Novel severe fever with thrombocytopenia syndrome virus vaccine

Abstract

The present invention relates to a novel vaccine against severe fever with thrombocytopenia syndrome virus (SFTSV). According to the invention, after the SFTSV vaccine is injected in vivo, the formation speed of Gn and Gc proteins can be obviously accelerated. Besides, the vaccine can effectively stimulate humoral immune response, for example, a specific neutralizing antibody aiming at a virus antigen is induced to generate, so that a remarkable protection effect aiming at SFTSV is provided for an individual.

Description

Technical Field

[0001] The present invention relates to a novel vaccine against fever with thrombocytopenia syndrome virus (SFTSV). Background Art

[0002] Vaccines are currently developed and produced in a variety of ways. Specific types of vaccines include: live attenuated vaccines, which provide immunity by injecting a weakened version of the pathogen; toxoid vaccines, which are prepared by inactivating pathogenic substances (such as toxins) rather than the pathogen itself; subunit vaccines, which are made by extracting and using separately the antigenic determinants that are recognized as antigens; and recombinant vaccines, which are prepared using genetic engineering technology.

[0003] Among the above vaccine types, recombinant vaccines have significant advantages due to their safety, while nucleic acid vaccines, especially those based on mRNA technology, have shown a series of advantages, including rapid production cycles, low costs, and the ability to effectively induce cell-mediated immunity and humoral immunity responses. In addition, the design of vaccine components is flexible, and different antigenic components can be targeted by selecting and using specific antigenic determinants that can induce immune responses. Nucleic acid vaccines have a wide range of applications. They can be used not only to prevent or treat relatively mild diseases such as colds, influenza, chickenpox, and cold sores, but also to prevent or treat serious diseases with serious side effects and sequelae such as rabies, Ebola virus, Marburg virus, Crimean-Congo hemorrhagic fever, AIDS, avian influenza, SARS, COVID-19, and cancer.

[0004] However, up to now, no vaccine has been successfully developed for Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV). SFTS, or fever with thrombocytopenia syndrome, is transmitted by the bite of ticks carrying SFTSV (SFTS virus). It is a zoonosis that can be transmitted between humans and animals. People may suddenly develop the disease during daily activities such as mountaineering, walking, camping, farming and forestry. The disease has a high mortality rate. There is currently no vaccine or effective treatment. To prevent SFTSV, the best strategy is to avoid contact with ticks. Therefore, the need to develop an SFTSV vaccine is very urgent. Summary of the invention

[0005] In order to develop a nucleic acid vaccine against the fever with thrombocytopenia syndrome virus (SFTSV), the inventors prepared nucleic acid molecules containing N-terminal glycoprotein (N-terminal glycoprotein, Gn) and nucleic acid molecules containing C-terminal glycoprotein (C-terminal glycoprotein, Gc), respectively, and successfully converted these nucleic acid molecules into vaccine candidates. In addition, the experimental results have confirmed that each nucleic acid molecule contained in the above-mentioned vaccine has an excellent protective effect against the fever with thrombocytopenia syndrome virus (SFTSV), and these nucleic acid molecules can be efficiently expressed as proteins and effectively induce individuals to produce a high-intensity immune response against the virus, which marks the successful completion of the present invention.

[0006] Each description and implementation disclosed in the present invention is equally applicable to other descriptions and implementations. In other words, any combination of different elements disclosed in the present invention should be deemed to be included in the protection scope of the present invention. In addition, the protection scope of the present invention should not be limited to the content described in the specific embodiments below.

[0007] In addition, the terms not clearly defined in this specification should be interpreted as the meanings generally understood in the technical field to which they belong. In addition, unless otherwise indicated in the context, the singular form in the specification should be understood to include the plural form, and the plural form should also be understood to include the singular form.

[0008] According to one aspect of the present invention, a SFTSV vaccine is provided, comprising: a first nucleic acid molecule comprising the N-terminal glycoprotein (Gn) of Severe fever with thrombocytopenia syndrome virus (SFTSV); and a second nucleic acid molecule comprising the C-terminal glycoprotein (Gc) of SFTSV.

[0009] In this specification, the term "Severe fever with thrombocytopenia syndrome (SFTS)" refers to a disease caused by infection with the severe fever with thrombocytopenia syndrome virus (SFTSV). SFTSV is mainly manifested by fever, diarrhea, abdominal pain, headache, muscle aches, fatigue and loss of appetite, and the mortality rate of this disease is high.

[0010] The term “Severe fever with thrombocytopenia syndrome virus (SFTSV)” used herein refers to Dabebanda virus.

