KR102135328B1 - Attenuated vaccinia virus expressing Mycobacterium tuberculosis divalent antigen and vaccine for preventing Mycobacterium tuberculosis comprising the same - Google Patents

Abstract

The present invention relates to an attenuated vaccinia virus strain comprising TB10.4 and HspX 2 antigen genes and a gene of the secretory signal peptide tPA, a vaccine composition for preventing tuberculosis comprising the same, and a method for preparing the vaccinia virus strain .

Description

Attenuated vaccinia virus expressing tuberculosis bivalent antigen and composition for preventing tuberculosis comprising the same {Attenuated vaccinia virus expressing Mycobacterium tuberculosis divalent antigen and vaccine for preventing Mycobacterium tuberculosis comprising the same}

The present invention is an attenuated recombinant vaccinia virus strain comprising TB10.4, an antigen of Mycobacterium tuberculosis, an HspX gene, and a tPA gene, a secretory signal peptide, and a vaccine composition for preventing tuberculosis comprising the same, and the recombinant It relates to a method for producing a vaccinia virus strain.

Tuberculosis is a disease caused by Mycobacterium tuberculosis infection and is known as the world's three major infectious diseases along with AIDS and malaria. According to the 2016 WHO Tuberculosis Analysis Report, it was estimated that in 2015, 1.4 million tuberculosis cases occurred worldwide, of which 1.4 million were killed. In recent years, 50% of AIDS patients have been reported as a double tuberculosis infection, and the mortality rate from tuberculosis has not decreased due to the increase in patients with refractory tuberculosis caused by multidrug-resistant bacteria. 146 people per 100,000 people worldwide are infected with tuberculosis, and the mortality rate from tuberculosis is a serious problem, with 49 people per 100,000 people. In Korea, the incidence and mortality rate of tuberculosis is the number one among OECD member countries. Since 2000, there have been 35,000 cases of tuberculosis patients each year. Currently, there are 32,181 new tuberculosis patients in Korea in 2015 (63.2 per 100,000 people), and as of 2015, the annual number of deaths from tuberculosis is 2,305, with the highest rate of TB incidence and mortality among OECD countries. high. This means that 60% of people who die from infectious diseases in Korea are due to tuberculosis, so the seriousness is rising in terms of national health.

As a method for preventing tuberculosis infection, other effective vaccines other than vaccinating the BCG vaccine have not been developed. However, the only tuberculosis vaccine, the BCG vaccine is a live vaccine, which can have side effects ranging from immunodeficiency patients to death, and lymph node lesions. In addition, although the effect of BCG is known to be effective in the early childhood period, it has been found through various studies that the vaccine has little effect after adolescence. Moreover, vaccine effects on infants and toddlers are also known to vary widely. Accordingly, there is a high need to develop effective vaccine candidates for the prevention of tuberculosis, and in particular, it is necessary to develop effective vaccines against infection after infancy and childhood, but the development of such vaccine candidates in Korea is still insufficient.

In particular, in the case of highly pathogenic Mycobacterium tuberculosis bacteria that have evolved to overcome immune stress against BCG vaccine, there is a problem that the existing BCG vaccine cannot be effectively prevented, and in Korea, 80% of clinical TB bacteria separated from patients Considering that the above is a highly pathogenic strain type, it is necessary to develop an effective vaccine for the highly pathogenic strain.

Registered Patent No. 10-1749993

Thus, the present inventors, while solving the side effects of the existing BCG vaccine, and the problem of preventing the tuberculosis prevention effect, in particular, while conducting research on vaccines having a preventive effect against the highly pathogenic tuberculosis strain, TB10, which is the main antigen of tuberculosis bacteria It was confirmed that the attenuated vaccinia virus in which .4 and HspX gene and secretion signal peptide were combined showed a high infection defense efficacy against not only standard tuberculosis but also highly pathogenic tuberculosis.

Accordingly, an object of the present invention is an attenuated vaccinia virus incorporating the TB10.4 and HspX gene of the Mycobacterium tuberculosis and tPA gene, which is a secretory signal peptide, into the vaccinia virus strain and the same. It is to provide a vaccine composition.

