KR102107332B1 - Cholera vaccine using CRISPR/Cas9 based Bacillus subtillis genome editing mechanism - Google Patents

Abstract

The present application relates to a composition for preventing or/and treating cholera, a method for manufacturing a vaccine, and a treatment method. An objective of the present application is to provide an antigenic substance, a CRISPR/Cas9 system, and a cholera vaccine composition containing the same. According to the present application, it is possible to provide the composition capable of preventing or/and treating cholera.

Description

Cholera vaccine using CRISPR / Cas9 based Bacillus subtillis genome editing mechanism

This application relates to antigenic substances targeting humans, CRISPR / Cas9 system and cholera vaccine composition comprising the same.

The present application relates to an antigenic substance targeting a human, a CRISPR / Cas9 system, and a method of manufacturing a cholera vaccine including the same.

The present application relates to a treatment method for treating cholera.

Vaccines are one of the most important measures you can take to prevent or / or treat disease.

Although various vaccines against various diseases have been developed to date, treatment or / and prevention are not possible only with the developed vaccine when mutation of the causative agent causing the disease occurs.

As such, it is necessary to develop a vaccine capable of rapidly responding to a mutation in a causative agent causing disease.

One task of the present application is to provide an antigenic substance, a CRISPR / Cas9 system and a cholera vaccine composition including the same.

Another object of the present application is to provide a vaccine against a causative agent causing cholera and a method for manufacturing the same.

Another object of the present application is to provide a use for preventing or / and treating cholera.

In order to solve the above-described problems of the present application, according to an aspect of the present application, i) bacteria-derived spores; At this time, the spore derived from bacteria is a spore having a sequence encoding an antigenic substance in genomic DNA,

ii) a spore coat protein present on the surface of the bacterial spore,

iii) cholera antigenic substance linked to the spore coat protein

A vaccine composition for preventing or treating cholera is provided.

In addition, the present application provides a vaccine composition for preventing or treating cholera, characterized in that the cholera antigenic substances are ctxA, ctxB, and tcpA.

In addition, the present application provides a vaccine composition for the prevention or treatment of cholera, characterized in that the concentration of cholera antigenic substance is 10 ¹² ~ 10 ¹⁴ / ml concentration based on the composition.

In addition, in order to solve the above-described problems of the present application, according to an aspect of the present application, i) knocking in an antigenic substance by using CRISPR / Cas9 in genomic DNA of bacteria (Knock-in) step;

ii) forming spores of the bacteria; And

iii) expressing an antigenic substance on the spore surface of the bacteria;

Provided is a method for producing a vaccine for preventing or treating cholera, which comprises a.

According to the present application, the following effects occur.

First, according to the present application, it is possible to provide a composition capable of preventing and / or treating cholera. In particular, it is possible to provide an antigenic substance, a CRISPR / Cas9 system and a composition for preventing or / and treating cholera including the same.

Second, by using the composition provided by the present application, it is possible to provide an effect capable of quickly responding when a mutation occurs in a causative agent causing cholera disease.

1 is a diagram showing a pHCas9 vector.
2 is a diagram showing a pJB vector containing CotG CDS containing a guide RNA and a promoter of B. subtilis Coat protein (CotG).
FIG. 3 is a diagram showing a pJB-GFP vector containing CotG CDS and GFP containing a guide RNA and a promoter of B. subtilis Coat protein (CotG).
FIG. 4 is a diagram showing a pJB001 vector containing CotG CDS and CtxA containing a guide RNA and a promoter of B. subtilis Coat protein (CotG).
5 is a diagram showing a pJB002 vector containing CotG CDS and CtxB containing a guide RNA and a promoter of B. subtilis Coat protein (CotG).
FIG. 6 is a diagram showing a pJB003 vector containing CotG CDS and TcpA containing a guide RNA and a promoter of B. subtilis Coat protein (CotG).
FIG. 7 is a diagram showing a pJB-sg02 vector containing a guide RNA targeting the ampicillin resistance gene for deletion of an antibiotic resistance gene.
8 is a diagram showing a pJB-sg03 vector containing a guide RNA targeting a neomycin resistance gene for deletion of an antibiotic resistance gene.
9 is a diagram showing the results of PCR analysis performed to confirm whether GFP is expressed on the spore surface of B. subtilis.
10 is a view showing the results of FACS analysis performed to confirm that GFP is expressed on the spore surface of B. subtilis.
11 is a diagram showing the results of PCR analysis performed to confirm whether CtxA, CtxB, and TcpA are expressed on the spore surface of B. subtilis.
12 is a diagram showing the results of ELISA confirming whether an antigen-specific antibody is formed.
13 is a view showing Western blot results confirming the formation of antigen-specific antibodies.

Terms or practices used in the techniques disclosed by this application are, unless defined otherwise, microbiology, virology, bacteriology, genetics, immunology, biochemistry, molecular biology, microbiology, cell biology, genomics and conventional practice within the skill of the art. It has the same meaning as commonly understood by a person skilled in the art using technology or the like.

1. Vaccine

One aspect of the present application relates to a vaccine.

"Vaccine" refers to a substance that produces protective immunity against disease. The protective immunity may include all that excludes pathogens from the living body as antibodies or T cells that specifically recognize pathogens such as viruses, bacteria, and parasites, but is not limited thereto. The vaccine may include, but is not limited to, conventional vaccines such as a killed vaccine, an attenuated vaccine, a toxoid vaccine, a subnunit vaccine, and a conjugated vaccine. . The terms "vaccine" or "prophylactic injection" are used interchangeably. To prevent illness, getting the above shot is called vaccination.

The target of the vaccine may be human, pig, chicken, dog, etc., but is not limited thereto.

For example, the subject of the vaccine in this application can be a human.

1-1. Spores

The vaccine may include spores.

The spores may include spores in their natural state and variations thereof.

For example, spores may be spores such as plants, yeast, fungi, bacteria, and the like.

In the spore of the bacteria, the bacteria may be Clostridium, Streptococcus, Bacillus, etc., but are not limited thereto.

The Clostridium genus may be, for example, C. difficile, C. botulinum, C. perfringens, but is not limited thereto.

The genus Bacillus may be, for example, B. subtilis, B. anthrasis, B. thuringiensis, B. cereus, but is not limited thereto.

For example, the present application may be a vaccine comprising spores of B. subtilis.

The bacteria may include bacteria, for example, B. subtilis, which are recognized as GRAS (generally recognized as safe).

In particular, when the present application is a vaccine containing spores, any bacteria recognized as GRAS can be used.

The spores may include exogenous spores formed on the outside of plants, yeasts, fungi, bacteria, etc. and endospores formed on the inside.

For example, the present application may be a vaccine comprising endospores.

The exogenous spores are made from the spores outside the sporangia and come out as they are, referring to spores involved in the reproductive function of the spores.

The endogenous spores refer to spores that form inside (specifically, inside a cell) spores, and refer to spores that are formed temporarily when conditions that have little metabolic activity and are difficult to survive.

For example, the present application may be a vaccine comprising endogenous spores of B. subtilis.

The conditions in which the endogenous spores are formed are under nutritional conditions where nutrients are depleted, temperature conditions at high or low temperatures, moisture conditions at high or low moisture, chemical conditions such as fungicides, preservatives, acidic or alkaline pH conditions, etc. It may be formed, but is not limited thereto.

As such, endogenous spores can be formed under difficult conditions, and thus can survive for a long time in various environments. In other words, endogenous spores can have tremendous resistance to harmful conditions such as heat, drying, toxic chemicals, depletion of nutrients, and ultraviolet irradiation.

The spore may include an extracellular spore protein, for example, the external spore protein may be spore exosporium protein, spore coat protein, transmembrane protein, spore appendage protein, spore associated enzyme, and the like.

For example, the present application may use each other's spore external protein between spores of microorganisms of the same genus.

The spore exosporium protein may be, for example, BclA, InhA, or the like.

The spore coat protein is, for example, CotA, CotB, CotC, CotD, CotE, CotF, CotG, CotN, CotS, CotT, CotV, CotW, CotX, CotY, CotZ, CotH, CotJA, CotJC, CotK , CotL, CotM, and the like.

The transmembrane protein may be, for example, prkC, OppA, or the like.

The spore appendage protein may be, for example, P29a, P29b, P85, and the like.

The spore associated enzyme may be, for example, CotA (laccase), oxdD (oxalate decarboxylase), CotQ (reticuline oxidase-like protein), tgI (transglutaminase), or the like.

The spores may be hydrophobic. At this time, the spore may be hydrophobic in all or part of it.

The spores can be a component of the spore system.

