Vaccine Composition for Preventing or Treating Diseases Caused by Severe Fever with Thrombocytopenia Syndrome (SFTS) Viral Infection

Abstract

The present disclosure relates to a vaccine composition for preventing or treating infectious diseases caused by severe fever with thrombocytopenia syndrome (SFTS) virus.

Claims

1. An antigenic composition comprising, as an active ingredient, any one or more selected from the group consisting of: a first recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 287, or which is encoded by a first recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 286; a second recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 289, or which is encoded by a second recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 288; a third recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 291, or which is encoded by a third recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 290; a fourth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 293, or which is encoded by a fourth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 292; and a fifth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 295, or which is encoded by a fifth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 294.

2. The antigenic composition of claim 1, which is injected in vivo through a route selected from among intramuscular, intradermal, subcutaneous, subepidermal, transdermal and intravenous routes.

3. The antigenic composition of claim 1, which is injected into a subject through intramuscular injection.

4. The antigenic composition of claim 1, which is injected into a subject through intradermal injection.

5. The antigenic composition of claim 2, wherein the in vivo injection of the antigenic composition into a subject is followed by electroporation.

6. A vaccine comprising, as an active ingredient, any one or more selected from the group consisting of: a first recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 287, or which is encoded by a first recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 286; a second recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 289, or which is encoded by a second recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 288; a third recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 291, or which is encoded by a third recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 290; a fourth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 293, or which is encoded by a fourth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 292; and a fifth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 295, or which is encoded by a fifth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 294.

7. The vaccine of claim 6, which is injected in vivo through a route selected from among intramuscular, intradermal, subcutaneous, subepidermal, transdermal and intravenous routes.

8. The vaccine of claim 6, which is injected into a subject through intramuscular injection.

9. The vaccine of claim 6, which is injected into a subject through intradermal injection.

10. The vaccine of claim 7, wherein the in vivo injection of the vaccine into a subject is followed by electroporation.

11. The vaccine of claim 6, further comprising an adjuvant.

12. The vaccine of claim 11, wherein the adjuvant is at least one of IL-7 and IL-33.

13. An expression vector comprising any one or more recombinant DNAs selected from the group consisting of: a first recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 286; a second recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 288; a third recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 290; a fourth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 292; and a fifth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 294.

14. A transformant obtained by introducing the expression vector of claim 13 into a host cell by transformation.

15. A method for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection, the method comprising a step of administering to a subject an effective amount of the antigenic composition of claim 1.

16. A pharmaceutical composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection, the pharmaceutical composition comprising, as an active ingredient, the antigenic composition of claim 1.

17. A method for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection, the method comprising a step of administering to a subject an effective amount of the expression vector of claim 13.

18. A method for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection, the method comprising a step of administering to a subject an effective amount of the transformant of claim 14.

19. The method of claim 15, wherein the antigenic composition is injected in vivo through a route selected from among intramuscular, intradermal, subcutaneous, subepidermal, transdermal and intravenous routes.

20. The method of claim 19, wherein the in vivo injection of the antigenic composition into a subject is followed by electroporation.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0105] FIG. 1 is a well image showing the results of ELISpot analysis performed to measure T-cell response specific to SFTS virus.

[0106] FIGS. 2A to 2E are graphs showing T-cell immune responses to SFTSV vaccine candidates.

[0107] FIGS. 3A to 3C are graphs showing the results of evaluating the multifunctionality of T cells by SFTSV DNA vaccine candidates.

[0108] FIG. 4 is a graph showing the multifuctionality of T-cells induced by vaccine candidates.

[0109] FIGS. 5A to 5F are graphs showing SFTSV-specific antibody production induced by vaccines.

[0110] FIG. 6 is a graph showing the results of quantitatively evaluating neutralizing antibody titers induced by SFTSV DNA vaccines.

[0111] FIG. 7 is a graph showing the results of validating the infection inhibitory effect of an SFTSV vaccine.

[0112] FIGS. 8A to 8D are graphs showing SFTSV-specific T cell immune responses induced by SFTSV vaccine candidates in medium-sized animals.

[0113] FIG. 9 is a graph showing the results of measuring, using ELISA assay, the formation of an SFTSV-specific reactive antibody formed by a DNA vaccine.

[0114] FIG. 10 is a graph showing the results of measuring, using PRNT50 assay, the neutralizing antibody titer of an antibody induced by a DNA vaccine.

[0115] FIG. 11 is a graph showing the survival rate in SFTSV-infected medium-sized animal models.

[0116] FIGS. 12A to 12C are graphs showing the results of measuring SFTSV virus load by real-time PCR.

[0117] FIGS. 13A to 13C are graphs showing the results of counting platelets.

[0118] FIGS. 14A and 14B are graphs showing the results of measuring body weight and body temperature after SFTSV infection.

[0119] FIGS. 15A and 15B are graphs showing the results of a PRNT50 test performed to evaluate the cross-reactivity of an SFTSV neutralizing antibody formed in mice after administration of SFTSV DNA vaccines.

[0120] FIG. 16 is a graph showing the results of identifying T-cell immune responses induced by vaccines in SFTSV-infected medium-sized models.

[0121] FIG. 17 is a graph showing the results of quantitatively evaluating antibody immune responses and neutralizing antibody titers induced by vaccines in SFTSV-infected medium-sized animal models.

[0122] FIG. 18 is a graph showing the results of evaluating the preventive effects of SFTSV-preventive DNA vaccines in SFTSV-infected medium-sized animal models.

[0123] FIG. 19 depicts graphs showing the results of measuring SFTSV virus load by real-time PCR.

[0124] FIG. 20 is a graph showing the results of measuring SFTSV virus load by real-time PCR

[0125] FIG. 21 depicts graphs showing the results of counting platelets after SFTSV infection.

[0126] FIG. 22 is a graph showing the results of counting platelets after SFTSV infection.

[0127] FIG. 23 depicts graphs showing the results of counting white blood cells.

[0128] FIG. 24 is a graph showing the results of counting white blood cells.

[0129] FIG. 25 depicts graphs showing the body weights of control animals after SFTSV infection.

[0130] FIG. 26 is a graph showing the body weights of control animals after SFTSV infection.

[0131] FIG. 27 depicts graphs showing the body temperatures of control animals after SFTSV infection.

[0132] FIG. 28 is a graph showing the body temperatures of control animals after SFTSV infection.

[0133] FIG. 29 depicts graphs showing serum ALT concentrations.

[0134] FIG. 30 is a graph showing serum ALT concentrations.

[0135] FIG. 31 depicts graphs showing serum AST concentrations.

[0136] FIG. 32 is a graph showing serum AST concentrations.

[0137] FIG. 33 is a view showing an SFTSV Gc expression plasmid (pGX-SFTSV Gc_hCO, 4635 bp).

[0138] FIG. 34 is a view showing an SFTSV Gn expression plasmid (pGX-SFTSV Gn_hCO, 4626 bp).

[0139] FIG. 35 is a view showing an SFTSV NP expression plasmid (pGX-SFTSV NP_hCO, 3756 bp).

