KR20230091734A - COVID-19 vaccine composition with increased immunogenicity - Google Patents

Abstract

The present invention relates to a coronavirus infection-19 (COVID-19) vaccine composition with increased immunogenicity, and more particularly, to a spike 1 recombinant antigen protein and a receptor binding domain (RBD) of the SARS-CoV-2 virus. ) an antigenic composition comprising a recombinant antigenic protein; And a COVID-19 vaccine composition with increased immunogenicity comprising Montanide Gel as an adjuvant.
It was confirmed that the COVID-19 vaccine composition of the present invention has increased antibody formation ability (immunogenicity) compared to the vaccine composition without Montanide gel, and by vaccination, CD4 ⁺ IL-5 ⁺ T Cells and CD4 ⁺ IL-17 ⁺ T cells were activated, and it was confirmed that IFN-γ and IL-5 cytokine production increased. In addition, since it was confirmed that the vaccine composition of the present invention shows high immunogenicity in the South African and British strains of the COVID-19 virus, it can be used as a vaccine composition for preventing or treating COVID-19.

Description

COVID-19 vaccine composition with increased immunogenicity {COVID-19 vaccine composition with increased immunogenicity}

The present invention relates to a coronavirus infection-19 (COVID-19) vaccine composition with increased immunogenicity, and more particularly, to a spike 1 recombinant antigen protein and a receptor binding domain (RBD) of the SARS-CoV-2 virus. ) an antigenic composition comprising a recombinant antigenic protein; And a COVID-19 vaccine composition with increased immunogenicity comprising Montanide Gel as an adjuvant.

Coronavirus is an RNA virus that causes respiratory diseases. In December 2019, global attention increased as cases of respiratory disease infections amplified in Wuhan, China. As a result of genome analysis of the coronavirus, it is SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2), a new virus that belongs to the beta corona virus and has never been found in humans or animals. was named Coronavirus Disease-2019 (COVID-19).

Main serious injuries that occur when infected with COVID-19 include symptoms such as lethargy, high fever of 37.5 degrees or higher, cough, sore throat, phlegm, muscle pain, headache, shortness of breath and pneumonia, respiratory failure due to lung damage, etc. However, it can lead to death in severe cases, so it is very urgent to develop a diagnosis, vaccine, and treatment for COVID-19. As of September 2021, 105 vaccines are in clinical trials, 35 are in final testing, and more than 75 vaccines are actively researched in preclinical animal studies. Currently, 8 vaccines have been approved, and 13 candidates have limited approval. It became.

Representative vaccines include mRNA vaccines produced by Pfizer and Moderna and adenovirus vaccines produced by AstraZeneca. In particular, mRNA vaccines have proven to be very effective in immunity against COVID-19, but have reduced preventive effects against side effects and variants. and problems such as a decrease in preventive effect over time.

On the other hand, SARS-CoV-2 has a 30 kb nucleotide and has four important structural proteins as follows; Nucleocapsid (N), Spike (S), Membrane (M), and Envelope (E) proteins. Among them, the S protein (spike protein) is an important site as it binds to the receptor of the host cell, and replication takes place while transferring the viral nucleocapsid into the cell.

In addition, SARS-CoV-2 is known to be capable of viral replication by binding to the ACE2 (angiotensin converting enzyme 2) receptor of human cells. The receptor-binding domain (RBD) included in the S protein (spike protein) of coronavirus is a key region that binds to the host cell's ACE2 receptor and induces strong neutralizing antibodies against SARS-CoV-2 infection. Because it contains multiple conformational-dependent epitopes, it can be a key target for COVID-19 treatment and vaccine development (J. Immunol., 2005;174:4908-4915).

Accordingly, in the present invention, as a result of diligent efforts to develop a coronavirus infection-19 (COVID-19) vaccine with increased immunogenicity, the spike 1 recombinant antigen protein and RBD (receptor binding) of the SARS-CoV-2 virus domain), when Montanide Gel was added as an immune enhancer to an antigen composition containing a recombinant antigen protein, it was confirmed that the immunogenicity against COVID-19 significantly increased, and the present invention was completed.

Accordingly, an object of the present invention is an antigen composition comprising Spike 1 recombinant antigen protein and RBD (receptor binding domain) recombinant antigen protein of SARS-CoV-2 virus; And to provide a COVID-19 vaccine composition with increased immunogenicity comprising Montanide Gel as an immune enhancer.

In order to achieve the above purpose,

The present invention relates to an antigen composition comprising spike 1 recombinant antigen protein and RBD (receptor binding domain) recombinant antigen protein of SARS-CoV-2 virus; and

Provided is a COVID-19 vaccine composition with increased immunogenicity, comprising Montanide Gel as an adjuvant.

In a preferred embodiment of the present invention, the Spike 1 recombinant antigen protein of the SARS-CoV-2 virus may be represented by the amino acid sequence of SEQ ID NO: 1.

In another preferred embodiment of the present invention, the RBD (receptor binding domain) recombinant antigen protein may be represented by the amino acid sequence of SEQ ID NO: 2.

In another preferred embodiment of the present invention, the amino acid of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 3, and the amino acid of SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 4.

In another preferred embodiment of the present invention, the immune enhancer may be included in 5 to 30% by weight of the total composition.

In another preferred embodiment of the present invention, the Montanide gel may be Montanide Gel PR02.

In another preferred embodiment of the present invention, the vaccine composition may be administered to a subject selected from the group consisting of mice, guinea pigs, cats and dogs.

