KR102619943B1 - Severe fever with thrombocytopenia syndrome model animal and method for producing the same - Google Patents

Abstract

The present invention relates to a severe febrile thrombocytopenia syndrome animal model and a method for producing the same. The severe febrile thrombocytopenia syndrome animal model was prepared by administering an antibody (IFNAR Ab) that inhibits the action of the interferon receptor and SFTSV under the conditions according to the present invention. Compared to WT mice, which previously did not show pathogenicity during SFTSV infection, susceptibility to SFTSV increased, and compared to the IFNAR ^-/- mouse, which died immediately upon SFTSV infection, it showed pathogenicity without causing death, and the pathology of SFTS was similar to that of humans. Because it exhibits clinical and clinical characteristics, it can be usefully used as an animal model for research on the pathological mechanism of SFTS and for screening treatments.

Description

Severe febrile thrombocytopenia syndrome animal model and method of manufacturing the same {SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME MODEL ANIMAL AND METHOD FOR PRODUCING THE SAME}

The present invention relates to an animal model of severe febrile thrombocytopenia syndrome and a method of producing the same.

Severe fever with thrombocytopenia syndrome (SFTS) is a serious disease accompanied by symptoms such as high fever, vomiting, diarrhea, thrombocytopenia, leukopenia, and multiple organ failure, and has 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). The causative agent of SFTS is SFTSV (severe fever thrombocytopenia syndrome virus) and belongs to the Bunyaviridae family. The Bunyavirus family is a negative-strand RNA virus containing three segments: Orthobunyavirus, Hantavirus, Nairovirus, Phlebovirus, and Tospovirus ( It consists of five genera, including Tospovirus. SFTSV belongs to the Phlebovirus genus, which also includes Rift valley fever virus. SFTSV was first reported in China in 2011 (Yu SFTSV is a ball-shaped virus with a diameter of 80-100 nm. This virus has three genes: a large (L) segment, a medium (M) segment, and a small (S) segment, which are single-stranded negative sense RNA segments. The SFTS virus is vectored by the small tick (Haemaphysalis longicornis), which is known to be widespread in Korea (Chae JS et al. J Vet Sci 2008; 9: 285-93; Kim CM et al. Appl Environ Microbiol 2006;72:5766-76). SFTS virus seroconversion and viremia have been found in farmed animals such as goats, sheep, cattle, pigs, and dogs, and these animals are also believed to act as intermediaries in areas where the SFTS virus is spreading (Zhao L et al. Emerg Infect Dis 2013; 18: 963-5; Niu G et al. Emerg Infect Dis 2013; 19: 756-63). SFTSV is detected in the patient's blood. In particular, the concentration of SFTSV is very high in the blood of critically ill patients, so human-to-human transmission is possible through blood (Tang X, Wu W, Wang H, et al. J Infect Dis 2013;207 :736-739.).

SFTS is a disease that can cause high fever, thrombocytopenia, multiple organ failure, and death upon infection in humans. However, since the exact pathological mechanism has not been revealed, there is a need for an animal model that shows symptoms similar to humans to study the pathological mechanism. has come to the fore. However, wild type (WT) mice do not show pathogenicity when infected with SFTSV, and transgenic mice lacking the genome of the type I interferon receptor, a key element in virus defense (Type I Interferon receptor Knockout, (IFNAR ^-/- ) dies immediately even if infected with a very small amount of SFTSV, and research on SFTSV in small animal models is very limited, so the establishment of an animal model is urgently needed.

The purpose of the present invention is to provide a composition for manufacturing a severe febrile thrombocytopenia syndrome model.

Additionally, the purpose of the present invention is to provide a method for producing an animal model of severe febrile thrombocytopenia syndrome.

Additionally, the purpose of the present invention is to provide an animal model for severe febrile thrombocytopenia syndrome.

Additionally, an object of the present invention is to provide a method for screening a treatment for severe fever with thrombocytopenia syndrome.

In addition, the purpose of the present invention is to provide a method for testing the effectiveness of a treatment for severe fever with thrombocytopenia syndrome.

In order to solve the above problems, the present invention provides a composition for preparing a SFTS model containing SFTSV and IFNAR as active ingredients.

