RU2766292C1 - Covid-19 vaccine composition - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: what is described is a pharmaceutical composition for inducing a specific immune response against the acute respiratory syndrome virus SARS-CoV-2, containing the nucleocapsid protein SARS-CoV-2. Vaccine has the following composition per 1 ml: nucleocapsid protein NSARS-CoV-2, having the amino acid sequence SEQ ID NO: 1 20–100 mcg; squalane 15–40 mg; α-tocopherol 5–15 mg; polysorbate 80 1–10 mg; disodium phosphate 0.1–2.0 mg; monopotassium phosphate 0.01–0.25 mg; potassium chloride 0.01–0.2 mg; sodium chloride 0.1–8.5 mg; water for injections up to 1 ml. Also disclosed is a method of producing said composition, which involves: providing a nucleocapsid protein NSARS-CoV-2, having the amino acid sequence SEQ ID NO: 1, in amount of 20 to 100 mcg/ml; dissolving the protein in the aqueous solvent and bringing the protein N content in the solution to the required concentration; preparation of an aqueous solution of an adjuvant; mixing an aqueous solution containing protein N and an aqueous solution of an adjuvant; homogenizing the obtained mixture until a color-uniform solution is obtained; adjusting the pH of the obtained composition to 7.2–7.6.

EFFECT: invention enables to obtain a stable composition which, when introduced into the body, can induce an immune response and induce a specific immune response against SARS-CoV-2.

10 cl, 3 dwg, 8 tbl, 7 ex

Description

Technical field

The invention relates to biotechnology, immunology and virology. A pharmaceutical composition for induction of specific immunity against SARS-CoV-2 acute respiratory syndrome virus has been created, containing the NSARS-CoV-2 nucleocapsid protein having the amino acid sequence of SEQ ID NO: 1, in an amount from 20 to
100 µg/ml and pharmaceutically acceptable excipients selected from the group consisting of buffer agents, surfactants, adjuvant, isotonic agent in pharmaceutically acceptable amounts.

Prior Art

In 2019, a new single-stranded RNA-containing virus belonging to the Coronaviridae family, to the Beta-CoV B lineage, was discovered in China. The new coronavirus quickly spread across all continents, causing severe conditions: viral pneumonia, vascular disorders, exacerbation of chronic diseases. In February 2020, the World Health Organization (WHO) officially named the new virus SARS-CoV-2, and the disease in the case of this virus was named COVID-19 (“Coronavirusdisease 2019”).

At present, the problem of preventing SARS-CoV-2 coronavirus infection is a big problem, because. effective drug has not yet been developed. The main prophylactic in the case of viral infections is a vaccine - a tool that includes an antigen that causes an immune response in the body and leads to the induction of a stronger secondary immune response. Thus, a vaccinated patient either does not become infected, or a reaction of rapid elimination of the virus occurs in the body, which in this case makes it possible to carry the disease in a milder form.

Various vaccine compositions are known for inducing an immune response in the case of novel coronavirus infections.

For example, according to patent RU 2709659, an immunobiological agent based on recombinant human adenovirus serotype 5 or recombinant human adenovirus serotype 26 is known, containing a consensus sequence of the full protective S antigen of coronavirus optimized for expression in mammalian cells based on the sequences of the S protein genes of modern virus strains 2015-2017 with a certain sequence.

Also known from US Pat. No. 1,0953,089 is a vaccine comprising nanoparticles with SARS-CoV-2 coronavirus glycoprotein S and a nonionic surfactant core, as well as a buffer and a saponin-based adjuvant. The proposed particles have stability and better presentation of the antigen epitope.

Patent CN111944064 describes a vaccine comprising S-S-RBD protein, assembly domain protein, signal proteins, and a squalene-based adjuvant. The resulting composition is a water-in-oil nanoemulsion.

Thus, the technical challenge is to develop and obtain a stable composition capable of inducing an immune response and inducing specific immunity against SARS-CoV-2 when administered to the body. The problem is solved by obtaining a composition containing the SARS-CoV-2 nucleocapsid protein N having the amino acid sequence of SEQ ID NO: 1, in an amount of 20 to 100 μg/ml and pharmaceutically acceptable excipients selected from the group including buffer agents, surface active substances, adjuvant, isotonic agents in pharmaceutically acceptable amounts. At the same time, the resulting composition has stability, the ability to induce a stable and long-lasting immune response, with a high titer of antibodies to SARS-CoV-2 coronavirus infection, and the composition can also be obtained quite simply from a technological point of view.

Description of figures

On FIG. 1 shows examples of detected changes in the lung: the development of severe interstitial pneumonia and epithelial necrosis are shown, in the study according to example 7, group 8.

On FIG. 2 shows examples of detected changes in the lungs: marked hyperplasia of type II alveolar epithelial cells, as well as mixed expressed mixed cell inflammatory infiltration, are shown, in the study according to example 7, group 8.

On FIG. 3 shows the normal structure of the lung, 2 weeks after the recovery of the animals, in the study according to example 7, group 7.

Detailed description of the invention

Vaccines, as a rule, are a composition that includes an antigen in the form of a protein or a mixture of proteins, as well as excipients.