In another preferred embodiment of the present invention, a single dose of each antigen protein in the vaccine composition may be 5 μg to 100 μg.

In another preferred embodiment of the present invention, the vaccine composition may be administered firstly to an individual and then secondly administered 10 to 30 days later.

In another preferred embodiment of the present invention, the vaccine composition can be administered through subcutaneous injection, intraperitoneal injection or intramuscular injection.

In another preferred embodiment of the present invention, the vaccine composition can activate CD4 ⁺ IL-5 ⁺ T cells (T helper 2) or CD4 ⁺ IL-17 ⁺ T cells (T helper 17).

In another preferred embodiment of the present invention, the vaccine composition can activate IFN-γ or IL-5 cytokines.

In another preferred embodiment of the present invention, the vaccine composition may have cross-protective efficacy against the South African or British variant COVID-19 virus.

The present invention also provides a pharmaceutical composition for preventing or treating COVID-19 with increased immunogenicity comprising the above vaccine composition.

The present invention also provides a method for preventing COVID-19 comprising administering the vaccine composition to a non-human subject.

It was confirmed that the COVID-19 vaccine composition of the present invention has increased antibody formation ability (immunogenicity) compared to the vaccine composition without Montanide gel, and by vaccination, CD4 ⁺ IL-5 ⁺ T It was confirmed that cells (T helper 2) and CD4 ⁺ IL-17 ⁺ T cells (T helper 17) were activated, and production of IFN-γ and IL-5 cytokines increased.

In addition, since it was confirmed that the vaccine composition of the present invention shows high immunogenicity in the South African and British strains of the COVID-19 virus, it can be used as a vaccine composition for preventing or treating COVID-19.

1 is data confirming the expression levels of S1 recombinant antigen protein (A) and RBD recombinant antigen protein (B) expressed from CHO cell lines.
2 is data confirming the degree of activation of (A) CD4 ⁺ IL-5 ⁺ T cells and CD4 ⁺ IL-17 ⁺ T cells according to the administration of the vaccine composition of the present invention and (B) the degree of IFN-γ or IL-5 production This is the data confirmed.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to an antigen composition comprising a spike 1 (Spike 1) recombinant antigen protein and RBD (receptor binding domain) recombinant antigen protein of SARS-CoV-2 virus; and a COVID-19 vaccine composition with increased immunogenicity, comprising Montanide Gel as an adjuvant.

In the present invention, the term "vaccine" is a biological preparation containing an antigen that induces an immune response in a living body, and is an immunogen that induces immunity in a living body by being injected or orally administered to humans or animals to prevent infections. say The animal is a human or non-human animal, and the non-human animal refers to pigs, cows, horses, dogs, goats, sheep, etc., but is not limited thereto.

In the present invention, the Spike 1 recombinant antigen protein of the SARS-CoV-2 virus may be represented by the amino acid sequence of SEQ ID NO: 1.

In the present invention, the RBD (receptor binding domain) recombinant antigen protein may be represented by the amino acid sequence of SEQ ID NO: 2.

In the present invention, the amino acid of SEQ ID NO: 1 may be encoded by the nucleotide sequence of SEQ ID NO: 3, and the amino acid of SEQ ID NO: 2 may be encoded by the nucleotide sequence of SEQ ID NO: 4.

In the present invention, the immune enhancer may be included in 5 to 30%, preferably 5 to 20% of the total composition, and the Montanide gel may be Montanide Gel PR02.

In the present invention, the vaccine composition comprises the Spike 1 recombinant antigen protein of SARS-CoV-2 virus comprising the amino acid sequence of SEQ ID NO: 1 and the receptor binding domain (RBD) comprising the amino acid sequence of SEQ ID NO: 2 It can be prepared by a method comprising mixing the recombinant antigen protein and Montanide Gel as an immunostimulant.

The Spike 1 recombinant antigen protein of the SARS-CoV-2 virus comprising the amino acid sequence of SEQ ID NO: 1 includes: (a) preparing a recombinant vector comprising the nucleotide sequence represented by SEQ ID NO: 3; (b) transforming cells with the vector and culturing the transformed cells; and obtaining the Spike 1 recombinant antigen protein of the SARS-CoV-2 virus from the cell supernatant cultured in step (b).

The RBD recombinant antigen protein comprising the amino acid sequence of SEQ ID NO: 2 comprises: (a) preparing a recombinant vector comprising the nucleotide sequence represented by SEQ ID NO: 4; (b) transforming cells with the vector and culturing the transformed cells; and obtaining the RBD recombinant antigen protein of the SARS-CoV-2 virus by disrupting the cells cultured in step (b).

In a specific embodiment of the present invention, the gene base sequence and amino acid sequence including Spike 1 (S1) and RBD (receptor binding domain) protein of SARS-CoV-2 isolated from Wuhan, China were obtained, which A vaccine composition containing the S1 recombinant antigen protein and the RBD recombinant antigen protein as antigen proteins was prepared using the vaccine composition.

A recombinant vector into which the S1 gene represented by the nucleotide sequence of SEQ ID NO: 3 of the present invention is inserted and a recombinant vector into which the RBD gene represented by the nucleotide sequence of SEQ ID NO: 4 is inserted are prepared, and then transformed into CHO cells to produce antigenic proteins. As a result of expression, as shown in FIG. 1, it was confirmed that both the S1 recombinant antigen protein and the RBD recombinant antigen protein were normally expressed.

In the present invention, the term "expression vector" is a gene product containing essential regulatory elements such as a promoter so that a target gene can be expressed in an appropriate host cell. Vectors may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors. Once transformed into a suitable host, the vector can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself.

