Modified NP peptide and use thereof

Abstract

The present invention relates to an NP antigen peptide of the SFTS virus, the NP antigen peptide including a novel mutation. NP protein sequences of 56 strains collected from the entire genotype were analyzed, consensus sequences of NP proteins were derived therefrom, and then an NP peptide antigen incorporating an amino acid substitution mutation and having improved structural stability was designed through structural analysis modeling. As a result of evaluating immunogenicity and protective ability using an SFTS virus vaccine composition prepared using the NP peptide antigen, it was found that a vaccine composition comprising the NP antigen of the present invention has an excellent ability to produce an immune response and an excellent ability to protect against SFTS virus infection, and thus the NP antigen peptide according to the present invention can be used in a vaccine composition for severe fever with thrombocytopenia syndrome.

Description

Modified NP Peptides and Their Uses

[0001] The present invention relates to a modified NP antigen peptide and a vaccine composition for preventing or treating infectious diseases caused by the virus disease of Severe Fever with Thrombocytopenia Syndrome (SFTS).

[0002]

[0003] Severe fever with thrombocytopenia syndrome (SFTS) is an emerging viral disease that is common in China, Korea, and Japan. Currently, there are no effective vaccines or specific treatments available for SFTS. SFTS is a serious disease accompanied by symptoms such as high fever, vomiting, diarrhea, thrombocytopenia, leukopenia, and multiple organ failure, with a mortality rate of 6–30% (Yu XJ et al., N. Engl. J. Med. 2011; 364:1523-32; Ding F et al Clin Infect Dis 2013; 56: 1682-3).

[0004] Although the SFTS virus was previously known to be primarily transmitted by ticks carrying the virus, human-to-human transmission through blood or bodily fluids has now also been reported. Additionally, seroconversion and viremia of the SFTS virus have been found in domesticated animals such as goats, sheep, cattle, pigs, and dogs, and these animals are also thought to act as intermediate vectors in areas where the SFTS virus has spread (Zhao L et al. Emerg Infect Dis 2013; 18: 963-5; Niu G et al. Emerg Infect Dis 2013; 19: 756-63).

[0005] Since 2013, cases of SFTS infection have continued to occur in Korea, and despite a mortality rate of nearly 20%, no clinically applicable treatments or preventive vaccines have been developed to date. The nucleotide sequences of the entire SFTS gene isolated from the serum of patients infected with the virus and from the small soft ticks inhabiting Korea have already been analyzed. As is already known, the SFTS virus is a typical virus belonging to the genus Phlebovirus, segmented into three genes. The genome is largely composed of the L segment, which is 6,368 nucleotides long; the M segment, which is 3,378 nucleotides long; and the S segment, which is 1,746 nucleotides long. The genotypes of the SFTS virus inhabiting Korea are known to be types A, B, D, and F, with type B being the most commonly found.

[0006] Chinese Patent Publication No. 102070704 discloses a kit for amplifying and detecting the SFTS virus using the SFTS virus, and Korean Patent Publication No. 10-2020-0001671 also discloses an effective vaccine against the SFTS virus. However, there is still a need for specific antigens with broad coverage against the SFTS virus, which possesses various genotypes and genetic mutations, and for vaccine compositions based on such antigens.

[0007] Accordingly, the inventors analyzed the NP protein sequences of 56 SFTS viruses of various genotypes to identify a consensus sequence and derived an antigen sequence capable of responding to various genotypes and mutations. By structurally modeling this, they designed an antigen with improved stability and developed a more effective vaccine composition against SFTS viruses.

[0008]

[0009] The objective of the present invention is to provide an SFTS antigen protein with improved stability and efficacy and an SFTS virus vaccine composition based on this antigen protein.

[0010] In order to achieve the above objective,

[0011] The present invention provides modified NP antigen peptides into which E13N, T45K, V51K, T54A, V69S, G73K, N77E, S78E, E88K, A117Q, N134D, L135S, E138Q, Q174L, D175V, V183P, K184N, A188K, T191E, V214Q, D228N, V230K, R233P, A235V, and V237A mutations are introduced relative to the wild type of severe fever with thrombocytopenia syndrome (SFTS) virus.

[0012] In addition, the present invention provides a nucleic acid encoding the antigen peptide.

[0013] In addition, the present invention provides an expression vector comprising the nucleic acid.

[0014] In addition, the present invention provides a cell transformed by the expression vector.

[0015] In addition, the present invention provides a vaccine composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection comprising the antigen peptide and a pharmaceutically acceptable carrier.

