown in FIG. 3. The molecular weight of the protein gradually increases as the concentration of added anhydride increases.

Table 1: arginine and lysine modification ratio and corresponding inhibition IC50 for EV-D68

Example 4: amplification and quantification of EV-D68, EV-A71 and CV-A16:

(1) RD cells were cultured and passaged with DMEM containing 10% fbs.

(2) When the cell density reached 80%, the culture supernatant was discarded, and DMEM containing 2% fbs was added. And simultaneously accessing each virus.

(3) After the cytopathy is completed in the next day, the cells are repeatedly frozen and thawed three times, and the supernatant is collected.

(4) The collected viral supernatants were concentrated by ultrafiltration using a 50kDa ultrafiltration tube.

(5) The concentrated virus was frozen in sub-packs at-80 ℃.

(6) The virus was quantified by the virus plaque method. The viral plaque method was performed with reference to the following plaque reduction experiments.

Example 5: detection of the Capacity of the anhydridized protein to inhibit EV-D68 infection of target cells

(1) Well-grown RD cells were passaged and plated in 96-well cell culture plates with 1000 cells per well.

(2) After 24h, when the cell density reached 80%, the anhydrified protein was diluted by a multiple of 10,5,2.5,1.25,0.625,0.3125,0.15625,0.078125,0.0390625. Mu.M to a concentration of 50. Mu.l per well, and then 50. Mu.l of EV-D68 at 0.5MOI was added, and after mixing, incubated at 37℃for 0.5h.

(3) The cell culture supernatant was discarded and the anhydridized protein was added to the RD cells along with the virus mixture. Cell controls without viral and protein addition and viral controls with viral and protein addition only were set simultaneously.

(4) After 24h, when the virus control lesions were apparent, the cell supernatant was discarded. CCK-8 solution was added.

(5) Incubate at 37℃for 2h.

(6) Determination of OD450 absorbance Using an enzyme-labeled Instrument

(7) The viral inhibition was calculated as follows: viral inhibition = (drug well-viral well) ×100%/(no drug well-viral well)

The above experiments were performed in triplicate. The results are shown in tables 2, 3 and 4, and 3HP-HSA and 3 HP-beta-LG were very effective in inhibiting EV-D68 infection. The median inhibitory concentration (IC 50) was 0.12 and 0.25. Mu.M. The effect of the two anhydridized proteins is different and is more efficient compared to 3 HP-HAS.

Table 2: EV-D68 Virus inhibition Rate of 3HP-HSA

Table 3: EV-D68 Virus inhibition Rate of 3 HP-beta-LG

Example 6: inhibition of EV-D68 by 3HP-HSA with different degrees of modification

(2) After 24h, when the cell density reached 80%, 3HP-HSA with different modification degrees was diluted by a multiple ratio to a concentration of 10,5,2.5,1.25,0.625,0.3125,0.15625,0.078125. Mu.M, 50. Mu.l per well, and then 50. Mu.l of EV-D68 with 0.5MOI was added, and after mixing, incubated at 37℃for 0.5h.

(5) Incubate at 37℃for 2h.

(6) Determination of OD450 absorbance Using an enzyme-labeled Instrument

(7) The viral inhibition was calculated as follows: viral inhibition = (drug well-viral well) x 100%/(no drug well-viral well)

The results are shown in Table 1, and demonstrate that the ability to inhibit EV-D68 gradually increases as the modification of 3-hydroxy-phthalic anhydride increases. Thus, the protein has antiviral effect, indeed due to anhydride modification. The more complete the anhydride modification, the better the antiviral effect.

Example 7 plaque reduction method 3HP-HSA was tested for inhibition of EV-D68, EV-A71 and CV-A16

(1) Well-grown RD cells were plated in 24-well plates.

(2) When RD cells grow into a monolayer, the whole wall bottom is paved. The 3HP-HSA protein was diluted. Then, the EV-D68, EV-A71 and CV-A16 viruses diluted in equal volumes were added.

