WO2026030473A1 - West nile virus neutralizing monoclonal antibodies - Google Patents

Abstract

Isolated monoclonal antibodies and antigen binding fragments thereof are disclosed that specifically binds to West Nile Virus. These monoclonal antibodies or antigen binding fragments can inhibit a West Nile Virus infection in a subject, and/or can be used to diagnose a West Nile Virus in a subject. Nucleic acid molecules encoding these antibodies and antigen binding fragments are also disclosed, as are vectors including these nucleic acid molecules, and host cells expressing these vectors.

Description

WEST NILE VIRUS NEUTRALIZING MONOCLONAL ANTIBODIES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This claims the benefit of U.S. Provisional Application No. 66/677,612, filed July 31, 2024, which is incorporated herein by reference in its entirety.

FIELD

[0002] This relates to monoclonal antibodies and antigen binding fragments that specifically bind West Nivel Virus and their use for inhibiting a West Nile Virus infection and detecting West Nile Virus in a biological sample.

SEQUENCE LISTING

[0003] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence. xml” (45,330 bytes), which was created on July 15, 2025, which is incorporated by reference herein.

PARTIES TO JOINT RESEARCH AGREEMENT

[0004] The U.S. Government and Sheba Impact LTD are parties to a joint research agreement.

BACKGROUND

[0005] West Nile virus is a positive-sense, single-stranded RNA virus that causes West Nile fever. It is a member of the family Flaviviridae, from the genus Flavivirus, which also contains the Zika virus, dengue virus, and yellow fever virus. The virus is primarily transmitted by mosquitoes, such as a species of Culex.

[0006] Like most other flaviviruses, West Nile Virus is an enveloped virus with icosahedral symmetry. Electron microscope studies reveal a 45-50 nm virion covered with a relatively smooth protein shell; this structure is similar to the dengue fever virus, another Flavivirus. The protein shell is made of two structural proteins: the glycoprotein E and the small membrane protein M. Protein E has numerous functions including receptor binding, viral attachment, and entry into the cell through membrane fusion. The RNA genome is bound to capsid (C) proteins, which is 105 amino-acid residues long, to form the nucleocapsid. The virus also includes nonstructural proteins, specifically NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 that mainly assist with viral replication or act as proteases.

[0007] West Nile fever is caused by infection with by West Nile Virus and is typically spread by mosquitoes. In about 80% of West Nile Virus infections there are few or no symptoms. However, about 20% of people develop a fever, headache, vomiting, or a rash. In some people, encephalitis or meningitis occurs, with associated neck stiffness, confusion, or seizures, and recovery can take weeks or months. The risk of death among those in whom the nervous system is affected is about 10 percent. Severe disease can also occur in horses. A need remains for agents that can be used to treat an infection with West Nile Virus.

SUMMARY

[0008] Isolated monoclonal antibodies or antigen binding fragments thereof are disclosed that specifically binds to West Nile Virus. These monoclonal antibodies or antigen binding fragments can neutralize West Nile Virus.

[0009] In some aspects, the antigen or antigen binding fragment includes a heavy chain variable (VH) region and a light chain variable region (VL) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL set forth as: a) SEQ ID NOs: 1 and 5, respectively (03012024 SPRI4-0008 1-FH); b) SEQ ID NOs: 9 and 13, respectively (03012024 SPRI4-0008 4-Al 1); c) SEQ ID NOs: 17 and 21, respectively (03012024 SPRI4-0007 1-C07); d) SEQ ID NOs: 25 and 29, respectively (03012024 SPRI4-0007 1-D01); or e) SEQ ID NOs: 33 and 37, respectively (03012024 SPRI4-0007 1 -F08), wherein the monoclonal antibody neutralizes West Nile Virus.

[0010] In some aspects, disclosed are nucleic acid molecules encoding these antibodies and antigen binding fragments, vectors including these nucleic acid molecules, and host cells including these vectors.

[0011] Pharmaceutical compositions including the antibodies, antigen binding fragments, nucleic acid molecules, and vectors are also disclosed. In more aspects, disclosed is the use of these pharmaceutical compositions to inhibit a West Nile Virus infection in a subject.

[0012] In yet other aspects, disclosed is the use of the disclosed antibodies and antigen binding fragments for the detection of a West Nile Virus in a biological sample.

[0013] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGs. 1A-1B. Flow cytometry results from sorted samples. FIG. 1 A shows the gating strategy to sort IgG⁺ B cells. FIG. IB shows antigen-specific B cells sorted from each PBMC sample. [0015] FIGs. 2A-2C. FIG. 2A:is a representative dose-response neutralization curves for each antibody. Each point represents the average of duplicate infections. FIG. 2B are half maximal effective concentration (EC50) values obtained from two independent neutralization assays. Horizontal black line represents the mean potency. FIG. 2C is a table of average EC50 values.

[0016] FIG. 3. Binding to West Nile Virus (WNV) E monomer, WNV E Domain III.

[0017] FIGs. 4A-4E. The heavy and light chain sequences of five neutralizing antibodies identified from the RATP-Ig supernatants were synthesized as human IgGl antibodies with sequences shown in FIGs. 4A-4E. The EC50 value is shown for each of the five antibodies.

[0018] FIGs. 5A-5B. Neutralization of the WNV. FIG. 5 A shows a circular phylogenetic tree of WNV strains with five concentric rings representing the EC50 neutralization values for the identified mAbs: mAb-198, mAb-209, mAb-211, mAb-213, and mAb-215. Each ring corresponds to one antibody, and each segment indicates the EC50 value against a given viral strain using a color scale. FIG. 5B shows neutralization EC50 values for the identified mAbs. Each dot represents an individual measurement against a WNV variant, and bars indicate the median EC50 value for each antibody. mAb-209 potently neutralized all WNV variants tested. The other antibodies were potent and broadly cross-neutralizing.

[0019] FIGs. 6A-6B. Cross-neutralization of Japanese Encephalitis Virus (JEV) serocomplex viruses. FIG. 6A provides a heat map showing neutralization EC50 values of monoclonal antibodies mAb-198 (198), mAb-209 (209), mAb-211 (211), mAb-213 (213), and mAb-215 (215) against West Nile Virus (WNV) and JEV strains. Shading represents potency on a logarithmic scale, with lower EC50 values indicated by dark shading. FIG. 6B provides neutralization curves for monoclonal antibodies mAb-198, mAb-209, and mAb-211 against WNV and JEV strains. Percent infection is plotted as a function of antibody concentration. Each curve represents an individual antibody- virus combination.

[0020] FIGS. 7A-7C. Neutralization curves. FIG. 7A shows neutralization curves comparing monoclonal antibody potency against mature and immature WNV particles. Percent infection is plotted as a function of antibody concentration. Symbols indicate distinct virion maturation states. There was equivalent neutralization of the virion maturation states. FIG. 7B and 7C provide neutralization curves for monoclonal antibodies against mature and immature West Nile Virus particles. Percent infection is plotted versus antibody concentration. Mature virions show reduced sensitivity to neutralization compared to immature virions. As in the exemplary results shown in FIG. 7B, most mAbs provide reduced neutralization of mature virions. FIG. 7C shows that mAbs 209, 211 and 213 (JEV cross-reactive) preferentially neutralize the mature virions.

[0021] FIGS. 8A-8E. Antibody-mediated protection from lethal WNV infection in mice. FIG. 8A is a schematic representation of a West Nile Virus (WNV) mouse challenge model. C57BL/6 mice were infected with WNV strain NY99 by footpad injection and treated with monoclonal antibodies at the indicated timepoints and doses. All antibodies were tested with the wild-type IgGl and with an IgG 1 containing the LALA mutation in the Fc region. FIG. 8B is a set of three graphs that shows survival and weight loss on Day -1. FIG. 8C is a set of three graphs that show survival and weight loss at Day +1. FIG. 8D is a set of three graphs that show survival and weight loss at Day +3. . FIG. 8E is a set of three graphs that show survival and weight loss at Day +5.

[0022] FIGs. 9A-9B. Break through protection. Viral RNA levels in mice treated with monoclonal antibodies following WNV infection. Plasma viral RNA was quantified from individual animals by qRT-PCR and reported as RNA copies per milliliter. Each dot represents an individual animal; dots with arrows indicate mice that survived the 21 -day observation period. FIG. 9A shows results on Day -1. FIG. 9B shows results at Day +1 (see FIG. 8A).

[0023] FIGs. 10A-10I. Down-dosing mAb-mediated protein from lethal infection.

[0024] FIG. 10A shows a schematic of the experimental design. mAbs were down-selected based on cross-reactivity, potency, in vivo protection and prevention of virus escape mutants. FIGs. 10B- 10E show Kaplan-Meier survival curves for C57BL/6 mice treated with decreasing doses of monoclonal antibodies following infection with WNV strain NY99 at the indicated doses. FIGs. 10F-10I are weight loss curves for the same treatment groups during the course of this experiment. All mAbs show protection against lethal WNV infection at low doses. mAb-21 showed the greatest protection at the lowest dose.

SEQUENCES

[0025] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The following sequences are provided:

SEQ ID NO 1: the amino acid sequence for 03012024 SPRI4-0008 1-F11 V_H.

SEQ ID NOs 2, 3, and 4: the amino acid sequences for the HCDR1, the HCDR2, and the HCDR3 of 03012024 SPRI4-0008 1-F11 V_H, respectively.

SEQ ID NO 5: the amino acid sequence for 03012024 SPRI4-0008 1-F11 V_L.

SEQ ID NOs 6, 7, and 8: the amino acid sequences for the LCDR1, the LCDR2, and the LCDR3 of 03012024 SPRI4-0008 1-F11 V_L, respectively.

SEQ ID NO 9: the amino acid sequence for 03012024 SPRI4-0008 4-Al 1 V_H.

SEQ ID NOs: 10, 11, and 12: the amino acid sequences for the HCDR1, the HCDR2, and the HCDR3 of 03012024 SPRI4-0008 4-A11 V_H, respectively.

SEQ ID NO 13: the amino acid sequence for 03012024 SPRI4-0008 4-A11 VL.

SEQ ID NOs 14, 15, and 16: the amino acid sequences for the LCDR1, the LCDR2, and the SEQ ID NO 17: the amino acid sequence for 03012024 SPRI4-0007 1-C07 V_H.

