Identification and use of nucleocapsid protein targeting molecules that protect animals against crimean-congo hemorrhagic fever virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is an endemic and emerging virus and a WHO priority pathogen, and thus medical countermeasures such as antibodies that broadly protect against heterologous strains of CCHFV are in need to mitigate a severe disease caused by CCHFV. The nucleocapsid (N) protein of CCHFV is highly conserved between CCHFV strains, and it can function as a target of therapeutic treatment. In this disclosure, a molecule, in particular a monoclonal antibody-based molecule, against the N protein of CCHFV is provided, comprising a polypeptide having a heavy chain variable region, a polypeptide having a light chain variable region and / or a polypeptide having a heavy chain variable region and a light chain variable region, or fragment or variant thereof as well as a nucleotide comprising a nucleic acid strand encoding a heavy chain variable region, a nucleotide comprising a nucleic acid strand encoding a light chain variable region and / or a nucleotide comprising a nucleic acid strand encoding a heavy chain variable region and a light chain variable region.

Description

Attorney Docket 15969-018PC0 IDENTIFICATION AND USE OF NUCLEOCAPSID PROTEIN TARGETING MOLECULES THAT PROTECT ANIMALS AGAINST CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support from the United States Army Medical Research Institute of Infectious Disease, an organization of the United States Army Medical Research and Materiel Command. The United States Government has certain rights in the invention. BACKGROUND

[0002] Crimean-Congo hemorrhagic fever virus (CCHFV) has a wide geographical distribution that is considered endemic. CCHFV is naturally spread through bites of ixodid ticks, primarily those of the genus Hyalomma, and due to the expansion of the tick, it is emerging in new areas and designated one of WHO priority pathogens.

[0003] CCHFV infects a large number of wild and domesticated mammalian species, including giraffe, buffalo, zebra, bovines and ovines, in addition to some avian species such as ostriches. However, infection in these animals is generally asymptomatic, at most producing a prolonged (> 5 days) viremia (Shepherd et al. Am. J. Trop. Med. Hyg. 40, 541–547 (1989); Spengler et al. Antivir. Res. 135, 31–47 (2016). In marked contrast, CCHFV infection in humans can lead to a severe and potentially life-threatening disease termed Crimean-Congo hemorrhagic fever (CCHF).

[0004] Human infections can result from occupational or ceremonial exposure to infected animals during slaughter of livestock such as ostriches, cattle, and sheep (Bente et al., Antivir. Res. 100, 159–189 (2013); Swanepoel et al. Epidemiol. Infect. 121, 427–432 (1998)). Hospital staff are also at a high risk of exposure to healthcare associated infections from infected patients, particularly in situations where CCHFV is unsuspected.

[0005] The mortality rate of CCHFV ranges from 3 – 30 % and is suspected to depend on multiple factors including viral strain, speed of diagnosis, and access to emergency healthcare.Attorney Docket 15969-018PC0 Currently, there are no licensed therapeutics or vaccines to prevent or treat CCHFV, although an antiviral drug ribavirin may provide some limited therapeutic benefit (Ergonul et al.,Clin. Infect. Dis. 39, 284–287 (2004)). Therefore, anti-CCHFV products that can be used both prophylactically and post-exposure are needed, and antibody based anti-CCHFV products may offer an important strategy to prevent spread of CCHFV and / or mitigate severe symptoms caused by the virus. SUMMARY

[0006] Embodiments of the present invention are based on results described herein, demonstrating that a molecule targeting the nucleocapsid (N protein) of CCHFV, in particular a non-neutralizing murine monoclonal antibody against the N protein of CCHFV, mAb-9D5, which was produced with strain IbAr 10200 using hybridoma cells, provides significant protection in two CCHFV adult mouse models, interferon knockout mice (IFNAR- / -) and type I interferon antibody blockaded mice (IS). Since CCHFV causes severe symptoms only in mice deficient in Type-1 interferon (IFN-I) signaling, these two IFN-I disrupted mouse systems are widely used for CCHFV studies. Alternatively, the molecule targeting the nucleocapsid of CCHFV may also be mAb-2B11.

[0007] Accordingly, in certain embodiments, a molecule, in particular a monoclonal antibody- based molecule, against the N protein of CCHFV is provided, comprising a polypeptide having a heavy chain variable region, a polypeptide having a light chain variable region and / or a polypeptide having a heavy chain variable region and a light chain variable region, or fragment or variant thereof as well as a nucleotide comprising a nucleic acid strand encoding a heavy chain variable region, a nucleotide comprising a nucleic acid strand encoding a light chain variable region and / or a nucleotide comprising a nucleic acid strand encoding a heavy chain variable region and a light chain variable region. The molecule confers protection against CCHFV.

[0008] The protective efficacy of mAb-9D5 was demonstrated in adult mice. It was shown that mAb-9D5 protected 50% of mice against lethal CCHFV infection, and that mAb-9D5 treatment resulted in a significant delay in mean time-to-death in mice that succumbed to disease compared with isotype control mice. It was shown that there was a delay in liver injury in mAb-9D5 treatedAttorney Docket 15969-018PC0 mice, an important tissue target of CCHFV, and that mAb-9D5 treatment delayed destruction of Kupffer cells and infiltration of inflammatory cells into the liver.

[0009] In another embodiment, it was shown that although mAb-9D5 protects against CCHFV when given prophylactically, it was not able to protect in a post-exposure setting. In another embodiment, this antibody-mediated protection was shown to be independent of Fc-receptor functionality and complement activity.

[0010] Further, in another embodiment, since mAb-9D5 was produced with strain IbAr 10200, the cross-protective efficacy of the mAb-9D5 was tested with another strain of CCHFV, Afg09- 2990 to prove that the protective efficacy of the antibody treatment was significant against both strains.

[0011] In summary, this disclosure demonstrates that the N protein is a viable target for antibody-based therapeutics and that mAb-9D5, a monoclonal antibody against the N protein of CCHFV can protect against heterologous CCHFV strains.

[0012] Further, it was previously discovered by our group that antibodies against the GP38 glycoprotein can protect against CCHFV prophylactically and post-exposure in murine systems 14. Thus, combination of N protein antibody and GP38 antibody can be explored to produce a synergistic product that functions as both a potent prophylaxis and post-exposure therapeutic.

[0013] These and other embodiments are described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Fig. 1: MAb-9D5 protects mice from CCHFV infection. FIG. 1A IFNAR− / −mice (N = 10 / group) were treated with mAb-9D5 (blue circles) or isotype control antibody (red squares) IP on days −1 and +3. Mice were challenged SC with CCHFV strain IbAr 10200 on day 0 and survival and group weight loss were monitored and plotted using Prism software. ****P < 0.0001. Source data is provided. FIG. 1B Representative H&E ISH staining of livers of CCHFV strain IbAr 10200-infected mice treated with mAb-9D5 (days 4, 8, and 12) or isotype control antibody (day 4). ISH-stained tissue was counterstained with hematoxylin. FIG. 1C Liver sections from CCHFV strain IbAr 10200-infected mice treated with anti-NP or isotype controlAttorney Docket 15969-018PC0 were stained with anti-CLEC4F (green) and anti-CCHFV NP antibodies (red). Cell nuclei were stained with DAPI (blue). FIG. 1D IFA straining for CD68+ macrophages (green) and CD45+ leukocytes (red) or Ki67+ proliferating cells (green) and MPO+ neutrophil granulocytes (green) in livers of anti-NP or isotype control-treated and -infected mice. Nuclei are stained with DAPI (blue). µM micrometer. FIG. 1B–D N = 5 mice / group for days 4 and 8, and N = 2 mice for day 12. Samples with the most severe pathology are shown.

[0015] FIG. 2: MAb-9D5 does not provide postexposure protection. FIG. 2A IFNAR− / −mice (N = 3) (circles, squares and right side up triangles) were euthanized four days post infection with strain IbAr 10200 or uninfected (N = 1) (upside down triangles), and the presence of NP, GC, and GP38 proteins in the sera was determined by MAGPIX assay. The line shows the mean of the two displayed replicates. FIG. 2B IFNAR− / −mice (N = 10 per group) were treated with two doses of the mAb-9D5 (1 mg / dose) on days −1 /  + 3 (black circle) or +1 /  + 4 (red square) or isotype control antibody days +1 /  + 3 (aqua triangle) and survival and group weight monitored. Mice were SC-infected with CCHFV strain IbAr 10200 on day 0 and survival and weight monitored. Log-rank test comparing each treatment group to the isotype control; ***P = 0.0004. Source data is provided.

[0016] FIG. 3: Surface localization of NP in CCHFV-infected cells. Non-permeabilized A549 cells infected with CCHFV strain IbAr 10200 were stained with the indicated antibodies against CCHFV viral proteins (NP; mAb-9D5 or Gc; mAb-8A1; green) and cell mask (red). Cell nuclei were stained with DAPI (blue). Representative images are shown from two experiments, each with six replicates. µM micrometer.