Another object of the present invention is to provide a composition for boosting a tuberculosis vaccine comprising the attenuated vaccinia virus.

In order to achieve the object of the present invention as described above, the present invention, TB10.4 of the Mycobacterium tuberculosis (Mycobacterium tuberculosis) and the HspX gene and the secreted signal peptide tPA gene is introduced, attenuated recombinant vaccination Provides a nia virus strain.

Vaccinia virus is a capsular DNA virus and has a double-stranded linear DNA genome of about 120-180 kb that encodes about 200 independent genes. Each gene consists of a short 5'-promoter, a single ORF encoding the protein without an intron, and a short 3'-polyadenylation site. These proteins are expressed dependently on the promoter in the viral infection stage of intermediate-early (IE), early (E) or L (late). The sequence of the early and late promoters is well established through functional experiments.

The vaccinia virus has become synonymous with immunomodulators to provide the root of the word vaccine after Edward Jenner first used it as a preventive vaccine against smallpox in the 18th century. Vaccinia virus, which was used in the early days, was a vaccine with excellent immune efficacy and contributed greatly to eradication of the smallpox, but the vaccine recipients sometimes showed serious side effects such as systemic infection or progressive infection. Accordingly, the attenuated vaccinia virus, which has been developed to reduce side effects, has attenuated virulence. Attenuation can be performed by passage over hundreds of times in eukaryotic cells.

The TB10.4 and HspX genes are one of the antigenic genes of M. tuberculosis. TB10.4 and HspX antigens are immune antigens present on the surface of malignant tuberculosis viruses and can strongly induce cell-mediated immune responses such as CD4+/CD8+ T-cells and IFN-γ.

According to one embodiment of the present application, the TB10.4 and HspX genes may be modified to have a gene sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2, but may not be limited thereto. The modified gene sequence represented by SEQ ID NO: 1 and SEQ ID NO: 2 was found to have about 81% homology with the gene sequence of the currently known Mycobacterium tuberculosis H37Rv strain as a result of NCBI genbank blast investigation. The modification may be by human codon optimization, and may be performed to facilitate expression in eukaryotic host cells.

According to one embodiment of the present application, the tPA gene may have a gene sequence represented by SEQ ID NO: 3, but may not be limited thereto. The SEQ ID NO: 3 was confirmed to have about 91% homology to the tPA secretion leader gene sequence of known pENTR4-Pfs25-SpyTag as a result of NCBI genbank blast investigation. The tPA gene is a sequence encoding a signal peptide inserted upstream of the gene to induce extracellular secretion after expressing the antigen gene as a protein. By inserting the signal sequence, it is possible to induce the expression and protective effect of the antigen gene.

According to one embodiment of the present application, the vaccinia virus strain may be deposited with accession number KCTC 13775BP, but may not be limited thereto.

In another aspect, the present invention can provide a vaccine composition for preventing tuberculosis comprising the recombinant vaccinia virus strain as an active ingredient.

The vaccine composition for preventing tuberculosis may be a vaccine composition inoculated alone or a booster vaccine composition having a tuberculosis prevention effect through co-administration with BCG.

In the present invention, the composition for vaccine booster refers to a composition capable of enhancing, extending or maintaining protective immunity to enhance the effectiveness of the first-administered vaccine treatment, and after the first vaccination, it is administered as needed at regular intervals. It is a composition that can.

As an aspect of the present invention, when a composition for vaccine booster is treated with BCG vaccination, it is possible to achieve excellent anti-tuberculosis prevention effect against highly pathogenic tuberculosis strains, such as HN878 strain, which have little effect in preventing tuberculosis by BCG. In particular, it has the advantage of effectively suppressing the re-activation of latent tuberculosis through the induction of an immune response.

Vaccine administration schedules, including primary vaccination and booster vaccination, can continue over the course of days, weeks, or years as long as the patient needs. In some aspects, the vaccine schedule is administered at the highest frequency at the start of the vaccine regimen, and administration of the booster vaccine for the booster effect can be administered gradually with reduced frequency.

