Novel Recombinant Protein Antigen Of Orientia Tsutsugamushi And Vaccine Composition Using The Same

Abstract

The present invention discloses a novel recombinant protein antigen and a vaccine composition using the same, in which the novel recombinant protein antigen is derived from the conserved sequence of a TSA56 antigen and can be useful in the diagnosis of infection with tsutsugamushi and as a vaccine for tsutsugamushi.

Claims

1. A recombinant protein antigen of Orientia tsutsugamushi, comprising any one amino acid sequence of SEQ ID NOS: 1 to 23, in which seven conserved block sequences of C1 to C7 are connected in order in each of 17 genotypes of FIGS. 3 to 8, 5 genogroups of FIGS. 9 and 10 and 1 representative sequence of FIG. 11.

2. A recombinant protein antigen of Orientia tsutsugamushi, comprising a sequence common to an amino acid sequence of SEQ ID NO: 23, in which seven conserved block sequences of C1 to C7 are connected in order in a representative sequence of FIG. 11, and having a sequence homology of 68.8% or more.

3. A gene encoding the recombinant protein antigen of claim 1.

4. A method of preparing the recombinant protein antigen of claim 1, the method comprising: (i) preparing an expression vector containing the gene of claim 3, (ii) transforming the expression vector into a host cell, (iii) culturing the transformed host cell, and (iv) isolating and purifying the recombinant protein antigen of claim 1 from a resultant culture broth.

5. A vaccine composition for Orientia tsutsugamushi, containing the recombinant protein antigen of claim 1 as an active ingredient.

6. The vaccine composition of claim 5, wherein the composition contains a pharmaceutically acceptable carrier.

7. The vaccine composition of claim 6, wherein the pharmaceutically acceptable carrier includes at least one selected from the group consisting of a diluent, an excipient, a stabilizer and a preservative.

8. The vaccine composition of claim 5, wherein the composition further contains an antigen adjuvant.

9. The vaccine composition of claim 9, wherein the antigen adjuvant is a gel-type aluminum salt.

10. A composition for detecting an Orientia tsutsugamushi-specific antibody, the composition comprising the recombinant protein antigen of claim 1.

11. The composition of claim 10, wherein the tsutsugamushi-specific antibody is a TSA56 antigen-specific antibody.

12. The composition of claim 10, wherein the composition is used in contact with serum as a biosample.

13. A kit for detecting an Orientia tsutsugamushi-specific antibody, the kit comprising the recombinant protein antigen of claim 1.

14. The kit of claim 13, wherein the kit further comprises a detection agent for detecting a complex of a tsutsugamushi-specific antibody in a biosample and the recombinant protein antigen of claim 1 specifically binding to the specific antibody.

15. The kit of claim 13, wherein the detection agent is a secondary antibody conjugated with a label or an enzyme.

16. The kit of claim 13, wherein the kit further comprises at least one selected from among a carrier, a washing buffer, a diluted sample solution, an enzyme substrate, a reaction stop solution and instructions to teach a method of use thereof.

17. A method of detecting an Orientia tsutsugamushi-specific antibody in a biosample, the method comprising: (a) reacting a biosample with a composition for detecting a tsutsugamushi-specific antibody including the recombinant protein antigen of claim 1 to afford a complex of the tsutsugamushi-specific antibody in the biosample and the recombinant protein antigen of claim 1 specifically binding to the specific antibody, and (b) detecting the complex.

18. The method of claim 17, wherein the tsutsugamushi-specific antibody is a TSA56 antigen-specific antibody.

19. The method of claim 17, wherein the biosample is serum.

20. The method of claim 17, wherein the detecting the complex in step (b) includes reacting a secondary antibody conjugated with a label or an enzyme capable of providing a detection signal with the complex and measuring an extent of reaction with the complex.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0086] FIG. 1 shows the results of phylogenetic tree analysis of 206 tsa56 genes collected and the genotype classification results depending thereon;

[0087] FIG. 2 shows seven conserved blocks (C1 to C7) identified through amino acid sequence analysis of 206 TSA56 proteins;

