WO2022216025A1 - Recombinant mycobacterium strain expressing sars-cov-2 antigen, and vaccine composition including same - Google Patents

Abstract

The present invention relates to a recombinant Mycobacterium paragordonae (Mpg) strain expressing a SARS-CoV-2 antigen, and a vaccine composition including same. The recombinant Mycobacterium paragordonae strain expressing the SARS-CoV-2 antigen of the present invention can generate remarkable immune responses, including neutralizing antibody activity, against SARS-CoV-2 in mice. Accordingly, the recombinant Mycobacterium paragordonae strain of the present invention can be effectively used as a vaccine for preventing or treating SARS-CoV-2 infection.

Description

Recombinant Mycobacterium strain expressing SARS-COV-2 antigen and vaccine composition comprising same

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.

Infection caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), first reported in Wuhan, China in 2019, is spreading around the world, facing a great health, social and economic crisis. have.

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.

As of January 17, 2021, the cumulative number of confirmed cases worldwide is 93,217,287, and the cumulative number of deaths is 2,014,957. In particular, the number of confirmed cases and deaths is increasing rapidly in the United States and Europe (World Health Organization. (https://covid19.who.int/)).

Currently, the cumulative number of confirmed cases in Korea is 74,262, and the number of deaths is known to be 1,328 (as of January 22, 2021) (Coronavirus Infectious Disease-19 (COVID-19). go.kr/)).

This SARS-CoV-2 virus can infect cells expressing angiotensin-converting enzyme 2 (ACE2) and TMPRSS2 on the cell surface. In particular, the viral site that binds to ACE2 in the host cell is the receptor binding domain (RBD) of the spike protein. 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]).

In order to overcome the SARS-CoV-2 virus, research on vaccines and therapeutics is being developed worldwide. Vaccine development studies for most SARS-CoV-2 viruses are being conducted targeting the spike protein. Spike protein is known to play an important role in viral entry and immune response. In addition, since it is known that the RBD of the spike protein contains a major antigenic determinant that can induce the formation of neutralizing antibodies, a protein containing RBD or a gene for it or a vector delivering them is utilized for effective vaccine development It is expected that this will be possible, and vaccines targeting them are being developed. Various researchers are developing vaccines that can control COVID-19, and some are being vaccinated, but vaccines with safety and efficacy have not yet been verified.

On the other hand, recombinant Mycobacterium strains such as recombinant BCG have attracted attention for the development of human immunodeficiency virus-1 (HIV-1) vaccines, and 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).

In addition, as for the Mpg strain, a study in which a recombinant Mpg strain expressing HIV-1 antigen p24 (rMpg_p24) increased p24-specific immunity compared to a recombinant BCG strain expressing the same antigen has also been reported (Kim BJ et al. al. Potential of recombinant Mycobacterium paragordonae expressing HIV-1 Gag as a prime vaccine for HIV-1 infection. Sci Rep. 2019 Oct 29;9(1):15515]).

Accordingly, the present inventors studied to develop a new vaccine against COVID-19. As a result, when 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.

In order to achieve the above object, 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 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.

Another aspect of the present invention, 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.

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 μg/ml kanamycin) after electroporation of the pMV306-Phsp:RBD vector into Mpg. .

6a and 6b show the colony pattern ( FIG. 6a ) and colony PCR results ( FIG. 6b ) formed on 7H10 solid medium (100 μg/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. Here, as a positive control, SARS-CoV spike S1 protein (Sino Biological, 40159-V08B1) was used.

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. Here, as a positive control, NP protein (Sino Biological, 40588-V08B) was used.

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. Here, 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. Here, as a positive control, RBD protein (Sino Biological, 40592-V08B) was used.

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. Here, statistical significance was tested by Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

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. Here, statistical significance was tested by Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

13A and 13B show RBD expression using RBD ELISA kit (FIG. 13A) and real-time PCR (FIG. 13b) shows the results confirmed. Here, statistical significance was tested by Student's t-test (**, p < 0.01; ***, p < 0.001).

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-γ ELISPOT assay using mouse splenocytes immunized with SC-immunized Mpg and rMpg_RBD (#5, #6, #7) strains. IFN by each rMpg_RBD strain. -γ ELISPOT results (FIG. 15A) and IFN-γ 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).

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-γ ( FIG. 16B ) levels were measured by ELISA, and the results of comparison are shown.

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. Here, 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. Here, rMpg-RBD 1 means #5; rMpg-RBD 2 means #6; rMpg-RBD 3 means #7.

19 shows an animal experiment schedule for evaluating RBD-specific immunity inducing ability after immunization with Mpg, rMpg_RBD and RBD proteins for mice.

20 is a graph showing the results of an IFN-γ 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-γ+ ( FIG. 21A ), CD3+ CD4+ TNF-α+ ( FIG. 21B ). ), CD3+ CD8+ IFN-γ+ ( FIG. 21C ), and CD3+ CD8+ TNF-α+ ( 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).

22A to 22E show the expression of cytokines IFN-γ (FIG. 22A) and TNF-α (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. Here, statistical significance was tested with Student's t-test (*, p < 0.05; **, p < 0.01; *** p <0.001).

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) The result of comparison by measuring the expression level of the antibody by absorbance is shown. Statistical significance was tested by Student's t-test (*, p < 0.05; **, p < 0.01; *** p <0.001).

24 shows the neutralizing antibody ability against live SARS-CoV-2 of mouse serum immunized with rMpg_RBD strain or RBD protein.

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.

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. Specifically, 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, and 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. Here, statistical significance was tested with Student's t-test (*, p < 0.05; **, p < 0.01; *** p < 0.001).

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. . Here, the control antibody is an anti-SARS-CoV-2 spike antibody (Cat#: 40150-T62-COV2).

30 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 live cell form. . Here, the 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. Here, 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.

33 shows a graph comparing the results for the rMpg_RBD strain among the results shown in FIGS. 29 to 31 as neutralizing antibody capacity according to the inoculation amount. Here, 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. means the serum obtained by subcutaneous injection into mice.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention provides a recombinant Mycobacterium paragordone strain expressing the SARS-CoV-2 antigen.

As used herein, the term "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

In one embodiment of the present invention, 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. Specifically, 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. . In addition, 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.