An antigen is a pharmaceutically active substance responsible for the development of an immune response in a patient's body. However, for various reasons, the injected antigen may be unstable, for example, it cannot be stored and processed without loss of activity for a certain time, under the influence of the environment. In addition, when introduced into the body, the antigen can be destroyed by the action of enzymes or other substances of the body (for example, the bloodstream environment).

According to the present invention, a pharmaceutical composition is proposed for inducing a specific immune response against the SARS-CoV-2 acute respiratory syndrome virus, containing the NSARS-CoV-2 nucleocapsid protein having the amino acid sequence SEQIDNO: 1, in an amount of 20 to 100 μg/ml and pharmaceutically acceptable excipients selected from the group consisting of buffering agents, surfactants, adjuvants, isotonic agents. These excipients are generally contained in the composition in pharmaceutically acceptable amounts. In one embodiment, the composition of the invention comprises the NSARS-CoV-2 nucleocapsid protein having the amino acid sequence of SEQ ID NO: 1 in an amount of 20 to 50 μg/ml.

In yet another embodiment, the composition according to the invention contains tocopherol, squalane, or a combination thereof as an adjuvant. In another embodiment, the composition according to the invention may contain as buffering agents any pharmaceutically acceptable buffer that can be used to maintain a pH of about 7, for example, phosphate-buffered saline, maleate buffer, tetrabutylammonium buffer solution, borate buffer, HEPES buffer, tris( hydroxymethyl)aminomethane buffer solution, or any other physiologically acceptable buffer used to maintain the pH in the range of 7-8, preferably in the range of 7.2-7.6.

Also, the composition according to the invention may contain pharmaceutically acceptable surfactants (surfactants), for example, polysorbates, tweens, sorbitans and other suitable surfactants, for example, sodium lauryl sulfate, sodium dioctylsulfosuccinate, sodium dioctylsulfonate, chenodeoxycholic acid, N-laurylsarcosine sodium salt, lithium dodecyl sulfate , sodium salt of 1-octane sulfonic acid, sodium cholate hydrate, sodium deoxycholate, sodium salt of glycodeoxycholic acid, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X- 114, lauromacrogol 400, polyoxyl-40-stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 20, 40, 60, 65, and 80, soy lecithin, DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. The preferred concentration of surfactant may be from 0.005 to 5% by weight, or, for example, 1-10 mg per 1 ml. The composition may also contain isotonic agents, for example, potassium chloride, sodium chloride. The isotonic agent acts not only to maintain a suitable osmotic pressure, but can also act as an auxiliary protein stabilizer in liquid or lyophilisate form. Examples of the isotonic agent include water-soluble inorganic salts such as sodium chloride, sodium sulfate, sodium citrate, calcium chloride, and combinations thereof. Most preferred is sodium chloride. Preferably the concentration of the isotonic agent is on the order of 5 to 200 mM, or for example 0.1 to 8.5 mg per ml. Within this range, the concentration of the isotonic agent can be adjusted according to the kinds and amounts of components contained so that the liquid composition is isotonic.

Adjuvants are components used in vaccine compositions that potentiate and/or modulate the immune response to an antigen in order to obtain the desired immune response. Mineral salts can be used as adjuvants, for example gels of aluminum hydroxide and aluminum or calcium phosphate; formulations based on oil emulsions and surfactants, e.g. MF59 (microfluidized detergent stabilized in oil-in-water emulsion), QS21 (purified saponin), AS02 [SBAS2] (oil-in-water emulsion + MPL + QS-21), montanide ISA-51 and ISA-720 (stabilized water-in-oil emulsion), particulate adjuvants, e.g. and lipids), polylactide co-glycolide (PLG), microbial derivatives (natural and synthetic), e.g. monophosphoryl lipid A (MPL), detox (MPL + M. phlei cell wall backbone), AGP [RC-529] ), DC_Chol (lipoid immunostimulants capable of self-assembling into liposomes), OM-174 (derivative of lipid A), CpG sequences (synthetic oligonucleotides containing CpG sequences with immunostimulatory activity), tocopherol, squalene and squalane and their derivatives, modifi LT and CT (genetically modified bacterial toxins to provide non-toxic adjuvant action), endogenous human immunomodulators, such as human GM-CSF and human IL-12 (cytokines that can be administered as a protein or encoded by plasmids), immudaptin (C3d tandem repeat ), inert carriers such as gold particles. In a preferred embodiment, the composition according to the invention comprises α-tocopherol, squalane, and combinations thereof as an adjuvant.

