KR20220155898A - Virus-like particle against SARS-CoV-2 and influenza H1N1 virus and use thereof - Google Patents

Abstract

The present invention relates to a virus-like particle against COVID-19 virus and influenza H1N1 virus and a use thereof, and more specifically, to: a virus-like particle containing COVID-19 virus spike and influenza H1N1 virus structural protein M1; and a vaccine composition comprising the same. The virus-like particle of the present invention is structurally similar to wild-type viruses, and thus high immunogenicity is expected.

Description

Virus-like particle against SARS-CoV-2 and influenza H1N1 virus and use thereof {Virus-like particle against SARS-CoV-2 and influenza H1N1 virus and use thereof}

The present invention relates to virus-like particles against COVID-19 virus and influenza H1N1 virus and uses thereof.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) first emerged in Wuhan, China in December 2019, then spread across China and around the world, causing a pandemic and causing a pandemic. 19, a new type of coronavirus that causes COVID-19).

SARS-CoV-2's spike (S) protein, like the S protein of other coronaviruses, exists as a trimer on the surface of the bias, and the receptor binding domain required for virus host cell invasion and cell invasion It has a fusion peptide that induces fusion between the viral membrane and organelle membrane, and is known to play a role such as inducing neutralizing antibodies against the S protein in the natural host. Therefore, the trimeric S protein is thought to act as the main antigen, and the trimeric structure is thought to play an important role in the induction of neutralizing antibodies.

In general, when a virus-like particle (VLP) of a virus having a double lipid membrane is produced, the surface protein of the virus (spike) and the protein necessary for the budding of the virus (matrix protein) are expressed in animal cells at the same time. In this case, the appearance or shape of the actual virus is similar to that of the mother virus, but it does not contain genetic material, so virus-like particles that do not multiply in the host are formed. These VLPs are known to exhibit higher immunogenicity than subunit vaccines by mimicking infection in the form of particles and capable of binding to host cell receptors, like wild-type vaccines.

Therefore, in an urgent situation of responding to COVID-19, which is spreading around the world, we are trying to develop a SARS-CoV-2 S antigen-based virus-like particle vaccine candidate to rapidly respond to the spread of infection in Korea and secure herd immunity.

Cell (2020) 181:914-921. Electrophoresis (2020) 41(13-14):1137-1151. Vaccine (2011) 29(38): 6606-6613. Virol Sin (2018) 33(5): 453-455.

The present invention was derived from the above needs, and the present inventors completed the present invention by confirming the immunogenicity of virus-like particles including spikes of the Corona 19 virus (SARS-CoV-2) and M1 of the influenza H1N1 virus. did

In order to solve the above problems, the present invention provides a virus-like particle comprising a corona 19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1.

In the present invention, the Corona 19 virus is BetaCoV/Korea/KCDC03/2020 and the influenza H1N1 virus is A/Korea/01/2009, or the Corona 19 virus spike is the amino acid of SEQ ID NO: 1, a nucleic acid encoding it, or The codon-optimized nucleic acid, and the influenza H1N1 viral structural protein M1 may be the amino acid of SEQ ID NO: 2, a nucleic acid encoding the same, or a codon-optimized nucleic acid.

As an example, the present invention provides a recombinant vector containing nucleic acids encoding virus-like particles including a corona 19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1.

In the present invention, the recombinant vector includes a pCAGEN vector, and may include 2X Flag and 6X His.

As another example, the present invention provides a vaccine composition comprising virus-like particles comprising spikes of SARS-CoV-2 and influenza H1N1 viral structural protein M1, and an immune adjuvant.

In the present invention, the immune adjuvant may be alum (aluminium hydroxide).

In addition, the vaccine composition of the present invention may be one in which both S1 and S2 specific antibodies, which are subdomains of spike, are induced, and may be administered twice or three times.

As another example, the present invention includes the steps of identifying the gene or amino acid sequence of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus; Preparing a recombinant vector of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus; It provides a method for producing virus-like particles comprising a.

As another example, the present invention relates to a vaccine composition comprising a virus-like particle comprising a COVID-19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1 and an adjuvant, and a COVID-19 virus including instructions for use. (SARS-CoV-2) infection (COVID-19) prevention kit is provided.

The virus-like particles containing the spike of the COVID-19 virus (SARS-CoV-2) and M1 of the influenza H1N1 virus according to the present invention are structurally similar to wild-type viruses, so high immunogenicity is expected, and antibodies induced according to actual viral infection are expected. Establishment of a rapid vaccine platform strategy technology that can design an immune antigen relatively quickly using only genetic information disclosed in the event of a new or variant infectious disease and evaluate its immunogenicity and efficacy as a vaccine candidate as an immune response can be induced similarly to can be useful for

1 shows a schematic view of the result of securing the Corona 19 virus spike and influenza H1N1 virus structural protein M1 gene and a recombinant vector for VLP vaccine production according to an embodiment of the present invention.
2 shows the results of codon optimization for the production of COVID-19 virus VLPs according to an embodiment of the present invention.
3 shows the results of the expression level according to the change in culture conditions for the production of COVID-19 virus VLPs according to an embodiment of the present invention.
4 shows protein expression results of COVID-19 virus spike and influenza H1N1 viral structural protein M1 in HEK293T cells according to an embodiment of the present invention.
5 shows an animal test for immunogenicity evaluation of a VLP vaccine candidate according to an embodiment of the present invention. (A) Experiment schedule, (B) Experiment population.
6 shows the results of analysis of the induction of specific antibodies against spike antigens in mouse serum of a VLP vaccine candidate according to an embodiment of the present invention.
FIG. 7 shows the results of analysis of neutralizing antibody induction in mouse serum of a VLP vaccine candidate according to an embodiment of the present invention.
8 shows the results of analysis of cell-mediated immune responses by immunization of VLP vaccine candidates according to an embodiment of the present invention.
9 shows an animal test in a golden Syrian hamster model for immunogenicity evaluation of a VLP vaccine candidate according to an embodiment of the present invention. (A) Experiment schedule, (B) Experiment population.
10 shows the results of analysis of the induction of specific antibodies against spike antigens in golden Syrian hamster serum of a VLP vaccine candidate according to an embodiment of the present invention.
FIG. 11 shows the results of analysis of neutralizing antibody induction in golden Syrian hamster serum of a VLP vaccine candidate according to an embodiment of the present invention.
12 shows weight changes of golden Syrian hamsters according to challenge inoculation after immunization with a VLP vaccine candidate according to an embodiment of the present invention.
13 shows the lung tissue analysis results of golden Syrian hamsters following challenge inoculation after immunization with a VLP vaccine candidate according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

