WO2018097603A2 - Middle east respiratory syndrome coronavirus s protein immunogenic composition and method for preparing same - Google Patents

Abstract

The present invention provides a MERS-CoV S immunogenic composition comprising at least one polypeptide selected from a group consisting of SEQ ID NOs: 2, 4, 6 and 8, and a method for preparing the same. The immunogenic composition prepared by the method of the present invention can be expected to have a high yield and can provide a composition for superior prevention of MERS-CoV.

Description

Middle East Respiratory Syndrome Coronavirus S Protein Immunogen Composition and Method of Making the Same

This application claims priority based on Korean Patent Application No. 10-2016-0156610, filed November 23, 2016, and all the contents disclosed in the specification and drawings of the application are incorporated in this application.

The present invention relates to a MERS-CoV spike (S) immunogen and its modifications, preparations, or uses thereof comprising an immunogenic composition for treating and / or preventing a Middle East Respiratory Syndrome (MERS-CoV) infection.

The Middle East Respiratory Syndrome (MERS), which first occurred in the Middle East in September 2012, causes severe acute respiratory syndrome such as acute pneumonia and acute renal failure. There were 1,800 MERS laboratory-confirmed cases (WHO standards) in 27 countries around the world since 2012 to September 2016, of which at least 640 people died, resulting in a severe infectious disease with an estimated 36% mortality. The clinical symptoms of MERS are respiratory symptoms with fever, and diarrhea and vomiting. The main subjects of MERS are the elderly and over 50 patients with underlying diseases and immune disorders, including lymphocytes and platelets, and acute renal failure. It was a pandemic in Korea in 2015, when an out-break occurred due to a second case of human infection.

The causative agent of MERS was first identified as the coronavirus family by Erasmus University Medical Center Rotterdam, the Netherlands in 2012, and as "MERS-Coronavirus" by the International Virus Classification Committee (ICTV) in May 2013. Unnamed: I was named. (Figure 1) Middle Respiratory Syndrome Coronavirus (MERS-CoV) belongs to beta coronavirus, positive sense single strand RNA virus (positive sense single strand RNA virus) virus genome (genome) has a size of about 30kb It encodes ten proteins. To date, no clear identification of human liver infection and propagation pathways has been made, but human DPP4 (Dipeptidyl peptidase 4) receptors are known as major viral receptors. In addition, the gene of MERS-CoV is known to have high gene homology with Bat-CoV-HKU4 and Bat-CoV-HKU5, which are the coronaviruses of bats. The discovery of neutralizing antibodies to MERS in Camel suggests that camels are a potential mediator of human transmission of MERS-CoV. Recently, full-length gene sequencing of viruses isolated from severe camels and those isolated from MERS deaths have been in agreement, and significant evidence of MERS-CoV infection in humans came from camels.

MERS-CoV encodes four structural proteins, and the structural proteins are spike (S), membrane (M), envelope (E) and nucleocapsid (N). The S protein is a type I trans-membrane glycoprotein that exists on the surface of MERS-CoV. It forms an envelope and an anchor and is trimeric. (Figure 2) MERS-CoV S protein plays an important role in determining host range and tropism, like most coronaviruses. The S protein of MERS-CoV consists of S1, the receptor binding region, and S2, the membrane fusion region.The receptor binding domain (RBD) in the S1 sub-domain of MERS-CoV is dipeptidyl peptidase-4 (known as CD-26). DPP4) is known to recognize and bind to receptors. COV-NL63 and SARS-COV bind to angiotensin-converting enzyme 2 (ACE2), while Porcine respiratory coronavirus and some COVs are known to recognize aminopeptidase N (APN). HR domains of the S2 subdomain are involved in mediated fusion between the envelope of the virus and the cell membrane of the target cell. This spike protein is a major antigen of coronaviruses and is targeted for vaccines and therapeutics because it induces high immunogenicity.

The present invention to solve the above problems, to obtain a high MERS-CoV antigen production capacity (yield), to provide an immunogen composition having a high immunogenicity and a method for producing the same.

The present invention seeks to provide a MERS-CoV vaccine having a high antibody titer and a method for preparing the same.

The inventors of the present invention seek to reduce the time and cost required to obtain a virus by improving MERS-CoV antigen yield.

Overview of the technology

The present invention relates to a Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, an immunogen composition comprising said immunogen, or a vaccine comprising said immunogen composition. More specifically, the present invention relates to a method for preparing and using a new MERS-CoV S protein expressing peptide or MERS-CoV antigen.

The immunogen secured by the present invention allows false induction of the immune response that occurs when an animal or human is actually infected with MERS-CoV. In addition, the present invention may induce and maintain such a false immune response and thus may neutralize MERS-CoV in the body when MERS-CoV is actually infected.

The present invention provides a suitable method for culturing and purifying the MERS-CoV S immunogen. MERS-CoV S immunogens produced by the culture and purification methods described herein can expect higher yields than the methods produced in media. In addition, the MERS-CoV S immunogen produced by the culture and purification methods presented herein provides high immunogenicity.

Each of the journal articles, patents, patent applications, publications, etc., described herein, are referenced in their entirety or in part, and it is to be understood that such references are also incorporated herein.

Justice

As used herein, the term "immunogen" refers to any substance that induces immunogenicity, including the term "antigen", means any substance that enters the body and induces an immune response. The term “immunogen” includes a protein or a large protein molecule composed of proteins or forms in which the protein is dissolved to form a fragment. Giant protein refers to a large number of protein sequences and protein assemblies, including polymeric immunogens and VLPs. Immunogenic induction here means both cellular and humoral immunity. The term “immunogen” includes, for example, all and part of foreign material that has penetrated into the body, and cellular immune induction by an immunogen may mean antigen specific antibody production. Antigen-specific antibodies include neutralizing antibodies that defend and neutralize re-penetrating foreign substances.

As used herein, the term "fragment" may be used in the same sense as fragments, fragments, etc., and may be used to include a form in which a portion of the protein is separated from the MERS-CoV S protein without losing immunogenic function. have. For example, when crushing and cleavage of the MERS-CoV S protein is induced by centrifugation in the process of separating from the cell membrane, the fragment of the MERS-CoV S protein of the present invention, unless the immunogenicity is lost, the present invention May be included in the MERS-CoV immunogen.

The term "vaccine" as used herein refers to a combination of derived antigenic determinants used to induce the formation or immunity of antibodies against dead or weakened pathogens or pathogens. Vaccines are provided to provide immunity against MERS caused by the MERS CoV virus. The term "vaccine" also refers to an immunogen suspension or solution that is administered to a vertebrate to produce protective immunity, i.e., immunity that reduces the severity of the disease associated with the infection.

As used herein, the term "comprising" means that the content and content together. The term "comprising" also means the content and the addition or inclusion of the content. It should be noted, however, that the term "comprising" may be used independently and in its own right.

"About" includes all values that provide the same effect and result as reference values. However, "about" gives meaning only for reference values. In addition, the range included by the term "about" may vary depending on the range or characteristics of the content (reference value) in which the term is included. Thus, depending on the content, "about" may mean, for example, less than ± 15%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1%, or less than 1%. Can be.

All numerical ranges described herein include all numerical values within the numerical range values. For example, it should be understood that the numerical range "1000-3000" includes all values between 1000 and 3000. In addition, all numerical values set forth herein may be regarded as independent numerical ranges, and may also be regarded as linkage-complex ranges between respective numerical values.