According to one embodiment of the present application, it may further include at least one pharmaceutically acceptable carrier or excipient, but may not be limited thereto.

For example, the carrier may be a solvent or dispersion medium containing water, ethanol, polyol (eg, glycerol, propylene glycol and/or liquid polyethylene glycol, etc.), suitable mixtures thereof and/or vegetable oils, It may not be limited. Moderate fluidity can be maintained, for example, by the use of coatings such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be desirable to include isotonic agents, such as sugars or sodium chloride, but may not be limited thereto.

According to one embodiment of the present application, it may further include at least one adjuvant, but may not be limited thereto. For example, the adjuvant may be aluminum hydroxide, and may include alhydrogel, but may not be limited thereto.

Pharmaceutically acceptable carriers and/or adjuvants mean that they do not cause allergies or similar inadequate reactions when administered to humans or animals, but may not be limited thereto.

According to one embodiment of the present application, the recombinant vaccinia virus included in the vaccine composition may be one that produces a tuberculosis defense antigen protein in vivo after being injected into the body, but may not be limited thereto. For example, the recombinant vaccinia virus included in the vaccine composition may be one that produces a tuberculosis defense antigen protein in vivo and secretes it out of the cell after being injected into the body, but may not be limited thereto. For example, the protective antigen protein may be produced in a cell and then transported outside the cell by a signal peptide, but may not be limited thereto. For example, the immune response of the living body may be induced by the tuberculosis defense antigen protein, and the antibody may be generated against the tuberculosis bacteria in the living body by the immune response, but may not be limited thereto.

According to one embodiment of the present application, the vaccine composition may be injected into a living body of an animal including a human to induce immunity against tuberculosis, but may not be limited thereto.

The vaccine composition of the present invention may be formulated for human or veterinary use, including, but not limited to, a pharmaceutically acceptable carrier and/or an adjuvant for administration. For oral administration, for example capsules or tablets; Powder or granules; The vaccine composition as separate units such as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; edible foams or whip formulations; or emulsions) , But may not be limited thereto. For example, upon parental administration, the vaccine composition may include aqueous and non-aqueous sterile injectable solvents, including antioxidants, buffers, bacteriostatic agents, and solutes that have been brought into isotonic state with the intended recipient's blood. In addition, aqueous and non-aqueous sterile suspending agents may include suspending agents and thickening agents, but may not be limited thereto. Additives useful for use as solvents for injection include, but are not limited to, water, alcohol, polyols, glycerin and vegetable oils, for example. The composition can be presented in unit dose (one serving) or multiple dose (several dose) containers. For example, it can be presented in sealed ampoules and vials, and can be stored under lyophilization conditions that require only the addition of water needed to make a sterile liquid carrier, e.g., an injection solution, just prior to use, , It may not be limited to this. Instantaneous injection solvents and suspensions may be prepared from sterile powders, granules and tablets, but may not be limited thereto.

For example, the administration may be by intramuscular injection, subcutaneous injection, or intraperitoneal injection, but may not be limited thereto.

The vaccine composition of the present invention can be administered by appropriately setting the dose according to the health condition, weight, sex and administration purpose of the recipient.

In another aspect, the present invention provides a method for producing a recombinant vaccinia virus strain comprising inserting a TB10.4 and HspX gene and a tPA gene, a secretory signal peptide, into a delivery vector and inserting the delivery vector into a vaccinia virus. can do.

Throughout this specification, the term "vector" can be used to refer to a carrier nucleic acid molecule capable of inserting an exogenous nucleotide sequence for introduction into a cell into which the vector can be replicated. The nucleotide sequence can be “exogenous,” meaning that it is external to the cell introducing the vector, or that the nucleic acid is located at a position in the host cell where the sequence is homologous to the sequence within the cell, but the sequence is not normally found. Can mean Vectors can include plasmids, cosmids, viruses (bacteriophages, animal viruses and plant viruses) and artificial chromosomes (eg YAC).

