Monoclonal antibodies neutralizing marburg and RAVN virus

Abstract

Embodiments of the invention provide a solution to the problems associated with the lack of treatments for MARV and RAVV. The inventors have generated neutralizing mAbs to MARV that neutralize MARY infection. Certain mAbs are cross-reactive to MARV and RAVV.

Description

[0001] MONOCLONAL ANTIBODIES NEUTRALIZING MARBURG AND RAVN VIRUS

[0002] RELATED APPLICATIONS

[0003]

[0001] This Application is an international application claiming priority to U.S. Provisional Patent Application 63 / 575,085 filed April 5, 2024 which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0005]

[0002] None.

[0006] REFERENCE TO SEQUENCE LISTING

[0007]

[0003] The present application contains a Sequence Listing which has been submitted in electronic format via EFS-Web (or other applicable filing system) and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on March 31, 2025, is named UTMBP0418 and is 158 kilobytes in size. The Sequence Listing complies with the requirements of WIPO Standard ST.26.

[0008] FIELD OF THE INVENTION

[0009]

[0004] Embodiments of the invention are directed generally to infectious medicine and virology, and particularly to methods and composition for detecting and treating filovirus infections.

[0010] BACKGROUND

[0011]

[0005] Marburg virus (MARV) and Ravn virus (RAW) belong to the family Filoviridae, genus Orthomarburgvirus, species Orthomarburgvirus marburgense . These filoviruses cause a lethal hemorrhagic fever, Marburg virus disease (MVD), in humans and nonhuman primates. The case fatality rate of MVD ranges from 24 to 88% depending on MARV variants and case management (Marburg virus disease at URL who.int / news-room / fact-sheets / detail / marburg- virus-disease; Asad et al., 2020, InfezMed 28, 332-345). The MARV infection was first reported in 1967 in Germany, and since then, sporadic outbreaks of MVD have been reported in the Democratic Republic of the Congo, Angola, Uganda, Guinea, Ghana, Equatorial Guinea, and Tanzania. Importantly, the recent outbreaks of MVD in Guinea, Ghana, Equatorial Guinea, and Tanzania, none of which had reported previous outbreaks until 2020, have emphasized the need for the development of prophylactic and therapeutic drugs for this deadly disease.

[0012]

[0006] Of the seven filovirus structural proteins, the glycoprotein (GP), which mediates both binding to cellular receptors and membrane fusion between viral envelope and cell membrane, is responsible for viral entry into cells and the only target of neutralizing antibodies. For Ebola virus disease, which is caused by Ebola virus (EBOV) belonging to another genus (Orthoebolavirus) in the same virus family, there are two FDA-approved GP-specific antibody therapies (monoclonal antibody cocktail REGN-EB3 and single monoclonal antibody (mAb) 114). However, these therapeutic antibodies are ineffective for the other human-pathogenic fdoviruses including MARV and RAW due to the antigenic differences. Although several broadly cross-reactive mAb to orthoebolavirus GPs have been reported (Furuyama et al., 2016, Sci Rep 6, 20514; Flyak et al., 2016 Cell 164, 392-405; King et al., 2019, Curr Opin Virol 34, 140-148), none of them efficiently neutralize MARV and RAW, and information on therapeutic mAbs and target B-cell epitopes for MVD are quite limited.

[0013]

[0007] Thus, there remains a need for additional compositions and methods for detection and treatment of MARV and / or RAW infection and their related disease.

[0014] SUMMARY

[0015]

[0008] Embodiments of the invention provide a solution to the problems associated with the lack of treatments for MARV and RAW. The inventors have generated neutralizing mAbs to MARV that neutralize MARV infection. Ten representative mAbs were produced and divided into three groups based on their putative epitopes and amino acid sequences of their variable regions of heavy and light chains. Five mAbs (AGP-R1, R7, R12, R15, and R17) were cross- reactive to MARV and RAW and most likely bind an epitope across the fusion loop and receptor binding domain of GP. On the other hand, AGP-R4, R5, R11, R20, and R22 neutralized MARV but not RAW and likely bind to an epitope on the head region of GP. The putative epitope of AGP-R4 and R5 was speculated to overlap with but slightly different from that of AGP-R11, R20, and R22. It was noted that all these mAbs showed neutralizing activity equivalent to or rather higher than a previously known neutralizing mAh MR78 The biological properties of the mAbs produced are described herein.

[0016]

[0009] Certain embodiments are directed to an antibody or antibody fragment that binds the MARV glycoprotein, the antibody or antibody fragment comprising (i) heavy chain CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO:42, and CDR3 of SEQ ID NO:43 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56; (ii) heavy chain CDR1 of SEQ ID NO: 44, CDR2 of SEQ ID NO: 45, and CDR3 of SEQ ID NO: 46 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:57; (iii) heavy chain CDR1 of SEQ ID NO:41, CDR2 of SEQ ID NO:42, and CDR3 of SEQ ID NO:47 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:58; (iv) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:49, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:61; (v) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:51, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:62; (vi) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:62; or (vii) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:63, and CDR3 of SEQ ID NO:62.

[0017]

[0010] In certain aspects the antibody or antibody fragment has a variable heavy chain / light chain pair having an amino acid sequence of (i) SEQ ID NO:2 and SEQ ID NO:4, (ii) SEQ ID NO:8 and SEQ ID NO: 10, (iii) SEQ ID NO: 18 and SEQ ID NO:20, (iv) SEQ ID NO:22 and SEQ ID NO:24, (v) SEQ ID NO:26 and SEQ ID NO:28, (vi) SEQ ID NO:30 and SEQ ID NO:32, or (vii) SEQ ID NO:34 and SEQ ID NO:36.

[0018]

[0011] Other embodiments are directed to a polynucleotide encoding the antibody or antibody fragment described herein or a cell or organism expressing the same.

[0019]

[0012] Embodiments are directed to methods of detecting MARV and RAW infection in a subject comprising: (a) contacting a sample from the patient with an antibody or antibody fragment described herein; and (b) detecting the binding of the antibody or antibody fragment to a component of the sample, binding being indicative of the presence of MARV and / or RAW.

[0020]

[0013] Certain embodiments are directed to treating a subject with a MARV or RAW infection or at risk of having a MARV or RAW infection by administering an antibody or antibody fragment described herein.

[0021]

[0014] Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. Each embodiment described herein is understood to be embodiments of the invention that are applicable to all aspects of the invention. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions and kits of the invention can be used to achieve methods of the invention.

[0022]

[0015] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0023]

[0016] Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0024]

[0017] The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or."

[0025]

[0018] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0026]

[0019] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0027] DESCRIPTION OF THE DRAWINGS

[0028]

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specification embodiments presented herein.

[0029]

[0021] FIG. 1 : Neutralizing activity of the mAbs against pseudotyped viruses. Neutralizing activities of mAbs were investigated using VSVAG*MARV-Angola, VSVAG*MARV-Musoke, VSVAG*-RAVV, and VSVAG*-EBOV. Averages and standard deviations of triplicated data are shown.

[0030]

[0022] FIG. 2: Neutralizing activity of the mAbs against MARV and RAW. Neutralizing activities of mAbs was investigated using infectious MARV (variant Angola) and RAW. Averages and standard deviations of triplicated data are shown.

[0031]

[0023] FIG. 3: Binding of the mAbs to purified GPs. Binding activities of mAbs were examined by ELISA using purified recombinant GPs of MARV (variant Angola), RAW, and EBOV as antigens.

[0032]

[0024] FIG. 4A-4C: Identification of putative epitopes of the mAbs. (A) Amino acid substitutions found in the MARV GP escape mutants selected by each mAb are shown. (B) The GP1 subunit contains the receptor binding domain (RBD), glycan cap, and mucin-like domain (MLD). The GP2 subunit contains the internal fusion loop (IFL), heptad repeats 1 and 2 (HR1 and HR2), transmembrane region (TM), and cytoplasmic tail (CT). Positions of the amino acid substitutions found in the escape mutants of R1 (red), R4 (green), and R11 (blue) group mAbs are shown. (C) Amino acid residues involved in the putative epitopes of the mAbs are mapped on the GP trimers (PDB code: 6BP2). Structures of amino acid positions 1-32, 181-468, 499-504, 630-681 are not available.