As used herein, an "effective amount" is generally a MERS-CoV S protein or its sufficient to induce immunity and to prevent and / or alleviate a viral infection or to reduce at least one symptom of an infection and / or to increase the effect of an immunogen. Refers to the amount of fragment or aggregate of MERS-CoV S protein. An effective amount may refer to an amount of MERS-CoV S protein or fragment thereof or aggregate of MERS-CoV S protein sufficient to delay or minimize the onset of infection. An effective amount may refer to the amount of MERS-CoV S protein or fragment thereof or aggregate of MERS-CoV S protein that provides a therapeutic benefit in the treatment or management of an infection. The effective amount may be an amount sufficient to enhance the subject's (eg, human) own immune response against subsequent exposure to the virus. The level of immunity can be observed, for example, by plaque neutralization, complement fixation, enzyme-linked immunoadhesion or microneutralization assays, eg, by measuring the amount of neutralizing secretion and / or serum antibodies. In the case of a vaccine, an "effective amount" is one that prevents a disease or reduces the severity of symptoms.

As used herein, baculovirus refers to a virus that infects insect cells. As used herein, baculovirus refers to alfalfa looper (or night moth known as Autographa californica-moth included in Noctuidae) nucleopolyhedrovirus strains (NPV strains) and modified viral strains. It should also be noted that the baculovirus as used herein also includes Bombyx mori (silk moth belonging to the silkworm moth-Bombycidae) nuclear polyhedron virus strain (NPV) and modified viruses.

As used herein, the MERS-CoV S recombinant baculovirus is constructed using homologous recombination. The resulting homologous recombinant baculovirus can produce recombinant MERS-CoV S foreign protein upon infection with the appropriate cell (eg insect cell line). Recombinant MERS-CoV S protein is overexpressed by polyhedrin promoter.

MERS-CoV immunogen

The present invention provides a MERS-CoV S immunogenic composition comprising any one or more polypeptides selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.

In one embodiment of the invention, the immunogenic composition comprises any MERS-CoV protein, including, but not limited to, spike (S) protein or fragment thereof of MERS-CoV or a spike protein combination of MERS-CoV. can do.

The MERS-COV S gene is GenBank accession No. Or derived from the strains KF186567 (Al-Hasa_1_2013) and KT029139 (MERS-COV / KOR / KNIH / 002_05_2015). DNA sequence of the gene can be optimized codon to facilitate expression in insect cells, Seq ID NO: 1 (Al-Hasa_1_2013, gene sequence), Seq ID NO: 2 (Al-Hasa_1_2013, amino acid sequence), Seq ID NO: 3 (KOR / KNIH / 002_05_2015, gene sequence), and Seq ID NO: 4 (KOR / KNIH / 002_05_2015, amino acid sequence).

In addition, Seq ID. MERS-CoV S protein was made to be secreted into the culture. No: 5, Seq ID. No: 6, Seq ID. No: 7, and / or Seq ID. The gene sequence and amino acid sequence of the MERS-CoV S partial protein of No: 8 can be provided.

The present invention provides a variant of the MERS-CoV protein. In particular, modifications of the amino acid sequence referred to in SEQ ID NO: of the present invention may comprise a change in the amino acid sequence of the constituent protein. The term "variant" in the context of a polypeptide refers to an amino acid sequence that has been changed by one or more amino acids in reference to a reference sequence. Variants may have “conservative” changes, and substituted amino acids have similar structural or chemical properties, eg, replacement of leucine by isoleucine. Optionally, the variant may have a “non conservative” change, eg, replacement of glycine by tryptophan. Similar micro modifications may also include amino acid deletions or insertions or both. Determination of which amino acid residues can be substituted, inserted or deleted without removing biological or immunological activity can be made using computer programs well known in the art, such as DNASTAR software.

The nucleic acids and polypeptides included in the MERS-CoV immunogenic composition of the present invention may be at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences shown in the sequence listing.

The inventors of the present invention provide a MERS-CoV immunogenic composition, in particular using all or part of the gene sequence numbers 1, 3, 5, 7 and / or amino acid sequence numbers 2, 4, 6, 8 of the MERS-CoV S moiety. . The inventors of the present invention provide particularly excellent antibody titers using the nucleic acids and / or polypeptides of SEQ ID NO: 2, 4, 6, 8, preferably the polypeptides of SEQ ID NO: 2 and / or 4, and improve immunogen yield. It provides an immunogenic composition that brings.

Pharmaceutical or Vaccine Preparations and Administration

One embodiment of the present invention provides a MERS-CoV prevention vaccine comprising any one of the MERS-CoV S polypeptide, selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.

The vaccine may further comprise an adjuvant or an immune enhancer.

The immunogenic composition of the present invention includes any pharmaceutical substance that does not itself cause an immune response harmful to the vertebrate receiving the composition, and may be administered without excessive toxicity with the MERS-CoV S polypeptide. Pharmaceutically acceptable carriers including suitable diluents or excipients. As used herein, the term "pharmaceutically acceptable" means to be listed in the US Pharmacopoeia, European Pharmacopoeia, or Vertebrate and more specifically other commonly recognized Pharmacopoeia for use in humans. MERS CoV S immunogens of the invention are administered in an effective amount or amount (as defined above) sufficient to stimulate an immune response against one or more strains of MERS CoV virus. Such compositions can be used as vaccines and / or immunogen compositions for inducing a protective immune response in vertebrates. The composition may contain other MERS-CoV S proteins or fragments thereof.

In one non-limiting embodiment, the concentration of immunogen is at least about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, About 100 μg / mL, about 200 μg / mL, or about 500 μg / mL. In certain embodiments, the concentration of the immunogen is about 10 μg / mL to about 1 mg / mL, or about 20 μg / mL to about 500 μg / mL, or about 30 μg / mL to about 100 μg / mL or about 30 μg / mL To about 50 μg / mL. In other embodiments the concentration of immunogen may be comprised between 10 μg / mL and 200 μg / mL.

In one embodiment, the pharmaceutical formulations disclosed herein comprise a MERS-CoV protein, primarily a spike protein; And pharmaceutically acceptable carriers or excipients.

In another embodiment, the pharmaceutical formulation comprises a purified, high affinity antibody produced in an animal to which the immunogen has been administered. Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffers, and combinations thereof. Pharmaceutically acceptable carriers, diluents and other excipients are provided in Remington's Pharmaceutical Sciences (Mack Pub. Co. N. J. current edition). The formulation should be suitable for the mode of administration. In a preferred embodiment, the formulation is suitable for administration to humans, preferably sterile, non-particulate and / or not pyrogenic. If desired, the composition may contain small amounts of wetting or emulsifying agents or pH buffers. The composition may be in solid form, such as a lyophilized powder suitable for recombination, liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained release preparations or powders. Oral formulations may include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and the like. The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of an immunogenic vaccine formulation. In a preferred embodiment, the kit comprises two containers, one containing a MERS CoV immunogen and the other containing an adjuvant. Notices in the form prescribed by government agencies governing the manufacture, use, or sale of a medicinal or biological product may be incorporated in such container (s), which notices may be made by an agency of manufacture, use, or sale for human administration. Indicates approval. The formulation may be packaged in a sealed container such as an ampoule or sachette indicating the amount of the composition. In one embodiment, the composition is supplied as a liquid, and in another embodiment, it is supplied as a dry sterile lyophilized powder or water removal concentrate in a sealed container, for example to administer to a subject in water or saline. Can be reconstituted to an appropriate concentration. Preferably, the composition is preferably about 1 μg, about 5 μg, about 10 μg, about 20 μg, about 25 μg, about 30 μg, about 50 μg, about 100 μg, about 125 μg, about 150 μg, or It is supplied as a dry sterile lyophilized powder in a container hermetically sealed at a unit dose of about 200 μg. Optionally, the unit dose of the composition is about 1 μg (eg, about 0.08 μg, about 0.04 μg, about 0.2 μg, about 0.4 μg, about 0.8 μg, about 0.5 μg or less, about 0.25 μg or less, or about 0.1 μg or less). ) Or greater than about 125 μg (eg, at least about 150 μg, at least about 250 μg, or at least about 500 μg). Such dosage can be measured as μg of total MERS-CoV protein (eg, spike protein or fragment thereof). The immunogen of the invention should be administered within about 12 hours, preferably within about 6 hours, within about 5 hours, within about 3 hours, or within about 1 hour after reconstitution of the lyophilized powder. In another embodiment, the MERS-CoV protein immunogenic composition is supplied in liquid form to a sealed container representing the amount and concentration of the MERS-CoV protein composition. Preferably, the liquid form of the immunogen composition of the present invention is at least about 50 μg / ml, more preferably at least about 100 μg / ml, at least about 200 μg / ml, at least 500 μg / ml, or at least 1 mg / ml It is supplied to a sealed container.