SEQ ID NOs 18, 19, and 20: the amino acid sequences for the HCDR1, the HCDR2, and the HCDR3 of 03012024 SPRI4-0007 1-C07 V_H, respectively.

SEQ ID NO 21 : the amino acid sequence for 03012024 SPRI4-0007 1-C07 V_L.

SEQ ID NOs 22, 23, and 24: the amino acid sequence for the LCDR1, the LCDR2, and the LCDR3 of 03012024 SPRI4-0007 1-C07 V_L, respectively.

SEQ ID NO 25: the amino acid sequence for 03012024 SPRI4-0007 1-D01 V_H.

SEQ ID NOs 26, 27, and 28: the amino acid s sequences for the HCDR1, the HCDR2, and the HCDR3 of 03012024 SPRI4-0007 1-D01 V_H, respectively.

SEQ ID NO 29: the amino acid sequence for 03012024 SPRI4-0007 1-D01 V_L.

SEQ ID NOs 30, 31, and 32: the amino acid sequences for the LCDR1, the LCDR2, and the LCDR3 of 03012024 SPR14-0007 1 -D01. respectively.

SEQ ID NO 33: the amino acid sequence for 03012024 SPRI4-0007 1-F08 VH.

SEQ ID NOs 34, 35, and 36: the amino acid sequences for the HCDR1, the HCDR2, and the HCDR3 of 03012024 SPRI4-0007 1-F08 V_H, respectively.

SEQ ID NO 37: the amino acid sequence for 03012024 SPRI4-0007 1-F08 VL.

SEQ ID NOs 38, 39, and 40: the amino acid sequences for the LCDR I , the LCDR2, and the LCDR3 of 03012024 SPRI4-0007 1-F08, respectively.

SEQ ID NOs 41 and 42: the amino acid sequences of the full length 03012024 SPRI4-0007 1- C07 heavy chain and light chain, respectively.

SEQ ID NOs 43 and 44: the amino acid sequences of the full length 03012024 SPRI4-0007 1- D01 heavy chain and light chain, respectively.

SEQ ID NOs 45 and 46: the amino acid sequences of the full length 03012024 SPRI4-0007 1- F08 heavy chain and light chain, respectively.

SEQ ID NOs: 47 and 48: the amino acid sequences of the full length 03012024 SPRI4-0008 4-A11 heavy chain and light chain, respectively.

SEQ ID NOs: 49 and 50: the amino acid sequences of the full length 03012024 SPRI4-0008 1-F11 heavy chain and light chain, respectively.

DETAILED DESCRIPTION

[0026] No vaccines targeting West Nile Virus are approved for use in humans. Prevention of West Nile Virus disease depends on community-level mosquito control programs, personal protective measures, and screening of blood and organ donors. Furthermore, as of July 2024, there was no clinically-approved treatment for West Nile Virus-induced disease; clinical management was supportive. Thus, a need remains for agents to prevent and treat West Nile Virus infections.

I. Summary of Terms [0027] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0028] About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

[0029] Administration: The introduction of an agent, such as a disclosed antibody, into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravascular, the agent (such as antibody) is administered by introducing the composition into a blood vessel of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

[0030] Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

[0031] Antibody and Antigen Binding Fragment: An immunoglobulin, antigen-binding fragment thereof, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as West Nile Virus, such as a dimer of E protein of West Nile Virus. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.

[0032] Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi-specific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Spring er- Verlag, 2010).

[0033] Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

[0034] An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

[0035] Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

[0036] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen.

[0037] References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0038] The Vn and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5^th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0039] The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5^th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927- 948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev.

Comp. Immunol., 27(1 ):55-77, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a VL CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0040] In some aspects, a disclosed antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutation) to increase halflife.

[0041] A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.)

[0042] A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one aspect, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences. [0043] A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.

[0044] A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some aspects, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory’ Manuel. l^sl Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0045] Antibody or antigen binding fragment that neutralizes West Nile Virus: An antibody or antigen binding fragment that specifically binds to a West Nile Virus antigen (such as the E protein) in such a way as to inhibit a biological function associated with West Nile Virus that inhibits infection. The antibody can neutralize the activity of West Nile Virus. For example, an antibody or antigen binding fragment that neutralizes West Nile Virus may interfere with the virus by binding it directly and limiting entry into cells. Alternately, an antibody may interfere with one or more postattachment interactions of the pathogen with a receptor, for example, by interfering with viral entry using the receptor. In some examples, an antibody that is specific for a dimer of E protein neutralizes the infectious titer of West Nile Virus.

[0046] In some aspects, an antibody or antigen binding fragment that specifically binds to West Nile Virus and neutralizes West Nile Virus inhibits infection of cells, for example, by at least 50% compared to a control antibody or antigen binding fragment.

[0047] A “broadly neutralizing antibody” is an antibody that binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity antigenic surface of antigen. With regard to an antigen from a pathogen, such as a virus, the antibody can bind to and inhibit the function of an antigen from more than one, lineage, or more than one clade of the pathogen. For example, with regards to West Nile Virus, the antibody can bind to and inhibit the function of an antigen, such as the E protein. The antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C.

[0048] Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having, a West Nile Virus infection.

[0049] Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.

[0050] Conditions sufficient to form an immune complex: Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a delectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological conditions” which include reference to conditions e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

[0051] The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scans, radiography, and affinity chromatography .

[0052] Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one aspect, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to West Nile Virus, covalently linked to an effector molecule, such as a detectable label. The linkage can be by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.” [0053] Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, a West Nile Virus-specific antibody can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for spike protein binding, and/or neutralization activity. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0054] Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

[0055] The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0056] Non-conservative substitutions are those that reduce an activity or function of the antibody, such as the ability to specifically bind to a West Nile Virus. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest. [0057] Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.

[0058] Control: A reference standard. In some aspects, the control is a negative control, such as sample obtained from a healthy patient not infected with a West Nile Virus, such as a West Nile Virus of lineage 2 or lineage 1, such as a West Nile Virus of clade A, clade B, or clade C. In other aspects, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with a West Nile Virus infection, such as an infection with a West Nile Virus of lineage 2 or lineage 1, such as an infection with a West Nile Virus of clade A, clade B, or clade C. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

[0059] A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

[0060] Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0061] Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel el al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

[0062] Detecting: To identify the existence, presence, or fact of something.

[0063] Effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit a West Nile Virus infection, or to measurably alter outward symptoms of such an infection. In aspects, the West Nile Virus can be a West Nile Virus of lineage 2 or lineage 1, such as a West Nile Virus of clade A, clade B, or clade C.

[0064] In one example, a desired response is to inhibit or reduce or prevent West Nile Virus infection. The West Nile Virus infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of can decrease the West Nile Virus infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the West Nile Virus by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable West Nile Virus infection)), as compared to a suitable control. [0065] In some aspects, administration of an effective amount of a disclosed antibody or antigen binding fragment that binds to a West Nile Virus can reduce or inhibit infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the West Nile Virus or by an increase in the survival time of infected subjects, or reduction in symptoms associated with the infection) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infection), as compared to a suitable control.

[0066] The effective amount of an antibody or antigen binding fragment that specifically binds the West Nile Virus that is administered to a subject to inhibit infection will vary depending upon a number of factors associated with that subject, for example the overall health and/or weight of the subject. An effective amount can be determined by varying the dosage and measuring the resulting response, such as, for example, a reduction in pathogen titer. Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays.

[0067] An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

[0068] Effector molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted, or a detectable marker. Effector molecules can include, for example, polypeptides and small molecules. Some effector molecules may have or produce more than one desired effect.

[0069] Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and/or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on a West Nile Virus E protein.

[0070] Expression: Transcription or translation of a nucleic acid sequence. For example, an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become mRNA. An encoding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.

[0071] Envelope glycoprotein (E protein): A West Nile structural protein that mediates binding of flavivirus virions to cellular receptors on host cells. The West Nile E protein is required for membrane fusion and is the primary antigen inducing protective immunity to a West Nile infection. West Nile Virus E protein affects host range, tissue tropism and viral virulence. The West Nile Virus E protein contains three structural and functional domains, DI-DIII. In mature virus particles the E protein forms head to tail homodimers lying flat and forming a dense lattice on the viral surface.

[0072] Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0073] Expression vector: A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0074] A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

[0075] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cy2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to EU numbering. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.

[0076] Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originated from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as an scFv, is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

[0077] Host cell: Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny is included when the term “host cell” is used. [0078] IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype comprises IgAi and IgA2. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C- terminus called the tail piece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.

[0079] IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgGi, IgGz, IgG?, and IgG4-

[0080] Immune complex: The binding of antibody or antigen binding fragment (such as a scFv) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

[0081] Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a West Nile Virus infection. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

[0082] The term “reduces” is a relative term, such that an agent reduces a disease or condition if the disease or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Similarly, the term “prevents” does not necessarily mean that an agent completely eliminates the disease or condition, so long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents an infection, can, but does not necessarily completely, eliminate such an infection, so long as the infection is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% the infection in the absence of the agent, or in comparison to a reference agent.

[0083] Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0084] Kabat position: A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. {Sequences of Proteins of Immunological Interest, 5^th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

[0085] Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0086] The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule: for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0087] Multispecific antibody: A recombinant molecule containing of two or more different variable fragments (Fv). The valency of a multispecific antibody is equal to the number of Fv fragments in the molecule. The specificity of a multispecific antibody is equal to the number of unique antigen-binding domains in the multispecific. In multispecific antibodies, specificity is limited by the number of total Fv domains available. Monospecific antibodies can be made using multispecific antibodies with a valency of 3, 4, 5 or more and in which all of the Fvs are identical. Bispecific antibodies can be made using multispecific antibodies with valency of 2, 3, 4 or more, where the Fv domains are made from two different antigen binding domains. In this instance the numbers of each antigen binding domain can be equal or different and can vary in position and location. Similarly, trispecific antibodies can be made with valency of 3, 4, 5 or more, where the Fv domains are made from three different antigen binding domains. In this instance the numbers of each antigen binding domain can be equal or different and can vary in position and location. Similarly, tetraspecific antibodies can be made with valency of 4, 5, 6 or more, where the Fv domains are made from four different antigen binding domains. In this instance the numbers of each antigen binding domain can be equal or different and can vary in position and location. Multispecific antibodies include chemically or genetically linked molecules of antigen binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. A multispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.