[0088] FIGS. 3, 4, 5, 6, 7, and 8 show respective amino acid sequences of seven conserved blocks derived from each of 17 genotypes;

[0089] FIGS. 9 and 10 show respective amino acid sequences of seven conserved blocks derived from each of 5 genogroups;

[0090] FIG. 11 shows respective amino acid sequences of seven conserved blocks of a representative sequence;

[0091] FIG. 12 shows the amino acid sequence of cTSA56_Boryong used as a vaccine antigen;

[0092] FIG. 13 shows the Coomassie blue staining results of a purified cTSA56_Boryong recombinant protein and a TSA56_Boryong recombinant protein and the western blotting results using the antiserum of a healthy person and a tsutsugamushi-infected patient;

[0093] FIG. 14 shows the results of IgG1 and IgG2c antibody titers specific to a TSA56_Boryong protein induced after immunization of mice with the recombinant protein three times; and

[0094] FIGS. 15 and 16 show the results of survival rate analysis of immunized mice after tsutsugamushi infection.

DETAILED DESCRIPTION

[0095] A better understanding of the present invention will be given through the following examples. However, these examples are not to be construed as limiting the scope of the present invention.

<Example 1> Identification of Conserved Blocks of TSA56 and Preparation of cTSA56 Recombinant Protein Antigen

[0096] 1030 tsa56 gene sequences published before Dec. 31, 2015 were collected from the base sequence database of the National Center for Biotechnology Information. Of these, 206 gene sequences, among 324 sequences including 85% or more of the site encoding the entire TSA56 protein, were selected (in the tsa56 gene sequences listed in the National Center for Bioinformatics, sequences containing the entire ORF and only a part thereof are present, and thus, among these gene sequences, genes including a site encoding an amino acid sequence having 85% or more correspondence with the entire TSA protein amino acid sequence of the corresponding strain are selected). The selected 206 gene sequences are shown in [Table 1] below (the following [Table 1] sets forth 206 gene sequences identified by Sequence ID, in which 206 genes are finally selected because gene sequences that are the same as each other are present in a total of 324 gene sequences).

