Low density lipoprotein receptor modulation for treatment of infections

Abstract

Compositions and methods for the prevention or treatment of Crimean-Congo hemorrhagic fever virus (CCHFV) infection, or for the prevention or treatment of Crimean-Congo hemorrhagic fever are provided. The methods comprise administering to a subject infected with or at risk of infection a virus an agent that reduces the expression or activity of LDLR, or inhibits the interaction between low density lipoprotein receptor (LDLR) and Gc glycoprotein (Gc) of CCHFV in the subject.

Description

Background Technology

[0001] Crimean-Congo hemorrhagic fever virus (CCHFV) is a widely distributed tick-borne zoonotic virus belonging to the genus Orthonairovirus of the family Nairoviridae in the order Bunyavirales. It has been reported in more than 30 countries in Africa, Europe, and Asia. Although CCHFV infection is asymptomatic in most vertebrates, it can cause severe viral hemorrhagic fever in humans, with a mortality rate as high as 40% in confirmed cases.

[0002] Currently, there are no approved vaccines or specific anti-CCHFV drugs, thus limiting treatment options for CCHFV infection. Due to its significant public health risks and inadequate response measures, CCHFV has long been listed by the World Health Organization (WHO) as a priority pathogen for development in the context of public health emergencies.

[0003] CCHFV possesses a negative-sense, three-segment RNA genome composed of S, M, and L segments, encoding a nucleoprotein (NP), a glycoprotein precursor (GPC), and an RNA-dependent RNA polymerase (RdRP), respectively. The GPC encoded by M is co-translated and cleaved by cellular proteases, producing two structural glycoproteins, Gc and Gn, and three non-structural proteins, mucin, GP38, and NSm. Gc and Gn glycoproteins form locally ordered heterodimeric lattices on the viral surface, responsible for binding to cellular receptors and subsequent fusion of the viral envelope with the host cell membrane. Gc is a class II membrane protein and the only known target for neutralizing antibodies against CCHFV.

[0004] Effective therapies are needed to prevent and treat CCHFV infection. Summary of the Invention

[0005] CCHFV entry into target cells has been shown to occur via receptor-mediated endocytosis. Multivesicular bodies are sites of virus-endosome membrane fusion. However, the cellular receptor for CCHFV infection has remained unknown, significantly hindering the understanding of CCHFV-host interactions and the development of effective therapeutic strategies against CCHF. The inventors of this invention have thus identified the low-density lipoprotein receptor (LDLR) as a crucial entry receptor for CCHFV infection.

[0006] Knockout of the LDLR gene impaired viral infection in various CCHFV-susceptible human, monkey, and mouse cells, while infection was restored upon reconstitution of ectopically expressed LDLR. Mutagenesis studies have shown that the ligand-binding domain (LBD) of LDLR is essential for CCHFV infection. The LDLR LBD binds directly to the CCHFV glycoprotein Gc with high affinity, thereby supporting viral attachment and internalization into host cells. Soluble sLDLR-Fc fusion protein or anti-LDLR blocking antibodies impaired CCHFV infection in various susceptible cells. Furthermore, knockout of the LDLR gene or administration of LDLR blocking antibodies significantly reduced viral load, pathological effects, and death following CCHFV infection in mice. Therefore, pharmacological targeting of LDLR provides an effective strategy for the prevention and treatment of CCHFV infection, a cause of Crimean-Congo hemorrhagic fever.

[0007] Therefore, one embodiment of this disclosure provides a method for preventing or treating Crimean-Congo hemorrhagic fever virus (CCHFV) infection in a subject, or for preventing or treating Crimean-Congo hemorrhagic fever, comprising administering to the subject an agent that reduces the expression or activity of LDLR, or inhibits the interaction between the low-density lipoprotein receptor (LDLR) in the subject and the Gc glycoprotein (Gc) of CCHFV.

[0008] In some embodiments, the agent is selected from the group consisting of: (a) Soluble LDLR protein; (b) Anti-LDLR antibody or antigen-binding fragment; (c) Anti-Gc antibody or antigen-binding fragment; (d) Small molecule inhibitors of LDLR or Gc; (e) Repressive RNA that inhibits LDLR expression; (f) Gene-editing agents that reduce LDLR expression or activity; (g) Rapamycin; (h) Liver X receptor (LXR) agonists, LXR proteins, or polynucleotides encoding LXR proteins; (i) Retinol X receptor (RXR) agonists, RXR proteins, or polynucleotides encoding RXR proteins; (j) ZFP36 ring finger protein-like 1 (ZFP36L1) agonist, ZFP36L1 protein or polynucleotide encoding ZFP36L1 protein; (k) ZFP36 ring finger protein-like 2 (ZFP36L2) agonist, ZFP36L2 protein or polynucleotide encoding ZFP36L2 protein; (l) Progestins and AdipoQ receptor family member 3 (PAQR3) agonists, PAQR3 protein or polynucleotides encoding PAQR3 protein; (m) proprotein convertase subtilisin / kexin type 9 (PCSK9) agonist, PCSK9 protein or polynucleotide encoding PCSK9 protein; (n) Low-density lipoprotein; and (o) Agents or molecules or combinations thereof that directly or indirectly cause downregulation of the level of LDLR or its soluble extracellular domain or CCHFV binding activity.

[0009] In some embodiments, the soluble LDLR protein does not contain the transmembrane domain of the LDLR protein and contains at least one LDLR type A repeat sequence in the ligand-binding domain (LBD). In some embodiments, the soluble LDLR protein contains at least LDLR type A repeat sequence 1, LDLR type A repeat sequence 2, LDLR type A repeat sequence 3, LDLR type A repeat sequence 4, LDLR type A repeat sequence 5, LDLR type A repeat sequence 6, or LDLR type A repeat sequence 7. In some embodiments, the soluble LDLR protein further contains an IgG Fc domain.

[0010] In some embodiments, the anti-LDLR antibody or antigen-binding fragment binds to at least one LDLR type A repeat sequence in the ligand-binding domain (LBD). In some embodiments, the anti-LDLR antibody or antigen-binding fragment does not bind to the EGF-like domain of the LDLR protein.

[0011] In some embodiments, the agent is an anti-Gc antibody or an antigen-binding fragment. In some embodiments, the agent is a small molecule inhibitor of LDLR or Gc.

[0012] In some implementations, the repressive RNA is selected from the group consisting of miRNA, siRNA, shRNA, piRNA, asRNA, and antisense RNA.

[0013] In some embodiments, the agent is a gene-editing agent that reduces LDLR expression or activity. In some embodiments, the gene-editing agent comprises a CRISPR / Cas agent, a TALEN agent, or a zinc finger nuclease.

[0014] In some implementations, the agent is rapamycin.

[0015] In some embodiments, the agent is a liver X receptor (LXR) agonist, an LXR protein, or a polynucleotide encoding an LXR protein. In some embodiments, the LXR agonist is selected from the group consisting of oxysterol, hypochondroitin, T0901317, GW3965, and N,N-dimethyl-3β-hydroxycholenamide (DMHCA). In some embodiments, the oxysterol is selected from the group consisting of 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholesteric acid.

[0016] In some embodiments, the agent is a retinol X receptor (RXR) agonist, an RXR protein, or a polynucleotide encoding an RXR protein. In some embodiments, the RXR agonist is 9-cis-13,14-dihydroretinoic acid.

[0017] In some embodiments, the agent is a protein selected from the group consisting of hepatic X receptor (LXR), retinol X receptor (RXR), ZFP36 ring finger protein-like 1 (ZFP36L1), ZFP36 ring finger protein-like 2 (ZFP36L2), progesterone and AdipoQ receptor family member 3 (PAQR3), and proprotein convertase subtilisin / kexin type 9 (PCSK9), or a polynucleotide encoding said protein. In some embodiments, said polynucleotide is cDNA or mRNA. In some embodiments, said mRNA is chemically modified. Attached Figure Description

[0018] Figures 1a-j demonstrate that LDLR is an important host factor for CCHFV infection: Figure 1a, screening for LDLR and LDLR-associated protein (LRP), which are crucial for CCHFV infection. 293T cells were edited with either control or sgRNAs targeting genes encoding LDLR and LRP (two sgRNAs per gene). After puromycin selection, the cell pool was infected with CCHFV (MOI=0.05) for 24 hours, followed by RT-qPCR. Data were normalized relative to CCHFV S mRNA levels in control sgRNA-edited cells. Figure 1b shows the surface expression of LDLR in different cell lines. The cell lines shown were evaluated by flow cytometry using an anti-LDLR mAb (R301-P). Figure 1c, CCHFV infectivity in different cell lines. The cell lines shown were inoculated with CCHFV (MOI=0.05) for 48 hours. CCHFV Gn-positive cells were examined by flow cytometry using anti-Gn antibody (clone 7A11, ABclonal). Figure 1d shows that LDLR overexpression enhances CCHFV infection in DLD1 cells. Control and LDLR-overexpressing DLD1 cells were infected with CCHFV (MOI=0.05) for 24 hours (left) or 48 hours (right). The levels of CCHFV S mRNA and NP protein were measured by RT-qPCR (left) and Western blotting (right), respectively. Figure 1e shows the effect of LDLR deficiency on CCHFV infection in SW13 cells. SW13 cells were edited with either a control (gNC) or three separate sgRNAs (gLDLR) targeting different regions of the LDLR coding sequence. Pools of SW13 cells edited with control and LDLR sgRNAs were infected with CCHFV (MOI=0.05). CCHFV NP expression (left panel, 48 hours post-infection), CCHFV S-segment mRNA levels (second panel, 24 hours post-infection), percentage of Gn-positive cells (third panel, 48 hours post-infection), and cytopathic effect (right panel, 72 hours post-infection) were measured by Western blotting, RT-qPCR, flow cytometry, and crystal violet staining, respectively. For bar graphs, data were normalized relative to data from control gRNA-edited cells. Figure 1f, Effect of LDLR deficiency on progeny virus production. SW13 cells were edited with either a control (gNC) or three separate sgRNAs (gLDLR) targeting different regions of the LDLR coding sequence. The sgRNA-edited SW13 cell pools were then infected with CCHFV (MOI=0.05) for 72 hours. The titer of progeny virus in the supernatant was measured by TCID50 assay. Data were normalized relative to data from control gRNA-edited cells. LOD, limit of detection. Figure 1g, Effect of LDLR deficiency on CCHFV infection in various cell types. Huh7, Vero E6, and Hepa1-6 cells were edited with control gRNA or gRNA targeting the LDLR gene as shown. Cells were infected with CCHFV (MOI=0.05) for 24 hours and then subjected to RT-qPCR. Data were normalized relative to CCHFV S mRNA levels in cells edited with control gRNA. Figure 1h, CCHFV infectivity in LDLR knockout SW13 and Huh7 cells. LDLR knockout SW13 and Huh7 monoclonal antibodies were isolated and confirmed by Western blotting (left panel). Control (gNC) or LDLR-deficient clones (gLDLR-C1) were infected with CCHFV (MOI=0.05) for 24 h, followed by RT-qPCR analysis. Data were normalized relative to data from each control gRNA-edited cell. Figure 1i, Ldlr - / - CCHFV infectivity in primary cells. From WT and Ldlr - / -Primary hepatocytes and lung fibroblasts (MLF) prepared from mice were incubated with CCHFV (MOI=0.05). The mRNA level of CCHFV S segment was measured by RT-qPCR (left panel, 48 hours post-infection) and the viral genome copy number in the supernatant (right panel, 72 hours post-infection). Figure 1j, Effect of LDLR deficiency on CCHFV, RVFV, EBIV, and VSV infection. Controls (gNC) or LDLR-deficient clones (gLDLR-C1) were inoculated with the virus shown 24 hours later, followed by RT-qPCR. Data are presented as mean ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0019] Figures 2a-f This indicates that LDLR is necessary for CCHFV infection: Figure 2a shows a schematic diagram of the full-length LDLR and its truncated mutants lacking the ligand-binding domain (ΔLBD) and epidermal growth factor-like domain (ΔEGF). Figures 2b-f show that the LBD of LDLR is important for CCHFV infection. Control (gNC) or LDLR-deficient (gLDLR-C1) SW13 cells were reconstructed using the control vector, full-length LDLR, or the truncated LDLR shown. Cells were seeded with CCHFV (MOI=0.05), and CCHFV NP expression (b, 48 h post-infection), CCHFV S-segment mRNA level (c, 24 h post-infection), percentage of CCHFV Gn-positive cells (d, 48 h post-infection), cell viability (e, 72 h post-infection), and CCHFV progeny virus production (f, 72 h post-infection) were measured by RT-qPCR, Western blotting, flow cytometry, crystal violet staining, and TCID50 assay. LOD is the limit of detection. Data are expressed as mean ± SEM. ****P < 0.0001; ns, not significant.

