KR100823866B1 - - -5 Modified flagellin improved Toll-like receptor 5 stimulating activity - Google Patents

Abstract

A modified flagellin is provided to improve toll-like receptor-5(TLR-5) stimulating activity by site directed mutagenesis of some amino acids of flagellin, which are TLR5 agonists, thereby enhancing unique activity of the flagellin. A modified flagellin which is a flagellin modified body of septic vibrio with enforced toll-like receptor-5(TLR-5) stimulus activity is described as SEQ ID : NO. 2, which is prepared by site directed mutagenesis of an aspartic acid, which is a 70th amino acid, by alanine, or prepared by site directed mutagenesis of an asparagine, which is a 135th amino acid, by an aspartic acid. A vaccine supplement agent comprises the modified flagellin.

Description

Modified flagellin improved Toll-like receptor 5 stimulating activity

Figure 1 compares the amino acid homology of Salmonella flagellin St-FliC, Escherichia coli flagellin Ec-FliC, and sepsis Vibrio flagellin Vv-FlaB, and the domains of flagellin proteins of each strain are boxed.

Figure 2 is a process for inducing a positional mutation in amino acid sequence 70 of the sepsis Vibrio flagellin gene flaB in the production of flagellin according to the invention from aspartic acid to alanine, 135 amino acid sequence from asparagine to aspartic acid It is a schematic diagram showing.

3 is a result showing the effect of the recombinant flagellin variant according to the invention on the flagellin polymerization (polymerization) in the aqueous solution.

4 is a graph showing the results of measuring the TLR5 stimulatory activity of the flagellin variant according to the present invention.

The present invention relates to a flagellin variant with enhanced toll-like receptor-5 (hereinafter, TLR5) stimulating activity, and more particularly, to TLR5 stimulating activity by point mutation of some amino acids of flagellin, which is a TLR5 agonist, to enhance TLR5 stimulatory activity. It relates to a flagellin variant with enhanced activity.

Flagella (flagella) is an important component that determines the motility of the bacteria and consists largely of hooks (basal body) and filaments (filament). Flagella is known to have a function of determining the swimming or swarming motility of bacteria, the taxis of bacteria, and forming a biofilm to determine the adhesion of pathogenic microorganisms. The structural unit protein constituting the flagella filaments is called flagellin, and flagellin is regularly assembled to form filaments. Hayashi et al. Reported that mammalian expression of TLR5 recognizes Gram-negative and Gram-positive bacteria flagellin and activates NF-κB (Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, Eng JK). , Akira S, Underhill DM, Aderem A: Nature 410: 1099-1103, 2001).

Toll-like receptors (TLR) are typical "pattern recognition receptors (P attern R ecognition R eceptor; PRR) '; a as it' (PAMP P athogen A ssociated M olecular P attern) Molecular structures associated with pathogens, present on pathogens It is a recognized receptor and is present on the surface of insects and plant cells as well as mammals. Thirteen types of TLRs have been identified so far, and studies on agonists of each TLR have been actively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124 (4): 783-801, 2006).

PRRs, such as TLRs, are distributed on the cell surface or cytoplasm of host cells, and induce 'innate immune response' by stimulating various PAMPs and further regulate 'adaptive immune response'. Thus, TLR agonists can be suitable targets for the development of various 'immunomodulators', particularly 'vaccine adjuvants'.

A vaccine adjuvant is a substance that can enhance, prolong or accelerate the antigen-specific immune response induced by the vaccine antigen when coadministration with the vaccine. It is called. Vaccine aids that have been approved for human use to date include 'alum' (aluminum phosphate and aluminum hydroxide) and 'squalene emulsion'. Vaccine adjuvant must meet at least one of the following five conditions: 1) Regulate co-stimulatory moleciule expression on the surface of antigen presenting cells, induce antigen-specific T lymphocyte response or cytosol Immunomodulation, such as regulation of cytokine secretion, 2) antigen presentation, 3) CD8 + T lymphocyte response, 4) targeting, 5) antigen generation.