In the present invention, the term "Corona 19 virus (SARS-CoV-2)" first occurred in Wuhan, China in December 2019, then spread throughout China and around the world, causing a pandemic, -19), it can be understood as equivalent to various terms including 'SARS coronavirus 2', 'SARS-CoV-2' and 'sever acute respiratory syndrome coronavirus 2'. .

The present invention provides a virus-like particle comprising a COVID-19 virus (SARS-CoV-2) spike and an influenza H1N1 viral structural protein M1.

As used herein, the term "virus like particle" refers to a non-infectious viral subunit with or without viral proteins. For example, a virus-like particle may completely lack a DNA or RNA genome. Additionally, virus-like particles comprising viral capsid proteins are capable of spontaneous self-assembly. In one embodiment, preparations of virus-like particles are contemplated, such preparations being purified to be free of infectious virions (or purified to be substantially free of infectious virions such that the preparation has an insufficient number of virions to be infectious).

The Corona 19 virus (SARS-CoV-2) and influenza H1N1 virus may include all or partial modifications of the corresponding virus, and preferably, BetaCoV/Korea/KCDC03/2020 and influenza H1N1 virus, respectively, are A/Korea/ 01/2009, but is not limited thereto.

The spike may include all or part of the sequence of the spike protein including various mutations of the Corona 19 virus (SARS-CoV-2), and part of the sequence of the wild strain is changed to increase the expression rate in mammalian cells ( codon optimization), preferably EPI_ISL_407193 deposited in GISAID, more preferably the amino acid of SEQ ID NO: 1, a nucleic acid encoding it, or a codon-optimized nucleic acid, more preferably SEQ ID NO: 1 The nucleic acid encoding the amino acid of is SEQ ID NO: 3, and the codon-optimized nucleic acid of SEQ ID NO: 1 may be SEQ ID NO: 5, but is not limited thereto.

In addition, the structural protein M1 of the influenza H1N1 virus may include the entire or partial sequence of the structural protein M1 including various mutations, and preferably the amino acid sequence of SEQ ID NO: 2, a nucleic acid encoding the same, or a codon-optimized nucleic acid More preferably, the nucleic acid encoding the amino acid of SEQ ID NO: 2 is SEQ ID NO: 4, and the codon-optimized nucleic acid of SEQ ID NO: 2 may be SEQ ID NO: 6, but is not limited thereto.

As an example, the present invention provides a recombinant vector containing nucleic acids encoding virus-like particles including a corona 19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1.

In the recombinant vector of the present invention, the nucleotide sequence is preferably present in a suitable expression construct. In the expression construct, the nucleotide sequence is preferably operably linked to a promoter. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and/or translation of said other nucleic acid sequence.

In the present invention, the recombinant vector includes all types of vectors capable of expressing a target gene, preferably including a pCAGEN vector, but is not limited thereto.

In addition, the recombinant vector may include various sites necessary for an additional vector preparation or production process including detection and purification, and preferably includes 2X Flag and 6X His, but is not limited thereto.

As another example, the present invention provides a vaccine composition comprising virus-like particles comprising spikes of SARS-CoV-2 and influenza H1N1 viral structural protein M1, and an immune adjuvant.

In addition, the vaccine composition of the present invention may be one in which both S1 and S2 specific antibodies, which are spike subdomains, are induced.

In one embodiment of the present invention, the vaccine composition is characterized by having a neutralizing ability specific to the Corona 19 virus, and is characterized in that it is for preventing Corona 19 virus infection (COVID-19), but is not limited thereto.

Carriers suitable for the vaccine composition of the present invention are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution or a non-aqueous solution, suspension or emulsion. Additionally, it may include a stabilizer, an inactivating agent, an antibiotic, a preservative, and the like, and may be used by mixing with distilled water or a buffer solution according to the route of administration of the vaccine composition.

A suitable dosage of the vaccine composition of the present invention may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. It means an amount sufficient to show the effect of the vaccine composition and an amount sufficient to not cause side effects or serious or excessive immune reactions. Various general matters to be considered in determining the effective amount of a vaccine composition can be considered matters well known to those skilled in the art.

The suitable frequency of administration of the vaccine composition of the present invention is not particularly limited as long as an immune response can occur, but may preferably be administered twice or three times.

The vaccine composition can be injected into a human or non-human animal to increase the rate of antibody production against the antigen, and the injection can be administered by subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration and It can be carried out by any one method selected from the group consisting of oral administration.

The vaccine composition may be administered individually or in combination, and may be administered sequentially or simultaneously with a conventional vaccine composition. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.

The vaccine composition according to one embodiment of the present invention may additionally include an adjuvant, an immune enhancer or an adjuvant to increase efficacy. Vaccine adjuvants are largely classified into three types according to their mechanism of action: antigen carriers, immune enhancers, and those that stimulate immune responses and act as matrices for antigens. Effective use of vaccine adjuvants can (1) increase the immunogenicity of recombinant antigens, (2) reduce antigen dose or reduce the number of immunizations, and (3) improve immunogenicity in immunocompromised infants and the elderly. A variety of effects can be obtained. As the vaccine adjuvant, adjuvant components known in the field can be used, preferably aluminum salt, MF59, AS03, AS04, etc. can be used alone or in combination, more preferably alum (aluminium hydroxide) It can be used, but is not limited thereto.