[0088] Nucleic acid (molecule or sequence): A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA or RNA. The nucleic acid can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (such as A, T/U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides.

[0089] “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

[0090] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0091] Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two proteincoding regions, in the same reading frame.

[0092] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

[0093] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as nonnatural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

[0094] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N- terminal) end and a carboxy-terminal end. In some aspects, the polypeptide is a disclosed antibody or a fragment thereof. [0095] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody ) is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0096] Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

[0097] Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the percentage identity between the sequences. Sequence identity can be measured in terms of percentage identity: the higher the percentage, the more identical the sequences. Homologs and variants of a VL or a VH of an antibody that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest.

[0098] Any suitable method may be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(1):237- 244, 1988; Higgins and Sharp, Bioinformatics, 5(2): 151-3, 1989; Corpet, Nucleic Acids Res.

16(22): 10881-10890, 1988; Huang et al. Bioinformatics, 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994., Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site. [0099] Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

[0100] Specifically bind: When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein, such as a West Nile Virus protein, in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example a E protein dimer, and does not bind in a significant amount to other proteins present in the sample or subject. With regards to an E protein, the epitope may be present on the E protein of more than one type of West Nile Virus, such that the antibody binds to the E protein on more than one types of West Nile Virus, but does not bind to other proteins, such as proteins from other viruses or other proteins (non-E) of West Nile Virus. Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manned, 2^nd ed., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0101] With reference to an antibody- antigen complex, specific binding of the antigen and antibody has a KD of less than about 10'⁷ Molar, such as less than about 10'⁸ Molar, 10'⁹, or even less than about 10'¹⁰ Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0102] An antibody that specifically binds to an epitope on a West Nile Virus E protein, can bind molecules/agents including this domain, including dimers of E protein, viruses, substrate to which the E protein (or dimer) is attached, or the protein (or dimer) in a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody and a nontarget. Typically, specific binding results in a much stronger association between the antibody and a spike protein than between the antibody other different West Nile Virus proteins (such as a non- structural or structural protein, such as M) or from non-West Nile Virus proteins. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid- phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

[0103] Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals, such as horses, non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like. In an example, a subject is a human. In another example, the subject is a horse. In an additional example, a subject is selected that needs inhibition of a West Nile Virus infection. For example, the subject is either uninfected and at risk of the West Nile Virus infection or is infected and in need of treatment.

[0104] Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

[0105] Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some aspects, a viral vector comprises a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment that specifically binds to a West Nile Virus and neutralizes the West Nile Virus. In some aspects, the viral vector can be an adeno-associated virus (AAV) vector.

[0106] Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

[0107] West Nile virus (WNV): A member of the virus family Flaviviridae and the genus Flavivirus. Other members of this genus include dengue virus, yellow fever virus, Japanese encephalitis virus (JEV), Zika virus and Spondweni virus. West Nile Virus was first isolated from a woman in the West Nile district of Uganda in 1937. The virus was later identified in birds in the Nile delta region in 1953. Human infections attributable to West Nile Virus have been reported in many countries for over 50 years. In 1999, a West Nile Virus circulating in Israel and Tunisia was imported into New York, producing a large and dramatic outbreak that spread throughout the continental United States in the following years. There are two main lineages of West Nile Virus. Lineage 1 is composed of West Nile Virus strains from different geographic regions, and it is subdivided into at least 3 clades. Clade A contains strains from Europe, Africa, the Middle East, and America; clade B represents the Australian (Kunjin) strains; and clade C contains Indian West Nile Virus isolates. Lineage 2 contains the B 956 prototype strain and other strains isolated exclusively found in sub- Saharan Africa and Madagascar thus far.

[0108] Human infection is most often the result of bites from infected mosquitoes but may also be transmitted through contact with other infection animals, their blood or other tissues. Infection with WNV is asymptomatic in about 80% of infected people, but about 20% develop West Nile fever. Symptoms include fever, headache, fatigue, body aches, nausea, vomiting, swollen lymph glands and in some cases, a skin rash. Approximately 1 in 150 of infected individuals develop severe, neuroinvasive disease, such as encephalitis, meningitis or poliomyelitis. Treatment of WNV infection is supportive, such as administration of intravenous fluids, respiratory support and prevention of secondary infections. There is currently no approved vaccine available for humans.

[0109] West Nile Virus non-structural protein: There are seven non-structural (NS) proteins of West Nile Virus, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, which are encoded by the portion of the flavivirus genome that is 3' to the structural proteins. NS1 has been implicated in RNA replication and has been shown to be secreted from infected mammalian cells (Post et al.. Virus Res. 18:291-302, 1991; Mackenzie et al.. Virology 220:232-240, 1996; Muylaert et al., Virology 222: 159- 168, 1996). NS1 can elicit strong humoral immune responses and is a potential vaccine candidate (Shlesinger et al., J. Virol. 60: 1153-1155, 1986; Qu et al., J. Gen. Virol. 74:89-97, 1993). NS2 is cleaved into NS2A and NS2B. NS2A is involved in RNA replication and virus particle assembly and secretion and NS2B forms a complex with NS3 and functions as a cofactor for the NS3 protease, which cleaves portions of the virus polyprotein. NS3 also functions as an RNA helicase and is used to unwind viral RNA during replication (Li et al., J. Virol. 73:3108-3116, 1999). NS4A and NS4B are thought to be involved in RNA replication and RNA trafficking (Lindenbach and Rice, In: Fields Virology, Knipe and Howley, eds., Lippincott, Williams, and Wilkins, 991-1041, 2001). Finally, the NS5 protein is an RNA-dependent RNA polymerase involved in genome replication (Rice et al., Science 229:726-733, 1985). NS5 also shows methyltransferase activity commonly found in RNA capping enzymes (Koonin, J. Gen. Virol. 74:733-740, 1993).

[0110] West Nile Virus structural protein: The capsid (C), pre-membrane (prM), and envelope (E) proteins of West Nile Virus are the viral structural proteins. West Nile Virus genomes consist of positive-sense RNAs that are roughly 11 kb in length. The genome has a 5' cap, but lacks a 3' polyadenylated tail (Wengler et al., Virology 89:423-437, 1978) and is translated into one polyprotein. The structural proteins (C, prM, and E) are at the amino-terminal end of the polyprotein followed by the non-structural proteins (NS 1-5). The polyprotein is cleaved by virus and host derived proteases into individual proteins. The C protein forms the viral capsid while the prM and E proteins are embedded in the surrounding envelope. The E protein functions in binding to host cell receptors resulting in receptor-mediated endocytosis. In the low pH of the endosome, the E protein undergoes a conformational change causing fusion between the viral envelope and the endosomal membranes.

The prM protein is believed to stabilize the E protein until the virus exits the infected cell, at which time prM is cleaved to the mature M protein.

II. Description of Several Aspects

[0111] Disclosed herein are 5 monoclonal antibodies that have high neutralization titers against West Nile Virus, do not bind E domain III, and show moderate to no binding against West Nile Virus E monomer, consistent with binding to epitopes specific to the E dimer or a quaternary structure on the virion. These antibodies have been synthesized as IgGl antibodies. In some aspects, isolated monoclonal antibodies and antigen binding fragments that specifically bind a West Nile Virus are provided. In more aspects, the monoclonal antibodies and antigen binding fragments specifically bind to E dimer or a quaternary structure on the virion.

[0112] The disclosed monoclonal antibodies and antigen binding fragments can neutralize West Nile Virus. In some aspects, the disclosed monoclonal antibodies can inhibit a West Nile Virus infection in vivo, and can be administered prior to, or after, an infection with a West Nile Virus. In aspects, the disclosed monoclonal antibody can bind, and inhibit, a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0113] Multispecific antibodies, such as bispecific antibodies, including the variable domains of these antibodies are also provided. In addition, disclosed herein are compositions comprising the antibodies and antigen binding fragments and a pharmaceutically acceptable carrier. Nucleic acids encoding the antibodies, antigen binding fragments, variable domains, and expression vectors (such as adeno-associated virus (AAV) viral vectors) comprising these nucleic acids are also provided. The antibodies, antigen binding fragments, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, treatment and prophylactic purposes. For example, the disclosed antibodies and antigen binding fragments can be used to diagnose a subject with a West Nile Virus infection or can be administered to inhibit a West Nile Virus infection in a subject.

A. Monoclonal Antibodies that Specifically Bind West Nile Virus and Antigen Binding Fragments Thereof

[0114] The discussion of monoclonal antibodies below refers to isolated monoclonal antibodies that include heavy and/or light chain variable domains (or antigen binding fragments thereof) comprising a CDR1, CDR2, and/or CDR3 with reference to the IMGT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions. The amino acid sequence and the CDRs of the heavy and light chain of the disclosed monoclonal antibody according to the IMGT numbering scheme are provided in the listing of sequences, but these are exemplary only. The disclosed monoclonal antibodies specifically bind . In more aspects, the monoclonal antibodies and antigen binding fragments specifically bind to E dimer or a quaternary structure on the virion.

[0115] The disclosed monoclonal antibodies and antigen binding fragments can neutralize West Nile Virus. In some aspects, the disclosed monoclonal antibodies can inhibit a West Nile Virus infection in vivo, and can be administered prior to, or after, an infection with a West Nile Virus. The antibody can bind to, and inhibit, a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to, and inhibit, a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to, and inhibit, a West Nile Virus of lineage 2.

/. Exemplary Monoclonal Antibodies

[0116] In some aspects, a monoclonal antibody is provided that comprises the heavy and light chain CDRs of any one of the antibodies described herein. In other aspects, a monoclonal antibody is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein. Antigen binding fragments of these monoclonal antibodies are also provided. In some aspects, this antibody, or an antigen binding fragment thereof, binds to, and neutralizes, West Nile Virus. In some aspects the monoclonal antibody binds E protein. In other aspects, the monoclonal antibody binds a dimer of E protein.