The ideal vaccine adjuvant should be 1) safe, 2) biodegradable in the body, 3) show higher protective or therapeutic immunity than when administered alone, 4) chemically or biologically identified, and 5 It is recommended to work at a lower concentration than the antigen and 6) to have a long half-life for commercial or clinical application.

Currently used or contemplated for use as a vaccine adjuvant include: 1) mineral salts such as aluminum hydroxide gels, 2) surfactants, 3) bacteria-derived materials, 4) cytokines or hormones, 6) polyanions (polyanion), 7) polyacryl, 8) carrier, 9) living vector with virus, 10) vehicle such as mineral oil or liposome ). Among these, active research is underway, and Vibrio , a protein-derived vaccine adjuvant, has attracted much attention. There are cholera toxin (CT) derived from cholerae and heat-labile toxin (LT) derived from E. coli. These vaccine adjuvants induce the production of antigen-specific antibodies in mucosal areas and serum and induce B7-2 expression on the surface of antigen presenting cells (APCs). It has been reported that it is a mechanism that promotes co-stimulatory signaling of CD4 + cells. However, since they are exotoxins with high enterotoxicity, research is being conducted to reduce toxicity and increase adjuvaticity.

In PCT International Patent Publication No. WO 2005/070455, the present inventors recently prepared a 'transposon mutant library' to screen for host adhesion and invasion factor of sepsis vibrio. Identifying two flagella related operons consisting of 56 genes during the analysis of three clones' Tn-flanking regions, which lost their adhesion and motility to cells. It was. In this process, sepsis vibrioviruses have a total of six flagellin genes ( flaA , flaB , flaF , flaC , flaD , flaE ), and the flaB gene is the major constituent of flagellin. The present inventors are pursuing the application of flagellin recombinant protein (FlaB), which is a component of the sepsis vibrio polar flagellin, as a 'component vaccine' against sepsis vibrio, We found that there was an adjuvant effect and we studied to find out. As a result, when the vaccine antigen tetanus toxicant is administered to the nasal cavity of experimental animals together with flagellin, flagellin signals the host's TLR5 to activate the immune system and amplifies the efficacy of the vaccine. Injecting lethal doses of tetanus toxin into mice immunized with JELIN proved to have an excellent 'mucosal vaccine adjuvant' efficacy by inducing 100% protective immunity (Lee SE, Kim SY, Jeong BC, Kim YR). , Bae SJ, Ahn OS, Lee JJ, Song HC, Kim JM, Choy HE, Chung SS, Kweon MN, Rhee JH .: Infect. Immun. 74: 694-702, 2006).

Flagellin, which stimulates TLR5 among various TLR agonists, is a protein component unlike other TLR agonists [CpG-DNA, MALP (mycoplasmal lipopeptide)], so it is not only possible to synthesize recombinant proteins for continuous quality control but also TLR5. Various recombinant fusion proteins with enhanced stimulatory activity can be produced.

According to a report on the three-dimensional structure of flagellin, polar monomers and charged amino acid residues react with each other in a polar-charge reaction between flagelone monomers. interaction) to form multimers to form the unique filament structure of Flagella (Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424 (6949): 643- 50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.Nature. 2001 410 (6826): 331-7). According to Smith et al., TLR5 was reported to recognize flagelin monomers rather than multimerized filament type flagelins (Smith KD, Andersen-Nissen E, Hayashi F, Strobe K, Bergman MA). , Barrett SL, Cookson BT, Aderem A. Toll-like receptor 5 recognizes a conserved site on flagellin required for protofilament formation and bacterial motility.Nat Immunol. 2003 4 (12): 1247-53).

A common problem observed in 'preventive and therapeutic vaccines' (infectious diseases, autoimmune diseases, allergic diseases, cancer vaccines) that are currently used in the clinic is that they are derived from vaccine supplements used to amplify the response. . That is, the development of safer and more efficient vaccine adjuvant is strongly desired.