As another example, the present invention includes the steps of identifying the gene or amino acid sequence of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus; Preparing a recombinant vector of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus; It provides a method for producing virus-like particles comprising a.

As another example, the present invention relates to a vaccine composition comprising a virus-like particle comprising a COVID-19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1 and an adjuvant, and a COVID-19 virus including instructions for use. (SARS-CoV-2) infection (COVID-19) prevention kit is provided.

The advantages and features of the present invention, and how to achieve them, will become clear with reference to the detailed description of the following embodiments. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in various different forms, and only the present embodiments will complete the disclosure of the present invention and allow common knowledge in the art to which the present invention belongs. It is provided to fully inform the holder of the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example 1> Cells and viruses

Vero-E6 and HEK293T cells for virus culture and preparation of virus analogues were cultured in DEME medium containing 10% FBS and 100 U/mL penicillin. SARS-CoV-2 was propagated and cultured in Vero-E6 cells, and all experiments, including virus culture and mouse and hamster experiments, were performed in a Biosafety level 3 (BSL3) facility of the Korea Centers for Disease Control and Prevention.

<Example 2> Production of virus-like particle (VLP)

When the VLP protein concentration produced after transfection was measured using the codon-optimized spike (SEQ ID NO: 5) and the original sequence spike (SEQ ID NO: 3), it was found that the concentration slightly increased in the codon-optimized VLP. It was confirmed, and as a result of Western blotting, it was overexpressed in the spike of the codon-optimized VLP (FIG. 2).

To prepare virus analogues, SARS-CoV-2 S antigen gene and influenza virus M1 antigen gene were cloned into pCAGEN (Addgene, Watertwon, MA, USA), an animal cell expression vector, and then cultured in large quantities in E. coli culture medium, followed by antigen gene In order to remove endotoxin, antigen genes were obtained using Endo-Free Maxi prep kit (Qiagen, Hilden, Germany). The prepared S and M1 gene expression vectors were transfected into HEK293T cells using Lipofectamine 2000 Transfection Reagent (Thermo Fisher, Waltham, MA, USA).

After transduction, VLP optimization conditions were set by varying the conditions according to the collection period and cultivation of the produced VLPs as follows. 1) Obtaining VLPs by collecting the supernatant after 3 days of culture in a volume of 50 mL; 2) Collecting the supernatant after culturing the 50 mL capacity for 1 day, exchanging the 30 mL medium and culturing again for 2 days to additionally collect the supernatant to obtain VLP; 3) Collect the supernatant after culturing for 2 days with a volume of 50 mL, exchange the medium for 30 mL, and re-culture for 1 day to obtain VLP by additionally collecting the supernatant.

After culture, the final protein concentration of VLPs produced under each condition according to the collection time and cultivation after culture was measured and the expression level was compared and analyzed through Western blot. , After exchanging the medium, culturing for 1 day and additionally collecting the supernatant was the optimal condition. Therefore, in subsequent experiments, culture was performed in a serum-free medium for 48 hours. After 48 hours, the supernatant was first collected and cultured for 24 hours by adding a serum-free medium, and then the collected supernatant was collected and centrifuged at 2,000 rpm for 10 minutes to separate only the culture medium. The final collected culture medium was centrifuged at 28,000 rpm for 3 hours using a 30% sucrose cushion method (30% sucrose 3 mL, VLP 32 mL) using an ultra-high-speed centrifuge (Beckman Coulter, SW32ti rotor, Brea, CA, USA) to isolate and concentrate only the VLPs (FIG. 3).

<Example 3> Mouse Immunization

For the mouse immune response, 6-week-old specific pathogen-free (SPF) BALB/c mice (Orient Bio, Seongnam, Korea) were inoculated with the immune antigen (VLP), and then the immune response was analyzed. Animals were acclimatized for one week prior to use, and were supplied with sterile feed and water. Animal experiments were approved by the Institutional Animal Care and Use Committee of the Korea Centers for Disease Control and Prevention, and all experiments were performed according to the guidelines of this committee.

For vaccination, 6- to 8-week-old female BALB/c mice were inoculated with VLP (5 μg, 15 μg, and 30 μg, n=5) intramuscularly three times at two-week intervals, 14 days and 28 days after the first immunization. On the day, two additional immunizations (Boost) were performed with the same dose of VLP. In addition, the experiment was performed by dividing into groups administered with the same dose of VLP immune antigen and adjuvant, and aluminum hydroxide (Alumhydroxyde, Alum; Invitrogen, Carlsbad, CA, USA) was used as the adjuvant. In order to detect SARS-CoV-2 S antigen-specific immune antibody (IgG) and analyze the virus neutralization reaction, blood was collected before vaccination, 13 days after the 1st and 2nd vaccination, and 20 days after the 3rd vaccination, and serum was obtained. separated. For the analysis of cellular immune response, splenocytes were obtained after spleen tissue was harvested and pulverized through autopsy on day 48 after the first inoculation.

<Example 4> Evaluation of virus-specific antibody production (Enzyme-linked immunosorbent assay, ELISA)

To detect SARS-CoV-2 S-specific antibody (IgG) after mouse immunization, S full, S1, and S2 of COVID-19 virus dissolved in 0.05 M carbonate buffer (pH 9.6) in a 96-well plate were added at 1 μg/mL, respectively. 100 μl was dispensed per well at each concentration, incubated at 4° C. for one day, and washed three times with PBST (PBS + 0.05% tween 20). Serum from each group obtained from mice was diluted stepwise by 2-fold starting with a concentration of 1/50, reacted for 2 hours at room temperature, and then anti-mouse-IgG (1:1,000, Southern Biotech, Birmingham, AL, USA) and reacted with the secondary antibody for 1 hour. For color development, the reaction was induced using an OPD substrate (0.5 mg/mL in 0.05 M phosphate citrate buffer, pH 5.0/0.03% sodium perborate, Sigma-aldrich, St. Louis, MO, USA), and after confirming color development, sulfuric acid ( 0.5 M) was added to stop the reaction. Then, the antibody titer was analyzed by measuring the absorbance (495 nm).

