阅读说明：本技术 诱导细胞性免疫的经鼻疫苗 (Nasal vaccine for inducing cellular immunity ) 是由 幸义和 中桥理佳 清野宏 于 2019-08-02 设计创作，主要内容包括：本发明提供一种诱导细胞性免疫的纳米凝胶经鼻疫苗。具体而言,本发明是一种疫苗制剂,是包含纳米凝胶、疫苗抗原和佐剂的复合体的疫苗制剂,能够有效地诱导细胞性免疫,并且诱导全身性和粘膜性免疫应答。(The invention provides a nanogel nasal vaccine for inducing cellular immunity. Specifically, the present invention is a vaccine formulation comprising a complex of nanogel, a vaccine antigen and an adjuvant, which is capable of effectively inducing cellular immunity and inducing systemic and mucosal immune responses.)

1. A vaccine formulation comprising a complex of nanogel, a vaccine antigen and an adjuvant.

2. The vaccine formulation of claim 1, wherein the adjuvant comprises 1 or more STING ligands.

3. The vaccine formulation of claim 2, wherein at least 1 of the STING ligands is a cyclic dinucleotide.

4. The vaccine formulation according to claim 3, wherein the cyclic dinucleotide is any one of cyclic guanylic acid-adenylic acid, cyclic di-adenylic acid, cyclic diguanylic acid, cyclic docylic acid, cyclic diuridylic acid, and cyclic diinosinic acid.

5. The vaccine formulation of any one of claims 1 to 4, wherein the vaccine antigen is an antigen from Mycobacterium tuberculosis.

6. The vaccine formulation of claim 5, wherein the antigen from Mycobacterium tuberculosis comprises at least the entirety of or a portion of the Ag85B gene product, the Rv2608 gene product, the Rv3619 gene product, the Rv3620 gene product, the Rv1813 gene product, the MTB32A gene product, the MTB39A gene product, and/or the MVA85A gene product.

7. The vaccine formulation of claim 5, wherein the antigen from Mycobacterium tuberculosis is a chimeric protein consisting of an Rv3875 gene product, an Rv0266 gene product, and an Rv0288 gene product.

8. The vaccine formulation according to any one of claims 1 to 4, wherein the vaccine antigen is an antigen from HPV, human papilloma virus.

9. The vaccine formulation of claim 8, wherein the antigen from HPV comprises at least the E6 gene product and/or the whole or part of the E7 gene product.

10. The vaccine formulation according to any one of claims 1 to 4, wherein the vaccine antigen is an antigen from RSV (respiratory syncytial virus).

11. The vaccine formulation of claim 10, wherein the antigen from RSV comprises at least the entirety of an SH peptide or a portion thereof.

Technical Field

The present invention relates to a nasal vaccine for inducing cellular immunity.

Background

Acquired immunity is responsible for two distinct mechanisms, humoral and cellular immunity.

Humoral immunity is mainly an immune system centered on antibodies, complement, and the like present in blood. When a foreign antigen invades into an organism, antigen-presenting cells such as dendritic cells absorb and fragment the foreign antigen, and then the foreign antigen is presented on the cell surface via MHC class II molecules. Thereafter, an antigen fragment present on B cells is recognized from Th2 cells stimulated from antigen presenting cells via a T cell antigen receptor (TCR), and release of Th2 cytokines and the like are performed. The B cells are exposed to the released Th2 cytokine to produce antibodies.

On the other hand, cellular immunity is an immune system in which foreign substances in an organism are eliminated by macrophages, cytotoxic T Cells (CTL), natural killer cells, and the like. If Th1 cells are activated by antigen fragments presented on antigen presenting cells via MHC class II molecules, IFN- γ is released, activating macrophages. In addition, it is also considered to induce ADCC (Antibody-Dependent-Cellular-cytoxicity) which induces an Antibody that binds to the cell surface, but not a neutralizing Antibody, activates macrophages and NK cells via Fc receptors of the Antibody, and attacks and destroys target cells. In addition, activated Th1 cells release IL-2, activating CTLs that recognize fragments of antigens presented with MHC class I molecules. Activated macrophages and CTLs attack and eliminate cells infected with viruses and the like, cancer cells, and the like. Since cellular immunity can also exclude infected cells, cancer cells, and the like, it is expected to exclude mycobacterium tuberculosis that can parasitize in cells, or to be applied to cancer immunotherapy.

The inventors have developed an effective vaccine delivery system using a self-aggregating nano-sized hydrogel (cCHP) composed of a cationic pullulan to which cholesterol is added (patent document 1 and non-patent document 1). In the chp nanogel, if a protein antigen is encapsulated inside its nanomatrix, it functions as an artificial partner, preventing aggregation and denaturation of the antigen, and assisting refolding after antigen release. The nanogel has a property of effectively adhering to a negatively charged mucosal surface, and induces an immune response by continuously releasing an antigen and delivering the antigen to an antigen-presenting cell (non-patent document 2, non-patent document 3, and patent document 2). In addition, in the mouse, the carrier [ 2 ] is administered through the nose¹¹¹In]The chp nanogel labeled with BoHc/a (the non-toxic region in the C-terminal region of the heavy chain of botulinum toxin type a) or the pneumococcal surface antigen PspA was not accumulated in the central nervous system such as olfactory bulb or brain (non-patent document 2), and the safety thereof was confirmed (non-patent document 4).

Nanogel vaccines suitable for nasal administration (nanogel nasal vaccines) are excellent in both safety and in inducing humoral immunity.

However, induction of cellular immunity has not been confirmed so far.

Documents of the prior art

Patent document

Patent document 1: WO00/12564

Patent document 2: japanese patent No. 5344558

Non-patent document

Non-patent document 1: ayame et al, bioconjugug Chem 19: 882-8902008

Non-patent document 2: nochi et al, Nat Mater 9: 572-Asonic 5782010

Non-patent document 3: yuki et al, Biotechnol Genet Eng Rev 29: 61-722013

Non-patent document 4: kong et al, feed Immun 81:1625-

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a nanogel nasal vaccine that induces cellular immunity.

In order to solve the above problems, the present inventors produced a vaccine in which a nanogel was encapsulated with a STING ligand as an adjuvant in addition to a vaccine antigen, and nasally administered the vaccine to mice, and as a result, succeeded in inducing antigen-specific Th1 cells.

That is, the present invention is (1) to (11) below.

(1) A vaccine formulation comprising a complex of nanogel, a vaccine antigen and an adjuvant.

(2) The vaccine formulation according to the above (1), wherein the adjuvant comprises 1 or more STING ligands.

(3) The vaccine preparation according to the above (2), wherein at least 1 of the STING ligands is a cyclic dinucleotide.