The vaccine can include a spore system.

The spore system refers to a system in which the system construct is spores or includes spores, antigenic substances and adjunct substances.

The adjunct substance refers to all substances capable of further enhancing the efficacy of the vaccine. For example, aluminum gel particles (aluminum salt), oil adjuvants containing mineral oil as a main component, surfactant-like adjuvants such as saponins purified from white hyacinth beans, toxins in bacteria ( LPS, etc.) derived TH1 inducible adjuvant, etc., but are not limited thereto.

1-2. Antigenic substance

The present application may be a vaccine containing an antigenic substance.

The term "antigen" as used in this application refers to a substance that causes an immune response. The antigen may include, for example, pathogens, viruses, proteins, polysaccharides, artificially synthesized substances, and substances having mutations in vivo, but is not limited thereto. The terms "antigen" or "immunogen" are used interchangeably.

The antigenic substance includes all substances that cause a protective immune response, and may be, for example, pathogens, epitopes, polypeptides, proteins, non-protein molecules and fragments thereof.

The antigenic substance may be of a wild type or a mutant type. At this time, the mutation may include natural manipulation or artificial manipulation, in which some or / and all of the antigenic substances are deleted, modified, and the like.

For example, it may be a vaccine containing a portion of the antigenic material.

For example, it may be a vaccine including all of the antigenic substances.

Pathogen refers to an individual that causes disease, including, for example, viruses, bacteria, bacteria, and the like.

For example, it may be a vaccine containing the pathogen itself as an antigenic substance.

For example, it may be a vaccine containing a part of the pathogen as an antigenic substance.

The pathogen may be a specific site modified or manipulated. The deformation and manipulation can be made naturally or artificially.

The pathogen may be a pathogen of a human target disease.

For example, the pathogen may be enterobacteriaceae.

For example, the pathogen may be vibrio.

For example, the pathogen may be Escherichia.

For example, the pathogen may be Salmonella.

For example, pathogens of human target disease may be Vibrio cholera, Vibrio parahemolyticus, Salmonella enterica enterica, serovar Typhi, Escherichia coli.

The vaccine of the present application may include a cholera pathogen.

Epitopes are called antigenic determinants and refer to specific regions of the antigen that allow it to be identified.

For example, it may be a vaccine containing the epitope itself as an antigenic substance.

For example, it may be a vaccine containing a portion of the epitope as an antigenic substance.

The epitope may be a specific site modified or manipulated. The deformation and manipulation can be made naturally or artificially.

The epitope may contain one or more toxins. Toxins can include exotoxins and endotoxins.

The toxins are Shiga toxin, heat-labile toxin, cholera toxin, Campylobacter jejuni enterotoxin, pertussis toxin, cigar-like Toxins (shiga-like toxin or verotoxin), but are not limited thereto.

For example, it may be a vaccine comprising one or more cholera toxin (CT). At this time, cholera toxin may include ctxA (cholera toxin subunit A), ctxB (cholera toxin subunit B), but is not limited thereto.

The epitope may also include multiple epitopes in which two or more are linked to each other by, for example, a single bond or a double bond.

For example, the vaccine of the present application may include cholera antigenic material.

This application can solve or minimize the limitations of conventional vaccines. In particular, it is possible to solve or minimize the limitations of the conventional cholera vaccine.

The present application is a vaccine containing one or more of the above antigenic substances, and can rapidly respond to mutations in the causative agent causing disease. In particular, since it contains at least one cholera toxin as an antigenic substance, it can be easily used for the prevention or / and treatment of cholera disease.

2. cholera

This application may be a vaccine targeting the prevention or / and treatment of cholera.

The cholera is a water-borne infectious disease that causes diarrhea and vomiting by toxins secreted by cholera bacteria (Vibrio cholera).

The cholera bacteria are curved Gram-negative rod bacteria belonging to the family Vibrionaceae. According to O antigen of lipid polysaccharide in the cell wall of cholera bacteria, it is divided into more than 200 serogroups.

For example, the cholera of the present application may be a V. cholerae O1 serogroup.

For example, the cholera of the present application may be a V. cholerae O27 serogroup.

For example, the cholera of the present application may be a V. cholerae O37 serogroup.

For example, the cholera of the present application may be the V. cholerae O139 serogroup.

The cholera is a disease caused by toxins. The cholera toxin is an exotoxin composed of one A subunit and five B subunits, and the B subunit binds to the GM1 ganglioside of the intestinal epithelial cell so that the A subunit penetrates well into the cytoplasm of the intestinal epithelial cell. The cholera toxin A subunit that has penetrated into the intestinal epithelial cells activates adenylatecyclase to increase intracellular cyclic AMP, thereby inhibiting the absorption of Na ⁺ and Cl ^- and promoting the secretion of Cl ^- and water, causing secretory diarrhea. The second virulence factor is toxin-coregulated philus (TCP), whose expression is regulated in parallel with the cholera toxin, and is essential for the colonization of the strain in the small intestine.

The cholera toxin may be a causative agent (or antigenic substance) causing cholera.

The gene associated with the cholera toxin may be, for example, ctxA, ctxB, etc., which are directly involved in pathogenicity, may be, for example, rstR, etc., which are involved in phage repressor, known as toxin-coregulated pilus (TCP) There may be tcpA involved in the adhesion of cholera bacteria to organs using the cilia, but this application is intended to include all factors related to cholera toxin.

For example, the cholera vaccine of the present application may include one or more selected from ctxA, ctxB, rstR, and tcpA.

2-1. Cholera vaccine

Currently, cholera vaccines include oral vaccines and injectable vaccines, but injection vaccines are not recommended by WHO because of their low defense and high incidence of severe adverse events. When the vaccine is administered orally, it is easy to administer, there is no risk of injection-related infection, and it has the advantage of stimulating the local immune system in the intestine in a manner very similar to the immune response induced by naturally occurring diseases. Vaccines have been developed. Oral vaccines include inactivated vaccines and live vaccines. Orochol (CVD 103-HgR), a live vaccine, has not been produced because it has not been sufficiently established or prevented for oral vaccines.

Vaccines targeting the prevention or / and treatment of cholera are killed vaccines, attenuated vaccines, component vaccines, DNA vaccines, DNA vaccines, and recombinant vaccines, depending on the method of manufacture. ), But any method capable of producing a vaccine is included, but is not limited thereto.

The four vaccines refer to a vaccine prepared by inactivating all or part of a virus or bacteria by using chemicals or heat.

The attenuated vaccine refers to a vaccine prepared by modifying a part of a virus or bacterium that causes disease, but has self-proliferation and immunity-inducing ability, but removes the ability to induce toxicity.

The component vaccine refers to a vaccine produced by heating a toxin produced by the metabolic process of a pathogen or possessing a pathogen itself, or treating a drug such as formalin. Component vaccines can also be referred to as subunit vaccines, subunit vaccines, and specific antigen extraction vaccines.

The DNA vaccine refers to a vaccine prepared by a method for delivering a gene using plasmid DNA. The plasmid DNA has various genetic elements for expressing a gene, which means that when entering into a cell, the antigen is expressed from the inserted gene.

The recombinant vaccine refers to a vaccine prepared by genetically engineering only a site that acts as an antigen in a pathogen. At this time, genetic manipulation refers to a technique of introducing and expressing the gene of an antigen into another cell using recombinant technology.

The recombination technique used when making the recombinant vaccine is not limited to those that can be genetically manipulated, such as a method using a vector, a method using a non-vector, a method using genome editing, and the like.

The vector may include a non-viral vector, a viral vector, and the like, all of which have functions capable of carrying a gene.

The virus of the viral vector may be a DNA virus or an RNA virus.

The DNA virus may be a double-stranded DNA (ds DAN) virus or a single-stranded DNA (ss DNA) virus.

The viral vector may be an adenovirus vector, a retrovirus vector, a lentivirus vector, an adeno-associated virus (AAV) vector, a vaccinia virus vector, a poxvirus vector, a herpes simplex virus, but is not limited thereto.

The viral vector has high gene transfer efficiency, but is a pathogenic virus, so it may be limited in terms of safety and the size of the gene that can be inserted into the vector.

The non-viral vector may be a plasmid, naked DNA, liposome, etc., but is not limited thereto.

Non-viral vectors do not introduce viral genetic material into human cells, and may have advantages such as ease of production, unlimited size of the inserted gene in the vector, and low side effects due to low immune response to the host. In comparison, there may be a disadvantage in that the efficiency of introduction into cells is significantly lower.

The non-vector may include methods such as electroporation, gene gun, ultrasonic perforation, self-injection, temporary cell compression or squeezing, lipid-mediated transfection, nanoparticles, silica, etc. If not, it is not limited to this.