Inserting the TB10.4 and HspX genes, which are the TB antigen protective antigen genes, into the delivery vector may be performed by a method selected from all genetic recombination methods known in the art, and the delivery vector is inserted into the vaccinia virus. Doing may be performed by a method selected from all transfection methods known in the art.

For example, the transfer vector may include a pVVT vector, but may not be limited thereto. The transfer vector may include pVVT-GFP, for example, pVVT1-GFP-C7L, pVVT1-GFP-K1L, and/or pVVT1-GFP-C7L-K1L, but may not be limited thereto.

According to one embodiment of the present application, after inserting the delivery vector into the vaccinia virus, it may further include confirming the production of TB10.4-HspX antigen by the prepared recombinant vaccinia virus, but is not limited thereto. Can.

For example, confirming the production of the tuberculosis defense antigen (TB10.4-HspX antigen) protein by the recombinant vaccinia virus may be performed by all protein detection and/or assay methods known in the art, eg For example, it may be by an immunological method, but may not be limited thereto. For example, Western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, aggregation reaction assay, complement -The generation of a tuberculosis protective antigen protein can be confirmed by a fixed assay, an immunoradiometric assay, a fluorescent immunoassay, or a protein A immune assay, or a fluorescent protein linked to the tuberculosis bacteria protective antigen is used to detect the protein. Generation can be confirmed, but may not be limited thereto.

According to the exemplary embodiment of the present application, the TB10.4 gene may be modified to have the gene sequence represented by SEQ ID NO: 1, and the HspX gene represented by SEQ ID NO: 2, but may not be limited thereto.

Further, according to one embodiment of the present application, the tPA gene may have a gene sequence represented by SEQ ID NO: 3, but may not be limited thereto.

The above-described problem solving means are merely exemplary and should not be construed as limiting the invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

By introducing the gene of TB10.4 and HspX as antigen genes and tPA as the secretory signal peptide gene into the attenuated vaccinia virus strain of the present invention, it has excellent anti-tuberculosis prevention and defense effects while alleviating side effects of BCG and preventing it. It can provide an attenuated recombinant vaccinia virus strain for use as a booster vaccine capable of increasing the

1 is a schematic diagram of a recombinant vaccinia virus production process according to an embodiment of the present application.
Figure 2 shows the sequence of the optimized TB10.4 and HspX genes according to an embodiment of the present application.
3 is a graph showing the results of humoral immune analysis of a recombinant vaccinia virus according to an embodiment of the present application.
4 is a graph showing the results of evaluating immunogenicity in a mouse model of a recombinant vaccinia virus according to an embodiment of the present application.
5 and 6 are graphs showing protective efficacy against spleen (FIG. 5) and lung (FIG. 6) infection in a mouse model immunized with a recombinant vaccinia virus according to an embodiment of the present application.
Figure 7 shows the results of evaluating the protection against infection by boosting inoculation of the recombinant vaccinia virus of the present application against the standard Mycobacterium tuberculosis (H37Rv) and highly pathogenic Mycobacterium tuberculosis (HN878).

Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present application pertains may easily practice. However, the present application may be implemented in various different forms and is not limited to the embodiments described herein. In addition, in order to clearly describe the present application in the drawings, parts irrelevant to the description are omitted.

Throughout the present specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless specifically stated to the contrary.

The terms "about", "substantially", and the like, as used throughout this specification, are used in or at a value close to that value when manufacturing and material tolerances specific to the stated meaning are given, and are understood herein. To help, accurate or absolute figures are used to prevent unconscionable abusers from unduly using the disclosed disclosure.

The term “~(steps)” or “steps of” to the extent used throughout this specification does not mean “steps for”.

Throughout the present specification, the term "combination of these" included in the expression of the marki form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the marki form, and the above component. It means to include one or more selected from the group consisting of.

Throughout this specification, the description of “A and/or B” means “A, B, or A and B”.

Hereinafter, the attenuated recombinant vaccinia virus strain of the present application, a vaccine composition comprising the same, and a method of manufacturing the vaccinia virus strain will be described in detail with reference to embodiments and examples and drawings. However, the present application is not limited to these embodiments and examples and drawings.

