KR101366702B1 - Vaccine composition for respiratory syncytial virus and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a vaccine composition for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV) and a method of manufacturing the same. In particular, the invention is induced by RSV, comprising a nucleic acid sequence encoding all or a portion of a peptide fragment comprising from 120 to 230 amino acids from the N-terminus of the respiratory Synsichia virus (RSV) G protein. Prokaryotic cell expression vector for vaccine preparation for preventing and treating respiratory diseases, Prokaryotic cell transformant for vaccine production for preventing and treating respiratory diseases caused by respiratory synsychia virus (RSV) transformed by the expression vector, Prokaryotic cell Method for producing peptide fragment of RSV G protein for vaccine for preventing and treating respiratory disease caused by RSV using transformant, 120th from N-terminus of RSV G protein By RSV comprising all or part of a peptide fragment comprising amino acids to 230 th amino acid The present invention relates to a vaccine composition for preventing and treating induced respiratory diseases, and a method for preventing and treating respiratory diseases caused by RSV infection, comprising administering the vaccine composition to a subject. The vaccine comprising the prokaryotic expressing RSV G protein fragment according to the present invention induces very high serum IgG and respiratory mucosal IgA even with two immunizations, and is effective against RSV replication without eosinophilic immunopathology due to the vaccine upon RSV infection. Indicates defense. In addition, it is economical as a vaccine using a purified protein from bacteria and by using the purified protein fragment as an antigenic vaccine, overcoming the stability problems that can be caused in the viral vaccine, which is the main target of RSV vaccine Given the newborns and infants, this is a beneficial effect. In addition, it has an effect of increasing the stability of vaccination because it shows sufficient immunity even in the sublingual administration excellent in stability.

Description

Vaccine composition for respiratory syncytial virus and manufacturing method

The present invention relates to a vaccine composition for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV) and a method of manufacturing the same. In particular, the invention is induced by RSV, comprising a nucleic acid sequence encoding all or a portion of a peptide fragment comprising from 120 to 230 amino acids from the N-terminus of the respiratory Synsichia virus (RSV) G protein. Prokaryotic cell expression vector for vaccine preparation for preventing and treating respiratory diseases, Prokaryotic cell transformant for vaccine production for preventing and treating respiratory diseases caused by respiratory synsychia virus (RSV) transformed by the expression vector, Prokaryotic cell Method for producing peptide fragment of RSV G protein for vaccine for preventing and treating respiratory disease caused by RSV using transformant, 120th from N-terminus of RSV G protein By RSV comprising all or part of a peptide fragment comprising amino acids to 230 th amino acid The present invention relates to a vaccine composition for preventing and treating induced respiratory diseases, and a method for preventing and treating respiratory diseases caused by RSV infection, comprising administering the vaccine composition to a subject.

Lower respiratory tract infections caused by Respiratory Syncytial Virus (RSV) cause pneumonia and bronchitis, and have long been considered a fatal problem worldwide, including in the United States, Europe, Australia and Japan. In particular, premature babies, children and the elderly, and eventually can cause problems in all individuals with a weakened immune system. It is estimated that about two-thirds of infants under 1 year of age and almost all infants between 1 and 4 years of age are infected with RSV at least once, and most recover without medical assistance. However, 5 to 10% of them persist in severe infections, particularly fatal to pulmonary dysplasia, congenital heart disease, cystic fibrosis, infants with cancer or various immunodeficiencies, and adults with hypoimmune conditions before bone marrow transplantation. It is a high risk group where infection can occur. In the elderly, the infection is similar to that of the influenza virus, and the excess mortality during the RSV epidemic was reported to be higher than during the influenza epidemic. There are 100,000 to 200,000 high-risk patients in the United States, with hospitalizations of more than 90,000 RSV infections each year, of which about 4,500 are reported to die. In Korea, RSV is prevalent every year, and a large number of patients are admitted to pediatric hospitals. 60% of all viral lower extremity infections isolated from Seoul National University Children's Hospital are caused by RSV. It is estimated that there will be many deaths.

Respiratory syncytial virus (RSV) is a genome of non-segmented RNA and belongs to paramyxoviridae. It is largely divided into A subtype and B subtype, and A subtype is generally dominant species. RSV synthesizes 10 proteins, including NS1, NS2, P, N, M, SH, G, F, M2 and L, in infected host cells. The N (nucleocapsid), P (phosphorus) and L (large polymerase subunit) proteins of RSV form the nucleoprotein core, which is considered the smallest infectious unit (Brown et al., J. Virol, 1). : 368-373, 1967), forming viral RNA dependent RNA transcriptases responsible for transcription and replication of the RSV genome (Yu et al., J. Virol. 69: 2412-2419, 1995; Grosfeld et al., J Virol. 69: 5677-5686, 1995). The M2 protein is known to induce an immune response by cytotoxic T lymphocytes involved in the removal of virus-infected cells. Recent studies have shown that M2 gene products (M2-1 and M 2-2) It has been shown to be involved in this transcription (Collins et al., 1996, Proc. Natl. Acad. Sci. 93: 81-85). Surface antigens include F protein (fusion protein), G protein, and SH protein. Among them, G protein and F protein, which are relatively high molecular weights and are present in the virus's outer membrane, are glycosylated glycosylated proteins. It plays the most important role in the variation of antigenicity. G proteins are involved in attachment to host cells to be infected, F proteins are involved in the formation of giant cells and invasion and attachment of viruses, and F and G proteins cause neutralizing antibodies.

The G protein does not have an MHC I species-restricted epitope and therefore does not induce an immune response by cytotoxic T lymphocytes (CTLs). The protein, however, has an IE ^d -restricted immunodominant epitope, an MHC II molecule from amino acid sequences 183 to 195, which induces many pathological CD4 T cells expressing Vβ4. Many studies have shown that immunization with RSV G protein and subsequent RSV infection enhances Th2 type immune responses with respiratory eosinophilia (Hancock et al., J Virol 70: 7783-91, 1996; J Virol et al., 72: 2871-80, 1998; Openshaw, et al., Int Immunol 4: 493-500, 1992; Tebbey, et al., J Exp Med 188: 1967-72, 1998).

