WO2022030702A1 - Vaccine composition for preventing and treating middle east respiratory syndrome-coronavirus - Google Patents

Abstract

The present invention relates to a method for producing an antigen for preventing MERS-CoV infections and a vaccine composition produced by method, more specifically to a vaccine composition comprising a protein in which n-Coronavirus spike protein or S1 domain is bound with human fragment crystallization (Fc) domain, and to inducing immune responses and preventing viral infections by means of the vaccine composition.

Description

Vaccine composition for prevention and treatment of Middle East respiratory syndrome coronavirus

The present invention relates to a method for preparing an antigen for preventing MERS-CoV infection and a vaccine composition using the same.

Middle East Respiratory Syndrome is an infectious disease caused by Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection. Middle East Respiratory Syndrome (Middle East Respiratory Syndrome) is a bat-derived beta-coronavirus, a viral disease that infects humans through camels and enables human-to-human transmission. The fatality rate due to MERS-CoV infection is over 35% (WHO statistics 27%), and the main symptoms are fever, cough, sputum, shortness of breath, muscle pain, diarrhea and abdominal pain. artificial respiration was required. The average incubation period was 5.5 days, and the incubation period of 1.9 to 14.7 days was more than 95% of the total infections. The main cause of death is pneumonia leading to acute respiratory distress syndrome, and kidney failure, disseminated vascular coagulation, and phlegmon have been reported in some cases. From the first case of infection on May 20, 2015 in Korea to July 16 of the same year, 186 people were infected with MERS-CoV, and 38 of them died. Even recently, it was reported that around 50,000 people are infected every year, mainly in Saudi Arabia in the Middle East, and 200 people die. As a result of analyzing the genome of MERS-CoV first isolated in 2012, it is a beta-coronavirus belonging to the same genus as the severe acute respiratory syndrome coronavirus (SARS-CoV). It has an RNA genome with a size of 30 kb. It was found to have the largest RNA genome of any virus. The genome of MERS-CoV consists of 10 frequent reading frames (ORFs), and 2/3 of the entire genome is ORF1ab, which is a gene of 16 non-structural proteins (NSP). The remaining 3' end gene has four structural protein genes, including spike (S), envelope (E), matrix (M) and nucleocapsid (N) protein genes. In addition, it has five accessory protein genes (ORF3, ORF4b, ORF5, ORF8b). It is speculated that the helper protein is related to viral infection and immune response rather than playing a role in genome replication or viral packaging of MERS-CoV. The helper protein is known to be involved in B cell activation by increasing the expression of NF-κB, which is mainly involved in the immune mechanism of the host cell.

The spike protein of MERS-CoV has a spike protein required for cell invasion of each virus on the surface of the envelope, such as SARS-CoV, infectious bronchitis coronavirus (IBV), and porcine epidemic diarrhea virus (PEDV). The spike protein is composed of the S1 domain that binds to the host cell receptor and the S2 domain that binds the cell membrane. During intracellular infection of the virus, the furin cleavage site is cut between the S1 and S2 domains and is separated into two domains. In the case of SARS-CoV, the spike protein binds to angiotensin converting enzyme 2 (ACE2) expressed on the surface of the host cell to cause intracellular infection. In the case of MERS-CoV, the spike protein binds to dipeptidyl peptidase 4 (DPP4) and infects the cell. this happens Two major domains of the S1 protein of coronavirus are S1, N-terminal domain (S1-NTD) and C-terminal domain (S1-CTD), which are known to play an important role in cell receptor binding, and the S1-NTD domain is mainly It is required for cell surface glycosylation and the S1-CTD domain is involved in ACE2, APN, and DPP4 binding. Therefore, a vaccine using the S1 domain of the Spike protein can be used as a very useful protein for the formation of virus neutralization (VN) antibodies that can prevent intracellular infection of viruses.

A treatment or vaccine for MERS-CoV has not yet been developed, but vaccine development using the receptor binding domain (RBD) of the spike protein as an antigen is underway. Various vaccine formulations such as vaccine development using viral vectors as antigen-expressing recombinant viruses, antigen genes or antigen carriers, and DNA vaccines are being studied. Among them, a subunit vaccine using a Spike protein using recombinant animal cells or a viromolecule capable of improving immunity and some antigenic sites is being developed. Monomeric vaccine development using recombinant animal cells has been used as a major protein expression system for antibody drug development and monomeric vaccine development since the 1980s. The biggest disadvantage of the unit vaccine using a recombinant protein is that it requires an immune enhancing substance such as an adjuvant due to its weak immunogenicity. Therefore, it is very important to find an antigen essential for the formation of a neutralizing antibody for suppressing viral infection, to express it as a recombinant protein without structural change, and to develop a safe and effective vaccine antigen manufacturing technology using an immune enhancing material.

Although MERS disease has recently been limited to the Middle East, it is a disease that has the potential to spread to the world again. Therefore, it is necessary to develop a vaccine that is safe and effective.

The present invention has completed the present invention by confirming the immune and therapeutic response to the Middle East Respiratory Syndrome coronavirus of the Middle East Respiratory Syndrome coronavirus spike protein or a protein combining the S1 region and the human Fc (fragment crystallisation) region.

Accordingly, an object of the present invention is to provide a technology for developing a subunit vaccine using a novel Middle East Respiratory Syndrome coronavirus spike protein.

In order to achieve the object of the present invention as described above, the present invention provides a gene encoding the MERS-CoV spike protein represented by the nucleotide sequence of SEQ ID NO: 1 or 2 and human IgG4 represented by the nucleotide sequence of SEQ ID NO: 3 or 4 A nucleic acid in which a gene encoding Fc is fused is provided.