In a preferred embodiment, the composition according to the invention has the following composition per 1 ml:

SARS-CoV-2 nucleocapsid protein having the amino acid sequence SEQIDNO: 1 20-100 mcg Squalane 15-40 mg α-tocopherol 5-15 mg Polysorbate 80 1-10 mg Sodium phosphate disubstituted 0.1-2.0 mg Potassium phosphate monosubstituted 0.01-0.25 mg Potassium chloride 0.01-0.2 mg Sodium chloride 0.1-8.5 mg Water for injections up to 1 ml

In a more preferred embodiment, the composition according to the invention has the following composition per 1 ml:

SARS-CoV-2 nucleocapsid protein having the amino acid sequence SEQIDNO: 1 50-100 mcg Squalane 24-36 mg α-tocopherol 8-12 mg Polysorbate 80 10-12 mg Sodium phosphate disubstituted 1.5-1.8 mg Potassium phosphate monosubstituted 0.1-0.122 mg Potassium chloride 0.1-0.21 mg Sodium chloride 4-8.1 mg Water for injections up to 1 ml

Compositions according to the invention may be a solution, emulsion or lyophilisate. The lyophilizate can be obtained from a solution or an emulsion by standard methods for this, for example, from a solution during lyophilization.

The composition according to the invention can be used to prevent or induce specific immunity against the acute respiratory syndrome virus SARS-CoV-2, i. be used as a vaccine. However, the content of protein N in the composition according to the invention may vary depending on the required dose for vaccination, the age of the patient and other clinical factors.

The use of said composition for prophylaxis against SARS-CoV-2 acute respiratory syndrome virus includes single or double administration of the composition. The decision on a single or double administration is made by the therapist, taking into account the age, patient history, the presence of chronic diseases and other clinical factors. In the case of double administration, the composition may be administered 2 times consecutively with an interval of at least 2 weeks, with the same or different content of nucleocapsid protein N in the composition.

Further, the invention also provides a process for preparing a composition containing protein N, which includes:

1) providing the NSARS-CoV-2 nucleocapsid protein having the amino acid sequence of SEQ ID NO: 1 in an amount of 20 to 100 μg/ml;

2) dissolving the protein in an aqueous solvent and bringing the protein content N in the solution to the required concentration;

3) preparation of an aqueous solution of the adjuvant;

4) mixing an aqueous solution containing protein N and an aqueous solution of an adjuvant;

5) homogenization of the resulting mixture until a homogeneous color solution is obtained;

6) regulation of the resulting composition pH to 7.2-7.6.

In one embodiment, the method for preparing the composition may further include lyophilizing the resulting composition to obtain a lyophilizate used for reconstitution with a solvent prior to administration to a patient. In some embodiments, the resulting composition is an emulsion, such as a water-in-oil or oil-in-water emulsion.

Further, the composition according to the invention will be described in examples, which are not intended to limit the scope of the present invention.

Examples

Example 1

Obtaining a plasmid containing the protein gene

A plasmid containing the N protein gene (Genbank YP_009724397.2) was used to obtain a modified codon-optimized nucleotide sequence using the CodonAdaptationIndex (http://genomes.urv.es/OPTIMIZER/). The codons are chosen in such a way that the most common codons found in E. coli are used whenever possible, and the content of nucleotides guanine and cytosine is in the range of 50-55%. The sequence at the 5' and 3' ends was flanked by sequences for recognition by NcoI (CCATGG) and XhoI (CTCGAG) endonucleases, respectively. Gene synthesis and cloning at the indicated restriction sites into the plasmid without pET-28a(+) insert were ordered commercially. The resulting plasmid was sequenced using primers T7_promotor (TAATACGACTCACTATAGGG), fwd_122 (GCCTTATGGTGCAAATAAGG) and fwd_300 (AAACATTGGCCGCAGATTGC) to confirm the nucleotide sequence and check for single nucleotide mutations.

The nucleotide sequence matched the sequence
SEQ ID NO:2.

Example 2

Obtaining an E. coli cell line modified with a plasmid

The E. coli cell line was transformed according to the following protocol. The tube with competent cells was removed from the freezer storage at -80°C. The ice was allowed to thaw for 10 min. Further contributed
1 µl containing 1 pg-100 ng of plasmid. The tube was gently mixed 4-5 times. The resulting mixture was placed on ice for 30 min. Next, the mixture was subjected to heat shock at 42°C for 30–60 s. The resulting treated mixture was placed on ice for 5 minutes. 950 µl of SOC medium was added at room temperature. Incubated at 30°C-37°C for 1 - 2 h with stirring 250 rpm. 10-50 μl were inoculated per plate with solid selective medium containing the antibiotic kanamycin. Incubated at 30°C-37°C overnight. The grown colonies contain E. coli cells transformed with a plasmid carrying an artificial gene for the nucleocapsid protein N.

Thus, an E. coli cell line was obtained with the incorporation of an artificial gene according to the invention.

This cell line was deposited at the All-Russian Research Institute of Agricultural Microbiology (FGBNU VNIISHM) under the number RCAM05390 (dated May 14, 2021).