(3) Incubate at 37℃for 0.5h.

(4) Cell supernatants were discarded and protein and virus mixtures were added to the cells, three replicates were set for each concentration. And a virus well control without adding only the virus and a cell control without adding the virus and the protein are arranged.

(5) Incubation was carried out at 37℃for 2h, with mixing every 0.5h, to allow uniform infection of the virus.

(6) After 2h, the cell supernatant was discarded and a solution containing 1% low melting agar prepared with DMEM was added.

(7) After the agar is solidified, the agar is placed in an incubator for 2-3d of inversion culture.

(8) When the plaques were apparent, a solution containing 4% formaldehyde and 1% crystal violet was added overnight for permanent staining.

(9) Agar was carefully washed with tap water, dried, and plaque was counted.

(10) The viral inhibition was calculated as follows: viral inhibition = 100% - (drug well-cell well) x 100%/(viral well-cell well).

Experiments were performed in triplicate and the results are shown in tables 4, 5, 6 and figures 5, 6 and 7. The 3HP-HSA and 3 HP-beta-LG have very good anti-EV-D68 activity as demonstrated by CCK-8 method. Wherein 3HP-HSA is slightly better than 3 HP-beta-LG. Thus, the activity of 3HP-HSA was further confirmed by a plaque reduction method; and further using a plaque reduction method to determine whether 3HP-HSA still has inhibitory activity against EV-A71 and CV-A16. The plaque reduction assay showed that 3HP-HSA did have the best activity for EV-D68. In addition, 3HP-HSA also showed good activity against EV-A71 and CV-A16. It is shown that 3HP-HSA is indeed a broad spectrum anti-enterovirus drug.

Table 4: EV-D68 Virus inhibition Rate of 3HP-HSA

Table 5: EV-A71 Virus inhibition ratio of 3HP-HSA

Table 6: CV-A16 Virus inhibition Rate of 3HP-HSA

Example 8: RT-qPCR method verification

The RT-qPCR method detects the activity of 3HP-HSA against EV-D68, EV-A71 and CV-A16.

(1) Well grown RD cells were plated in 96 well plates, 10000 cells per well.

(2) 3HP-HSA at different concentrations was mixed with each virus at 0.5MOI and incubated at 37℃for 0.5h.

(3) Added to RD cells.

(4) After 24 hours, the viral supernatant was taken and viral RNA was extracted using the viral RNA extraction kit from all gold company.

(5) The detection was performed using Takara RT-qPCR kit, incorporated by reference.

The results are shown in FIGS. 8,9 and 10, and the 3HP-HSA can effectively reduce the viral copy number of EV-D68, EV-A71 and CV-A16 by taking the viral RNA copy number in the viral wells without protein drugs as 100%.

Example 9: detection of inhibition of EV-A71-GFP virus by 3HP-HSA

(1) Well grown RD cells are plated in 96 well plates, 10000/well.

(2) Various concentrations of 3HP-HSA were diluted and then EV-A71-GFP (the viral genome harbored the GFP gene and expressing GFP green fluorescence upon replication) was added. Incubate at 37℃for 0.5h.

(3) The protein and virus mixture was added to RD cells.

(4) After 24h, cells were fixed 15min with 4% paraformaldehyde.

(5) Cells were lysed by adding 0.1% Triton for 10min.

(6) DAPI was added for nuclear staining.

(7) Photographs were taken under a fluorescence microscope.

The results are shown in FIG. 11: as the concentration of 3HP-HSA increased, GFP fluorescence was less and less, indicating that 3HP-HSA did inhibit EV-A71 infection.

Example 10: detection of 3HP-HSA cytotoxicity

(1) Well grown RD cells were plated in 96 well plates with 15000 cells per well.

(2) After 24h, 3HP-HSA was added at various concentrations. Three duplicate wells were set for each concentration.