[0017] FIG. 4: Fc-domains do not impact the protective efficacy of mAb-9D5 in IFN-I antibody-blockaded mice. FcR− / −, C3− / −, or B6:129 (wild-type) mice (N = 10 / group) were injected SC with two doses of the mAb-9D5 (black squares) or an isotype control antibody (red circle) (1 mg / dose) onD-1 /  + 3. IFN-I was blocked on day +1 using mAb-5A3 (2.5 mg) injected IP. Mice were challenged with CCHFV strain IbAr 10200 by the IP route. Survival and group weights were plotted. Log-rank test; ****P < 0.0001, ***P = 0.0004, **P = 0.0012. Source data is provided.

[0018] FIG. 5: MAb-9D5 binding kinetics of on NP of representative CCHFV and nearAttorney Docket 15969-018PC0 neighbor species.. FIG. 5 BLI binding kinetics of mAb-9D5 hybridoma with nairovirus NPs. BLI SA biosensors were loaded with NP (antigen) at concentration of 500 nM. Anti-NP mAb was evaluated in triplicate at concentrations of 700, 350, and 100 nM. NB not binding. Source data is provided. IbAr 10200 (accession #MH483987.1), Afg09 (accession #HM452305.1), Kosova Hoti (accession #JN173797.1), Oman (accession #DQ211645.1), Semunya (accession #DQ076413), Senegal (accession #DQ211640), Aigai virus (accession #NC_078226), Hazara virus (accession #NC_038711) and Erve virus (accession #JF911699)

[0019] FIG. 6: Heterologous protection of mice by mAb-9D5. IFNAR− / −(N = 10) mice were treated with two doses of the mAb-9D5 (blue circles; green triangles) or an isotype control antibody (red squares; purple triangles) on day −1 /  + 3, and challenged SC with either IbAr 10200 (blue circles, red squares) or Afg09 (green triangles, purple triangles). Survival and percent group weight change were monitored. Log-rank test; **P = 0.0055, *P = 0.011. Source data is provided.

[0020] FIG. 7 Histopathology and ISH of CCHFV infected mouse liver. Representative H&E ISH staining of livers of CCHFV strain IbAr 10200 infected mice treated with mAb-9D5 (days 4, 8 and 12) or isotype control antibody (day 4). Red box denotes the magnified regions shown in Figure 1B. Day 4 N=5 mice / group, day 8 N=5 mice mAb 9D5 treated, day 12 N=2 mAb 9D5 treated were evaluated and representative images depicting the most severe pathology are shown.

[0021] FIG. 8 Histopathology and ISH of CCHFV infected mouse spleen. H&E and ISH comparison of spleen between mice treated with mAb-9D5 (days 4, 8 and 12) or isotype control animals (day 4 only) (4x magnification). Day 4, MAb-9D5 treated mice had mild ISH labeling corresponding with to areas of necrosis / inflammation. Day 8 animals had moderate lymphoid depletion and apoptosis / necrosis, indicated by areas of pallor in the white pulp, especially in the marginal zone, imparting a “moth eaten” appearance to the white pulp; ISH labeling was minimal. mAb-9D5 day 12 spleens showed mild lymphoid depletion in the white pulp with foci of inflammation and necrosis in the red pulp (and / or at the junction with the white pulp), and mild ISH labeling. Isotype controls spleens had marked lymphoid depletion with apoptosis / necrosis and hemorrhage in the white pulp, and inflammation in the red pulp. ThereAttorney Docket 15969-018PC0 were also marked ISH labeling. ISH stained spleens were counterstained with hematoxylin. Day 4 N=5 mice / group, day 8 N=5 mice mAb 9D5 treated, day 12 N=2 m and representative images depicting the most severe pathology are shown.

[0022] FIG. 9 Surface localization of NP in CCHFV infected cells. Permeabilized and non-permeabilized A549 cells infected with CCHFV strain IbAr10200 were stained with the indicated antibodies against CCHFV viral proteins (NP;MAb-9D5 or Gc; mAb-8A1; green) and cell mask (red). Cell nuclei were stained with DAPI (blue). Two experiments were conducted with six replicates per experiment.

[0023] FIG. 10. Mouse ascite-mAbs binding affinity screening to NP. BLI SA biosensors were loaded with NP (antigen) at concentrations of 500 nM and a single dilution (1:64) of the ascite- mAbs was employed in triplicate. Wavelength shift (nm) correspond to a semi- quantitative measurement of the binding affinities between the mAbs and NP. Erve-virus did not bind to any of the tested mAbs.

[0024] FIG. 11. IFNAR− / −mice (N = 8 / group) were treated with mAb-2B11 (triangles) or isotype control antibody (squares) IP on days −1 and +3. Mice were challenged SC with CCHFV strain Afg09-2990 on day 0 and survival was monitored and plotted using Prism software. ****P = 0.0017. DETAILED DESCRIPTION

[0025] Based on the discoveries presented herein, one embodiment pertains to a molecule targeting nucleocapsid protein of Crimean-Congo Hemorrhagic fever virus (CCHFV) and methods thereof involving administering such molecule for treating a subject who has CCHFV infection. Provided in the antibodies section are sequences pertaining to the protective molecule targeting the N-protein of CCHFV. In one example, the molecule includes (a) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2, or fragment or variant thereof; (b) a polypeptide comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 4, or fragment or variant thereof; (c) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence ofAttorney Docket 15969-018PC0 SEQ ID NO: 2, or fragment or variant thereof, and a polypeptide comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 4, or fragment or variant thereof; or (d) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence of SEQ ID NO: 4. In one example, the molecule targeting nucleocapsid protein of CCHFV is mAb-9D5 (BEI resources, cat no. NR-40270) or mAb-2B11 (BEI resources, cat no. NR-40257).

[0026] In other examples, the molecule targeting nucleocapsid protein of CCHFV may include one or more portions having sequences related to SEQ ID NOs: 2 and 4. In specific examples, the molecule may include (a) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide comprising a light chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4; (c) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and a polypeptide comprising a light chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (d) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4.

[0027] In a specific example, the molecule targeting nucleocapsid protein of CCHFV of claim 1 or 2, wherein the molecule is a single chain polypeptide. In a more specific example, the molecule comprises two single chain polypeptides. The molecule may be a monoclonal antibody.

[0028] In other embodiments, the molecule targeting nucleocapsid protein of CCHFV includes an immunoglobulin constant domain, wherein the constant domain is selected from an IgGl or aAttorney Docket 15969-018PC0 variant thereof, an IgG2 or a variant thereof, IgG3 or a variant thereof, an IgG4 or a variant thereof, an IgA or a variant thereof, an IgE or a variant thereof, an IgM or a variant thereof, and / or an IgD or a variant thereof.

[0029] In yet other embodiments, the molecule targeting nucleocapsid protein of CCHFV is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, or a diabody.

[0030] Other embodiments pertain to a nucleic acid strand encoding a molecule targeting nucleocapsid protein of CCHFV of claim 1, comprising SEQ ID NO: 1 and / or SEQ ID NO: 3. Also provided is a recombinant vector comprising one or both of the aforementioned nucleic acid strand(s). Also provided herein is host cell comprising the aforementioned nucleic acid strand(s), wherein the host cell is selected from bacterial, yeast, insect or mammalian cell. The host cell may be selected from the group of CHO cell, 293 cell, or hybridoma.

[0031] Also provided is a pharmaceutical composition comprising a molecule targeting nucleocapsid protein of CCHFV. The pharmaceutical composition may include on or more of (a) the molecule targeting nucleocapsid protein of CCHFV, the nucleic acid strand, the recombinant vector, or the host cell as described herein; and, optionally, (b) a pharmaceutically acceptable additive, excipient and / or carrier.

[0032] In other embodiments, provided is a method of treating a subject in need so as to confer protection against CCHFV. The method may involve administering to the subject a composition that comprises the molecule targeting nucleocapsid protein of CCHFV, a nucleic acid strand or a recombinant vector as described herein. In an alternative embodiment, administered is one or more additional molecules targeting other CCHFV proteins such as antibodies against GP38 before, concomitantly, or after the administration of the molecule targeting nucleocapsid protein of CCHFV, a nucleic acid strand or a recombinant vector for prophylactic and / or post-exposure treatment against CCHFV. Definitions

[0033] The preferred materials and methods are described herein; any methods and materials similar or equivalent to those described herein can be used in the practice of or testing of theAttorney Docket 15969-018PC0 invention. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to be limiting.

[0034] The articles "a," "an," "the" and the like are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article, unless specifically noted otherwise. By way of example, "an element" means one element or more than one element. Unless otherwise indicated, “or” encompasses “and.” To illustrate, “A, B, or C” means A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C, unless otherwise illustrated.