[0025] FIG. 5: The epitopes recognized by the mAbs conserved among MARV strains. The amino acid sequences around the epitope recognized by the mAbs were compared among MARV strains. GenBank accession numbers of Angola-2005, Ci67-200, Musoke-1980, Ravn- 1987, Durba-09DRC99, Uganda 200703648, and 02Uga07 are ABE27015.1, AB SI 7558.1, CAA78117.1, ABE27071.1, ABE27092, AMZ00488.1, and ACT79201.1, respectively.

[0033]

[0026] FIG. 6A-6C. Mouse survival study as assessed by (A) survival, (B) body temperature, and (C) weight change. All mice treated with control IgG succumbed to the disease at 7 d.p.i., all mice treated with each mAb, excepting one mouse treated with AGP-R5, survived from lethal MARV infection with showing only minor weight loss.

[0034] DESCRIPTION

[0035]

[0027] Described herein are highly potent mAbs that neutralize MARV infection. Ten representative mAbs were produced and divided into three groups based on their putative epitopes and amino acid sequences of their variable regions of heavy and light chains. Five mAbs (i.e., R1 group: AGP-R1, R7, R12, R15, and R17) were cross-reactive to MARV and RAW that most likely recognize an epitope across the fusion loop and receptor binding domain of GP. On the other hand, AGP-R4, R5, R11, R20, and R22 neutralized MARV but not RAW and likely bind to an epitope on the head region of GP. The putative epitope of AGP-R4 and R5 (i.e., the R4 group) was speculated to overlap with but slightly different from that of AGP-R11, R20, and R22 (i.e., Rl l group). It was noted that all these mAbs showed neutralizing activity equivalent to or rather higher than a previously known neutralizing mAb MR78. The sequences and biological properties of the representative antibodies are described herein.

[0036]

[0028] An AGP-R1 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:4 and can be encoded by the nucleic acid sequence of SEQ ID NO:3, and having the following CDRs: CDR1 TDIDDD (SEQ ID NO:54), CDR2 EGN (SEQ ID NO:55), and CDR3 LQSDNLPLT (SEQ ID NO: 56); and a heavy chain variable region (VH) as set forth in SEQ ID NO:2 and can be encoded by the nucleic acid of SEQ ID NO: 1, and having the following CDRs: CDR1 GYTFTNFG (SEQ ID NO:41), CDR2 INTYTGEP (SEQ ID NO:42), and CDR3 ARERAGYYVDS (SEQ ID NO:43).

[0029] An AGP-R7 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:8 and can be encoded by the nucleic acid sequence of SEQ ID NO:7, and having the following CDRs: CDR1 TDIDDD (SEQ ID NO:54), CDR2 EGN (SEQ ID NO:55), and CDR3 LQSDDLPLT (SEQ ID NO: 57); and a heavy chain variable region (VH) as set forth in SEQ ID NO:6 and can be encoded by the nucleic acid of SEQ ID NO:5, and having the following CDRs: CDR1 GYTFTNYG (SEQ ID NO:44), CDR2 INTYSGEA (SEQ ID NO:45), and CDR3 ARERAGYYVDY (SEQ ID NO: 46).

[0037]

[0030] An AGP-R12 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO: 12 and can be encoded by the nucleic acid sequence of SEQ ID NO:11, and having the following CDRs: CDR1 TDIDDD (SEQ ID NO: 54), CDR2 EGN (SEQ ID NO: 55), and CDR3 LQSDNLPLT (SEQ ID NO:56); and a heavy chain variable region (VH) as set forth in SEQ ID NO: 10 and can be encoded by the nucleic acid of SEQ ID NO:8, and having the following CDRs: CDR1 GYTFTNFG (SEQ ID NO:41), CDR2 INTYTGEP (SEQ ID NO:42), and CDR3 ARERAGYYVDS (SEQ ID NO:43).

[0038]

[0031] An AGP-R15 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:16 and can be encoded by the nucleic acid sequence of SEQ ID NO:15, and having the following CDRs: CDR1 TDIDDD (SEQ ID NO: 54), CDR2 EGN (SEQ ID NO: 55), and CDR3 LQSDNLPLT (SEQ ID NO:56); and a heavy chain variable region (VH) as set forth in SEQ ID NO: 14 and can be encoded by the nucleic acid of SEQ ID NO: 13, and having the following CDRs: CDR1 GYTFTNFG (SEQ ID NO:41), CDR2 INTYTGEP (SEQ ID NO:42), and CDR3 ARERAGYYVDS (SEQ ID NO:43).

[0039]

[0032] An AGP-R17 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:20 and can be encoded by the nucleic acid sequence of SEQ ID NO: 19, and having the following CDRs: CDR1 TDIDDD (SEQ ID NO: 54), CDR2 EGN (SEQ ID NO: 55), and CDR3 LESDNLPLT (SEQ ID NO: 58); and a heavy chain variable region (VH) as set forth in SEQ ID NO: 18 and can be encoded by the nucleic acid of SEQ ID NO: 17, and having the following CDRs: CDR1 GYTFTNFG (SEQ ID NO:41), CDR2 INTYTGEP (SEQ ID NO:42), and CDR3 ARERAGYFVDS (SEQ ID NO:47).

[0033] An AGP-R4 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:24 and can be encoded by the nucleic acid sequence of SEQ ID NO:23, and having the following CDRs: CDR1 QSLVHSNGNTY (SEQ ID NO:59), CDR2 KVS (SEQ ID NO:60), and CDR3 SQSTHIPRT (SEQ ID NO:61); and a heavy chain variable region (VH) as set forth in SEQ ID NO:22 and can be encoded by the nucleic acid of SEQ ID NO:21, and having the following CDRs: CDR1 GYTFTDYA (SEQ ID NO:48), CDR2 ISLYYDKT (SEQ ID NO:49), and CDR3 ARSDYRYGDYYAMDY (SEQ ID NO: 50).

[0040]

[0034] An AGP-R5 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:28 and can be encoded by the nucleic acid sequence of SEQ ID NO:27, and having the following CDRs: CDR1 QSLVHSNGNTY (SEQ ID NO:59), CDR2 KVS (SEQ ID NO:60), and CDR3 SQSTHFPRT (SEQ ID NO:62); and a heavy chain variable region (VH) as set forth in SEQ ID NO:26 and can be encoded by the nucleic acid of SEQ ID NO:25, and having the following CDRs: CDR1 GYTFTDYA (SEQ ID NO:48), CDR2 ISTYYDKT (SEQ ID NO:51), and CDR3 ARSDYRYGDYYAMDY (SEQ ID NO: 50).

[0041]

[0035] An AGP-R11 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:32 and can be encoded by the nucleic acid sequence of SEQ ID NO:31, and having the following CDRs: CDR1 QSLVHSNGNTY (SEQ ID NO:59), CDR2 KVS (SEQ ID NO:60), and CDR3 SQSTHFPRT (SEQ ID NO:62); and a heavy chain variable region (VH) as set forth in SEQ ID NO:30 and can be encoded by the nucleic acid of SEQ ID NO:29, and having the following CDRs: CDR1 GYTFTDYA (SEQ ID NO:48), CDR2 MNIYYDRR (SEQ ID NO:52), and CDR3 ARSDYRYGDYYAMDY (SEQ ID NO: 50).

[0042]

[0036] An AGP-R20 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:36 and can be encoded by the nucleic acid sequence of SEQ ID NO:35, and having the following CDRs: CDR1 QSLVHSNGNTY (SEQ ID NO:59), CDR2 KIS (SEQ ID NO:63), and CDR3 SQSTHFPRT (SEQ ID NO:62); and a heavy chain variable region (VH) as set forth in SEQ ID NO:34 and can be encoded by the nucleic acid of SEQ ID NO:33, and having the following CDRs: CDR1 GYTFTDYA (SEQ ID NO:48), CDR2 MNIYYDRR (SEQ ID NO: 52), and CDR3 ARSDYRFGDYYAMDY (SEQ ID NO: 53).