Currently no vaccines or specific therapies have been developed for RSV infection. In particular, formalin-inactivated RSV vaccine (FI-RSV) was developed in the past and clinical trials were conducted.However, 80% of children who received FI-RSV were hospitalized due to RSV infection. And severe lower respiratory tract disease in the lungs. Currently, the approved treatment is Ribavirin (Ribavirin, Virazole) is used, but the clinical efficacy is low, high toxicity, and the inconvenience of spraying in the form of aerosol (aerosol) is used only in limited cases. In addition, several countries, including the United States, use palivizumab (synagis), a monoclonal antibody to RSV intravenous immunoglobulins (RSV-IGIV, Respigam) or RSV F protein, to prevent severe RSV infection in high-risk groups. U.S. Patent Publication 2006/0018925 describes and claims antibodies and small peptides that can block the interaction of G proteins with the CX3C region of their receptors, but because they are prophylactic and expensive rather than therapeutic treatments of RSV, Not available to many people. Therefore, the development of a treatment for RSV is very urgent.

There are several important points to consider when developing an RSV vaccine. Firstly, the main target of the RSV vaccine is infants, which should be safe. Second, simple administration should be possible for infants who are the main targets of the vaccine, and third, they should not be neutralized by the parental antibody.

The present inventors have tried to develop a safer, more economical, and more effective RSV vaccine. As a result, when some sequences of RSV G protein are expressed and purified in prokaryotic cells, especially Escherichia coli, they do not cause immunopathy such as eosinophilia. The present invention has been completed and found to be an effective defense against RSV infection and increased safety compared to infection with viral vector conditions. In addition, when administered using a sublingual administration method that is commonly used to induce immune tolerance and prevent an immune response such as an allergic reaction, the stability is increased, the administration is easy for infants, and effective immunity against RSV infection. It was found that the reaction can be completed to complete the present invention.

One object of the present invention For the prevention and treatment of RSV-induced respiratory diseases comprising a nucleic acid sequence encoding all or part of a peptide fragment comprising the 120th to 230th amino acids from the N-terminus of the respiratory synsychia virus (RSV) G protein It is to provide a prokaryotic expression vector for vaccine production.

Another object of the present invention is to provide a prokaryotic transformant for the production of a vaccine for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV), which is transformed by the expression vector.

Another object of the present invention is to culture the prokaryotic transformant; And it provides a method for producing a peptide fragment of RSV G protein for vaccines for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV) comprising the step of separating and purifying the peptide expressed from the transformant.

Another object of the present invention is to prevent respiratory diseases caused by RSV comprising all or part of a peptide fragment comprising 120 to 230 amino acids from the N-terminus of the respiratory Synsichia virus (RSV) G protein. And it provides a therapeutic vaccine composition.

It is another object of the present invention to provide a method for preventing and treating respiratory diseases caused by RSV infection, comprising administering the vaccine composition to a subject.

As one embodiment for achieving the above object, the present invention provides a nucleic acid sequence encoding all or part of a peptide fragment comprising 120 to amino acid from the N- terminus of the respiratory Synsichia virus (RSV) G protein Prokaryotic expression vector for the production of vaccines for preventing and treating respiratory diseases caused by RSV to provide.

In the present invention, the G protein of RSV is a major adhesion protein of RSV, and as a protein important for RSV infection defense, wild type nucleotides (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of RSV G protein are known in the art. (Wertz et al., Proc. Natl. Acad. Sci. USA 92: 4075-4079, 1985; Satake et al., Nucl.Acids Res. 13 (21): 7795-7810, 1985). The G protein does not have an MHC I species-restricted epitope and therefore does not induce an immune response by cytotoxic T lymphocytes. The protein, however, has an IE ^d -limited immunodominant epitope from amino acids 183 to 195, which induces many CD4 T cells expressing Vβ4.

In addition, the RSV G protein used by this invention contains the variant of G protein as well as the protein which has a wild-type amino acid sequence. Variant of a G protein means a protein having a sequence different from the native wild type amino acid sequence by deletion, insertion, non-conservative or conservative substitution, or a combination of one or more amino acid residues from the native wild type amino acid sequence of the G protein. In addition, RSV G proteins may be derivatized to include molecules that enhance antigen presentation and enhance antigen targeting to antigen presenting cells.

As used herein, "G protein fragment of RSV" refers to all or a portion of a peptide fragment comprising amino acids 120 to 230 from the N-terminus of the respiratory synshichia virus (RSV) G protein, and refers to "respiratory synshichia virus." Some of the peptide fragments comprising amino acids 120 to 230 from the N-terminus of the (RSV) G protein are non-contiguous or consecutive portions of the sequence from amino acids 120 to 230 of the N-terminus of the RSV G protein. It means a peptide fragment comprising a. The RSV G protein fragment used in the embodiment of the present invention comprises amino acids 120 to 230 amino acids from the N-terminus (SEQ ID NO: 3), comprising 131 amino acids to 230 amino acids (SEQ ID NO: 4). May be used, but is not limited thereto, and all peptide fragments including some sequences which are non-contiguous or consecutive among the 120-amino acid sequence from the N-terminus of the RSV G protein may be used. The RSV G protein fragment may be in the form of monomers of all or a portion of a peptide fragment comprising amino acids 120-230 from the N-terminus, but a large amount of two or more of the peptide fragments linked in whole or in part to further enhance immunogenicity. Sieve form.

The nucleic acid sequence encoding the RSV G protein fragment of the present invention can optimize the amino acid translation codons in order to increase the expression of the RSV G protein fragment, and in embodiments of the present invention, the nucleic acid sequence of SEQ ID NO: 5 or 6 It may have, but is not limited thereto.