[0117] In some aspects, the monoclonal antibody is 03012024 SPRI4-0008 1-F11. In other aspects, the monoclonal antibody is 03012024 SPRI4-0008 4-Al 1. In more aspects, the monoclonal antibody is 03012024 SPRI4-0007 1-C07. In further aspects, the monoclonal antibody is 03012024 SPRI4- 0007 1-D01. In some aspects, the monoclonal antibody is 03012024 SPRI4-0007 1-F08.

[0118] Table A provides the antibody names, sequences contained in the VH, HCDR1, HCDR2, HCDR3, VL, LCDR1, LCDR2, and LCDR3, and some position information, for the antibodies disclosed herein.

[0119] Table A. IMGT sequences VH and VL CDRS of Isolated Antibodies and SEQ ID NOs. a. Monoclonal antibody 03012024 SPRI4-0008 1-F11 (also named hAIS-215)

[0120] In some aspects, the monoclonal antibody or antigen binding fragment is based on or derived from the 03012024 SPRI4-0008 1 -Fl 1 antibody, and specifically binds to a West Nile Virus.

[0121] In some examples, the monoclonal antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 03012024 SPRI4-0008 1- F11 antibody, and specifically binds to a West Nile Virus.

[0122] In aspects, the monoclonal antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0123] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the monoclonal antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In additional aspects, the monoclonal antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus [0124] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7 (DDS), and 8, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus. [0125] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7 (DDS), and 8 respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and wherein the V comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the monoclonal antibody or antigen binding fragment specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In this aspect, variations due to sequence identify fall outside the CDRs.

[0126] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus.

[0127] In some aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 49. In more aspects, the monoclonal antibody includes a light chain comprising the amino acid sequence set forth as SEQ ID NO: 50. In further aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 49 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 50.

[0128] In some aspects, the disclosed antibodies inhibit viral entry and/or replication. b. Monoclonal antibody 03012024 SPRI4-00084-A11 (also named hAIS-213 )

[0129] In some aspects, the monoclonal antibody or antigen binding fragment is based on or derived from the 03012024 SPRI4-0008 4-Al 1 antibody, and specifically binds to a West Nile Virus.

[0130] In some examples, the monoclonal antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 03012024 SPRI4-0008 4- A11 antibody, and specifically binds to a West Nile Virus.

[0131] In aspects, the monoclonal antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0132] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the monoclonal antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In additional aspects, the monoclonal antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus

[0133] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, and/or a V comprising a LCDR I , a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15 (LDN), and 16, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus.

[0134] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15 (LDN), and 16, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and the monoclonal antibody or antigen binding fragment specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In this aspect, variations due to sequence identify fall outside the CDRs.

[0135] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. [0136] In some aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 47. In more aspects, the monoclonal antibody includes a light chain comprising the amino acid sequence set forth as SEQ ID NO: 48. In further aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 47 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 48.

[0137] In some aspects, the disclosed antibodies inhibit viral entry and/or replication. c. Monoclonal antibody 03012024 SPRI4-00071-C07 (also named hAIS-198)

[0138] In some aspects, the monoclonal antibody or antigen binding fragment is based on or derived from the 03012024 SPRI4-0007 1-C07 antibody, and specifically binds to a West Nile Virus.

[0139] In some examples, the monoclonal antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 03012024 SPRI4-0007 1- C07 antibody, and specifically binds to a West Nile Virus.

[0140] In aspects, the monoclonal antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can hind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0141] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the monoclonal antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In additional aspects, the monoclonal antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus

[0142] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19, and 20, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23 (AAS) and 24, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus.

[0143] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19, and 20, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23 (AAS), and 24, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, and the monoclonal antibody or antigen binding fragment specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In this aspect, variations due to sequence identify fall outside the CDRs.

[0144] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the antibody or antigen binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus.

[0145] In some aspects, the monoclonal antibody includes a heavy chain set forth comprising the amino acid sequence as SEQ ID NO: 41 . In more aspects, the monoclonal antibody includes a light chain comprising the amino acid sequence set forth as SEQ ID NO: 42. In further aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 41 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 42.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. d. Monoclonal antibody 03012024 SPRI4-00071-D01(also named hAIS-209)

[0146] In some aspects, the monoclonal antibody or antigen binding fragment is based on or derived from the 03012024 SPRI4-0007 1-D01 antibody, and specifically binds to a West Nile Virus.

[0147] In some examples, the monoclonal antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 03012024 SPRI4-0007 1- D01 antibody, and specifically binds to a West Nile Virus.

[0148] In aspects, the monoclonal antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0149] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the monoclonal antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In additional aspects, the monoclonal antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus

[0150] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28, respectively, and/or a V comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31 (QDT), and 32, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus.

[0151] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31 (QDT), and 32, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the monoclonal antibody or antigen binding fragment specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In this aspect, variations due to sequence identify fall outside the CDRs.

[0152] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus.

[0153] In some aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 43. In more aspects, the monoclonal antibody includes a light chain comprising the amino acid sequence set forth as SEQ ID NO: 44. In further aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 43 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 44.

[0154] In some aspects, the disclosed antibodies inhibit viral entry and/or replication. e. Monoclonal antibody 03012024 SPRI4-00071-F08 (also named hAIS-211)

[0155] In some aspects, the monoclonal antibody or antigen binding fragment is based on or derived from the 03012024 SPRI4-0007 1-F08 antibody, and specifically binds to a West Nile Virus.

[0156] In some examples, the monoclonal antibody or antigen binding fragment comprises a VH and a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the 03012024 SPRI4-0007 1- F08 antibody, and specifically binds to a West Nile Virus.

[0157] In aspects, the monoclonal antibody can bind a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2.

[0158] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the monoclonal antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In additional aspects, the monoclonal antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus

[0159] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39 (QDN), and 40, respectively, and specifically binds to a West Nile Virus and neutralizes the West Nile Virus.

[0160] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs:38, 39 (QDN), and 40, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 33, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and the monoclonal antibody or antigen binding fragment specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In this aspect, variations due to sequence identify fall outside the CDRs. [0161] In some aspects, the monoclonal antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a West Nile Virus, and neutralizes the West Nile Virus.

[0162] In some aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 45. In more aspects, the monoclonal antibody includes a light chain comprising the amino acid sequence set forth as SEQ ID NO: 46. In further aspects, the monoclonal antibody includes a heavy chain comprising the amino acid sequence set forth as SEQ ID NO: 45 and a light chain comprising the amino acid sequence set forth as SEQ ID NO: 46.

[0163] In some aspects, the disclosed antibodies inhibit viral entry and/or replication.

2. Additional Description of Antibodies and Antigen Binding Fragments

[0164] An antibody or antigen binding fragment of the antibodies disclosed herein can be a human antibody or fragment thereof. Chimeric antibodies are also provided. The antibody or antigen binding fragment can include any suitable framework region, such as (but not limited to) a human framework region from another source, or an optimized framework region. Alternatively, a heterologous framework region, such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies.

[0165] The antibody can be of any isotype. The antibody can be, for example, an IgA, IgM or an IgG antibody, such as IgGi, IgGz, IgGi, or IgG , . The class of an antibody that specifically binds to a West Nile Virus can be switched with another. In one aspect, a nucleic acid molecule encoding V or VH is isolated such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic acid moleculeB8 encoding VL or VH is then operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved, for example, using a vector or nucleic acid molecule that comprises a CL or CH chain. For example, an antibody that specifically binds the spike protein, that was originally IgG may be class switched to an IgA. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG2, IgG s. or IgG4.

[0166] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers and so on.

[0167] The antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein). In general, the antibody or antigen binding fragment is derivatized such that the binding to the spike protein is not affected adversely by the derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a poly histidine tag).

(a) Binding affinity

[0168] In several aspects, the antibody or antigen binding fragment specifically binds the West Nile Virus, such as a dimer of E protein, with an affinity (e.g., measured by KD) of no more than 1.0 x 10‘⁸ M, no more than 5.0 x 10'⁸ M, no more than 1.0 x 10'⁹ M, no more than 5.0 x 10'⁹ M, no more than 1.0 x 10 ¹⁰ M, no more than 5.0 x 10 ¹⁰ M, or no more than 1.0 x 10 ¹¹ M. KD can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody- coated plate (see, e.g., Chen et al., J. Mol. Biol. 293(4):865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg/ ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (NUNC™ Catalog #269620), 100 pM or 26 pM [¹²⁵I]- antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57(20):4593-4599, 1997). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pl/ we 11 of scintillant (MICROSCINT™-20; PerkinElmer) is added, and the plates are counted on a TOPCOUNT™ gamma counter (PerkinElmer) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays.

[0169] In another assay, KD can be measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (-0.2 pM) before injection at a flow rate of 5 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C at a flow rate of approximately 25 1/min. Association rates (k_On) and dissociation rates (k_Off) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k_off/k_on. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M^-1 s^-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. Affinity can also be measured by high throughput SPR using the Carterra LSA.

(h) Multispecific antibodies

[0170] In some aspects, a multi-specific antibody, or a bi-specific antibody, such as a dual variable domain antibody (DVD-IG™) is provided that comprises an antibody or antigen binding fragment that specifically binds a West Nile Virus, as provided herein. Multi-specific antibodies formats that can be produced using the presently disclosed antibody and antigen binding fragments are disclosed, for example, in Misasi et al., doi.org/10.1101/2022.07.29.502029, biorxiv.org/content/10.1101/2022.07.29.502029v3, November 21, 2022, which is incorporated herein by reference.

[0171] The multispecific antibody can be, for example, a bispecific, or trispecific antibody. In some aspects, the multi-valent antibody is a monospecific antibody (for example, trivalent but one specificity). In some aspects, the multispecific antibody can include 03012024 SPRI4-0008 1-F11, or an antigen binding fragment thereof. In other aspects, the multispecific antibody can include 03012024 SPRI4-0008 4-A11 or an antigen binding fragment thereof. In more aspects, the multispecific antibody can include 03012024 SPRI4-0007 1-C07 or an antigen binding fragment thereof. In further aspects, the multispecific antibody an include 03012024 SPRI4-0007 1-D01 or an antigen binding fragment thereof. In some aspects, the monoclonal antibody can include 03012024 SPRI4-0007 1-F08 or an antigen binding fragment thereof.