In addition, the present invention provides a recombinant expression vector comprising the nucleic acid according to the present invention.

In addition, the present invention provides a cell line transformed with the recombinant expression vector according to the present invention.

In one embodiment of the present invention, the cell line may be a Chinese hamster ovary cell LV-MS1-Fc cell (KCTC14342BP).

In addition, the present invention provides a recombinant protein produced from the cell line according to the present invention.

In addition, the present invention comprises the steps of transforming a cell with the recombinant expression vector according to the present invention; recovering the recombinant protein from the cell or culture thereof; And it provides a method for producing a recombinant protein, comprising the step of purifying the recombinant protein.

In addition, the present invention provides a vaccine composition for preventing or treating Middle East respiratory syndrome coronavirus comprising the combination protein according to the present invention as an active ingredient.

In one embodiment of the present invention, the vaccine may be an inactivated vaccine.

In one embodiment of the present invention, the vaccine may further include an immune enhancing material or adjuvant.

In addition, the present invention provides a method for preventing or treating Middle East respiratory syndrome coronavirus infection comprising administering the vaccine composition to an individual.

In addition, the present invention provides a method for evaluating an immune response in an animal, comprising administering the recombinant expression vector or the recombinant protein to the animal.

In one embodiment of the present invention, the method can evaluate the immune response by measuring the IgG antibody titer from the animal's serum.

The use of the MERS LV-MS1-Fc antigen containing the spike protein of the Middle East Respiratory Syndrome coronavirus of the present invention can be usefully used as a vaccine for prevention and improvement of inducing an immune response that can fight MERS-CoV infection. .

1 illustrates the structure of a recombinant expression vector.

2 illustrates a genetic schematic diagram of a recombinantly expressed protein.

3 shows the result of confirming the expression of MERS-CoV S recombinant protein (LV-MS1-Fc) by Western blot method.

4 depicts MERS-CoV S protein specific antibody production and ELISA titers.

5 shows neutralizing antibody titers against MERS-CoV.

Figure 6 depicts a mouse model for the evaluation of vaccine efficacy.

7 shows the viral titers of the right lung of mice after challenge inoculation test.

8 shows the histopathological H&E staining results of the left lung of a mouse after the challenge inoculation test.

Figure 9 depicts body weight, food intake, water intake and anatomical observations of mice in the vaccinated group.

In the present invention, the spike protein gene is a coronavirus gene having DPP4 binding ability, preferably a spike protein gene of the Middle East Respiratory Syndrome coronavirus consisting of the nucleotide sequence shown in SEQ ID NO: 1, and the 2015 Korean MERS-CoV It is a spike protein gene established based on genetic information (GeneBank accession No. KT029139, Middle East respiratory syndrome coronavirus isolate MERS-CoV/KOR/KNIH/002 05 2015). More preferably, it is a gene including the S1-CTD gene including the receptor binding domain (RBD) among the spike protein genes. More preferably, it is a gene comprising the spike amino acid residues 19 to 647 of the Middle East respiratory syndrome coronavirus represented by SEQ ID NO: 2. More preferably, it is a nucleotide sequence that has undergone codon optimization in mammalian cells. In addition, the gene of the Fc protein is a gene of the Fc region of human Immunoglobulin G represented by SEQ ID NO: 3. Preferably, it is an Fc gene of subtype IgG4 of Immunoglobulin G. More preferably, it is a gene encoding the 238th to 457th amino acid sequence of the amino acid sequence represented by SEQ ID NO: 4. The gene encoding the Spike protein 19-647 amino acid sequence is artificially synthesized using a gene synthesis device, or an oligonucleotide primer (oligo nucleotide primer) and can be produced using the PCR method. On the other hand, since there may be differences from the spike gene of the Middle East Respiratory Syndrome coronavirus due to codon-optimization according to the expression system, the recombinant spike LV-MS1-Fc gene is the spike amino acid residue 19 of the Middle East Respiratory Syndrome coronavirus. It may exist in the form of various nucleotide sequences including 647.

For the LV-MS1-Fc gene synthesized through codon optimization, a recombinant protein expression system was used for protein production using the optimal protein expression system. In addition, the gene includes the Fc domain of human IgG. The recombinant protein expression system should be easy to produce while maintaining the immunogenicity of the vaccine antigen, and it should be of a low risk factor to the human body. In addition, it should be selected in consideration of various factors such as the ease of production method and production cost. The host cell of the present invention may be a prokaryotic or eukaryotic cell having a high DNA introduction rate and a high expression rate of the introduced DNA. For example, eukaryotic and prokaryotic hosts such as, but not limited to, Escherichia coli, mammalian cell lines, insect cell lines, fungi, and yeast can be used. As for the recombinant protein expression system, four methods are mainly used. Mammalian cells, insect cells, yeast. And bacterial systems are being used. Each recombinant protein expression system has advantages and disadvantages, and depending on the type of protein, the optimal system should be considered as an optimal system. If posttranslational modification affects the protein properties, the expression system should be selected. The LV-MS1-Fc antigen protein expression system may specifically use E. coli. The E. coli expression system is a system capable of mass production at low cost by easily introducing protein genes. However, there is a risk of structural denaturation or, in severe cases, the formation of insoluble precipitates (inclusion bodies) due to misfolding during expression fixation of viral proteins. In addition, there is a difficult problem in the process of removing lipopolysaccharide (LPS) derived from E. coli cells. A second recombinant baculovirus can also be used to produce protein antigens using the insect cell Spodoptera frugiperda (Sf) cell line. It is a method widely used for viral proteins and has the advantage of good expression, but has the disadvantage that recombinant virus seeds and cell lines must be prepared simultaneously according to GMP regulations. Among the expression systems, the most used and verified (or commercialized product) recombinant protein expression system is a protein expression system using recombinant animal cells. It is a method to create a recombinant cell line by inserting a protein gene into a specific expression vector and transfecting the cell. The mainly used cell lines are 293, 293T, BHK21, CHO, NS0 and Sp2 cell lines. Preferably, a recombinant cell line is prepared using a BHK21 or CHO cell line among cell lines, and more preferably, a recombinant cell line is prepared using a CHO cell line.