Example 3

Obtaining recombinant protein N SARS-CoV-2

The E. coli biomass containing the expressed N protein was resuspended in lysis buffer (Tris-HCl 50 mM, pH 9) and homogenized using a laboratory homogenizer, e.g. GEA Panda 1000. All buffers except for chromatography buffers in the third purification step contained EDTA and PMSF protease inhibitors. Then, the insoluble fraction of biomolecules was precipitated by centrifugation at 20,000 x g, 4°C, 30 min. The soluble fraction was transferred to a clean dish and 20% (w/v) ammonium sulfate was added to it to precipitate protein N. The precipitated fraction was collected by centrifugation at 20,000 xg, 4°C, 30 min, followed by dissolution in Tris-HCl 50 buffer. mM, pH 9. After filtration through a membrane with a pore diameter of 0.22 μm, the solution was used for chromatographic purification. The first stage of purification on anion-exchange chromatography was carried out in the breakthrough mode (Tris-HCl buffer 50 mm, pH 9). The second purification step on cation-exchange chromatography was carried out in capture mode eluting with a NaCl gradient from 0 M to 1 M (Tris-HCl buffer 50 mM, pH 9). The third stage of purification on hydrophobic chromatography was carried out in capture mode with elution with a gradient of ammonium sulfate from 1 M to 0 M (NaH2PO4 buffer 50 mM, pH 7.5).

Next, the process of dialysis was carried out in dialysis cassettes in 5 l of phosphate-buffered saline. The process took place at room temperature for 2 hours. Then the buffer was replaced with 5 L of fresh buffer and the process was carried out at +4°C for 10 hours.

After the end of dialysis, the protein was removed from the dialysis cassettes and filtered. The resulting precipitate was dissolved in water for injection to obtain a lyophilisate, or the resulting protein solution was adjusted to the desired protein concentration by adding the previously obtained lyophilisate.

The resulting solution was examined for the following parameters: protein content, pH, mechanical inclusions, transparency, sterility, particle size. For this, standard methods of analysis were used.

Example 4

Preparation of the composition according to the invention

An aqueous solution was obtained containing protein N, obtained according to example 3, with different concentrations of protein: 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 60 μg/ml, 70 μg/ml, 80 μg /ml, 90 µg/ml, 100 µg/ml.

Depending on the protein content, the calculated amount of tocopherol, squalane, and polysorbate 80 was placed in a separate container; phosphate-buffered saline was added in parts, with constant stirring. For example, at 20 μg/ml, 45 g of tocopherol, 15 g of polysorbate 80, and 15 g of squalane and 750 ml of buffer were used. After dissolution, the resulting solution (adjuvant solution) was divided into equal parts and sonicated. Then the treated solutions were combined into one beaker and mixed. Next, the solution was filtered through a 0.22 μm membrane. The resulting filtered solution was combined with protein N solutions and stirred until a homogeneous solution.

In some cases a white or yellowish emulsion was formed.

The resulting solution containing protein N and adjuvant was adjusted to
pH 7.2-7.6 with sodium hydroxide solution or hydrochloric acid. Further, the resulting solution was poured into sterile vials and sealed. If necessary, a lyophilizate was obtained from the solution by lyophilization in the apparatus. Water for injection or sterile saline was used to reconstitute the vaccination solution.

Example 5

Analysis of the properties of compositions according to the invention

To study the properties of the composition according to the invention, the following compositions were obtained (shown in the table below).

Table 1

composition number Content Protein, mcg squalane, mg tocopherol, mg polysorbate 80, mg Na ₂ HPO _4, mg KH ₂ PO ₄ , mg KCl, mg NaCl, mg water for injection one twenty 15 five one 0.1 0.01 0.01 0.1 Up to 1 ml 2 fifty 25 10 five 0.71 0.122 0.101 4.003 Up to 1 ml 3 one hundred 40 10 10 1.42 0.244 0.2 8.005 Up to 1 ml 4 0 25 10 five 0.71 0.212 0.101 4.003 Up to 1 ml

Compositions with different protein content, obtained according to example 5, were examined for stability before the start of the study and after 6 weeks of storage under stress conditions ("accelerated storage" conditions). Before the start of the study, pH, protein content were measured, transparency, homogeneity were visually assessed if the composition was an emulsion. Compositions with different contents of nucleocapsid protein N were filled into sterile vials, stoppered and placed in an accelerated storage chamber for stability testing. The chamber was maintained at a temperature of +20°C for 6 weeks. After the time had elapsed, the vials were removed and analyzed for pH and nucleocapsid N protein content. pH was measured directly in solution using a pH meter. Analysis of the protein content was carried out by spectrophotometry, at a wavelength of 280 nm. Further, the decrease in the concentration of the nucleocapsid protein N in the test solution after storage for 6 weeks was calculated relative to the initial value.

The results are presented in the table below.

table 2

composition number Stability for 2 weeks pH Protein content (% of original content) conclusions one Stable, no precipitation or discoloration detected 7.5 99.25% stable 2 Stable, no precipitation or discoloration detected 7.4 99.13% stable 3 Stable, no precipitation or discoloration detected 7.5 99.37% stable 4 Stable, no precipitation or discoloration detected 7.3 99.27% stable

Thus, based on the results obtained, it can be concluded that the resulting compositions containing the nucleocapsid protein N at a concentration of 20 μg/ml to 100 μg/ml are stable.

Example 6

Study of the immunogenic properties of the resulting compositions

The resulting compositions were investigated for the possibility of induction of specific immunity (immunogenicity)

Animals of the Syrian hamster species (females) in the amount of 240 pcs. randomly divided into the following equal groups:

Group No. 1 - a composition containing 20 μg/ml of protein was administered.