[0035] The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0036] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, a quantum of measurement, and the like, is meant to encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%, even more preferably .+-.1%, and still more preferably .+-.0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0037] The terms "administration" or “administering” of an antibody or a polypeptide to a subject refers to introducing or delivering to a subject a substance in order to perform its intended function. The administering or administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administering or administration includes self- administration and the administration by another.

[0038] The terms “co-administration” or “co-administering” as used herein refer to the administration of a substance before, concurrently, or after the administration of anotherAttorney Docket 15969-018PC0 substance such that the biological effects of either substance synergistically overlap.

[0039] The term "antigen" or "Ag" refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells (e.g. through T cell receptor activation), or both. Any macromolecule, including virtually all proteins, peptides, carbohydrate, nucleotide, viruses, or fragments thereof can serve as an antigen. Furthermore, antigens can be generated from recombinant or genomic DNA or from RNA or DNA from a virus. Any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. An antigen need not be encoded solely by a full-length nucleotide sequence (e.g., a full gene). An antigen need not be encoded by a "gene" at all, and may comprise, for example, sugars or non-coding nucleic acids, alone or in combination with a protein or peptide. An antigen can be generated, synthesized, or originate from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a cell, or a biological fluid. For example, a biological sample can comprise a producing cell line, a virus, and culture media, wherein the producing cell line produces the virus, and wherein the virus may or may not be released into the culture media.

[0040] As used herein, the term "antibody", unless otherwise indicated, refers to a monoclonal antibody (mAb), or an immunologically effective fragment thereof, such as a Fab, Fab', or F(ab')2 fragment. In some contexts, regardless of whether fragments are specified, the term "antibody" includes such fragments as well as single-chain forms. As long as the protein retains the ability specifically to bind its intended target, it is included within the term "antibody."

[0041] The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). Each heavy chain consists of at least four domains (each about 100 to 110 amino acids long)- an amino-terminal variable (VH) domain, which is primarily responsible for antigen recognition, followed by three constant domains: CH1, CH2, and the carboxy-terminal CH3. The carboxy-terminal portion (Fc) including CH2 and CH3 domains defines a region primarily responsible for effector function. The "hinge" connects CH2 and CH3 domains in the central part of the heavy chains. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. NaturallyAttorney Docket 15969-018PC0 produced antibodies are also glycosylated, typically on the CH2 domain. Each light chain consists of two domains - an amino-terminal variable (VL) domain, which is primarily responsible for antigen recognition, followed by a carboxy-terminal constant (CL) domain. Within light and heavy chains, the variable region and constant region are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 3 or more amino acids, consequently forming a V(D)J segment.

[0042] Light chains are classified as kappa and lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within each isotype, there may be subtypes, such as IgG1, IgG2, IgG3, IgG4, etc. The variable regions of each light / heavy chain pair form the antibody binding site, and two binding sites exist in an intact antibody. In the variable regions, both chains all exhibit the same general structure of relatively conserved four framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs in both chains are aligned between the framework regions. From N- terminal to C-terminal of the variable region, both light and heavy chains comprise the domains of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0043] The present disclosure also contemplates isotype modification. By modifying the Fc region to have a different isotype, different functionalities can be achieved. For example, recombinant full length IgG antibodies can be generated by subcloning the heavy chain DNA of this disclosure from the cloning vector into an IgG plasmid vector, transfected into 293 Freestyle cells or CHO cells, and antibodies were collected a purified from the 293 or CHO cell supernatant.

[0044] Modified antibodies may be made by any technique known to those of skill in the art, including expression through standard molecular biological techniques, or the chemical synthesis of polypeptides.

[0045] As used herein, the term “antibody fragment” or “antigen binding fragment” refers to a polypeptide containing fragments of a full-length antibody, maintaining the ability to bind specifically to the same antigen, and / or to compete with the full length antibody to bind to the antigen, which is also called “the antigen binding portion”. See Fundamental Immunology, Ch.Attorney Docket 15969-018PC0 7 (Paul, W., ed. 2, Raven Press, N.Y. (1989)), including the entire article and references in this invention for all purposes.

[0046] Antibody fragment or antigen binding fragment can be generated by recombinant DNA techniques or by cleaving intact antibodies with proteolytic enzymes or chemicals. In some cases, the antibody fragment or antigen binding fragment include Fab, Fab′, F(ab)2, Fd, Fv, dAb, and CDR fragments, single chain antibodies (e.g., scFv), chimeric antibody, humanized antibody, diabody, and the polypeptide that at least contains an antibody portion which is sufficient to confer a specific antigen binding capacity to the polypeptides.

[0047] As used herein, the term “immunoreactive fragment” or “immunologically effective fragment” refers in this context to an antibody fragment reduced in length compared with the full length wild-type or parent antibody, which retains an acceptable degree or percentage of binding capacity to the target antigen. As will be appreciated by one of skill in the art, what is an acceptable degree will depend on the intended use.

[0048] As used herein, the term “epitope” refers to any moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. An epitope can be composed of a plurality of chemical atoms or groups on an antigen, which are surface-exposed when the antigen adopts a relevant three-dimensional conformation. Such chemical atoms or groups can be physically near to each other in space when the antigen adopts such a conformation, or at least some of such chemical atoms or groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

[0049] The term “hybridoma” refers to the cells formed via fusion between a short-lived antibody-producing B cell and an immortal myeloma cell. Each hybridoma constitutively expresses a large amount of one specific mAb, and favored hybridoma cell lines can be cryopreserved for long-lasting mAb production.

[0050] The antibody-producing B lymphocytes (B cells), obtained from biopsied spleens or lymph nodes or from circulating blood of the immunized animal such as mice immunized with IbAr10200 CCHFV strain, are then fused with immortal myeloma cells, generally one of the same species as the animal that was immunized or human or human / mouse chimeric cells. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-Attorney Docket 15969-018PC0 antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art.

[0051] For culturing, the selection medium may be HAT or HAT with ouabain. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a line of EBV -transformed B cells, ouabain is also used for drug selection of hybrids as EBV-transformed B cells are susceptible to drug killing, whereas the myeloma partner used is chosen to be ouabain resistant.

[0052] Culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radio-immunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays dot immunobinding assays, and the like. The selected hybridomas are then serially diluted or single- cell sorted by flow cytometric sorting and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to produce mAbs.

[0053] The hybridoma cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into an animal, preferably immunocompromised mice, such as SCID mice, to prevent tumor rejection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to obtain mAbs in high concentration. Alternatively, hybridoma cells lines can be cultured in vitro to produce mAb in cell supernatant. The cell lines can be adapted for growth in serum-free medium to optimize the ability to recover human monoclonal antibodies of high purity.Attorney Docket 15969-018PC0

[0054] Monoclonal antibodies produced by either ways may be purified, if desired, using filtration, centrifugation and various chromatographic methods such as Protein G or A affinity chromatography or FPLC, using assay of binding and neutralization.

[0055] Generally, complete antibodies are fractionated utilizing agents (i.e., protein A or protein G) that bind the Fc portion of the antibody. Alternatively, antigens fixed to a support, such as beads or resins packed in a column may be used to simultaneously purify and select appropriate antibodies. After antibody binding to the protein A / G or antigen fixed to a support, contaminants are removed (e.g., washed away), and the antibodies are released by applying conditions (salt, heat, etc.).

[0056] The fragments of monoclonal antibody of the present disclosure can be obtained from the purified monoclonal antibodies by methods that include digestion with enzymes, such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, the fragments of monoclonal antibody encompassed by the present disclosure can be synthesized using an automated peptide synthesizer.

[0057] It also is contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. For this, hybridomas may be cultured, then cells lysed, and total RNA extracted. Random hexamers may be used with RT to generate cDNA copies of RNA, and then PCR performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. PCR product can be cloned into pGEM-T Easy vector, then sequenced by automated DNA sequencing using standard vector primers. Alternatively, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the cell lines and phagemids expressing appropriate antibodies are selected by panning using viral antigens.

[0058] Monoclonal antibodies according to the present disclosure may be defined, in the first instance, by their binding specificity which in this case is for CCHFV N protein. Those of skill in the art, by assessing the binding specificity / affinity of a given antibody using techniques well known to those of skill in the art, can determine whether such antibodies fall within the scope of the instant claims.

[0059] The term “immune response” as used herein, unless otherwise indicated, refers to humoral immune response, which produces antigen-specific antibodies and is primarily drivenAttorney Docket 15969-018PC0 by B cells, and / or cell-mediated immune response, which does not depend on antibodies for its adaptive immune responses and is primarily driven by T cells, antigen-presenting macrophages and the release of cytokines in response to an antigen. “Adaptive immune response” or “acquired immune response”, unless otherwise indicated, refers to antigen-specific immune responses including a humoral immune response and / or cell-mediated immune response leading to activation and proliferation of B- and / or T-lymphocytes and, though not necessarily, activation and proliferation of antigen presenting cells, but can include it. “Innate immune response” refers to a nonspecific, non-B and / or T-lymphocyte mediated immune response, such as inflammatory responses and phagocytosis by cells such as neutrophils and macrophages.