The G protein fragment of RSV of the present invention can be expressed using prokaryotic cells, preferably E. coli as a host. Generally, since protein post-translational modifications affect the immunogenicity of proteins, antigen protein expression has been performed in eukaryotic cells such as animal cells. However, the inventors have found that the RSV G protein fragment of the present invention has excellent immunogenicity even when expressed in prokaryotic cells, preferably E. coli. Thus, vaccines can be produced economically and are safer than infection with viral vector conditions because they are administered in the form of purified proteins.

As used herein, the term “expression vector” refers to a recombinant vector capable of expressing a protein of interest in a suitable host cell, wherein the gene construct includes essential regulatory elements operably linked to express the gene insert. Standard recombinant DNA techniques can be used for the preparation. As used herein, the term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein to perform a general function. For example, a nucleic acid sequence that encodes a promoter and a protein may affect the expression of a nucleic acid sequence that is operably linked. The type of recombinant vector is not particularly limited as long as it functions to express a gene of interest in various host cells of prokaryotic cells and to produce a protein of interest, but a form similar to that of a natural state with a strong promoter and strong expression ability Vectors capable of producing large amounts of foreign proteins of are preferred. The recombinant vector preferably contains at least a promoter, an initiation codon, a gene encoding a protein of interest, and a termination codon terminator. In addition, a DNA encoding an signal peptide, an operator sequence, a non-translated region, a selectable marker region, or a replicable unit on the 5 'and 3' sides of the gene of interest may be appropriately included. The initiation codon and the termination codon can generally be considered part of the nucleic acid sequence encoding the immunogenic target protein and must be functional when the gene construct is introduced and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

In the present invention, a nucleic acid sequence encoding a G protein fragment in which neutralizing antibody epitopes are concentrated in order to induce RSV-specific mid-reverse reaction in mucosa and serum is inserted into an E. coli expression vector and 3'-end of the nucleic acid sequence for ease of purification. The nucleic acid sequence encoding the tag may be further included.

As used herein, the term "tag" refers to a molecule that exhibits quantifiable activity or properties, and refers to a polypeptide fluorescent substance such as Fluorescein, Fluorescent Protein (GFP) or related protein. It may be a fluorescent molecule included, or may be an epitope tag, such as a Myc tag, a Flag tag, a Histidine tag, a Leucine tag, an IgG tag, and a Strepptavidin tag. . Suitable tag polypeptides generally have 6 or more amino acid residues and typically have about 8 to 50 amino acid residues, wherein 6 histidine tags were used in the present invention. Preferably, the tag is at the C terminal portion of the G protein fragment.

In addition, the vector of the present invention may further include a stop codon at the C-terminal portion of the G protein fragment, a preferred vector may be a vector shown in the cleavage map of FIG.

As another aspect, the present invention is a prokaryotic transformant for the production of a vaccine for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV) transformed by the expression vector of the present invention, and the prokaryotic transformation Culturing the transformants; And it provides a method for producing a peptide fragment of RSV G protein for vaccines for the prevention and treatment of respiratory diseases caused by respiratory synsychia virus (RSV) comprising the step of separating and purifying the peptide expressed from the transformant.

In the present invention, the G protein fragment can be obtained by transforming a host cell using the expression vector, followed by culturing. Methods for preparing transformants by introducing expression vectors into host cells include Sambrook, J., et al., Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, 1. 74, 1989 The calcium phosphate method or the calcium chloride / rubidium chloride method, the electroporation method, the electroinjection method, the chemical treatment method, such as PEG, the method using a gene gun, etc. are described. When the transformant expressing the vector is cultured in a nutrient medium, a large amount of useful protein can be prepared and separated. The medium and culture conditions may be appropriately selected depending on the host cell. In culture, conditions such as temperature, pH of the medium, and incubation time should be appropriately adjusted to be suitable for cell growth and mass production of proteins. Host cells that can be transformed with the expression vector according to the present invention include prokaryotic cells, and a host with high expression efficiency of introduced DNA and a high expression efficiency of introduced DNA is usually used. Bacteria, for example, known prokaryotic hosts such as Escherichia, Pseudomonas, Bacillus, Streptomyces, are examples of host cells that can be used. Preferably, Escherichia coli can be used. Expression of the peptide can be induced using inducer IPTG and induction time can be controlled to maximize the amount of protein. In the present invention, recombinantly produced peptides can be recovered from medium or cell lysate. If membrane bound, it can be liberated from the membrane by using a suitable surfactant solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used for peptide expression can be disrupted by various physical or chemical means, such as freeze-thaw repetition, sonication, mechanical disruption or cell digestion, and are isolated by conventional biochemical separation techniques. Purification is possible (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989; Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA, 1990). Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent affinity chromatography, reverse phase HPLC, gel permeation HPLC), isoelectric focus and various variations and combinations thereof Including but not limited to.

The prokaryotic cells of the present invention, preferably G protein fragments expressed and purified by an E. coli expression vector, may have immunogenicity and thus may be included in vaccine compositions for the prevention and treatment of respiratory diseases caused by RSV infection. As used herein, the term "immunogenic" refers to the ability to induce both cellular and humoral immune responses to cope with foreign substances, and the substance that induces this immune response is called an immunogen.

In another aspect, the present invention provides a method for preventing respiratory disease caused by RSV comprising all or a portion of peptide fragments comprising 120 to 230 amino acids from the N-terminus of the respiratory Synsichia virus (RSV) G protein. And a therapeutic vaccine composition, preferably a vaccine composition for preventing and treating respiratory diseases caused by RSV, comprising a peptide fragment of RSV G protein prepared by the above method.

As used herein, the term "prevention" means any action that inhibits or delays the development of a respiratory disease caused by RSV infection by administration of a composition.