(4) The vaccine preparation according to item (3), wherein the cyclic dinucleotide is any one of cyclic guanylic acid-adenylic acid (cGAMP), cyclic di-adenylic acid (cyclic-di AMP), cyclic digylic acid (cyclic-di GMP), cyclic dicytylic acid (cyclic-di CMP), cyclic diuridylic acid (cyclic-di UMP), or cyclic diinosinic acid (cyclic-di IMP).

(5) The vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from Mycobacterium tuberculosis.

(6) The vaccine preparation according to the above (5), wherein the antigen derived from Mycobacterium tuberculosis comprises at least the entirety of or a part of the Ag85B gene product, the Rv2608 gene product, the Rv3619 gene product, the Rv3620 gene product, the Rv1813 gene product, the MTB32A gene product, the MTB39A gene product and/or the MVA85A gene product.

(7) The vaccine preparation according to the above (5), wherein the antigen derived from Mycobacterium tuberculosis is a chimeric protein consisting of Rv3875 gene product, Rv0266 gene product and Rv0288 gene product.

(8) The vaccine preparation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from HPV (human papilloma virus).

(9) The vaccine preparation according to (8) above, wherein the antigen derived from HPV comprises at least the E6 gene product and/or the E7 gene product in whole or in part.

(10) The vaccine formulation according to any one of the above (1) to (4), wherein the vaccine antigen is an antigen derived from RSV (respiratory syncytial virus).

(11) The vaccine formulation according to (10) above, wherein the antigen derived from RSV comprises at least the entirety of an SH peptide or a part thereof.

By administering the nanogel vaccine of the invention, cellular immunity can be induced.

By administering the nanogel vaccine of the invention, both systemic immune response and mucosal immune response can be induced efficiently.

Drawings

FIG. 1 is the result of detecting Th1 cell response induced by Nanogel Mycobacterium transnasal vaccine and STING ligand. cGMP, cGAMP, and cAMP denote cyclic diguanylic acid, cyclic guanylic acid-adenylic acid, and cyclic digenylic acid, respectively. -: vaccine-free chp: cationic cholesterol-substituted pullulan (cationic cholesterol-group-bearing pulullan).

FIG. 2 is the result of detection of Th1 cell response induced by Nanogel Mycobacterium tuberculosis transnasal vaccine.

FIG. 3 is the result of detection of Th17 cell response induced by Nanogel Mycobacterium tuberculosis transnasal vaccine.

FIG. 4 is a study of the protective immune effect of the Nanogel Mycobacterium tuberculosis via nasal vaccine. (A) The survival rate is shown, and the number of tubercle bacillus detected from the lung and spleen is shown. The 'control' is an unimmunized mouse group, the 'BCG' is a BCG vaccinating group, and the 'nanogel' is a cCHP-Ag85B + cyclic diguanylic acid vaccinating group.

FIG. 5 is the result of detection of Th1 cell response induced by Nanogel Mycobacterium transnasal vaccine (chimeric antigen).

Fig. 6 is the detection result of CTL cell response induced by nanogel HPV nasal vaccine.

Fig. 7 is the detection result of Th1 cell response induced by nanogel HPV nasal vaccine.

Fig. 8 is a comparison of CTL cell responses (left) and Th1 cell responses (right) induced by nanogel HPV nasal vaccine using 3 STING ligands as adjuvants.

Figure 9 is the detection of immune responses induced by nanogel RSV nasal vaccine.

Figure 10 is the detection results of IgG subtypes induced by nanogel RSV nasal vaccine.

Detailed Description

Embodiment 1 of the present invention is a vaccine preparation containing a complex of a nanogel, a vaccine antigen, and an adjuvant (hereinafter also referred to as "vaccine preparation of the present invention").

In the present invention, the nanogel refers to polymer gel nanoparticles in which a hydrophilic polysaccharide (for example, pullulan) is added with hydrophobic cholesterol as a side chain. The nanogel can be produced, for example, by a known method, for example, a method described in International publication No. WO 00/12564.

Specifically, a hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms is first reacted with a diisocyanate compound represented by OCN-R1 NCO (wherein R1 is a hydrocarbon group having 1 to 50 carbon atoms) to produce an isocyanate group-containing hydrophobic compound obtained by reacting 1 molecule of the hydroxyl group-containing hydrocarbon or sterol having 12 to 50 carbon atoms. The obtained hydrophobic compound containing isocyanate groups is reacted with a polysaccharide to produce a polysaccharide containing hydrophobic groups and hydrocarbon groups or sterol groups having 12 to 50 carbon atoms. Then, the obtained product is purified by a ketone solvent, whereby a hydrophobic group-containing polysaccharide having high purity can be produced.

Here, as the polysaccharide, pullulan, amylose, dextran, hydroxyethyl dextran, mannan, polyfructose, inulin, chitin, chitosan, xyloglucan, water-soluble cellulose or the like can be used, and pullulan is particularly preferable.

Examples of the nanogel used in embodiment 1 of the present invention include cationic cholesterol-substituted pullulan (chp) and derivatives thereof. The cCHP has a structure in which 1 to 10, preferably 1 to several cholesterols are substituted for 100 monosaccharides in pullulan having a molecular weight of 3 to 20 ten thousand, for example, a molecular weight of 100000. The amount of the substituted cholesterol may be appropriately changed depending on the size of the antigen and the degree of hydrophobicity. In addition, an alkyl group (about 10 to 30 carbon atoms, preferably about 12 to 20 carbon atoms) may be added to the CHP in order to change the degree of hydrophobicity. The particle size of the nanogel used in the invention is 10 to 40nm, preferably 20 to 30 nm. Nanogels are widely commercially available, and therefore, these commercially available products can also be used.

The nanogel used in the embodiment of the present invention is a nanogel in which a functional group having a positive charge, for example, an amino group, is introduced so that a vaccine can invade a negatively charged nasal mucosal surface. Examples of a method for introducing an amino group into a nanogel include the use of cholesterol pullulan (CHPNH) to which an amino group is added₂) The method of (1). Specifically, CHP dried under reduced pressure was dissolved in dimethyl sulfoxide (DMSO), 1' carbonyldiimidazole was added thereto under a nitrogen gas flow, and reacted at room temperature for several hours. Ethylenediamine is slowly added to the reaction solution, and the mixture is stirred for several hours to several tens of hours. The resulting reaction solution was dialyzed against distilled water for several days. The dialyzed reaction solution was freeze-dried to obtain a milky white solid. The degree of substitution with ethylenediamine can be evaluated by elemental analysis, H-NMR, or the like.

The vaccine antigen is not particularly limited, and may be arbitrarily selected depending on the use of the vaccine preparation. In particular, the vaccine preparation of the present invention can efficiently induce cellular immunity, and is therefore very suitable for activation of the cellular immune system in prevention or treatment of diseases and the like. Examples of such diseases include tuberculosis in which a vaccine effective for adults does not exist, an acapsular haemophilus influenzae (NTHi), rsv (respiratory synthetic virus) or hsv (herpes simplex virus) infectious disease in which a vaccine itself does not exist, an hpv (human papilloma virus) infectious disease which is considered to be important for the treatment thereof and which is infected with such an infectious disease, and cervical cancer which develops from such an infectious disease.

