# Patent

## Title
Modified flagellin improved toll-like receptor 5 stimulating activity

## Abstract

Disclosed herein are flagellin mutants having an enhanced activity of stimulating the toll-like receptor-5 (hereinafter referred to as “TLR5”). More specifically, disclosed are flagellin mutants, prepared by point-mutating some of the amino acids of a TRL5 agonist flagellin so as to enhance the TRL-stimulating activity of the flagellin.

## Background
This application is a Divisional of U.S. patent application Ser. No. 12/526,187, filed Aug. 6, 2009, issued as U.S. Pat. No. 8,236,327, which is a National Stage application of PCT/KR2008/000709, filed Feb. 5, 2008, which claims priority from Korean patent Application No. 10-2007-0013846, filed Feb. 9, 2007, and from Korean patent Application No. 10-2008-0011330, filed Feb. 4, 2008 in the Korean Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.
Sequence Listing
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Feb. 15, 2012, is named Q114416.txt and is 71,196 bytes in size.


TECHNICAL FIELD
The present invention relates to flagellin mutants having an enhanced activity of stimulating the toll-like receptor-5 (hereinafter referred to as “TLR5”), and more particularly to flagellin mutants, prepared by point-mutating some of the amino acids of a TRL5 agonist flagellin so as to enhance the TRL5-stimulating activity of the flagellin.
BACKGROUND ART
Flagella are important structural elements determining the motility of bacteria and are generally composed of a hook, a basal body and a filament. It is known that flagella contribute to the swimming or swarming motility, the taxis of bacteria, the adhesion of pathogenic microorganisms to host cells, and formation of biofilms. A subunit protein forming the flagellar filament is referred as flagellin, and flagellins are regularly assembled to form the filament. Hayashi et al. reported that TLR5 expressed on mammalian cells recognize the flagellin of gram-negative and gram-positive bacteria to activate NF-κB (Hayashi F, Smith K D, Ozinsky A, Hawn T R, Yi E C, Goodlett D R, Eng J K, Akira S, Underhill D M, Aderem A: Nature 410:1099-1103, 2001).
Toll-like receptors (TLRs) are typical “Pattern Recognition Receptors (PRRs)”, which recognize “Pathogen Associated Molecular Patterns (PAMPs)” present in pathogens and are found not only in mammals, but also on the surfaces of insects and plant cells. Thirteen kinds of TLRs have been found to date, and studies on agonists of TLRs have been actively conducted (Akira S, Uematsu S, Takeuchi O: Cell 124 (4): 783-801, 2006).
PRRs such as TLRs are distributed on the surface or in the cytoplasm of host cells, induce innate immune responses after being stimulate with various PAMPs, and furthermore, regulate adaptive immune responses. Thus, TLR agonists can serve targets suitable for the development of various immune regulators, particularly vaccine adjuvants.
As used herein, the term “vaccine adjuvants” refers to substances which can enhance, prolong or accelerate Ag-specific immune responses induced by vaccine antigens, when they are co-administered with vaccines. Vaccine adjuvants approved for use in the human body include aluminum phosphate, aluminum hydroxide, and squalene emulsion. Vaccine adjuvants must satisfy at least one of the following five requirements: 1) regulation of expression of co-stimulatory molecules on the surface of antigen-presenting cells, induction of antigen-specific T-lymphocyte responses, or immunomodulation such as the modulation of cytokine secretion; 2) antigen presentation; 3) induction of CD8+ cytotoxic T lymphocyte responses; 4) targeting; and 5) depot generation.
Ideal vaccine adjuvants: 1) must be safe; 2) must be biodegraded in vivo; 3) must show potent protective or therapeutic immune responses compared to when antigens are administered alone; 4) must be chemically or biologically verified substances; 5) preferably act at concentrations lower than antigens; and 6) must have a long half life, such that they can be readily applied commercially or clinically.
Substances, which are currently used as vaccine adjuvants or considered for use as vaccine adjuvants, include: 1) mineral salts such as aluminum hydroxide gel; 2) surfactant substances; 3) bacteria-derived substances; 4) cytokines or hormones; 5) polyanions; 6) polyacryl; 7) carriers; 8) living vectors comprising virus; and 9) vehicles, such as mineral oil liposomes. Among them, protein-derived vaccine adjuvants, which are currently actively studied and receive great attention, include Vibrio cholerae-derived cholera toxin (CT) and Escherichia coli-derived heat-labile toxin (LT). It was reported that these vaccine adjuvants induce the production of antigen-specific antibodies in mucosal areas and sera and induce the expression of B7-2 on the surface of antigen-presenting cells (APCs) to stimulate the co-stimulatory signaling of CD4+ helper T cells. However, these adjuvants are exotoxins having high enterotoxicity, and thus studies focused on reducing the toxicity thereof and increasing the adjuvanticity thereof, are in progress.