[ Example ]

1. Construction of recombinant virus

1-1. Construction of transfer vectors

Sequence modification was performed to increase the expression rate in cells using the TB10.4 and HspX gene sequences of the M. tuberculosis H37Rv strain. After humanized codon optimization using M. tuberculosis TB10.4 and HspX gene sequences, gene synthesis with tPA (tissue plasmodium activator) sequence (SEQ ID NO: 3), an intracellular secretory signal peptide upstream of the gene Proceeded. As a result, the codon optimized TB10.4 and HspX gene sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2, the tPA gene sequence is shown in SEQ ID NO: 3, and the original and optimized sequences of TB10.4 and HspX genes are shown. It is shown in 2. The synthesized gene was ligated to pVVT1-GFP-C7L plasmid, a vaccinia virus delivery vector after cleavage using Sfi1 restriction enzyme, to produce pVVT1-C7L-tPA-TB10.4-HspX, which was E. coli DH5α Was transformed. The structure of the constructed pVVT1-C7L-tPA-TB10.4-HspX vector is shown in FIG. 1. The cloned plasmid confirmed the nucleotide sequence of the inserted gene through gene sequencing using VVTK-F and VVTK-R primers, mass cultured in medium containing ampicillin, and mass separated using a midiprep kit Did. The sequencing primer sequences are shown in Table 1 below.

primer order VVTK-F 5`-TTTGAAGCATTGGAAGCAACT-3` (SEQ ID NO: 4) VVTK-R 5`-ACGTTGAAATGTCCCATCGACT-3` (SEQ ID NO: 5)

1-2. Transfection to produce recombinant virus

The vaccinia virus strain KVAC103 and the cloned transfer vector were co-transfected into vero cells. Vero cells were seeded at 1×10 5 cells/well using OPTI-MEM (2% FBS) medium in 12-well plates one day prior to transfection. After infecting the parent virus KVAC103 strain at a rate of 0.02 MOI for 2 hours, after spraying the mixed solution of lipofectin 2000 and the cloned transfer vector 1.5 µg into the pre-infected Vero cells, infected for 4 hours, 5% CO2 CPE (cytopathic effect) was confirmed while incubating for 3 to 4 days in an incubator. After the recombinant virus is passaged 2-3 times using Vero cells cultured in 5% FBS DMEM medium, the selective cultured virus is diluted in concentrations of 10 ^-5 , 10 ^-6 , 10 ^-7 to isolate only pure recombinant viruses. Re-infection with Vero cell by using 1.5% low melting agarose and conducting two or more plaque isolation experiments using an overlay method, the plaques identified as CPE were PCR using the VVTK-F/VVTK-R primer of the transfer vector. It was confirmed whether or not the recombinant virus was successfully produced. As a result of the Western blot experiment, the recombinant virus was confirmed to have protein expression of the antigen gene. 1 is a schematic diagram of a recombinant vaccinia virus (rVV-tPA-TB2) production process of the present invention.

1-3. Culture and concentration of recombinant virus

After infecting the recombinant virus produced in Vero cells (SFM, OptiMEM), CPE was confirmed for 2 to 3 days. When CPE was confirmed, freezing and thawing were repeated 2-3 times to break the cells and recover the medium and cells. After centrifugation at 4000 rpm for 30 minutes to recover only the supernatant from which the cell residue was removed, the recombinant virus was concentrated using an amicon filter of pore size 100,000 NMWL.

2. Evaluation of immunogenicity

2-1. Mouse immunization

Mice were immunized with vaccine candidates to analyze in vivo efficacy. As a laboratory animal, female C57BL/6N mice without specific pathogens 5 to 6 weeks old were used, and the prepared tuberculosis candidates were administered to mice. To evaluate the immune efficacy as a booster vaccine, the BCG vaccine was injected subcutaneously. Then, after 6 weeks of immunization, the recombinant virus of the present invention was administered. The 5 × 10 ⁷ PFU virus was inoculated twice every three weeks and blood, spleen and lung tissues were collected from mice 2 weeks after the last inoculation. The mouse was anesthetized with ketamine and rumpoon, and then a heart was drawn. After the blood was left at 4°C for 12 hours or more, the serum was separated by centrifugation and the separated serum was stored at -70°C.

