RU2810740C1 - Method of obtaining inactivated vaccine against covid-19 and vaccine obtained by method - Google Patents

Abstract

FIELD: medical biotechnology and immunology.

SUBSTANCE: vaccine against COVID-19 and a method of its preparation by cultivating the viral strain nCoV-19/Russia/SP48-1339/2021 of SARS-Cov-2 virus deposited in a specialized collection of reference cultures of strains of pathogenicity groups II–IV (arboviruses and others) are described. FSBI "48 Central Research Institute" of the Russian Ministry of Defense under registration No. 1339, with subsequent inactivation and purification of the vaccine antigen.

EFFECT: increase in the degree of purification of the viral antigen of the SARS-Cov-2 virus. The use of a highly virulent strain, highly pathogenic for humans, ensures high immunogenicity and effectiveness of the vaccine, and the use of a highly purified vaccine preparation based on a viral antigen reduces the risks of unwanted side effects by increasing the degree of its purification.

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Description

Field of technology to which the invention relates

The invention relates to the field of medical biotechnology and immunology, specifically to an inactivated whole-virion vaccine against COVID-19 and a method for its production by cultivating the causative viral strain with subsequent inactivation and purification of the viral antigen. The vaccine obtained by the proposed method can be used to prevent COVID-19 infection.

Prior Art

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused the global COVID-19 pandemic, was first detected in China in late 2019. The SARS-CoV-2 virus, also called 2019-nCoV (hereinafter referred to as the virus), belongs to the family Coronaviridae, genus Betacoronavirus (betaCoV), and its natural reservoir is bats. The genome of the virus is represented by single-stranded RNA, consisting of 29903 bp. (the reference full-length genome sequence of the Wuhan-Hu-1 virus strain is located in the Genbank Database under number NC_045512.2), which encodes a polypeptide of 9860 amino acids, including 25 non-structural proteins and 4 structural proteins, such as spike (S) proteins, envelope ( E), membrane (M) and nucleocapsid (N). The nucleotide sequence of the viral genome is 89% identical to the SARS-like CoVZXC21 bat virus and 82% identical to the human SARS-CoV virus (Chan J.F.-W. et al., Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan, 2020, Emerg Microbes Infect,., 2020 Jan 28;9(1):221-236). The virus is sensitive to ultraviolet radiation, heat and lipid solvents.

Transmission of SARS-CoV-2 infection occurs through airborne droplets, airborne dust and contact. The leading route of transmission of SARS-CoV-2 is airborne, which occurs when coughing, sneezing and talking at close range. A contact route of transmission is possible, which occurs during handshakes and other types of direct contact with an infected person, as well as through surfaces and objects contaminated with the virus. In addition, the virus can be transmitted through blood transfusion, transplacentally and sexually. Although SARS-CoV-2 infection may only present with mild symptoms, typically fever and cough, or even be asymptomatic; at the other extreme it can be fatal. The main symptoms are usually fever, cough and difficulty breathing.

SARS-CoV-2 currently poses a significant threat to public health. Since 2019, the virus has rapidly spread throughout the world and, according to WHO, at the end of February 2023 has already led to more than 757 million confirmed cases of COVID-19, including almost 7 million deaths (https://covid19.who.int /). In this regard, the search continues for therapeutic agents that could finally stop the transition from infection to the development of a severe form of COVID-19.

Researchers are focusing on developing various therapeutic strategies, including preventing SARS-CoV-2 from binding to receptors on human cells, blocking viral ribonucleic acid (RNA) synthesis and replication, restoring host innate immunity, and regulating specific host receptors or enzymes. However, the most promising approach to containing the pandemic is considered to be vaccines that prevent infection by SARS-CoV-2 (Vaccines against SARS-CoV-2: bright and dark sides, translation by Savitskaya R., posted 08/09/2021 at https://medach.pro /post/2680).

To date, various types of COVID-19 vaccines have been developed, including whole-virion attenuated and inactivated vaccines, peptide and vector vaccines, DNA and RNA vaccines, which are aimed at stimulating the body's immune response to neutralize the virus. According to WHO, at the end of February 2023, it is known that 199 vaccine candidates against COVID-19 are in preclinical testing and 180 vaccines are in clinical trials, of which only 22 are inactivated vaccines (https://www .who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines).

At least six whole virion inactivated vaccines are available for use for the prevention of COVID-19, five of which are used for humans and one for carnivores, such as: Russian vaccines for humans - CoviVac (“Chumakov Institute of Poliomyelitis”) and for animals - Carnivac-Cove (FSBI "ARRIAH"), Chinese vaccines - CoronaVac and PiCoVacc (Sinovac), as well as BBIBP-CorV/COVILO (Sinopharm) and the Indian vaccine Covaxin/BBV152 (Bharat Biotech).

The following documents relating to whole-virion vaccine preparations against COVID-19 and methods for their production based on inactivated viral strains can be considered as analogues of the proposed invention.

International patent application WO 2022038642 A1, publ. 02/24/2022, which discloses a method for producing a whole-virion inactivated vaccine based on a viral antigen from the NIV-2020-770 strain of the SARS-CoV-2 virus and a vaccine preparation obtained by the proposed method.

The known method involves culturing the SARS-CoV-2 strain (NIV-2020-770) in Vero, Vero E6 or Vero-TMPRSS2 cells, followed by inactivation of the virus, purification using size exclusion or affinity chromatography (using Capto core 700, Sepharose CL-4B etc.), carrying out diafiltration and ultracentrifugation, concentrating the viral antigen using tangential flow filtration (TFF) and diluting with phosphate buffer with the addition of an adjuvant.

International patent application WO 2021209060 A1, publ. 10.21.2021, relating to the inactivated PiCoVacc vaccine against SARS-Cov-2 based on the viral antigen from the CN3 strain of the SARS-CoV-2 virus and the method for its production. To produce PiCoVacc, virus strain CN3 is cultured in Vero cells and inactivated with β-propiolactone, the viral antigen is purified using depth filtration or centrifugation and two optimized steps of ion exchange chromatography (IEC) and/or size exclusion chromatography (SEC), before size exclusion chromatography the inactivated virus SARS-Cov-2 is treated with non-restriction endonuclease, and before mixing with adjuvants, filtration through a membrane with a pore diameter of 0.2-0.25 μm is used.

International patent application WO 2021176434 A1, publ. 09/10/2021, discloses a method for obtaining a vaccine against SARS-CoV-2 and a vaccine obtained by this method. Whole virion inactivated COVID-19 vaccine is produced by culturing the virus in Vero/E6 cells followed by inactivation, for example with β-propiolactone, batch adsorption purification using Capto™ Core 700 (CC700) or CC400 chromatography media and ultra/diafiltration (TFF), after which adjuvants are added. The known vaccine contains:

(i) an inactivated SARS-CoV-2 particle, wherein the amount of inactivated SARS-CoV-2 virus per dose in the vaccine is from about 0.01 to 25 mAU (milliabsorptive units x minutes), preferably from about 0.25 to 2.5 mAE as assessed by SEC-HPLC;

(ii) CpG-containing oligodeoxynucleotide (CpG-ODN) from 0.25 to 6 mg/ml, preferably about 2 mg/ml, i.e. 1 mg/dose; And

(iii) aluminum hydroxide 0.1 to 2 mg/ml, preferably 1 mg/ml, i.e. 0.5 mg/dose, with the vaccine delivered in a volume of 0.5 ml.

An effective amount of SARS-CoV-2 vaccine per dose is from 0.05 to 50 mg total protein, preferably about 5 μg total protein/dose, i.e. as measured by (p)BCA. The vaccine provides prevention of SARS-CoV-2 infection without inducing antibody-dependent enhancement (ADE) of SARS-CoV-2-associated disease (COVID-19) or immunopathology in the subject.

