WO2022216025A1 - Souche de mycobacterium recombinée exprimant un antigène du sars-cov-2, et composition vaccinale la contenant - Google Patents

Souche de mycobacterium recombinée exprimant un antigène du sars-cov-2, et composition vaccinale la contenant Download PDF

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WO2022216025A1
WO2022216025A1 PCT/KR2022/004915 KR2022004915W WO2022216025A1 WO 2022216025 A1 WO2022216025 A1 WO 2022216025A1 KR 2022004915 W KR2022004915 W KR 2022004915W WO 2022216025 A1 WO2022216025 A1 WO 2022216025A1
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rbd
strain
cov
sars
rmpg
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김범준
김병준
정도현
신주엽
진주영
이문수
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주식회사 라파스
서울대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora

Definitions

  • the present invention relates to a recombinant Mycobacterium strain expressing the SARS-CoV-2 antigen and a vaccine composition comprising the same as an active ingredient.
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • SARS-CoV-2 has a higher infectivity than the existing coronavirus or flu virus, and is known to show various symptoms ranging from asymptomatic to mild respiratory symptoms, acute respiratory distress syndrome, and death. Due to the worldwide spread of SARS-CoV-2, the World Health Organization (WHO) declared a pandemic on March 11, 2020.
  • WHO World Health Organization
  • the cumulative number of confirmed cases worldwide is 93,217,287, and the cumulative number of deaths is 2,014,957.
  • the number of confirmed cases and deaths is increasing rapidly in the United States and Europe (World Health Organization. (https://covid19.who.int/)).
  • This SARS-CoV-2 virus can infect cells expressing angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 on the cell surface.
  • ACE2 angiotensin-converting enzyme 2
  • RBD receptor binding domain
  • Known to increase infectivity Cannalire R, et al. SARS-CoV-2 Entry Inhibitors: Small Molecules and Peptides Targeting Virus or Host Cells. Int J Mol Sci. 2020; 21(16): 5707; Tay MZ, et al. The trinity of COVID-19: immunity, inflammation and intervention, Nat Rev Immunol. 2020; 20(6):363-374; and Saha RP, et al. Repurposing Drugs, Ongoing Vaccine, and New Therapeutic Development Initiatives against COVID-19. Front Pharmacol. 2020;11:1258]).
  • recombinant Mycobacterium strains such as recombinant BCG have attracted attention for the development of human immunodeficiency virus-1 (HIV-1) vaccines
  • Mycobacterium paragordonae Mpg is a temperature-sensitive strain that stimulates the immune response by proliferating on the skin and subcutaneously when inoculated, but inhibits proliferation due to the characteristic that it can grow only at a temperature lower than body temperature in the center of the body, so the possibility of infection is low. It is known that it can be used for prophylactic vaccines and immunotherapy for Korea (Korean Patent Publication Nos. 2018-0080999 and 2016-0021933).
  • the present inventors studied to develop a new vaccine against COVID-19.
  • mice were immunized using a recombinant Mycobacterium paragordone strain transformed with a vector expressing the SARS-CoV-2 antigen.
  • the present invention was completed by confirming that a significant immune response including neutralizing antibody ability was generated in mice against SARS-CoV-2.
  • one aspect of the present invention provides a recombinant Mycobacterium paragordone strain expressing the SARS-CoV-2 antigen.
  • Another aspect of the present invention is a pharmaceutical composition for the treatment or prevention of SARS-CoV-2 infection
  • a pharmaceutical composition for the treatment or prevention of SARS-CoV-2 infection comprising the recombinant Mycobacterium paragordone strain as an active ingredient, SARS of the recombinant Mycobacterium paragordone strain -Use for the treatment or prevention of CoV-2 infection, or provides a method for the treatment or prevention of SARS-CoV-2 infection using the recombinant Mycobacterium paragordone strain.
  • a first composition comprising the recombinant Mycobacterium paragordone strain as an active ingredient; And a kit for treating or preventing SARS-CoV-2 infection, comprising a second composition comprising a receptor binding domain of SARS-CoV-2 or a fragment thereof, alum, or a combination thereof as an active ingredient, or provide a way
  • Another aspect of the present invention provides a vector comprising a nucleic acid molecule in which a polynucleotide encoding a receptor binding domain of SARS-CoV-2 or a fragment thereof is operably linked to an hsp gene promoter.
  • Another aspect of the present invention provides an isolated cell comprising the vector.
  • Another aspect of the present invention provides a method for producing a recombinant Mycobacterium paragordone strain expressing a SARS-CoV-2 antigen, comprising transforming the Mycobacterium paragordone strain with the vector do.
  • the recombinant Mycobacterium paragordone strain expressing the SARS-CoV-2 antigen of the present invention can induce a remarkable immune response including neutralizing antibody ability against SARS-CoV-2 in mice. Therefore, the recombinant Mycobacterium paragordone strain of the present invention can be usefully used as a vaccine for preventing or treating SARS-CoV-2 infection.
  • FIG. 1 shows a schematic diagram of the insertion sequence production using the RBD portion of SARS-CoV-2.
  • Figure 2 shows a schematic diagram of the insertion sequence using the NP (nucleocapside phosphoprotein) portion of SARS-CoV-2.
  • 3A and 3B show a vector map obtained by cloning a Phsp:RBD sequence (pMV306-Phsp:RBD) and a Phsp:NP sequence (pMV306-Phsp:NP) into the pMV306 shuttle vector, respectively.
  • 4A and 4B show the results of confirming the presence or absence of an insertion sequence through colony PCR in E. coli colonies transformed with pMV306-Phsp:RBD vector and pMV306-Phsp:NP vector, respectively.
  • Figures 5a and 5b show the colony pattern (Figure 5a) and colony PCR results (Figure 5b) formed on 7H10 solid medium (100 ⁇ g/ml kanamycin) after electroporation of the pMV306-Phsp:RBD vector into Mpg. .
  • FIG. 6a and 6b show the colony pattern ( FIG. 6a ) and colony PCR results ( FIG. 6b ) formed on 7H10 solid medium (100 ⁇ g/ml kanamycin) after electroporation of the pMV306-Phsp:NP vector into Mpg.
  • Figure 7 shows the pattern of the bands confirmed when an antibody (Sino Biological, 40591-T62) against the SARS-CoV-2 spike protein was extracted from the wild-type Mpg strain and the three rMpg_RBD strains.
  • an antibody Sino Biological, 40591-T62
  • SARS-CoV spike S1 protein Sino Biological, 40159-V08B1
  • FIG. 8 shows the patterns of the bands identified when an antibody (Thermo Fisher, MA5-29981) against the SARS-CoV-2 nucleocapsid protein was extracted by extracting the proteins of wild-type Mpg and two rMpg_NP strains.
  • NP protein Session Biological, 40588-V08B
  • Figure 9 shows the expression patterns after infecting dendritic cells with a wild-type Mpg strain (10 M.O.I.) and rMpg_RBD strain (1 or 10 M.O.I.), extracting RNA from the cells, performing real-time PCR with RBD gene-specific primers, and A comparison graph is shown.
  • statistical significance was tested by Student's t-test (**, p ⁇ 0.01).
  • Figure 10 shows that the protein of the wild-type Mpg strain and the rMpg_RBD strain (P1, 1st generation; P3, 3rd generation; P5, 5th generation) was extracted and an antibody against SARS-CoV-2 spike protein (Sino Biological, 40591-T62) was attached. When, it shows the confirmed band pattern.
  • RBD protein (Sino Biological, 40592-V08B) was used as a positive control.