2-2. Humoral immunoassay (analysis of blood IgG titer)

In order to analyze the humoral immune response of the prepared tuberculosis candidates alone or by booster inoculation with BCG, the antigen-specific blood IgG antibody titers of mice were measured by ELISA. The vaccine candidate was suspended in a carbonate buffer (pH 9.6) and then added to a 96-well plate at 100 μl/well and coated at 4° C. for 12 hours. The next day, 300 μl of PBS solution containing 1% fetal bovine serum albumin (BSA) was added and blocked, followed by washing with PBS (PBST) containing 0.5% tween 20. 100 µl of serially diluted mouse serum was added with a blocking solution and reacted at 37°C for 2 hours, and then the wells were washed with PBST. After reacting with horseradish peroxidase-conjugated goat anti-mouse (IgG), IgG1 and IgG2a isotype antibody at 37°C for 1 hour, TMB solution was added for color development, and then 50 N of 0.1 N sulfuric acid solution was added to react. After stopping, absorbance was measured at 490 nm (Figure 3). As a result, it was observed that the antibody response was higher in the group inoculated with boost after the initial inoculation of rVV-tPA-TB2 than in the case where only the BCG vaccine was administered as a control group.

2-3. Evaluation of immunogenicity in splenocytes and lung cells of mouse model (IFN-γ)

For the immunoassay of the tuberculosis vaccine candidate substance of the present invention, rVV-tPA-TB2, splenocytes and lung cells of the BCG-only inoculation group and boosting inoculation group were separated, and the spleen cells and lung cells each had IFN-γ secretion activity. Cell numbers were analyzed by ELISpot (Figures 4A and 4B). As a result, it was confirmed that the number of cells secreting IFN-γ specific to the tuberculosis antigen in both the spleen and lung of the boosted inoculated group was significantly increased compared to the group inoculated only with the control group BCG vaccine.

In addition, the distribution of multifunctional T-cells secreting cytokines such as IFN-γ, IL-2, and TNF-α was examined using flow cytometry of isolated immune cells (CD4 and CD8 T-cells) (FIG. 5 and 6). As a result, as shown in FIG. 6, it was confirmed that induction of multifunctional T cells in the lung tissue of the group inoculated with rVV-tPA-TB2 was significantly increased.

2-4. Verification of multifunctional T-cell activity in spleen cells of mouse model

Polyfunctional CD4+T cells secrete all of IFN-γ, IL-2 and TNF-α, and are known as important host factors for defense against tuberculosis. In order to confirm that the recombinant virus of the present invention induces such multifunctional T cells, the antigen-specific multifunctional T cell distribution of the immune cells (CD4 and CD8 T-cells) isolated from the spleen of the immunized mouse using a flow cytometer Was confirmed (FIGS. 5 and 6 ). As a result, as shown in Figure 5, it was confirmed that the relatively large increase only in multifunctional CD4 + T cells secreting all of IFN-γ, IL-2 and TNF-α.

2-5. Mycobacterium tuberculosis Defense ability evaluation

To confirm the protective efficacy of the tuberculosis vaccine candidate, the recombinant virus of the present invention (rVV-tPA-TB2) was boosted twice in BCG immunized mice. The standard tuberculosis bacterium H37Rv and the highly pathogenic tuberculosis bacterium HN878 were respectively infected 3 weeks after the last inoculation, and 4 to 12 weeks later, the protective ability of tuberculosis in mouse lung and spleen was confirmed. As a result, both types of Mycobacterium tuberculosis bacteria showed a significant decrease in Mycobacterium tuberculosis when boosted with rVV-tPA-TB2 in the lungs compared to the BCG-only treatment group, and inhibited inflammatory lesions and granuloma formation in the lungs than the BCG alone group I could confirm. (Fig. 7)

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Korea Research Institute of Bioscience and Biotechnology KCTC13775BP 20181214

