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ARENAVIRUS MONOCLONAL ANTIBODIES AND USES

20240352095 ยท 2024-10-24

    Inventors

    Cpc classification

    International classification

    Abstract

    Disclosed herein are compositions comprising recombinant arenavirus monoclonal antibodies and antigen-binding fragments thereof, as well as therapeutic methods using the antibodies. In some embodiments, the antibodies provide pan-arenavirus protection against a number of arenavirus types and strains.

    Claims

    1-27. (canceled)

    28. An antigen-binding composition comprising a combination of three recombinant monoclonal neutralizing antibodies or neutralizing antigen-binding antibody fragments thereof, which are specific to Lassa virus glycoprotein, wherein the composition comprises: a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H CDR1 of SEQ ID NO: 83, a V.sub.H CDR2 of SEQ ID NO: 84, a V.sub.H CDR3 of SEQ ID NO: 85, a V.sub.L CDR1 of SEQ ID NO: 125, a V.sub.L CDR2 of sequence Gly Ala Ser, and a V.sub.L CDR3 of SEQ ID NO: 126; a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H CDR1 of SEQ ID NO: 92, a V.sub.H CDR2 of SEQ ID NO: 93, a V.sub.H CDR3 of SEQ ID NO: 94, a V.sub.L CDR1 of SEQ ID NO: 131, a V.sub.L CDR2 of sequence Glu Val Ser, and a V.sub.L CDR3 of SEQ ID NO: 132; and a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H CDR1 of SEQ ID NO: 98, a V.sub.H CDR2 of SEQ ID NO: 99, a V.sub.H CDR3 of SEQ ID NO: 100, a V.sub.L CDR1 of SEQ ID NO: 135, a V.sub.L CDR2 of sequence Gly Ala Ser, and a V.sub.L CDR3 of SEQ ID NO: 136.

    29. The composition of claim 28, wherein each antigen-binding antibody fragment is selected from the group consisting of a Fab, a Fab, and a F(ab).sub.2 fragment.

    30. A pharmaceutical composition for treating a Lassa virus or a lymphocytic choriomeningitis virus infection comprising the composition of claim 28 and a pharmaceutically acceptable carrier.

    31. The composition of claim 28 wherein the composition comprises: a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H of SEQ ID NO: 39 and a V.sub.L of SEQ ID NO: 55; a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H of SEQ ID NO: 42 and a V.sub.L of SEQ ID NO: 58; and a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H of SEQ ID NO: 44 and a V.sub.L of SEQ ID NO: 60.

    32. The composition of claim 31, wherein each antigen-binding antibody fragment is selected from the group consisting of a Fab, a Fab, and a F(ab).sub.2 fragment.

    33. A pharmaceutical composition for treating infection by a Lassa virus or a lymphocytic choriomeningitis virus comprising the composition of claim 31 and a pharmaceutically acceptable carrier.

    34. An antigen-binding composition comprising a recombinant human monoclonal neutralizing antibody or a neutralizing antigen-binding antibody fragment thereof, which is specific for Lassa virus glycoprotein; the antibody or antibody fragment thereof comprising a V.sub.H CDR1 of SEQ ID NO: 83, a V.sub.H CDR2 of SEQ ID NO: 84, a V.sub.H CDR3 of SEQ ID NO: 85, a V.sub.L CDR1 of SEQ ID NO: 125, a V.sub.L CDR2 of sequence Gly Ala Ser, and a V.sub.L CDR3 of SEQ ID NO: 126.

    35. The composition of claim 34, wherein the antigen-binding antibody fragment is selected from the group consisting of a Fab, a Fab, and a F(ab).sub.2 fragment.

    36. The composition of claim 34 wherein the composition comprises a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H of SEQ ID NO: 39 and a V.sub.L of SEQ ID NO: 55.

    37. A pharmaceutical composition for treating infection by a Lassa virus or a lymphocytic choriomeningitis virus comprising the composition of claim 34 and a pharmaceutically acceptable carrier.

    38. An antigen-binding composition comprising a recombinant human monoclonal neutralizing antibody or a neutralizing antigen-binding antibody fragment thereof, which is specific for Lassa virus glycoprotein; the antibody or antibody fragment thereof comprising a V.sub.H CDR1 of SEQ ID NO: 92, a V.sub.H CDR2 of SEQ ID NO: 93, a V.sub.H CDR3 of SEQ ID NO: 94, a V.sub.L CDR1 of SEQ ID NO: 131, a V.sub.L CDR2 of sequence Glu Val Ser, and a V.sub.L CDR3 of SEQ ID NO: 132.

    39. The composition of claim 38, wherein the antigen-binding antibody fragment is selected from the group consisting of a Fab, a Fab, and a F(ab).sub.2 fragment.

    40. The composition of claim 38, wherein the composition comprises a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising a V.sub.H of SEQ ID NO: 42 and a V.sub.L of SEQ ID NO: 58.

    41. A pharmaceutical composition for treating infection by a Lassa virus or a lymphocytic choriomeningitis virus comprising the composition of claim 38 and a pharmaceutically acceptable carrier.

    42. A method of treating or preventing a Lassa virus infection or a lymphocytic choriomeningitis virus infection in a subject comprising administering the composition of claim 28 to the subject.

    43. A method of treating or preventing a Lassa virus infection or a lymphocytic choriomeningitis virus infection in a subject comprising administering the composition of claim 31 to the subject.

    44. A method of treating or preventing a Lassa virus infection or a lymphocytic choriomeningitis virus infection in a subject comprising administering the composition of claim 34 to the subject.

    45. A method of treating or preventing a Lassa virus infection or a lymphocytic choriomeningitis virus infection in a subject comprising administering the composition of claim 36 to the subject.

    46. A method of treating or preventing a Lassa virus infection in a subject comprising administering the composition of claim 38 to the subject.

    47. A method of treating or preventing a Lassa virus infection in a subject comprising administering the composition of claim 40 to the subject.

    Description

    DESCRIPTION OF THE FIGURES

    [0098] FIG. 1 depicts (A) Schematic representation of LASV GP; (B) Arenavirus GP complex; and (C) Recognition of different LASV GP species by LASV hMAbs. (A) LASV GP is synthesized as the precursor protein GPC. Signal peptidase (Spase) cleaves the small stable signal peptide (SSP) that remains associated with GP1 and GP2 to form the GP complex (FIG. 1, Panel B). The cellular protease SK1/S1P cleaves GPC into GP1 and GP2. Construct rGPe corresponds to a recombinant LASV GPC ectodomain lacking GP2 and with a non-cleavable linker replacing the SK1/S1P cleavage recognition site. Constructs expressing recombinant SSP-GP1 (rGP1) and SSP fused to GP2 (rGP2) were also generated. (B) GP-1 forms the globular head subunit that interact with the cellular receptor whereas GP2 mediates the fusion of the viral envelop with the cell membrane. SSP remains associated with both GP1 and GP2 and plays critical roles in the biology of the GP complex. (C) 293T cells were transfected with pCAGGS expressing plasmids encoding LASV rGP1, rGP2 and GPC and the reactivity of LASV hMAbs evaluated at 48 h post-transfection by immunofluorescence. The distribution of LASV GP-specific hMAbs by subunit specificity, neutralizing activity and reactivity to linear epitopes is indicated.

    [0099] FIG. 2 depicts in vitro neutralization of LCMV ARM with the 15 LASV GP-specific neutralizing hMAbs: LASV Josiah (squares) and LCMV ARM (triangles) GP-pseudotyped rLCMVGP/GFP viruses were incubated for 90 min at 37 C. with a 2-fold dilution of the indicated LASV GP-specific BNhMAb before infecting LCMV GP-expressing Vero cells (96 plate format, triplicates). Virus neutralization was determined under a fluorescent microscope and quantified using a GFP microplate reader at 72 hours post-infection. Results are presented as percent inhibition after normalizing to respective viral infections in the absence of hMAbs. Virus infection in the absence of hMAbs was used as internal control. Mean values and standard deviation are shown. Standard error was calculated based on 2-6 replicates.

    [0100] FIG. 3 depicts in vivo neutralization of the 6 LCMV neutralizing antibodies (12.1F, 9.8A, 37.2D, 36.9F, 37.2G, and 18.5C) using the non-crossreactive antibodies 19.7E and 8.9F as internal controls. Mice were infected with rCl-13 (2106 pfu; i.v.) and treated with the indicated hMAb (20 mg/kg; i.p.), as well as an isotype hMAb control (20 mg/kg, i.p.) or vehicle. At days 4 and 21, post inoculation (p.i.) viremia (i.e., the presence of viruses in the blood) was determined. Results correspond to the average and standard deviation (SD) of four mice/group; LoD=limit of detection.

    [0101] FIG. 4 illustrates Viremia data from treated and control GP plasma on days 7 and 14 PI. Viremia levels for day 7 treatment groups 37.7H, 12.1F, and 25.6A as well as day 14 12.1F, 37.2D, 19.7E, and 10.4B were below the limit of detection (LOD). Error bars represent standard deviation from mean values. *denotes P_0.05. **denotes P_0.001. ***denotes P<0.0001.

    [0102] FIG. 5 depicts clinical scores of HuMAb treated and untreated guinea pigs. HuMAbs 8.9F and 12.1 treated GP showed no variation in clinical score from baseline (data not shown). Error bars (thin lines) represent standard deviation from mean values.

    [0103] FIG. 6 illustrates the effect of antibodies on rVSV-LASV GP infection and fusion. Antibody-mediated neutralization of (A) rVSV-LASV GP or (B) rVSV-VSV-G. The antibody 9.7A is non-neutralizing and in the same competition group as 37.7H (GPC-B); 13.4E binds to a linear epitope in the T-loop of GP2; 12.1F binds to the GP1 subunit of LASV. Error bars indicate the standard deviation of at least six (two biological replicates, each having three or more technical replicates). (C) Antibody-mediated inhibition of rVSVLASV GP fusion at the cell surface. Error bars indicate the standard error of the mean of six (except 37.7H, where N=9). (D) Fab 37.7H reduces binding of a LAMP1-Fc fusion protein to LASV GPCysR4. Error bars indicate the standard deviation of six and three technical replicates.

    [0104] FIG. 7 provides a sequence alignment prepared using CLUSTAL OMEGA (1.2.4) multiple sequence alignment (from EMBL-EBI, a part of the European Molecular Biology Laboratory) for the heavy chain variable region amino acid sequences, with CDRs highlighted in bold typeface: CDR1 (marked with +), CDR 2 (marked with {circumflex over ()}), and CDR3 (marked with #). The sequences shown include 10.4B (SEQ ID NO: 33), 19.7E (SEQ ID NO: 34), 2.9D (SEQ ID NO: 35), 25.6A (SEQ ID NO: 36), 36.1F (SEQ ID NO: 37), 36.9F (SEQ ID NO: 38), 37.2D (SEQ ID NO: 39), 37.2G (SEQ ID NO: 40), 37.7H (SEQ ID NO: 41), 8.9F (SEQ ID NO: 42), NE13 (SEQ ID NO: 43), 12.1F (SEQ ID NO: 44), 9.8A (SEQ ID NO: 45), 18.5C (SEQ ID NO: 46), 8.11G (SEQ ID NO: 47), and 25.10C (SEQ ID NO: 48).

    [0105] FIG. 8 provides a sequence alignment prepared using using CLUSTAL OMEGA (1.2.4) multiple sequence alignment for the light chain variable region amino acid sequences, with CDRs highlighted in bold typeface: CDR1 (marked with +), CDR 2 (marked with {circumflex over ()}), and CDR3 (marked with #). The sequences shown include 10.4B (SEQ ID NO: 49), 19.7E (SEQ ID NO: 50), 2.9D (SEQ ID NO: 51), 25.6A (SEQ ID NO: 52), 36.1F (SEQ ID NO: 53), 36.9F (SEQ ID NO: 54), 37.2D (SEQ ID NO: 55), 37.2G (SEQ ID NO: 56), 37.7H (SEQ ID NO: 57), 8.9F (SEQ ID NO: 58), NE13 (SEQ ID NO: 59), 12.1F (SEQ ID NO: 60), 9.8A (SEQ ID NO: 61), 18.5C (SEQ ID NO: 62), 8.11G (SEQ ID NO: 63), and 25.10C (SEQ ID NO: 64).

    DETAILED DESCRIPTION

    General Techniques

    [0106] The practice of the materials and methods described herein will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are all within the normal skill of the art. Such techniques are fully explained in the literature, such as, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (I. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Cabs, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et aL, eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds., Harwood Academic Publishers, 1995).

    [0107] As used herein, the singular form a, an, and the includes plural references unless indicated otherwise. For example, a monoclonal antibody includes one or more monoclonal antibodies.

    [0108] Generally, monoclonal antibodies specific for LASV, monoclonal antibodies specific for LCMV, the polynucleotides encoding the antibodies, and methods for using these antibodies in prevention, diagnosis, detection, and treatment are described herein. Specifically, human monoclonal antibodies specific for LASV, human monoclonal antibodies specific for LCMV, and combinations thereof for development and production of diagnostics, vaccines, therapeutics, and screening tools are provided. Generally, B cell clones producing specific IgG to GP of any Lassa virus isolate or strain may be utilized to derive the antibodies described herein.

    Polynucleotides

    [0109] The term polynucleotide is used broadly and refers to polymeric nucleotides of any length (e.g., oligonucleotides, genes, small inhibiting RNA, fragments of polynucleotides encoding a protein, etc.). By way of example and not limitation, the polynucleotides of the invention may comprise a sequence encoding all or part of the ectodomain and part of the transmembrane domain. The polynucleotide of the invention may be, for example, linear, circular, supercoiled, single-stranded, double-stranded, branched, partially double-stranded or partially single-stranded. The nucleotides comprised within the polynucleotide may be naturally occurring nucleotides or modified nucleotides.