It should be noted that the advantages of inactivated vaccines over live ones are that there is no risk of infection, since killed viruses cannot mutate; therefore, they do not have the potential danger of live vaccines, during the replication of which in the host’s body a return to the wild type or recombination with the virus is possible wild type. Compared to vector and mRNA vaccines, the advantages of inactivated vaccines are that the patient is injected with the entire viral capsid, which leads to the formation of immunity against the entire complex of viral proteins, and not just a fragment of the S-protein, previously considered conservative in mutated strains, but real pandemic conditions have shown otherwise, leading to decreased effectiveness of both vector and mRNA vaccines targeting the S protein alone.

Known inactivated vaccines also have disadvantages associated with insufficient immunogenic activity, namely, insufficient formation of T-cell antiviral immunity, which requires the use of adjuvants to increase their effectiveness, difficulties in their production and undesirable side effects.

In this regard, there is a need to develop technology for obtaining new vaccine drugs effective against COVID-19. An ideal COVID-19 vaccine candidate should be highly immunogenic, reliably able to elicit an effective immune response with neutralizing antibodies, provide virtually complete protection against severe forms of COVID-19, and be safe, i.e., without significant side effects.

As the closest analogue (prototype) of the proposed invention, the method for producing a whole-virion inactivated vaccine against SARS-CoV-2, disclosed in WO 2021176434 A1, 09.10.2021, can be selected.

The disadvantages of the prototype include the following:

- use of the original Wuhan strain of coronavirus, which is no longer relevant from an epidemic point of view;

- insufficient purification of the viral antigen, which can lead to undesirable side effects when using a vaccine product containing it;

- the amount of active substance - inactivated viral antigen in the vaccine preparation is expressed in arbitrary units, which do not reflect the absolute amount of antigen and prevent the reproducibility and scaling of the process under GMP conditions.

Thus, in this field of technology, there is a problem in the lack of high-purity effective vaccine preparations against COVID-19 on the pharmaceutical market, obtained by inactivating the viral strain that causes COVID-19.

The objective of the present invention is to develop a method for producing a vaccine effective against coronavirus infection caused by the pathogen COVID-19.

Disclosure of the invention

Unless otherwise defined, all terms and concepts used in the disclosure of the present invention, including technical and scientific terms, have a meaning that is commonly understood by one of ordinary skill in the technical field to which the present invention belongs. In general, the classification used and the methods of cellular and molecular biology, immunology, microbiology, virology, analytical, preparative, medicinal and pharmaceutical chemistry described herein are well known to those skilled in the art and are widely used in the art. Procedures for obtaining viruses and culturing producer cells, isolating the target protein, purifying and testing the activity of the target protein are carried out in accordance with the manufacturer's instructions, as is usually carried out in the art, or as described in detail herein.

All documents cited or referenced in this specification, and all documents cited or referenced in documents cited in this specification, together with any manufacturer's instructions, descriptions and modes of use of the method or products mentioned herein document or any document incorporated by reference herein are incorporated by reference herein and may be used in practicing the proposed invention.

The technical result of the proposed invention is to increase the degree of purification of the viral antigen of the whole-virion inactivated vaccine and the proposed composition of the vaccine based on the purified viral antigen from the unique strain nCoV-19/Russia/SP48-1339/2021 of the SARS-Cov-2 virus deposited in a specialized collection of reference cultures strains of pathogenicity groups II-IV (arboviruses and others) of the Federal State Budgetary Institution “48 Central Research Institute” of the Russian Ministry of Defense under registration No. 1339.

It should also be noted additional advantages of the proposed invention over the closest analogue, which are:

- the use of a highly virulent strain, which remains the same among known variants of the virus and is highly pathogenic for humans, which ensures high immunogenicity and effectiveness of the vaccine, including cross-validation;

- reducing the risk of unwanted side effects when using a vaccine preparation based on an inactivated viral antigen by increasing the degree of its purification;

- unambiguous determination of the dose of the inactivated virus, which allows for research and development (GLP), as well as scaling up production (GMP);

- the use of optimally low concentrations of adjuvants in combination, primarily CpG-ODN, which significantly reduces the safety risks of the drug during mass use.

The specified technical result is achieved through the proposed method of obtaining a vaccine against COVID-19, which includes cultivating a strain of the causative agent of this infection, namely, nCoV-19/Russia/SP48-1339/2021 of the SARS-Cov-2 virus in a monolayer of Vero cells, followed by inactivation virus, purifying the viral antigen and supplementing the vaccine with pharmaceutically acceptable excipients, thereby obtaining a vaccine against said infection.

To obtain the vaccine according to this document, a unique strain nCoV-19/Russia/SP48-1339/2021 is used, which is isolated from a biosample from a patient at the Federal State Budgetary Institution “48 Central Research Institute” of the Russian Ministry of Defense and deposited in a specialized collection of reference cultures of strains of pathogenicity groups II-IV (arboviruses and others) FSBI “48 Central Research Institute” of the Russian Ministry of Defense under registration No. 1339.

In accordance with the present invention, the term "purification of viral antigen" means the main purification methods or main purification steps, purification methods such as chromatography or ultrafiltration/diafiltration, if necessary, various concentration and purification methods can be used, for example, sucrose density gradient centrifugation (batch centrifugation). The chromatography used may be ion exchange chromatography, for example, anion exchange or cation exchange chromatography, multimodal chromatography (chromatography on multimodal matrices), hydrophobic interaction chromatography, combination chromatography, pseudoaffinity chromatography, affinity chromatography, liquid-liquid chromatography and the like.

As used herein, the term "ultrafiltration" or "UF" means a method in which a solution or suspension is filtered through a semi-permeable membrane that retains macromolecules while allowing solvent and small solute molecules to pass through. Ultrafiltration can be used to increase the concentration of macromolecules in a solution or suspension.

As used herein, the term "diafiltration" or "DF" refers to a special class of filtration in which the retained substance is diluted with a solvent and filtered again, thereby reducing the concentration of the soluble components of the permeate. Diafiltration may or may not cause an increase in the concentration of retained components, including, for example, proteins. For example, in continuous diafiltration, solvent is continuously added to the retained material at the rate of filtrate formation. In this case, the volume of the retained substance and the concentration of the retained components do not change during this process. On the other hand, in the case of batch diafiltration or diafiltration followed by dilution, the ultrafiltration step is followed by the addition of solvent on the retained side; if the volume of solvent added from the retained substance side is not equal to or greater than the volume of the formed filtrate, then the concentration of retained components increases. Diafiltration can be used to change the pH value, ionic strength, salt composition, buffer composition, or other properties of a solution or suspension of macromolecules.

The term "ultrafiltration/diafiltration" or "UF/DF" means any process, method or combination of methods of performing ultrafiltration and/or diafiltration sequentially or simultaneously.

In a preferred embodiment of the invention, the method involves purifying the viral antigen using anion exchange and multimodal chromatography with additional ultrafiltration/diafiltration. Most preferably, viral antigen purification is accomplished by sequentially performing an initial ultrafiltration/diafiltration step, anion exchange and multimodal chromatography, and a final ultrafiltration/diafiltration step.

Also, the specified technical result is achieved due to the vaccine against COVID-19 infection, caused by the severe acute respiratory syndrome virus SARS-Cov-2, obtained by the proposed method, based on purified inactivated viral antigen of the causative agent of this infection in an effective amount with pharmaceutically acceptable excipients.

As a purified inactivated viral antigen of the COVID-19 pathogen, a vaccine antigen from the unique strain nCoV-19/Russia/SP48-1339/2021 of the SARS-Cov-2 virus, deposited in a specialized collection of reference cultures of strains of pathogenicity groups II-IV (arboviruses and others) is used ) Federal State Budgetary Institution “48 Central Research Institute” of the Russian Ministry of Defense under registration No. 1339.

The viral antigen is used in an effective amount as part of a pharmaceutical composition, in particular a vaccine, which is a means of inducing a specific immune response against the SARS-Cov-2 virus.