[0016]

[0017] The present invention relates to an NP antigen peptide of the SFTS virus into which multiple mutations are introduced relative to the NP protein of the wild-type SFTS virus. By analyzing common sequences derived from the NP protein sequences of 56 strains, an NP peptide antigen was designed by introducing amino acid substitution mutations with improved structural stability. As a result of evaluating immunogenicity and protective ability using an SFTS virus vaccine composition prepared using this, it was confirmed that the vaccine composition containing the NP antigen of the present invention has excellent immune response generation ability and excellent protective ability against SFTS virus infection.

[0018]

[0019] Figure 1 shows the tertiary structure of the NP protein modeled for the SFTS virus and the mutation region designed to improve structural stability.

[0020] Figure 2 is a schematic diagram showing the process of an inoculation experiment performed to confirm the immune response-inducing ability of a modified NP peptide (antigen).

[0021] Figure 3 is a graph analyzing the levels of antigen-specific binding antibodies against NP antigens before, after the first dose (prime), and after the second dose (boost) of a vaccine containing nucleic acid (mRNA) encoding a modified NP peptide (hereinafter abbreviated as 'NP peptide-based vaccine').

[0022] Figure 4 is a diagram showing the results of analyzing antibody titers according to the isotype of the antibody, Figure 4a shows the analysis of IgG1 antibody titers specific to NP, Figure 4b shows the analysis of IgG2c antibody titers specific to NP, and Figure 4c is a graph showing the ratio between IgG1 and IgG2c.

[0023] Figure 5 is a graph analyzing the number of IFN-γ-secreting T cells among spleen cells after the second vaccination.

[0024] Figure 6 is a schematic diagram of the experimental method performed using IFNAR- / - mice to confirm protective ability against attack infection.

[0025] Figure 7 is a graph analyzing the binding antibody titers of the modified NP peptide-based vaccine before (pre) and after (boost) the second dose.

[0026] Figure 8 is a graph summarizing the survival rate of mice after attack infection.

[0027] Figure 9 is a graph summarizing the changes in body weight of mice after attack infection.

[0028] Figure 10 shows the results of analyzing the copy number of the SFTS virus in the serum after an attack infection.

[0029] Figure 11 shows the results of analyzing changes in the cell composition in the blood before and after an attack infection, Figure 11a shows the results of measuring the platelet count and Figure 11b shows the results of measuring the white blood cell count.

[0030] Figure 12 shows the results of a pathological analysis of the liver after an attack infection, Figure 12a shows the results of hematoxylin & eosin (H&E) staining of liver tissue, Figure 12b shows the results of pathological score analysis, Figure 12c shows the results of observing the inflammatory response within the liver, Figure 12d shows the results of observing vascular degeneration, and Figure 12e shows the results of observing tissue necrosis.

[0031] Figure 13 shows the results of a pathological analysis of the spleen after an attack infection, Figure 13a shows the results of H&E staining of the spleen tissue, Figure 13b shows the results of analyzing the lymphocyte depletion level, and Figure 13c shows the results of measuring the number of megakaryocytes in the spleen.

[0032] Figure 14 shows the results of a pathological analysis of cervical lymph nodes after an attack infection, Figure 14a shows the results of H&E staining of cervical lymph tissue, and Figure 14b shows the results of an analysis of the lymphocyte depletion level within the lymph nodes.

[0033]

[0034] The present invention will be described in detail below.

[0035] The present invention provides modified NP peptides into which E13N, T45K, V51K, T54A, V69S, G73K, N77E, S78E, E88K, A117Q, N134D, L135S, E138Q, Q174L, D175V, V183P, K184N, A188K, T191E, V214Q, D228N, V230K, R233P, A235V, and V237A mutations are introduced relative to the wild type of NP protein of the severe fever with thrombocytopenia syndrome (SFTS) virus.

[0036] The above SFTS virus is a negative single-stranded RNA virus belonging to the family Bunyaviridae and the genus Phlebovirus. The virus is a spherical virus with a diameter of 80 to 100 nm and is transmitted by ticks. The genome of the virus consists of large (L), medium (M), and small (S) segments, which code for six proteins: RNA-dependent RNA polymerase (RdRp), glycoprotein precursor (M), glycoprotein N (NP), glycoprotein C (Gc), nucleocapsid protein (NP), and non-structural protein (NSs).