Group No. 2 - a composition containing 50 μg/ml of protein was administered.

Group No. 3 - a composition containing 100 μg/ml of protein was administered.

Group No. 4 - a composition that did not contain protein (placebo) was administered.

More detailed data on the composition are presented in the table below.

Table 3

Group Content Protein, mcg squalane, mg tocopherol, mg poly-sorbate 80, mg Na ₂ HPO _4, mg KH ₂ PO ₄ , mg KCl, mg NaCl, mg water for injection one twenty 15 five one 0.1 0.01 0.01 0.1 Up to 1 ml 2 fifty 25 10 five 0.71 0.122 0.101 4.003 Up to 1 ml 3 one hundred 40 10 10 1.42 0.244 0.2 8.005 Up to 1 ml 4 0 25 10 five 0.71 0.212 0.101 4.003 Up to 1 ml

* - all values are given per 1 ml of the composition.

At the beginning of the experiment, the age of the animals was 6-8 weeks, the weight range was 56.37-149.28 g. The adaptation period was 5 days, with daily examination of the animals (condition, temperature measurement). The animals were kept under standard conditions in accordance with Directive 2010/63/EU of the European Parliament and of the Council of the European Union of September 22, 2010 on the protection of animals used for scientific purposes.

The compositions were administered intramuscularly, at a dose of 0.5 ml per animal, using fractional administration, with a break of 14 days (administration on day 1 and day 15).

The scheme of the experiment is presented in the table below.

Table 4

manipulation Experiment Days Intramuscular fractional introduction of the studied objects 1st and 15th day Registration of body weight of animals 1st (before the introduction of the objects under study), 15th, 29th, 43rd, 57th, 71st, 85th, 99th, 113th, 127th, 141st, 155th, 169th, 183rd, 197th, 211th, 225th, 239th, 253rd, 267th, 281st, 295th, 309th, 323rd th, 337th, 351st, 365th, 379th Registration of deviations in the general condition of animals and mortality Daily Clinical examination of animals 2nd, 15th, 29th, 43rd, 57th, 71st, 85th, 99th, 113th, 127th, 141st, 155th, 169th th, 183rd, 197th, 211th, 225th, 239th, 253rd, 267th, 281st, 295th, 309th, 323rd, 337th, 351st, 365th, 379th Euthanasia of 5 animals of each group, followed by taking a blood sample from the heart for enzyme immunoassay (ELISA) 1st (before the introduction of the studied objects), then - to the 8th, 15th, 22nd, 29th, 43rd, 162nd and 386th.

ELISA analysis for specific antibodies was performed according to the following method.

After immunization with compositions 1-4 Balb/c mice, aged 6-8 weeks, with a dose of 500 µl, 250 µl in each hind thigh.

Next, 42 days after the second dose, the mice were euthanized in a chamber filled with CO ₂ gas. Further, blood was taken for the content of specific antibodies and internal organs for histological analysis.

Blood was placed into Eppendorf tubes containing EDTA at a concentration of 1–2 mg/mL. Tubes with blood and EDTA were centrifuged at +4°C for 20 minutes. The supernatant was transferred into clean tubes in a laminar flow hood. The resulting plasma was frozen.

Procedure: enzyme immunoassay (ELISA)

The following reagents were used:

planting antigens, 1 µg/ml, 100 µl/well; blocking with 0.5% milk in wash buffer (20 mMTris, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4 ± 0.1), 300 µl/well.

Washing was carried out as follows: the contents of the plate were poured out, the plate was washed 4 times with 300 μl per well with wash buffer (20 mM Tris, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4 ± 0.1), the wells were thoroughly dried, avoiding the formation of bubbles, by tapping the tablet on a tissue.

When applying the sera, the titer was selected for each individual serum, 100 μl/well, thermostatically controlled and centrifuged.

Application of secondary antibodies was carried out at an initial dilution of 1:64000, 100 μl/well, with thermostating and stirring.

Washing was carried out as follows: the contents of the plate were poured out, the plate was washed 4 times with 300 μl per well with wash buffer (20 mM Tris, 150 mM NaCl, 0.05% polysorbate 20, pH 7.4 ± 0.1), the wells were thoroughly dried, avoiding the formation of bubbles, by tapping the tablet on a tissue.

Chromogen solution, 100 μl/well, was added for 12 minutes in a place protected from light. Next, a stop solution was added, 50 μl/well. The reading of the results was carried out at a wavelength of 450 nm, reference 620 nm. The results were considered acceptable if the eligibility and acceptance criteria were met.

According to IUPAC, the limit of detection is calculated from the standard deviation of a blank sample. To do this, measure the value of the analytical signal for a sufficient number of blank samples and calculate the standard deviation of their values.

As a rule, the formula is used (Kaiser H. Zurdefinitiondernachweisgrenze, dergarantiegrenzeundderdabeibenutztenbegriffe, Dörffel K. Statistics in analytical chemistry):

LOD = Average+k*SD ,

where k = 3

The limit of detection (LOD) is calculated from the optical density of the blocking buffer using the formula:

LOD = Average+10*SD ,

where average is the average value of the optical density of the blocking buffer,

SD is the standard deviation of the optical density of the blocking buffer,

It is assumed that specific antibodies are present in the sample if the average optical density of the sample is greater than Сut-off, while Сut-off = LOD + 20% (20% correspond to the ELISA system suitability criterion for optical density at the absorption level of the blocking buffer (blank) ):

Cut-off = 1.2* LOD = 1.2*(Average+10*SD).