The terms "pharmaceutical composition", "vaccine composition", "vaccine" and the like are used interchangeably herein and refer to a formulation containing a therapeutically effective agent, preferably together with pharmaceutically acceptable excipients. Said pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering said pharmaceutical composition to a subject, preferably for preventing, preventing or preventing the occurrence of coronavirus disease caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by inducing specific immunity .

The terms "subject", "patient", "individual" and the like are used interchangeably herein and refer to any animal amenable to the proposed means and methods. Preferably, the subject, patient or individual is a person of any gender and any age.

The term “prevention,” “avoidance,” or “warning,” and the like, means slowing or preventing the onset of symptoms of a disease or infection, preferably symptoms of COVID-19 disease or infection caused by the SARS-Cov-2 virus.

The term "immune induction" or "induction of an immune response" as used in the present invention refers to the specific control or influence on the activity of an immune response and includes activation of an immune response, stimulation of an immune response, enhancement of an immune response.

The term “specific immunity” as used herein refers to a state of immunity to disease due to the induction of an immune response, preferably immunity to COVID-19 due to the induction of an immune response against the SARS-Cov-2 virus.

The term "disease" refers to a state of health of a subject in which the functions of maintaining homeostasis are impaired and, if the disease is not alleviated, the subject's health continues to deteriorate or does not improve.

The term "infection" as defined herein refers to the infection of a living organism by an infectious agent, the process of its reproduction or development in the body, and the body's response to the presence of an infectious agent in it and to the toxins produced by it, preferably the infection is caused by the SARS-Cov virus -2 and represents COVID-19 infection.

The term “infectious disease” as used herein refers to the development of a disease as a result of an infection, preferably the infectious disease is a coronavirus disease, namely COVID-19, caused by an infectious agent such as the SARS-Cov-2 virus.

The term "effective amount", including a "pharmacologically effective amount", "prophylactically effective amount" or "therapeutically effective amount" refers to an amount that provides a desired response or effect, alone or together with additional doses, in particular, which will relieve to a certain extent from one or more symptoms of the disease for which treatment is being carried out.

The desired response is preferably associated with the prevention of coronavirus disease caused by the SARS-Cov-2 virus, namely, inhibiting or preventing the occurrence of coronavirus disease caused by the SARS-Cov-2 virus. The desired effect may be to delay the onset or prevent the onset of COVID-19.

Also, the desired reaction is associated with the treatment of coronavirus disease caused by the SARS-Cov-2 virus, namely with inhibiting the course of the disease, alleviating the severity of the infection, reducing one or more symptoms of the disease, accelerating the recovery process, reducing complications of the underlying disease and reducing the severity of concomitant pathological conditions. Desired effects include slowing disease progression, such as disease resolution or asymptomatic progression of COVID-19.

The effective amount of the vaccine compositions described herein may depend on the condition being treated, the severity of the disease, individual patient characteristics including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy, if any, specific route of administration and other factors. . Accordingly, the administered doses of the vaccine compositions described herein may depend on a variety of such parameters. In the event that the patient's response or effect is insufficient at the initial dose, higher doses or effectively higher doses achieved through another more precisely localized route of administration may be used.

Preferably, the effective amount herein is an amount sufficient to produce virus-neutralizing antigen-specific antibodies, namely anti-SARS-CoV-2 antibodies, in the subject.

This amount is calculated per dose of the vaccine, which is preferably from 0.5 to 10 ml, most preferably 0.5 ml per administration to the subject.

The amount of said viral antigen in the vaccine composition is from 3 to 60 μg per dose, preferably 7.5-30 μg per dose, most preferably 15 μg per dose.

The concentration of the specified viral antigen in the vaccine composition is from 6 to 120 μg/ml, preferably 15-60 μg/ml, most preferably 30 μg/ml.

The pharmaceutically acceptable excipients used in the pharmaceutical composition of the present invention are one or more adjuvants and a pharmaceutically acceptable diluent, excipient or carrier. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers.

Pharmaceutical compositions may contain one or more adjuvants or may be administered with one or more adjuvants.

The term "adjuvant" refers to a compound that prolongs, enhances or accelerates the immune response. Adjuvants include a heterogeneous group of compounds such as oil emulsions, e.g. Freund's adjuvants, mineral compounds such as alum, e.g. aluminum hydroxide (e.g., but not limited to, commercially available aluminum hydroxide gels - Alhydrogel™ (Brenntag Biosector), Rehydragel™ ( Reheis), Rehsorptar™ (Armour Pharmaceutical), etc.), bacterial products such as Bordetella pertussis toxin or immunostimulating complexes. Examples of adjuvants include, but are not limited to: LPS, GP96, CpG-containing oligodeoxynucleotides (CpG-ODN, for example, but not limited to, such as CPG-ODN 1018 (Dynavax Technologies), CpG-ODN 1826 (Synthol), CpG-ODN 2006 (Invivogen), CpG-ODN 2216 (Invivogen), etc.), lipopeptides such as Pam3Cys, growth factors and cytokines such as monokines, lymphokines, interleukins, chemokines such as IL-1, IL-2, IL-3 , IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN-γ, GM-CSF and others.

The term "excipient" means an agent that can be added to a composition to provide a desired consistency, such as changing bulk properties, improving stability and/or adjusting osmolality. Examples of commonly used excipients include, but are not limited to, sodium chloride; sugars, such as sucrose; polyols, eg mannitol; amino acids, for example, histidine, arginine; surfactants such as polysorbate 80 and polymers.

The term "carrier" refers to a component, which may be natural, synthetic, organic, inorganic, in which the active component is combined to facilitate, enhance or enable administration of the pharmaceutical composition. The carrier used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating agents that are suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "diluent" refers to a diluting and/or liquefying agent. Moreover, the term "diluent" includes any one or more of a liquid, liquid or solid suspension and/or mixing medium. Examples of suitable diluents include a buffer solution such as phosphate buffered saline (PBS), NaCl solution (0.9%), ethanol, glycerol or water.

The pharmaceutical compositions of the present invention may contain suitable additives, salts, buffers, antioxidants, preservatives, osmolality adjusting agents and pH adjusting agents, and optionally other therapeutic agents, for example other immunoregulatory agents.

Suitable additives include chelating agents, e.g. diethylenetriaminepentaacetic acid (DTPA), DTPA-bisamide and others, or calcium chelating complexes, e.g. calcium DTPA, CaNaDTPA-bisamide and others, or optionally calcium or sodium salt additives, e.g. calcium chloride, calcium ascorbate, calcium gluconate, calcium lactate and others.

Suitable buffers for use in the pharmaceutical compositions of the present invention include physiologically biocompatible buffer solutions, for example, PBS and others.

Phosphate-buffered saline (PBS) is a physiologically acceptable water-based buffered saline solution (pH ~ 7.4) containing disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate, which helps maintain a constant pH. A typical 1X PBS chemistry has a final concentration of NaCl - 137 mM/L, KCl - 2.7 mM/L, Na ₂ HPO ₄ - 10 mM/L, KH ₂ PO ₄ - 1.8 mM/L.

If necessary, the composition of the pharmaceutical compositions of the present invention can be supplemented with suitable preservatives, for example, but not limited to, benzalkonium chloride, chlorobutanol, paraben and thimerosal and others.

Pharmaceutically acceptable excipients may be selected based on the intended route of administration and standard pharmaceutical practice.

The pharmaceutical compositions described herein can be administered subcutaneously, intradermally, or intramuscularly. In a preferred embodiment, the pharmaceutical compositions are formulated for systemic administration by intramuscular injection.