As disclosed in PCT International Patent Publication No. WO 2005/070455, the present inventors constructed a transposon mutant library in order to screen adhesion and invasion factors of Vibrio vulnificus, and analyzed the Tn-flanking regions in three clones, which lost their adhesion to host cells and their motility, and, as a result, the present inventors identified two flagella operons consisting of 56 genes. In this analysis process, it was found that V. vulnificus has a total of six flagellin genes (flaA, flaB, flaF, flaC, flaD and flaE), and among them, the flaB gene is the major component of the flagellins. The present inventors studied the possibility that the flagellin recombinant protein (FlaB), the component of the polar flagellin of V. vulnificus, can be applied as a component vaccine against V. vulnificus, and, as a result, the present inventors found that the flagellin recombinant protein (FlaB), besides high antigenicity, also has a strong vaccine adjuvant effect. To elucidate this finding, the present inventors conducted further studies. As a result, it was demonstrated that, when a vaccine antigen tetanus toxoid was administered to the nasal cavity of test animals together with flagellin, the flagellin amplified the effect of the vaccine by transmitting a signal to the TLR5 of host cells to activate the immune system, and when a lethal dose of tetanus toxin was challenged to mice immunized by administering tetanus toxoid and flagellin, the flagellin induced complete defense immunity to the toxin, suggesting that the flagellin had an excellent mucosal vaccine adjuvant effect (Lee S E, Kim S Y, Jeong B C, Kim Y R, Bae S J, Ahn O S, Lee J J, Song H C, Kim J M, Choy H E, Chung S S, Kweon M N, Rhee J H.: Infect. Immun. 74: 694-702, 2006).
Among various TLR agonists, flagellin stimulating TLR5 is a protein component, unlike other TLR agonists [CpG-DNA, MLP (mycoplasmal lipopeptide)]. Thus, it is possible to synthesize recombinant flagellin proteins, the quality of which can be continuously controlled, and in addition, it is possible to construct various recombinant fusion proteins having an enhanced activity of stimulating TLR5.
According to the results of studies on the three-dimensional structure of flagellin, polar amino acid residues and charged amino acid residues in a flagellin monomer react with those in another monomer, so that the axial interaction between the monomers occurs to form a polymer, thus forming the characteristic filament structure of flagellin (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 F A, 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 the study of Smith et al., it was reported that TLR5 does not recognize a filament-type flagellin polymer, but recognizes flagellin monomers (Smith K D, Andersen-Nissen E, Hayashi F, Strobe K, Bergman M A, Barrett S L, Cookson B T, 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 (vaccines for infectious diseases, autoimmune diseases, allergic diseases and cancers), which are currently used in clinical applications, is that these vaccines lack effective vaccine adjuvants, which can specifically amplify relevant responses. Thus, there is a strong need to develop safer and more effective vaccine adjuvants.
DISCLOSURE
Technical Problem
Accordingly, the present inventor have prepared recombinant flagellin mutants, which can suppress polar-charge reactions that are involved in the axial interaction between flagellin monomers, of the flagellin gene flaB of V. vulnificus, by changing amino acid residues anticipated to be involved in the axial interaction, and have found that the prepared flagellin mutants have significantly enhanced TLR5-stimulating activity compared to that of prior (? wild type?) flagellin proteins, thereby completing the present invention.
Therefore, it is an object of the present invention to provide flagellin mutants having an enhanced activity of stimulating TLR5.
It is another object of the present invention to provide a vaccine adjuvant containing at least one of said flagellin mutants as an active ingredient.
Technical Solution
To achieve the above objects, the present invention provides flagellin mutants for suppressing the interaction between flagellin monomers in the TLR5 agonist flagellin.
Also, the present invention provides a vaccine adjuvant containing at least one of said flagellin mutants as an active ingredient.
Advantageous Effects
In the present invention, more improved flagellin vaccine adjuvants were developed by providing flagellin mutants having enhanced TLR-stimulating activity compared to that of a prior flagellin, which was found to show a potent mucosal vaccine adjuvant effect by stimulating TLR5 as disclosed in PCT International Patent Publication No. WO 2005/070455. The flagellin mutants according to the present invention will be applied for the treatment of infectious diseases, autoimmune diseases, allergic diseases and the like, and furthermore, in anticancer therapy, and will provide an important ring connecting basic research with bedside clinical studies. Thus, the application of the inventive flagellin mutants to a variety of vaccine formulations can create very high added-value.