MERS-CoV recombinant expression vector can be prepared by cloning into pcDNA3.4 TOPO expression vector having CMV promoter for expression in CHO cell line. Finally, various signal peptide genes for secretion out of cells after protein synthesis can be introduced at the 5' end of the recombinant protein LV-S1SheFc gene. Preferably, NP-000468, AAA52897, AAA59018, NP-001691, NP-001890, AAC32752, BR14604, AAS93426, Alfa-galactosidase (mutant m3), CAA03658, Q766C3, ABF74624, 2205370A, AAM54023, which are known as signal peptides can be introduced More preferably, the signal peptide gp64 may be introduced. In introducing the Fc gene, a linker peptide can be inserted between the spike protein gene and the Fc gene. Linker peptides can be designed by various amino acid combinations. In the present invention, a linker peptide designed to prevent structural changes between protein domains was introduced. Preferably, it is designed as an amino acid sequence that is unlikely to affect the protein structure and is composed of cysteine, glycine, proline, and alanine. More preferably, a linker made of the GlyGlyGlyProGLyGlyGly amino acid sequence may be introduced.

The gene structure of the entire expression vector is shown in FIG. 1 .

Conventional transformation methods may be used for transformation into recombinant protein-expressing cells. The transformation method includes an electroporation method and a heat shock method, and transformation is preferably performed using a heat shock method.

In the present invention, the recombinant expression vector means that a foreign gene can be introduced to express a recombinant spike gene consisting of the nucleotide sequence shown in SEQ ID NO: 1 and can operate protein synthesis. It can be a plasmid or a potential genomic insertion. A variety of expression vectors are commercially available that effectively induce the expression of a foreign gene in a particular host cell. In the present invention, pcDNA3.4 TOPO vector was used. The recombinant spike gene LV-MS1-Fc of the present invention was operatively linked to the promoter region of the expression vector to obtain a recombinant expression vector. The host cell that can be transformed with this vector is preferably a mammalian cell, and the pcDNA3.4 TOPO vector was transfected into ExpiCHO-S cells derived from the mammalian Chinese Hamster Ovary to produce a protein with a high protein expression rate. to provide. The cell line is LV-MS1-Fc (Accession No.: KCTC 14342BP).

Next, a method for culturing the recombinant cell line Chinese hamster ovary cell LV-MS1-Fc (recombinant cell line) and the expression of the spike protein LV-MS1-Fc (recombinant antigen) produced during the culture will be described.

The present invention also provides a recombinant spike protein LV-MS1-Fc of Middle East Respiratory Syndrome coronavirus produced by the cell line.

The recombinant spike protein LV-MS1-Fc includes a recombinant spike protein of Middle East Respiratory Syndrome coronavirus having an amino acid sequence represented by SEQ ID NO: 5 or a material functionally equivalent thereto. The "functionally equivalent substance" means at least 70% or more, preferably 80% or more, more preferably 90% or more, and most preferably the amino acid sequence represented by SEQ ID NO: 5 as a result of amino acid substitution, deletion and addition. is 95% or more of sequence homology, and refers to a protein that exhibits substantially the same physiological activity as the protein having the amino acid sequence shown in SEQ ID NO: 5. In addition, the human IgG4 Fc protein is linked to the S1Sh2 antigen through a peptide bond through an amino acid linker. The IgG4 Fc protein consists of a sequence that is preferably 80% or more, more preferably 90% or more, and most preferably 95% or more identical to the amino acid sequence of a normal human IgG4 Fc protein. Finally, it refers to the amino acid sequence from 99 to 326 of the IgG4 Fc (GeneBank accession No. AAB59394.1, immunoglobulin gamma-4 heavy chain, partial [Homo sapiens]) domain.

In the present invention, the recombinant spike protein LV-MS1-Fc can be isolated using a conventional protein isolation method. For example, the recombinant spike protein LV-MS1-Fc can be isolated from other cultures according to a variety of methods including, but not limited to, centrifugation, filtration, and precipitation. Recombinant spike protein LV-MS1-Fc can be separated and precipitated from other cultures according to the fractional solubility of various salts. The precipitation of a specific protein is the concentration of the protein to be separated from the culture medium, or the most optimal chemical and the chemical used to create a condition in which the protein to be separated is maximally dissolved or other proteins are precipitated. It depends on the optimum concentration. The type of chemical used may include ammonium sulfate, acid for isoelectric precipitation, addition of a base, ethanol, polyethylene glycol, polyvalently charged ions, and the like. For example, ammonium sulfate can be used.