[0060] The term “immunogenic” refers to a substance used to stimulate the immune system of a living organism, so that one or more functions of the immune system are induced, increased and directed towards the immunogenic substance. An “immunogenic polypeptide”, “immunogenic protein”, “immunogenic peptide” is a polypeptide, protein, or peptide that elicits a cell-mediated and / or humoral immune response, whether alone or linked to a carrier in the presence or absence of an adjuvant. Preferably, antigen presenting cells may be activated.

[0061] As used herein, the term “passive transfer of antibodies”, known as artificially acquired passive immunity, refers to injection of antibodies through intravenous or intramuscular injections. The forms of antibody can be human or animal blood plasma or serum, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, as high-titer human IVIG or IG from immunized or from donors recovering from disease, and as monoclonal antibodies (mAb). Such immunity generally lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non- human origin. However, passive immunity provides immediate protection. The antibodies will be formulated in a carrier suitable for injection, i.e., sterile and syringeable.

[0062] As used herein, the term "pharmaceutical composition" refers to a composition in which an active substance is formulated together with one or more pharmaceutically acceptable carriers. The composition is suitable for administration to a human or animal subject. The active substance is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.Attorney Docket 15969-018PC0

[0063] The composition of the present invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the antibody or fragment thereof, preferably in purified form, together with a suitable amount of additive, excipient and / or carrier so as to provide the form for proper administration to the patient. The composition should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, or delivered by mechanical ventilation.

[0064] The term “pharmaceutically acceptable”, when used within the context of a carrier, additive, or excipient, is one that is not unacceptably toxic to the subject to which it is administered. Pharmaceutically acceptable carriers, additives, or excipients can comprise: (1) fillers or extenders including, but not limited to, as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders including, but not limited to, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or acacia; (3) humectants including, but not limited to, glycerol; (4) disintegrating agents including, but not limited to, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators including, but not limited to, quaternary ammonium compounds; (7) wetting agents including, but not limited to, cetyl alcohol and glycerol monostearate; (8) absorbents including, but not limited to, kaolin and bentonite clay; (9) lubricants including, but not limited to, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. By way of example, a pharmaceutically acceptable carrier, additive, or excipient can include sodium citrate or calcium carbonate.Attorney Docket 15969-018PC0

[0065] The term “prophylactic” or “preventive” as used herein refers to the use of an antibody or a substance in order to reduce the likelihood of the occurrence of symptoms of condition, disease, or disorder, to delay the onset, and / or to reduce the risk of the development, frequency, and / or severity of symptoms of the disease, disorder or condition in a patient or subject. In this regard, “prevent” or “prophylactic” should not be understood to absolutely prevent the disease in the entire population, as it is epidemiologically impossible to attribute “prevent” to mean “absolute prevention.” The term “reducing the likelihood” refers to the fact that in a given population of subjects, the embodiments herein may be used to reduce the likelihood of an occurrence or recurrence of symptoms of the disease, disorder or condition in one or more subjects within that population of all subjects, rather than prevent, in all subjects, the occurrence or recurrence of a disease or each and every symptom.

[0066] The term “post-exposure” is used to describe the use of the antibody described herein to prevent or reduce CCHF symptoms after a possible exposure.

[0067] The term “sequence identity” or “identity,” as used herein in the context of two polynucleotides or polypeptides, refers to the residues in the sequences of the two molecules that are the same when aligned for maximum correspondence over a specified comparison window. As used herein, the term “percentage of sequence identity” or “% sequence identity” refers to the value determined by comparing two optimally aligned sequences (e.g., nucleic acid sequences or polypeptide sequences) of a molecule over a comparison window, wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleotide or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. A sequence that is identical at every position in comparison to a reference sequence is said to be 100% identical to the reference sequence, and vice-versa.

[0068] As used herein, the term “subject” means a human or an animal to whom the antibody is administered, treated or prophylactically treated and / or to whom methods of administration, treatment, and / or prophylactic treatment are directed. A subject can be a mammal, includingAttorney Docket 15969-018PC0 human, a domesticated animal, including bovine, ovine, and porcine, wild animals including giraffe, buffalo, zebra , and laboratory test animal, including a mouse or a non-human primate. A “subject in need” is a subject who has been exposed or is at risk of exposure to CCHFV. A risk of exposure is created when a subject has either been contact or proximity to a person with CCHFV, or has been contacted or bitten by an arthopod (e.g. tick) carrying CCHFV, such that transmission of CCHFV to the subject is possible or likely.

[0069] As used herein, the term "treat" refers to therapeutic application of a substance, where a condition to be treated is already known to be present and prophylaxis - i.e., prevention of, or amelioration of, the possible future onset of a condition.

[0070] Within the context of an amount, “effective” – as in “effective amount” – describes an amount of a compound, composition, or component, which is used to produce or effect an intended result, whether that result relates to eliciting an immunogenic response, elucidating a specific immune response to the administered antigen or nucleic acid encoding the antigen, or whether that result relates to the treatment or prevention / prophylaxis of a disorder or condition associated with the present invention or alternatively, is used to produce another compound, agent, or composition (i.e. an antibody). This term subsumes all other effective amount or effective concentration terms which are otherwise described herein.

[0071] Effective amount also encompasses an amount sufficient to be biologically active for a sufficient duration to produce or effect the intended result, such as an immunogenic response, in the intended or target tissue. A compound is subject to pharmacokinetics (administration, e.g. adherence and route; bioavailability, e.g. the production of an antigen from a nucleic acid, absorption and first pass metabolism and activation including conversion from “pro” forms; and distribution, e.g. diffusion and transport; and clearance, e.g. metabolism and clearance) and pharmacodynamics (concentration at target, affinity to target, molecular effects, physiological effects), and accordingly the “effective amount” accounts for the effects of pharmacodynamics at the target tissue and pharmacokinetics of the modality of administration to the target tissue in achieving the intended result.

[0072] As defined herein, a "therapeutically effective" treatment refers a treatment that is capable of producing a desired effect. Such effects include, but are not limited to, enhancedAttorney Docket 15969-018PC0 survival, reduction in presence or severity of symptoms, reduced time to recovery, and prevention of initial infection.

[0073] As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. A variant can also differ functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence. A variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. A variant polypeptide or nucleic acid may not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. When a reference polypeptide or nucleic acid has one or more biological activities, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.

[0074] As used herein, “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid', which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into whichAttorney Docket 15969-018PC0 they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors " Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual β α ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.(1989)), which is incorporated herein by reference for any purpose. Overview

[0075] CCHFV is an enveloped virus and a member of the genus Orthonairovirus in the Nairoviridae family in the order Bunyavirales. CCHFV has a tripartite, negative-sense RNA genome comprising small (S), medium (M) and large (L) segments. The S segment encodes the nucleocapsid (N) protein, the M segment encodes the glycoprotein precursor complex (GPC) from the glycoprotein open reading frame (ORF) that is co-translationally cleaved into two structural glycoproteins (GNand GC) as well as several non-structural proteins (mucin-like domain, GP38, GP160, GP85, and NSM), and the L segment encodes the RNA-dependent RNA polymerase.

[0076] Although the M-segment has the largest divergence, most efforts have focused on targeting the viral M-segment glycoproteins, including GP38 and GC, as the glycoproteins are accessible on the virion surface and are the target of neutralizing and non-neutralizing antibodies. Several vaccines targeting the M-segment glycoproteins has been reported to protect animals against severe disease (Buttigieg et al., PLoS ONE 9, e91516 (2014); Dowall et al. PLoS ONE 11, e0156637 (2016); Garrison et al. PLoS ONE 11, e0156637 (2016)).Attorney Docket 15969-018PC0

[0077] Recent work also revealed that antibodies targeting a specific glycoprotein protect the animal against lethal CCHFV infection in murine models. In particular, it has been discovered that the GP38 viral protein is an important target of antibody against CCHFV (Golden et al. ci. Adv. 5, eaaw9535 (2019)). A non-neutralizing monoclonal antibody (mAb) targeting this protein protects against lethal infection in a post-exposure environment and protects against different strains of CCHFV (Durie et al. Nat. Commun. 13, 7298 (2022)).

[0078] However, glycoproteins are poorly conserved among geographically separated viral strains. Accordingly, identification of antibodies that broadly protect against heterologous strains of CCHFV are in need.

[0079] N protein encoded by the S segment exhibits high homology between CCHFV strains in comparison with the proteins encoded by the M segment, and in several other virus groups including influenza, arenaviruses and coronaviruses, N protein targeting antibodies have been shown to protect against those viruses. Additionally, several N-based vaccine studies indicated that immune responses against N protein confer protection and proved that the N protein functions as a viable vaccine target (Appelberg et al., PLoS ONE 11, e0156637 (2016)).

[0080] Because N is an internal viral protein in both infected cells and within viral particles45and is not generally thought to be exposed on the viral surface to the outside environment, it was presumed that vaccine-facilitated protection was predominately mediated by a T-cell response, and the ability of N protein to function as a protective antibody target was overlooked.