[0140] FIG. 36 is a view showing an SFTSV NS expression plasmid (pGX-SFTSV NS_hCO, 3900 bp).

[0141] FIG. 37 is a view showing an SFTSV RdRp expression plasmid (pGX-SFTSV RdRp_hCO, 9273 bp).

[0142] FIG. 38 is a view showing a mouse IL-7 expression plasmid (pGX-mIL-7_mCO, 3483 bp).

[0143] FIG. 39 is a view showing an IL-33 expression plasmid (pGX-mIL-33_mCO, 3819 bp).

BEST MODE

[0144] According to one embodiment of the present disclosure, there is provided an antigenic composition or vaccine comprising, as an active ingredient, any one or more recombinant peptides selected from the group consisting of: a first recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 287, or which is encoded by a first recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 286; a second recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 289, or which is encoded by a second recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 288; a third recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 291, or which is encoded by a third recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 290; a fourth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 293, or which is encoded by a fourth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 292: and a fifth recombinant peptide which comprises an amino acid sequence represented by SEQ ID NO: 295, or which is encoded by a fifth recombinant DNA comprising a nucleotide sequence represented by SEQ ID NO: 294.

[0145] The antigenic composition or vaccine of the present disclosure may further comprise an adjuvant. In this case, the adjuvant may be at least one of IL-7 and IL-33. preferably IL-33, but is not limited thereto.

Mode for Invention Hereinafter, the present disclosure will be described in detail with reference to examples. However, the following examples serve merely to illustrate the present disclosure, and the scope of the present disclosure is not limited by the following examples.

[0146] Synthesis of Five SFTS Virus Antigen Genes and Two Adjuvant Genes

[0147] IgE leader and Kozak sequences were inserted into the 5′ end of each target gene (SFTSV antigen or adjuvant gene), and a termination codon was inserted into the 3′ end of the target gene. Finally, restriction enzyme sequences (5′ BamHI and 3′ Not) were inserted into both ends of the gene, followed by gene synthesis.

[0148] Cloning into High-Efficiency Backbone Plasmid (pGX0001)

[0149] After completion of synthesis of each insertion gene, each insertion gene was cleaved with BamHI and NotI and inserted into a high-efficiency backbone plasmid (pGX0001) cleaved with the same restriction enzymes, thereby constructing candidate plasmids (see FIGS. 33 to 39). The results of gene synthesis and cloning were confirmed by nucleotide sequencing.

[0150] Optimization of Sequence of DNA Vaccine Highly Expressing Antigen In Vivo

[0151] As five optimal antigens. Gc (glycoprotein C). Gn (glycoprotein N). NP (nucleocapsid protein), NS (non-structural protein) and RdRp (RNA dependent RNA polymerase) were selected, and as two optimal adjuvants, IL-7 and IL-33 were selected. For the five antigens, a consensus sequence derived from 27 to 32 SFTS virus strains isolated from Korean. Chinese and Japanese was secured. The consensus sequence was designed as a universal antigen sequence with cross-immunity using the amino acid sequence of an antigen common to various SFTS virus subtypes and variants. Major human MHC class I and II epitopes present in various SFTS virus subtypes and variants were identified by using in silico immunoinformatics techniques, and antigen sequences were designed to contain these epitopes. Thereafter, based on the optimized amino acid sequences of the SFTS virus antigens, nucleotide sequences for DNA vaccines were finally derived.

[0152] Expression cassette structures according to one embodiment of the present disclosure, which are used in the following experiment, were configured to contain a high-expression promoter (plasmid backbone sequence), a Kozak sequence, an IgE leader sequence, and a poly-A signal sequence (plasmid backbone sequence). At this time, the Kozak and IgE leader sequences were inserted upstream of the target gene (SFTSV antigen or adjuvant) in order to increase the expression level of the gene in vivo. Meanwhile, in order to increase the expression level of the antigen gene in vivo, the sequences of five SFTS virus antigens (Gc, Gn, NP, NS, and RdRp) were optimized with human codons.

[0153] Generation of DNA Vaccine Lead Candidates Expressing SFTS Virus Antigen

[0154] An IgE leader sequence and a Kozak sequence were inserted into 5′ end of each target gene (SFTSV antigen or adjuvant gene), and a stop codon was inserted into the 3′ end thereof. Finally, restriction enzyme sequences (5′ BamHI and 3′ NotI) were inserted into both ends of each gene, followed by gene synthesis. After completion of synthesis of each insertion gene, each insertion gene was cleaved with BamHI and NotI and inserted into a high-efficiency backbone plasmid (pGX0001) cleaved with the same restriction enzymes, thereby constructing candidate plasmids. The results of gene synthesis and cloning were confirmed by gene sequencing.

[0155] Evaluation of Immunogenicity of SFTS Virus Antigen-Expressing DNA Vaccines Using Mouse Models

[0156] Overlapping peptide (OLP) pools for immunogenicity evaluation were created.

[0157] Specifically, in order to evaluate the immunogenicity of the five SFTS virus antigens, the sequence of each antigen was fragmented into 15-mer peptides overlapping 8 amino acids. The purity of each of the peptides was qualitatively and quantitatively analyzed using high performance liquid chromatography and electrospray mass spectrometry in the production process. Through this process, a total of 76 peptides were obtained from the Gn antigen, and 38 of these peptides were mixed together to prepare OLP1 and OLP2 (25 μg/ml each peptide) for Gn (Table 1). From the Gc antigen, a total of 76 peptides were obtained and 38 of these peptides were mixed together to prepare OLP3 and OLP4 (Table 2). From the NP antigen, a total of 34 peptides were obtained and mixed together to prepare OLP5 (Table 3). From the NS antigen, a total of 41 peptides were obtained and mixed together to prepare OLP6 (Table 4). From the RdRp antigen, a total of 58 peptides were obtained, and 29 of these peptides mixed together to prepare OLP7 and OLP8 (Table 5).

[0158] More specifically, as shown in Tables 1 to 5 below, the mixture of SEQ ID NO: 1 to SEQ ID NO: 38 is OLP1. In addition, the mixture of SEQ ID NO: 39 to SEQ ID NO: 76 is OLP2. The mixture of SEQ ID NO: 77 to SEQ ID NO: 114 is OLP3. The mixture of SEQ ID NO: 115 to SEQ ID NO: 152 is OLP4. The mixture of SEQ ID NO: 153 to SEQ ID NO: 186 is OLP5. The mixture of SEQ ID NO: 187 to SEQ ID NO: 227 is OLP6. The mixture of SEQ ID NO: 228 to SEQ ID NO: 256 is OLP7. The mixture of SEQ ID NO: 257 to SEQ ID NO: 285 is OLP8.

[0159] The OLP pools created as described above were used for evaluation of T-cell immune responses in the following experiment.