The vaccine antigen for tuberculosis is not particularly limited, and may be derived from, for example, Mycobacterium tuberculosis: (A), (B), and (C)Mycobacterium tuberculosis) The Ag85B (Rv1886) gene product, the ESAT6(Rv3875) gene product, the Rv2660 gene product, the Rv2608 gene product, the Rv3619 gene product, the Rv3620 gene product, the Rv1813 gene product, the MTB32A (Rv0125) gene product, the MTB39A (Rv1196) gene product, the MVA85A gene product or the entirety or a portion thereof of the Rv0288 gene product of (i.e. a chimeric protein of an ESAT6-Rv2660-Rv0288 gene product) or a fusion protein selected from a plurality of these proteins.

As a vaccine antigen of non-capsular Haemophilus influenzae (NTHi), D15, P1, P2, P4, P5, P6, Hmw/hia, Hap, protein E, protein F, protein D, Pil A, NucA, HtrA, OMP26, PCP, TbpB, LOS as a whole or a part thereof, or a fusion protein selected from a plurality of these proteins may be mentioned.

The vaccine antigen of RSV is not particularly limited, and may be, for example, the whole or a part of F protein (fusion protein) or SH protein derived from RSV, or a fusion protein selected from a plurality of these proteins.

The vaccine antigen for HSV is not particularly limited, and may be, for example, a gD gene product, a gB gene product, a gC gene product, a gE gene product, capsid protein UL19, tegument protein UL47 or a part thereof, or a fusion protein selected from a plurality of these proteins, from HSV.

The HPV vaccine antigen is not particularly limited, and may be, for example, a mutation or defect product of the E6 gene product derived from HPV, particularly the E6 binding site of the oncogene suppressor product P53, a mutation or defect product of the E7 gene product derived from HPV, particularly the E7 binding site of the oncogene suppressor product Rb, or the like, more specifically, may be the whole or part of HPV 6E 7(23-27 defect), HPV 11E 7(23-27 defect), HPV 16E 7(D21G, C24G, E26G variant) or HPV 16E 7(21-24 defect), HPV 18E 7(24-27 defect), HPV 31E 7(22-26 defect), HPV 33E 7(22-26 defect), HPV 45E 7(26-30 defect), HPV 52E 7(22-26 defect) or HPV 52E 7(22-26 defect) or HPV 58E 7(22-26 defect), or a fusion protein selected from a plurality of these proteins.

The adjuvant used in the embodiment of the present invention is synonymous with a case called an antigenic potentiator, an immune activator, or the like, and is used for general purposes of use of these agents in this field. The active ingredient of the adjuvant used in the embodiment of the present invention is not particularly limited, and examples thereof include STING ligands (e.g., cyclic dinucleotides such as cyclic guanosine-adenosine, cyclic guanosine, cyclic dinucleotides, cyclic diuridylic acid, and cyclic diinosinic acid), xanthone (xanthone) derivatives such as DMXAA (5, 6-dimethyl XAA (xanthone-4-acetic acid), Vadimezan, and ASA404), poly-IC, and CpG ODN) that activate STING (stimulator of interferon gene stimulators). The adjuvant may further contain pharmaceutically acceptable carriers or other ingredients (e.g., stabilizers, pH adjusters, preservatives, buffers, and the like). Pharmaceutically acceptable carriers and other ingredients need to be substances that do not adversely affect the health of the animal to which the vaccine is administered.

The complex of nanogel, vaccine antigen and adjuvant (or an active ingredient of adjuvant, the same applies hereinafter) can be prepared by allowing nanogel, vaccine antigen and adjuvant to coexist and interact with each other, and incorporating antigen and adjuvant into nanogel. In this case, the mixing ratio of the nanogel to the vaccine antigen and the nanogel to the adjuvant is not particularly limited, and can be easily determined by preliminary experiments as long as it is a person skilled in the art. If the criteria are hard to be enumerated, the vaccine antigen: the nanogel is, for example, about 0.1:10, 1:5, 1:2, or 1:1 in terms of molar ratio. The content of the adjuvant may be about 0.01 to 99.99% by weight based on 100% by weight of the vaccine, and may be about 0.01 to 10% by weight based on 1% by weight of the antigen, for example.

The complex of the nanogel, the vaccine antigen and the adjuvant can be formed by mixing the nanogel, the vaccine antigen and the adjuvant and allowing the mixture to stand at 4 to 50 ℃, for example, 40 ℃ for 30 minutes to 48 hours, for example, about 1 hour. The buffer used for forming a complex of a nanogel, a vaccine antigen and an adjuvant is not particularly limited, and examples thereof include Tris-HCl buffer and the like, if given by way of hard example.

The vaccine preparation of the present invention may contain a pharmacologically allowable additive as a composition (the vaccine composition of the present invention). The vaccine preparation of the present invention is suitable for nasal administration, and as a dosage form, a dosage form capable of nasal administration is also preferable, and examples thereof include liquid preparations (nasal drops, injections, and the like).

When the vaccine preparation of the present invention is a liquid preparation, the active ingredient may be dissolved in distilled water for preparation, together with a pH adjuster such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, or the like, or a tonicity adjuster such as sodium chloride, glucose, or the like, as required, and the resulting solution may be filled into an ampoule after aseptic filtration, or may be further added with mannitol, dextrin, cyclodextrin, gelatin, or the like and freeze-dried under vacuum to prepare a ready-to-use dissolution type preparation. The liquid preparation may contain pharmaceutically acceptable known stabilizers, preservatives, antioxidants and the like, examples of the stabilizers include gelatin, dextran, sorbitol and the like, examples of the preservatives include thimerosal, beta propiolactone and the like, and examples of the antioxidants include alpha-tocopherol and the like.

Embodiment 2 of the present invention is a method for preventing and/or treating a disease, comprising nasally administering to a patient a vaccine preparation comprising a complex of a nanogel, a vaccine antigen and an adjuvant (embodiment 1).

The disease to be treated or prevented according to embodiment 2 is not particularly limited depending on the vaccine antigen used, and may be any disease including cancer (e.g., cervical cancer) and the like, including diseases expected to be cured by cellular immunity, in addition to infectious diseases caused by pathogens (e.g., tuberculosis, HSV, RSV, and the like).

The vaccine formulation of the present invention may be administered via the nasal mucosa. Examples of the method include a method of administering the drug into the nasal cavity by spraying, smearing, dropping, etc. the nasal mucosa.