[0037] The above NP peptide is an antigen peptide designed by obtaining and analyzing 56 full-length sequences of NP domains of various genotypes such as types A, B, D, and F to derive a consensus sequence, and using the derived sequence for structural modeling to introduce a mutation that improves structural stability in the tertiary structure.

[0038] The NP antigen peptide into which a mutation for structural stabilization has been introduced may be composed of the amino acid sequence of SEQ ID NO. 1 or may be a peptide variant containing one or more amino acid variations based on the amino acid sequence of SEQ ID NO. 1. The above peptide variant refers to a peptide in which a substitution, deletion, addition, and / or insertion mutation has occurred in the amino acid sequence of the peptide, and which exhibits almost the same biological function as the peptide composed of the original amino acid sequence. The peptide variant may have an identity of 70% or more, preferably 85% or more, with the amino acid sequence of SEQ ID NO. 1.

[0039] Name Sequence Sequence Number NP Peptide MSEWSRIAVEFGNQQLNLTELEDFARELAYEGLDPALIIKKLKEKGGDDWKKDAKFIIVFALTRGNKISKASKKMSEEGSKRLMALQKKYGLVERAETRLSITPVRVAQSLPTWTCQAAAALKEYLPVGPAVMDSKVQNYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWLVAFTKTINPNMRGKSKEEVYNSFRDPLHAAVNSVFFPNDQRVKWLKAKGILGPNGKPSPAVEAAAAAYRNL1

[0040] In addition, the present invention provides a nucleic acid encoding a modified NP peptide.

[0041] In the present invention, the nucleic acid may be a codon-optimized polynucleotide sequence for expression in a host or target. The codon-optimization refers to modifying the coding region of the nucleic acid or the codons within the gene to reflect the typical codon utilization of the host or target without altering the polypeptide encoded by the nucleic acid for the transformation of various hosts or targets. Codon-optimization may involve synthesizing all (or a portion thereof) of DNA to remove all destabilized sequences or regions of the transcribed mRNA that may exist in the secondary structure, or synthesizing all (or a portion thereof) of DNA to modify the base composition to be more preferred in the target host or target cell.

[0042] The above nucleic acid can be selected without limitation as long as it is a sequence encoding the above NP peptide, and as an example, any one of the nucleotide sequences listed in Table 2 can be selected.

[0043] 명칭서열서열번호NP펩타이드의 핵산 서열 -1ATGAGCGAGTGGTCTAGAATCGCCGTCGAGTTCGGCAACCAGCAGCTGAACCTGACCGAGCTGGAAGATTTCGCCAGAGAGCTGGCCTACGAGGGCCTGGACCCCGCCCTCATCATTAAGAAGCTGAAGGAGAAGGGAGGAGATGATTGGAAGAAGGACGCCAAGTTCATCATCGTGTTTGCCCTGACACGGGGCAACAAGATCAGCAAGGCCAGCAAGAAAATGAGCGAGGAAGGCTCTAAAAGACTGATGGCCCTGCAGAAAAAGTACGGCCTGGTCGAGCGGGCCGAAACCAGACTGAGCATCACCCCTGTGCGGGTGGCCCAGAGCCTCCCCACATGGACCTGTCAGGCCGCTGCTGCCCTGAAGGAATACCTGCCTGTGGGCCCTGCCGTGATGGACAGCAAAGTGCAGAACTACCCTCCTGAGATGATGTGCATGGCTTTTGGCAGCCTGATCCCTACCGCAGGCGTGTCCGAGGCCACAACCAAGACCCTGATGGAAGCCTACTCCCTGTGGCTGGTGGCCTTCACCAAGACAATCAACCCCAATATGAGAGGAAAAAGCAAGGAAGAAGTGTACAACTCCTTCCGCGACCCTCTGCACGCCGCTGTGAACAGCGTGTTCTTCCCCAACGACCAAAGAGTGAAGTGGCTGAAAGCCAAGGGCATCCTGGGCCCAAACGGCAAGCCTTCTCCAGCTGTTGAGGCCGCCGCCGCCGCTTATAGAAATCTG2NP 펩타이드의 핵산 서열-

[0044] In addition, the present invention provides an expression vector encoding an NP peptide.

[0045] The above expression refers to the production of proteins or nucleic acids in cells.

[0046] The above expression vector refers to a vector capable of expressing a target protein or target nucleic acid in a suitable host cell, and means a gene construct comprising an essential regulatory element operably linked to enable the expression of a gene insert.