[0020] Figures 3a-e show that LDLR is crucial for CCHFV binding to cells: Figure 3a, Effect of LDLR on CCHFV attachment and internalization. Control (gNC), LDLR-deficient (gLDLR-C1), or LDLRAP1-deficient (gLDLRAP1) SW13 cells were incubated with CCHFV at 4°C for 1 hour (for binding assay), or subsequently at 37°C for 1 hour (for internalization assay). Cells were collected, and CCHFV S mRNA levels were measured by RT-qPCR. Data were normalized relative to CCHFV S mRNA levels in control gRNA-edited cells. Figure 3b shows the effect of LDLR blocking antibodies on CCHFV infection. SW13, Huh7, Vero E6, and Hepa1-6 cells were pre-incubated for 1 hour with rabbit anti-hLDLR monoclonal antibody (R301), goat anti-hLDLR polyclonal antibody (#AF2148), rabbit anti-mLDLR monoclonal antibody (R004), or their respective control IgG as shown, and then infected with CCHFV (MOI=0.05). Twenty-four hours post-infection, cells were collected for RT-qPCR analysis of CCHFV S mRNA levels. Data were normalized relative to cells treated with 0 µg / ml of their respective control IgG. Figure 3c shows the effect of LDLR blocking antibodies on RVFV, EBIV, and VSV entry. SW13 cells were pre-incubated for 1 hour with control rIgG or rabbit anti-hLDLR monoclonal antibody (R301) at the concentrations shown, and then infected with RVFV, EBIV, or VSV. Twenty-four hours post-infection, mRNA levels of the RVFV M segment, EBIV M segment, or VSV L gene were measured by RT-qPCR analysis. Data were normalized relative to data from cells treated with 0 µg / ml of their respective control IgG. Figure 3d shows that soluble human LDLR-Fc fusion protein (sohLDLR-Fc) inhibits CCHFV infection. CCHFV (MOI=0.05) was pre-incubated with the indicated concentration of Fc or sohLDLR-Fc for 1 h before infecting SW13 and Huh7 cells. CCHFV S mRNA levels were analyzed by RT-qPCR 24 h post-infection. Data were normalized relative to data from cells infected with untreated virus. Figure 3e shows the effect of sohLDLR-Fc on RVFV, EBIV, and VSV infection in SW13 cells. RVFV (MOI=0.1), EBIV (MOI=0.5), or VSV (MOI=0.1) were pre-incubated with the indicated concentrations of sohLDLR-Fc or Fc for 1 hour before infecting SW13 cells. Twenty-four hours post-infection, the mRNA levels of the RVFV M segment, EBIV M segment, or VSV L gene were measured by RT-qPCR analysis. Data were normalized relative to data from cells infected with their respective untreated viruses. Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ns, not significant.

[0021] Figures 4a-d show the direct binding of LDLR to CCHFV's Gc: Figure 4a shows the pull-down of CCHFV viral particles using shLDLR. CCHFV, biotinylated shLDLR, and magnetic streptavidin beads were co-incubated as shown. The precipitate was pulled down using a magnet and analyzed by RT-qPCR. Data are presented as mean ± SEM. Figure 4b shows monoclonal antibodies against CCHFV Gc (ADI 36121), Gn (JE12), or their respective control IgGs immobilized on a plate. Binding assays were performed using CCHFV, biotinylated shLDLR, and avidin-HRP based ELISA. Data are presented as mean ± SEM. Figure 4c shows the co-incubation of recombinant Gc from CCHFV YL16070 and IbAr 10200 strains with biotinylated shLDLR as indicated. A pull-down assay was performed using magnetic streptavidin beads, and the precipitate was immunoblotted with the indicated antibody. Figure 4d Biotinylated shLDLR was immobilized on a streptavidin biosensor. The binding parameters of recombinant Gc or Gn (YL16070 and IbAr 10200 strains) and VSV-G to LDLR were measured in PBS as shown by biolayer interferometry (BLI).

[0022] Figures 5a-d This indicates that LDLR is essential for the pathogenesis of CCHFV in mice. Figures 5a and 5b show that WT (n=11) and LDLR knockout (n=11) mice were pretreated with the anti-IFNAR1 monoclonal antibody MAR1-5A3 (200 µg / mouse) 24 hours before infection, and then infected with CCHFV (10 TCID50) via intraperitoneal route. Forty-eight hours post-infection, mice were administered 200 µg of MAR1-5A3. Body weight (a) and survival rate were monitored daily (b). Figures 5c and 5d show WT (n=7) and LDLR knockout (n=7) mice pretreated with anti-IFNAR1 monoclonal antibody MAR1-5A3 (200 µg / mouse) 24 hours before infection, followed by intraperitoneal infection with CCHFV (10 TCID50). Forty-eight hours post-infection, mice were administered 200 µg of MAR1-5A3. Necropsy was performed on days 3 and 5 post-infection, and livers and spleens were collected. Viral load was quantified by RT-qPCR, shown as viral RNA copies per microgram of organ or per milliliter of serum (c). H&E staining and anti-Gn mAb (7A11) immunostaining were performed (d). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0023] Figure 6a-e The results showed that anti-LDLR treatment could protect mice from the pathogenesis of CCHFV: Figure 6a, experimental flowchart. C57BL / 6 mice were pretreated intraperitoneally 24 hours before infection with anti-IFNAR1 monoclonal antibody MAR1-5A3 (300 µg / mouse) plus control rIgG (n=10, 100 µg / mouse) or LDLR blocking antibody (R004) (n=7, 100 µg / mouse). One hour before infection, a second dose of rIgG or R004 mAb (100 µg) was administered. Mice were challenged with CCHFV (100 TCID). 50 / animal, subcutaneously). Administer MAR1-5A3 (200 µg / animal) 24 hours after infection. Administer rIgG or R004 mAb 100 µg daily for 5 days post-infection. Figures 6b and 6c show the protective effect of LDLR blocking antibody against CCHFV-induced death. As shown in Figure a, C57BL / 6 mice were treated with rIgG (n=10) or LDLR blocking antibody (R004) (n=7) and challenged with CCHFV. The body weight (b) and survival rate of the mice were monitored daily (c). Figures 6d and 6e illustrate the protective effect of LDLR blocking antibodies against the pathogenesis of CCHFV. As shown in Figure a, C57BL / 6 mice were treated with rIgG (n=6) or LDLR blocking antibody (R004) (n=6) and challenged with CCHFV. On day 5 post-infection, mice were necropsies performed, and livers and spleens were collected. Viral load was quantified by qRT-PCR, shown as viral RNA copies per microgram of organ or per milliliter of serum (d). H&E staining and anti-Gn mAb (7A11) immunostaining were performed (e). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01.

[0024] Figures 7a-c The effect of LRP on CCHFV infection is shown: Figure 7a shows the effect of LRP knockdown on CCHFV infection in LDLR-deficient Huh7 cells. LDLR-deficient or control Huh7 cells were transfected with siRNA targeting LDLR or a member of the LRP family shown for 48 hours, then infected with CCHFV (MOI=0.05) for 24 hours, followed by RT-qPCR to measure CCHFV S mRNA levels. Figure 7b shows the effect of ectopic expression of LDLR and LRP on CCHFV infection in DLD1 cells. DLD1 cells were transiently transfected with LDLR and LRP family members shown for 24 hours, then infected with CCHFV (MOI=0.05) for 24 hours, followed by RT-qPCR to measure CCHFV S mRNA levels. Figure 7c shows the effect of soluble human RAP protein on CCHFV and SFV infection. LDLR-deficient or control Huh7 cells were pre-incubated with the indicated concentrations of RAP or Fc for 1 hour, followed by CCHFV or SFV infection. Twenty-four hours post-infection, cells were collected for RT-qPCR analysis of CCHFV S mRNA or SFV NSP1 mRNA levels. Relative infectivity was calculated by normalizing to mRNA levels in untreated cells.

[0025] Figures 8a-b The presence of LDL inhibits CCHFV infection in SW13 cells (a) and Huh7 cells (b). SW13 cells (Fig. 8a) and Huh7 cells (Fig. 8b) were infected with CCHFV (MOI = 0.05) or not in the presence of the indicated LDL concentration. CCHFV S mRNA levels were analyzed by RT-qPCR 24 hours post-infection. Data were normalized relative to those from cells infected with CCHFV in the presence of 0 µg / ml LDL. Detailed Implementation definition

[0026] It should be noted that the terms “a” or “an” entity refer to one or more of the same entity; for example, “an antibody” should be understood to represent one or more antibodies. Therefore, the terms “a” (or “an”), “one or more”, and “at least one” are used interchangeably herein.