[0081] However, the N protein of CCHFV is highly abundant and highly immunogenic, and anti-N protein antibodies are reported to be readily produced during infection in survivors (Papa et al. . Clin. Virol. 64, 137–143 (2015); Emmerich et al. PLoS Negl. Trop. Dis. 12, e0006366 (2018)). A more recent study using B-cell deficient mice suggested that antibodies were important for N protein targeting vaccine-mediated protection with a vaccine including both the glycoproteins and N protein (Karaasian et al., PLoS Negl. Trop. Dis. 15, e0009973 (2021)). However, direct evidence of anti-N protein antibodies protecting against CCHFV infection have not been reported. Antibody(ies) and Sequences thereofAttorney Docket 15969-018PC0

[0082] In another embodiment, the amino acid sequence of the N protein of IbAr 10200 CCHFV strain, which is structurally composed of a globular head domain and a flexible arm, was shown, and the amino acid sequence of the epitope region to which mAb-9D5 binds were identified by mass spectrometry and protease protection analysis. The epitope is found to be located within a region of the N protein containing high structural flexibility51, and it is highly conserved between the N proteins of various CCHFV strains.

[0083] Amino acid sequences and nucleotide sequences of mAb-9D5 heavy chain variable region and light chain variable region were identified (SQ1-SQ4). SQ1. mAb-9D5 Heavy chain variable region nucleotide sequence SEQ ID NO: 1 H- GAGGTCCAGCTGCAGCAGTCTGGAGCTGAACTGGTGAAACCCGGGGTATCAGTGAAGTTGTCCTGCAA GGCTTCTGGCTACACCTTCACTGAGCATTTTATACACTGGGTAAACCAGAGGTCTGGACAGGGTCTTGA GTGGATCGGGTGGCTTTCCCCTGGAAGTGATAATATGAAGTATAATGAGAAATTCAAGGACAAGGCCA CATTGACTGCGGACAAATCCTCCAACACAGTCTATTTGGAGCTTAGTAGATTGACATCTGAAGACTCTGC GGTCTATTTCTGTGCAAGACACGAAAGGGGAAAAACTTCCTGGTTTGCTTACTGGGGCCAAGGGACTCT GGTCACTGTCTCTGCA SQ2. MAb-9D5 Heavy chain variable region translated amino acid sequence SEQ ID NO: 2 EVQLQQSGAELVKPGVSVKLSCKASGYTFTEHFIHWVNQRSGQGLEWIGWLSPGSDNMKYNEKFKDKA TLTADKSSNTVYLELSRLTSEDSAVYFCARHERGKTSWFAYWGQGTLVTVSA SQ3. MAb-9D5 Light chain variable region nucleotide sequence SEQ ID NO: 3 K- GACATCCTGATGACCCAATCTCCATCCTCCATGTCTGTATCTCTGGGAGACACAGTCAGCATCACTTGCCA TGCAAGTCAGGGCATTTACAGTAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCC TGATCTATCTTGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAG ATTATTCTCTCACCATCAGCGGCCTGGAATCTGAAGATTTTGCTGACTATTACTGTGTACAGTATGCTCAG TTTCCTCCCACGTTCGGAGGGGGGACCAAGCTGGAGATAAAACGGAttorney Docket 15969-018PC0 SQ4. MAb-9D5 Light chain variable region amino acid sequence SEQ ID NO: 4 DILMTQSPSSMSVSLGDTVSITCHASQGIYSNIGWLQQKPGKSFKGLIYLGTNLEDGVPSRFSGSGSGADYSLT ISGLESEDFADYYCVQYAQFPPTFGGGTKLEIKR EXAMPLES

[0084] Example 1: A monoclonal antibody targeting CCHFV NP protects mice against infection

[0085] To determine if NP-targeting antibodies could provide protection against CCHFV, two groups of 10 IFNAR− / −mice were injected with an anti-NP monoclonal antibody (mAb-9D5) or an isotype control antibody via the intraperitoneal (IP) route on day −1 and day +3 relative to challenge. On day 0, all mice were infected with 100 plaque-forming units (PFU) of CCHFV strain IbAr 10200 by the subcutaneous (SC) route. The mice were monitored for signs of disease, and group weights were taken daily. All isotype control group mice succumbed to infection by day 5 (Fig. 1A). In contrast, mice treated with the mAb-9D5 antibody were significantly protected with 50% survival. In addition to the increase in percent surviving in the mAb-9D5- treated group (50%) versus the control group, there was also a delay in death in the treated group, with a median survival of 14 days compared to 5 days in the isotype control group. These findings indicated that NP-targeting antibodies can protect mice from CCHFV infection. Example 2: MAb-9D5 limits early viral spread to the liver and spleen and delays lesion development

[0086] To more critically assess the protective efficacy of mAb-9D5, we conducted a serial time course study in mice treated with isotype control antibody or mAb-9D5. Liver samples from five mice were collected from both groups on day 4 (isotype control and mAb-9D5-treated) and because mAb-9D5 treated mice survived, samples from this group were also collected on days 8 and 12. Generally, mAb-9D5 delayed liver injury in infected mice compared to isotype control- treated mice on day 4 (FIG. 1B and FIG. 7). Histopathological lesions in livers of isotype control CCHFV-infected mice taken on day 4 included multifocal areas of lytic necrosis, characterized by shrunken hepatocytes with hypereosinophilic cytoplasm and pyknotic and / or karyorrhectic nuclei. The pyknotic / karyorrhectic nuclei were arranged either as individualized cells (i.e., “single-cell necrosis”) in clusters, or as large aggregates of cellular debris. The necrosis often associated with areas of inflammation. All mAb-9D5-treated mice had a clinical score of 0 onAttorney Docket 15969-018PC0 day 4 dpi and one animal in the control group had a clinical score of 1 (slightly ruffled appearance), on day 8 dpi one mAb-9D5-treated mouse had a clinical score of 1 (slightly ruffled appearance) and the remaining mice scored 0. On day 4, livers from mAb-9D5 mice had markedly reduced liver injury compared to the isotype control antibody-treated mice. However, by day 8, hepatic damage in mAb-9D5 mice was mostly indistinguishable from isotype control mice on day 4. We also observed hepatocyte vacuolation located diffusely across regions (i.e., portal to central veins) and consistent with microvesicular (small vacuoles) in day 8 mAb-9D5 treated mice, and, to a lesser extent in a single isotype control liver from day 4. Vacuolation was absent in mice treated with mAb-9D5 on days 4 and 12. Viral RNA indicated by in situ hybridization (ISH) was present in all mice; however, staining was more intense in day 4 isotype controls and days 8 and 12 mAb-9D5-treated mice, compared to day 4 mAb-9D5-treated animals (Fig. 1B).

[0087] Spleens of isotype control-treated infected mice had a depletion of lymphocytes and areas of lymphocyte apoptosis / necrosis, inflammation of red pulp, histiocyte infiltration, and fibrin deposition (FIG. 8). In contrast, splenic lesions were largely absent in day 4 infected, mAb-9D5 treated mice. However, by day 8 and day 12 splenic lesions in mAb-9D5 treated mice were equivalent to or higher than those of the isotype control animals on day 4. Among those more severe was histiocyte infiltration in the red pulp of day 8 mAb-9D5 treated animals. In the spleen, ISH signal was mostly absent, except for a single mAb-9D5 mouse on day 4, but similar to control mice, viral RNA was prevalent at later time points (FIG. 8).

[0088] Our work has previously shown that mice infected with CCHFV and lacking IFN-I activity have nearly a complete loss of Kupffer cells, indicated by an absence ofCLEC4F+staining on day 4 post infectionhttps: / / www.nature.com / articles / s41467-024-46110-4 -ref-CR41. Here, we also observed an extensive loss of CLEC4F+staining on day 4 in CCHFV- infected mice treated with the isotype control antibody, which was associated with intense staining for the viral NP (Fig. 1C). However, Kupffer cells were not lost on day 4 in mAb-9D5 treated mice, and NP staining was only minor. On day 8, Kupffer cells were mostly absent in mAb-9D5-treated mice, which coincided with high levels of NP. By day 12, the Kupffer cell population was restored, but viral antigen was still present, albeit in lower amounts compared to day 8. We also observed that mAb-9D5 treatment delayed the infiltration of CD68+Attorney Docket 15969-018PC0 macrophages and CD45+ immune cells (Fig. 1D). Similar delays were seen with MPO+ granulocytes and Ki67+ proliferating cells (Fig. 1D). Example 3: MAb-9D5 does not protect against postexposure