In one embodiment of the present invention, the SARS-CoV-2 antigen may be expressed from a polynucleotide encoding the SARS-CoV-2 antigen operably linked to a promoter. Specifically, the polynucleotide encoding the SARS-CoV-2 antigen may be the nucleotide sequence of SEQ ID NO: 2. In addition, 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.

In one embodiment of the present invention, the recombinant Mycobacterium paragordone strain may be a wild-type Mycobacterium paragordone strain transformed to express the SARS-CoV-2 antigen. Specifically, in the recombinant Mycobacterium paragordone strain, 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. In addition, the SARS-CoV-2 antigen may be a receptor binding domain.

According to an embodiment of the present invention, 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 ℃, it is a temperature-sensitive strain that does not grow at 37 ℃, the temperature at which general bacteria grow well. Due to these characteristics, safety can be secured when applied to the human body. For the 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.

In the recombinant Mpg strain (rMpg_RBD) into which the receptor binding domain (RBD) gene constituting the spike protein among the SARS-CoV-2 antigens was introduced according to an embodiment of the present invention, many colonies were secured during transformation, and many of them were It was confirmed that the number had RBD, and in fact, a band specific for the RBD antibody was detected by western blot (see FIGS. 5 and 7 ). In addition, it was confirmed that RBD was also detected in intracellular RNA during dendritic cell infection (see FIG. 9 ). Furthermore, when mice were immunized with rMpg_RBD, it was confirmed that rMpg_RBD exhibited excellent immunogenicity against SARS-CoV-2 pseudovirus (see FIGS. 15 to 18 ).

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.

In one embodiment of the present invention, the pharmaceutical composition may be a vaccine.

In one embodiment of the present invention, the pharmaceutical composition may be administered in a single dose or in multiple doses. The multiple administration may be two administrations.

In another embodiment of the present invention, 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. In addition, the pharmaceutical composition may be for humans.

In addition, as 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. , in the case of injections, 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. For example, 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. In addition, 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.

Meanwhile, examples of 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. In addition, 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.

According to one embodiment of the present invention, the vaccine may be administered two or more times in a normal prime-boost form. In this case, the same vaccine may be administered several times, or different types of vaccines containing the same antigen may be administered. For example, 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.

According to another embodiment of the present invention, the recombinant Mycobacterium paragordone strain may be used in live, attenuated or inactivated form.

Another aspect of the present invention, 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.

According to one embodiment of the present invention, the kit may be for prime-boost inoculation. Upon prime-boost inoculation, the first composition may be prime-administered and the second composition may be boost-administered.

In one embodiment of the present invention, when RBD protein was administered after administration with rMpg_RBD, the production of antibodies against live SARS-CoV-2 in mouse serum increased and the neutralizing antibody ability was excellent compared to the case where only rMpg_RBD was administered. was confirmed (see FIGS. 24 and 25a to 25d).

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.

In one embodiment of the present invention, the hsp gene promoter may be a heat shock protein 65 (hsp65) gene promoter derived from Mycobacterium bovis BCG.

In one embodiment of the present invention, the vector may be for Mycobacterium strain expression, specifically Mycobacterium-E. coli shuttle vector. In addition, the vector may be pMV306-Pshp:RBD having the structure shown in FIG. 3A.

Another aspect of the present invention provides an isolated cell comprising the vector described above. 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

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

I. Construction of a recombinant strain expressing SARS-CoV-2 antigen using a temperature-sensitive Mycobacterium paragordonae (Mpg) strain and evaluation of its immunogenicity

Example 1. Construction of mycobacteria-E. coli shuttle vector for SARS-CoV-2 antigen expression

Based on the nucleotide sequence of the receptor binding domain (RBD) and nucleocapsid phosphoprotein (NP) constituting the spike protein among SARS-CoV-2 antigens, a sequence optimized for the codon of Mycobacterium tuberculosis ( Using the codon optimization web tool: http://www.jcat.de/) was obtained through gene synthesis (Bionics: http://www.bionicsro.co.kr/). The RBD base sequence consists of 675 nucleotides (SEQ ID NO: 2) and encodes 224 amino acids (SEQ ID NO: 1) corresponding to amino acids 319 to 541 of the total spike protein (FIG. 1). 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.

Primer sets (SEQ ID NOs: 6 to 9) and temperature conditions used for Phsp:RBD and Phsp:NP sequence amplification

Primers	Sequences (5' to 3')	Tm (℃)	Product
NotI_Phsp_F	AAA GCG GCC GCG GTG ACC ACA ACG ACG C	56	Phsp:RBD (1,050bp)
RBD_TB_XbaI_R	AAA TCT AGA TTA GAA GTT CAC GCA CTT GTT CTT C
Phsp_EcoRV_F	TTT GAT ATC GGT GAC CAC AAC GAC GCG C	62	Phsp:NP (1,029bp)
NP_XbaI_R	AAA TCT AGA TTA GGC CTG AGT TGA GTC AGC ACT G

Subsequently, the 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. Construction of SARS-CoV-2 RBD-expressing Mpg and NP-expressing Mpg

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.

Mpg transformation conditions

Plasmid DNA concentration	1-2 μg/5 μl
Voltage	2.5 kV
Capacitance	25 μF
Resistance	1,000 Ω
Cuvette (mm)	2
Volume	200 μl

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 μg/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. When transformed with pMV306-Phsp:RBD, a significant number of colonies were secured ( FIG. 5A ). 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 ).

On the other hand, in the case of pMV306-Phsp:NP, two transformation attempts were made, no colonies were formed in the first time, and a small number of colonies were formed in the second time (level of 6 to 10 colonies per plate) (Fig. 6a). As a result of colony PCR, a positive result was confirmed only in 2 colonies out of 8 colonies (FIG. 6b).

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) Thus, positively confirmed colonies, randomly selected among positively confirmed colonies), inoculated into 7H9 liquid medium (containing 100 μg/ml of kanamycin), and cultured at 30° C. for about 21 days, then some was prepared as a stock (20% glycerol, stored at -70°C), some were subcultured, and the remaining culture was used for analysis to confirm the expression pattern of RBD and NP in the strain.

Experimental Example 1. Confirmation of RBD and NP expression of recombinant Mpg

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 μg/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.

