Aarenavirus monoclonal antibody and application

Abstract

The invention relates to an arenavirus monoclonal antibody and application thereof. Disclosed herein are compositions comprising arenavirus monoclonal antibodies, as well as therapeutic, diagnostic and prophylactic methods using the novel antibodies. The prophylaxis methods include the preparation of vaccines, and factors (e.g., small molecules, peptides) that inhibit the infection of old world arenavirus, including LASV and LCMV. In some embodiments, the antibodies provide pan arenavirus protection against a number of arenavirus types and strains. Also disclosed are diagnostic and therapeutic antibodies including neutralizing antibodies for the prevention and treatment of LASV and other arenavirus infections, as well as novel tools and methods for designing, producing, and using arenavirus monoclonal antibodies, including expression in engineered bacterial-based and mammalian-based systems.

Description

[0001] This application is a divisional application of Chinese patent application 201780085558.6 “Monoclonal antibodies to arenaviruses and uses” filed on December 5, 2017. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Serial No. 62 / 430,225, filed December 5, 2016, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made in part with U.S. government support under Grant Nos. U19AI109762, 1R01AI104621, R43 AI120472 awarded by the National Institutes of Health, and NIAID Project No. 272200900049C-0-0-1. The government may have certain rights in this invention. Technical Field

[0003] The present invention relates to novel arenavirus monoclonal antibodies, compositions comprising arenavirus monoclonal antibodies, and methods comprising the same.

[0004] Incorporation of Sequence Listing The biological sequence information of the present application is included in an ASCII text file with the file name "TU439SEQ.txt", created on December 4, 2017 and having a file size of 120,885 bytes, which is incorporated herein by reference. Background Art Lassa virus (LASV) and several other members of the Arenaviridae family are classified as Biosafety Level 4 and NIAID Biodefense Category A pathogens. The present invention will meet important biodefense needs for rapid multi-reagent immunodiagnostic assays for arenaviruses and for effective therapeutics against arenavirus diseases, and will provide a major advance in the public health management of this important family of viral pathogens. Several arenaviruses, primarily the West African Lassa virus (LASV), cause hemorrhagic fever (HF) disease in humans and pose a serious public health concern in areas where they are endemic. The prototypical arenavirus, lymphocytic choriomeningitis virus (LCMV), is not the causative agent of HF, but there is growing evidence that LCMV is a neglected clinically significant human pathogen that can cause neurological disease in the fetal, pediatric, and adult stages. In addition, LCMV poses a particular threat in immunocompromised individuals, as tragically exemplified by recent cases of transplant-associated infection with LCMV in the United States and Australia with fatal outcomes. Furthermore, the high seroprevalence of LCMV within various urban populations around the world, including the United States, has raised the question of whether LCMV may contribute to the many undiagnosed cases of aseptic meningitis reported each year.

[0006] Lassa fever. The most prevalent arenavirus disease is Lassa fever (LF), an often fatal hemorrhagic fever named after a town in Nigeria where the first described case occurred in 1969 (Buckley and Casals, 1970). Guinea, Sierra Leone, Nigeria, and parts of Liberia are endemic for the pathogen LASV (Birmingham and Kenyon, 2001). Although detailed surveillance of LASV is hampered by many factors, including the lack of widely available diagnostic tests, it is clear that the public health impact is enormous. There are as many as 300,000 cases of Lassa fever and 5,000 deaths each year in West Africa (see the CDC website www (dot) cdc (dot) gov / ncidod / dvrd / spb / mnpages / dispages / lassaf (dot) htm). In some areas of Sierra Leone, 10-16% of all patients admitted to hospitals have Lassa fever. The case fatality rate for Lassa fever has been generally reported as 15% to 20%, and as high as 45% during epidemics, with a recent multi-year study in Sierra Leone reporting a 69% rate (Schaffer et al., 2014). LASV has been associated with severe nosocomial outbreaks involving health care workers and laboratory personnel (Fisher-Hoch et al., 1995). The mortality rate for women in the last month of pregnancy is always high, approximately 90%, and LASV infection causes high fetal mortality at all stages of pregnancy (Walls, 1985). The mortality rate for Lassa fever appears to be higher in non-Africans, which is important because Lassa fever is the most commonly exported hemorrhagic fever (Haas et al., 2003; Holmes et al., 1990).

