WO2021212755A1 - Anti-novel coronavirus antibody and preparation method therefor and use thereof - Google Patents

Abstract

Provided is an anti-novel coronavirus antibody. The antibody is a specific antibody extracted from chicken egg yolk. Also provided is an auxiliary drug using the anti-novel coronavirus antibody as a main component for preventing and controlling the novel coronavirus. The auxiliary drug can specifically bind to novel coronavirus in the oropharynx and nasal cavity and be used for preventing and blocking novel coronavirus infections and the auxiliary treatment of early infections.

Description

An antibody against novel coronavirus and its preparation method and application Technical field

The invention relates to the technical field of biological antibody immunization, and specifically refers to an antibody against a novel coronavirus and its preparation method and application.

Background technique

Coronavirus belongs to the order Nidovirales, Coronaviridae, and Coronavirus. It is an important pathogen of many domestic animals, pets, including human diseases, and causes a variety of acute and chronic diseases. The ninth report of the International Committee on Virology Classification divided the coronavirus family into three genera, namely α, β and γ. The new coronavirus belongs to the Sarbecovirus subgenus of β-coronavirus.

The new type of coronavirus pneumonia is a new infectious disease. The population has no immunity and is generally susceptible. Pneumonia caused by the new coronavirus infection can occur in people with low immune function and normal immune function, and it has a certain relationship with the amount of exposure to the virus. For people with poor immune function, such as the elderly, pregnant women, or people with abnormal liver and kidney function, and people with chronic diseases, the condition will be more severe after infection. According to WHO's latest evidence analysis on the transmission route of the new coronavirus, there are three transmission routes including symptomatic, pre-symptomatic and asymptomatic.

Since the spread of the new coronavirus around the world, the number of cases has continued to rise. Countries have adopted a series of measures such as isolation, personal protection, travel restrictions, and cessation of non-essential economic activities to control the development of the epidemic. At present, there are no specific drugs or vaccines for the prevention, treatment, and control of the new coronavirus, and the spread of the epidemic cannot be truly controlled. It can only be dealt with through symptomatic treatment and strict monitoring. New Coronavirus Pneumonia Diagnosis and Treatment Plan (Seventh Edition). We urgently need to find drugs that can treat the cause of the new coronavirus; there is currently no specific drug for the treatment of new coronavirus pneumonia, and the treatment of the disease is limited to symptomatic treatment. After some antiviral drugs are used, the clinical results are not clear, and the drugs The use of pathogens cannot be specifically killed, and the side effects are large.

In the past, vaccines were considered the most effective means of prevention and control of herd immunity. At present, there is no new coronavirus vaccine on the market. The most effective way to prevent and control the virus is isolation. This measure has brought many inconveniences and economic losses to people's production and life. The world is also vigorously developing vaccines. There are 115 candidate vaccine research projects for COVID-2019 worldwide. Vaccine technologies currently under study include: nucleic acids (DNA and RNA), virus-like particles, synthetic peptides, viral vectors (replicating and non-replicating), recombinant proteins, attenuated viruses, and inactivated viruses. The fastest time to market the vaccine is more than 18 months. Vaccine research and development, the market has a long time, and it is difficult to cover a large area of vaccination in a short period of time, which conflicts with people's urgent need for group protection.

At present, the main methods of killing the new coronavirus are chemical killing and physical killing. The chemical killing method is to kill the virus by using a broad-spectrum chemical to kill bacteria and viruses, such as 84 disinfectant, medical alcohol, etc., chemical agents are generally toxic to the human body, so they are mostly used as environmental disinfectants. However, it cannot be directly applied to the surface of human sensitive organs, such as eyes, ears, mouth and nose. Some chemically toxic chemicals cannot even come into excessive contact with human skin, so the protection effect on the human body is very limited; while the physical killing method requires high temperature , High pressure, ultraviolet rays and other physical means to kill viruses, and this method is highly dependent on the external environment, and it cannot be directly applied to the protection of the human body surface. No matter whether it is chemical killing or physical killing, it is impossible to achieve pathogens. Specific killing, large side effects, and limited auxiliary effect on the prevention and control of mass viruses. Therefore, there is an urgent need for effective methods that can be applied to the surface of the human body without adversely affecting the human body to prevent and urgently block the new coronavirus infection.