TABLE-US-00001 TABLE 1 Selected 206 gene sequences ue Strain name Matsuzawa yeo-joo CBNU-20 Matsuzawa Mori Okazaki Kamimoto 402I Hirahata R9 Hirahata R9 R39 R39 CMM1 KNP1 KNP2 TD-17 5-05 Sato SH234 je-cheon TW261 UT213 UT336 UT169 UT395 UT221 FPW2031 UT219 Taitung-7 TP0607a KHC0606a MZ01-1 MZ01-2 OI06-2 OI07 Inha-Kp241680-1 Inha-Kp241680-2 Inha-Kp1186344 Inha-Kp155080 O2 O3 Karp Hualien-12 UT176 UT177 KHC0609c HL05 KM16-1 04QNg_VN LA-1 T0224198_KH TW73R KM0607h TW45R KM01 KM05 KM11-1 KM18 KM19-1 TW201 TWyu81 PH01 PH02 PH04 Taitung-6 Taitung-3 UT76 T1118373_KH UT167 UT316 UT332 UT150 T1121050_KH UT418 Lc-1 KM0605a KM07 KM09 KM13 TY0610a TP0708a HL02-1 HL04 KM11-2 KM12 KM17-1 KM17-2 OI05-2 OI08 S0902151_KH S0915092_KH S1007358_KH S1020210_KH T0727121_KH T0122244_KH T0925265Lu_KH T0928182Li_KH T1008243Sp_KH U0215166_KH 01QNg_VN 05QNg_VN 05QN_VN 07QN_VN 06QN_VN 11QY87_VN 15QY87_VN 13QY87_VN 33BVKH_VN Jin/2012 Jin/2013 HSB1 HSB2 FAR1 UAP7 UAP1 UAP2 UAP4 young-worl pa-joo TW441 TW141 TW121 CH0711a SH245 Nishino TPC0701a KOR 03-S47 KOR 03-S45 Inha-B201883-1 Inha-B201883-2 Inha-B201883-3 Inha-B1009202-1 Inha-B1009202-2 Inha-B1341342-1 Inha-B1341342-2 Inha-B201883-4 Inha-B203537-1 Inha-B203537-2 Inha-B697253-1 CBNU-15 CBNU-24 CBNU-25 CBNU-38 CBNU-42 CBNU-44 CBNU-3 CBNU-4 CBNU-5 CBNU-8 CBNU-35 CBNU-7 CBNU-9 CBNU-10 CBNU-21 CBNU-40 CBNU-43 CBNU-12 CBNU-18 CBNU-26 CBNU-27 CBNU-29 CBNU-31 CBNU-33 CBNU-36 CBNU-37 CBNU-39 CBNU-16 CBNU-22 CBNU-32 CBNU-41 CBNU-45 B119 Kuroki TW461 Taitung-2 Hualien-2 Taitung-5 Hualien-10 KM0606a TT05 0Hualien-7 0Hualien-9 0Hualien-8 0Hualien-13 0KHC0707a 0MZ02 0T1015340Sp_KH 0UT196 0UT144 0UT125 0FPW2016 0FPW2049 0UT329 0TPC0707a 0KHC0706a 1TT01-2 1TT02-1 1TT02-2 1KM14 1KM16-2 1OI02 1S1006257_KH 1S1213056_KH 1S1227072_KH 1T1015340_KH 2T1019165_KH 2T1116018_KH 2T1116116_KH 261QN_VN 2Liu/2011 2Mu/2013 2Oishi 2Taguchi 2CBNU-2 2CBNU-6 2CBNU-17 2Kanda 32Kawasaki 2CBNU-11 2CBNU-34 2PT0712b 3CBNU-1 3CBNU-13 3CBNU-14 3CBNU-19 3CBNU-28 3CBNU-30 3Hualien-1 3NT0711a 3TT0711a 3Sxh951 4Ikeda 4LP-1 4Iwataki-1 4HSB3 4FAR2 4Kaisei 4KOR 03-S17 4405S 4UAP6 4Inha-W351003-2 4SH216 94Yonchon 4Taiwan CDC Gilliam 4Neimeng-65 4KM02 4S0617100_KH 4TT0705a 4TT03-1 5KM15-1 5KM15-2 5Taitung-4 5Hualien-11 5UT302 5KM04 5KM08 5TT06-1 5KM20 5KM21-3 5PH03 5PH05 5TT06-6 6KM10-1 6KM10-2 6KM19-2 6OI05-1 645QN_VN 6TW521 6TW381 6Hualien-14 6KHC0704a 6HL03-1 6NT0707a 6HL01 6HL02-2 7KM21-2 7OI10 7OI011 702QNg_VN 67TA763 7Omagari 37Kato 7LF-1 7Hualien-3 7Hualien-5 7Hualien-6 8HC0605a 8KM0607b 8HL03-2 8KM03 8TT03-2 8TT04 8OI01-2 8OI03-1 8OI03-2 8OI04 99TA678 9TW62R 9TW44R 9KM06 9KM21-1 9TWyu11 9Hualien-4 9KHC0606b 9FPW1038 9TT01-1 9OI06-1 9OI09 9S0522327_KH 9T1009163Lu_KH 9S0923259_KH 9S0923262_KH 9T0928133_KH 9T1027262_KH 90TA716 0N.A. 080-Yamagata-201 30Shimokoshi 0R08-m133 0LX-1 0Fuji

[0097] The selected 206 tsa56 genes were converted to amino acid sequences, followed by multiple sequence alignment using an MAFFT algorithm program (Multiple Alignment using Fast Fourier Transform; Molecular Biology and Evolution, 2013, 30, 772-780). The protein-coding sections, which are contained in common in the 206 genes, were converted to base sequences and used to construct the phylogenetic tree.