[0172] In some aspects, the antibody can be a bispecific antibody. Any suitable method can be used to design and produce a bispecific antibody, such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies, such as bispecific antibodies, include those described in PCT Pub. No. WO2013/163427, which is incorporated by reference herein in its entirety. Non-limiting examples of suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).

[0173] The multispecific antibody may have any suitable format that allows for binding to the West Nile Virus by the antibody or antigen binding fragment as provided herein. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are provided in U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538. Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack et al., J. Immunol., 158(8):3965-3970, 1997; Mack et al., Proc. Natl. Acad. Sci. U.S.A., 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother., 45(3-4): 193-197, 1997; Lbffler et al. , Blood, 95(6):2098-2103, 2000; and Briihl et al., J. Immunol., 166(4):2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans et al. (J. Immunol., 165(12):7050- 7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2): 161- 176, 2003). For bibodies, a scFv molecule can be fused to one of the VL -CL (L) or VH -CHI chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.

[0174] The bispecific tetravalent immunoglobulin known as the dual variable domain immunoglobulin or DVD-immunoglobulin molecule is disclosed in Wu et al., MAbs. 2009;1:339-47, doi: 10.4161/mabs.l.4.8755, incorporated herein by reference. See also Nat Biotechnol. 2007 Nov;25(ll):1290-7. doi: 10.1038/nbtl345. Epub 2007 Oct 14., also incorporated herein by reference. A DVD-immunoglobulin molecule includes two heavy chains and two light chains. Unlike IgG, however, both heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and VL of an existing monoclonal antibody (mAb). Thus, when the heavy and the light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, incorporated herein by reference, see Fig. 1 for schematic and space-filling diagrams. A DVD-immunoglobulin molecule functions to bind two different antigens on each DFab simultaneously.

[0175] The outermost or N-terminal variable domain is termed VD1 and the innermost variable domain is termed VD2; the VD2 is proximal to the C-terminal CHI or CL. As disclosed in Jakob et al., supra, DVD-immunoglobulin molecules can be manufactured and purified to homogeneity in large quantities, have pharmacological properties similar to those of a conventional IgGi, and show in vivo efficacy. Any of the disclosed monoclonal antibodies can be included in a DVD- immunoglobulin format. (c) Antigen Binding Fragments

[0176] Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab')2, and Fv which include a heavy chain and VL and specifically bind a West Nile Virus, such as a dimer of the E protein. These antibody fragments retain the ability to selectively bind with the antigen and are “antigen-binding” fragments. Non-limiting examples of such fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain;

(3) (Fab'H the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; Ffab'E is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the Vi. and Vi. expressed as two chains; and

(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). The intramolecular orientation of the VH - domain and the V -domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (VH-domain-linker domain- V -domain; V -domain-linker domain-Vn-domain) may be used.

(6) A dimer of a single chain antibody (scFVz), defined as a dimer of a scFV. This has also been termed a “miniantibody.”

[0177] Any suitable method of producing the above-discussed antigen binding fragments may be used. Non-limiting examples are provided in Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013.

[0178] Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.

[0179] Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

(d) Variants

[0180] In some aspects, amino acid sequence variants of the antibodies and multispecific antibodies, such as bispecific antibodies, provided herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody or bispecific antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody VH domain and/or VL domain, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

[0181] In some aspects, variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0182] The variants typically retain amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the V regions to increase yield.

[0183] In some aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 1. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 5.

[0184] In more aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 9. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 13. [0185] In more aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 17. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 21.

[0186] In more aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 25. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 29.

[0187] In more aspects, the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 33. In some aspects, the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 35.

[0188] In some aspects, the antibody or antigen binding fragment can include up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) in the framework regions of the heavy chain of a disclosed antibody/multispecific antibody, or the light chain of a disclosed antibody/multipecific antibody, or the heavy and light chains of the antibody/multispecific antibody, compared to known framework regions, or compared to the framework regions of the antibody, and maintain the specific binding activity for the epitope of the spike protein. In these aspects, the substitutions are not in the CDRs.

[0189] In some aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In some aspects of the variant VH and VL sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions. In some aspects of the variant VH and VL sequences provided above, only the framework residues are modified so the CDRs are unchanged.

[0190] To increase binding affinity of the antibody, the VL and VH segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the HCDR3 or LCDR3, respectively. In this process, the primers have been “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode Vnand VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be tested to determine the binding affinity for the spike protein. In particular examples, the VH amino acid sequence is one of SEQ ID NOs: 1, 9, 17, 25 or 33. In other examples, the VL amino acid sequence is SEQ ID NOs: 5, 13, 21, 29, or 37, respectively.

[0191] In some aspects, an antibody disclosed herein, an antigen binding fragment, or bispecific antibody is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.

[0192] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CFL domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(l):26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.

[0193] In one aspect, variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; WO 2002/031140; Okazaki etal., J. Mol. Biol., 336(5): 1239-1249, 2004; Yamane-Ohnuki et al., Bioteclmol. Bioeng. 87(5):614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; US Pat. Appl. No. US 2003/0157108 and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Bioteclmol. Bioeng., 87(5): 614-622, 2004; Kanda et al., Bioteclmol. Bioeng., 94(4):680-688, 2006; and W02003/085107).

[0194] Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.y U.S. Pat. No. 6,602,684 (Umana et al.y and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764.

[0195] In other aspects, mutations can also be included in the constant domain that reduce or eliminate Fc-mediated effector functions, such as ADCC and CDC. In some aspects, mutations can be introduced into the constant region of an IgGi heavy chain in which the leucine residue at positions 234 and 235 (per EU numbering) are substituted with alanine (L234A and L235A, respectively). This mutation is termed “LALA.” Without being bound by theory, these engineered modifications disrupt binding to Fc gamma receptors (FcyRs) and complement protein Clq, thereby minimizing antibodydependent effector functions (Armour et al., Eur. J. Immunol. 29:2613-2624, 1999). Monoclonal antibodies containing the LALA mutation exhibit reduced or eliminate binding to FcyRs and Clq, and can reduce or eliminate Fc-mediated effector functions, such as ADCC and CDC (Leo et al., J. Biol. Chem. 292:3900-3908, 2017). The LALA mutation can be included in the constant domain of any of the antibodies disclosed herein.

[0196] In several aspects, the constant region of the antibody or bispecific antibody comprises one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several aspects, the antibody comprises an amino acid substitution that increases binding to the FcRn. Non-limiting examples of such substitutions include substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176(1 ):346-356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Bioteclmol., 28(2): 157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol.

Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen binding fragments can be linked to or comprise an Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions.

[0197] In some aspects, an antibody or multispecific (such as bispecific) antibody provided herein may be further modified to contain additional nonproteinaceous moieties. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly -1,3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.

B. Conjugates

[0198] The antibodies, antigen binding fragments, and multispecific (such as bispecific) antibodies that specifically bind to a West Nile Virus, as disclosed herein, can be conjugated to an agent, such as an effector molecule or detectable marker. Both covalent and noncovalent attachment means may be used. Various effector molecules and detectable markers can be used, including (but not limited to) toxins and radioactive agents such as ¹²⁵1, ³²P, ¹⁴C, ³H and ³⁵S and other labels, target moieties, enzymes and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.

[0199] The procedure for attaching a detectable marker to an antibody, antigen binding fragment, or bispecific antibody, varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxyl (-COOH), free amine (-NH2) or sulfhydryl (- SH) groups, which are available for reaction with a suitable functional group on a polypeptide to result in the binding of the effector molecule or detectable marker. Alternatively, the antibody, antigen binding fragment, or bispecific antibody, is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any suitable linker molecule. The linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody, antigen binding fragment, or bispecific antibody, and the effector molecule or detectable marker are polypeptides, the linkers may be joined to the constituent amino acids through their side chains (such as through a disulfide linkage to cysteine) or the alpha carbon, or through the amino, and/or carboxyl groups of the terminal amino acids.

[0200] In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, a suitable method for attaching a given agent to an antibody or antigen binding fragment or bispecific antibody can be determined.

[0201] The antibody, antigen binding fragment or multispecific (such as bispecific) antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT, computed axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1 -napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). An antibody, antigen binding fragment, or multispecific (such as bispecific) antibody, can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, [3- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. An antibody, antigen binding fragment, or multispecific (such as bispecific) antibody, may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

[0202] The antibody, antigen binding fragment or bispecific antibody, can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels. Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese. An antibody, antigen binding fragment, or multispecific (such as bispecific) antibody, may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). [0203] The antibody, antigen binding fragment or multispecific (such as bispecific) antibody, can also be conjugated with a radiolabeled amino acid, for example, for diagnostic purposes. For instance, the radiolabel may be used to detect the presence of West Nile Virus by radiography, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes: ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ^99mTc, ^{l n}In, ¹²⁵I, ¹³¹I. The radiolabels may be detected, for example, using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0204] The average number of detectable marker moieties per antibody, antigen binding fragment, or bispecific antibody in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment. In some aspects, the average number of effector molecules or detectable marker moieties per antibody or antigen binding fragment in a conjugate range from about 1 to about 2, from about 1 to about 3, about 1 to about 8; from about 2 to about 6; from about 3 to about 5; or from about 3 to about 4. The loading (for example, effector molecule per antibody ratio) of a conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments.

C. Polynucleotides and Expression

[0205] Nucleic acid molecules (for example, cDNA or RNA molecules) encoding the amino acid sequences of antibodies, antigen binding fragments, bispecific antibodies, and conjugates that specifically bind to a West Nile Virus, as disclosed herein, are provided. Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and VH and VL sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several aspects, nucleic acid molecules can encode the VH, the VL, or both the VH and V (for example in a bicistronic expression vector) of a disclosed antibody or antigen binding fragment. In some aspects, the nucleic acid molecules encode an scFv. In several aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment. Nucleic acid molecules encoding an scFv are provided. [0206] The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids which differ in their sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the VL and/or VH nucleic acid sequence. [0207] In a non-limiting example, an isolated nucleic acid molecule encodes the VH of a disclosed antibody. In another non-limiting example, the nucleic acid molecule encodes the VL of a disclosed antibody. In further non-limiting examples, the nucleic acid molecule can encode a bi-specific antibody, such as in DVD-immunoglobulin format.