[0089] NP appears to be highly abundant during CCHFV infection in cell culture Sanchez et al. J. Virol. 76, 7263–7275 (2002)). Therefore, we examined the day 4 serial time point isotype control animals (Fig. 1B) to determine the extent to which cell-free NP was present in the circulation. For this, we used a sensitive antigen capture system using MAGPIX to examine the presence of NP in the cell-free serum (Fig. 2A). This study revealed that secreted NP was readily detected in the serum of infected animals in addition to glycoproteins Gc and GP38. Of these proteins, levels of NP appeared to be the highest. Because NP was detected at high levels in the serum of all three infected mice, we wanted to determine if this may confound the post-challenge protective efficacy of the anti-NP antibody. Mice (N = 10 / group) were treated with two doses of mAb-9D5 either on days −1 /  + 3 or days +1 /  + 4 relative to infection, and a control group was treated on day +1 /  + 4 with a nonspecific antibody (isotype control) (Fig. 2B). Isotype control- treated mice succumbed to infection by day 5 and exhibited precipitous weight loss throughout infection (Fig. 2B). As above, mice treated with mAb-9D5 on days −1 /  + 3 exhibited a delayed weight loss compared to control mice and 50% of the mice survived, which compared to the isotype control group was significant (log rank; P = 0.0004). Mice treated with mAb-9D5 post infection had less weight loss compared to control mice but were poorly protected from lethality and succumbed by day 8. These findings indicated that while mAb-9D5 protects against CCHFV when given prophylactically, postexposure protection was limited. Example 4: CCHFV NP was detected on the surface of infected cells in vitro

[0090] To gain a mechanistic understanding of how NP-targeting antibodies protect against CCHFV, we first demonstrated that mAb-9D5 does not neutralize virus in a plaque reduction assay, in contrast to an anti-GC antibody, a well-established neutralizing antibody target (Bertolotti-ciarlet et al. J. Virol. 79, 6152–6161 (2005)) (Table 1). We then investigated if the NP could localize to the surface of CCHFV-infected cells. A549 cells were infected with an MOI of 1 with CCHFV IbAr 10200 for 24 h and stained for NP or GCunder non-permeabilizing conditions and compared to staining under permeabilizing conditions (Fig. 3 and Fig. 9). NP andAttorney Docket 15969-018PC0 GCwere detected under both conditions. The NP pattern in the non-permeabilized cells was distinct from the permeabilized cells, the latter maintained the perinuclear staining. Example 5: mAb-9D5-mediated protection does not require Fc functionality

[0091] Given that NP was detected on the surface of CCHFV-infected cells in vitro, we next evaluated if Fc-mediated processes, such as antibody-mediated cytotoxicity (ADCC) or complement-mediated functions, were important for protection conferred by mAb-9D5. For this experiment, we evaluated protection using Fc-receptor-deficient (FcR− / −) and C3 deficient (C3− / −) mice or wild-type control mice (Fig. 4). FcR− / −and C3− / −mice are unable to facilitate Fc- receptor function or complement-mediated activity, respectively. Because CCHFV only causes severe disease in mice deficient in IFN-I signaling, infected mice were treated with an antibody to block IFN-I signaling. In this system, the kinetics of disease is identical to IFNAR− / −mice. Mice (N = 10 per group) were challenged with 100 PFU of CCHFV strain IbAr 10200 by the IP route. Mice were treated with mAb-9D5 or an isotype control antibody on days −1 /  + 3 by the SC route. On day +1 post infection, mice were injected IP with mAb-5A3 to disrupt IFN-I activity. The majority of FcR− / −mice were significantly protected by mAb-9D5 (log rank; P < 0.0001), but not by the isotype control antibody. Mice lacking C3− / −(which were on a C57BL / B6:129 background) were also protected from CCHFV infection to the same degree as wild-type control mice (log rank; P = 0.0201). These data suggest that ADCC or complement are not major contributors to protection. Example 6: Identification of the mAb-9D5 epitope and cross-reactivity against other CCHFV strains

[0092] MAb-9D5 was produced against strain IbAr 10200, a laboratory-adapted strain isolated from a tick feeding on a camel (Bertolotti-Ciarlet et al.. J.Virol. 79, 6152–6161 (2005)), and our mouse studies above examined protection against this homologous strain. To evaluate the cross- protective potential of mAb-9D5, BioLayer Interferometry (BLI) was used to determine the binding affinity of mAb-9D5 for NP originated from a broad range of CCHFV strains. Afg09- 2990 (Afg09) and Kosova Hoti (Clades IV and V) presented the highest nanomolar binding affinities (KD values), followed by Semunya (Clade II) (Fig. 5C). NP from IbAr 10200 and Senegal strains (Clades III and I) showed lower KD values. The protein originating from the closely related Aigai virus possessed a KD with mAb-9D5 similar to that observed from the Semunya strain.Attorney Docket 15969-018PC0

[0093] To reveal the impact of strain-strain differences of other identified NP antibodies compared to mAb-9D5, several anti-NP mouse ascites-mAbs were screened against a similar array of NP using BLI (Fig. 10 and Table 2). We used a recently established methodology Dzimianski et al., Sci Rep. 10, 21738 (2020) that allows for rapid semi-quantitative screening of non-purified mAbs from animal fluid to assess antibody-antigen interactions. This methodology focuses on the measurement of wavelength changes (nm) during the association step and allowed insights into binding similarities within this broad range of mAbs and NPs. Binding magnitudes for NPs from CCHFV stains, or the Aigai virus towards mAb-9D5, mAb-9A1, and mAb-5F4 were consistent. mAb-7E8 and mAb-2G9 had marked differences in binding between the same NPs. While mAb-7E8 bound to all CCHFV strains, no measurable binding event occurred with the Aigai NP. Also, mAb-2G9 only showed a relatively strong binding event with the NP from the Senegal strain of CCHFV. As expected, due to its evolutionary distance from CCHFV (Wang et al. J. Virol. 89, 11740–11749 (2015)) , Erve virus NP did not bind to any of the tested mAbs and functions as a negative control (FIG. 10 and Table 2). These results indicated that NP- targeting CCHFV mAbs have broad spectrum cross-binding ability. Example 7: MAb-9D5 protects against heterologous strains of CCHFV

[0094] Because mAb-9D5 bound Afg09 with the highest affinity, we evaluated its ability to protect mice against this strain. Both strains IbAr 10200 and Afg09 are lethal in IFN-I deficient mice, and kinetics of infection are identical (Golden et al. ci. Adv. 5, eaaw9535 (2019)). Ten mice per group were treated IP with 1 mg of mAb-9D5, or isotype control, on day −1 and day +3 relative to SC challenge with 100 PFU of CCHFV strain IbAr 10200 or Afg09. The antibody treatment against IbAr 10200 challenge resulted in 40% survival and 80% survival against Afg09, compared to the isotype controls for each virus strain (Fig. 6). Both mAb-9D5 treatment groups had similar weight loss curves, which was delayed compared to the isotype control groups (Fig. 6). The protective efficacy of the antibody treatment was significant against both strains. These findings indicated that mAb-9D5 has the ability to protect against heterologous CCHFV strains, and protection may be enhanced due to higher binding affinity to Afg09 NP. FIG. 11 shows a graph demonstrating protective effects of mAb-2B11.Attorney Docket 15969-018PC0 Example 8: Materials and Methods related to Examples 1-7 Mice

[0095] C57BL / 6 (BL6), IFNAR KO mice (B6.129S2-Ifnar1tm1Agt / Mmjax), Bl6;129 mice, and C3 knockout mice (B6;129S4-C3tm1Crr / J) were purchased from The Jackson Laboratory. Fc- receptor KO mice (C.129P2(B6)-Fcer1gtm1RavN12) were obtained from Taconic. Mice were all female and 7–9 weeks in age at the time of challenge. Mice were maintained in an environment with 12 h light / dark cycle, 68–79 °F (set point: 74.5 °F), and 30–70% humidity. Virus and cells

[0096] HepG2 cells (ATCC; HB-8065) were propagated in Modified Eagle’s Medium with Earl’s salts (MEM)(Corning) and Huh7 (Texas Biomedical Research Institute), and SW13 (ATCC; #CCL−105) cells were propagated in Dulbecco’s Modified Eagles Medium with Earle’s Salts (DMEM) (Corning). Media were supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin / streptomycin (Gibco), 1% sodium pyruvate (Sigma), 1% l-glutamine (HyClone), and 1% HEPES (Gibco). A549 cells (ATCC’; #CCL-185) were propagated in Hams F-12 media supplemented with 10% FBS, 1% glutamax, 1% penicillin / streptomycin, and 1% nonessential amino acids. Minimally passaged CCHFV strain Afg09-2990 (Afg09) (Olschlager et al. J. Clin. Virol. 50, 90–92 (2011)) or strain IbAr 10200 (USAMRIID collection) were used for all experiments as indicated. Afg09-2990 was previously passaged three times in Vero cells and propagated two times in Huh7 cells, and IbAr 10200 was previously passaged nine times in suckling mouse brain and then propagated three times in HepG2 cells. The viruses were collected from clarified cell culture supernatants, sequenced to confirm identity and free of contamination, and stored at −80 °C. All CCHFV work was handled in BSL-4 containment at USAMRIID. Anti-CCHFV and isotype antibodies