DESCRIPTION OF DRAWINGS
FIG. 1 shows the comparison of amino acid homology between the Salmonella flagellin St-FliC (SEQ ID NO: 45), the E. coli flagellin Ec-FliC (SEQ ID NO: 46) and the V. vulnificus flagellin Vv-FlaB (SEQ ID NO: 47), in which the domain of the flagellin protein of each bacterial strain is indicated as a box (Samatey F A, et al., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7).
FIG. 2 is a schematic diagram showing a strategy inducing site-directed mutations in of the V. vulnificus flagellin gene flaB in the construction of the flagellin mutant according to the present invention (Samatey F A, et al., Structure of the bacterial flagellar protofilament and implications for a switch for supercoiling. Nature. 2001 410(6826):331-7).
FIGS. 3 and 4 show the effect of the inventive recombinant flagellin mutant on flagellin polymerization in an aqueous solution.
FIGS. 5 and 6 are graphic diagrams showing the measurement results of TLR-stimulating activity of the flagellin mutant according to the present invention.



BEST MODE
Hereinafter, the present invention will be described in further detail.
The present invention relates to flagellin mutants, prepared by point-mutating some of the amino acids of a TLR5 agonist flagellin, such that flagellin mutation suppress the multimerization of flagellin monomers, thus showing an enhanced activity of stimulating TLR5.
The present invention relates to: a flagellin mutant (D70A) of SEQ ID NO: 2, prepared by site-directed mutagenesis of aspartic acid (D70) to alanine (A) at position 70 of the wild-type V. vulnificus flagellin FlaB analyzed to be involved in the axial interaction between flagellin monomers in the TLR5 agonist flagellin; a flagellin mutant (R93A) of SEQ ID NO: 4, prepared by site-directed mutagenesis of arginine (R93) to alanine (A) at position 93; a flagellin mutant (L97W) of SEQ ID NO: 6, prepared by site-directed mutagenesis of leucine (L97) to tryptophan (W) at position 97; a flagellin mutant (N101A) of SEQ ID NO: 8, prepared by site-directed mutagenesis of asparagine (N101) to alanine (A) at position 101; a flagellin mutant (S103W) of SEQ ID NO: 10, prepared by site-directed mutagenesis of serine (S103) to tryptophan (W) at position 103; a flagellin mutant (E108A) of SEQ ID NO: 12, prepared by site-directed mutagenesis of glutamic acid (E108) to alanine (A) at position 108; a flagellin mutant (N135D) of SEQ ID NO: 14, prepared by site-directed mutagenesis of asparagine (N135) to aspartic acid (D) at position 135; a flagellin mutant (A151W) of SEQ ID NO: 16, prepared by site-directed mutagenesis of alanine (A151) to tryptophan (W) at position 151; a flagellin mutant (N153W) of SEQ ID NO: 18, prepared by site-directed mutagenesis of aspargine (N153) to tryptophan (W) at position 153; and a flagellin mutant (V291W) of SEQ ID NO: 20, prepared by site-directed mutagenesis of valine (V291) to tryptophan (W) at position 291. Also, among the flagellin mutants, flagellin mutants having two or more amino acid substitutions fall within the scope of the present invention, and examples thereof may include a flagellin mutant (D70A/N135D) of SEQ ID NO: 44, prepared by site-directed mutagenesis of aspartic acid (D70) to alanine (A) at position 70 of the wild-type V. vulnificus flagellin FlaB, and asparagine (N135) to aspartic acid (D) at position 135, but the scope of the present invention is not limited thereto.
As used herein, the term “flagellins” refers to single molecules constituting flagella that determine the mobility of bacteria.
The flagellin mutants, provided according to the present invention, have a significantly enhanced activity of stimulating TLR5, and this phenomenon is because the flagellin mutants significantly reduce flagellin multimerization compared to prior flagellin mutants, thus suppressing flagellin polymerization.
Accordingly, the flagellin mutants having significantly enhanced TLR5-stimulating activity, prepared according to the present invention, can provide more potent TLR agonists as substitutes for the prior flagellins, thus providing vaccine adjuvants having increased efficiency compared to that of the prior flagellins.
A process of demonstrating the enhanced TLR5-stimulating activity of the flagellin mutants according to the present invention comprises the steps of:
1) preparing flagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44;
2) confirming that the flagellin mutants suppress flagellin polymerization in an aqueous solution; and
3) confirming the TLR5 activation of each of recombinant flagellin mutants induced in E. coli. 
[Mode for Invention]
Hereinafter, the present invention will be described in further detail with reference to examples and test examples. It is to be understood, however, that these examples and test examples are illustrative only, and the scope of the present invention is not limited thereto.
The characteristics of strains and plasmids, used in the present invention, are summarized in Table 1 below. A method of preparing each of flagellin mutants, and the detailed characteristics of each flagellin mutant, are shown in examples and test examples.