[0037] An AGP-R22 antibody can include a light chain variable region (VL) as set forth in SEQ ID NO:40 and can be encoded by the nucleic acid sequence of SEQ ID NO:39, and having the following CDRs: CDR1 QSLVHSNGNTY (SEQ ID NO:59), CDR2 KVS (SEQ ID NO:60), and CDR3 SQSTHFPRT (SEQ ID NO:62); and a heavy chain variable region (VH) as set forth in SEQ ID NO:38 and can be encoded by the nucleic acid of SEQ ID NO: 37, and having the following CDRs: CDR1 GYTFTDYA (SEQ ID NO:48), CDR2 MNIYYDRR (SEQ ID NO:52), and CDR3 ARSDYRYGDYYAMDY (SEQ ID NO: 50).

[0043]

[0038] An MARV or MARV / RAVV antibody includes an antibody comprising a (1) heavy chain variable region having CDRs with a consensus amino acid sequence of CDR1 = GYTFTX1X2X3 wherein Xi is N or D, X2is F or Y, X3is G or A (SEQ ID NO:64); CDR2 = IX4TYX5GEX6wherein X4is N or S, and X5is S, T, or Y, X6is P, A, T, or R (SEQ ID NO: 65) or ISX7YYDKT wherein X7is L or T (SEQ ID NO: 66) or MNIYYDRR (SEQ ID NO: 52); CDR3 = ARERGYX8VDX9wherein X8is Y or F, X9is S or Y (SEQ ID NO:67) or ARSDYRX10GDYYAMDY wherein X10 is Y or F (SEQ ID NO:68) and (2) light chain variable region having CDRs with a consensus amino acid sequence of CDR1 = TDIDDD (SEQ ID NO:54) or QSLVHSNGNFY (SEQ ID NO:59); CDR2= EGN (SEQ ID NO:55) or KX11S wherein Xu is V or I; CDR3= LXi2SDXi3LPLT(SEQ ID NO:69) wherein X12 is Q or E, Xi3is N or D or SQSTHXI4PRT wherein Xi4is I or F (SEQ ID NO: 70).

[0044]

[0039] The term “Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE, IgM, IgD, and IgY) including various monomeric and polymeric forms of each isotype, unless otherwise specified. The term “antibody” or “immunoglobulin” is used to include intact antibodies and binding fragments / segments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to an antigen. Fragments include separate heavy chains, light chains, Fab, Fab' F(ab')2, Fabc, and Fv. Fragments / segments are produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. The term “antibody” also includes bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. A bispecific antibody of can include any two heavy / light chain pairs described herein (e.g., (AGP-R1 paired with AGP-R7, AGP-R12, AGP-R15, AGP-R17 AGP-R4, AGP-R5, AGP-R11 , AGP-R20, or AGP-R22; AGP-R7 paired with AGP-R1, AGP-R12, AGP-R15, AGP-R17 AGP- R4, AGP-R5, AGP-R11, AGP-R20, or AGP-R22; AGP-R12 paired with AGP-R1, AGP-R7, AGP-R15, AGP-R17 AGP-R4, AGP-R5, AGP-R11, AGP-R20, or AGP-R22; AGP-R15 paired with AGP-R1, AGP-R7, AGP-R12, AGP-R17 AGP-R4, AGP-R5, AGP-R11, AGP-R20, or AGP-R22; AGP-R17 paired with AGP-R1, AGP-R7, AGP-R12, AGP-R15 AGP-R4, AGP-R5, AGP-R11, AGP-R20, or AGP-R22; AGP-R4 paired with AGP-R1, AGP-R7, AGP-R12, AGP- R15 AGP-R17, AGP-R5, AGP-R11, AGP-R20, or AGP-R22; AGP-R5 paired with AGP-R1, AGP-R7, AGP-R12, AGP-R15 AGP-R17, AGP-R4, AGP-R11, AGP-R20, or AGP-R22; AGP- Rl l paired with AGP-R1, AGP-R7, AGP-R12, AGP-R15 AGP-R17, AGP-R4, AGP-R5, AGP- R20, or AGP-R22; AGP-R20 paired with AGP-R1, AGP-R7, AGP-R12, AGP-R15 AGP-R17, AGP-R4, AGP-R5, AGP-R11, or AGP-R22; AGP-R22 paired with AGP-R1, AGP-R7, AGP- R12, AGP-R15 AGP-R17, AGP-R4, AGP-R5, AGP-R11, or AGP-R20). Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin Exp Immunol 79 :315-21, 1990; Kostelny et al., J. Immunol. 148:1547-53, 1992.

[0045]

[0040] “Functional fragments” of such antibodies comprise portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, functional fragments can comprise at least the CDRs of either the heavy chain and / or light chain variable region. Functional fragments can also comprise the heavy chain or light chain variable region, or sequences that are substantially similar to the heavy or light chain variable region. Further suitable functional fragments include, without limitation, antibodies with multiple epitope specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab, F(abz)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies (also called ScFv), individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes can be used to produce functional fragments of the antibodies herein. Functional fragments can be recombinantly or synthetically produced, with natural or unnatural nucleic acid or amino acid molecules.

[0041] The antibodies or functional fragments thereof of the disclosed subject matter can be generated from any species. The antibodies or functional fragments thereof described herein can be labeled or otherwise conjugated to various chemical or biomolecule moieties, for example, for therapeutic or diagnostic or detection or treatment applications. The moieties can be detectable labels, for example, fluorescent labels, radiolabels, biotin, and the like, which are known in the art. When the antibodies of the present invention are used, e.g., for flow cytometric detection, for scanning laser cytometric detection, or for fluorescent immunoassay, they can usefully be labeled with fluorophores. There are a wide variety of fluorophore labels that can usefully be attached to the antibodies of the present invention. For detection applications, common useful fluorophores can be fluorescein isothiocyanate (FITC), allophycocyanin (APC), R-phycoerythrin (PE), peridinin chlorophyll protein (PerCP), Texas Red, Cy3, Cy5, fluorescence resonance energy tandem fluorophores such as PerCPCy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, and APC-Cy7. Other fluorophores include, inter alia, Alexa Fluor® 350, Alexa Fluor® 488, Alexa 25 Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647 (monoclonal antibody labeling kits available from Molecular Probes, Inc., Eugene, OR, USA), BODIPY dyes, such as BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, lissamine rhodamine B, Marina Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, rhodamine 6G, rhodamine green, rhodamine red, tetramethyl rhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR, USA), and Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, all of which are also useful for fluorescently labeling the antibodies of the present invention. For secondary detection using labeled avidin, streptavidin, captavidin or neutravidin, the antibodies of the present invention can usefully be labeled with biotin. When the antibodies of the present invention are used, e.g., for western blotting applications, they can usefully be labeled with radioisotopes, such as 33P, 32P, 35S, 3H, and 1251. As another example, when the antibodies of the present invention are used for radioimmunotherapy, the label can usefully be 3H, 228Th, 227 Ac, 225Ac, 223Ra, 213Bi, 212Pb, 212Bi, 211At, 203Pb, 1940s, 188Re, 186Re, 153Sm, 149Tb, 1311, 1251, U lin, 105Rh, 99mTc, 97Ru, 90Y, 90Sr, 88Y, 72Se, 67Cu, or 47Sc.

[0046]

[0042] Modifications and / or changes may be made in the amino acid composition of polypeptides, and thus the present invention contemplates variation in sequences of the polypeptides, and nucleic acids coding therefor, where they are nonetheless able retain substantial activity with respect to the therapeutic, preventative, and curative aspects of the present invention.

[0047]

[0043] The biological functional equivalent may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the original or standard peptide. This can be accomplished through the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. In one example, one of skill in the art may wish to introduce a restriction enzyme recognition sequence into a polynucleotide while not disturbing the ability of that polynucleotide to encode a protein.

[0048]

[0044] “Substantially similar” with respect to nucleic acid or amino acid sequences, means at least about 80% identity, more preferably at least about 85% identity, more preferably at least about 90% identity, more preferably at least about 91% identity, more preferably at least about 92% identity, more preferably at least about 93% identity, more preferably at least about 94% identity, more preferably at least about 95% identity, more preferably at least about 96% identity, more preferably at least about 97% identity, more preferably at least about 98% identity, and more preferably at least about 99% or greater identity. Such identity can be determined using algorithms known in the art, such as the mBLAST algorithm. In certain aspects the CDR regions can have 1, 2, 3, 4, or 5 amino acid substitution and retain binding activity. In particular the amino acid substitutions are conservative substitutions. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called “conservative” changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the protein's ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.