[0011] The terms “N-terminal glycoprotein (Gn)” and “C-terminal glycoprotein (Gc)” used herein refer to the structural domains of the glycoprotein (Glycoprotein) constituting SFTSV.

[0012] In this specification, the term glycoprotein may be used interchangeably with M segment, structural protein, Gn and Gc dimer, and Gn / Gc dimer.

[0013] The SFTSV vaccine according to the present invention is characterized in that it comprises a nucleic acid molecule comprising Gn and a nucleic acid molecule comprising Gc, respectively.

[0014] Specifically, the Gn of the SFTSV may include the amino acid sequence of sequence number 1, or consist of the amino acid sequence of sequence number 1. In addition, the Gc of the SFTSV may include the amino acid sequence of sequence number 2, or consist of the amino acid sequence of sequence number 2.

[0015] In the present invention, the first nucleic acid molecule may include an open reading frame (ORF) encoding Gn. In addition, the second nucleic acid molecule may include an ORF encoding Gc.

[0016] As used herein, the term "open reading frame (ORF)" refers to a nucleotide sequence located between a start codon and a stop codon.

[0017] In this specification, the terms "ORF encoding Gn" and "ORF encoding Gc" used refer to nucleotide coding sequences encoding Gn or Gc proteins. The coding sequence includes a start signal and a stop signal capable of directing the expression of nucleic acid molecules in individual cells or mammalian cells, and these signals can be operably connected to regulatory elements including a promoter and a polyadenylation signal. In addition, the coding sequence may further include a sequence encoding a signal peptide.

[0018] Specifically, the Gn of the SFTSV includes a nucleotide sequence of sequence number 3, or consists of a nucleotide sequence of sequence number 3. In addition, the Gc of the SFTSV may include a nucleotide sequence of sequence number 4, or consists of a nucleotide sequence of sequence number 4. In addition, the ORF encoding the SFTSV Gn may include a nucleotide sequence of sequence number 3, or consists of a nucleotide sequence of sequence number 3. In addition, the ORF encoding the SFTSV Gc may include a nucleotide sequence of sequence number 4, or consists of a nucleotide sequence of sequence number 4.

[0019] In this specification, the term "nucleic acid molecule" used refers to a molecule composed of a single or multiple nucleotides, and the term comprehensively includes DNA and RNA. Nucleotides are the basic building blocks of the nucleic acid molecules, including not only natural nucleotides, but also analogs modified by sugar or base. Examples of the modified analogs include, but are not limited to, pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, or a combination thereof. The sequence of the nucleic acid molecule of the present invention is allowed to vary, and the variation includes the addition, deletion, non-conservative substitution or modification of one or more nucleotides.

[0020] In the present invention, the first nucleic acid molecule and the second nucleic acid molecule may further include a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR) and a poly (A) tail.

[0021] In this specification, the term "5' untranslated region (5' untranslated region, 5' UTR)" used refers to the mRNA region immediately upstream of the start codon, which does not encode a polypeptide. In this specification, the term "3' untranslated region (3' UTR)" used refers to the mRNA region located downstream of the stop codon, which does not encode a polypeptide. In this specification, the term "poly (A) tail" used refers to a string of continuous adenine nucleotides attached to the 3' end of the mRNA. For example, the length of the poly (A) tail is 50 to 250 nucleotides.

[0022] Specifically, the first nucleic acid molecule comprising the 5'UTR, the ORF encoding SFTSV Gn, the 3'UTR and the poly(A) tail may include the nucleotide sequence of sequence number 5, or consist of the nucleotide sequence of sequence number 5. In addition, the second nucleic acid molecule comprising the 5'UTR, the ORF encoding SFTSV Gc, the 3'UTR and the poly(A) tail may include the nucleotide sequence of sequence number 6, or consist of the nucleotide sequence of sequence number 6.

[0023] In the present invention, considering the possible existence of variants with the same biological activity, all sequences mentioned in the present invention, including base sequences and amino acid sequences, should be understood to include those sequences having substantial identity with the sequences recorded in the Sequence Listing. The term "substantial identity" means that when the sequence of the present invention is compared with any other sequence to achieve the maximum match and the compared sequences are analyzed using an algorithm recognized in the art, the homology revealed is at least 60%, more specifically at least 70%, even more specifically at least 80%, and most specifically, the homology can reach 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In addition, in the electronic version of the sequence listing, the letter "t" represents thymine in DNA sequences and uracil in RNA sequences.

[0024] Therefore, any sequence having a high degree of homology with the sequences shown in sequence numbers 1 to 8 of the present invention, including but not limited to those having at least 70%, more specifically at least 80%, and even more specifically at least 90% homology with the sequences shown in sequence numbers 1 to 8, should be deemed to fall within the scope of protection of the present invention.