  • FIG. 11A to 11D show the percentage of MHCII+ (FIG. 11A), CD40+ (FIG. 11B), CD80+ (FIG. 11C), and CD86+ (FIG. 11D) cell populations by FACS analysis of dendritic cell maturation markers upon infection with Mpg and rMpg_RBD. The result of comparing (%) is shown.
  • statistical significance was tested by Student's t-test (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
  • FIG. 12A and 12B show the results of measuring and comparing the expression levels of cytokines IL-10 (FIG. 12A) and IL-12 (FIG. 12B) in dendritic cell culture medium during infection with Mpg and rMpg_RBD by ELISA.
  • cytokines IL-10 FIG. 12A
  • IL-12 FIG. 12B
  • Student's t-test *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
  • FIG. 13A and 13B show RBD expression using RBD ELISA kit (FIG. 13A) and real-time PCR (FIG. 13b) shows the results confirmed.
  • FIG. 13A shows RBD expression using RBD ELISA kit
  • FIG. 13b shows the results confirmed.
  • statistical significance was tested by Student's t-test (**, p ⁇ 0.01; ***, p ⁇ 0.001).
  • FIG. 14 shows a schematic diagram of the Mpg and rMpg_RBD strain inoculation schedule for mice.
  • 15A and 15B are graphs showing the results of IFN- ⁇ ELISPOT assay using mouse splenocytes immunized with SC-immunized Mpg and rMpg_RBD (#5, #6, #7) strains. IFN by each rMpg_RBD strain. - ⁇ ELISPOT results (FIG. 15A) and IFN- ⁇ ELISPOT results by all rMpg_RBD strains (FIG. 15B) are shown. 15A shows a photograph of a representative membrane. Here, statistical significance was tested by Student's t-test (*, p ⁇ 0.05; ***, p ⁇ 0.001).
  • FIG. 16A and 16B show the expression of cytokine IL-12 (FIG. 16A) and splenocytes of mice immunized with SC-immunized Mpg and rMpg_RBD strains (#5, #6, #7) with S1 protein.
  • IFN- ⁇ ( FIG. 16B ) levels were measured by ELISA, and the results of comparison are shown.
  • FIG. 17A to 17C show serum samples of mice immunized with Mpg and rMpg_RBD strains (#5, #6, #7) with RBD protein after stimulation with RBD protein, IgG2 ( FIG. 17A ), IgG1 ( FIG. 17B ) and total IgG.
  • FIG. 17c shows the result of comparing the antibody expression level by measuring the absorbance.
  • statistical significance was tested by Student's t-test (**, p ⁇ 0.01).
  • 18A to 18D show the SARS-CoV-2 pseudovirus neutralizing antibody ability of mouse serum immunized with wild-type Mpg wild-type and three rMpg_RBD strains.
  • rMpg-RBD 1 means #5
  • rMpg-RBD 2 means #6
  • rMpg-RBD 3 means #7.
  • FIG. 20 is a graph showing the results of an IFN- ⁇ ELISPOT assay using mouse splenocytes immunized with SC-immunized Mpg, rMpg_RBD and RBD proteins. At the bottom, a photograph of a representative membrane is shown. Here, statistical significance was tested by Student's t-test (**, p ⁇ 0.01).
  • 21A to 21D show mouse splenocytes immunized with SC-immunized Mpg, rMpg_RBD and RBD proteins with RBD protein, followed by CD3+ CD4+ IFN- ⁇ + ( FIG. 21A ), CD3+ CD4+ TNF- ⁇ + ( FIG. 21B ). ), CD3+ CD8+ IFN- ⁇ + ( FIG. 21C ), and CD3+ CD8+ TNF- ⁇ + ( FIG. 21A ) T cell populations analyzed by FACS are shown. Here, statistical significance was tested with Student's t-test (*, p ⁇ 0.05; **, p ⁇ 0.01; *** p ⁇ 0.001).
  • FIG. 22A to 22E show the expression of cytokines IFN- ⁇ (FIG. 22A) and TNF- ⁇ (FIG. 22B) after stimulation of splenocytes of mice immunized with Mpg, rMpg_RBD and RBD protein with RBD protein. ), IL-2 (FIG. 22C), IL-10 (FIG. 22D), and IL-12 (FIG. 22E) levels were measured and compared by ELISA.
  • IL-2 FIG. 22C
  • IL-10 FIG. 22D
  • FIG. 22E IL-12
  • FIG. 23A to 23C show serum samples of mice immunized with Mpg, rMpg_RBD, and RBD protein with RBD protein after stimulation with RBD protein, IgG2 (FIG. 23A), IgG1 (FIG. 23B), and total IgG (FIG. 23C)
  • IgG2 FIG. 23A
  • IgG1 FIG. 23B
  • total IgG FIG. 23C
  • 25A to 25D show the ability of neutralizing antibody against SARS-CoV-2 pseudovirus of mouse serum immunized with rMpg_RBD strain or RBD protein.
  • FIGS. 26A and 26B show the results of an ACE2-RBD binding assay for evaluating the effect of PBS, Mpg, and rMpg_RBD-immunized mouse serum on the binding of ACE2 to RBD.
  • Figure 26a shows the absorbance measurement and analysis result of the amount of HRP-IgG binding to the Fc tag of the RBD protein bound to ACE2
  • Figure 26b is the absorbance result normalized (the highest value is 100%, the most A graph is shown in which ACE2-RBD binding is converted to % by converting low values to 0%).
  • the p value was expressed by analyzing the results of PBS and Mpg and rMpg_RBD by Student's t-test (***, p ⁇ 0.001).
  • FIG. 27 shows the results of comparing BALF samples of mice immunized with Mpg, rMpg_RBD and RBD proteins with the RBD protein by measuring the absorbance of the expression level of the IgA antibody.
  • statistical significance was tested with Student's t-test (*, p ⁇ 0.05; **, p ⁇ 0.01; *** p ⁇ 0.001).
  • FIG. 28 shows a host cell molecule used when SARS-CoV-2 virus infects a host cell and a schematic diagram thereof.
  • Figure 29 shows the SARS-CoV-2 Vero p2 neutralizing antibody ability of serum obtained from mice at 2 weeks and 2 weeks and 5 days after subcutaneous injection of rMpg_RBD strain (#7 strain) into mice at 1 ⁇ 10 6 CFU in live cell form.
  • the control antibody is an anti-SARS-CoV-2 spike antibody (Cat#: 40150-T62-COV2).
  • control antibody is an anti-SARS-CoV-2 spike antibody (Cat#: 40150-T62-COV2).
  • Figure 31 shows the SARS-CoV-2 Vero p2 neutralizing antibody ability of serum obtained from mice at 2 weeks and 2 weeks and 5 days by subcutaneously injecting the rMpg_RBD strain (#7 strain) into mice at 1 ⁇ 10 7 CFU in an inactivated form. indicates.
  • the control antibody is an anti-SARS-CoV-2 spike antibody (Cat#: 40150-T62-COV2).
  • FIG. 32 shows a graph in which the results for the rMpg_RBD strain among the results shown in FIGS. 29 to 31 are expressed as changes according to the blood sampling period after inoculation.
  • Live ⁇ 6, Live ⁇ 7 and Inactivation ⁇ 7 are 1 ⁇ 10 6 CFU of live cell form, 1 ⁇ 10 7 CFU of live cell form and 1 ⁇ 10 6 CFU of inactivated form of the rMpg_RBD strain (#7 strain), respectively.
  • One aspect of the present invention provides a recombinant Mycobacterium paragordone strain expressing the SARS-CoV-2 antigen.