[0049]

[0045] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein and / or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in selected amino acids (or nucleotides) may be substituted. In certain aspects, a polypeptide is 80, 85, 90, 92, 94, 96, 98, or 100% identical to the wildtype form of the polypeptide. In certain aspects, polypeptide(s) 80, 85, 90, 92, 94, 96, 98, or 100% identical to, or 1, 2, 3, 4, or 5, amino acid substitutions in the CDR1, CDR2, CDR3, CDR1 and CDR2, CDR1 and CDR3, or CDR2 and CDR3 regions of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 are used or nucleic acids encoding the same.

[0050]

[0046] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and / or the like. An analysis of the size, shape and / or type of the amino acid side-chain substituents reveals that arginine, lysine, and / or histidine are all positively charged residues; that alanine, glycine, and / or serine are all a similar size; and / or that phenylalanine, tryptophan, and / or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine, and / or histidine; alanine, glycine, and / or serine; and / or phenylalanine, tryptophan, and / or tyrosine are defined herein as biologically functional equivalents.

[0051]

[0047] To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and / or charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (_0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and / or arginine (-4.5).

[0052]

[0048] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index and / or score and / or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0053]

[0049] It also is understood that the substitution of like amino acids can be made effectively based on hydrophilicity. As detailed in U.S. Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (±3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (_0.5); histidine (_0.5); cysteine (_1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). In making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred, those that are within ±1 are particularly preferred, and / or those within ±0.5 are even more particularly preferred.

[0054]

[0050] A biological sample from a patient means a sample from a subject suspected to be affected by a disease. As used herein, the term “subject” refers to any mammal, including both human and other mammals. Preferably, the methods of the present invention are applied to human subjects.

[0055]

[0051] The term “providing” is used according to its ordinary meaning “to supply or furnish for use.” In some embodiments, the protein is provided directly by administering the protein, while in other embodiments, the protein is effectively provided by administering a nucleic acid that encodes the protein. In certain aspects the invention contemplates compositions comprising various combinations of nucleic acid, antigens, peptides, and / or epitopes.

[0056]

[0052] The phrase “specifically binds” or “specifically immunoreactive” to a target refers to a binding reaction that is determinative of the presence of the molecule in the presence of a heterogeneous population of other biologies. Thus, under designated immunoassay conditions, a specified molecule binds preferentially to a particular target and does not bind in a significant amount to other biologies present in the sample. Specific binding of an antibody to a target under such conditions requires the antibody be selected for its specificity to the target. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0057] I. Nucleic Acid Compositions

[0058]

[0053] The term “nucleic acid vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated, transcribed, and / or translated (i.e., expressed). A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced. Nucleic acid vectors include plasmids, cosmids, viral genomes, and other expression vectors (bacteriophage, animal viruses, and plant viruses), artificial chromosomes (e.g., YACs), and the like. Given the current disclosure, one of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory, New York., 1989; Ausubel et al., Current Protocols in Molecular Biology, New York City, NY, John Wiley & Sons, Inc., 1994, both incorporated herein by reference).

[0059]

[0054] The term “Polynucleotide,” synonymously referred to as “nucleic acid molecule” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and doublestranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be singlestranded or, double-stranded, or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0055] The term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of inhibitory RNA, antisense molecules, or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.

[0060]

[0056] Certain aspects involve the use of nucleic acids encoding antibodies or antibody fragments as described herein. Examples of nucleic acids include antibody coding regions provided in SEQ ID NO: 1, 3, 4, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, and / or 39 or the equivalent as would be recognized by one skilled in the art. In certain aspects the nucleic acid comprise a nucleotide sequence that is 80, 85, 90, 95, 98, or 100% identical to SEQ ID NO: 1, 3, 4, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, or 39. In certain embodiments, nucleic acids of the invention encode proteins that are 80, 85, 90, 95, 98, or 100% identical to the proteins of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 and maintain the appropriate activity.

[0061]

[0057] The sequences may be modified, given the ability of several different codons to encode a single amino acid, while still encoding for the same protein or polypeptide. Optimization of codon selection can also be undertaken in light of the particular organism used for expression.

[0062] II. Pharmaceutical Compositions

[0063]

[0058] In light of the current specification, the determination of an appropriate treatment regimen (e.g., dosage, frequency of administration, systemic vs. local, etc.) is within the skill of the art. For administration, one or more antibody or antibody fragments described herein will be formulated in a unit dosage form (solution, suspension, emulsion, etc.) in association with a pharmaceutically acceptable carrier. Such vehicles are usually nontoxic and non-therapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used. A preferred vehicle is 5% (w / w) human albumin in saline. The vehicle may contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0064]

[0059] The terms “treating” or “treatment” refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the injury, pathology, or condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, neurological examination, and / or psychiatric evaluations.

[0065]

[0060] “Effective amount” and “therapeutically effective amount” are used interchangeably and refer to an amount of an antibody or antibody fragment, as described herein, effective to achieve a particular biological or therapeutic result such as, but not limited to, the biological or therapeutic results disclosed herein. A therapeutically effective amount of the antibody or antibody fragment thereof may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or functional fragment thereof to elicit a desired response in the individual. Such results may include, but are not limited to, the treatment of MARV or RAW infections, as determined by any means suitable in the art.

[0066]

[0061] The therapeutic compositions described herein, as well as their biological equivalents, can be administered independently or in combination by any suitable route. Examples of parenteral administration include intravenous, intraarterial, intramuscular, intraperitoneal, and the like. The routes of administration described herein are merely an example and in no way limiting.

[0067]

[0062] The dose of the therapeutic compositions administered to a subject or an animal, particularly in a human, in accordance with embodiments of the invention, should be sufficient to result in a desired response in the subject over a reasonable time frame. It is known that the dosage of therapeutic compositions depends upon a variety of factors, including the strength of the particular therapeutic composition employed, the age, species, condition or disease state, and the body weight of the animal.

[0068]

[0063] Therefore, the amount of the therapeutic composition must be effective to achieve an enhanced therapeutic index. If multiple doses are employed, the frequency of administration will depend, for example, on the type of subject. One skilled in the art can ascertain upon routine experimentation the appropriate route and frequency of administration in a given subject that are most effective in any particular case. Suitable doses and dosage regimens can be determined by conventionally known range-finding techniques. Generally, treatment is initiated with smaller dosages, which are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect under the circumstances is obtained.

[0069]

[0064] The therapeutic compositions for use in embodiments of the invention generally include carriers. These carriers may be any of those conventionally used and are limited only by the route of administration and chemical and physical considerations, such as solubility and reactivity with the therapeutic agent. In addition, the therapeutic composition may be formulated as polymeric compositions, inclusion complexes, such as cyclodextrin inclusion complexes, liposomes, microspheres, microcapsules, and the like, without limitation.

[0070]

[0065] The pharmaceutically acceptable excipients described herein, for example, vehicles, adjuvants, carriers, or diluents, are well known and readily available. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert with respect to the therapeutic composition and one that has no detrimental side effects or toxicity under the conditions of use.

[0071]

[0066] The choice of excipient will be determined, in part, by the particular therapeutic composition, as well as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of the pharmaceutical composition used in the embodiments of the invention. For example, the non-limiting formulations can be injectable formulations such as, but not limited to, those for intravenous, subcutaneous, intramuscular, intraperitoneal injection, and the like, and oral formulations such as, but not limited to, liquid solutions, including suspensions and emulsions, capsules, sachets, tablets, lozenges, and the like. Non-limiting formulations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, including non-active ingredients such as antioxidants, buffers, bacteriostats, solubilizers, thickening agents, stabilizers, preservatives, surfactants, and the like. The solutions can include oils, fatty acids, including detergents and the like, as well as other well known and common ingredients in such compositions, without limitation.