<110> REPUBLIC OF KOREA (Korea Centers for disease control and prevention) <120> Attenuated vaccinia virus expressing Mycobacterium tuberculosis divalent antigen and vaccine for preventing Mycobacterium tuberculosis comprising the same <130> pn1812-505 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 288 <212> DNA <213> Mycobacterium tuberculosis <400> 1 atgtcccaga ttatgtacaa ctaccccgca atgctgggcc acgccggtga catggctggc 60 tacgctggaa cactgcagtc cctgggtgca gagatagccg tcgagcaggc cgctctgcag 120 agtgcctggc agggcgacac agggatcact tatcaggcct ggcaggctca gtggaatcag 180 gctatggaag atctggtgcg cgcctatcac gcaatgtcat ccacccacga ggccaacacc 240 atggctatga tggccaggga tacagcagag gctgcaaaat ggggcggt 288 <210> 2 <211> 435 <212> DNA <213> Mycobacterium tuberculosis <400> 2 atggctacaa cactgcccgt acagcgacac ccccgctccc tgtttcccga attctcagag 60 ctatttgctg ccttcccctc ttttgctggc ctgcgaccca cttttgacac ccgactgatg 120 cgacttgaag acgaaatgaa ggagggccgc tatgaggtgc gcgctgagct tccaggcgtc 180 gaccctgata aggatgtaga cattatggtg cgggatggcc agctgacaat caaggccgaa 240 cggaccgaac aaaaggattt cgacggtagg tccgaatttg cttacgggtc tttcgtcaga 300 accgtgtccc tgcccgtcgg ggccgatgaa gatgatatca aggcaaccta tgataaggga 360 atactgaccg tgtctgtggc tgtctccgag ggaaagccaa ccgagaagca cattcaaatt 420 cggagcacta attga 435 <210> 3 <211> 69 <212> DNA <213> Mycobacterium tuberculosis <400> 3 atggacgcca tgaagcgggg tctgtgttgt gtgctgctgt tgtgcggggc tgttttcgtc 60 agtccctct 69 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Vaccinia virus shuttle vector VVTK-F primer <400> 4 tttgaagcat tggaagcaac t 21 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Vaccinia virus shuttle vector VVTK-R primer <400> 5 acgttgaaat gtcccatcga ct 22 <210> 6 <211> 9 <212> DNA <213> Artificial Sequence <220> <223> optimized linker sequence of TB10.4-HspX gene <400> 6 gggtccggc 9

Claims (11)

A bivalent antigen comprising a TB10.4 gene consisting of a codon-optimized sequence represented by SEQ ID NO: 1 of Mycobacterium tuberculosis H37Rv and an HspX gene consisting of a codon-optimized sequence represented by SEQ ID NO: 2 gene; And
An attenuated recombinant vaccinia virus strain (KCTC13775BP) comprising a tPA (tissue plasminogen activator) gene of a secretion signal peptide consisting of a codon optimized nucleotide sequence shown in SEQ ID NO: 3. delete delete delete delete A vaccine composition for preventing tuberculosis, comprising the recombinant vaccinia virus strain of claim 1 (KCTC13775BP) as an active ingredient. The method of claim 6,
The vaccine composition is a booster vaccine for tuberculosis vaccine, characterized in that the vaccine composition for preventing tuberculosis. A bivalent antigen gene comprising a TB10.4 gene consisting of a codon optimized nucleotide sequence represented by SEQ ID NO: 1 of H37Rv and an HspX gene consisting of a codon optimized nucleotide sequence represented by SEQ ID NO: 2; And inserting the tPA gene of the secretion signal peptide consisting of the codon optimized nucleotide sequence shown in SEQ ID NO: 3 into the delivery vector; And
A method of preparing a recombinant vaccinia virus strain (KCTC13775BP), comprising inserting the delivery vector into a vaccinia virus. The method of claim 8,
After inserting the transfer vector into the vaccinia virus, further comprising the step of confirming the production of TB10.4 and HspX antigens by the prepared recombinant vaccinia virus, recombinant vaccinia virus strain (KCTC13775BP) production method. delete delete

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