The vaccine or immunogen composition of the invention can be administered to an animal to induce an immune response against MERS-CoV. In one embodiment, the animal is susceptible to MERS-CoV infection. In one embodiment, the animal is a human. Preferably, administration of the immunogen induces substantial immunity against at least one MERS-CoV strain, isolate, clade and / or species. In one embodiment, administration of the immunogen induces substantial immunity against at least two MERS-CoV strains, isolates, clades and / or species. Typically, the dosage can be adjusted within this range based on, for example, age, physical condition, weight, age, food, time of administration and other clinical factors. Accordingly, the present invention encompasses a method of formulating a vaccine or immunogen composition that induces substantial immunity against an infection or at least one symptom thereof in a subject, comprising adding an effective amount of an immunogen to said formulation. Stimulation of substantial immunity by a single dose is preferred, but to achieve the desired effect, additional doses may be administered via the same or different routes. In newborns and infants, for example, multiple doses may be necessary to elicit sufficient levels of immunity. Dosing can be continued at intervals throughout childhood, if necessary to maintain a sufficient level of protection against infection. Similarly, adults who are particularly susceptible to repeated or severe influenza infections, such as, for example, health workers, daycare teachers, family members of children, elderly people and individuals with impaired cardiopulmonary resuscitation, develop protective immune responses and / or Or multiple immunity may be required to maintain. The level of immunity induced can be observed by measuring, for example, neutralizing gland and serum antibodies and the amount of controlled dose or repeated vaccination as needed to elicit and maintain the desired level of protection. Thus, in one embodiment, a method of inducing substantial immunity against a viral infection or at least one symptom thereof in a subject comprises administering at least one effective amount of MERS CoV Spike protein or fragment or aggregate thereof. Methods of administering vaccines and / or immunogen preparations include parenteral administration (eg, endothelial, intramuscular, intravenous and subcutaneous), epidural and mucosal (eg nasal and oral or pulmonary routes or suppositories) It is not limited to this. In certain embodiments, the composition is administered intramuscularly, intravenously, subcutaneously, orally or intradermally. The composition may be administered, for example, by infusion or bolus injection, by absorption through the epithelium or inside the mucosa (eg, oral mucosa, colon, conjunctiva, nasopharynx, central pharynx, vagina, urinary tract, bladder, intestinal mucosa, etc.). It may be administered by any convenient route and may be administered with other biologically active substances. In some embodiments, administration via the nasal or other mucosal route can induce an antibody or other immune response substantially higher than other routes of administration. In other embodiments, the nasal or other mucosal route of administration of the immunogenic composition and / or vaccine can induce an antibody or other immune response that will induce cross protection against other strains of the virus. Administration can be systemic or local. Prophylactic vaccine formulations are administered systemically by subcutaneous or intramuscular injection or needleless injection devices using needles and injections. Optionally, the vaccine formulation is administered nasal by drops into the upper airway, large particle aerosols (greater than about 10 microns) or spraying. Any of these pathways of delivery give rise to an immune response, whereas nasal administration provides an increased effect of inducing mucosal immunity at the site of infiltration of the virus. In another embodiment, the vaccine and / or immunogenic agent is administered in a manner that targets mucosal tissue to elicit an immune response at the site of immunization. For example, mucosal tissue, such as gut associated lymphoid tissue (GALT), can be targeted for immunization by using oral administration of a composition containing an immunogen adjuvant with specific targeting properties. Other mucosal tissues may also be targeted, such as nasopharyngeal lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT). Vaccines and / or immunogenic agents may be administered according to a dosing regime such as administering the original vaccine composition followed by augmentation of administration. In certain embodiments, the second dose of the composition is administered at any time between two weeks and one year after the initial administration, preferably about 1, about 2, about 3, about 4, about 5 to about 6 months. In addition, the third dose may be administered about 3 months to about 2 years or more, preferably about 4, about 5, or about 6 months or about 7 months to about 1 year after the second dose and the first dose. The third dose can be administered orally after the second dose when the subject's serum and / or urine or mucosal secretions are absent or a small amount of specific immunoglobulin is detected. In a preferred embodiment, the second dose is administered about 1 month after the first dose and the third dose is administered about 6 months after the first dose. In another embodiment, the second dose is administered about 6 months after the first administration. In other embodiments, an immunogen comprising a MERS-CoV protein can be administered as part of a combination therapy. For example, the MERS-CoV protein or fragment thereof or aggregate thereof may be formulated with other immunogenic compositions and / or antiviral agents. Dosages of pharmaceutical agents may be administered first in an effective dose to elicit a prophylactic or therapeutic immune response, for example, by measuring the serum titer of virus specific immunoglobulins or by measuring the inhibition rate of antibodies in serum samples or urine samples or mucosal secretions. It can be easily determined by one skilled in the art by confirming. In addition, human clinical studies can be conducted by those skilled in the art to determine the desired effective amount for a human. Such clinical studies are routine and well known in the art. The exact dosage to be used will depend on the route of administration. Effective amounts can be estimated from dose-response curves derived from ex vivo or animal testing systems. As is well known in the art, the immunity of certain compositions can be enhanced by using nonspecific stimulants of the immune response, known as adjuvant. Antigen adjuvant was used to experimentally enhance the general increase in immunity against unknown immunogens (eg US Pat. No. 4,877,611). Immunization protocols have used adjuvant for stimulating reactions for many years, which are well known to those skilled in the art. Some antigen adjuvant affects the way antigens are present. For example, the immune response increases when protein antigens are submerged by alum. Emulsification of the antigen extends the antigen presentation period. Antigen adjuvant may be included. Suitable adjuvant agents include those described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients (2nd Edition), incorporated by reference in its entirety for all purposes. Other exemplary, adjuvant agents are complete Freund's adjuvant. (Nonspecific stimulants of immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant and aluminum hydroxide adjuvant. Other antigen adjuvant include GMCSP, BCG, aluminum hydroxide, thur-MDP and nor- MDP compounds such as MDP, CGP (MTP-PE), Lipid A, Montanide ISA 206 and Monophosphoryl Lipid A (MPL) Bacteria, MPL, Trehalose Dimycolate (TDM) and (CWS) RIBIs containing three components extracted from the cell wall backbone (CWS) in a 2% squalene / twin 80 emulsion are contemplated, MF-59, Novasome®, MHC antigens may also be used. In an embodiment, the adjuvant is a paucilamellar lipid vesicle with two to ten bilayers arranged in a substantially spherical sheath form separated by an aqueous layer surrounding the large amorphous central cavity from which the lipid bilayer has been removed. Pausilamela lipid vesicles can play a role in stimulating the immune response in many ways, such as nonspecific stimulants, carriers for antigens, carriers of other adjuvants, and combinations thereof. When prepared by mixing antigens, the antigen acts as a nonspecific immune enhancer, leaving the antigen extracellular to the vesicle, by encapsulating the antigen in the central cavity of the vesicle, the vesicle acts as an immune enhancer and a carrier for the antigen. In an embodiment, the vesicles are made predominantly of non-phospholipid vesicles. A basom. NOVA ® is a cotton pouch sila melanoma non-phospholipid vesicles of about 100nm to about 500nm. These include Brij 72, cholesterol, oleic acid and squalene. Novasome has proven to be an effective adjuvant for antigens (see US Pat. Nos. 5,629,021, 6,387,373 and 4,911,928, incorporated herein by reference in their entirety for all purposes). In one embodiment, the adjuvant effect is achieved by the use of a substance such as alum and is used in a solution of about 0.05 to about 0.1% in phosphate buffered saline. Optionally, the immunogen can be prepared from a premix with sugar's synthetic polymer (Carbopol®) used in about 0.25% solution. Some antigen adjuvant such as certain organic molecules obtained from bacteria act on the host rather than the antigen. Examples are muramyl dipeptides (N-acetylmuramil-L-alanyl-D-isoglutamine [MDP]), bacterial peptidoglycan. In other embodiments, hemocyanin and hemoerythrin can be used. Although mollusc gates and arthropod hemocyanin and hemoerythrin can be used, hemocyanin from keyhole limpet (KLH) is preferred in certain embodiments. Various polysaccharide antigen adjuvant can be used. For example, the use of pneumococcal polysaccharide antigen adjuvant for antibody response in mice is disclosed (Yin et al, 1989). Doses that do not generate optimal response or that do not cause inhibition should be used as directed (Yin et al, 1989). Polyamine variants of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin. In another embodiment, the muramyl dipeptide lipophilic disaccharide-tripeptide derivatives disclosed for use in artificial liposomes were formed from phosphatidyl choline and phosphatidyl glycerol. Other suitable antigen adjuvant include surface active agents that are bipolar such as saponins and derivatives such as QS21 (Cambridge Biotech). Saponin-based antigen adjuvant includes those containing substrate A and substrate C alone and in combination.