[0098] In order to construct the phylogenetic tree, base sequences were corrected with an optimized substitution matrix using a jModelTest 2.0 program (Darriba D, Taboada G L, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9(8), 772; Guindon S and Gascuel O (2003). A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood, Systematic Biology 52: 696-704). The gene phylogenetic tree was constructed through a SeaView 4.5.1 program based on, as a kind of Maximum Likelihood, RaxML (Randomized Axelerated Maximum Likelihood, BIOINFORMATICS APPLICATIONS NOTE Vol. 22 no. 21 2006, pages 2688-690), and these sequences were classified into 17 genotypes with a statistically significant difference (support value=0.9) through a Shimodaira-Hasegawa-like (SH-like) test (Molecular Biology and Evolution, 2010, 27, 221-224), which were then further classified into 5 genogroups based on the distances therebetween in the phylogenetic tree [FIG. 1]. In order to define the sections in which amino acid sequences of the 206 genes were conserved, the arithmetic average value of amino acid mutations of genes for each section sequence comprising 10 consecutive amino acids in individual positions of the amino acid sequences (e.g. the first amino acid forms a section with the next 9 amino acids, and the second amino acid forms a section with the next 9 amino acids) was calculated, thus obtaining the overall average value of 3.3 amino acid mutations/site, and the sections in which the number of amino acid mutations was kept below the above average value were determined among sections comprising 10 consecutive amino acids in individual positions of the amino acid sequences. Next, using a Gblocks program (Systematic biology, 2007, 56, 564-577), conserved sites having a small difference in consecutive amino acid sequences among genes and having high consistency were determined. Using these two methods, seven conserved blocks having a relatively small difference in the genes were determined [FIG. 2]. The amino acid sequences of the conserved blocks of 17 genotypes and 5 genogroups derived from the main amino acid conserved sequences (major consensus sequences) of 206 genes and a representative sequence (universal conserved sequence) representing the entire sequence are summarized in [FIG. 3] to [FIG. 11].

[0099] The results of comparison of sequence homology of the representative sequence (the sequence in which C1 to C7 are connected in order) and the remaining 22 sequences are shown in [Table 2] below.

TABLE-US-00002 TABLE 2 Results of comparison of sequence homology of representative sequence and remaining sequences Sequence homology with Genotype representative sequence (%) Karp_C con 94.5 Karp_B con 91.9 Karp_A con 92.3 Saitama con 85.6 Boryong con 88.2 JG_C con 92.3 Kawasaki con 89.7 JG_B con 89.3 JG_A con 92.3 Gilliam con 92.3 TD con 87.1 TA763_B con 88.9 TA763_A con 91.9 Kato_B con 91.9 Kato_A con 90 Shimokoshi con 72.7 TA686 con 87.5 Karp group con 94.5 Gilliam group con 94.1 TA763 group con 92.6 Kato group con 93.4 Shimokoshi group con 76.4 Universal TSA56 con 100

[0100] In order to evaluate the likelihood of use of the recombinant protein antigens comprising the amino acid sequences of a total of 23 conserved blocks (protein antigens comprising conserved block sequences connected in order) for diagnosis and as a vaccine, the protein of the sequence resulting from connecting seven conserved block sequences of the Boryong genotype as a typical example, as shown in [FIG. 4] (cTSA56_Boryong) and the protein of the representative sequence (universal conserved TSA56; ucTSA56) were used for experiments. The sequences of C1 to C7 of cTSA56_Boryong were obtained by extracting the sequences of amino acids having the highest frequency from amino acid sequences corresponding to the seven conserved sites of 17 genes identified as Boryong genotype among 206 genes. The sequence of cTSA56_Boryong is shown in [FIG. 12] (and SEQ ID NO: 24), and the amino acid sequence of cTSA56_Boryong shown in [FIG. 12] resulted from removing the amino acid sequence of LSLTTGLPFGGTLAAGMTIA from the Boryong genotype conserved block 1 (C1) amino acid sequence of [FIG. 4], and this amino acid sequence was excluded because it was predicted to constitute a transmembrane motif and also because it was confirmed to inhibit the production of the recombinant protein, thereby preparing a recombinant antigen protein. ucTSA56 is a sequence composed of amino acids at positions having the highest frequency among the amino acid sequences included in seven conserved blocks of the genes included in 17 genotypes, and the corresponding sequence is shown in [FIG. 11] and SEQ ID NO: 23.