[0027] As used herein, the term "peptide" is intended to encompass both single "peptides" and multiple "peptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "peptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Therefore, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to one or more chains of two or more amino acids are included within the definition of "peptide," and the term "peptide" may be used in place of or interchangeably with any of these terms. The term "peptide" is also intended to refer to products modified after peptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by amino acids not naturally occurring. Peptides may be derived from natural biological sources or produced through recombinant technologies, but are not necessarily translated from a specified nucleic acid sequence. They can be produced in any manner, including by chemical synthesis.

[0028] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be compared for comparative purposes. When positions in the compared sequences are occupied by the same bases or amino acids, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of shared matching or homologous positions. "Unrelated" or "non-homologous" sequences share less than 40% identity with a sequence of this disclosure, but preferably less than 25%.

[0029] "Sequence identity" of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that when the two sequences are compared, that percentage of bases (or amino acids) are the same.

[0030] The term "equivalent nucleic acid or polynucleotide" refers to a nucleic acid having a nucleotide sequence that has a degree of homology or sequence identity with the nucleotide sequence of said nucleic acid or its complementary sequence. Homologs of double-stranded nucleic acids are intended to include nucleic acids having a nucleotide sequence that has a degree of homology or identity with the nucleic acid or its complementary sequence. In one aspect, the homolog of the nucleic acid is capable of hybridizing with said nucleic acid or its complementary sequence. Similarly, "equivalent polypeptide" refers to a polypeptide having a degree of homology or sequence identity with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, or combinations thereof compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0031] As used herein, "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment or single chain thereof. Therefore, the term "antibody" includes any protein or peptide molecule comprising at least a portion of an immunoglobulin molecule that has the biological activity of binding an antigen. Such examples include, but are not limited to, the complementarity-determining region (CDR) or ligand-binding portion of the heavy or light chain, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame (FR) region or any portion thereof, or at least a portion of a binding protein.

[0032] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to a part of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that functions like an antibody by binding to a specific antigen to form a complex.

[0033] "Single-chain variable fragment" or "scFv" refers to the immunoglobulin heavy chain (V). H ) and light chains (V L A fusion protein with a variable region. In some aspects, the region is linked by a short linker peptide of about 10 to about 25 amino acids. The linker may be enriched with glycine to provide flexibility, and serine or threonine to provide solubility, and may also allow V... H N-terminus and V LThe C-terminus is linked, and vice versa. Despite the removal of the constant region and the introduction of a linker, the protein retains the specificity of the original immunoglobulin. ScFv molecules are known in the art and described, for example, in U.S. Patent 5,892,019.

[0034] The term antibody encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chains are classified as γ, μ, α, δ, or ε (γ, μ, α, δ, ε), with several subclasses (e.g., γ1-γ4). It is the nature of this chain that determines the "class" of the antibody, such as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and are known to confer functional specialization. Given this disclosure, modified versions of each of these classes and isotypes are readily identifiable to those skilled in the art and are therefore within the scope of this disclosure. All immunoglobulin classes are obviously within the scope of this disclosure, and the following discussion will generally refer to the IgG class of immunoglobulin molecules. Regarding IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with molecular weights of 53,000-70,000. The four chains are typically connected by disulfide bonds in a “Y” configuration, with the light chain starting from the opening of the “Y” and continuing through the variable region to support the heavy chain.

[0035] The antibodies, antigen-binding peptides, variants, or derivatives thereof disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab, Fab', and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VK or VH domains, fragments generated from Fab expression libraries, and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules.

[0036] "Specific binding" or "specific to" generally refers to an antibody binding to an epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to it more readily through its antigen-binding domain than to a random, unrelated epitope. The term "specific" is used in this document to define the relative affinity of a particular antibody for a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" may be considered to bind to epitope "C" with higher specificity than to related epitope "D."

[0037] As used herein, the terms “treatment” or “management” refer to therapeutic treatments and preventative or safeguarding measures aimed at preventing or slowing (mitigating) undesirable physiological changes or conditions, such as the progression of cancer. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, decrease in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” can also refer to an extension of life expectancy compared to the expected lifespan without treatment. Those requiring treatment include those who already have a condition or disorder, those who are susceptible to a condition or disorder, or those for whom prevention of a condition or disorder is desired.

[0038] "Subject" or "individual" or "animal" or "patient" or "mammal" refers to any subject, especially a mammalian subject, for whom a diagnosis, prognosis, or treatment is expected. Mammal subjects include humans, domestic animals, farm animals, and zoo, sporting, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, etc.

[0039] As used herein, phrases such as “for patients in need of treatment” or “subjects in need of treatment” include subjects who will benefit from the administration of the antibodies or compositions disclosed herein, such as mammalian subjects, for example, for detection, diagnostic procedures and / or treatment. Regulation of low-density lipoprotein receptor (LDLR)

[0040] Crimean-Congo hemorrhagic fever virus (CCHFV) is the most widespread tick-borne zoonotic Bunyavirus, causing severe hemorrhagic fever and death in humans. CCHFV enters cells via clathrin-mediated endocytosis, which depends on its surface glycoproteins. CCHFV was isolated in 1956 and identified as the pathogen of CCHF in 1969. Unfortunately, the true entry receptor for CCHFV was only identified in this study, which has hindered the understanding of CCHFV-host interactions and the development of effective treatments for CCHFV infection.

[0041] Through unexpected discoveries and detailed verification studies, the inventors of this invention demonstrate that LDLR is a universal entry receptor for CCHFV infection in mammals. First, the surface level of LDLR in different cell types is positively correlated with CCHFV infectivity. Second, LDLR knockout impairs CCHFV infection in multiple cell types from mice to humans. Third, LDLR blocking antibodies or soluble LDLR-Fc fusion proteins impair CCHFV infection in multiple cell types in a dose-dependent manner. Fourth, LDLR knockout in susceptible cells impairs CCHFV binding to cells and its internalization, while knockout of the LDLR adaptor protein LDLRAP1, which is crucial for LDLR-mediated endocytosis, impairs CCHFV internalization but not its binding to cells. Fifth, mutagenesis and reconstitution experiments show that LBD in the extracellular fragment of LDLR is essential for CCHFV infection. In vitro biochemical experiments show that CCHFV Gc, rather than Gn, enters the cell via Ca2+. 2+ The LDLR interacts directly with high affinity via a dependent pathway. The affinity between shLDLR and CCHFVGc is comparable to that between shLDLR and VSV G. Finally, knockout of the LDLR gene or administration of LDLR-blocking antibodies significantly reduced CCHFV infection and pathogenesis in mice. In conclusion, these results establish LDLR as a universal entry receptor for CCHFV infection across a wide range of cell types, from mice to humans.

[0042] LDLR is expressed in almost all human tissues, which is related to the extensive tissue tropism of CCHFV in vivo. In addition, LDLR is conserved across different species, making it an ideal receptor for the transmission of CCHFV from intermediate animal hosts (such as cattle, sheep, goats, camels, hares, and mice) to humans.

[0043] Interestingly, this study suggests that LDLR is not used for entry by other detected Bunyaviruses, including RVFV and EBIV. LDLR deficiency or treatment with shLDLR or LDLR blocking antibodies had no significant effect on RVFV or EBIV infection in SW13 cells. These results indicate that LDLR is specifically used for entry by CCHFV, but is not universally used by all Bunyaviruses.

[0044] According to one embodiment of this disclosure, a method for preventing or treating Crimean-Congo hemorrhagic fever virus (CCHFV) infection, or for preventing or treating Crimean-Congo hemorrhagic fever, is provided. In some embodiments, the method includes administering to a subject an effective amount of an agent that inhibits the biological activity or expression of low-density lipoprotein receptor (LDLR). In some embodiments, the agent inhibits the binding between the ligand-binding domain (LBD) and the Gc protein of CCHFV. Drugs that inhibit LDLR activity or expression

[0045] Agents that can inhibit LDLR activity or reduce its expression are known in the art, as discussed further below. Furthermore, agents capable of inhibiting the biological activity or expression of target proteins are readily available. One such example is an antibody or antigen-binding fragment. Methods for obtaining antibodies and fragments are known in the art.

[0046] In one embodiment, the agent is an anti-LDLR antibody or an antigen-binding fragment. In another embodiment, the agent is an anti-Gc antibody or an antigen-binding fragment. In yet another embodiment, the agent is a soluble LDLR protein or fragment containing at least one LDLR type A repeat sequence from the LBD, optionally fused to another peptide. A. Soluble LDLR protein and low-density lipoprotein (LDL)

[0047] As demonstrated in the experimental example, the soluble LDLR-Fc fusion protein impairs CCHFV infection in a dose-dependent manner. This fusion protein contains an extracellular fragment of the LDLR protein, thus enabling it to competitively bind to the viral Gc protein against the native LDLR protein.

[0048] As shown in Figure 2a and Table 1, the LDLR protein (SEQ ID NO:1) comprises a signal peptide (residues 1-21), followed by seven LDLR type A repeat sequences in the ligand-binding domain (LBD). LDLR type A repeat sequence 1 includes residues 25 to 58. LDLR type A repeat sequence 2 includes residues 66 to 104. LDLR type A repeat sequence 3 includes residues 107 to 143. LDLR type A repeat sequence 4 includes residues 147 to 179. LDLR type A repeat sequence 5 includes residues 195 to 231. LDLR type A repeat sequence 6 includes residues 235 to 270. LDLR type A repeat sequence 7 includes residues 278 to 308. The extracellular portion of the LDLR protein also contains an EGF-like domain with a β-propeller module and a proximal membrane O-linked sugar domain. As shown, these C-terminal domains do not participate in Gc binding.

[0049] Therefore, the soluble LDLR protein or fragment applicable to this technology does not contain transmembrane domains of LDLR or other proteins, and contains at least one LDLR type A repeat sequence in LBD.

[0050] In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequence 1 (residues 25 to 58 of SEQ ID NO:1) or a biological variant thereof. As used throughout this disclosure, a “biological variant” of the reference protein is a protein having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with the reference protein. In some embodiments, the biological variant retains the desired activity of the reference protein (e.g., binding to Gc of CCHFV).

[0051] In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 2 (residues 66 to 104 of SEQ ID NO:1) or a biological variant thereof. In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 3 (residues 107 to 143 of SEQ ID NO:1) or a biological variant thereof. In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 4 (residues 147 to 179 of SEQ ID NO:1) or a biological variant thereof. In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 5 (residues 195 to 231 of SEQ ID NO:1) or a biological variant thereof. In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 6 (residues 235 to 270 of SEQ ID NO:1) or a biological variant thereof. In one embodiment, the soluble LDLR protein contains at least LDLR type A repeat sequence 7 (residues 278 to 308 of SEQ ID NO:1) or a biological variant thereof.