Therefore, the present inventors mutated an amino acid sequence predicted to be involved in the axial interaction between the flagellin monomers of the sepsis Vibrio flagellin gene flaB and involved in the axial interaction. By preparing a recombinant flagellin variant capable of inhibiting charge reaction, the present inventors have found that a flagellin variant with significantly improved TLR5 stimulating activity compared to conventional flagellin can be prepared.

Accordingly, it is an object of the present invention to provide flagellin variants with enhanced TLR5 stimulatory activity.

It is also an object of the present invention to provide a vaccine adjuvant containing the flagellin variant as an active ingredient.

In order to achieve the above object, the present invention provides a flagellin variant that suppresses the interaction between flagellin monomer (flameline) monomer of flagellin TLR5 agonist.

In another aspect, the present invention provides a vaccine adjuvant containing the improved flagellin variant as an active ingredient.

Hereinafter, the present invention will be described in more detail.

The present invention relates to flagellin variants having enhanced TLR5 stimulation activity by inhibiting multimerization of flagellin monomers by point mutation of some amino acids of flagellin, a TLR5 agonist.

The present invention provides a sequence number prepared by mutating the 70th amino acid aparteic acid (D70), which is analyzed to be involved in the axial interaction between flagellin monomers of flagellin, a TLR5 agonist, with alanine (A). It relates to the flagellin variant (N135D) of SEQ ID NO: 4 prepared by positional mutation of flagellin variant (D70A) and 135th amino acid asparagine (N135) with aspartic acid (D). In addition, the flagellin variant according to the present invention is alanine (A) as the aspartic acid (aparteic acid; D70), the 70th amino acid, asparagine (A135) as the aspartic acid (aspartic acid; D), 135th amino acid It relates to a flagellin variant (D70A / N135D) of SEQ ID NO: 6 prepared by concomitant positional mutation.

Flagellin as referred to herein is a unit molecule constituting flagella, the cilia that determine the motility of bacteria.

The flagellin variants provided in the present invention have significantly improved TLR5 stimulating activity, and this phenomenon shows that the flagellin variant is significantly reduced in multimerization compared to conventional flagellin in aqueous solution, indicating that flagellin polymerization is suppressed. Could know.

Therefore, the flagellin variant with improved TLR5 stimulatory activity produced by the present invention can provide a more potent TLR5 agonist by replacing the existing flagellin, thereby providing a vaccine adjuvant enhanced than the existing flagellin.

The process of demonstrating enhanced TLR5 stimulation activity using the flagellin variant according to the present invention is as follows:

1) preparing flagellin variants of SEQ ID NOs: 2, 4 and 6;

2) confirming that the flagellin variant inhibits flagellin polymerization in an aqueous solution; And

3) confirming TLR5 activation of each recombinant flagellin variant derived from E. coli;

It includes.

Hereinafter, an Example and a test example are given and this invention is demonstrated in detail. However, these examples or test examples are intended to describe the present invention in detail, and the scope of the present invention is not limited by these examples or test examples.

The characteristics of the strains and plasmids used in the present invention are summarized in Table 1. Each manufacturing method and detailed characteristics are indicated in the corresponding examples and test examples.

Strain or Plasmid Characteristic source Esherichia coli DH5α F- recA 1; restriction negative Existing lab ER2566 F- λ- fhuA2 [ lon ] ompT lacZ :: T7 gene1 gal sulA1 1 (mcrC - mrr ) 114 :: IS10 R (mcr -73 :: miniTn10 - TetS) 2 R (zgb-210 :: Tn10) (TetS) endA1 [dcm] New england biolab BL21 (DE3) F- ompT hsdSB (rB - mB - ) gal dcm (DE3) Novagen Plasmids pCR2.1 TOPO PCR TA Cloning Vector, AmpR, KmR Invitrogen pTYB12 Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT) expression vector, AmpR New england biolab

<Cultivation and storage of each strain>

E. coli strains used in the following examples and test examples were cultured in Luria Bertani medium (Difco Co.). The used strains were added to glycerol 30% after incubation and stored in -80 ℃ cryogenic freezer.

