阅读说明：本技术 一种抗人新型冠状病毒肺炎的抗体及其制备方法 (Antibody for resisting novel human coronavirus pneumonia and preparation method thereof ) 是由 刘会芳 宋勤叶 刘朋朋 左广双 张鹤 宋辉格 陈桂清 智健飞 王银钱 王春涛 朱 于 2020-04-07 设计创作，主要内容包括：本发明涉及免疫学和抗体制备技术领域,尤其涉及一种抗人新型冠状病毒肺炎的抗体及其制备方法。该抗体是通过用新冠肺炎病毒灭活疫苗免疫健康禽类动物,当被免疫的所述禽类动物血清或卵黄内抗新冠肺炎抗体滴度达到要求时,收集血清或卵黄中的水溶性组分,纯化后得到。所得产物还可通过喷雾、冻干等处理获得相应的抗体干粉。本发明所得抗体能够用于新冠肺炎早期、中期患者的治疗和后期重症患者的救治,也可用于新冠肺炎的预防,还能配合新冠肺炎疫苗使用,增强新冠肺炎的防控效果。(The invention relates to the technical field of immunology and antibody preparation, in particular to an anti-human novel coronavirus pneumonia antibody and a preparation method thereof. The antibody is obtained by immunizing healthy poultry with the Newcrown pneumonia virus inactivated vaccine, collecting water-soluble components in serum or yolk and purifying when the titer of the anti-Newcrown pneumonia antibody in the serum or the yolk of the immunized poultry meets the requirement. The obtained product can also be processed by spraying, freeze-drying and the like to obtain corresponding antibody dry powder. The antibody obtained by the invention can be used for treating early and middle stage patients of new coronary pneumonia and treating late stage severe patients, can also be used for preventing the new coronary pneumonia, and can be used together with a new coronary pneumonia vaccine to enhance the prevention and control effect of the new coronary pneumonia.)

1. The antibody for resisting the novel human coronavirus pneumonia is obtained by immunizing healthy poultry with a novel coronavirus inactivated vaccine, and collecting water-soluble components in serum or yolk when the OD value of the anti-novel coronavirus pneumonia antibody in the serum or the yolk of the immunized poultry is more than 0.2 than the OD value of a positive control antibody, and purifying the water-soluble components.

2. The antibody against human novel coronavirus pneumonia according to claim 1, wherein said novel coronavirus inactivated vaccine comprises at least one of an L-subtype vaccine and an S-subtype vaccine; and/or

The avian animal is poultry; and/or

The virus content in the new coronavirus inactivated vaccine is more than or equal to 10⁵TCID₅₀/mL。

3. The method for producing an antibody against human novel coronavirus pneumonia according to claim 1 or 2, comprising the steps of:

immunizing healthy poultry by using the Newcastle pneumonia virus inactivated vaccine once every 5-7 days, wherein the immunization dose is 0.5-1 mL, measuring the anti-Newcastle pneumonia antibody in serum or yolk by using an indirect ELISA method, collecting the serum when the OD value of the anti-Newcastle pneumonia antibody in the serum is more than 0.2 of the OD value of a positive control antibody, and collecting the water-soluble component in the yolk when the OD value of the anti-Newcastle pneumonia antibody in the yolk is more than 0.2 of the OD value of the positive control antibody.

4. The method of claim 3, further comprising boosting the immune response with the inactivated vaccine against neocoronary pneumonia every 15-20 days when the OD of the serum anti-neocoronary pneumonia antibody is greater than the OD of the positive control antibody by 0.2 or when the OD of the yolk anti-neocoronary pneumonia antibody is greater than the OD of the positive control antibody by 0.2.

5. The method of producing an antibody against human novel coronavirus pneumonia according to claim 3, wherein the method of collecting serum comprises: collecting poultry blood, standing for 0.5-1 hour, centrifuging for 5-15 minutes at 4000-8000 rpm, extracting supernatant, filtering, and collecting filtrate to obtain the serum.

6. The method for producing an antibody against human novel coronavirus pneumonia according to claim 3, wherein the method for collecting water-soluble components in yolk comprises: collecting yolk, adding purified water with the volume of 3-5 times of that of the yolk, stirring for demulsification, standing for 2-6 hours, collecting supernatant, filtering, and collecting filtrate to obtain the yolk; the pure water contains a demulsifier.