    [0110] Functional equivalents of these polynucleotides are also intended to be encompassed by this invention. By way of example and not limitation, functionally equivalent polynucleotides are those that possess one or more of the following characteristics: the ability to generate antibodies (including, but not limited to, viral neutralizing antibodies) capable of recognizing LASV GP or the ability to generate antibodies specific to LASV GP that show neutralizing activity against LASV lineages I-IV, and proposed new lineages (e.g. lineage V from Mali, lineage VI from Togo and Benin.

    [0111] Polynucleotide sequences that are functionally equivalent may also be identified by methods known in the art. A variety of sequence alignment software programs are available to facilitate determination of homology or equivalence. Non-limiting examples of these programs are BLAST family programs including BLASTN, BLASTP, BLASTX, TBLASTN, and TBLASTX (BLAST is available from the National Institutes of Health website), FASTA, COMPARE, DOTPLOT, BESTFIT GAP FRAMEALIGN, CLUSTALW, and PILEUP. Other similar analysis and alignment programs can be purchased from various providers such as DNA Star's MEGALIGN, or the alignment programs in GENEJOCKEY. Alternatively, sequence analysis and alignment programs can be accessed through the world wide web at sites such as the CMS Molecular Biology Resource at San Diego Supercomuter Center (SDSC) website; and the Swiss Institute of Bioinformatics SIB Bioinformatics Resource Portal website ExPASy Proteomics Server. Any sequence database that contains DNA or protein sequences corresponding to a gene or a segment thereof can be used for sequence analysis. Commonly employed databases include but are not limited to GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS.

    [0112] Parameters for determining the extent of homology set forth by one or more of the aforementioned alignment programs are well established in the art. They include but are not limited to p value, percent sequence identity and the percent sequence similarity. P value is the probability that the alignment is produced by chance. For a single alignment, the p value can be calculated according to Karlin et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 2246. For multiple alignments, the p value can be calculated using a heuristic approach such as the one programmed in BLAST. Percent sequence identify is defined by the ratio of the number of nucleotide or amino acid matches between the query sequence and the known sequence when the two are optimally aligned. The percent sequence similarity is calculated in the same way as percent identity except one scores amino acids that are different but similar as positive when calculating the percent similarity. Thus, conservative changes that occur frequently without altering function, such as a change from one basic amino acid to another or a change from one hydrophobic amino acid to another are scored as if they were identical.

    [0113] The term analog includes any polypeptide having an amino acid residue sequence substantially identical to a polypeptide of the invention in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the functional aspects of the polypeptides as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; and the substitution of one acidic residue, such as aspartic acid or glutamic acid or another.

    [0114] The phrase conservative substitution also includes the use of a chemically derivatized residue in place of a non-derivatized residue. Chemical derivative refers to a subject polypeptide having one or more amino acid residues chemically derivatized by reaction of a functional side group. Examples of such derivatized amino acids include for example, those amino acids in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Also, the free carboxyl groups of amino acids may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Also, the free hydroxyl groups of certain amino acids may be derivatized to form 0-acyl or 0-alkyl derivatives. Also, the imidazole nitrogen of histidine may be derivatized to form N-imbenzylhistidine. Also included as chemical derivatives are those proteins or peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline, 5-hydroxylysine may be substituted for lysine, 3-methylhistidine may be substituted for histidine, homoserine may be substituted for serine, and ornithine may be substituted for lysine. Polypeptides of the present invention also include any polypeptide having one or more additions and/or deletions of residues relative to the sequence of any one of the polypeptides whose sequence is described herein.

    [0115] Two polynucleotide or polypeptide sequences are said to be identical if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. A comparison window as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75 contiguous positions, or 40 to about 50 contiguous positions, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

    [0116] Optimal alignment of sequences for comparison may be conducted using the MEGALIGN program in the LASERGENE suite of bioinformatics software (DNASTAR, Inc., Madison, WI), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteinsMatrices for detecting distant relationships in Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358 (1978); Hem J., Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA (1990); Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

    [0117] Preferably, the percentage of sequence identity is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

    Expression Vectors

    [0118] Expression vectors comprising at least one polynucleotide encoding an antibody or antibody fragment protein also are described herein. Expression vectors are well known in the art and include, but are not limited to viral vectors or plasmids. Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus), Ross River virus, adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655), vaccinia virus (e.g., Modified Vaccinia virus Ankara (MVA) or fowlpox), Baculovirus recombinant system and herpes virus.

    [0119] Nonviral vectors, such as plasmids, are also well known in the art and include, but are not limited to, yeast- and bacteria-based plasmids.

    [0120] Methods of introducing the vectors into a host cell and isolating and purifying the expressed protein are also well known in the art (e.g., Molecular Cloning: A Laboratory Manual, second edition, Sambrook, et al., 1989, Cold Spring Harbor Press). Examples of host cells include, but are not limited to, mammalian cells such as NS0 and CHO cells.

    [0121] By way of example, vectors comprising the polynucleotides described herein may further comprise a tag polynucleotide sequence to facilitate protein isolation and/or purification. Examples of tags include but are not limited to the myc-epitope, S-tag, his-tag, HSV epitope, V5-epitope, FLAG and CBP (calmodulin binding protein). Such tags are commercially available or readily made by methods known to the art.

    [0122] The vector may further comprise a polynucleotide sequence encoding a linker sequence. Generally, the linking sequence is positioned in the vector between the antibody polynucleotide sequence and the polynucleotide tag sequence. Linking sequences can encode random amino acids or can contain functional sites. Examples of linking sequences containing functional sites include but are not limited to, sequences containing the Factor Xa cleavage site, the thrombin cleavage site, or the enterokinase cleavage site.

    [0123] By way of example, and not limitation, an antibody specific for LASV may be generated as described herein using mammalian expression vectors in mammalian cell culture systems or bacterial expression vectors in bacterial culture systems. By way of example, and not limitation, an antibody specific for LCMV may be generated as described herein using mammalian expression vectors in mammalian cell culture systems or bacterial expression vectors in bacterial culture systems.

    Antibodies

    [0124] Examples of antibodies disclosed herein, include, but are not limited to, antibodies specific for LASV or LCMV, antibodies that cross react with native Lassa virus antigens and/or native lymphocytic choriomeningitis virus antigens, and neutralizing antibodies. By way of example, a characteristic of a neutralizing antibody includes the ability to block or prevent infection of a host cell. The antibodies may be characterized using methods well known in the art.

    [0125] The antibodies useful in the compositions and methods described herein can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab, F(ab)2, Fv, Fc, etc.), chimeric antibodies, bi-specific antibodies, heteroconjugate antibodies, single-chain fragments (e.g. ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human, or of any other origin (including chimeric or humanized antibodies).

    [0126] Methods of preparing monoclonal and polyclonal antibodies are well known in the art. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired an adjuvant. Examples of adjuvants include, but are not limited to, keyhole limpet hemocyanin (KLH), serum albumin, bovine thryoglobulin, soybean trypsin inhibitor, complete Freund adjuvant (CFA), and MPL-TDM adjuvant. The immunization protocol can be determined by one of skill in the art.

    [0127] The antibodies may alternatively be monoclonal antibodies. Monoclonal antibodies may be produced using hybridoma methods (see, e.g., Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D. W., et al., In Vitro, 18:377-381(1982).

    [0128] If desired, the antibody of interest may be sequenced and the polynucleotide sequence may then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest may be maintained in the vector in a host cell, and the host cell can then be expanded and frozen for future use. In an alternative embodiment, the polynucleotide sequence may be used for genetic manipulation to humanize the antibody or to improve the affinity, or other characteristics of the antibody (e.g., genetically manipulate the antibody sequence to obtain greater affinity to LASV and/or LCMV glycoprotein and/or greater efficacy in inhibiting the fusion of LASV and/or LCMV to the host cell).

    [0129] The antibodies may also be humanized by methods known in the art (See, for example, U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370). In yet another alternative, human antibodies may be obtained by using mice that have been engineered to express specific human immunoglobulin proteins.

    [0130] In another alternative embodiment, antibodies may be made recombinantly and expressed using any method known in the art. By way of example, antibodies may be made recombinantly by phage display technology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et at., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro. Phage display can be performed in a variety of formats; for review, see Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). By way of example, LASV and/or LCMV glycoprotein as described herein may be used as an antigen for the purposes of isolating recombinant antibodies by these techniques.

    [0131] Antibodies may be made recombinantly by first isolating the antibodies and antibody producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that may be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for expressing antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65 (1995); and Pollock, et al., J. Immunol. Methods 231:147 (1999). Methods for making derivatives of antibodies (e.g. humanized and single-chain antibodies, etc.) are known in the art.

    [0132] The antibodies described herein can be bound to a carrier by conventional methods for use in, for example, isolating or purifying LASV and/or LCMV glycoprotein or detecting LASV and/or LCMV glycoproteins, antigens, or particles in a biological sample or specimen. Alternatively, by way of example, the neutralizing antibodies of the invention may be administered as a therapeutic treatment to a subject infected with or suspected of being infected with LASV or LCMV. A subject, includes but is not limited to humans, simians, farm animals, sport animals, and pets. Veterinary uses are also encompassed by methods described herein. For diagnostic purposes, the antibodies can be labeled, e.g., bound to a detectable labelling group such as a fluorescent dye (e.g., a ALEXA FLUOR dye), a quantum dot label (e.g., a QDOT label), R-phycoerythrin, streptavidin, biotin, an enzyme (e.g., Glucose Oxidase, Horseradish Peroxidase or Alkaline Phosphatase), a radioiosotope (e.g., iodine-125, indium-111), and the like. Such labelling techniques are well known in the antibody art.

    Antibody DNA Sequences

    [0133] Sixteen neutralizing antibodies against LASV were identified, which are designated herein as 10.4B, 19.7E, 2.9D, 25.6A, 36.1F, 36.9F, 37.2D, 37.2G, 37.7H, 8.9F, NE13, 12.1F, 9.8A, 18.5C, 8.11G, and 25.10C. Nucleotide sequences (cDNA) encoding portions of heavy chain (HC) and light chain (LC) of each antibody are shown below. The illustrated nucleotide sequences encode portions of the HC and LC encompassing the variable regions thereof, i.e., the V.sub.H and V.sub.L regions, respectively, along with portions of vector sequences.