In addition, vectors may contain expression control elements that allow for correct expression of the coding region in a suitable host. Such regulatory elements are well known to those skilled in the art and include, for example, promoters, ribosome-binding sites, enhancers and other regulatory elements for regulating gene transcription or mRNA translation. can do. The specific structure of the expression control sequence may vary depending on the function of the species or cell type, but generally includes 5' ratios that participate in transcription initiation and translation initiation, such as TATA boxes, capped sequences, CAAT sequences, etc., respectively. -contains a transcribed sequence, and a 5' or 3' non-translated sequence. For example, a 5' non-transcribed expression control sequence can include a promoter region that can include promoter sequences for transcribing and regulating functionally linked nucleic acids.

In the present invention, the term "promoter" refers to a minimal sequence sufficient to direct transcription. In addition, promoter constructs sufficient to allow expression of a regulatable promoter dependent gene induced by cell type specific or external signals or agents may be included, and such constructs may be located on the 5' or 3' portion of the gene. . Both conserved promoters and inducible promoters are included. Promoter sequences can be derived from prokaryotes, eukaryotes or viruses.

In the present invention, the term "transformation" refers to introducing a vector having a polynucleotide encoding one or more target proteins into a host cell, to prepare a transformant (transformed cell) into which the expression vector is introduced into the host cell. Methods for this include the calcium phosphate method or calcium chloride/rubidium chloride method described in the literature (Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989), Examples include electroporation, electroinjection, chemical treatment methods such as PEG, and methods using a gene gun.

When the transformant expressing the vector is cultured in a nutrient medium, antibody protein can be produced and isolated in large quantities. Media and culture conditions can be appropriately selected and used according to the host cell. Conditions such as temperature, medium pH, and incubation time should be appropriately adjusted so as to be suitable for cell growth and mass production of proteins during culture.

The vector according to the present invention can be transformed into a host cell, preferably a mammalian cell, for antibody production. A number of suitable host cell lines capable of expressing fully glycosylated proteins have been developed in the art and include COS-1 (eg ATCC CRL 1650), COS-7 (eg ATCC CRL-1651), HEK293 , BHK21 (eg ATCC CRL-10), CHO (eg ATCC CRL 1610) and BSC-1 (eg ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells , P3X63Ag8653, SP2/0-Agl4, 293 cells, HeLa cells, etc., and these cells are readily available, for example, from the American Type Culture Collection (ATCC, USA).

In the present invention, the vaccine composition may be administered to a subject selected from the group consisting of mice, guinea pigs, cats and dogs.

In the present invention, a single dose of each antigen protein in the vaccine composition may be 5 μg to 100 μg.

In the present invention, the vaccine composition may be administered firstly to an individual and then secondly administered 10 to 30 days later.

In the present invention, the vaccine composition is administered parenterally, for example, rectal, transdermal, intravenous injection, intraarterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, intraventricular injection, etc. It may be administered, preferably via subcutaneous injection, intraperitoneal injection or intramuscular injection.

In the present invention, the vaccine composition can activate CD4 ⁺ IL-5 ⁺ T cells (T helper 2) or CD4 ⁺ IL-17 ⁺ T cells (T helper 17), IFN-γ or IL-5 cyto Cain expression may be increased.

In the present invention, the vaccine composition may have cross-protective efficacy against the South African or British variant COVID-19 virus.

In another specific embodiment of the present invention, an antigen composition comprising SARS-CoV-2 virus spike 1 (Spike 1) recombinant antigen protein and RBD (receptor binding domain) recombinant antigen protein; And a COVID-19 vaccine composition with increased immunogenicity, including Montanide Gel as an adjuvant, was prepared, and the stability of the vaccine composition was confirmed in mice, guinea pigs, cats, and dogs.

As a result of confirming the immunogenicity between the vaccine composition containing Montanide gel as an immunity enhancer (Experimental Example) and the vaccine composition without Montanide gel (Comparative Example), the immune It was confirmed that the rawness increased remarkably. In addition, it was confirmed that the vaccine composition of the present invention shows cross-protective efficacy against the South African or British variant COVID-19 virus.

In another specific embodiment of the present invention, when the vaccine composition of the present invention is administered, as a result of confirming whether in vivo T cell activity is induced, CD4 ⁺ IL-5 ⁺ T cells or CD4 ⁺ IL by administration of the vaccine composition -17 ⁺ T cells were activated, and it was confirmed that IFN-γ or IL-5 cytokine production increased (FIG. 2).

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating COVID-19 with increased immunogenicity, including the vaccine composition of the present invention.

In another aspect, the present invention relates to a method for preventing COVID-19 comprising administering the vaccine composition of the present invention to a non-human subject.

The vaccine composition of the present invention may be a non-human subject, preferably a mouse, guinea pig, cat or dog, and can prevent or treat COVID-19 by administering the vaccine composition of the present invention.

The vaccine composition of the present invention can be prepared in any suitable, pharmaceutically acceptable formulation. For example, it may be in the form of a solution or suspension for extemporaneous administration, a concentrated stock solution suitable for dilution prior to administration, or prepared in a reconstitutable form such as a lyophilized, freeze-dried, or frozen formulation.

The vaccine composition of the present invention may be formulated by including a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers that can be used for formulation of vaccine compositions are listed and regulated in the Korean Pharmacopoeia or other pharmacopeias, particularly in the US, Japan, and European pharmacopeias, and reference may be made to these pharmacopeias.