7. The method for producing an antibody against human novel coronavirus pneumonia according to claim 5 or 6, wherein the filtration is performed by using a filter having a pore size of 0.22 μm.

8. The method for producing an antibody against human novel coronavirus pneumonia according to claim 5 or 6, wherein the method further comprises concentrating the filtrate at 55 to 65 ℃ to obtain a concentrated solution.

9. The method of producing an antibody against human novel coronavirus pneumonia according to claim 8, wherein the method further comprises drying the concentrated solution.

10. The method for producing an antibody against human novel coronavirus pneumonia according to claim 9, wherein the drying is high-temperature centrifugal spray drying at 155 to 165 ℃ and at 8000 to 25000 rpm; or

The drying is freeze drying; or

The drying is low-temperature centrifugal vacuum spraying, the drying temperature is 55-65 ℃, and the rotating speed of a centrifugal spraying head is 8000-25000 rpm.

Technical Field

The invention relates to the technical field of immunology and antibody preparation, in particular to an anti-human novel coronavirus pneumonia antibody and a preparation method thereof.

Background

The pathogen SARS-COV-2 of new type coronavirus pneumonia (COVID-19, Chinese short for new type coronary pneumonia) is different coronavirus from SARS and MARS, although the disease condition of patient is similar, they are also obviously different, and their respective vaccines are different, and their antibodies have no cross-protection action. SARS-COV-2 is less virulent but more sterically transmitting than SARS and MARS. The research finds that SARS-COV-2 is mainly applied to L subtype and S subtype strains, wherein, the L subtype virus has stronger virulence than the S subtype virus, but the S subtype virus has stronger inactivation resistance and is easier to spread over a long distance. In actual work, medical workers and researchers find that asymptomatic infectors (without presenting ocular symptoms such as fever and dry cough) or novel coronavirus carriers with unobvious ocular symptoms and no hospitalization account for a high proportion of infected people, so that experts predict that the disease can exist in global people for a long time, even continuously infect for ten years, annual infection can reach 5% of global population, and the disease can not be controlled until reaching about 70% of the global population, namely nearly 50 hundred million people. If not prevented as soon as possible, the disease may end up as a background existing disease in the population, so there is a great need to research and develop relevant vaccines and antagonists.

Among the drugs for resisting the new coronavirus, the anti-new coronavirus (polyclonal) antibody has the fastest effect and the best effect, and has better effect than that of the recommended western medicines of Reidesvir and hydroxychloroquine sulfate, and the single Chinese medicine and the compound Chinese medicine thereof cannot give a clear and effective result in a certain replication step of a virus cell level protection test, a target site determination test or an impedance virus, so that most of the countries do not consider using the anti-new coronavirus (polyclonal) antibody. However, because the recovered human serum is not easy to obtain and the actually available serum is few, and the recovered human serum possibly still contains the new coronary pneumonia virus and needs further virus inactivation or removal work, the safe humanized serum antibody for resisting the new coronary pneumonia virus is precious and cannot meet the requirements of a large number of patients at all. Since the blood of ordinary people cannot be used for preparing serum, the number of simians close to the human blood is small, and the simians often carry pathogens with great threat to human beings and are not suitable for large-scale use, the humanized and simian anti-new crown pneumonia serum antibody is prepared and cannot meet the emergency use of millions, millions or hundreds of millions of people in the future at all.

Disclosure of Invention

Aiming at the problem that the existing human-derived and simian-derived anti-new coronary pneumonia serum antibody can not meet the actual requirement, the invention provides an anti-human novel coronavirus pneumonia antibody.

The invention also provides a preparation method for resisting the novel human coronavirus pneumonia.

In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:

an antibody for resisting the pneumonia of the novel human coronavirus is obtained by immunizing healthy poultry with a virus inactivated vaccine (including an aqueous agent inactivated vaccine and an oil emulsion inactivated vaccine) for resisting the pneumonia of the novel human coronavirus, collecting water-soluble components in serum or yolk of the immunized poultry when the OD value of the antibody for resisting the novel human coronavirus in the serum or the yolk of the immunized poultry is more than 0.2 of the OD value of a positive control antibody, and purifying the water-soluble components.

The OD value of the anti-new crown pneumonia antibody is larger than that of the positive control antibody, namely the S/P value. The positive control antibody OD value in the ELISA detection system is 1.8.