    TABLE-US-00001 (10.4BV.sub.H) SEQIDNO:1 tgcgcgttacngatccaagctgtgaccggcgcctacctgagatcaccggtgctagcacca 60 tggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtg 120 accaggtgcagctggtacagtctgggggaggcgtggtccagcctgggaggtccctgagag 180 tctcctgtgttacgtctggattcaatttcagagcctacggcatgcactgggtccgccaga 240 ttccaggcaagggactggagtgggtggcagatatttggtctgccgagactaatagacact 300 atgcagattccgtgaagggccgattcaccatctccagagacaactccaagagcacactgt 360 atctgcaaatgaacagcctgagagccgaggacacgggcgtatatttctgtgccaaagcgc 420 gaccaggctatgattatgtcgttgacttatggggccagggaacgctggtcatcgtctcct 480 cagcttccaccaagggcccatcggtcttccccctggcgccctgctccaggagcacctctg 540 ggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgt 600 cgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcct 660 caggactcta 670 (19.7EV.sub.H) SEQIDNO:2 atccagctgtgaccggcgcctacctgagatcaccggtgctagcaccatggagacagacac 60 actcctgctatgggtactgctgctctgggttccaggttccactggtgacgaggtgcagct 120 ggtggagtctgggggaggcttagttcggcctggggggtccctgagactctcctgtgcagc 180 ctctggattctccttcagtagctactcgatgcactgggtccgccatgttcctgggaaggg 240 gctggtgtgggtctcatatattaatagtgatgggagtactaaaatctacgcggactccgt 300 gaagggccgattctccatctccagagacaatgccaagaacaagctctatctgcaaatgga 360 cagtttgagagtcgaggacacggctgtatattcgtgtgtaaggcttgtacattacgactg 420 gtccccattcgtgtggggccagggaaccctggtcaccgtctcctcagcctccaccaaggg 480 cccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccct 540 gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgc 600 cctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccct 660 cagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgt 720 gaatcacaagcccagcaacaccaaggtggacaagaaagttgagccccaatcttgtgacaa 780 aactcacacatgcccaccgtgcccagcacctgaactcct 819 (2.9DV.sub.H) SEQIDNO:3 gtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgcta 60 tgggtactgctgctctgggttccaggttccactggtgacgaggtgcagctggtggagtct 120 gggggaggcctggtcaagcctggggggtcccttagactctcctgtgcagcctctggattc 180 accttcactagatttactttgacctgggtccgccaggctccagggaaggggctggagtgg 240 gtctcatccattagtagtgggagtagtgacataaactacgcagactcagtgaagggccga 300 ttcaccatatccagagacaacgccaggaactccctgttcctgcaaatgagcagcctgaga 360 gtcgacgacacggctgtgtattactgtgcgaaagatccccggtcggggatctctggtcgc 420 tacgggatggacgtctggggccaagggaccacggtcatcgtctcctcagcttccaccaag 480 ggcccatcggtcttccccctggcgccctgctccaggagcacctctgggggcacagcggcc 540 ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggc 600 gccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactcc 660 ctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaac 720 gtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgac 780 aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc 840 ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc 900 gtggtggtggacgtgagcca 920 (25.6AV.sub.H) SEQIDNO:4 acctcggttcttcgattggctagcaccatggagacagacacactcctgctatgggtactg 60 ctgctctgggttccaggttccactggtgaccaggtgcagctgcaggagtcaggaggaggc 120 ctggtcaaggctggggggtccctgagactctcctgtgcagcctctggattcatgttcgag 180 agatatagccttcactgggtccgtcagactccaggcaaggggctggagtgggtctcatcc 240 attagtagtcttagtggcagtcacataaactacgcagactcagtgaagggccgattcacc 300 atctccagagacaacgccaagaattcactgtctctgcaaatgaacagcctgagagtcgaa 360 gacacggctatatattattgtgcgagagatcgacgttcggggagttcccccgtccccttg 420 gacgtctggggccaagggaccacggtcaccgtctcctctgcctccaccaagggcccatcg 480 gtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgc 540 (36.1FV.sub.H) SEQIDNO:5 gtcactgccctcggttctatcgattggctagcaccatggagacagacacactcctgctat 60 gggtactgctgctctgggttccaggttccactggtgaccaggtgcagctgcaggagtcgg 120 gcgcgggactggtgaagccttcggagaccctgtccctcacctgcgctgtctcaggtggac 180 ccttcagcggtgcctactggacgtggatccgccaaactccagggaaggggctggagtgga 240 ttggagaggccggtcggagtggaaccaccaactacaatccgtccctcaagagtcgagtca 300 ccatatcactggacacgtccaagagccagttttccctgaagctgacttccgtgaccgccg 360 cggacacggctgtttacttctgtgggagacgccaaataatgtctttgagtaatctttata 420 agagacccgttgactcttggggccggggaaccccggtcatcgtctcctcagcctccacca 480 agggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcgg 540 ccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcag 600 gcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctact 660 ccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgca 720 acgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtg 780 acaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtct 840 tcctcttccccccaa 855 (36.9FV.sub.H) SEQIDNO:6 gtcactgccctcggttctatcgattggctagcaccatggagacagacacactcctgctat 60 gggtactgctgctctgggttccaggttccactggtgacgaggtgcagctggtgcagtctg 120 gaggaggcctggtcaaggcgggggggtccctgaaactctcctgtggagcctctggattca 180 ccttcagtagttatagcatgagctgggtccgccaggctccagggaaggggctggagtggg 240 tctcatacattagtagtggtgggagttctatacactacgcagactcagtgaagggccgat 300 tcaccatctccagagacaacgccaagaattcactgtatctgcaaatgaagaacctgaggg 360 tcgacgacacgggtcggtattattgtgtgagagatccccgatcggggatctctggtcggt 420 acggtatggacgtctggggtcaagggaccacggtcaccgtctcctcagcctccaccaagg 480 gcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccc 540 tgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcg 600 ccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccc 660 tcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacg 720 tgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgaca 780 aaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcc 840 tcttccccccaaacccaaggacaccctcatgatc 874 (37.2DV.sub.H) SEQIDNO:7 tcactgccctcggttctatcgattggctagcaccatggagacagacacactcctgctatg 60 ggtactgctgctctgggttccaggttccactggtgacgaagtgcagctggtgcagtctgg 120 agctgaggtgaagaagcctggggcttcagtgaaggtgtcctgcaaggcctctggttacac 180 ctttacgaaatacggaatcagctgggtgcgacaggcccctggacaagggcttgagtggat 240 gggatggatcagcgcgtttaatggttacacaaggtatggtcagagattccagggcaaagt 300 caccatgaccacagacacatccacgaacacagcctctttggaggtgaggaccctgacatc 360 taacgacacggccgtctattactgtgcgagacaatatcccgaccaatatagtagcagcgg 420 ttggccccgcctcttcgccatggacgtctggggccaagggaccacggtcatcgtctcccc 480 agcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgg 540 gggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtc 600 gtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctc 660 aggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagac 720 ctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcc 780 caaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggggg 840 accgtcagtcttcctcttc 859 (37.2GV.sub.H) SEQIDNO:8 tcactgccctcggttctatcgattggctagcaccatggagacagacacactcctgctatg 60 ggtactgctgctctgggttccaggttccactggtgacgaggtgcagctggtggagtctgg 120 gggaggcctggtcaagccgggggggtcccggagactctcctgtgctgcctctggattcac 180 cttcagtagagataccatgacctgggtccgccaggctccagggaaggggctggagtgggt 240 cgcatccataagtagtggtagcagtgacataaactacgcagactcagtgaagggccgatt 300 caccatctccagagacaacggcaagaactcactgtatctgcacatgaacagcctgagagc 360 cgacgacacggctatatattactgtgcgagagatccccggtcgggaatctctggtcggta 420 tggtatggacgtctggggccaagggaccacggtcaccgtctcctcagcctccaccaaggg 480 cccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccct 540 gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgc 600 cctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccct 660 cagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgt 720 gaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaa 780 aactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcct 840 cttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgt 900 ggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgt 960 (37.7HV.sub.H) SEQIDNO:9 gtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgcta 60 tgggtactgctgctctgggttccaggttccactggtgacgaggtgcagctggtgcagtct 120 ggaggaggcctggtcaaggcgggggggtccctgaggctctcctgtgcagcctccggattc 180 acattcagcacctacagtatgaactggatccgccaggctccagggaaggggctggagtgg 240 gtcgcttccattagtagtcgaagtggcagtcacataaactacgtagactcagtgaaggga 300 cgattcaccatctccagagacaacgccagggacttattgtatctgcaaatgaacagcctg 360 agagtcgacgactcggctctctattactgtgcgagagatcgccgttcggggacttctccc 420 ctccccttggacgtctggggccaagggaccacggtcaccgtcttctcagcctccaccaag 480 ggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggcc 540 ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggc 600 gccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactcc 660 ctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaac 720 gtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgac 780 aaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttc 840 ctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc 900 gtggtggtggacgtgagccacgaa 924 (8.9FV.sub.H) SEQIDNO:10 cctcggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactg 60 ctgctctgggttccaggttccactggtgaccagggcaccttgagggagtctggtccagga 120 ctggtgaggccttcggagaccctgtccctcacctgcggtgtctctggttattccatcagt 180 agtggttactactggggctggatccggcagcccccagggaaggggctggagtggattggg 240 aatatctatcgtagtgggagcacctactacaacccgtccctcaagagtcgagtcaccgtc 300 tcaatagacacgtccaaaaaccagttctccctgaagttgaattctgtgaccgccgcagac 360 acggccgtgtattactgtgcgagatcgggtataaaagtggctgacgactattactacgaa 420 atggacgtctggggccaagggaccgacgactactcttacgctatggacgtctggggccaa 480 gggaccacggtcaccgtctcctcagcctccaccaagggcccatcggtcttccccctggca 540 ccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactac 600 ttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacacc 660 ttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccc 720 tccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacacc 780 aaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgc 840 ccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggac 900 accctcatgat 911 (NE13V.sub.H) SEQIDNO:11 actgcacctcggttctatcgattggctagcaccatggagacagacacactcctgctatgg 60 gtactgctgctctgggttccaggttccactggtgacgaggttcagctggtggagtctggg 120 ggaggcctggtcaagcctggggggtccctgagactctcctgtgtagcctctggattcacc 180 ttcagttcctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtc 240 tcatccattagtagtggtagtagttacatagagtacgcagactcagtgaagggccgactc 300 accatctccagagacaacgccaagaagtcactgtatctgcaactgaacagcctgagagcc 360 gaggacacggctgtgtattactgtgcgagacacacagctcgaatcgactcttaccacggt 420 atggacgtctggggccaagggaccacagtcaccgtctcctcagcctccaccaagggccca 480 tcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggc 540 tgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctg 600 accagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagc 660 agcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaat 720 cacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaact 780 cacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttc 840 cccccaaaacccaaggacaccctcatgatctcccggacccc 881 (12.1FV.sub.H) SEQIDNO:12 gtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgcta 60 tgggtactgctgctctgggttccaggttccactggtgaccaggtgcagctgcaggagtcg 120 ggcgcaggactgttgaagccttcggagaccctgtccctcagttgcactgtcgatggtgag 180 tccttcaatggtttcttctggacgtggatccgccagcccccagggaagggtctggagtgg 240 attggagaaatcaatcatcttgcaagcaccggctacaacccgtccctcaagagtcgagtc 300 accatttcagtagacacgtccaagaaccagttctctttgaagttgacctctgtgaccgcc 360 gcggacacggctgtgtattactgtgcgagaggatacagctatggttttgcatggcccaac 420 taccactatttggacgtctggggcaaagggaccacggtcaccgtctcctcagcctccacc 480 aagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcg 540 gccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactca 600 ggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctac 660 tccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgc 720 aacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgt 780 gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtc 840 ttcctcttncccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcaca 900 tgcgtggtggtggacgtgagc 921 (9.8AV.sub.H) SEQIDNO:13 ttctatcgatttggctagcaccatggagac9.8Aagacacactcctgctatgggtactgctgct 60 ctgggttccaggttccactggtgacgaggtgcagctggtgcagtctggaggacgcttggt 120 acagcctggggggtccctgagactctcctgtgtagcctctggattcacctttagcagcca 180 tgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcaggttttag 240 tggtagtagtggtaccacaaagtacgcagactccgtgaagggccggttcaccatctccag 300 agacaattccaagaaaacgctgtatctgcaaatgaacagcctgagagccgaggacacggc 360 cgtatattactgtgcgaaaggcttctccccatttcggggagtacaattcccctactttga 420 ctactggggccagggaacgctggtcaccgtctcctcagcctccaccaagggcccatcggt 480 cttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcct 540 ggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccag 600 cggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgt 660 ggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaa 720 gcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacac 780 atgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccccc 840 aaaacccaggacaccctcatgatctcccggaccc 874 (18.5CV.sub.H) SEQIDNO:14 gtccactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgct 60 atgggtactgctgctctgggttccaggttccactggtgacgaggttcagctggtggagtc 120 tgggggaggcctggtcaggccgggggggtcccttagactctcctgtgcagccgctggatt 180 cactttcaagagttatagcatgaattgggtccgccaggctccagggaggggcctggagtg 240 ggtctcatctatcactagtggtggtagtaagacatactatgcagacgtagtgaagggccg 300 attcaccgtctccagagacaacgccaagcagtcgctctatctgcaaatgaacagcctgag 360 agccgaggacacggctatatacttctgtgcgagatccctacatagtaccagccagcctag 420 ctacatggacgtctggggcagaaagatcacggtcatcgtctcctcagcctccaccaaggg 480 cccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccct 540 gggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgc 600 cctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccct 660 cagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgt 720 gaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaa 780 aactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcct 840 cttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgc 898 (8.11GV.sub.H) SEQIDNO:15 tgcacctcggttctatcgattggctagcaccatggagacagacacactcctgctatgggt 60 actgctgctctgggttccaggttccactggtgaccaggtgcagctgcaggagtcgggtcc 120 aggactggtgaagccttcggagaccctgtccctcacctgcagtatttctggtgtgtccac 180 cagaaattattattggagctggatccgccagtccccagggaagggactggagtggattgg 240 atatatctttaacattgggaccaccaactacaatccgtccctcaagagtcgactcaccat 300 atctgtagacacgtcgaagaaccagttctccctgaagatcacctctgtgaccgctgcgga 360 cacggccgtctattactgtgcgagtggatttgagtacggtgactataccttcgactactg 420 gggccagggaaccccggtcaccgtctcctcagcctccaccaagggcccatcggtcttccc 480 cctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaa 540 ggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgt 600 gcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgac 660 cgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccag 720 caacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgccc 780 accgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacc 840 caaggacaccctcatgatcttccggacccctgaggtcacatgcgtggtggtggacgtgag 900 cca 903 (25.10CV.sub.H) SEQIDNO:16 ctcggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactgc 60 tgctctgggttccaggttccactggtgaccaggtgcagctgcaggagtctgggggaggcc 120 tggtcaagcctggggggtccctgagactctcctgtacagcctctggattcaacttcaata 180 aatataacatgaactgggtccgccaggctccagggaaggggctggagtgggtctcatcca 240 ttagtgctcttagcacttacatctattatgcagactcgctgaagggccgattcaccgtct 300 ccagagacaacgccaagaactcactgtttctgcaaatgaacagcctgagagacgacgaca 360 cggctgtttattactgtgcgagagaaatacgacgtgccagtacctggtccgccgacctct 420 ggggccgtggcactctggtcactgtctcctcagcctccaccaagggcccatcggtcttcc 480 ccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtca 540 aggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcg 600 tgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtga 660 ccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagccca 720 gcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcc 780 caccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccctccaaacc 840 caaggacaccctcatgatct 860 (10.4BV.sub.L) SEQIDNO:17 agctgtgaccggcgcctacctgagatcaccggtgctagcaccatggagacagacacactc 60 ctgctatgggtactgctgctctgggttccaggttccactggtgacgaaattgtgttgaca 120 cagtctccatcctcactgtctgcgtctgtaggagacagagtcaccatcacttgtcgggcg 180 agtcgggacatcaatacttatttaggttggtttcagcagagaccagggaaagcccctaag 240 tccctgatctatggtgcatctaatttgcaaaatggggtcccatcaaggttcagcggcagt 300 ggatctgggacgtattttactctcaccatcaacggcctgcagactgaagactttgcgact 