The dose of the mucosal vaccine preparation can be appropriately determined depending on the age, body weight, and the like of a subject to be administered, but the mucosal vaccine preparation contains a pharmaceutically effective amount of the vaccine antigen. A pharmaceutically effective amount refers to the amount of antigen required to induce an immune response to the vaccine antigen. For example, the vaccine antigen may be administered in an amount of several μ g to 10mg for 1 time, 1 time to several times for 1 day, or a total of several times at intervals of 1 to several weeks, for example, about 1 to 5 times.

The disclosures of all documents cited in this specification are incorporated in their entirety by reference into the specification. In addition, in the entire specification, when words such as "a", "an", and "the" are included in the singular form, the singular form includes not only the singular form but also the plural form as long as it is not explicitly stated otherwise from the context.

The present invention will be further described with reference to the following examples, which are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention.

Examples

Method

1. Mycobacterium tuberculosis vaccine

1-1. preparation of antigenic proteins

Ag85B gene (987bp) (SEQ ID NO: 1) derived from Mycobacterium tuberculosis (ATCC25618) was artificially synthesized and inserted into EcoRI-HinIII (TAKARABIO Co.) site of pET-20b (+) vector (Novagen) having a gene of His-tag sequence. The prepared expression vector was transformed into Rosetta2(DE3) pLysS-E.coli by a conventional method. The resulting transformant was cultured at 37 ℃ in a medium containing 100. mu.g/mL ampicillin (ampicillin) and 34. mu.g/mL chloramphenicol (chloremphenicol) until OD600nm became 0.5-0.8. Thereafter, 1.0mM isopropyl-. beta. -D-1-thiogalactopyranoside (and Wako pure chemical industries) was added and cultured for 4 hours. The cultured E.coli was recovered by centrifugation (5000rpm, 15 minutes). The recovered E.coli was washed with a solution containing 10mM imidazole and protease inhibitors (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20mM Tris-HCl, 500mM NaCl, 10mM imidazole and 6M urea. The extracted protein fraction was packed in a nickel affinity column (GE Healthcare Bio-Sciences Co.), washed with an adsorption buffer until OD280nm became 0.01 or less, and the protein was dissolved in a solution containing 20mM Tris-HCl, 500mM NaCl, 500mM imidazole and 6M urea. Subsequently, the eluate was concentrated by AMICON, gel-filtered through Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-urea PBS to collect the Ag85B fraction, and dialyzed stepwise against 4M-urea PBS, 2M-urea PBS, 1M-urea PBS, and PBS to prepare natural Ag 85B. 50mg of Ag85B (SEQ ID NO: 2) was recovered from 12L of E.coli culture and the purity was 95% on SDS-PAGE.

1-2 nano-gelation of antigen (preparation of vaccine)

The chp nanogel was prepared according to the method already reported (non-patent document 2).

Mixing the prepared cCHP nanogel with purified Ag85B protein according to the molecular ratio of 1:1, 3 kinds of STING ligands (cyclic diguanylic acid, cyclic diguanylic acid and cyclic guanylic acid-adenylic acid) were further added as adjuvants, respectively, and thereafter, incubated for 1 hour by a heating block at 40 ℃.

In addition, the cCHP nanogel and chimeric purified protein (ESAT6-Rv2660c-Rv0288) (amino acid sequence: SEQ ID NO: 8, nucleic acid sequence: SEQ ID NO: 9) were mixed at a molecular ratio of 1:1, and a STING ligand (cyclodialenylic acid) was further added as a mucosal adjuvant, followed by incubation with a heating block at 40 ℃ for 1 hour.

1-3 nasal immunization of mice

The mixed solution of cCHP-Ag85B + STING ligand was nasally administered to 7-week-old female Balb/c mice. The amount of antigen administered was 10. mu.g per dose, converted to the amount of Ag85B protein. In addition, STING ligands were prepared and administered in the range of 1 μ g to 10 μ g per dose. The nasal immunization was performed 3 times at 1 week intervals.

In addition, 7-week-old female Balb/c mice were nasally administered a solution of the cCHP-chimera + STING ligand. For the amount of antigen administered, 10. mu.g of chimeric protein and 10. mu.g of STING ligand were administered per dose. The nasal immunization was performed 3 times at 1 week intervals.

1-4 purification and enumeration of antigen-specific T cells

(1) Ag85B antigen

Antigen-specific Th1 cells (IFN γ -producing cells) or Th17 cells (IL-17-producing cells) were counted by the ELISPOT method from week 2 after final administration of the vaccine. Systemic immune responses were evaluated in the spleen and mucosal surface by antigen-specific T cells generated in lung tissue.

After the mice were euthanized, the lungs and spleen were removed to prepare cell suspensions. CD4 positive T cells were purified from the prepared cell suspension using the MACS system (miltenyi biotec). On the other hand, CD 90.2-negative cells were purified from the spleen of an unimmunized mouse in the same manner as antigen-presenting cells. CD4 positive T cells and gamma irradiated antigen presenting cells were co-cultured for 48-72 hours under stimulation of purified Ag85B antigen. Here, an anti-IFN γ or anti-IL-17 antibody is adsorbed in advance as a capture antibody at the bottom of the culture well.

The culture supernatant and cells were removed, and after washing the wells, biotin-labeled anti-IFN γ antibody or anti-IL-17 antibody was added and reacted at room temperature for 2 hours. Thereafter, the wells were washed, streptavidin HRP was reacted, 3-amino-9-ethylcarbazole (AEC) as a substrate of HRP was added after washing to develop color, and antigen-specific Th1 cells or Th17 cells were detected as spots. The number of spots was measured using an Elispot counter.

(2) ESAT6-Rv2660c-Rv0288 chimeric antigen

Antigen-specific Th1 cells (IFN γ -producing cells) were counted by the ELISPOT method 2 weeks after the final dosing. Systemic immune responses were evaluated in the spleen and mucosal surface by antigen-specific T cells generated in lung tissue.

After the mice were euthanized, the lungs and spleen were removed and cell suspensions were prepared. CD4 positive T cells were purified therefrom using magnetic beads. On the other hand, CD 90.2-negative cells were similarly purified from the spleen of an unimmunized mouse as antigen-presenting cells. CD4 positive T cells and gamma-irradiated antigen presenting cells were co-cultured for 48 to 72 hours under stimulation with purified chimeric antigen or recombinant ESAT6(Abcam Co.). anti-IFN gamma was plated on the bottom of the culture wells as a capture antibody, and the resulting cells were detected.

The culture supernatant was removed, and the wells were washed to allow the biotin-labeled anti-IFN γ antibody to react. After further washing, streptavidin HRP was reacted, and after washing, 3-amino-9-ethylcarbazole (AEC) as a substrate of HRP was reacted to develop color, and antigen-specific Th1 was detected as a spot. Spots were measured using an Elispot counter.