  • SARS-CoV-2 antigen refers to a polypeptide or polypeptide fragment capable of inducing an immune response, such as a humoral and/or cellular mediated immune response, against SARS-CoV-2 in a subject. it means.
  • the antigen is a protein, fragment or epitope thereof, or several SARS-CoV-2 proteins or these capable of inducing an immune response against SARS-CoV-2 in a subject or exhibiting a protective immune effect. It may be a combination of some of the
  • the SARS-CoV-2 antigen may be a spike (S) protein or a fragment thereof that forms a protruding spike on the surface of SARS-CoV-2.
  • the SARS-CoV-2 antigen may be an S1 subunit or a fragment thereof comprising a receptor binding domain (RBD) of a spike protein that binds to angiotensin-converting enzyme 2 (ACE2) of a host cell.
  • the SARS-CoV-2 antigen may be a receptor binding domain or a fragment thereof.
  • the receptor binding domain or fragment thereof may have the amino acid sequence of SEQ ID NO: 1.
  • the SARS-CoV-2 antigen may be expressed from a polynucleotide encoding the SARS-CoV-2 antigen operably linked to a promoter.
  • the polynucleotide encoding the SARS-CoV-2 antigen may be the nucleotide sequence of SEQ ID NO: 2.
  • the promoter may be a heat shock protein (hsp) promoter. More specifically, the promoter may be a heat shock protein 65 (hsp65) gene promoter derived from Mycobacterium bovis BCG.
  • the recombinant Mycobacterium paragordone strain may be a wild-type Mycobacterium paragordone strain transformed to express the SARS-CoV-2 antigen.
  • the polynucleotide encoding the SARS-CoV-2 antigen is operable on the Hsp65 (heat shock protein 65) gene promoter derived from Mycobacterium bovis BCG. It may be transformed with a tightly linked nucleic acid molecule.
  • the SARS-CoV-2 antigen may be a spike (S) protein, which is a surface antigen of SARS-CoV-2, and more specifically, may be an S1 subunit.
  • the SARS-CoV-2 antigen may be a receptor binding domain.
  • the recombinant Mycobacterium paragordone strain may be transformed with the pMV306-Pshp:RBD vector having the structure shown in FIG. 3A.
  • the (wild type) Mycobacterium paragordone (Mpg) strain used in the embodiment of the present invention is a naturally occurring strain, morphologically similar to Mycobacterium gordonae, but the optimum growth temperature is 25 to 30 °C, it is a temperature-sensitive strain that does not grow at 37 °C, the temperature at which general bacteria grow well. Due to these characteristics, safety can be secured when applied to the human body.
  • Mpg strain reference may be made to Korean Patent Application Laid-Open No. 2016-0021933 and the like. This document is considered a part of this specification. Specifically, the Mpg strain may be deposited at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center under the accession number KCTC 12628BP.
  • Another aspect of the present invention provides a pharmaceutical composition for the treatment or prevention of SARS-CoV-2 infection, comprising the recombinant Mycobacterium paragordone strain as an active ingredient.
  • the pharmaceutical composition may be a vaccine.
  • the pharmaceutical composition may be administered in a single dose or in multiple doses.
  • the multiple administration may be two administrations.
  • the pharmaceutical composition may be used as a priming vaccine in a prime-boosting inoculation method.
  • the boosting inoculation may be used together with the receptor binding domain of SARS-CoV-2, alum, or a combination thereof.
  • the pharmaceutical composition may be in the form of a microneedle or microarray formulation, powder, capsule, tablet, granule, aqueous suspension, inhalant, suppository, injection, ointment, patch, powder or beverage.
  • the pharmaceutical composition may be for humans.
  • a pharmaceutically acceptable excipient used in the pharmaceutical composition in the case of oral administration, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a pigment, a fragrance, etc. may be used.
  • a binder a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a pigment, a fragrance, etc.
  • buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used.
  • the dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable excipients as described above.
  • the pharmaceutical composition of the present invention may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. in the case of oral administration, and in the case of injections in the form of unit dose ampoules or multiple doses. can be manufactured.
  • the pharmaceutical composition of the present invention may be formulated as a solution, sustained release preparation, pill, dragee, liquid, gel or slurry. In the case of a patch, it may be formulated in the form of microneedles or microarrays.
  • suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate , cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.
  • Routes of administration of the pharmaceutical composition include, but are not limited to, subcutaneous, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. Routes are included, and subcutaneous or transdermal routes may be preferred.
  • the dosage of the pharmaceutical composition may vary depending on a number of factors including the subject's age, weight, general health, sex, diet, administration frequency, administration period, administration route, excretion rate, and the severity of the specific disease to be treated or prevented. and may be appropriately selected by those skilled in the art. Typically, the dosage may be 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg per day. Administration may be performed once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.
  • the vaccine may be administered two or more times in a normal prime-boost form.
  • the same vaccine may be administered several times, or different types of vaccines containing the same antigen may be administered.
  • the vaccine of the present invention may be used as a priming vaccine, and a receptor binding domain of SARS-CoV-2 or a fragment thereof, alum, or a combination thereof may be used in a boosting inoculation.
  • the recombinant Mycobacterium paragordone strain may be used in live, attenuated or inactivated form.
  • a first composition comprising the above-described recombinant Mycobacterium paragordone strain as an active ingredient; And it provides a kit for the treatment or prevention of SARS-CoV-2 infection, comprising a second composition comprising a receptor binding domain of SARS-CoV-2 or a fragment thereof, alum, or a combination thereof as an active ingredient do.
  • the kit may be for prime-boost inoculation.
  • the first composition may be prime-administered and the second composition may be boost-administered.
  • Another aspect of the present invention provides a vector comprising a recombinant nucleic acid molecule in which a polynucleotide encoding a receptor binding domain of SARS-CoV-2 is operably linked to an hsp gene promoter.
  • the hsp gene promoter may be a heat shock protein 65 (hsp65) gene promoter derived from Mycobacterium bovis BCG.
  • the vector may be for Mycobacterium strain expression, specifically Mycobacterium-E. coli shuttle vector.
  • the vector may be pMV306-Pshp:RBD having the structure shown in FIG. 3A.
  • the cell may be a Mycobacterium strain, specifically, a Mycobacterium paragordone strain.
  • Another aspect of the present invention provides a method for producing a recombinant Mycobacterium paragordone strain expressing a SARS-CoV-2 antigen, comprising transforming the above-described vector into the Mycobacterium paragordone strain. to provide.
  • the transformation can be performed by various methods known in the art, for example, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, Agrobacterium-mediated transfection, DEAE-dextran treatment, and gene balm. It may be performed using a badment or the like. In an embodiment of the present invention, transformation was performed using electroporation.
  • Example 1 Construction of mycobacteria-E. coli shuttle vector for SARS-CoV-2 antigen expression
  • RBD receptor binding domain
  • NP nucleocapsid phosphoprotein
  • the NP sequence consists of 654 nucleotides (SEQ ID NO: 4) and encodes 217 amino acids (SEQ ID NO: 3) corresponding to amino acids 138 to 353 of the entire nucleocapsid phosphoprotein (FIG. 2).
  • the hsp65 (heat shock protein 65) gene promoter (Phsp) portion (375 bp) (SEQ ID NO: 5) was amplified through PCR from the genomic DNA of Mycobacterium bovis BCG (The Tuberculosis Research Institute), and the synthesized Phsp:RBD (1,050 bp) and Phsp:NP (1,029 bp) sequences were prepared by linking each gene sequence and overlapping PCR. Table 1 shows the primer sets and temperature conditions used for the overlapping PCR.