Nonionic block copolymer surfactants (Rabinovich et al, 1994) can be used. Oligonucleotides are another useful group of antigen adjuvant (Yamamoto et al, 1988). Another group of adjuvant agents are detoxified endotoxins, such as purified detoxified endotoxins of US Pat. No. 4,866,034. These purified detoxified endotoxins are effective in eliciting adjuvant responses in vertebrates. Of course, the detoxified endotoxin can be combined with other antigen adjuvant to prepare multi-antigen adjuvant formulations. For example, the combination of detoxified endotoxin and trehalose dimycolate is described in US Pat. Particularly contemplated, as disclosed in 4,435,386. The binding of detoxified endotoxin with trehalose dimycolate and endotoxin glycolipid is also contemplated (US Pat. No. 4,505,899), and the binding of detoxified endotoxin with cell wall backbone (CWS) or CWS and trehalose dimycolate is described in US Pat. Are considered as disclosed in 4,436,727, 4,436,728 and 4,505,900. Without detoxified endotoxin, the binding of CWS to trehalose dimycolate is also described in US Pat. It is believed to be effective as disclosed in 4,520,019. Alkyl lysophospholipids (ALP); BCG; And other kinds of antigen adjuvant capable of conjugation with vaccines, including biotin (including biotinylated derivatives). Particular antigen adjuvant that particularly considers use is teichoic acid derived from Gram cells. This includes lipoteichoic acid (LTA), ribitol teichoic acid (RTA) and glycerol teichoic acid (GTA). Active forms of such synthetic counterparts may also be used (Takada et al, 1995).

Various antigen adjuvant that is not commonly used in humans can still be used in other vertebrates, for example, when generating antibodies or subsequently obtaining active T cells. Toxicity or other adverse effects that may arise from cells, such as those that may occur using antigen adjuvant or non-irradiated tumor cells, are irrelevant to this environment. Other methods of inducing an immune response can be completed by formulating an immunogen of the present invention with an "immune enhancer". These are the body's own chemical messengers (cytokines) to increase the response of the immune system. Immune enhancers are various cytokines with immunostimulatory activity, immune enhancing activity and inflammation-inducing activity such as interleukin (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13). Caine, lymphokine and chemokine; Growth factors (eg granulocyte-macrophage (GM) -colony stimulating factor (CSF)); and other immune stimuli such as macrophage inflammatory factors, Flt3 ligands, B7.1, B7.2, etc. Molecules, including but not limited to, immune stimulatory molecules may be administered as the immunogen to the same agent or may be administered separately The protein or expression vector encoding the protein may be administered to produce an immunostimulatory effect. May be present in a range with the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg , About 7 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, or about 150 μg. Can be: about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 μg, about 80 Μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg or about 200 μg.In certain embodiments, the alum ranges from about 80 μg to about 120 μg or about 100 μg to about 120 μg Saponin-based antigen adjuvant may be present in a range with the following lower limits: about 0.2 μg, about 0.4 μg, about 0.6 μg, about 0.8 μg, about 1 μg, about 2 μg, about 3 Μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 9 μg, about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg.Saponin-based antigen adjuvant May be present in the range of: about 10 μg, about 15 μg, about 20 μg, about 25 μg, about 30 μg, about 35 μg, about 40 μg, about 45 μg, about 50 μg, about 60 μg, about 70 Μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg or about 200 μg. In certain embodiments, the saponin-based antigen adjuvant is about 5 μg to about 20 μg or about 1 μg to about 10 μg. Such dosages are particularly appropriate in mice and can be adjusted for human use based on a normal mouse weight of 20 g versus a human weight of about 60 kg.

In one embodiment of the present invention, MERS-CoV S protein, fragments or aggregates thereof, more preferably MERS-CoV S immunity comprising any one or more polypeptides selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8 Provided is a method of inducing prophylactic immunity against MERS-CoV infection comprising administering a native composition or a vaccine comprising the same.

MERS-CoV S Protein Immunogen Preparation

Production of Recombinant baculovirus Introduced with MERS-CoV S Gene

In one embodiment of the present invention, (a) introducing one or more MERS-COV S protein gene selected from the group consisting of SEQ ID NO: 1, 3, 5 and 7 to prepare a recombinant viral vector, and (b) the Provided is a method for producing a MERS-COV S protein antigen comprising the step of inoculating a recombinant virus vector into a host cell to culture the host cell to obtain a culture in which the MERS-COV S protein antigen is expressed.

The method may further comprise purifying the expressed MERS-COV S protein or fragment thereof.

The recombinant viral vector can be, for example, a phage, plasmid, virus or retroviral vector, preferably a viral vector can be used.

In one embodiment, the vector is a recombinant baculovirus vector. Constructs and / or vectors encoding genes should be operably linked to appropriate promoters, such as the AcMNPV polyhedrin promoter (or other baculovirus), the Faji Lambda PL promoter, the E. coli lac, phoA, and tac promoters. Preferably overexpressed by a polyhedrin promoter.