[0052] In some embodiments, the soluble LDLR protein comprises at least two adjacent LDLR type A repeat sequences or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 1 and 2 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 2 and 3 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 3 and 4 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 4 and 5 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 5 and 6 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 6 and 7 or their biological variants.

[0053] In some embodiments, the soluble LDLR protein comprises at least three adjacent LDLR type A repeat sequences or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 1 to 3 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 2 to 4 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 3 to 5 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 4 to 6 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 5 to 7 or their biological variants.

[0054] In some embodiments, the soluble LDLR protein comprises at least four adjacent LDLR type A repeat sequences or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 1 to 4 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 2 to 5 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 3 to 6 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 4 to 7 or their biological variants.

[0055] In some embodiments, the soluble LDLR protein comprises at least five adjacent LDLR type A repeat sequences or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 1 to 5 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 2 to 6 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 3 to 7 or their biological variants.

[0056] In some embodiments, the soluble LDLR protein comprises at least six adjacent LDLR type A repeat sequences or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 1 to 6 or their biological variants. In one embodiment, the soluble LDLR protein comprises at least LDLR type A repeat sequences 2 to 7 or their biological variants. In some embodiments, the soluble LDLR protein comprises all seven LDLR type A repeat sequences 1 to 7 or their biological variants.

[0057] In some embodiments, the soluble LDLR protein does not contain an EGF-like domain (residues 354 to 388 of SEQ ID NO:1). In some embodiments, the soluble LDLR protein does not contain a proximal membrane O-linked sugar domain (residues 721 to 768 of SEQ ID NO:1). In some embodiments, the soluble LDLR protein does not contain a transmembrane domain or intracellular portion of the LDLR protein.

[0058] In some embodiments, the soluble LDLR protein also contains peptides that help improve exposure, stability, or cell penetration. Exemplary peptides are immunoglobulin Fc fragments. Another example is albumin proteins. Yet another example is cell-penetrating peptides (CPPs), such as ZFP2 (tandem zinc finger peptide), TAT (transcriptional transactivator peptide), and Pep-1. In some embodiments, additional peptides are fused to the C-terminus of the LDLR fragment.

[0059] In some embodiments, the soluble LDLR protein is provided as a protein, optionally in a pharmaceutically acceptable formulation. In some embodiments, the soluble LDLR protein is provided as encoding a polynucleotide, such as mRNA or cDNA. If provided as cDNA, the cDNA can be introduced onto a vector. Each of these is described in more detail below.

[0060] In some embodiments, the agent is low-density lipoprotein (LDL) or a composition containing LDL or capable of being converted into LDL in vivo. B. Anti-LDLR or anti-Gc antibodies

[0061] Antibodies or antigen-binding fragments that bind to the LDLR protein or the viral Gc protein can block the binding between them, thereby inhibiting viral entry into target cells. Such antibodies have been tested. For example, the anti-LDLR monoclonal antibody #10231-R301-P and the anti-Gc antibody ADI 36121 from SinoBiological (based on Fels et al. Cell 184, 3486-3501e3421, 389 (2021)) have shown efficacy in blocking viral entry.

[0062] Other antibodies against LDLR or viral Gc proteins are publicly available or readily available from public sources. For example, PCT application WO2001068710A1 discloses a monoclonal antibody against human LDLR. In WO2001068710A1, five clones (12, 28, 29, 30, and 50) and 29 subclones were identified that exhibited high affinity for the human LDLR protein. It also demonstrates that obtaining novel anti-LDLR antibodies is routine, as commonly understood by those skilled in the art. Other commercially available versions of anti-LDLR antibodies include, for example, sc-11824 from Santa Cruz Biotechnology.

[0063] In some embodiments, the anti-LDLR antibody or antigen-binding fragment of this disclosure interacts with one or more residues of a ligand-binding domain (LBD) or one or more LDLR type A repeat sequences, said LBD comprising residues 25 to 308 of SEQ ID NO:1. In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 1 of the LDLR protein (residues 25 to 58 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 2 of the LDLR protein (residues 66 to 104 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 3 of the LDLR protein (residues 107 to 143 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 4 of the LDLR protein (residues 147 to 179 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 5 of the LDLR protein (residues 195 to 231 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 6 of the LDLR protein (residues 235 to 270 of SEQ ID NO:1). In one embodiment, the anti-LDLR antibody or antigen-binding fragment binds at least LDLR type A repeat sequence 7 of the LDLR protein (residues 278 to 308 of SEQ ID NO:1).

[0064] In some embodiments, the anti-LDLR antibody or antigen-binding fragment does not interact with residues in the EGF-like domain (residues 354 to 388 of SEQ ID NO:1) or the proximal membrane O-linked sugar domain (residues 721 to 768 of SEQ ID NO:1).

[0065] In some embodiments, the anti-Gc antibody or antigen-binding fragment binds to any residue of the Gc protein (SEQ ID NO:3). In some embodiments, the anti-Gc antibody or antigen-binding fragment competes with LDLR for binding to the Gc protein. In some embodiments, the anti-Gc antibody or antigen-binding fragment inhibits the binding between LDLR and the Gc protein. C. Repressive RNA molecules

[0066] Another example of such agents is repressive RNA. Repressive RNA is an RNA molecule that can suppress gene expression at the posttranscriptional level of the LDLR gene. There are several types of repressive RNA, which will be further described below.

[0067] MicroRNAs (miRNAs): miRNAs are small non-coding RNAs that regulate gene expression by targeting specific mRNAs for degradation or translational repression. miRNAs are transcribed from DNA and then processed by the cell into mature miRNAs, which can recognize and bind to complementary sequences on target mRNAs, leading to their degradation or translational repression.

[0068] Small interfering RNA (siRNA): siRNA is another type of small non-coding RNA that can induce gene silencing by targeting specific mRNAs for degradation or translational repression. siRNA is typically introduced into cells via transfection or viral transduction and can be used for research or therapeutic purposes.

[0069] Short hairpin RNA (shRNA): shRNA is an RNA molecule that induces gene silencing by mimicking the structure of miRNA precursors. They are typically introduced into cells via transfection or viral transduction and can be used for research or therapeutic purposes.

[0070] Piwi-interacting RNAs (piRNAs): piRNAs are a class of small non-coding RNAs that play a role in regulating transposons and maintaining the stability of germ cell genomes. piRNAs interact with a class of proteins called Piwi proteins and can induce gene silencing through epigenetic mechanisms such as DNA methylation or histone modification.

[0071] Antisense RNA (asRNA): asRNA is an RNA molecule that is complementary to a specific mRNA and can induce gene silencing by hybridizing with the mRNA and preventing its translation or promoting its degradation. D. Gene or RNA editing

[0072] Targeted editing of genes or transcripts has also proven to be an effective means of reducing the expression or activity of target genes, such as LDLR. For example, to reduce LDLR expression, the promoter region of the LDLR gene can be edited using techniques such as TALEN, CRISPR, or zinc finger nucleases, thereby reducing its efficiency. In another instance, the coding sequence of the LDLR gene can be edited to express a mutant LDLR protein with reduced activity.

[0073] CRISPR / Cas-mediated genome editing technology has been widely used since its invention due to its simplicity and efficiency. In two classes of CRISPR-Cas systems, targeting and cleavage are achieved by a Cas protein bound to a single CRISPR RNA (“crRNA”). These two systems can be further subdivided into type II Cas9 and type V Cas12, which target DNA, and type VI Cas13, which targets RNA. Cas9 and Cas12a / Cpf1 have been extensively studied and widely used for gene editing in various cell types and organisms in prokaryotes and eukaryotes. Both the Cas9 and Cas12 systems utilize guide RNA to recognize target sites, use prototypical spacer adjacent motifs (PAMs) to determine cleavage sites, and generate double-strand breaks. Cas9 recognizes 3'-G-rich PAMs and generates blunt ends cleaved by RuvC and HNH domains, while Cas12 recognizes 5'-T-rich PAMs and generates sticky ends cleaved only by the RuvC domain. Pre-assembled Cas13 and crRNA recognize the target RNA. Upon binding to RNA, Cas13 undergoes a conformational change and induces the catalytic activity of its nuclease domain, leading to the cleavage of the target transcript. In some embodiments, gRNA or crRNA may be programmed to target DNA sequences in the genome. In some embodiments, the Cas protein contains an inactive nuclease domain. In some embodiments, the Cas protein does not contain an active nuclease domain.

[0074] In some embodiments, the guide RNA (“gRNA”) comprises two short non-coding RNA species, referred to as crRNA and trans-activating RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with the Cas protein of this disclosure. gRNA: The Cas protein complex binds to a target polynucleotide sequence in the genome.

[0075] Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific DNA sequences. They are created by fusing a TAL effector DNA-binding domain with a DNA-cutting domain (a nuclease that cuts the DNA strand). Transcription activator-like effectors (TALEs) can be engineered to bind to virtually any desired DNA sequence, so when combined with a nuclease, the DNA can be cleaved at a specific location. Restriction enzymes can be introduced into cells for gene editing or in situ genome editing. E. Regulatory molecules

[0076] The expression or activity of LDLR can also be indirectly regulated. For example, rapamycin (Sirolimus®) can inhibit the gene expression and protein level of LDLR (Liu et al., “Rapamycin inhibits peritoneal fibrosis by modifying lipid homeostasis in the peritoneum,” Am J Transl Res. 2019; 11(3):1473-1485).

[0077] LDLR expression is also regulated by the LXR-IDOL pathway, which is involved in LDLR protein degradation. Agonists of the LXR (hepatic X receptor) or LXR / RXR (retinol X receptor) heterodimer have been shown to reduce LDLR expression (see, for example, Zelcer et al., “LXR Regulates Cholesterol Uptake Through Idol-Dependent Ubiquitination of the LDL Receptor,” Science, 2009, Vol 325, Issue 5936, pp. 100-104). Examples of LXR agonists, without limitation, include oxosterols, hypocholesterol, T0901317, GW3965, or N,N-dimethyl-3β-hydroxycholenic acid (DMHCA). Oxosterols are oxidized derivatives of cholesterol, such as 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholesteric acid, which are natural ligands of LXR.

[0078] Non-limiting examples of RXR agonists include 9-cis-13,14-dihydroretinoic acid.

[0079] Furthermore, LDLR mRNA is known to be unstable, but upon activation of extracellular signal-regulated kinase (ERK), it is stabilized by the binding of certain proteins to the 3'-untranslated region (UTR) of LDLR mRNA. Protein agents, such as ZFP36L1 (ZFP36 ring finger protein-like 1) and ZFP36L2 (ZFP36 ring finger protein-like 2), specifically bind to the 3'-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylate enzyme complex, leading to mRNA instability (Adachi et al., “ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway,” Nucleic Acids Res, 2014Sep;42(15):10037-49. doi: 10.1093 / nar / gku652. Epub 2014 Aug 8).