After loading the protein (60 μg) extracted from each recombinant strain on an SDS-PAGE gel, anti-SARS-CoV-2 spike protein antibody (Sino Biological, 40591-T62; 1:2,000) and anti-SARS-CoV-2 nucleoside By attaching a capsid protein antibody (Thermo Fisher, MA5-29981; 1:1,000), it was confirmed whether RBD and NP were properly expressed. In addition, as an internal control, an antibody (Santa Cruz, sc-57842; 1:200) capable of binding to hsp65 (heat shock protein 65) protein of Mycobacterium tuberculosis was used. In this case, as positive controls, 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.

As a result, an RBD-specific band was detected in the rMpg_RBD strain ( FIG. 7 ). On the other hand, no NP-specific band was observed in the rMpg_NP strain even when the same experiment was repeated ( FIG. 8 ).

Experimental Example 2. Measurement of RBD expression level by rMpg_RBD during dendritic cell infection

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 μM), 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.

In order to exclude DNA contamination from the obtained RNA, DNaseI (Promega, Cat: M6101) was treated and reacted at 37° C. for 30 minutes, and then RNA was extracted by a general EtOH precipitation method. Real-time PCR (CFX Connect™ Real-Time System, Bio-RAD) was performed on the extracted RNA with a SYBR kit (SensiFAST™ 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 (℃)
RBD_RT_F1	ACC GAG TCG ATC GTG CGC TTC	60
RBD_RT_R1	GAA GCA CAG GTC GTT CAG CTT

As a result, compared to the uninfected cells and the cells infected with the Mpg wild-type strain, a relatively three-fold higher expression of RBD was confirmed at the mRNA level in the cells infected with rMpg_RBD (10 M.O.I.). 1 M.O.I. Although RBD expression was detected even during infection, the level was insignificant ( FIG. 9 ).

Experimental Example 3. Immunogenicity evaluation of recombinant Mpg strain

In order to confirm whether the immune-inducing ability of the recombinant Mpg strain (rMpg_RBD) prepared in Example 2 is 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.

First, 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 μM) 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).

Comparison of expression levels of maturation markers in dendritic cells

Cell population (%)
	MHCII+	CD40+	CD80+	CD86+
no treat	1.66±0.81	11.22±1.97	6.81±0.89	14.33±0.50
mpg	4.32±1.41	41.20±2.65	33.83±1.42	29.03±0.46
rMpg_RBD (1 MOI)	1.19±0.33	11.96±1.76	10.60±0.52	15.73±0.31
rMpg_RBD (10 MOI)	3.37±1.10	30.07±1.14	25.53±0.81	28.90±0.56
LPS	5.00±1.04	29.55±0.64	22.33±3.26	27.25±1.20

As a result, 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. . After each cytokine-specific antibody was coated on a 96-well plate (4° C., O/N), 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

As a result, similar to the dendritic cell maturation pattern, the highest level of cytokine was expressed when infected with Mpg, and a relatively high level of cytokine expression was also exhibited when infected with rMpg_RBD (Figs. 12a and 12b, Table 5). rMpg_RBD 1 M.O.I. Upon infection, the expression of 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. That is, 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.

However, when comparing the infection dose, 1 M.O.I. Upon infection, the ability to induce immunity in dendritic cells was insignificant.

Example 3. Subculture of Recombinant Mpg Strain

The rMpg_RBD strain prepared in Example 2 was subcultured in 7H9 liquid medium (containing 100 μg/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.

Experimental Example 4. Confirmation of RBD expression in the subcultured rMpg_RBD strain

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 μg/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.

Using the obtained protein (100 μg), the expression of RBD in the protein was confirmed with the S1RBD ELISA kit (ELV-COVID19S1-1) provided by RayBiotech. First, 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. Then, the color was developed and the absorbance was measured with an ELISA reader device (Tecan Sunrise) at OD 450 nm, and the protein expression level was quantified based on the standard. As a result, it was confirmed that RBD was highly expressed in the recombinant rMpg_RBD strains of each generation (73.4, 82.9, and 65.3 pg/ml in the 1st, 5th and 10th generations, respectively) compared to the Mpg wild type (17.2 pg/ml). (Fig. 13a).

In addition, 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. The obtained pellet was dissolved in DEPC-treated water to obtain RNA. In order to exclude DNA contamination from the obtained RNA, it was treated with DNaseI (Promega, Cat: M6101) and reacted at 37° C. for 30 minutes. Then, RNA was extracted through a general EtOH precipitation method. Real-time PCR (CFX Connect™ Real-Time System, Bio-RAD) was performed on the extracted RNA with a SYBR kit (SensiFAST™ 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 (℃)
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

As a result, similar to the RBD ELISA results, it was confirmed that the expression of RBD increased at the mRNA level in each generation of the rMpg_RBD strain (1st, 5th, and 10th generation) compared to the Mpg wild type (FIG. 13b). As such, Example 2 When the rMpg_RBD strain (#7 strain) prepared in

II. Evaluation of RBD-specific immune response induction ability by recombinant Mpg strain (rMpg_RBD) in mouse model

Experimental Example 5. Evaluation of immune response inducing ability of rMpg_RBD strain

In order to evaluate the immune response inducing ability of the rMpg_RBD strains (#5, #6, #7 strains) prepared in Example 2, an in vivo experiment was performed according to the inoculation schedule shown in FIG. 14 as follows.

Three 8-week-old BALB/c female mice were configured in each group, and rMpg_RBD strains (#5, #6, #7 strains) were inoculated into each group by subcutaneous (SC) route, and the inoculation dose was 1×10 ⁶ .

The immunized mice were sacrificed to obtain splenocytes. After the obtained splenocytes were stimulated with S1 protein, i) IFN-γ ELISPOT and ii) IFN-γ, 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.

Experimental Example 5.1. IFN-γ ELISPOT assay

ELISPOT assay for IFN-γ 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-γ antibody (anti-mouse IFN-γ, clone: AN-18, 3 μg/ml), and SARS- Splenocytes were stimulated for 24 hours by treatment with CoV-2 spike (S1) protein at 5 μg/ml. Then, each well was washed 3 times with PBST and PBS, respectively. Then, the detection antibody (anti-mouse-IFN-γ biotin, clone: XMG1.2, 3 μg/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).