[0025] One or more nucleic acid molecules selected from the group consisting of the first nucleic acid molecule and the second nucleic acid molecule are contained in a vector or connected to a vector. The "vector" refers to a material that can appropriately deliver the mRNA vaccine to a designated location in vivo or ex vivo. The vector may include, but is not limited to, an immune adjuvant, an excipient, a lipid nanoparticle, and the like.

[0026] In this specification, the term "lipid nanoparticle" used refers to a particle having a nanometer-scale (e.g., 1 to 1,000 nanometers) diameter. Specifically, the nucleic acid molecules of the present invention can be encapsulated in lipid nanoparticles. Therefore, the nucleic acid molecules are protected from degradation by enzymes and can resist other adverse effects (e.g., adverse immune responses) that may be induced by the host organism or cellular mechanisms.

[0027] In the present invention, the lipid nanoparticles can be used to deliver nucleic acid molecules to target locations.

[0028] In addition, the composition of the lipid nanoparticles may include cationic lipids, non-cationic lipids, sterols, and PEG-modified lipids. As a specific example, the cationic lipids may be one or more selected from DLin-KC2-DMA (2,2-dilinoleyl-4-dimethylaminoethyl [1,3] -dioxolane), DLin-MC3-DMA (dilinoleyl-methyl-4-dimethylaminobutyrate), and L319 (di ((Z) -non-en-1-yl) 9- ((4- (dimethylamino) butanoyl) oxy) heptadecanedioate)), the non-cationic lipids may be one or more neutral lipids selected from DSPC, DPPC, POPC, DOPE, and SM, the sterol may be cholesterol, and the PEG-modified lipids may be one or more selected from PEG-DMG and PEG-cDMA, but are not limited thereto.

[0029] In addition, the composition of the lipid nanoparticles may include cationic lipids, neutral lipids, sterols and PEG-modified lipids, and the molar ratios thereof are approximately 20% to 60%, 5% to 25%, 25% to 55% and 0.5% to 15%, respectively, but this ratio range is not limiting. As a specific example, the lipid nanoparticles may contain the following molar ratios of components: 25% to about 75% of cationic lipids, 0.5 to 15% of neutral lipids, 5 to 50% of sterols and 0.5 to 20% of PEG or PEG-modified lipids; 35% to about 65% of cationic lipids, 3 to 12% of neutral lipids, 15 to 45% of sterols and 0.5 to 10% of PEG or PEG-modified lipids; 45% to about 65% of cationic lipids, 5 to 10% of neutral lipids, 25 to 40% of sterols and 0.5 to 10% of PEG or PEG-modified lipids. % PEG or PEG-modified lipids; about 60% cationic lipids, about 7.5% neutral lipids, about 31% sterols and about 1.5% PEG or PEG-modified lipids; about 50% cationic lipids, about 10% neutral lipids, about 38.5% sterols and about 1.5% PEG or PEG-modified lipids; about 50% cationic lipids, about 10% neutral lipids, about 35% sterols and about 4.5% or about 5% PEG or PEG-modified lipids, and about 0.5% targeting lipids; 40% cationic lipids, about 15% neutral lipids, about 40% sterols and about 5% PEG or PEG-modified lipids, but this ratio range is not limiting.

[0030] In the present invention, the lipid nanoparticles can be used for imaging. For example, the lipid nanoparticles can be liposomes, and the liposomes can contain gadolinium (III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-aminomethyl]-1,4,7,10-tetraazacyclodode-1-yl}-acetic acid, 7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-aminomethyl]-1,4,7,10-tetraazacyclodode-1-yl}-acetic acid, and neutral saturated phospholipids and other ingredients, but are not limited thereto.

[0031] In the present invention, the lipid nanoparticles may further contain nutrients, the deficiency of which may lead to health problems such as anemia, neural tube defects, etc. Specifically, the nutrients contained in the lipid nanoparticles may include, but are not limited to, ferrous iron, ferrous salts or elemental iron, iodine, folic acid, vitamins, and iron in the form of micronutrients.

[0032] In the present invention, the lipid nanoparticles can be in various forms, including but not limited to swellable nanoparticles, polyanhydride nanoparticles, water-soluble nanoparticles, inorganic nanoparticles, stealth nanoparticles or stealth nanoparticles with target specificity. As a specific example, the inorganic nanoparticles can have zwitterionic ligands, but are not limited thereto. In addition, the stealth nanoparticles or target-specific stealth nanoparticles may include a polymeric matrix, which includes but is not limited to polyethylene, polycarbonate, polyanhydride, polyhydroxyacid, polypropylfumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyphosphazene, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or a combination thereof.

