WO2022101805A1 - Hyperimmune globulins for treatment of chikungunya virus infections - Google Patents

Abstract

The present disclosure is directed to methods of using mixtures of anti-Chikungunya virus (CHIKV) antibodies and/or antigen-binding fragments thereof to treat CHIKV infections in human patients.

Description

HYPERIMMUNE GLOBULINS FOR TREATMENT OF CHIKUNGUNYA VIRUS INFECTIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This applications claims the priority benefit of U.S. Provisional Application No. 63/113,753, filed on November 13, 2020, and U.S. Provisional Application No. 63/227,492, filed on July 30, 2021, each of which is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Chikungunya virus (CHIKV) causes a mosquito-borne disease endemic to parts of sub-Saharan Africa, Southeast Asia, tropical areas of the Indian subcontinent, and islands in the southwestern Indian Ocean, and Latin America. CHIKV has been identified in over 60 countries in Europe, the Americas, Asia ,and Africa. Large outbreaks have been recorded in Democratic Republic of Vietnam (50,000 cases); on Lamu Island in Kenya (13,500 cases); on La Reunion Island (266,000 cases), and in India (1,300,000 cases).

[0003] The infection causes debilitating disease with long-term consequences, including arthralgia-like symptoms. Severe chikungunya fever can manifest as encephalopathy and encephalitis, myocarditis, hepatitis, and multi-organ failure, in multi-morbid patients with other disorders, diabetes, neonates, young children and elderly. Persistent pain and chronic musculoskeletal complaints are critical sequelae of CHIKV infection.

[0004] There are no approved drugs or vaccines for the control of CHIKV infections, so there is a need for compositions and methods that can improve control of CHIKV infections.

BRIEF SUMMARY OF THE INVENTION

[0005] Provided herein are methods for treating, preventing, or reducing the risk of a Chikungunya virus (CHIKV) infection in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. [0006] Provided herein are methods for treating, preventing, or reducing the risk of CHIKV infection-associated arthritis in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0007] Provided herein are methods for treating, preventing, or reducing the risk of CHIKV infection-associated arthritis-like symptoms in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0008] Provided herein are methods for treating, preventing, or reducing the risk of CHIKV infection-associated arthralgia in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0009] Provided herein are methods for reducing viral load of CHIKV in a bodily fluid, tissue, or cell of a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0010] Provided herein are methods for increasing antibody or antigen-binding fragment thereof titers to CHIKV in a bodily fluid, tissue, or cell of a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. Provided herein are methods for passively increasing antibody or antigen-binding fragment thereof titers to CHIKV in a bodily fluid, tissue, or cell of a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0011] Provided herein are methods of eliciting an immune response against CHIKV in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. Provided herein are methods of passively eliciting an immune response against CHIKV in a subject. In some aspects, the method comprises administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0012] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from pooled plasma from birds, optionally wherein the birds are geese, ostriches or chickens. In some aspects, the birds were immunized with CHIKV, a CHIKV protein, or a chimeric CHIKV. In some aspects, an adjuvant was administered with the CHIKV, CHIKV protein, or chimeric CHIKV. In some aspects, the chimeric CHIKV is an Eilat/Chikungunya chimeric virus. In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El and/or a polynucleotide encoding CHIKV Envelope glycoprotein E2.

[0013] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from pooled plasma from mammals. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from serum from mammals. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from pooled plasma and/or serum from mammals. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from pooled milk and/or colostrum from mammals.

[0014] In some aspects, the pool is from at least two mammals. In some aspects, the pool is from at least three mammals.

[0015] In some aspects, the mammals are horses, cows, or sheep. In some aspects, the mammals are horses. In some aspects, the mammal was immunized with CHIKV, a CHIKV protein, or a chimeric CHIKV. In some aspects, an adjuvant was administered with the CHIKV, CHIKV protein, or chimeric CHIKV. In some aspects, the chimeric CHIKV is an Eilat/Chikungunya chimeric virus. In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El and/or a polynucleotide encoding CHIKV Envelope glycoprotein E2. In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El, a polynucleotide encoding a subdomain of CHIKV Envelope glycoprotein El (e.g., wherein the subdomain comprises a consensus or modified amino acid polypeptide, optionally wherein the subdomain comprises >10 amino acids), a polynucleotide encoding CHIKV Envelope glycoprotein E2, and/or a polynucleotide encoding a subdomain of CHIKV Envelope glycoprotein E2 (e.g., wherein the subdomain comprises a consensus or modified amino acid polypeptide, optionally wherein the subdomain comprises >10aa).

[0016] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are pooled from the egg yolks of birds (e.g., geese, ostriches or chickens). In some aspects, the bird was immunized with CHIKV, a CHIKV protein, or a chimeric CHIKV. In some aspects, an adjuvant was administered with the CHIKV, CHIKV protein, or chimeric CHIKV. In some aspects, the chimeric CHIKV is an Eilat/Chikungunya chimeric virus. In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El and/or a polynucleotide encoding CHIKV Envelope glycoprotein E2.

[0017] In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El, a polynucleotide encoding a domain of CHIKV Envelope glycoprotein El, a polynucleotide encoding CHIKV Envelope glycoprotein E2, and/or a polynucleotide encoding a domain of CHIKV Envelope glycoprotein E2.

[0018] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise antibodies or antigen-binding fragments thereof that are CHIKV-virus neutralizing antibodies or antigen-binding fragments thereof, e.g., as measured by Plaque Reduction Neutralization Test (PRNT).

[0019] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof do not contain a Fragment, crystallizable (Fc) domain. In some aspects, polyclonal antibodies or antigen-binding fragments thereof do not contain a Fc domain cause less inflammation when administered to a subject than polyclonal antibodies or antigenbinding fragments thereof do contain a Fc domain.

[0020] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein El and/or E2.

[0021] In some aspects, the polyclonal antibodies or antigen-bindings comprise F(ab’)2 fragments. In some aspects, the polyclonal antibodies or antigen-bindings comprise F(ab) fragments. In some aspects, the polyclonal antibodies or antigen-bindings comprise F(ab’)2 fragments and F(ab) fragments.

[0022] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise immunoglobulin G (IgG) antibodies or antigen-binding fragments thereof.

[0023] In some aspects, the composition comprises a purified gamma globulin fraction of equine plasma.

[0024] In some aspects, the composition comprises about 30-70 milligrams (mg) of protein /milliliter (mL).

[0025] In some aspects, the composition comprises about 49 milligrams (mg) of protein /milliliter (mL). [0026] In some aspects, the composition is a liquid. In some aspects, the composition is a filtered sterile solution. In some aspects, the composition was obtained by pepsin and/or papain digestion. In some aspects, the composition was treated to reduce procoagulation activity. In some aspects, the composition was subjected to virus filtration. In some aspects, the composition is lyophilized. In some aspects, the composition is reconstituted from a lyophilized composition.

[0027] In some aspects, the composition is administered intravenously.

[0028] In some aspects, the administration increases the anti-CHIKV antibodies or antigen-binding fragments thereof in the subject by at least 2-fold, by at least 3 -fold, by at least 4-fold, or by at least 5-fold.

[0029] In some aspects, the CHIKV infection is acute. In some aspects, the CHIKV infection is an Asian CHIKV infection. In some aspects, the CHIKV infection is a West African CHIKV infection. In some aspects, the CHIKV infection is an East Central South African CHIKV infection.

[0030] In some aspects, the subject is human.

[0031] In some aspects, the method further comprises administering a non-steroidal antiinflammatory.

[0032] In some aspects, the polycloncal CHIKV antibodies or antigen-binding binding fragments thereof are capable of neutralizing more than 80% of CHIKV at a concentration of 0.02 mg/mL as determined by plaque reduction neutralization test (PRNT). In some aspects, the polyclonal CHIKV antibodies or antigen-binding binding fragments thereof are capable of neutralizing o'nyong-nyong virus (ONNV) in vitro.