TABLE-US-00001 TABLE 1 SFTSV consensus glycoprotein Gn sequence Gn (535 aa) MMKVIWFSSLICLVIQCSGDTGPIICAGPIHSNKSANIPHLLGYSEKI CQIDRLIHVSSWLRNHSQFQGYVGQRGGRSQVSYYPAENSYSRWSGLL SPCDADWLGMLVVKKAKGSDMIVPGPSYKGKVFFERPTFDGYVGWGCG SGKSRTESGELCSSDSGTSSGLLPSDRVLWIGDVACQPMTPIPEETFL ELKSFSQSEFPDICKIDGIVFNQCEGESLPQPFDVAWMDVGHSHKIIM REHKTKWVQESSSKDFVCYKEGTGPCSESEEKTCKTSGSCRGDMQFCK VAGCEHGEEASEAKCRCSLVHKPGEVVVSYGGMRVRPKCYGFSRMMAT LEVNPPEQRIGQCTGCHLECINGGVRLITLTSELKSATVCASHFCSSA TSGKKSTEIQFHSGSLVGKTAIHVKGALVDGTEFTFEGGSCMFPDGCD AVDCTFCREFLKNPQCYPAKKWLFIIIVILLGYAGLMLLTNVLKAIGV WGSWVIAPVKLMFAIIKKLMRSVSCLMGKLMDRGRQVIHEEIGENREG NQDDVRIE* (SEQ ID NO: 298) SEQ SEQ ID NO sequence ID NO sequence  1 MMKVIWFSSLICLVI 39 SESEEKTCKTSGSCR  2 SSLICLVIQCSGDTG 40 CKTSGSCRGDMQFCK  3 IQCSGDTGPIICAGP 41 RGDMQFCKVAGCEHG  4 GPIICAGPIHSNKSA 42 KVAGCEHGEEASEAK  5 PIHSNKSANIPHLLG 43 GEEASEAKCRCSLVH  6 ANIPHLLGYSEKICQ 44 KCRCSLVHKPGEVVV  7 GYSEKICQIDRLIHV 45 HKPGEVVVSYGGMRV  8 QIDRLIHVSSWLRNH 46 VSYGGMRVRPKCYGF  9 VSSWLRNHSQFQGYV 47 VRPKCYGFSRMMATL 10 HSQFQGYVGQRGGRS 48 FSRMMATLEVNPPEQ 11 VGQRGGRSWVSYYPA 49 LEVNPPEQRIGQCTG 12 SQVSYYPAENSYSRW 50 QRIGQCTGCHLECIN 13 AENSYSRWSGLLSPC 51 GCHLECINGGVRLIT 14 WSGLLSPCDADWLGM 52 NGGVRLITLTSELKS 15 CDADWLGMLVVKKAK 53 TLTSELKSATVCASH 16 MLVVKKAKGSDMIVP 54 SATVCASHGCSSATS 17 KGSDMIVPGPSYKGK 55 HFCSSATSGKKSTEI 18 PGPSYKGKVFFERPT 56 SGKKSTEIQFHSGSL 19 KVFFERPTFDGYVGW 57 IQFHSGSLVGKTAIH 20 TFDGYVGWGCGSGKS 58 LVGKTAIHVKGALVD 21 WGCGSGKSRTESGEL 59 HVKGALVDGTEFTFE 22 SRTESGELCSSDSGT 60 DGTEFTFEGSCMFPD 23 LCSSDSGTSSGLLPS 61 EGSCMFPDGCDAVDC 24 TSSGLLPSDRVLWIG 62 DGCDAVDCTFCREFL 25 SDRVLWIGDVACQPM 63 CTFCREFLKNPQCYP 26 GDVACQPMTPIPEET 64 LKNPQCYPAKKWLFI 27 MTPIPEETFLELKSF 65 PAKKWLFIIIVILLG 28 TFLELKSFSQSEFPD 66 IIIVILLGYAGLMLL 29 FSQSEFPDICKIDGI 67 GYAGLMLLTNVLKAI 30 DICKIDGIVFNQCEG 68 LTNVLKAIGVWGSWV 31 IVFNQCEGESLPQPF 69 IGVWGSWVIAPVKLM 32 GESLPQPFDVAWMDV 70 VIAPVKLMFAIIKKL 33 FDVAWMDVGHSHKII 71 MFAIIKKLMRSVSCL 34 VGHSHKIIMREHKTK 72 LMRSVSCLMGKLMDR 35 IMREHKTKWVQESSS 73 LMGKLMDRGRQVIHE 36 KWVQESSSKDFVCYK 74 RGRQVIHEEIGENRE 37 SKDFVCYKEGTGPCS 75 EEIGENREGNQDDVR 38 KEGTGPCSESEEKTC 76 EGNQDDVRIE

TABLE-US-00002 TABLE 2 SFTSV consensus glycoprotein Gc sequence Gc (538 aa) MARPRRVRHWMYSPVILTILAIGLAEGCDEMVHADSKLVSCRQGSGNM KECVTTGRALLPAVNPGQEACLHFTAPGSPDSKCLKIKVKRINLKCKK SSSYFPVPDARSRCTSVRRCRWAGDCQSGCPPHFTSNSFSDDWAGKMD RAGLGFSGCSDGCGGAACGCFNAAPSCIFWRKWVENPHGIIWKVSPCA AWVPSAVIELTMPSGEVRTFHPMSGIPTQVFKGVSVTYLGSDMEVSGL TDLCEIEELKSKKLALAPCNQAGMGVVGKVGEIQCSSEESARTIKKDG CIWNADLVGIELRVDDAVCYSKITSVEAVANYSAIPTTIGGLRFERSH DSQGKISGSPLDITAIRGSFSVNYRGLRLSLSEITATCTGEVTNVSGC YSCMTGAKVSIKLHSSKNSTAHVRCKGDETAFSVLEGVHSYTVSLSFD HAVVDEQCQLNCGGESQVTLKGNLIFLDVPKFVDGSYMQTYHSTVPTG ANIPSPTDWLNALFGNGLSRWILGVIGVLLGGLALFFLIMSLFKLGTK QVFRSRTKLA* (SEQ ID NO: 289) SEQ SEQ ID NO sequence ID NO sequence  77 MARPRRVRHWMYSPV 115 GKVGEIQCSSEESAR  78 RHWMYSPVILTILAI 116 CSSEESARTIKKDGC  79 VILTILAIGLAEGCD 117 RTIKKDGCIWNADLV  80 IGLAEGCDEMVHADS 118 CIWNADLVGIELRVD  81 DEMVHADSKLVSCRQ 119 VGIELRVDDAVCYSK  82 SKLVSCRQGSGNMKE 120 DDAVCYSKITSVEAV  83 QGSGNMKECVTTGRA 121 KITSVEAVANYSAIP  84 ECVTTGRALLPAVNP 122 VANYSAIPTTIGGLR  85 ALLPAVNPGQEACLH 123 PTTIGGLRFERSHDS  86 PGQEACLHFTAPGSP 124 RFERSHDSQGKISGS  87 HFTAPGSPDSKCLKI 125 SQGKISGSPLDITAI  88 PDSKCLKIKVKRINL 126 SPLDITAIRGSFSVN  89 IKVKRINLKCKKSSS 127 IRGSFSVNYRGLRLS  90 LKCKKSSSYFVPDAR 128 NYRGLRLSLSEITAT  91 SYFVPDARSRCTSVR 129 SLSEITATCTGEVTN  92 RSRCTSVRRCRWAGD 130 TCTGEVTNVSGCYSC  93 RRCRWAGDCQSGCPP 131 NVSGCYSCMTGAKVS  94 DCQSGCPPHFTSNSF 132 CMTGAKVSIKLHSSK  95 PHFTSNSFSDDWAGK 133 SIKLHSSKNSTAHVR  96 FSDDWAGKMDRAGLG 134 KNSTAHVRCKGDETA  97 KMDRAGLGFSGCSDG 135 RCKGDETAFSVLEGV  98 GFSGCSDGCGGAACG 136 AFSVLEGVHSYTVSL  99 GCGGAACGCFNAAPS 137 VHSYTVSLSFDHAVV 100 GCFNAAPSCIFWRKW 138 LSFDHAVVDEQCQLN 101 SCIFWRKWVENPHGI 139 VDEQCQLNCGGHESQ 102 WVENPHGIIWKVSPC 140 NCGGHESQVTLKGNL 103 IIWKVSPCAAWVPSA 141 QVTLKGNLIFLDVPK 104 CAAWVPSAVIELTMP 142 LIFLDVPKFVDGSYM 105 AVIELTMPSGEVRTF 143 KFVDGSYMQTYHSTV 106 PSGEVRTFHPMSGIP 144 MQTYHSTVPTGANIP 107 FHPMSGIPTQVFKGV 145 VPTGANIPSPTDWLN 108 PTQVFKGVSVTYLGS 146 PSPTDWLNALGFNGL 109 VSVTYLGSDMEVSGL 147 NALFGNGLSRWILGV 110 SDMEVSGLTDLCEIE 148 LSRWILGVIGVLLGG 111 LTDLCEIEELKSKKL 149 VIGVLLGGLALFFLI 112 EELKSKKLALAPCNQ 150 GLALFFLIMSLFKLG 113 LALAPCNQAGMGVVG 151 IMSLFKLGTKQVFRS 114 QAGMGVVGKVGEIQC 152 GTKQVFRSRTKLA