The source of the poultry is rich, the serum antibody is easy to obtain, the egg yield of the female poultry is also large, and the price is low. The avian animals are not infected with the human new coronavirus, and the antibody preparation uses a complete inactivated vaccine, so that the possibility of virus dispersion does not exist. According to the application, the poultry is immunized by using the Newcrown pneumonia virus inactivated vaccine, so that antiserum for resisting the Newcrown pneumonia is generated in the blood of the poultry, or yolk antibody for resisting the Newcrown pneumonia is generated in a poultry egg produced by the poultry, the antibody cannot generate drug resistance, the administration method is safe and effective, and oral administration or injection can be selected according to clinical needs.

The new coronary pneumonia antibody can be widely applied to early and middle stage patients, after the antibody is absorbed, the in vivo virus can be quickly neutralized, the condition that the patient turns to severe illness is avoided, the organism of the patient can have the capability and generate normal antibody immune response within a long time without entering severe illness, once the antibody of a human individual is generated, the virus can be easily cleared and immune memory can be established, and the immunity and the anti-capacity can be generated to the virus within a long time or even years.

The severe symptoms of the patient caused by the new coronary pneumonia mainly come from two parts, wherein firstly, the virus damages the organism, secondly, the cytokine storm (inflammatory storm) caused by the virus often causes the function reduction, apoptosis and even rupture death of a large number of normal functional cells, so that the organism has no capacity and can generate antibody immune response in time, which is an important reason that the common patient turns to severe cases to death, if the anti-new coronary pneumonia antiserum or the yolk antibody can be used for neutralizing the new coronary pneumonia virus in the early stage of the severe cases, generally, after dozens of hours, the lethal cytokine storm in the patient can be greatly reduced because of the great reduction or disappearance of infection sources, thereby playing the role of saving life.

For early and middle stage patients, the application of the antiserum or the yolk antibody for resisting the new coronary pneumonia can lead the infected people to neutralize the virus in the early stage and avoid the virus from being converted into severe patients. The anti-neocoronarism antiserum or yolk antibody can eliminate viruses in vivo for asymptomatic infected persons or latent infected persons carrying viruses. The antibody is used by people in a large-scale preventive manner, so that the infection rate can be greatly reduced, the number of infected people is reduced, virus diffusion is restrained, secondary or multiple outbreaks of epidemic caused by recessive transmission are reduced, the development of the epidemic is resisted, and large-area market-crossing, provincial-crossing, even country-crossing and continent-crossing transmission is avoided.

The antiserum or the yolk antibody for resisting the new coronary pneumonia can be matched with a new coronary pneumonia vaccine for use, so that the prevention and control effects of the new coronary pneumonia are enhanced: on one hand, the antibody can be used for neutralizing most latent new coronary pneumonia virus wild strains of a human body before the new coronary vaccine is immunized by the human body, so that the stress of vaccine immunization and the effect of exciting the latent wild strains by live vaccines in the vaccine are reduced; on the other hand, when the antibody level in the body of a vaccine vaccinee is low and is not enough to resist virus invasion or the virus breaks through the immune barrier of a human body, the use of the antibody can prevent the wild strain from being massively copied, and effective immune time and safety guarantee are provided for reseeding vaccines.

Because the poultry animals are cheap and easy to obtain, the method is simple and easy to operate, the cost for obtaining the antibody can be greatly reduced, the expensive treatment cost of a patient can be reduced from thousands of yuan to millions of yuan to several yuan to dozens of yuan, the current heavy medical burden is greatly reduced, and the method is suitable for the prevention and treatment of the ordinary people occupying the majority of people.

Even if a new epidemic wild strain appears along with natural or non-natural variation of the virus and affects the common people, the method of the invention can be applied to select the epidemic strain to prepare the vaccine for immunizing poultry animals for multiple times, and antiserum or yolk antibody can be prepared in a large amount in one half month.

Preferably, the novel inactivated coronavirus vaccine comprises at least one of an L subtype vaccine and an S subtype vaccine. The L subtype strain and the S subtype strain of the main epidemic strain of the new coronavirus can be prepared into an L subtype inactivated vaccine and an S subtype inactivated vaccine respectively, and can also be directly prepared into an L subtype and S subtype bivalent vaccine.