Such carriers will usually include diluents, excipients, stabilizers, preservatives, and the like. Suitable diluents include propylene glycol, polyethylene glycol, non-aqueous solvents such as vegetable oils such as olive oil and peanut oil, saline (preferably 0.8% saline), water containing a buffer medium (preferably 0.05M phosphate buffer), etc. suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk , glycerol, propylene, glycol, water, ethanol, and the like, and suitable stabilizers include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, and glucose, or animal and vegetable substances such as milk powder, serum albumin, and casein. or proteins such as microbial proteins. Suitable preservatives include thimerosal, merthiolate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B and the like.

The vaccine composition of the present invention may further include an adjuvant other than Montanide gel. An adjuvant may be composed of one or more substances that enhance an immune response to an antigen. Immunostimulants such as complete Freund's, incomplete Freund's, saponins, gelatinous aluminum adjuvants, surface active substances (e.g. lysolecithin, pluronic glycol, polyanions, peptides, oils or hydrocarbon emulsions, etc.), vegetable oils (cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, and the like.

The vaccine composition of the present invention can be prepared in arbitrary unit doses. A unit dose refers to the amount of an active ingredient and a pharmaceutically acceptable carrier contained in an individual product packaged for use at least once in humans, and the appropriate amount of such active ingredient and carrier is An amount capable of functioning as a vaccine when administered one or more times can be determined nonclinically and clinically within the ordinary skill of a person skilled in the art.

In the present invention, the pharmaceutical composition is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, and activity of the drug in the patient. , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Production of SARS-CoV-2 S1 recombinant antigen protein and RBD recombinant antigen protein

1-1: Base sequence and amino acid sequence secured

In the present invention, gene sequences including spike 1 (Spike 1, S1) and RBD (receptor binding domain) proteins of SARS-CoV-2 isolated from Wuhan, China were obtained.

The SARS-CoV-2 S1 recombinant antigen protein comprising the amino acid sequence of SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 3, and the RBD recombinant antigen protein comprising the amino acid sequence of SEQ ID NO: 2 is encoded by the nucleotide sequence of SEQ ID NO: 4. It was confirmed that it was encoded, and antigen proteins for vaccine production were prepared using these sequences. Positions 670 to 675 in the amino acid sequence of SEQ ID NO: 1 and positions 224 to 229 in the amino acid sequence of SEQ ID NO: 2 correspond to the His-Tag portion.

1-2: Preparation of recombinant vector into which S1 gene and RBD gene are inserted

Using a method known in the art, the SARS-CoV-2 S1 recombinant antigen protein gene having the nucleotide sequence of SEQ ID NO: 3 and the RBD recombinant antigen protein having the nucleotide sequence of SEQ ID NO: 4 were transferred to the pcDNA™® (Thermo Fisher) vector. Recombinant vectors into which each gene was inserted were prepared, and as a result of sequence analysis of the gene products of the recombinant vectors, it was confirmed that they matched 100% with the template.

1-3: Recombinant vector transformation

The S1 recombinant antigen protein-expressing recombinant vector and the RBD recombinant antigen protein-expressing recombinant vector prepared in Example 1-2 were each transformed into CHO cells using a method known in the art.

After culturing the transformed CHO cells, S1 recombinant antigen protein (C-His) and RBD recombinant antigen protein (C-His) were respectively obtained from the culture supernatant.

Western blot was performed to confirm the expression of SARS-CoV-2 recombinant S1 (C-His) antigen protein and SARS-CoV-2 recombinant RBD (C-His) antigen protein.

First, S1 (C-His) antigen protein and recombinant RBD (C-His) antigen protein expressed in cells transfected with a recombinant vector were purified, and then each antigen was 2 μg/8 μl/well by SDS After -PAGE was lowered, anti-COVID19 spike glycoprotein antibody (HRP) and anti-COVID19 RBD antibody (anti-COVID19 RBD antibody (HRP)) were treated and reacted as primary antibodies. Then, the secondary antibody (anti-mouse) is treated and reacted, and the reaction-completed membrane is developed with CN/DAB substrate to detect recombinant S1 (C-His) antigen protein and recombinant RBD (C-His) antigen protein. Expression was confirmed.

As a result, as shown in FIG. 1, it was confirmed that both the recombinant S1 (C-His) antigen protein and the recombinant RBD (C-His) antigen protein were normally expressed.

Animal test evaluation of SARS-CoV-2 S1 recombinant antigen protein and RBD recombinant antigen protein

2-1: Preparation of test vaccine

In the present invention, a test vaccine was prepared as shown in Table 1 below using the recombinant S1 (C-His) antigen and the recombinant RBD (C-His) antigen prepared in Example 1, and as an immune enhancer, Montanide Gel ) was added.

Test vaccine composition division effective antigen Content (μg/dose) immune booster preservative Test vaccine composition S1 60 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 60

2-2: Confirmation of test vaccine stability in experimental animals

The safety of the test vaccine by determining the presence or absence of pathogenicity of the test vaccine prepared in Example 2-1 using a mouse (7 weeks old, C57BL/6 female) and a guinea pig (300-350 g, Hlakoat: Ha, female) as experimental animals. confirmed. As an experimental group, it was divided into an inoculation group inoculated with the test vaccine prepared with the composition of the test vaccine in Table 1 and a negative control group that was not inoculated.