(3) After 12h, cell exchange was performed with DMEM containing 2% fbs.

(4) After 12h, CCK-8 solution was added.

(5) Calculation of cell viability

The results are shown in Table 7 and FIG. 12, which show that 3HP-HSA does not have significant toxicity to cells at high concentrations of 200. Mu.M. It was demonstrated that 3HP-HSA is a very safe drug.

Table 7: effect of 3HP-HSA on cell viability

Example 11: CCK8 method for detecting activity of 3 HP-beta-LG in inhibiting EV-A71

(1) Well grown RD cells were plated in 96 well plates 10000 per well.

(2) Diluted 3 HP-beta-LG was mixed with EV-A71 virus at 0.5MOI and incubated at 37℃for 0.5h.

(3) The above mixture was added to the cells and incubated for 24h.

(3) And adding CCK-8 when the virus control hole lesion is obvious.

(4) And calculating the virus inhibition rate.

As shown in Table 8 and FIG. 13, 3HP- β -LG was also effective in inhibiting EV-A71 infection.

Table 8: EV-A71 Virus inhibition Rate of 3 HP-beta-LG

Example 12: CCK8 method for detecting activity of 3 HP-beta-LG for inhibiting CV-A16

(1) Well grown RD cells were plated in 96 well plates 10000 per well.

(2) Diluted 3 HP-beta-LG was mixed with CV-A16 virus at 0.5MOI and incubated at 37℃for 0.5h.

(3) The above mixture was added to the cells and incubated for 24h.

(3) When the lesions of the virus control hole are obvious, adding CCK-8

(4) And calculating the virus inhibition rate.

The results are shown in Table 9 and FIG. 14, 3HP- β -LG was effective in inhibiting CV-A16 infection.

TABLE 9

Example 13: time-of-addition experiment

To investigate at what stage 3HP-HSA exerts antiviral effect, we used different time points to add 3HP-HSA and then to examine whether antiviral effect was present.

(1) Well grown RD cells were plated in 96 well plates 10000 per well.

(2) 1.25. Mu.M 3HP-HSA was added 0.5h before and 0.5,1,1.5,2,4,8h after viral addition, respectively.

(3) And adding CCK-8 when the virus control hole lesion is obvious.

(4) And calculating the virus inhibition rate.

As a result, as shown in FIG. 15, 3HP-HSA was added at 4 hours after the addition of the virus, and the antiviral activity was significantly decreased, and the activity was poor at 8 hours. The 3HP-HSA was shown to exert antiviral effects at an early stage, the earlier the 3HP-HSA addition, the more effective the EV-D68 infection inhibition.

Example 14: time-of-remote experiment

(1) Well grown RD cells were plated in 96 well plates 10000 per well.

(2) 1.25. Mu.M 3HP-HSA was added to the cells, incubated for 0.5h,

(3) The 3 HP-HSA-added cell wells were then washed three times with DMEM to remove the 3HP-HSA completely.

(4) EV-D68 was added at 0.5 MOI.

(5) After 24h, CCK-8 was added.

(6) And calculating the inhibition rate.

As a result, as shown in FIG. 16, 3HP-HSA was washed away, and the antiviral activity was lost, indicating that it exerted an antiviral effect not on host cells but on viruses.

Example 15: detection of anti-EV-D68 Activity of 3HP-HSA in vivo.

(1) 10A 129 mice were randomly divided into two groups of 5 mice.

(2) The mice were subjected to nasal drip and toxin-attacking EV-D68 with a toxin-attacking dose of 10 ⁷ PFU。

(3) After 0.5h, 5 mice of the treatment group were given nasal drops to 100mg/kg of 3HP-HSA.

(4) On day 4 of challenge, mice were euthanized. Mouse lungs were taken. Trizol homogenate was added.

(5) RNA in the lung was extracted and RT-qPCR was used to detect viral RNA copy number.