Preferably, the vaccine contains adjuvants such as aluminum hydroxide and/or CpG-containing oligodeoxynucleotide (CpG-ODN) in a pharmaceutically acceptable diluent, preferably a complex adjuvant of aluminum hydroxide and CpG-ODN in phosphate-buffered saline (PBS). In this case, aluminum hydroxide is preferably used in an amount of 100 - 1000 μg per dose (at a concentration of 200 - 2000 μg/ml), and CpG-ODN in an amount of 1 - 100 μg per dose (at a concentration of 2-200 μg/ml), most preferably aluminum hydroxide in an amount of 500 μg per dose (at a concentration of 1000 μg/ml) and CpG-ODN in an amount of 10 μg per dose (at a concentration of 20 μg/ml).

In the most preferred embodiment of the invention, the proposed vaccine composition contains a viral antigen at a concentration of 30 μg/ml in combination with aluminum hydroxide at a concentration of 1000 μg/ml and CpG-ODN at a concentration of 20 μg/ml, adjusting the volume of the solution with phosphate-buffered saline (PBS) up to 1 ml, and is used at a dose of 0.5 ml per administration to the subject.

Per one dose for administration to a subject, for example, at a dose of 0.5 ml per intramuscular injection to a subject, the proposed vaccine composition contains 15 μg of viral antigen; 500 mcg aluminum hydroxide; 10 µg CpG-ODN and PBS to 0.5 ml.

The resulting vaccine is packaged in vials for freeze-drying or sale in liquid form. In liquid form, the vaccine preparation is preferably a suspension for intramuscular administration and has the following ratio of components:

- inactivated purified viral antigen of the strain “nCoV-19/Russia/SP48-1339/2021” in an amount of 3 - 60 μg per dose (at a concentration of 6-120 μg/ml), most preferably in an amount of 15 μg per dose (in concentration 30 µg/ml);

- aluminum hydroxide in an amount of 100 - 1000 μg per dose (at a concentration of 200 - 2000 μg/ml), preferably in an amount of 500 μg per dose (at a concentration of 1000 μg/ml);

- CpG-ODN in an amount of 1 - 100 μg per dose (at a concentration of 2-200 μg/ml), most preferably in an amount of 10 μg per dose (at a concentration of 20 μg/ml);

- PBS (sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, water for injection) - the rest.

The inactivated purified viral antigen of the strain “nCoV-19/Russia/SP48-1339/2021” is a suspension containing purified whole-virion capsids of the COVID-19 pathogen, which have completely retained their protein and antigenic structure, but as a result of the destruction of the genetic apparatus have lost their virulence and ability to reproduction.

To prepare a dry preparation, the liquid vaccine is dried by vacuum lyophilization. The finished vaccine is controlled for physical properties, sterility, immunogenicity, harmlessness, avirulence, etc.

The vaccine containing inactivated viral material of the COVID-19 pathogen is stored at a temperature of 2-8°C.

The proposed use of the vaccine ensures the induction of a sustainable immune response against COVID-19, which makes it possible to prevent the development of infection caused by the SARS-Cov-2 virus or reduce at least one of its symptoms.

A method of using the proposed vaccine to induce a specific immune response against an infection involves single or multiple administration of a vaccine composition at a dose effective for the treatment of COVID-19 to a subject in need.

Although it is preferred to stimulate sustained immunity by a single or double dose, additional doses may be administered by the same or a different route of administration to achieve the desired effect. For example, in children, achieving sufficient levels of immunity may require multiple doses of the vaccine at regular intervals throughout childhood, as necessary, maintaining sufficient levels of protection against infection. Similarly, for adults who are particularly susceptible to re-emergence or severe coronavirus infection, such as health or social care workers, the elderly and those with chronic diseases, multiple immunizations may be required to establish and/or maintain protective immune responses. Levels of induced immunity can be monitored, for example, by measuring levels of neutralizing secretory and serum antibodies, and, if necessary, dosage adjustments or repeat vaccinations can be made to induce or maintain required levels of protection against COVID-19.

The proposed use of the vaccine consists of a single or multiple administration, preferably two or three times, dose of the vaccine to induce durable immunity sufficient to prevent the development of the infection or reduce at least one of its symptoms.

Vaccine compositions can be administered on a multiple-dose schedule, for example, by administering a primary administration of the vaccine composition followed by booster administrations. A repeat dose of the composition is administered within a range of two weeks to one year, preferably two weeks to six months, most preferably two to three weeks after the initial administration. If necessary, after the second dose, a third and subsequent doses may be administered at intervals of three weeks to three years, most preferably three weeks to one year after the initial administration.

A method for preventing or treating COVID-19 may involve combination therapy, for example, using the proposed vaccine composition together with other active agents, for example, immunogenic compositions and/or antiviral drugs.

Carrying out the invention

Before describing specific embodiments of the invention, it should be noted that the present invention is not limited to the specific methods, means and methods, products or combinations used, since such methods, means and methods, products or combinations, of course, may vary. It should also be noted that the terminology used in this application is not intended to be limiting, since the scope of the present invention will be limited only by the proposed claims.

Example 1. Preparation of an inactivated viral antigen included in the proposed composition of the vaccine against COVID-19

1.1. Preparation of cell culture

This example shows the use of Vero (B) cell lines and specially selected conditions for this, but other mammalian cells suitable for cultivating coronaviruses can be used, for example, Vero cell lines, Vero C1008, Vero E6, Vero-TMPRSS2, GMK, SPEV , MDCK, COS, VNK-21 and other suitable conditions (see Guide to Virology: Viruses and Viral Infections of Humans and Animals / ed. D. K. Lvov, Moscow: Medical Information Agency, 2013. - 1197 pp.; Medical microbiology, virology and immunology: textbook in 2 volumes / edited by V. V. Zverev, M. N. Boychenko, Moscow, GEOTAR-Media, 2014. - T. 1 - 447 pp.; T. 2 - 477 pp.; Medical microbiology, virology and immunology: textbook / edited by A. A. Vorobyov, 2nd ed., revised and supplemented - Moscow: MIA, 2006. - 702 pp.).

Obtaining a monolayer cell culture of the Vero (B) line includes the following stages: re-preservation of the cell culture, obtaining a seed culture, reseeding the cell culture into a plastic roller bottle.

As the starting object, a Vero (B) cell culture is used, stored in ampoules at a temperature of minus 196°C (liquid nitrogen), in the form of a suspension of frozen cells in fetal calf serum (FCS) with the addition of a preservative - DMSO.

1.1.1. To dilute the thawed cell suspension, nutrient medium 199 with Earle's salts supplemented with FBS (Gibco), 200 mM glutamine solution and gentamicin sulfate solution is used. The ampoule with frozen seed material of Vero cells (B) is thawed in a water bath at a temperature of 35 to 37 ° C until the entire cell suspension passes into the liquid phase. After this, the ampoule is opened under aseptic conditions in a laminar flow hood and its contents are transferred into a plastic tube with a volume of 50 cm ³ with the gradual addition of a nutrient medium. No later than 20 minutes after dilution, a sample is taken from a test tube with a re-preserved cell suspension, in which the proportion of viable cells and microbiological purity are determined by microscopy. The re-preserved culture must be microbiologically pure and contain at least 85% viable cells.

1.1.2. Obtaining seed culture

To grow cells in plastic flasks and rollers, medium 199 with Earle's salts supplemented with fetal bovine serum (FCS) (Gibco), 200 mM glutamine solution and gentamicin sulfate solution is also used. The unpreserved cell suspension is transferred into a plastic bottle with a working surface area of 75 cm ² in a seed concentration of 4.0 * 10 ⁴ cells * cm ² . The bottles are placed in a horizontal position in a CO ² incubator and cultured for 4 days at a temperature of about 37°C, replacing the nutrient medium with a fresh one 24 hours after adding the cell suspension to a plastic bottle. The state of the developing monolayer is monitored by viewing it under a microscope. During the passage, an even, continuous monolayer should form without signs of cell degeneration.