TABLE-US-00003 TABLE 3 SFTSV consensus nuclear protein NP sequence NP (245 aa) MSEWSRIAVEFGEQQLNLTELEDFARELAVEGLDPALIIKKLKETGG WVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERA ETRLSITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPE MMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASK TEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAGKILGPDGVPSRAAEV AAAAYRNL* (SEQ ID NO: 291) SEQ ID NO sequence 153 MSEWSRIAVEFGEQQ 154 AVEFGQEELNLTLEL 155 QLNLTELEDFARELA 156 EDFARELAYEGLDPA 157 AYEGLDPALIIKKLK 158 ALIIKKLKETGGDDW 159 KETGGDDWVKDTKFI 160 WVKDTKFIIVFALTR 161 IIVFALTRGNKIVKA 162 RGNKIVKASGKMSNS 163 ASGKMSNSGSKRLMA 164 SGSKRLMALQEKYGL 165 ALQEKYGLVERAETR 166 LVERAETRLSITPVR 167 RLSITPVRVAQSLPT 168 RVAQSLPTWTCAAAA 169 TWTCAAAAALKEYLP 170 AALKEYLPVGPAVMN 171 PVGPAVMNLKVENYP 172 NLKVENYPPEMMCMA 173 PPEMMCMAFGSLIPT 174 AFGSLIPTAGVSEAT 175 TAGVSEATTKTLMEA 176 TTKTLMEAYSLWQDA 177 AYSLWQDAFTKTINV 178 AFTKTINVKMRGASK 179 VKMRGASKTEVYNSF 180 KTEVYNSFRDPLHAA 181 FRDPLHAAVNSVFFP 182 AVNSVFFPNDVRVKW 183 PNDVRVKWLKAKGIL 184 WLKAKGILGPDGPPS 185 LGPDGVPSRAAEVAA 186 SRAAEVAAAAYRNL

TABLE-US-00004 TABLE 4 SFTSV consensus non-structural protein NS sequence NS (293 aa) MSLSKCSNVDLKSVAMNANTVRLEPSLGEYPTLRRDLVECSCSVLTLS MVKRMGKMTNTVWLFGNPKNPLHQLEPGLEQLLDMYYKDMRCYSQREL SALRWPSGKPSVWFLQAAHMFFSIKNSWAMETGRENWRGLFHRITKGQ KYLFEGDMILDSLEAIEKRRLRGLPEILITGLSPILDVAAQIESLARL GMSLNHHLFTSSSLRKPLLDCWDFFIPIRKKKTDGSYSVLDEDDEPGV LQGYPYLMAHYLNRCPFHNLIRFDEELRTAALNTIWGRDWPAIGDLPK EV* (SEQ ID NO: 239) SEQ SEQ ID NO sequence ID NO sequence 187 MSLSKCSNVDLKSVA 208 FEGDMILDSLEAIEK 188 NVDLKSVAMNANTVR 209 DSLEAIEKRRLRLGL 189 AMNANTVRLEPSLGE 210 KRRLRLGLPEILITG 190 RLEPSLGEYPTLRRD 211 LPEILITGLSPILDV 191 EYPTLRRDLVECSCS 212 GLSPILDVALLQIES 192 DLVECSCSVLTLSMV 213 VALLQIESLARLRGM 193 SVLTLSMVKRMGKMT 214 SLARLRGMSLNHHLF 194 VKRMGKMTNTVWLFG 215 MSLNHHLFTSSSLRK 195 TNTVWLFGNPKNPLH 216 FTSSSLRKPLLDCWD 196 GNPKNPLHQLEPGLE 217 KPLLDCWDFFIPIRK 197 HQLEPGLEQLLDMYY 218 DFFIPIRKKKTDGSY 198 EQLLDMYYKDMRCYS 219 KKKTDGSYSVLDEDD 199 YKDMRCYSQRELSAL 220 YSVLDEDDEPGVLQG 200 SQRELSALRWPSGKP 221 DEPGVLQGYPYLMAH 201 LRWPSGKPSVWFLQA 222 GYPYLMAHYLNRCPF 202 PSVWFLQAAHMFFSI 223 HYLNRCPFHNLIRFD 203 AAHMFFSIKNSWAME 224 FHNLIRFDEELRTAA 204 IKNSWAMETGRENWR 225 DEELRTAALNTIWGR 205 ETGRENWRGLFHRIT 226 ALNTIWGRDWPAIGD 206 RGLFHRITKGQKYLF 227 RDWPAIGDLPKEV 207 TKGQKYLFEGDMILD