[0007] Old and New World Arenaviruses. Arenaviruses are enveloped viruses with a two-segment negative-strand (NS) RNA genome. Each genomic RNA segment, L (about 7.3 kb) and S (about 3.5 kb), uses an ambisense coding strategy to direct the synthesis of two polypeptides in opposite directions separated by a noncoding intergenic region. S RNA encodes the viral glycoprotein precursor (GPC) and the nucleoprotein (NP). GPC is co-translationally and post-translationally processed to give two mature virion surface glycoproteins, GP1 and GP2, which together with a stable signal peptide (SSP) form the GP complex, which decorates the viral surface and directs viral cellular entry via receptor-mediated endocytosis. L RNA encodes the viral RNA-dependent RNA polymerase (L polymerase) and the small RING finger protein Z that has the function of a true matrix protein. The structure of arenavirus GP2 appears to be a class I fusion protein, which is common to the envelope glycoproteins of myxoviruses, retroviruses, and filoviruses (Gallaher, DiSimone and Buchmeier, 2001). When observed by transmission electron microscopy, the enveloped spherical virus particles (diameter: 110-130 nm) show granular particles, which are ribosomes obtained from the host cell (Murphy and Whitfield, 1975), thus the basis of the family name from the Latin word "arena", which means "sandy". Arenaviruses are divided into the Old World or lymphocytic choriomeningitis virus (LCMV) / LASV complex and the New World or Tacaribe complex (Bowen, Peters and Nichol, 1997). There is considerable diversity among the members of the Arenaviridae family ( Figure 1), and there is considerable variation even within the same virus species (Bowen et al., 2000). In addition to LASV, other arenaviruses that cause severe disease in humans and are classified as BSL-4 and NIAIDA class pathogens include the New World Arenaviruses Machupo virus (MACV, Bolivian hemorrhagic fever), Junin virus (JUNV, Argentine hemorrhagic fever), Guanarito virus (GUAV, Venezuelan hemorrhagic fever) and Sabia virus (SABV, Brazilian hemorrhagic fever). Arenaviruses are zoonotic; each virus is associated with a specific species of rodents (Bowen, Peters and Nichol, 1997). The LCMV / LASV complex is associated with Old World rats and mice (Muridae, Murinae). The Tacaribe complex is usually associated with New World rats and mice (Muridae, Cotton Ratinae); however, the reservoir of the Tacaribe virus itself appears to be bats (Bowen, Peters and Nichol, 1996). The reservoir of LASV is the "polymammate rat" of the genus Rattus (Monath et al., 1974). Rat species are ubiquitous in sub-Saharan Africa (Demby et al., 2001) and are known to be peridomestic, often living in human homes; however, many questions about the taxonomy, geographic distribution, and ecology of Rattus species remain unanswered. Like the natural hosts of other arenaviruses, Rattus does not show symptoms of LASV infection but sheds virus in saliva, urine, and feces. Eradication of widespread rodent reservoirs of LASV and other arenaviruses is impractical and ecologically undesirable.

[0008] Arenaviruses cause chronic infections of rodents worldwide, with human infection occurring primarily through mucous membrane exposure or direct contact with infectious material through abraded skin. Arenaviruses are easily transmitted from rodents to humans via direct contact with rodent excreta or contact with excreta-contaminated material or its ingestion (Bausch et al., 2001; Demby et al., 2001). In the case of species of the genus Rattus, infection may also occur when the animals are captured, prepared as a food source, and eaten. Most arenaviruses, including LASV, are easily transmitted between humans, making hospital infections another issue of great concern. Human-to-human transmission can occur via exposure to blood or body fluids. LASV can also be transmitted to the sexual partners of convalescent males via semen up to six weeks after infection.