1-5 Studies of the Effect of the defense against immunity

(1) Administration of vaccines to mice

Mice were treated with 7-week-old female Balb/c. The positive control BCG vaccine was suspended in PBS solution and administered subcutaneously 1 time for primary immunization of mice. The mixed solution of cCHP-Ag85B + cyclic diguanylic acid is administered nasally 3 times at 1-week intervals in a total amount of 10 μ g per Ag85B protein. The non-immunized control mice were given 3 times nasal PBS every 1 week and 1 time subcutaneous at the time of primary immunization.

(2) Respiratory tract infection of virulent strain of tubercle bacillus

After 8 weeks from the final immunization of the vaccine, each was infected with 100CFU of a virulent strain Erdman of mycobacterium tuberculosis via the respiratory tract.

(3) Measurement of Mycobacterium tuberculosis count in spleen and Lung tissues

Mice were euthanized at 12 weeks post-infection, lungs and spleen were removed, tissues were disrupted and suspended in PBS, and 6 dilution series were prepared, each inoculated in agar medium. Culturing for 4 weeks in anaerobic environment, measuring bacterial colony, and calculating the number of Mycobacterium tuberculosis in each tissue.

Preparation of HPV vaccine

2-1 preparation of antigenic proteins

The 3 amino acid D21G, C24G and E26G variant E7(Van der Burg SH et al. vaccine 19:3652-3660,2001) genes (307bp) (SEQ ID NO: 3) of the oncosuppressor gene product of HPV16 virus were artificially synthesized, and inserted into EcoRI-HinIII (TAKARA BIO) site of pET-20b (+) vector (Novagen) having the gene of His-tag sequence. The prepared expression vector was transformed into Rosetta2(DE3) pLysS-E.coli by a conventional method. The resulting transformant was cultured at 37 ℃ in a medium containing 100. mu.g/mL ampicillin and 34. mu.g/mL chloramphenicol until OD600nm became 0.5 to 0.8. Thereafter, 1.0mM isopropyl-. beta. -D-1-thiogalactopyranoside (and Wako pure chemical industries) was added and cultured for 4 hours. The cultured E.coli was recovered by centrifugation (5000rpm, 15 minutes). The recovered E.coli was washed with a solution containing 10mM imidazole and protease inhibitors (Roche Diagnostics), and the protein was extracted with an adsorption buffer containing 20mM Tris-HCl, 500mM NaCl, 10mM imidazole and 6M urea. The extracted protein fraction was packed in a nickel affinity column (GE Healthcare Bio-Sciences Co.), washed with an adsorption buffer until OD280nm became 0.01 or less, and the protein was dissolved in a solution containing 20mM Tris-HCl, 500mM NaCl, 500mM imidazole and 6M urea. Subsequently, the eluate was dialyzed against 6M urea-PBS (0.15M NaCl), adsorbed on a DEAE-Sepharose column (GE Healthcare Bio-Sciences K.K) equilibrated with the same buffer solution, and eluted with a solution containing 0.5M NaCl-PBS-6M urea. This eluate was concentrated by AMICON, gel-filtered through Sephacryl S-100 column (GE Healthcare Bio-Sciences) equilibrated with 6M-urea PBS to collect a mutant E7 fraction, and dialyzed stepwise against 4M-urea PBS, 2M-urea PBS, 1M-urea PBS, and PBS to prepare a natural mutant E7 (SEQ ID NO: 4). 60mg of variant E7 was recovered from 12L of E.coli culture and was 95% pure in SDS-PAGE.

2-2 nano-gelation of antigen (preparation of vaccine)

The chp nanogel was prepared according to the method already reported (non-patent document 2).

The prepared chp nanogel and the purified variant E7 protein were mixed in a molecular ratio of 1:1, and further adding cyclodialkynate alone, 3 types of STING ligands (cyclodialkynate, cyclic guanylate-adenylate) or poly I: C, CpG ODN type K3 or D35, respectively, as adjuvants, followed by incubation for 1 hour with a heating block at 40 ℃.

2-3 nasal immunization of mice

The mixed solution of chp-variant E7+ each mucosal adjuvant was nasally administered to 7-week-old female Balb/c mice. The amount of antigen administered was 10. mu.g per dose, converted to the amount of mutant E7 protein. In addition, 5. mu.g or 10. mu.g of each mucosal adjuvant was administered. The nasal immunization was performed 3 times in total at 1-week intervals.

2-4 purification and enumeration of antigen-specific T cells

(1) The case of cyclic AMP as an adjuvant

Antigen-specific CTL cells (granzyme B-producing cells) or Th1 cells (IFN γ -producing cells) were counted by the ELISPOT method at 1 week after final administration of the vaccine. Systemic immune responses were evaluated by spleen and immune responses in the genital mucosa by antigen-specific T cells induced to the cervical part.

After the mice were euthanized, the spleen and cervix uteri were removed and cell suspensions were prepared. T cells (CD90.2 positive) were purified from the prepared cell suspension using the MACS system (miltenyi biotec). On the other hand, CD 90.2-negative cells were similarly purified from the spleen of an unimmunized mouse as antigen-presenting cells. The purified T cells and gamma-irradiated antigen presenting cells were co-cultured for 48-72 hours under the stimulation of purified variant E7 antigen. Here, an anti-granzyme B antibody or an anti-IFN γ antibody is adsorbed in advance as a capture antibody at the bottom of the culture well.

The culture supernatant and cells were removed, and after washing the wells, biotin-labeled anti-granzyme B antibody or anti-IFN γ antibody was added and reacted at room temperature for 2 hours. Thereafter, the wells were washed, streptavidin HRP was reacted, 3-amino-9-ethylcarbazole (AEC) as a substrate of HRP was added after washing to develop color, and antigen-specific CTL cells or Th1 cells were detected as spots. The number of spots was measured using an Elispot counter.

(2) Cases where 3 types of STING ligands (cyclic diguanylic acid, cyclic guanylic acid-adenylic acid) are used as adjuvants

Antigen-specific Th1 cells (IFN γ -producing cells) and CTLs (granzyme B-producing cells) in the cervix were counted by the ELISPOT method 1 week after the final administration. After the mice were euthanized, the cervical part of the uterus was removed and a cell suspension was prepared. T cells (CD90.2 positive) were purified therefrom using magnetic beads. On the other hand, CD 90.2-negative cells were similarly purified from the spleen of an unimmunized mouse as antigen-presenting cells. The purified T cells and the gamma-irradiated antigen presenting cells were co-cultured for 48-72 hours under the stimulation of the purified variant E7 antigen. The bottom of the culture well is paved with anti-IFN gamma antibody or anti-granzyme B antibody as capture antibody, and the generated cells are detected.

The culture supernatant was removed, and the wells were washed to allow the biotin-labeled anti-IFN γ antibody or anti-granzyme B antibody to react. After further washing, streptavidin HRP was reacted, and after washing, AEC as a substrate of HRP was reacted and developed, and antigen-specific Th1 or CTL was detected as a spot. Spots were measured using an Elispot counter.