[0097] Anti-CCHFV murine mAbs are part of the USAMRIID hybridoma collection produced by Jonathan F. Smith. As previously described, the USAMRIID anti-CCHFV murine mAb were developed by immunizing BALB / c mice with CCHFV-infected suckling mouse brain homogenates, and the B-cell hybridomas were generated by the fusion of Sp2 / 0 mouse myeloma cells with the splenocytes from the immunized mice Betolotti-Ciarlet J. Virol. 79, 6152–6161Attorney Docket 15969-018PC0 (2005)). The mAb-9D5 is an isotype IgG2a. Antibodies for murine challenges were purified in- house using the USAMRIID hybridoma facility. Murine isotype control antibodies were also provided by the USAMRIID hybridoma facility. Passive protection experiments

[0098] Mice were challenged with 100 PFU of CCHFV strain IbAr 10200 or Afg09 by the subcutaneous (SC) (IFNAR− / −) or intraperitoneal (IP) (all other mice) route as indicated. Virus was diluted in a total volume of 0.2 ml PBS. All mice except IFNAR− / −were IP injected with 2.5 mg of anti-IFNR1 (mAb-5A3) (Leinco Technologies, Inc; I-401) diluted in PBS 24 h post infection in a total volume of 0.4 ml. For mAb-9D5 antibody injections, mice were injected SC or IP with 1 mg / antibody / dose in a total volume of 0.2 ml diluted in PBS, as indicated. Histology

[0099] Necropsy was performed on the liver and spleen. Tissues were immersed in 10% neutral buffered formalin for 30 days. Tissues were then trimmed and processed according to standard protocolshttps: / / www.nature.com / articles / s41467-024-46110-4 - ref-CR81. Histology sections were cut at 5–6 µM on a rotary microtome, mounted onto glass slides and stained with hematoxylin and eosin (H&E). Examination of the tissue was performed by a board-certified veterinary pathologist. In situ hybridization

[0100] CCHFV was detected in infected liver samples by ISH probes targeting IbAr 10200 or Afg09 M-segment of CCHFV as previously reported (Lindquist et al. J. Virol. https: / / doi.org / 10.1128 / JVI.01083-18 (2018)). Formalin-fixed paraffin-embedded (FFPE) liver sections were deparaffinized and peroxidase blocked. Sections were then incubated with ISH probes at 40 °C for 2 h, rinsed, and the signal amplified by applying Pre-amplifier and Amplifier conjugated with HRP. A red substrate-chromogen solution was applied for 10 m at ambient temperature. The slides were further stained with hematoxylin. Images were captured on a Zeiss LSM 880 confocal system and processed using ImageJ software. IFA of tissuesAttorney Docket 15969-018PC0

[0101] FFPE tissue sections were deparaffinized using xylene and a series of ethanol washes. The sections were heated in Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA Solution, 0.05% Tween-20, pH 9.0) for 15 min to reverse formaldehyde crosslinks. After rinses with PBS (pH 7.4), the section was blocked with PBT (PBS + 0.1% Tween-20) containing 5% normal goat serum overnight at 4 °C. Then the sections were incubated with primary antibodies: rabbit polyclonal anti-myeloperoxidase (MPO) at a dilution of 1:200 (A039829-2, Dako Agilent Pathology Solutions), rat monoclonal anti-CD45 antibody at a dilution of 1:100 (05-1416, Millipore Sigma), rabbit polyclonal anti-CD68 at a dilution of 1:200 (ab125212, Abcam), and mouse monoclonal anti-Ki67 at a dilution of 1:200 (clone B56, BD Biosciences) for 2 h at room temperature. For CLEC4F and CCHFV NP detection, samples were incubated with a polyclonal goat anti-CLEC4F antibody at 1:20 dilution (PA5-47396; Thermo Fisher Scientific) and the anti- CCHFV NP murine monoclonal antibody MAb-9D5 protein at 1:500 dilution overnight at 4 °C. After rinses with PBT, the sections were incubated with secondary goat anti-rabbit or anti- chicken Alexa Fluor 488 at dilution of 1:500 (Thermo Fisher Scientific) and goat anti-mouse or anti-rat Alexa Fluor 568 at a dilution of 1:500 (Thermo Fisher Scientific) antibodies, for 1 h at room temperature. Sections were coverslipped using the Prolong Diamond mounting medium with DAPI (ThermFisher). Images were captured on a Zeiss LSM 880 or LSM700 (CLEC4F staining) confocal system (Zeiss) and processed using ImageJ software (National Institutes of Health). MAGPIX assay Capture bead preparation

[0102] CCHFV monoclonal antibodies (mAbs) were covalently linked to magnetic microspheres following the manufacturer’s instructions (Luminex) to capture NP (12E10), GC(11E7), and GP38 (13G8). Antibodies were from the USAMRIID monoclonal antibody repository. Briefly, 12.5 million microspheres were washed three times with 500 µL of activation buffer and resuspended in 274.5 µL of activation buffer. Next, 144.0 µL of sulfo-N- hydroxysulfosuccinimide and 81.5 µL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride solutions were added and tubes were gently rotated for 20 min. After activation, microspheres were washed three times with coupling buffer, and antibody was added at 4 µg per million microspheres. The reaction was allowed to incubate for 2 h, after which the microspheresAttorney Docket 15969-018PC0 were washed three times with 500 µL of PBS-T (phosphate buffered saline with 0.05% Tween- 20), resuspended at 12.5 million microspheres per mL in PBS-T, and stored at 4 °C. Each mAb was coupled to a spectrally distinct microsphere for ease of multiplexing. Detector antibody labeling

[0103] CCHFV mAbs to detect NP (5G2), GC (8A8), and GP38 (9C6) were covalently labeled with biotin according to the manufacturer’s instructions. Biotinylation of mAbs was achieved with the EZ-link™ Sulfo-NHS-LC-Biotin, No-Weigh™ Format kit (Pierce). Briefly, 50–60 µg of mAbs were reacted with a 20-fold molar excess of freshly prepared sulfo-NHS-LC-biotin (sulfosuccinimidyl-6-[biotin-amido]hexanoate) at room temperature for 30 m. After the reaction, excess biotin was removed by dialyzing against PBS-T. General assay procedure

[0104] Assays were developed on the Magpix® platform using white, Costar, round-bottom 96- well plates. Plates were loaded with 2500 microspheres per well of each capture bead to create a triplex, placed on a magnetic block for 1 m, and manually decanted. Samples were diluted 1:20 in 5% skim milk in PBS-T (SM), and 50 µL was applied to each well in triplicate. Plates were then covered and allowed to incubate with 450 rpm shaking for 1 h. After incubation, microspheres in each well were washed three times with 100 µL of PBS-T. Biotinylated detector mAbs were diluted to 4 µg / mL in SM and 50 µL was added to the appropriate wells. Plates were then covered and allowed to incubate with 450 rpm shaking for an additional hour. After incubation, microspheres were washed three times with PBS-T. For the fluorescent reporter, streptavidin phycoerythrin (SAPE; Thermo Fisher) was diluted to 10 µg / mL in SM and 50 µL was added per well before covering and incubating with shaking at 450 rpm for 30 m. After the final incubation, microspheres were washed three times with PBS-T, suspended in 100 µL of PBS-T, and read by the Magpix® instrument. Two replicates were run per sample. BLI assays mAbs binding to NP NP expression and purificationAttorney Docket 15969-018PC0

[0105] Plasmid constructs expressing full-length NP from five CCHFV clades (Afg09, Hoti, IbAr 10200, Senegal, and Semunya), and the related viruses (Aigai-Pentalofos and Erve) were cloned into pET-28a (+) vector (GenScript). NP coding sequences were added downstream to the 8X His and glutathione S-transferase (GST) tags sequences and HRV3C protease cleavage site to allow the release of the NP from the fused tags. The plasmid inserts were codon-optimized for expression in BL21 (DE3) Escherichia coli competent cells (New England BioLabs). Protein expression and purification consisted of several steps, including immobilized metal affinity (IMAC) and size-exclusion (Superdex 200—Cytiva) chromatography and were performed as previously described (Carter et al. J. Virol. 86, 10914–10923 (2012)). BLI kinetics assay mAb-9D5 to NP

[0106] BLI assay was performed using GatorPrime (GatorBio) instrument at 30 °C with shaking at 1000 rpm. Streptavidin biosensors (Flex SA) were pre-hydrated in priming buffer assay (GatorBio) for 600 s and dipped into baseline assay buffer (K buffer) for 120 s. SA biosensors were then loaded with biotinylated NP (for all strains tested) at a concentration of 500 nM for 500 s, followed by a second baseline step (K buffer) for 240 s. For the association step, the loaded biosensors were dipped into purified anti-NP mAb-9D5 (hybridoma) at concentrations of 700, 350, and 100 nM in triplicates for 1000 s, followed by a dissociation step (K buffer) for 4000 s. mAb-9D5 binding kinetic values (KD) were obtained by fitting the association and dissociation curves of all concentrations tested (global fit) after subtracting controls (unloaded biosensors) using Gator Analysis software (v 2.7.31013). Mouse ascites-mAbs binding to different NPs