After harvesting the recombinant cell line culture, purification is performed by centrifugation. Purification may be performed by centrifugation or micro filtration. Centrifugation conditions were performed at 10,000 g, 10 minutes, and 4° C. so that the supernatant could be used for the next purification process. In the case of the microfiltration method, a filter having a pore size between 1.0 μm and 0.2 μm may be used. Preferably, a filter having a pore size of 0.2 μm may be used. As the filtration method, a dead end filtration method or a cross flow filtration method can be used, and both methods are applicable. Centrifugation supernatant or filtrate (permeate) can be separated from other proteins through precipitation. The ammonium sulfate concentration used may be 30-90%. Preferably, it may be 40 to 80%. More preferably, it may be 50 to 70%. Most preferably, it may be 70%. Ammonium sulfate precipitation can be left at 4° C. for 1 to 8 hours, preferably 2 to 7 hours, more preferably 3 to 6 hours, more preferably 4 to 5 hours until the supernatant and the precipitate are sufficiently separated. have. Most preferably, it can be left for 5 hours. Centrifuge (4,500g, 30 minutes, 4℃) to separate the precipitate containing the precipitated recombinant antigen, discard the supernatant, and resuspend the precipitate with 5 mL of 20 mM sodium phosphate buffer (pH 7.4). After that, it can be dialyzed against 20 mM sodium phosphate buffer containing 0.15 M NaCl in NMWCO 20 kDa pore size dialysis bag at 4°C. Dialysis is stirred using a magnetic bar for 3 to 4 hours. Dialysis can be performed using a total of 1L buffer, and after dialysis, for the next chromatography purification process, known methods including extraction through chromatography (affinity, ion exchange, size exclusion, hydrophobicity) or ultrafiltration method It can be purified through

The present invention comprises the steps of transforming the recombinant expression vector into a cell; recovering the recombinant protein from the cell or culture thereof; and purifying the recombinant protein; provides a method for producing a recombinant protein, including.

Since the method of the present invention includes the above-described recombinant expression vector and recombinant protein, the description of the content overlapping with the vector and protein of the present invention is omitted in order to avoid excessive complexity of the present specification due to the description of the overlapped content. do.

The present invention provides a vaccine composition for preventing or treating Middle East respiratory syndrome coronavirus comprising a recombinant protein as an active ingredient.

In addition, the vaccine may be an inactivated vaccine.

The inactivated vaccine composition may be a fusion protein consisting of a protein among the spike proteins of the purified Middle East Respiratory Syndrome virus and a human IgG Fc protein and an appropriate immune activation agent. A vaccine comprising the composition may be prepared by a method known in the art. For example, after obtaining purified antigens, they are treated with formalin, betapropriolactone (BPL), binary ethylenimine (BEI) or gamma rays, or inactivated by other methods known to those skilled in the art. The inactivated virus is then mixed with a pharmaceutically acceptable carrier (eg saline solution) and optional adjuvants. Preferably, it may be inactivated at a final concentration of 0.1% formalin.

In addition, the vaccine may further include an immune enhancing material or an adjuvant.

Immunopotentiator or adjuvant refers to a compound or mixture that enhances the immune response and promotes the rate of absorption after inoculation, including, but not limited to, any absorption-promoting agent. For example, it may contain auxiliary molecules added to vaccines or oils such as aluminum hydroxide, mineral oil or generated by the body after each induction by these additional ingredients. The auxiliary molecule includes interferon, interleukin, growth factor, and the like.

The vaccine composition of the present invention refers to a pharmaceutical composition containing at least one immunologically active ingredient that induces an immunological response in humans. The immunologically active component of the vaccine is the known LV-MS1-Fc recombinant protein. In addition, one or more additional antigens may be included to enhance the efficacy of the vaccine. It can also be prepared (polynucleotide vaccination) by induction of the above synthetic process in an animal. A vaccine may comprise one or more than one of the elements described above at the same time.

The vaccine composition may be in any form known in the art, for example, in the form of solutions and injections, or in solid form suitable for suspension, but is not limited thereto. Such formulations may also be emulsified or encapsulated in liposomes or soluble glass, or may be prepared in the form of an aerosol or spray. They may also be incorporated into transdermal patches. Liquids or injectables may contain propylene glycol if necessary and sodium chloride in an amount sufficient to prevent hemolysis (eg, about 1%).

The vaccine composition of the present invention may further include a pharmaceutically acceptable carrier or diluent. In the above, "pharmaceutically acceptable" means a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to pigs and does not normally cause gastrointestinal disorders, allergic reactions such as dizziness, or similar reactions. say

Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. Such carriers may be aqueous or non-aqueous solutions, suspensions or emulsions. As an adjuvant for increasing immunogenicity, a regular or atypical organic or inorganic polymer may be used. Adjuvants are generally known to promote immune responses through chemical and physical binding to antigens. For example, as an adjuvant, an atypical aluminum gel, an oil emulsion, or a double oil emulsion and an immunosol, etc. may be used. In addition, various plant-derived saponins, levamisole, CpG dinucleotides, RNA, DNA, LPS, various types of cytokines, etc. may be used to promote the immune response. The above immune composition can be used as a composition for inducing an optimal immune response by a combination of various adjuvants and immune response promoting additives. In addition, as the composition to be added to the vaccine, a stabilizer, an inactivating agent, an antibiotic, a preservative, and the like may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water or a buffer solution and used.

The vaccine composition may be administered through an oral, intramuscular, subcutaneous, etc. administration route, but is not limited thereto, and may preferably be administered via an intramuscular route.

The present invention provides a method for preventing or treating Middle East Respiratory Syndrome coronavirus infection comprising administering a vaccine composition to an individual.

The "subject" of the present invention means a subject in need of a method for preventing, controlling or treating a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat , which means mammals such as horses and cattle. In some cases, humans may be excluded.

"Prevention" of the present invention means any action that suppresses or delays the onset of Middle East Respiratory Syndrome coronavirus infection by administration of the composition according to the present invention.

"Treatment" of the present invention means any action in which the symptoms for Middle East Respiratory Syndrome coronavirus infection are improved or changed advantageously by administration of the composition according to the present invention.

Since the method of the present invention includes the above-described vaccine composition, descriptions of overlapping contents with the above-described vaccine composition of the present invention are omitted in order to avoid excessive complexity of the present specification due to description of overlapping contents.