[0009] Natural history of Lassa fever. The signs and symptoms of Lassa fever vary widely, occurring 1-3 weeks after viral exposure, but generally begin with an insidious onset of fever and other nonspecific symptoms (such as headache, general weakness and malaise), followed by sore throat, retrosternal pain, conjunctival injection, abdominal pain and diarrhea within a few days. LASV infects endothelial cells, resulting in increased capillary permeability, which can produce a decrease in effective circulating volume (Peters et al., 1989). Severe cases progress to swelling of the face and neck, shock and multi-organ system failure. Frank's bleeding, usually mucous membranes (gingivae, etc.), occurs in less than one-third of cases, but confers a poor prognosis. Neurological problems have also been described, including hearing loss, tremors and encephalitis. Surviving patients begin to defervesce 2-3 weeks after the onset of the disease. Transient or permanent unilateral or bilateral deafness that occurs in one-third of Lassa fever patients during recovery is not related to the severity of the acute illness (Cummins et al., 1990; Rybak, 1990; Hartnet et al., 2015; Anderson et al., 2015; Branco et al., October 2011; Grove et al., 2011; Branco et al., 2010; Branco et al., August 2011).

[0010] Potential for using arenaviruses as biological weapons. In addition to high case fatality rates, arenaviruses have a number of features that enhance their potential as biological weapons. Arenaviruses have relatively stable virions, do not require passage via insect vectors, are easily transmitted by person-to-person contact, and are capable of aerosol diffusion or other simple modes of dispersion. The high prevalence of Lassa fever in West Africa, together with the ease of travel to and from that region and to areas where MACV, JUNV, GUAV, SABV, and other highly pathogenic arenaviruses are endemic, allows for easy access to these viruses for use as biological weapons. A cluster of hemorrhagic fever cases in the United States caused by any arenavirus would be a major public health event. Because febrile illnesses are common and the availability of reliable arenavirus diagnostic tests is not common, an initial cluster of undiagnosed cases would greatly increase the impact of an attack and allow for more widespread spread via person-to-person contact. The potential use of LASV and other arenaviruses as biological weapons against civilian or military targets has been demonstrated through the marketing of an immunodiagnostic test for the rapid detection of LASV infection (the ReLASV Rapid Diagnostic Test [RDT]). TM ,RePanLASV RDT TM ) and companion ELISA diagnostics for detecting antigenemia and immunoglobulin (Ig) M (IgM) and G (IgG) responses to infection, complementing the commercial development of validated diagnostics already completed by VHFC (www(dot)zalgenlabs(dot)com / products).

[0011] Treatment / Prevention of Arenavirus Infections. There is no Food and Drug Administration (FDA)-approved arenavirus vaccine, and current anti-arenavirus therapy is limited to the off-label use of the antiviral drug ribavirin, which is only partially effective and can cause significant side effects. Ribavirin is effective in treating Lassa fever only when administered early in the course of the disease (Johnson et al., 1987; McCormick et al., 1986). Ribavirin administered within the first six days of illness to patients with high viral loads (and therefore high risk of death) reduced the case fatality rate from 55% to 5% (McCormick et al., 1986). Several anecdotal reports suggest that the drug is also effective against other arenavirus hemorrhagic fevers (Barry et al., 1995; Kilgore et al., 1997; Weissenbacher et al., 1986a; Weissenbacher et al., 1986b). The efficacy of preventive treatment of Lassa fever is unknown, although oral ribavirin treatment has been proposed for people with high risk exposure. The control of LCMV infection is mainly mediated by cellular immune response, and significant titers of neutralizing antibodies against LCMV usually only appear after the patient has clinically recovered. However, passive antibody transfer has been shown to confer protection in animal models of LCMV infection (Enria et al., 1984; Frame et al., 1984; Jahrling, 1983; Jahrling and Peters, 1984; Jahrling, Peters and Stephen, 1984; Weissenbacher et al., 1986a). Therefore, antibody-based therapy can provide a safer alternative for the treatment of LCMV based on a predetermined protective correlation. Previous studies of passive transfer of serum to treat Argentine hemorrhagic fever (AHF) and Lassa fever provide a strong rationale for the method disclosed herein. Although the passive transfer of serum has been shown to be effective for New World and Old World viruses, this method cannot be expanded to protect a large number of people under the assumption that these viruses are released. Another concern is the safety of transfused serum or plasma, particularly for those living in areas where circulating unknown pathogens are a concern. Recombinant, neutralizing human antibodies have never been tested as potential therapeutics in arenavirus-induced HF, but these limitations may be overcome. No arenavirus vaccines are currently available, although vaccines against LASV and JUNV are in development. Effective diagnostic assays are absolutely necessary for the development and field testing of arenavirus vaccines.