The method of using the genome editing may include Zinc (Zinc-finger nucleases), TALEN (transcription activator-like effector nucleases), CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated sequences), etc. Any technique capable of editing a target site of a gene using an artificial enzyme used to cut a DNA site is not limited thereto.

The ZFN refers to a genome editing technique consisting of a zinc finger protein capable of recognizing and binding a specific DNA sequence and a FokI restriction endonuclease domainin capable of cleaving DNA.

The TALEN refers to a genome editing technology composed of a DNA binding domain and a FokI nuclease domain.

The CRISPR / Cas is also called RGEN (RNA-guided endonuclease) and refers to a genome editing technique in which DNA sequence specificity is induced by RNA, unlike ZFN and TALEN.

The vaccine disclosed in the present application may be a cholera vaccine made using CRISPR / Cas.

In particular, the vaccine disclosed in the present application may be a cholera vaccine made using spCas9.

CRISPR / Cas refers to the combination of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated sequences (cas).

The CRISPR / Cas system consists of guide RNA and Cas protein.

The guide RNA refers to RNA capable of recognizing a target sequence, and refers to one having a function capable of guiding a Cas protein to a target sequence by binding to a Cas protein.

The guide RNA may include crRNA (CRISPR RNA) or / and tracrRNA (trans-activating crRNA). The form in which a specific site of the crRNA and the tracrRNA is fused may be referred to as sgRNA (sinlge-chain guide RNA).

The Cas protein refers to a protein that performs a function capable of cleaving a target sequence or gene, and the Cas protein may include an enzyme, and the enzyme may include nucleases, proteases, restriction enzymes, etc. It is not limited.

The gene scissor protein may include a form of an artificially generated enzyme or protein that is present or not present in nature, or a part thereof.

The enzyme may be a CRISPR enzyme.

The CRISPR enzyme is a protein constituting the CRISPR-Cas system, and may bind a guide nucleic acid to form a complex to cut a target sequence or modify a position.

The CRISPR enzyme may include a form modified to have incomplete or partial activity by changing the activity of the enzyme.

The CRISPR enzymes are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2 , Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx4, Csxf, Csxf , Cpf1 or the like, or may include artificially modified forms, but is not limited thereto.

The CRISPR enzyme can be largely divided into six types according to the type of the CRISPR system. Type I may consist of Cas1, Cas2, Cas3, Cas8, etc .; Type II may consist of Cas1, Cas2, Cas2 / Cas4, Cas9, etc .; Type III may consist of Cas5, Cas6, Cmr1, Cmr3, Cmr4, Cmr5, Cas5 / Cmr1, Cam2, Cmr5, etc .; Type IV may consist of Csf1, Csf2, Csf3, etc .; Type V may be composed of Cas1, Cas2, Cas1, Cas2, Cas4, Cpf1, Cpf2, etc .; Type VI may be composed of C2C2, Cas1, Cas2, etc., but is not limited thereto.

Among the types of the CRISPR enzyme, Cas9 of Type II and Cpf1 of Type V are generally used the most.

In the CRISPR / Cas system of the present application, the CRISPR enzyme can use Cas9.

The Cas9 is Streptococcus pyogenes , Streptococcus thermophilus , Streptococcus sp., Staphylococcus aureus , Nocardiopsis dassoniville , Streptomyces pristinaespiralis , Streptomyces viridochromogenes , Streptomyces viridochromogenes , Streptosporangium roseum , Streptosporangium roseum , AlicyclobacHlus acidocaldarius , Bacillus pseudomycoides , Bacillus selenitireduces , Bacillus selenitireducens Ricum ( Exiguobacterium sibiric um ), Lactobacillus delbrueckii , Lactobacillus salivarius, Microscilla marina , Burkholderiales bacterium , Polaromonas naphthaleni (Polaromonas naphthalenivorans), Paula to Pseudomonas genus (Polaromonas sp.), Black COSPA Era watts soniyi (Crocosphaera watsonii), cyano tese in (Cyanothece sp.), micro-hour seutiseu Oh rugi labor (Microcystis aeruginosa), a cine Coco Synechococcus sp., Acetohalobium arabaticum , Ammonifex degensii , Caldicelulosiruptor bescii , Candidatus Desulforudis , Candidatus Desulfordi Clostridium botulinum , Clostridium difficile , Finegoldi a magna ), Natranaerobius thermophilus , Pelotomaculum thermopropionicum , Acidithiobacillus caldus , Acidithiobacillus ferrooxidans ), Allochromatium vinosum , Marinobacter sp., Nitrosococcus halophilus , Nitrosococcus watsoni , Pseudoaltero monas haloplantis (Pseudoalteromonas haloplanktis), keute also Novak Hotel racemic Pere (Ktedonobacter racemifer), metadata is Bruno Avenue Stevenage Away with you Tomb (Methanohalobium evestigatum), Ana Buena Varia Billy's (Anabaena variabilis), rowing dulra Leah's pumi dehydrogenase (Nodularia spumigena ), Nostoc sp., Arthrospira maxima , Arthrospira platensis ( Arthrospira platensis ), Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes , Oscillatoria sp., Petrotoga mobilis ( Petrotoga mobilis ), Thermosipho africanus or Acaryochloris marina , such as Cas9 from various microorganisms.

In addition, the Cpf1 is Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacterdium, Listeria, , Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus derived Cpf1.

The CRISPR enzyme interacts with the guide nucleic acid to form a complex to perform a function of cutting or modifying a target sequence of a target nucleic acid or gene. At this time, the function should include a PAM (protospacer adjacent modif) recognized directly by the CRISPR enzyme. That is, the base sequence of the PAM depends on the species of the CRISPR enzyme. For example, in the case of Cas9 protein derived from Streptococcus pyogenes , which is commonly used, 5'-NGG-3 'becomes a PAM sequence. For example, Cas9 protein derived from Staphylococcus aureus has 5'-NNGRRT-3 'as the PMA base sequence. As a result, when the target sequence of the target nucleic acid or gene is cleaved or the position is modified, the base sequence of PAM is essentially changed according to the selection of the species of the CRISPR enzyme, thereby cleaving the target sequence or modifying the position. Is decided.

The CRISPR / Cas system of the present application can be used to manipulate a target.

The operation may be any one or more selected from knock-out, knock-in, and knock-down.

For example, the manipulation can be knock-in.

For example, the manipulation can be knockout.

The target may be an organism containing a target nucleic acid or gene.

The organism can be a cell, tissue, plant, animal or human.

For example, the organism can be human.

The cells may be prokaryotic or eukaryotic cells.

Therefore, the vaccine composition and manufacturing method targeting the prevention or / and treatment of cholera disclosed by the present application may be superior in effect compared to the existing cholera vaccine, and are expected to be able to respond quickly to mutations in the cholera causative agent. .

3. Cholera vaccine composition

In order to solve the above-described problems of the present application, according to an aspect of the present application, a cholera vaccine composition is provided.

The cholera vaccine composition comprises: i) spores derived from bacteria; In this case, the bacterial-derived spore is a spore having a sequence encoding an antigenic substance in genomic DNA, ii) a spore coat protein present on the surface of the bacterial spore, and iii) cholera antigenicity linked to the spore coat protein. It may contain substances.

The cholera vaccine composition disclosed by the present application can quickly respond to mutations in pathogens.

The bacterial spore of the cholera vaccine composition of the present application may be a spore selected from B. subtilis, B. anthrasis, B. thuringiensis, and B. cereus.

For example, the bacterial spores of the cholera vaccine composition of the present application may be spores of B. subtilis.

Spore coat proteins of the cholera vaccine composition of the present application are CotA, CotB, CotC, CotD, CotE, CotF, CotG, CotN, CotS, CotT, CotV, CotW, CotX, CotY, CotZ, CotH, CotJA, CotJC, CotK, CotL , It may be a spore coat protein selected from CotM.

For example, the spore coat protein of the cholera vaccine composition of the present application may be a spore coat protein selected from CotB, CotC, and CotG.

The antigenic substance of the cholera vaccine composition of the present application may be an antigenic substance selected from ctxA, ctxB, rstR, and tcpA.

The cholera vaccine composition of the present application may include bacterial spores, 1 spore coat protein and 1 antigenic substance.

The cholera vaccine composition of the present application may include bacterial spores, one spore coat protein, and two or more antigenic substances.

The cholera vaccine composition of the present application may include bacterial spores, two or more spore coat proteins, and one antigenic substance.

The cholera vaccine composition of the present application may include bacterial spores, two or more spore coat proteins, and two or more antigenic substances.