Additionally, the present invention provides a method for producing a severe febrile thrombocytopenia syndrome animal model.

Additionally, the present invention provides an animal model for severe febrile thrombocytopenia syndrome.

Additionally, the present invention provides a method for screening for a treatment for severe fever with thrombocytopenia syndrome.

In addition, the present invention provides a method for testing the effectiveness of a treatment for severe fever with thrombocytopenia syndrome.

The severe febrile thrombocytopenia syndrome animal model prepared by administering SFTSV and an antibody (IFNAR Ab) that inhibits the action of the interferon receptor under the conditions according to the present invention is susceptible to SFTSV compared to WT mice, which did not show pathogenicity during SFTSV infection. Compared to the IFNAR ^-/- mouse, which dies immediately upon SFTSV infection, it shows pathogenicity without causing death and shows pathological and clinical characteristics of SFTS similar to those of humans, so this can be used to study the pathological mechanism of SFTS and screen for therapeutic agents. It can be useful as an animal model.

Figure 1 is a graph showing the survival rate, weight loss, and body temperature of three groups of mice with different type I interferon susceptibility after SFTSV infection (inoculation):
A: Survival rate of mice by infection concentration of SFTSV; and
B: Graph of body temperature and weight changes _during ⁵
Figure 2 shows white blood cells (WBC) (A), platelets (B), red blood cells (RBC) (C), and hematocrit (D) of three groups of mice with different type I interferon susceptibility after SFTSV infection. ) This is a diagram showing the change graph.
Figure 3 is a graph showing the SFTSV virus copy number measurement in each organ of three groups of mice with different type I interferon susceptibility after SFTSV infection.
Figure 4 is a diagram confirming the viral load detected in oral and eye swabs, and urine and stool samples collected from SFTSV-infected mice.
Figure 5 shows the results of histological observation of pathological changes in the liver (A) and small intestine (B) and the ratio of villi to crypts (C) in three groups of mice with different type I interferon susceptibility after SFTSV infection. This is a diagram showing .
Figure 6 shows the results of histopathological changes (A) and immunohistochemical staining analysis (B) of SFTSV NP antigen-positive cells in the spleens of three groups of mice with different type I interferon susceptibility after SFTSV infection. .

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

As used herein, the terms “step of” or “step of” do not mean “step for.”

In this specification, the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more components selected from the group consisting of the components described in the Markushi format expression, It means including one or more selected from the group consisting of.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.

In one aspect, the present invention provides a severe fever with thrombocytopenia syndrome (SFTS) animal model comprising severe fever with thrombocytopenia syndrome virus (SFTSV) and interferon (IFN)-α receptor (IFNAR) inhibitor as active ingredients. It relates to a composition for manufacturing.

In one embodiment, the interferon-α receptor (IFNAR) may be a type I interferon (type I IFN)-α receptor.

In one embodiment, the interferon-α receptor (IFNAR) inhibitor may be an antibody or fragment thereof.

In one embodiment, the antibody or fragment thereof may be selected from the group consisting of recombinant antibodies, antibodies with reduced effector function, chimeric antibodies, humanized antibodies, and human antibodies.

In one embodiment, the antibody may be a monoclonal antibody.

In one embodiment, the fragment is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region It may be any one selected from the group consisting of fragments, humanized antibodies, chimeric antibodies, and diabodies.

In one embodiment, it may include 5 × 10 ⁵ to 5 × 10 ² FAID ₅₀ SFTSV, and when the animal model is a mouse, it is more preferable to include 5 × 10 ² FAID ₅₀ SFTSV.

In one embodiment, the composition can be administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously or as an inhalant, with intraperitoneal administration being more preferred.

In one embodiment, the animal may be one or more non-human animals selected from the group consisting of rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig, and goat, and is more preferably a mouse.

In one embodiment, the SFTSV and the IFNAR inhibitor may be administered simultaneously, separately, or sequentially, and it is more preferable to administer sequentially in the order of the IFNAR inhibitor, SFTSV, and the IFNAR inhibitor, and the animal If the model is a mouse, it is most preferable to infect with SFTSV 1 day after administering the IFNAR inhibitor and administer the IFNAR inhibitor 2 days later.