  • Phsp:RBD and Phsp:NP sequences were cloned into a mycobacteria-E. coli shuttle vector, pMV306, using a combination of NotI and XbaI or EcoRV and XbaI restriction enzymes to construct a vector ( FIGS. 3A and 3B ), and the vector was transformed into E. coli. Thereafter, PCR was performed on some of the colonies formed through kanamycin selection to select colonies in which the insert sequence was strongly amplified ( FIGS. 4A and 4B ). Plasmid DNA was extracted and plasmid DNA identical to the inserted sequence was obtained through sequencing.
  • Example 2 Each of the plasmid DNAs (pMV306-Phsp:RBD and -Phsp:NP) obtained in Example 1 was subjected to electroporation (Gene Pulser Xcell TM , Bio-RAD) into competent Mpg prepared in-house. Transformed under the conditions described in Table 2.
  • the Mpg generated after electroporation was inoculated into 7H9 liquid medium (Becton Dickinson) without antibiotics and incubated at 30°C for 24 hours, and then 1/10 to 1/2 dilutions of 7H10 solids containing kanamycin (100 ⁇ g/ml) It was plated on medium (Becton Dickinson). Thereafter, it was cultured at 30° C. for 14 to 21 days, and colony formation was checked.
  • pMV306-Phsp:RBD a significant number of colonies were secured ( FIG. 5A ).
  • FIG. 5b As a result of confirming some of the obtained colonies by PCR, it was confirmed that most colonies showed a positive reaction (RBD expression: amplification of the transformed RBD gene was confirmed) ( FIG. 5b ).
  • Colonies were selected for each prepared recombinant Mpg strain (rMpg_RBD and rMpg_NP) (colonies corresponding to #5, #6, and #7 among several colonies were selected, and the colony was subjected to colony PCR targeting RBD)
  • rMpg_RBD and rMpg_NP colonnies corresponding to #5, #6, and #7 among several colonies were selected, and the colony was subjected to colony PCR targeting RBD
  • Each recombinant Mpg (rMpg_RBD and rMpg_NP) strain culture was centrifuged (2,500 rpm, 10 min) to obtain a pellet, and then B-PER buffer (Thermo Scientific; 100 ⁇ g/ml lysozyme, 5 U/ml DNase) , and supplemented with proteinase inhibitor) and sonicated on ice (2 reactions 5 min; pulse: 0.3 sec, stop: 0.7 sec).
  • the pulverized solution was centrifuged (13,000 rpm, 15 min, 4° C.) to take the supernatant, and western blot was performed.
  • anti-SARS-CoV-2 spike protein antibody (Sino Biological, 40591-T62; 1:2,000) and anti-SARS-CoV-2 nucleoside
  • a capsid protein antibody (Thermo Fisher, MA5-29981; 1:1,000)
  • an antibody (Santa Cruz, sc-57842; 1:200) capable of binding to hsp65 (heat shock protein 65) protein of Mycobacterium tuberculosis was used.
  • SARS-CoV-2 spike S1-His recombinant protein (Sino Biological, 40159-V08B1) and SARS-CoV-2 nucleocapsid-His recombinant protein (Sino Biological, 40588-V08B) were used.
  • Dendritic cells (Huiying Bio. Tech) were seeded in a 24-well plate (5 x 10 5 cells/well) and 24 hours later, the rMpg_RBD strain was infected at 1 or 10 MOI (multiplicity of infection). After 4 hours, the infected cells were washed with PBS to remove extracellular bacteria, replaced with a medium containing amikacin (10 ⁇ M), and cultured at 37° C. for 24 hours. Uninfected dendritic cells and cells infected with the Mpg wild-type strain were prepared in the same manner as controls.
  • the obtained cell culture was stored at -20°C for immune cytokine analysis.
  • Cells were washed with PBS and then mixed with 1 ml Trizol (TRIzol® Reagent, ThermoFisher Scientific, REF: 15596018). Then, chloroform was added, mixed well, and incubated for 3 minutes at room temperature. The supernatant was centrifuged (12,000 x g, 15 min, 4°C), mixed with the same amount of isopropanol, and centrifuged again. After washing with 75% EtOH, the pellet was dried in air by centrifugation. Thereafter, it was dissolved in an appropriate volume of DEPC-treated water to obtain total RNA.
  • Real-time PCR (CFX ConnectTM Real-Time System, Bio-RAD) was performed on the extracted RNA with a SYBR kit (SensiFASTTM SYBR® Lo-ROX One-Step kit, Bioline), and based on the obtained Cq value, intracellular RBD The expression level was relatively quantified.
  • Table 3 shows the primer sets and temperature conditions for real-time PCR used for RBD detection.
  • Primer sets (SEQ ID NOs: 10 and 11) and temperature conditions used for RBD detection Primers Sequences (5' to 3') Tm (°C) RBD_RT_F1 ACC GAG TCG ATC GTG CGC TTC 60 RBD_RT_R1 GAA GCA CAG GTC GTT CAG CTT
  • the recombinant Mpg strain (rMpg_RBD) prepared in Example 2 was similar to that of the wild-type Mpg strain, the recombinant Mpg strain and the wild-type Mpg strain were each infected with dendritic cells, and then the expression pattern of dendritic cell maturation markers. was confirmed, and the expression pattern of IL-10 and IL-12 cytokines was confirmed from the cell culture medium. A group untreated and a group treated with LPS were used as negative and positive controls, respectively.
  • dendritic cells were seeded in 24-well plates (5 x 10 5 cells/well) and 24 hours later, they were infected with Mpg (10 MOI) and rMpg_RBD strains (1 or 10 MOI). After 4 hours, the infected cells were washed with PBS to remove extracellular bacteria. Then, the medium was replaced with a medium containing amikacin (10 ⁇ M) and cultured at 37° C. for 24 hours.
  • the obtained cell culture was stored at -20°C for cytokine analysis. After washing with PBS, cells were removed and blocked with CD16/32 antibody (Biolegend, Cat: 101301) for 30 minutes, BV605 conjugated anti-CD86, PE conjugated anti-CD40, FITC conjugated anti-MHCII, APC conjugated anti -CD80 antibody (BD Biosciences) was stained at 4°C for 30 minutes under light-blocked conditions. Then, the expression pattern of the maturation marker of dendritic cells was confirmed by FACS (BD LSRFortessa).
  • the expression rate of dendritic cell maturation markers (MHCII, CD40, CD80, CD86) was relatively high when Mpg-infected, and in the case of rMpg_RBD strain infection (10 M.O.I.), It was confirmed that the maturation marker expression pattern was increased to a similar level ( FIGS. 11a to 11d , Table 4). However, rMpg_RBD 1 M.O.I. Upon infection, the maturation marker hardly increased as in the untreated group ( FIGS. 11A to 11D , Table 4). In addition, the expression patterns of cytokines IL-10 and IL-12 in cell culture were analyzed by ELISA. .
  • each well was washed 3 times with wash buffer and blocked with assay buffer at room temperature for 1 hour. After washing each well again, the obtained culture solution was added and reacted at room temperature for 2 hours. After washing each well, a detection antibody for each cytokine was attached and reacted at room temperature for 1 hour. Then, after each well was developed with a color reagent, absorbance was measured with an ELISA reader device (Tecan Sunrise) at OD 450 nm, and the expression level of IL-10 and IL-12 cytokines was measured based on the standard value. did.