[0047] In the present invention, the term "operably linked" refers to a functional linkage between a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to influence the expression of the coding nucleic acid sequence. Operable linkage with a recombinant vector can be prepared using gene recombination techniques well known in the art, and site-specific DNA cleavage and linkage may be performed using enzymes generally known in the art. The vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable expression vectors may include expression regulatory elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, and may be prepared in various ways depending on the purpose; the promoter of the vector may be constitutive or inducible.

[0048] In addition, the present invention provides a cell transformed by the expression vector.

[0049] The above expression vector can be performed by selecting a standard technique suitable for the host cell as known in the art, such as conventional transfection methods, e.g., DEAE-dextran, calcium phosphate method, microinjection method, DNA-containing liposome method, lipofectamine-DNA complex method (Molecular Cloning, Cold Spring Harbor, New York, Cold Spring Harbor Laboratory Press (1989)).

[0050] The cells capable of transformation are not particularly limited, but it is preferable to use COS-7 cells, 293T cells, HEK293T, CHO and HeLa, etc., and more preferably COS-7 cells and 293T cells.

[0051]

[0052] In addition, the present invention provides a vaccine composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection comprising a modified NP peptide and a pharmaceutically acceptable carrier.

[0053] The above-mentioned vaccine implies the prevention of infection or reinfection by the said pathogen, reduction of the severity of symptoms caused by the said pathogen or elimination of symptoms, or substantial or complete elimination of the said pathogen or the disease caused by said pathogen, by inducing an immune response against the said pathogen in a subject, primarily a human. Accordingly, the vaccine composition of the present invention may be administered to a human prophylactically before infection by the said pathogen or therapeutically after infection by the said pathogen. Here, the above-mentioned immune response includes humoral immune response, cellular immune response, or both.

[0054] The above prevention refers to any act of delaying the growth, proliferation, infiltrativeness, or infectivity of the virus through the administration of the vaccine composition of the present invention.

[0055] The above treatment refers to any act in which the growth, proliferation, or infectivity of the virus is suppressed through the administration of the vaccine composition of the present invention, thereby improving or beneficially altering SFTS virus-related diseases.

[0056] The vaccine composition of the present invention may be prepared in any suitable, pharmaceutically acceptable formulation. For example, it may be in the form of an immediate-administration solution or suspension, a concentrated stock solution suitable for dilution prior to administration, or a reconstituteable form such as a lyophilized, freeze-dried, or frozen formulation.

[0057] Pharmaceutically acceptable carriers that can be used for the formulation of vaccine compositions are listed and specified in the Korean Pharmacopoeia or the pharmacopoeias of other countries, particularly those of the United States, Japan, and Europe, and these pharmacopoeias may be referenced.

[0058] These carriers will typically include diluents, excipients, stabilizers, preservatives, etc. Suitable diluents may include non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil and peanut oil, or aqueous solvents such as brine (preferably 0.8% brine) and water containing a buffer medium (preferably 0.05M phosphate buffer). Suitable excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, anhydrous skim milk, glycerol, propylene glycol, water, ethanol, etc. Suitable stabilizers may include carbohydrates such as sorbitol, mannitol, starch, sucrose, dextran, glutamate, glucose, etc., or proteins such as milk powder, serum albumin, casein, etc., animal, plant, or microbial proteins. Suitable preservatives include thimerosal, merthioleate, gentamicin, neomycin, nystatin, amphotericin B, tetracycline, penicillin, streptomycin, polymyxin B, etc.

[0059] The above vaccine composition may further include an immune adjuvant.

[0060] The above-mentioned adjuvant is also called an antigen adjuvant or adjuvant and may be a substance that enhances the immune response to an antigen.

[0061] The above-mentioned immune enhancers may be, for example, complete Freund, incomplete Freund, saponins, gel-like aluminum adjuvants, surfactants (e.g., lysolecithin, fluron glycol, polyanionic, peptide, oil or hydrocarbon emulsion, etc.), vegetable oils (cottonseed oil, peanut oil, corn oil, etc.), vitamin E acetate, etc.

[0062] The above vaccine composition may be administered to an individual at an immunologically effective amount. The above immunologically effective amount refers to a sufficient amount to produce a preventive or therapeutic effect against Severe Fever with Thrombocytopenia Syndrome (SFTS) virus or SFTS virus, and an amount that does not cause side effects or severe or excessive immune responses. The exact dosage concentration varies depending on the specific immunogen to be administered and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the age, weight, health, gender, sensitivity of the individual to drugs, route of administration, and method of administration of the subject for prevention or treatment, and may be administered one to several times.