[0033] In the present invention, the designed nanoparticles can be optimized by geometric engineering to adjust their targeting efficiency and / or immune response. By changing the shape, size and / or surface charge of the geometrically engineered particles, the nucleic acid molecules of the present invention can be effectively encapsulated to achieve targeted delivery, such as pulmonary delivery. The geometrically engineered particles have a variety of physical properties, including but not limited to fenestration, angled arms, morphological asymmetry, surface roughness, and charge that can adjust the ability to interact with cells and tissues.

[0034] In addition, the nanoparticle can be a nucleic acid hybrid structure with a high-density nucleic acid layer, which can not only be embedded in the core of the nanoparticle, but also coated on a low-density porous three-dimensional structure, in which its coating can carry or couple at least one payload and position it inside or on the surface of the nanostructure, but is not limited to this.

[0035] In this specification, the term "vaccine" refers to an immunogenic or antigenic substance, which, as a biological preparation, contains an antigen that can stimulate an individual's immune response. The vaccine is administered to an individual by injection or oral administration, etc., and is intended to prevent specific infectious diseases. The vaccine may be an RNA vaccine, specifically, an mRNA vaccine. The term "mRNA vaccine" refers to a vaccine that induces an immune response by artificially replicating part of the gene of the antigen and injecting it into the body. This mRNA vaccine has several advantages over existing protein vaccines: first, it can be synthesized only through the genetic information of the target antigen, so there is no need to directly contact dangerous pathogens; second, it only uses part of the gene required to induce an immune response, so there is no need to worry about special toxicity after injection; finally, due to its simple structure and consisting only of mRNA, vaccines for emerging infectious diseases or different mutations can be quickly developed.

[0036] The vaccine of the present invention may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" covers any solvent, dispersion medium, coating agent, antigen enhancer, stabilizer, excipient, diluent, preservative, antibacterial agent, antifungal agent, isotonic agent, delayed adsorbent, etc. Specific examples of the carrier include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerol, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0037] In addition, the vaccine of the present invention can be prepared into a variety of oral dosage forms according to conventional pharmaceutical methods, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., and can also be prepared into sterile injections. During preparation, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation.

[0038] The vaccine can be administered to an individual in a variety of ways. The “administration” can be administered subcutaneously, intramuscularly, intradermally, intraperitoneally, intravenously, nasally, transdermally, parenterally, orally, but is not limited thereto.

[0039] The vaccine may contain one or more adjuvants to improve or enhance the immune response. Suitable adjuvants include synthetic analogs of double-stranded RNA (dsRNA), unmethylated cytidine guanidine oligonucleotides, peptides, aluminum hydroxide, aluminum phosphate, aluminum oxide, mineral oil or vegetable oil such as Marcol 52, and a composition composed of one or more emulsifiers, lysolecithin, multivalent cations, multivalent anions and other surface active substances.

[0040] Another aspect of the present invention provides a method for eliciting an immune response against fever with thrombocytopenia syndrome virus in an animal, the method comprising the step of administering the SFTSV vaccine to a subject.

[0041] In the method according to the present invention, unless explicitly stated otherwise, the meanings of the terms are as defined above.

[0042] In this specification, the term "individual" refers to any entity that may be infected with a virus or has been infected with a virus, including but not limited to humans, non-human animals, fish, and plants, etc. The non-human animals may be vertebrates, such as primates, dogs, cows, horses, pigs, and rodents, such as mice, rats, hamsters, guinea pigs, etc. In this specification, the term "individual" may be used interchangeably with "subject" or "patient".

[0043] As used herein, the term "immune response" refers to the process by which an individual's immune system responds to the introduction of an antigen and is subsequently activated. In this case, the immune response may be in the form of cell-mediated immunity, humoral immunity, or both.

[0044] In the method of generating an immune response, the SFTSV vaccine of the present invention may comprise an effective dose of an active ingredient, i.e., a nucleic acid molecule of the present invention, and a pharmaceutically acceptable carrier and adjuvant. The term "effective dose" refers to the necessary dose that can induce a specific immune response against viral infection in a vaccinated individual. Those skilled in the art can easily determine the effective dose, for example, by conducting standard animal experiments.

[0045] In this specification, the term "generating an immune response against SFTSV" refers to the process of providing protective immunity and / or vaccination to a subject in order to prevent SFTSV infection. The term also includes stimulating an expected immune response or immune effect against SFTSV infection in an individual in need for preventive purposes. In this specification, "generating an immune response" and "inducing an immune response" can be used interchangeably. The "protective immunity" or "protective immune response" means that after vaccination, an individual is able to effectively control an infection caused by the pathogen targeted by the vaccine. Generally speaking, when an individual develops a protective immune response, they usually only experience mild to moderate clinical symptoms, or even show no symptoms.