The expression constructs will further comprise ribosome binding sites for translation, at sites for transcription initiation, termination and transcribed regions. The coding portion of the transcripts expressed by the constructs will preferably initially comprise a translation initiation codon and a termination codon suitably located at the end of the polypeptide to be translated. Expression vectors will preferably comprise at least one selectable marker. Such markers include dihydrofolate reductase, G418 or neomycin resistance gene for eukaryotic cell culture and tetracycline, kanamicin, or impicillin resistance gene for E. coli and other bacterial cultures. . Among the vectors, Baculoviridae (eg, Autographa californica nucleopolyhedrovirus), Adenoviridae (eg, canine adenovirus), Hepadnavirus family (Hepadnaviridae (e.g., Avi hepadna virus), Vacciniaviridae (e.g., modified vaccinia Ankara virus), and Parvoviridae (e.g. Autono) Preference is given to any one or more viral vectors selected from the group consisting of mus parvovirus (Autonomous Parvovirus). The baculovirus may be selected from: Autographa californica nuclear polyhedral disease virus strains or modified virus strains thereof; Or Bombyx mori nuclear polyhedral disease virus strains or modified strains thereof. Bacterial vectors can also be used. Exemplary bacterial vectors include pQE70, pQE60 and pQE-9, p BlueScript vectors, phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5. Among the eukaryotic vectors, pFastBac1 pWINEO, pSV2CAT, pOG44, pXTl, pSG, pSVK3, pBPV, pMSG, pSVL and the like are preferred.

Recombinant vectors such as those described above can be used for transfection, infection or transformation and can express proteins in eukaryotic and / or prokaryotic cells. Eukaryotic host cells can include yeasts, insects, birds, plants, nymphs (or nematodes) and mammalian host cells. Non-limiting examples of insect cells are, for example, Spodoptera frugiperda (Sf) cells such as Sf9, Sf21, Trichoplusiani cells such as high five cells, and Drosophila S2 cells. Examples of fungal (including yeast) host cells include S. cerevisiae, Kluyveromyces lactis (K. lactis), C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica Candida species. Examples of mammalian cells include 293 cell line (human embryonic kidney lineage), CHO cell line (Chinese hamster ovary cell lineage), Vero cell line (African green monkey lineage), MRC cell line (human lung fibroblast cell lineage), and MDCK cell Madin-darby canine kidney cell lineage cells. Xenopus laevis oocytes or other cells from amphibian origin may also be used. Prokaryotic host cells include bacterial cells such as, for example, E. coli, B. subtilis, and mycobacteria.

Expression, isolation of MERS-CoV S protein, fragments or combinations thereof

MERS-CoV S proteins, fragments or combinations thereof, which act primarily as MERS-CoV immunogens, are produced from recombinant cell lines that are processed to produce proteins when the host cells grow in cell culture.

The cells are infected with the recombinant baculovirus vector at the most efficient multiplicity of infection. The most efficient multiplicity of infection is preferably between 0.001 and 5.0 MOI, more preferably between 0.002 and 4.0 MOI, between 0.003 and 3.0 MOI, between 0.004 and 2.0 MOI, even more preferably between 0.005 and 1.0 MOI, most preferably between 0.01 and 0.8 MOI. Can be.

The infection of the recombinant baculovirus can occur effectively when the cells are in the early-log phase of growth and in the 1.0E5-7.0E6 cell / ml concentration, preferably in the range of 5.00E5-1.50E6 cells / ml concentration.

Harvesting the cells after infecting the recombinant baculovirus preferably means that the viability of the cells is at least 1%, at most 99%, preferably at 50-98.9%, preferably at 60-98.5%, more preferably at 75-90%. Harvesting at 98% is effective.

Infectious conditions of the recombinant baculovirus can minimize the cleavage of the target protein by proteases flowing out of the cells in the killing step. In addition, the use of less virus inoculum can improve the yield of the culture.

The method for preparing a MERS-CoV S protein immunogen of the present invention may further comprise a step for isolation of the protein.

Specifically (C) separating the supernatant and cells by centrifuging the culture at 3000 to 8000xg for 1-30 minutes to obtain precipitated Sf9 cells, (d) lysing the precipitated Sf9 cells at pH 6.5-8.5 Cell lysis to obtain a lysate; And (e) centrifuging the lysate to remove the supernatant of the lysate to obtain only components of cells comprising the MERS-CoV antigen consisting of MERS-CoV S protein or fragments thereof. (F) The MERS- And selectively extracting only a portion of the cell component including the CoV antigen including the antigen including the MERS-CoV.

As mentioned above (e) 'mers of cells containing the MERS-CoV antigen' means a plasma membrane, rough ER, smooth ER, Golgi and the like.

The culture can be centrifuged to improve production yield by using precipitated Sf9 cells other than the medium. That is, without using the culture medium containing the culture as it is, the step of removing the supernatant after centrifugation, can lead to an increase in protein production.

Preferably the centrifugation of step (c) is carried out at a rate of 1000 to 20000xg, preferably at a rate of 1500 to 15000xg, more preferably at a rate of 2000 to 10000xg, most preferably at 3000 to 8000xg for 1-30 minutes. Can be.

Sequential centrifugation of step (e) may be performed through at least two steps.

In the first centrifugation, (i) the cell lysate can be centrifuged at a rate of 500 to 20,000 × g for 1-30 minutes to remove settled precipitates and take supernatant including cell membranes. Preferably the centrifugation of step (i) of (e) is at a speed of 700 to 18,000xg, preferably at a rate of 1,000 to 15,000xg, more preferably at a speed of 1,500 to 10,000xg, most preferably 2,000 to 7,000 This can be done for 1-30 minutes at xg.

In the second centrifugation, (ii) the supernatant of step (i) was ultracentrifuged at 4-10 ° C. at 10,000 to 400,000 × g for 60-420 minutes to obtain a precipitate, and the supernatant was removed to remove MERS-CoV S protein or fragment thereof. Only components of these contained cells can be taken. Preferably the centrifugation of step (ii) of (e) is at a rate of 10,000 to 400,000xg, preferably at a rate of 20,000 to 350,000xg, more preferably at a rate of 35,000 to 300,000xg, most preferably 50,000 to 250,000 60-420 minutes at xg.

The extraction process of step (f) may be performed through at least two steps.

(I) MERS-CoV S protein by adding an extraction buffer from the components of the cell containing the MERS-CoV antigen consisting of the MERS-CoV S protein or fragments thereof obtained after completion of forward dimensional separation in step (e) Extract only the part that contains a fragment of it,

(ii) the contents secured by the extraction of (i) were ultracentrifuged at 5,000 to 300,000 × g at 4-10 ° C. to discard the supernatant to ensure only precipitate. Preferably the centrifugation of step (ii) of (f) is at a rate of 7,000 to 200,000xg, preferably at a rate of 10,000 to 250,000xg, more preferably at a rate of 15,000 to 200,000xg, most preferably of 30,000 to 150,000 It can be done in xg.

The antigen production method may further comprise the step of purifying the MERS-COV antigen consisting of (g) MERS-CoV S protein or fragments thereof. Purifying the antigen may include at least two purification steps.

The purification is carried out by (i) cation exchange chromatography (anion exchange chromatography),

(ii) Purification can be carried out by glucose affinity chromatography.

The purification method using the chromatography may use a method commonly used in the industry to purify a protein. For example, 1: Robert K. Scopes, Protein Purification: Principles and Practice, 1993, Springer Science & Business Media, or 2: Coelho, LCBB et al., Protein Purification (Edited by Rizwan Ahmad): chapter 3 affinity chromatography, 2012, Intech, DOI: 10.5772 / 1287 is available.

The present invention provides a MERS-CoV antigen composition having high production capacity (yield) and a method for preparing the same.

The MERS-CoV vaccine of the present invention provides high antibody titers.

The present invention can improve the yield of the antigen produced by the MERS-CoV virus, it is possible to optimize the process efficiency by minimizing the amount of virus inoculum.