TABLE-US-00005 TABLE 5 SFTSV consensus RNA dependent RNA polymerase RdRp sequence RdRp (2085 aa) MNLEVLCGRINVENGLSLGEPGLYDQIYDRPGLPDLDVTVDATGVTVD IGAVPDSASQLGSSINAGLITIQLSEAYKINHDFTFSGLSKTTDRRLS EVFPITHDGSDGMTPDVIHTRLDGTIVVVEFSTTRSHNIGGLEAAYRT KIEKVYRDPISRRVDIMENPRVFFGVIVVSSGGVLSNMPLTQDEAEEL MYRFCIANEIYTKARSMDADIELQKSEEELEAISRALSFFSLFEPNIE RVEGTFPNSEIEMLEQFLSTPADVDFITKTLKAKEVEAYADLCDSHYL KPEKTIQERLEINRCEAIDKTQDLLALHARSNKQTSLNRGTVKLPPWL PKPSSESIDIKTDSGFGSLMDHGAYGELWAKCLLDVSLGNVEGVVSDP AKELDIAISDDPEKDTPKEAKITYRRFKPALSSSARQEFSLQGVEGKK WKRMAANQKKESESHETLSPFLDVEIDIGFLTFNNLLADSRYGDESVQ RAVSILLEAKASAMQDTELTHALNDSFKRNLSSNVVQWSLWVSCLAQE LASALKQHCRAGEFIIKKLKFWPIYVIIKPTKSSSHIFYSLGIRKADV TRRLTGRVFSDTIDAGEWELTEFKSLKTCKLTNLVNLPCTMLNSIAFW REKLGVAPWLVRKPCSELREQVGLTFLISLEDKSKTEEIITLTRYTQM EGFVSPPMLPKPQKMLGKLDGPLRTKLQVYLLRKHLDCMVRIASQPFS LIPREGRVEWGGTFHAISGRSTNLENMVNSWYIGYYKNKEESTELNAL GEMYKKIVEMEEDKPSSPEFLGWGDTDSPKKHEFSRSFLRAACSSLER EIAQRHGRQWKQNLEERVLREIGTKNILDLASMKATSNFSKDWELYSE VQTKEYHRSKLLEKMATLIEKGVMWYIDAVGQAWKAVLDDGCMRICLF KKNQHGGRLEIYVMDANARLVQFGVETMARCVCELSPHETVANPRLKN SIIENHGLKSARSLGPGSININSSNDAKKWNQGHYTTKLALVLCWFMP AKFHRFIWAAISMFRRKKMMVDLRFLAHLSSKSESRSSDPFREAMTDA FHGNREVSWMDKGRTYIKTETGMMQGLILHFTSSLLHSCVQSFYKSYF VSKLKEGYMGESISGVVDVIEGSDDSAIMISTRPKSDMDEVRSRFFVA NLLHSVKFLNPLFGIYSSEKSTVNTVYCVEYNSEFHFHRHLVRPTLRW IAASHQISETEALASRQEDYSNLLTQCLEGGASFSLTYLIQCAQLLHH YMLLGLCLHPLFGTFMGMLISDPDPALGFFLMDNPAFAGGAGFRFNLW RACKTTDLGRKYAYYFNEIQGKTKGDEDYRALDATSGGTLSHSVMVYW GDRKKYQALLNRMGLPEDWVEQIDENPGVLYRRAANKKELLLKLAEKV HSPGVTSSLSKGHVVPRVVAAGVYLLSRHCFRFSSSIHGRGSAQKASL IKLLMNSSISAMKHGGSLNPNQERMLFPQAQEYDRVCTLLEEVEHLTG KFVVRERNIVRSRIDLFQEPVDLRCKAEDLVSEVWFGLKRTKLGPRLL KEEWDKLRASFAWLSTDPSETLRDGPFLSHVQFRNFIAHVDAKSRSVR LLGAPVKKSGGVTTISQVVRMNFFPGFSLEAEKSLDNQERLESISILK HVLFMVLNGPYTEEYKLEMIIEAFSTLVIPQPSEVIRKSRTMTLCLLS NYLSSRGGSILDQIERAQSGTLGGFSKPQKTFIRPGGGIGYKGKGVWT GVMEDTHVQILIDGDGTSNWLEEIRLSSDARLYDVIESIRRLCDDLGI NNRVASAYRGHCMVRLSGFKIKPASRTDGCPVRIMERGFRIRELQNPD EVKMRVRGDILNLSTIQEGRVMNILSYRPRDTDISESAAAYLWSNRDL FSFGKKEPSCSWICLKTLDNWAWSHASVLLANDRKTQGTDNRAMGNIF RDCLEGSLRKQGLMRSKLTEMVEKNVVPLTTQELVDILEEDIDFSDVI AVELSEGSLDIESIFDGAPILWSAEVEEFGEGVVAVSYSSYYHLTLMD QAAITMCAIMGKEGCRGLLTEKRCMAAIREQVRPFLIFLQIPEDSISW VSDQFCDSRGLDEESTIMG* (SEQ ID NO: 295) SEQ SEQ ID NO sequence ID NO sequence 228 MNLEVLCGRINVENG 257 KARSMDADIELQKSE 229 GRINVENGLSLGEPG 258 DIELQKSEEELEAIS 230 GLSLGEPGLYDQIYD 259 EEELEAISRALSFFS 231 GLYDQIYDRPGLPDL 260 SRALSFFSLFEPNIE 232 DRPGLPDLDVTVDAT 261 SLFEPNIERVEGTFP 233 LDVTVDATGVTVDIG 262 ERVEGTFPNSEIEML 234 TGVTVDIGAVPDSAS 263 PNSEIEMLEQFLSTP 235 GAVPDSASQLGSSIN 264 LEQFLSTPADVDFIT 236 SQLGSSINAGLITIQ 265 PADVDFITKTLKAKE 237 NAGLITIQLSEAYKI 266 TKTLKAKEVEAYADL 238 QLSEAYKINHDFTFS 267 EVEAYADLCDSHYLK 239 INHDFTFSGLSKTTD 268 LCDSHYLKPEKTIQE 240 SGLSKTTDRRLSEVF 269 KPEKTIQERLEINRC 241 DRRLSEVFPITHDGS 270 ERLEINCREAIDKTQ 242 FPITHDGSDGMTPDV 271 CEAIDKTQDLLAGLH 243 SDGMTPDVIHTRLDG 272 QDLLAGLHARSNKQT 244 VIHTRLDGTIVVVEF 273 HARSNKQTSLNRGTV 245 GTIVVVEFSTTRSHN 274 TSLNRGTVKLPPWLP 246 FSTTRSHNIGGLEAA 275 VKLPPWLPKPSSESI 247 NIGGLEAAYRTKIEK 276 PKPSSESIDIKTDSG 248 AYRKTIEKYRDPSIR 277 IDIKTDSGFGSLMDH 249 KYRDPISRRVDIMEN 278 GFGSLMDHGAVGELW 250 RRVDIMENPRVFFGV 279 HGAYGELWAKCLLDV 251 NPRVFFGVIVVSSGG 280 WAKCLLDVSLGNVEG 252 VIVVSSGGVLSNMPL 281 VSLGNVEGVVSDPAK 253 GVLSNMPLTQDEAEE 282 GVVSDPAKELDIAIS 254 LTQDEAEELMYRFCI 283 KELDIAISDDPEKDT 255 ELMYRFCIANEIYTK 284 SDDPEKDTPKEAKIT 256 IANEIYTKARSMDAD 285 TPKEAKITYRRFKPA

[0160] Validation of Immunogenicity of Five SFTS Virus Antigens and Two Adjuvants