In the present invention, the term “injection” can be used interchangeably with “introduction”, “injection”, or “infection”.

In the present invention, the term “animal” or “laboratory animal” means any mammalian animal other than humans. The animals include animals of all ages, including embryos, fetuses, neonates, and adults. Animals for use in the present invention are available, for example, from commercial sources. These animals include laboratory animals or other animals, rabbits, rodents (e.g. mice, rats, hamsters, gerbils and guinea pigs), cattle, sheep, pigs, goats, horses, dogs, cats and birds (e.g. chickens, turkeys, ducks, geese), and primates (e.g., chimpanzees, monkeys, rhesus monkeys).

In the present invention, the term "animal model" refers to an animal model with a disease that is very similar to a human disease. The significance of disease animal models in research on human diseases is due to the physiological or genetic similarities between humans and animals. In disease research, biomedical animal models provide materials for research into various causes, pathogenesis, and diagnosis of diseases, enable understanding of disease-related pathogenic mechanisms through research on disease animal models, and provide information on the development of new drug candidates. Through actual efficacy and toxicity tests, basic data can be obtained to determine whether commercialization is possible.

In the present invention, the term “SFTS animal model” is not particularly limited and can be used in any form as long as it is suitable for evaluating the efficacy of SFTS test drugs. For example, any non-human animal such as a monkey, dog, cat, rabbit, rat, mouse, cow, sheep, pig, goat, etc. can be used, and for the purpose of the present invention, it can be a non-human animal in particular. Mammals such as monkeys, dogs, cats, rabbits, rats, mice, cows, sheep, pigs, goats, etc. have a stable supply of individuals with certain characteristics and are easy to raise and handle during experiments. Therefore, it is desirable as a model animal. Although a mouse is used in a specific embodiment of the present invention, it is not limited thereto. Breeding conditions before producing model animals can be selected according to conventional methods and are not particularly limited, but in order to obtain homogeneous model animals, it is desirable to breed each individual under the same conditions as possible.

In the present invention, the antibody is not only in the form of a whole antibody but also includes functional fragments of the antibody molecule. A full antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment that possesses an antigen-binding function. Examples of antibody fragments include (i) the variable region (VL) of the light chain, the variable region (VH) of the heavy chain, the constant region (CL) of the light chain, and Fab fragment consisting of the first constant region (CH1) of the heavy chain; (ii) Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. F(ab')2 fragment; (vii) single chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and VL domain to form an antigen binding site; (viii) bispecific single chain Fv dimer (PCT/US92/09965) and (ix) diabody WO94/13804, which is a multivalent or multispecific fragment produced by gene fusion.

The antibody or fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments thereof having immunological activity. The antibody may be recombinantly or synthetically produced.

A chimeric antibody is one in which the constant region of an animal-derived antibody, which causes an anti-isotype reaction, is replaced with the constant region of a human antibody using genetic engineering methods. Chimeric antibodies have significantly improved anti-isotype responses compared to animal-derived antibodies, but animal-derived amino acids still exist in the variable region, resulting in potential side effects on anti-idiotypic responses. I'm doing it. Humanized antibodies were developed to improve these side effects. It is produced by transplanting the CDR (complementary determining regions) region, which plays an important role in antigen binding, among the variable regions of a chimeric antibody, into the human antibody framework. The most important thing in CDR grafting technology to produce humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. To this end, use of an antibody database and crystal structure (crystal structure) Structure analysis, molecular modeling technology, etc. are used. However, even when the CDR region of an animal-derived antibody is transplanted onto an optimized human antibody framework, there are cases where amino acids that affect antigen binding are present in the framework of the animal-derived antibody, so antigen-binding ability is not preserved in many cases. Therefore, the application of additional antibody engineering technology to restore antigen binding ability can be said to be essential.

The antibody or fragment thereof may be isolated from a living body (not present in the living body) or non-naturally occurring, for example, may be produced synthetically or recombinantly.