The present invention provides a method for evaluating an immune response in an animal, comprising administering the recombinant expression vector or the recombinant protein to the animal.

In addition, the present invention provides a method for evaluating the immune response in small animals of the recombinant spike protein LV-MS1-Fc of Middle East Respiratory Syndrome coronavirus.

The animal is preferably a mammal, and the present invention includes the evaluation of immunogenicity in mice. Inoculation may be performed within two times at regular time intervals through the muscle or subcutaneous or oral or nasal skin of the immunogen animal, and preferably, two inoculations may be performed with an interval of two weeks through intramuscular inoculation. Two weeks after immunization of the immunized animal, mouse serum is obtained through tail blood or cardiac blood collection, and the effect on immune induction can be confirmed by measuring the IgG antibody titer through the ELISA system. Preferably, viral infection A protective antibody titer can be measured by measuring a virus neutralization antibody titer that can inhibit .

The present invention also includes a method for measuring Middle East Respiratory Syndrome virus neutralizing antibody. We provide a method for indirectly measuring neutralizing antibody titer by measuring luciferase activity by virus neutralizing antibody by making Vesicular stomatitis virus luciferase (VSVluc)-MERSS pseudovirus. Through the above method, the virus neutralizing antibody titer of the vaccine tested in the present invention can be 12 to 512 times. Preferably, it may be 128 times to 256 times. More preferably, a neutralizing antibody titer of 256 fold or more should be measured.

The present invention may provide methods for evaluating the efficacy of vaccines in mammals. The test method may be a transgenic animal (DPP4 knock in) expressing a human DPP4 virus receptor susceptible to the Middle East Respiratory Syndrome virus. Preferably, it may be a human DPP4 expressing mouse. To be precise, it is known that mice are generally not susceptible to MERS-CoV, so infection is impossible. In addition, it is known that mice expressing Human DPP4 (hDPP4 knock-in mice) are susceptible to MERS-CoV and can be infected, but human MERS-CoV does not cause disease in human DPP4 knock-in mice. However, it was confirmed that the mouse-adapted MERS-CoV produced as a result of passage more than 30 times in hDPP4 knock-in mice caused infection in hDPP4 knock-in mice and a disease similar to that in humans. In this study, hDPP4 knock-in mouse and mouse-adapted MERS-CoV were used as mouse models to evaluate vaccine efficacy.

The present invention provides a method for inducing a protective immune response comprising administering a vaccine composition to an animal.

Specifically, the present invention provides a method of inducing a protective immune response against Middle East Respiratory Syndrome virus in an animal, the method comprising administering the vaccine composition to a human in an immune effective amount of the vaccine. The vaccine may be an inactivated vaccine.

The animal may be a mammal such as a human or a non-human primate, a mouse, a rat, a dog, a cat, a horse, a cow, and preferably a human.

The immune response is a vaccine composition or an antigen or antigens contained in a vaccine containing the same, specifically directed to antibodies, B cells, helper T cells, suppressor T cells, cytotoxic T cells and gamma-delta T cells production or activation, exhibiting a therapeutic or protective immunological response in the host, thereby enhancing resistance to new infections or reducing the clinical severity of the disease. Preferably, it may be a protective immune response.

Such protection is evidenced by a reduction or absence of clinical signs normally exhibited by an infected host, a faster recovery time or lower duration, or a lower viral titer in the tissues or body fluids or feces of the infected host.

The immune effective amount means an amount of vaccine capable of inducing an immune response to reduce the frequency or severity of Middle East Respiratory Syndrome virus infection in humans, and those skilled in the art can appropriately select it. For example, the effective amount may be 100 μg to 10 μg of the purified antigen protein when the vaccine is an inactivated vaccine. More preferably, it may be 50 μg to 20 μg, and more preferably, it may be 20 μg or more.

The method for inducing an immune response is not limited thereto, but may be vaccination by oral, transdermal, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. Preferably, the vaccine may be intramuscularly inoculated during the first and second inoculations.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1: Use of spike protein for MERS-CoV vaccine

MERS-CoV has about 30 kb of positive-sense single-stranded RNA as genetic information. It is composed of 11 ORFs, and S gene, E gene, M gene, N gene, etc., which are proteins constituting the structure of the virus, are coded. Here, the S gene encodes a spike protein among proteins constituting the structure of MERS-CoV. The spike protein binds to DPP4 (Dipeptidyl-peptidase 4) of the host cell and is involved in membrane fusion between the virus and the host cell. In particular, the RBD inside the S gene plays an important role in the binding of the viral spike protein to the host cell. In addition, since the RBD is not pathogenic on its own, it is evaluated as an important target for the treatment and vaccine development of MERS-CoV. In the present invention, MERS-CoV spike protein was used as a vaccine to develop a prevention strategy that can control the further spread of MERS-CoV.

Example 2: Preparation and securing of a recombinant expression vector capable of protein expression in mammalian cells

The sequence of the spike protein was determined based on the 2015 Korean MERS gene information (GeneBank accession No. KT029139, Middle East respiratory syndrome coronavirus isolate MERS-CoV/KOR/KNIH/002 05 2015). MERS LV-MS1-Fc, a fusion protein in which the Fc domain of human IgG4 was tagged with the gene, was designed as a MERS recombinant vaccine antigen. The finally expressed recombinant vaccine antigen was designed to have a human Fc domain at the 3' end of the gene. In order to express it in mammalian cells, the following experiment was performed. Through the following experimental procedure, the codon-optimized MERS LV-MS1-Fc gene was cloned into pcDNA3.4 TOPO vector to construct a MERS recombinant expression vector and named MERS LV-MS1-Fc.