[0033] In some aspects, the disclosure provides a method of detecting a CHIKV and/or Semliki Forest protein in a sample comprising contacting the sample with a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

[0034] In some aspects, the disclosure provides a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof for use in detecting a CHIKV and/or Semliki Forest protein.

[0035] In some aspects, the polyclonal CHIKV antibodies or antigen-binding fragments were produced by immunized a mammal or bird with CHIKV, a CHIKV protein, a CHIKV protein domain, or a chimeric CHIKV. In some aspects, the polyclonal CHIKV antibodies or antigen-binding fragments are equine antibodies or antigen-binding fragments. In some aspects, the composition comprises a purified gamma globulin fraction of equine plasma.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0036] FIG. 1 shows CHIKV-EIG potency and cross-reactivity against CHIKV lineages (Asian represented by 181/25 and 99659 strains, African represented by 37997 strain, and Indian Ocean represented by LR strain). (See Example 1.)

[0037] FIG. 2 shows that all doses of CHIKV-EIG completely prevented CHIKV-induced footpad swelling in comparison to methotrexate (MTX) or phosphate buffered saline (PBS) diluent alone. *** indicates p<0.001. (See Example 2.)

[0038] FIG. 3 shows that all doses of CHIKV-EIG reduced serum and tissue CHIKV viral loads in comparison to MTX or PBS diluent alone. ** indicates P<0.05 (See Example 2.)

[0039] FIG. 4 shows that all doses of CHIKV-EIG significantly reduced CHIKV-induced pro-arthralgia cytokines in comparison to MTX or PBS diluent alone. * indicates p< 0.05. (See Example 2.)

[0040] FIG. 5 shows that CHIKV-EIG enhanced the survival, reduced viremia, and reduced body weight loss associated with CHIKV infection in comparison to MTX or PBS diluent alone. (See Example 3.)

[0041] FIG. 6 shows CHIKV-EIG potency against the CHIKV 181/25 strain. (See Example 4.)

[0042] FIG. 7 shows CHIKV-EIG potency against the CHIKV 99659 strain. (See Example 4.)

[0043] FIG. 8 shows CHIKV-EIG potency against the CHIKV LR strain. (See Example 4.)

[0044] FIG. 9 shows CHIKV-EIG potency against the CHIKV 37997 strain. (See Example 4.)

[0045] FIG. 10A shows CHIKV-EIG potency against the CHIKV 99659, SFV, and ONNV strains. (See Example 5.)

[0046] FIG. 10B shows CHIKV-EIG potency against the CHIKV 99659, MAYV FMD3212, MAYV INHRR1 la-10, MAYV BeAR505411, MAYV TRVL15537, and RRV strains. (See Example 5.) [0047] FIGS. 11 A-l 1C show the immunization scheme for a chimeric Eilat virus containing Chikungunya virus genomic material (EILV/CHIKV) used as an immunogen to hyperimmunize three horses, according to aspects of the disclosure. (See Example 1.) FIG. 11 A shows the results of a MagPix screening of antibodies binding to CHIKV A226V El protein. FIG. 1 IB shows the results of PRNT assay testing of antibodies neutralizing CHIKV 181/25. FIG. 11C shows PRNT titers from previous study of Cynomolgus macaques that were vaccinated with 1.3 x 10⁸ PFU of EILV/CHIKV. Horses generated higher titers (>2560).

DETAILED DESCRIPTION

[0048] The present disclosure provides method of controlling (e.g., treating, preventing, and/or reducing the risk of) CHIKV infections using compositions comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof.

Definitions

[0049] The terms “antibody,” “immune globulin,” and “immunoglobulin” refer to a protein that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the terms “immune globulin” and “antibody” encompasses intact polyclonal antibodies, human antibodies, and other modified antibodies so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three- dimensional configurations.

[0050] The term “monoclonal antibodies,” as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, the term “polyclonal antibodies” refers to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen (e.g., different epitopes of CHIKV or different epitopes of CHIKV proteins such as Envelope glycoprotein El and/or E2).

[0051] The term “mixture” as used herein refers to a combination of at least two different components, e.g., a mixture of antibodies refers to at least two unique antibodies. The antibodies can differ e.g., based on their sequence, the target to which they bind, and/or the epitope to which they bind within the target.

[0052] The term “antibody fragment” or “immune globulin fragment” refers to a portion of an intact antibody or immune globulin. An “antigen-binding fragment,” “antigenbinding domain,” or “antigen-binding region,” refers to a portion of an intact antibody or immune globulin that binds to an antigen. An antigen-binding fragment can contain the antigenic determining regions of an intact antibody or immune globulin (e.g., the complementarity determining regions (CDR)). Examples of antigen-binding fragments of antibodies or immune globulins include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single chain antibodies. An antigen-binding fragment of an antibody or immune globulin can be derived from any animal species, including humans, or can be artificially produced.

[0053] As used herein, the term “constant region” or “constant domain” are interchangeable and have its meaning common in the art. The constant region is an antibody portion, e.g., a carboxyl terminal portion of a light and/or heavy chain which is not directly involved in binding of an antibody to antigen but which can exhibit various effector functions, such as interaction with the Fc receptor. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain. The “fragment crystallizable region” or “Fc region” contains only constant regions from the heavy chain.

[0054] The terms "anti-CHIKV antibody," "CHIKV antibody" and "antibody that binds to CHIKV" are used interchangeably herein to refer to an antibody that is capable of binding to CHIKV. The extent of binding of a CHIKV antibody to an unrelated, non- CHIKV virus can be less than about 10% of the binding of the antibody to CHIKV as measured. A CHIKV antibody can be capable of binding to one or more CHIKVs (e.g., an Asian CHIKV. A West African CHIKV, and/or an East Central South African CHIKV).

[0055] As used herein, the terms “immunospecifically binds,” “immunospecifically recognizes,” “specifically binds,” and “specifically recognizes” are analogous terms in the context of antibodies or antigen-binding fragments thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain and that the binding entails some complementarity between the antigen binding domain and the epitope. Accordingly, for example, an antibody or immune globulin that “specifically binds” CHIKV can bind to CHIKV, but the extent of binding to an unrelated virus is less than about 10% of the binding of the antibody or immune globulin to CHIKV, e.g., as determined by BiaCore.

[0056] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between the binding sites of molecules (e.g., antibodies or antigen-binding fragment thereofs) and their binding partners (e.g., antigens). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody or antigen-binding fragment thereof and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured and/or expressed in a number of ways known in the art, including, but not limited to, equilibrium dissociation constant (KD), and equilibrium association constant (KA). The KD is calculated from the quotient of k_Off/k_On, whereas KA is calculated from the quotient of kon/koff. k_On refers to the association rate constant of, e.g., an antibody or antigenbinding fragment thereof to an antigen, and k_Off refers to the dissociation of, e.g., an antibody or antigen-binding fragment thereof from an antigen. The kon and k_Off can be determined by techniques known to one of ordinary skill in the art, such as BIAcore® or KinExA.

[0057] The term "purified," as used herein, is intended to refer to a composition, isolated from other components, wherein the protein is purified to any degree relative to its naturally-obtainable state. A purified protein therefore also refers to a protein, free from the environment in which it naturally occurs.

[0058] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. In one aspect, the effect is therapeutic, i.e., the effect partially or completely cures an infection and/or adverse symptom attributable to the infection. In one aspect, the effect is preventing an increase in severity of an infection and/or adverse symptom attributable to the infection. [0059] A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result (e.g., treatment of an infection).

[0060] Alternatively, the pharmacologic and/or physiologic effect may be prophylactic, i.e., the effect completely or partially prevents a disease or symptom thereof. In this respect, the disclosed method comprises administering a “prophylactically effective amount” of a drug (e.g., polyclonal antibodies or antigen-binding fragments thereof). A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic result (e.g., prevention of a CHIKV infection).