Preferably, the virus content in the new coronavirus inactivated vaccine is more than or equal to 10⁵TCID₅₀and/mL. The antibody titer in the serum and the yolk can reach the expected requirement through four to six times of immunization. The virus content of each feather of the inactivated vaccine of the new coronavirus is not less than 10 no matter the vaccine is monovalent or bivalent⁵TCID₅₀/mL。

Preferably, the avian animal is poultry. The poultry in China are billions in number, the sources are rich, the serum and the poultry eggs are easy to obtain, and the cost is low. The inactivated vaccine of neocoronarism can be used for immunizing specific pathogen-free chickens (SPF chickens) or healthy laying hens. When the egg yolk antibodies are to be collected, laying hens in the egg laying peak period are preferably immunized.

The embodiment of the invention also provides a preparation method of the anti-human novel coronavirus pneumonia antibody, which specifically comprises the following operations:

immunizing healthy poultry by using the Newcastle pneumonia virus inactivated vaccine once every 5-7 days, wherein the immunization dose is 0.5-1 mL, measuring an anti-Newcastle pneumonia antibody in serum or yolk by using an indirect ELISA method, collecting the serum when the OD value of the anti-Newcastle pneumonia antibody in the serum is more than 0.2 than the OD value of a positive control antibody, and collecting water-soluble components in the yolk when the OD value of the anti-Newcastle pneumonia antibody in the yolk is more than 0.2 than the OD value of the positive control antibody to obtain the vaccine.

The preparation method provided by the invention can obtain a large amount of anti-neocoronary pneumonia antiserum or anti-neocoronary pneumonia yolk antibody within about one and a half months.

Preferably, the preparation method further comprises the step of boosting immunization with the new coronavirus inactivated vaccine every 15-20 days after the OD value of the anti-new coronavirus antibody in serum is greater than the OD value of the positive control antibody by more than 0.2 or the OD value of the anti-new coronavirus antibody in yolk is greater than the OD value of the positive control antibody by more than 0.2 so as to maintain high-level antibodies.

Preferably, the method for collecting serum comprises: collecting poultry blood, standing for 0.5-1 hour, centrifuging for 5-15 minutes at 4000-8000 rpm, extracting supernatant, filtering, and collecting filtrate to obtain the serum. After blood is kept still for 0.5-1 hour, the blood is coagulated, faint yellow liquid appears on the upper layer, and the centrifugation is to carry out the common centrifugation on the coagulated blood, the faint yellow liquid and the components between the coagulated blood and the faint yellow liquid.

Preferably, the method for collecting the water-soluble components in the yolk is as follows: collecting yolk, adding purified water with the volume of 3-5 times of that of the yolk, stirring for demulsification, standing for 2-6 hours, collecting supernatant, filtering, and collecting filtrate to obtain the yolk; the pure water contains a demulsifier to promote yolk demulsification. The filtrate contains yolk antibody against neocoronary pneumonia. The demulsifier can be selected from commercially available products with medical standards, such as polyethylene glycol 6000, polyoxyethylene polyoxypropylene and the like, and the dosage of the demulsifier is 1-3% of the mass of the purified water.

Preferably, the filtration is performed by using a filter membrane with a pore size of 0.22 μm.

Preferably, the preparation method further comprises the step of concentrating the filtrate at 55-65 ℃ to obtain a concentrated solution. For example, the filtrate can be concentrated by a low-temperature vacuum concentration tank to 1/3-1/4 of the original volume. The concentrated filtrate is convenient for further processing into clinically required products, such as drinking water agents, injection and the like.

Preferably, the preparation method further comprises drying the concentrated solution. Drying, pulverizing, sieving to obtain antibody powder, and collecting the antibody powder to make into tablet, capsule, granule, powder and injectable sterile powder for clinical application.

Preferably, the drying is high-temperature centrifugal spray drying, the drying temperature is 155-165 ℃, and the rotating speed of a centrifugal spray head is 8000-25000 rpm.

Preferably, the drying is freeze drying. The freeze drying can reduce the loss of the antibody in the high-temperature drying process and improve the content of the effective antibody in the product.

Preferably, the drying is low-temperature centrifugal vacuum spraying, the drying temperature is 55-65 ℃, and the rotating speed of a centrifugal spraying head is 8000-25000 rpm. The antibody loss in the drying method is similar to that of freeze drying, the antibody is less damaged, and the time cost and the power cost of production are lower than those of vacuum freeze drying.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.