  • Cytokine expression level comparison result Concentration (pg/ml) IL-10 IL-12 no treat 70.02 ⁇ 1.82 3.50 ⁇ 0.41 mpg 384.49 ⁇ 4.98 90.27 ⁇ 1.91 rMpg_RBD (1 M.O.I.) 86.48 ⁇ 2.06 7.66 ⁇ 1.95 rMpg_RBD (10 M.O.I.) 267.91 ⁇ 40.13 61.61 ⁇ 4.17 LPS 750.27 ⁇ 7.39 125.76 ⁇ 4.07
  • rMpg_RBD 1 M.O.I.
  • IL-10 and IL-12 showed a statistically significant increase compared to the untreated group, but the expression level was significantly lower than that of the other groups.
  • Example 2 prepared in Example 2 When one rMpg_RBD strain was infected with dendritic cells with 10 M.O.I., the expression of dendritic cell maturation markers (MHCII, CD40, CD80, CD86) was increased to a level similar to that of Mpg infection, and IL-10 and IL- It was confirmed that the expression of cytokines such as 12 is also increased.
  • MHCII dendritic cell maturation markers
  • the rMpg_RBD strain prepared in Example 2 was subcultured in 7H9 liquid medium (containing 100 ⁇ g/ml of kanamycin), the protein of the 5th generation cultured strain was extracted, and RBD western blot was performed to confirm RBD expression (FIG. 10). ).
  • the culture of the strain was completed up to 10 generations, and each cultured strain was suspended in 20% glycerol and stored at -70°C.
  • the culture solution of the subcultured rMpg_RBD strain (strain #7) was centrifuged (2,500 rpm, 10 min) to obtain a pellet, and then B-PER buffer (Thermo Scientific; 100 ⁇ g/ml lysozyme, 5 U/ml DNase, and proteinase inhibitor) and sonication on ice (5 min in 2 reactions; pulse: 0.3 sec, stop: 0.7 sec) on ice.
  • the pulverized solution was centrifuged (13,000 rpm, 15 min, 4°C), and the supernatant was taken to secure the protein.
  • the expression of RBD in the protein was confirmed with the S1RBD ELISA kit (ELV-COVID19S1-1) provided by RayBiotech.
  • the extracted protein was put in a 96-well plate coated with an S1 RBD-specific antibody and reacted at room temperature for 2.5 hours. Then, each well was washed 3 to 4 times with the wash buffer provided by the kit, and the S1 RBD detection antibody was put into each well and reacted at room temperature for 1 hour. After washing each well again, HRP-streptavidin was added to each well and reacted at room temperature for 45 minutes.
  • the rMpg_RBD strain culture pellet was mixed with Trizol (TRIzol® Reagent, ThermoFisher Scientific, REF: 15596018), transferred to a tube containing a glass bead, and pulverized 3 times for 30 seconds each with a Mini BeadBeater device (BioSpec). Then, chloroform/isoamyl alcohol (29:1) was added and mixed well. Then, centrifugation (16,000 x g, 5 min) was performed, and the obtained supernatant was treated with isopropanol and 3 M sodium acetate, and precipitated at -20°C. The precipitated RNA was washed with 75% EtOH and then dried in air.
  • Trizol Trizol® Reagent, ThermoFisher Scientific, REF: 15596018
  • RNA was extracted through a general EtOH precipitation method.
  • Real-time PCR (CFX ConnectTM Real-Time System, Bio-RAD) was performed on the extracted RNA with a SYBR kit (SensiFASTTM SYBR® Lo-ROX One-Step kit, Bioline), and based on the obtained Cq value, RBD in the strain The expression level was relatively quantified. At this time, the relative expression level was calculated by correcting each RBD Cq value with the Cq value for hsp65.
  • Table 6 shows the primer sets and temperature conditions for real-time PCR used for RBD detection.
  • Primer set (SEQ ID NOs: 12-15) and temperature conditions used for RBD detection Primers Sequences (5' to 3') Tm (°C) RBD_RT_F1 ACC GAG TCG ATC GTG CGC TTC 60 RBD_RT_R1 GAA GCA CAG GTC GTT CAG CTT Mpg_hsp_RT_F GTC GAG GAG TCC AAC ACC TT 60 Mpg_hsp_RT_R CTG GAG ACC AGC AGG ATG TA
  • rMpg_RBD strains #5, #6, #7 strains
  • SC subcutaneous
  • mice were sacrificed to obtain splenocytes. After the obtained splenocytes were stimulated with S1 protein, i) IFN- ⁇ ELISPOT and ii) IFN- ⁇ , IL-12 immune cytokines were analyzed by ELISA. In addition, iii) expression of S1-specific IgG (IgG1, 2 and total IgG) and iv) neutralizing antibody ability against SARS-CoV-2 pseudovirus were analyzed in serum samples.
  • S1-specific IgG IgG1, 2 and total IgG
  • neutralizing antibody ability against SARS-CoV-2 pseudovirus were analyzed in serum samples.
  • ELISPOT assay for IFN- ⁇ was performed on splenocytes isolated from immunized mice. First, splenocytes of each group were seeded at 1 x 10 6 cells/well on a PVDF membrane plate coated with IFN- ⁇ antibody (anti-mouse IFN- ⁇ , clone: AN-18, 3 ⁇ g/ml), and SARS- Splenocytes were stimulated for 24 hours by treatment with CoV-2 spike (S1) protein at 5 ⁇ g/ml. Then, each well was washed 3 times with PBST and PBS, respectively.
  • IFN- ⁇ antibody anti-mouse IFN- ⁇ , clone: AN-18, 3 ⁇ g/ml
  • SARS- Splenocytes were stimulated for 24 hours by treatment with CoV-2 spike (S1) protein at 5 ⁇ g/ml. Then, each well was washed 3 times with PBST and PBS, respectively.
  • the detection antibody (anti-mouse-IFN- ⁇ biotin, clone: XMG1.2, 3 ⁇ g/ml) was treated and reacted at 4° C. for 24 hours. After washing each well again, streptavidin-HRP was treated at room temperature for 2 hours. Thereafter, color was developed using an AEC substrate kit (within 10 minutes), and spots on each membrane were counted with an ELISPOT reader device (AID EliSpot Reader).
  • Splenocytes isolated from immunized mice were cultured in vitro with S1 protein (5 ⁇ g/ml) for 3 days, then the culture medium was collected and stored at -70°C. Thereafter, ELISA was performed for IFN- ⁇ and IL-12 cytokines to compare and analyze their expression patterns. ELISA was performed using mouse IFN- ⁇ and IL-12 ELISA kit (Invitrogen).
  • the expression of IL-12 and IFN- ⁇ cytokines specific for S1 protein in splenocytes immunized with the rMpg_RBD strain was increased compared to the case of immunization with the Mpg strain.
  • the rMpg_RBD #7 strain showed the highest IL-12 and IFN- ⁇ cytokine expression levels were confirmed (Fig. 16, Table 8).
  • RBD protein (Sino Biological) was coated (5 ⁇ g/ml) on an ELISA plate and reacted at 4° C. for 24 hours. Thereafter, each well was washed with wash buffer (0.05% Tween 20), and then each well was blocked with 5% BSA. Serum samples isolated from mice were diluted (1:10), put into each well, and reacted at room temperature for 2 hours.