TABLE 1







Characteristics
Sources


















Strains or




plasmids


Escherichia


coli


DH5α
F recA1; restriction negative
Our




laboratory


ER2566
F-λ-fhuA2[lon] ompT lacZ::T7 gene1 gal
New



sulA11 (mcrC-mrr)114:IS10R(mcr-
England



73::miniTn10-TetS)2 R(zgb-
Biolab



210::Tn10)(TetS) endA1 [dcm]


BL21(DE3)
F-ompT hsdSB(rB-mB-) gal dcm (DE3)
Novagen


Plasmids


pCR2.1 TOPO
PCR TA cloning vector, AmpR, KmR
Invitrogen


pTYB12
IMPACT (Intein Mediated Purification with
New



an Affinity Chitin-binding Tag) expression
England



vector, AmpR
Biolab









<Culture and Storage of Each Strain>
E. coli strains, used in the following examples and test examples, were cultured in Luria Bertani media (Difco Co.). The cultured strains were stored in 30% glycerol in a freezer at −80° C.
EXAMPLE 1
Preparation of Recombinant Flagellin Mutants by Site-directed Mutagenesis
According to the previously reported data of analysis for the three-dimensional structure of Salmonella flagellin (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 F A, 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 the bioinformatics analysis conducted by the present inventors, amino acid residues required for the axial interaction between V. vulnificus flagellin FlaB monomers are as follows. That is, it was analyzed that aspartic acid (D70) at position 70 of the upper subunit of each monomer, which is involved in the axial interaction, asparagine (D135) at position 135 of the upper subunit, and alanine (A151) at position 153 of the upper subunit, form multimers through polar-charge reactions with asparagines (N100) at position 100 of the lower subunit of each monomer, glutamic acid (E108) at position 108 of the lower subunit, arginine (R93) at position 93 of the lower subunit and alanine (A292) at position of the lower subunit, respectively. That is, the polar-charge reactions occur between the following amino acid residues: upper flagellin D70 and lower flagellin N100; upper flagellin N135 and lower flagellin E107; upper flagellin A151 and lower flagellin R93; and upper flagellin N153 and lower flagellin A292.
In the present invention, the following flagellin mutants were prepared by site-directed mutagenesis of the following amino acid residues analyzed to be involved in the axial interaction between flagellin subunits: a flagellin mutant (D70A) of SEQ ID NO: 2, prepared by site-directed mutagenesis of aspartic acid (D70) to alanine (A) at position 70; a flagellin mutant (R93A) of SEQ ID NO: 4, prepared by site-directed mutagenesis of arginine (R93) to alanine (A) at position 93; a flagellin mutant (N101A) of SEQ ID NO: 8, prepared by site-directed mutagenesis of asparagine (N101) to alanine (A) at position 101; a flagellin mutant (E108A) of SEQ ID NO: 12, prepared by site-directed mutagenesis of glutamic acid (E108) to alanine (A) at position 108; a flagellin mutant (N135D) of SEQ ID NO: 14, prepared by site-directed mutagenesis of asparagine (N135) to