The present invention can save the time and cost required to obtain the MERS-CoV antigen.

1 is a detailed virological classification of MERS-CoV.

2 is a diagram illustrating a gene encoding the MERS-CoV S protein. Where SP stands for signal peptide. Here, S1 and S2 mean S1 and S2 sub-domains, respectively. In addition, RBD means receptor binding domain and TM means trans-membrane domain.

Figure 3 is a WB result used in the first purification and confirmation of MERS-CoV S by Anion exchange chromatography. Anion exchange chromatography used here is TMAE.

Figure 4 shows the results of performing UD / DF with a filter membrane after performing the secondary purification using affinity chromatography. Affinity chromatography here is Lentil Lectin resin chromatography.

5 is the DLS Zeta-average value of purified MERS-CoV S antigen.

6 is a transmission electron microscopy photograph of the purified MERS-CoV S antigen.

7A and 7B are diagrams illustrating an animal experiment schedule. The animal used here is mouse. Injection means the administration of the MERS-CoV S antigen. The antigen administered may include an immune enhancer. Where W represents one week.

Figure 8 is a graph of the results of the measurement of total antibody titers to determine the mouse immunogenic induction of MERS-COV S antigen according to the presence and dosage of aluminum-based adjuvant. Total antibody titer is the value confirmed by ELISA analysis. Where GMT is Geometric Mean Titer. Here, "2.5ug + Adjuvant", "8ug + Adjuvant", and "25ug + Adjuvant" are substances which adsorb 2.5 ug, 8 ug, and 25 ug of MERS-COV S antigen to the aluminum adjuvant, respectively. Here, "PBS + Adjuvant" is a material obtained by mixing aluminum adjuvant with sterile phosphate buffer saline.

Figure 9 is a graph of the results of the measurement of total antibody titers to determine the mouse immunogenic induction of MERS-CoV S antigen according to the number of administration and the blood collection time. Total antibody titer is the value confirmed by ELISA analysis. Where GMT is Geometric Mean Titer. "2.5ug + Adjuvant" and "8ug + Adjuvant" are the substances which adsorbed 2.5ug and 8ug of MERS-CoV S antigen to the aluminum adjuvant, respectively. Here, "PBS + Adjuvant" is a material obtained by mixing aluminum adjuvant with sterile phosphate buffer saline. "1st boosting" herein means a second administration after the first MERS-CoV S antigen. "2nd boosting" here means a third dose. "Week 2" herein means blood collection two weeks after administration. “Week 4” herein means blood collection 4 weeks after administration.

Figure 10 is a graph of the results of the antigen-RBD immunospecific IgG antibody titer measurement experiment to confirm that the antibody induced after administration of the expressed / purified immunogen in this example shows immunogenicity. The antigen-RBD immunospecific IgG antibody titer is a value identified through a specific antibody sandwich ELISA method. Wherein the specific antibody is a 1E9 monoclonal antibody that specifically binds to RBD. "8ug + Adjuvant" are substances that adsorb 8ug of MERS-CoV S antigen to aluminum adjuvant. Here, "PBS + Adjuvant" is a material obtained by mixing aluminum adjuvant with sterile phosphate buffer saline.

11-18 show the MERS-CoV S gene and polypeptide sequences of the present invention.

Hereinafter, examples and the like will be described in detail to help understand the present invention. However, embodiments according to the present invention can be modified in many different forms, the scope of the invention should not be construed as limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

Example  One. MERS - CoV  S gene introduction recombination baculovirus  production

Accession No. The antigen obtained by optimizing the codon of KF186567 (Al-Hasa_1_2013) MERS-CoV S protein was prepared. The MERS-CoV S gene described is cloned to make MERS-CoV S protein recombinant baculovirus (AcMNPV) and then inoculated into insect cell, Sf9.

The usual cell concentration of Sf9 insect cells should not exceed 3.00E6 / ml. Sf9 insect cells are cultured in sterile 125ml / 250ml / 500ml / 1L spinner flasks or 5L / 50L / 100L bioreactor. The medium used for culturing in this example uses serum-free insect cell specialized medium without serum. Here, the serum-free insect cell specialized medium may include Insect Express (Lonza). In this example, the MERS-CoV S recombinant baculovirus for MERS-CoV S production is inoculated between 0.01-0.8 MOI (Multiplicity Of Infection), where the concentration of Sf9 insect cells is 5.00E5-1.50, where the growth phase is the initial phase of the exponential phase. Use concentration of E6. The MERS-CoV S recombinant baculovirus used in this example is harvested at 60-90 hours post inoculation, with viability of 75-98%. The reason for inoculating low growth cells into low growth cells and harvesting at relatively high viability in this example is to minimize target protein cleavage by proteases exiting the killing cells and reduce the amount of virus inoculum used. This is to minimize.

Example  2. MERS - CoV  Expression, Purification and Analysis of S Antigens

The polypeptide immunogen of SEQ ID NO: 4 is obtained by the following method.

First, the Sf9 insect cells obtained after recombinant baculovirus inoculation in Example 1 are centrifuged at 3000-8000xg for 1-30 minutes to separate the cell sedimentation layer and the media supernatant. Remove the separated media supernatant and secure the precipitated cells. Precipitated Sf9 cells are dissolved by adding lysis buffer. The lysis buffer contains a suitable buffer and salt, and the pH is between 6.5 and 8.5.

The lysed lysate is subjected to sequential centrifugation to separate the cell membrane containing the MERS-CoV S protein. In this example, the sequential centrifugation method is as follows. The dissolved and crushed cell lysate is centrifuged at 2000-7000xg for 1-30 minutes to remove precipitates and take the supernatant. The sediment contains nuclei, some mitochondria, lysosomes, peroxisomes, and the supernatant contains residual lysosomes and peroxisomes, mitochondria and cytosolic proteins, microsomes, ER, Golgi, and cell membranes. The supernatant is again ultracentrifuged at 50,000 ° C. to 50,000-250,000 × g for 60-420 minutes at 4 ° C. to take the precipitate and remove the supernatant.

The separated cell membrane was added to the extraction buffer to crush the cell membrane and centrifuged at 30,000-150,000 × g using an ultracentrifuge to extract the stock solution containing the MERS-CoV S protein. Extraction buffers contain appropriate buffers, non-ionic detergents and salts, and have a pH between 6.5 and 8.5.

Extraction stock containing MERS-CoV S protein is subjected to the first purification by anion exchange chromatography. In the present application, primary purification was performed using TMAE chromatography and AKTA explorer (HPLC, BD). In this example, TMAE used Fractogel TMAE Hicap (M) resin (Merck), and loaded with the extracted crude sample at a pressure not exceeding 0.3 megapascals (MPa). The sample was loaded and washed with a loading buffer, and the solution was irradiated with 7 CV by the illution buffer. The loading buffer and the elution buffer include Tris, Triton X-100, NaCl, etc., and the pH is between 7.0-8.5. (Figure 3) Secondary purification of the purified crude solution subjected to primary purification by glucose affinity chromatography. In this example, the second tablet was made of lentil lectin resin (lentil lectin sepharose 4B), and loading and reduction were performed at a pressure of less than 0.3 megapascals (MPa). The loading buffer contains Tris, NaCl, CaCl 2, tween80, MnCl 2, and the pH is between 7.0-8.5. Illution buffer is composed of borate, methyl mannoside, methyl glucoside, and the pH is between 5.0-6.5. After the second purification, the stock solution is filtered and concentrated by ultrafiltration and diafiltration using a filter membrane. (FIG. 4) The Zeta-average value measured by DLS of the finally filtered and concentrated antigen is 20-100 nm (FIG. 5), and the antigen is observed using an electron microscope. (Figure 6)