The results are shown in FIG. 17, in which the number of viruses in the mice was significantly reduced by 3HP-HSA with the number of viral copies of the virus control group being 100%.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited by the foregoing description, but is set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to the present description may be made without departing from the spirit or scope of the invention as defined in the appended claims.

Claims

1. A method of inhibiting enterovirus growth, preventing or controlling enterovirus-infected cells in vitro with an anhydride modified protein or a composition comprising an anhydride modified protein, the method comprising the step of contacting the anhydride modified protein or the composition comprising an anhydride modified protein with an article or sample containing, suspected of containing, or at risk of containing enterovirus; wherein the protein is selected from albumin and beta lactoglobulin and the anhydride is 3-hydroxyphthalic anhydride.

2. The method of claim 1, wherein the protein is fully anhydrified as determined by the lysine and arginine modification ratio of the protein.

3. The method of claim 1, wherein the sample is selected from the group consisting of stool, saliva, blood, and serum.

4. The method of claim 1, wherein the article is selected from the group consisting of sanitary napkins, pantiliners, wet wipes, and tissues; the paper towel comprises handkerchiefs, removable toilet paper, napkin or hand towel.

5. The method of claim 1, wherein the article or sample further comprises, is suspected of comprising, or is at risk of comprising a virus selected from the group consisting of: human papilloma virus, herpes simplex virus, respiratory syncytial virus, and ebola virus.

6. The method of any one of claims 1-5, wherein the enterovirus is selected from enterovirus type 68, enterovirus type 71, and coxsackievirus.

7. The method of claim 6, wherein the coxsackievirus is coxsackievirus type 16.

8. The method of any one of claims 1-5, wherein the albumin is selected from serum albumin or egg white albumin; and/or the beta lactoglobulin is bovine beta lactoglobulin.

9. The method of claim 8, wherein the albumin is selected from human serum albumin, bovine serum albumin, or chicken serum albumin.

10. The method of any one of claims 1-5, wherein the composition is in the form of a tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel, or aerosol.

11. The method of any one of claims 1-5, wherein the anhydride modified protein is prepared by mixing the anhydride with the protein.

12. Use of an anhydride modified protein or a composition comprising an anhydride modified protein for the preparation of a medicament for the treatment or prevention of an enterovirus infection or a disease caused by an enterovirus infection in a subject, wherein the protein is selected from albumin and beta lactoglobulin and the anhydride is 3-hydroxyphthalic anhydride.

13. The use according to claim 12, wherein the protein is fully anhydrified as determined by the lysine and arginine modification ratio of the protein.

14. The use according to claim 12, wherein the medicament is in the form of a tablet, capsule, powder, nasal drops or aerosol, or in the form of a tincture, spirit, powder, lotion, oil, emulsion, ointment, paste, plaster, film, gel or aerosol.

15. The use of claim 12, wherein the subject is further suffering from or at risk of suffering from a viral infection selected from the group consisting of: human papilloma virus, herpes simplex virus, respiratory syncytial virus, and ebola virus.

16. The use according to claim 12, wherein the disease is hand-foot-and-mouth disease.

17. The use of claim 12, wherein the subject is a mammal.

18. The use of claim 17, wherein the mammal is a human.

19. The use according to any one of claims 12-18, wherein the enterovirus is selected from enterovirus type 68, enterovirus type 71 and coxsackievirus.

20. The use according to claim 19, wherein the coxsackievirus is coxsackievirus type 16.

21. The use according to any one of claims 12-18, wherein the albumin is selected from serum albumin or ovalbumin; and/or the beta lactoglobulin is bovine beta lactoglobulin.

22. The use according to claim 21, wherein the albumin is selected from human serum albumin, bovine serum albumin or chicken serum albumin.

23. The use according to any one of claims 12-18, wherein the anhydride modified protein is prepared by mixing the anhydride with the protein.