  • IgG1 with HRP goat anti-mouse IgG1, HRP conjugated; ThermoFisher Scientific
  • IgG2a goat anti-mouse IgG2, HRP conjugated; Invitrogen
  • total IgG anti mouse IgG, biotinylated; eBioScinece
  • IgG production pattern for RBD was measured by absorbance Absorbance (OD 450nm) Stimulated with RBD IgG2 IgG1 Total IgG mpg 0.087 ⁇ 0.003 0.555 ⁇ 0.046 0.384 ⁇ 0.005 rMpg_RBD #5 0.09 0.526 0.513 rMpg_RBD #6 0.137 0.692 0.537 rMpg_RBD #7 0.188 0.775 0.619
  • pCAGGS NR-52310, BEI Resources
  • SARS-CoV-2 spike glycoprotein S sequence was co-transfected into human embryonic kidney 293T cells to obtain a SARS-CoV-2 pseudovirus pellet.
  • -transfection was carried out.
  • 5X PEG-it LV810A-1-SBI
  • 5X PEG-it LV810A-1-SBI
  • rMpg_RBD 1, #5; rMpg_RBD 2, #6; rMpg_RBD 3, #7) and a wild-type Mpg strain were subcutaneously injected into BALB/c mice twice at 2-week intervals. Two weeks after the last injection, they were euthanized and serum was obtained from each mouse. Each serum is diluted in RPMI 1640 medium in multiples of 1:10, 1:20, 1:50, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 in a 96-well plate to prepare 100 ⁇ l each. did.
  • SARS-CoV-2 pseudovirus pellet was diluted in PRMI 1640 and mixed with serum by pipetting. Then, it was cultured in an incubator at 37° C. for 2 hours. After 2 hours of reaction, all of the culture medium was treated with Huh7 cells (96-well plate, 5 ⁇ 10 4 cells/well), which is a human liver cancer cell line, and cultured in an incubator at 37° C. for 48 hours. Then, the supernatant was removed and the cells were lysed with a cell lysis buffer (Promega, USA). Then, immediately after reaction with a luciferase substrate (Promega, USA), luciferase color development was confirmed with a Luminometer (Tecan) ( FIGS. 18A to 18B ).
  • NT50 value is the serum dilution factor when the color of luciferase is reduced by 50% based on the pseudovirus infection without mixing the serum, and means a neutralizing antibody titer of 50.
  • the rMpg_RBD strain 3 showed the highest NT50 value.
  • the rMpg_RBD strain 3 (#7 strain) It is expected that the neutralizing antibody ability against SARS-CoV-2 will be the best. Even when the same neutralizing antibody ability test was performed on Calu-3 cells, it was confirmed that the neutralizing antibody ability of the mouse serum immunized with the rMpg_RBD 3 (#7) strain was the best.
  • RBD protein and alum were used, RBD protein was adjusted to a concentration of 10 ⁇ g/ml, and alum was adjusted to a concentration of 100 ⁇ g/ml, and immunization was performed after mixing them together. All inoculation volumes were adjusted to 100 ⁇ l.
  • mice 8-week-old BALB/c female mice were immunized with each strain, and each group was composed of 5 mice.
  • the immunized mice were sacrificed to obtain splenocytes, and the obtained splenocytes were stimulated with SARS-CoV-2 RBD protein.
  • IFN- ⁇ ELISPOT assay was performed as follows, ii) CD4, CD8 T cell populations expressing IFN- ⁇ or TNF- ⁇ were analyzed by FACS, iii) immune cytokines (IFN) - ⁇ , TNF- ⁇ , IL-2, IL-10 and IL-12) were analyzed by ELISA, iv) the expression of RBD specific IgG (IgG1, IgG2 and total IgG) in serum samples was analyzed, v) Comparing the neutralizing antibody ability against live SARS-CoV-2 virus, vi) comparing the neutralizing antibody ability against SARS-CoV-2 pseudovirus, vii) performing an ACE2-RBD binding assay using serum , viii) IgA expression specific for SARS-CoV-2 RBD was confirmed in bronchoalveolar lavage fluid (BALF) of mice.
  • BALF bronchoalveolar lavage fluid
  • ELISPOT for IFN- ⁇ was performed on splenocytes isolated from immunized mice.
  • splenocytes of each group were seeded at 1 x 10 6 cells/well on a PVDF membrane plate coated with IFN- ⁇ (anti-mouse IFN- ⁇ , clone: AN-18, 3 ⁇ g/ml), and SARS-CoV Splenocytes were stimulated for 24 hours by treatment with -2 RBD protein at 5 ⁇ g/ml. Then, each well was washed three times with PBST and PBS, respectively, and then treated with a detection antibody (anti-mouse-IFN- ⁇ biotin, clone: XMG1.2, 3 ⁇ g/ml). Then, it was made to react at 4 degreeC for 24 hours.
  • streptavidin-HRP was treated at room temperature for 2 hours. Thereafter, color was developed using an AEC substrate kit (within 10 minutes), and the spot of each membrane was counted with an ELISPOT reader device (AID EliSpot Reader).
  • the splenocytes of each immunized mouse were stimulated with RBD protein for 18 hours, and then treated with brefeldin A (eBioscience TM BrefeldinA, Invitrogen) for 4 to 6 hours to capture intracellular cytokines. Thereafter, surface molecular staining (4°C, 30 min) was first performed using FITC-conjugated anti-CD3, V500-conjugated anti-CD4, and PE-conjugated anti-CD8 antibodies (BD BioSciences). Then, cells were fixed with 1% paraformaldehyde (PFA) at room temperature for 20 minutes, and treated with 0.1% Triton X-100 (in FACS buffer) (RT, 30 minutes) to increase cell permeabilization.
  • PFA paraformaldehyde
  • CD4 and CD8 T cell populations secreting IFN- ⁇ showed statistical significance and increased compared to other immunized groups (FIG. 21, Table 11).
  • CD4+ IFN- ⁇ + T cells showed the highest aspect in the group immunized with rMpg_RBD once, which was determined to be correlated with the IFN- ⁇ ELISPOT results (Fig. 20, Fig. 21, Table 10, Table). 11).
  • TNF- ⁇ -secreting CD4 and CD8 T cell populations In the case of TNF- ⁇ -secreting CD4 and CD8 T cell populations, the rMpg_RBD immunized group (1 or 2 immunizations) showed a high pattern, and the rMpg_RBD immunized group showed a high pattern in the 2 immunized group.
  • TNF- ⁇ -secreting CD4 and CD8 T cell populations tended to increase compared to the group.
  • the TNF- ⁇ -secreting T cell population In particular, in the group immunized with rMpg_RBD prime-RBD protein boosting, the TNF- ⁇ -secreting T cell population showed the highest pattern (FIG. 21, Table 11).
  • Splenocytes isolated from immunized mice were incubated with SARS-CoV-2 RBD protein (5 ⁇ g/ml) in vitro for 3 days, then the culture medium was collected and stored at -70°C. Thereafter, ELISA was performed for IFN- ⁇ , TNF- ⁇ , IL-2, IL-10 and IL-12 cytokines to compare and analyze their expression patterns.
  • ELISA was mouse IFN- ⁇ , TNF- ⁇ , IL-2.
  • IL-10 and IL-12 ELISA kits (Invitrogen) were used.
  • an antibody specific for each cytokine was coated on a 96-well plate (4° C., O/N), and then each well was washed 3 times with wash buffer and blocked with assay buffer at room temperature for 1 hour. After washing each well again, the obtained culture solution was added and reacted at room temperature for 2 hours. After washing each well, a detection antibody for each cytokine was attached and reacted at room temperature for 1 hour. Then, after each well was developed with a color reagent, absorbance was measured with an ELISA reader device (Tecan Sunrise) at OD 450 nm, and based on the standard values, IFN- ⁇ , TNF- ⁇ , IL-2. The expression levels of IL-10 and IL-12 cytokines were measured.