In this example, the protein is diluted with 2xSDS sample buffer containing βME (beta-mercaptoethanol) in each purification process, filled with 15-20 μl of each well of SDS-gel, followed by electrophoresis. Total proteins were stained by In addition, SDS-gel was transferred to the membrane and further analyzed by WB (Western blot) using an anti-single or complex MERS-COV S specific antibody. WB incubated the protein-transferred membrane with buffer solution (PBS) containing 5% skim milk (Sigma) at 4 ° C for 4 to 24 hours, washed with buffer, and diluted 1: 2000 after MERS-COV S specificity. The antibody is added and incubated at 4 ° C. for 4-24 hours. After incubation, washed three times with a buffer solution containing 0.1% tween20, added mustard peroxidase (HRP) binding Goat anti-rabbit IgG antibody (Invitrogen, US) diluted 1: 5000, and at room temperature for 1-2 hours. Incubate during. After incubation, the membrane is washed three times with 0.1% tween20 buffer solution, developed with ECL detection reagent (Amersham), and photographed by film.

Example  3. MERS - CoV  Animal Administration / Immune Induction with S Antigen

Animal experiments using mice were performed with the antigens secured in this example. The mouse used in the experiment can be used by itself as the antigen used in animal experiments, and may be used with an immune enhancer such as an aluminum adjuvant. The adjuvant may include, for example, aluminum or calcium salts, specifically inorganic salts such as hydroxide, phosphoric acid, calcium phosphate and the like.

The adjuvant of the aluminum (Aluminum or Alum) family is the most commonly used adjuvant in human vaccines, so the adjuvant has proven its stability and effectiveness to be used as a standard for developing and evaluating new adjuvants. to be. The most commonly used alum adjuvant is aluminum hydroxide (Al (OH) 3) and aluminum phosphate (AlPO4). The two alum adjuvant have different physical and adjuvant properties. In particular, aluminum hydroxide is the most widely used in the aluminum adsorption vaccine, in this example, aluminum hydroxide was used as an immune enhancer, where aluminum hydroxide was used at a level of 180 ug / 1 dose.

In this example, the concentrations of MERS-CoV S antigen administered were 2.5 ug / ml, 8 ug / ml, 25 ug / ml, and the number of administrations was differently applied to the two-dose group and the three-dose group. It was. In this example, the administration schedule was performed by two administrations after two weeks of first administration and three administrations after two weeks of two administrations. Administration group only). (FIG. 7A, FIG. 7B)

Example  4. MERS - CoV  Identification of immunogenicity in animals administered S antigen

The MERS-CoV S antigen obtained in this example was confirmed to be immunogenic induction effect depending on the presence or absence of aluminum hydroxide adjuvant, dosage, frequency of administration, and blood collection time. Confirmation of the immunogenicity induced in this example was carried out through the measurement of total antibody and neutralizing antibody.

In this example, the total antibody titer to confirm antigen-specific IgG antibody titers was performed using the ELISA method. MERS-CoV S antigen was coated on a 96-well plate by 100ng per well and incubated overnight at 4 ° C. After incubation, the plate was washed three times with 0.1% tween20 buffer solution, incubated with 5% skim milk buffer solution for 4 hours, and washed again with 0.1 tween20 buffer solution three times. The obtained mouse blood is centrifuged to obtain a serum sample, and the prepared serum sample is diluted to an initial concentration of 1/20 and then serially diluted four times to make a final concentration of 1/36980 times. Diluted serum samples were incubated at room temperature for 2 hours at 100 µl each in a washed 96-well plate, and washed three times with a buffer solution containing 0.1% tween20. 100 μl of mustard peroxidase (HRP) -binding Goat anti-mouse IgG antibody (Invitrogen, US) diluted 1: 3000 was added to the washed plate, incubated at room temperature for 2 hours, and buffered with 0.1% tween20. Wash three times with solution. 3,3A, 5,5A-tetramethylbenzidine substrate (TMB substrate, KPL) was added to each well of the washed plate and reacted with HRP for 10 minutes, and then TMB stop solution (KPL) was added to stop the reaction. . The absorbance 450 nm value for each well was measured using a Microtiter plate reader (Molecular Devices), and the total antibody titer measured in this example was confirmed that the expressed / purified MERS-COV S antigen induces high immunogenicity. (FIG. 8A, FIG. 9)

In this example, the antigen-RBD immunospecific IgG antibody titer was used to confirm that the MERS-COV S immunogen expressed / purified exactly contains a receptor binding domain (RBD) that is known to bind to the cell's DPP4 receptor and cause a neutralizing immune response. Was measured by a specific antibody sandwich ELISA method. 100 ng of 1E9 RBD specific antibody that specifically binds to the MERS-CoV S RBD portion of the 96-well plate was coated and incubated overnight at 4. After incubation, the plate was washed three times with 0.1% tween20 buffer solution, incubated with 5% skim milk buffer solution for 4 hours, and washed again with 0.1 tween20 buffer solution three times. Subsequently, MERS-COV S antigen was added to a plate coated with 1E9 RBD specific antibody (Xian-Chun Tang et al. PNAS 2014; 111: E2018-E2026) at 100ng / well, followed by incubation at room temperature for 2 hours, and 0.1% tween20. Wash three times with the included buffer solution. The prepared serum samples are diluted to the initial concentration of 1/20 and then serially diluted 4 times to make the final concentration 1/36980 times. Diluted serum samples were incubated at room temperature for 2 hours at 100 µl each in a washed 96-well plate, and washed three times with a buffer solution containing 0.1% tween20. 100 μl of mustard peroxidase (HRP) -binding Goat anti-mouse IgG antibody (Invitrogen, US) diluted 1: 3000 was added to the washed plate, incubated at room temperature for 2 hours, and buffered with 0.1% tween20. Wash three times with solution. 3,3A, 5,5A-tetramethylbenzidine substrate (TMB substrate, KPL) was added to each well of the washed plate and reacted with HRP for 10 times. Then, TMB stop solution (KPL) was added to stop the reaction. . The absorbance 450 nm values for each well were measured using a Microtiter plate reader (Molecular Devices), and the antigen-RBD immunospecific IgG antibody titers measured in this example were expressed / purified by the MERS-COV S immunogen. This results in an effective labeling of the functioning RBD moiety and induction of antibodies to the RBD moiety upon administration of an immunogen. (Figure 10)

Similar results were obtained in an immune induction reaction using the polypeptide antigen of SEQ ID NO: 2.

The present invention can be used as a MERS-CoV vaccine having a high antibody titer.

The present invention provides a MERS-CoV vaccine with reduced time and cost required for antigen acquisition.

Claims (34)

1. MERS-CoV S immunogenic composition comprising any one or more polypeptides selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.
2. The MERS-CoV S immunogenic composition of claim 1, wherein the immunogenic composition comprises a polypeptide of SEQ ID NO: 2 or 4. 3.
3. A MERS-CoV preventive vaccine comprising an immunogenic composition comprising any one of the MERS-CoV S polypeptides, selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8.
4. The vaccine for preventing MERS-CoV according to claim 3, wherein the vaccine further comprises an adjuvant or an immune enhancer.
5. Preventing prophylactic immunity against MERS-CoV infection, comprising administering a MERS-CoV S immunogenic composition comprising at least one polypeptide selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 8 or a vaccine comprising the same How to induce.
6. (a) preparing a recombinant viral vector by introducing any one or more MERS-CoV S protein genes selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7; And

   (b) inoculating the recombinant viral vector into a host cell to culture the host cell to obtain a culture in which the MERS-CoV S protein immunogen is expressed.