In the present invention, the term “antibody” refers to a substance produced by stimulation of an antigen within the immune system, the type of which is not particularly limited, and can be obtained naturally or unnaturally (e.g., synthetically or recombinantly). there is. Antibodies are very stable not only in vitro but also in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, so their adhesion ability (avidity) is very high. A complete antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses. It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term "heavy chain" refers to a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to include both the full-length heavy chain including the hinge and fragments thereof. Additionally, the term "light chain" refers to both a full-length light chain and fragments thereof comprising a variable region domain V _L and a constant region domain C _L comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to mean inclusive.

In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and the variable region has complementary There are crystalline regions CDR1, CDR2 and CDR3. A framework region (FR) exists between the CDRs and serves to support the CDR ring. The “complementarity determining region” is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.

In the present invention, the term "scFv (single chain fragment variable)" refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single-chain antibody made by connecting the VH region and VL region of the antibody with a short peptide chain. refers to antibodies The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or understood from context. This is self-evident to those skilled in the art.

In the present invention, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope.

In the present invention, the term "specifically binds" or "specifically recognizes" has the same meaning commonly known to those skilled in the art, and means that an antigen and an antibody specifically interact to produce an immunological reaction. .

In the present invention, the term “antigen-binding fragment” refers to a fragment of the entire structure of an immunoglobulin and a portion of a polypeptide containing a portion to which an antigen can bind. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab' or F(ab') 2, but is not limited thereto. Among the antigen-binding fragments, Fab has one antigen-binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (C _H1 ) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv (two-chain Fv) is a non-covalent bond in which the heavy chain variable region and a light chain variable region are connected, while a single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the short chain variable region through a peptide linker. They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" refers to a region contained in the heavy chain of an antibody, which exists between the C _H1 and C _H2 regions and functions to provide flexibility of the antigen binding site in the antibody. It means area. For example, the hinge may be derived from a human antibody, specifically, IgA, IgE, or IgG, such as IgG1, IgG2, IgG 3, or IgG4.

In one aspect, the present invention includes the steps of (a) administering an interferon-α receptor (IFNAR) inhibitor to a model animal for severe febrile thrombocytopenia syndrome, excluding humans; (b) infecting with 5 × 10 ⁵ to 5 × 10 FAID ₅₀ SFTSV; and (c) administering an interferon-α receptor (IFNAR) inhibitor. It relates to a method for producing a severe febrile thrombocytopenia syndrome animal model.

In one embodiment, the IFNAR inhibitor may be an antibody or fragment thereof.

In one embodiment, the animal may be one or more non-human animals selected from the group consisting of rat, mouse, guinea pig, monkey, dog, cat, rabbit, cow, sheep, pig, and goat, and is more preferably a mouse.

In one embodiment, when the severe febrile thrombocytopenia syndrome model animal is a mouse, it can be infected with 5 × 10 ² FAID ₅₀ SFTSV.

In one embodiment, SFTSV infection can be performed one day after administration of the IFNAR inhibitor.

In one embodiment, the administration of the IFNAR inhibitor in step (c) may be performed 2 days after the administration of the IFNAR inhibitor.

In one aspect, the present invention relates to an animal model of severe febrile thrombocytopenia syndrome produced by the method of the present invention.

In one embodiment, the severe febrile thrombocytopenia syndrome animal model of the present invention has a higher susceptibility to SFTSV than WT and a lower susceptibility to SFTSV than IFNAR knockout mice, showing the pathogenicity of SFTSV without mortality, and showing the pathological characteristics and clinical characteristics of SFTS similar to humans. characteristics can be expressed.

In one aspect, the present invention includes the steps of (a) injecting a severe febrile thrombocytopenia syndrome treatment candidate for testing purposes into the severe febrile thrombocytopenia syndrome animal model of the present invention; (b) evaluating the degree of relief or treatment of severe febrile thrombocytopenia syndrome in experimental group animals injected with the severe febrile thrombocytopenia syndrome treatment candidate for the purpose of the test and control animals not administered the severe febrile thrombocytopenia syndrome treatment candidate; and (c) when the degree of relief or treatment of severe febrile thrombocytopenia syndrome in the experimental group animals is increased compared to control animals, determining the treatment candidate as a treatment for severe febrile thrombocytopenia syndrome, severe febrile thrombocytopenia It is about a method of screening for a treatment for the syndrome.