At the end of the passage, the formed monolayer of cells is transferred to a suspended state using a sterile Versene solution and a chymopsin solution. The number of cells and the proportion of viable cells in the suspension are determined. The final cell concentration must be at least 0.9*10 ⁶ cells*cm ³ . The suspension in a seed concentration of at least 4*10 ⁴ cells*cm ² is transferred to a plastic mattress (175 cm2) and the volume of the medium is adjusted to 75 cm ³ . After this, the second passage of growing a monolayer culture begins with a cultivation duration of 3 days.

1.1.3. Reseeding the cell culture into a plastic roller bottle

Growing a monolayer cell culture is carried out in plastic roller bottles with a working surface area of 800 cm ² and 4250 cm ² , respectively, into which cells from the previous stage are transferred by sequential subculture in a seed concentration of at least 4 * 10 ⁴ cells * cm ² . First, cultivation is carried out for 3 days in roller bottles with a working surface area of 800 cm ² , which are placed in a horizontal position on a roller unit in a CO ₂ incubator at a temperature of about 37°C with a rotation speed of 0.4 revolutions per minute.

The state of the developing monolayer is monitored by viewing it under a microscope. During the passage, an even, continuous monolayer should form without signs of cell degeneration. Microbiological purity is controlled by microscopy.

Then the cells are reseeded at a seeding concentration of 1.4 * 10 ⁴ cells * cm ² - 1.6 * 10 ⁴ cells * cm ² on ribbed rollers with a surface area of 4250 cm ² and incubated for 1 day.

1.2. Obtaining virus biomass

Obtaining virus biomass includes the following stages: obtaining virus biomass, assessing the quality of the resulting biomass and inactivating the virus biomass.

1.2.1. To obtain biomass, the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-CoV-2 virus belonging to the genetic line B.1.617.3 is used (Deposited in the Specialized Collection of Reference Cultures of Strains of II-IV Pathogenicity Groups (arboviruses and others) of the Federal State Budgetary Institution "48 Central Research Institute" of the Russian Ministry of Defense, inventory number of strain 1339).

A working (seed) culture of the virus is obtained by cultivating on a daily monolayer of Vero (B) cells in plastic bottles with a surface area of 25 cm ² . At an infectious dose of 0.1 PFU*cell ^-1 , the cultivation time is 24 hours (controlled by the onset of a cytopathic effect). The biological activity of the SARS-CoV-2 virus in the resulting biomass is at least 6.0 lg PFU*ml ^-1 , in the absence of foreign microflora.

Then, to obtain virus biomass, ribbed rollers with a surface area of 4250 cm ² are used with a monolayer of Vero (B) cells 1 day old. To infect the monolayer, maintenance medium 199 with Hanks salts supplemented with FBS (Gibco), 200 mM glutamine solution and Antibiotic-antimecotic solution (100x) (Gibco) is used. The filling volume of the roller is 600 ml; a virus culture with an infectious dose of 0.1 PFU*cell ^-1 is added directly to the medium. The growth medium is drained from rollers with a daily monolayer and the supporting medium is filled with a virus culture, cultivated at a temperature of about 37°C and a rotation speed of 0.4 revolutions per minute for approximately 50 hours (monitored by the onset of a cytopathic effect by microscopy). Once the virus activity reaches at least 6.0 lg PFU*ml ^-1, a culture liquid containing biomass of the SARS-CoV-2 virus in a concentration of at least 10 ⁸ viral particles/ml is extracted.

Next, the resulting biomass of the virus from the rollers is poured into a separate container, samples are taken for quality control and transferred to the inactivation stage.

1.2.2. Assessment of the quality of the obtained biomass

Determination of biological activity is carried out by the formation of negative colonies under an agar coating on Vero C1008 cell culture. From the sample taken after completion of cultivation, successive tenfold dilutions are prepared in sterile Hanks' saline solution with 2% FCS and antibiotics. The three highest dilutions are used to infect a monolayer of Vero C1008 cell culture.

Vials with a one-day monolayer of Vero C1008 cell culture, selected for the experiment, are removed from the thermostat and the growth medium is removed from them. Add 0.5 ml of the appropriate dilution to each vial, the calculated concentration of the virus in which is 1.0; 10.0; 100.0 PFU*ml ^-1 , and left in a thermostat at a temperature of about 37° C for virus adsorption. After one hour, 10 ml of primary agar coating is added to each bottle and incubation is continued. On the 3rd day of incubation, for the purpose of staining, a secondary agar coating containing neutral red is added to the vials. The results are recorded after 1 day of incubation at the same temperature.

Determination of microbiological purity is carried out by inoculation on a thioglycollate medium; for this, a sample of the virus biomass taken after completion of cultivation is added to 3 glass test tubes with a liquid thioglycollate medium, then incubated at a temperature of about 37 ° C. A conclusion about microbiological purity is made if there is no growth in test tubes with thioglycollate medium after 7 days.

1.2.3. Inactivation of virus biomass

Beta-propiolactone is diluted with saline solution in a ratio of 1:10. Diluted beta-propiolactone is added with a pipette to the biomass of the SARS-CoV-2 virus to a final inactivating concentration of 0.0167% (dilution 1:6000). The inactivation process is carried out by mixing the biomass of the SARS-CoV-2 virus in a container at a temperature of 20 to 25°C for 4 hours and then 2 hours at a temperature of 37°C. The inactivated biomass of the SARS-CoV-2 virus for transfer to the purification stage is stored at a temperature of 2 to 6°C under conditions that prevent accidental opening of the container.

Example 2 Purification of an inactivated viral antigen included in the proposed composition of a vaccine against COVID-19

This example shows the use of a sequential initial step of ultrafiltration/diafiltration, anion exchange and multimodal chromatography and a final step of ultrafiltration/diafiltration to purify a viral antigen, but purification can also be carried out using a different sequence of stages, other modes, reagents, materials and equipment (see Komissarov A.V., Aleshina Yu.A. and others, The use of ultrafiltration for the concentration and purification of antigens, Problems of especially dangerous infections, issue 1, 2015, pp. 79-84; international application WO 2013/010797, published 24.01 .2013; EAPO patent EA001414B1, published 02.26.2001).

2.1. Ultrafiltration/diafiltration stage 1 (UF/DF 1)

The material of the initial inactivated virus-containing liquid (VCL) is filtered before the process using a system of sequentially installed Opticap filters and a sterilizing filter. The resulting VSG material is concentrated on a SARTOCON Slice Cassette (Sartorius) with a cutoff of 300 kDa by a total of 25 times. After concentration is complete, the concentrate is dialyzed against solution A (15 mM Na-P, 600 mM sodium chloride, 0.05% polysorbate 80, pH 7.5 ± 0.1, with specific electrical conductivity (SEC) 55.5 ± 3.5 mS /cm) in a volume of 10 diafiltration volumes (DV). The next step is to dialyze the concentrate against solution B (15 mM Na-P, 600 mM sodium chloride, pH 7.5 ± 0.1, SEP 55.5 ± 3.5 mS/cm) in a volume of 10 DV. The final stage of the UF/DF stage is reconcentration to 70% of the retentate volume and displacement of up to 100% of the retentate volume from the cassette with solution B. This eliminates the loss of intermediate product in the tangential filtration cassette. At the end of the stage, the intermediate product is filtered through a filter with a pore size of 0.22 microns and stored at a temperature of 2-8°C. The yield is 80%. At this stage, the material is concentrated, all low-molecular impurities (less than 300 kDa) are removed, the dye is removed from the VSG (due to dialysis with a buffer containing polysorbate) and the material is prepared for the subsequent stages of chromatographic purification.