[0061] The term “pharmaceutical composition” or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0062] The terms “administer,” “administering,” “administration,” and the like, as used herein, refer to methods that can be used to enable delivery of a composition (e.g., a therapeutic composition comprising polyclonal CHIKV antibodies and/or antigen-binding fragments thereof) to the desired site of biological action (e.g., intravenous administration). Administration techniques that can be employed with the agents and methods described herein are found in e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington’s, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0063] As used herein, the terms “subject” and “patient” are used interchangeably. The subject can be an animal. In some aspects, the subject is a mammal such as a non-human animal (e.g., cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a human.

[0064] As used herein, the term “increasing the level of anti-CHIKV antibodies in a subject” can be used interchangeably with the term “increasing the concentration of anti- CHIKV antibodies in a subject.”

[0065] As used herein, the terms “combination” and “administered in combination” refer to the administration of one active agent (e.g., polyclonal CHIKV antibodies or antigenbinding fragments) with another active agent (e.g., a non-steroidal anti-inflammatory agent). Active agents “administered in combination” can be administered simultaneously or sequentially. Active agents “administered in combination” can be administered in the same or in different compositions.

[0066] As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0067] It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and/or “consisting essentially of’ are also provided. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can mean "includes," "including," and the like; "consisting essentially of' or "consists essentially" are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art aspects.

[0068] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0069] As used herein, the terms “about” and “approximately,” when used to modify a numeric value or numeric range, indicate that deviations of up to 10% above and down to 10% below the value or range remain within the intended meaning of the recited value or range. It is understood that wherever aspects are described herein with the language “about” or “approximately” a numeric value or range, otherwise analogous aspects referring to the specific numeric value or range (without “about”) are also provided

[0070] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

I. Compositions Comprising Polyclonal CHIKV Antibodies and/or Antigen- Binding Fragments Thereof

[0071] As provided herein, a composition (e.g., a pharmaceutical composition composition) can comprise polyclonal CHIKV antibodies and/or antigen-binding fragments thereof. Accordingly, in some aspects, a composition comprises polyclonal CHIKV antibodies. In some aspects, a composition comprises polyclonal CHIKV antigen-binding fragments of antibodies. In some aspects, a composition comprises polyclonal CHIKV antibodies and antigen-binding fragments thereof.

[0072] In some aspects, a composition (e.g., a pharmaceutical composition composition) can comprise a combination of CHIKV antibodies and/or antigen-binding fragments thereof from different B cell clones. In some aspects, a composition comprises oligoclonal CHIKV antibodies and antigen-binding fragments thereof.

[0073] A composition comprising polyclonal CHIKV antibodies and/or antigen-binding fragments thereof can be a hyperimmune globulin composition.

[0074] Such polyclonal CHIKV antibodies and antigen-binding fragments thereof can be capable of neutralizing CHIKV, e.g., CHIKV attenuated strain 181/25, CHIKV strain 99659 (Asian/ American lineage), CHIKV strain 37997 (West African lineage); and/or CHIKV strain LR (Indian Ocean lineage). Assays for determining the ability of an agent (e.g., polyclonal CHIKV antibodies and antigen-binding fragments thereof) to neutralize CHIKV are known in the art and have been described. For example, Azami et al. Methods Mol Biol 14262^r13-'&2 (2016) provides a Plaque Reduction Neutralization Test (PRNT), Nasar et al., PNAS 109 14622-7 (2012) provides a plaque assay on mosquito cells, and Beaty et al., Diagnostic procedures for viral, rickettsial and chlamydial infections. Washington DC: American Public Health Association; 1989. p. 797-855 provides a plaque assay on Vero cells, each of which is herein incorporated by reference in its entirety. For example, such neutralization assays can comprise incubating the polyclonal antibodies or antigen-binding fragments thereof (e.g., optionally serial dilutions of the polyclonal antibodies or antigen-binding fragments thereof) with CHIKV, optionally in the presence of media (e.g., minimal essential medium media). The incubation can be e.g., for about an hour. The incubation can be e.g., at about 37°C. The incubation can be e.g., for about an hour at about 37°C. The incubated mixture can be added to cells that are capable of being infected by CHIKV, e.g., VERO cells and further incubated. This incubation can be e.g., in the presence of minimal essential medium with agarose (e.g., 0.4% agarose). This incubation can be e.g., until plaques appear or about 2 days. This incubation can be e.g., at about 37°C. This incubation can be e.g., for about 2 days at about 37°C. The cells can be fixed, e.g., with 10% formaldehyde and stained, e.g., with 0.5% crystal violate (in 20% ethanol). The cells can be stained, e.g., for about 3 minutes. The number of plaques can then be detected and compared, e.g., to the number of plaques that form in the absence of the polyclonal antibodies or antigen-binding fragments thereof. Accordingly, polyclonal CHIKV antibodies and antigen-binding fragments thereof can be capable of neutralizing CHIKV (e.g., CHIKV attenuated strain 181/25, CHIKV strain 99659 (Asian/ American lineage), CHIKV strain 37997 (West African lineage); and/or CHIKV strain LR (Indian Ocean lineage)) as determined using a plaque assay.

[0075] In some aspects, the ability of polyclonal CHIKV antibodies or antigen-binding fragments thereof to neutralize CHIKV can be determined by Plaque Reduction Neutralization Test (PRNT).

[0076] In some aspects, the ability of polyclonal CHIKV antibodies or antigen-binding fragments thereof to neutralize CHIKV can be determined by Cytopathic Effect (CPE) Inhibition Assay.

[0077] In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 80% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.01 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 80% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.02 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 80% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.03 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 80% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.04 mg/mL (e.g., as determined by PRNT).

[0078] In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 85% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.01 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 85% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.02 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 85% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.03 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 85% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.04 mg/mL (e.g., as determined by PRNT).

[0079] In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 90% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.01 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 90% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.02 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 90% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.03 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 90% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.04 mg/mL (e.g., as determined by PRNT).

[0080] In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 95% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.01 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 95% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.02 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 95% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.03 mg/mL (e.g., as determined by PRNT). In some aspects, a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof is capable of neutralizing more than 95% of CHIKV (e.g., strain 99659 and/or strain 37997) at a concentration of 0.04 mg/mL (e.g., as determined by PRNT).

[0081] In some aspects, the polyclonal CHIKV antibodies or antigen-binding fragments thereof are mammalian (e.g., equine, bovine, ovine), or avian, (e.g., anserine) antibodies or antigen-binding fragments thereof. Avian (e.g., anserine) antibodies and antigenbinding fragments thereof, their production, and use are discussed, for example, in Fink et al. PLOS Neglected Tropical Diseases 77 7):e0005721 (2017), which is herein incorporated by reference in its entirety. The polyclonal antibodies or antigen-binding fragments thereof can be from plasma and/or serum from a mammal (e.g., a horse, cow, sheep, or goose). The polyclonal antibodies or antigen-binding fragments thereof can be from pooled plasma and/or serum, i.e., plasma and/or serum from more than one individual mammal. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof are from pooled from the milk or colostrum of mammals. The mammal can be mammal that was immunized or infected with CHIKV or with an Eilat/Chikungunya chimeric virus (EILV/CHIKV).

[0082] In some aspects, the mammal can be mammal that was immunized with an adjuvant. An adjuvant is used to enhance the mammal’s immune response.

[0083] In some aspects, the polyclonal CHIKV antigen-binding antibody fragments comprise F(ab’)2 fragments. In some aspects, the polyclonal CHIKV antigen-binding antibody fragments comprise F(ab) fragments In some aspects, the polyclonal CHIKV antigen-binding antibody fragments comprise F(ab’)2 fragments and F(ab) fragments. Such fragments can be generated e.g., by pepsin and/or papain digestion.

[0084] The polyclonal CHIKV antibodies or antigen-binding fragments thereof can be IgG antibodies or antigen-binding fragments thereof.