Summary of the invention

The purpose of the present invention is to provide an antibody against the novel coronavirus.

Another object of the present invention is to provide a method for preparing the antibody against the novel coronavirus.

Another object of the present invention is to provide the anti-new coronavirus antibody prepared for topical application to the nasal cavity and oropharyngeal mucosa, without the limitations and side effects of systemic medication, but also with fast onset of local medication, less dosage, and curative effect. Good; and the antibody is not easy to be absorbed by the human body, is not easy to destroy the ecological environment of the normal flora in the nasal cavity and oropharynx, does not cause local flora imbalance and other effects, has no toxic side effects; and does not activate the complement system, and is not easy to interact with rheumatoid factors Produce non-specific binding, not easy to fix Staphylococcus aureus A protein, not easy to bind to protein G and human cell FC receptor, so it is not prone to allergies, without the limitations and side effects of systemic medication, so that it can be used for the prevention of new coronaviruses. Control adjuvant medication.

The present invention is realized by the following technical solution: an antibody against the novel coronavirus, characterized in that the antibody is a high-titer specific antibody extracted from chicken egg yolk, and the chicken egg yolk is mainly obtained from immunized hens. Obtained from the laid eggs, the immunized hens are healthy hens injected with the new coronavirus vaccine.

Wherein, the novel coronavirus vaccine is mainly prepared by inactivated novel coronavirus or recombinant novel coronavirus antigen.

The spike protein of the new coronavirus invading cells can only use ACE2 as a designated spot to infect cells. Birds have not been tested for ACE2 protease in large numbers. Currently this site is mainly contained in mammalian cells. Therefore, compared with mammals, fish, birds and reptiles are very unlikely to become intermediate hosts of new coronary pneumonia. Therefore, by immunizing the hens and then extracting and preparing antibodies against the new coronavirus from the yolk of the chickens, there is obvious chance. Vaccines are prepared from inactivated new coronavirus (SARS-CoV-2) or recombinant new coronavirus (SARS-CoV-2) S antigen to immunize hens, and produce high-titer specific antibodies (ie specific IgY) in the yolk of chickens ), using biotechnology to separate and purify the specific egg yolk antibody, and use enzyme-linked immunosorbent assay to detect the titer of the finished egg yolk antibody. The titer is greater than or equal to 1:5000, which has unexpected technical effects.

The preparation process of the antibody against the new coronavirus is as follows:

Use recombinantly expressed new coronavirus (SARS-CoV-2) S antigen or virus-inactivated new coronavirus (SARS-CoV-2) antigen, diluted and emulsified with adjuvant to prepare vaccines for immunization; use this Vaccine the laying hens, obtain the immunized eggs, sterilize, separate the yolk and the egg white, take the yolk, extract with water dilution, and centrifuge to obtain the water-soluble components, and extract with ammonium sulfate to obtain the crude extract of the new coronavirus antibody; The extract is dissolved in a buffer solution and then inactivated and purified by ultrafiltration; according to the antibody titer test results, the new coronavirus egg yolk antibody is prepared, and after sterilization and filtration, an appropriate amount of stabilizer is added to make the new coronavirus egg yolk antibody product.

The present invention is an auxiliary drug for the prevention and control of a new coronavirus. The drug uses an antibody against the new coronavirus as a main component and adds a biopharmaceutically acceptable auxiliary component to prepare a pharmaceutical composition. The pharmaceutical composition is in liquid form, wherein the liquid form is in the form of an aqueous solution. The prevention and control auxiliary medication can directly act on the oropharynx and nasal cavity, and can specifically bind to the new coronavirus in the oropharynx and nasal cavity in a short time. , And can stay on the active surface for a long time, effectively neutralize the new coronavirus, and the whole process of acting on the pathogen is painless and non-irritating to the oropharyngeal and nasal mucosa.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The antibodies against the new coronavirus described in the present invention can bind to the specific pathogen of the new coronavirus in a short time, and the antibody stays on the surface for a long time, which can effectively neutralize the new coronavirus and affect the oropharynx during the whole process of the pathogen. And the nasal mucosa is painless and non-irritating, and the protection range is wider;