aspartic acid (D) at position 135; a flagellin mutant (A151W) of SEQ ID NO: 16, prepared by site-directed mutagenesis of alanine (A151) to tryptophan (W) at position 151; and a flagellin mutant (N153W) of SEQ ID NO: 18, prepared by site-directed mutagenesis of aspargine (N153) to tryptophan (W) at position 153. However, alanine (A292) at position 292, analyzed to be involved in the axial interaction between flagellin monomers, was excluded from the construction of mutants, because it was thought to not be changed even after it would be mutated.
In addition, the following mutants were prepared by site-directed mutagenesis of the following amino acid residues inferred to be involved in the axial interaction between flagellin monomers: a flagellin mutant (L97W) of SEQ ID NO: 6, prepared by site-directed mutagenesis of leucine (L97) to tryptophan (W) at position 97; a flagellin mutant (S103W) of SEQ ID NO: 10, prepared by site-directed mutagenesis of serine (S103) to tryptophan (W) at position 103; and a flagellin mutant (V291W) of SEQ ID NO: 20, prepared by site-directed mutagenesis of valine (V291) to tryptophan (W) at position 291.
First, in order to obtain template DNA for site-directed mutagenesis, a 1,142-bp DNA fragment containing the flaB gene ORF was amplified by PCR using a PCR primer FlaB-V5 of SEQ ID NO: 21 (5′-gcggccgcatggcagtgaatgtaaatgtaaatacaaac-3′; underlined portion: NotI restriction enzyme recognition site for cloning) and a PCR primer FlaB-V4 of SEQ ID NO: 22 (5′-cccggggcctagtagacttagcgctga-3′; underlined portion: SmaI restriction enzyme recognition site for cloning. The PCR amplification was performed using the primers FlaB-V5 and FlaB-V4 in the following conditions: initial denaturation at 95° C. for 1 min, and then 30 cycles of denaturation at 95° C. for 30 sec, annealing at 70° C. for 30 sec and extension at 72° C. for 1 min, followed by final extension at 72° C. for 10 min. The amplified flaB DNA fragment was cloned into a PCR 2.1-TOPO cloning vector (Invitrogen Co.), and the resulting vector was named “pCMM270”. The pCMM270 plasmid was used as template DNA for subsequent site-directed mutagenesis.
PCR conditions and cloned plasmids for the construction of site-directed mutants of the V. vulnificus flagellin gene flaB are summarized in Table 2 below.











TABLE 2








Name of
Name of


Mutation

PCR annealing
pCR2.1-TOPO
pTYB12


position
Primer (SEQ ID NO)
temperature
clone
clone







D70A
FlaB-SD1 (SEQ ID NO: 23)
63° C.
pCMM272
pCMM276



5′-gtacgtaacgccaacgcaggtatctcaatc-3′






FlaB-SD2 (SEQ ID NO: 24)






5′-gattgagatacctgcgttggcgttacgtac-3′





 


R93A
FlaB-SD13-2 (SEQ ID NO: 25)
63° C. 
pCMM281
pCMM291



5′-catcctacaacgtatggctgacctatctctacaatc-3′






FlaB-SD14-2 (SEQ ID NO: 26)






5′-gattgtagagataggtcagccatacgttgtaggatg-3′





 


L97W
FlaB-SD17 (SEQ ID NO: 27)
66° C. 
pCMM282
pCMM292



5′-gcgtgacctatcttggcaatccgcgaacgg-3′






FlaB-SD18 (SEQ ID NO: 28)






5′-ccgttcgcggattgccaagataggtcacgc-3′





 