<Example 5> Virus neutralization test (Plaque reduction neutralization assay, PRNT)

To confirm the production of virus-neutralizing antibodies, Vero-E6 cells were cultured for 16 to 20 hours by adding 200,000 cells per well to a 12-well cell culture plate. Mouse serum was inactivated at 56°C for 30 minutes. It was diluted 10-fold using DMEM containing 2% FBS (Fetal bovine serum), mixed with the virus suspension, and allowed to stand at 37°C for one hour. The virus solution mixed with serum was aliquoted into cells prepared the day before and incubated at 37° C. for one hour. The mixture was removed, and the surface of the cell layer was covered with an agar or carboxymethylcellulose layer to prevent indiscriminate spread of the virus, followed by further incubation at 37°C for 3 days. After culturing for 3 days, the cells were fixed with 4% paraformaldehyde, stained with crystal violet, and the number of plaques was measured. The neutralization titer was calculated as half-water neutralization titer (50% neutralization titer, PRNT ₅₀ ) using the Reed and Muench method.

<Example 6> Evaluation of cellular immunity (Enzyme-linked immunosorbent spot, ELISPOT)

Cellular immunity was evaluated using the IFN-γ ELISPOT kit (R&D Systems, Minneapolis, MN, USA). Twenty days after the third mouse immunization, the excised mouse spleen was disrupted with a tissue crusher and immune cells were isolated using a 70 μm cell strainer. After putting the isolated splenocytes into each well at 1X10 ⁶ cells/well, the peptide (5 μg/mL) that can confirm the Corona 19 virus spike CD8 ⁺ T cell response, negative control DMSO, and positive control Concanavalin A Each was added to make the final 200 μl, and cultured for 48 hours in an incubator at 37° C. and 5% CO ₂ . After incubation, each well was reacted with a secondary antibody, biotinylated anti-mouse IFN-γ, reacted with enzyme conjugated streptavidin-AP, and then BCIP/NBT and AEC were sequentially colored to confirm spots. The number of T cells secreting IFN-γ was measured using an ELISPOT reader system (CTL analyzer, Cellular Technology, Cleveland, OH, USA).

<Example 7> Evaluation of protective efficacy in golden Syrian hamsters

7- to 10-week-old male SPF golden Syrian hamsters (Coatek, Pyeongtaek, Korea) were inoculated with the immune antigen, and then the immune response was analyzed and challenge inoculation experiments were conducted. All experiments were performed at the Korea Centers for Disease Control and Prevention's BSL-3 facility. For immunization, a 15 μg dose was inoculated by intramuscular injection three times at 2-week intervals. 11 mice were immunized per group, and the experiment was conducted by dividing into a group administered with VLP immune antigen alone and a group administered with alum, an adjuvant, simultaneously with VLP immune antigen. As a negative control, only PBS was inoculated. For the detection of SARS-CoV-2 specific IgG and the analysis of the antibody immune response, blood was collected before inoculation, 13 days after the 1st and 2nd inoculations, and 20 days after the 3rd inoculations, and serum was separated.

48 days after the first immunization, challenge inoculation was performed as follows to confirm SARS-CoV-2 susceptibility in golden Syrian hamsters. Golden Syrian hamsters were anesthetized by intramuscular injection with ketamine, and then infected with SARS-CoV-2 (1X10 ⁶ /pfu) intranasally. Weight measurement and clinical symptoms were observed for 14 days after infection. In order to confirm the virus proliferation patterns and histopathological findings in tissues, euthanasia and autopsy were performed on day 3 after infection, and lung tissues were extracted, and tissue slides were prepared for virus titer measurement and lung tissue pathology examination.

<Example 8> Measurement of golden Syrian hamster lung tissue titer

Three days after virus challenge inoculation, infected hamsters were necropsied by 3 animals per immune group, and lung tissue was removed and weighed. The extracted lungs were put in DMEM medium containing antibiotics (1% penicillin), the tissue was disrupted, and the supernatant was recovered by centrifugation, and the diluted supernatant was inoculated into Vero-E6 cells to measure the virus titer. .

<Test Example 1> Production of COVID-19 virus analogue (VLP) vaccine candidate

1. Antigen cloning and synthesis for the production of S antigen-based VLP vaccine candidates

In the present invention, in order to improve the low immunogenicity of antigens, which are disadvantages of subunit and DNA vaccines, and to prepare a vaccine against SARS-CoV-2 based on VLPs known to have high immunogenicity in vivo in laboratory animals. The SARS-CoV-2 S gene was designed. SARS-CoV-2 glycoprotein antigen (S) genetic information is a VLP vaccine candidate using the gene sequence of BetaCoV/Korea/KCDC03/2020 virus isolated from a domestic patient registered in GISAID (Global Initiative on Sharing All Influenza Data) material was designed. To prepare a VLP composed of M1 proteins of SARS-CoV-2 S and influenza virus (H1N1), a 2X Flag tag and a 6X Histidine tag were inserted into the C-terminal of the antigen gene and designed (Fig. 1A).

Genes for VLP production are based on the S of domestic isolate BetaCoV/Korea/KCDC03/2020 virus and the M1 sequence of A/Korea/01/2009 virus, genetic code optimization for optimal expression in host cells (animal cells) (codon optimization) and synthesized. The synthesized gene was inserted into pCAGEN vector (Addgene), an animal cell expression vector for VLP production, and finally pCAGEN/SARS-CoV-2 spike and pCAGEN/H1N1 M1 antigen gene were obtained.