In one embodiment, assessing the degree of relief or treatment of severe febrile thrombocytopenia syndrome may include evaluating clinical symptoms of severe febrile thrombocytopenia syndrome, viral load and viral copy number of organs, and level of viral antigen shedding.

In the present invention, “screening” refers to selecting a substance with a specific target property from a candidate group consisting of various substances using a specific manipulation or evaluation method. For the purpose of the present invention, the screening of the present invention involves treating the severe febrile thrombocytopenia syndrome animal model according to the present invention with a candidate substance for preventing or treating severe febrile thrombocytopenia syndrome, and determining the severity of severe febrile thrombocytopenia syndrome by the candidate substance. If the candidate substance is prevented, treated, or has an improved prognosis compared to the control substance, the candidate substance is determined as a preventive or therapeutic agent for severe febrile thrombocytopenia syndrome.

In the present invention, “treatment” or “alleviation” means any action that treats, improves, or beneficially changes the symptoms of severe febrile thrombocytopenia syndrome by administering a test substance or candidate substance. Specifically, “treatment” refers to an approach for obtaining a beneficial or desirable clinical outcome. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of disease extent, stabilization of the disease state (i.e., not worsening), delay or reduction in the rate of disease progression, disease state, etc. Improvement or temporary relief and alleviation (partial or total) of , including whether or not it is detectable. “Treatment” refers to both therapeutic treatment and prophylactic or preventive measures. The treatments include treatment required for disorders that have already occurred as well as disorders that are being prevented. “Palliating” a disease means reducing the extent and/or undesirable clinical signs of the condition and/or slowing or prolonging the time course of its progression compared to no treatment. It means losing.

Candidate compounds (or test substances) for the treatment of severe febrile thrombocytopenia syndrome analyzed by the screening method of the present invention may be single compounds, mixtures of compounds (e.g., natural extracts or cell or tissue cultures), or biopharmaceuticals (e.g., proteins). , antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes and DNAzymes). The test substances can be obtained from libraries of synthetic or natural compounds and methods for obtaining libraries of such compounds are known in the art.

In one aspect, the present invention includes the steps of (a) injecting a severe febrile thrombocytopenia syndrome treatment agent for testing purposes into the severe febrile thrombocytopenia syndrome animal model of the present invention; (b) evaluating the degree of relief or treatment of feverish thrombocytopenia syndrome in experimental group animals injected with a treatment for severe fever with thrombocytopenia syndrome for the purpose of the test and positive control animals administered with an existing treatment for severe fever with thrombocytopenia syndrome; and (c) comparing the degree of relief or treatment of feverish thrombocytopenia syndrome in the experimental group animals with that of positive control animals.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Construction of SFTSV pathogenic mouse model and confirmation of mortality rate

Mice were divided into three groups: WT, IFNAR Ab, and IFNAR ^-/- and administered SFTSV (Severe fever with thrombocytopenia syndrome virus) at four doses (KH1, 5 × 10 ⁵ to 5 × 10 ² FAID (fluorescence active infectious dose) ₅₀ ) was injected intraperitoneally. Specifically, the WT group used 6-week-old C57BL/6 female mice (WT, Samtako, Kyoung Gi-do, Korea), and the IFNAR ^-/- group used type 1 IFN receptor knockout (IFNAR ^-/- ) female mice. In the IFNAR Ab group, MAR1-5A3 (mouse anti-mouse IFNAR, IgG1) monoclonal antibody (MAb) was administered to C57BL/6 mice one day before SFTSV infection and 2 days post-infection (dpi) per mouse. 100 μg was injected intraperitoneally (IP). Body temperature was measured using rectal probes. Additionally, the body weight of each group of mice was measured and the survival rate was confirmed.