More specifically, the pcDNA3.4 TOPO vector was inserted into the gene through TA cloning, and the inserted gene with 'A' added to the 3' end was secured through PCR amplification using taq polymerase. The ratio of inserted gene: vector was 3 : 1, and TOPO cloning reaction was performed. E. coli strain TOP10 strain was used as a host for general cloning, and transformation was performed. Thereafter, the strain into which the recombinant gene was inserted was selected using the ampicillin marker. From the selected strain, DNA was obtained by plasmid purification, and the final gene was confirmed through sequencing analysis. A vector map corresponding to this is shown in FIG. 1 . The final gene schematic diagram obtained through cloning is shown in FIG. 2 . The base sequence of the final gene is shown in SEQ ID NO: 6.

For the confirmed MERS LV-MS1-Fc-TOPO expression gene, sufficient genetic resources were obtained through plasmid midi scale purification. Genetic resources were used for transfection (transfection) of 1 μg of genes per ml into CHO-S cells.

Example 3: Transient transfection to express MERS LV-MS1-Fc in CHO cells

(1) cell culture

ExpiCHO-S cell derived from CHO-S (Chinese Hamster Ovary) cell is a CHO cell line with high protein expression efficiency. ExpiCHO-S cells were used for transient transfection. ExpiCHO-S cells were used for culture and transfection in ExpiCHO expression medium. A 125-mL or 250-mL Erlenmeyer flask was used as a periodic CHO-S cell culture flask. CHO-S cells were seeded in Erlenmeyer flask at a density of 0.5 x 10 ⁶ cells/mL and grown in a suspension form in 8% CO ₂ , orbital shaker platform conditions at 37° C. in a cell incubator. Cell growth was maintained by subculture when the cells reached a density of 3 to 4 x 10 ⁶ cells/mL.

(2) MERS LV-MS1-Fc transfection (Transient Transfection)

In order to express the MERS LV-MS1-Fc antigen in CHO cells, transfection was performed on a 250-ml Erlenmeyer flask scale as follows. The day before transfection, CHO-S cells were stained with trypan blue for pre-seeding, and then the cell density was measured by dispensing in a hemocytometer. CHO-S cells have a doubling time of approximately 17 hours, and seeded at 3 x 10 ⁶ ~4 x 10 ⁶ cells/mL to secure a cell density of 3.0 x 10 ⁸ cells/50 mL during transfection and 7 the next day. It was made to reach a cell density of x 10 ⁶ ~ 10 x 10 ⁶ cells/mL. On the day of transfection, cell density was measured in the same manner using trypan blue staining. After that, seeding was performed at 6 x 10 ⁶ cells/mL to secure a cell density of 3.0 x 10 ⁸ cells/50mL in total, and transfection was performed. The reagent used for transfection was ExpiFectamine CHO reagent. 2 mL of mixture A was prepared by mixing 160 μL of ExpiFectamine CHO reagent and 1.84 mL of OptiPRO SFM. 50 μg of the MERS LV-MS1-Fc-TOPO expression gene was mixed with OptiPRO SFM to prepare 2 mL of mixture B. Mixture A and mixture B prepared through the above method were mixed to make a MERS LV-MS1-Fc expression gene transfection mixture and incubated at room temperature for 5 minutes. The cultured MERS LV-MS1-Fc expression gene transfection mixture was slowly aliquoted into CHO-S cells seeded in a 250-ml Erlenmeyer flask in advance and mixed well. After transfection, CHO-S cells were cultured on an orbital shaker platform at 8% CO ₂ , 37°C. Twenty hours after transfection, 12 ml of ExpiCHO Feed and 300 μL of ExpiCHO Enhancer were added and cultured until day 8 to express MERS LV-MS1-Fc antigen.

The cell line transfected with the LV-MS1-Fc gene was named Chinese hamster ovary cell LV-MS1-Fc cell, and it was deposited with the Center for Biological Resources at the Korea Research Institute of Bioscience and Biotechnology and was given accession number KCTC14342BP on October 22, 2020.

Example 4: Confirmation of LV-MS1-Fc antigen expression

(1) Securing the expressed LV-MS1-Fc antigen

On the 8th day of transfection, the cultured cells were centrifuged at 1,500 rpm for 5 minutes to separate the cells and the expression solution. The separated cells were washed twice with 1 X DPBS 20 mL. After that, 5 mL of 1 X DPBS and protease inhibitor were added, followed by 10 min ~ 30 sec on/off disruption by sonication to obtain a cell lysate.

(2) Identification of the expressed MERS LV-MS1-Fc antigen

In order to confirm the expression of the MERS LV-MS1-Fc antigen in the obtained expression solution and the cell lysate, the following experiment was performed.

After measuring the protein concentration using BCA protein analysis, 20 μg of protein from the expression solution and the cell lysate was separated through 10% SDS-PAGE and electrophoresed on a nitrocellulose membrane. The membrane was prepared using an α-MERS spike protein polyclonal antibody-specific reaction that specifically binds to amino acid residues 381-505 of the MERS spike protein and a goat anti-human IgG-HRP (goat anti-human IgG-HRP) antibody. Protein expression verification was performed. The results of confirming the protein are shown in FIG. 3 .

As shown in FIG. 3 , expression of recombinant MERS LV-MS1-Fc protein with a size of about 130-kDa was confirmed in both the CHO-S cell culture medium and the lysate. The prepared MERS LV-MS1-Fc expression gene was prepared to include a signal peptide. In addition, the gene was induced to be secreted after the protein was expressed in CHO-S cells, and it was confirmed that the MERS LV-MS1-Fc antigen was also present in the expression solution.

Example 5: MERS LV-MS1-Fc antigen purification

Protein purification was performed on the MERS LV-MS1-Fc antigen expression solution obtained in Example 3 using affinity chromatography as follows.