Example 1 Preparation of Positional Mutant Recombinant Flagellin Variant

Previously reported flagellin three-dimensional structural analysis data of Salmonella (Yonekura K, Maki-Yonekura S, Namba K: Complete atomic model of the bacterial flagellar filament by electron cryomicroscopy. Nature. 2003 424 (6949): 643-50; Samatey FA, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto M, Namba K: Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling.Nature. 2001 410 (6826): 331-7) and inventors According to the bioinformatics analysis, amino acid residues required for axial interaction between sepsis Vibrio flagellin FlaB monomers are as follows. That is, aspartic acid (D70), the 70th amino acid of the upper subunit, and asparagine (D77), the 135th amino acid, the lower monomer of each monomer involved in the axial reaction. Asparagine (N101), the 101st amino acid, and glutamic acid (E108), the 108th amino acid of the subunit, were analyzed to form multimers through a polar-charge reaction. (Ie, upper flagellin D70: lower flagellin N101; upper flagellin N135: lower flagellin E108).

In the present invention, aspartic acid (aparteic acid; D70) and 135th amino acid asparagine (N135), which are analyzed to be involved in the axial interaction between flagellin monomers, are respectively alanine (A) and aspartic acid. (aspartic acid; D), respectively, by site-directed mutation, D70A of SEQ ID NO: 2 and N135D flagellin variant of SEQ ID NO: 4 were prepared, respectively.

PCR primer FlaB-V5 of SEQ ID NO: 7 (5'- gcggccgc atggcagtgaatgtaaatgtaaatacaaac-3 '; restriction enzyme Not I restriction enzyme inserted for cloning first to obtain template DNA for site-directed mutagenesis) Recognition sites are underlined) and PCR primer FlaB-V4 (5'- cccggg gcctagtagacttagcgctga-3 ': underlined with restriction enzyme Sma I restriction enzyme insertion site for cloning ) FlaB via PCR using DNA fragments of 1,142 bp containing the ORF of the gene were amplified. In other words, PCR reaction using primers FlaB-V5 and FlaB-V4 was repeated at 95 ° C. for 1 minute after initial denaturation at 95 ° C. for 30 seconds, annealing at 70 ° C. for 30 seconds, extended 72 ° C. for 1 minute, and repeated 30 times at the last 72 ° C. It carried out on the conditions made to react for 10 minutes. The flaB DNA fragment thus amplified was cloned into PCR 2.1-TOPO cloning vector (Invitrogen Co.) and named pCMM270. The pCMM270 plasmid was used as template DNA for the following positional mutations.

Sepsis Vibrio flagellin gene flaB PCR primer FlaB-SD1 (5'-gtacgtaacgccaacgcaggtatctcaatc-3 ') of SEQ ID NO: 9 and PCR primer FlaB- of SEQ ID NO: 10 to replace the 70th amino acid aspartic acid (D70) with alanine (A) SD2 (5'-gattgagatacctgcgttggcgttacgtac-3 '), PCR template pCMM270, unique Pfu with proof reading activity Polymerase (Stratagene Co.) was used to synthesize mutant strands (D70A) according to the manufacturer's instructions. In other words, PCR reaction using FlaB-SD1 and FlaB-SD2 primers was repeated after initial denaturation at 95 ° C for 45 seconds, denaturation at 95 ° C for 45 seconds, annealing at 63 ° C for 1 minute, extended 68 ° C for 10 minutes, and 16 times after the last 68 ° C. It carried out on the conditions made to react for 10 minutes. The amplified D70A positional mutant flaB DNA of SEQ ID NO: 1 was cloned into a PCR 2.1-TOPO cloning vector and named pCMM272.