[0161] For vaccination. BALB/c mice were divided into the following five groups, each consisting of 6 mice: a naïve group; a group injected intramuscularly with a DNA vaccine: a group injected intramuscularly with a DNA vaccine and then subjected to electroporation: a group injected intramuscularly with a DNA vaccine and an IL-7 adjuvant and then subjected to electroporation; and a group injected intramuscularly with a DNA vaccine and an IL-33 adjuvant and then subjected to electroporation. Each mouse of the naïve group was vaccinated with 200 μg of a plasmid into which no SFTS virus gene was inserted, and each mouse of each of the other four groups was vaccinated with a total of 200 μg (40 μg for each DNA) of five SFTS virus antigen-expressing DNAs (DNA sequences of Gn, Gc, NP, NS and RdRp, which correspond to the DNA sequences of SEQ ID NOs: 286, 288, 290, 292 and 294, respectively, and to the amino acid sequences of SEQ ID NOs: 287, 289, 291, 293 and 295, respectively). In addition, each mouse of the groups to be vaccinated with IL-7 and IL-33 adjuvants was vaccinated with 50 μg of each adjuvant in addition to each DNA. For the mice of three groups, except the naïve group and the group injected intramuscularly with the DNA vaccine, the vaccination site of each mouse was subjected to electroporation using an electroporator at 0.2 A immediately after intramuscular injection. 21 days after the first vaccination, the second vaccination was performed using the same amount of the DNA. 21 days after the second vaccination, the mice were sacrificed, and the spleens and inguinal lymph nodes were isolated and used for immunogenicity evaluation.

[0162] Validation of T-Cell Immune Response to SFTSV Vaccine Candidate

[0163] ELISpot assay was performed to measure T-cell response specific to SFTS virus (FIG. 1). 100 μl of an anti-human IFN-γ antibody (2 μg/ml; endogen) diluted in PBS was dispensed into each well of a 96-well filtration plate and incubated overnight at 4° C. Then, mouse spleen cells (5×10.sup.1 cells/well) were incubated at 37° C. for 24 hours while the cells were stimulated with eight OLPs prepared from the peptides of SFTS virus. Then, the plate was washed, and 100 μl of biotinylated anti-human IFN-γ antibody (0.5 μg/ml; endogen) diluted in PBS/Tween 20/1% BSA was dispensed into each well and incubated overnight at 4° C. After washing four times, 100 μl of streptavidin-alkaline phosphatase (BD) diluted at 1:5.000 in PBS/Tween 20/1% BSA was dispensed into each well and incubated at 37° C. for 1 hour. Using an AP conjugate substrate kit (BIO-RAD), a reaction was performed for 10 minutes and stopped by washing, and then SFTS virus-specific production of IFN-γ by T-cells in response to the SFTS virus antigen was detected by ImmimoSpot (Cellular Technology Limited). Thereby, it was confirmed that T-cell immune response specific to SFTS virus was successfully induced in the mouse models after DNA vaccination. This immune response could be clearly observed in the group subjected to electroporation after intramuscular injection of the DNA vaccine and the group injected with the IL-33 adjuvant in addition to the DNA vaccine. In particular, it was confirmed that the immune response of T cells significantly increased in the group injected with the IL-33 adjuvant together with the SFTS virus antigen-expressing DNA vaccine (FIGS. 2A to 2E).

[0164] Evaluation of Multifunctionality of T Cells Induced by SFTSV DNA Vaccine Candidate

[0165] The spleen cells isolated from the vaccinated mice were stimulated with each of the SFTS virus OLPs. and then analyzed by intracellular cytokine staining (ICS) using multicolor.

[0166] The cells were stimulated with each of the eight SFTS virus OLPs shown in Tables to 5 above, and were sorted into T-cell subsets secreting IFN-γ, TNF-α and IL-2, respectively. The proportion of T-cells secreting each cytokine was determined and the results are shown in FIGS. 3A to 3C.

[0167] As shown in FIGS. 3A to 3C, it was confirmed that a higher immune response generally occurred in the group (IMEP) subjected to both intramuscular injection and electroporation than in the group (IM) subjected to intramuscular injection alone. In addition, it was confirmed that the strongest immune response tended to occur in the group (IL-33) injected with IL-33 as an adjuvant in addition to being subjected to electroporation. This tendency was better identified in CD8+ T cells (FIGS. 3A to 3C).

[0168] It was confirmed that OLP6, an OLP corresponding to the NS protein of SFTS virus, induced the strongest immune response, and in particular, a very strong immune response appeared in cells secreting IFN-γ and TNF-α. In addition, it could be very clearly confirmed in the CD8+ T cells treated with OLP6 that the IMEP group showed a stronger immune response than the IM group, and that the strongest immune response was induced when IL-33 was used as an adjuvant.

[0169] This means that, when electroporation and IL-33 are used, the proportion of SFTS virus-specific T-cells induced by the vaccine is further increased.

[0170] Analysis of Multifunctionality of T-Cells Induced by Vaccine Candidate

[0171] Based on FACS data, the multifunctionality of T-cells in each group was analyzed. Based on the results of FACS, the multifunctionality of CD8+ T cells stimulated with OLP6 inducing the strongest immune response was summarized for each group and the results were recorded. The results indicated that the proportion of mulifunctional T cells was higher in the IMEP group than in the IM group and was the highest in the group injected with IL-33 as an adjuvant in addition to being subjected to electroporation. This means that the T-cell immune response induced by the vaccine was qualitatively better when electroporation and IL-33 are used. This tendency also appeared in the proportion of multifunctional T cells among total effector T cells (FIG. 4). In addition. CD4+ T cells showed no significant difference in multifunctionality.

[0172] Evaluation of Antibody Formation Ability of SFTSV Vaccine Candidate

[0173] Verification of SFTSV-Specific Antibody Production Response Induced by Vaccine

[0174] Enzyme-linked immunosorbent assay (ELISA) was performed to measure an SFTS virus-specific antibody production response induced by SFTSV vaccine. In order to establish an ELISA technique for the recombinant SFTSV NP antigen protein, an experiment was conducted using the serum of mice evaluated to have vaccine-induced immunogenicity. Using the ELISA assay technique as described above, the antibody immune response induced by the vaccine was quantitatively analyzed. As shown in FIGS. 5A to 5F, it was confirmed that the antibody immune response generated in the mice of the intramuscular injection+electroporation (IMEP) group was stronger than those in other groups. In FIGS. 5A to 5F, CrMN represents a nano-pattern formed on the surface of a microneedle (MN) by treating the microneedle surface with a chromium precursor.

[0175] Quantitative Evaluation of Neutralizing Antibody Titer Induced by SFTSV DNA Vaccine

[0176] The neutralizing antibody titers of 33 antibodies produced in the mice by the DNA vaccine candidates and various adjuvants were measured by PRNT50 assay.

[0177] The mouse standard antibody developed by the present inventors was used as a positive control. The experimental results indicated that the animals of the test groups showed an SN titer of 20 to 160, suggesting that a neutralizing antibody was formed. In particular, a stronger neutralizing antibody response was observed in the group (IMEP) injected with the DNA vaccine by intramuscular injection+electroporation, and a very weak neutralizing antibody titer was detected in the microneedle group (Microneedle) (FIG. 6).