As used herein, the term "treatment" means any action by which administration of the composition improves or benefits the condition already caused by RSV infection. Respiratory diseases caused by respiratory syncytial virus (RSV) infection include cough, sneezing, high fever, wheezing, bronchitis, bronchiolitis, pneumonia, asthma, croup and respiratory failure.

The vaccine composition of the present invention may also comprise a pharmaceutically acceptable carrier. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvant, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in the composition for vaccines include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In addition, the vaccine composition of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, and sterile injectable solutions, respectively, according to a conventional method. In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate and sucrose in the lecithin-like emulsifier. (sucrose), lactose (lactose), gelatin, etc. can be mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. As a liquid preparation for oral administration, suspending agents, liquid solutions, emulsions, syrups, etc. may be used, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. Can be. Formulations for non-oral administration include sterile aqueous solutions, water insolubles, suspensions, emulsions, lyophilized agents. As the non-aqueous preparation and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used.

In another aspect, the present invention provides a method for the prevention and treatment of respiratory diseases caused by RSV infection, comprising administering the vaccine composition to a subject.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. As used herein, the term "administration" means introducing a substance into a patient in any suitable manner. For example, nasal administration has an advantage of high immunogenicity, but there is a concern that the drug may move to the central nervous system through an olfactory bulb in the nasal cavity and may cause side effects due to an inflammatory response. There is a weak point. On the contrary, sublingual administration has not been reported and has been used as an immunotherapy method for allergy for a long time, so there is an advantage in terms of safety because the concern of side effects on the human body is very low. As used herein, the term "sublingual administration" refers to the route of administration in which the dosage form is applied to the lower part of the tongue to obtain a local or systemic effect of the active ingredient, and "intranasal administration" means By means of a route of administration in which the dosage form is applied under the nasal cavity to be absorbed into the nasal mucosa to obtain a local or systemic effect of the active ingredient. The vaccine composition of the present invention may be administered by sublingual administration or nasal administration, but is not limited thereto. The vaccine composition of the present invention has an excellent effect of inducing local mucosal immunity compared to systemic vaccination in the case of sublingual administration and nasal administration, and especially in the case of sublingual administration, it shows an advantageous immunogenicity in terms of safety. Formulations suitable for sublingual or nasal administration may be prepared using methods known to those skilled in the art.

The vaccine composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to exhibit a vaccine effect and an amount not to cause side effects or serious or excessive immune responses, and an effective dose level refers to the disorder or disorder to be treated. Severity, activity of a particular compound, route of administration, rate of elimination, duration of treatment, drug used in combination or concurrently, age, weight, sex, diet, general health of the subject, and factors known in the pharmaceutical and medical arts. It will depend on a variety of factors.

Various general considerations that are considered in determining “pharmaceutically effective amount” are known to those of skill in the art and are described, for example, in Gilman et al., Eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press , 1990, and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And single or multiple administrations. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art. The composition may also be administered by any device in which the active agent may migrate to the target cell.

Vaccine compositions of the present invention may include one or more adjuvants and the like to improve or enhance an immune response. Suitable auxiliaries include compositions consisting of mineral oils or vegetable oils such as peptides, aluminum hydroxides, aluminum phosphates, aluminum oxides and Marcol 52 and one or more emulsifiers or surface actives such as lysolececitin, polyvalent cations, polyvalent anions and the like. .

The vaccine composition of the present invention may further comprise one or more immunostimulants, preferably cholera toxin (CT), aluminum hydroxide, carbopol, mineral oil or Biodegradable Oil may be included.

The term “immune enhancer” in the present invention generally refers to any substance that increases the humoral and / or cellular immune response to the antigen. Traditional vaccines consist of unprocessed preparations of dead pathogenic microorganisms, while impurities associated with cultures of pathogenic microorganisms can act as an adjuvant to enhance the immune response, but homogeneous preparations of purified protein subunits can be used as antigens for vaccination. In use, the immunity triggered by such antigens is insufficient, requiring the addition of some foreign substance as an adjuvant. By using an adjuvant, smaller doses of antigen may be required to stimulate an immune response, thereby reducing the cost of vaccine production. These adjuvants are classified according to their raw materials (minerals, bacteria, plants) and components (emulsion suspensions). Commercially available adjuvants include aluminum hydroxide, carbopol, mineral oil (mineral oil) or biodegradable oil.

The term “cholera toxin (CT)” in the present invention is known as the A and B subunits and refers to a structure composed of two protein subunits as shown in toxins produced from dysentery, toxin-producing E. coli, and intestinal hemorrhagic E. coli. Have

In a specific embodiment of the present invention, E. coli expression vector pET-21d (+)-RSV Gcf was prepared to express the RSV G protein fragment (FIG. 1) transformed to E. coli to induce the expression of ~ 17 kDa size Overexpressed RSV G protein fragments (RSV-Gcf) could be observed and purified to high purity via metal ion chromatography (FIG. 2).

The effect of the G protein fragment expressed by the prokaryotic expression vector according to the present invention on the prevention and treatment of respiratory diseases caused by RSV was examined. In vivo (in mice for In vivo ), 20 μg of purified RSV G protein fragment without endotoxin alone or in combination with an immunopotentiator CT (Cholera toxin) was immunized to mice via sublingual or nasal administration, and very high serum levels after two doses. It was confirmed to elicit a total IgG (Immunoglobulin G) response (FIG. 3). In particular, it was confirmed that RSV G protein specific total IgG antibody response was generated even when sublingually or nasal administration of RSV G protein fragment containing no adjuvant alone. After measurement of the degree of RSV G protein specific secretion IgA (Immunoglobulin A) present in saliva after two sublingual or nasal administrations, the saliva of the RSV G protein fragment alone group and the CT-administered adjuvant group was measured. Secretory IgA response was observed in saliva (FIG. 4). As a result, it has been confirmed that sublingual or nasal immunity of the purified G protein fragment of the present invention induces strong serum IgG and mucosal IgA responses, and in particular safely and effectively induces immune responses even when no immunopotentiator is included. In this regard, the inventors further confirmed that the G protein fragment has potent chemotactic activity in vitro, and the four cysteine residues of the G protein fragment (Cys-173, Cys-176, Cys-182 and Cys-186) The mutant Gcf (GcfΔCys4) substituted with alanine confirmed that the chemotactic activity was significantly reduced, confirming that the chemotactic activity was due to the four cysteine residues. Therefore, it was confirmed that the purified G protein fragment of the present invention exhibited autoimmune-enhancing properties without an immune enhancer, thereby effectively inducing an immune response.