(2) The anti-new coronavirus antibody as the main component of the auxiliary drug for prevention and control of the present invention can be locally applied to the nasal cavity, oral cavity, pharynx, larynx and bronchial mucosa, without the limitations and side effects of systemic drugs, but also Topical medication has a quick onset, small dosage, good curative effect, is not easily absorbed by the body, does not damage the ecological environment of the normal flora in the nasal cavity, oral cavity, pharynx, throat, and bronchus, and does not cause local flora imbalance;

(3) The anti-coronavirus antibody as the main component of the auxiliary drug for prevention and control of the present invention is not easy to activate the complement system, not easy to produce non-specific binding with rheumatoid factors, not easy to fix Staphylococcus aureus A protein, and not easy to interact with protein G and FC receptors of human cells bind, so allergic reactions are not easy to occur;

(4) The prevention and control supplementary drugs described in the present invention with antibodies against the new coronavirus as the main component do not produce acute toxic reactions, are actually non-toxic, and can independently neutralize the new coronavirus and block the virus from passing through the nose. The effect of oropharyngeal infection does not require the cooperation of complement and other immune cells;

(5) The anti-new coronavirus antibody product prepared in the present invention has stable biological activity and can be stored at room temperature for a long time. The anti-new coronavirus antibody contains the corresponding specific pathogen antibody titer not less than 1:5000 per milliliter. With more antigen binding sites, it can effectively neutralize the new coronavirus and block the spread of the virus.

Description of the drawings

Figure 1 is an SDS-PAGE analysis diagram of the purified antibody (chicken egg yolk antibody IgY) in the present invention;

Figure 2 is a Western Blot analysis diagram of the purified antibody (chicken egg yolk antibody IgY) in the present invention;

Figure 3 is a diagram of the antibody titer of the finished product detected by the enzyme-linked immunosorbent method in the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the examples, but the implementation of the present invention is not limited to this. Without departing from the above technical ideas of the present invention, various substitutions and changes can be made based on common technical knowledge and conventional means in the field. , Should be included in the scope of the present invention.

In order to make the purpose, process conditions and advantages of the present invention clearer, the present invention will be further described in detail in conjunction with the following embodiments. The specific embodiments described here are only used to explain the present invention, and are not intended to limit the present invention.

Example 1:

The present invention specifically provides an antibody against the novel coronavirus, which is characterized in that the antibody is a high-titer specific antibody extracted from chicken egg yolk, and the chicken egg yolk is mainly derived from eggs laid by immunized hens. Obtained, the immunized hen is a healthy hen injected with a new coronavirus vaccine.

Wherein, the novel coronavirus vaccine is mainly prepared by recombinantly expressed novel coronavirus (SARS-CoV-2) S antigen or virus-inactivated novel coronavirus (SARS-CoV-2) antigen.

The specific preparation method includes the following steps:

1. Antibody preparation:

S1. Vaccine preparation:

Take the recombinantly expressed novel coronavirus (SARS-CoV-2) S antigen or the virus-inactivated novel coronavirus (SARS-CoV-2) antigen, and add the antigen to Freund's complete adjuvant in a ratio of 1:1 (preliminary) Exempt) or Freund's incomplete adjuvant (enhanced immunity), emulsify on a tissue dissector at 20-25 Hz resonance for 3 to 5 minutes to prepare a vaccine. After the physical properties are qualified, it can be used for immune healthy laying hens.

S2. Immunization program

Select healthy hens from 90 to 120 days old, and take the prepared 5μg/ml and 10μg/ml vaccines for subcutaneous and intramuscular multi-point injections, 0.25～1.0ml/bird; after the initial immunization, the vaccine will be given at an interval of 2-8 weeks Boost immunity, 0.25～1.0ml/feather subcutaneous and intramuscular injections at multiple points.

S3. Egg collection: After the initial immunization, eggs are taken regularly to test the growth of the yolk antibody titer. After the booster immunization, the yolk antibody titer is greater than or equal to 1:5000 (detected by ELISA method), and then the eggs are collected.

According to the production of egg yolk antibodies in two different immunization dose groups, the antigen immunization dose is preferred.

2. The crude extraction and purification of new coronavirus antibodies.

S1. Wash the eggs collected after immunization, break the shells, separate the egg whites and yolks, remove the egg whites, collect the yolks and weigh them.