[0101] The nucleic acid sequence encoding cTSA56_Boryong or ucTSA56 was chemically synthesized, amplified through PCR, cloned into a pET-28a(+) plasmid as an Escherichia coli expression vector, and introduced into Escherichia coli BL21(DE3). The recombinant Escherichia coli was cultured in a Kanamycin (50 g/mL)-containing LB broth until OD600 nm (Optical Density 600 nm) reached 0.6-0.8. Then, a 0.1 mM isopropyl -D-thiogalactoside (IPTG) was added thereto and then cultured at 16 C. for 18 hr, thus inducing the expression of a protein.

[0102] After termination of induction of expression, the bacteria were centrifuged at 1,000g for 10 min, suspended in a Ni-nitrilotriacetic acid (NTA) His-binding buffer solution (300 mM NaCl, 50 mM sodium phosphate buffer, 10 mM imidazole) containing 1 mg/mL lysozyme, and reacted at 4 C. for 30 min. Thereafter, sonication on ice was performed for 5 min, and the resulting lysate was centrifuged at 1,600g at 4 C. for 20 min.

[0103] The supernatant was collected and reacted at 4 C. for 60 min with a Ni-nitrilotriacetic acid (NTA) His-binding resin pre-equilibrated with a binding buffer solution.

[0104] The resin was washed with a binding buffer solution containing 50 mM imidazole, and the protein was then purified with a binding buffer solution containing 250 mM imidazole. Thereafter, in order to remove free imidazole, dialysis was conducted at 4 C. for 18 hr in a phosphate buffer solution (pH 7.4), and in order to remove endotoxins from the purified protein, an endotoxin removal resin was used. The removal of the endotoxin to the level of EU<0.05/dose from the purified protein solution was confirmed through LAL (limulus amebocyte lysate) assay.

[0105] The results of Coomassie blue staining after electrophoresis of the extracted protein and the results of western blotting for the reaction of the tsutsugamushi disease patient serum and the anti-His antibody are shown in [FIG. 13]. As is apparent from the results of [FIG. 13], a cTSA56_Boryong recombinant protein having a molecular weight of 30 kDa was obtained as expected, and the cTSA56_Boryong recombinant protein did not react with the healthy serum but reacted with the tsutsugamushi-infected patient (OT patient) serum and thereby was useful in the diagnosis of a tsutsugamushi-infected patient.

<Example 2> Verification of Immunogenicity and Vaccine Efficacy Using cTSA56_Boryong Recombinant Protein Antigen

[0106] 1. Recombinant Protein Antigen Immunization and Blood Collection

[0107] 20 g of the purified cTSA56_Boryong recombinant protein, 20 g of TSA56_Boryong (a control, excluding an extracellular site of Boryong genotype TSA56 protein, namely a signal peptide and a transmembrane domain), and a phosphate buffer solution as a negative control were used for immunization. Each vaccine formulation was mixed with a phosphate buffer solution so that the total amount was 80 L, and was added with 20 L of 2% Alhydrogel as an immune adjuvant so that the final volume ratio was 4:1 (antigen:immune adjuvant), followed by reaction at room temperature for 15 min.

[0108] Each vaccine formulation thus obtained was subcutaneously injected into C57BL/6 (6- to 8-week-old female) mice, and immunization was carried out a total of three times at intervals of two weeks.

[0109] Seven days after each immunization, blood was collected through orbital blood collection, and the serum was separated through centrifugation for 5 min at 2500g.

[0110] 2. ELISA for Quantifying Specific Antibody

[0111] The purified TSA56_Boryong protein was diluted to a concentration of 1 g/mL with a 0.05 M bicarbonate buffer (pH 9.5) and an immunoassay plate was coated with 100 L thereof per well at 4 C. for 18 hr.

[0112] The coated wells were washed using a washing solution (0.05% Phosphate-Buffered Saline Tween-20, PBST), and blocked using 3% BSA (bovine serum albumin) at room temperature for 2 hr.

[0113] A 100-fold-diluted solution of the mouse serum was subjected to 2-fold serial dilution, added in an amount of 100 L/well, and reacted at room temperature for 1 hr.