N101A
FlaB-SD9-3 (SEQ ID NO: 29)
64° C. 
pCMM283
pCMM293



5′-ctacaatccgcggccggctcaaactcaaaatc-3′






FlaB-SD10-3 (SEQ ID NO: 30)






5′-gattttgagtttgagccggccgcggattgtag-3′





 


S103W
FlaB-SD15 (SEQ ID NO: 31)
63° C. 
pCMM284
pCMM294



5′-caatccgcgaacggctggaactcaaaatcag-3′






FlaB-SD16 (SEQ ID NO: 32)






5′-ctgattttgagttccagccgttcgcggattg-3′





 


E108A
FlaB-SD11 (SEQ ID NO: 33)
63° C. 
pCMM285
pCMM295



5′-caaactcaaaatcagcgcgcgtggcgattc-3′






FlaB-SD12 (SEQ ID NO: 34)






5′-gaatcgccacgcgcgctgattttgagtttg-3′





 


N135D
FlaB-SD3 (SEQ ID NO: 35)
63° C. 
pCMM273
pCMM277



5′-cgtcttttggtggtgacaagctgctaaacg-3′






FlaB-SD4 (SEQ ID NO: 36)






5′-cgtttagcagcttgtcaccaccaaaagacg-3′





 


A151W
FlaB-SD5 (SEQ ID NO: 37)
63° C. 
pCMM286
pCMM296



5′-gcaatgcaaattggttgggataacggtgaagcg-3′






FlaB-SD6 (SEQ ID NO: 38)






5′-cgcttcaccgttatcccaaccaatttgcattgc-3′





 


N153W
FlaB-SD7 (SEQ ID NO: 39)
67° C. 
pCMM287
pCMM297



5′-caaattggtgcggattggggtgaagcggtcatg-3′






FlaB-SD8 (  40)






5′-catgaccgcttcaccccaatccgcaccaatttg-3′





 


V291W
FlaB-SD19 (SEQ ID NO: 41)
67° C. 
pCMM288
pCMM298



5′-gcgcaagagtcgtgggcgattgtggatgcg-3′






FlaB-SD20 (SEQ ID NO: 42)






5′-cgcatccacaatcgcccacgactcttgcgc-3′





 


D70A/N13 
FlaB-SD1 (SEQ ID NO: 23)
63° C. 
pCMM274
pCMM278


5D
5′-gtacgtaacg ccaacgcagg tatctcaatc-3′






FlaB-SD2 (SEQ ID NO: 23)