S2. Add purified water to dilute the homogenate in step S1 at a ratio of 1:6-9, adjust the pH to 4.8-6.0 with 1 mol/L hydrochloric acid solution, and let it stand at 2-8°C for 1-24 hours.

The liquid obtained in step S3.S2 is centrifuged at 4000 to 20000 r/min to remove the precipitate and take the supernatant.

Add 10%-35% ammonium sulfate to the supernatant prepared in steps S4.S3 to salt out, mix well, and settle overnight.

S5.S4 steps to prepare the precipitate 4000 to 20000r/min centrifugal desalting, take the precipitate to prepare the crude egg yolk antibody extract, and remove the ammonium sulfate supernatant.

The precipitate prepared in step S6.S5 is reconstituted with 1/150～1/300 volume weak acid buffer, adjusted to pH 4.8～6.0, fully dissolved at 2～8℃ overnight, and the supernatant is taken.

The supernatant obtained in steps S7.S6 is inactivated at 54-60°C for 8-20 hours and the virus is inactivated.

In steps S8.S7, the inactivated supernatant is subjected to ultrafiltration with an ultrafiltration membrane with a relative molecular weight cut-off of 50-100kD.

The ultrafiltrate prepared in steps S9.S8 is sterilized and filtered into IgY stock solution.

S10. The IgY stock solution prepared in step S9 is made into an egg-yolk antibody preparation according to the detected antibody titer plus a stabilizer.

3. The extraction effect of yolk antibody, as shown in Figure 1.

Among them, 1 and 2 are IgY purified by ultrafiltration tube after ammonium sulfate precipitation.

It can be seen from Figure 1 that IgY can be further purified by ultrafiltration after extraction by ammonium sulfate method to reduce contaminant proteins. The purified IgY has high purity and few miscellaneous bands; SDS-PAGE non-reducing electrophoresis shows that the extracted anti-neovirus IgY has a molecular weight of about 180kD, and the gel imaging software analyzes the IgY antibody purity to be more than 80%.

It can be seen from Figure 2 that according to Western blot analysis, the molecular weights of IgY heavy chain and light chain are 65kD and 25kD, respectively.

Enzyme-linked detection method was used to detect the titer of finished egg yolk antibody, as shown in Figure 3.

The titer of the anti-new coronavirus IgY: ≥1:64000.

Example 2:

This example compares the production of egg yolk antibodies with different antigen immunization doses in two groups, and the preferred antigen immunization dose is shown in the following table:

Table 1 Growth and decline of yolk antibody titers with different antigen immunization doses

Immunization dose	4 weeks after initial exemption	5 weeks after initial exemption	6 weeks after initial exemption	8 weeks after initial exemption	10 weeks after initial exemption
5μg/feather	1:16000	1:80000	1:80000	1:80000	1:64000
10μg/feather	NT	1:40000	1:40000	1:80000	1:32000

(NT: Because the hen did not lay eggs after being immunized, it was not tested.)

It can be seen from the above table that the 5μg/feather antigen dose group is preferred, and the yolk antibody titers produced after 6, 8, and 10 weeks of primary immunization in this dose group are not lower than the 10μg/feather antigen dose group.

Example 3:

In this example, to verify the virus neutralization performance of the anti-new coronavirus egg yolk antibody, the following experiments were performed:

Take 10 ⁵ ～10 ⁷ TCID ₅₀ /ml of the new coronavirus liquid according to the volume ratio of 1:1 and the concentration of 1mg/ml, 2mg/ml, 3mg/ml of the new coronavirus egg yolk antibody samples and mix them evenly, and place 25～37. Neutralize at ℃ for 1 to 2 hours, add it to a 96-well plate containing cells, and incubate in a carbon dioxide incubator at 33 to 35°C. Observe cytopathic changes daily under a microscope.

Under the condition of 25～37℃, the concentration of 2mg/ml, 3mg/ml of the new coronavirus egg yolk antibody is mixed with 105～107TCID50/ml of the new coronavirus liquid, and the cells have no pathological changes. Positive control, the new coronavirus liquid can cause cell pathological changes.