As a result of confirming the expression pattern of IFN-γ for S1 protein after immunization with ELISPOT

Stimulated with RBD	Spot formin units (SFUs)
mpg	3.00 ± 2.94
rMpg_RBD #5	53.50 ± 44.55
rMpg_RBD #6	161.00 ± 29.70
rMpg_RBD #7	300.50 ± 65.76

As a result, few spots were observed in the group immunized with Mpg, but relatively many spots were confirmed in the group immunized with rMpg_RBD. In particular, compared to other rMpg_RBD strains, S1 protein in splenocytes immunized with rMpg_RBD #7 strain. It was confirmed that the highest expression of IFN-γ specific to (Fig. 15, Table 7). Experimental Example 5.2. Cytokine expression level measurement

Splenocytes isolated from immunized mice were cultured in vitro with S1 protein (5 μg/ml) for 3 days, then the culture medium was collected and stored at -70°C. Thereafter, ELISA was performed for IFN-γ and IL-12 cytokines to compare and analyze their expression patterns. ELISA was performed using mouse IFN-γ and IL-12 ELISA kit (Invitrogen).

First, antibodies specific for IFN-γ and IL-12 were coated on 96-well plates (4°C, O/N). Then, each well was washed 3 times with wash buffer, and blocked with assay buffer for 1 hour at room temperature. 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. After that, each well was developed with a color reagent, and absorbance was measured with an ELISA reader device (Tecan Sunrise) at OD 450 nm, and the expression level of IFN-γ and IL-12 cytokines was measured based on the standard value. did.

After immunization with Mpg and rMpg_RBD strains (#5, #6, #7), cytokine level comparison results for S1 protein

Concentration (pg/ml)
	IL-12	IFN-γ
mpg	19.141 ± 8.567	31.736 ± 10.557
rMpg_RBD #5	31.960	39.201
rMpg_RBD #6	55.378	406.327
rMpg_RBD #7	149.943	469.912

As a result, the expression of IL-12 and IFN-γ 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. In particular, the rMpg_RBD #7 strain showed the highest IL-12 and IFN-γ cytokine expression levels were confirmed (Fig. 16, Table 8).

Experimental Example 5.3. Checking the IgG level in the serum

In this experimental example, using serum isolated from immunized mice, it was attempted to analyze and evaluate the IgG expression pattern by RBD in the serum.

For this, RBD protein (Sino Biological) was coated (5 μg/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. Then, IgG1 with HRP (goat anti-mouse IgG1, HRP conjugated; ThermoFisher Scientific), IgG2a (goat anti-mouse IgG2, HRP conjugated; Invitrogen) and total IgG (anti mouse IgG, biotinylated; eBioScinece) Antibodies were added and reacted at room temperature for 1 hour, followed by color development. Thereafter, the absorbance was measured at a wavelength of 450 nm with an ELISA reader device (Tecan Sunrise), and the OD value for each antibody isotype was evaluated.

When Mpg and rMpg_RBD strains (#5, #6, #7) were immunized with SC, 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

As a result, it was confirmed that immunization with the rMpg_RBD strain increased the production of RBD-specific IgG2, IgG1, and total IgG compared to the immunization with the Mpg strain. In addition, it was confirmed that the highest level of IgG was produced by the rMpg_RBD #7 strain (FIG. 17, Table 9).

Experimental Example 5.4. Confirmation of neutralizing antibody ability

A pseudovirus neutralizing antibody ability experiment of the recombinant rMpg_RBD strain was conducted.

First, pCAGGS (NR-52310, BEI Resources) containing pNL4-3.luc.RE and the 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. After 72 hours, 5X PEG-it (LV810A-1-SBI) was added to the supernatant and centrifuged (1,500 g, 30 min) to obtain a lentivirus pellet.

Meanwhile, three recombinant rMpg_RBD strains (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 μl each. did. To react the diluted serum with the virus, 100 μl of 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 ).

The same SARS-Cov-2 pseudovirus neutralizing antibody ability test was also performed on Calu-3 cells (Korea Cell Line Bank), a human lung cancer cell line, and DMEM was used as the medium ( FIGS. 18c to 18d ).

As a result, both the wild-type Mpg and mouse sera immunized with the three rMpg-RBD strains exhibited neutralizing antibody ability against SARS-CoV-2 pseudovirus from the decrease in the SARS-CoV-2 pseudovirus infected with cells as the dilution factor increased. was confirmed ( FIGS. 18A to 18D ). Here, the relative luciferase unit (RLU) 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.

In addition, all three rMpg_RBD strains exhibited higher NT50 values than wild-type Mpg, confirming excellent neutralizing antibody ability against SARS-CoV-2 pseudovirus ( FIGS. 18A to 18D ). Here, 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.

Although it was not possible to confirm the statistical significance between the NT50 values of the three strains, the rMpg_RBD strain 3 showed the highest NT50 value. Among the three recombinant rMpg_RBD strains, 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.

Experimental Example 6. Confirmation of SARS-CoV-2 RBD protein-specific immune response of rMpg_RBD strain in mice

An experiment to evaluate whether SARS-CoV-2 RBD protein-specific immune response is induced in mice using the rMpg_RBD strain (#7 strain) selected based on the results of Experimental Example 5 was performed according to the administration schedule shown in FIG. 19 . did.

All strains were inoculated by the subcutaneous route (SC), and the inoculation dose was 1×10 ⁶ .

Experimental group: i) PBS (2 inoculations, S.C.); ii) Mpg (2 doses, S.C.); iii) rMpg_RBD (single inoculation, S.C.); iv) rMpg_RBD (2 doses, S.C.); v) rMpg_RBD (single dose, S.C.) + RBD protein and alum (single dose, S.C.); vi) RBD protein and alum (2 doses, S.C.). Here, when RBD protein and alum were used, RBD protein was adjusted to a concentration of 10 μg/ml, and alum was adjusted to a concentration of 100 μg/ml, and immunization was performed after mixing them together. All inoculation volumes were adjusted to 100 μl.

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. Thereafter, i) IFN-γ ELISPOT assay was performed as follows, ii) CD4, CD8 T cell populations expressing IFN-γ or TNF-α were analyzed by FACS, iii) immune cytokines (IFN) -γ, TNF-α, 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.