2. Production of S antigen-based VLP vaccine candidates in animal cell expression system

After cloning the SARS-CoV-2 S antigen gene into an animal cell expression vector, the plasmid was purified using Endo-Free Maxi Prep kit to remove endotoxin. The S and M1 genes (vectors) prepared for VLP vaccine production were simultaneously transfected into HEK293T cells, cultured for 48 hours, cultured for 24 hours, cultured for a total of 72 hours, purified by centrifugation, and Vaccine candidates were prepared by isolation.

<Test Example 2> Mass production and purification of COVID-19 virus VLP vaccine candidate

1. Establishment and purification of conditions optimized for mass expression of VLP vaccine candidates

After transforming the antigen gene into E. coli (DH5α) for mass production of the prepared VLP vaccine candidate, it was mass-cultured in E. coli culture medium, and to remove the endotoxin of the antigen gene, an Endo-Free Maxi Prep kit was used. Genes were obtained. For optimal VLP production, HEK293T cells were transduced with the S and M1 genes (vector) at the same time, cultured in serum-free medium for 48 hours, and after collecting the supernatant, serum-free medium was added and cultivated for 24 hours. The amount was collected, and the collected supernatant was centrifuged at 2,000 rpm for 10 minutes to separate only the culture medium. Finally, the collected culture medium was centrifuged at 28,000 rpm for 3 hours in a 30% sucorse cushion method using an ultra-high-speed centrifuge to concentrate the VLP, and the pellet from which the supernatant was removed was dissolved in 500 μl of PBS to measure the concentration.

2. Characterization of VLP vaccine candidates in animal cell expression systems

Transmission electron microscopy (TEM) analysis and protein to verify structural stability after self-assembly of VLPs produced by overexpression and transduction of SARS-CoV-2 S and influenza virus (H1N1) M1 expression vectors in human fetal kidney cells (HEK293T) Western blot was performed to confirm expression. As a result of TEM analysis, it was confirmed that VLPs of about 100 to 200 nm in size, which are similar in size to the actual wild-type virus, were formed, and it was confirmed that the S and M1 genes were expressed through Western blotting (FIG. 4).

<Test Example 3> Evaluation of immunogenicity of COVID-19 virus vaccine candidates in experimental animals

1. Evaluation of immunogenicity of VLP vaccine candidates in experimental animals (mouse, Balb/c)

Animal experiments to evaluate the immunogenicity of the prepared VLPs were performed using 6-week-old female mice (Balb/c), 5 animals per experimental group, and immunization was performed at three doses (5, 10, and 30 μg). Each immune antigen was immunized three times at 2-week intervals by the intramuscular route, and blood was collected 2 weeks after immunization, 1st immunization, 2nd immunization, and 3rd immunization to confirm the production of antibodies to the immune antigen, respectively. Immunogenicity was evaluated, such as antibody (IgG) production and neutralizing antibody production. At this time, PBS was used as the negative control group (FIG. 5).

1-1. Humoral immune response analysis of VLP vaccine candidates

The immunogenicity of the prepared VLP was evaluated by ELISA to determine whether antigen-specific antibodies were produced in serum. After 2 or 3 immunizations, it was confirmed that a sufficient IgG titer appeared in the mouse, and it was confirmed that both S1 and S2 specific antibodies, which are subdomains of the S antigen, were produced by the vaccine of the present invention. In particular, it was confirmed that the titer for S2 was generated high. In addition, it was confirmed that the group immunized with an adjuvant (alum hydroxide) showed higher IgG titers and neutralizing antibody titers than the group not concurrently immunized (FIG. 6).

1-2. Analysis of humoral neutralizing antibody induction of VLP vaccine candidates

After isolating serum from the blood of mice immunized with the prepared VLP immune antigen, the Plaque reduction neutralization test (PRNT) method, commonly known as the "gold standard" among methods for measuring virus neutralizing antibodies, is used to detect SARS-CoV-2 The neutralizing ability of the antibody against was confirmed. To analyze the neutralizing antibody titer, a virus (BetaCoV/Korea/KCDC03/2020) distributed from the National Culture Collection for Pathogens was used, and the change in infection rate after treatment with mouse serum (antibody) was measured. Confirmed.

Neutralizing antibody induction showed a higher neutralizing antibody titer in the group simultaneously immunized with adjuvants than in the group injected with VLP immune antigen alone, similar to the results of IgG production, and compared to serum immunized with a low concentration, the neutralizing antibody titer was higher in the group immunized with the adjuvant. It was confirmed that a higher neutralizing antibody titer appeared in the serum collected after the treatment. In addition, it was confirmed that neutralizing antibodies were not induced in the negative control PBS group (FIG. 7).

1-3. Analysis of cell-mediated immune response of VLP vaccine candidates

Differences in cell-mediated immune responses to immunity of the VLP immune antigens were analyzed using the ELISPOT test method. For ELISPOT analysis, IFN-γ ELISPOT kit was used. As for the cellular immune response, it was confirmed that the number of T cells secreting IFN-γ increased in the group immunized with VLP compared to the PBS group, which was a negative control group, and increased in proportion to the antigen concentration. However, the increasing effect according to the adjuvant was not shown. In conclusion, it was confirmed that both humoral and cellular immune responses were induced by VLP immunization (FIG. 8).

2. Analysis of protective efficacy in experimental animal (Golden Syrian hamster) model of VLP vaccine candidates

Through immunogenicity confirmation experiments in mice, it was confirmed that the VLP immune antigen induces viral S antigen-specific antibodies and induces neutralizing antibodies against SARS-CoV-2. Accordingly, the protective ability of VLP vaccine candidates was evaluated using a golden Syrian hamster model known to be susceptible to SARS-CoV-2. To evaluate the protective ability of the candidate substance, animal experiments were conducted with 7-week-old Golden Syrian hamster males. Experimental animals were conducted with 11 animals per group, and the experiment was performed using alum, which is expected to exhibit higher immunogenicity compared to immunization with the VLP immune antigen alone, as an adjuvant for the VLP immune antigen. As an experimental negative control group, only PBS was inoculated.