[0063] The vaccine composition of the present invention may preferably be administered parenterally, for example, by rectal, percutaneous, intravenous injection, intra-arterial injection, intramuscular injection, intradermal injection, subcutaneous injection, intraperitoneal injection, intraventricular injection, etc., but is not limited thereto.

[0064] In addition, the present invention provides a vaccine composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection comprising the above vector.

[0065] In a specific embodiment of the present invention, the sequences of 56 NP peptides were analyzed to derive a common sequence, and then modified NP peptide sequences were designed by introducing mutations that enhance structural stability through tertiary structural modeling (Fig. 1). After manufacturing a vaccine (i.e., a modified NP peptide-based vaccine) containing nucleic acid (mRNA) encoding the designed modified NP peptide, mice were inoculated to confirm the immune response-inducing effect of the designed NP antigen. As a result, it was confirmed that the antigen-binding antibody titer was high after only the first inoculation, and the binding antibody titer was even higher after the second inoculation, and it was confirmed that immune responses by Th1 and Th2 cells were evenly induced (Figs. 2 to 4). In addition, it was confirmed that T cell activation occurred, indicating that a cellular immune response was also induced (Fig. 5).

[0066] To evaluate the protective ability against challenge infection, experiments using an animal model were conducted, and blood and tissue samples were collected and analyzed (Fig. 6). Similar to the previous results, the group administered the modified NP peptide-based vaccine showed high levels of binding antibody titers after the second dose. In the PBS-administered group, weight loss occurred after challenge infection and all individuals died within 5 days, whereas in the group administered the modified NP peptide-based vaccine, there was almost no change in body weight even after challenge infection, and the survival rate was maintained for 14 days (Figs. 7 to 9).

[0067] When evaluating viremia by measuring viral gene levels in serum collected after challenge infection, generally low viral copy numbers were observed when the modified NP peptide-based vaccine was administered, and it was observed that the number of platelets and white blood cells, which are major symptoms of SFTS virus infection, did not decrease (Figs. 10 and 11). Pathological symptoms of the liver, spleen, and cervical lymph tissues were also relatively mild when the modified NP peptide-based vaccine was administered, confirming that symptoms caused by infection were almost non-existent (Figs. 12 to 14).

[0068] Through this, it can be seen that using the modified NP antigen peptide of the present invention can effectively prevent SFTS virus infection by inducing an immune response and can be effectively used in vaccine manufacturing.

[0069]

[0070] The present invention will be explained in more detail below through specific embodiments.

[0071] However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0072]

[0073] <Example 1> Design of NP Antigen through Modeling Structure Analysis

[0074] Based on previously published references, SFTS viruses were selected to include various genotypes, and NP protein sequences were collected from NCBI Genbank for the selected viruses. By aligning a total of 56 sequences, a consensus sequence of NP proteins identified from various genotypes and strains was derived. Based on the derived sequences, structural modeling was performed using AlphaFold 2 developed by Google DeepMind. Using the predicted 3D structure as input, atomic-level Rosetta energy scores were calculated via pSUFER and FuncLib servers, and modified NP peptides were designed by introducing amino acid mutations to enhance the stability of the protein structure. The designed structure is shown in Figure 1, and the amino acid sequence of the modified NP peptide is listed as SEQ ID NO. 1.

[0075]

[0076] <Example 2> Preparation of NP antigen and preparation of vaccine composition containing the same

[0077] The nucleic acid sequence encoding the NP antigen designed in Example 1 was inserted into an RNA expression vector (in vitro transcription vector, IVT vector), and transcription was performed to produce and purify RNA. The purified RNA was encapsulated in a drug delivery vehicle (lipid nanoparticle, LNP) to produce a modified NP peptide-based vaccine. Although any nucleic acid sequence encoding the amino acid sequence of SEQ ID NO. 1 can be used without restriction for the NP antigen, in this example, the sequence of SEQ ID NO. 3 was selected to produce a modified NP peptide-based vaccine.

[0078]

[0079] <Example 3> Evaluation of Immunogenicity of Modified NP Peptide-Based Vaccine

[0080] To evaluate the immunogenicity of the modified NP peptide-based vaccine using a mouse model, C57BL / 6 mice (6 weeks old) were administered the modified NP peptide-based vaccine (containing 1.25 µg of antigen) twice intramuscularly (IM) at 3-week intervals, and serum and splenocytes were obtained (Fig. 2).