[0085] In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein El. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein E2. In some aspects, the polyclonal antibodies or antigen-binding fragments thereof comprise antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein El and antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein E2.

[0086] In some aspects, the polyclonal CHIKV antigen-binding antibody fragments do not contain an Fc region. The lack of an Fc region can result in less inflammation in a subject as compared to an antibody or antigen-binding fragment comprising an Fc region. In some aspects, polyclonal antibodies or antigen-binding fragments thereof that do not contain a Fc domain avoid Antibody-Dependent Enhancement (ADE). In some aspects, polyclonal antibodies or antigen-binding fragments thereof that do not contain a Fc domain avoid complement activation.

[0087] In some aspects, the polyclonal CHIKV antigen-binding antibody fragments do not contain a CH2 constant domain. In some aspects, the polyclonal CHIKV antigenbinding antibody fragments do not contain a CH3 constant domain. In some aspects, the polyclonal CHIKV antigen-binding antibody fragments do not contain a CH2 constant domain or a CH3 constant domain.

[0088] A composition (e.g., a pharmaceutical composition) comprising polyclonal anti- CHIKV antibodies or antigen-binding fragments thereof can be a liquid. Such a liquid can comprise about 40-60 milligrams of protein per milliliter (mg/mL), about 45-55 mg/mL, or about 49 mg/mL. In some aspects, such a liquid can comprise about 30-70 mg/mL. A composition (e.g., a pharmaceutical composition) comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof can be a filtered sterile solution. Such compositions can be formulated for intravenous administration.

[0089] Compositions (e.g., a pharmaceutical compositions) comprising a polyclonal CHIKV antibodies or antigen-binding fragments thereof can be made using any method known in the art and/or provided herein.

[0090] Methods of making such compositions can comprise purifying the mixture of polyclonal CHIKV antibodies or antigen-binding fragments thereof (e.g., from plasma and/or serum). The plasma and/or serum can be pooled. The plasma and/or serum can be from a mammal. The plasma and/or serum can be from horses, cows, sheep, or geese. The plasma and/or serum can be from a mammal that was immunized with CHIKV. The plasma and/or serum can be from a mammal that was immunized with a chimeric CHIKV (e.g., an Eilat/Chikungunya chimeric virus). The CHIKV or chimeric CHIKV can comprise a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein El, a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein El, or a polynucleotide encoding CHIKV Envelope glycoprotein El and a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein EL The plasma and/or serum can be from a mammal that was immunized with a CHIKV protein, e.g., CHIKV Envelope glycoprotein El and/or E2.

[0091] Methods of making such compositions can comprise purifying the mixture of polyclonal CHIKV antibodies or antigen-binding fragments thereof (e.g., from the egg yolk of birds, the plasma of birds, or from plasma, colostrum, or serum of mammals). The egg yolk and/or plasma can be from a bird that was immunized with CHIKV. The egg yolk and/or plasma can be from a bird that was immunized with a chimeric CHIKV (e.g., an Eilat/Chikungunya chimeric virus). The egg yolk of birds, the plasma of birds, or the plasma, milk and/or colostrum, or serum of mammals can be pooled.

[0092] The plasma, milk, colostrum, and/or serum can be from a mammal. The plasma, milk, colostrum, and/or serum can be from horses, cows, sheep, or geese, ostriches or chickens. The plasma, milk, colostrum, and/or serum can be from a mammal that was immunized with CHIKV. The plasma milk, colostrum, and/or serum can be from a mammal that was immunized with a chimeric CHIKV (e.g., an Eilat/Chikungunya chimeric virus). The CHIKV or chimeric CHIKV can comprise a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein El, a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein E2, or a polynucleotide encoding CHIKV Envelope glycoprotein El and a polynucleotide comprising a nucleic acid sequence encoding CHIKV Envelope glycoprotein E2.

[0093] In some aspects, the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El, a polynucleotide encoding a subdomain of CHIKV Envelope glycoprotein El (e.g., wherein the subdomain comprises a consensus or modified amino acid polypeptide, optionally wherein the subdomain comprises of >10 amino acids), a polynucleotide encoding CHIKV Envelope glycoprotein E2, and/or a polynucleotide encoding a subdomain of CHIKV Envelope glycoprotein E2 (e.g., wherein the subdomain consistscomprises ofa consensus or modified amino acid polypeptide, optionally wherein the subdomain comprises of >10 amino acids).

[0094] The plasma, milk, colostrum, and/or serumcan be from a mammal that was immunized with a CHIKV protein, e.g., CHIKV Envelope glycoprotein El and/or E2.

[0095] Methods of making such compositions can comprise subjecting polyclonal CHIKV antibodies or antigen-binding fragments thereof (e.g., from plasma and/or serum) to viral filtration.

[0096] In some aspects, methods of making such compositions can comprise lyophilizing a liquid composition. In such a case, the lyophilized composition is reconstituted in liquid prior to administration. [0097] Methods of making such compositions can comprise digesting polyclonal CHIKV antibodies or antigen-binding fragments thereof (e.g., from plasma and/or serum) to form F(ab’)2 and F(ab’)2-related antigen-binding fragments of antibodies.

IL Methods of Using Compositions Comprising Polyclonal CHIKV Antibodies and/or Antigen-Binding Fragments Thereof

[0098] Provided herein are methods of using compositions comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof. The compositions can be used therapeutically, e.g., to treat a CHIKV infection in a patient (e.g., a human patient). The compositions can also be used prophylactically, e.g., to prevent a CHIKV infection in a patient (e.g., a human patient). The composition can also be used to reduce the risk of CHIKV infection. Such methods can comprise administering such a composition to a subject in need thereof. Accordingly, also provided herein are compositions comprising polyclonal CHIKV antibodies or antigen-binding fragments for treating, preventing, or reducing the risk of CHIKV infection or for use in manufacturing a medicament for treating, preventing, or reducing the risk of CHIKV infection.

[0099] Also provided herein are compositions comprising a combination of CHIKV antibodies and/or antigen-binding fragments thereof from different B cell clones for treating, preventing, or reducing the risk of CHIKV infection or for use in manufacturing a medicament for treating, preventing, or reducing the risk of CHIKV infection. In some aspects, the disclosure provides compositions comprising oligoclonal CHIKV antibodies and antigen-binding fragments thereof for treating, preventing, or reducing the risk of CHIKV infection or for use in manufacturing a medicament for treating, preventing, or reducing the risk of CHIKV infection.

[0100] Also provided herein are methods of reducing the risk of CHIKV infection- associated arthralgia, arthritis, or arthritis-like symptoms. Arthralgia refers to joint pain and can include polyarthralgia (pain in multiple joints). A diagnosis of arthritis requires physical signs of articular inflammation or the physical or roentgenograph! c signs of osteoarthritis. The risk of CHIKV infection-associated arthralgia, arthritis, or arthritislike symptoms can be reduced by administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof to a subject in need thereof. Accordingly, also provided herein are compositions comprising polyclonal CHIKV antibodies or antigen-binding fragments for reducing the risk of CHIKV infection- associated arthralgia, arthritis, or arthritis-like symptoms or for use in manufacturing a medicament for reducing the risk of CHIKV infection-associated arthralgia, arthritis, or arthritis-like symptoms.

[0101] Also provided herein are methods of reducing CHIKV viral load. The viral load can be reduced in a subject or in a bodily fluid, tissue, and/or cell of a subject. The viral load can be reduced by administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof to a subject in need thereof. Accordingly, also provided herein are compositions comprising polyclonal CHIKV antibodies or antigen-binding fragments for reducing CHIKV viral load or for use in manufacturing a medicament for reducing CHIKV viral load.