2.2. Anion exchange chromatography

The sterile intermediate after stage UF/DF 1 is checked for pH and SEP, which should be in the range of 7.5±0.1 and 53-57 mS/cm, respectively. UEP is titrated with 1 M sodium chloride solution. An XK 26 column (GE Healthcare) with Q Sepharose FF sorbent (GE Healthcare) is sanitized with 1 M sodium hydroxide solution and the sorbent is recharged with 1 M sodium chloride. Next, equilibrating buffer C (15 mM Na-P, 600 mM sodium chloride, pH 7.5 ± 0.1, SEP 55.5 ± 3.5 mS/cm) is passed through the column with the sorbent. speed 170 cm/h. The next step after the pre-equilibrated sorbent is the application of the prepared material at a speed of 170 cm/h, with a contact time of about 10 minutes with the sorbent. Fraction collection begins immediately after the optical density increases to more than 2 mOU (optical density units). Upon completion of application, the sorbent is washed with equilibrating buffer solution C at a speed of 170 cm/h until the optical density decreases to less than 2 mFU, at which point the collection of the target fraction is completed. The intermediate product from the anion exchange stage of chromatography is sterilely filtered through a filter with a pore size of 0.22 microns and stored at a temperature of 2-8°C. This stage is carried out in filtration mode, which allows the material to be purified from free DNA that is retained on the sorbent.

2.3. Multimodal chromatography

The sterile intermediate after the anion exchange chromatography stage is checked for pH and SEP, which should be in the range of 7.5±0.1 and 54-57 mS/cm, respectively. If necessary, the UEP is titrated using a solution of 1 M sodium chloride. An ECO PLUSE TAC 35*500 (YMC) column with Capto Core 700 sorbent (Cytiva) is sanitized with 0.5 M sodium hydroxide, followed by passing 30% isopropyl alcohol through the sorbent. Next, equilibrating buffer D (15 mM sodium phosphate, 600 mM sodium chloride, pH 7.50±0.1, SEP 55.5±3.5 mS/cm) is passed through the column with the sorbent for at least 5 column volumes at speed 124 cm/h. The next step, after equilibrating the sorbent, is to apply the prepared material at a speed of 124 cm/h, while the contact time with the sorbent should be at least 10 minutes. Fraction collection begins immediately after the optical density increases to more than 2 mFU. Upon completion of application, the sorbent is washed with equilibrating buffer solution D at a speed of 124 cm/h until the optical density decreases to less than 2 mFU, at which point the collection of the target fraction is completed. The intermediate product from the multimodal chromatography stage is sterilely filtered through a filter with a pore size of 0.22 microns and stored at a temperature of 2-8°C. At this stage, fragments of the S- and N-proteins of the viral envelope are removed, which makes it possible to obtain whole-capsid viral material.

2.4. Ultrafiltration/diafiltration stage 2 (UF/DF 2)

The filtrate from the previous stage, pre-warmed to 18-25°C, is concentrated 2 times. Then the material is dialyzed against solution E (15 mM Na-P, 140 mM sodium chloride, pH 7.2 ± 0.1 UEP 15.0 ± 2.0 mS/cm) in a volume of 10 DV. Next, the material, if necessary, is concentrated to a volume equal to 1/50 of the initial volume of the VSL entering the process. To displace material from the Pellicon 2 MAXI (MERCK) cassette, the active pharmaceutical substance (API) is reconcentrated to 70% of the retentate volume and displaced from the cassette with solution E to 100% of the retentate volume. This eliminates the loss of intermediate product in the tangential filtration cassette. Upon completion of the stage, the intermediate product is filtered through a filter with a pore size of 0.22 μm to obtain a purified viral antigen in a concentration of at least 30 μg/ml and stored at a temperature of 2-8 ^° C. At this stage, the concentration of the antigen in the API is adjusted, and formulation is also carried out product into the final storage buffer, which is used to obtain finished forms of the vaccine composition in Example 3.

Example 3 Analysis of the resulting product and formulation of the finished vaccine composition

3.1. Conduct an analytical assessment of the product obtained according to Example 2

3.1.1. “Protein Quantification” method based on the ELISA method using monoclonal antibodies to the SARS-CoV-2 spike protein (CR3022, GTX01555, GeneTex). According to SDS-PAGE data, the API was characterized by a high purity of the product - more than 96% and according to SE-HPLC data by an insignificant amount of aggregates - less than 4%.

3.1.2. Methodology “Determination of authenticity”, based on the ELISA method. The product induced the formation of specific antibodies to the SARS-CoV-2 coronavirus when immunizing Balb/c mice. A pronounced immune response was recorded with single and double intramuscular administration of the compositions at a dose of 0.5 ml/animal with a peak titer of specific antibodies on the 18th day of the experiment and persisting until the 40th day. The appearance of specific antibodies was recorded 14 days after the first vaccination of animals.

3.1.3. Method “Determination of residual proteins of cell culture” using F975 kits (Cygnus Technologies). At the moment, purification achieves a residual content of impurity proteins of less than 200 ng/ml (100 ng/dose).

3.1.4. Method “Determination of residual DNA of cell culture” using Vero Host Cell DNA Residue Detection Kits (Yeasen Biotechnology). At the moment, purification achieves a residual contaminant DNA content of less than 20 ng/ml (10 ng/dose).

3.1.5. Methodology “Specific safety (completeness of inactivation) in vitro.” Vero cells are seeded into 6 well plates/culture dishes. When a monolayer is reached, the nutrient medium is changed and various concentrations of inactivated viral suspension are added to the wells with cells. Each well is then coated with carboxymethylcellulose and incubated for 3 days. At the end of incubation, the coating mixture is removed from all wells, the cells are fixed with 4% paraformaldehyde and stained with crystal violet dye solution. After staining, the cells are washed with purified water and the PFU is counted for each concentration of the inactivated viral suspension. Based on the test results, there are no PFUs available for each concentration.

3.1.6. Methodology “Determination of toxicity in vivo”.

Based on the results of two preclinical studies (PCT) “Preclinical study of the toxicity, safety and immunogenicity of the vaccine preparation after double intramuscular injection in rhesus monkeys (GLP)” and “Study of the toxicity of the inactivated sorbed vaccine against the new coronavirus infection COVID-19 with repeated administration to rabbits” were obtained results confirming the safety of the vaccine, since no immunotoxicity or allergenicity of the vaccine was detected.

3.2. Obtaining finished forms of the vaccine composition

Before filling the finished product into vials, under aseptic conditions, the API is mixed with a complex adjuvant and diluted with a formulation buffer solution to obtain the target concentration (see Tables 1-3). If necessary, the drug is freeze-dried; the concentration of the components in Tables 1-3 is indicated before drying.

Example 4 Evaluation of the immunogenic and protective properties of samples of inactivated purified sorbed Covid vaccine containing various doses of SARS-CoV-2 virus antigens and various adjuvants.

I. Materials used:

1. Vaccine samples: 30/0/0; 30/500/0; 30/500/10; 15/0/0; 15/500/0; 15/500/10; 7.5/500/10, where the first number is the concentration of the viral antigen (µg per dose), the second is the concentration of aluminum hydroxide (µg per dose) and the third is the concentration of CpG-ODN in the vaccine samples (µg per dose).

2. Samples for adjuvant control: 0/0/0; 0/500/0; 0/500/10, where the first number is the concentration of the viral antigen (µg per dose), the second is the concentration of aluminum hydroxide (µg per dose) and the third is the concentration of CpG-ODN in the vaccine samples (µg per dose).

3. Syrian hamsters weighing 40-60 g - 200 heads;

4. Infectious material: SARS-CoV-2 virus, variant A, strain nCoV-19/Russia/SP48-1339/2021, strain culture passed 3 passages on Vero B cell culture, accumulation substrate - Vero B cell culture, stored in cryovials at temperatures from minus 18°C to minus 22°C, biological activity - 6.8 1g PFU/ml.

II. Methodology for conducting the experiment

1. Immunization

Vaccine samples are prepared based on different doses of the SARS-CoV-2 virus antigen: 30, 15 and 7.5 μg of protein per dose. In addition, samples with doses of 30 and 15 μg of protein are presented in three variants: without adjuvants, with aluminum hydroxide (500 μg), with aluminum hydroxide (500 μg) in combination with CpG (10 μg).