360 tattattgccaacaatatagcatctacccgctcagtctcggcggagggaccaaggcggac 420 atgaagcgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttg 480 aaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaa 540 gtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagag 600 caggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagac 660 tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcc 716 (19.7EV.sub.L) SEQIDNO:18 tcagctgtgaccggcgcctacctgagatcaccggtgctagcaccatggagacagacacac 60 tcctgctatggctcctgctgctctgggttccaggttccactggtgacgaaattgtgttga 120 cacagtctccttccaccctgtctgcatctgtgggagacagagtcaccatcacttgccggg 180 ccagtcagagtattaataattggttggcctggtatcaggagaaaccagggaaagccccta 240 agctcctgataaataaggcgtctagtttagaaagtggggtcccatcaaggttcagcggca 300 gtggatctgggacagaattcactctcaccatcaccagcctgcagcctgatgattttgcaa 360 cttattactgccaacaatataatagtaattcgtggacgttcggccaagggaccaaggtgg 420 acatgaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagt 480 tgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggcca 540 aagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacag 600 agcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcag 660 actacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccg 720 tcacaaagagcttcaacaggggagagtgttagagggagctagctcgacatgataagatac 780 attgatgagtttgggacaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaa 840 atttgtgatgctattgcttttattgtgaaatttgtgatgctattgctttatttgtaacca 900 ttataa 906 (2.9DV.sub.L) SEQIDNO:19 actgcacctcggttctatcgattggctagcaccatgaagacagacacactcctgctatgg 60 gtactgctgctctgggttccaggttccactggtgacgacattgtgctgacccagtctcca 120 gactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagccagagt 180 gttttatacagctccaacaataagaactacttagcttggtaccagcagaagccaggacag 240 cctcctaagctgctcatttactgggcatctacccgggaatccggggtccctgaccgattc 300 agtggcagcgggtctgggacagatttcactctcaccatcagcagcctgcaggctgaagat 360 gtggcagtttattactgtcagcaatattatagtactcctccgacgttcggccaagggacc 420 aaggtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgat 480 gagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccaga 540 gaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagt 600 gtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagc 660 aaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagc 720 tcgcccgtcacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgcc 780 atggcccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaa 840 tttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaactc 894 (25.6AV.sub.L) SEQIDNO:20 ctcggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactgc 60 tgctctgggttccaggttccactggtgacctgcctgtgctgactcagcctgcctccgtgt 120 ctgggtctcctggacagtcgatcaccatctcctgcactggaaccagcagtgacgttggtg 180 cttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcataattt 240 atgaagtcaagattcggccgtcaggggtgtctaatcgtttctctggctccaagtctggca 300 acacggcctccctgaccatctctgggctccaggctgaggacgaggctgattatttttgca 360 gctcatattcaaccaacagcccttgggtgttcggcggagggacgaaggtgaccgtcctac 420 gtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgaggagcttcaag 480 ccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtgg 540 cctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccctccaaac 600 aaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcagtggaagt 660 cccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtgg 720 cccctacagaatgttcatgagcggccgcaagcttggccgccatggcccaacttgtttatt 780 gcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcattt 840 ttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgg 900 atc 903 (36.1FV.sub.L) SEQIDNO:21 tccaggtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcc 60 tgctatgggtactgctgctctgggttccaggttccactggtgacgaaattgtgctgacac 120 agtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcca 180 gtcagagtgttactaaaaactacttagcctggtaccagcagaaacctggccaggctccca 240 ccctcgtcatctatgatgcatccaccagggccagtggcatcccagacaggttcattggca 300 gtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcag 360 tatattactgccaccagtatggcagctcacctccgtacacttttggccgggggaccaagc 420 tggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc 480 agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg 540 ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca 600 cagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag 660 cagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgc 720 ccgtcacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgccatgg 780 cccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc 840 acaaataaagcatttttttcactgcattctagttgtgggttgtccaaactcatcaatgta 900 (36.9FV.sub.L) SEQIDNO:22 aggtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgc 60 tatgggtactgctgctctgggttccaggttccactggtgacgacatcgtgatgacccagt 120 ctccagactccctggctgtgtctctgggcgagagggccaccatcaactgcaagtccagcc 180 agactgttttgttcacctcctattacgtagcttggtatcaacaaaagccagggcagccgc 240 ctaagttgctcttttccggggcctcttctcgggaatccggggtccctgaccgattcagtg 300 ccggcgggtctgggacagatttctatctcaccatcaacagcctgcaggctgaagatgtgg 360 cagattactattgtcagcaatatcatactcctcctttcactttcggcggagggaccaagc 420 tggagatcagacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagc 480 agttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagagg 540 ccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtca 600 cagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaag 660 cagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgc 720 ccgtcacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgccatgg 780 cccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc 840 acaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgta 900 tcttatcatgtctggatcggga 922 (37.2DV.sub.L) SEQIDNO:23 tcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgctat 60 gggtactgctgctctgggttccaggttccactggtgacgaaacgacactcacgcagtctc 120 cagccaccctgtctgtgtctccaggggaaacagccaccctctcctgcagggccagtcaaa 180 atgttatcaacaacttagcctggtaccagcagaaacctggccaggctcccaggctcctca 240 tttatggtgcatccaccagggccactggtatcccagccaggttcagtggcagtgggtctg 300 ggacagagttcactctcaccatcagcagcatgcagtctgaagattttgcagtttattact 360 gtcagcaatataatgactggcctcgaagttttggccaggggaccaggctggacatcagac 420 gaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctg 480 gaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagt 540 ggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggaca 600 gcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgaga 660 aacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaaga 720 gcttcaacaggggagagtgttaggcggccgcaagcttggccgccatggcccaacttgttt 780 attgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagca 840 tttttttcactgcattct 858 (37.2GV.sub.L) SEQIDNO:24 tccaggtcactgccctcggttctatcgattggctagcaccatggagacagacacactcct 60 gctatgggtactgctgctctgggttccaggttccactggtgacgacattgtgctgaccca 120 gtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccag 180 tcagagtgtgaacagcatcttcttagcctggtaccagcagaaacctggccaggctcccag 240 gctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcag 300 tgggtctgggacagacttcactctcaccatcagcagactggagcctgaggattttgcagt 360 gtattactgtcagcagtatcatagctcacctaagctcactttcggcggagggaccaaggt 420 ggagatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctggtgagca 480 gttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggc 540 caaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcac 600 agagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagc 660 agactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcc 720 cgtcacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgccatggc 780 ccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttca 840 caaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtat 900 cttatcatgtctggatcgggaattaattcggcgcagcaccatggcctgaaataacctc 958 (37.7HV.sub.L) SEQIDNO:25 tcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgctat 60 gggtactgctgctctgggttccaggttccactggtgaccagtctgccctgactcagcctg 120 cctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaaccggcagtg 180 acattggtggttataactttgtctcctggtaccaacagtatcccggcaaagcccccaaac 240 tcattatttatgaggtccgtattcgggcctcaggggtttccaatcgcttctctggctcca 300 agtctggcaacacggcctccctgaccatctctggactccaggctgaggacgaggctgatt 360 attactgcaactcatattcaatccacagcccttgggtgttcggcggagggaccaagttga 420 ccgtcctgcgtcagcccaaggctgccccctcggtcactctgttcccaccctcctctgagg 480 agcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccg 540 tgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacac 600 cctccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagc 660 agtgggagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggaga 720 agacagtggcccctacagaatgttcatgagcggccgcaagcttggccgccatggcccaac 780 ttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaat 840 aaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttat 900 catgtctggatcgggaattaattcggcgcagcaccatggcctgaaataccctctgaaaga 960 ggaacttggttaggtaccttctgaggcggaaagaaccatctgtggaatgtgtgtc 1015 (8.9FV.sub.L) SEQIDNO:26 cactgccctcggttctatcgattggctagcaccatggagacagacacactcctgctatgg 60 gtactgctgctctgggttccaggttccactggtgaccaggcagggctgactcagcctgcc 120 tccgtgtctgggtctcctggacagtcgatcaccatctcctgcactgcagccaacagtgac 180 attggtgattttaactttgtctcctggtaccaacagcgcccagacaaagcccccaaactc 240 atggtttatgaggtcagcagtcggccctcaggggtttctaatcgcttctctggctccaag 300 tctggcaacacggcctccctgaccatctctgggctccaggctgaggacgaggctgattat 360 tactgcacctcatatacaagcagcagcacttttgtcttcggaactgggaccaaggtcacc 420 gtcctaggtcagcccaaggccaaccccactgtcactctgttcccgccctcctctgaggag 480 cttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtg 540 acagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagaccaccacaccc 600 tccaaacaaagcaacaacaagtacgcggccagcagctacctgagcctgacgcctgagcag 660 tggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaag 720 acagtggcccctacagaatgttcatgagcggccgcaagcttggccgccatggcccaactt 780 gtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataa 840 agcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatca 900 tgtctggatc 910 (NE13V.sub.L) SEQIDNO:27 ctcccaggtcactgcacctcggttctatcgattggctagcaccatggagacagacacact 60 cctgctatgggtactgctgctctgggttccaggttccactggtgacgaaacgacactcac 120 gcagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggc 180 cagtcagagtgttagcagcacctacttagcctggtaccagcagaaacctggccagtctcc 240 caggctcctcatttatggtgcatccagtagggccactggcatcccagacaggttcagtgg 300 cagtgggtctgggacacagttcactctcaccatcaacagactggagcctgaagattttgc 360 agtgtattactgtcagcagtttggtagcccgtggacattcggccaagggaccaaggtgga 420 aatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagtt 480 gaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaa 540 agtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacaga 600 gcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcaga 660 ctacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgt 720 cacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgccatggccca 780 acttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaa 840 ataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatctt 900 atcatgtc 908 (12.1FV.sub.L) SEQIDNO:28 gtcactgcacctcggttctatcgattggctagcaccatggagacagacacactcctgcta 60 tgggtactgctgctctgggttccaggttccactggtgacgaaacgacactcacgcagtct 120 ccagccaccctgtctttgtctccaggggagagagccaccctctcctgtagggccagtcag 180 agtgttagcagctacttagcctggtaccaacacaaacctggccaggctcccaggctcctc 240 atctatggtgcatcaaagagggccactggcatcccgtccaggttcagtggcagtgggtct 300 gggacagacttcagtctcaccatcagcagcctagagcctgaagattttgcagtttactac 360 tgtcagcaccgaagcgactggcggactaccttcggccaagggacacgactggagattaaa 420 cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatct 480 ggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacag 540 tggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggac 600 agcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgag 660 aaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaag 720 agcttcaacaggggagagtgttaggcggccgcaagcttggccgccatggcccaacttgtt 780 tattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagc 840 atttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgt 900 ctggatcgggaaattaatcggcgcagcaccat 932 (9.8AV.sub.L) SEQIDNO:29 ggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactgctgc 60 tctgggttccaggttccactggtgacgacatcgtgatgacccagtctccttccaccctgt 120 ctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattgataggt 180 ggttggcctggtatcagcagaaaccagggaaagcccctaagctcctgatctatcaggcat 240 ctagtttagaaagaggggtcccatcaaggttcagcggcagtggatctgggacagaattca 300 ctctcaccatcagcagcctgcagcccgatgattttgcaacttattactgccaacagtata 360 atggttaccctctcactttcggcggagggaccaaggtggagatcaaacgaactgtggctg 420 caccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctg 480 ttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggata 540 acgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagca 600 cctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtct 660 acgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggg 720 gagagtgttaggcggccgcaagcttggccgccatggcccaacttgtttattgcagcttat 780 aatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactg 840 cattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcg 895 (18.5CV.sub.L) SEQIDNO:30 tccaggtccactgcacctcggttctatcgattggctagcaccatggagacagacacactc 60 ctgctatgggtactgctgctctgggttccaggttccactggtgacgacatccagatgacc 120 cagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggcc 180 agtcagagtgttatcagttactacgtagcctggtaccagcacaaaggtggccaggctccc 240 aggctcctcatttatggtgcatccagcagggccactggcgtcccagacaggttcagtggc 300 agtgggtctgggacagacttcactctcaccatcagcagcctggagcctgaagattttgca 360 ctgtattactgtcagtactatgggagctcacctctgtgggcgttcggccaagggaccaag 420 gtggaaatcaaacgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgag 480 cagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagag 540 gccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtc 600 acagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaa 660 gcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcg 720 cccgtcacaaagagcttcaacaggggagagtgttaggcggccgcaagcttggccgccatg 780 gccc 784 (8.11GV.sub.L) SEQIDNO:31 cggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactgctg 60 ctctgggttccaggttccactggtgacgaaattgtgctgactcagtctccagccaccctg 120 tctgtgtctccagggggtagggcctccctctcctgccgggccagtcagagtattggcgac 180 aagttatcctggtatcagcagaaacctgggcaggctcccaggctcgtcatctatggtgca 240 tataccagggccactgatatctcacccaggttcagtggcagtaggtctgggacagacttc 300 aatctcaccatcagcagaatgcagtctggagactttgcagtttatttctgtcagcagtat 360 gaaaactggcctcggacttttggccaggggaccaagctggagatcaaacgaactgtggct 420 gcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctct 480 gttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggat 540 aacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagc 600 acctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtc 660 tacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagg 720 ggagagtgttaggcggccgcaagcttggccgccatggcccaacttgtttattgcagctta 780 taatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcact 840 gcatt 845 (25.10CV.sub.L) SEQIDNO:32 cctcggttctatcgattggctagcaccatggagacagacacactcctgctatgggtactg 60 ctgctctgggttccaggttccactggtgacgacatccagatgacccagtctccatcctcc 120 ctgtctgcatctgttggagacagagtcatcatcacttgccgggcaagtcagagcatcagc 180 agctctttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctatgct 240 gcagtcaatttggagactggggtcccgtcaaggttcagtggcagtggatttgggacagat 300 ttcactctcgccatcagcaatgtgcaacctgaagattttgcaacttactactgtcaacag 360 agcgatactcggacttttggccgggggaccaagctggacgtcaaacgaactgtggctgca 420 ccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgtt 480 gtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataac 540 gccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacc 600 tacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctac 660 gcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacagggga 720 gaagtgttaggcggccgcaagcttggccgccatggcccaacttgtttattgcagcttata 780 atggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgc 840 attctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcgggaatt 900