[0102] Also provided herein are methods of increasing antibody or antigen-biding fragment thereof titers to CHIKV. Also provided herein are methods of passively increasing antibody or antigen-binding fragment thereof titers to CHIKV. The antibody or antigen-biding fragment thereof titers can be reduced in a subject or in a bodily fluid, tissue, and/or cell of a subject. The antibody or antigen-biding fragment thereof titers can be increased by administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof to a subject in need thereof. Accordingly, also provided herein are compositions comprising polyclonal CHIKV antibodies or antigenbinding fragments for increasing antibody or antigen-biding fragment thereof titers to CHIKV or for use in manufacturing a medicament for increasing antibody or antigenbiding fragment thereof titers to CHIKV.

[0103] Also provided herein are methods of eliciting an immune response against CHIKV in a subject. Also provided herein are methods of passively increasing antibody or antigen-binding fragment thereof titers to CHIKV. The immune response can be elicited by administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof to a subject in need thereof. Accordingly, also provided herein are compositions comprising polyclonal CHIKV antibodies or antigenbinding fragments for eliciting an immune response against CHIKV or for use in manufacturing a medicament for eliciting an immune response against CHIKV.

[0104] The CHIKV can be an Asian/ American lineage CHIKV (e.g., strain 99659). The CHIKV can be a West African lineage CHIKV (e.g., strain 37997). The CHIKV can be an Indian Ocean lineage CHIKV (e.g., strain LR). [0105] The methods described herein, while not bound by theory, are believed to exert their effects by increasing the level of anti-CHIKV antibodies or antigen-binding fragments thereof in a subject. In some aspects, the level of anti- CHIKV antibodies or antigen-binding fragments thereof in a subject is increased as compared to the natural level of anti-CHIKV antibodies or antigen-binding fragments in the subject. In some aspects, as compared to the natural level of anti-CHIKV antibodies or antigen-binding fragments thereof in a subject, the level of anti-CHIKV antibodies or antigen-binding fragments thereof is increased about 2 times, about 3 times, about 4 times, or about 5 times, the natural levels of anti-CHIKV antibodies or antigen-binding fragments thereof in a subject. In some aspects, the level of anti-CHIKV antibodies or antigen-binding fragments thereof increases within 12 hours, within 1 day, or within 2 days of administration of a composition comprising polyclonal CHIKV antibodies or antigenbinding fragments thereof.

[0106] In some aspects, provided herein are methods of treating CHIKV infection in a patient, e.g., a human patient. In some aspects, the methods of treating a CHIKV infection in a patient provided herein comprise administering to a patient an effective amount of a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof. Suitable compositions comprising polyclonal CHIKV antibodies or antigenbinding fragments thereof are described elsewhere herein and can be administered, e.g., intravenously.

[0107] In some aspects, administration of a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof can reduce one or more symptoms of CHIKV infection. Symptoms of CHIKV infections included, e.g., fever, joint pain, muscle pain, joint swelling, headache, nausea, fatigue, and rash.

[0108] In some aspects, a CHIKV infection is an acute infection.

[0109] In some aspects, a CHIKV infection is an Asian CHIKV infection, a West African

CHIKV infection, or an East Central South African CHIKV infection.

[0110] In some aspects, the methods provided herein comprise administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof and standard of care.

[OHl] In some aspects, the methods provided herein comprise administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof and an anti-pyretic, analgesic, paracetamol, acetaminophen and/or non-steroidal anti-inflammatory (NSAID). The composition comprising the polyclonal CHIKV antibodies or antigen-binding fragments thereof and the anti-pyretic, analgesic, paracetamol, acetaminophen and/or NSAID can be administered on the same day or on different days. In some aspects, the anti-pyretic, analgesic, paracetamol, acetaminophen and/or NSAID is administered within two weeks, within one week, within 5 days, within 3 days, within 2 days, or within 1 day of the composition comprising the polyclonal CHIKV antibodies or antigen-binding fragments thereof.

[0112] In some aspects, the methods provided herein comprise administering a composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof and a NSAID. The composition comprising the polyclonal CHIKV antibodies or antigen-binding fragments thereof and the NSAID can be administered on the same day or on different days. In some aspects, the NSAID is administered within two weeks, within one week, within 5 days, within 3 days, within 2 days, or within 1 day of the composition comprising the polyclonal CHIKV antibodies or antigen-binding fragments thereof.

[0113] In some aspects of the compositions comprising polyclonal CHIKV antibodies or antigen-binding binding fragments thereof disclosed herein, the antibodies or antigenbinding binding fragments cross-react with pathogen glycoproteins across Semliki forest complex. As such, the compositions disclosed herein are useful as detection and/or diagnostic compositions. In some aspects, the antibodies or antigen-binding binding fragments thereof for detection and/or diagnostic uses are derived from mammals, e.g., horses.

[0114] In some aspects, the disclosure provides a method of detecting a CHIKV protein in a sample comprising contacting the sample with a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof.

Examples

[0115] The examples in this Examples Section are offered by way of illustration, and not by way of limitation. Example 1: CHIKV-EIG

[0116] A chimeric Eilat virus containing Chikungunya virus genomic material (EILV/CHIKV) was used as immunogen to hyperimmunize three horses (FIGS. 11 A- 11C). The EILV/CHIKV chimeric virus genome encoded genes for EILV nonstructural proteins (nsPs) and CHIKV structural proteins (sPs) and had a particle structure identical to wild-type CHIKV but is incapable of infecting vertebrate cells. (See Erasmus et al., Nat. Med 23: 192-199 (2017), which is herein incorporated by reference in its entirety). The immunizations were done by the subcutaneous (s.c.) route. In some aspects, an adjuvant was used when immunizing the horses.

[0117] Plasma was pooled from immunized horses and an CHIKV-EIG product containing predominantly F(ab’)2 and F(ab) fragments was prepared.

[0118] Antibodies from the horses were screened and showed cross-reactivity against multiple CHIKV lineages as assessed by Plaque Reduction Neutralization Test (PRNT), as previously described (Erasmus et al 2017). (Figure 11). The antibodies had high potency against CHIKV attenuated strain 181/25 and against three wild type strains: strain 99659 (Asian/ American lineage); strain 37997 (West African lineage); and strain LR (Indian Ocean lineage). (Figures 1, 6-9, and Example 4.)

Example 2: Efficacy of CHIKV-EIG Against CHIKV Infection in Immunocompetent Mice

[0119] A model of Chikungunya virus (CHIKV) in the DBA/1 J mouse strain was used to assess the efficacy of CHIKV-EIG against CHIKV infection in immunocompetent mice. This is a non-lethal model that recapitulates many features of CHIKV infection in humans, a high rate of symptomatic disease, and arthralgia. Immunocompetent DBA1/J mice (Jackson labs) were exposed to CHIKV LR06 strain. A challenge dose of 10^{5 5} CCIDso was administered in a total volume of 0.1 ml, 0.05 ml via s.c. injection into the right hind footpad and 0.05 ml via s.c. hock injection into the right hind leg.

[0120] Mice were treated with CHIKV-EIG at doses of 100, 50, or 25 mg/kg/day via i.p. injection beginning 4 hours prior to virus challenge. Treatments were administered daily for 3 or 5 days post-virus inoculation (dpi). A group of mice was treated with 2 mg/kg of methotrexate (as a positive anti-inflammatory control) via s.c. injection for five days beginning -1 dpi. [0121] Sham-infected, placebo, and normal control groups were included. The study design is detailed below in Table 1.

Table 1 : Study Design for CHIKV-EIG Against CHIKV Infection in Immunocompetent Mice

[0122] Mice were monitored daily for mortality and disease signs; n = 10 for all infected groups. Individual weights were recorded daily from 0-7 dpi, and on 9, 11, 14, and 21 dpi. Serum was collected on 2 dpi for virus titration. Footpad swelling, viral load in serum and tissue, and pro-arthralgia cytokines in tissue were evaluated. A caliper was used to measure footpad thickness of the right and left hind legs daily from 5-9 dpi, after which the percent increase of the footpad at the site of footpad inoculation was calculated using the contralateral foot as the baseline. A cohort of mice was necropsied on 6 dpi to assess virus titer and cytokines of the hind limbs.