[0107] A total of five mouse ascites-mAb (9D5, 9A1, 7E8, 5F4, and 2G9) binding to different NP strains were analyzed by BLI employing 1:64 dilutions (within the mAb-9D5 hybridoma range of concentration) in K buffer. The assay conditions were the same as mentioned above except for the second baseline step which consisted of 2% BSA in K buffer, as a blocking step for unspecific binding. Ascites-mAb binding to NP-loaded biosensors were analyzed in triplicate, including a reference control of unloaded biosensor (no NP). The values for ascites- mAb NP binding are expressed as wavelength shift values (nm) which are directly proportional to the amount of binding within a range of linearity. The average nm values at the endpoint association step for each NP were subtracted from the values at the beginning (2 and 4 s) afterAttorney Docket 15969-018PC0 reference control subtraction and association curve fitting (Dzimianski et al. Sci. Rep. 10, 21738 (2020)). Plaque reduction neutralization test

[0108] A dilution series from 1:20 to 1:640 of mAb-9D5, or the positive control mAb−11E7, or the negative control mAb-6D8 (anti-Ebola GP antibody) were incubated with either CCHFV strain Afg09 or IbAr 10200, with or without the addition of human complement (5% final concentration) (Cederlane) at 4 °C overnight. Monoclonal antibodies were from the USAMRIID repository. After the incubation, the antibody and virus mixtures were then added to 80–90% confluent SW13 cells in duplicate in six-well plates after the cell media was removed (100 µl of sample was added per well), and the plates were incubated at 37 °C with 5% CO2for 1 h and rocked every 15 m to prevent drying of the monolayer. A 2 ml per well primary overlay was added with a final concentration of 1× phenol red free EMEM (Quality Biologics), 5% FBS (HyClone), and 1% Glutamax (Gibco), and 0.5% SeaKem ME agarose (Kemp) and the plates were incubated for 3 days at 37 °C with 5% CO2.On the third day 2 ml of a 5% neutral red solution (SIGMA) in PBS was added to each well and incubated for 1–2 h before counting the plaques. A 50% reduction in plaques was calculated from the mAb-6D8 negative control. Surface presence of NP

[0109] A549 cells were infected with CCHFV strain IbAr 10200 for 48 h at an MOI of 1 in black 96-half-well optical plates (Corning). Following infection cells were fixed with methanol- free formaldehyde for one hour at room temperature. The cells were blocked with either solutions of 3% BSA (Sigma) and 0.1% Triton X-100 (Sigma) in PBS (Corning) to permeabilize, or 3% BSA in PBS without Triton x-100 to not permeabilize. The cells were then stained overnight at 4 °C, in either the permeabilizing or non-permeabilizing buffers described previously, with mAb-9D5 anti-CCHFV N, mAb-8A11 anti-Gc, or mAb-7D11 at a 1:200 dilution. These antibodies are from the USAMRIID monoclonal repository. The cells were then stained with Alexa 488 goat anti-mouse secondary Ab (Invitrogen; A-11001) at a dilution of 1:1000 in the same buffers as the primary antibody for one hour in the dark. After staining, all cells were fully submerged in 10% neutral buffered formalin (VAL Tech) for 24 h minimum, at +4 °C in the dark, to inactivate virus. Prior to imaging cells were additionally stained withAttorney Docket 15969-018PC0 Hoechst (Invitrogen) and Cell Mask Deep Red (Invitrogen; C10046) in PBS, at dilutions of 1:10,000 and 1:50,000, respectively. Two experiments were conducted, each with six replicates. Statistical analysis

[0110] Survival statistics utilized the log-rank test. Significance levels were set at a P value less than 0.05. All analyses were performed using GraphPad Prism 7 software.

Claims

Attorney Docket 15969-018PC0 CLAIMS What is claimed is:

1. A molecule targeting nucleocapsid protein of Crimean-Congo Hemorrhagic fever virus (CCHFV) comprising (a) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2, or fragment or variant thereof; (b) a polypeptide comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 4, or fragment or variant thereof; (c) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2, or fragment or variant thereof, and a polypeptide comprising a light chain variable region comprising an amino acid sequence of SEQ ID NO: 4, or fragment or variant thereof; or (d) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence of SEQ ID NO:

4.

2. The molecule targeting nucleocapsid protein of CCHFV of claim 1, comprising (a) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide comprising a light chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4; (c) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, and a polypeptide comprising a light chain variable region comprising an amino acid sequence with at leastAttorney Docket 15969-018PC0 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 4; or (d) a polypeptide comprising a heavy chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:

4.

3. The molecule targeting nucleocapsid protein of CCHFV of claim 1 or 2, wherein the molecule is a single chain polypeptide.

4. The molecule targeting nucleocapsid protein of CCHFV of claim 1 or 2, wherein the molecule comprises two single chain polypeptides.

5. The molecule targeting nucleocapsid protein of CCHFV of any one of preceding claims, wherein the molecule is a monoclonal antibody.

6. The molecule targeting nucleocapsid protein of CCHFV of any one of preceding claims, wherein the molecule includes an immunoglobulin constant domain, wherein the constant domain is selected from an IgGl or a variant thereof, an IgG2 or a variant thereof, IgG3 or a variant thereof, an IgG4 or a variant thereof, an IgA or a variant thereof, an IgE or a variant thereof, an IgM or a variant thereof, and an IgD or a variant thereof.

7. The molecule targeting nucleocapsid protein of CCHFV of any one of preceding claims, wherein the molecule is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, or a diabody.

8. A nucleic acid strand encoding a molecule targeting nucleocapsid protein of CCHFV of claim 1, comprising SEQ ID NO: 1 and / or SEQ ID NO: 3 9. A recombinant vector comprising the nucleic acid strand of claim 8.

10. A host cell comprising the nucleic acid strand of claim 8, wherein the host cell is selected from bacterial, yeast, insect or mammalian cell.Attorney Docket 15969-018PC0 11. The host cell of claim 10, wherein the host cell is selected from the group of CHO cell, 293 cell, or hybridoma.

12. A pharmaceutical composition comprising: (a) the molecule targeting nucleocapsid protein of CCHFV of any one of claims 1 to 7, the nucleic acid strand of claim 8, the recombinant vector of claim 9, or the host cell of claim 10; and (b) a pharmaceutically acceptable additive, excipient and / or carrier.

13. A method of treating a subject in need, comprising the steps of: administering to the subject in need a therapeutically effective amount of a composition that comprises the molecule targeting nucleocapsid protein of CCHFV of any one of claims 1 to 7, a nucleic acid strand of claim 8 or a recombinant vector of claim 9.

14. The method of claim 13, further comprising administering one or more additional molecules targeting other CCHFV proteins, said other CCHFV proteins optionally being antibodies against GP38, before, concomitantly, or after the administration of the molecule targeting nucleocapsid protein of CCHFV of any one of claims 1 to 7, a nucleic acid strand of claim 8 or a recombinant vector of claim 9 for prophylactic and / or post-exposure treatment against CCHFV.

15. The method of claim 13 or 14, wherein a subject includes human, primate, dog, cat, rabbit, rodent, a wild animal such as camel, giraffe, buffalo, zebra, and a domestic animal such as cattle, sheep, goat, horse, pig, chicken, turkey and ostriches.

16. The molecule targeting nucleocapsid protein of CCHFV of any one of claims 1 to 7, wherein the epitope within the nucleocapsid protein of CCHFV comprises an amino acid sequence of SEQ ID NO:

5.

17. A method of treating a subject in need comprising the steps of: administering to the subject in need a therapeutically effective amount of a composition that comprises a molecule targeting nucleocapsid protein of CCHFV.

18. The method of claim 17, wherein the molecule targeting nucleocapsid protein of CCHFV is selected from a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-Attorney Docket 15969-018PC0 bonded Fv fragment, a scFv fragment, a single domain antibody, humabody, nanobody, or a diabody.

19. The method of claim 17 or 18, wherein the molecule targeting nucleocapsid protein of CCHFV is an antibody.

20. The method of claim 19, wherein the antibody is or comprises mAb-9D5 and / or mAb-2B11 21. The method of any of claims 17-20, further comprising administering one or more additional molecules targeting other CCHFV proteins, said other CCHFV proteins optionally being antibodies against GP38, before, concomitantly, or after the administration of the molecule targeting nucleocapsid protein of CCHFV.

22. The method of any of claims 17-21, wherein a subject is a human or non-human mammal.

23. The method of claim 22, wherein the subject is a human.

24. The molecule of any of claims 1-6, wherein the molecule targeting nucleocapsid of CCHFC is a humanized antibody.