    Antibody Amino Acid Sequences

    [0134] The V.sub.H and V.sub.L amino acid sequences of the antibodies and complementarity determining regions (CDR) of the V.sub.H and V.sub.L sequences are shown and discussed below.

    TABLE-US-00002 (10.4BV.sub.H) SEQIDNO:33 METDTLLLWVLLLWVPGSTGDQVQLVQSGGGVVQPGRSLRVSCVISGFNF RAYGMHWVRQIPGKGLEWVADIWSAETNRHYADSVKGRFTISRDNSKSTL YLQMNSIRAEDIGVYFCAKARPGYDYVVDLWGQGTLVIVSSASTKGPSVF PLAPCSRSTSGGTAPLLGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGL (19.7EV.sub.H) SEQIDNO:34 METDTLLLWVLLLWVPGSTGDEVQLVESGGGIVRPGGSLRLSCAASGYSF ESYSMHWVREVPGKGINWVSYINSDGSTKIYADSVKGRFSISRDNAKNKL YLQMDSLRVEDTAVYSCVRLVHYDWSPFVWGQGTLVTVSSASTKGPSVFP LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPQSCDKTHTCP PCPAPELL (2.9DV.sub.H) SEQIDNO:35 METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVKPGGSLRLSCAASGFTF TRFTLTWVRQAPGKGLEWVSSISSGSSDINYADSVKGRFTISRDNARNSL FLQMSSLRVDDTAVYYCAKDPRSGISGRYGMDVWGQGTTVIVSSASTKGP SVFPLAPCSRSTSGGIAALGCLVKDYFPEPVTVEWNSGALTSGVHTYPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVS (25.6AV.sub.H) SEQIDNO:36 METDTLLLWVLLLWVPGSTGDQVQLQESGGGLVKAGGSLRLSCAASGFMF ERYSLHWVRQTPGKGLEWVSSISSLSGSHINYADSVKGRFTISRDNAKNS LSLQMNSLBVEDTAIYYCARDRRSGSSPVPLDVWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGC (36.1FV.sub.H) SEQIDNO:37 METDTLLLWVLLLWVPGSTGDQVQLQESGAGIVKPSETLSLTCAVSGGPF SGAYWTWIRQTPGKGLEWIGEAGRSGTTNYNPSLKSRVTISLDISKSQFS LKLTSVTAADTAVYFCGRRQIMSLSNLYKRPVDSWGRGTPVIVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTYPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLFPPX (36.9FV.sub.H) SEQIDNO:38 METDTLLLTWVLLLWVPGSTGDEVQLVQSGGGLVKAGGSLKLSCGASGFT FSSYSMSWVRQAPGKGLEWVSYISSGGSSIHYADSVKGRFTISRDNAKNS LYLQMKNLRVDDTGRYYCVRDPRSGISGRYGMDVWGQGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVETFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCDAPELLGGPSVFLPPNPRTPS*S (37.2DV.sub.H) SEQIDNO:39 METDTLLLWVLLLWVPGSTGDEVQLVQSGAEVKKPGASVKVSCKASGYTF TKYGISWVRQAPGQGLEWMGWISAFNGYTRYGQRFQGKVTMTTDTSTNTA SLEVRTLTSNDTAVYYCARQYPDQYSSSGWPRIFAMDVWGQGTTVIVSPA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVEWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPK SCDKTHTCPPCPAPELLGGPSVFLF (37.2GV.sub.H) SEQIDNO:40 METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVKPGGSRRLSCAASGFTF SRDTMTWVRQAPGKGLEWVASISSGSSDINYADSVKGRFTISRDNGKNSL YLHMNSLRADDTAIYYCARDPRSGISGRYGMDVWGQGTTVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGV (37.7HV.sub.H) SEQIDNO:41 METDTLLLWVLLLWVPGSTGDEVQLVQSGGGLVKAGGSLRLSCAASGFTF STYSMNWIRQAPGKGLEWVASISSRSGSHINYVDSVKGRFTISRDNARDL LYLQMNSLRVDDSALYYCARDRRSGTSPLPLDVWGQGTTVTVFSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE (8.9FV.sub.H) SEQIDNO:42 METDTLLLWVLLLWVPGSTGDQGTLRESGPGLVRPSETLSLTCGVSGYSI SSGYYNGWIRQPPGKGLEWIGNIYRSGSTYYNPSLKERVTVSIDTSKNQF SLKLNSVTAADTAVYYCARSGIKVADDYYYEMDVWGQGTDDYSYAMDVWG QGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYETEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSN TKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMX (NE13V.sub.H) SEQIDNO:43 METDTLLLWVLLLWVPGSTGDEVQLVESGGGLVKPGGSLRLSCVASGFTF SSYSMNWVRQAPGKGLEWVSSISSGSSYIEYADSVKGRLTISRDNAKKSL YLQLNSLRAEDTAVYYCARHTARIDSYHGMDVWGQGTTVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNIKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP (12.1FV.sub.H) SEQIDNO:44 METDTLLLWVLLLWVPGSTGDQVQLQESGAGLLKPSETLSLSCTVDGESF NGFFWTWIRQPPGKGLEWIGEINHLASTGYNPSLKSRVTISVDTSKNQFS LKLTSVTAADTAVYYCARGYSYCFAWPNYHYLDVWGKGTTVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYYPEPVTVWNSGALTSGVHTFPAV ILQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPELLGGPSVFLXDPKPKDTLMISRTPEVTCVVVDVS (9.8AV.sub.H) SEQIDNO:45 METDTDLLLWVLLLWVPGSTGDEVQLVQSGGRLVQPGGSLRLSCVASGFT FSSHAMSWVRQAPGKGLEWVSGFSGSSGTIKYADSVKGRFTISRDNSKKT LYLQMNSLRAEDTAVYYCAKGFSPFRGVQFPYFDYWGQGTLVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYTPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPELLGGPSVFLFDPKPRIPS*SPGP (18.5CV.sub.H) SEQIDNO:46 METDTMLLWVLLLWVPGSTGDRVQLVESGGCLVRPGGSLRLSCAAAGFTF KSYSMNWVRQAPGRGLEWVSSITSGGSKTYYADVVKGRFTVSRDNAKQSL YLQMNSLRAEDTAIYFCARSLHSTSQPSYMDVWGRKITVIVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPRVTC (8.11GV.sub.H) SEQIDNO:47 METDTLLLWVLLLWVPGSTGDQVQLQESGPGLVKPSETLSLTCSISGVST RNYYWSWIRQSPGKGLEWIGYIFNIGTTNYNPSLKSRLTISVDTSKNQFS LKITSVTAADTAVYYCASGFEYGDYTFDYWGQGTPVTVSSASTKGPSVET LAPSSKSTSGGTAALGCLVKDYFPEPVTVEWNSGALTSGVHTYPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMIFRTPEVTCVVVDVS (25.10CV.sub.H) SEQIDNO:48 METDTLLLWVLLLWVPGSTGDTQLQESGGGLVKPGGSLRLSCTASGFNFN KYNKNWVRQAPGKGLEWVSSISALSTYIYYADSLKGRFTVSRDNAKNSLF LQMNSLRDDDTAVYYCAREIRRASTWSADLWGRGTLVTVSSASTKGPSVF PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPNPRTPS (10.4BV.sub.L) SEQIDNO:49 METDTLLLWVLLLWVPGSTGDEIVLTQSPSSLSASVGDRVTITCRASRDI NTYLGWFQQRPGKAPKSLIYGASNLQNGVPSRFSGSGSGTYFTLTINGLQ TEDFATYYCQQYSIYPLSLGGGTKADMKRTVAAPSVFIFPPSDEQLKEGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYFKHKVYACEVTHQGLSSP (19.7EV.sub.L) SEQIDNO:50 METDTLLLWLLLLWVPGSTGDEIVLTQPSTLSASVGDRVTITCRASQSIN NWLAWYQEKPGKAPKLLINKASSLESGVPSRFSGSGSGTEFTLTITSLQP DDFATYYCQQYNSNSWTFGQGTKVDMKRTVAAPSVFIFPPSDEQLKSGTA SVVCLLNNFYPREAKVQWKVDNALQSGNESQESVTEQSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (2.9DV.sub.L) SEQIDNO:51 MKTDTLLLWVLLLWVPGSTGDDIVLTQSPDSLAVSLGERATINCKSSQSV LYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTL TISSLQAEDVAVYYCQQYYSTPPTFGQGTKVEIKRTVAAPSVFIFPPSDE QLKSGTASVVCLLNNTYPREAKVQWKVDNALQSGNSQESVTFQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (25.6AV.sub.L) SEQIDNO:52 METDTLLLWVLLLWVPGSTGDLPVLTQPASVSGSPGQSITISCTGTSSDV GAYNYVSWYQQHPGKAPKLIIYEVKIRPSGVSNRFSGSKSGNTASLTISG LQAEDEADYFCSSYSTNSPWVFCGGTKVTVLRQPKAAPSVTLFPPSSEEL QANKATLVCLISDFYPCAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* (36.1FV.sub.L) SEQIDNO:53 METDTLLLWVLLLWVPGSTGDEIVLTQSPGTLSLSPGERATLSCRASQSV TKNYLAWYQQKPGQAPTLVIYDASTRASGIPDRFIGSGSGTDFTLTISRL EPEDFAVYYCHQYGSSPPYTFGRGTKLEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (36.9FV.sub.L) SEQIDNO:54 METDTLLLWVLLLWVPGSTGDDIVMTQSPDSLAVSLGERATINCKSSQTV LFTSYYVAWYQQKPGQPPKLLFSGASSRESGVPDRESAGGSGTDFYLTIN SLQAEDVADYYCQQYHTPPFTFGGGTKLEIRRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS SILTLSKADYEKHKVYACEVTHQGLSSPVTKEFNRGEC* (37.2DV.sub.L) SEQIDNO:55 METDTLLLWVLLLWVPGSTGDETTLTQSPATLSVSPGETATISCRASQNV INNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSMQ SEDFAVYYCQQYNDWPRSFGQGTRLDIRRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (37.2GV.sub.L) SEQIDNO:56 METDTLLLWLLLWVPGSTGDDIVLTQSPGTLSLSPGERATLSCRASQSVN SIFLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLE PEDFAVYYCQQYHSSPKLTFGGGTKVEIKRTVAAPSVFIFPPSGEQLKSG TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (37.7HV.sub.L) SEQIDNO:57 METDTLLLWVLLLWVPGSTGDQSALTQPASVSGSPGQSITISCTGTGSDI GGYNFVSWYQQYPGKAPKLIIYEVRIRASGVSNRFSGSKSGNTASLTISG LQAEDEADYYCNSYSIHSPWVFGGGTKLTVLRQPKAAPSVTLFPPSSEEL QANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWESHRSYSCQVTHEGSTVEKTVAPTECS* (8.9FV.sub.L) SEQIDNO:58 METDTLLLWVLLLWVPGSTGDQAGLTQPASVSGSPGQSITISCTAANSDI GDFNFVSWYQQRPDKAPKLMVYEVSSRPSGVSNRFSGSKSGNTASLTISG LQAEDEADYYCTSYTSSSTFVFGTGTKVTVLGQPKANPTVTLFPPSSEEL QANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS* (NE13V.sub.L) SEQIDNO:59 TMETDTLLLWVLLLWVPGSTGDETTLTQSPGTLSLSPGERATLSCRASQS VSSTYLAWYQQKPGQSPRLLIYGASSRATGIPDRFSGSGSGTQFTLTINR LEPEDFAVYYCQQFGSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG TASVVCLLNNEYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (12.1FV.sub.L) SEQIDNO:60 METDTLLLWVLLLWVPGSTGDETTLTQSPATLSLSPGERATLSCRASQSV SSYLAWYQHKPGQAPRLLIYGASKRATGIPSRFSGSGSGTDFSLTISSLE PEDFAVYYCQHRSDWRTTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFKRGEC* (9.8AV.sub.L) SEQIDNO:61 METDTLLLWVLLLWVPGSTGDDIVMTQSPSTLSASVGDRVTITCRASQSI DRWLAWYQQKPGKAPKLLIYQASSLERGVPSRFSGSGSGTEFTLTISSLQ PDDFATYYCQQYNGYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSEFRGEC* (18.5CV.sub.L) SEQIDNO:62 METDTLLLWVLLLWVPGSTGDDIQMTQSPGTLSLSPGERATLSCRASQSV ISYYVAWYQHKGGQAPRLLIYGASSRATGVPDRFSGSGSGTDFTLTISSL EPEDFALYYCQYYGSSPLWAFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESV7EQDSKD5TYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (8.11GV.sub.L) SEQIDNO:63 METDTLLLWVLLLWVPGSTGDEIVLTQSPATLSVSPGGRASLSCRASQSI GDKLSWYQQKPGQAPRLVIYGAYTRATDISPRFSGSRSGTDFNLTISRMQ SGDFAVYFCQQYENWPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGT ASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (25.10CV.sub.L) SEQIDNO:64 METDTLLLWVLLLWVPGSTGDDIQMTQSPSSLSASVGDRVIITCRASQSI SSSLNWYQQKPGKAPKLLIYAAVNLETGVPSRFSGSGFGTDFTLAISNVQ PEDFATTYCQQSDTRTFGRGTKLDVKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNAIQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVIKSENRGEVLGGRKLGBEGPTCLLQL TMVTNKAIASQISQIKHFFHCILVVVCPNSSMYLIMSGSGI

    [0135] FIG. 7 provides a sequence alignment prepared using CLUSTAL OMEGA (1.2.4) multiple sequence alignment (from EMBL-EBI, a part of the European Molecular Biology Laboratory) for the heavy chain variable region amino acid sequences, with CDRs highlighted in bold typeface: CDR1 (marked with +), CDR 2 (marked with {circumflex over ()}), and CDR3 (marked with #).