The serum titer is considered to be the extreme dilution at which the optical density exceeds the signal of the discrimination level (cut-off), the average optical density is used.

The results for determining the titer of specific IgG protein antibodies are presented in the table below.

Table 5

group number Average antibody titer per 1st* 8th 15th 22nd 29th 43rd one Nd 1:16000 1:16000 1:16000 1:32000 1:32000 2 Nd 1:64000 1:64000 1:64000 1:64000 1:64000 3 Nd 1:64000 1:64000 1:64000 1:128000 1:128000 4 Nd 2 out of 5 positive
(1 case of titer 800)** 2 out of 5 positive
(1 case of titer 800)** 2 out of 5 positive
(1 case of titer 800)** 2 out of 5 positive
(1 case of titer 800)** 2 out of 5 positive
(1 case of titer 800)**

* - measurement before injection.

** - detected titers of non-specific antibodies characteristic of this animal species.

Nd - not determined, no titer.

A pronounced immune response was recorded with a single and double intramuscular injection of the compositions in groups Nos. 1-3 at a dose of 0.5 ml/animal with a peak titer of specific antibodies on the 22nd day of the experiment and maintaining up to the 43rd day in the range of 1:32000 -1:17800. The appearance of specific antibodies was recorded 15 days after the first vaccination of animals.

Example 7

Study of the immunogenic properties of the obtained compositions (protectiveness)

The resulting compositions were investigated for the possibility of induction of specific immunity (protectiveness).

The evaluation of the protective properties of the compositions was evaluated after two vaccinations and infection with a culture of coronavirus.

For the development of immunity, the compositions and the vaccination scheme were used, as described in example 5. For immunization, compositions containing protein N in various concentrations were used, as well as a placebo (composition containing no protein and containing only a solvent and excipients).

Compositions for vaccination are presented in the table below.

Table 6

Group Content Protein, mcg squalane, mg tocopherol, mg poly-sorbate 80, mg Na ₂ HPO _4, mg KH ₂ PO ₄ , mg KCl, mg NaCl, mg water for injection five twenty 15 five one 0.1 0.01 0.01 0.1 Up to 1 ml 6 fifty 25 10 five 0.71 0.122 0.101 4.003 Up to 1 ml 7 one hundred 40 10 10 1.42 0.244 0.2 8.005 Up to 1 ml 8 0 25 10 five 0.71 0.212 0.101 4.003 Up to 1 ml

* - all values are given per 1 ml of the composition.

Female Syrian hamsters in the amount of 50 pcs. randomly divided into 5 groups containing 10 individuals. Groups of animals were first immunized on the 1st and 15th day with compositions with different protein content. Then, on the 29th day, the animals were infected with the SARS-CoV-2 virus and the clinical manifestations of the disease and recovery were recorded.

The designation of the group, depending on the composition entered and the infection with the virus, is given in the table below.

Table 7

Group No. Composition Introduction Infection five Administration of 20 μg/ml of protein 50 µl at a dose of 5 lgTCID50 per animal 6 Administration of 50 µg/ml of protein 7 Administration of 100 µg/ml protein 8 Administration of 0 µg/ml protein nine Intact group (did not administer composition or placebo), did not infect with infection

In order to develop pathology, the animals under general anesthesia were intranasally injected once with the SARS-CoV-2 virus (certified strain of the Federal State Budgetary Scientific Institution “FNTsIRIP named after M.P. Chumakov RAS” No. PIK35 GISAID ID EPI_ISL_428852) in a volume of 50 μl at a dose of 5 lgTCID50 per animal. During the experiment, the animals were monitored for general condition and body weight. Animals were euthanized on the 3rd and 7th days after infection. The lungs were photographed and weighed for further evaluation of the mass factor. Macroscopically, changes in the color and structure of the lung tissue were assessed. The right lung was used to assess microscopic changes. The histological analysis included evaluation of three features: pneumonia, cellular infiltrate, and edema. The left lung and turbinate tissues were used to assess the amount of SARS-CoV-2 virus RNA by RT-PCR in terms of Ct.

During the immunization period, no pathological changes in the general condition of the experimental animals and weight loss were noted.

Pathology photographs are shown in Fig. 1-2.

The overall results are presented in the table below.

Table 8

Group No. Lung mass ratio (average) Recovery five 0.81±0.0.092 On the 5th day 6 0.83±0.0121 On the 5th day 7 0.80±0.0.097 On the 5th day 8 0.91±0.192 On the 18th day nine 0.75±0.046 On the 20th day

After infection with the virus in animals, the following was noted: a decrease in weight by 10-15% at the entrance of the infectious process, the presence of clinical symptoms, the accumulation of RNA virus in the lower and upper respiratory tract on the 3rd day of infection, macro- and microscopic changes in the lungs, most pronounced on the 7th day of development of pathology, an increase in the mass coefficient of the lungs by 1.5 times.