[0081]

[0082] <3-1> Confirmation of antigen-specific antibodies

[0083] To measure the titer of the binding antibody that specifically binds to the antigen, an enzyme-linked immunosorbent assay (ELISA) was performed using serum.

[0084] Specifically, a 96-well plate was coated overnight with SFTS virus NP protein (100 ng / well) and then blocked with 5% skim milk at 37°C for 1 hour. After washing with washing buffer (PBS + 0.05% Tween-20, PBST), serum serially diluted twofold with 3% skim milk was dispensed onto the coated plate and incubated at 37°C for 2 hours. After washing with washing buffer, a secondary antibody (Goat anti-Mouse IgG, HRP conjugate) diluted 1:50,000 was dispensed and incubated at 37°C for 1 hour. After washing again, TMB substrate solution was added and incubated at room temperature for 10 minutes, after which the reaction was stopped by adding stop solution (2M H2SO4). The level of antigen-specific antibody formation was measured by measuring the absorbance at a wavelength of 450 nm using a microplate reader.

[0085] As a result, as shown in Figure 3, it can be confirmed that a high NP antigen-specific binding antibody titer is obtained with only one dose (prime), and a significant increase in antibodies is observed through boosting.

[0086]

[0087] <3-2> Confirmation of binding antibodies according to IgG isotype

[0088] ELISA was performed, but the experiment was conducted using a secondary antibody against IgG1 or an antibody against IgG2, respectively, to determine the titers of the IgG1 antibody (Fig. 4a) or IgG2 antibody (Fig. 4b) against the NP antigen and to analyze the ratio of the two isoform antibodies (Fig. 4c).

[0089] As a result, it was confirmed that using a vaccine composition containing the NP antigen of the present invention improves the ability to produce anti-NP antibodies against different isoforms, and that a balanced humoral immune response through Th1 and Th2 cells can be induced, as the ratio of the two isoforms is close to 1.

[0090]

[0091] <3-3> Confirmation of Cellular Immune Response

[0092] To evaluate whether it induces a cellular immune response, ELISpot was performed to measure T cells secreting virus-specific IFN-γ in splenocytes of mice isolated from immune cells.

[0093] Specifically, spleens from vaccine-immunized mice were isolated in RPMI medium containing 5% FBS and 1% Penicillin-Streptomycin (P / S), and then pulverized into single cells using a gentleMACS instrument. The splenocytes were filtered through a 70 μm strainer and centrifuged (2,000 rpm, 5 min, 4°C). The supernatant was removed, and 5 ml of ACK Lysing buffer was added to the remaining pellet, vortexed, and incubated at 37°C for 5 minutes. An additional 10 ml of RPMI medium containing 5% FBS and 1% P / S was added, and the mixture was centrifuged under the same conditions. The supernatant was discarded, and 1–3 ml of RPMI medium containing 10% FBS and 1% P / S was added to thoroughly loosen the pellet, after which the splenocytes were counted using a LUNA cell counter. Splenocytes 5 x 10⁶ in a 96-well plate in the Mouse IFN-γ ELISpot plus kit (MABTECH) 5 Cells were dispensed per well. As a stimulant, 100 ng / well of NP overlap peptide of the SFTS B genotype was finally dispensed, and the cells were incubated for 18–24 hours under 5% CO2 at 37°C. After incubation, the supernatant was removed, and the cells were washed with PBS. The primary antibody was diluted in PBS-0.5% FBS solution and dispensed, followed by incubation at room temperature for 2 hours. After washing as described above, the ALP (alkaline phosphatase)-labeled secondary antibody was diluted in PBS-0.5% FBS solution and dispensed, followed by incubation at room temperature for 1 hour. After washing again, filter-sterilized substrate solution (BCIP / NBT-plus) was added, and the reaction was carried out for 10–30 minutes until spots appeared. To stop the reaction, the cells were washed with distilled water and thoroughly dried. The generated spots were counted using an automated CTL instrument.

[0094] As a result, as shown in Figure 5, more T cells secreting IFN-γ were observed in the group administered the vaccine composition containing the NP antigen of the present invention, so it can be seen that the vaccine composition containing the NP antigen can induce a cellular immune response.

[0095]

[0096] <Example 4> Evaluation of Infection Protection Ability of Modified NP Peptide-Based Vaccine

[0097] The infection-protective ability of a modified NP peptide-based vaccine was evaluated using a mouse model.