PCR primer FlaB-SD3 (5'-cgtcttttggtggtgacaagctgctaaacg-3 ') of SEQ ID NO: 11 and PCR primer FlaB- of SEQ ID NO: 12 to replace 135th amino acid asparagine (N135) with aspartic acid (D) Mutant strand (N135D) according to the manufacturer's instructions using SD4 (5'-cgtttagcagcttgtcaccaccaaaagacg-3 '), PCR template pCMM270, plasmid and unique Pfu polymerase (Stratagene Co.) with proof reading activity Was synthesized. In other words, the PCR reaction using the SEQ ID NO: 11 and 12 primers after the initial denaturation 95 ℃ 45 seconds, denaturation 95 ℃ 45 seconds, annealing 63 ℃ 1 minutes, expansion 68 ℃ 10 minutes, repeated 16 times, the last 10 minutes at 68 ℃ It carried out on the conditions made to react. This amplified N135D positional mutant flaB DNA of SEQ ID NO: 3 was named pCMM273.

To prepare the D70A / N135D double-positioning mutant flagellin variant of SEQ ID NO: 6, pCMM273 having N135D-positioning mutant flaB DNA was used as a template of the PCR reaction. Mutagen strand according to manufacturer's instructions using PCR primers FlaB-SD1 of SEQ ID NO: 9 and PCR primer FlaB-SD2 plasmid of SEQ ID NO: 10 and a unique Pfu polymerase (Stratagene Co.) with proof reading activity (D70A) was synthesized. The PCR reaction was carried out under the conditions of initial denaturation at 95 ° C. for 45 seconds, denaturation at 95 ° C. for 45 seconds, annealing at 63 ° C. for 1 minute, expansion at 68 ° C. for 10 minutes, and repeated 16 times, followed by reaction at 68 ° C. for 10 minutes. The amplified D70A / N135D double-positioning mutant flaB DNA of SEQ ID NO: 5 was named pCMM274.

DNA fragments of SEQ ID NOs: 1, 3, and 5 containing the positional mutant flaB (D70A, N135D or D70A / N135D) gene were cut with restriction enzymes Not I and Sma I and pTYB12 plasmid cut with the same restriction enzyme (New England Biolabs) And cloned into pCMM276, pCMM277 and pCMM278 respectively. These plasmids were placed in ER2566 Escherichia coli by electroporation and 0.5 mM of 5-bromo-indole-3-chloro-isopropyl-β-D-galactopyranoside (IPTG) was added to induce expression. Point mutant flagellin (D70A, N135D, D70A /) from intein fusion proteins using chitin bead columns and 1,4-dithiothreitol (1,4-DTT) according to the manufacturer's (New England Biolabs Inc.) instructions N135D) only proteins were obtained. Endotoxins contained in the isolated point-mutated flagellin protein were removed using AffinityPak Detoxi Gel Endotoxin Removing gel (Pirece). The recombinant protein thus separated was subjected to gel-filtration (using Amersham's AKTA-Prime) using a Superdex120 (Amersham) column to separate and purify the recombinant purelin variant of high purity.

[Test Example 1] Flagelin Modification Production of recombinant flagellin variant and biochemical properties in aqueous solution

Flageline variants To prepare recombinant flageline variants, the genes of SEQ ID NOs: 1, 3 and 5 encoding flageline variants were cloned into pTYB12 plasmids, respectively, and then transformed into ER2566 Escherichia coli and 0.3 mM IPTG (Isopropyl-β) in the medium. -D-1-thiogalactoside) was added to induce the recombinant flagellin variant, followed by SDS-polyacrylamide gel electrophoresis and native-gel electrophoresis, and the results are shown in FIG. 3.