As a result, the WT mouse group did not show mortality at any dose of SFTSV infection, whereas the 5 × 10 ⁵ , 5 × 10 ⁴ and 5 × 10 ³ FAID ₅₀ groups of IFNAR Ab mice infected with SFTSV had mortality rates of 4 and 5, respectively. and showed a mortality rate of 100% at 7 dpi, with a steady weight loss until just before death (Figure 1a). However, even in the IFNAR Ab mouse group, when infected with 5 × 10 ² FAID ₅₀ SFTSV, mortality caused by SFTSV was not observed, and weight loss was recovered after 7 dpi (Fig. 1b). It is inferred that the results of such low-dose SFTSV infection are due to the half-life (5.2 days) of the IFNAR Ab. In addition, the IFNAR ^−/− group appeared to be immediately killed by SFTSV infection within 4 dpi, regardless of the dose of SFTSV (5 × 10 ⁵ to 5 × 10 ² FAID ₅₀ ) (Figure 1a). Although SFTSV-infected mice did not have high fever (Figure 1b), all mice that died showed decreased body temperature, wrinkled fur, anorexia, depression, gastrointestinal symptoms, and weight loss after virus infection. Through this, it was confirmed that the virus titer for producing the SFTSV pathogenic animal model was 5 × 10 ² FAID ₅₀ .

Example 2. Confirmation of hematological SFTSV susceptibility of SFTSV pathogenic mouse model

Referring to the results of Example 1, each group _of mice (20 each) was infected with ⁵ Hematological changes were confirmed in mice. Specifically, blood samples were collected by retro-orbital plexus puncture every 2 days and a complete blood count (CBC) was performed using an automatic blood cell counter (Exigo-Vet., Boule Medical AB Inc., Stockholm, Sweden). ) was analyzed.

As a result, the number of WBC (white blood cells) was within the standard range (2600-10,100/μL) in the WT group, but increased significantly between 4 and 10 dpi in the IFNAR Ab group and recovered after 12 dpi (Figure 2a). Additionally, severe thrombocytopenia was not found in any mice after SFTSV infection (Figure 2b). A pattern of decline in RBCs and hematocrit levels was observed in both groups by 12 dpi, with levels appearing significantly lower in the IFNAR Ab group compared to the WT group from 4 to 10 dpi (Figures 2c and d).

Example 3. Confirmation of viral load in SFTSV pathogenic mouse model organs

To confirm the pathogenesis of SFTSV, 5 × 10 ² SFTSV-infected mice (n = 4) from each group were necropsied at 1, 3, 7, 12, and 17 dpi, and the viral load in each organ was measured at RT- Evaluation was performed by real-time PCR (PCR). Specifically, SFTSV-infected mice were euthanized, organs (brain, lung, liver, spleen, kidney, small intestine (S.Intestine), and large intestine (L.Intestine)) were removed aseptically, and Hybrid-R™ (GeneAll, Seoul, Korea) was used to isolate total RNA from each organ. The isolated RNA was reverse transcribed into cDNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan), L-segment-based SFTSV-specific primers shown in Table 1 below, and qPCR SyGreen Mix (PCRbiosystems, Seoul, Korea). And the copy number of the virus was confirmed by RT-PCR using CFX96 Real-time PCR (Bio-Rad Laboratories, Hercules, CA).

Primer name Sequence (5'->3') SFTSV-L-F AACATCCTGGACCTTGCATC SFTSV-L-R CAATGTGGCCATCTTCTCCA

As a result, SFTSV RNA was detected in all organs at 1 dpi, but there was no significant difference in viral copy number between mouse groups (Figure 3). However, at 3 dpi, SFTSV RNA levels were significantly higher in all organs of mice in the IFNAR ^−/− group compared to the other two groups (p<0.05). Among organs in the IFNAR ^−/− group, the spleen showed the highest level of SFTSV RNA. SFTSV RNA virus levels were similarly detected in all organs of the WT and IFNAR Ab mouse groups. SFTSV RNA was not detectable after 12 dpi in the brain and lungs of the WT and IFNAR Ab mouse groups, and was not detectable after 17 dpi in the liver, kidney, small intestine, and large intestine. In serum samples, SFTSV RNA was significantly higher in the IFNAR ^−/− group compared to the IFNAR Ab group at 2 dpi, and SFTSV RNA was consistently detected in the WT and IFNAR Ab groups until 14 dpi.