[0098] Specifically, IFNAR - / - Mice (4 to 8 weeks old) were administered two doses of PBS or a modified NP peptide-based vaccine (containing 1.25 µg of NP antigen) at 3-week intervals, and challenge infections were performed by intramuscularly inoculating genotype B SFTS virus at a lethal dose (100×LD50(4 FFU)) at 7 weeks after the first injection. The body weight and survival rate of the mice were recorded daily for 14 days, and blood and / or tissues were collected 1 and 4 days after the challenge infection (Fig. 6).

[0099]

[0100] <4-1> Check survival rate after attack infection

[0101] As a result of measuring the binding antibody titer using the method described in Example 3 with blood collected after challenge infection, it was confirmed that the binding antibody titer appeared at a high level when the modified NP peptide-based vaccine was administered as a second dose (Fig. 7).

[0102] As a result of observing the body weight and survival rate of mice for 14 days after challenge infection, all individuals in the control group challenged after PBS inoculation died within 5 days of challenge infection, whereas all individuals in the group administered the modified NP peptide-based vaccine survived (Table 3 and Fig. 8). Regarding the average body weight, a decrease in body weight was observed starting from day 2 after challenge infection in the control group challenged after PBS inoculation, whereas no decrease in body weight was observed in the experimental group challenged after inoculation with the modified NP peptide-based vaccine (Fig. 9).

[0103]

[0104] Experimental Group Experimental Group Survival Rate 1 PBS Inoculation 100% (8 / 8) 2 Challenge Infection 0% (0 / 15) after PBS Inoculation 3 Challenge Infection 100% (15 / 15) after NP Composition Inoculation

[0105] <4-2> Confirmation of Infection Levels Through Viral Gene Detection

[0106] As described in Example <4-1>, IFNAR - / - Mice (4 to 8 weeks old) were administered two doses of PBS or a modified NP peptide-based vaccine via intramuscular injection. A challenge infection was performed at week 7 following the first injection, and mouse body weight and survival rates were recorded daily for 14 days. Blood samples were collected 1 and 4 days after the challenge infection. RNA was extracted from serum isolated from the collected blood. Additionally, PowerChek was used to detect the SFTS virus gene. TM The amount of viral genome was measured using the SFTSV Real-Time PCR Kit Ver.1.0 (cat #: IR5010S).

[0107] As a result, there was no numerical difference on day 1 post infection (1 DPI), but on day 4 (4 DPI), fewer viral genes were detected in the modified NP peptide-based vaccine group compared to the PBS group (Fig. 10).

[0108] Through this, it can be seen that the modified NP peptide-based vaccine can prevent viremia.

[0109]

[0110] <4-3> Checking Platelet and White Blood Cell Counts

[0111] Since a well-known symptom of SFTS virus infection is a decrease in platelet and white blood cell counts, a complete blood count (CBC) was performed using collected blood to measure platelet and white blood cell counts in order to determine whether these symptoms could be suppressed in an actual animal model.

[0112] As a result, there was no significant difference in platelet and leukocyte counts before and after the attack infection in the control group, but in the group that was attacked after PBS inoculation, it was confirmed that platelet and leukocyte counts in the blood on day 4 after the attack infection were significantly reduced compared to day 0 before the attack infection (Figs. 11a and 11b).

[0113] In contrast, in the group administered the modified NP peptide-based vaccine, no decrease in platelet and leukocyte counts was observed (Figs. 11a and 11b).

[0114] In other words, it was confirmed that the NP antigen of the present invention has an excellent protective effect against the SFTS virus, as it was observed that platelet and leukocyte depletion caused by SFTS virus infection did not occur when the modified NP peptide-based vaccine was administered.

[0115]

[0116] <Example 5> Pathological analysis of the efficacy of a modified NP peptide-based vaccine

[0117] Since a decrease in lymphocytes is commonly observed in cases of SFTS virus infection, we wanted to determine if a modified NP peptide-based vaccine could also help prevent these symptoms.

[0118] As described in Example 4, mice were inoculated twice with PBS or a modified NP peptide-based vaccine, and after challenge infection, liver, spleen, and cervical lymph nodes were collected and each tissue was stained with Hematoxylin and eosin (H&E) and then histologically analyzed.

[0119] Specifically, tissues fixed in 10% formalin solution were paraffin-embedded to form blocks, sectioned to a thickness of 3 μm, and stained with H&E. The stained tissues were observed under a light microscope, and scores were assigned based on the severity. The major lesions observed in the tissues were inflammation, lymphocyte depletion, and necrosis. Scores were assigned based on the frequency and severity of each lesion, and the combined values ​​were compared.