More specifically, MERS LV-MS1-Fc antigen protein precipitation was performed through a pretreatment process of adding 90% ammonium sulfate prior to affinity chromatography. MERS LV-MS1-Fc antigen protein was precipitated by adding 662 g/L of ammonium sulfate to the expression solution. The precipitated protein was dissolved in 20 mM sodium phosphate buffer (pH 7.4) to obtain a pellet at 13,000 rpm, 30 min, 4°C. The dissolved protein was placed in a cellulose membrane and dialysis was performed twice for 2 hours in 20 mM sodium phosphate buffer (pH 7.4). Finally, purified protein was obtained through 0.45 μm filtration after centrifugation at 13,000 rpm, 30 min, and 4°C. The column used for purification was HiTrap™ protein A HP. During elution, neutralization was performed with 0.1 M citric acid (pH 3.0) and 1 M Tris-H Cl (pH 9.0). The obtained protein was measured for protein concentration using BCA protein analysis. After separating the protein through 10% SDS-PAGE, the protein was confirmed through Coomassie blue staining. In addition, Western blotting was performed through the membrane electrophoresed with a nitrocellulose membrane. The results are shown in FIG. 3 . It was confirmed that the MERS LV-MS1-Fc antigen of approximately 130 kDa was secured through affinity chromatography.

Example 6: Confirmation of mouse immunogenicity of MERS antigen

The MERS LV-MS1-Fc antigen protein obtained by the above method was immunized with 5-week-old female BALB/c mice as follows. More specifically, as a group, the PBS group (negative control group), LV-MS1-Fc 0.5 μg, 2 μg, and 5 μg were divided into 4 groups, and immunization was carried out by 5 animals each. Inoculation was performed subcutaneously twice at an interval of 2 weeks at an amount of 100 μL/animal for each group. Antisera were obtained through blood sampling at the 2nd and 4th weeks of immunization from the day of the first inoculation. In order to confirm the antibody reaction of the obtained mouse antiserum and MERS spike protein, the following binding reaction was performed. To measure the antibody titer of MERS spike protein by MERS LV-MS1-Fc, 200 ng/100 μL of commercially purchased MERS Spike protein (Sino) was coated on a 96-well plate for ELISA measurement, followed by a general ELISA measurement method. Antibody titers were measured. The results are shown in FIG. 4 .

As shown in FIG. 4 , it was confirmed that the MERS Spike protein IgG antibody titer of the group inoculated with the LV-MS1-Fc antigen protein showed a significant difference from the control group. In addition, it was confirmed that the antibody titer was further increased at the antigen concentration of 5 μg than at the antigen concentration of 2 μg. Through these results, it was confirmed that the mouse IgG antibody formation against the MERS Spike protein was effective through the MERS LV-MS1-Fc antigen protein.

Example 7: Evaluation of neutralizing antibodies against Middle East Respiratory Syndrome coronavirus spike protein

For the neutralizing antibody against the MERS LV-MS1-Fc antigen obtained in Example 5 and the challenge test, most mice generally do not have a sensitivity to MERS-CoV, so infection is impossible. Mice expressing Human DPP4 (hDPP4 knock-in mice) are susceptible to MERS-CoV and are susceptible to infection. However, human MERS-CoV does not cause disease in human DPP4 knock-in mice. As shown in FIG. 6 , the mouse-adapted MERS-CoV produced as a result of passage more than 30 times in hDPP4 knock-in mice causes infection in hDPP4 knock-in mice and a disease similar to that in humans. Therefore, in this study, hDPP4 knock-in mouse and mouse-adapted MERS-CoV were used as mouse models to evaluate vaccine efficacy. The above mouse model and virus were developed at the University of Iowa, and mice and viruses were acquired from Seoul National University. Four weeks after vaccination, blood was collected using cardiac bleed, and serum was separated by centrifugation at 2,000 g for 10 minutes. The separated serum was incubated at 56°C for 30 minutes. To measure the neutralizing antibody titer, Huh7 cells were prepared in a 96-well microplate. Thereafter, the serum was diluted 1:10 and serially diluted two-fold. After the same amount of VSVluc-MERS was added, it was incubated at 37°C for 1 hour. Next, the virus and serum mixture was inoculated into Huh7 and incubated for 1 hour. After removing the virus and serum mixture, a fresh medium was added, and incubated at 37°C for 18 hours. After removing the medium, 50 μL of 1X luciferase lysis buffer was added to each well and stored at -80°C. The luciferase activity was measured using a luciferase substrate, and high neutralizing antibody results were confirmed as shown in FIG. 5 .

Example 8: Conducting an attack inoculation test against the Middle East Respiratory Syndrome coronavirus spike protein

To evaluate the efficacy of the tested vaccine, transgenic mice expressing DPP4, known as an infection receptor of MERS-CoV, were vaccinated ( FIG. 6 ). After vaccination, neutralizing antibodies were measured and the vaccine's defense was evaluated through a challenge test using a pathogenic virus.

Mouse-adapted MERS-CoV culture was performed in the BL3 laboratory of Chungbuk National University. After preparing VeroE6 cells in T-75, when the cells were attached to the flask, 10 ⁶ PFU Mouse-adapted MERS-CoV was diluted in 2 mL media and inoculated. One hour after inoculation, the virus was removed and new media (10 mL) was added. It was cultured in an incubator at 37° C. until a cytopathic effect was observed. When 80-90% of the cytopathic effect was observed, the virus was released from the cells by Freeze-Thawing. The supernatant was centrifuged at 10,000 g for 10 minutes to remove cell suspension. The supernatant was aliquoted by 1 mL and stored in a -80°C ultra-low temperature freezer. VeroE6 cells were prepared in a 6-well plate to measure the virus titer (performed in the BL3 laboratory of Chonbuk National University), and then the virus was diluted 10-fold. 200 μL of the serially diluted virus was inoculated into VeroE6 cells. One hour after inoculation, the virus was removed and a medium containing 0.8% agarose was added and cultured for 3 days. The virus concentration was calculated by counting the number of plaques generated after 3 days.