[0080] Furthermore, PAQR3 (a member of the progesterone and AdipoQ receptor family 3) plays an important role in controlling the degradation of LDLR in the liver. PAQR3 overexpression can reduce LDLR protein (see, for example, Huang et al., “PAQR3 modulates blood cholesterol level by facilitating interaction between LDLR and PCSK9,” Metabolism, 2019 May;94:88-95. doi: 10.1016 / j.metabol.2019.02.005. Epub 2019Mar 1).

[0081] Similarly, overexpression of PCSK9 (proprotein convertase subtilisin / kexin type 9) can reduce LDLR expression because PCSK9 binds to and degrades LDLR.

[0082] For any of the protein agents described above, it is readily understood that they may be delivered as a protein or biological variant, or as a polynucleotide (cDNA or mRNA) encoding that protein. Agonists capable of increasing the expression or activity of these protein agents are also within the scope of this disclosure.

[0083] In some embodiments, each mRNA is a linear or circular mRNA. In some embodiments, each mRNA also contains a miRNA binding site. In some embodiments, each mRNA does not contain any chemical modifications that reduce immunogenicity. In some embodiments, the mRNA does not contain any chemical modifications to the backbone. In some embodiments, each mRNA contains only natural nucleosides.

[0084] In some embodiments, at least one uridine nucleoside in the mRNA is chemically modified. In some embodiments, the chemically modified uridine nucleoside is N1-methylpseudouridine. In some embodiments, the first and second mRNAs are formulated with pharmaceutically acceptable vectors.

[0085] In some embodiments, the carrier comprises lipid nanoparticles (LNPs). In some embodiments, the LNPs comprise (a) 40-60 molar ratio of ionizable amino lipids, 8-16 molar ratio of phospholipids, 30-45 molar ratio of sterols, and 1-5 molar ratio of PEG-modified lipids; (b) 45-65 molar ratio of ionizable amino lipids, 5-10 molar ratio of phospholipids, 25-40 molar ratio of sterols, and 0.5-5 molar ratio of PEG-modified lipids; and (c) 40-60 molar ratio of ionizable amino lipids, 8-16 molar ratio of phospholipids, 30-45 molar ratio of sterols, and 1-5 molar ratio of PEG. Modified lipids, (d) 45-65% molar ratio of ionizable amino lipids, 5-10% molar ratio of phospholipids, 25-40% molar ratio of sterols and 0.5-5% molar ratio of PEG-modified lipids, (e) 40-60% molar ratio of ionizable amino lipids, 8-16% molar ratio of phospholipids, 30-45% molar ratio of sterols and 1-5% molar ratio of PEG-modified lipids, or (f) 45-65% molar ratio of ionizable amino lipids, 5-10% molar ratio of phospholipids, 25-40% molar ratio of sterols and 0.5-5% molar ratio of PEG-modified lipids.

[0086] In some embodiments, each mRNA is packaged in a liposome. In some embodiments, the liposome comprises cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids.

[0087] In some embodiments, the cationic lipid is selected from the group consisting of: 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecane-2-ol) (C12-200), (6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (MC3), N,N-dimethyl-2,3-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)prop-1-amine (DLinDMA), 2-(2,2-di((9Z,12Z)-octadec-9 ,12-dien-1-yl)-1,3-dioxolane-4-yl)-N,N-dimethylethylamine (DLinKC2DMA, [XTC2]), 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12), 10,13-dimethyl-17-(6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentaphenanthrene-3-yl 3-(1H-imidazol-5-yl)propionate (ICE), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadecano-9,12-dien-1-yl)tetracosyl C15,18-dien-1-amine (HGT5000), (4Z,15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadec-9,12-dien-1-yl)tetracos-4,15,18-trien-1-amine (HGT5001), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N,N-distearate-N,N-dimethylammonium bromide (DDAB), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), dioleoyloxy-N-[2-sperminecarboxamido]ethyl]-N,N-dimethyl-1-propanetrimonium trifluoroacetate (DOSPA), bis(octadecylamidoglycylspermine) (DOGS), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA) and N,N-dimethyl-(2,3-dimyristyloxy)propylamine (DMDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA), (2S)-2-(4-((10,13-dimethyl-17-(6-methylhept-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecanoyl-1H-cyclopenta[a]phenanthrene-3-yl)oxy)butoxy)-N,N-dimethyl-3-((9Z,12Z)-octadecano-9,12-Dien-1-yloxy)prop-1-amine (CLinDMA), 2-[5'-(cholest-5-en-3[β]-oxy)-3'-oxaproxy)-3-dimethyl-1-1(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoxybenzylamine (DMOBA), 1,2-N,N'-dioleocarbamoyl-3-dimethylaminopropane (DOcarbDAP), (9Z,9'Z,12Z,12'Z)- 3-(dimethylamino)propane-1,2-diylbis(octadec-9,12-dienoate) (DLinDAP), 1,2-dilinylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2-((2,3-bis((9Z,12Z)-octadec-9,12-dien-1-yloxy)propyl)dithioalkyl)-N,N-dimethylethylamine (HGT4003) and combinations thereof.

[0088] In some embodiments, the cholesterol-based lipid is cholesterol or PEGylated cholesterol. In some embodiments, the cationic lipid comprises about 30-50% of the liposome molar ratio. In some embodiments, the ratio of cationic lipid:non-cationic lipid:cholesterol lipid:PEGylated lipid is about 40:30:25:5. In some embodiments, the liposome comprises a combination selected from the group consisting of: cKK-E12, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol and 1,2-dimyristoyl-sn-glycerol, methoxy polyethylene glycol (DMG-PEG2K); C12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; or ICE, DOPE, cholesterol and DMG-PEG2K. Pharmaceutical compositions and combinations

[0089] The specific dosage and treatment regimen for any given patient will depend on a number of factors, including the specific drug used, the patient's age, weight, general health condition, sex, and diet, as well as the timing of administration, excretion rate, drug combination, and the severity of the specific disease being treated. The healthcare professional's judgment regarding these factors is within the scope of ordinary skill in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0090] The methods of administration of the pharmaceutical preparation include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptide or composition can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal skin linings (e.g., oral mucosa, rectal and intestinal mucosa), and can be administered together with other bioactive agents. Therefore, pharmaceutical compositions containing the antigen-binding polypeptide of this disclosure can be administered orally, rectally, parenterally, intracerebrospinal, intravaginally, intraperitoneally, topically (as a powder, ointment, drops, or transdermal patch), orally, or as an oral or nasal spray.

[0091] As used in this article, the term "parenteral" includes administration methods such as intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0092] Administration can be systemic or localized. Furthermore, it may be desirable to introduce the agents of this disclosure into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter (e.g., connected to a reservoir, such as the Ommaya reservoir). Lung administration may also be employed, for example, by using an inhaler or nebulizer and formulated as an aerosol.

[0093] It may be desirable to apply the agents or compositions of this disclosure topically to the area requiring treatment; this can be achieved, for example, but not limited to, local infusion, local application (e.g., in combination with a wound dressing after surgery), injection, via a catheter, via a suppository, or via an implant that is a porous, non-porous, or gel-like material, including membranes (such as silicone rubber membranes) or fibers. Preferably, when applying proteins (including the agents of this disclosure), care must be taken to use materials that are not absorbed by the protein.

[0094] As described above, a pharmaceutical composition is described comprising an agent that reduces LDLR expression or activity, or inhibits the interaction between low-density lipoprotein receptor (LDLR) and Gc glycoprotein (Gc) of CCHFV in a subject.

[0095] Such compositions are suitable for oral, parenteral, topical, or inhalation administration. Therefore, a pharmaceutical composition comprising at least one agent according to this disclosure can be administered parenterally, such as intravenously, intramuscularly, or subcutaneously. Alternatively, the agent can be administered via a non-parenteral route, such as oral or topical administration. In a preferred embodiment, the pharmaceutical composition comprising an agent according to this disclosure is administered intravenously or subcutaneously.

[0096] In one embodiment, this disclosure provides a pharmaceutical composition comprising one or more agents according to this disclosure for the prevention and / or treatment of a disease. In one embodiment, this disclosure provides a pharmaceutical composition comprising one or more agents according to this disclosure for use as a medicine. In one embodiment, this disclosure provides a pharmaceutical composition comprising one or more agents according to this disclosure for the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases and / or cancer.

[0097] The pharmaceutical agents described herein can be used in combination as a single composition. However, in some embodiments, they are administered separately to the same patient. Example Example 1: LDLR is the entry receptor for Crimean-Congo hemorrhagic fever virus.

[0098] The cellular receptors necessary for CCHFV entry are not yet known. This example demonstrates that the low-density lipoprotein receptor (LDLR) is the entry receptor for CCHFV. method mice

[0099] Wild-type and LDLR knockout (T001464) C57BL / 6J mice were purchased from Gempharmatech Co., Ltd. Mice were housed in groups under a 12-hour dark / light cycle with free access to food and water. All animal experiments were conducted in accordance with the policies of the Wuhan Institute of Virology, Chinese Academy of Sciences, and were approved by the Institutional Animal Care and Use Committee (IACUC) (ethics number: WIVA31202301). cell

[0100] SW13, Huh7, A549, HEK293T, Vero E6 (all from ATCC), and DLD1 cells were cultured at 37°C in Dulbecco modified Eagle medium (DMEM) (GIBCO) supplemented with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin (Hyclone). Primary mouse hepatocytes and MLF cells were isolated and cultured. FreeStyle 293F cells were cultured at 37°C on a track-shaking platform (120 rpm) in SMM293-TII expression medium (Sino Biological). All cell lines were tested using the MycoBlue Mycoplasma Detection Kit (#D101, Vazyme), and all results showed no mycoplasma contamination. Virus

[0101] The CCHFV (YL16070 strain, GenBank accession number: KY354082) used in this study was provided by the National Virus Resource Center (Wuhan, China). The NSs-deficient RVFV (RVFV-r∆NSs-eGFP, strain BJ01) and EBIV (Cu-XJ20 isolate) were also provided by them. CCHFV was propagated in Vero E6 cells, and TCID50 was measured in SW13 cells. RVFV-r∆NSs-eGFP and EBIV were propagated, and viral titers were determined by plaque assay. All experiments involving live CCHFV were performed at the BSL-3 facility of the Wuhan Institute of Virology, Chinese Academy of Sciences. CRISPR-Cas9 knockout

[0102] Gene editing was performed using the CRISPR / Cas9 system. In short, a double-stranded oligonucleotide corresponding to the target sequence was cloned into the lenti-CRISPR-V2 vector, which was co-transfected into HEK293 cells along with the packaging plasmids psPAX2 and pMD2.G. Two days after transfection, the virus was harvested and used to infect target cells. Infected cells were selected with puromycin (1 μg / ml) for at least 7 days. LDLR-deficient clones were obtained through limiting dilutions. The LDLR gene mutation and its expression defect were confirmed by Sanger sequencing and Western blotting, respectively. RT-qPCR

[0103] Total RNA was isolated from cells using RNAiso Plus (#9109, TaKaRa), and 1 µg of RNA was reverse transcribed using a cDNA synthesis kit (#R212, Vazyme) according to the manufacturer's instructions. Viral RNA was extracted from cell culture supernatant using the TaKaRa MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0 (#9766, TaKaRa). Threshold cycles (Ct) for each gene were normalized against the housekeeping gene GAPDH and are shown as relative mRNA levels. Flow cytometry

[0104] Cells were seeded overnight in six-well plates, scraped off the plates, and washed with PBS. To detect LDLR surface expression, cells were suspended in 200 µl PBS and incubated on ice for 1 h with 2 µg / ml phycoerythrin (PE)-conjugated anti-LDLR monoclonal antibody (#10231-R301-P, SinoBiological), followed by fixation with 4% paraformaldehyde for 15 min. After staining, cells were washed with PBS and analyzed by flow cytometry.