The cholera vaccine composition may further include a spore coat protein and a linker connecting an antigenic substance.

The linker may be an enzyme, a cleavage enzyme, a polypeptide, a protein, a base sequence, an artificial base sequence that forms an artificial chemical bond, and the like, but is not limited to, as long as it has a function of connecting the spore coat protein and an antigenic substance. .

The cholera vaccine composition is a stabilizer, emulsifier, aluminum hydroxide, aluminum phosphate, pH adjuster, surfactant, liposome, iscom adjuvant, synthetic glycopeptide, extender, carboxypolymethylene, bacterial cell wall, bacterial cell wall derivative, bacterial vaccine , Animal poxvirus protein, subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyl Dioctadecyl-ammonium bromide and mixtures thereof may further include any one or more second adjuvants selected from the group.

In addition, the cholera vaccine composition, medically acceptable carriers, diluents, binders, disintegrants, lubricants, pH adjusters, antioxidants, dissolution aids, antigen adjuvants, stabilizers, preservatives, antibacterial agents, antifungal agents, adsorption retardants, etc. It may further include.

The carrier is defined as a compound that facilitates the addition of a compound into cells or tissues. For example, dimethyl sulfoxide (DMSO) can be used.

The diluent (buffer solution) is, for example, sugar, starch, microcrystalline cellulose, lactose (lactose hydrate), glucose, di-mannitol, alginate, alkaline earth metal salts, clay, polyethylene glycol, calcium hydrogen phosphate anhydrous, or Mixtures of these and the like can be used; Binders are starch, microcrystalline cellulose, highly dispersible silica, mannitol, di-mannitol, sucrose, lactose hydrate, polyethylene glycol, polyvinylpyrrolidone (povidone), polyvinylpyrrolidone copolymer (copovidone), hypromellose , Hydroxypropyl cellulose, natural gums, synthetic gums, copovidone, gelatin, or mixtures thereof.

The disintegrating agent may be, for example, starch or modified starch such as sodium starch glycolate, corn starch, potato starch or pregelatinized starch; Clays such as bentonite, montmorillonite, or veegum; Cellulose such as microcrystalline cellulose, hydroxypropyl cellulose or carboxymethyl cellulose; Algins such as sodium alginate or alginic acid; Cross-linked celluloses such as croscarmellose sodium; Gums such as guar gum and xanthan gum; Crosslinked polymers such as crosslinked polyvinylpyrrolidone (crospovidone); Effervescent agents, such as sodium bicarbonate and citric acid, or mixtures thereof can be used.

The lubricant is, for example, talc, stearic acid, magnesium stearate, calcium stearate, sodium lauryl sulfate, hydrogenated vegetable oil, sodium benzoate, sodium stearyl fumarate, glyceryl behenate, glyceryl monorate, glyceryl monostea Rate, glyceryl palmitostearate, colloidal silicon dioxide, or mixtures thereof.

The pH adjusting agent is, for example, acidifying agents such as acetic acid, adipic acid, ascorbic acid, sodium ascorbate, sodium etherate, malic acid, succinic acid, tartaric acid, fumaric acid, citric acid (citric acid), precipitated calcium carbonate, ammonia water, Basic agents such as meglumine, sodium carbonate, magnesium oxide, magnesium carbonate, sodium citrate, and tribasic calcium phosphate can be used.

The antioxidant may be, for example, dibutyl hydroxy toluene, butylated hydroxyanisole, tocopherol acetate, tocopherol, propyl gallate, sodium hydrogen sulfite, sodium pyrosulfite, and the like.

The solubilizer may be, for example, sodium lauryl sulfate, polyoxyethylene sorbitan fatty acid esters such as polysorbate, docusate sodium, poloxamer, or the like.

In addition, an enteric polymer, a water-insoluble polymer, a hydrophobic compound, and a hydrophilic polymer may be included to make a delayed-release preparation.

The enteric polymer is, for example, hypromellose acetate succinate, hypromellose phthalate (hydroxypropylmethylcellulose phthalate), hydroxymethylethylcellulose phthalate, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate maleate , Enteric cellulose derivatives such as cellulose benzoate phthalate, cellulose propionate phthalate, methylcellulose phthalate, carboxymethylethylcellulose, ethylhydroxyethylcellulose phthalate and methylhydroxyethylcellulose; Styrene-acrylic acid cellulose, methyl acrylate-acrylic acid cellulose, methyl methacrylate acrylic acid cellulose (e.g., acrylic-is), butyl acrylate-styrene-acrylic acid cellulose, and methyl acrylate-methacrylic acid-octyl acrylate The enteric acrylic acid-based cellulose such as; Poly (methyl methacrylate) cellulose (e.g. Eudragit L, Eudragit S, Evocelgert, Kar (ethyl methacrylate)) cellulose (e.g. Eudragit L100-55 Enteric carmethacrylate cellulose such as); vinyl acetate-tyl maleate cellulose, cellulose, styrene-maleate maleate cellulose, styrene-maleic acid monoester cellulose, vinyl methyl ether-maleic acid maleate cellulose cellulose, Enteric properties such as ethylene-butyl maleate cellulose, vinylbutyl ether-maleate maleate cellulose, acrylonitrile-methyl acrylate, maleate maleate cellulose, and butyl styrene-maleate maleate cellulose Maleic acid-based cellulose; and nitrile alcohol phthalate, nitrile acetal phthalate, polyvinyl butylate phthalate, and polyvinyl acetal phthalate. There are enteric polyvinyl derivatives.

The water-insoluble polymer refers to a polymer that is not soluble in pharmaceutically acceptable water that controls drug release. For example, the water-insoluble polymer is polyvinyl acetate (eg, Colicoat SR30D), a water-insoluble polymethacrylate copolymer [eg, poly (ethylacrylate-methyl methacrylate) copolymer (eg, Eudragit NE30D) , Poly (ethyl acrylate-methyl methacrylate-trimethylaminoethyl methacrylate) copolymer (e.g. Eudragit RSPO), etc., ethyl cellulose, cellulose ester, cellulose ether, cellulose acylate, cellulose diacylate, Cellulose triacylate, cellulose acetate, cellulose diacetate and cellulose triacetate.

The hydrophobic compounds include, for example, fatty acid and fatty acid esters such as glyceryl palmitostearate, glyceryl stearate, glyceryl bihenate, cetyl palmitate, glyceryl mono oleate and sleaic acid; Fatty alcohols such as cetostearyl alcohol, cetyl alcohol and stearyl alcohol; Waxes such as carnauba wax, beeswax, and microcrystalline wax; Mineral materials such as talc, precipitated calcium carbonate, calcium monohydrogen phosphate, zinc oxide, titanium oxide, kaolin, bentonite, montmorillonite, and arsenic.

The hydrophilic polymers include, for example, dextrin, polydextrin, dextran, pectin and pectin derivatives, alginates, polygalacturonic acid, xylan, arabinoxylan, arabinogalactan, starch, hydroxypropyl starch, amylose, And sugars such as amylopectin; Cellulose derivatives such as hypromellose, hydroxypropylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, methylcellulose and sodium carboxymethylcellulose; Gums such as guar gum, locust bean gum, tragacanta, carrageenan, acacia gum, gum arabic, gellan gum, and xanthan gum; Proteins such as gelatin, casein, and zein; Polyvinyl derivatives such as polyvinyl alcohol, polyvinyl pyrrolidone, and polyvinyl acetal diethyl amino acetate; Poly (butyl methacrylate- (2-dimethylaminoethyl) methacrylate-methylmethacrylate) copolymer (e.g. Eudragit E100, Evonik, Germany), poly (ethyl acrylate-methyl methacrylate) Hydrophilic polymethacrylate copolymers such as triethylaminoethyl-methacrylate chloride) copolymers (eg Eudragit RL, RS, Evonik, Germany); Polyethylene derivatives such as polyethylene glycol and polyethylene oxide; And carbomers.

In addition, pharmaceutically acceptable additives may be selected and used as various additives selected from colorants and fragrances to formulate the formulations of the present application.

In the present application, the range of additives is not limited to the use of the additives, and the above-described additives can be formulated by containing a dose in a normal range by selection.

In addition, the cholera vaccine composition can be used by formulating in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols 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, disintegrating agents, surfactants, etc., which are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose in the lecithin-like emulsifier. , Gelatin, etc. can be prepared by mixing. It is also possible to use lubricants, such as magnesium stearate, in addition to simple excipients. As a liquid preparation for oral administration, suspending agents, intravenous solutions, emulsions, syrups, etc. can be used. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc. Can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, and lyophilized preparations. Non-aqueous preparations, suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate, but are not limited thereto.