   Method for producing a MERS-CoV S protein immunogen comprising.
7. The method of claim 6, wherein the method further comprises purifying the expressed MERS-CoV S protein or fragment thereof.
8. The method of claim 6, wherein the recombinant viral vector is any one selected from the group consisting of Baculoviridae, Adenoviridae, Hepadnaviridae, Vacciniaviridae, and Parvooviridae. Method of producing a MERS-CoV S protein immunogen, characterized in that the above viral vector.
9. 10. The method of claim 8, wherein said recombinant viral vector is a baculovirus vector.
10. 10. The method of claim 9, wherein the baculovirus comprises: Autographa californica nuclear polyhedral disease virus strains or modified virus strains thereof; or

    A method for producing a MERS-CoV S protein immunogen, comprising Bombyx mori nuclear polyhedral disease virus strain or modified virus strain thereof.
11. The method of claim 6, wherein the host cell is E. coli , Bacillus subtilis (B. subtilis ) and one of the prokaryotic host cells selected from the group consisting of mycobacteria;

    Human embryonic kidney lineage, Chinese hamster ovary cell lineage, African green monkey lineage, Vero cell line, human lung fibroblast cell lineage, and MDCK cell line (madin-darby) any one mammalian host cell selected from the group consisting of canine kidney cell lineage; And

    Sf9, Sf21 cells, and high-five (High-Five) cells, characterized in that any one or more selected from the group of insect host cells selected from the group consisting of, MERS-CoV S protein immunogen production method.
12. The method of claim 6,

    Step (a) is a step of preparing a recombinant baculovirus vector by introducing at least one MERS-CoV S protein gene selected from the group consisting of SEQ ID NOs: 1, 3, 5, and 7,

    The step (b) is a method for producing a MERS-CoV S protein immunogen, characterized in that the recombinant baculovirus vector infects Sf9 insect cells at a concentration of 5.00E5-1.50E6 at 0.001 to 5 MOI.
13. The method of claim 12, wherein the recombinant baculovirus vector is infected with Sf9 insect cells at 0.002 to 4.0 MOI.
14. The method of claim 12, wherein the recombinant baculovirus vector is infected with Sf9 insect cells at 0.003 to 3.0 MOI.
15. The method of claim 12, wherein the recombinant baculovirus vector is infected with Sf9 insect cells at 0.004 to 2.0 MOI.
16. The method of claim 12, wherein the recombinant baculovirus vector is infected with Sf9 insect cells at 0.005 to 1.0 MOI.
17. The method of claim 12, wherein the recombinant baculovirus vector is infected with Sf9 insect cells at 0.01 to 0.8 MOI.
18. The method of claim 12, wherein the MERS-CoV S protein antigen is obtained in a state in which the survival rate of Sf9 cells is 1 to 99%.
19. 19. The method of claim 18, wherein the MERS-CoV S protein antigen is obtained in a state where the survival rate of Sf9 cells is 75 to 98%.
20. The method of claim 12, wherein the immunogen manufacturing method

    (c) centrifuging the culture at 1,000 to 20,000 × g for 1-30 minutes to separate supernatant and cells to obtain precipitated Sf9 cells;

    (d) cell lysis of the precipitated Sf9 cells with a lysis buffer at pH 6.5-8.5 to obtain a lysate;

    (e) centrifuging the lysate sequentially to remove the supernatant of the lysate to obtain only components of cells containing MERS-CoV S protein or fragments thereof; And

    (f) selectively extracting only a portion of the cell component including the MERS-CoV antigen-containing antigen containing MERS-CoV, MERS-CoV S protein immunogen.
21. The method of claim 20,

    Step (c) is characterized in that the culture is centrifuged at a rate of 1,500 to 15,000xg, MERS-CoV S protein immunogen production method.
22. The method of claim 20,

    Step (c) is characterized in that the culture is centrifuged at a rate of 2,000 to 10,000xg, MERS-CoV S protein immunogen production method.
23. The method of claim 20,

    Step (c) is characterized in that the culture is centrifuged at a rate of 3,000 to 8,000xg, MERS-CoV S protein immunogen production method.
24. The method of claim 20, wherein the step (e) sequential centrifugation

    (i) centrifuging the lysate at 500-20,000 × g for 1-30 minutes to remove precipitates and take supernatant including cell membranes; And

    (ii) the supernatant of step (i) was ultracentrifuged at 50,000-250,000 × g for 60-420 minutes at 4-10 ° C. to obtain a precipitate, and the supernatant was removed to form a cell containing MERS-CoV S protein or fragment thereof. A method of making a MERS-CoV S protein antigen, comprising taking only urea.
25. The method of claim 24,

    Step (i) of the sequential centrifugation of step (e) is a step of centrifuging the lysate at 500 to 20000xg for 1-30 minutes to remove precipitates and take supernatant including cell membranes. Method for preparing a MERS-CoV S protein immunogen.
26. The method of claim 24,

    Step (i) of the sequential centrifugation of step (e) is a step of centrifuging the lysate at 1,000 to 15,000 × g for 1-30 minutes to remove precipitates and take supernatant including cell membranes. To prepare a MERS-CoV S protein immunogen.
27. The method of claim 24,

    Step (i) of the sequential centrifugation of step (e) is a step of centrifuging the lysate at 1,500 to 10,000xg for 1-30 minutes to remove precipitates and take supernatant including cell membranes. To prepare a MERS-CoV S protein immunogen.
28. The method of claim 24,

    Step (i) of the sequential centrifugation of step (e) is a step of centrifuging the lysate at 2,000 to 7,000xg for 1-30 minutes to remove precipitates and take supernatant including cell membranes. To prepare a MERS-CoV S protein immunogen.
29. The method of claim 24,

    Step (e) of step (e) is a method for producing a MERS-CoV S protein immunogen, characterized in that the supernatant of step (i) is ultracentrifuged at a rate of 10,000 to 400,000xg to take a precipitate.
30. The method of claim 24,

    Step (ii) of step (e) is a method for producing a MERS-CoV S protein immunogen, characterized in that the supernatant of step (i) is ultracentrifuged at a rate of 20,000 to 350,000xg to take a precipitate.
31. The method of claim 24,

    Step (ii) of step (e) is a method for producing a MERS-CoV S protein immunogen, characterized in that the supernatant of step (i) is ultracentrifuged at a rate of 35,000 to 300,000xg to take a precipitate.
32. The method of claim 24,

    Step (ii) of step (e) is the method of producing a MERS-CoV S protein immunogen, characterized in that the supernatant of step (i) is ultracentrifuged at a rate of 50,000 to 250,000xg to take a precipitate.
33. The method of claim 20, wherein the extracting of the step (f)

    (i) adding MERS-CoV S protein or fragment thereof by adding an extraction buffer from a cell component comprising a MERS-CoV antigen obtained from the obtained MERS-CoV S protein or fragment thereof Selectively extracting portions;

    (ii) preparing the MERS-CoV S protein immunogen, comprising the step of discarding the supernatant by ultracentrifuging the contents obtained by the extraction of (i) at 5,000 to 300,000 × g at 4-10 ° C. .
34. The method of claim 20, wherein the immunogen manufacturing method

    (g) purifying the MERS-CoV antigen consisting of the MERS-CoV S protein or fragment thereof,

    The purification is performed by (i) primary purification by cation exchange chromatography,

    (ii) A method for producing a MERS-CoV S protein immunogen, characterized in that secondary purification is performed by glucose affinity chromatography.

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