Experimental Example 6.1. IFN-γ ELISPOT assay

ELISPOT for IFN-γ 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-γ (anti-mouse IFN-γ, clone: AN-18, 3 μg/ml), and SARS-CoV Splenocytes were stimulated for 24 hours by treatment with -2 RBD protein at 5 μg/ml. Then, each well was washed three times with PBST and PBS, respectively, and then treated with a detection antibody (anti-mouse-IFN-γ biotin, clone: XMG1.2, 3 μg/ml). Then, it was made to react at 4 degreeC 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 the spot of each membrane was counted with an ELISPOT reader device (AID EliSpot Reader).

As a result of confirming the expression pattern of IFN-γ for RBD protein after immunization with ELISPOT

Stimulated with RBD	Spot formin units (SFUs)
PBS	1.00 ± 1.26
mpg	1.00 ± 1.67
Immune to rMpg_RBD 1 time	7.50 ± 4.72
Immune to rMpg_RBD 2 times	2.83 ± 2.14
rMpg_RBD + RBD protein	5.50 ± 3.51
RBD protein	4.17 ± 3.43

As a result, in the group immunized with the rMpg_RBD strain (once, twice, and rMpg_RBD + RBD protein immunity), relatively many IFN-γ spots were confirmed compared to the PBS and Mpg strain immunization groups. However, only the group immunized with the rMpg_RBD strain once and the group immunized with RBD protein boosting after primed with rMpg_RBD showed significant differences from the PBS and Mpg immunization groups. Among them, the most IFN-γ spots were identified in the group immunized with the rMpg_RBD strain once ( FIG. 20 , Table 10).

Experimental Example 6.2. FACS analysis

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. Differences in CD4 and CD8 T cell populations expressing IFN-γ or TNF-α (using APC-conjugated anti-IFN-γ, APC_Cy7 conjugated anti-TNF-α antibody) (BD BioSciences) by intracellular staining thereafter were analyzed by FACS (FACS LSRFortessa).

As a result of FACS analysis of CD4 and CD8 T cell populations expressing RBD-specific IFN-γ and TNF-α after immunization

Cell population (%)
	CD3+CD4+IFNγ+	CD3+CD4+TNFα+	CD3+CD8+IFNγ+	CD3+CD8+TNFα+
no treat	0.537±0.021	0.510±0.035	0.630±0.276	1.017±0.311
Mpg (2 times immunity)	1.090±0.290	0.293±0.021	0.960±0.071	1.305±0.163
rMpg_RBD (1 time)	2.305±0.262	0.497±0.177	1.355±0.290	2.150±0.509
rMpg_RBD (2 times)	1.490±0.198	0.990±0.495	1.460±0.226	2.817±0.601
rMpg_RBD+RBD protein	0.693±0.233	2.355±1.407	0.897±0.156	3.267±0.955
RBD protein (2 times)	0.333±0.212	0.530±0.113	0.187±0.120	1.710±0.064

As a result, in the group immunized with rMpg_RBD, the CD4 and CD8 T cell populations secreting IFN-γ showed statistical significance and increased compared to other immunized groups (FIG. 21, Table 11). In particular, CD4+ IFN-γ+ T cells showed the highest aspect in the group immunized with rMpg_RBD once, which was determined to be correlated with the IFN-γ ELISPOT results (Fig. 20, Fig. 21, Table 10, Table). 11). In the case of TNF-α-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-α-secreting CD4 and CD8 T cell populations tended to increase compared to the group. In particular, in the group immunized with rMpg_RBD prime-RBD protein boosting, the TNF-α-secreting T cell population showed the highest pattern (FIG. 21, Table 11).

However, in the group immunized with only RBD protein, the T cell population expressing IFN-γ and TNF-α was low ( FIG. 21 , Table 11).

Experimental Example 6.3. Cytokine expression level measurement

Splenocytes isolated from immunized mice were incubated with SARS-CoV-2 RBD protein (5 μg/ml) in vitro for 3 days, then the culture medium was collected and stored at -70°C. Thereafter, ELISA was performed for IFN-γ, TNF-α, IL-2, IL-10 and IL-12 cytokines to compare and analyze their expression patterns. ELISA was mouse IFN-γ, TNF-α, IL-2. IL-10 and IL-12 ELISA kits (Invitrogen) were used.

First, 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-γ, TNF-α, IL-2. The expression levels of IL-10 and IL-12 cytokines were measured.

Comparison of RBD-specific cytokine expression levels after immunization

Concentration (pg/ml)
	IFN-γ	TNF-α	IL-2	IL-10	IL-12
no treat	11.821±0.344	1.971±0.091	69.707±18.908	60.563±7.421	43.534±8.123
Mpg (2 times immunity)	14.201±2.436	2.564±0.504	379.699±70.316	123.755±27.244	50.168±32.984
rMpg_RBD (1 time)	2823.047±361.184	16.883±2.230	607.431±53.096	63.073±8.877	123.311±9.823
rMpg_RBD (2 times)	326.993±215.479	20.163±5.373	4272.077±605.079	118.317±36.668	241.710±95.435
rMpg_RBD+RBD protein	31.683±20.169	3.444±1.514	449.954±66.844	115.781±14.654	120.982±22.163
RBD protein (2 times)	52.190±13.942	4.771±1.063	1728.377±328.780	1052.501±283.557	19.985±2.602

As a result, as with the previous IFN-γ ELISPOT and FACS results (Fig. 20, Fig. 21; Table 10, Table 11), the highest expression of IFN-γ was confirmed in the group immunized with the rMpg_RBD strain once (Fig. 22a). , Table 12). The group immunized with the rMpg_RBD strain twice also increased the expression of IFN-γ statistically significantly compared to the other immunization groups (Fig. 22a, Table 12). However, the expression level of IFN-γ was found to be low in the group immunized with RBD protein boosting after primed with rMpg_RBD and in the group immunized with RBD protein alone. For IL-2, IL-12 and TNF-α, 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. In the case of TNF-α, 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). ).

In the case of the RBD protein-immunized group, the expression levels of cytokines (TNF-α, IFN-γ, 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).