For oral dosage forms, the release site can be the stomach, small intestine (duodenum, jejunum, ileum) or large intestine. Those skilled in the art can use formulations that do not dissolve in the stomach but release the substance into the duodenum or elsewhere in the intestine, for example by using an enteric coating. Examples of more common inert ingredients used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit (Eudragit) ) L30D, Aquateric, Cellulose Acetate Phthalate (CAP), Eudragit L, Eudragit S and Shellac. These coatings can be used as mixed films.

In addition, coatings or mixtures of coatings can be used on tablets that are not intended to protect the stomach. This can include sugar coatings or coatings that make the tablet easier to swallow. The capsule may be composed of a hard shell (eg, gelatin) for transport of the dried therapeutic agent (ie, powder), or in the case of liquid form, a soft gelatin shell may be used. The shell material of the case can be thick starch or other edible paper. For tablets, lozenges, molded tablets or tablet abrasives, a moist massing technique can be used. The formulation of the substance for capsule administration may also be in the form of a powder, a lightly compressed plug or even a tablet. The therapeutic agent can be prepared by compression.

4. Method for manufacturing cholera vaccine

In order to solve the above-described problems of the present application, according to an aspect of the present application, a method for manufacturing a cholera vaccine is provided.

For example, the method for preparing the cholera vaccine of the present application includes: i) knocking-in an antigenic substance using CRISPR / Cas9 in genomic DNA of bacteria (Knock-in); ii) forming spores of the bacteria; And iii) expressing an antigenic substance on the spore surface of the bacteria.

The method for preparing the cholera vaccine may include inserting a foreign material as a donor into the bacterial genomic DNA.

The “donor” means an exogenous nucleotide capable of expressing a specific peptide or protein, and can be inserted into genomic DNA using, for example, an HDR mechanism. At this time, the donor may include a specific nucleic acid encoding a specific peptide or protein.

The donor may be double-stranded nucleic acid or single-stranded nucleic acid.

The donor can be linear or circular.

The donor may include a nucleic acid sequence having homology to the target gene or nucleic acid.

The donor may be a nucleic acid encoding an antigenic substance.

For example, the donor may be a nucleic acid encoding a cholera antigenic substance. For example, it may be a nucleic acid encoding any one or more selected from ctxA, ctxB, rstR and tcpA. As another example, the donor may be a nucleic acid encoding all of the cholera antigenic substances ctxA, ctxB, and tcpA.

The donor may be a cholera antigenic substance coupled with a nucleic acid sequence having homology to a specific region of a bacterial genomic DNA sequence (eg, a homology arm).

The method for preparing the cholera vaccine of the present application may be modified in a genomic DNA of bacteria using a specific example, CRISPR / Cas9.

Modification to the genomic DNA of bacteria using the CRISPR / Cas9 may be at least one selected from knock-in, knock-out, and knock-down.

For example, CRISPR / Cas9 can be used to target a specific region of the bacterial genomic DNA to knock out the foreign antigenic material.

For example, CRISPR / Cas9 can be used to knock out specific genes of bacterial genomic DNA.

For example, CRISPR / Cas9 can be used to knock down specific genes of bacterial genomic DNA.

Hereinafter, a process of inserting (knocking in) a nucleic acid encoding a cholera antigenic substance by targeting a specific region of bacterial genomic DNA using CRISPR / Cas9 will be described.

To this end, in order to knock in the cholera antigenic substance, the target site sequence or the base sequence of the target gene in the bacterial genomic DNA is determined. In this case, the target sequence may be a CRISPR enzyme, that is, a nucleotide sequence located close to a PAM sequence recognizable by Cas9 or Cpf1. The target sequence may have or be complementary to the guide RNA.

For example, target sequencing data is collected using a known database (eg, NCBI), and PAM sequence (eg, 5'-NGG-3) which is a site recognized by CRISPR / Cas9 among the sequencing. Guide RNA can be designed considering ').

The CRISPR / Cas9 Cas9 protein can be derived from the genus Streptococcus spp. For example, Streptococcus pyogenes Cas9 (spCas9) can be used, but is not limited thereto. At this time, the PAM sequence may be 5'-NGG-3 '(N is A, T, G, or C).

The target gene or nucleic acid can be cleaved using CRISPR / Cas9. The cleaved target nucleic acid site can include modifications on the bacterial genomic DNA via NHEJ or HDR mechanisms.

Since CRISPR / Cas9 used as an embodiment in the present application is a prokaryotic-derived system, it has an advantage of having high efficiency in the modification of bacterial genomic DNA.

In particular, compared to eukaryotic cells, the HDR mechanism works better in bacteria, so that certain nucleic acids can be inserted on the bacterial genomic DNA with very high efficiency.

When an antigenic substance is inserted (transformed) into the bacterial genomic DNA, it uses the bacterial genomic DNA's own expression system, so that the antigenic substance can be continuously expressed and transformed every time the vaccine is prepared. It has the advantage of omitting the process of performing.

In one embodiment of the present application, at least one cholera antigenic material among ctxA, ctxB, and tcpA was inserted on the genomic DNA in Bacillus with high efficiency.

Meanwhile, in addition to the knock-in method using the CRISPR / Cas9, if it is a knock-in technique such as a method using zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), it is not limited thereto.

Vectors can be used for the knock-in.

Vectors can be transformed by preparing a vector to knock in bacteria using guide RNA, Cas9, and antigenic substances. The CRISPR / Cas9 vector and the antigenic material vector may include Cas9, guide RNA, and antigenic material in one vector, or Cas9, guide RNA, and antigenic material may be independently included in the same or different vectors, respectively. .

The vector may be a viral vector or a non-viral vector.

The viral vector may be a viral vector such as retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, but is not limited thereto.

The non-viral vector may be plasmid, naked DNA, etc., but is not limited thereto.

In addition, in addition to a method using a vector, an electroporation method, a gene gun, a self-injection method, an ultrasonic perforation method, etc. may be used, but is not limited thereto.

After inserting a donor encoding a cholera antigenic substance into the bacterial genomic DNA, a step of forming spores from the bacteria is performed to use a spore display system.

The step ii) of forming the spores of the bacteria may include nutrient-depleted nutritional conditions, high-temperature or low-temperature temperature conditions, high- or low-moisture moisture conditions, chemical conditions such as fungicides, preservatives, acidic or alkaline Spores may be formed using pH conditions, but are not limited thereto.

For example, when preparing the cholera vaccine of the present application, the step of forming spores of bacteria may be made of any one or more of nutrient-depleted pharmacologic conditions, high temperature or low temperature conditions.

Next, in the spores of the formed bacteria, the step of expressing an antigenic substance on the surface of the spores is performed (step iii)).

The antigenic substance may be expressed by a) directly linked to a spore coat protein expressed on the surface of a bacterial spore, or b) indirectly linked.

The a) direct expression refers to a form in which a spore coat protein and an antigenic substance present on a bacterial spore surface are directly connected without a mediator (eg, a linker).

The b) indirect expression refers to a form indirectly linked by a linker connecting a spore coat protein and an antigenic substance present on the surface of a spore of bacteria.

In one embodiment of the present application, the spore coat protein and the cholera antigenic substance may be expressed by linking with a linker.

The method for preparing the cholera vaccine may further include a curing step.

The curing step may be to obtain only the desired substance or remove unwanted substances from the composition of the cholera vaccine.

For example, the curing step may be to remove antibiotic resistance-causing substances.

For example, the curing step may be to remove the foreign plasmid added during the vaccine manufacturing process.

The curing step may be performed by two or more passages, but the conventional curing method is not limited thereto.

The method for preparing the cholera vaccine may further include adding an additional substance.

In this case, the additional substance is considered to include all of the pharmaceutically acceptable additives in the manufacture of the vaccine.

The cholera vaccine is formulated for intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery. do.

The route of administration of the vaccine is selected from intramuscular, intranasal, oral, intraperitoneal, subcutaneous, topical, intradermal and transdermal delivery.

5. How to treat cholera

In order to solve the above-described problems of the present application, according to an aspect of the present application, a cholera treatment method is provided.

The cholera treatment method provides a method of treating cholera by administering a composition comprising the composition as an active ingredient.

The term "administration" in this application means to introduce the pharmaceutical composition of the present application to a mammal in any suitable way, and the route of administration of the pharmaceutical composition of the present application is administered through any general route as long as it can reach the target tissue. Can be. Oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endothelial administration, intranasal administration, intrapulmonary administration, rectal administration, intranasal administration, intraperitoneal administration, intrathecal administration, but are not limited thereto. In addition, the mammal may include humans, puppies, mice, and the like, but is not limited thereto.