    [0136] FIG. 8 provides a sequence alignment prepared using using CLUSTAL OMEGA (1.2.4) multiple sequence alignment for the light chain variable region amino acid sequences, with CDRs highlighted in bold typeface: CDR1 (marked with +), CDR 2 (marked with {circumflex over ()}), and CDR3 (marked with #).

    [0137] The HC CDR Sequence Table below lists the sequences of CDR1, CDR 2, and CDR3 of the V.sub.H of each of the 16 neutralizing antibodies described herein. The LC CDR Sequence Table below lists the sequences of CDR1, CDR 2, and CDR3 of the V.sub.L of each of the 16 neutralizing antibodies described herein.

    TABLE-US-00003 HCCDRSequenceTable. Antibody HCCDR1 HCCDR2 HCCDR3 10.4B GFNFRAYG IWSAETNRH AKARPGYDYVVDL (SEQIDNO:65) (SEQIDNO:66) (SEQIDNO:67) 19.7E GFSFSSYS INSDGSTKI VRLVHYDWSPFV (SEQIDNO:68) (SEQIDNO:69) (SEQIDNO:70) 2.9D GFTFTRFT ISSGSSDIN AKDPRSGISGRYGMDV (SEQIDNO:71) (SEQIDNO:72) (SEQIDNO:73) 25.6A GFMFERYS ISSLSGSHIN ARDRRSGSSPVPLDV (SEQIDNO:74) (SEQIDNO:75) (SEQIDNO:76) 36.1F GGPFSGAY AGRSGTTN GRRQIMSLSNLYKRPVDS (SEQIDNO:77) (SEQIDNO:78) (SEQIDNO:79) 36.9F GFTFSSYS ISSGGSSIH VRDPRSGISGRYGMDV (SEQIDNO:80) (SEQIDNO:81) (SEQIDNO:82) 37.2D GYTFTKYG ISAFNGYTR ARQYPDQYSSSGWPRLFAMDV (SEQIDNO:83) (SEQIDNO:84) (SEQIDNO:85) 37.2G GFTFSRDT ISSGSSDIN ARDPRSGISGRYGMDV (SEQIDNO:86) (SEQIDNO:87) (SEQIDNO:88) 37.7H GFTFSTYS ISSRSGSHIN ARDRRSGTSPLPLDV (SEQIDNO:89) (SEQIDNO:90) (SEQIDNO:91) 8.9F GYSISSGYY IYRSGSTY ARSGIKVADDYYYEMD (SEQIDNO:92) (SEQIDNO:93) VWGQGTDDYSYAMDV (SEQIDNO:94) NE13 GFTFSSYS ISSGSSYIE ARHTARIDSYHGMDV (SEQIDNO:95) (SEQIDNO:96) (SEQIDNO:97) 12.1F GESFNGFF INHLASTG ARGYSYGFAWPNYHYLDV (SEQIDNO:98) (SEQIDNO:99) (SEQIDNO:100) 9.8A GFTFSSHA FSGSSGTTK AKGFSPFRGVQFPYFDY (SEQIDNO:101) (SEQIDNO:102) (SEQIDNO:103) 18.5C GFTFKSYS ITSGGSKTY ARSLHSTSQPSYMDV (SEQIDNO:104) (SEQIDNO:105) (SEQIDNO:106) 8.11G GVSTRNYY IFNIGTTN ASGFEYGDYTFDY (SEQIDNO:107) (SEQIDNO:108) (SEQIDNO:109) 25.10C GFNFNKYN ISALSTYIY AREIRRASTWSADL (SEQIDNO:110) (SEQIDNO:111) (SEQIDNO:112)

    TABLE-US-00004 LCCDRSequenceTable. Antibody LCCDR1 LCCDR2 LCCDR3 10.4B RDINTY GAS QQYSIYPLS (SEQIDNO:113) (SEQIDNO:114) 19.7E QSINNW KAS QQYNSNSWT (SEQIDNO:115) (SEQIDNO:116) 2.9D QSVLYSSNNKNY WAS QQYYSTPPT (SEQIDNO:117) (SEQIDNO:118 25.6A SSDVGAYNY EVK SSYSTNSPWV (SEQIDNO:119) (SEQIDNO:120) 36.1F QSVTKNY DAS HQYGSSPPYT (SEQIDNO:121) (SEQIDNO:122) 36.9F QTVLFTSYY GAS QQYHTPPFT (SEQIDNO:123) (SEQIDNO:124) 37.2D QNVINN GAS QQYNDWPRS (SEQIDNO:125) (SEQIDNO:126) 37.2G QSVNSIF GAS QQYHSSPKLT (SEQIDNO:127 (SEQIDNO:128) 37.7H GSDIGGYNF EVR NSYSIHSPWV (SEQIDNO:129) (SEQIDNO:130) 8.9F NSDIGDFNF EVS TSYTSSSTFV (SEQIDNO:131) (SEQIDNO:132) NE13 QSVSSTY GAS QQFGSPWT (SEQIDNO:133) (SEQIDNO:134) 12.1F QSVSSY GAS QHRSDWRTT (SEQIDNO:135) (SEQIDNO:136) 9.8A QSIDRW QAS QQYNGYPLT (SEQIDNO:137) (SEQIDNO:138) 18.5C QSVISYY GAS QYYGSSPLWA (SEQIDNO:139) (SEQIDNO:140) 8.11G QSIGDK GAY QQYENWPRT (SEQIDNO:141) (SEQIDNO:142) 25.10C QSISSS AAV QQSDTRT (SEQIDNO:143) (SEQIDNO:144)

    Diagnostics

    [0138] The antibodies described herein may be used in a variety of immunoassays for LASV, LCMV, and other arenaviruses. The antibodies of the invention can be produced with high quality control and are suitable as reagents for the purposes of detecting antigen in biological samples. By way of example and not limitation, antibodies of the invention could be used as reagents in an ELISA assay to detect Lassa antigen in a biological sample from a subject. The antibodies can be labeled, e.g., bound to a detectable labelling group such as a fluorescent dye, a quantum dot label, R-phycoerythrin, streptavidin, biotin, an enzyme, a radioisotope, and the like. Such labelling techniques are well known in the antibody art.

    Vaccines

    [0139] Vaccines for LASV, LCMV, and other arenaviruses also are described herein. In one aspect the vaccines are DNA-based vaccines. One skilled in the art is familiar with administration of expression vectors to obtain expression of an exogenous protein in vivo. See, e.g., U.S. Pat. Nos. 6,436,908; 6,413,942; and 6,376,471. Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art and non-limiting examples are described herein.

    [0140] Administration of expression vectors includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Targeted delivery of therapeutic compositions containing an expression vector or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

    [0141] Non-viral delivery vehicles and methods can also be employed, including but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Cunel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles (see, e.g., U.S. Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338); and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and U.S. Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95/13796, WO 94/23697, WO 9 1/14445; and EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

    [0142] For human administration, the codons comprising the polynucleotide encoding one or more antibodies specific for LASV glycoprotein and/or LCMV glycoprotein may be optimized for human use, a process that is standard in the art.

    [0143] In another aspect, one or more antibodies specific to LASV and/or LCMV or combinations thereof is used as a vaccine. The one or more antibodies or combination thereof may be administered by itself or in combination with an adjuvant. Examples of adjuvants include, but are not limited to, aluminum salts, water-in-soil emulsions, oil-in-water emulsions, saponin, QuilA and derivatives, iscoms, liposomes, cytokines including gamma-interferon or interleukin 12, DNA (e.g. unmethylated poly-CpG), microencapsulation in a solid or semi-solid particle, Freunds complete and incomplete adjuvant or active ingredients thereof including muramyl dipeptide and analogues, DEAE dextrarilmineral oil, Alhydrogel, Auspharm adjuvant, and Algammulin.

    [0144] The antibody vaccine comprising one or more antibodies specific to LASV and/or LCMV or combinations thereof can be administered orally or by any parenteral route such as intravenously, subcutaneously, intraarterially, intramuscularly, intracardially, intraspinally, intrathoracically, intraperitoneally, intraventricularly, sublingually, and/or transdermally.

    [0145] Dosage and schedule of administration can be determined by methods known in the art. Efficacy of the one or more antibodies specific to LASV and/or LCMV or combinations thereof as a vaccine for Lassa virus, lymphocytic choriomeningitis virus, or related arenaviruses may also be evaluated by methods known in the art.

    Pharmaceutical Compositions

    [0146] The polynucleotides, polypeptides, and antibodies described herein can further comprise pharmaceutically acceptable carriers, excipients, or stabilizers known in the art (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the employed dosages and concentrations, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, marmose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN, PLURONICS or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

    [0147] The compositions used in the methods described herein generally comprise, by way of example and not limitation, an effective amount of a polynucleotide or polypeptide (e.g., an amount sufficient to induce an immune response) of the invention or antibody of the invention (e.g., an amount of a neutralizing antibody sufficient to mitigate infection, alleviate a symptom of infection and/or prevent infection).

    [0148] The pharmaceutical composition can further comprise additional agents that serve to enhance and/or complement the desired effect. By way of example, to enhance the efficacy of the one or more antibodies specific to LASV and/or LCMV or combinations thereof administered as a pharmaceutical composition, the pharmaceutical composition may further comprise an adjuvant. Examples of adjuvants are provided herein.

    [0149] Also by way of example and not limitation, if the one or more antibodies specific to LASV and/or LCMV or combinations thereof of the invention is being administered to augment the immune response in a subject infected with or suspected of being infected with LASV or LCMV and/or if antibodies of the present invention are being administered as a form of passive immunotherapy, the composition can further comprise other therapeutic agents (e.g., anti-viral agents).

    Kits

    [0150] Kits for use in the instant methods also are described. Kits include one or more containers comprising by way of example, and not limitation, polynucleotides encoding one or more antibodies specific to LASV and/or LCMV or combinations thereof or fragments thereof of the invention and instructions for use in accordance with any of the methods of the invention described herein. In some embodiments of the kit, the antibodies are bound to a detectable label as discussed above.

    [0151] Generally, instructions comprise a description of administration or instructions for performance of an assay. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the invention are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

    [0152] The kits are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. A kit may have a sterile access port (e.g. the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g. the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Kits may optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container.

    [0153] The following non-limiting examples are provided to illustrate certain aspects and features of the materials and methods described herein.

    EXAMPLES

    Example 1: LCMV Infection of the Mouse and Recombinant Arenaviruses are a Powerful Experimental System to Assess the Potency and Breath of LCMV Neutralizing mMAbs In Vivo

    [0154] Both host and viral factors, as well as route of infection and dose of virus influence the outcome of LCMV infection of the mouse. Thus, intravenous (i.v.) inoculation of adult immune competent mice with LCMV Armstrong (ARM) strain results in an acute infection that induces a protective immune response that mediates virus clearance in 10 to 14 days, a process predominantly mediated by virus-specific CD8+ cytotoxic T lymphocytes (CTL). In contrast, i.v. inoculation with a high dose of the immunosuppressive clone 13 (Cl-13) strain of LCMV causes a persistent infection associated with sustained viremia and generalized immune suppression that can last for 60 to 100 days. This model is robust and has clear outcomes, which provide a valid and cost effective experimental system for initial evaluation of the efficacy of antibody-based strategies to control and clear a LCMV infection. In this regard, the use of Cl-13 based recombinant viruses expressing GPs of interest allows assessment of the safety and in vivo neutralizing activity of GP-specific BNmMAbs. This approach is feasible using state-of-the-art arenavirus reverse genetics that allows rescue of infectious recombinant LCM viruses with predetermined mutations of interest, as well as expressing heterologous either viral or non-viral genes of interest. A single-cycle infectious, reporter expressing, recombinant LCMV in which the GP ORF is replaced by GFP (rLCMVGP/GFP) was generated. Genetic complementation with plasmids or stable cell lines expressing arenavirus GPs of interest produces the corresponding GP-pseudotyped rLCMVGP/GFP that can be used to evaluate antibody responses to HF arenaviruses using a BSL2 platform.

    Example 2: Identification of LASV (Josiah Strain) GP-Specific hMAbs that Cross-React with the GP of LCMV ARM Strain

    [0155] Generation of LASV GP-specific hMAbs: Peripheral blood mononuclear cells (PBMCs) isolated from 17 different LF survivors in Sierra Leone and Nigeria were used to identify B cell clones producing specific IgG to LASV GP. RNA from these B cell clones was used to clone the light chain (LC) and heavy chain (HC) genes. Paired LC and HC were expressed in human 293T cells to generate a collection of 120 LASV GP-specific hMAbs. These hMAbs arose from different germline genes and were likely independently derived. All but one (8.9F) of the hMAbs reacted in ELISA with GP from Josiah strain of LASV (lineage IV), which is closely related to the currently circulating LASV strains in Sierra Leone. LASV GP consists of a SSP and GP1 and GP2 subunits, as shown in FIG. 1, Panels A and B. To define the GP subunits recognized by the LASV hMAbs, an immunofluorescence assay was used to test the recognition by hMAbs of human 293T cells expressing either rGP1 or rGP2 alone, or full-length GP, shown in FIG. 1, Panel C. Twenty-nine hMAbs, including three with neutralizing activity, reacted with LASV rGP1, shown by FIG. 1, Panel C, left and Table 1. Fifty-seven hMAbs recognized LASV rGP2 but none of these exhibited neutralizing activity, as shown by FIG. 1, Panel C, middle and Table 1. Seven hMAbs reacted with peptides representing three linear epitopes in GP2, whereas the remaining hMAbs appeared to recognize conformational epitopes. Twenty-seven hMAbs reacted with cells expressing full-length GP but did not react with either rGP1 or rGP2 expressed individually. Remarkably, thirteen of these hMAbs were neutralizing, as shown by FIG. 1, Panel C, right and Table 1. Inhibitory concentration 50 (IC50) and 80 (IC80) neutralizing activity of LASV GP-specific hMAbs was evaluated using lentivirus particles pseudotyped with the different lineage I-IV LASV GPs. Results are shown in Table 1. Based on the data in Table 1, the antibodies can be classified as most potent (hMAbs exhibiting IC values of <1 g/mL); potent (hMAbs exhibiting IC values in the range of 1 to 2.5 g/mL), weak (hMAbs exhibiting IC values of >3 and <20 g/mL); non-neutralizing (hMAbs exhibiting IC values >20 g/mL).