It can be known from the test results that the anti-new coronavirus IgY prepared in the present invention has the effect of neutralizing the new coronavirus.

Example 4:

In order to verify the long residence time of specific egg yolk antibodies in different parts of living organisms, a small animal imaging experiment of drug residence time was performed on the obtained egg yolk antibodies. The specific process is as follows:

Adjust the pH of the buffer system (sodium bicarbonate buffer system) to 8.5-10.0, add a small amount of fluorescent dye to the egg yolk antibody solution at a temperature of 4°C, and mix vertically overnight. Then ultrafiltration centrifugation (5000-6000g, centrifugation time 4-6min) to remove unreacted fluorescent molecules.

Test method: Make a yolk antibody stock solution containing fluorescent molecules into an oral spray. Divide into three experimental groups: group a: water-free for 8 hours after spraying, no fasting; group b: fasting for 8 hours after spraying, no watering; group c: water-free and fasting for 8 hours after spraying; skin spraying: back removal Spray after part of the fur.

Detect the fluorescence intensity of the spray site through small animal imaging, and judge the residence time of the egg yolk antibody stock solution at the spray site by the weakening of the fluorescence intensity. After the oral spray, the fluorescence of the egg yolk antibody stock solution of different groups weakens with time. After the oral spray, different tests The stay time of the groups in the oral cavity can reach 8.5 hours, and the effect of the fasting and water-free experimental group is better. The egg yolk antibody stock solution containing fluorescent molecules can stay on the skin for more than 70 hours.

Example 5:

In this example, in order to verify the safety of the antibodies against the novel coronavirus on the surface of the eyeballs of living organisms, an acute eye irritation test was performed on the obtained antibacterial chicken egg yolk composite antibodies. The specific process is as follows:

0.1ml of the test substance was dropped into the rabbit's right eye conjunctival sac and passively closed for 4s. After 30s, it was rinsed with a sufficient amount of normal saline with a fast flow rate but not causing eye damage for 30s. The left eye was a blank control. Record the local reactions at 1h, 24h, 48h, 72h, 7d, 14d and 21d after eye instillation. If there is no irritation response within 72 hours, or the eye irritation response is fully recovered on the 7th and 14th days, the test can be terminated early. The test results are shown in Table 2.

Table 2 Eye irritation test results of anti-new coronavirus antibodies on rabbits

*Average score refers to the sum of 24h, 48h, and 72h scores divided by the number of observation periods 3.

The test results showed that after the test substance was instilled into the rabbit's right eye conjunctival sac, there were no abnormalities in the conjunctiva, cornea, and iris of the 3 rabbits. The anti-new coronavirus antibody described in the present invention has no irritation to the results of acute eye irritation test in rabbits.

Example 6:

In this example, in order to verify the safety of the antibodies against the novel coronavirus on the surface of living organisms, multiple complete skin irritation tests were performed on the obtained antibacterial chicken egg-yolk composite antibodies, as follows:

Twenty-four hours before the test, the hair on both sides of the rabbit's back spine was cut off, and the hair removal range was about 3cm×3cm on the left and right. During the test, 0.5ml of the stock solution of the test substance was applied to the skin of 2.5cm×2.5cm on one side, and the skin on the other side was used as a blank control. Remove the test substance 4 hours after application, apply it once a day, and apply it continuously for 14 days. The results were observed 24 hours after each application, and the results of repeated intact skin irritation tests on rabbits were non-irritating.

After the test substance was applied to the skin of rabbits, there was no erythema, edema and inflammatory reaction in the skin of the animals. The average irritation response score of each animal per day was 0, as shown in Table 3.

Table 3 Results of multiple intact skin irritation tests on rabbits with antibodies against the new coronavirus

It can be seen from the above table that the sample is non-irritating to rabbits after repeated intact skin irritation tests.

Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and modifications can be made to these embodiments without departing from the principle and purpose of the present invention. The scope of is defined by the claims and their equivalents.