Experimental Example 6.4. IgG analysis in serum

Based on the selected recombinant strain, the experiment was conducted with a total of 30 mice, in total of 6 groups: 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.

Using serum isolated from immunized mice, we tried to analyze and evaluate the IgG expression pattern by SARS-CoV-2 RBD in the serum. For this, the RBD protein was coated on an ELISA plate (5 μg/ml) 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 each mouse were diluted (1:10), put into each well, and reacted at room temperature for 2 hours. Then, IgG1 with HRP (goat anti-mouse IgG1, HRP conjugated; ThermoFisher Scientific), IgG2a (goat anti-mouse IgG2, HRP conjugated; Invitrogen) and total IgG (anti mouse IgG, biotinylated; eBioScinece) Antibodies were added and reacted at room temperature for 1 hour, followed by color development. By measuring the absorbance at a wavelength of 450 nm with an ELISA reader device (Tecan Sunrise), the OD value for each antibody isotype was evaluated.

After immunization, 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

As a result, it was confirmed that the group inoculated with rMpg_RBD twice had the highest expression of IgG2, which is known to be involved in cell-mediated Th1 immunity ( FIG. 23A , Table 13). The group inoculated with rMpg_RBD showed a similar level of IgG2 expression to the Mpg wild-type and RBD protein immune groups. In the case of IgG1, high expression was shown in the group inoculated with rMpg_RBD twice, but it was confirmed that the expression of IgG1 was increased to a similar level in the group immunized with the Mpg strain (FIG. 23b, Table 13). RBD-specific Contrary to the results of cell-mediated immune response, 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).

Experimental Example 6.5. Evaluation of neutralizing antibody ability against live SARS-CoV-2 in mouse serum immunized with rMpg_RBD

In order to evaluate the neutralizing antibody ability against live SARS-CoV-2 of mouse sera obtained from each group, an experiment was conducted in the Biosafety Level 3 laboratory.

First, for culturing SARS-CoV-2, 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. In this way, 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.

Meanwhile, 150 μl 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 μl 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 μl/well and incubated for 2 days in a 37° C. incubator.

After 3 days, in order to develop a plaque, 1 ml (1:2 volume)/well of 100% methanol was treated and reacted at room temperature for 15 minutes to dissolve 0.8% methylcellulose. Methylcellulose was removed, washed with PBS, and then treated with 4% paraformaldehyde 500 μl/well, and the cells were fixed at room temperature for 15 minutes. After that, the sample was inactivated by irradiating UV for 1 hour with the plate cover open. After 1 hour, the solution was removed, and 100% methanol was treated with 500 μl/well, followed by fixation/permeation for 15 minutes. After washing with PBS, the primary antibody against the nucleocapsid (NP) of SARS-CoV2 was diluted 1:1000 in 2% BSA (in PBS) and treated with 200 μl/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 μl/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 μl/well. When plaque development progressed, the substrate solution was removed, washed with DW, and water was removed.

As a result, as the dilution factor of serum increased in all groups except for the serum of untreated mice, plaque formation of SARS-CoV-2 was reduced. Confirmed. At this time, the reduction in the number of plaques was calculated as the neutralization rate (%) based on the virus infection that did not react with the serum.

In addition, the titer of the Plaque Reduction Neutralization Test 50 (PRNT50) 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). When comparing the PRNT50 values, in the case of mice injected with the strain or protein, 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. In addition, it was confirmed that 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.

When 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.

From this result, it was found that the production of antibodies against live SARS-CoV-2 in mouse serum increased when RBD protein was injected after one injection of rMpg_RBD than when recombinant rMpg_RBD was injected once.

Experimental Example 6.6. Comparison of neutralizing antibody ability against SARS-CoV-2 pseudovirus of mouse serum immunized with rMpg_RBD

In order to confirm the neutralizing antibody ability of the mouse serum injected with recombinant rMpg_RBD or RBD protein against SARS-CoV-2 pseudovirus, it was carried out in the same manner as the neutralizing antibody activity confirmation experiment of Experimental Example 5.4.

First, 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 ).

As a result, in all of the mouse serum immunized with the strain or protein, except for the case where the mice were not treated at all, as the dilution factor of the serum increased, the pseudovirus infection in Calu-3 cells was reduced, so SARS-CoV-2 It was found that there is neutralizing antibody ability against pseudovirus. Here, the relative luciferase unit (RLU) 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.

In addition, 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. Here, 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.

On the other hand, when rMpg_RBD was injected twice, when rMpg_RBD and RBD protein were injected, when wild-type Mpg was injected into mice, when only RBD was injected, the NT50 value of serum in the Calu-3 cell experiment was statistically It was confirmed that the neutralizing antibody ability against SARS-CoV-2 pseudovirus was excellent as it was found to be high at a significant level. This difference was confirmed not only in Calu-3 but also in Huh7, and in the Huh cell experiment, it was confirmed that the efficiency of the serum SARS-CoV-2 pseudovirus neutralizing antibody was superior in the case of single injection of rMpg_RBD than in the case of injection of wild-type Mpg.

In addition, when rMpg_RBD was injected twice or rMpg_RBD was injected once and RBD protein was injected, 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. In addition, in preparation for the double injection of rMpg_RBD, when RBD protein was injected after rMpg_RBD was injected once, 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

Based on these results, it was found that the production of antibodies against SARS-CoV-2 pseudovirus in serum increased when the recombinant rMpg_RBD was injected twice into mice than when the recombinant rMpg_RBD was injected once. In addition, it was found that the production of antibodies against SARS-CoV-2 pseudovirus in serum increased when RBD protein was injected into mice after rMpg_RBD was injected once more than when rMpg_RBD was injected twice.

Experimental Example 6.7. Evaluation of the Effect of Mouse Serum Immunized with rMpg_RBD on ACE2-RBD Binding

Through Experimental Examples 6.5 and 6.6, it was experimentally confirmed that rMpg_RBD-immunized mouse serum had neutralizing antibody ability against live SARS-CoV-2 and SARS-CoV-2 pseudovirus. Therefore, in order to confirm whether the antibodies in the immunized mouse serum actually interfere with binding to ACE2, a molecule that RBD recognizes and binds, an ACE2-RBD binding assay was performed.