Preparation of RSV vaccine

3-1 preparation of antigenic proteins

A DNA sequence (1172bp) obtained by repeating 3 PspA's through a linker (GGGGS) (SEQ ID NO: 7) to an SH peptide (SEQ ID NO: 5) of RSV virus was artificially synthesized, and inserted into a pET-20b (+) vector (Novagen) having a gene with a His-tag sequence using restriction enzymes EcoRV and NotI (TAKARA BIO). This plasmid was transformed into Rosetta2(DE3) pLysS-E.coli by conventional methods. The Escherichia coli was cultured at 37 ℃ in a medium containing 100. mu.g/mL ampicillin and 34. mu.g/mL chloramphenicol until OD600nm became 0.5 to 0.8. Thereafter, 1.0mM isopropyl-. beta. -D-1-thiogalactopyranoside (Wako pure chemical industries, Ltd.) was added thereto and cultured for 4 hours, and then Escherichia coli was recovered by centrifugation (5000rpm, 15 minutes). The bacteria were washed with liquid containing 20mM imidazole and protease inhibitors (Roche Diagnostics) and the proteins were extracted with adsorption buffer containing 20mM Tris-HCl, 500mM NaCl, 10mM imidazole. Saturated ammonium sulfate solution was added to the extract to reach 80% saturation, and ammonium sulfate precipitation was performed. The precipitate was collected by centrifugation, and the same buffer as that used in the extraction was used as an external solution for dialysis. The dialyzed solution was packed into a nickel affinity column (GE Healthcare Bio-Sciences Co.), washed with an adsorption buffer until OD280nm became 0.01 or less, and eluted with a solution containing 20mM Tris-HCl, 500mM NaCl and 500mM imidazole. The eluate was concentrated by AMICON, gel-filtered through Sephadex G-100 column (GE Healthcare Bio-Sciences) equilibrated with PBS, and the fraction Psp A-SH 3 was recovered, concentrated and purified. 70mg of Psp A-SH 3 were recovered in 20L of E.coli culture with a purity of 95% in SDS-PAGE.

3-2 nano-gelation of antigen (preparation of vaccine)

The chp nanogel and psppa-SH 3 purified protein (sequence No. 6) were mixed at a molecular ratio of 1:1, and cyclodicacid was further added as a mucosal adjuvant, and thereafter, the mixture was incubated for 1 hour by a heat block at 40 ℃.

3-3 nasal immunization of mice

A7-week-old female Balb/c mouse was nasally administered with a mixed solution of cCHP-PspA-SH 3+ cyclic AMP. For the amount of antigen administered, 10 μ g of each was administered in an amount of Psp A-SH 3 protein. In addition, 10ug of cyclic AMP was administered. Nasal immunization was performed 3 times at 1-week intervals, then 1 time at 4-week intervals, and further 1 time at 4-week intervals for a total of 5 times.

3-4. determination of antibody titer

About 100. mu.l of blood was collected from the inferior palate vein every week, and the serum was recovered by centrifugation at 15000rpm at 4 ℃.

Measurement of IgG antibody titer and measurement of IgG subtype in PspA or SH-specific serum were carried out by ELISA method. One day before ELISA was performed, PspA or BSA coupled SH was diluted to 1. mu.g/ml with PBS, dispensed to a 96-well plate (Thermo scientific, 3355) at 100. mu.l per well as a capture antibody, and incubated at 4 ℃ for evening primrose. The plates were washed 4 times with 300. mu.l PBS (PBS-T) containing 0.05% Tween (nacalai tesque, 28353-85) using a plate washer, PBS-T containing 1% BSA (nacalai tesque, 01863-48) was added at 200. mu.l/well, incubated at room temperature for 1 hour, and the wells were blocked. Next, the plate was washed 3 times with 300. mu.l of PBS-T using a plate washer. Each sample will be diluted 2 with 1% BSA in PBS-T⁸The diluted sample obtained by the doubling was placed in a well at one end of the plate, diluted 2-fold to the other end, and a stepwise dilution series was prepared and incubated at room temperature for 2 hours. Blank was set to 1% BSA in PBS-T. After the incubation was completed, the plate was washed 4 times with 300. mu.l of PBS-T using a plate washer. Subsequently, a diluted sample obtained by diluting 6 of Goat anti-Human (Goat anti-Human) IgG, IgG1, IgG2a, IgG2b, IgG2c, IgG3(Southern Biotech) with PBS-T containing 1% BSA was added to 100. mu.l/well by 4000-fold, and the mixture was incubated at room temperature for 1.5 hours. Thereafter, the plate was washed 4 times with 300. mu.l of PBS-T using a plate washer. A mixture of TMB substrate and TMB solution (seracare, 5120-0050) was added in an amount of 100. mu.l/well, and after 30 minutes of color development reaction, 50. mu.l of 2N H was added₂SO₄(nacalai tesque, 32520-55), the reaction was stopped. The OD450 values were measured by a plate reader, and the log2 titer values were calculated. The threshold value was set as the average of the blank wells + 0.1.

Results

1. Mycobacterium tuberculosis vaccine

(1) Ag85B antigen

Compared with the case of adding a mixture of 310. mu.g of CpGK known to exert adjuvant activity and 1. mu.g of cyclic guanosine monophosphate as 1 kind of STING ligand, induction of the same degree of antigen-specific Th1 cellular immunity was observed at 10. mu.g of cyclic diguanylic acid (FIG. 1). Cyclic-di-adenylate (cAMP) is considered relatively effective in comparison of STING ligands alone (cAMP, GMP and cGAMP).

Next, the induction effects of Th1 cells and Th17 cells were examined in the case of using STING ligand as an adjuvant. Cyclic diguanylic acid is used as STING ligand. It was found that antigen-specific Th1 cells and Th17 cells were hardly induced in mice administered with the vaccine antigen without cyclodiguanylic acid (FIGS. 2 and 3, "cCHP-Ag 85B"). In addition, T cells are hardly induced without stimulating antigen-presenting cells by antigen. On the other hand, it was found that antigen-specific Th1 and Th17 were significantly induced and both systemic immune response and mucosal immune response were efficiently induced in the lung and spleen by nasal administration of chpp-Ag 85B + cyclic diguanylic acid (fig. 2 and 3).

The effect of the nanogel nasal vaccine of the present invention using STING ligand as an adjuvant on the survival rate and proliferation of mycobacterium tuberculosis when administered to mice was examined using BCG vaccine as a positive control. As a result of calculating the survival rate of several cases of death from the time period from 12 weeks after infection, the nonimmunized mice (negative control) was 56%, the BCG vaccine group (positive control) was 67%, and the nanogel group (administered chp-Ag85B + cyclic diguanylic acid) was 89% compared to the positive control and showed resistance to infection (fig. 4A). In addition, the number of mycobacterium tuberculosis in the spleen was equally significantly inhibited by the BCG and nanogel vaccine groups from the proliferation of the bacteria compared to the non-immunized mice, and the same trend was observed in the lung (fig. 4B).