[0178] Establishment of Medium-Sized Animal Models as SFTSV-Infected Animal Models

[0179] New animal models for validating the infection inhibitory effect of the SFTSV vaccine were developed. As a result of infecting medium-sized animals with SFTSV isolated from an SFTS patient, it was observed that the virus was detected in the blood and that the platelet count continued to decrease up to day 8 after infection (FIG. 7). In addition, it was confirmed that the body temperature increased by 2° C. or more on day 4, and as this symptom persisted, and all the infected animals died about 9 days after infection. These results were very similar to the clinical courses of the patient. When the animals do not enter the recovery phase after SFTSV infection, they tended to die after about 10 days. The medium-sized animal models established by the present inventors showed very similar clinical findings to those of SFTS patients, such as high fever, increased viral load, platelets and changes in blood components when infected with SFTSV, and thus were determined to show excellent suitability as SFTSV-infected animal models for vaccine efficacy validation.

[0180] Validation of Immunogenicity of SFTSV Vaccine Using SFTSV-Infected Animal Models

[0181] The present inventors used the established SFTSV-infected medium-sized animal models as models to validate the infection inhibitory effect of the SFTSV vaccine. Each animal of a vaccination group (N=6) was vaccinated with a total of 1 mg (200 μg for each DNA) of the five SFTS virus antigen-expressing DNAs (see Table 6 below) by intradermally injecting the DNAs into both femurs in an amount of 500 μg for each femur. Each animal of a control group was vaccinated with 1 mg of a mock plasmid (derived from pVax-1), into which no SFTS virus gene was inserted, by intradermally injecting the DNAs into both femurs in an amount of 500 μg for each femur. Both the two groups were subjected to electrophoresis using an electroporator at 0.2 A immediately after intradermal injection. The vaccination was performed a total of 5 times, once every two weeks (vaccinated on days 0, 14, 28, 42 and 56).

TABLE-US-00006 TABLE 6 Control Vaccination pVax1 1 mg pGX27-Gn 200 ug pGX27-Gc 200 ug pGX27-NP 200 ug pGX27-NSs 200 ug pGX27-RdRp 200 ug

[0182] Evaluation of T-Cell Immune Response Induced by Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0183] Before vaccination and 2 weeks after each of 2.sup.nd vaccination, 4.sup.th vaccination and 5.sup.th vaccination, 5 ml of blood was sampled, PBMCs and serum were isolated therefrom, and SFTSV-specific T cell immune response (ELISpot assay) and antibody immune response (ELISA and neutralizing antibody assay) were measured. SFTSV-specific T cell immune response induced by the vaccine was evaluated by ELISpot assay. As shown in FIGS. 8A to 8D, it was confirmed that the SFTSV vaccine candidate induced very strong SFTSV-specific T cell immune response in the medium-sized animals, and that stable immune response was observed after 2.sup.nd vaccination.

[0184] Quantitative Evaluation of Antibody Immune Response and Neutralizing Antibody Titer Induced by Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0185] The formation of SFTSV-specific reactive antibody by the DNA vaccine was measured and evaluated by ELISA assay. As shown in FIG. 9, it was confirmed that the mock group (n=6) vaccinated with the empty vector vaccine showed an ELISA value similar to that of the serum of the negative control, indicating that no specific antibody was formed in the mock group. However, it was observed that the production of SFTSV-specific reactive antibody in the group (n=6) vaccinated with the SFTSV DNA vaccine was similar to or higher than that in the serum of the positive control. This result suggests that the vaccine candidate can effectively induce antibody immune response in medium-sized animals.

[0186] The neutralizing antibody titer of the antibody induced by the DNA vaccine was measured by PRNT50 assay. As shown in FIG. 10, it was confirmed that the titer of neutralizing antibody produced in the six animals vaccinated with the SFTSV DNA vaccine was similar to that in the positive control group. In contrast, it could be confirmed that no neutralizing antibody was produced in the group (n=6) vaccinated with the empty vector vaccine. These results suggest that SFTSV-specific neutralizing antibody can be effectively induced by the DNA vaccine candidate.

[0187] Evaluation of Preventive Effect of SFTSV Preventive DNA Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0188] To evaluate the preventive effect of the SFTSV vaccine the animals vaccinated with SFTSV vaccine were infected with a lethal dose of SFTSV, and then evaluated for clinical symptoms such as survival rate, SFTSV viral load, platelet count, and body temperature and body weight changes. As shown in FIG. 11, as a result of evaluating survival rate, it was confirmed that the six control animals all died (2 animals on day 7, 3 animals on day 8, and 1 animal on day 9 after infection), whereas the six animals vaccinated with the SFTSV vaccine all survived.

[0189] SFTSV viral load was measured by real-time PCR. As shown in FIGS. 12A to 12C, it was observed that the viral load in the control group increased on day 2 after infection and was the highest on day 4 after infection. In comparison with this, no viral load was detected in four medium-sized animals of the vaccinated group. It was confirmed that, in one animal of the vaccinated group, a viral load similar to that in the control group was detected, but decreased on day 4 after infection and then was completely removed on day 6 after infection.

[0190] It was observed that this animal was the same animal as the animal whose platelets have increased, and as the viral load therein decreased, the platelet count thereof was also returned to normal.

[0191] As a result of counting platelets, it was observed that the platelet count of the control group was decreased rapidly by SFTSV infection (FIGS. 13A to 13C). In comparison with this, it was confirmed that the platelet count of the vaccinated group was maintained normally. It was observed that, in one animal of the vaccinated group, the platelet count decreased to about 120×10.sup.3/μl on day 4 after infection, but it was confirmed that the platelet count was returned to normal on day 6 after infection.

[0192] Meanwhile, as shown in FIG. 14A, it was confirmed that the animals of the control group showed a significant loss in body weight after SFTSV infection, but the animals of the vaccinated group showed no loss in body weight. In addition, as shown in FIG. 14B, the animals of the control group showed an increase in body temperature of about 2° C. after SFTSV infection, but no apparent change in body temperature was observed in the animals of the vaccinated group.

[0193] Taking these results together, it could be seen that the SFTSV-preventive DNA vaccine candidate developed by the present inventors could effectively prevent SFTSV infection, as confirmed through verification of various clinical indicators (survival rate, platelet count, body temperature, and body weight) in the medium-sized animal models.

[0194] Evaluation of Cross-Reactivity of Neutralizing Antibody Induced in Mice and Medium-Sized Animals

[0195] To evaluate the cross-reactivity of SFTSV neutralizing antibody produced in mice after SFTSV DNA vaccination, a PRNT50 test was performed using other SFTS viruses. As shown in FIG. 15A, a neutralizing antibody against SFTSV/2014 virus was produced at a titer of about 40 to 80, whereas the production of a neutralizing antibody against another virus SFTSV/2015 did not clearly appear.

[0196] In addition, to evaluate the cross-reactivity of SFTSV neutralizing antibody produced in medium-sized animals after SFTSV DNA vaccination, a PRNT50 test was performed using other SFTS viruses. As shown in FIG. 15B, a neutralizing antibody against SFTSV/2014 virus was produced at a titer of about 160 to 320, which was about two times higher than the titer value against another virus SFTSV/2015.

