Recombinant protein antigen of <i>Orientia tsutsugamushi </i>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 having a sequence homology of 90% or more with SEQ ID NO 23.

2. A recombinant protein antigen of claim 1, the antigen having the sequence of SEQ ID NO: 23.

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

4. A method of preparing a recombinant protein antigen of Orientia tsutsugamushi having a sequence homology of 90% or more with SEQ ID NO: 23, 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 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 8, 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 Orientia tsutsugamushi-specific antibody is a TSA56 antigen-specific antibody.

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

13. The kit of claim 12, wherein the kit further comprises a detection agent for detecting a complex of a Orientia tsutsugamushi-specific antibody in a biosample and the recombinant protein antigen of claim 1 specifically binding to the specific antibody.

14. The kit of claim 12, wherein the detection agent is a secondary antibody conjugated with a label or an enzyme.

15. The kit of claim 12, 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.

16. 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 Orientia tsutsugamushi-specific antibody including the recombinant protein antigen of claim 1 to afford a complex of the Orientia 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.

17. The method of claim 16, wherein the Orientia tsutsugamushi-specific antibody is a TSA56 antigen-specific antibody.

18. The method of claim 16, wherein the biosample is serum.

19. The method of claim 16, 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

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

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

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

(4) FIGS. 9 and 10 show respective amino acid sequences of seven conserved blocks derived from each of 5 genogroups;

(5) FIG. 11 shows respective amino acid sequences of seven conserved blocks of a representative sequence;

(6) FIG. 12 shows the amino acid sequence of cTSA56_Boryong used as a vaccine antigen;

(7) 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;

(8) 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

(9) FIGS. 15 and 16 show the results of survival rate analysis of immunized mice after tsutsugamushi infection.

DETAILED DESCRIPTION

(10) 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

(11) 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).

(12) 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.

(13) 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 GL, 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].

(14) 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 1] below.

(15) TABLE-US-00001 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

(16) 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 (SEQ ID NO: 25) 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.

(17) 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.

(18) 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.

(19) 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.

(20) 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.

(21) 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

(22) 1. Recombinant Protein Antigen Immunization and Blood Collection

(23) 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.

(24) 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.

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

(26) 2. ELISA for Quantifying Specific Antibody

(27) 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.

(28) 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.

(29) 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.

(30) 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.

(31) 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.

(32) 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.

(33) 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].

(34) 3. Mouse Infection Test

(35) 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.

(36) 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].

(37) <cTSA56_Boryong Recombinant Protein Antigen>

(38) 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.

(39) 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].

(40) <ucTSA56 Recombinant Protein Antigen>

(41) 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.

(42) 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].

(43) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. 1.52(e).