[0105] To detect Gn in cells after CCHFV infection, cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with Perm / Wash buffer (#554723, BD) for 15 minutes, and then stained with anti-Gn monoclonal antibody (clone 7A11, Abclonal) for 1 hour. After washing three times with Perm / Wash buffer, cells were stained with FITC-conjugated anti-mouse IgG for 30 minutes. After staining, cells were washed with PBS and analyzed by flow cytometry. Crystal violet staining

[0106] LDLR-edited and control SW13 cells were infected with CCHFV at the indicated MOI for 72 hours. The cells were then fixed with 4% paraformaldehyde for 15 minutes and stained with 1% crystal violet for 30 minutes before being photographed. TCID50 determination

[0107] Cells were seeded in 96-well plates overnight, followed by TCID50 assay. In short, the CCHFV stock solution was diluted with DMEM from 1:10 to 1:10. 6 Serial dilutions were performed. SW13 cells were incubated with 100 µl of each dilution stock solution for 1 hour. Virus was then removed, and 150 µl of DMEM containing 2% FBS was added to the cells to maintain cell growth for 5 days. Cytopathic effects were observed, and viral titers were calculated using the Reed-Muench method. Reconstruction of LDLR-deficient cells

[0108] The full-length human LDLR (NM_000527.5) cDNA encoding the C-terminal Flag tag, or a truncated cDNA lacking the LBD (aa24-313) or EFG-like domain (aa314-712), was cloned into the retroviral vector pMSCV. The sgRNA targeting sequence in the LDLR cDNA was synonymously mutated to avoid Cas9 editing of the reconstructed cDNA.

[0109] LDLR or its mutants were reconstituted into LDLR-deficient cells via retroviral-mediated transduction. In short, HEK293T cells plated in 100 mm culture dishes were transfected with a retroviral plasmid (10 μg) along with pGag-pol (10 μg) and pVSV-G (3 μg). Two days after transfection, the virus was harvested and used to infect LDLR-deficient SW13 cells in the presence of polybrene (8 μg / ml). Infected cells were selected with blastomycin (1 μg / ml) for at least 7 days. The expression of LDLR or its truncated variant in the reconstituted cells was assessed by Western blotting using an anti-LDLR rabbit polyclonal antibody (#A14996, Abclonal). Virus attachment and internalization assay

[0110] For the virus attachment assay, WT and LDLR-deficient SW13 cells were seeded overnight in 12-well plates. Cells were incubated with CCHFV (MOI=5) on ice for 1 hour. After washing five times with ice-cold PBS, cells were collected and RNA was extracted for RT-qPCR analysis. For the internalization assay, after incubation and washing on ice, the plates were placed at 37°C. One hour later, cells were washed once with PBS and treated with 500 ng / mL proteinase K on ice for 1 hour to stop endocytosis and degrade uninternalized viruses. Cells were then washed three times with PBS and collected for RT-qPCR analysis. Blocking assay using anti-LDLR antibody or soluble LDLR-Fc fusion protein

[0111] Cells were seeded in 12-well plates 12 hours prior to treatment. For antibody blocking assays, cells were pre-incubated at 37°C for 1 hour with serially diluted anti-hLDLR (#10231-R301, SinoBiological), anti-mLDLR (#50305-R004, SinoBiological), or control IgG (#CR1, SinoBiological), and then infected with CCHFV (MOI=0.05), RVFV (MOI=0.1), EBIV (MOI=0.5), or VSV (MOI=0.1). Twenty-four hours post-infection, cells were collected for RT-qPCR detection of CCHFV S segment, RVFV M segment, EBIV S segment, and VSV L segment, respectively.

[0112] For the blocking of soluble LDLR protein, serially diluted shLDLR-Fc (#10231-H05H, SinoBiological) or control Fc (#10690-MNAH, SinoBiological) was pre-incubated with CCHFV (MOI=0.05), RVFV (MOI=0.1), EBIV (MOI=0.5), or VSV (MOI=0.1) in 100 μl at 37°C for 1 h. Cells were then seeded with this mixture. Twenty-four hours post-infection, cells were collected for RT-qPCR detection of CCHFV S segment, RVFV M segment, EBIV S segment, and VSV L segment, respectively. Preparation of CCHFV Gc and VSV-G proteins

[0113] pCAGGS-GPC10200 (a codon-optimized GPC expression plasmid for the CCHFV IbAr 10200 strain for human cells) was used as a template for constructing the Gc expression plasmid. In short, a pCAGGS-based Gc expression plasmid for the CCHFV IbAr 10200 strain (pCAGGS-Gc10200) was constructed, in which a furin cleavage site (RSKR) is inserted into the 3' end of the cDNA encoding GPC10200 aa1-515, followed by the cDNA encoding GPC10200 aa1041–1579 and a 6xHis tag at the C-terminus. To express Gc of CCHFV YL16070 strain, the coding sequence for GPC10200 aa1041–1579 in pCAGGS-Gc10200 was replaced with the sequence encoding GPC aa1054–1592 of CCHFV YL16070 strain. To express VSV G protein, cDNA encoding VSV (Indiana strain) G was fused with a 6xHis tag and cloned into the pCAGGS vector. FreeStyle 293F cells were cultured at 5 × 10⁻⁶ cells per cell line the day before transfection. 6 Seed at a density of cells / ml. Dilute 200 μg of plasmid with Opti-MEM (ThermoFisher) and mix with PEI MAX. ® Transfection reagent (Polysciences, Inc.) was compounded and then added to cells. Four days after transfection, the supernatant was collected, centrifuged at 3,000g for 15 minutes, and purified by Ni-NTA Sepharose (GE) chromatography. To purify VSV-G, transfected cells were collected and sonicated with PBS. Cell lysates were centrifuged at 14,000g for 15 minutes and subjected to Ni-NTA Sepharose chromatography. The eluted proteins were then purified using SEC on a Superdex 200 column (GE). The purified proteins were dialyzed against PBS, filtered through a 0.20-μm filter, and stored at -80°C. The purity of each protein was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining. Pull-down measurement

[0114] 2 μg of biotinylated LDLR (#10231-H08H-B, SinoBiological) was mixed with 500 μl of CCHFV virus stock solution or 2 μg of CCHFV Gc protein in 1 ml PBS at 4°C for 2 hours in the presence of 0.5 mM CaCl2. Then, 30 μl of streptavidin magnetic beads (#HY-K0208, MedChemExpress) was added and the mixture was vortexed at 4°C for 30 minutes. The magnetic beads were collected and washed three times with PBS containing 0.5 mM CaCl2. For RT-qPCR experiments, RNA was extracted from the magnetic beads using 500 μl of RNAiso Plus (#9109, TaKaRa). For Western blotting, the magnetic beads were treated with 80 μl of 2xSDS loading buffer and then subjected to SDS-PAGE. ELISA-based binding assay

[0115] The binding of shLDLR to CCHFV viral particles was determined by ELISA. Monoclonal antibodies against CCHFV Gc (ADI 36121, Felset al. Cell 184, 3486-3501 e3421, 389 (2021)), Gn (JE12) (#MAB12317, The Native Antigen Company), or their respective control IgG (#12000C, Invitrogen; #I5381, Sigma) were fixed in PBS (50 μl, 0.5 μg / ml) overnight at 4°C on Maxisorp ELISA plates (Thermo Fisher) at 4°C. The plates were washed four times with PBS and blocked with PBS supplemented with 4% BSA for 1 hour at room temperature. Then, CCHFV virus stock solution (50 μl / well) was added to the plates and incubated for 1 hour at room temperature, followed by washing three times with PBS. Then, biotinylated LDLR (#10231-H08H-B, SinoBiological) diluted with PBS supplemented with 2% BSA at the indicated concentration was added, and the plate was incubated at room temperature for 1 hour. After washing 5 times with PBS, the plate was incubated with avidin-HRP (1:1000 dilution, Cat. 405103, Biolegend) at room temperature for 1 hour, followed by washing 5 times with PBS. Finally, TMB (3,3',5,5'-tetramethylbenzidine) was added, and the reaction was terminated with 2N H2SO4. The absorbance was read at 450 nm using a microplate reader (Biotech). ForteBio Octet Red Biolayer Interferometry

[0116] The binding affinity of shLDLR to CCHFV YL16070 strain Gc, CCHFV IbAr 10200 strain Gc or Gn (#REC31615, The Native Antigen Company) was measured using the ForteBio Octet Red system (ForteBio, Inc.). Biotinylated shLDLR (25 μg / ml) was immobilized on a streptavidin biosensor, and binding and dissociation were monitored in 200 μl of PBS containing 0.02% Tween 20, 0.5 mM CaCl2, and 1 mg / ml BSA. The dissociation constant was calculated using Octet RED software. mouse CCHFV infection

[0117] (1) To assess the effect of LDLR deficiency on CCHFV infection, 8-week-old female WT and LDLR-deficient mice were anesthetized with isoflurane and given anti-IFNAR1 monoclonal antibody MAR1-5A3 (#BE0241, BioXCell) (300 µg / mouse) 24 hours before viral infection. CCHFV (10 TCID50 / mouse) was injected intraperitoneally, and 200 µg of anti-IFNAR1 monoclonal antibody was given 24 hours after infection.

[0118] (2) To evaluate the protective effect of LDLR blocking antibodies, 8-week-old female C57BL / 6J mice were anesthetized with isoflurane and administered intraperitoneally 300 µg of anti-IFNAR1 monoclonal antibody MAR1-5A3 (#BE0241, BioXCell) plus 100 µg of LDLR blocking antibody (#50305-R004, SinoBiological) or control IgG (#CR1, SinoBiological) 24 hours before challenge. CCHFV (100 TCID50 / mouse) was subcutaneously inoculated, and anti-IFNAR1 monoclonal antibody (200 µg / mouse) was administered 24 hours after challenge. After infection, anti-LDLR or control IgG 100 µg was administered intraperitoneally daily for 5 days.