In addition, a method of administering a composition containing the composition as an active ingredient may be administered as in the following examples, but is not limited thereto.

For example, when administering a composition containing the composition as an active ingredient, the dosage for the human body may vary depending on the patient's age, weight, gender, dosage form, health status and disease level, and an adult patient with a body weight of 70 kg Based on the, it is generally 0.01 mg to 5000 mg per day, and may be dividedly administered once to several times a day at regular time intervals according to the judgment of a doctor or pharmacist.

The dose of the cholera vaccine is preferably 1 µg to 100 µg of the derivative per animal, and this dose may vary depending on the size of the animal. For pigs, for example, a very suitable dosage is 20 μg to 80 μg. For example, for humans, a very suitable dosage is 10 μg to 100 μg.

The method of administration of the cholera vaccine is intramuscular administration, intranasal administration, oral administration, intraperitoneal administration, subcutaneous administration, topical administration, intradermal It may be administered by a method such as administration or transdermal administration, but any conventional administration method may be used.

For example, the cholera vaccine of the present application may use an intramuscular administration method.

For example, the cholera vaccine of the present application may use an intranasal administration method.

For example, the cholera vaccine of the present application may use an oral administration method.

For example, the cholera vaccine of the present application may use an intraperitoneal administration method.

For example, the cholera vaccine of the present application may use a subcutaneous administration method.

For example, the cholera vaccine of the present application can use a method of topical administration.

For example, the cholera vaccine of the present application may use an intradermal administration method.

For example, the cholera vaccine of the present application may use a method of transdermal administration.

When the cholera vaccine composition comprising the composition disclosed by the present application as an active ingredient is administered to a mammal, the concentration may be administered at 1 mg / kg to 100 mg / kg.

The present application can provide a method of treating cholera by administering a composition containing the composition as an active ingredient to a human.

For example, when a composition containing the composition as an active ingredient is administered to a human from 1 mg / kg to 100 mg / kg, a method of treating cholera may be provided.

Therefore, treatment of cholera with the composition disclosed by the present application may not only be able to respond quickly even if a mutation in the pathogen of the hospital occurs, but also may be more effective than the existing cholera vaccine.

[Form for conduct of application]

Hereinafter, the present application will be described in more detail through examples.

The following examples of the present application relate to the production of antigens, antibodies and vaccines for cholera vaccines, but these examples are only intended to illustrate the present application and the scope of the present application is not limited by these examples. It will be apparent to those of ordinary skill in the art.

Example 1: Bacillus experimental species and vector production

i) Bacillus test species

Bacillus subtilis 168 used in the experiment was pre-sale from Bacillus Genetic Stock Center and cultured at 37 ° C using Luria-Bertani (LB) broth and LB plate medium. The pre-distributed cell stock was subjected to primary screening (18 hs) in an LB solid medium, and secondary colony (18 hs) was performed by securing one colony.

ii) B. subtilis sporulation

B. sporulation a salt subtillis (1 M CaCl ₂ 1ml / L, 0.1 M MnCl ₂ 1 ml / L, 0.01 M FeSO ₄ 1 ml / L) of NB medium (nutrient broth 8 g / L, MgSO 4 contain - 0.25 g / L, KCl-1 g / L), followed by shaking culture at 37 ° C (48 h), centrifugation at 8,000 rpm for 15 min, and harvesting.

It was resuspended by concentrating 60% using cold water (D.W.) and reacted overnight at 4 ℃ (18 h). Then, heat inactivation is performed at 80 ° C for 30 minutes. After centrifugation at 8,000 rpm 15 min and resuspension with 1 X PBS after harvest, the resulting spores were stored and used at 4 ° C.

iii) Vector production

(a) pHCas9 expression vector (Figure 1)

As a pHCas9 vector for expression of S. pyogenes Cas9 (SpCas9) protein in B. subtilis , the pHCas9 expression vector was used by the Korea Institute of Bioscience and Biotechnology.

(b) epitope vector

A vector containing a donor DNA, a gene complex containing a CotG CDS containing a guide RNA and a promoter of B. subtilis Coat protein (CotG) and an antigenic determinant, was prepared.

Guide RNA production program CCTOP (https://crispr.cos.uni-heidelberg.de) from the B. subtilis genome was used to construct guide RNA (SEQ ID NO. 1: TACGTTCATCCTTAACATATTTTTAAAAGGA) in the CotG region, with the restriction enzyme NcoI It was introduced into the pHY300PLK vector using EcoRV and named pJB (FIG. 2).

At this time, a vector containing GFP and Cholera Toxin, CtxA, CtxB, and TcpA, was prepared as antigenic determinants.

ㆍ pJB-GFP (Fig. 3)

To confirm that the desired epitope was located on the surface of B. subtilis spores, an expression vector in which green fluorescent protein (eGFP) was inserted was constructed. The GFP gene was amplified through PCR by constructing a primer from the MDH1-PGK-GFP_2.0 vector (SEQ ID NO. 2, 3 in Table 1), a restriction enzyme PstI at the 5 ′ end, and BamHI at the 3 ′ end. Did. The amplified GFP DNA was prepared by removing and introducing CtA from the pJB001 vector using restriction enzymes PstI and BamHI to construct pJB-GFP.

ㆍ pJB-Cholera Toxin (pJB001 to pJB003) (FIGS. 4 to 6)

B. subtilis spore surface to raise the antigenic determinant site B. subtilis gDNA using CotG-Full_F and CotG-Full_R primers (Table 1; SEQ ID NO. 4, 5) promoter and CDS portion of Coat protein (CotG) Was amplified, and a restriction enzyme PstI containing a restriction enzyme HindIII at the 5 'end and a 15 bp linker at the 3' end was produced. In addition, the antigen-determining site identified cholera toxin A, B, and TcpA regions in Vibrio cholerae gDNA, and CtxA_F, CtxB_F, TcpA_F containing restriction enzymes PstI, and CtxA_R, CtxB_R, and TcpA_R containing BamHI were used to obtain cholera toxin genes. (Table 1; SEQ ID NO. 6 to 11)

The obtained coat protein and cholera toxin genes were cut using PstI restriction enzyme, and donor DNA was prepared using T4 ligase. The produced donor DNA was introduced into the pJB vector using restriction enzymes HindIII and BamHI to produce pJB001 with cholera toxin CtA inserted, pJB002 with CtB inserted, and pJB003 with TcpA inserted.

No. Name Sequence (5 '→ 3') SEQ ID NO. 2 GFP_F ATAAAGCTTATGGTGAGCAAG SEQ ID NO. 3 GFP_R TATGGATCCTTACTTGTACAG SEQ ID NO. 4 CotG-Full_F ATAAAGCTTAGTGTCCCTAGCTCCG SEQ ID NO. 5 CotG-Full_R TATCTGCAGTGAACCCCCACCTCCTTTGTATTTCTTTTTG SEQ ID NO. 6 CtxA_F ATACTGCAGATGGTAAAGATA SEQ ID NO. 7 CtxA_R TATGGATCCGGATGAATTATGA SEQ ID NO. 8 CtxB_F ATACTGCAGATGATTAAATTAA SEQ ID NO. 9 CtxB_R TATGGATCCTATGGCAAATTAA SEQ ID NO. 10 TcpA_F ATACTGCAGATGACATTACTCGAAG SEQ ID NO. 11 TcpA_R TATGGATCCGTAACAGTTAA

(c) Antibiotic deficiency vector (pJB-sg02 to pJB-sg03) (FIGS. 7 to 8)

A guide RNA (SEQ ID NO. 12: CGATACGTGCTCCGGTTCCCAACGATCAAGGA) targeting the ampicillin resistance gene and a guide RNA targeting the neomycin resistance gene (SEQ ID NO.13: GACGAGT TATTAATAGCTGAATAAGAACGGT) were prepared for the deletion of the antibiotic resistance gene, and limited The enzymes NcoI and EcoRV were introduced into the pJB vector, and pJB-sg02 (amp deletion) and pJB-sg03 (neo deletion) vectors were prepared using the donor DNA, which lacked the base sequence of the antibiotic resistance gene.

Example 2: Bacillus soluble cell production (B. Subtilis soluble cell (Competent cell) production)

B. Subtilis cells were carried out as follows to introduce foreign substances (plasmid, phage DNA, etc.) into the cells.

For B. subtilis soluble cells, one B. subtilis colony was primary cultured (18 hs) in a 37 ° C shake incubator using growth medium (LB broth + 0.5M Sorbitol) and diluted 1/16 in growth medium. Secondary incubation was performed from .D600 to 0.85-0.95.