Referring to FIG. 3, SDS-PAGE of flagellin (FlaB) and flagellin variant (mutated FlaB) resulted in point mutations of the SEQ ID NOS: 2, 4, and 6 flagellin (D70A, N135D, D70A / N135D) and the conventional wild-type flagella. Jelly (FlaB) all showed major bands at 41.5 KDa. Native-gel electrophoresis shows that FlaB forms very large multimers, so most of the protein remains in the stacking gel and some FlaB proteins entering the resolving gel are 66Kda and 140 It was observed that the distribution was blurred between KDa. On the other hand, the point mutant flagellin variant (D70A, N135D, D70A / N135D) protein according to the present invention formed a multimer near 140KDa. The above results indicate that FlaB protein forms a very large multimer in aqueous solution, but the point-mutated flagellin variants according to the present invention (D70A, N135D, D70A / N135D) are smaller multimers. I knew it existed. That is, it was confirmed that the flagellin variants (D70A, N135D, D70A / N135D) of SEQ ID NOs: 2, 4, and 6 according to the present invention inhibit the polymerization of flagellin.

Test Example 2 TLR5 Stimulating Activity of the Flagellin Variant

24-well to measure the transcriptional activity of NF-κB, a major factor involved in TLR5-mediated signaling, in order to measure TLR5 stimulatory activity of wild-type FlaB and flagellin variants of SEQ ID NOs: 2, 4 and 6 according to the present invention 293T cells were plated in a plate incubator and cultured overnight, followed by NF-κB-Luc, a reporter plasmid that can observe NK-κB transcriptional activity using FuGENE6 (Roche). P3xFlag-hTLR5 plasmid cloned from Professor Jung Mok Kim and TLR5 gene (obtained from Steven B. Mizel of Wake Forest University School of Medicine, Departments of Microbiology and Immunology, USA) and beta-galactosidase A control plasmid (Clontech) was introduced into the cell at the same time. After 24 hours of incubation, fresh medium was replaced and luciferase (luciferase) after 18-24 hours of flageline variants (D70A, N135D, D70A / N135D) of SEQ ID NOs: 2, 4 and 6 isolated in Example 1 ) NF-κB transcription was measured using a luminometer (luminometer, Berthold) and the results are shown in FIG. 4. Referring to FIG. 4, the group treated with 50 ng / ml FlaB recombinant protein increased the TLR5-mediated NF-κB transcriptional activity by about 1.8 times compared to the control group treated with only phosphate buffer. On the other hand, the group treated with 50 ng / ml of D70A FlaB, N135D FlaB and D70A / N135D FlaB recombinant flagellin variants showed approximately 5.2-fold, 5.6-fold and TLR5-mediated NF-κB transcriptional activity, respectively, compared to the control treated with impression buffer alone. 4.4-fold increase.

In the present invention, it is further upgraded by providing a flagellin variant which further improves the TLR5 stimulating activity of flagellin, which was found to be a potent mucosal vaccine adjuvant by stimulating TLR5 by PCT WO 2005/070455. Flagellin vaccine adjuvant was developed. This is the new paradigm's treatment of 'infectious diseases', 'autoimmune diseases' and 'allergic diseases', and 'applied science research' such as 'anti-cancer immunity treatment' is the next generation's attention research theme. It will provide an important link between bed-side clinical research. Therefore, the application of the results of the present invention to various vaccine preparations can create tremendous added value.

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Claims (4)

Flagellin variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulatory activity, prepared by site-directed mutation of the 70th amino acid, aspartic acid (D70), with alanine (A) Flageline variant of SEQ ID NO: 2. Flagellin variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulatory activity, prepared by site-mutation of the 135th amino acid asparagine (N135) with aspartic acid (D) Flageline variant of SEQ ID NO: 4. Flageline variant of sepsis vibrio with enhanced toll-like receptor-5 (TLR-5) stimulatory activity, wherein the 70th amino acid, aspartic acid (D70) is alanine (A), and the 135th amino acid is D70A / N135D flagellin variant of SEQ ID NO: 6 prepared by simultaneous positional mutation of asparagine (N135) with aspartic acid (D). A vaccine adjuvant comprising the flagellin variant according to claim 1 as an active ingredient.

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