Laboratory animal safety assessment laboratory animal test group inoculation route Inoculation amount (ml) Injection site side effects and clinical symptoms dead head/
public announcement mouse control group - - doesn't exist 0/8 Inoculation group abdominal cavity 0.5 doesn't exist 0/8 Guinea pig control group - - doesn't exist 0/2 Inoculation group muscle One doesn't exist 0/2

As a result, the safety of the vaccine composition of the present invention was confirmed in mice and guinea pigs, which are experimental animals.

2-3: Confirmation of test vaccine stability in target animals

The safety of the test vaccine was confirmed by determining the pathogenicity of the test vaccine prepared in Example 2-1 using a cat (3-5 months old, female). As an experimental group, it was divided into an inoculation group inoculated with the test vaccine prepared with the composition of the test vaccine in Table 1 and a negative control group that was not inoculated.

Target animal safety evaluation laboratory animal test group inoculation route Inoculation amount (ml) Injection site side effects and clinical symptoms dead head/
public announcement cat control group - - doesn't exist 0/2 Inoculation group abdominal cavity 2 (oligarch) doesn't exist 0/3

As a result, the safety of the vaccine composition of the present invention was confirmed in cats as well as experimental animals.

Immunogenicity Assessment Following Administration of COVID-19 Vaccine Compositions in Cats

In the present invention, when the COVID-19 vaccine composition additionally containing Montanide gel as an adjuvant of the present invention was administered to a cat, which is a target animal, it was attempted to confirm the immunogenicity and the degree of determination of antigen amount.

Immunogenicity (long-term immunogenicity) evaluation group of the test vaccine in cats division public announcement effective antigen Content (μg/dose) immune booster preservative Experimental Example 1 2 S1 10 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 10 Experimental Example 2 2 S1 30 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 30 Experimental Example 3 2 S1 60 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 60 Comparative Example 1 2 S1 30 Rehydra gel (10%) Thimerosal
(0.01%) RBD 30 control group 2 PBS - - -

As shown in Table 4, the test vaccine was administered to each individual, and boosting was performed 3 weeks after inoculation. Before vaccination, 3 weeks after vaccination (before Boosting), 5 weeks after vaccination (2 weeks after Boosting), 9 weeks after vaccination (6 weeks after Boosting), 13 weeks after vaccination (10 weeks after Boosting) and 21 weeks after vaccination (After 18 weeks of boosting) After blood was collected, serum was separated and tested for neutralizing antibodies against COVID-19 to evaluate long-term immunogenicity.

Long-term immunogenicity evaluation Montanide gel PR02 (10%) Rehydra gel (10%) No adjuvant Experimental Example 1 Experimental Example 2 Experimental Example 3 comparative example control group One 2 One 2 One 2 One 2 One 2 Before inoculation 0 0 0 0 0 0 0 0 0 0 3 weeks after inoculation
(Before Boosting) 0 0 0 0 0 0 0 0 0 0 5 weeks after inoculation
(2 weeks after boosting) 20 80 640 80 160 640 80 10 0 0 9 weeks after inoculation
(After 6 weeks of Boosting) 20 80 320 320 640 640 20 40 0 0 13 weeks after inoculation
(After 10 weeks of Boosting) 10 80 340 320 640 160 10 80 0 0 21 weeks after inoculation
(After 18 weeks of Boosting) 0 80 160 80 160 80 0 20 0 0

As shown in Table 5, compared to Comparative Example in which Rehydra gel was added as an immune enhancer, the vaccine composition in which Montanide gel was added as an immune enhancer of the present invention, especially Experimental Example 2 and Experimental Example 3 In the case of , it was confirmed that long-term immunogenicity was maintained high.

About the COVID-19 virus mutation Confirmation of cross-protective ability of COVID-19 vaccine composition

In the present invention, it was attempted to confirm whether the vaccine composition of the present invention maintains immunogenicity even against COVID-19 virus mutation.

The COVID-19 vaccine composition to which Rehydra gel was added as an immune enhancer and the COVID-19 vaccine composition to which Montanide gel was added were administered to cats (3 to 5 months old, female). Inoculated subcutaneously twice at 3-week intervals, and blood was collected as shown in Table 6. Serum was separated from the collected blood samples, and neutralizing antibodies were tested using each mutant strain.

Evaluation of protective ability through challenge inoculation in cats Montanide gel PR02 (10%) Rehydra gel (10%) Experimental Example 2 (30 μg/dose) Experimental Example 3 (60 μg/dose) Comparative Example (30 μg/dose) One 2 One 2 One 2 Wuhan south africa uk Wuhan south africa uk Wuhan south africa uk Wuhan south africa uk Wuhan south africa uk Wuhan south africa uk Before inoculation 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 weeks after inoculation
(Before Boosting) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 5 weeks after inoculation
(2 weeks after boosting) 1280 640 320 640 320 160 640 160 80 1280 160 320 160 80 80 10 20 10 9 weeks after inoculation
(After 6 weeks of Boosting) 1280 640 320 640 320 160 1280 320 160 640 40 160 40 20 10 40 160 20

As a result, as shown in Table 6, the comparative example in which Rehydra gel was added as an immunostimulant showed low immunogenicity to the South African mutant virus and the British mutant virus. On the other hand, it was confirmed that the COVID-19 vaccine composition to which Montanide gel was added as an immune enhancer of the present invention showed high immunogenicity against the South African mutant virus and the British mutant virus.

Immunogenicity Assessment Following Administration of COVID-19 Vaccine Compositions in Dogs

5-1: Vaccination

It was intended to confirm whether immunogenicity against the COVID-19 virus is formed in dogs (beagles) by the COVID-19 vaccine composition of the present invention.

As shown in Table 7 below, the experiment was conducted by dividing the groups into Experimental Example 3 of Example 3 and the control group.