 The culture solution was reacted on ice for 10 minutes to cool, and centrifugation was performed at 4 ° C and 5000 X g for 5 minutes to discard the supernatant and ice-cold Wash buffer (0.5 M sorbitol, 0.5 M mannitol, 10%) glycerol) and washed 4 times. After diluting the electroporation solution (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol) to 1-1.3 * 1010 cfu / mL, water-soluble cells were prepared and then aliquoted at 60 μl and stored at -80 ° C.

Example 3: B. Subtilis transformant production

Each of the vectors prepared in Example 1 was introduced into B. Subtilis soluble cells (Competent cell) prepared in Example 2 and transformed as follows.

Transformation of B. subtilis was performed by Electroporation method (Xue. Et al. 1999) using Gene Pulser System (BIO-RAD. USA). To the prepared B. subtilis soluble cells (60 μl), 5 μl (200 ng / μl) of the transformed expression vector is added, mixed, and reacted on ice for 3 minutes, and then placed in an electroporation cuvette (0.1 cm gap). voltage; 2,100 V, time constant; 5 ms, No. of pulses; Under the condition of 1, an electric shock is given to induce the transformation expression vector to be introduced into the cell. Thereafter, the cells are cultured at 37 ° C for 3 hours to induce recovery of transformed cells, and plated on an antibiotic selective medium to culture at 37 ° C.

(a) B. Subtilis transformant introduced with pHCas9 vector

B. Subtilis transformants introduced with pHCas9 vector were cultured by adding Neomycin (10 μg / mL).

(b) B. Subtilis transformant introduced with pJB-GFP vector

B. Subtilis transformants introduced with pJB-GFP vector were cultured in LB solid medium with Ampicillin (100 μg / mL) and Neomycin (10 μg / mL).

(c) pJB-Cholera Toxin (pJB001 to pJB003) vector introduced B. Subtilis transformant

B. Subtilis transformants introduced with pJB001 to pJB003 vectors were cultured in LB solid medium containing Ampicillin (100 μg / mL) and Neomycin (10 μg / mL).

(d) B. Subtilis transformants introduced with antibiotic deletion vectors (pJB-sg02 to pJB-sg03)

B. Subtilis transformants introduced with pJB-sg02 or pJB-sg03 vectors for the deletion of antibiotic resistance genes were first cultured in LB solid medium without antibiotics, and then transferred to each colony to the antibiotic medium. Colonies that did not grow were selected.

Example 4: Confirmation of vector introduction in B. subtilis transformants

In order to confirm that GFP is expressed on the spore surface of the spore by introducing the GFP expression vector into the spores of B. subtilis, it was confirmed through PCR analysis and flow cytometry.

ㆍ PCR analysis

Colony of transformant B. subtilis was cultured in LB broth to isolate gDNA. Primer was prepared (SEQ ID NO. 14, 15 in Table 2; In-GFP_F, In-GFP_R) to perform PCR to confirm the presence of transgenes in transformants and wild-type DNA.

No. Name Sequence (5 '→ 3') SEQ ID NO. 14 In-GFP_F ATCATGGCCGACAAGCAGAA SEQ ID NO. 15 In-GFP_R TCTCGTTGGGGTCTTTGCTC SEQ ID NO. 16 In-CtxA_F ATACTGCAGATGGTAAAGATA SEQ ID NO. 17 In-CtxA_R TATGGATCCTCATAATTCATCC SEQ ID NO. 18 In-CtxB_F ATACTGCAGATGATTAAATTAA SEQ ID NO. 19 In-CtxB_R TATGGATCCTTAATTTGCCATA SEQ ID NO. 20 In-TcpA_F ATACTGCAGATGACATTACTCGAAG SEQ ID NO. 21 In-TcpA_R TATGGATCCTTAACTGTTAC

The results of PCR performed to confirm the expression of GFP on the surface of B. subtilis spores by introducing a GFP expression vector into the genome of B. subtilis cells are shown in FIG. 9.

As shown in Figure 9, the band of GFP was confirmed, it was found that GFP is expressed on the surface of B. subtilis cell spores.

ㆍ Flow cytometry (FACS)

To verify the surface expression of the introduced gene protein in the transformant of B. subtilis, it was confirmed by performing flow cytometry using the pJB-GFP vector.

For analysis by flow cytometry, B. subtilis transformed with pJB-GFP vector was washed with 0.22 μm filtered 1.5 × PBS and diluted. The concentration of microspheres was calculated in the range of 106 beads ^-1 , and B. subtilis cells were injected into the flow cytometer within minutes after resuspension. Analysis was performed using a FACScalibur (Becton Dickinson, Oxford, UK) instrument.

Fluorescence in the range of 515-545nm was detected through a fluorescence detector set at a voltage of 600V. Forward scattering (FS) was collected with a diode with an amplification factor of 10 and calculated as a logarithmic value. The side scatter (SS) was detected at the logarithmic value by a photomultiplier tube set to 366V. For all output signals [Fluorescence (FL), FS and SS], the logarithmic channel number is converted to linear intensity and the geometric mean with 'G average' is defined as: G mean = 10ΣlogX (i) / n; 10,000 signals were measured for each measurement.

The results of flow cytometry performed to confirm whether B. subtilis cells were introduced into the pJB-GFP expression vector are shown in FIG. 10.

In FIG. 10, compared to the wild type graph in which the pJB-GFP expression vector was not introduced, the graph of GFP in the CotG-GFP, CotC-GFP, and CotB-GFP graphs into which the pJB-GFP expression vector was introduced was confirmed, and the PCR result and Similarly, it was found that GFP is expressed on the surface of B. subtilis cell spores.

Example 5: Identification of target antigenic determinants on the surface of B. subtilis spores

In Example 4, the GFP expression vector was introduced into the spores of B. subtilis to confirm whether GFP was expressed on the spore surface, and to confirm whether the desired antigen expression vector was expressed on the surface of the spore instead of the GFP expression vector. Expression of (pJB001 to pJB003) was confirmed by PCR analysis.

ㆍ PCR analysis

Colony of transformant B. subtilis was cultured in LB broth to isolate gDNA. Primer was prepared (SEQ ID NO. 16 to 21 in Table 2; In-CtA_F, In-CtA_R, In-CtB_F, In-CtB_R, In-TcpA_2F, In-TcpA_2R) to perform PCR with transformants and wild type It was confirmed whether or not the transgene was introduced from DNA.

The PCR results performed to confirm the expression vector of the desired antigen on the surface of B. subtilis spores are shown in FIG. 11.

In FIG. 11, amplification bands of CtxA, CtxB, and TcpA were identified, and it was found that the desired antigen was expressed on the spore surface of B. subtilis.

Example 7: Confirmation of antibody formation

In order to remove the antibiotic resistance gene (eg, neo, amp, etc.) inserted into the genome of B. subtilis spores, the B. subtilis transformant into which Cholera toxin expression vector was introduced was transformed into pJB-sg02, 03 vector Was performed to induce a deletion of the antibiotic resistance gene. After transforming the transformant onto LB plate medium without antibiotics, each single colony formed was negatively selected and cultured on the antibiotic LB plate medium, and colonies that did not grow on the antibiotic LB plate medium were screened for antibiotic gene defect B. subtilis. Secured.

B. subtilis spores were subjected to high-temperature treatment at 80 ° C for 20 minutes and inactivated, and then used in animal experiments to confirm immunity using B. subtilis transformants expressing the target antigen with the antibiotic gene deletion target antigen. . C57BL / 6, female, 8 weeks of age were used for the mouse, and 5 mice were set as a group.

The route of administration was directly administered in the stomach using a sonde for mouse to confirm immunity through the mucous membrane, and the dose was 5x10 ^ 10 spore / 0.2ml. The administration was conducted three times in total, and when administered once, it was administered continuously for 3 days, and the interval between each cycle was set to 2 weeks.

The antibody formation confirmation experiment was analyzed by ELISA and western blot using animal experiments.

Serum required for ELISA and western blot was performed by orbital blood collection after light anesthesia one day prior to administration, followed by inactivation at room temperature for 30-60 minutes, followed by centrifugation at 6000 rpm, 7 min, 4 ° C to separate only the supernatant.

ㆍ Indirect ELISA analysis

The method of coating the antigen on the plate was used, and the purified cholera toxin peptide was also coated at a concentration of 100 ng / well to detect cholera toxin specific antibody formation. Carbonate / bicarbonate buffer (pH 9.6) of 0.05M was used as the coating solution.