Example 4. Confirmation of SFTS virus shedding in feces of SFTSV pathogenic mouse model

After infection of each group of mice with 5 × 10 ² FAID ₅₀ SFTSV, serum, eye and oral swabs, stool and urine samples were collected at 1, 3, 5, 7, 9, 11, 13, 15 and 17 dpi. , viral antigen shedding and viral load were evaluated. As a result, WT mice did not show clinical symptoms after SFTSV infection, but SFTS viral RNA was detected in eye and oral swabs, and urine and stool samples at 5 dpi (Figure 4). For the IFNAR Ab and IFNAR ^−/− groups, SFTSV RNA was detected in eye and oral swabs, and urine earlier (after 3 dpi) compared to WT mice, and the IFNAR Ab mice showed longer viral shedding compared to WT mice ( up to 7 dpi). The results show that SFTSV-infected mice can shed SFTS viral RNA regardless of clinical symptoms and that SFTS viral RNA can be released for a longer time in mice with downregulated IFN signaling.

Example 5. Pathological confirmation of SFTSV pathogenic mouse model

To determine the progression of SFTSV over time, mice from each group infected with 5 × 10 ² FAID ₅₀ SFTSV were necropsied at 1, 3, 7, 12, and 17 dpi, and each organ was subjected to histopathological and IHC examination. did. Specifically, the brain, lung, liver, spleen, kidney, small intestine, and large intestine of mice in each group were removed, and the tissues of each organ were fixed with 10% formalin and embedded in paraffin. Afterwards, the tissue was sectioned to a thickness of 6 μm with a microtome (HM-340E; Thermo Fisher Scientific Inc., Waltham, MA, USA) and placed on a slide. Sectioned tissues were stained with hematoxylin and eosin (H&E), and viral antigens were detected by immunohistochemistry (IHC) using a mouse monoclonal antibody against SFTSV N protein (NP) as the primary antibody. In addition, one of the main clinical symptoms of SFTS is gastrointestinal symptoms, and since gastrointestinal symptoms appeared in the SFTSV-infected IFNAR Ab group and the IFNAR ^-/- group, to histopathologically evaluate the small intestine of the two groups, The ratio of more than 20 villus/crypt heights (villus/crypt, V/C) was measured in each group of mice.

As a result, no histological abnormalities were found in WT mice, and lesions were observed more quickly in the IFNAR ^-/- mouse group among the two mouse groups in which IFN signaling was controlled. At 3 dpi, coagulation necrosis was observed in the liver, and in particular, white pulp atrophy was observed in the marginal zone of the spleen of IFNAR ^−/− mice (FIGS. 5A and 6A). At 7 dpi, coagulative necrosis and mononuclear inflammatory cell infiltration were observed around necrotic lesions in the liver of mice in the IFNAR Ab group, and white pulp atrophy was observed in the spleen. In addition, as a result of V/C confirmation, WT mice were found to have long, intact, and regular villi at all dpi, no histological changes in villi were observed, and the V/C ratio did not change statistically significantly (Figures 5b and c). However, mice in the IFNAR Ab group and the IFNAR ^−/− group showed shorter, damaged, and blunt villi at 3 dpi, and the V/C ratio was significantly reduced from 3 dpi. Unlike the IFNAR ^-/- mice, the villous morphology and V/C ratio of the IFNAR Ab mice recovered at 12 dpi similar to those at 1 dpi. In addition, as a result of immunohistochemistry (IHC) analysis performed at 3 dpi using spleen sections, which are the primary target organ of SFTSV and in which the largest amount of SFTS RNA was detected, the frequency of SFTSV NP antigen-positive cells was appeared in the order of IFNAR ^−/− , IFNAR Ab and WT groups, consistent with the qPCR results (Figure 6b). SFTSV N protein antigen-positive cells were rarely found in the white medulla regardless of mouse group, but were observed inside mononuclear cells in the marginal area of the spleen.