Immunogenicity evaluation group of test vaccine in dogs division public announcement effective antigen content
(μg/dose) immune booster preservative Experimental Example 3 3 S1 60 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 60 control group 3 PBS - - -

First, 6 healthy beagle dogs with COVID-19 antibody negative were prepared, and the test vaccine was dosed subcutaneously according to the instructions, and additional vaccination was performed 3 weeks later (hair removal was administered to the vaccination area to observe abnormalities in the vaccination area) .

5-2: Clinical Observation

Weight gain was checked for 5 weeks after the first vaccination, and rectal body temperature was measured before vaccination, 1, 2, and 3 days after vaccination, and 2 weeks, 3 weeks, 4 weeks, and 5 weeks after vaccination. In addition, to observe side effects at the injection site, side effects such as inflammatory reaction, granuloma or purulent formation at the injection site were checked for 5 weeks after the first inoculation.

Observe weight change 1st vaccine day 0 1st vaccine day 1 1st vaccine day 2 1st vaccine day 3 Day 7 of the 1st vaccine Day 14 of the 1st vaccine 2nd vaccine day 0
(21) 2nd vaccine day 1
(22) 2nd vaccine day 2
(23) 2nd vaccine day 3
(24) 2nd vaccine day 7
(28) 2nd vaccine Day 14
(35) Experimental Example 3 One 5.15 5.15 5.2 5.2 5.25 5.5 5.95 5.85 5.95 5.95 5.85 6.3 2 4.95 5.05 4.95 5.15 5.1 5.35 5.4 5.4 5.75 5.7 5.55 5.7 3 4.9 5.1 5.0 5.2 5.1 5.35 5.65 5.45 5.15 5.2 5.7 5.85 control group One 5.45 5.45 5.45 5.35 5.5 5.55 5.7 5.8 5.95 5.95 5.95 6.05 2 5.25 5.35 5.35 5.3 5.3 5.35 5.65 5.6 5.7 5.6 5.9 6.05 3 5.10 5.15 5.15 5.2 5.25 5.4 5.8 5.7 5.9 5.85 5.95 6.25

Observation of body temperature changes 1st vaccine day 0 1st vaccine day 1 1st vaccine day 2 1st vaccine day 3 Day 7 of the 1st vaccine 1st vaccine day 14 2nd vaccine day 0
(21) 2nd vaccine day 1
(22) 2nd vaccine day 2
(23) 2nd vaccine day 3
(24) 2nd vaccine day 7
(28) 2nd vaccine Day 14
(35) Experimental Example 3 One 38.4 38.1 38.2 38.4 38.7 38.6 38.5 38.6 38.3 38.6 38.8 38.7 2 38.4 38.2 37.8 38.0 38.2 38.3 38.1 38.4 38.2 38.5 38.8 39.0 3 38.4 38.5 38.4 38.6 38.9 38.6 38.9 39.0 38.9 38.4 39.0 39.3 control group One 38.1 38.0 37.9 38.5 38.4 38.5 38.5 39.3 38.8 38.4 38.8 38.8 2 38.6 38.4 38.7 38.8 38.9 38.6 39.2 38.3 38.8 39.0 39.0 39.4 3 38.4 38.1 38.5 38.6 38.9 38.7 39.0 38.5 39.0 38.8 39.2 38.8

Observation of side effects - Check for lethargy and anaphylaxis 1st vaccine day 0 1st vaccine day 1 1st vaccine day 2 1st vaccine day 3 Day 7 of the 1st vaccine Day 14 of the 1st vaccine 2nd vaccine day 0
(21) 2nd vaccine day 1
(22) 2nd vaccine day 2
(23) 2nd vaccine day 3
(24) 2nd vaccine day 7
(28) 2nd vaccine Day 14
(35) Experimental Example 3 One X X X X X X X X X X X X 2 X X X X X loss of appetite loss of appetite loss of appetite loss of appetite X X X 3 X X X X X X X X X X X X control group One X X X X X X X X X X X X 2 X X loss of appetite X X X X X X X X X 3 X X loss of appetite X X X X X X X X X

Observation of side effects - Check for swelling, redness, and granuloma at the injection site 1st vaccine day 0 1st vaccine day 1 1st vaccine day 2 1st vaccine day 3 Day 7 of the 1st vaccine Day 14 of the 1st vaccine 2nd vaccine day 0
(21) 2nd vaccine day 1
(22) 2nd vaccine day 2
(23) 2nd vaccine day 3
(24) 2nd vaccine day 7
(28) 2nd vaccine Day 14
(35) Experimental Example 3 One X X X X X X X X X X X X 2 X X X X X X X X X X X X 3 swelling X swelling X X X X X X X X X control group One X X X X X X X X X X X X 2 X X X X X X X X X X X X 3 X X X X X X X X X X X X

Experimental Example 3 Both the administration group and the control group showed normal weight gain (Table 8), and it was confirmed that normal body temperature was maintained without side effects (Table 9). In addition, mild lethargy of the degree of anorexia was observed, but it was evaluated as temporary and insignificant, and although swelling and redness at the injection site occurred, it was evaluated as temporary and insignificant (Table 10 and Table 11).

5-3: Evaluation of antibody formation ability

In order to confirm the immunogenicity of the COVID-19 vaccine composition of the present invention, serum was separated from the blood samples and neutralizing antibodies against COVID-19 were tested to evaluate the immunogenicity.