5′-gattgagata cctgcgttgg cgttacgtac-3′









In order to construct each of the site-directed mutant flaB DNAs (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D), a mutant strand was synthesized by PCR reaction using each of the primer pairs shown in Table 2 above, pCMM270 as a PCR template, and a proofreading Pfu polymerase in accordance with the manufacturer's instruction (Stratagene Co.). Each of the PCR reactions was performed in the following conditions: initial denaturation at 95° C. for 45 sec, and then 16 cycles of denaturation at 95° C. for 45 sec, annealing for 1 min at the temperature shown in Table 2, and extension at 68° C. for min, followed by final extension at 68° C. for 10 min. Each of the amplified site-directed mutant flaB DNAs of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 was cloned into a PCR 2.1-TOPO cloning vector, and the resulting vectors were named “pCMM272”, “pCMM281”, “pCMM282”, “pCMM283”, “pCMM284”, “pCMM285”, “pCMM273”, “pCMM286”, “pCMM287” and “pCMM288” (Table 2).
To prepare a D70A/N135D dual site-directed mutant flagellin D70A/N135D of SEQ ID NO: 44, pCMM273 with flaB DNA was used as a template for PCR reaction. The mutant strand (D70A) was synthesized by PCR using a PCR primer FlaB-SD1 of SEQ ID NO: 23, a PCR primer FlaB-SD2 of SEQ ID NO: 24 and a proofreading Pfu polymerase in accordance with the manufacturer's instruction (Stratagene Co.). The PCR reaction was performed in the following condition: initial denaturation at 95° C. for 45 sec, and then 16 cycles of denaturation at 95° C. for 45 sec, annealing at 63° C. for 1 min and extension at 68° C. for 10 min, followed by final extension at 68° C. for 10 min. The amplified D70A/N135D dual site-directed mutant flaB DNA of SEQ ID NO: 43 was named “pCMM274”.
Each of the DNA fragments of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, containing the site-directed mutant flaB genes (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D), were digested with restriction enzymes NotI and SmaI, and then cloned into a pTYB12 plasmid (New England Biolabs) digested with the same restriction enzymes. The resulting plasmids were named “pCMM276”, “pCMM291”, “pCMM292”, “pCMM293”, “pCMM293”, “pCMM294”, “pCMM295”, “pCMM277”, “pCMM296”, “pCMM297”, “pCMM298” and “pCMM278”, respectively. These plasmids were electroporated into E. coli ER2566, and 0.5 mM of 5-bromo-indole-3-chloro-isopropyl-β-D-glactopyranoside (IPTG) was added thereto to induce the expression of the mutant flagellin genes. Point mutant flagellin (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) proteins were obtained from intein fusion proteins using 1,4-dithiothreitol (1,4-DTT) and a chitin bead column according to the manufacturer's instruction. Before use, endotoxin contained in the isolated point mutant proteins was removed using AffinityPakTM Detoxi GelTM Endotoxin Removing gel (Pirece). The isolated recombinant proteins were subjected to gel filtration using a Superdex 120 column (AKTA-Prime, Amersham), thus purifying the recombinant flagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44 with high purity.
Test Example 1
Production of Recombinant Flagellin Mutants and Biochemical Properties thereof in Aqueous Solution
In order to prepare recombinant flagellin mutants, each of the genes of SEQ ID NOS: SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 43, encoding the flagellin mutants, was cloned into a pTYB12 plasmid, and then transfected into E. coli ER2566. 0.3 mM IPTG (isopropyl-β-D-1-thiogalactoside) was added to the media to induce the production of recombinant flagellin mutants, and then, the resulting proteins were analyzed by SDS-polyacrylamide gel electrophoresis and native-gel electrophoresis. The analysis results are shown in FIGS. 3 and 4.
As shown in FIGS. 3 and 4, which show the results of SDS-PAGE analysis of the wild-type flagellin (FlaB) and the mutated flagellin FlaB, the point-mutated flagellin proteins (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44, and the wild-type flagellin (FlaB) protein, all showed a major band at 41.5 kDa. In the results of native-gel electrophoresis, it was observed that the wild-type FlaB protein formed a huge multimer, so that most of the protein remained in the stacking gel, and some of the FlaB protein, introduced into the resolving gel, was distributed between 66 kDa and 140 kDa. However, the point-mutated flagellin proteins (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) according to the present invention formed multimers at about 140 kDa. The above results suggest that the wild-type FlaB protein formed a huge multimer in an aqueous solution, but the point-mutated flagellin mutants (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) according to the present invention were present as multimers having a size smaller than that of the wild-type flagellin protein. Accordingly, it could be confirmed that the inventive flagellin mutants (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44 suppressed the polymerization of flagellin.
Test Example 2
TLR5-stimulating Activity of Flagellin Mutants