  • the rMpg_RBD strain was The immunized group showed a higher cytokine expression level than the Mpg wild-type immunized group, and in particular, the group immunized with the rMpg_RBD strain twice showed the highest expression level.
  • TNF- ⁇ it was correlated with the results of FACS analysis (Fig. 21, Table 11), and the group immunized with the rMpg_RBD strain twice showed the highest expression level (Fig. 22b, Fig. 22c, Fig. 22e, Table 12). ).
  • the expression levels of cytokines (TNF- ⁇ , IFN- ⁇ , IL-12, etc.) important for the Th1-cell-mediated immune response were significantly lower than those of the other immune groups, but Th2 immunity It was confirmed that the expression level of IL-10 associated with the response was significantly higher than that of other immune groups (Fig. 22d, Table 11).
  • mice were tested for the following treatments: no treat, wild-type Mpg, rMpg_RBD(1), rMpg_RBD(2), rMpg_RBD + RBD, and RBD.
  • Serum was obtained from mice two weeks after the last injection of the strain or protein, and the obtained serum was stored at -20°C.
  • IgG1 with HRP goat anti-mouse IgG1, HRP conjugated; ThermoFisher Scientific
  • IgG2a goat anti-mouse IgG2, HRP conjugated; Invitrogen
  • total IgG anti mouse IgG, biotinylated; eBioScinece
  • RBD-specific IgG production pattern was measured by absorbance Absorbance (OD 450nm) IgG2 IgG1 Total IgG no treat 0.082 ⁇ 0.006 0.053 ⁇ 0.003 0.259 ⁇ 0.034 Mpg (2 times immunity) 0.124 ⁇ 0.021 0.583 ⁇ 0.055 0.448 ⁇ 0.032 rMpg_RBD (1 time) 0.136 ⁇ 0.039 0.286 ⁇ 0.165 0.366 ⁇ 0.047 rMpg_RBD (2 times) 0.197 ⁇ 0.050 0.564 ⁇ 0.058 0.470 ⁇ 0.027 rMpg_RBD+RBD protein 0.090 ⁇ 0.009 0.315 ⁇ 0.159 0.381 ⁇ 0.060 RBD protein (2 times) 0.113 ⁇ 0.018 0.344 ⁇ 0.046 0.395 ⁇ 0.044
  • the group immunized with rMpg_RBD showed a similar level of IgG1 expression to the rMpg_RBD prime-RBD boosting group and the RBD protein immune group.
  • the expression of total IgG seemed to reflect the high expression of IgG1, showing the highest pattern in the group immunized with the Mpg strain and the group immunized with the rMpg_RBD strain twice (FIG. 23c, Table 13).
  • the monkey kidney cell line Vero E6 was cultured in a T75 flask (5 ⁇ 10 6 in DMEM (supplemented with 10% FBS)) for 24 hours, and then in a 37°C water bath (water bath). bath) dissolved in the SARS-CoV-2 stock was dissolved in 20 ml DMEM (supplemented with 10% FBS) to replace the medium for Vero E6.
  • the infection was carried out in an incubator at 37° C. for 1 hour, and the flask was lightly shaken from side to side so that the virus could be uniformly infected at intervals of 10 minutes. After 1 hour, the supernatant was removed, replaced with a virus-free medium, and cultured at 37°C for 3 days. Thereafter, the supernatant was collected and aliquoted and stored in a -80°C deepfreezer.
  • 150 ⁇ l of mouse serum was prepared by diluting it in DMEM medium at dilution concentrations of 1:2, 1:10, 1:20, 1:50, 1:100, 1:1000, and 1:10000. Thereafter, 150 ⁇ l of SARS-CoV-2 of 100 to 150 pfu was reacted at 37° C. for 1 hour.
  • Each well of Vero E6 cells 24 well plate, 5 ⁇ 10 5 cells/well prepared the day before was infected with the reacted serum-virus mixture, and then reacted at 37° C. for 1 hour. At this time, the plate was lightly shaken at 10-minute intervals so that the virus could be uniformly infected. After 1 hour of infection, the medium was removed and washed with PBS. Then, DMEM (supplemented with 10% FBS and 0.8% methylcellulose) was replaced with 500 ⁇ l/well and incubated for 2 days in a 37° C. incubator.
  • the primary antibody against the nucleocapsid (NP) of SARS-CoV2 was diluted 1:1000 in 2% BSA (in PBS) and treated with 200 ⁇ l/well, followed by reaction at 4° C. overnight. Thereafter, the primary antibody was removed, and washing was performed three times with PBS. Then, the secondary antibody to which alkaline phosphatase (AP) is attached was diluted 1:5000 in 2% BSA (in PBS) and treated with 200 ⁇ l/ml, followed by reaction at room temperature for 2 hours. The secondary antibody was removed, washed with PBS, and the substrate NBT/BCIP was diluted 1:10000 in 2% BSA (in PBS) and treated with 200 ⁇ l/well. When plaque development progressed, the substrate solution was removed, washed with DW, and water was removed.
  • AP alkaline phosphatase
  • the titer of the Plaque Reduction Neutralization Test 50 refers to the dilution factor of the serum when the virus plaque formation is reduced by 50% compared to the case of no treatment. can be (Fig. 24).
  • PRNT50 Plaque Reduction Neutralization Test 50
  • the serum showed an excellent neutralizing antibody ability against SARS-CoV-2 at a statistically significant level compared to the case where the mice were not treated at all.
  • the serum in the case of treatment with a recombinant strain or a combination of a recombinant strain and a protein has excellent neutralizing antibody ability compared to the case of treatment with wild-type Mpg.
  • mice were immunized with recombinant rMpg_RBD once and immunized twice at 2-week intervals, it was confirmed that there was no difference in serum SARS-CoV-2 neutralizing antibody ability. However, compared to the case of immunization with rMpg_RBD once, it was confirmed that the neutralizing antibody ability of mouse serum was excellent at a statistically significant level when RBD protein was injected after immunization with rMpg_RBD once.
  • the mouse serum was diluted in multiples of 1:10, 1:20, 1:50, 1:100, 1:1000, 1:10000, 1:100000, 1:1000000 and reacted with SARS-CoV-2 at 37°C. did it Then, the serum-virus culture was infected with Calu-3 and Huh7 cells. At 48 hours after infection, inhibition of intracellular pseudovirus infection was confirmed through luciferase color development ( FIGS. 25A to 25D ).
  • the relative luciferase unit is the amount of luciferase expressed by SARS-CoV-2 pseudovirus infecting cells, and it can be said that the smaller the value, the more inhibited the infection.
  • the serum when comparing NT50 values, in the case of mice injected with the rest of the strains or proteins in preparation for no treatment, the serum was statistically significant for SARS-CoV-2 pseudovirus in both Calu-3 and Huh7 experiments. It showed excellent neutralizing antibody ability.
  • the NT50 value is the serum dilution factor when the color of luciferase is reduced by 50% based on the pseudovirus infection without mixing the serum, and means a neutralizing antibody titer of 50.
  • NT50 values were high in both Calu-3 and Huh7 experiments. It was confirmed that the pseudovirus neutralizing antibody ability was excellent at a statistically significant level.
  • the NT50 value of the mouse serum was found to be statistically significant, indicating that the mouse serum has excellent pseudovirus neutralizing antibody ability. that has been confirmed
  • HRP-conjugated IgG was attached to each well and reacted at room temperature for 1 hour. After washing each well again, a coloring reagent was added and allowed to develop color for up to 30 minutes. Then, the reaction was stopped, and absorbance was measured at 450 nm with an ELISA reader device (Tecan Sunrise). At this time, it can be determined that the lower the absorbance, the more inhibiting the binding of ACE2 and RBD.