(2) ESAT6-Rv2660c-Rv0288 chimeric antigen

It was found that antigen-specific Th1 cells were induced in the spleen and cervix by nasal administration of the cCHP-chimera + cyclic AMP (FIG. 5). In addition, when only the chp-chimera was administered, since antigen-specific Th1 was not induced in any organ, cyclic adenosine monophosphate was considered to be essential for Th 1.

HPV vaccine

(1) The case of cyclic AMP as an adjuvant

The nanogel transnasal vaccine of the present invention was prepared using HPV variant E7 protein as an antigen, and the effect of inducing T cells and the like in the vaccine was examined.

It was found that antigen-specific CTL was induced in the spleen and cervix by nasal administration of cCHP-variant E7+ cyclodextrana (FIG. 6). Furthermore, it was found that antigen-specific Th1 cells were induced in the spleen and cervix by nasal administration of cCHP-variant E7+ cyclic AMP (FIG. 7).

(2) Cases where 3 types of STING ligands (cyclic diguanylic acid, cyclic guanylic acid-adenylic acid) are used as adjuvants

It was found that antigen-specific Th1 (right in FIG. 8) and CTL (left in FIG. 8) were induced in the cervical region in mucosal adjuvants combined with the cCHP-variant E7 protein antigen, particularly in nasal immunization combined with 3 types of STING ligands. In Th1 induction, no major difference was observed between STING ligands, and in CTL, induction by cyclic-adenosine was clearly observed.

RSV vaccine

It was found that both the antibody against the SH peptide and the antibody against PspA as a carrier protein increased with time with the number of nasal immunizations (fig. 9). In addition, in SH peptide-specific immune response, IgG induction was more significant in the group to which cyclic adenosine was added (fig. 9 left), but in the group to which chp-psppa-SH 3 alone was administered without cyclic adenosine, induction of specific antibodies depending on the number of immunizations was also observed.

In the IgG subclass, IgG1 predominated in the absence of cyclic adenosine adjuvant and IgG1 and IgG2b were induced in the presence of adjuvant in either of the anti-SH peptide and anti-PspA antibodies (fig. 10).

As described above, when a nanogel vaccine is administered with an adjuvant (STING ligand in this example), T cells having cellular immunity characteristics such as Th1 cells and CLT cells are induced. In addition, it was also clarified that mucosal immunity in genital mucosal tissue is induced in addition to systemic immunity and upper respiratory tract and lower respiratory tract mucosal tissue.

Industrial applicability

The nanogel nasal vaccine of the present invention can induce cellular immunity, and thus is expected to be used in the medical field such as immunocytotherapy.

Sequence listing

<110> national university of Law Tongjing university

Hena Bios Ltd

<120> nasal vaccine for inducing cellular immunity

<130> TPC0283UVT

<150> 2018-146519

<151> 2018-08-03

<160> 9

<170> PatentIn version 3.5

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ttctcccggc cggggctgcc ggtcgagtac ctgcaggtgc cgtcgccgtc gatgggccgc 180

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ggcctgcgcg cccaagacga ctacaacggc tgggatatca acaccccggc gttcgagtgg 300

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ctgaccagcg agctgccgca atggttgtcc gccaacaggg ccgtgaagcc caccggcagc 480

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cagcagttca tctacgccgg ctcgctgtcg gccctgctgg acccctctca ggggatgggg 600

cctagcctga tcggcctcgc gatgggtgac gccggcggtt acaaggccgc agacatgtgg 660

ggtccctcga gtgacccggc atgggagcgc aacgacccta cgcagcagat ccccaagctg 720

gtcgcaaaca acacccggct atgggtttat tgcgggaacg gcaccccgaa cgagttgggc 780

ggtgccaaca tacccgccga gttcttggag aacttcgttc gtagcagcaa cctgaagttc 840

caggatgcgt acaacgccgc gggcgggcac aacgccgtgt tcaacttccc gcccaacggc 900

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Met Thr Asp Val Ser Arg Lys Ile Arg Ala Trp Gly Arg Arg Leu Met

1 5 10 15

Ile Gly Thr Ala Ala Ala Val Val Leu Pro Gly Leu Val Gly Leu Ala

20 25 30

Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser Arg Pro Gly Leu Pro Val

35 40 45

Glu Tyr Leu Gln Val Pro Ser Pro Ser Met Gly Arg Asp Ile Lys Val

50 55 60

Gln Phe Gln Ser Gly Gly Asn Asn Ser Pro Ala Val Tyr Leu Leu Asp

65 70 75 80

Gly Leu Arg Ala Gln Asp Asp Tyr Asn Gly Trp Asp Ile Asn Thr Pro

85 90 95

Ala Phe Glu Trp Tyr Tyr Gln Ser Gly Leu Ser Ile Val Met Pro Val

100 105 110

Gly Gly Gln Ser Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys Gly

115 120 125

Lys Ala Gly Cys Gln Thr Tyr Lys Trp Glu Thr Phe Leu Thr Ser Glu

130 135 140

Leu Pro Gln Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly Ser

145 150 155 160

Ala Ala Ile Gly Leu Ser Met Ala Gly Ser Ser Ala Met Ile Leu Ala

165 170 175

Ala Tyr His Pro Gln Gln Phe Ile Tyr Ala Gly Ser Leu Ser Ala Leu

180 185 190

Leu Asp Pro Ser Gln Gly Met Gly Pro Ser Leu Ile Gly Leu Ala Met

195 200 205

Gly Asp Ala Gly Gly Tyr Lys Ala Ala Asp Met Trp Gly Pro Ser Ser

210 215 220

Asp Pro Ala Trp Glu Arg Asn Asp Pro Thr Gln Gln Ile Pro Lys Leu

225 230 235 240

Val Ala Asn Asn Thr Arg Leu Trp Val Tyr Cys Gly Asn Gly Thr Pro

245 250 255

Asn Glu Leu Gly Gly Ala Asn Ile Pro Ala Glu Phe Leu Glu Asn Phe

260 265 270

Val Arg Ser Ser Asn Leu Lys Phe Gln Asp Ala Tyr Asn Ala Ala Gly

275 280 285

Gly His Asn Ala Val Phe Asn Phe Pro Pro Asn Gly Thr His Ser Trp

290 295 300

Glu Tyr Trp Gly Ala Gln Leu Asn Ala Met Lys Gly Asp Leu Gln Ser

305 310 315 320

Ser Leu Gly Ala Gly

325

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Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln

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Pro Glu Thr Thr Gly Leu Tyr Gly Tyr Gly Gln Leu Ser Asp Ser Ser

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Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala Gly Gln Ala Glu Pro Asp

35 40 45

Arg Ala His Tyr Asn Ile Val Thr Phe Cys Cys Lys Cys Asp Ser Thr

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Leu Arg Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu Glu