Claims (10)

1. An antibody against a novel coronavirus, characterized in that the antibody is a high-titer specific antibody extracted from chicken egg yolk, which is mainly obtained from eggs laid by immunized hens. The hens are healthy hens that have been vaccinated against the new coronavirus.
2. The antibody against the novel coronavirus according to claim 1, wherein the vaccine for the novel coronavirus is mainly prepared from inactivated novel coronavirus or recombinant novel coronavirus antigen.
3. The method for preparing an antibody against the novel coronavirus according to claim 1 or 2, characterized in that it comprises the following steps:

   (1) Preparation of a new coronavirus vaccine;

   (2) Use the prepared new coronavirus vaccine to immunize healthy laying hens;

   (3) Collect immunized eggs laid by immunized hens;

   (4) Crude extraction and purification of new coronavirus antibodies from immune eggs;

   (5) Detect the titer of the antibody stock solution and add a stabilizer to obtain an antibody preparation against the new coronavirus.
4. The method for preparing an antibody against the novel coronavirus according to claim 3, characterized in that, in the step (1), the specific process of preparing a novel coronavirus vaccine is: taking a recombinantly expressed novel coronavirus antigen or The virus-inactivated novel coronavirus antigen is added with an equal volume of Freund’s complete adjuvant or Freund’s incomplete adjuvant, and emulsified on a tissue lysator at 20～25Hz resonance for 3～5min. After passing the physical property inspection, supply it Healthy laying hens are immune to use.
5. The method for preparing an antibody against the novel coronavirus according to claim 3 or 4, characterized in that, in the step (2), the specific process of immunizing the hens is: taking healthy egg-laying mothers of appropriate age For chickens, take the prepared new coronavirus vaccine into the deltoid or pectoral muscles of the wing base of the laying hens to inject intramuscularly and subcutaneously, the dosage is 0.25-1.0ml/feather; after the initial immunization, it will be administered at an interval of 2-8 weeks. Vaccine booster, that is, multiple injections under the skin and muscle of laying hens, the dosage is 0.25～1.0ml/feather.
6. The method for preparing an antibody against the novel coronavirus according to claim 3 or 4, characterized in that, in the step (4), the specific process of crude extraction and purification of the antibody against the novel coronavirus from immunized eggs includes The following steps:

   (4.1) Clean the eggs collected after immunization, break the shells, separate the egg whites and yolks, remove the egg whites, collect the yolks and weigh them;

   (4.2) Add the egg yolk in a ratio of 1:6-9 with purified water to dilute the homogenate, adjust the pH to 4.8-6.0 with 1mol/L hydrochloric acid solution, and let it stand at 2-8℃ for 1-24 hours;

   (4.3) Centrifuge the liquid after standing to remove the sediment and take the supernatant;

   (4.4) Add 10% to 35% ammonium sulfate to the obtained supernatant for salting out, mix well, and then settle overnight;

   (4.5) After the precipitation is completed, perform centrifugal desalting, obtain the precipitate to prepare the crude egg yolk antibody extract, and remove the ammonium sulfate supernatant;

   (4.6) The obtained crude egg yolk antibody extract was reconstituted with 1/150～1/300 volume weak acid buffer, adjusted the pH to 4.8～6.0 to make it fully dissolved, and left overnight at 2～8℃, afterwards Obtain the supernatant;

   (4.7) Virus inactivation of the obtained supernatant;

   (4.8) Ultrafiltration of the supernatant after virus inactivation is performed with an ultrafiltration membrane;

   (4.9) Sterilize the ultrafiltration solution to obtain the antibody stock solution.
7. The method for preparing an antibody against the novel coronavirus according to claim 6, wherein the centrifugation parameter of the centrifugation process in the preparation method is 4000 to 20000 r/min.
8. The method for preparing an antibody against the novel coronavirus according to claim 6, characterized in that the relative molecular weight cut-off of the ultrafiltration membrane used in the ultrafiltration in the step (4.6) is 50-100 kD.
9. An auxiliary drug for the prevention and control of a new type of coronavirus, which is characterized in that it is a pharmaceutical composition prepared by taking the antibody of claim 1 or 2 as the main component and adding biopharmaceutically acceptable auxiliary components.
10. An auxiliary drug for the prevention and control of a novel coronavirus according to claim 9, wherein the pharmaceutical composition is in liquid form, wherein the liquid form is in the form of an aqueous solution, and the auxiliary drug for prevention and control can directly act on The oropharynx and nasal cavity of organisms neutralize the new coronavirus.

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