[0046] According to the present invention, an effective dose of the SFTSV vaccine can be administered by intramuscular or subcutaneous injection, and can also be administered by intravenous, intradermal, dermal or intranasal administration. For example, for intramuscular injection, the SFTSV vaccine can be administered by conventional needle injection, or a needle-free injection device (such as a biological syringe) can be used as an alternative.

[0047] The SFTSV vaccine can be formulated into preparations suitable for different routes of administration, and can be formulated without adding an immune adjuvant as needed. For example, a person skilled in the art can use an isotonic vehicle, such as sodium chloride injection, Ringer's injection or lactated Ringer's injection, to prepare a suitable solution, and if necessary, preservatives, stabilizers, buffers, antioxidants and other additives can also be added. As a specific example, the SFTSV vaccine may include the above-mentioned first nucleic acid molecule and the second nucleic acid molecule, and can be formulated into lipid nanoparticles containing MC3, cholesterol, DSPC and PEG2000-DMG, as well as a preparation of trisodium citrate buffer, sucrose and water for easy administration. As another specific example, the formulation of the SFTSV vaccine may include the following ingredients, 2.0 mg / mL of each nucleic acid molecule (i.e., a nucleic acid molecule comprising an ORF encoding SFTSV Gn and a nucleic acid molecule comprising an ORF encoding SFTSV Gc), 21.8 mg / mL of MC3, 10.1 mg / mL of cholesterol, 5.4 mg / mL of DSPC, 2.7 mg / mL of PEG2000-DMG, 5.16 mg / mL of trisodium citrate buffer, 71 mg / mL of sucrose, and 1.0 mL of water, for preparing a formulation for administration.

[0048] In the present invention, the SFTSV vaccine can be injected in multiple doses, specifically, including priming immunization and / or boosting administration. There is no particular restriction on the time and number of priming immunization and boosting immunization. For example, the SFTSV vaccine can be used for priming immunization, and before the boosting immunization vaccine is injected, the priming immunization can be performed again. In addition, it is also possible to consider additional inoculation of the SFTSV vaccine to achieve further immune enhancement. As a specific embodiment, the boosting immunization can be performed within 1 to 12 weeks after the priming immunization (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks), and the boosting immunization can also be performed within 12 to 52 weeks after the first vaccination (for example, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 48, 50 or 52 weeks). Those skilled in the art can appropriately adjust the timing, frequency and dosage of the initial immunization and the booster immunization based on the technical content disclosed in the present invention and existing technical knowledge.

[0049] Another aspect of the present invention provides a method for treating or preventing fever with thrombocytopenia syndrome virus, the method comprising the step of administering the SFTSV vaccine to a subject.

[0050] In the method according to the present invention, unless explicitly stated otherwise, the meanings of the terms are as defined above.

[0051] In the present specification, the term "treatment" used refers to any measure to alleviate or cure the symptoms caused by the SFTSV virus by administering the SFTSV vaccine of the present invention.

[0052] In the present specification, the term "prevention" used refers to any measure to prevent or delay the occurrence of fever with thrombocytopenia syndrome by vaccinating the SFTSV vaccine of the present invention.

[0053] According to the present invention, the SFTSV vaccine can significantly accelerate the formation rate of Gn and Gc proteins after in vivo injection. In addition, the vaccine can effectively stimulate humoral immune response, such as inducing the production of specific neutralizing antibodies against viral antigens, thereby providing individuals with significant protection against SFTSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The results of electrophoresis analysis of mRNA containing N-terminal glycoprotein (N-terminal glycoprotein, Gn) and mRNA containing C-terminal glycoprotein (C-terminal glycoprotein, Gc) are shown.

[0055] Figure 2a Western blot analysis results of proteins expressed by mRNA containing Gn are shown. The position of Gn protein is indicated by an arrow.

[0056] Figure 2b The results of Western blot analysis of proteins expressed by mRNA containing Gc are shown. The position of the Gc protein is indicated by an arrow.

[0057] Figure 3 It is a schematic diagram of the immunization experiment of the SFTSV vaccine of the present invention.

[0058] Figure 4A graph showing the measurement of neutralizing antibodies produced against SFTSV after vaccination with the SFTSV vaccine of the present invention (named "Ver1") is shown, wherein the PRNT (Plaque Reduction Neutralization Test) 50 value has been converted to a logarithmic value. These results represent experimental data 2 weeks after the first vaccination (recorded as 1st 2w), 2 weeks after the second vaccination (recorded as 2nd 2w), and 4 weeks after the second vaccination (recorded as 2nd 4w).