Immunization was inoculated with a dose of 15 μg per animal through the intramuscular injection route three times at 2-week intervals. was isolated (FIG. 9). After separating the serum from the blood of the immunized hamster, the immunogenicity of the VLP immune antigen was confirmed using ELISA and PRNT methods.

2-1. Analysis of antibody induction of VLP vaccine candidates

After separating serum from the blood of hamsters immunized with the prepared VLP immune antigen, it was confirmed by ELISA whether antigen-specific antibodies were produced in the serum. It was confirmed that sufficient IgG titers appeared after a total of 2 or 3 mouse immunizations, and both S1 and S2 specific antibodies, which are subdomains of the S antigen, were produced by the vaccine of the present invention. In addition, the group immunized with the adjuvant alum at the same time showed somewhat higher IgG titers and neutralizing antibody titers than the group not co-administered (FIG. 10).

2-2. Analysis of neutralizing antibody induction of VLP vaccine candidates

After separating serum from the blood of hamsters immunized with the prepared VLP immune antigen, the neutralizing ability of the antibody against SARS-CoV-2 was confirmed using the PRNT method. As a result of neutralizing antibody titer analysis, similar to the results of IgG production, compared to the group injected with only VLP immune antigen, the group immunized with adjuvant showed higher neutralizing antibody titer, and higher neutralizing antibody was induced in tertiary immunization serum. It became. In addition, neutralizing antibodies were not induced in the negative control PBS group (FIG. 11).

2-3. Hamster survival rate and body weight analysis according to VLP vaccine candidate administration

48 days after the first immunization, 7-week-old golden Syrian hamster males were anesthetized with Ketamine intramuscular injection to confirm the protective ability against SARS-CoV-2 infection in golden Syrian hamsters whose immunogenicity was confirmed. Virus and negative control group (PBS) were infected intranasally, and body weight, body temperature and clinical symptoms were observed for 14 days after infection.

There was no death of hamsters for 2 weeks after infection, and hamsters immunized with VLP immune antigen showed lower weight loss compared to negative controls. In particular, the group immunized with VLP immune antigen and alum at the same time showed the lowest weight change (FIG. 12). These results confirm that the protective effect against SARS-CoV-2 virus attack appears according to VLP vaccination.

2-4. Analysis of hamster lung tissue according to VLP vaccine candidate administration

After immunization with the VLP vaccine, an autopsy was performed on the 3rd day after infection to confirm the virus proliferation pattern and histopathological findings in the lung tissue following challenge, and the virus titer in the autopsied lung tissue was measured.

After SARS-CoV-2 challenge, immunization with VLP immune antigen alone showed some reduced viral titers compared to the negative control group, but the group immunized with VLP immune antigen and adjuvant (alum) was comparable to the negative control group. 91% low virus titer was shown (FIG. 13).