[0072]

[0067] The terms “single chain antibody” (SCA), “single-chain antigen-binding molecule or antibody” or “single-chain Fv” (sFv) are used interchangeably. The single chain antibody has binding affinity for the antigen. Single chain antibody (SCA) or single-chain Fvs can and have been constructed in several ways. A description of the theory and production of single-chain antigen-binding proteins is found in commonly assigned U.S. patent application Ser. No. 10 / 915,069 and U.S. Pat. No. 6,824,782, the contents of each of which are incorporated by reference herein.

[0073] III. Kits

[0074]

[0068] In another aspect, the present invention provides kits for treating and / or detecting MARV and / or RAW infection. In one embodiment, the kit comprises a solid support, such as a chip, a microtiter plate or a bead or resin having a capture reagent (MARV or RAW binding antibody) attached thereon, wherein the capture reagent binds a MARV or RAW component. In the case of biospecfic capture reagents, the kit can comprise a solid support with a reactive surface, and a container comprising the biospecific capture reagent.

[0075]

[0069] The kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the capture reagent and the washing solution allows capture of the MARV or RAW component or antigen on the solid support for subsequent detection. The kit may include more than one type of adsorbent, each present on a different solid support.

[0076] IV. Examples

[0077]

[0070] The following examples as well as the figures are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples or figures represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0078] EXAMPLE 1

[0079] PRODUCTION OF NOVEL NEUTRALIZING ANTIBODIES FOR MARBURG VIRUS DISEASE THERAPY

[0080]

[0071] In the study described below the inventors generated highly potent mAbs that neutralize MARV infection. Ten representative mAbs were produced and divided into three groups based on their putative epitopes and amino acid sequences of their variable regions of heavy and light chains. Five mAbs (i.e., R1 group: AGP-R1, R7, R12, R15, and R17) were cross- reactive to MARV and RAW that most likely recognize an epitope across the fusion loop and receptor binding domain of GP. On the other hand, AGP-R4, R5, R11, R20, and R22 neutralized MARV but not RAW and likely bind to an epitope on the head region of GP. The putative epitope of AGP-R4 and R5 (i.e., R4 group) was speculated to be overlapped with but slightly different from that of AGP-R11, R20, and R22 (i.e., R11 group). It was noted that all these mAbs showed neutralizing activity equivalent to or rather higher than a previously known neutralizing mAb MR78. The inventors describe in detail the biological property of the mAbs produced in this study.

[0081] I. RESULTS

[0082]

[0072] Generation of mAbs against MARV GP. To obtain mAbs against MARV GP, hybridomas were generated using spleen cells of mice infected with recombinant replication- competent vesicular stomatitis Indiana virus carrying the MARV GP gene (rVSV / MARV- Angola) instead of the G gene of the parent virus (Takada et al., 2003, J Virol 77, 1069-74; Saito et al., 2020, Viruses 12, 923), followed by the booster injection with purified soluble form of recombinant MARV GP (Furuyama et al., 2016, Sci Rep 6, 20514; Nakayama et al., 2010, Clin Vaccine Immunol 17, 1723-28). The hybridoma supernatants were screened for the neutralizing activity to replication-incompetent VSV pseudotyped with MARV GP (VSVAG*MARV- Angola) (Furuyama et al., 2016, Sci Rep 6, 20514; Maruyama et al., 2014, J Virol 88, 99-109; Takada et al., 1997, PNAS USA 94, 14764-69), and 10 mAb clones (mAbs AGP-R1, R4, R5, R7, R11, R12, R15, R17, R20, and R22) were identified that had distinct amino acid sequences in the variable regions of heavy and light chains (Tables 1 and 2). All of these mAbs had a kappa light chain and an IgG2a heavy chain, except AGP-R7 (IgG3).

[0083]

[0073] Leader and FR Light chain sequences include: AGP-R1 Leader MAWVWTLLFLMAAAQSAQA (SEQ ID NO:71), FR1

[0084] QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 72), FR2 MNWVKQAPGKGLKWMGW (SEQ ID NO:73), FR3 TYADDFKGRFAFSLETSASTAYLQINNLKSEDTATYFC (SEQ ID NO:74), FR4 WGQGTTLTVSS (SEQ ID NO: 75); AGP-R7 Leader MAWVWTLLFLMAAAQSAQA (SEQ ID NO:76), FR1

[0085] QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 77), FR2 MNWVKQAPGKDLQWMGW (SEQ ID NO: 78), FR3 TYADDFRGRFAFSLETSASTAYLQINNLKNEDTATYFC (SEQ ID NO:79), FR4 WGQGTTLTVSS (SEQ ID NO:80); AGP-R12 Leader MAWVWTLLFLMAAAQSAQA (SEQ ID NO:81), FR1

[0086] QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 82), FR2 MNWVKQAPGKGLKWMGW (SEQ ID NO:83), FR3 TYADDFKGRFAFSLETSASTAYLQINNLKSEDTATYFC (SEQ ID NO:84), FR4 WGQGTTLTVSS (SEQ ID NO: 85); AGP-R15 Leader MAWVWTLLFLMAAAQSAQA (SEQ ID NO:86), FR1

[0087] QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 87), FR2 MNWVKQAPGKALKWMGW (SEQ ID NO:88), FR3 TYADDFKGRFAFSLETSASTAYLQINNLKSEDTATYFC (SEQ ID NO: 89), FR4 WGQGTTLTVSS (SEQ ID NO: 90); AGP-R17 Leader MAWVWTLLFLMAAAQSAQA (SEQ ID NO:91), FR1

[0088] QIQLVQSGPELKKPGETVKISCKAS (SEQ ID NO: 92), FR2 MNWVKQAPGKGLKWMGW (SEQ ID NO:93), FR3 TYADDFKGRFAFSLETSASTAYLQINNLKSEDTATYFC (SEQ ID NO:94), FR4 WGQGTTLTVSS (SEQ ID NO:95); AGP-R4 Leader MGWSCIIFFLVATATGVHS (SEQ ID NO:96), FR1 QVQLQQSGPELVRPGESVKISCKGS (SEQ ID NO:97), FR2 MYWVKQSHAKSLEWIGL (SEQ ID NO:98), FR3 NYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYC (SEQ ID NO:99), FR4 WGQGTSVTVSS (SEQ ID NO: 100); AGP-R5 Leader MGWSCIIFFLVAIATGVHS (SEQ ID NO: 101), FR1 QVQLQQSGPELVRPGESVKISCKGS (SEQ ID NO: 102), FR2 MHWVKQSHAKSLEWIGL (SEQ ID NO: 103), FR3

[0089] NHNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYC (SEQ ID NO: 104), FR4 WGQGTSVTVSS (SEQ ID NO: 105); AGP-R11 Leader MGWSCIIFFLVATATGVHS (SEQ ID NO: 106), FR1 QVQLQQSGPELVRPGESVKISCKGS (SEQ ID NO: 107), FR2 IHWVKQSHAKSLEWIGL (SEQ ID NO: 108), FR3

[0090] NHNQKFRGKATMTVDKSSSTAYMELARLTSEDSAIYYC (SEQ ID NO: 109), FR4 WGQGTSVTVSS (SEQ ID NO: 110); AGP-R20 Leader MGWSCIIFFLVATATGVHS (SEQ ID NO: 111), FR1 QVQLQQSGPELVRPGESVKISCKGS (SEQ ID NO: 112), FR2 IHWVKQSHTKSLEWIGL (SEQ ID NO: 113), FR3

[0091] NHNQKFRGKATMTADKSSSTAYMELARLTSEDSAIYYC (SEQ ID NO: 114), FR4 WGQGTSVTVSS (SEQ ID NO: 115); AGP-R22 Leader MGWSCIIFFLVATATGVHS (SEQ ID NO: 116), FR1 QVQLQQSGPELVRPGESVKISCKGS (SEQ ID NO: 117), FR2 IHWVKQSHAKSLEWIGL (SEQ ID NO: 118), FR3

[0092] NHNQKFRGKATMTVDKSSNTAYMELARLTSEDSAIYYC (SEQ ID NO: 119), FR4 WGQGTSVTVSS (SEQ ID NO: 120).