Blood collection was performed while the dogs were safely calibrated, and 2 to 3 ml of blood was collected before vaccination, 3 weeks after the first vaccination, and 2 weeks after the second vaccination.

The neutralizing antibody test was performed with a virus titer of 3.5 TCID ₅₀ / ml, and the detection limit was performed at a dilution factor of 10.

Confirmation of antibody formation ability DAY
(repeat 2) 0 0 0
average 21 21 21
average 35 35 35
average Experimental Example 3 One 10 10 10 10 10 10 320 320 320 2 10 10 10 10 10 10 160 160 160 3 10 10 10 10 10 10 640 640 640 control group One 10 10 10 10 10 10 10 10 10 2 10 10 10 10 10 10 10 10 10 3 10 10 10 10 10 10 10 10 10

As shown in Table 12, antibody formation was not observed in the control group, and it was confirmed that the group administered with the vaccine composition (Experimental Example 3) to which Montanide gel was added as an immune enhancer of the present invention showed a high antibody formation rate.

Confirmation of induction of T cell activity according to COVID-19 vaccine composition administration

6-1: Confirmation of induction of T cell activity

In the present invention, it was attempted to confirm whether in vivo T cell activity is induced by the administration of the COVID-19 vaccine composition.

A mouse was used as the experimental animal, and the experiment was conducted by dividing the groups like Experimental Example 3 and the control group of Example 3 as shown in Table 13 below.

T cell activity induction evaluation group of test vaccine in mice division public announcement effective antigen content
(μg/dose) immune booster preservative Experimental Example 3 10 S1 60 Montanide gel PR02 (10%) Thimerosal
(0.01%) RBD 60 control group 4 PBS - - -

Specifically, the COVID-19 vaccine and control group of Experimental Example 3 were administered to 7-week-old mice (C57BL/6 female) twice at 2-week intervals, and 2 weeks after the last vaccine administration, mouse spleen cells were isolated and SARS- CoV-2 S1 recombinant antigen protein and RBD recombinant antigen protein were reprocessed (10 μg/ml or 20 μg/ml antigen, respectively). Next, the isolated splenocytes were treated with fluorescently labeled CD4, IL-5, and IL-17 antibodies, and then the activity of CD4 ⁺ T cells was measured using a flow cytometer.

As a result, as shown in FIG. 6, the COVID-19 vaccine composition in which the SARS-CoV-2 S1 recombinant antigen protein and RBD recombinant antigen protein of the present invention and Montanide gel as an immune enhancer were added showed CD4 ⁺ IL-5 ⁺ T It was confirmed that the cells (T helper 2) and CD4 ⁺ IL-17 ⁺ T cells (T helper 17) were effectively induced.

Since the activity of CD4 ⁺ T cells is involved in the production of specific antibodies to antigens and the enhancement of mucosal immunity, the vaccine composition of the present invention means that it can effectively increase the efficacy of immune activity against the COVID-19 virus.

6-2: Confirmation of induction of cytokine production

In the present invention, it was attempted to confirm whether in vivo cytokine production is induced by the administration of the COVID-19 vaccine composition.

A supernatant of the mouse splenocytes isolated in Example 6-1 was obtained, and the levels of IFN-γ and IL-5 production were confirmed through ELISA analysis.

As a result, as shown in FIG. 7, the COVID-19 vaccine composition to which the SARS-CoV-2 S1 recombinant antigen protein and RBD recombinant antigen protein of the present invention and Montanide gel as an immune enhancer were added showed IFN-γ and IL-5 It was confirmed that the production was effectively induced.