Syrian hamsters were divided into 10 groups of 20 animals each. Animals in each group were administered one of the vaccine samples intramuscularly, 0.5 ml, twice with an interval of 21 days. 21 days after the second immunization, 5 animals from each group were immunized with the same samples in the same doses and in the same way for the third time.

2 Assessment of the protective properties of antigens

21 days after the second immunization, 10 individuals from each group were infected with the virulent strain intranasally at a dose of 1 ⋅ 10 ⁵ PFU/individual. On the 2nd day (3 heads), 4th day (4 heads each) and 6th day (3 heads each), the animals in each group were sacrificed and lung samples were taken in compliance with aseptic rules. Lung samples from each group for each period were pooled, and the tissues were then homogenized in sterilized chilled porcelain mortars with the addition of sterilized river sand. From the resulting homogenate, a 10% suspension was prepared in Hanks' solution with the addition of 2% FBS and antibiotics (streptomycin sulfate and benzylpenicillin sodium salt, 200 U/ml). For titration, a daily monolayer of Vero B cells was used, which, after removing the growth medium, was infected with successive 10-fold dilutions of virus-containing material, adding 0.5 ml of the appropriate dilution to 2 bottles of cell culture. By rocking the bottle, the inoculum was evenly distributed throughout the entire monolayer. The vials were laid horizontally, with the surface with the infected cell monolayer at the bottom. The bottles were incubated in a thermostat at a temperature of 37.0±0.5°C for 60 minutes. After incubation, the inoculum was removed by pipetting and 10 ml of primary agar coating was added to each bottle. Next, the vials were again laid horizontally (the surface with the infected monolayer was at the bottom). After the coating hardened, the bottles were turned over with the monolayer up and placed in a thermostat at a temperature of 37.0 ± 0.5 ° C for 48 hours. After the incubation period for the purpose of staining the cell culture monolayer, 10 ml of secondary agar coating containing neutral red was added to the vials and continued to incubate at a temperature of 37.0±0.5°C for 24 hours, after which the number of negative colonies was counted.

Protective properties against coronavirus were assessed by the concentration of the virus in the lungs of vaccinated Syrian hamsters on days 2, 4 and 6 after intranasal infection with the virulent strain at a dose of 10 ⁵ PFU/individual. The protection of animals was assessed using the virological lung protection index on days 2, 4 and 6 after infection by the decrease in the concentration of coronavirus in the lungs of immunized Syrian hamsters compared to the concentration in the lungs of non-immunized animals.

3 Assessment of immunogenicity of antigens

21 days after the second immunization and 21 days after the third immunization, total blood was taken from 5 animals from each group, from which blood sera were obtained to determine the titer of specific virus-neutralizing antibodies in the neutralization reaction with the virulent SARS-CoV-2 virus, to reduce plaque-forming activity virus on a monolayer culture of Vero cells under an agar coating (a variant of the reaction is a constant dose of the virus and serum dilution).

We studied the blood serum of experimental animals, as well as a positive control sample (blood serum of a recovered person, known to contain specific antibodies to the SARS-CoV-2 virus in a titer of 1:40).

Serum dilutions were prepared using Hanks solution with 2% FBS and antibiotics (streptomycin sulfate and benzylpenicillin sodium salt) at 200 U/ml in twofold steps, starting with a dilution of 1:10. A working dilution of the virus was prepared using the same dilution liquid. In the prepared dilution, the concentration of the SARS-CoV-2 virus was 200 PFU/ml.

A mixture of equal volumes of serum and a working dilution of the SARS-CoV-2 virus culture was incubated in test tubes for 60 minutes at a temperature of 36.5 to 37.5°C, then 0.5 ml was added to vials with a monolayer of Vero B cells, previously by removing the growth medium from them. After adsorption of the AG+AT complex on the cells for 60 minutes at a temperature of 36.5 to 37.5°C, the inoculum was decanted, then a primary agar coating developed for the SARS-CoV-2 virus was applied, and the monolayer was further incubated at a temperature of 36.5 to 37.5°C for two days.

After three days, a secondary agar coating was applied to the infected monolayer of cells for staining with a 0.1% neutral red solution and incubated for 24 hours at a temperature of 36.5 to 37.5°C, then a visual count of negative colonies in the vials was carried out.

The antibody titer of the test serum was taken to be the highest dilution of blood serum, in which suppression of negative colonies formed by the SARS-CoV-2 virus was detected by 50% or more compared to the negative control (virus + dilution liquid).

III. Experiment results

The results of assessing the protective and immunogenic properties of antigens are presented in Tables 4 and 5.

Vaccine samples 30/500/10 and 15/500/10 had the most pronounced protective activity. After infection of hamsters immunized twice with these samples with a virulent strain of the SARS-CoV-2 virus at a dose of 10 ⁵ PFU/individual, the virus was not isolated in the lungs of the animals throughout the entire observation period: 2, 4 and 6 days after infection. Antibody titers in most animals were >1:40, with seroconversion rates ranging from 80 to 100%.

Vaccine samples without adjuvants had the least protective activity. The addition of aluminum hydroxide enhanced the protective properties of antigens at doses of 15 and 30 μg per dose. Antibody titers did not exceed 1:20, seroconversion ranged from 60 to 100%. Both adjuvants did not affect the level of virus accumulation in the lungs, that is, they did not aggravate the process.

According to the results of this experiment, a dose of viral antigen of 15 mcg per dose is optimal in terms of protective and immunogenic properties.

The immunogenic properties of the study vaccine against different strains of the SARS-CoV-2 virus, known as “Delta” and “Omicron”, were also studied in monkeys based on the level of virus-neutralizing antibodies. According to the data obtained from the results of the DCT “Study of the immunogenicity and safety of the inactivated sorbed vaccine against the new coronavirus infection COVID-19 when modeling the SARS-CoV-2 disease in rhesus monkeys,” the proposed vaccine has high immunogenicity and effectiveness, including crossover.

Tables

Table 1 - Composition of the vaccine composition per 1 ml, dose is 0.5 ml (option 1) Vaccine components Concentration, µg/ml Purified inactivated viral antigen from the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-Cov-2 virus 6 aluminum hydroxide 200 CpG-containing oligodeoxynucleotide (CpG-ODN) 2 Phosphate-buffered saline (PBS) rest

Table 2 - Composition of the vaccine composition per 1 ml, dose is 0.5 ml (option 2) Vaccine components Concentration, µg/ml Purified inactivated viral antigen from the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-Cov-2 virus thirty aluminum hydroxide 1000 CpG-containing oligodeoxynucleotide (CpG-ODN) 20 Phosphate-buffered saline (PBS) rest

Table 3 - Composition of the vaccine composition per 1 ml, dose is 0.5 ml (option 3) Vaccine components Concentration, µg/ml Purified inactivated viral antigen from the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-Cov-2 virus 120 aluminum hydroxide 2000 CpG-containing oligodeoxynucleotide (CpG-ODN) 200 Phosphate-buffered saline (PBS) rest

Table 4 - Results of assessing the protective properties of vaccine samples against coronavirus infection with double immunization according to the scheme (0+21) Vaccine sample code Amount of antigen per dose, mcg Presence of adjuvants, dose, mcg Number of animals Level of virus accumulation in the lungs, 1g PFU/ml Lung protection index on ... day after infection 2 4 6 2 4 6 0/0/0 ¹ 0 No 20 5.3 4.7 3.6 - - - 0/500/0 A1(OH) _3,500 20 5.1 5.1 3.7 - - - 0/500/10 A1(OH) ₃ , 500 + CpG, 10 20 3.7 5.0 3.1 - - - 30/0/0 thirty No 20 4.3 4.8 2.3 1.0 0 1.3 30/500/0 A1(ON)z, 500 20 3.4 1.8 1.2 1.9 2.9 2.4 30/500/10 A1(OH)z, 500 + CpG, 10 20 0 0 0 >5.3 >4.7 >3.6 15/0/0 15 No 20 5.3 4.2 1.4 0 0.5 2.2 15/500/0 A1(OH) _3,500 20 2.9 2.0 0 2.4 2.7 >3.6 15/500/10 A1(OH)3, 500 + CpG, 10 20 0 0 0 >5.3 >4.7 >3.6 7.5/500/10 7.5 A1(OH)3, 500 +CpG, 10 20 3.3 1.0 0 2.0 3.7 >3.6 Herd control
(not immunized, without infection) 6 0 0 0 - - -

¹ Note. The control group was taken to be 0/0/0 - animals that were injected with a two-fold dilution liquid without adjuvants.