In addition, in order to determine the protective properties of the induced immune response, the protective efficiency after infection was determined by measuring the titer of RSV. When the vaccine composition containing 20 μg of purified RSV G protein fragment was immunized with two sublingual administrations, In addition, the protective immunity was greatly increased, and it was observed that almost no virus was detected in the lung tissue even after 4 days of the highest replication level after the live RSV test infection (FIG. 5). In particular, even when immunized with the vaccine composition containing only the RSV G protein fragments showed a similar immunodeficiency effect similar to the live RSV immunization group.

In addition, to determine whether RSV G protein fragment administration is likely to induce G protein specific CD4 T cells, resulting in pulmonary eosinophilia, RSV G protein fragment or RSV inactivated with formalin (FI-RSV). Immunized mice were infected with RSV after 4 weeks and lung cells were isolated after 4 days, and stimulated with G protein IE ^d -immunodominant epitope (183-195) peptide followed by IFN-γ (interferon). -gamma production showed little IFN-γ production in mice immunized with the sublingual route of RSV G protein fragments (less than about 2.0% of the total CD4 T cells in the lungs). However, in the group immunized with live RSV, more than 3% of the total CD4 T cells in the lung showed CD4 T cell responses (FIG. 6). Mice immunized with live RSV, FI-RSV, or RSV G protein fragments, respectively, were infected with RSV after 3 weeks, and the number of eosinophils contained in the bronchoalveolar lavage fluid was measured by the CD11c ^- Siglec-F ⁺ phenotype. In the group immunized with FI-RSV, a marked increase in CD11c ^- Siglec-F ⁺ eosinophils was seen (Fig. 7, 45% of all BAL cells), whereas in the group immunized with sublingual administration of RSV-G protein fragments. Eosinophils were observed in less than 1% in total BAL (Bronchoalveolar lavage) cells (FIG. 7). This was about the same as when immunizing live RSV. Therefore, it was found that when the RSV G protein fragment of the present invention was used for sublingual administration, little pathology of the lung due to eosinophil expansion occurred (FIG. 7). Therefore, the present invention is a very effective vaccine that can induce an effective protective immune response with a single administration at the early stage of birth of infants, which is the main target of RSV vaccine, without causing secondary immunopatholgy such as eosinophilia. Able to know.

The vaccine comprising the prokaryotic expressing RSV G protein fragment according to the present invention induces very high serum IgG and respiratory mucosal IgA even with two immunizations, and is effective against RSV replication without eosinophilic immunopathology due to the vaccine upon RSV infection. Indicates defense. In addition, it is economical as a vaccine using a purified protein from bacteria and by using the purified protein fragment as an antigenic vaccine, overcoming the stability problems that can be caused in the viral vaccine, which is the main target of RSV vaccine Given the newborns and infants, this is a beneficial effect. In addition, it has an effect of increasing the stability of vaccination because it shows sufficient immunity even in the sublingual administration excellent in stability.

1 shows a cleavage map of the whole RSV G protein fragment expression vector according to the present invention.
Figure 2 shows the results confirmed by SDS-PAGE (A) and Western blotting (B) for the expression and purification of RSV G protein fragment in E. coli.
Figure 3 shows RSV G protein fragment (Gcf) sublingual administration (sl), RSV-Gcf / CT (Cholera toxin) sublingual administration, intramuscular administration of FI-RSV through the plantar (im), RSV-Gcf nasal administration (in) In the case of immunization through RSV-Gcf / CT nasal administration, the IgG titer was measured by ELISA for the degree of IgG induction in serum compared to the control (PBS).
Figure 4 shows the results of confirming the degree of IgA induction of the respiratory tract under the same conditions as in Figure 3 by ELISA method.
Figure 5 shows the results of measuring the RSV titer in the lung tissue by plaque assay method 4 days after RSV A2 5 X 10 ⁶ PFU test infection for the control group (PBS) and immunization group. .
FIG. 6 shows the results of confirming whether G protein-specific CD4 T cell responses are induced through IFN-γ staining for the control group (PBS) and the immunization group.
Figure 7 shows the results measured by flow cytometry after the CD11c / Siglec-F staining eosinophil count contained in bronchoalveolar lavage fluid (BAL) for the control group (PBS) and immunization group.
FIG. 8A shows the results of measuring chemotactic activity mediated by Gcf by measuring cell migration for wild-type Gcf and mutant Gcf (GcfΔCys4) in vitro, and B is a chemotaxis upon nasal administration in vivo. The result of measuring activity is shown.
9 shows the results of measuring specific serum antibody titers for wild type Gcf and mutant Gcf (GcfΔCys4).

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Example  One : RSV stock  Ready

RSV A2 strain contains 3% heat-inactivated fetal calf Serum (FCS), 2 mM glutamine, 20 mM 2- [4- (2-hydrocyethyl) -1-piperazinyl] ethane Cultured in HEp-2 cells (ATCC, Manassas, VA) in DMEM medium (Dulbecco's Modified Eagle Medium; Life Technologies, Gaithersburg, MD) to which sulfonic acid (HEPES) and non-essential amino acids were added. Specifically, the RSV A2 virus diluted in HEp-2 cells of about 70% confluency is infected with a frequency of MOI = 0.01 and 72 ~ 96, the most cytopathic effect due to virus replication. After time, the culture and the cells were recovered. After crushing the cells by repeated freeze-thaw, the supernatant was collected, the virus was precipitated by ultra-high-speed centrifugation, and suspended in an appropriate amount of DMEM medium to prepare a stock.