[0012] Antibody-based therapy against human viral infection. Viral antigen variability can pose a major obstacle to the development of effective vaccines against human viral infection, as exemplified by the cases of HIV and influenza virus. It is noteworthy that recent findings have shown that some infected individuals produce broadly neutralizing monoclonal antibodies (BNhMAbs) that target the conserved domains in the stem region of the viral surface envelope (Env) glycoprotein of HIV-1, and can block infection by many phylogenetic unique isolates. Similarly, it has been shown that many BNhMAbs target the conserved domains in the proximal membrane stem domain of influenza virus hemagglutinin (HA), and when passively administered in a mouse model of influenza virus infection, several BnhMAbs such as MAbF16 and MAb 5A7 prove to be protective. Antibodies generally exhibit desired pharmacological characteristics, including long serum half-life, high efficacy and limited off-target toxicity. Therefore, the latest developments in the field of BnhMAbs have generated great interest in exploring their development as viable antiviral therapies. Furthermore, because BNhMAbs often recognize conserved epitopes within regions of viral glycoproteins that mediate fusion between viral and cellular membranes, they may also facilitate the identification and structural characterization of highly conserved viral epitopes, knowledge that could be used to generate universal vaccines and broad-spectrum antiviral drugs against these viral pathogens. As with HIV-1 and influenza viruses, arenavirus cell entry requires a pH-dependent fusion event mediated by the fusion domain of GP2. The identification and characterization of LCMVGP-specific BNhMAbs will facilitate the development of novel antibody-based therapeutics to treat LASV- and LCMV-induced disease in humans. Furthermore, this work could generate valuable information for the design of immunogens to facilitate the development of universal arenavirus vaccines, as well as broad-spectrum antiviral drugs that target the conserved structural and functional motifs identified by the BNhMAbs.

[0013] Need for the invention. The work described herein combines the use of prior art arenavirus reverse genetics and the acquisition of a unique collection of LASV GP-specific human monoclonal antibodies (hMAbs) that have been shown to cross-react and neutralize different strains of LCMV, including isolates from human cases of LASV- and LCMV-induced disease, and WE strains that cause LF-like disease in non-human primates. The present disclosure provides antibody-based therapies to treat human cases of LCMV-induced disease. Unlike vaccines that rely on the host's ability to generate an effective immune response, this novel approach can provide protection in immunosuppressed individuals, including cases of LASV and LCMV infection associated with severe clinical symptoms in individuals undergoing transplant surgery. In addition, detailed characterization of conserved epitopes within the LCMV GPC recognized by these BNhMAbs can help design immunogens aimed at developing vaccines capable of conferring protection against all LASV and LCMV strains associated with disease in humans. In addition, information obtained from the identification and characterization of LASV BNhMAbs will help identify broad-spectrum anti-LASV and LCMV drugs by targeting conserved epitopes identified by these BNhMAbs. The experimental approach described herein involves the use of unique reagents and assays to identify and characterize LASV and LCMV BNhMAbs and their targets.