<110> CTC VAC Co.,Ltd <120> COVID-19 vaccine composition with increased immunogenicity <130> PDPC214659 <150> KR 10-2021-0181112 <151> 2021-12-16 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 675 <212> PRT <213> artificial sequence <220> <223> SARS-CoV-2 spike 1 <400> 1 Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser 1 5 10 15 Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val 20 25 30 Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr 35 40 45 Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe 50 55 60 Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr 65 70 75 80 Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp 85 90 95 Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val 100 105 110 Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val 115 120 125 Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val 130 135 140 Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe 145 150 155 160 Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu 165 170 175 Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His 180 185 190 Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu 195 200 205 Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln 210 215 220 Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser 225 230 235 240 Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln 245 250 255 Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp 260 265 270 Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu 275 280 285 Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg 290 295 300 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 305 310 315 320 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 325 330 335 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 340 345 350 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 355 360 365 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 370 375 380 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 385 390 395 400 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 405 410 415 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 420 425 430 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 435 440 445 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 450 455 460 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 465 470 475 480 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 485 490 495 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 500 505 510 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe 515 520 525 Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe 530 535 540 Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala 545 550 555 560 Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser 565 570 575 Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln 580 585 590 Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala 595 600 605 Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly 610 615 620 Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His 625 630 635 640 Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys 645 650 655 Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala His His His 660 665 670 His His His 675 <210> 2 <211> 229 <212> PRT <213> artificial sequence <220> <223> SARS-CoV-2 RBD <400> 2 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe His 210 215 220 His His His His 225 <210> 3 <211> 2103 <212> DNA <213> artificial sequence <220> <223> SARS-CoV-2 Spike 1 <400> 3 aagcttgcca ccatggagtt tgggctgagc tgggttttcc tcgttgctct ttttagaggt 60 gtccagtgtg tgaacctgac caccagaaca cagctgcctc cagcctacac caacagcttt 120 accagaggcg tgtactaccc cgacaaggtg ttcagatcct ccgtgctgca cagcacccag 180 gacctgtttc tgcccttctt cagcaacgtg acctggttcc acgccatcca cgtgtccggc 240 accaatggca ccaagagatt cgacaacccc gtgctgcctt tcaacgacgg ggtgtacttt 300 gcctccaccg agaagtccaa catcatcaga ggctggatct tcggcaccac actggacagc 360 aagacccaga gcctgctgat cgtgaacaac gccaccaacg tggtcatcaa agtgtgcgag 420 ttccagttct gcaacgaccc cttcctgggc gtctactacc acaagaacaa caagtcctgg 480 atggaatccg agttccgggt gtactcctcc gccaacaact gcaccttcga gtacgtgtcc 540 cagcctttcc tgatggacct ggaaggcaag cagggcaact tcaagaacct gcgcgagttc 600 gtgttcaaga acatcgacgg ctacttcaag atctactcca agcacacccc tatcaacctc 660 gtgcgggatc tgcctcaggg cttctctgct ctggaacctc tggtggatct gcccatcggc 720 atcaaca cccggtttca gaccctgctg gccctgcaca gatctttct gacccctggc 780 gactcctctt ctggatggac tgctggcgct gctgcttact acgtgggcta tctgcagccc 840 agaaccttcc tgctgaagta caacgagaac ggcaccatca ccgacgccgt ggattgtgct 900 cttgaccctc tgtccgagac aaagtgcacc ctgaagtcct tcaccgtgga aaagggcatc 960 taccagacct ccaacttccg ggtgcagcct acagagtcta tcgtgcggtt ccctaacatc 1020 accaacctgt gtcctttcgg cgaggtgttc aatgccacca gattcgcctc tgtgtacgcc 1080 tggaaccgga agcggatctc taactgcgtg gccgactaca gcgtgctgta taactccgcc 1140 tccttcagca ccttcaagtg ctatggcgtg tcccctacca agctgaacga cctgtgcttt 1200 accaacgtgt acgccgactc tttcgtgatc agaggcgacg aagtgcggca gatcgctcct 1260 ggacagaccg gcaagatcgc cgattacaac tacaagctgc ccgacgactt caccggctgc 1320 gtgatcgcct ggaatagcaa caacctggac tccaaagtcg gcggcaacta caactacctg 1380 taccggctgt tccggaagtc taacctgaag cctttcgagc gggacatcag caccgagatc 1440 taccaggctg gctctacccc ttgtaacggc gtggaaggct tcaactgcta cttcccactg 1500 cagtcctacg gctttcagcc taccaatggc gtgggctacc agccttatag agtggtggtg 1560 ctgtccttcg agctgctgca tgctcctgct accgtgtgcg gccctaagaa gtctaccaac 1620 ctggtcaaga acaaatgcgt gaacttcaac ttcaacggcc tgaccggcac aggcgtgctg 1680 accgagtcta acaagaagtt cctgcctttc cagcagttcg gccgggatat cgctgatacc 1740 accgatgccg tcagagatcc ccagacactg gaaatcctgg acatcacccc ttgttccttt 1800 ggcggcgtgt ccgtgatcac ccctggcacc aatacttcta accaggtggc cgtgctgtac 1860 caggacgtga actgtactga ggtgccagtg gctatccacg ccgatcagct gacacccact 1920 tggagagtgt actccaccgg ctccaacgtg ttccagacaa gagctggctg tctgatcgga 1980 gccgagcacg tgaacaattc ctacgagtgc gacatcccca tcggcgctgg catctgtgcc 2040 tctttatcaga cccagaccaa ctctccccgt cgtgctcacc atcaccacca ccattgagga 2100 tcc 2103 <210> 4 <211> 747 <212> DNA <213> artificial sequence <220> <223> SARS-CoV-2 RBD <400> 4 atggagtttg ggctgagctg ggttttcctc gttgctcttt ttagaggtgt ccagtgtcgg 60 gtgcagccta cagagtctat cgtgcggttc cctaacatca ccaacctgtg tcctttcggc 120 gaggtgttca atgccaccag attcgcctct gtgtacgcct ggaaccggaa gcggatctct 180 aactgcgtgg ccgactacag cgtgctgtat aactccgcct ccttcagcac cttcaagtgc 240 tatggcgtgt cccctaccaa gctgaacgac ctgtgcttta ccaacgtgta cgccgactct 300 ttcgtgatca gaggcgacga agtgcggcag atcgctcctg gacagaccgg caagatcgcc 360 gattacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg gatagcaac 420 aacctggact ccaaagtcgg cggcaactac aactacctgt accggctgtt ccggaagtct 480 aacctgaagc ctttcgagcg ggacatcagc accgagatct accaggctgg ctctacccct 540 tgtaacggcg tggaaggctt caactgctac ttccccactgc agtcctacgg ctttcagcct 600 accaatggcg tgggctacca gccttataga gtggtggtgc tgtccttcga gctgctgcat 660 gctcctgcta ccgtgtgcgg ccctaagaag tctaccaacc tggtcaagaa caaatgcgtg 720 aacttccacc atcaccacca ccattga 747

Claims (3)

an antigen composition comprising spike 1 recombinant antigen protein and receptor binding domain (RBD) recombinant antigen protein of SARS-CoV-2 virus; and
A COVID-19 vaccine composition with increased immunogenicity, comprising Montanide Gel as an adjuvant.
A pharmaceutical composition for preventing or treating COVID-19 comprising the COVID-19 vaccine composition having increased immunogenicity according to claim 1.
A method for preventing COVID-19 comprising administering the COVID-19 vaccine composition having increased immunogenicity according to claim 1 to a non-human subject.

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