[0059] Figure 5a A graph showing the level of binding antibodies induced against the Gn protein of SFTSV after vaccination with the SFTSV vaccine of the present invention (named "Ver1") is shown. These results represent experimental data 2 weeks after the first vaccination (recorded as 1st 2w), 2 weeks after the second vaccination (recorded as 2nd 2w), and 4 weeks after the second vaccination (recorded as 2nd 4w).

[0060] Figure 5b A graph showing the level of binding antibodies induced against the Gc protein of SFTSV after vaccination with the SFTSV vaccine of the present invention (named "Ver1") is shown. These results represent experimental data 2 weeks after the first vaccination (recorded as 1st 2w), 2 weeks after the second vaccination (recorded as 2nd 2w), and 4 weeks after the second vaccination (recorded as 2nd 4w).

[0061] Figure 6 It is a schematic diagram of the immune effect of the SFTSV vaccine of the present invention and the SFTSV aggressive vaccination experiment.

[0062] Figure 7 A graph showing the survival rate of mice after vaccination with the SFTSV vaccine of the present invention (designated as "Ver1") and after SFTSV challenge vaccination is shown. "N" represents the result of the non-vaccinated group.

[0063] Figure 8 The graph shows the change in body weight of mice after vaccination with the SFTSV vaccine of the present invention and after SFTSV challenge vaccination. "N" represents the result of the non-vaccinated group.

[0064] Fig. 9 A graph showing the number of viral copies in the organs of mice after vaccination with the SFTSV vaccine of the present invention and after SFTSV challenge vaccination is shown. "Empty LNP" represents the result of the unvaccinated group. DETAILED DESCRIPTION

[0065] The present invention will be described in more detail below by the following examples, but these examples are not intended to limit the scope of protection of the present invention. In addition, those skilled in the art can make various modifications and variations to the present invention without departing from the basic spirit and essence of the present invention.

[0066] Example 1. Production of vaccines Example 1-1. Preparation of DNA template for in vitro transcription (IVT) Two mRNAs were prepared by in vitro transcription (IVT): the first mRNA contained the N-terminal glycoprotein (Gn) of severe fever with thrombocytopenia syndrome virus (SFTSV); the second mRNA contained the C-terminal glycoprotein (Gc) of SFTSV. The DNA sequences of Gn and Gc of SFTSV were determined based on public information in the NCBI GenBank database. The first and second mRNAs were specifically designed and prepared to ensure that they contained an open reading frame (ORF) encoding the N-terminal glycoprotein (Gn) and an ORF encoding the C-terminal glycoprotein (Gc).

[0067] Specifically, the DNA template for in vitro transcription (IVT) was designed to contain the following elements: 1) T7 promoter; 2) 5' untranslated region (5'UTR); 3) open reading frame (ORF) of Gn or Gc derived from SFTSV glycoprotein; 4) 3' untranslated region (3'UTR); and 5) a 120-base poly A tail (poly A). The nucleotide sequence of the DNA template containing SFTSV Gn protein is shown in sequence number 7 in the sequence list, while the nucleotide sequence of the DNA template containing SFTSV Gc protein is shown in sequence number 8 in the sequence list. The 5'UTR and 3'UTR sequences are derived from betaglobin of African clawed frog (Xenopus laevis), and these sequences have been confirmed through public data on NCBI GenBank.

[0068] The DNA template was synthesized by GenScript and successfully subcloned into the pUC57-Kan plasmid vector. Further, to confirm the expression of mRNA, we performed a standard PCR amplification method using Q5 high-fidelity DNA polymerase (New England Biolabs) and inserted a DNA fragment with a FLAG tag (DYKDDDDK) into the 3' end of each ORF (Gn, Gc, or GFP).

[0069] Example 1-2. In vitro transcription (IVT) The plasmid vector prepared in Example 1-1 was linearized using restriction enzyme BspQI (New England Biolabs). Subsequently, for in vitro transcription (IVT), the following mixture was prepared: 5 mM ATP, 5 mM CTP, 5 mM GTP, 5 mM m1Ψ, 4 mM SmartCap (SC101, STPharm), 1 μg of linearized plasmid vector (i.e., linearized template DNA) or 0.5 μg of PCR product, and 2 μL of T7 RNA polymerase mixture. 20 μL of the reaction solution was incubated at 37°C for 2 hours for in vitro transcription (IVT). 2 units of DNase I (ThermoFisher) were used for treatment at 37°C for 15 minutes to remove the template DNA remaining after the in vitro transcription reaction, followed by column purification.

[0070] The in vitro transcription (IVT) products of Gn and Gc were analyzed by agarose gel electrophoresis. The base length analysis results of Gn and Gc mRNA are detailed in Figure 1 .