<110> KCDC <120> Virus-like particle against SARS-CoV-2 and influenza H1N1 virus and use its <130> M21-0000 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 1273 <212> PRT <213> Artificial Sequence <220> <223> SARS-CoV-2 S full <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Al a Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 70 5 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pr o Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Se r Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu 1010 1015 1020 Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val 1025 1030 1035 1040 Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala 1045 1050 1055 Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu 1060 1065 1070 Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His 1075 1080 1085 Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val 1090 1095 1100 Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr 1105 1110 1115 1120 Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr 1125 1130 1135 Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu 1140 1145 1150 Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp 1155 1160 1165 Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp 1170 1175 1180 Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu 1185 1190 1195 1200 G ln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile 1205 1210 1215 Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile 1220 1225 1230 Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val 1250 1255 1260 Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 2 <211> 252 <212> PRT < 213> Artificial Sequence <220> <223> Influenza H1N1 M1 <400> 2 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro 1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Ser Val Phe 20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr 35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Lys Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ser Leu Ser Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Thr Val Thr Thr Glu Ala Ala Phe 130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Ala Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Asn Gln 195 200 205 Thr Arg Gln Met Val His Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220 Ser Ser Ala Gly Leu Lys Asp Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245 250 <210> 3 <211> 3819 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV-2 S full <400> 3 atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60 agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120 aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180 aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240 aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300 ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360 aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420 ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480 tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540 ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600 tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660 tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720 ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagggt 78 tgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840 gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900 tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960 caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020 gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080 tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140 ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200 gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260 tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320 cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380 ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440 aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500 aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560 ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620 ttcaacttca atg gtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680 cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740 acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800 ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860 cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920 aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980 gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040 cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100 gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160 agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220 tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280 acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340 gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400 aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460 ctacttttca acaaagtga c acttgcagat gctggcttca tcaaacaata tggtgattgc 2520 cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580 ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640 acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700 caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760 aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820 acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880 acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940 ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000 cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060 tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120 gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180 gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240 atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300 cactggtttg taacacaaag gaat ttttat gaaccacaaa tcattactac agacaacaca 3360 tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420 ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480 tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540 aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600 caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660 atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720 tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780 tctgagccag tgctcaaagg agtcaaatta cattacaca 3819 <210> 4 <211> 756 <212> DNA <213> Artificial Sequence <220> <223> Influenza H1N1 M1 <400> 4 atgagtcttc taaccgaggt cgaaacgtac gttctttcta tcatcccgtc aggccccctc 60 aaagccgaga tcgcgcagag actggaaagt gtctttgcag gaaagaacac agatcttgag 120 gctctcatgg aatggctaaa gacaagacca atcttgtcac ctctgactaa gggaatttta 180 ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc taaatgggaa tggggacccg aacaac atgg atagagcagt taaactatac 300 aagaagctca aaagagaaat aacgttccat ggggccaagg aggtgtcact aagctattca 360 actggtgcac ttgccagttg catgggcctc atatacaaca ggatgggaac agtgaccaca 420 gaagctgctt ttggtctagt gtgtgccact tgtgaacaga ttgctgattc acagcatcgg 480 tctcacagac agatggctac taccaccaat ccactaatca ggcatgaaaa cagaatggtg 540 ctggctagca ctacggcaaa ggctatggaa cagatggctg gatcgagtga acaggcagcg 600 gaggccatgg aggttgctaa tcagactagg cagatggtac atgcaatgag aactattggg 660 actcatccta gctccagtgc tggtctgaaa gatgaccttc ttgaaaattt gcaggcctac 720 cagaagcgaa tgggagtgca gatgcagcga ttcaag 756 <210> 5 <211> 3888 <212> DNA <213> Artificial Sequence <220> <223> SARS-CoV2 S full(Codon optimization) <400> 5 atgtttgttt ttcttgtgct gctcccactg gtttcctctc agtgtgttaa cctgaccact 60 cgaacccaat tgcctccggc gtacactaac agctttacca ggggggtgta ctaccccgat 120 aaagttttca ggagttccgt cctccactca acccaggacc tgtttttgcc gtttttcagt 180 aacgtgactt ggtttcatgc aatacacgta agcgggacca acggtaccaa gagattcgat 240 aaccctgtcc tcccctttaa tgatggcgta tacttcgcct ccacagagaa gagcaacatt 300 atcagaggct ggatcttcgg gacaactctc gatagcaaga cccagtctct cctgattgtg 360 aacaatgcca ccaatgtggt gataaaagtt tgcgagttcc agttttgtaa tgaccccttt 420 ctgggcgtat actaccataa ataataacaaa agttattact aggcgtg 420 ctgggcgtat agttggatgg agtcgtg agttcagcca acaactgcac ctttgagtac gtgtctcagc ccttcctgat ggacctggag 540 gggaaacagg ggaacttcaa gaacctgagg gagttcgttt tcaagaacat cgacggctac 600 ttcaaaattt acagcaagca cacaccaatt aaccttgtga gagatttgcc ccaagggttt 660 agcgccttgg agccgctggt ggacctgccc atcggcatca atataaccag gttccagacg 720 ttgctggcac tgcatcgatc ttacctgacg ccaggcgata gtagttctgg gtggacggcc 780 ggggccgcgg cttactatgt cggctacctt cagcccagga cctttttgct caagtataat 840 gagaacggga caatcacgga cgctgttgat tgcgctctgg acccgctgtc tgagaccaag 900 tgcaccttga agagtttcac cgttgaaaag ggcatttacc agacctctaa ctttagagtg 960 cagcctaccg agagtattgt gcgattcccc aacatcacca acctttgtcc ttttggggag 1020 gtttttaacg cgacgaggtt tgcgtcagtg tatgcttgga acaggaaacg catcagcaac 1080 tgtgtggccg actactcagt gctttataat tccgctagct ttagtacctt caagtgctac 1140 ggggtgtctc cgactaagct gaatgatctc tgcttcacta atgtttatgc ggacagcttc 1200 gttattaggg gtgacgaagt acgacaaata gctcctggtc aaacaggcaa gatcgcagac 1260 tataactaca agctccccga tgacttcaca ggctgtgtaa tcgcgtggaa cagcaacaac 1320 cttgattcca aag tgggtgg gaactacaac tacctgtacc ggcttttccg aaagagcaat 1380 ctgaagcctt tcgaacgcga catctccaca gaaatatatc aggcagggag cacgccttgt 1440 aacggggtgg aagggttcaa ctgctacttt ccgctgcaga gttatgggtt ccagcccacc 1500 aatggcgtcg ggtaccagcc ctacagagtg gtggtgctct catttgagct gctgcacgcc 1560 ccagccacgg tgtgcgggcc gaaaaaatct accaaccttg tgaagaataa gtgtgtcaat 1620 tttaacttca atggcttgac tgggactggc gtactgactg agtccaataa aaaatttctc 1680 ccattccagc agttcggacg agacatcgct gatacaacgg acgcagttcg agacccacag 1740 acactggaga tcctcgatat aactccatgt agtttcggag gtgtctctgt aatcactccc 1800 ggaacgaaca cctcaaacca ggtggcggta ctttaccagg atgtcaattg taccgaggtg 1860 cccgtggcga tacatgctga tcaactgacc ccgacctggc gggtttatag caccgggagc 1920 aatgtttttc agacgcgggc cggctgtctg attggcgccg aacacgtgaa caacagttat 1980 gagtgcgata tccccattgg cgctggtatc tgcgccagct accaaactca aacaaatagt 2040 ccccgccgcg cccgctccgt agcctcccag agcattatcg cttacactat gtcactcggt 2100 gcggaaaact cagtggctta cagtaacaat tccattgcga ttccaactaa tttcactatt 2160 agcgtcacaa cagaaatac t tcctgtctcc atgaccaaaa caagtgttga ttgcacgatg 2220 tatatttgtg gcgattctac tgaatgctct aacttgctcc ttcagtatgg ttccttctgc 2280 acccagctga accgagcatt gactggcatc gctgtggaac aggataagaa tacacaggag 2340 gtttttgctc aagttaagca gatttataag acccccccta ttaaggattt cggaggcttt 2400 aacttcagcc agatacttcc agatccctct aagccctcaa aacgctcctt tattgaggat 2460 ttgcttttta ataaggttac cctggccgat gctggtttca ttaagcagta cggggattgt 2520 cttggcgaca ttgcggcgcg cgatctgatt tgcgcacaga agtttaatgg cctgacggtg 2580 cttccgcccc tccttaccga cgaaatgata gcgcagtata cctccgctct gttggcaggc 2640 accataacgt ccggctggac ctttggagcc ggggccgcct tgcagatccc ctttgcgatg 2700 caaatggcct atcggtttaa cggcataggt gtcactcaga acgtactgta tgaaaatcaa 2760 aaacttatcg ctaaccagtt caactccgcg attggaaaaa ttcaagattc ccttagctcc 2820 acggcaagtg ccctgggtaa gttgcaagac gtagtgaacc agaatgcaca agccctgaat 2880 acgctcgtca agcagctttc atccaatttc ggtgcaatat caagtgtttt gaacgatatt 2940 ctctccaggc tcgataaagt cgaggctgag gtacagatcg acaggctgat cactggcagg 3000 ttgcagagct tgcagactta tgtc actcag caactgatcc gcgctgctga aatcagggcc 3060 agtgcgaacc tcgccgcgac aaagatgtcc gagtgtgtac tcggtcagag taaaagagtc 3120 gatttctgcg gtaaaggata tcatcttatg tctttcccgc aaagtgcccc tcatggggtg 3180 gttttcctcc acgtgaccta tgtccccgct caagagaaga attttacgac ggctcctgcc 3240 atctgccacg acgggaaggc acacttccct agagaggggg tattcgtgag caatggcact 3300 cactggttcg tgacacaaag gaatttctat gagccccaga tcataaccac ggataacact 3360 ttcgtcagcg gtaactgtga tgtggtcatt ggcatcgtga ataacacagt ctacgaccca 3420 ctgcagcctg aattggacag ttttaaagag gaactggata aatacttcaa gaatcacacc 3480 tctcctgacg tggacctcgg cgatatctcc gggattaatg ctagcgtagt caacattcag 3540 aaagaaatag acaggctgaa tgaggtggcc aaaaatctga acgaatccct catcgacctc 3600 caagaactgg gtaaatatga gcaatacatc aagtggcctt ggtatatttg gctcgggttt 3660 attgctggac tgatcgctat tgtaatggtt accatcatgc tgtgctgcat gaccagctgc 3720 tgtagctgtc tgaaggggtg ttgtagctgt ggttcatgtt gcaagtttga cgaggacgac 3780 agtgaacctg ttcttaaggg tgttaaattg cactatactg attataagga cgatgacgac 3840 aaggactata aggacgacga cgacaagcat catcatcacc atcactga 3888 <210> 6 <211> 825 <212> DNA <213> Artificial Sequence <220> <223> Infulenza H1N1 M1(Codon optimization) <400> 6 atgagcctgc tgaccgaggt ggaaacatac gtgctgagca tcatccccag cggacctctg 60 aaagccgaga tcgcccagag actggaatct gtgttcgccg gcaagaacac cgacctggaa 120 gccctgatgg aatggctgaa aacccggcct atcctgtctc ctctgacaaa gggcatcctg 180 ggcttcgtgt ttaccctgac cgtgccttct gagagaggcc tgcagcggag aagattcgtg 240 cagaacgctc tgaacggcaa cggcgacccc aacaacatgg atagagccgt gaagctgtac 300 aagaagctga agagagagat caccttccac ggcgccaaag aggtgtccct gagctattct 360 acaggcgccc tggcctcttg catgggcctg atctacaata gaatgggcac cgtgaccacc 420 gaggccgcct ttggacttgt gtgtgccaca tgcgagcaga tcgccgatag ccagcacaga 480 tctcacagac agatggccac caccaccaat cctctgatcc ggcacgagaa cagaatggtg 540 ctggcctcta caaccgccaa ggccatggaa cagatggctg gatcttctga acaggccgcc 600 gaagctatgg aagtggccaa tcagacaagg cagatggtgc acgccatgcg gacaatcgga 660 acacaccctt ctagctctgc cggcctgaag gacgacctgc tggaaaacct gcaggcttaga 7 gacgagcttagag atgcagcgg ttcaaggatt ataaagatga tgatgataaa 780gattataaag atgatgatga taaacaccac caccaccacc actaa 825