[0123] Mice infected with CHIKV LR06 and treated with CHIKV-EIG had similar weight change curves as compared with sham-infected controls. A single mouse died on 1 dpi, despite appearing normal prior to mortality. The cause of death was not determined but was likely due to technician error.

[0124] Footpad swelling was measured between 5 and 9 dpi. (Figure 2.) Infection controls had significant right hind footpad swelling with a peak over 50% on 7 dpi compared to the contralateral (left hind) footpad. Animals treated with methotrexate (MTX) had significantly (P<0.01) lower swelling on 7 dpi, but had a similar curve as the infection control group that was delayed by a day, which is consistent with previous studies. All groups treated with CHIKV-EIG had footpad swelling similar to sham- infected controls. Footpad swelling was significantly (P<0.001) lower than infection controls. Treatment for 3 days was also as effective as 5 days of treatment for reducing footpad swelling. These results indicate that CHIKV-EIG treatment administered beginning 4 hours prior to virus challenge can prevent or reduce footpad swelling of infected mice.

[0125] Hind limbs were collected on 6 dpi from 7 mice from each group and evaluated for virus titer. (Figure 3.) Virus titers were elevated above background in the majority of animals in the infection control and methotrexate treatment groups. Virus titers were reduced to background levels in groups treated with CHIKV-EIG, aside from a few animals in the 50 mg/kg treatment group as well as in some of the animals treated with 100 mg/kg of CHIKV-EIG for 3 days. Due to the high variability, there were no significant differences between groups. Virus titers in the left hind limb of animals treated with MTX, contralateral to the site of virus challenge, were significantly (P<0.01) elevated as compared with placebo-treated, infection controls, which was similar to previous studies. Virus titers of all other animals, including the placebo-treated infection controls, were not elevated above background. Serum was collected 2 dpi from all animals within the study. Virus titers were significantly (P<0.05) reduced in all groups treated with CHIKV-EIG. Virus was also observed in 2 animals treated with Methotrexate.

[0126] In general, cytokines were typically not significantly different from controls on 6 dpi, except for monocyte chemotactic protein- l(MCP-l) and regulated upon activation normal T cell expressed and presumably secreted (RANTES). All animals treated with placebo had elevated levels of MCP-1 in the right hind limb at the site of virus challenge. Elevations were also observed in animals treated with methotrexate, and there was no significant difference in MCP-1 levels between this group and the placebo control group. All CHIKV-EIG groups had significant reductions in MCP-1 on 6 dpi. One animal treated with 50 mg/kg had an elevated level of this cytokine, but the rest of the animals had baseline levels. Significant (*P<0.05) reductions in RANTES was also observed in animals treated with CHIKV-EIG, while levels in mice treated with MTX were not significantly different from placebo-treated controls. (Figure 4.)

[0127] In summary, the results, shown in Figures 2-4 demonstrate that immunocompetent mice treated with CHIKV-EIG showed significantly better outcomes than vehicle controls after infection with the wild-type CHIKV-LR06 strain, including complete prevention of footpad swelling at the site of infection, weight loss and virus burden.

Example 3: Efficacy of CHIKV-EIG Against CHIKV Infection in Immunocompromised Mice

[0128] Immunocompromised A129 interferon a/p receptor-knockout (IFNaPR-/-) mice were exposed to CHIKV 9965 strain by the intradermal (i.d.) route and treated once a day for 5 days with doses of 100 mg/kg CHIKV-EIG starting with 1 dpi, or with 100 mg/kg daily starting with 0 dpi (same day as infection).

[0129] The study was performed as a single cohort. After randomization into treatment groups, mice were i.d. challenged in the rear footpad with IxlO³ PFU CHIKV-99659. Animals receiving CHIKV-EIG were then administered five consecutive daily doses of 100 mg/kg CHIKV-EIG, beginning either on the day of infection (4-6 hours postinfection (p.i.); Group 2) or one day post infection (20-24 hours) (1 dpi; Group 3). Control animals (Group 1) were administered a matching dose volume of PBS for five consecutive daily doses beginning at 1 dpi. The study design is detailed below in Table 2.

Table 2: Study Design for CHIKV-EIG Against CHIKV Infection in Immunocompromised Mice

[0130] Survival, viremia, and body weight were evaluated, and the results are shown in Figure 5.

[0131] A statistically significant improvement in survival was observed in both CHIKV- EIG treatment groups compared to controls; n = 10 for all infected groups. Asingle Group 2 animal succumbing at 4 dpi compared to 100% mortality in control animals. Median time to death was also significantly greater in Group 2 and Group 3 compared to controls (p < 0.001 for both comparisons).

[0132] Viremia peaked for both Group 2 and Group 3 on Day 2 (median of 4x10⁴ and IxlO⁵ PFU/mL, respectively), and was eliminated by 9 dpi. In contrast, untreated animals exhibited a peak viremia approximately two orders of magnitude greater than treated animals (median of 7xl0⁶ PFU/mL).

[0133] Body weight loss was severe in control animals to 5 dpi when the animals were euthanized. Treated animals showed a similar rate of weight loss as controls starting with the day after CHIKV-EIG treatment completed. The peak weight loss observed at approximately 7 dpi (Group 2) or 11 dpi (Group 3); however, treated animals eventually recovered to pre-study weight levels by 21 dpi. The greatest average weight loss was 9.53% of baseline weight (observed at Day 11 in animals treated beginning 24 hrs after infection).

[0134] Clinical scores for treated animals were low and never exceeded a score of 3 out of 4. In contrast, all surviving control animals met euthanasia criteria at 5 dpi with clinical scores of 4. Footpad size in both treated groups increased to 4 dpi, plateauing at approximately 3.5 mm compared to approximately 1.5 mm on the day of infection. Footpad size in treated animals then decreased from 7 to 21 dpi. Control animal footpad size followed a similar pattern, but maximum swelling was not more than approximately 2.6 mm prior to death at 5 dpi, substantially lower than that of treated animals. In sum, the control group unexpectedly had less footpad swelling than the treated groups.

[0135] The results, shown in Figure 5, demonstrate that treatment of immunocompromised (IFNAR1 -/-) mice with CHIKV-EIG after infection resulted in significantly enhanced survival and reduced virus burden and disease severity compared to controls.

[0136] These results indicate that CHIKV-EIG is an effective therapy against CHIKV infection.

Example 4: In Vitro Neutralization Potency of CHIKV-EIG by Plaque Reduction Neutralization Test (PRNT)

[0137] The in vitro neutralization potency of CHIKV-EIG against CHIKV as well as cross-neutralization ability against different CHIKV strains from different lineages was determined by Plaque Reduction Neutralization Test (PRNT).

PRNT Procedure

[0138] Virus stock capable of 800 PFU/mL was prepared, and 225 pL of MEM media was added to the first wells (Al to Hl) of plate. 125 pL of MEM media was added to the remaining wells. 25 pL of CHIKV-EIG was added to the first well (each sample has one row) until rows Al to Hl were filled. A multi-pipettor was used to mix samples 15 times, then 125 pL of sample was removed and added to the next column of wells. At each dilution the samples were mixed 15 times, and new tips were changed. Then 125 pL was removed and discarded. When all wells had 125 pL of sample, 125 pL of diluted virus was added to each well. Serial 2-fold dilutions of virus stock were made in MEM to back- titrate the virus. The sample/virus mixture and virus dilutions were incubated at 37°C for 1 hour. 100 pL of each sample/virus mixture and virus titrations were added to a well of 12-well plate of VERO cells and incubated at 37°C for 1 hour with rocking every 15 minutes. Each well was overlay ed with 2 mL of MEM with 0.4% agarose and incubated at 37°C until plaques appeared (about 48 hours). Plates were fixed with 10% formaldehyde and stained with 0.5% Crystal Violet (in 20% ethanol) for 3 minutes. The plaque numbers present in 1 :2 virus titration wells were averaged and the PRNTxo cut-off plaque numbers were calculated. The PRNT titer was read as the highest dilution of sample that inhibits 80% of plaques. [0139] Samples were tested in duplicate with 2-fold dilutions out to 1 :40960. Assay positive control and negative control were included for each batch of cells tested together on a given day. The controls were run with 6 dilutions for positive control, and 3 dilutions for negative control.