[0014] There is a need to address LASV and LCMV infections from natural sources, as well as weaponized versions of these viruses. There is also a need for neutralizing antibodies against LASV and LCMV for diagnostic and analytical purposes. The materials (e.g., antibodies and fragments thereof) and methods described herein address these needs. Summary of the invention A single-cycle infectious, GFP-expressing rLCMV (rLCMVΔGP / GFP) in which GP is replaced by GFP has been generated. Genetic complementation with a plasmid or stable cell line expressing the target arenavirus GP results in the production of the corresponding GP-pseudotype rLCMVΔGP / GFP, which is used to evaluate neutralizing antibody responses to different LCMV strains using a novel GFP-based microneutralization assay. A three-segment LCMV platform has been developed within the backbone of the ARM or Cl-13 LCMV strains, which allows the co-expression of the selected arenavirus GP and appropriate reporter genes (e.g., fluorescence and luciferase proteins) for easy identification of LCMV BNhMAbs and monitoring the emergence of BNhMAb LCMV escape mutants. A reverse genetic approach has been developed that allows the generation of rLCM viruses within the backbone of the immunosuppressive Cl-13 LCMV strain expressing the target GP, which can be used to characterize the in vivo therapeutic value of these BNhMAbs. Highly specific anti-idiotypic antibodies were generated to individually detect and characterize the PK, concentration, and clearance from the circulation of each MAb used in combination therapy to enhance neutralization efficacy while minimizing the emergence of escape mutants. A panel of anti-idiotypic antibodies (anti-ids) against 37.2D specifically detected this BNhMAb when spiked into human serum and did not capture or detect any other arenavirus BNhMAb tested to date, or any other IgG specifically present in human serum in ELISA- and SPR-based studies.

[0016] Disclosed herein are compositions comprising monoclonal antibodies to arenaviruses (e.g., fully human monoclonal antibodies), and therapeutic, diagnostic, and prophylactic methods using the antibodies. Prophylactic methods include the preparation of vaccines, as well as factors (e.g., small molecules, peptides) that inhibit infectivity of Old World arenaviruses (including LASV and LCMV). Also disclosed are diagnostic and therapeutic antibodies, including neutralizing antibodies for the prevention and treatment of LASV and other arenavirus infections, and new tools and methods for the design, production, and use of monoclonal antibodies to arenaviruses, including expression in engineered bacterial-based systems and mammalian-based systems.

[0017] One embodiment of the materials and methods described herein relates to monoclonal antibodies and fragments thereof effective against LASV.

[0018] Another embodiment of the materials and methods described herein relates to monoclonal antibodies or fragments thereof effective against LCMV.

[0019] Another embodiment of the materials and methods described herein relates to methods of generating monoclonal antibody forms effective against LASV and / or LCMV.

[0020] Another embodiment of the materials and methods described herein relates to an expression vector comprising a polynucleotide encoding a LASV or LCMV GP-specific hMAb form.

[0021] One embodiment of the materials and methods described herein relates to the diagnostic use of antibodies or fragments thereof, such as neutralizing antibodies, specific for LASV or LCMV.

[0022] Another embodiment of the materials and methods described herein relates to a diagnostic agent comprising an antibody or fragment thereof specific for LASV or LCMV, including a labeled antibody or fragment thereof of the invention.

[0023] Another embodiment of the materials and methods described herein relates to a kit comprising an antibody of the invention.

[0024] The following non-limiting embodiments are provided to illustrate certain aspects and features of the materials and methods described herein.