Claims (12)

Virus-like particle containing COVID-19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1
The virus-like particle according to claim 1, wherein the Corona 19 virus is BetaCoV/Korea/KCDC03/2020 and the influenza H1N1 virus is A/Korea/01/2009
The method of claim 1, wherein the Corona 19 virus spike is an amino acid of SEQ ID NO: 1, a nucleic acid encoding the same, or a codon-optimized nucleic acid, and the influenza H1N1 viral structural protein M1 is an amino acid of SEQ ID NO: 2, a nucleic acid encoding the same, or a codon-optimized nucleic acid. Virus-like particles, characterized in that
Recombinant vectors containing nucleic acids encoding virus-like particles comprising the COVID-19 virus (SARS-CoV-2) spike and influenza H1N1 viral structural protein M1
The recombinant vector according to claim 4, wherein the recombinant vector comprises a pCAGEN vector.
The recombinant vector according to claim 4, characterized in that the recombinant vector comprises 2X Flag and 6X His.
A vaccine composition comprising a spike of COVID-19 virus (SARS-CoV-2) and virus-like particles comprising influenza H1N1 viral structural protein M1 and an immune adjuvant
The vaccine composition according to claim 7, wherein the adjuvant is aluminum hydroxide.
The vaccine composition according to claim 7, wherein both S1 and S2 specific antibodies, which are subdomains of Spike, are induced.
The vaccine composition according to claim 7, characterized in that it is administered twice or three times.
Identifying the gene or amino acid sequence of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus;
Preparing a recombinant vector of SARS-CoV-2 spike antigen and structural protein M1 of influenza H1N1 virus; Method for producing virus-like particles comprising
Corona 19 virus (SARS-CoV-2) vaccine composition including virus-like particles and immune adjuvants including spike and influenza H1N1 viral structural protein M1 and instructions for use ) Infectious disease (COVID-19) prevention kit

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