[0208] Nucleic acid molecules encoding the antibodies, antigen binding fragments, multispecific (such as bispecific) antibodies, and conjugates that specifically bind to a West Nile Virus can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0209] Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements).

[0210] Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).

[0211] The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be expressed as individual proteins including the VH and/or VL (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; nonlimiting examples are provided in ALRubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011). An immunoadhesin can also be expressed. Thus, in some examples, nucleic acids encoding a VH and VL, and immunoadhesin are provided. The nucleic acid sequences can optionally encode a leader sequence.

[0212] To create a scFv the VH- and VL-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH domains joined by the flexible linker (see, e.g.. Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Spring er- Verlag, 2010; Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

[0213] The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to a West Nile Virus, such as a dimer of E protein, and another antigen. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains. Linkers can also be encoded, such as when the nucleic acid molecule encodes a bi-specific antibody in DVD-IG™ format.

[0214] One or more DNA sequences encoding the antibodies, antigen binding fragments, bispecific antibodies, or conjugates can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host may be used. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

[0215] The expression of nucleic acids encoding the antibodies, antigen binding fragments, and multispecific antibodies (such as bispecific antibodies) described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (z.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

[0216] To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lamda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

[0217] Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.

[0218] Once expressed, the antibodies, antigen binding fragments, multispecific (such as bispecific) antibodies, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The antibodies, antigen binding fragment, and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylatically, the polypeptides should be substantially free of endotoxin.

[0219] Methods for expression of antibodies, antigen binding fragments, bispecific antibodies, and conjugates, and/or refolding to an appropriate active form, from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242): 544-546, 1989.

D. Methods and Compositions

1. Inhibiting a West Nile Virus infection

[0220] Methods are disclosed herein for the inhibition of a West Nile Virus infection in a subject, In some aspects, the disclosed monoclonal antibodies can inhibit a West Nile Virus infection in vivo, and can be administered prior to, or after, an infection with a West Nile Virus. In aspects, the disclosed monoclonal- antibody can bind, and inhibit, a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and inhibit, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to and inhibit a West Nile Virus of lineage 2. [0221] Methods are disclosed herein for the inhibition of a West Nile Virus. The methods include administering to the subject an effective amount (that is, an amount effective to inhibit replication in the subject) of a disclosed antibody, antigen binding fragment, bispecific antibody, or a nucleic acid encoding such an antibody, antigen binding fragment, or bispecific antibody, to a subject at risk of a West Nile Virus infection or having a West Nile Virus infection. The methods can be used preexposure or post-exposure.

[0222] Methods are disclosed for treating a West Nile Virus infection in a subject. Methods are also disclosed for preventing a West Nile Virus infection in a subject. These methods include administering one or more of the disclosed antibodies, antigen binding fragments, bispecific antibodies, or nucleic acid molecule encoding such molecules, or a composition including such molecules, as disclosed herein.

[0223] The methods include administering to the subject an effective amount (that is, an amount effective to inhibit the infection in the subject) of a disclosed monoclonal antibody, antigen binding fragment, or multispecific (such as bispecific) antibody, or a nucleic acid encoding such an antibody, antigen binding fragment, or multispecific antibody (such as bispecific antibody), to a subject at risk of a West Nile Virus infection or having the West Nile Virus infection. The methods can be used preexposure or post-exposure. The methods can be used prior to travel to an area where West Nile Virus is present. The methods can be used following diagnosis of a West Nile Virus infection.

[0224] The subject can be a human. The subject can be immunocompromised. The subject can be elderly, such as greater than about 65, about 70, about 75, about 80, about 85 or about 90 years of age. The subject can be an infant or a child, such as a subject of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years of age. The subject can be a teenager, such as a subject of about 13, 14, 15, 16, 17 or 18 years of age. The method can be used in a subject with encephalitis due to a West Nile Virus infection.

The method can be used in a subject with Guillain Barre syndrome. The method can be used in a subject with a West Nile Virus infection, to inhibit development of encephalitis or Guillain Barre syndrome. The subject can be a veterinary subject, such as a horse.

[0225] The infection does not need to be completely eliminated or inhibited for the method to be effective. For example, the method can decrease the infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable West Nile Virus infection) as compared to the West Nile Virus infection in the absence of the treatment. In some aspects, the subject can also be treated with an effective amount of an additional agent.

[0226] In some aspects, administration of an effective amount of a disclosed antibody, antigen binding fragment, bispecific antibody, or nucleic acid molecule, inhibits the establishment of an infection and/or subsequent disease progression in a subject, which can encompass any statistically significant reduction in activity (for example, growth or invasion) or symptoms of the West Nile Virus infection in the subject. In some aspects, the method reduces encephalitis in the subject, or the symptoms of Guillain-Barre syndrome.

[0227] Antibodies, antigen binding fragments thereof, and bispecific antibodies can be administered by intravenous infusion. Doses of the antibody, antigen binding fragment, or bispecific antibody vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the antibody, antigen binding fragment or bispecific antibody can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg. The antibody, antigen binding fragment, or bispecific antibody is administered according to a dosing schedule determined by a medical practitioner. In some examples, the antibody, antigen binding fragment or bispecific antibody is administered weekly, every two weeks, every three weeks or every four weeks.

[0228] In some aspects, the method of inhibiting the infection in a subject further comprises administration of one or more additional agents to the subject. Additional agents of interest include, but are not limited to, anti-viral agents.

[0229] In some aspects, the method comprises administration of a first antibody that specifically binds to a West Nile Virus as disclosed herein and a second antibody that also specifically binds to a West Nile Virus. Combinations of antibodies are of use. The method can include administering at least 2, 3, 4, or 5 monoclonal antibodies or antigen binding fragments.

[0230] In some aspects, the method can include administration of an effective amount of 03012024 SPRI4-0008 1-F11, or an antigen binding fragment thereof. In other aspects, the method can include administration of an effective amount of 03012024 SPRI4-0008 4-A11 or an antigen binding fragment thereof. In more aspects the method can include administration of an effective amount of 03012024 SPRI4-0007 1-C07 or an antigen binding fragment thereof. In further aspects, the method can include administration of an effective amount of 03012024 SPRI4-0007 1-D01 or an antigen binding fragment thereof. In some aspects, the method can include administration of a an effective amount of 03012024 SPRI4-0007 1-F08 or an antigen binding fragment thereof.

[0231] The disclosed methods can include the administration of one or more multispecific antibodies. Combinations of these multispecific antibodies, and combinations of bispecific antibodies, such as one, two, three, four, or five of these multispecific antibodies, such as bispecific antibodies, can be administered to the subject. Nucleic acid molecules are also of use in these aspects.

[0232] In some aspects, a subject is administered DNA or RNA encoding a disclosed antibody, antigen binding fragment, or multispecific antibody [such as a bispecific antibody ), to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed antibody, antigen binding fragments thereof, or multispecific antibody (such as a bispecific antibody), can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof. In some aspects, a disclosed antibody or antigen binding fragment is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005, which is incorporated by reference herein).

[0233] In several aspects, a subject (such as a human subject at risk of a West Nile Virus infection or having a West Nile Virus infection) can be administered an effective amount of an AAV viral vector that comprises one or more nucleic acid molecules encoding a disclosed antibody, antigen binding fragment, or multispecific (such as bispecific) antibody. The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody, antigen binding fragment, or bispecific antibody, and administration of the effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antibody, antigen binding fragment, or bispecific antibody in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed antibody, antigen binding fragment, or bispecific antibody in a subject include those provided in Johnson et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541): 87- 91, 2015, each of which is incorporated by reference herein in its entirety.

[0234] In one aspect, a nucleic acid encoding a disclosed antibody, antigen binding fragment, or multispecific antibody (such as a bispecific antibody), is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOSa Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

[0235] Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 mg/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

[0236] Single or multiple administrations of a composition including a disclosed antibody, antigen binding fragment, or multispecific antibody (such as a bispecific antibody), conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once, but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit a West Nile Virus infection without producing unacceptable toxicity to the patient.

[0237] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The effective dose can be determined from cell culture assays and animal studies.

[0238] The West Nile Virus-specific monoclonal antibody, antigen binding fragment, multispecific antibody (such as a bispecific antibody), or nucleic acid molecule encoding such molecules, or a composition including such molecules, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intraarterially, intraperitoneally, intramuscularly, intradermally, or intrathecally. In an aspect, the antibody, antigen binding fragment, multispecific antibody (such as a bispecific antibody), or nucleic acid molecule encoding such molecules, or a composition including such molecules, is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day. The antibody, antigen binding fragment, multispecific antibody (such as a bispecific antibody), conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, can also be administered by direct injection at or near the site of a particular symptom. A further method of administration is by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre-determined period. The osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.

2. Compositions

[0239] Compositions are provided that include one or more of the West Nile Virus-specific monoclonal antibodies, antigen binding fragments, multispecific antibodies (such as a bispecific antibody), conjugates, or nucleic acid molecules encoding such molecules, that are disclosed herein in a pharmaceutically acceptable carrier. In some aspects, the composition comprises two, three, four or more antibodies, antigen binding fragments, or bispecific antibodies, that specifically bind a West Nile Virus, such as a dimer of E protein. The compositions are useful, for example, for example, for the inhibition or detection of a West Nile Virus infection.

[0240] In some aspects, the compositions includes at least one monoclonal antibody disclosed herein, or an antigen binding fragment thereof, or a multispecific antibody thereof. In some aspects, the composition comprises more than one antibody that specifically bind a West Nile Virus. The compositions are useful, for example, for example, for the inhibition or detection of a West Nile Virus infection. In some aspects, the monoclonal antibody is 03012024 SPRI4-0008 1-F11. In other aspects, the monoclonal antibody is 03012024 SPRI4-0008 4-A11. In more aspects, the monoclonal antibody is 03012024 SPRI4-0007 1-C07. In further aspects, the monoclonal antibody is 03012024 SPRI4-0007 1-D01. In some aspects, the monoclonal antibody is 03012024 SPRI4-0007 1-F08 [0241] The compositions can be prepared in unit dosage forms, such as in a kit, for administration to a subject. The amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes. The antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules can be formulated for systemic or local administration. In one example, the, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, is formulated for parenteral administration, such as intravenous administration.