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Asp Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln

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Lys Pro

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Asn Lys Leu Ser Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu

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Pro Arg Ala Arg Val Asn Thr

20

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<223> PspA-SH fusion protein

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Glu Glu Ser Pro Val Ala Ser Gln Ser Lys Ala Glu Lys Asp Tyr Asp

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Ala Ala Lys Lys Asp Ala Lys Asn Ala Lys Lys Ala Val Glu Asp Ala

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Gln Lys Ala Leu Asp Asp Ala Lys Ala Ala Gln Lys Lys Tyr Asp Glu

35 40 45

Asp Gln Lys Lys Thr Glu Glu Lys Ala Ala Leu Glu Lys Ala Ala Ser

50 55 60

Glu Glu Met Asp Lys Ala Val Ala Ala Val Gln Gln Ala Tyr Leu Ala

65 70 75 80

Tyr Gln Gln Ala Thr Asp Lys Ala Ala Lys Asp Ala Ala Asp Lys Met

85 90 95

Ile Asp Glu Ala Lys Lys Arg Glu Glu Glu Ala Lys Thr Lys Phe Asn

100 105 110

Thr Val Arg Ala Met Val Val Pro Glu Pro Glu Gln Leu Ala Glu Thr

115 120 125

Lys Lys Lys Ser Glu Glu Ala Lys Gln Lys Ala Pro Glu Leu Thr Lys

130 135 140

Lys Leu Glu Glu Ala Lys Ala Lys Leu Glu Glu Ala Glu Lys Lys Ala

145 150 155 160

Thr Glu Ala Lys Gln Lys Val Asp Ala Glu Glu Val Ala Pro Gln Ala

165 170 175

Lys Ile Ala Glu Leu Glu Asn Gln Val His Arg Leu Glu Gln Glu Leu

180 185 190

Lys Glu Ile Asp Glu Ser Glu Ser Glu Asp Tyr Ala Lys Glu Gly Phe

195 200 205

Arg Ala Pro Leu Gln Ser Lys Leu Asp Ala Lys Lys Ala Lys Leu Ser

210 215 220

Lys Leu Glu Glu Leu Ser Asp Lys Ile Asp Glu Leu Asp Ala Glu Ile

225 230 235 240

Ala Lys Leu Glu Asp Gln Leu Lys Ala Ala Glu Glu Asn Asn Asn Val

245 250 255

Glu Asp Tyr Phe Lys Glu Gly Leu Glu Lys Thr Ile Ala Ala Lys Lys

260 265 270

Ala Glu Leu Glu Lys Thr Glu Ala Asp Leu Lys Lys Ala Val Asn Glu

275 280 285

Pro Glu Lys Pro Ala Pro Ala Pro Glu Thr Pro Ala Pro Glu Gly Gly

290 295 300

Gly Gly Ser Asn Lys Leu Ser Glu Tyr Asn Val Phe His Asn Lys Thr

305 310 315 320

Phe Glu Leu Pro Arg Ala Arg Val Asn Thr Gly Gly Gly Gly Ser Asn

325 330 335

Lys Leu Ser Glu Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu Pro

340 345 350

Arg Ala Arg Val Asn Thr Gly Gly Gly Gly Ser Asn Lys Leu Ser Glu

355 360 365

Tyr Asn Val Phe His Asn Lys Thr Phe Glu Leu Pro Arg Ala Arg Val

370 375 380

Asn Thr

385

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<213> Artificial sequence

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Gly Gly Gly Gly Ser

1 5

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<220>

<223> ESAT & -Rv2660-Rv0288 fusion

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Met Thr Glu Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser

1 5 10 15

Ala Ile Gln Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly

20 25 30

Lys Gln Ser Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser

35 40 45

Glu Ala Tyr Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu

50 55 60

Leu Asn Asn Ala Leu Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly

65 70 75 80

Gln Ala Met Ala Ser Thr Glu Gly Asn Val Thr Gly Met Phe Ala Gly

85 90 95

Pro Gly Pro Gly Ile Ala Gly Val Asp Gln Ala Leu Ala Ala Thr Gly

100 105 110

Gln Ala Ser Gln Arg Ala Ala Gly Ala Ser Gly Gly Val Thr Val Gly

115 120 125

Val Gly Val Gly Thr Glu Gln Arg Asn Leu Ser Val Val Ala Pro Ser

130 135 140

Gln Phe Thr Phe Ser Ser Arg Ser Pro Asp Phe Val Asp Glu Thr Ala

145 150 155 160

Gly Gln Ser Trp Cys Ala Ile Leu Gly Leu Asn Gln Phe His Gly Pro

165 170 175

Gly Pro Gly Ser Gln Ile Met Tyr Asn Tyr Pro Ala Met Leu Gly His

180 185 190

Ala Gly Asp Met Ala Gly Tyr Ala Gly Thr Leu Gln Ser Leu Gly Ala

195 200 205

Glu Ile Ala Val Glu Gln Ala Ala Leu Gln Ser Ala Trp Gln Gly Asp

210 215 220

Thr Gly Ile Thr Tyr Gln Ala Trp Gln Ala Gln Trp Asn Gln Ala Met

225 230 235 240

Glu Asp Leu Val Arg Ala Tyr His Ala Met Ser Ser Thr His Glu Ala

245 250 255

Asn Thr Met Ala Met Met Ala Arg Asp Thr Ala Glu Ala Ala Lys Trp

260 265 270

Gly Gly

<210> 9

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<223> synthetic nucleotide

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gcggcctggg gcggtagcgg ttcggaggcg taccagggtg tccagcaaaa atgggacgcc 180

acggctaccg agctgaacaa cgcgctgcag aacctggcgc ggacgatcag cgaagccggt 240

caggcaatgg cttcgaccga aggcaacgtc actgggatgt tcgcaggacc aggtcctgga 300

atagcgggcg tcgaccaggc gcttgcagca acaggccagg ctagccagcg ggcggcaggc 360

gcatctggtg gggtcaccgt cggtgtcggc gtgggcacgg aacagaggaa cctttcggtg 420

gttgcaccga gtcagttcac atttagttca cgcagcccag attttgtgga tgaaaccgca 480

ggtcaatcgt ggtgcgcgat actgggattg aaccagtttc acggaccagg tcctggatcg 540

caaatcatgt acaactaccc cgcgatgttg ggtcacgccg gggatatggc cggatatgcc 600

ggcacgctgc agagcttggg tgccgagatc gccgtggagc aggccgcgtt gcagagtgcg 660

tggcagggcg ataccgggat cacgtatcag gcgtggcagg cacagtggaa ccaggccatg 720

gaagatttgg tgcgggccta tcatgcgatg tccagcaccc atgaagccaa caccatggcg 780

atgatggccc gcgacacggc cgaagccgcc aaatggggcg gc 822