[0119] For (1) and (2), the weight changes of mice were monitored daily after CCHFV challenge. Mice were euthanized when they lost more than 20% of their body weight, had difficulty crawling, and / or became unresponsive to touch. A subset of mice were euthanized on day 5 post-infection, and livers and spleens were collected for further viral load measurement and pathological analysis. As previously described, tissue RNA was extracted and RT-qPCR was performed to quantify the amount of vRNA in each tissue. statistics.

[0120] Statistical analysis was performed using Prism version 8.0 (GraphPad). Statistical significance was analyzed using a two-way ANOVA followed by the Dunnett test. If only two conditions were being compared, a two-tailed unpaired (Student) t-test was performed. For animal survival studies, Kaplan-Meier survival curves were generated and analyzed using the Log-Rank test. A p-value < 0.05 was considered statistically significant. Unless otherwise stated, error bars show the mean and standard deviation (Mean ± SD). All data represent at least two independent experiments with similar results. result Identification of LDLR as a candidate host factor for CCHFV infection

[0121] This study investigated whether LDLR and its family members play a role in CCHFV infection. Using the CRISPR-Cas9 system to target two independent sites for each gene, we generated 293T cells deficient in LDLR family members. These edited cell lines were inoculated with CCHFV (MOI=0.05) for 24 hours, and the expression of the viral S-segment was then examined by reverse transcription quantitative PCR (RT-qPCR).

[0122] As shown in Figure 1a, knockout of LDLR and LDLR adaptor protein 1 (LDLRAP1), rather than knockout of the other 18 LDLR family members or related signaling components, significantly suppressed the mRNA level of the S segment after CCHFV infection (Figure 1a). These results indicate that LDLR and LDLRAP1 (rather than the other proteins detected) are important for CCHFV infection. LDLR levels are associated with CCHFV infectivity

[0123] LDLR (e.g., GenBank Gene ID: 3949) is a cell membrane glycoprotein that plays a role in binding and internalizing cholesterol-containing lipoprotein particles, while LDLRAP1 is a cytoplasmic adaptor protein that interacts with the cytoplasmic tail of LDLR and promotes its endocytosis. Therefore, we subsequently focused on LDLR and attempted to determine whether it is a cell entry receptor for CCHFV. We first analyzed the correlation between LDLR expression and CCHFV infectivity using a group of human cell lines, including human adrenocortical carcinoma SW13, human hepatocellular carcinoma Huh7, human embryonic kidney 293T, human colorectal carcinoma DLD1 cells, and monkey kidney Vero E6 cells. Flow cytometry analysis of membrane LDLR levels using an anti-LDLR monoclonal antibody showed that LDLR was highly expressed in SW13 and Huh7 cells, moderately expressed in VeroE6 cells, lowly expressed in 293T cells, and almost undetectable in DLD1 cells (Figure 1b). Correspondingly, flow cytometry analysis showed that the percentage of Gn-positive cells after CCHFV infection was high in SW13 and Huh7 cells (>50%), moderate in VeroE6 cells (~10%), low in 293T cells (0.6%), and almost undetectable in DLD1 cells (0.027%) (Fig. 1c). Ectopic expression of LDLR in DLD1 cells significantly enhanced CCHFV infection, as indicated by increased CCHFV S mRNA and NP protein (Fig. 1d). These results suggest a positive correlation between membrane LDLR levels and CCHFV infectivity. LDLR deficiency impairs CCHFV infectivity in different cell types

[0124] Because the SW13 cell line expresses high levels of LDLR and is highly susceptible to CCHFV infection, we further edited LDLR in these cells using CRISPR-Cas9 with three separate sgRNAs targeting different sites in the LDLR coding sequence to confirm its function in CCHFV infection. As shown in Figure 1e, CCHFV infectivity was impaired in LDLR-edited SW13 cells, as determined by a significant reduction in CCHFV NP protein levels, S-segment mRNA levels, the percentage of Gn-positive cells, and the cytopathic effect on infected cells. Consistently, the progeny virus titer in the cell culture supernatant of LDLR-edited SW13 cells was also reduced (Figure 1f). In these experiments, the degree of inhibition of CCHFV infectivity correlated with the knockdown efficiency of the three LDLR sgRNAs (Figures 1e and 1f). These results indicate that LDLR is an important cytokine for CCHFV infection and replication.

[0125] We further confirmed the function of LDLR in CCHFV infection in Huh7, Vero E6, and mouse hepatocellular carcinoma Hepa1-6 cells by editing LDLR with two or three different sgRNAs. Similarly, LDLR deficiency impaired CCHFV infection, as shown by the significant reduction in S-segment mRNA levels in all tested cell lines (Fig. 1g), indicating that LDLR plays a conserved role in mediating CCHFV infection in human, monkey, and mouse cells.

[0126] In the experiments described above, we used LDLR-edited cell pools where LDLR expression was significantly reduced but not completely eliminated. CCHFV infection was also significantly reduced but not completely eliminated in these cells. To determine whether the remaining infectivity was due to residual LDLR expression or redundant cytokines, we isolated monoclonal LDLR-edited SW13 and Huh7 cells with completely deficient LDLR expression and infected these cells with CCHFV (Fig. 1h). The results showed that CCHFV infection was completely eliminated in LDLR-deficient SW13 cells, but in LDLR-deficient Huh7 cells, approximately 10% of infectivity remained based on CCHFV S mRNA levels (Fig. 1h). Furthermore, we examined samples from WT and Ldlr... - / - CCHFV infectivity in primary mouse hepatocytes and lung fibroblasts (MLF). Results confirmed that, despite low levels of residual infectivity, LDLR-deficient cells exhibited significantly reduced mRNA levels of the CCHFV S segment and progeny virus production (Fig. 1i). These results suggest that LDLR is essential for CCHFV infection in SW13 cells, while cytokines other than LDLR may inefficiently support CCHFV infection in Huh7 cells, as well as in primary mouse hepatocytes and MLF.

[0127] Therefore, we tested whether other LRPs were functionally redundant with LDLR to promote CCHFV infection in Huh7 cells. To this end, we knocked down 18 known LRPs using siRNA in LDLR-deficient Huh7 cells and examined CCHFV infectivity in these cells (Fig. 7). Furthermore, we ectopically expressed 15 LRPs in DLD1 cells and examined their effects on CCHFV infectivity (Fig. 7b). The results showed that none of the examined LRPs functioned as cytokines supporting CCHFV infection; that is, CCHFV infection should be impaired when depleted by siRNA in LDLR-deficient Huh7 cells and / or promoted when overexpressed in DLD1 cells. Moreover, pre-incubation of LDLR-deficient Huh7 cells with soluble human receptor-associated protein (RAP) (a universal molecular chaperone that can block ligand binding to all LRP family members) had no significant effect on CCHFV infection (Fig. 7c). In similar experiments, RAP completely eliminated Semliki Forest Virus (SFV) infection in Huh7 cells (Figure 7c), as SFV is known to utilize VLDLR and ApoER2 (also known as LRP8) of the LRP family as entry receptors. In summary, these results indicate that LDLR (rather than the known LRPs) is crucial for CCHFV infection. LDLR is required for CCHFV infection, but not for other detected Bunyavirus infections.

[0128] Next, we investigated whether LDLR is essential for infection with other Bunyaviruses, including Rift Valley Fever Virus (RVFV) and Lake Ebinur Virus (EBIV), by examining the infectivity of RVFV and EBIV in LDLR-deficient SW13 cells. The results showed that LDLR deficiency had no significant effect on RVFV or EBIV infection in SW13 cells (Figure 1j). In the same experiment, LDLR deficiency reduced VSV infection. In conclusion, these results indicate that LDLR is specifically required for CCHFV infection in SW13 cells, but not for infection with the other Bunyaviruses detected. The ligand-binding domain of LDLR is essential for CCHFV infection.

[0129] Because LDLR is a membrane protein located on the cell surface, we hypothesized that LDLR functions as an entry receptor for CCHFV. We first investigated whether the extracellular domain of LDLR is essential for CCHFV infection. LDLR contains a signal peptide, a ligand-binding domain (LBD) with seven LDLR type A repeat sequences, followed by an EGF-like domain containing a β-propeller module, a proximal membrane O-linking glycoside domain, a transmembrane anchoring region, and a cytoplasmic domain (Fig. 2a, Table 1). We reconstituted LDLR-deficient SW13 cells with full-length LDLR and two truncated mutants lacking either the LBD (ΔLBD) or the EGF-like domain (ΔEGF), respectively (Fig. 2a). CCHFV infection of these reconstructed cells showed that full-length LDLR restored CCHFV infectivity, as determined by NP expression levels (Fig. 2b), S-segment mRNA levels (Fig. 2c), percentage of Gn-positive cells (Fig. 2d), cytopathic effect (Fig. 2e), and progeny virus production (Fig. 2f) comparable to those of the control SW13 cells expressing endogenous LDLR. In these experiments, LDLR-deficient SW13 cells reconstructed with LDLR (ΔEGF) partially supported CCHFV infection, while reconstructed with LDLR (ΔLBD) failed to support CCHFV infection (Fig. 2b-f). These results suggest that LBD of LDLR is essential for LDLR-mediated CCHFV infection. Table 1. LDLR sequences and domains LDLR is essential for the binding of CCHFV to cells.

[0130] We then investigated whether LDLR is essential for CCHFV binding to the cell surface and its internalization. We found that, compared to control cells, CCHFV binding at 4°C and internalization at 37°C were significantly reduced in LDLR-deficient cells (Fig. 3a). In similar experiments, CCHFV internalization (rather than its binding to the cell surface) was significantly affected in LDLRAP1-deficient SW13 cells (Fig. 3a), LDLRAP1 being an adaptor protein involved in LDLR internalization after its ligand binding. These results indicate that LDLR is important for both CCHFV cell binding and internalization.