In order to measure the transcriptional activity of NF-κB, involved in TLR5-mediated signaling, so as to determine the TLR5-stimulating activities of the wild-type FlaB and the inventive flagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44, 293T cells were dispensed into each well of 24-well plates at a density of 1×105 cells/well and cultured overnight. Then, a reporter plasmid NF-κB-Luc (provided by professor Jeong-Mok Kim, Institute of Biomedical Science, Hanyang University College of Medicine, Korea) enabling transcriptional activity to be observed, a TLR5 gene-cloned p3xFlag-hTLR5 plasmid (provided by Dr. Steven B. Mizel, Departments of Microbiology and Immunology, Wake Forest University School of Medicine, USA) and a beta-galactosidase expression plasmid (Clontech) were simultaneously introduced into the cells using FuGENE6 (Roche). After the cells were additionally cultured for 24 hours, the cultures were replaced with fresh media, and the cells were treated with the flagellin mutants (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44, isolated in Example 1, for 18-24 hours. Then, the transcription of NF-κB in the cells was assayed by measuring the luciferase activity using a luminometer (Berthold), and the measurement results are shown in FIGS. 5 and 6. As shown in FIGS. 5 and 6, the group treated with 20 ng/ml of the wild-type FlaB protein showed an increase in TLR5-mediated NF-κB transcription activity of about 6.6 fold compared to the control group treated only with phosphate-buffered solution. However, the groups treated with 20 ng/ml of each of the recombinant flagellin FlaB mutants (D70A, R93A, L97W, N101A, S103W, E108A, N135D, A151W, N153W, V291W and D70A/N135D) showed increases in TLR5-mediated NF-κB transcription activity of about 24.6 fold, 14 fold, 25 fold, 22.6 fold, 23.7 fold, 21 fold, 21.7 fold, 22.5 fold, 24.5 fold, 16.6 fold and 14.9 fold, respectively, compared to the control group treated only with phosphate-buffered solution.
[Sequence List Text]
SEQ ID NOS: 1 and 2 are the gene and amino acid sequences of a flagellin mutant (D70A), prepared by site-directed mutagenesis of aspartic acid (D70) to alanine (A) at position 70 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 3 and 4 are the gene and amino acid sequences of a flagellin mutant (R93A), prepared by site-directed mutagenesis of arginine (R93) to alanine (A) at position 93 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 5 and 6 are the gene and amino acid sequences of a flagellin mutant (L97W), prepared by site-directed mutagenesis of leucine (L97) to tryptophan (W) at position 97 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 7 and 8 are the gene and amino acid sequences of a flagellin mutant (N101A), prepared by site-directed mutagenesis of asparagine (N101) to alanine (A) at position 101 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 9 and 10 are the gene and amino acid sequences of a flagellin mutant (S103W), prepared by site-directed mutagenesis of serine (S103) to tryptophan (W) at position 103 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 11 and 12 are the gene and amino acid sequences of a flagellin mutant (E108A), prepared by site-directed mutagenesis of glutamic acid (E108) to alanine (A) at position 108 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 13 and 14 are the gene and amino acid sequences of a flagellin mutant (N135D), prepared by site-directed mutagenesis of asparagine (N135) to aspartic acid (D) at position 135 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 15 and 16 are the gene and amino acid sequences of a flagellin mutant (A151W), prepared by site-directed mutagenesis of alanine (A151) to tryptophan (W) at position 151 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 17 and 18 are the gene and amino acid sequences of a flagellin mutant (N153W), prepared by site-directed mutagenesis of asparagine (N153) to tryptophan (W) at position 153 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 19 and 20 are the gene and amino acid sequences of a flagellin mutant (V291W), prepared by site-directed mutagenesis of valine (V291) to tryptophan (W) at position 291 of the V. vulnificus flagellin FlaB.
SEQ ID NOS: 21 to 42 are primer sequences used in the flagellin mutants of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 44.
SEQ ID NOS: 43 and 44 are the gene and amino acid sequences of a flagellin mutant (D70A/N135D), prepared by site-directed mutagenesis of aspartic acid (D70) to alanine (A) at position 70 of the V. vulnificus flagellin FlaB, and asparagine (N135) to aspartic acid (D) at position 135 of the V. vulnificus flagellin FlaB.

## Claims

1. An isolated Vibrio vulnificus flagellin mutant having enhanced toll-like receptor (TLR-5)-stimulating activity, wherein the mutant is the flagellin mutant (E108A) of SEQ ID NO: 12, prepared by site-directed mutagenesis of glutamic acid (E108) to alanine (A) at position 108 of the V. vulnificus flagellin FlaB.

2. A vaccine adjuvant containing, as an active ingredient, the isolated flagellin mutant of SEQ ID NO: 12.

3. A method for stimulating the activity of a toll-like receptor-5 (TLR5), comprising contacting a host cell with the isolated flagellen mutant of SEQ ID NO: 12, thereby stimulating TLR5 activity.

4. The method of claim 3, wherein the contacting comprises introducing the flagellin mutant into the host cell.

5. A method for stimulating the activity of a toll-like receptor-5 (TLR5) in a subject, comprising administering a composition comprising the isolated flagellin mutant of SEQ ID NO: 12 to the subject.