Specifically, using the COVID-19 Spike-ACE2 Binding Assay Kit (CoV-ACE2S2-1) provided by RayBiotech, it was evaluated whether the antibody in the serum of mice immunized with rMpg_RBD affects the binding of ACE2 and RBD. First, the serum of mice immunized with PBS, Mpg, rMpg_RBD (once or twice) was diluted 1:10, 1:100, 1:1000, 1:10000 and mixed with RBD protein. Then, it was put in a 96-well plate coated with ACE2 protein and reacted at room temperature for 3 hours. After washing each well 4 times with the wash buffer provided by the kit, 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.

Absorbance results of an ACE2-RBD binding assay to evaluate the effect of serum from mice immunized with PBS, Mpg and rMpg_RB on the binding of ACE2 and RBD

	Absorbance (OD 450nm)
	1:10	1:100	1:1000	1:10000
PBS	0.482±0.017	0.493±0.004	0.515±0.009	0.547±0.039
mpg	0.464±0.010	0.471±0.018	0.501±0.019	0.528±0.027
rMpg_RBD(1)	0.316±0.013	0.347±0.004	0.416±0.022	0.478±0.006
rMpg_RBD(2)	0.334±0.011	0.351±0.020	0.399±0.009	0.483±0.011

As a result, when the serum of the group immunized with PBS and Mpg was mixed with RBD protein and reacted with ACE2, it was confirmed that the absorbance was maintained at a similar level regardless of the dilution factor. However, in the case of mouse serum samples immunized with rMpg_RBD, there was no difference between the 1 or 2 immunization groups, but compared to the PBS and Mpg immunization groups, the binding of ACE2 and RBD was inhibited. This trend showed a low absorbance at a statistically significant level compared to the PBS and Mpg immune groups even when the serum was diluted ( FIG. 26 , Table 14). However, when the serum was diluted about 1:10000, the absorbance significantly increased. In addition, the 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.

Experimental Example 6.8. Confirmation of IgA expression in BALF

To determine the expression level of IgA specifically responding to SARS-CoV-2 RBD in bronchoalveolar lavage (BALF) of mice immunized with rMpg_RBD, IgA ELISA was performed.

First, the immunized 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 μl 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 μl of BALF was obtained, and stored at -20°C until used in the experiment.

Using BALF isolated from immunized mice, the IgA expression pattern by SARS-CoV-2 RBD in BALF was confirmed by ELISA. To this end, RBD protein was coated on an ELISA plate (5 μg/ml) and reacted at 4° C. for 24 hours, and then each well was washed with a wash buffer (0.05% Tween 20). Then, each well was blocked with 5% BSA. After washing each well again, the BALF sample isolated from the mouse was put into each well and reacted at room temperature for 2 hours. After that, 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. By measuring the absorbance at a wavelength of 450 nm with an ELISA reader device (Tecan Sunrise), the OD value for IgA was measured.

The results of comparing BALF samples of mice immunized with Mpg, rMpg_RBD, and RBD proteins with SC were stimulated with RBD protein, and then the expression level of IgA antibody was measured by absorbance and compared.

	Absorbance (OD 450nm)
PBS	0.078±0.012
mpg	0.1024±0.009
rMpg_RBD (1)	0.124±0.012
rMpg_RBD (2)	0.129±0.010
rMpg_RBD + RBD	0.088±0.013
RBD	0.099±0.016

As a result, although a certain level of IgA expression in BALF was confirmed even during Mpg immunization, 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. In addition, 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).

Summary and consideration of the results of Experimental Examples 6.1 to 6.8

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. ); ii) Mpg (2 doses, S.C.); iii) rMpg_RBD (single inoculation, S.C.); iv) rMpg_RBD (2 doses, S.C.); v) rMpg_RBD (single dose, S.C.) + RBD protein and alum (single dose, S.C.); vi) RBD protein and Alum (2 doses, S.C.)] to summarize the results of comparison and evaluation.

First, as a result of performing IFN-γ ELISPOT using spleen cells stimulated with RBD protein, in the group immunized with the rMpg_RBD strain (1 time, 2 times, or rMpg_RBD prime-RBD boosting immune group), the PBS and Mpg strain immune group A higher IFN-γ spot could be confirmed compared to . Among them, the highest IFN-γ spot was confirmed in the group immunized with the rMpg_RBD strain once ( FIG. 20 , Table 10).

In addition, as a result of FACS analysis of IFN-γ and TNF-α-secreting T cell populations, IFN-γ-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-γ showed the tendency to be most induced. Also, in the case of TNF-α-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 α showed the highest pattern (FIG. 21, Table 11).

In addition, after each immunized mouse spleen cells were stimulated with RBD protein, immune cytokine expression was evaluated in the cell culture medium. As a result, the expression of IFN-γ was highest in the group immunized with the rMpg_RBD strain once, similar to the results of ELISPOT and FACS analysis (Fig. 20, Fig. 21, Fig. 22a; Table 10, Table 11, Table 12), The expression of cytokines (TNF-α, IL-2, and IL-12) important for the remaining Th1 immune response showed the highest aspect in the group immunized with the rMpg_RBD strain twice ( FIGS. 22b, 22c, 22e, Table 12). 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. In the RBD-only immune group, only 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.

Also, the expression of IgG2, which is known to be involved in cell-mediated Th1 immunity, 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. Specifically, it was confirmed that the expression of 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).

In addition, the neutralizing antibody ability against live SARS-CoV-2 and SARS-CoV-2 pseudovirus was evaluated using the sera of each immunized mouse. As a result, when the rMpg_RBD strain was immunized once or twice, or when immunized with rMpg_RBD prime-RBD boosting, a neutralizing antibody ability was relatively higher than that of the PBS, Mpg, and RBD alone immunization groups ( FIGS. 24 and 25A to 25D ). . However, after immunization with the recombinant strain once, the serum immunized with the RBD protein showed the highest neutralizing antibody ability ( FIGS. 24 and 25A to 25D ). rMpg_RBD prime-RBD boosting immunity had no significant effect on RBD-specific cell-mediated immune responses (IFN-γ ELISPOT, IFN-γ or TN F-α expressing T cell assay and immune cytokine measurement), and RBD-specific antibody There was no significant effect on production.