Example 2 Preparation, Expression and Purification of Escherichia Coli Expression Vectors Expressing RSV G Protein Fragments

A 333-mer-length base nucleotide DNA was prepared by replacing the DNA nucleic acid sequence corresponding to amino acid sequences 131 to 230 of the RSV G protein (RSV A2 strain) with the most frequently used codon (SEQ ID NO: 5). Bioneer Corp., Daejeon, Korea). PCR using 5 ': GGG CCC GAA TTC CCG TCA AGA CCA AAA ACA CAA CA (SEQ ID NO: 7), 3': GGG CCC AAG CTT GGG CTT GGT GGT GGG TAC TTC (SEQ ID NO: 8) primers After amplification through EcoR I and Hind III (New England Biolabs, Beverly, MA) restriction enzymes were cut and inserted into the pET21d vector (Novagen, Madison, WI) cut with the same restriction enzyme (Fig. 1). In addition, mutant DNA in which four cysteine residues (Cys-173, Cys-176, Cys-182, Cys-186) were substituted with alanine was produced by the mega-PCR method using mutant primers. The prepared pET-21d (+)-RSV G was transformed into E. coli BL21 (DE3). After addition to (Novagen), protein expression was induced with 0.5M IPTG (Isopropyl β-D-1-thiogalactopyranoside, Takara, Shiga, Japan). After centrifugation, bacterial cells were suspended in binding buffer (20 mM Tris, 0.5 M NaCl, pH 8.0) and sonicated. Soluble and insoluble proteins were separated by centrifugation at 40,000 x g for 20 minutes and the clear supernatant was used for further purification. Expressed RSV G protein fragments (Gcf) and mutant Gcfs (GcfΔCys4) were purified by Ni-NTA affinity chromatography (Peptron, Daejeon, Korea) using HisTrap column (GE healthcare). After washing with binding buffer containing 20 mM imidazole, the loaded protein was eluted with elution buffer (500 mM imidazole, 20 mM Tris, 0.5 NaCl, pH 7.4). The eluted protein fractions were equilibrated with PBS (GE healthcare) and then further purified using a Superdex-75 column. In order to remove endotoxin, Triton X-114 was added to 1% of purified RSV G protein, strongly vortexed, and incubated for 30 minutes while shaking at 4 ° C. Next, incubated for 20 minutes in a water bath (37 ℃), centrifuged for 5 minutes at 14,000 rpm to separate the layers. The supernatant was then transferred to a new tube. The above procedure was repeated five times, and 100 μg of SM-2 beads (Bio-Rad, Hercules, CA) washed with methanol was placed in a stomach tube and incubated in a rotator for 2 hours. Centrifugation was performed at 14,000 rpm for 5 minutes, and the supernatant was filtered using Spin-X (Corning Costar, Lowell, MA). Endotoxin levels were measured using the following biological endotoxin test (limulus amebocyte lysate (LAL) assay kit (Lonza, Switzerland). Endotoxin levels were less than 5EU / mg.

Protein concentration was measured by Bio-Rad Protein Assay (Bio-Rad Laboratories). Centrifugal filter, Amicon® ultra (Millipore, Bedford, Mass.) Was used for further concentration. Purified protein was confirmed by Coomassie Brilliant Blue staining after 15% SDS-PAGE. Purity of the Gcf vaccine was confirmed by Western blotting using RSV-specific goat polyclonal antibody (US Biological) and HRP-binding anti-chlorine Ig antibody (Zymed Laboratories, San Francisco, Calif.). Purified protein was stored in fractions at -80 ° C until use.

As a result, it was possible to confirm the overexpressed RSV G protein fragment (Gcf) of ˜17 KDa size, which shows the expression level and purification process of the RSV G protein fragment in E. coli (FIG. 2).

Example  3: immunization ( Immunization )Wow RSV  infection

To determine whether a vaccine composition consisting of fragments of RSV G protein induces an antigen-specific immune response in vivo when immunized, female BALB / C mice from 6 to 8 weeks old (Charles River Laboratories Inc. Yokohama, Japan) 20 μg of purified G protein fragments were immunized via two (day 0 and 14), sublingual (sl) or nasal (intranasal, in) routes, respectively. For sublingual immunization, mice are intraperitoneally injected with ketamine and anesthetized and 15 μl of RSV G protein fragments are administered alone through the sublingual site (hereinafter referred to as RSV-Gcf), or the stimulator cholera toxin ( 2 µg of CT (Cholera toxin) was mixed and administered. For nasal immunization, mice were anesthetized with isoflurane and 50 μl of vaccine or PBS was administered to the left nostril. Anesthetized mice with formalin-inactivated RSV (FI-RSV) (1 × 10 ⁵ pfu in 50 μl) inactivated with formalin, including aluminum hydroxide (Sigma-Aldrich, Seoul, Korea) It was administered through the foot pad of the. As a positive control, live RSV (1 × 10 ⁵ pfu) was administered nasal. Three to four weeks after the last immunization mice were treated nasal with either 1 × 10 ⁶ or 2 × 10 ⁶ PFU of live RSV A2.