[0242] In some aspects, the antibody, antigen binding fragment, bispecific antibody, or conjugate thereof, in the composition is at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) pure. In some aspects, the composition contains less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0243] The compositions for administration can include a solution of the monoclonal antibody, antigen binding fragment, multispecific antibody (such as a bispecific antibody), conjugate, or nucleic acid molecule encoding such molecules, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by any suitable technique. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of antibody in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

[0244] A typical composition for intravenous administration comprises about 0.01 to about 30 mg/kg of the monoclonal antibody, antigen binding fragment, bispecific antibody, or conjugate per subject per day (or the corresponding dose of a conjugate including the antibody or antigen binding fragment). Any suitable method may be used for preparing administrable compositions; non-limiting examples are provided in such publications as Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013. In some aspects, the composition can be a liquid formulation including one or more antibodies, antigen binding fragments, or bispecific antibodies, in a concentration range from about 0.1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.

[0245] Monoclonal antibodies, an antigen binding fragment thereof, a multispecific antibody (such as a bispecific antibody), or a nucleic acid encoding such molecules, can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. A solution including the antibody, antigen binding fragment, bispecific antibody, or a nucleic acid encoding such molecules, can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art in the administration of antibody drugs, which have been marketed in the U.S. since the approval of Rituximab in 1997. Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules, can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.

[0246] Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the active protein agent, such as a cytotoxin or a drug, as a central core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 mm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 mm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 mm in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

[0247] Polymers can be used for ion-controlled release of the compositions disclosed herein. Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug.

2. Methods of detection and diagnosis [0248] Methods are also provided for the detection of the presence of a West Nile Virus in vitro or in vivo. In one example, the presence of a West Nile Virus is detected in a biological sample from a subject and can be used to identify a subject with a West Nile Virus infection. In some aspects, the antibody can bind to, and be used to detect, a West Nile Virus of lineage 1 and/or lineage 2. In aspects, the antibody can bind to, and be used to detect, a West Nile Virus of lineage 1. In further aspects, the antibody can bind to and be used to detect, West Nile Viruses of clade A, clade B, and/or clade C. In aspects, the antibody can bind to, and be used to detect, a West Nile Virus of lineage 2. [0249] The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. The method of detection can include contacting a cell or sample, with an antibody, antigen binding fragment, or multispecific antibody (such as a bispecific antibody), that specifically binds to a West Nile Virus, such as a dimer of an E protein, or conjugate thereof (e.g., a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment.

[0250] In one aspect, the antibody, antigen binding fragment or multispecific antibody (such as a bispecific antibody), is directly labeled with a detectable marker. In another aspect, the antibody (or antigen binding fragment or bispecific antibody) that binds the West Nile Virus (the primary antibody) is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection. The secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody. For example, if the first antibody is a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the antibody, antigen binding fragment, bispecific antibody or secondary antibody are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.

[0251] In some aspects, the disclosed antibodies, antigen binding fragments thereof, or multispecific antibody (such as a bispecific antibody), are used to test vaccines. For example, to test if a vaccine composition including a West Nile Virus E protein or fragment thereof assumes a conformation including the epitope of a disclosed antibody. Thus, provided herein is a method for testing a vaccine, wherein the method comprises contacting a sample containing the vaccine, such as a West Nile Virus E protein immunogen, with a disclosed antibody, antigen binding fragment, or multispecific (such as bispecific) antibody, under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the vaccine including the epitope of interest in the sample. In one example, the detection of the immune complex in the sample indicates that vaccine component, such as the immunogen assumes a conformation capable of binding the antibody or antigen binding fragment. Examples

Example 1: West Nile Virus-specific single B cell sorting

[0252] Cryopreserved PBMC samples from two West Nile Virus convalescent individuals were thawed and cells were stained with a cocktail consisting of antibodies and probes shown in the Table B below. The probes correspond to West Nile Virus and the related Usutu Virus E and NS1 proteins. Antigen-specific IgG⁺ B cells were sorted at a single-cell per well into 96-well plates. Sort gating strategy and frequency results are shown in FIGS. 1 A-1B.

[0253] Table B: Stain cocktail

Example 2; Rapid Assembly, Transfection and Production of Immunoglobulins (RATP-Ig)

[0254] A method for rapid assembly, transfection, and production of immunoglobulins (abbreviated to

RATP-Ig) was developed from single-sorted B cells. RATP-Ig relies on 5’ -RACE and high-fidelity DNA assembly to produce recombinant heavy and light chain-expressing linear DNA cassettes, which can be directly transfected into 96 deep- well mammalian cell culture plates. Resulting culture supernatants containing the expressed mAbs at high concentrations can then be tested for functionality.

[0255] Single-cell cDNA synthesis: Antigen-specific single B cells were sorted by flow cytometry into 96- or 384-well plates. Full-length cDNA was then synthesized using 5’ RACE reversetranscription, adding distinct 3’ and 5’ template switch oligo adapters. cDNA was subsequently amplified.

[0256] Immunoglobulin enrichment and sequencing: The product was then split into two reactions and heavy and light chain variable regions were enriched by amplifying with gene-specific primers. An aliquot of enriched product was reserved for sequencing which was performed simultaneously. [0257] Expression Cassette assembly: Each cassette component (CMV promoter, heavy and light chain constant regions, polyA signal and 1GV enrichment products from above) contains overlapping 5’ and/or 3’ ends that allow for assembly into a single linear strands of DNA. Overlapping sequences facilitate precision ligation prior to the final cassette amplification to produce large quantities of DNA for transfection into 96 deep-well mammalian cell culture plates. Separate IgH and IgL expression cassettes for each mAb were produced.

[0258] Transfection and mAb production: The expression cassette amplified products were then directly transfected into 293F cells for Ig expression. After 3-5 days, each transfection yielded 700 microliters mAb at a concentration of about 50 micrograms/ml that was used available for testing in binding and functional assays.

Example 3: Antibody Synthesis and Function

[0259] All mAb supernatants from the RATP-Ig pipeline were initially screened for binding and neutralization. The heavy and light chain sequences of five potently neutralizing antibodies identified from the RATP-Ig supernatants were synthesized as human IgGl antibodies, shown in FIGS. 4A-4E. Serial dilutions of each antibody were incubated with GFP-expressing WNV strain NY99 Reporter Virus Particles (RVPs). Antibody-RVP complexes were used to infect a Raji B cell line that expresses the flavivirus attachment factor DC-SIGN-R. Two days later, flow cytometry was used to quantitate the number of infected (GFP+) cells. The resulting data was analyzed by non-linear regression analysis to estimate the concentration of antibody that results in half maximal infectivity (EC50) (FIGS. 2A-2C). The ability of the antibodies to bind the WNV E monomer and WNV E Domain HI was determined by ELISA. E16 is a previously described potently neutralizing WNV-specific antibody that binds to E domain in, used here as a control. The newly identified antibodies do not bind E domain in, and show moderate to no binding against WNV E monomer, consistent with binding to epitopes specific to the E dimer or a quaternary structure on the virion (FIG. 3). Additional results and sequences are provided in FIGS. 4A-4E. Example 4: Neutralization of diverse West Nile Virus variants

[0260] Monoclonal antibodies were tested against a panel of 27 WNV reporter viral particles (RVPs) representing lineage I and lineage II strains. For these studies, the antibodies are named as shown below:

[0261] WNV mAb ID naming:

03012024 SPRI4-0007 1-C07 is hAIS-198

03012024 SPRI4-0007 l-D01is hAIS-209 03012024 SPRI4-0007 1-F08 is hAIS-211 03012024 SPRI4-0008 4-Al lis hAIS-213 03012024 SPRI4-0008 1-Fl lis hAIS-215

[0262] For these studies, RVPs were produced by co-transfection of HEK-293T cells with a GFP -expressing WNV sub-genomic replicon and an expression vector encoding the structural genes of the indicated flavivirus strain. RVP-containing supernatants were collected on day 3-7 post-transfection for use in downstream assays. For the neutralization assay, the monoclonal antibodies were serially diluted and incubated with a panel of WNV RVPs representing 27 lineage I and lineage II strains. RVPs were sufficiently diluted to ensure antibody excess at informative points of the dose-response curve. The antibody: antigen immune complexes were incubated at 37°C for 1 hour to ensure steady-state binding, followed by infection of Raji cells expressing the flavivirus attachment factor DC-SIGNR. GFP -expressing, infected cells were quantified 36-48h post-infection using flow cytometry. The resulting data was analyzed by nonlinear regression analysis (GraphPad PRISM) to estimate the EC50 neutralizing titer. A circular phylogenetic tree of WNV diversity is shown in FIG. 5A, with EC50 values overlaid as a heatmap, and a graph showing the neuralization EC50 (Ng/ml) is shown in FIG. 5B. mAb-209 equivalently and potently neutralized all WNV variants tested. The additional monoclonal antibodies disclosed herein also showed broad cross-neutralization.

Example 5: Cross- neutralization of Japanese Encephalitis Virus (JEV) serocomplex viruses [0263] Neutralization assays were performed using RVPs incorporating structural proteins from West Nile Virus (WNV), Japanese Encephalitis Virus (JEV), Usutu Virus (USUV), Murray Valley Encephalitis Virus (MVEV), Saint Louis Encephalitis Virus (SLEV), Dengue Virus (DENV1), Zika Virus (ZIKV), and Yellow Fever Virus (YFV). The monoclonal antibodies were serially diluted and incubated with RVPs incorporating the structural proteins of the indicated flaviviruses. The antibody: ntigen immune complexes were incubated at 37°C for 1 hour to ensure steady-state binding, followed by infection of Raji-DCSIGNR cells. Green fluorescent protein (GFP)-expressing, infected cells were quantified 36-48h post-infection using flow cytometry. The resulting data was analyzed by nonlinear regression analysis (GraphPad PRISM) to estimate the EC50 neutralizing titer. Neutralization potency is shown in FIG. 6 A. mAbs 209, 211, and 213 cross-neutralized WNV and JEV. Neutralization curves for mAb-213 against WNV NY99, JEV, MVEV, and SLEV are shown in FIG. 6B. mAb-213 was shown to neutralize WNV, JEV, and MVEV, but not SLEV.