[0093]

[0074] Leader and FR Heavy chain sequences include: AGP-R1 Leader MTMFSLALLLSLLLLCVSDSRA (SEQ ID NO: 121), FR1

[0094] ETTVTQSPASLSMAIGEKVTIRCITS (SEQ ID NO: 122), FR2 MNWYQQKPGEPPKLLIS (SEQ ID NO: 123), FR3 TLRPGVPSRFSSGGYGADFVFTIENMLLEDVADYYC (SEQ ID NO: 124), FR4 FGAGTKLELK (SEQ ID NO: 125); AGP-R7 Leader MTMFSLALLLSLLLLCVSDSRA (SEQ ID NO: 126), FR1

[0095] ETTVTQSPASLSMAIGEKVTIRCITS (SEQ ID NO: 127), FR2 MNWYQMKPGEPPQLLIS (SEQ ID NO: 128), FR3 TLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYC (SEQ ID NO: 129), FR4 FGAGTKLELK (SEQ ID NO: 130); AGP-R12 Leader MTMFSLALLLSLLLLCVSDSGA (SEQ ID NO: 131), FR1

[0096] ETTVTQSPASLSMAIGEKVTIRCITS (SEQ ID NO: 132), FR2 MNWYQQKPGEPPKLLIS (SEQ ID NO: 133), FR3 TLRPGVPSRFSSSGYGADFVFTIENMLSEDVADYYC (SEQ ID NO: 134), FR4 FGAGTKLELK (SEQ ID NO: 135); AGP-R15 Leader MTMFSLALLLSLLLLCVSDSRA (SEQ ID NO: 136), FR1

[0097] ETTVTQSPASLSMAIGEKVTIRCITS (SEQ ID NO: 137), FR2 MNWYQQKPGEPPKLLIS (SEQ ID NO: 138), FR3 TLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYC (SEQ ID NO: 139), FR4 FGAGTKLDLK (SEQ ID NO: 140); AGP-R17 Leader MTMFSLALLLSLLLLCVSDSRA (SEQ ID NO: 141), FR1

[0098] ETTVTQSPASLSMAIGEKVTIRCKTS (SEQ ID NO: 142), FR2 MNWYQQKPGEPPKLLIS (SEQ ID NO: 143), FR3 TLRPGVPSRFSSSGYGTDFVFTIENMLSEDVADYYC (SEQ ID NO: 144), FR4 FGAGTKLELK (SEQ ID NO: 145); AGP-R4 Leader MKLPVRLLVLMFWIPASSS (SEQ ID NO: 146), FR1

[0099] DVVMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO: 147), FR2 LHWYLQKPGQSPKLLIY (SEQ ID NO: 148), FR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 149), FR4 FGGGTKLEIK (SEQ ID NO: 150); AGP-R5 Leader MKLPVRLLVLMFWIPASSS (SEQ ID NO: 151), FR1

[0100] DVVMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO: 152), FR2 LHWYLQKPGQSPKLLIY (SEQ ID NO: 153), FR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 154), FR4 FGGGTKLEIK (SEQ ID NO: 155); AGP-R11 Leader MKLPVRLLVLMFWIPASSS (SEQ ID NO: 156), FR1

[0101] DVVMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO: 157), FR2 LHWFLQKPGQSPKLLIY (SEQ ID NO: 158), FR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO:159), FR4 FGGGTKLEIK (SEQ ID NO: 160); AGP-R20 Leader MKLPVRLLVLMFWIPASSS (SEQ ID NO: 161), FR1

[0102] DVVMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO: 162), FR2 LHWFLQKPGQSPKLLIY (SEQ ID NO: 163), FR3 NRFSGVPDRFSGSGSGTNFTLKISRVEAEDLGVYFC (SEQ ID NO: 164), FR4 FGGGTKLEIK (SEQ ID NO: 165); AGP-R22 Leader MKLPVRLLVLMFWIPASSS (SEQ ID NO: 166), FR1

[0103] DVVMTQTPLSLPVSLGDQASISCRSS (SEQ ID NO: 167), FR2 LHWFLQKPGQSPKLLIY (SEQ ID NO: 168), FR3 NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO: 169), FR4 FGGGTKLEIK (SEQ ID NO: 170).

[0104]

[0075] In vitro properties of the AGP-R mAbs. These 10 mAbs were categorized into three groups, R1 (AGP-R1, 7, 12, 15, and 17), R4 (AGP-R4 and 5), and Rl l groups (AGP-R11, 20, and 22), based on the amino acid sequences of their complementarity-determining regions (CDRs) and neutralization profiles against pseudotyped viruses (VSVAG*MARV-Angola, VSVAG*MARV-Musoke, and VSVAG*RAVV) (FIG. 1, Tables 3 and 4). The R1 group mAbs efficiently neutralized VSVAG*MARV-Angola, VSVAG*MARV-Musoke, and VSVAG*RAVV, and AGP-R7 showed the highest neutralizing activity in the R1 group with the 50% inhibitory concentrations (IC50) value of 0.59, 0.60, and 0.75 pg / ml against VS VAG*MARV- Angola, VSVAG*MARV-Musoke, and VSVAG*RAVV, respectively. The R4 and Rl l group mAbs showed high neutralizing activity against VSVAG*MARV-Angola and VSVAG*MARV-Musoke, but none of them neutralized VSVAG*RAVV (FIG. 1 and Table 4). Then, neutralizing activities of the mAbs against authentic MARV and RAW were assessed in a plaque reduction neutralization test (FIG. 2). The R1 group mAbs, except for AGP-R17, exhibited over 80% neutralizing activity against MARV (variant Angola) and RAW at concentration of 100 pg / ml, representing the highest neutralizing activity against infectious MARVs among mAbs tested to date (Flyak et al., 2015, Cell 160, 893-903; Bezhanova et al., 2020, PNAS USA 117, 31142-48; Kajihara et al., 2012, Journal of Virology 86, 13467-74; Zhang et al., 2023, eLife 12; Ilinykh et al., 2020, Cell Host & Microbe 27, 976-91; Marceau et al., 2014. Trials in Vaccinology 3, 89-94). Particularly, AGP-R7 achieved more than 90% and 80% neutralization against both MARV and RAW at 100 pg / ml and 25 pg / ml, respectively. Interestingly, MR78 and the R4 and R11 group mAbs, all of which showed high neutralizing activity against pseudotyped viruses having MARV GPs, only partially neutralized authentic MARVs. Consistent with the neutralizing activity, mAbs of the R1 group bound to both MARV and RAW GPs with a slight preference to MARV GPs, whereas the R4 and R11 group mAbs only bound to MARV GP (FIG. 3). MR78 showed affinity to both GPs despite the inability to neutralize RAW.

[0105] Table 3

[0106] Table 4

[0107]

[0076] Identification of putative epitopes of the mAbs. To obtain information on the putative epitopes of the mAbs, GP escape mutants were selected using rVSV / MARV-Angola. rVSV / MARV-Angola was propagated in the presence of each mAh and escape mutants were plaque-cloned. Then full-length GP genes were sequenced and compared with the parent GP to identify amino acid substitutions (FIG. 4). The escape mutants selected by the R1 group mAbs had amino acid substitutions at K58, K90, K120, D459, N508, E509, N510, A514, and N564 of GP. These amino acid residues are in the receptor binding domain (RBD) or the internal fusion loop. On the surface model of the GP molecule, these amino acid positions are in close proximity at the lateral portion of the MARV GP trimer. In contrast, the escape mutants selected by the R4 group mAbs had mutations at amino acids in the mucin-like domain (MLD); F447, L448, L451, L452, P455, 1456, F458, and D459. Since crystal structures of amino acid positions 1-32, 181- 468, 499-504, 630-681 are not available, these mutations could not be mapped on the 3D structure. The amino acid mutations found in the escape mutants of the R4 group mAbs were in RBD (Q126, H131, Y197, and T199) and MLD and partly overlapped with those of the R1 and R4 group mutants. While the mutations in MLR were unable to be mapped on the structure, some unique positions in RBD (i.e., Q126 and H131) were likely near the glycan cap at the head region of the GP. Comparison of the putative epitope regions among several MARV variants showed that the amino acid residues involved in the epitope of the R1 group mAbs are highly conserved among MARV variants and RAW (FIG. 5). The amino acid residues involved in the putative epitopes of the R4 and Rl l group mAbs were also generally conserved, which was inconsistent with the results of the neutralization and binding tests. However, these amino acid positions are mainly present in MLD, which is variable among MARVs and RAW, suggesting that some amino acid residues at other positions are also important for the epitope formation for these MARV-specific mAbs.