In addition, when an ACE2-RBD binding assay was performed using rMpg_RBD-immunized mouse serum, there was no difference between the rMpg_RBD-immunized mouse serum samples between the 1st or 2nd immunization groups, but there was no difference between the PBS and Mpg immunization groups. In comparison, it showed an aspect that interfered with the binding of ACE2 and RBD (FIG. 26, Table 14). In addition, the 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.

In addition, it was confirmed that 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).

In addition, through immunization of the rMpg_RBD strain, RBD-specific cell-mediated immune response, neutralizing antibody ability against SARS-CoV-2, and antibody formed in serum induce inhibition of binding of ACE2 and RBD, and RBD-specific in BALF. It was confirmed that it induces the expression of specific IgA. In the case of rMpg_RBD prime-RBD boosting immunity, induction of RBD-specific immune cytokine expression, IFN-γ and TNF-α 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.

Finally, although 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.

In Table 16 below, the results of Experimental Examples 6.1 to 6.8 are qualitatively shown in terms of materials to be evaluated.

Group	tIgG	IFN-r	IgA (BALF)	nIgGs (IC50)	IFN-r CD4+Tcell	TNF-a CD4+Tcell	IFN-r CD8+Tcell	TNF-a CD8+Tcell
r-Mpg Prime	++	++++	+++	1:762	++++	++	+++	++
r-Mpg Prime/boosting	+++	++	++++	1:6,008	+++	+++	++++	+++
r-Mpg Prime+RBD	++	+++	++		++	++++	++	++++
RBD (Alum)	++	++	++		+	++	+	+

Experimental Example 7. Additional confirmation of neutralizing antibody ability of mouse serum immunized with rMpg_RBD strain SARS-CoV-2 RBD protein-specific immune response in mice using the rMpg_RBD strain (#7 strain) selected based on the results of Experimental Example 5 An experiment was further conducted to evaluate whether this was induced. BALB/c 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.

The obtained mouse 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. prepared, 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 .

As a result, it was confirmed that in the group subcutaneously injected with 1×10 ⁶ CFU in live cell form, TCID ₅₀ or higher in serum diluted to 1/320 ( FIGS. 29, 32 and 33 ), and 1× in live cell form In the group subcutaneously injected with 10 ⁷ CFU, it was confirmed that the serum diluted at 1/640 showed a value of ₅₀ or more TCID ( FIGS. 30 , 32 and 33 ). This means that the neutralizing antibody capacity can be increased in a dose-dependent manner when inoculated in the form of live cells. In addition, it was confirmed that the group subcutaneously injected with 1×10 ⁷ CFU in the inactivated form also exhibited a value of ₅₀ or more TCID at a 1/160 dilution, confirming the neutralizing ability even in the inactivated state ( FIGS. 31 , 32 and 33 ) ). This example means that the recombinant Mycobacterium paragordone strain has neutralizing antibody ability against SARS-CoV-2 virus regardless of live cells and inactivated state.

Claims (22)

1. A recombinant Mycobacterium paragordonae strain expressing the SARS-CoV-2 antigen.
2. The method of claim 1,

   The SARS-CoV-2 antigen is a spike protein, recombinant Mycobacterium paragordone strain.
3. 3. The method of claim 2,

   The spike protein is a receptor binding domain (Receptor binding domain) or a fragment thereof, recombinant Mycobacterium paragordone strain.
4. 4. The method of claim 3,

   The SARS-CoV-2 antigen is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, recombinant Mycobacterium paragordone strain.
5. 4. The method of claim 3,

   The strain was transformed with a nucleic acid molecule in which a polynucleotide encoding a receptor binding domain of SARS-CoV-2 is operably linked to a heat shock protein 65 (hsp65) gene promoter derived from Mycobacterium bovis BCG. That is, the recombinant Mycobacterium paragordone strain.
6. 4. The method of claim 3,

   The strain was transformed with the pMV306-Pshp:RBD vector having the structure shown in FIG. 3 , the recombinant Mycobacterium paragordone strain.
7. A pharmaceutical composition for the treatment or prevention of SARS-CoV-2 infection, comprising the strain of any one of claims 1 to 6 as an active ingredient.
8. 8. The method of claim 7,

   The pharmaceutical composition is a vaccine, pharmaceutical composition.
9. 8. The method of claim 7,

   The pharmaceutical composition is administered in a single dose or twice, the pharmaceutical composition.
10. 8. The method of claim 7,

    The pharmaceutical composition is used as a priming vaccine in the prime-boosting inoculation method, pharmaceutical composition.
11. 11. The method of claim 10,

    In the boosting inoculation, the receptor binding domain of SARS-CoV-2, alum, or a combination thereof is used, a pharmaceutical composition.
12. 8. The method of claim 7,

    The strain is a live cell, pharmaceutical composition.
13. 8. The method of claim 7,

    The strain is attenuated or inactivated, the pharmaceutical composition.
14. A first composition comprising the strain of any one of claims 1 to 6 as an active ingredient; and a second composition comprising a receptor binding domain of SARS-CoV-2, alum, or a combination thereof as an active ingredient, a kit for treating or preventing SARS-CoV-2 infection.
15. 15. The method of claim 14,

    The kit is for prime-boost inoculation.
16. 16. The method of claim 15,

    wherein the first composition is primed and the second composition is boost administered.
17. A vector comprising a recombinant nucleic acid molecule wherein a polynucleotide encoding the receptor binding domain of SARS-CoV-2 is operably linked to an hsp gene promoter.
18. 18. The method of claim 17,

    The hsp gene promoter is mycobacterium bovis (Mycobacterium bovis) hsp65 (heat shock protein 65) gene promoter derived from BCG, vector.
19. 18. The method of claim 17,

    The vector is for expression of the Mycobacterium strain, the vector.
20. 18. The method of claim 17,

    The vector is pMV306-Pshp:RBD having the structure shown in FIG. 3 .
21. 21. An isolated cell comprising the vector according to any one of claims 17 to 20.
22. Claims 17 to 20 of the recombinant Mycobacterium paragordone strain expressing the SARS-CoV-2 antigen, comprising transforming the Mycobacterium paragordone strain with the vector according to any one of claims 17 to 20. manufacturing method.

Images