For vaccination, 2.5 μg of antigen (PBS, LV-MS1-Fc) was intramuscularly inoculated twice with alum at an interval of 2 weeks. The mice vaccinated 1 day before challenge inoculation were transferred to the BL3 laboratory of Chonbuk National University. To perform challenge inoculation (conducted at Chonbuk National University BL3 laboratory) - After anesthetizing the mice using Isofluorane, vaccinated mice were intranasally inoculated with 10 ⁴ pfu/50 μL of the virus suspension. For 7 days after virus inoculation, the weight and clinical symptoms of mice were observed. On the 7th day after challenge inoculation, euthanasia was induced by cervical dislocation after anesthesia. Euthanized mice were subjected to autopsy and lungs were removed. After the right lung was disrupted for virus isolation, only the supernatant was separated by centrifugation at 1,000 g for 10 minutes. The supernatant was assayed for virus titer by plaque assay. The left lung was fixed in 10% neutral formalin for more than 24 hours for histopathological examination. After that, it was transferred to the College of Veterinary Medicine, Chungnam National University, and H&E staining was performed.

As a result of the challenge inoculation, the mice inoculated with PBS started to gradually lose weight from 1 day after the virus challenge inoculation, and showed a decrease of 25% or more of the body weight before inoculation. However, there was no change in body weight in mice vaccinated with LV-MS1-Fc. Of the 4 mice inoculated with PBS, one died 4 days after challenge inoculation. Then, 6 days after challenge inoculation, two animals died (mortality rate 75%). Mortality was not observed in mice vaccinated with LV-MS1-Fc (mortality rate 0%). In mice inoculated with PBS, activity decreased sharply from 1 day after challenge inoculation, and the amount of drinking water and feed intake significantly decreased. No specific clinical symptoms were observed in mice vaccinated with LV-MS1-Fc. As shown in FIG. 7 , as a result of measuring the virus titer in the left lung after challenge inoculation, the PBS inoculation group showed 1X10 ^4.2 PFU/mL, and the LV-MS1-Fc inoculation group showed 1X10 ² PFU/mL. It was confirmed that the amount of virus detected in the LV-MS1-Fc inoculated group was lower. As shown in FIG. 8, histopathological symptoms in the lung tissue of mice inoculated with PBS were inflammatory cell infiltration, edema, exudate, and the like. However, no specific findings were observed in the lungs of mice vaccinated with LV-MS1-Fc.

Example 9: Antigen toxicity and safety evaluation

For antigen toxicity and safety evaluation, 20 μg of LV-MS1-Fc antigen in 50 μL of PBS was mixed with the same amount of alum, and then inoculated twice at 2-week intervals into the femoral muscle of C57BL/6 mice and body weight for 4 weeks Changes, feed intake, and drinking water were measured. The negative control group was inoculated with the same amount of PBS in the same way. Ten mice per each test group were run. The results are shown in FIG. 9 . As shown in FIG. 9, it was confirmed that there was no significant difference in body weight change, feed intake and drinking water between the PBS inoculation group and the LV-MS1-Fc antigen inoculation group.

As described above, although specific embodiments of the present invention have been described in detail, those skilled in the art who understand the spirit of the present invention may add, change, delete, etc. other components within the scope of the same spirit, and other degenerate inventions However, other embodiments included within the scope of the present invention may be easily proposed. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

[Certificate of deposit of microorganisms]

Name of depositary institution: Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC)

Accession number: KCTC 14342BP

Deposit date: 20201022

Claims (12)

1. A nucleic acid in which a gene encoding a MERS-CoV spike protein represented by the nucleotide sequence of SEQ ID NO: 1 or 2 and a gene encoding a human IgG4 Fc represented by the nucleotide sequence of SEQ ID NO: 3 or 4 are fused.
2. A recombinant expression vector comprising the nucleic acid of claim 1.
3. A cell line transformed with the recombinant expression vector of claim 2.
4. 4. The method of claim 3,

   The cell line is a Chinese hamster ovary cell LV-MS1-Fc cell (KCTC 14342BP), the cell line.
5. A recombinant protein produced from the cell line of claim 3 .
6. transforming the cells with the recombinant expression vector of claim 2;

   recovering the recombinant protein from the cell or culture thereof; and

   Purifying the recombinant protein; comprising, a method for producing a recombinant protein.
7. A vaccine composition for preventing or treating Middle East respiratory syndrome coronavirus, comprising the recombinant protein of claim 5 as an active ingredient.
8. 8. The method of claim 7,

   The vaccine is a vaccine composition for preventing or treating Middle East respiratory syndrome coronavirus, characterized in that it is an inactivated vaccine.
9. 8. The method of claim 7,

   The vaccine is a vaccine composition for preventing or treating Middle East respiratory syndrome coronavirus, which further comprises an immune enhancing material or adjuvant.
10. A method for preventing or treating Middle East respiratory syndrome coronavirus infection, comprising administering the vaccine composition of claim 7 to an individual.
11. A method for evaluating an immune response in an animal, comprising administering the recombinant expression vector of claim 2 or the recombinant protein of claim 5 to the animal.
12. 12. The method of claim 11,

    The method is to evaluate the immune response by measuring the antibody titer (antibody titer) from the animal's serum, immune response evaluation method in animals.

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