[0197] Evaluation of Immunogenicity of SFTSV Preventive DNA Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0198] Using the SFTSV-infected medium-sized animal models established as described above, the infection preventive effect of the SFTSV vaccine was evaluated. To evaluate the preventive effect of each vaccine, SFTSV-vaccinated groups (N=4, N=3, N=3, and N=3, respectively) were vaccinated respectively with 1 mg of a Gn/Gc vaccine, an NP vaccine, an NS vaccine and an RdRp vaccine, which are SFTS virus antigen-expressing DNAs, by intradermally injecting each DNA into both femurs. A control group was vaccinated with 1 mg of a mock plasmid (derived from pVax-1), into which no SFTS virus gene was inserted, by intradermally injecting the plasmid into both femurs (500 μg for each femur). Both the vaccinated groups and the control group were subjected to electroporation using an electroporator at 0.2 A immediately after intradermal injection. The vaccination was performed a total of three times, once every two weeks (vaccinated on days 0, 14 and 28).

[0199] Evaluation of T-Cell Immune Response Induced by Vaccine In SFTSV-Infected Medium-Sized Animal Models

[0200] Before vaccination and 2 weeks after 3.sub.rd vaccination, 5 ml of blood was sampled. PBMCs and serum were isolated therefrom, and SFTSV-specific T cell immune response (ELISpot assay) and antibody immune response (ELISA and neutralizing antibody assay) were measured. SFTSV-specific T cell immune response induced by the vaccine was evaluated by ELISpot assay. It was confirmed that a very strong SFTSV-specific T cell immune response against each SFTSV antigen depending on the kind of vaccine was induced by the SFTSV vaccine candidate in the medium-sized animal models. As shown in FIG. 16, the highest SFTSV-specific immune response could be detected in the group treated with the Ga/Gc SFTSV vaccine.

[0201] Quantitative Evaluation of Antibody Immune Response and Neutralizing Antibody Titer Induced by Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0202] The neutralizing antibody titer of antibody induced by the DNA vaccine was measured by PRNT50 assay. As shown in FIG. 17, it was confirmed that neutralizing antibody titer was effectively produced in the four animals vaccinated with the DNA vaccine against SFTSV Gu/Gc. In contrast, it could be confirmed that no neutralizing antibody was produced in the animals of the groups vaccinated with the vaccines against NP, NS and RdRp, respectively, including the group (n=6) vaccinated with the empty vector vaccine. These results mean that SFTSV-specific neutralizing antibody can be effectively induced by the DNA vaccine candidate against Gn/Gc.

[0203] Evaluation of Preventive Effect of SFTSV Preventive DNA Vaccine in SFTSV-Infected Medium-Sized Animal Models

[0204] To evaluate the preventive effect of each vaccine, the animals vaccinated with SFTSV vaccines against Gn/Gc, NP, NS and RdRp, respectively, were infected with a lethal dose of SFTSV, and then evaluated for clinical symptoms such as survival rate, SFTSV viral load, platelet count, body temperature and body weight changes, and ALT and AST changes.

[0205] As shown in FIG. 18, as a result of evaluating survival rate, it was confirmed that, in the control group, the six animals all died after infection, and in each of the three groups vaccinated with the SFTSV vaccines against NP, NS and RdRp, respectively, only one of the three animals survived, whereas the four animals vaccinated with the SFTSV vaccine against Gn/Gc all survived.

[0206] In addition, SFTSV viral load was measured by real-time PCR. The results are shown in FIGS. 19 and 20. It was observed that the viral load in the control group increased on day 2 after infection and was the highest on day 6 after infection. In comparison with this, no viral load was detected in four medium-sized animals of the Gn/Gc vaccine group. It was confirmed that, in one animal of the G/Gc vaccine group, a very low viral load was detected on day 2 after infection, but was completely removed on day 4 after infection. However, it was confirmed that the viral loads in the three groups vaccinated with the SFTSV vaccines against NP. NS and RdRp, respectively, increased to levels similar to the viral load of the control group.

[0207] In addition, as shown in FIGS. 21 and 22, as a result of counting platelets, it was observed that the platelet count of the control group was decreased rapidly by SFTSV infection. In comparison with this, it was confirmed that the platelet count of the Gn/Gc vaccine group was maintained normally. However, in the three groups vaccinated with the SFTSV vaccines against NP, NS and RdRp, respectively, it was observed that the platelet count decreased to about 300×10.sup.3/μl up to day 6 after infection.

[0208] As shown in FIGS. 23 and 24, as a result of counting white blood cells, it was observed that white blood cell count of the control group was decreased rapidly by SFTSV infection. In comparison with this, it was confirmed that the white blood cell count of the Gn/Gc vaccine group was maintained normally. On the other hand, it was observed that, in the three groups vaccinated with the SFTSV vaccines against NP, NS and RdRp, respectively, the white blood cell count decreased up to days 6 or 4 after infection.

[0209] Meanwhile, it was confirmed that, after SFTSV infection, the animals of the control group showed a significant loss in body weight (relative body weight of 80% or less), whereas the animals of the vaccine groups showed no loss in body weight (relative body weight of 90% or less) (see FIGS. 25 and 26).

[0210] In addition, as shown in FIGS. 27 and 28, after SFTSV infection, the animals of the control group showed an increase in body temperature of about 2° C., but no apparent change in body temperature was observed in the animals of the Gn/Gc vaccine group. However, it was observed that the three groups vaccinated with the SFTSV vaccines against NP, NS and RdRp, respectively, showed an increase in body temperature of about 0.5 to 1° C.

[0211] As shown in FIGS. 29 and 30, as a result of measuring serum ALT concentration, it was observed that the ALT concentration in the control group was increased rapidly by SFTSV infection. In comparison with this, the ALT concentration in the Gn/Gc vaccine group was maintained normally. On the other hand, it was observed that, in the three groups vaccinated with the SFTSV vaccines against NP. NS and RdRp, respectively, the ALT concentration increased rapidly up to day 6 after infection.

[0212] In addition, as shown in FIGS. 31 and 32, as a result of measuring serum AST concentration, it was observed that the AST concentration in the control group was increased rapidly by SFTSV infection. In comparison with this, it was confirmed that the AST concentration in the Gn/Gc vaccine group was maintained normally. On the other hand, it was observed that, in the three groups vaccinated with the SFTSV vaccines against NP. NS and RdRp, respectively, the AST concentration increased rapidly up to day 6 after infection.

[0213] As a result of evaluating the preventive effect of each of the Gn/Gc, NP, NS and RdRp antigens in the infected medium-sized animal (ferret) models as described above, it could be confirmed that the Gn/Gc DNA vaccine exhibited a significantly higher protective effect than other antigen-expressing DNA vaccines.

[0214] Although the present disclosure has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this detailed description is only of a preferred embodiment thereof, and does not limit the scope of the present disclosure. Thus, the substantial scope of the present disclosure will be defined by the appended claims and equivalents thereto.

INDUSTRIAL APPLICABILITY

[0215] The present disclosure relates to a vaccine composition for preventing or treating an infectious disease caused by severe fever with thrombocytopenia syndrome (SFTS) virus.