[0108]

[0077] Neutralization mechanism of AGP-R mAbs. Exact mechanisms of neutralization by these mAb specific antibodies have not been clarified. It is speculated that these mAbs inhibit receptor binding, membrane fusion, conformational change, and / or proteolytic processing of the MARV and RAW glycoproteins.

[0109]

[0078] Efficacy of AGP-R mAbs in mouse models for prevention and treatment. To evaluate the treatment efficacy of mAbs against MARV infection, the mice was inoculated with 100 plaque forming units (PFU), approximately 100 50% lethal dose (LD50), of mouse-adapted MARV Angola and intraperitoneally treated with 100 pg of mAbs (AGP-R1, R7, R17, R4, R5, Rl l, or R20) at 1 day post-inoculation (d.p.i.). Whereas all mice treated with control IgG succumbed to the disease at 7 d.p.i., all mice treated with each mAb, excepting one mouse treated with AGP-R5, survived from lethal MARV infection with showing only minor weight loss (FIG. 6). These results indicate that our mAbs are promising candidates of anti-Marburg virus disease treatment.

[0110] II. Materials and Methods

[0111]

[0079] Cells and Viruses. African green monkey kidney Vero E6 and human embryonic kidney HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Coming) supplemented with 10% fetal bovine serum (FBS) (Gibco), 100 U / mL penicillin, and 0.1 mg / mL streptomycin (Gibco). Mouse myeloma P3U1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS, 4 mM Glutamine (Gibco), 100 U / mL penicillin, and 0.1 mg / mL streptomycin. Expi293F cells were maintained in Expi293F expression medium (Thermo Fisher Scientific), according to the manufacture’s instruction. These cells were cultured at 37°C with 5% CO2.

[0112]

[0080] Pseudotyped vesicular stomatitis Indiana virus containing the green fluorescent protein (GFP) gene instead of the G gene bearing GPs of MARV variants Angola and Musoke, RAW, or EBOV variant Mayinga (VSVAG*MARV-Angola, VSVAG*MARV-Musoke, VSVAG*RAVV, and VSVAG*EBOV, respectively) were generated as described previously (Furuyama et al., 2016, Sci Rep 6, 20514; Maruyama et al., 2014, J Virol 88, 99-109; Takada et al., 1997, PNAS USA 94, 14764-69). Briefly, HEK293T cells were transfected with the expression plasmid pCAGGS containing GP genes, and 24 hours later, the cells were infected with VSVAG*-G at a multiplicity of infection of 1.0. After a 16 hours incubation, the supernatants were collected and centrifuged to remove cell debris. Infectious units (IU) of the pseudotyped viruses were determined as described previously (Furuyama et al., 2016, Sci Rep 6, 20514; Maruyama et al., 2014, J Virol 88, 99-109; Takada et al., 1997, PNAS USA 94, 14764- 69). Briefly, 10-fold serially diluted pseudotyped viruses were inoculated into confluent Vero E6 cell monolayers seeded on 96-well plates, and the IU was determined 24 hours later by counting GFP-expressing cells using an In Cell analyzer 2500 system (GE Healthcare). To reduce the background infectivity of the residual parent VSVAG*-G, each pseudotyped virus stock was treated with a neutralizing monoclonal antibody specific for the G protein (VSV-G[N]l-9) (Nakayama et al., 201 1, J Infect Dis 204, S978-S985) before use.

[0113]

[0081] Replication-competent recombinant vesicular stomatitis Indiana virus carrying the MARV (Angola) GP gene (rVSV / MARV-Angola) were generated as described previously (Takada et al., 2003, J Virol 77, 1069-74; Saito et al., 2020, Viruses 12, 923). rVSV / MARV- Angola was propagated in Vero E6 cells and stored at -80°C until use. Virus titers were determined by a plaque assay. Briefly, 10-fold serially diluted viruses were inoculated into confluent Vero E6 cells in 12-well plates and inoculated for 1 hour at 37°C with 5% CO2. After the inoculum was removed, the cells were washed with DMEM and overlaid with Eagle’s minimum essential medium (EMEM) containing 0.8% Bacto Agar (Becton Dickinson), 0.3% bovine serum albumin, lOO U / ml penicillin, and O.l mg / ml streptomycin. After 48 hours of incubation, cells were fixed with 10% formalin and stained with crystal violet. Viral titers were represented as plaque forming units (PFU).

[0082] Infectious MARV variant Angola (Angola 200501379) (Towner et al., 2006, J Virol 80, 6497-6516) and RAW (Kenya 1987) (Johnson et al., 1996, Arch Virol Suppl 11, 101-114) were originally obtained from the Special Pathogens Branch, U.S. Centers for Disease Control (CDC), and deposited at the World Reference Center for Emerging Viruses and Arboviruses (WRECEVA) housed at the University of Texas Medical Branch (UTMB). MARVs and RAW were propagated in Vero E6 cells and stored at -80°C until use. All work with infectious MARV and RAW was performed in biosafety level 4 (BSL-4) facilities in the Galveston National Laboratory (GNL) at UTMB in accordance with institutional guidelines. Virus titers were determined by a plaque assay as described previously with slight modification and represented as PFU (Flyak et al., 2015, Cell 160, 893-903). Briefly, confluent Vero E6 cells in 12-well plates were infected with 10-fold serial dilutions of virus stocks and incubated for 30 minutes at 37°C with 5% CO2. After the inoculum was removed, the cells were washed, and overlaid with EMEM containing 2% FBS, 1% penicillin-streptomycin, and 0.6% tragacanth (Sigma). After 9 days of incubation, cells were fixed with 10% formalin and stained with 0.25% crystal violet.

[0114]

[0083] Expression and purification of His-MARV, His-RAVV, and His-EBOV GPs. Expi293F cells were transfected with expression plasmids encoding histidine-tagged soluble forms of GP, His-MARV (Angola), RAW, and EBOV GPs (Nakayama et al., 2010, Clin Vaccine Immunol 17, 1723-28) using Expi293 Expression System (Thermo Fisher Scientific) according to the manufacture’s instruction After 96 hours of transfection, the culture supernatant was harvested and centrifuged at 3,000 rpm for 15 minutes at 4°C. The recombinant His-MARV, RAW, and EBOV GPs were purified from the supernatant by using the Ni-nitriiotriacetic acid (NT A) purification system (Invitrogen) according to the manufacturer's instructions and used as antigen. Purified His-MARV, RAW, and EBOV GPs were used for booster immunization of mice and for antigen coating in ELISA.

[0115]

[0084] Generation of anti-MARV neutralizing antibodies. Animal studies for generation of hybridomas were carried out in strict accordance with the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan. The protocol was approved (18-0026) by the Hokkaido University Animal Care and Use Committee. Six-week-old female BALB / c mice were purchased from Sankyo Lab Service. All animals were housed in animal biosafety level-2 (BSL-2) facilities in the International Institute for Zoonosis Control, Hokkaido University. The mice were infected intraperitoneally with 106PFU of rVSV / MARV-Angola. At 4 weeks after immunization, the animals were boosted with 100 pg of purified His-MARV GP intravenously. Three days later, the mice were euthanized, and the spleen cells and P3U1 cells were fused and maintained according to a standard procedure (Furuyama et al., 2016, Sci Rep 6, 20514; Shahhosseini et al., 2007, J Virol Methods 143, 29-37). Hybridomas were screened for secretion of neutralizing mAbs using VSVAG*MARV-Angola and hybridomas producing mAbs were cloned twice by limiting dilution of the cells. Neutralizing mAbs were then purified from mouse ascites. The isotype and subclass of mAbs were determined using the Mouse Isotyping Kit (Biorad). Mouse monoclonal antibody MR78 (Flyak et al., 2015, Cell 160, 893-903) was obtained from Absolute Antibody (Drydock Avenue, BOS, USA) and used to compare neutralizing activity.