[0131] To further confirm the effect of LDLR on CCHFV binding and entry, we investigated whether LDLR-specific antibodies could block CCHFV infection. Pretreatment of SW13 cells with anti-human LDLR mAb (R301) or pAb (#AF2148) reduced CCHFV infection in a dose-dependent manner (Fig. 3b). Similarly, pretreatment with anti-human LDLR mAb (R301) reduced CCHFV infection in Huh7 and Vero E6 cells, while pretreatment with an anti-mouse LDLR antibody reduced CCHFV infection in mouse Hepa1-6 cells (Fig. 3b). In similar experiments, pretreatment of SW13 cells with the same anti-human LDLR mAb had no significant effect on RVFV, EBIV, or VSV infection (Fig. 3c). We also generated an Fc fusion protein with a soluble extracellular domain of human LDLR (shLDLR-Fc) and evaluated its ability to inhibit CCHFV infection. Pre-incubation of CCHFV with shLDLR-Fc (but not control Fc) reduced viral infection in SW13, Huh7, 293T, Vero E6, and Hepa1-6 cells in a dose-dependent manner (Fig. 3d). Furthermore, pre-incubation with shLDLR-Fc also reduced VSV infection, but had no significant effect on RVFV and EBIV infection (Fig. 3e). In summary, these results indicate that LDLR mediates CCHFV entry and internalization. LDLR directly combines with CCHFV's Gc

[0132] Next, we determined whether LDLR could directly bind to CCHFV viral particles. We incubated CCHFV with biotinylated shLDLR and captured LDLR using magnetic streptavidin beads. The bound CCHFV viral particles were detected by RT-qPCR. As shown in Figure 4a, biotinylated LDLR could pull down CCHFV viral particles. Furthermore, we performed ELISA-based binding assays using immobilized anti-Gc mAb, anti-Gn mAb, or their respective control IgGs, CCHFV, incrementing concentrations of biotinylated shLDLR, and horseradish peroxidase-conjugated avidin. As shown in Figure 4b, anti-Gc or anti-Gn mAb (but not their respective control IgGs) captured CCHFV viral particles, which interacted with biotinylated shLDLR, as evidenced by an increase in OD450 absorbance.

[0133] Since Gc is responsible for CCHFV entry, we then examined whether LDLR could directly interact with Gc. In vitro pull-down assays showed that recombinant shLDLR directly binds to the extracellular domain of Gc in CCHFV strains YL16070 and IbAr 10200, but not to Gn (Fig. 4c). Octet biolayer interferometry showed that recombinant shLDLR effectively binds to the extracellular domain of Gc, with a KD of 32.6 nM for Gc in YL16070 and 42.6 nM for Gc in IbAr 10200, comparable to the binding between shLDLR and VSV G protein (KD = 54.3 nM). The binding of shLDLR to Gc is Ca2+-dependent. 2+ Because EDTA eliminates their binding ( Figure 4d ), which is similar to LDLR in Ca 2+ This is consistent with the view that shLDLR binds to its physiological ligand (LDL) in a dependent manner. In these experiments, shLDLR could not bind to Gn ( Figure 4d In summary, these results indicate that LDLR binds directly to Gc of CCHFV with high affinity. Table 2. CCHFV GPC Sequences and Domains LDLR is essential for the pathogenesis of CCHFV in mice.

[0134] To assess the physiological importance of LDLR as a CCHFV entry receptor in vivo, we utilized LDLR-deficient mice. Since immunocompetent mice are resistant to CCHFV infection, we used an anti-IFNAR1 monoclonal antibody (MAR1-5A3) to transiently inhibit the type I IFN-triggered antiviral effect in CCHFV infection, as previously described. Eight-week-old female WT and LDLR mice were administered... - / - Mice were administered MAR1-5A3 for 24 hours, followed by intraperitoneal challenge with CCHFV (10 TCID50 / mouse), and monitored daily thereafter. As shown in Figures 5a and 5b, LDLR-deficient mice exhibited significantly less weight loss and higher survival rates. Furthermore, viral load in the liver, spleen, and serum of LDLR knockout mice was significantly reduced (Figure 5c). Histopathological analysis revealed extensive necrosis and congestion in the liver of CCHFV-infected WT mice, while the degree of liver damage in LDLR knockout mice was much lower. The spleen of CCHFV-infected WT mice showed extensive necrosis with blurred boundaries between the white and red pulp, while the white and red pulp boundaries of the spleen in LDLR knockout mice were clearly visible, with significantly reduced necrosis (Figure 5d). Immunohistochemical analysis showed a significant reduction in CCHFV Gn-positive cells in the liver and spleen of LDLR-deficient mice (Figure 5d).

[0135] Next, we evaluated whether LDLR blocking antibodies impaired CCHFV infection in C57BL / 6 mice. Wild-type C57BL / 6 mice were pretreated intraperitoneally 24 hours before infection with anti-IFNAR1 antibody (MAR1-5A3) plus anti-mLDLR antibody (50305-R004) or control IgG. To avoid the potential impact of antibodies administered via the same route on viral infection, mice were challenged subcutaneously with CCHFV in this experiment. After CCHFV infection (100 TCID50 / mouse, a dose that can cause death via subcutaneous administration), mice were administered anti-mLDLR antibody (50305-R004) or control IgG daily for 5 days (Fig. 6a). As shown in Figs. 6b and 6c, treatment with anti-mLDLR antibody significantly reduced weight loss and mortality in CCHFV-infected mice. Consistently, viral load (Fig. 6d), pathological damage, and CCHFV Gn-positive cells (Fig. 6e) in the liver and spleen of mice treated with anti-mLDLR antibody were significantly reduced. These results indicate that LDLR plays an important role in the infection and pathogenesis of CCHFV in mice.

[0136] This example then examined whether LDL could inhibit CCHFV infection. SW13 and Huh7 cells were infected with CCHFV (MOI=0.05) or not in the presence of the indicated LDL concentration. CCHFV S mRNA levels were analyzed by RT-qPCR twenty-four hours post-infection. Data were normalized relative to data from cells infected with CCHFV in the presence of 0 µg / ml LDL. Figures 8a-b As shown, LDL inhibited CCHFV infection in both cell types in a dose-dependent manner. * * *

[0137] This disclosure is not limited to the scope described by the specific embodiments, which are intended as individual illustrations of various aspects of this disclosure, and any functionally equivalent compositions or methods are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the methods and compositions of this disclosure without departing from the spirit or scope of this disclosure. Therefore, this disclosure is intended to cover modifications and variations thereof, provided they fall within the scope of the appended claims and their equivalents.

[0138] All publications and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

Claims

1. A method for preventing or treating Crimean-Congo hemorrhagic fever virus (CCHFV) infection in a subject, comprising administering to the subject an agent that reduces the expression or activity of LDLR, or inhibits the interaction between the low-density lipoprotein receptor (LDLR) and the Gc glycoprotein (Gc) of CCHFV.

2. The method according to claim 1, wherein the agent is selected from the group consisting of: (a) Soluble LDLR protein; (b) Anti-LDLR antibody or antigen-binding fragment; (c) Anti-Gc antibody or antigen-binding fragment; (d) Small molecule inhibitors of LDLR or Gc; (e) Repressive RNA that inhibits LDLR expression; (f) Gene-editing agents that reduce LDLR expression or activity; (g) Rapamycin; (h) Liver X receptor (LXR) agonists, LXR proteins, or polynucleotides encoding LXR proteins; (i) Retinol X receptor (RXR) agonists, RXR proteins, or polynucleotides encoding RXR proteins; (j) ZFP36 ring finger protein-like 1 (ZFP36L1) agonist, ZFP36L1 protein or polynucleotide encoding ZFP36L1 protein; (k) ZFP36 ring finger protein-like 2 (ZFP36L2) agonist, ZFP36L2 protein or polynucleotide encoding ZFP36L2 protein; (l) Progestins and AdipoQ receptor family member 3 (PAQR3) agonists, PAQR3 protein or polynucleotides encoding PAQR3 protein; (m) proprotein convertase subtilisin / kexin type 9 (PCSK9) agonist, PCSK9 protein or polynucleotide encoding PCSK9 protein; (n) Low-density lipoprotein; and (o) Agents or molecules or combinations thereof that directly or indirectly cause downregulation of the level of LDLR or its soluble extracellular domain or CCHFV binding activity.

3. The method according to claim 2, wherein the agent is a soluble LDLR protein, preferably not containing a transmembrane domain of the LDLR protein and containing at least one LDLR type A repeat sequence in a ligand-binding domain (LBD).

4. The method according to claim 3, wherein the soluble LDLR protein comprises at least LDLR A-type repeat sequence 1, LDLR A-type repeat sequence 2, LDLR A-type repeat sequence 3, LDLR A-type repeat sequence 4, LDLR A-type repeat sequence 5, LDLR A-type repeat sequence 6 or LDLR A-type repeat sequence 7.

5. The method of claim 3, wherein the soluble LDLR protein further comprises an IgG Fc domain.

6. The method according to claim 2, wherein the agent is an anti-LDLR antibody or an antigen-binding fragment, preferably binding to at least one LDLR type A repeat sequence in a ligand-binding domain (LBD).

7. The method of claim 6, wherein the anti-LDLR antibody or antigen-binding fragment does not bind to the EGF-like domain of the LDLR protein.

8. The method according to claim 2, wherein the agent is an anti-Gc antibody or an antigen-binding fragment.

9. The method according to claim 2, wherein the agent is a small molecule inhibitor of LDLR or Gc.

10. The method of claim 2, wherein the agent is a repressive RNA that inhibits LDLR expression.

11. The method of claim 10, wherein the repressive RNA is selected from the group consisting of miRNA, siRNA, shRNA, piRNA, asRNA, and antisense RNA.

12. The method of claim 2, wherein the agent is a gene-editing agent that reduces LDLR expression or activity.

13. The method of claim 12, wherein the gene-editing agent comprises a CRISPR / Cas agent, a TALEN agent, or a zinc finger nuclease.

14. The method according to claim 2, wherein the agent is rapamycin.

15. The method of claim 2, wherein the agent is a liver X receptor (LXR) agonist, an LXR protein, or a polynucleotide encoding an LXR protein.

16. The method of claim 15, wherein the LXR agonist is selected from the group consisting of oxosterol, hypochondramide, T0901317, GW3965 and N,N-dimethyl-3β-hydroxycholenic acid (DMHCA).

17. The method of claim 16, wherein the oxosterol is selected from the group consisting of 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol and cholesteric acid.

18. The method of claim 2, wherein the agent is a retinol X receptor (RXR) agonist, an RXR protein, or a polynucleotide encoding an RXR protein.

19. The method of claim 18, wherein the RXR agonist is 9-cis-13,14-dihydroretinoic acid.

20. The method of claim 2, wherein the agent is a protein selected from the group consisting of hepatic X receptor (LXR), retinol X receptor (RXR), ZFP36 ring finger protein-like 1 (ZFP36L1), ZFP36 ring finger protein-like 2 (ZFP36L2), progesterone and AdipoQ receptor family member 3 (PAQR3) and proprotein convertase subtilisin / kexin type 9 (PCSK9), or a polynucleotide encoding said protein.

21. The method of claim 20, wherein the polynucleotide is cDNA or mRNA.

22. The method of claim 21, wherein the mRNA is chemically modified.

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