Through the results of the above examples, it was confirmed that no SFTSV-specific lesions were observed in WT mice during SFTSV infection experiments, and that death of IFNAR ^-/- mice occurred too quickly without sufficient time to study the disease. It was confirmed that in mice injected twice with the present invention's IFNAR Ab before and after infection, death or recovery patterns could be controlled depending on the inoculated virus concentration, and lesions similar to those in humans were observed. Therefore, it was confirmed that mice injected with IFNAR Ab twice before and after infection were conditions in which infection experiments could be maintained and additional mechanistic studies could be conducted.

Claims (14)

A composition for producing a severe fever with thrombocytopenia syndrome (SFTS) animal model containing severe fever with thrombocytopenia syndrome virus (SFTSV) and interferon (IFN)-α receptor (IFNAR) inhibitor as active ingredients. The composition for preparing an animal model of severe febrile thrombocytopenia syndrome according to claim 1, wherein the interferon-α receptor (IFNAR) inhibitor is an antibody or fragment thereof. The composition for producing a severe fever thrombocytopenia syndrome animal model according to claim 2, wherein the antibody or fragment thereof is selected from the group consisting of recombinant antibodies, antibodies with reduced effector functions, chimeric antibodies, humanized antibodies, and human antibodies. The composition for producing a severe febrile thrombocytopenia syndrome animal model according to claim 1, comprising 5 × 10 ⁵ to 5 × 10 ² SFTSV of FAID ₅₀ . The composition for producing a severe febrile thrombocytopenia syndrome animal model according to claim 1, wherein the composition is administered subcutaneously, intraperitoneally, intramuscularly, intraarterially, intravenously or as an inhalant. (a) administering an interferon-α receptor (IFNAR) inhibitor to a non-human model animal for severe febrile thrombocytopenia syndrome;
(b) infecting with 5 × 10 ⁵ to 5 × 10 ² SFTSV of FAID ₅₀ ; and
(c) A method for producing a severe febrile thrombocytopenia syndrome animal model comprising administering an interferon-α receptor (IFNAR) inhibitor. The method of claim 6, wherein the IFNAR inhibitor is an antibody or fragment thereof. The method of claim 6, wherein, when the model animal for severe febrile thrombocytopenia syndrome is a mouse, it is infected with 5 × 10 ² FAID ₅₀ SFTSV. The method of claim 6, wherein SFTSV infection is performed one day after administration of the IFNAR inhibitor. The method of claim 6, wherein the administration of the IFNAR inhibitor in step (c) is performed 2 days after the administration of the IFNAR inhibitor. Severe febrile thrombocytopenia syndrome animal model prepared by the method of claim 6. (a) injecting a severe febrile thrombocytopenia syndrome treatment candidate for testing purposes into the severe febrile thrombocytopenia syndrome animal model of claim 11;
(b) evaluating the degree of relief or treatment of severe febrile thrombocytopenia syndrome in experimental group animals injected with the severe febrile thrombocytopenia syndrome treatment candidate for the purpose of the test and control animals not administered the severe febrile thrombocytopenia syndrome treatment candidate; and
(c) Severe febrile thrombocytopenia syndrome, comprising the step of determining the treatment candidate as a treatment for severe febrile thrombocytopenia syndrome when the degree of relief or treatment of severe febrile thrombocytopenia syndrome in the experimental group animals is increased compared to control animals. Methods for screening therapeutics. The method of claim 12, wherein the evaluation of the degree of relief or treatment of severe fever thrombocytopenia syndrome evaluates the clinical symptoms of severe fever thrombocytopenia syndrome, the viral load and viral copy number of the organ, and the level of viral antigen excretion. Methods for screening for syndrome treatments. (a) injecting a treatment for severe febrile thrombocytopenia syndrome for testing purposes into the severe febrile thrombocytopenia syndrome animal model of Article 11;
(b) evaluating the degree of relief or treatment of feverish thrombocytopenia syndrome in experimental group animals injected with a treatment for severe fever with thrombocytopenia syndrome for the purpose of the test and positive control animals administered with an existing treatment for severe fever with thrombocytopenia syndrome; and
(c) A method of testing the effect of a treatment for severe fever with thrombocytopenia syndrome, comprising the step of comparing the degree of relief or treatment of fever with thrombocytopenia syndrome in the experimental group animals with that of the positive control animals.