[0120] First, the results of staining the liver tissue are shown in Fig. 12a, and the results of pathological analysis based on the tissue analysis results, including the calculation of scores according to the level of inflammation and the degree of damage, are shown in Figs. 12b to 12e.

[0121] As a result, severe vacuolar degeneration and mild to severe necrosis occurred in the PBS administration group, but mild vacuolar degeneration was confirmed in only 3 individuals in the modified NP peptide-based vaccine administration group.

[0122] The results of spleen staining are shown in Fig. 13a, and the levels of lymphocyte deficiency and megakaryocytes, which are the progenitor cells of platelets, are shown in Figs. 13b and 13c.

[0123] As a result, severe lymphocyte deficiency was observed in the white and red medulla of all individuals in the PBS administration group (Fig. 13b), and the number of megakaryocytes increased significantly, indicating that the immune response was greatly activated due to the infection (Fig. 13c).

[0124] However, in the modified NP peptide-based vaccine administration group, mild to severe lymphocyte deficiency was confirmed in the spleen of only one individual (Fig. 13b), and megakaryocytes in the spleen were observed at a level similar to the control (Fig. 13c).

[0125] The staining results of the cervical lymph nodes are presented in Fig. 14a, and the lymphocyte deficiency levels within the cervical lymph nodes are presented in Fig. 14b.

[0126] Severe lymphocyte deficiency was observed in all individuals in the PBS administration group, but severe lymphocyte deficiency was observed in only one individual in the modified NP peptide-based vaccine administration group (Fig. 14b).

[0127] In other words, it can be seen that administering a modified NP peptide-based vaccine alleviates symptoms caused by SFTS virus infection and causes almost no pathological symptoms.

[0128] Through this, it was confirmed that inoculating with the modified NP peptide-based vaccine of the present invention enhances the immune response against the SFTS virus, thereby providing an infection prevention effect, and also helps prevent symptoms such as lymphopenia. Therefore, by using the NP antigen of the present invention, a vaccine composition having a preventive or therapeutic effect against SFTS virus infection can be prepared.

Claims

1. Modified NP peptides including E13N, T45K, V51K, T54A, V69S, G73K, N77E, S78E, E88K, A117Q, N134D, L135S, E138Q, Q174L, D175V, V183P, K184N, A188K, T191E, V214Q, D228N, V230K, R233P, A235V, and V237A mutations relative to the wild-type NP protein of the severe fever with thrombocytopenia syndrome (SFTS) virus.

2. In Paragraph 1, The above mutation is a modified NP peptide that improves protein structural stability.

3. In Paragraph 1, The above peptide is a modified NP peptide composed of the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having at least 90% identity therewith.

4. A nucleic acid encoding the peptide of paragraph 1.

5. An expression vector comprising the nucleic acid of paragraph 4.

6. Cells transformed by the expression vector of paragraph 5.

7. A vaccine composition for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection comprising a modified NP peptide of claim 1, a nucleic acid encoding the same, an expression vector comprising said nucleic acid, or a cell transformed by said expression vector.

8. In Paragraph 7, The above vaccine composition is a vaccine composition that induces the production of IgG antibodies.

9. In Paragraph 8, A vaccine composition in which the IgG is IgG1, IgG2c, or both of the above.

10. In Paragraph 7, The above vaccine composition is a vaccine composition that induces cellular immunity, humoral immunity, or both cellular and humoral immunity.

11. In Paragraph 7, The above vaccine composition further comprises an adjuvant for immune response activity.

12. In Paragraph 7, The above vaccine composition is a vaccine composition that is injected into the body through a selected route, either intramuscularly, intradermally, subcutaneously, subcutaneously, transdermally, or intravenously.

13. A pharmaceutical composition for the prevention or treatment of severe fever with thrombocytopenia syndrome (SFTS) virus infection comprising a modified NP peptide of claim 1, a nucleic acid encoding the same, an expression vector comprising said nucleic acid, or a cell transformed by said expression vector.

14. In Paragraph 13, The above vaccine composition is a method for preventing or treating severe fever with thrombocytopenia syndrome (SFTS) virus infection, comprising intramuscular, subcutaneous, intradermal, subcutaneous, transdermal, or intravenous administration steps.

15. Use of the modified NP peptide of claim 1, the nucleic acid encoding the same, the expression vector comprising the nucleic acid, or cells transformed by the expression vector for the prevention or treatment of severe fever with thrombocytopenia syndrome (SFTS) virus infection.

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