Example  4 : IgG  Induction effect measurement

Blood was collected through a heparinized capillary tube from the retroorbital plexus of each immunized mouse by the method of Example 3 to obtain serum through centrifugation, and anti-specific to RSV G protein. RSV IgG titers were confirmed through an enzyme-linked immunosorbent assay (ELISA). 96-well plates were coated overnight at 100 μl per well with 2 × 10 ³ PFU of purified RSV A2 virus diluted in BS and blocked with PBS containing 2% BSA and 0.05% Tween 20. Thereafter, serum or BAL solution was added to serial dilutions and incubated for 2 hours. Next, the plate was washed 5 times with 0.05% PBST (Phosphate Buffered Saline Tween 20), and then the rabbit-derived anti-mouse secondary antibody (HRP-conjugated affinity-) conjugated with HRP (horseradish-peroxidase) at various concentrations. Purified rabbit anti-mouse antibody) IgG (Zymed Laboratories, San Francisco, CA) was added for 1 hour. After washing three times, the color was developed with 3,3 ', 5,5'-tetramethylbenzidine, and the color was stopped with 1M H ₃ PO ₄ and the absorbance was measured at 450 nm (Thermo ELISA plate reader) ( 3).

As described above, two weeks after the immunization measures, the degree of induction of G-specific IgG was measured using ELISA, RSV G protein fragment (RSV-Gcf) sublingual administration, RSV-Gcf / CT (Cholera toxin) sublingual administration, Intramuscular administration of FI-RSV through the sole of the foot, RSV-Gcf nasal administration, and RSV-Gcf / CT nasal administration, respectively, compared to the control group (PBS), each of the groups immunized with serum showed higher IgG induction. Surprisingly, it was confirmed that RSV G-specific IgG antibody responses were generated by two administrations in the RSV-Gcf-only sublingual or nasal-administered group without the adjuvant (CT) (FIG. 3).

Thus, it can be seen that the RSV G protein fragment vaccine composition according to the present invention induces an IgG response by sublingual or nasal administration.

Example  5: IgA  Induction effect measurement

Secretory IgA present in the mucosa is the first foremost mechanism by which the host defends against foreign pathogens invading through the mucosa. Therefore, an effective RSV vaccine should be able to induce virus specific secretory IgA in the respiratory mucus region. In order to investigate whether the RSV G protein fragment vaccine composition induces an IgA response, the following experiment was performed.

In the method of Example 3, 5 days after RSV infection, the mice were sacrificed via anesthesia, the trachea was dissected, a tube connected to a 1 ml syringe was inserted, and the lungs and bronchus were washed three times with 0.8 ml PBS. . Bronchoalveolar lavage fluid; Cells contained in BAL) were collected by centrifugation, and secretory IgA titers were measured by ELISA as described above (FIG. 4).

As a result of measuring specific IgA induction using ELISA, RSV G protein fragment (RSV-Gcf) sublingual administration, RSV-Gcf / CT (Cholera toxin) sublingual administration, FI-RSV muscles through the sole In each of the groups immunized by intranasal administration, RSV-Gcf nasal administration, and RSV-Gcf / CT nasal administration, all of them showed higher IgA induction in the respiratory tract compared to the control group (PBS). RSV-Gcf alone Secretory IgA response was also observed in sublingually or nasally administered groups (FIG. 4).

Thus, it can be seen that the RSV G protein fragment vaccine composition induces a secretory IgA response by sublingual or nasal administration in the respiratory mucus region.

Example  6: mouse Lung tissue  undergarment RSV Potency  Measure

In order to investigate the protective properties of the induced immune response, the immunized mice were infected with RSV A2 at 5 X 10 ⁶ PFU after 4 weeks in the respiratory tract, and their defense efficiency was examined. Four days later, the mice were anesthetized and killed, and the lung tissue was removed and placed in Eagle's modified essential medium. The tissues were then ground to steel screens into single cell suspensions and filtered through a 70 μm cell strainer (BD Labware, Franklin Lakes, NJ). Supernatants were collected and RSV titers were measured by plaque assay (FIG. 5).

As a result, live RSV was found in the lung tissue of control mice, whereas RSV replication was suppressed in the group immunized with sublingual or nasal passages of RSV G protein fragments, and almost no virus was detected (FIG. 5). The RSV-treated group showed the same protective effect after FI-RSV or live RSV A2 infection, but the FI-RSV group showed significant weight loss and disease. These results show that immunization of RSV G protein fragment vaccines through sublingual or nasal mucosa induces stable and effective protective immunity against RSV infection.

Example  7: Measurement of response of G protein specific T cells

Since the RSV G protein fragment used for sublingual administration has a known IE ^d -restricted CD4 T-cell epitope, we investigated whether administration of RSV G protein fragment induced G protein-specific CD4 T cells and caused pulmonary eosinophilia. It was. Mice immunized with RSV-Gcf or FI-RSV were infected with RSV after 4 weeks and the immune cells of the lung were isolated after 4 days and stimulated with G protein IE ^d -restricted immunodominant epitope (183-195) peptide. IFN-γ production was examined by staining.

The method for isolating immune cells is as follows. 5 ml PBS containing 10 U / ml heparin (Sigma-Aldrich, St. Louis, Mo.) was perfused through the syringe of a 25-gauge needle into the right ventricle of the mouse heart. The lungs were extracted and ground on a RPMI medium steel screen containing 10% FBS (HyClone, Logan, UT) to form a single cell suspension, followed by a 70 μm cell strainer (BD Labware). , Franklin Lakes, NJ). Cells were then separated by centrifugation and PBS / 3% FBS / 0.09% NaN ₃ Staining was performed with appropriate fluorochrome-conjugated antibodies in the buffer. The antibody used was anti-CD3e (clone 145-2C11), anti-CD4 (clone RM4-5), anti-CD11b (clone M1 / 70), anti-CD11c (HL3), anti-CD14 (rmC5-3), anti-CD44 (clone IM7), anti-CD80 (clone 16-10A1), and All antibodies were purchased from BD PharMingen (San Diego, CA) with anti-Siglec F (clone E50-2440). After staining, cells were fixed with PBS / 2% (wt / vol) paraformaldehyde and analyzed using a FACSCalibur ^™ flow cytometer (BD Biosciences, San Diego, Calif.).