Example 6: Sensitivity of neutralization to virion maturation state.

[0264] WNV RVPs (strain NY99) were produced under conditions that increase (furin- overexpressing HEK-293T cell line; mature RVPs) or decrease (furin-knockout HEK-293T cell line; immature RVPs) the efficiency of prM cleavage during virion maturation. RVP-containing supernatants were collected on day 3-7 post-transfection. For these neutralization assays, the monoclonal antibodies were serially diluted and incubated with WNV RVPs that retain high (immature) or low (mature) levels of uncleaved prM. The antibody: antigen immune complexes were incubated at 37°C for 1 hour to ensure steady-state binding, followed by infection of Raji-DCSIGNR cells. GFP-expressing, infected cells were quantified 36-48h post-infection using flow cytometry. The resulting data was analyzed by nonlinear regression analysis (GraphPad PRISM) to estimate the EC50 neutralizing titer.

[0265] Neutralization curves showing neutralization of mature and immature viral particles by mAb- 198 are shown in FIG. 7 A. Equivalent neutralization of mature and immature virions was documented. A control antibody (E53) showed reduced potency against mature virions, consistent with observations for previously described WNV-specific mAbs (FIG. 7B). FIG. 7C presents EC50 values for each mAb against both virion forms (mature and immature). JEV cross-reactive mAbs (209, 211, and 213) preferentially neutralized mature virions All five disclosed mAbs were insensitive to virion maturation state. Specifically, JEV cross-reactive mAbs (209, 211, and 213) preferentially neutralized mature virions.

Example 7: mAb-mediated protection from lethal WNV infection in mice

[0266] The efficacy of the five antibodies including a LALA mutation in their Fc domain was assessed in the WNV challenge model. A schematic of this study is shown in FIG. 8A. An antibody is administered one day prior to infection to test prophylactic efficacy, or administered 1, 3, or 5 days post-infection to test therapeutic efficacy. For the lethal WNV challenge model, wildtype C57B1/6 mice are infected subcutaneously (via footpad inoculation) with WNV strain NY99, and subsequently monitored. For these studies, C57BL/6 mice were infected via footpad injection with 100 PFU WNVNY99 and antibody was administered via intraperitoneal (IP) injection with the indicated dose (n=5 mice/prophylaxis; n=10 mice/therapeutic group). Mice were monitored for 21 days postinfection and weight loss and survival were measured daily. A simple survival analysis was performed using the Kaplan-Meier estimator.

[0267] Results are shown in FIGs. 8B-8E. All mAbs provided protection against lethal WNV infection when administered either prophylactically or therapeutically. No differences in protection were observed between antibodies with a wild-type Fc domain, or antibodies including an Fc domain with the LALA mutation .

[0268] Viral RNA was quantified from plasma samples using qRT-PCR.As shown in FIGs. 9A and 9B, low levels of breakthrough WNV infection were detected in at least one mouse from the mAb-198 WT and LALA groups.

Example 8: Down-Dosing mAb-mediated protection from lethal infection

[0269] The monoclonal antibodies were down-selected based on cross-reactivity, potency, in vivo protection and prevention of virus escape mutants. For these studies, C57BL/6 Mice were infected via footpad injection with 100PFU WNVNY99 and antibody was administered via intraperitoneal (IP) injection with the indicated dose (n=10 mice/group). Mice were monitored for 21 days post-infection and weight loss and survival were measured daily. A simple survival analysis was performed using the Kaplan-Meier estimator.

[0270] The antibodies were administered intraperitoneally on day 3 post-infection at of 50 pg, 10 pg, 2 pg, or 0.4 pg , and the mice were monitored for 21 days to assess survival and weight loss.

[0271] [0272] Results are shown in FIGs. 10B-10I. All of the monoclonal antibodies provided protection against lethal WNV infection at low doses. Among them, mAb-21 exhibited the greatest protective activity at the lowest dose.

[0272] In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. An isolated monoclonal antibody or antigen binding fragment thereof, comprising a heavy chain variable (VH) region and a light chain variable region (V ) comprising a heavy chain complementarity determining region (HCDR)l, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)l, a LCDR2, and a LCDR3 of the VH and VL set forth as: a) SEQ ID NOs: 9 and 13, respectively (03012024 SPRI4-0008 4-A11; hAIS-213); b) SEQ ID NOs: 1 and 5, respectively (03012024 SPRI4-0008 1-F11; hAIS-215); c) SEQ ID NOs: 17 and 21, respectively (03012024 SPRI4-0007 1-C07; hAIS-198); d) SEQ ID NOs: 25 and 29, respectively (03012024 SPRI4-0007 1-D01; hAIS-209); or e) SEQ ID NOs: 33 and 37, respectively (03012024 SPRI4-0007 1-F08; hAIS-211), wherein the monoclonal antibody or antigen binding fragment specifically binds West Nile

Virus.

2. The isolated monoclonal antibody or antigen binding fragment of claim 1, wherein the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acids sequences set forth as: a) SEQ ID NOs: 10, 11, 12, 14, 15, and 16, respectively; b) SEQ ID NOs: 2, 3, 4, 6, 7, and 8; respectively, c) SEQ ID NOs: 18, 19, 20, 22, 23, and 24, respectively; d) SEQ ID NOs: 26, 27, 28, 30, 31, and 32, respectively; or e) SEQ ID NOs: 34, 35, 36, 38, 39, and 40, respectively.

3. The isolated monoclonal antibody or antigen binding fragment of claim 1 or claim 2, wherein the VH and the V comprise the amino acid sequences at least 90% identical to the amino acid sequences set forth as: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 1 and 5, respectively; c) SEQ ID NOs: 17 and 21, respectively; d) SEQ ID NOs: 25 and 29, respectively; or e) SEQ ID NOs: 33 and 37, respectively.

4. The isolated monoclonal antibody or antigen binding fragment of any one of the prior claims, comprising a human framework region.

5. The isolated monoclonal antibody or antigen binding fragment of any one of the prior claims, wherein the VH and the VL comprise the amino acid sequences set forth as: a) SEQ ID NOs: 9 and 13, respectively; b) SEQ ID NOs: 1 and 5, respectively; c) SEQ ID NOs: 17 and 21, respectively; d) SEQ ID NOs: 25 and 29, respectively; or e) SEQ ID NOs: 33 and 37, respectively.

6. The isolated monoclonal antibody of any one of claims 1-5, wherein the monoclonal antibody comprises a human constant domain.

7. The isolated monoclonal antibody of any one of claims 1-6, wherein the monoclonal antibody is a human antibody.

8. The isolated monoclonal antibody of any one of claims 1-7, wherein the monoclonal antibody is an IgGl.

9. The isolated monoclonal antibody of claim 8, wherein the monoclonal antibody comprises a heavy chain and a light chain comprising the amino acid sequences set forth as: a) SEQ ID NOs: 47 and 48, respectively; b) SEQ ID NOs: 49 and 50, respectively; c) SEQ ID NOs: 41 and 42, respectively; d) SEQ ID NOs: 43 and 44, respectively; or e) SEQ ID NOs: 45 and 46, respectively.

10. The isolated monoclonal antibody of any one of claims 1-8, comprising a recombinant constant domain comprising a modification that increases the half-life of the monoclonal antibody.

11. The isolated monoclonal antibody of claim 10, wherein the modification increases binding to the neonatal Fc receptor.

12. The isolated monoclonal antibody of any one of claims 1-9, comprising a recombinant constant domain, wherein the recombinant constant domain comprises L234A and L235A according to EU numbering.

13. The isolated monoclonal antibody or antigen binding fragment of any one of claims 1-12, wherein the monoclonal antibody binds E protein.

14. The isolated monoclonal antibody or antigen binding fragment of any one of claims 1-12, wherein the monoclonal antibody binds E dimer.

15. The antigen binding fragment of any one of claims 1-5 or 13-14.

16. The antigen binding fragment of claim 15, wherein the antigen binding fragment is a Fv, Fab, F(ab')2, scFV or a scFV2 fragment.

17. The isolated monoclonal antibody or antigen binding fragment of any one of claims 1-16, conjugated to a detectable marker.

18. A multi-specific antibody comprising the monoclonal antibody or antigen binding fragment of any one of claims 1-17.

19. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment of any one of claims 1-18, or a VH or VL of the monoclonal antibody.

20. The nucleic acid molecule of claim 19, wherein the nucleic acid molecule is a cDNA sequence encoding the VH and the VL-

21. The nucleic acid molecule of claim 19 or 20, operably linked to a promoter.

22. A vector comprising the nucleic acid molecule of any one of claims 19-21.

23. A host cell comprising the nucleic acid molecule of any one of claims 19-21, or the vector of claim 22.

24. A pharmaceutical composition for use in inhibiting a West Nile virus infection, comprising an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1-17, the multispecific antibody of claim 18, the nucleic acid molecule of any one of claims 19-21, or the vector of claim 22; and a pharmaceutically acceptable carrier.

25. A method of producing an antibody or antigen binding fragment that specifically binds to West Nile Virus, comprising: expressing one or more nucleic acid molecules encoding the monoclonal antibody, antigen binding fragment of any one of claims 1-17 in a host cell; and purifying the antibody or antigen binding fragment.

26. A method of detecting the presence of West Nile Virus in a biological sample from a subject, comprising: contacting the biological sample with an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1-17 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of the West Nile Virus in the sample.

27. The method of claim 25, wherein detecting the presence of the immune complex in the biological sample indicates that the subject has a West Nile Virus infection.

28. A method of inhibiting a West Nile Virus infection in a subject, comprising administering an effective amount of the monoclonal antibody or antigen binding fragment of any one of claims 1 -17, the multispecific antibody of claim 18, the nucleic acid molecule of any one of claims 19-21, the vector of claim 22, or the pharmaceutical composition of claim 24 to the subject, wherein the subject has or is at risk of a West Nile Virus infection.

29. Use of the monoclonal antibody, antigen binding fragment, multispecific antibody, nucleic acid molecule, vector, or pharmaceutical composition of any one of claims 1-24 to inhibit a West Nile Virus infection in a subject or to detect the presence of West Nile Virus in a biological sample.