Dilution fold of antibody and dilution of secondary antibody were determined and analyzed using immobilized solution (1% BSA) to establish ELISA assay conditions. The dilution factor of the primary antibody was 1:10 to 1: 10,000, and the secondary antibody was Goat-anti-mouse IgG-HRP, and the dilution factor was set to a range of 1: 5,000 to 1: 160,000 and analyzed. The concentration (1: 10,000) at which the value of the reaction degree (B _T / B ₀ ) maintains 50% was selected and determined as the optimal concentration used in the assay.

As a washing solution, PBS buffer containing 0.05% Tween 20 was used, and the reaction of each step was incubated at room temperature for 2 hours to react. TMB was used as the substrate solution, and the reaction was terminated by injecting 100 ul of 0.16 MH ₂ SO ₄ (stop solution), and after stopping the reaction, the OD value was measured at a wavelength of 450 nm in an ELISAreader (TECAN SUNRISE, BioSurplus) (FIG. 12).

12 shows the results of the Indirect ELISA analysis.

Through the ELISA results of FIG. 12, it can be seen from the results of FIG. 12 that CtxA is generated at week 2 and CtxB and TcpA are generated at week 4 among antigen-specific antibodies in the blood of rats administered the antigen.

ㆍ Western blot analysis

Protein was isolated by loading 500 ng / ml of cholera toxin peptide onto 10% SDS-PAGE gel. The isolated protein was transferred to a polyvinylidene difluoride membrane (PVDF, Bio-rad) for 2 hours in a cold room at 4 ° C for 2 hours, followed by a TBST (Tris buffered saline with Tween 20, pH 7.5) solution containing 5% skim milk. By blocking at room temperature for 1 hour. As the primary antibody, Anti-Ovalbumin antibody (abcam) was diluted 1: 5 in 5% skim milk, and in order to examine the amount of Ovalbumin antibody expression in the mouse, serum of blood collected from the mouse was 5% skim. Diluted in milk with 1:10 or 1: 100 was used. Each was reacted at 4 ° C. for 18 hours, and then washed three times with TBST for 5 minutes. As a secondary antibody, goat anti-mouse IgG H & L (abcam) bound with horse radish peroxidase (HRP) was diluted to 1: 20,000 in 5% skim milk, reacted for 1 hour at room temperature, and washed 3 times with TBST. After reacting with ECL (Thermo Scientific, Rockford, IL, USA) substrate for 1-3 minutes, each protein band was visualized with LAS-3000 (FIG. 13).

13 shows the results of Western blot analysis.

Through the results of FIG. 13, it can be confirmed through FIG. 13 that CtxA is generated at week 2 and CtxB and TcpA are generated at week 4 among antigen-specific antibodies in the blood of mice to which the antigen has been administered.

The CRISPR / Cas9 based Bacillus subtillis spores produced by the method of the present application through the Indirect ELISA and Western blot analysis of FIGS. 12 and 13 can express the desired antigen cholera antigen on the surface of the spores of B. subtilis to form an antibody. It was confirmed that there was.

<110> JBBiotech <120> Cholera vaccine using CRISPR / Cas9 based Bacillus subtillis genome          editing mechanism <130> CP19-055 <160> 21 <170> KoPatentIn 3.0 <210> 1 <211> 31 <212> RNA <213> Artificial Sequence <220> <223> CotG_gRNA <400> 1 tacgttcatc cttaacatat ttttaaaagg a 31 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFP_F <400> 2 ataaagctta tggtgagcaa g 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> GFP_R <400> 3 tatggatcct tacttgtaca g 21 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CotG-Full_F <400> 4 ataaagctta gtgtccctag ctccg 25 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> CotG-Full_R <400> 5 tatctgcagt gaacccccac ctcctttgta tttctttttg 40 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CtxA_F <400> 6 atactgcaga tggtaaagat a 21 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CtxA_R <400> 7 tatggatccg gatgaattat ga 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CtxB_F <400> 8 atactgcaga tgattaaatt aa 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CtxB_R <400> 9 tatggatcct atggcaaatt aa 22 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> TcpA_F <400> 10 atactgcaga tgacattact cgaag 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TcpA_R <400> 11 tatggatccg taacagttaa 20 <210> 12 <211> 32 <212> RNA <213> Artificial Sequence <220> <223> ampicillin_gRNA <400> 12 cgatacgtgc tccggttccc aacgatcaag ga 32 <210> 13 <211> 31 <212> RNA <213> Artificial Sequence <220> <223> neomycin_gRNA <400> 13 gacgagttat taatagctga ataagaacgg t 31 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> In-GFP_F <400> 14 atcatggccg acaagcagaa 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> In-GFP_R <400> 15 tctcgttggg gtctttgctc 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> In-CtxA_F <400> 16 atactgcaga tggtaaagat a 21 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> In-CtxA_R <400> 17 tatggatcct cataattcat cc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> In-CtxB_F <400> 18 atactgcaga tgattaaatt aa 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> In-CtxB_R <400> 19 tatggatcct taatttgcca ta 22 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> In-TcpA_F <400> 20 atactgcaga tgacattact cgaag 25 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> In-TcpA_R <400> 21 tatggatcct taactgttac 20

Claims (17)

i) spores of Bacillus subtilis;
ii) CotG protein, a spore coat protein present on the surface of the Bacillus subtilis spore;
iii) a linker composed of a peptide linked to the spore coat protein;
iv) any one or more human-derived cholera antigenic substances selected from CtxA, CtxB, and TcpA linked to the linker;
Including,
Among the Bacillus subtilis spore's genomic DNA sequences, a sequence encoding an exogenous CotG, a sequence encoding a linker, and a sequence encoding a human-derived cholera antigenic substance are sequentially inserted in a sequence encoding a CotG region. Characterized by a spore having a genomic DNA,
Vaccine composition for preventing or treating human cholera.
delete delete delete delete delete delete delete According to claim 1,
The concentration of the cholera antigenic substance in the composition is a vaccine composition for preventing or treating human cholera, characterized in that 10 ¹² ~ 10 ¹⁴ pieces / ml concentration based on the composition.
delete delete According to claim 1,
The vaccine composition is for oral administration, for intraperitoneal administration, for intravenous administration, for intramuscular administration, for subcutaneous administration, for endothelial administration, for intranasal administration, for intrapulmonary administration, for rectal administration, for intranasal administration, intraperitoneally. A vaccine composition for the prevention or treatment of human cholera, characterized in that it is at least one selected from among administration and intrathecal administration.
According to claim 1,
Stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, extenders, carboxypolymethylene, bacterial cell walls, bacterial cell wall derivatives, bacterial vaccines, animal poxvirus proteins , Subviral particle adjuvant, cholera toxin, N, N-dioctadecyl-N ', N'-bis (2-hydroxyethyl) -propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium A vaccine composition for preventing or treating human cholera, further comprising any one or more selected from bromide.
Steps to introduce the following into the Bacillus subtilis
i) Cas9 protein or nucleic acid encoding it;
ii) a nucleic acid encoded by SEQ ID NO: 1 or guide RNA transcribed therefrom;
-At this time, the guide RNA targets a part of the sequence encoding CotG among the genomic DNA sequences of the Bacillus subtilis-;
iii) a donor comprising a nucleic acid encoding an exogenous CotG, a nucleic acid encoding a linker, a nucleic acid encoding at least one of the cholera antigenic substances CtxA, CtxB, and TcpA, and a nucleic acid encoding an antibiotic resistance gene ;
Selecting a Bacillus subtilis having genomic DNA in which the donor is inserted in a sequence encoding CotG among the genomic DNA sequences of the Bacillus subtilis;
Knocking out the antibiotic resistance gene inserted into the genome of the selected Bacillus subtilis;
Selecting a Bacillus subtilis knocked out by the antibiotic resistance gene;
Forming spores with the selected Bacillus subtilis;
Method for producing a vaccine for preventing or treating cholera, comprising a.
The method of claim 14,
The introduction is a method for producing a vaccine for preventing or treating cholera, characterized by using a vector.
The method of claim 14,
The step of forming spores includes a method using nutrient-depleted pharmacologic conditions; And
A method using high temperature or low temperature conditions;
Method for producing a vaccine for preventing or treating cholera, characterized in that it is carried out with any one or more selected from.
The method of claim 14,
Step of knocking out the antibiotic resistance gene,
Characterized in that the guide RNA targeting the antibiotic resistance gene region further comprises a vector comprising any one of SEQ ID NO: 12 or SEQ ID NO: 13 to form an indel within the nucleic acid sequence encoding the antibiotic resistance gene. Method for preparing a vaccine for the prevention or treatment of cholera.

Images