Histological studies of the lungs are shown in Fig. 1-3.

In accordance with the data obtained, it was found that the introduction of composition No. 8 did not lead to the development of pharmacological effects on the course of coronavirus infection in Syrian hamsters. The introduction of compositions containing protein, Nos. 5, 6, 7 had a positive effect on the infectious process in the form of a tendency to increase body weight and improve the general well-being of animals on the 5th day of pathology, and a decrease in the severity of histological changes in the lung tissue on the 7th day after infection.

In the course of this study, it was shown that compositions containing N protein at various concentrations and an adjuvant have protective properties when infected with SARS-CoV-2 infection. The discovered properties are manifested in the form of a reduction in the period of the disease due to the complete absence of clinical signs of infection by the 5th day after infection, a significant increase in body weight on the 6-7th day of infection with almost complete recovery to the initial level, a tendency to increase Ct in the lung tissue on the 3rd day, reducing the severity of macro- and microscopic changes in the lung tissue.

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SEQUENCE LIST

<110> FSUE SPbNIIVS FMBA of Russia

<120> COMPOSITION OF THE COVID-19 VACCINE

<160> NUMBER OF SEQ ID NOS: 3

<210> SEQIDNO 1

<211> 420

<212> artificial sequence

<213> artificial amino acid sequence

<400> SEQUENCE1:

MGSDNGPQNQRNAPRITFGGPSDSTGSNQN 30

GERSGARSKQRRPQGLPNNTASWFTALTQH 60

GKEDLKFPRGQGVPINTNSSPDDQIGYYRR 90

ATRRIRGGDGKMKDLSPRWYFYYLGTGPEA 120

GLPYGANKDGIIWVATEGALNTPKDHIGTR 150

NPANNAAIVLQLPQGTTLPKGFYAEGSRGG 180

SQASSRSSRSRNSSRNSTPGSSRGTSPAR 210

MAGNGGDAALALLLLDRLNQLESKMSGKGQ 240

QQQGQTVTKKSAAEASKKPRQKRTATKAYN 270

VTQAFGRRGPEQTQGNFGDQELIRQGTDYK 300

HWPQIAQFAPSASAFFGMSRIGMEVTPSGT 330

WLTYTGAIKLDDKDPNFKDQVILLNKHIDA 360

YKTFPPTEPKKDKKKKADETQALPQRQKKQ 3900

QTVTLLPAADLDDFSKQLQQSMSSADSTQA 420

<160> SEQIDNO: 2

<210> 3

<211> 6496

<212> DNA

<213> artificial sequence

<400> artificial nucleotide sequence

tggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcg 60

120

180

gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttc 240

300

360

ttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgattta 420

480

tcggggaaatgtgcgcggaacccctatttgttttatttttctaaatacattcaaatatgta 540

tccgctcatgaattaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttat 600

tcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaa 660

actcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactc 720

gtccaacatcaatacaacctattaatttcccctcgtcaaaaataaggttatcaagtgaga 780

aatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttcc 840

agacttgttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaac 900

960

aattacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatat 1020

tttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcag 1080

1140

taaattccgtcagccagtttagtctgaccatctcatctgtaacatcattggcaacgctac 1200

1260

1320

1380

1440

1500

gatccttttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg 1560

1620

agagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaag 1680

aactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcc 1740

1800

cagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctac 1860

accgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggaga 1920

1980

ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgag 2040

2100

gcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgtta 2160

2220

2280

2340

caatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactg 2400

ggtcatggctgcgccccgacaccgccaacacccgctgacgcgccctgacgggcttgtct 2460

gctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagag 2520

gttttcaccgtcatcaccgaaacgcgcgaggcagctgcggtaaagctcatcagcgtggtc 2580

2640

aagcgttaatgtctggcttctgataaagcgggccatgttaagggcggttttttcctgttt 2700

ggtcactgatgcctccgtgtaagggggatttctgttcatgggggtaatgataccgatgaa 2760

acgagagaggatgctcacgatacgggttactgatgatgaacatgcccggttactggaacg 2820

ttgtgagggtaaacaactggcggtatggatgcggcgggaccagagaaaaatcactcaggg 2880

tcaatgccagcgcttcgttaatacagatgtaggtgttccacagggtagccagcagcatcc 2940

tgcgatgcagatccggaacataatggtgcagggcgctgacttccgcgtttccagacttta 3000

3060

gcagtcgcttcacgttcgctcgcgtatcggtgattcattctgctaaccagtaaggcaacc 3120

3180

catgccggcgataatggcctgcttctcgccgaaacgtttggtggcgggaccagtgacgaa 3240

ggcttgagcgagggcgtgcaagattccgaataccgcaagcgacaggccgatcatcgtcgc 3300

3360

gagttgcatgataaagaagacagtcataagtgcggcgacgatagtcatgccccgcgccca 3420

ccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgccta 3480

3540

cctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtat 3600

3660

3720

3780

3840

3900

3960

4020

agacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagat 4080

4140

ggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttccacagcaatgg 4200

4260

tgtgcaccgccgctttacaggcttcgacgccgcttcgttctaccatcgacaccaccacgc 4320

4380

4440

ccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttttcccgcgttt 4500

4560

catactctgcgacatcgtataacgttactggtttcacattcaccaccctgaattgactct 4620

4680

tctcgacgctctcccttatgcgactcctgcattaggaagcagcccagtagtaggttgagg 4740

4800

4860

4920

4980

aattaatacgactcactataggggaattgtgagcggataacaattcccctctagaaataa 5040

ttttgtttaactttaagaaggagatatataccatgggcagcgataatggcccgcagaatcag 5100

5160

5220

gccagttggtttaccgccctgacccagcatggcaaagaagatctgaaatttccgcgcggt 5280

cagggcgtgccgattaataccaatagcagtccggatgatcagattggctattatcgccgc 5340

gccacccgccgcattcgcggtggtgacggtaaaatgaaagatctgagcccgcgttggtat 5400

ttttattatctgggcaccggcccggaagccggtctgccttatggtgcaaataaggatggt 5460

attatttgggtggcaaccgaaggtgcactgaataccccgaaagatcatattggtacccgt 5520

aatccggcaaataatgcagcaattgttctgcagctgccgcagggtaccaccctgccgaaa 5580

ggcttttatgccgaaggcagtcgcggcggcagtcaggctagtagccgtagcagtagccgt 5640

agtcgcaatagcagtcgcaatagtaccccgggcagcagccgtggcaccagtcctgctcgt 5700

atggcaggtaatggtggtgacgccgccctggcactgctgctgctggatcgcctgaatcag 5760

ctggaaagcaaaatgagtggtaaaggccagcagcagcagggccagaccgtgaccaaaaaa 5820

tctgcagcagaagcaagcaaaaaaccgcgccagaaacgtaccgcaaccaaagcatataat 5880

5940

6000

agcgcaagcgcatttttcggcatgagtcgcattggcatggaagttaccccgagcggtacc 6060

tggctgacctataccggtgcaattaagctggatgataaagatccgaattttaaagatcag 6120

6180

6240

cagaccgtgacactgctgccggcagccgatctggatgattttagtaaacagctgcagcag 6300

agtatgagtagcgcagatagtacccaggcataactcgagcaccaccaccaccaccactga 6360

gatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaa 6420

taactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaagga 6480

ggaactatatccggat 6496

<---

Claims (18)

1. Pharmaceutical composition intended to induce a specific immune response against the SARS-CoV-2 acute respiratory syndrome virus, containing the SARS-CoV-2 nucleocapsid protein having the amino acid sequence SEQIDNO: 1, and having the following composition per 1 ml: SARS-CoV-2 nucleocapsid protein N having the amino acid sequence SEQIDNO: 1 20-100 mcg Squalane 15-40 mg α-tocopherol 5-15 mg Polysorbate 80 1-10 mg Sodium phosphate disubstituted 0.1-2.0 mg Potassium phosphate monosubstituted 0.01-0.25 mg Potassium chloride 0.01-0.2 mg Sodium chloride 0.1-8.5 mg Water for injections up to 1 ml 2. Composition according to claim 1, characterized in that it has a pH of 7.2 to 7.6. 3. Composition according to paragraphs. 1, 2, characterized in that it has the following composition per 1 ml: SARS-CoV-2 nucleocapsid protein having the amino acid sequence SEQIDNO: 1 50-100 mg Squalane 24-36 mg α-tocopherol 8-12 mg Polysorbate 80 10-12 mg Sodium phosphate disubstituted 1.5-1.8 mg Potassium phosphate monosubstituted 0.1-0.122 mg Potassium chloride 0.1-0.21 mg Sodium chloride 4-8.1 mg Water for injections up to 1 ml 4. Composition according to paragraphs. 1-3, characterized in that it is a solution, emulsion or lyophilisate. 5. Composition according to claim 4, characterized in that it is an emulsion. 6. The use of the composition according to paragraphs. 1-5 for the prevention or induction of specific immunity against acute respiratory syndrome virus SARS-CoV-2. 7. Use according to claim 6, including the administration of the composition 2 times consecutively with an interval of at least 2 weeks. 8. The method of obtaining the composition according to paragraphs. 1-5 which includes: 1) providing SARS-CoV-2 nucleocapsid protein N having the amino acid sequence of SEQ ID NO: 1 in an amount of 20 to 100 μg/ml; 2) dissolving the protein in an aqueous solvent and bringing the protein content N in the solution to the required concentration; 3) preparation of an aqueous solution of the adjuvant; 4) mixing an aqueous solution containing protein N and an aqueous solution of an adjuvant; 5) homogenization of the resulting mixture until a homogeneous color solution is obtained; 6) regulation of the resulting composition pH to 7.2-7.6. 9. The method according to claim 8, further comprising lyophilizing the resulting solution to obtain a lyophilizate used for reconstitution with a solvent prior to administration to a patient. 10. The method according to p. 9, characterized in that the resulting composition is an emulsion.

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