    TABLE-US-00005 TABLE 1 Neutralizing activity of LASV GP-specific hMAbs against LASV lineages I-IV. LASV Josiah (IV) LASV 237 (III) LASV A19 (II) LASV Pinneo(I) Mab IC50 IC80 IC50 IC80 IC50 IC80 IC50 IC80 25.10C 0.094 0.174 0.058 0.180 0.104 0.364 0.226 0.564 12.1F 0.158 0.562 0.146 0.458 0.463 2.266 0.285 0.692 8.9F 0.126 1.604 0.182 3.097 0.125 0.467 0.403 2.210 37.2D 0.559 1.983 0.256 0.844 0.469 1.154 0.537 1.861 37.7H 0.191 0.532 0.077 0.202 0.255 0.537 0.301 1.658 25.6A 0.743 1.999 0.169 0.603 0.483 3.509 1.826 3.114 9.8A 1.309 2.423 0.193 0.494 0.150 0.527 1.003 2.587 18.5C 1.935 3.985 0.621 3.231 1.200 4.633 6.111 12.170 8.11G 0.361 1.736 1.166 3.637 1.481 4.591 3.245 10.540 37.2G 5.599 16.000 2.020 5.231 1.100 10.000 >20 >20 2.9D 6.895 16.700 1.582 5.511 3.072 14.780 10.130 >20 NE13 10.680 19.500 2.136 7.000 5.409 13.180 >20 >20 19.7E 5.908 >20 1.062 15.000 >20 >20 1.558 3.273 36.9F 18.000 >20 4.687 19.350 13.000 >20 6.984 >20 36.1F 0.248 0.755 >20 >20 >20 >20 >20 >20 10.4B >20 >20 >20 >20 >20 >20 >20 >20

    [0156] Neutralizing properties of LASV GP-specific hMAbs: The neutralizing properties of the LASV GP-specific hMAbs were evaluated using envelope-deficient core HIV-1 pseudotyped with LASV GP (LASVpp) (shown in Table 1) and standard plaque reduction neutralization test (PRNT) with authentic LASV. Fifteen of the 120 hMAbs neutralized LASVpp expressing GP from Josiah strain of LASV lineage IV, as shown in Table 1. These neutralizing GP-specific hMAbs were also tested against LASVpp containing GP of the three other LASV lineages I-III (shown in Table 1). The IC50 and IC80 values showed that those with the greatest potency and breadth against all four LASV linages were 25.10C, 12.1F, 8.9F, 37.2D, 37.7H, 25.6A and 8.11G (Table 1). The remaining hMAbs showed weaker and variable potency. Neutralization activity of these GP-specific hMAbs was further confirmed for LASV Josiah strain using a LCMV-based pseudovirus assay. These results revealed that out of the 120 tested LASV GP-specific hMAbs, 15 neutralized to different degrees LASV Josiah strain, and some of them exhibited broad neutralizing activity against representative strains from LASV lineages I-II.

    [0157] Cross-reactivity of LASV GP-specific hMAbs with LCMV ARM: The 16 LASV GP-specific hMAbs with neutralizing activity (as shown in Table 1) were characterized with respect their ability to recognize LCMV ARM strain GP expressed in human 293T cells transfected with GP-expressing plasmids by immunofluorescence. Human 293T cells transfected with LASV GPs from linages I-IV were included as controls. Nine of the LASV GP-specific neutralizing hMAbs (12.1F, 37.7H, 37.2D, 25.6A, 9.8A, 18.5C, 37.2G, 2.9D and 36.9F) cross-reacted with LCMV ARM GP.

    Example 3: Identification of LASV GP-Specific hMAbs with Broad Cross-Reactivity Against GPs from Different LCMV Strains

    [0158] The ability of LASV GP-specific neutralizing hMAbs (as shown by FIG. 1 and Table 1) to recognize GPs from five LCMV strains associated with human cases of LCMV-induced disease was examined. These strains corresponded to the WE strain that caused a zoonotic infection in New York in 1935; Rhode Island (RI) strain, responsible for four human cases and three fatalities from a transplant case in 2005; Ohio (OH) strain that is similar to the Michigan LCMV strain responsible for a human case in 2005; Wisconsin (WI) strain responsible for four human deaths in 2003; and Massachusetts (MA) strain, responsible for two human deaths in 2008. LASV GP-specific neutralizing hMAbs 12.1F, 37.2D, 9.8A, 18.5C and 36.9F recognized all five LCMV GP strains. LASV GP-specific neutralizing hMAbs 37.7H, 25.6A, 37.2G and 2.9D recognized four LCMV strains (ARM, WE, WI, and MA, but not RI or AH). The rest of the hMAbs did not cross-react with any of the LCMV strains tested.

    Example 4: Identification of LASV GP-Specific hMAbs with Strong Broadly Neutralizing Activity (BNhMAbs) Against GPs from Different LCMV Strains in Cell-Based Assays

    [0159] A validated cell-based microneutralization assay was used to identify LASV GP-specific hMAbs that not only cross-reacted with different LCMV GPs, but also neutralized LCMV ARM, as they would represent primary candidates to display broadly antiviral activity in vivo against LCMV strains previously associated with disease cases in humans. From the 15 LASV GP-specific neutralizing hMAbs, six of them (12.1F, 37.2D, 9.8A, 18.5C, 37.2G and 36.9F) neutralized LCMV ARM, as shown in FIG. 2, with IC50<1 g/mL, with the exception of 18.5C that exhibited a higher (>10 g/mL) IC50. Results are displayed in Table 2, which shows the neutralizing activity of the 15 LASV GP-specific neutralizing hMAbs against LCMV ARM, and in particular, the IC50 and IC80 values of the 15 LASV GP-specific neutralizing hMAbs against LCMV ARM. Values were obtained from the cell-based microneutralization assay (shown in FIG. 2) using LASV or LCMV GP-pseudotyped rLCMVGP/GFP viruses. Grey indicates LASV GP-specific neutralizing hMAbs that neutralized LCMV GP ARM. Neutralization of LASV GP-pseudotyped rLCMVGP/GFP was similar to neutralization results obtained using the LASV GP-pseudotyped lentivirus particles shown in Table 1.

    TABLE-US-00006 TABLE 2 Neutralizing activity of 15 LASV GP-specific neutralizing hMAbs against LCMV Armstrong strain (ARM). LASV Josiah (IV) LCMV ARM hMAb IC50 IC80 IC50 IC80 25.10C 0.160 0.247 >10 >10 12.1F 0.172 0.258 0.167 0.265 8.9F 0.134 >10 >10 >10 37.2D 0.137 0.260 0.518 2.358 37.7H 0.134 0.214 >10 >10 25.6A 0.188 0.300 >10 >10 9.8A 0.139 0.253 0.112 0.248 18.5C >10 >10 2.207 4.83 8.11G >10 >10 >10 >10 37.2G 0.405 1.776 0.525 2.461 2.9D 0.942 2.706 >10 >10 NE13 0.567 1.763 >10 >10 19.7E 1.189 >10 >10 >10 36.9F 0.570 2.228 0.591 3.328 36.1F 0.132 0.206 >10 >10 10.4B >10 >10 >10 >10

    Example 5: In Vivo Characterization of Selected GP-Specific BNhMAb

    [0160] The well-characterized mouse model of LCMV infection was used to test whether LASV GP-specific neutralizing hMAbs with broadly neutralizing activity against LCMV (shown in FIG. 2 and Table 2) also exhibited in vivo neutralizing activity. The immunosuppressive Clone 13 (Cl-13) strain of LCMV was used. Infection (i.v.) of B6 WT mice with a high dose (10.sup.6 PFU) of Cl-13 results in transient generalized immunosuppression and establishment of a persistent infection with well-established parameters. Virus clearance takes place between days 60 to 100 (post inoculation (p.i.). However, treatment of Cl-13 infected mice that results in reduced viral load accelerates Cl-13 clearance. Therefore, it was predicted that LASV GP-specific neutralizing hMAbs exhibiting in vivo neutralizing activity would either prevent the establishment of Cl-13 persistence or accelerate its clearance. Mice were treated with the indicated hMAbs at 20 mg/Kg intraperitoneally (i.p.) and were infected with either rCl-13/WT or rCl-13/LASV-GP(mCD). rCl-13/LASV-GP(mCD) was used because it contains mutations C459K and K461G within the cytosolic domain of GP that enhance persistence in mice. The in vivo results are shown in FIG. 3 and correlate with those previously documented in cultured cells (shown in FIG. 2 and Table 2). Mice treated with hMAbs 12.1F, 37.2D, 9.8A and 36.9F prevented persistence of rCl-13/WT. Unexpectedly, hMAbs 37.2G and 18.5C did not prevent Cl-13 persistence in vivo. As expected, based on cross-reactivity and neutralization results in cultured cells, hMAbs 19.7E and 8.9F did not prevent persistence of rCl-13.

    [0161] Table 3 displays a summary of the cross-reactivity and neutralizing activity in vitro and in vivo of LASV GP-specific hMAbs against six LCMV strains (ARM, WE, RI, OH, WI, and MA) tested.

    TABLE-US-00007 TABLE 3 Summary of the cross-reactivity and neutralizing activity in vitro and in vivo of LASV GP-specific hMAbs against LCMV. Cross-reactivity Neutralizing activity LCMV.sub.ARM LCMV.sub.WE LCMV.sub.RI LCMV.sub.OH LCMV.sub.WI LCMV.sub.MA In vitro In vivo 25.10C 8.9F 12.1F + + + + + + + + 37.7H + + + + 36.1F 8.11G 37.2D + + + + + + + + 25.6A + + + + 9.8A + + + + + + + + 18.5C + 37.2G + + + + + 19.7E 2.9D + + + + NE13 36.9F + + + + + + + + 10.4B

    Example 6: Assay Development

    [0162] A panel of murine antibodies against Fab or F(ab)2 fragments of leading candidate therapeutic BNhMAbs was derived for isolation of highly specific anti-idiotypic reagents for assay development. In order to develop a highly protective therapeutic BNhMAb cocktail containing two to four antibodies that together confer maximum pre- and post-exposure protection against LCMV infections, while minimizing the emergence of escape mutants, it is important to characterize the PK of each antibody when administered in a cocktail form. To distinguish between all BNhMAbs included in the cocktail after administration, highly specific anti-idiotypic antibodies are the best tool available to rapidly determine concentration and clearance of individual hMAbs from the blood. A panel of anti-idiotypic antibodies to 37.2D and 12.1F has been developed. Anti-idiotypic mMAbs to 37.2D have specifically detected this BNhMAb when spiked into human serum. The anti-idiotypic antibodies do not capture or detect any other arenaviral BNhMAb tested or any other IgG specificity present in human serum on both ELISA and SPR based studies, and thus are useful for assaying 37.2D.

    Example 7: Therapeutic Efficacy of First-In-Class Human LASV-Specific Antibodies in Guinea Pig (GP) and Cynomolgus Macaque (CM) Models of Lassa Fever

    [0163] These studies were done under BSL-4 biocontainment at the Galveston National Laboratory. Outbred Hartley strain GP were challenged i.p. with 1,000 pfu of GP adapted (GPA) LASV Josiah strain (N=5/group). This model has been described recently for testing therapeutics against LASV. The advantage of using outbred animals to model human infection is inferred from the higher variability of immune responses inherent in outbred populations. Viremia was compared by Kruskal-Wallis test supported by Dunn's Multiple comparison posttest (PRISM 5 software available from GraphPad Software, La Jolla, CA) to detect differences from the control group for time points relevant to onset (day 7) or peak viremia (day 14) as determined from historical data.

    [0164] Eleven LASV hMAbs tested in a Hartley GP model of LF segregated into three distinct protection groups: (1) 25.6A, 2.9D, 8.9F, 12.1F, and 37.7H conferred 100% protection and no change in clinical score in GPs. (2) 37.2D, 19.7E, and 37.2G protected 80 to 90% of animals. (3) 10.4B, 25.10C, and 36.1F, conferred 40%, 30%, and 20% protection, respectively. An irrelevant recombinant human isotype control (IgG1) Ab did not confer protection (0% survival).

    [0165] With respect to viremia, untreated control animals averaged 3.5 and 4.5 Log PFU/mL on days 7 and 14, respectively, as shown in FIG. 4. Despite 100% protection at the study endpoint, some animals from treatment group 8.9F or 37.7H, 2.9D, and 25.6A had low level viremia on day 7 or 14, respectively. Treatment groups where 90% protection was afforded (37.2D and 19.7E) had reduced mean viremia titers and minimal clinical score values. Treatment groups with 80% or less survival had comparable mean viremia titers to control animals on day 7, but by day 14 mean viremia was markedly lower than control animals. Groups with 80 to 90% survival exhibited relatively low mean clinical scores (FIG. 5) and all remaining treatment groups exhibited concomitant increases in mean clinical scores with decreases in survival per group. Endpoint viremia was not determined for these studies as survival was the primary metric of interest, though all surviving animals demonstrated no clinical signs.