  • ACE2-RBD binding inhibitory concentration (inhibitory concentration 50, IC50) by the rMpg_RBD immune serum was equal to 1 :762.2
  • rMpg_RBD (two immunizations) was 1:6008, indicating that serum immunized with rMpg_RBD twice had an excellent effect on inhibiting ACE2-RBD binding even at low concentrations.
  • IgA ELISA was performed.
  • mice were dissected to expose the bronchi. Thereafter, a 22-gauge catheter (BD BioSciences) was intubated into the bronchus, and then replaced with a 1 ml syringe containing 800 ⁇ l of PBS and injected. At this time, when injecting or withdrawing, visually confirm whether the lungs swell and subside again. The operation of inserting and withdrawing PBS with a syringe was repeated 3 to 4 times, and finally about 500 to 600 ⁇ l of BALF was obtained, and stored at -20°C until used in the experiment.
  • BD BioSciences BD BioSciences
  • an HRP-attached IgA antibody (goat anti-mouse IgA secondary antibody, HRP conjugated; Invitrogen) was added and reacted at room temperature for 1 hour, followed by color development.
  • HRP conjugated HRP conjugated; Invitrogen
  • the IgA expression pattern in BALF of the rMpg_RBD immunized group was statistically significant compared to the rMpg_RBD prime-RBD boosting and RBD immunization groups including Mpg. An increasing trend could be observed.
  • the group immunized with rMpg_RBD twice showed the highest expression of IgA, which was higher than the group immunized with rMpg_RBD once ( FIG. 27 , Table 15).
  • mice were immunized with the rMpg_RBD #7 strain according to an embodiment of the present invention according to the dosing schedule of FIG. 19, and the formation of SARS-CoV-2 RBD-specific immune-inducing ability was confirmed by several immune combinations [i) PBS (twice inoculation, S.C.
  • the PBS and Mpg strain immune group A higher IFN- ⁇ spot could be confirmed compared to .
  • the highest IFN- ⁇ spot was confirmed in the group immunized with the rMpg_RBD strain once ( FIG. 20 , Table 10).
  • IFN- ⁇ -secreting CD4 and CD8 T cell populations were different in the group immunized with rMpg_RBD (once or twice). It showed a statistically significant increase compared to the group (FIG. 21, Table 11). In particular, in the group immunized with the rMpg_RBD strain once, the CD4 T cell population expressing IFN- ⁇ showed the tendency to be most induced.
  • TNF- ⁇ -secreting CD4 and CD8 T cell populations the group immunized with rMpg_RBD (1 or 2 immunizations) showed a high pattern, but in the group immunized with rMpg_RBD prime-RBD protein boosting, TNF- The T cell population secreting ⁇ showed the highest pattern (FIG. 21, Table 11).
  • the immune cytokine expression pattern of the rMpg_RBD prime-RBD boosting immunization group was lower than that of the rMpg_RBD 1 or 2 immunization group.
  • the expression of IL-10, an anti-inflammatory cytokine was significantly higher than in the rest of the immune group, and the expression of immune cytokines important for the rest of the vaccine immunity showed a low tendency compared to other groups.
  • IgG2 which is known to be involved in cell-mediated Th1 immunity
  • the expression of IgG2 in the serum of each immunized mouse was confirmed to be the highest in the group immunized with rMpg_RBD twice, and the group immunized with rMpg_RBD once or rMpg_RBD prime-RBD boosting. showed similar or lower IgG2 expression patterns to those of the Mpg immune group.
  • IgG1 and total IgG1 in the group inoculated with Mpg bacteria was high without much difference from the rMpg_RBD 2 immunization group. It is considered that it should be considered (FIG. 23, Table 13).
  • rMpg_RBD prime-RBD boosting immunity had no significant effect on RBD-specific cell-mediated immune responses (IFN- ⁇ ELISPOT, IFN- ⁇ or TN F- ⁇ expressing T cell assay and immune cytokine measurement), and RBD-specific antibody There was no significant effect on production.
  • ACE2-RBD binding inhibitory concentration (IC50) by rMpg_RBD immune serum was 1:762.2 for rMpg_RBD (single immunization) and 1:6008 for rMpg_RBD (two immunizations), indicating that the serum immunized with rMpg_RBD twice had a lower concentration (IC50). It means that even at the concentration, it has an excellent effect on inhibiting the binding between ACE2-RBD.
  • the IgA expression pattern in the BALF of the group immunized with rMpg_RBD increased to a statistically significant level compared to the rMpg_RBD prime-RBD boosting and RBD immunization groups including Mpg. It showed a high expression pattern of IgA (Fig. 27, Table 15).
  • rMpg_RBD prime-RBD boosting immunity induction of RBD-specific immune cytokine expression, IFN- ⁇ and TNF- ⁇ expressing T cells, and RBD-specific IgG and IgA expression induction, rMpg_RBD strain immune group (1 or 2 times) immune group), but showed the greatest effect compared to the rMpg_RBD immune group in the induction of neutralizing antibodies against live SARS-CoV-2 and SARS-CoV-2 pseudovirus.
  • the rMpg_RBD strain alone induced RBD-specific immunity well
  • the rMpg_RBD prime-RBD boosting immunization induced the formation of neutralizing antibodies well. is expected to be usable.
  • mice were divided into three groups and the rMpg_RBD strain was injected subcutaneously at 1 ⁇ 10 6 CFU and 1 ⁇ 10 7 CFU in live cell form, and 1 ⁇ 10 7 CFU in inactivated form, respectively, at 2 weeks and 2 weeks and 5 days. Serum was obtained from mice.
  • mice serum was diluted to a dilution concentration of 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1280, 1:2560, 1:5120, 1:10240.
  • an anti-SARS-CoV-2 spike antibody (Cat#: 40150-T62-COV2) (1 mg/ml) was diluted to the same dilution concentration to prepare a control antibody.
  • SARS-CoV-2 Vero p2 (CoV/Korea/KCDC/2020 NCCP43326), a virus distributed from the Korea Centers for Disease Control and Prevention, was mixed at a concentration of 100 TCID 50 in the same volume as the serum and control antibody, and at 37 °C. Incubated for 30 minutes. Then, it was infected with the monkey kidney cell line Vero E6 (96 well plate, 1.5x10 ⁇ 6 cells/well) seeded 24 hours before the experiment to measure the degree of neutralization. Readings were confirmed by tissue culture infectious dose (TCID 50 ) format. In general, the TCID 50 method represents the dilution factor of the virus that infects 50 % of the cells by inoculating the virus suspension. The degree of neutralization was confirmed in a way that the experimental group and the control group lowered the infectivity of the virus. The results are shown in FIGS. 29 to 33 .

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Abstract

La présente invention concerne une souche recombinée de Mycobacterium paragordonae (Mpg) exprimant un antigène du SARS-CoV-2, et une composition de vaccin la comprenant. La souche recombinée de Mycobacterium paragordonae exprimant un antigène du SARS-CoV-2 de la présente invention peut générer des réponses immunitaires considérables, y compris une activité d'anticorps neutralisants, contre le SARS-CoV-2 chez les souris. Par conséquent, la souche recombinée de Mycobacterium paragordonae de la présente invention peut être utilisée efficacement comme vaccin pour prévenir ou traiter une infection à SARS-CoV-2.
PCT/KR2022/004915 2021-04-06 2022-04-06 Souche de mycobacterium recombinée exprimant un antigène du sars-cov-2, et composition vaccinale la contenant WO2022216025A1 (fr)

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