[0071] Example 1-3. Transfection and Western Blotting The mRNA prepared in the above Example 1-2 was transfected into 293FT cells or SJCRH30 cells (CRL-2061, ATCC). During transfection, mRNA was mixed with Lipofectamine MessengerMax (ThermoFisher) at a ratio of 1:2, i.e., 1 μg mRNA with 2 μl Lipofectamine MessengerMax, or 2 μg mRNA with 4 μl Lipofectamine MessengerMax. 24 hours after transfection, cell lysates were prepared using NP-40 cell lysis buffer (150 mM sodium chloride, 1% NP-40 / 50 mM Tris pH 8.0).

[0072] Protein analysis was performed using rabbit anti-Gn antibody (NBP2-41153, Novus Biologicals), rabbit anti-Gc antibody (NBP2-41156, Novus Biologicals), mouse anti-FLAG antibody (MA1-91878, ThermoFisher), HRP-labeled secondary antibodies (HAF007 and HAF008, R&D Systems), and substrate (SuperSignal West Pico Plus Chemiluminescent, ThermoFisher).

[0073] The expression of Gn protein was analyzed in 293FT cells transfected with Gn mRNA, Gc mRNA, or a mixture of Gn and Gc mRNA (Gn+Gc mRNA) and FLAG-tagged Gn mRNA (Gn-FLAG mRNA). Figure 2a The arrow in the figure indicates the expressed Gn protein. At this time, as a control group, purified Gn protein (Gn purified, PV-GP-40p, eEnzyme) was used.

[0074] In addition, the expression of Gc protein was analyzed in 293FT cells transfected with FLAG-tagged Gc mRNA (Gc-FLAG mRNA). Figure 2b The arrow in the figure indicates the expressed Gc protein. As a control group, the cell lysate obtained by transfecting GFP mRNA with a FLAG tag (i.e., GFP-FLAG mRNA) was used.

[0075] Example 2. Effect of vaccine Example 2-1. Immunogenicity The immunogenicity of the SFTSV vaccine containing the mRNA of SFTSV Gn and the mRNA of SFTSV Gc prepared in Example 1 above was evaluated. Immunogenicity refers to the ability of an antigen to induce an immune response in vivo. In order to analyze the immunogenicity of the SFTSV vaccine, this example detected whether the vaccine can produce neutralizing antibodies or binding antibodies against SFTSV in vivo. Neutralizing antibodies can neutralize antigens before they infect host cells without the assistance of other immune cells, thereby preventing reinfection. Binding antibodies cannot neutralize antigens, but they can induce immune responses in immune cells after infection, thereby destroying antigens.

[0076] Eight-week-old female C57BL / 6 mice (Narabiotech, Seoul, Korea) received low-dose (2 μg), medium-dose (10 μg), and high-dose (20 μg) SFTSV vaccines by intramuscular injection, with vaccinations every three weeks, at week 0 and week 3 from the start of the experiment, for a total of two immunizations. After each immunization, serum was collected three times, at week 2, week 5, and week 7 from the start of the experiment. Schematic diagram of this experiment is shown in Figure 3 The antibody titers against SFTSV Gn and Gc in the serum were analyzed by enzyme-linked immunosorbent assay (ELISA) or plaque reduction neutralizing test (PRNT).

[0077] like Figure 4 As shown in the figure, mice vaccinated with the SFTSV vaccine produced higher neutralizing antibody titers against Gn and Gc proteins. Specifically, after only one vaccination with the SFTSV vaccine, high neutralizing antibody titers were shown, and after the second vaccination, the neutralizing antibody titers remained at a similar level as after the first vaccination. Except for the low dose used for the first time, the neutralizing antibody titers at the medium and high doses were maintained at similar levels.

[0078] In addition, if Figure 5a and Figure 5b As shown, mice vaccinated with the SFTSV vaccine produced higher binding antibody titers to Gn and Gc proteins. Specifically, mice showed higher binding antibody titers after a single SFTSV vaccination, and after the second vaccination, the binding antibody titers increased significantly, 10 to 100 times or more than the first vaccination. In particular, after the second vaccination, the binding antibody titers remained at similar levels, whether 2 weeks after vaccination (5 weeks after the start of the experiment) or 4 weeks after vaccination (7 weeks after the start of the experiment), and remained at similar levels when using medium and high doses of the vaccine, except when using low doses of the vaccine.

[0079] The results showed that after vaccination with the SFTSV vaccine of the present invention, neutralizing antibodies and binding antibodies against SFTSV can be rapidly induced within two weeks, and these antibodies are maintained at a high level for a long time after vaccination, showing an excellent humoral immune response, which indicates that the SFTSV vaccine can not only induce an immune response to SFTSV, but also help prevent reinfection with SFTSV.