Results

[0140] CHIKV-EIG was tested for PRNTso titers against CHIKV attenuated strain 181/25 and three wild type strains: strain 99659 (Asian/ American lineage); strain 37997 (West African lineage); and strain LR (Indian Ocean lineage). The PRNTso results for these strains are provided below in Table 3.

Table 3. RNTso Titers Against CHIKV Strains

[0141] PRNTso assays using CHIKV attenuated strain 181/25 were performed. Under the same assay condition without neutralizing antibodies, CHIKV 181/25 virus produced an average of 40 plaques. The sample plaque numbers were converted to percentage of Plaque Reduction/Neutralization using the following calculation:

(40 - average plaque number) 40 x 100% = % of Plaque Reduction/Neutralization [0142] The potency of CHIKV-EIG against the 181/25 strain is shown in Figure 6 as a plot of the sample concentration and % of Plaque Reduction/Neutralization.

[0143] Strain 99659 was also used for testing CHIKV-EIG neutralization potency.

Briefly, CHIKV 99659 virus stock was diluted to target 800 PFU/mL as working stock, and three 2-fold dilutions of working stock were made in MEM and back-titrated on Vero cells, in order to provide the baseline plaque numbers for the PRNTso calculation. The average virus plaque number for the 1 :2 dilution was 22, and the PRNTso cut off point was set up at 4 plaques. The sample plaque numbers were converted to percentage of Plaque Reduction/Neutralization using the following calculation:

(22 - average plaque number) 22 x 100% = % of Plaque Reduction/Neutralization. [0144] The potency of CHIKV-EIG against strain 99659 is shown in Figure 7 as a plot of the sample concentration and % of Plaque Reduction/Neutralization.

[0145] To test the cross-neutralization of CHIKV-EIG against strains representing lineages other than Asian lineage (immunogen strain), the CHIKLR strain (Indian Ocean Lineage) and CHIKV-37997 (West African Lineage) strains were selected for PRNT assay testing.

[0146] Briefly, CHIK-LR virus stock was diluted to 800 PFU/mL and set up to provide the baseline plaque numbers for the PRNTxo calculation. The average virus plaque number for the 1 :2 dilution was 32, and PRNTso cut off point was set up at 6 plaques. The sample plaque numbers were converted to percentage of Plaque Reduction/Neutralization using the following calculation:

(32 - average plaque number) 32 x 100% = % of Plaque Reduction/Neutralization.

[0147] The potency of CHIKV-EIG against the CHIK-LR virus sample is shown in Figure 8 as a plot of the sample concentration and % of Plaque Reduction/Neutralization.

[0148] Strain 37997 was also used for testing CHIKV-EIG neutralization potency. Briefly, CHIKV strain 37997 virus stock was diluted to 800 PFU/mL and tested to provide the baseline plaque numbers for PRNT80 calculation. The average virus plaque number for the 1 :2 dilution was 19, and PRNTso cut off point was set up at 4 plaques. The sample plaque numbers were converted to percentage of Plaque Reduction/Neutralization using the following calculation:

(19 - average plaque number) 19 x 100% = % of Plaque Reduction/Neutralization.

[0149] The potency of CHIKV-EIG against strain 37997 is shown in Figure 9 as a plot of the sample concentration and % of Plaque Reduction/Neutralization.

[0150] CHIKV-EIG showed high neutralization potency against all the strains tested. The PRNT assay results indicated that CHIKV-EIG has high potency against parental strain 99659, providing 100% neutralization up to 1 : 1280 dilution. CHIKV-EIG also has high potency against 181/25 attenuated strain, which was derived from AF15561 and belongs to the same Asian lineage as 99659. CHIKV-EIG has similar potency against West African lineage strain 37997, and comparable potency against Indian Ocean lineage strain LR.

[0151] The results indicate that CHIKV-EIG can neutralize diverse CHIKV strains. Example 5: CHIKV-EIG Neutralization of Viruses of the Semliki Forest Antigenic Complex

[0152] As CHIKV-EIG showed efficacy against multiple wild-type CHIKV strains (see e.g., Example 4 above,) the in vitro neutralization potency of CHIKV-EIG against phylogenetically related viruses (CHIKV) of the Semliki Forest antigenic complex was studied. The phylogenetically related viruses included Mayaro virus (MAYV), Ross River virus (RRV), O’nyong-nyong virus (ONNV) and Semliki Forest virus (SFV).

[0153] The PRNT procedure was followed generally as set forth in Example 4, except that CHIKV-EIG was tested in duplicate with the 2-fold dilutions from 1 :20 to 1 :20480. Assay positive controls and negative controls were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), and antibody-positive controls were generated against each individual virus. The controls were run with at least 4 dilutions for positive control.

[0154] CHIKV-EIG was tested for PRNTso and PRNTxo titers against the SFV antigenic complex viruses listed below in Table 4. PRNT titers were read as the highest dilution of sample that inhibits 50% or 80% of plaques (average plaque number present in the 1 :2 virus titration). Assay positive controls and negative controls were included for each virus, and each batch of cells tested together on a given date. The results are provided in Table 4.

Table 4. PRNT Titers Against SFV Antigenic Complex Viruses

[0155] CHIKV-EIG has a protein concentration of 49 mg/mL. In a 1 :20 diluted sample, the CHIKV-EIG concentration is 49 mg/mL x 1/20 = 2.45 mg/mL. Similar calculations were used to convert other dilutions to sample concentrations. The sample plaque numbers were converted to percentage of Plaque Reduction/Neutralization using the following calculation:

(virus Ctrl plaque number - plaque numbers at each dilution) virus Ctrl plaque number x 100% = % of Plaque Reduction/Neutralization/

[0156] The potency of CHIKV-EIG against the SFV Antigenic Complex Viruses is shown in Figures 10A and 10B as plots of the sample concentration and % of Plaque Reducti on/N eutralizati on .

[0157] These results indicate that CHIKV-EIG has cross-neutralization potency against distinct alphaviruses within the SFV complex, and the greatest cross-neutralization by CHIKV-EIG was against o'nyong-nyong virus (ONNV) (see Table 4 and Figures 10A and 10B).

Example 6: Neutralization Titers of CHIKV-EIG Against Five Different Alpha Viruses by Cytopathic Effect (CPE) Inhibition Assay

[0158] CPE inhibition assays were performed as follows. Confluent or near-confluent cell culture monolayers in 96-well disposable microplates were prepared. Cells were maintained in minimum essential medium (MEM) or Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with fetal bovine serum (FBS) as required for each cell line. For antiviral assays, the same medium was used but with FBS reduced to 2% or less and supplemented with 50 pg/ml gentamicin. The test compound was prepared at four final concentrations: 0.1, 1.0, 10, and 100 pg/ml or pM. Lower concentrations were used when insufficient compound is supplied for the usual concentrations. Five microwells were used per dilution: three for infected cultures and two for uninfected toxicity controls. Controls for the experiment consisted of six microwells that were infected but not treated (virus controls) and six that were untreated (cell controls). The virus control and cell control wells are on every microplate. A known active drug (Infergen (synthetic type-I interferon) was tested in parallel with each assay as a positive control, using the same method as is applied for test compounds.