[0114] Washing was performed using a washing solution (0.05% Phosphate-Buffered Saline Tween-20, PBST), after which each of 10000-fold-diluted anti-mouse-IgG1 and IgG2c HRP conjugates was added in an amount of 100 L/well, and reacted at room temperature for 1 hr.

[0115] Washing was performed using a washing solution (0.05% Phosphate-Buffered Saline Tween-20, PBST), after which a color-developing agent 3,3,5,5-tetramethylbenzidine (TMB) solution was added in an amount of 100 L/well, and reacted at room temperature for 7 min.

[0116] A reaction stop solution (1N H.sub.2SO.sub.4) was added in an amount of 100 L/well, after which the absorbance was measured at 450 nm using a microplate reader.

[0117] In the cTSA56_Boryong recombinant protein and TSA56_Boryong protein immunization test groups, except for the phosphate buffer group as the negative control, the IgG1 and IgG2c antibody titers specific to TSA56_Boryong were confirmed to be increased almost identically [FIG. 14].

[0118] 3. Mouse Infection Test

[0119] As described above, each vaccine formulation was subcutaneously injected into C57BL/6 (6- to 8-weak-old female) mice (n=5/group), and immunization was carried out a total of three times at intervals of two weeks. After seven days, the immunized mice were intraperitoneally infected with Boryong or Karp genotype tsutsugamushi in an amount corresponding to 100 times the half-lethal dose (100LD50). After infection, the survival rate of the mice was observed for 30 days.

[0120] All of the mice immunized with TSA56_Boryong or cTSA56_Boryong recombinant protein survived after Boryong genotype infection. In the case of Karp genotype infection, only 40% of the mice immunized with TSA56_Boryong survived, but all of the mice immunized with cTSA56_Boryong survived. All mice died in the negative control not immunized with the antigen. Therefore, it can be confirmed that protective immunity was induced for the same genotype through cTSA56_Boryong recombinant protein immunization and also that further improved protective immunity was provided for other genotypes. The results are shown in [FIG. 15].

[0121] <cTSA56_Boryong Recombinant Protein Antigen>

[0122] The cTSA56_Boryong or TSA56_Boryong vaccine formulation of Example was subcutaneously injected into C57BL/6 (6- to 8-week-old, female) mice (n=5/group), and immunization was carried out a total of three times at intervals of two weeks. After seven days, the immunized mice were intraperitoneally infected with Boryong or Karp genotype tsutsugamushi in an amount corresponding to 100 times the half-lethal dose (100LD50). After infection, the survival rate of the mice was observed for 30 days.

[0123] All of the mice immunized with TSA56_Boryong or cTSA56_Boryong recombinant protein survived after Boryong genotype infection. In the case of Karp genotype infection, only 40% of the mice immunized with TSA56_Boryong survived, but all of the mice immunized with cTSA56_Boryong survived. All mice died in the negative control not immunized with the antigen. Therefore, it can be confirmed that protective immunity was induced for the same genotype through cTSA56_Boryong recombinant protein immunization and also that further improved protective immunity was provided for other genotypes. The results are shown in [FIG. 15].

[0124] <ucTSA56 Recombinant Protein Antigen>

[0125] A ucTSA56 recombinant protein antigen was injected into C57BL/6 (6- to 8-week-old, female) mice (n=5/group), and the effect thereof on the mouse survival rate was evaluated in the same manner as in Example above. As a control, TSA56_Boryong was used.

[0126] Consequently, all of the mice immunized with TSA56_Boryong or ucTSA56 recombinant protein survived after Boryong genotype infection. In the case of Karp genotype infection, only 20% of the mice immunized with TSA56_Boryong survived, but all of the mice immunized with ucTSA56 survived. In the case of Kato genotype infection, all of the mice immunized with TSA56_Boryong died, but 40% of the mice immunized with ucTSA56 survived. All mice died in the negative control not immunized with the antigen. Therefore, it can be confirmed that further improved protective immunity was provided for a variety of genotypes through ucTSA56 recombinant protein immunization. The results are shown in [FIG. 16].