[0080] Example 2-2. Protective effect against SFTSV infection The protective effect against SFTSV infection was evaluated in mice vaccinated with the SFTSV vaccine comprising the first mRNA of SFTSV Gn and the second mRNA of SFTSV Gc prepared in Example 1 above.

[0081] Eight-week-old female and male IFNAR- / -C57BL / 6 mice (Jacksonlab, Barharbor, Marine, USA) received low-dose (2 μg) and high-dose (20 μg) SFTSV vaccines by intramuscular (IM) injection, with vaccination every three weeks, at week 0 and week 3 from the start of the experiment, for a total of two immunizations. At week 5 after each immunization (i.e., week 5 after the start of the experiment), the mice were challenged with 1,000 pfu of the 2015-JJ01 (Bgenotype) virus strain. After infection, the survival rate and body weight changes of the mice were monitored daily, and the mice were killed at week 7 after infection, and their livers, kidneys, and spleens were subsequently harvested. For a schematic diagram of this experiment, see Figure 6 .

[0082] The experimental results show that Figure 7 As shown, mice that were not vaccinated with the SFTSV vaccine all died within 7 days of infection, while mice that were vaccinated with the SFTSV vaccine all survived within 15 days of infection. The survival rate of mice was the same whether they received a low dose or a high dose of the SFTSV vaccine.

[0083] In addition, if Figure 8 As shown in the figure, mice that were not vaccinated with SFTSV continued to lose weight after infection until they eventually died; in contrast, mice that were vaccinated with SFTSV maintained their weight at the same level before infection 15 days after infection. Whether they received a low or high dose of SFTSV vaccine, the weight loss of mice was equally suppressed.

[0084] In addition, if Fig. 9 As shown in the figure, the SFTSV viral load in the liver and spleen of mice not vaccinated with SFTSV vaccine was significantly above the detection limit, while the SFTSV viral load in the liver, kidney and spleen of mice vaccinated with SFTSV vaccine was almost undetectable. Whether receiving a low dose or a high dose of SFTSV vaccine, the inhibitory effect on viral proliferation was the same.

[0085] The results show that vaccination with the SFTSV vaccine of the present invention can quickly inhibit the proliferation and replication of the virus in the body, thereby producing a significant protective effect against SFTSV infection. It is worth noting that the vaccine can quickly achieve this effect with only one vaccination, which indicates that the SFTSV vaccine can effectively prevent the occurrence of SFTSV infection.

[0086] [Sequence Listing] Electronic file of sequence listing attached to the attachment (C:\KipoNet\NKEditor\appfile\DP23318KR.xml).

Claims

1. A SFTSV vaccine comprising: A first nucleic acid molecule comprising an N-terminal glycoprotein (Gn) of Severefever with thrombocytopenia syndrome virus (SFTSV); and The second nucleic acid molecule comprises the C-terminal glycoprotein (Gc) of SFTSV.

2. The SFTSV vaccine according to claim 1, wherein The Gn of the SFTSV comprises the amino acid sequence of sequence number 1.

3. The SFTSV vaccine according to claim 1, wherein The Gc of the SFTSV comprises the amino acid sequence of sequence number 2.

4. The SFTSV vaccine according to claim 1, wherein The Gn of the SFTSV comprises a nucleotide sequence of sequence number 3.

5. The SFTSV vaccine according to claim 1, wherein The Gc of the SFTSV comprises the nucleotide sequence of sequence number 4.

6. The SFTSV vaccine according to claim 1, wherein The first nucleic acid molecule includes an open reading frame (ORF) encoding Gn.

7. The SFTSV vaccine according to claim 1, wherein The second nucleic acid molecule includes an ORF encoding Gc.

8. The SFTSV vaccine according to claim 1, wherein The first nucleic acid molecule and the second nucleic acid molecule comprise one or more additional nucleic acid molecules selected from the group consisting of a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR) and a poly(A) tail.

9. The SFTSV vaccine according to claim 8, wherein The poly(A) tail is 50 to 250 nucleotides in length.

10. The SFTSV vaccine according to claim 1, wherein The first nucleic acid molecule comprises the nucleotide sequence of sequence number 5.

11. The SFTSV vaccine according to claim 1, wherein The second nucleic acid molecule comprises the nucleotide sequence of sequence number 6.

12. The SFTSV vaccine according to claim 1, wherein One or more nucleic acid molecules selected from the group consisting of the first nucleic acid molecule and the second nucleic acid molecule are contained in the vector or are linked to the vector.

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