    [0166] Results from the guinea pig studies informed studies for the Cynomolgus macaque (CM) model of LF. These studies demonstrated that several of the antibodies with high potency in the GP model also protected 100% of the CMs when administered on the day of challenge. 19.7E protected 75% of CMs. Notably a treatment dose as low as 6 mg/kg of hMAb 37.2D provided 100% protection in CMs, whereas 19.7E protected 75% of CMs. A cocktail of three human MAbs (37.2D, 12.1F, and 8.9F at 15 mg/kg each) rescued 100% of CMs even after delay in the start of treatment to 3, 6, or 8 days post-infection (therapeutic walk-out studies). At 8 days post-infection, untreated CMs had developed high viral loads and were extremely ill. CM also were protected from lethal LF induced by challenge with either strain Josiah (lineage IV) or a contemporary lineage II strain derived from a lethal case of LF in Nigeria, both with the first treatment administered at 8 days post-infection.

    Example 8: Structural Definition of the Anti-LASV 37.7H Epitope

    [0167] Monomeric GPCysR4 was incubated with excess Fab 37.7H and subjected to SEC-MALS analysis. SEC-MALS indicated the formation of trimeric GP-Fab complexes in addition to monomeric GP-Fab complexes. Crystals of both the monomeric and trimeric fractions of the GPCysR4-Fab 37.7H complex formed in space group P6122 and diffract to 3.2 with a trimer of GP bound to three Fabs in the asymmetric unit. Phases were determined with an iterative approach by using molecular replacement with a related Fab structure and the LCMV GP crystal structure.

    [0168] The antibody 37.7H against LASV neutralizes viruses representing all four known lineages of LASV in vitro and offers protection from lethal LASV challenge in guinea pig and nonhuman primates. The antibody simultaneously binds two GP monomers at the base of the GP trimer, where it engages four discontinuous regions of LASV GP, two in site A and two in site B. Site A contains residues 62 to 63 of the N-terminal loop of GP1 and residues 387 to 408 in the T-loop and HR2 of GP2. Site B contains residues 269 to 275 of the fusion peptide and residues 324 to 325 of HR1 of GP2. In total, 37.7H buries about 1620 .sup.2 of GP: about 1000 .sup.2 of GP at site A and about 620 .sup.2 of GP at site B. Although nearly the entire surface buried on GP belongs to GP2, the presence of both GP1 and GP2 is critical for 37.7H recognition, likely because GP1 is required to maintain the proper prefusion conformation of GP2 for 37.7H binding.

    [0169] The antibody 37.7H also recognizes the GPC of LCMV but does not recognize the GPC of the more distantly related Old World arenavirus LUJV nor the GPC of New World arenaviruses. A sequence comparison among these arenaviruses demonstrates nearly complete sequence conservation throughout the 37.7H epitope for all LASV lineages and LCMV. However, the sequences of LUJO, JUNV, and MACV GPCs are far more divergent, particularly in HR2 of GP2, which is heavily involved in binding to 37.7H. The 37.7H antibody neutralizes by stabilizing the prefusion GP.

    [0170] The quaternary nature and the involvement of the fusion peptide in the 37.7H epitope suggest that this antibody neutralizes the virus by stabilizing GPC in the prefusion conformation, thereby preventing the conformational changes required for infection. This was verified by analyzing the ability of LASV GP-pseudotyped recombinant vesicular stomatitis virus (rVSV-LASV GP) to mediate fusion with cell membranes.

    [0171] First the ability of 37.7H to neutralize rVSVLASV GP was determined. FIG. 6 shows the effect of antibodies on rVSV-LASV GP infection and fusion. Antibody-mediated neutralization of rVSV-LASV GP is shown in FIG. 6, Panel A. Antibody-mediated neutralization of rVSV-VSV-G is shown in FIG. 6, Panel B. The antibody 9.7A is non-neutralizing antibody and in the same competition group as 37.7H (GPC-B); 13.4E binds to a linear epitope in the T-loop of GP2; 12.1F binds to the GP1 subunit of LASV. Error bars indicate the standard deviation of at least six (two biological replicates, each having three or more technical replicates). FIG. 6, Panel C shows antibody-mediated inhibition of rVSVLASV GP fusion at the cell surface. Error bars indicate the standard error of the mean of six (except 37.7H, where N=9). FIG. 6, Panel D shows Fab 37.7H reduces binding of a LAMP1-Fc fusion protein to LASV GPCysR4. Error bars indicate the standard deviation of six and three technical replicates.

    [0172] 37.7H effectively prevented cellular infection by rVSV-LASV GP, as did the antibody 12.1F, which binds to the upper, 3-sheet face of LASV GP1 and is presumed to block cell attachment. In contrast, antibodies 13.4E, which binds a linear epitope in the T-loop, and 9.7A, which is a non-neutralizing GPC-B antibody, did not prevent viral infection (FIG. 6, Panels A and B).

    [0173] Next, the ability of 37.7H to prevent fusion of rVSV-LASV GP with cell membranes when exposed to low pH was examined. Unlike the non-neutralizing antibodies 9.7A and 13.4E, which were not effective in preventing fusion, 37.7H reduced fusion by nearly 80% compared with rVSV-LASV GP alone (FIG. 6, Panel C). In contrast, the neutralizing antibody against GP1 (anti-GP1), 12.1F, showed only a slight reduction in infectivity, suggesting that the effect of 37.7H was strictly due to disruption in fusogenicity of the GPC and not attachment to cells.

    [0174] Before exposure of the GP2 fusion peptide and loop and subsequent fusion of the viral and host cell membranes, LASV GP1 engages LAMP1. Engagement of this receptor is thought to require conformational changes in GP1 that are triggered by exposure to the low pH in the endosome. Tomography of LASV spikes in the presence of low pH and LAMP1 shows an opening of the trimer compared with its neutral pH conformation. To determine whether 37.7H could prevent these conformational changes, the ability of GPCysR4 to bind to a soluble LAMP1-Fc fusion alone and when bound to Fab 37.7H was analyzed. In the absence of Fab 37.7H, GPCysR4 effectively bound to LAMP1 when exposed to low pH. In the presence of Fab 37.7H, however, interaction between GPCysR4 and LAMP1 was markedly reduced (FIG. 6, Panel D).

    [0175] Based on crystallographic data, the footprint of 37.7H and the footprint of LAMP1 are separated by about 50 , and the angle adopted by the bound Fab fragments of 37.7H suggests that it is unlikely to sterically interfere with LAMP1. Thus, there are likely to be conformational changes in GP1 required for LAMP1 binding that are prevented by this human survivor antibody. Taken together, these results demonstrate that the probable mechanism of action for 37.7H and probably for other antibodies in its potent GPC-B competition group is stabilization of the prefusion GPC trimer and prevention of the conformational changes required for binding of LAMP1 and triggering of the GP2 fusion peptide and fusion loop in the endosome.

    [0176] Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the claims.

    [0177] The following reference articles are incorporated herein by reference.

    REFERENCES

    [0178] 1. Auperin, D. D., Sasso, D. R. and McCormick, J. B. (1986). Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA. Virology 154, 155-167. [0179] 2. Beyer, W. R., Popplau, D., Garten, W., von Laer, and Lenz O. (2003). Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the sibtilase SKI-1/S1P. J. Virol. 77, 2866-2872. [0180] 3. Buchmeier, M. J. (2002). Arenaviruses: protein structure and function. Curr. Top. Microbiol. Immunol. 262, 259-173. [0181] 4. Buchmeier, M. J., and Parekh, B. S. (1987). Protein structure and expression among arenaviruses. Current Topics in Microbiology and Immunology 133, 41-57. [0182] 5. Buchmeier, M. J., Lewicki, H. A., Tomor, O., and Jonhson, K. M. (1980). Monoclonal antibodies to lymphocytic choriomeningitis virus reacts with pathogenic arenaviruses. Nature, London 288, 4876-4877. [0183] 6. Burnette, W. N. (1981). Western Blotting: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112, 195-203. [0184] 7. Clegg, J. C. and Lloyd, G. (1983). Structureal and cell-associated proteins of Lassa virus. Journal of General Virology 64, 1127-1136. [0185] 8. Eichler, R., Lenz, O., Strecker, T., Eickmann, M., Klenk, H. D., and Garten, W. (2004). Lassa virus glycoprotein signal peptide displays a novel topology with an extended ER-luminal region. J. Biol. Chem. 279, 12293-12299. [0186] 9. Eichler, R., Lenz, O., Strecker, T., Eickmann, M., Klenk, H. D., and Garten, W. (2003). Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor. EMBO Rep. 4, 1084-1088. [0187] 10. Eichler, R., Lenz, O., Strecker, T., Eickmann, and Garten, W. (2003). Signal peptide of Lassa virus glycoprotein GP-C exhibits an unusual length. FEBS Lett. 538, 203-206. [0188] 11. Elagoz, A., Benjannet, S., Mammarbassi, A., Wickham, L., and Seidah, N. G. (2002). Biosynthesis and cellular trafficking of the convertase SKI-1/S1P: ectodomain shedding requires SKI-1 activity. J. Biol. Chem. 277, 11265-11275. [0189] 12. Hufert, F. T., Ludke, W., and Schmitz, H. (1989). Epitope mapping of the Lassa virus nucleocapsid protein using monoclonal anti-nucleocapsid antibodies. Archives of Virology 106, 201-212. [0190] 13. Lenz, O., ter Meulen, J., Feldmann, H., Lenk, H.-D., and Garten, W. (2000). Identification of a novel consensus sequence at the cleavage site of the Lassa virus glycoprotein. J. Virol. 74, 11418-11421. [0191] 14. Lukashevich L. S., Clegg J. C., and Sidibe K. (1993). Lassa virus activity in Guinea: distribution of human antiviral antibody defined using enzyme-linked immunosorbent assay with recombinant antigen. J Med Virol. 40, 210-7. [0192] 15. McCormick, J. B., and Fisher-Hoch, S. P. (2002). Lassa Fever. Curr. Top. Microbiol. Immunol. 262, 75-109. [0193] 16. Ruo, S. L., Mitchell, S. W., Killey, M. P., Roumillat, L. F., Fisher-Hoch, S. P., and McCormick, J. B. (1991). Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies. Journal of General Virology 72, 549-555. [0194] 17. Sanchez, A., Pifat, D. Y., Kenyon, R. H., Peters, C. J., McCormick, J. B., and Kiley, M. P. (1989). Junin virus monoclonal antibodies: characterization and cross-reactivity with other arenaviruses. J. Gen. Virol. 70, 1125-1132. [0195] 18. Spiropoulou, C. F., Kunz, S., Rollin, P. E., Campbell, K. P., and Oldstone, M. B. A. (2002). New World arenavirus clade C, but not clade A and B viruses, utilizes a-dystroglycan as its major receptor. J. Virol. 76, 5140-5146. [0196] 19. ter Meulen J., Badusche M., Kuhnt K., Doetze A., Satoguina J., Marti T., Loeliger C., Koulemou K., Koivogui L., Schmitz H., Fleischer B., and Hoerauf A. (2000). Characterization of human CD4(+) T-cell clones recognizing conserved and variable epitopes of the Lassa virus nucleoprotein. J. Virol. 74, 2186-92. [0197] 20. ter Meulen, J., Koulemou K., Wittekindt T., Windisch K., Strigl S., Conde S., and Schmitz H. (1998). Detection of Lassa Virus Antinucleoprotein Immunoglobulin G (IgG) and IgM Antibodies by a Simple Recombinant Immunoblot Assay for Field Use. J. Clin. Microbiol. 36, 3143-3148. [0198] 21. York, J., Agnihothram, S. S., Ronamowski, V., and Nunberg, J. H. (2005). Genetic analysis of heptad-repeat regions in the G2 fusion subunit of the Junin arenavirus envelope glycoprotein. Virology 343, 267-279. [0199] 22. York, J., Ronamowski, V., Lu, M., and Nunberg, J. H. (2004). The signal peptide of the Junin arenavirus envelope glycoprotein is myristoylated and forms an essential subunit of the mature G1-G2 complex. J. Virol. 78, 10783-10792. [0200] 23. Shaffer J G, Grant D S, Schieffelin J S, Boisen M L, Goba A, et al. 2014. Lassa Fever in Post-Conflict Sierra Leone. PLoS Negl Trop Dis 8: e2748. [0201] 24. Hartnett J N, Boisen M L, Oottamasathien D, Jones A B, Millett M M, . . . Garry R F, Branco L M & the VHFC (2015). Current and emerging strategies for the diagnosis, prevention and treatment of Lassa fever. Future Virology. Review. Vol. 10, No. 5, Pages 559-584. [0202] 25. Andersen K G, Shapiro B J, Matranga C B, Sealfon R, Lin A E, . . . Branco L M, Gire S K, Phelan E, Tariyal R, Tewhey R, . . . Garry R F, Sabeti P C. Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus. Cell. 2015 Aug. 13; 162(4):738-50. [0203] 26. Luis M Branco, Jessica N Grove, Matt L Boisen, Jeffrey G Shaffer, Augustine Goba, . . . Robert F Garry. Emerging trends in Lassa fever: redefining the role of immunoglobulin M and inflammation in diagnosing acute infection. Virology Journal 2011, 8:478 (24 Oct. 2011). [0204] 27. Jessica N Grove, Luis M Branco, Matt L Boisen, Ivana J Muncy, Lee A Henderson, . . . Robert F Garry. Capacity building permitting comprehensive monitoring of a severe case of Lassa hemorrhagic fever in Sierra Leone with a positive outcome: Case Report. Virology Journal 2011, 8:314 (20 Jun. 2011). [0205] 28. Luis M Branco, Jessica N Grove, Frederick J Geske, Matt L Boisen, Ivana J Muncy, . . . Robert F Garry. Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever. Virology Journal 2010, 7:279 (20 Oct. 2010). [0206] 29. Luis M Branco, Matt L Boisen, Kristian G Andersen, Jessica N Grove, Lina M Moses, . . . Robert F Garry. Lassa Hemorrhagic Fever in a Late Term Pregnancy from Northern Sierra Leone with a Positive Maternal Outcome: Case Report. Virology Journal 2011, 8:404 (15 Aug. 2011).