In order to analyze the cells secreting IFN-γ, intracellular staining was performed. 2 x 10 ⁶ lung cells were placed in a culture tube and 10 μM G protein immunodominant epitope peptide (WAICKRIPNKKPG) was added and 37 ° C 5% CO ₂ for 5 hours. And cultured in an incubator. 5 μg / ml of Brefeldin A (Sigma-Aldrich) was also added during incubation to allow IFN-γ to accumulate in the cells. The cell surface markers were stained, washed and fixed, infiltrated with FACS buffer containing 0.5% saponin (Sigma-Aldrich, Seoul, Korea) and stained with IFN-γ (clone XMG1.2) antibody. Dead cells were excluded, the data collected through the CELLQuest ^TM software (BD Biosciences), CELLQuest ^TM and WinMDI version 2.9 software (The Scripps Research Institute, La Jolla, Calif.) (FIG. 6).

As a result, the response of G protein-specific CD4 T cells as seen in FIG. 6 was rarely seen in mice immunized via sublingual administration route using RSV G protein fragments when viewed through intracellular IFN-γ staining (lung 1.4% of the total CD4 T cells). In contrast, the group immunized with Gcf / CT and live RSV showed a CD4 T cell response of 2.5-3% or more of the total CD4 T cells in the lung.

Example  8: eosinophils ( eosinophil Number measurement

In mice immunized with FI-RSV, the phenomenon of respiratory eosinophilia due to subsequent RSV infection is well known. Indeed, to investigate whether sublingual administration of RSV G protein fragments causes eosinophilia, a secondary immunopathology phenomenon, the mice were anesthetized three weeks after RSV infection and five days after the mice were anesthetized. After sacrifice, the trachea was dissected and a tube connected to a 1 ml syringe was inserted to wash the lungs and bronchus three times with 0.8 ml PBS. The cells contained in the bronchoalveolar lavage fluid were collected by centrifugation, stained with fluorescent antibodies against CD45R, CD11c, and Siglec-F, and the number of eosinophils of the CD45R + CD11c-Siglec-F + phenotype was measured by flow cytometry. 7).

As a result, as expected, a significantly increased eosinophil was seen in the group immunized with FI-RSV (Fig. 7, ~ 45% of all CD45R + BAL cells), whereas in the group immunized with RSV G protein fragments in whole BAL cells. Less than about 2% was observed as eosinophils. This is about the same as when immunizing live RSV. Therefore, sublingual pathway immunization of RSV G protein fragment vaccine hardly causes lung pathology due to eosinophilia.

Example  9: recombination Gcf Chemotactic activity of

Given that Gcf's mucosal immunity produces a strong humoral response without an immune enhancer, the in vitro chemotaxis assay of Gcf with the CX3C motif examined whether purified Gcf exhibited chemotactic activity. It was. As a control, a mutant Gcf (GcfΔCys4) in which four cysteines were substituted with alanine was used.

Chemotaxis was analyzed using a 24-well tissue-culture-treated transwell insert plate (Costar, Corning, NY) with a 5 μm pore size. 10 μg of Gcf or GcfΔCys4 in DMEM medium containing 1% BSA was added to the lower chamber and a total of 5 × 10 ⁵ THP-1 cells were added to the upper chamber of the plate. Plates were incubated at 37 ° C. for 4 hours, and cells transferred to the lower chambers were harvested and counted in a blind manner. Three experiments were conducted to show the mean value.

In culture of THP-1 cells, the number of migrating cells increased 2.5- to 3-fold in the wild-type Gcf and 10% FBS (positive control) groups, respectively, but in the mutant Gcf group, significantly reduced compared to wild-type Gcf. It showed chemical activity. This suggests that cysteine residues of Gcf are required for Gcf-mediated chemotactic activity.

Next, in administration of Gcf alone without an immune enhancer, in order to confirm in vivo chemotaxis, it was confirmed whether in vivo administration of Gcf alone introduced immune cells to the site. As shown in B of FIG. 8, nasal administration of Gcf significantly increased infiltration of CD11c ^hi CD80 ⁺ , CD11b ^hi CD14 ⁺ , and CD3 ⁺ cells into the lung, but not mutant GcfΔCys4.

Finally, to determine whether chemotactic activity of recombinant Gcfs is required for induction of specific immune responses of Gcf alone without immune enhancers, mice were immunized with wild-type Gcf or mutant GcfΔCys4 by nasal administration. The reaction was confirmed. As shown in FIG. 9, specific serum antibody responses were observed at lower levels in the group of mutant GcfΔCys4 than in the negative control. These results indicate that high Gcf immunogenicity can be maintained in the absence of immune enhancers due to cysteine residues and chemotactic activity of Gcf.

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Claims (15)

delete delete delete delete delete delete delete delete A peptide vaccine composition for the prophylaxis or treatment of respiratory diseases caused by respiratory synsychia virus (RSV), comprising a peptide fragment expressed in prokaryotic cells as an immunogen,
The peptide fragment is composed of 131 to 230 amino acids from the N-terminal end of the RSV G protein represented by SEQ ID NO: 2, characterized in that it is not expressed in the form of a fusion protein,
The composition, characterized in that the immunogenic agent does not contain an immunogenic agent, vaccine composition.
10. The composition of claim 9, wherein the composition induces an immune response in the muscle, nasal or sublingual route, and does not cause eosinophilia, even without an adjuvant.
The composition of claim 9, wherein the peptide fragment is cysteine at the 173th, 176th, 182th and 186th amino acid residues from the N-terminus of the RSV G protein represented by SEQ ID NO: 2.
delete The composition of claim 9, wherein the vaccine composition is in a formulation suitable for sublingual administration, nasal administration or intramuscular administration.
10. The composition of claim 9, wherein the vaccine composition further comprises one or more immunopotentiators.
The composition of claim 14, wherein the adjuvant is cholera toxin, aluminum hydroxide, carbopol, mineral oil or Biodegradable oil.

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