[0159] Growth medium was removed from the 96-well plates of cells, then the test compound was applied in 0.1 ml volume to wells at 2X concentration. Chikungunya virus strain S27 (African lineage prototype originally isolated from serum collected from a patient in Tanzania in 1953), normally <100 CCID50 (50% cell culture infectious doses) in 0.1 ml volume, was added to wells designated for virus infection. Medium devoid of virus was placed in toxicity control wells and cell control wells. Virus control wells were treated similarly with virus.

[0160] Plates were incubated at 37 °C with 5% CO2 until maximum CPE was observed microscopically in virus control wells. Plates were then stained with 0.011% neutral red for approximately two hours at 37 °C in a 5% CO2 incubator. The neutral red medium was removed by complete aspiration, and the cells were rinsed with phosphate buffered saline (PBS) to remove residual dye. The PBS was completely removed and the incorporated neutral red was eluted with 50% Sorensen’s citrate buffer/50% ethanol for at least 30 minutes. Neutral red dye penetrates into living cells, thus, the more intense the red color, the larger the number of viable cells present in the wells. The dye content in each well was quantified using a spectrophotometer at 540 nm wavelength. The dye content in each set of wells was converted to a percentage of dye present in untreated control wells using a Microsoft Excel™ computer-based spreadsheet. The 50% effective (EC50, virus-inhibitory) concentrations and 50% cytotoxic (CC50, cell-inhibitory) concentrations were then calculated by regression analysis. The quotient of CC50 divided by EC50 gives the selectivity index (SI50) value. Compounds showing SI50 values >10 are considered active and are merit further investigation.

[0161] CHIKV-EIG neutralization titers were assessed using the CPE assays, with output expressed as the 50% effective (EC50, virus inhibitory) and 50% cytotoxic (CC50, cell- inhibitory) concentrations calculated by regression analysis. Infergen (synthetic type-I interferon) was used as a positive control compound in the study. The finalized data is tabulated below in Table 5for Infergen (positive control) and CHIKV-EIG (test article),.

Table 5: Chikungunya Virus

[0162] These results demonstrate that CHIKV-EIG effectively neutralized CHIKV with an SI50 value of >370 (neutral red test).

* * *

[0163] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes

Claims

- 34 -

WHAT IS CLAIMED IS: A method for treating, preventing, or reducing the risk of a Chikungunya virus (CHIKV) infection in a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A method for treating, preventing, or reducing the risk of CHIKV infection-associated arthritis in a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A method for treating, preventing, or reducing the risk of CHIKV infection-associated arthralgia in a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A method for reducing viral load of CHIKV in a bodily fluid, tissue, or cell of a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A method for increasing antibody or antigen-binding fragment thereof titers to CHIKV in a bodily fluid, tissue, or cell of a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A method of eliciting an immune response against CHIKV in a subject, the method comprising administering to the subject an effective amount of a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. - 35 - The method of any one of claims 1-6, wherein the polyclonal antibodies or antigenbinding fragments thereof are from pooled plasma and/or serum from mammals and/or birds. The method of claim 7, wherein the mammals are horses, cows, or sheep, optionally wherein the birds are geese, ostriches or chickens. The method of claim 8, wherein the mammals are horses. The method of any one of claims 7-9, wherein the mammals and/or birds were immunized with CHIKV, a CHIKV protein, a CHIKV protein domain, or a chimeric CHIKV. The method of claim 10, wherein the chimeric CHIKV is an Eilat/Chikungunya chimeric virus. The method of claims 10-12, wherein an adjuvant was administered with the CHIKV, CHIKV protein, or chimeric CHIKV. The method of any one of claims 10-12, wherein the chimeric CHIKV comprises a polynucleotide encoding CHIKV Envelope glycoprotein El, a polynucleotide encoding a subdomain of CHIKV Envelope glycoprotein El, a polynucleotide encoding CHIKV Envelope glycoprotein E2, and/or a polynucleotide encoding a domain of CHIKV Envelope glycoprotein E2. The method of any one of claims 1-13, wherein the polyclonal antibodies or antigenbinding fragments thereof comprise antibodies or antigen-binding fragments thereof that are CHIKV -virus neutralizing antibodies or antigen-binding fragments thereof. The method of any one of claims 1-14, wherein the polyclonal antibodies or antigenbinding fragments thereof do not contain a Fragment, crystallizable (Fc) domain, optionally wherein the polyclonal antibodies or antigen-binding fragments thereof cause less inflammation-than polyclonal antibodies or antigen-binding fragments thereof comprising an Fc domain. The method of any one of claims 1-15, wherein the polyclonal antibodies or antigenbinding fragments thereof comprise antibodies or antigen-binding fragments thereof that bind to CHIKV Envelope glycoprotein El and/or E2. The method of any one of claims 1-16, wherein the polyclonal antibodies or antigenbindings comprise F(ab’)2 fragments. The method of any one of claims 1-16, wherein the polyclonal antibodies or antigenbindings comprise F(ab) fragments. The method of any one of claims 1-16, wherein the polyclonal antibodies or antigenbindings comprise F(ab’)2 fragments and F(ab) fragments. The method of any one of claims 1-19, wherein the polyclonal antibodies or antigenbinding fragments thereof comprise immunoglobulin G (IgG) antibodies or antigenbinding fragments thereof. The method of any one of claims 1-20, wherein the composition comprises a purified gamma globulin fraction of equine plasma. The method of any one of claims 1-21, wherein the composition comprises about 30-70 milligrams (mg) of protein /milliliter (mL). The method of any one of claims 1-21, wherein the composition comprises about 49 milligrams (mg) of protein /milliliter (mL). The method of any one of claims 1-23, wherein the composition is a liquid. The method of any one of claims 1-21, wherein the composition is lyophilized. The method of any one of claims 1-23, wherein the composition is a filtered sterile solution. The method of any one of claims 1-26, wherein the composition was obtained by pepsin and/or papain digestion. The method of any one of claims 1-27, wherein the composition was treated to reduce procoagulation activity. The method of any one of claims 1-28, wherein the composition was subjected to virus filtration. The method of any one of claims 1-29, wherein the composition is administered intravenously. The method of any one of claims 1-30, wherein the administration increases the anti- CHIKV antibodies or antigen-binding fragments thereof in the subject by at least 2-fold, by at least 3-fold, by at least 4-fold, or by at least 5-fold. The method of any one of claims 1-31, wherein the CHIKV infection is acute. The method of any one of claims 1-32, wherein the CHIKV infection is an Asian CHIKV infection. The method of any one of claims 1-32, wherein the CHIKV infection is a West African CHIKV infection. The method of any one of claims 1-32, wherein the CHIKV infection is an East Central

South African CHIKV infection. - 38 - The method of any one of claims 1-35, wherein the subject is human. The method of any one of claims 1-36, further comprising administering a non-steroidal anti-inflammatory drug. The method of any one of claims 1-37, wherein the polycloncal CHIKV antibodies or antigen-binding binding fragments thereof are capable of neutralizing more than 80% of CHIKV at a concentration of 0.02 mg/mL as determined by plaque reduction neutralization test (PRNT). The method of any one of claims 1-38, wherein the polyclonal CHIKV antibodies or antigen-binding binding fragments thereof are capable of neutralizing o'nyong-nyong virus (ONNV) in vitro. A method of detecting a CHIKV and/or Semliki Forest protein in a sample comprising contacting the sample with a composition comprising CHIKV polyclonal antibodies or antigen-binding fragments thereof. A composition comprising polyclonal CHIKV antibodies or antigen-binding fragments thereof for use in detecting a CHIKV and/or Semliki Forest protein. The method of claim 40 or 41, wherein the polyclonal CHIKV antibodies or antigenbinding fragments were produced by immunized a mammal or bird with CHIKV, a CHIKV protein, a CHIKV protein domain, or a chimeric CHIKV. The method of any one of claims 40-42, wherein the polyclonal CHIKV antibodies or antigen-binding fragments are equine antibodies or antigen-binding fragments. The method of any one of claims 40-43, wherein the composition comprises a purified gamma globulin fraction of equine plasma.