[0116]

[0085] 5’RACE to determine variable region of mAbs. Total RNA was extracted from the hybridoma producing mAbs using TRIzol™ Reagent (Thermo Fisher Scientific) and reverse- transcribed with a SMARTer RACE 573’ kit (Clontech) using reverse transcription primers (Table 6) (Meyer et al., 2019, PLoS One 14, e0218717). Subsequently, the variable gene segments for the heavy chain (VH) and light chain (VL) were amplified by PCR with the primer sets (Meyer et al., 2019, PLoS One 14, e0218717) specific to VH and VL (Table 7) and Universal Primer A Mix (Clontech) using KOD One (TOYOBO) according to the manufacturer’s instructions. The PCR products for the VH and VL genes were cloned into linearized pRACE vectors (Clontech) using an In-Fusion HD Cloning Kit (Clontech), and subjected to nucleotide sequencing. The nucleotide sequences were determined by Sanger sequencing at GENEWIZ (South Plainfield, NJ, USA).

[0117]

[0086] Neutralization tests. VSVAG*MARV-Angola, VSVAG* ARV-Musoke, VSVAG*RAVV, and VSVAG*EBOV were appropriately diluted to yield 500 to 3,000 lU / well and mixed with serially diluted mAbs for 1 hour at room temperature and inoculated into confluent Vero E6 cells grown in 96-well plates. Twenty hours post inoculation, GFP -positive cells were counted. The relative percentage of infectivity was calculated by setting the number of cells infected in the absence of mAbs to 100%. Neutralizing activities against MARV (Angola), and RAW were determined by a Plaque reduction neutralizing test. MARV and RAW were diluted with 2% FBS DMEM to yield 100 to 150 PFU / well and mixed with serially diluted mAbs. After 30 minute-incubation in 37°C, the mixture was inoculated into monolayer of Vero E6 cells in 6-well plates, and incubated in a CO2 incubator for 30 minutes at 37°C. After washing inoculum out, EMEM with 0.6% Tragacanth and 5% FBS was added as overlay. After incubation for 7 days, cells were fixed with 10% formalin and plaques were visualized by crystal violet staining. The relative percentage of infectivity was calculated by setting the number of plaques in the absence of mAbs to 100%.

[0118]

[0087] Enzyme-linked immunosorbent assay (ELISA). Filovirus GP -based ELISA was performed as described previously (Furuyama et al., 2016, Sci Rep 6, 20514; Nakayama et al., 2010, Clin Vaccine Immunol 17, 1723-28). Briefly, ELISA plates (Thermo Fisher Scientific) were coated with purified His-MARV, His-RAVV, and His-EBOV GPs (50 ng / 50 pl / well) in PBS at 4°C overnight and then washed with PBS containing 0.05% Tween 20 (PBST). The plates were blocked with 3% skim milk (180 pl / well) for 1 hour at room temperature and serially diluted purified mAbs with PBST containing 1% skim milk were added and incubated at 4°C overnight. After washing three times with PBST, bound antibodies were visualized with horseradish peroxi dase-conjugated goat anti-mouse IgG (H + L) (invitrogen) and 3,3’, 5,5’- tetramethylbenzidine (Sigma). The reaction was stopped by adding 1 N phosphoric acid and the optical density at 450 nm (OD450) was measured using Biotek ELx808 Microplate Reader (Agilent).

[0119]

[0088] Selection of rVSV / MARV-Angola escape mutants and identification of putative epitopes. Selection of escape mutants and identification of the putative epitopes on the MARV GP molecule were performed as described previously (Furuyama et al., 2016, Sci Rep 6, 20514). Ten-fold serial dilutions of rVSV / MARV-Angola were incubated with 10 pg / ml of each mAb for 1 hour at room temperature and inoculated into confluent Vero E6 cells grown in 6-well plates. After adsorption for 1 hour, the inoculum was removed, and the cells were overlaid with EMEM containing 0.8% Bacto Agar, 0.3% bovine serum albumin, lOO U / ml penicillin, O. l mg / ml streptomycin, and 10 pg / ml of each mAb and then incubated for 2 days at 37°C. Mutant viruses growing in the presence of mAbs were purified from single isolated plaques at the highest dilution of the virus and propagated in Vero E6 cells. Viral RNAs were extracted from the supernatant and purified using Viral RNA Mini Kit (QIAGEN). The nucleotide sequences of the GP genes of the parent virus and the escape mutants were determined by using a BigDye Terminator sequencing kit, version 3.1 (Applied Biosystems), and an Applied Biosystems 3130x1 genetic analyzer (Applied Biosystems). The deduced amino acid sequences were compared among the viruses. The amino acid sequences of MAR V (Angola), were obtained from GenBank (Accession numbers, KY047763.1). The substituted amino acid positions were mapped on the trimeric structure of GPs constructed using Discovery Studio 4.1 (Biovia) based on the crystal structure of RAW GP (PDB code: 6BP2).

[0120]

[0089] Investigation of therapeutic effect of the mAh in mouse models. The animal protocol to investigate therapeutic effect of the mAb in mice was reviewed and approved by the Institutional Animal Care and Use Committee of UTMB (IACUC2306037). The animal studies were conducted according to the National Institutes of Health guidelines. Five- to 7-week-old BALB / c mice were purchased from Charles River. All animals were housed in animal biosafety level 2 (ABSL-2) and ABSL-4 facilities in the GNL at UTMB.

[0121]

[0090] Statistical Analysis. Statistical analyses were performed with GraphPad Prism Software (ver. 9.1.2). Statistical differences were calculated by 2-way ANOVA followed by Dunnett’s post.

Claims

CLAIMS1. An antibody or antibody fragment that binds Marburg virus (MARV) and / or Ravn virus (RAW) comprising (i) heavy chain CDR1 of SEQ ID NO: 41, CDR2 of SEQ ID NO: 42, and CDR3 of SEQ ID NO:43 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; (ii) heavy chain CDR1 of SEQ ID NO:44, CDR2 of SEQ ID NO:45, and CDR3 of SEQ ID NO:46 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:57; (iii) heavy chain CDR1 of SEQ ID NO:41, CDR2 of SEQ ID NO:42, and CDR3 of SEQ ID NO:47 and light chain CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:58; (iv) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:49, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:61; (v) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO: 51, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:62; (vi) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:50 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:60, and CDR3 of SEQ ID NO:62; or (vii) heavy chain CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53 and light chain CDR1 of SEQ ID NO: 59, CDR2 of SEQ ID NO:63, and CDR3 of SEQ ID NO:62.

2. The antibody or antibody fragment of claim 1, comprising a variable heavy chain / light chain pair having an amino acid sequence of (i) SEQ ID NO:2 and SEQ ID NO:4, (ii) SEQ ID NO: 8 and SEQ ID NO: 10, (iii) SEQ ID NO: 18 and SEQ ID NO: 20, (iv) SEQ ID NO: 22 and SEQ ID NO:24, (v) SEQ ID NO:26 and SEQ ID NO:28, (vi) SEQ ID NO:30 and SEQ ID NO:32, or (vii) SEQ ID NO:34 and SEQ ID NO:36.

3. A polynucleotide encoding the antibody or antibody fragment of claim 1 or claim 2.

4. A method of detecting Marburg virus (MARV) and / or Ravn virus (RAW) infection in a subject comprising:(a) contacting a sample from the subject with an antibody or antibody fragment of claim 1; and(b) detecting the binding of the antibody or antibody fragment to a component of the sample.

5. A method of inhibiting Marburg virus (MARV) or / and Ravn virus (RAW) in a subject infected with MARV or / and RAW, or inhibiting infection of a subject at risk of contractingMARV or / and RAW, comprising administering to said subject an effective amount of an antibody or antibody fragment of claim 1 or claim 2.

6. A method of preventing and / or treating a subject having or suspected of having a Marburg virus (MARV) and / or Ravn virus (RAW) infection comprising administering a therapeutic amount of an antibody or antibody fragment of claim 1 or claim 2.

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