Table 5 - Results of assessing the titers of virus-neutralizing antibodies in the blood serum of immunized hamsters Antigen code Hamster blood serum no. Serum dilution Number of bottles Number of PFU/
bottle Antibody titer Seroconversion, % 1 2 3 4 5 6 7 8 0/0/0 1 1:10 2 116 120 <1:10 0% 2 1:10 2 118 124 <1:10 3 1:10 2 127 120 <1:10 4 1:10 2 116 119 <1:10 5 1:10 2 121 117 <1:10 0/500/0 6 1:10 2 123 126 <1:10 0% 7 1:10 2 114 127 <1:10 8 1:10 2 124 122 <1:10 9 1:10 2 125 128 <1:10 10 1:10 2 116 120 <1:10 0/500/10 eleven 1:10 2 112 125 <1:10 0% 12 1:10 2 116 127 <1:10 13 1:10 2 111 123 <1:10 14 1:10 2 119 130 <1:10 15 1:10 2 121 128 <1:10 30/0/0 16 1:10 2 114 119 <1:10 0% 1:20 2 118 113 1:40 2 112 115 17 1:10 2 123 115 <1:10 1:20 2 116 111 1:40 2 114 108 30/0/0 18 1:10 2 116 122 <1:10 1:20 2 104 109 1:40 2 118 113 19 1:10 2 109 117 <1:10 1:20 2 118 115 1:40 2 120 115 20 1:10 2 117 105 <1:10 1:20 2 111 108 1:40 2 114 112 30/500/0 21 1:10 2 0 0 >1:40 100% 1:20 2 3 3 1:40 2 17 15 22 1:10 2 54 56 1:10 1:20 2 77 79 1:40 2 126 120 23 1:10 2 52 56 1:10 1:20 2 80 75 1:40 2 119 114 24 1:10 2 60 53 1:10 1:20 2 115 117 1:40 2 118 120 25 1:10 2 55 52 1:10 1:20 2 90 82 1:40 2 116 123 30/500/10 26 1:10 2 0 0 >1:40 80% 1:20 2 0 0 1:40 2 0 0 30/500/10 27 1:10 2 0 0 >1:40 1:20 2 0 0 1:40 2 0 0 28 1:10 2 71 69 <1:10 1:20 2 118 116 1:40 2 121 126 29 1:10 2 0 0 >1:40 1:20 2 0 0 1:40 2 0 0 thirty 1:10 2 0 0 >1:40 1:20 2 0 0 1:40 2 5 2 15/0/0 31 1:10 2 94 79 <1:10 0% 1:20 2 101 102 1:40 2 >100 >100 32 1:10 2 81 92 <1:10 1:20 2 >100 >100 1:40 2 >100 >100 33 1:10 2 >100 >100 <1:10 1:20 2 >100 >100 1:40 2 >100 >100 34 1:10 2 >100 >100 <1:10 1:20 2 >100 >100 1:40 2 >100 >100 35 1:10 2 >100 >100 <1:10< 1:20 2 >100 >100 1:40 2 >100 >100 15/500/0 36 1:10 2 4 3 1:20(1:30) 60% 1:20 2 17 28 1:40 2 58 59 37 1:10 2 89 78 <1:10 1:20 2 >100 >100 1:40 2 >100 >100 38 1:10 2 8 10 1:20 1:20 2 29 31 1:40 2 80 62 39 1:10 2 >100 >100 <1:10 1:20 2 >100 >100 1:40 2 >100 >100 40 1:10 2 eleven eleven 1:20 1:20 2 38 35 1:40 2 75 85 15/500/10 41 1:10 2 0 0 >1:40 100% 1:20 2 0 0 1:40 2 3 5 42 1:10 2 0 1 >1:40 1:20 2 2 2 1:40 2 13 15 43 1:10 2 0 0 >1:40 1:20 2 0 0 1:40 2 1 1 44 1:10 2 23 24 1:10 1:20 2 57 58 1:40 2 92 95 15/500/10 45 1:10 2 0 0 >1:40 1:20 2 0 0 1:40 2 0 0 7.5/500/10 46 1:10 2 29 14 1:20 20% 1:20 2 40 thirty 47 1:10 2 >100 >100 <1:10 1:20 2 >100 >100 48 1:10 2 76 76 <1:10 1:20 2 98 95 49 1:10 2 >100 >100 <1:10 1:20 2 >100 >100 50 1:10 2 75 74 <1:10 1:20 2 94 >100 Control of positive serum with antibody titer 1:40 1:20 2 thirty 22 1:40 - 1:40 2 40 35 Control (working virus dilution + dilution liquid 1:1) 3 108 115 74 Average number of BFU-132 Serum control (pure), dilution 1:2 0.5 ml dil. aliquots of serum + 0.5 ml of medium without serum (incubated at 37C, 60 min.) 0
No plaques Note. Estimated dose of virus: 100 PFU (200 PFU 1 ml + 1 ml dilution liquid) kept at 37 ° C for 60 minutes.

Claims (7)

1. A method for producing a vaccine against COVID-19, including cultivating the pathogen strain in a monolayer of Vero cells, followed by inactivation of the virus, purification of the viral antigen and addition of pharmaceutically acceptable excipients to the vaccine composition, thus obtaining a vaccine against COVID-19, characterized in that provides for the use of the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-Cov-2 virus, deposited in a specialized collection of reference cultures of strains of pathogenicity groups II-IV (arboviruses and others) of the Federal State Budgetary Institution "48 Central Research Institute" of the Russian Ministry of Defense under registration No. 1339, and performing viral antigen purification using anion exchange and multimodal chromatography with additional ultrafiltration/diafiltration. 2. The method according to claim 1, characterized in that the purification of the viral antigen is carried out by sequentially carrying out the initial stage of ultrafiltration/diafiltration, anion exchange and multimodal chromatography and the final stage of ultrafiltration/diafiltration. 3. Vaccine against COVID-19 based on inactivated viral antigen from the nCoV-19/Russia/SP48-1339/2021 strain of the SARS-Cov-2 virus, deposited in a specialized collection of reference cultures of strains of pathogenicity groups II-IV (arboviruses and others) of the Federal State Budgetary Institution "48 Central Research Institute" of the Russian Ministry of Defense under registration No. 1339, in an effective amount with pharmaceutically acceptable excipients. 4. The vaccine according to claim 3, characterized in that the pharmaceutically acceptable excipients are adjuvants and a pharmaceutically acceptable diluent, excipient or carrier. 5. The vaccine according to claim 3, characterized in that aluminum hydroxide and a CpG-containing oligodeoxynucleotide (CpG-ODN) are used as an adjuvant. 6. The vaccine according to any one of claims 3-5, characterized in that the vaccine contains a viral antigen and a complex adjuvant - aluminum hydroxide and CpG-ODN in a pharmaceutically acceptable diluent - phosphate-buffered saline (PBS). 7. The vaccine according to claim 6, characterized in that the vaccine contains a viral antigen at a concentration of 30 μg/ml in combination with aluminum hydroxide at a concentration of 1000 μg/ml and CpG-ODN at a concentration of 20 μg/ml in PBS.