[0025] Embodiment 1 is an antigen-binding composition comprising a neutralizing antibody or a neutralizing antigen-binding antibody fragment thereof specific for glycoprotein 1 (GP1), glycoprotein 2 (GP2), glycoprotein precursor (GPC) or full-length glycoprotein (GP) of Lassa virus (LASV), wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) and light chain variable region (V L ), the V H and V L Each comprises a complementarity determining region CDR1, CDR2 and CDR3 selected from the group consisting of: (a) V of SEQ ID NO: 65 H CDR1, V of SEQ ID NO:66 H CDR2, V of SEQ ID NO:67 H CDR3, V of SEQ ID NO: 113 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 114 L CDR3 (from MAb 10.4B); (b) V of SEQ ID NO: 68 H CDR1, V of SEQ ID NO:69 H CDR2, V of SEQ ID NO:70 H CDR3, V of SEQ ID NO: 115 L CDR1, sequence Lys Ala Ser V LCDR2 and V of SEQ ID NO: 116 L CDR3 (from MAb 19.7E); (c) V of SEQ ID NO: 71 H CDR1, V of SEQ ID NO:72 H CDR2, V of SEQ ID NO:73 H CDR3, V of SEQ ID NO: 117 L CDR1, sequence Trp Ala Ser V L CDR2 and V of SEQ ID NO: 118 L CDR3 (from MAb 2.9D); (d) V of SEQ ID NO: 74 H CDR1, V of SEQ ID NO:75 H CDR2, V of SEQ ID NO:76 H CDR3, V of SEQ ID NO: 119 L CDR1, sequence Glu Val Lys V L CDR2 and V of SEQ ID NO: 120 L CDR3 (from MAb 25.6A); (e) V of SEQ ID NO: 77 H CDR1, V of SEQ ID NO:78 H CDR2, V of SEQ ID NO: 79 H CDR3, V of SEQ ID NO: 121 L CDR1, sequence Asp Ala Ser V L CDR2 and V of SEQ ID NO: 122 L CDR3 (from MAb 36.1F); (f) V of SEQ ID NO: 80 H CDR1, V of SEQ ID NO:81 H CDR2, V of SEQ ID NO:82 H CDR3, V of SEQ ID NO: 123 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 124 L CDR3 (from MAb 36.9F); (g) V of SEQ ID NO: 83 H CDR1, V of SEQ ID NO:84 H CDR2, V of SEQ ID NO:85 H CDR3, V of SEQ ID NO: 125 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 126 L CDR3 (from MAb 37.2D); (h) V of SEQ ID NO: 86 H CDR1, V of SEQ ID NO:87 H CDR2, V of SEQ ID NO:88 H CDR3, V of SEQ ID NO: 127 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 128 L CDR3 (from MAb 37.2G); (i) V of SEQ ID NO: 89 H CDR1, V of SEQ ID NO:90 H CDR2, V of SEQ ID NO:91 H CDR3, V of SEQ ID NO: 129 L CDR1, sequence Glu Val Arg V L CDR2 and V of SEQ ID NO: 130 L CDR3 (from MAb 37.7H); (j) V of SEQ ID NO:92 H CDR1, V of SEQ ID NO:93 H CDR2, V of SEQ ID NO:94 H CDR3, V of SEQ ID NO: 131 L CDR1, sequence Glu Val Ser V L CDR2 and V of SEQ ID NO: 132 L CDR3 (from MAb 8.9F); (k) V of SEQ ID NO:95 H CDR1, V of SEQ ID NO:96 HCDR2, V of SEQ ID NO:97 H CDR3, V of SEQ ID NO: 133 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 134 L CDR3 (from MAb NE13); (1) V of SEQ ID NO:98 H CDR1, V of SEQ ID NO:99 H CDR2, V of SEQ ID NO: 100 H CDR3, V of SEQ ID NO: 135 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 136 L CDR3 (from MAb 12.1F); (m) V of SEQ ID NO: 101 H CDR1, V of SEQ ID NO: 102 H CDR2, V of SEQ ID NO: 103 H CDR3, V of SEQ ID NO: 137 L CDR1, sequence Gln Ala Ser V L CDR2 and V of SEQ ID NO: 138 L CDR3 (from MAb 9.8A); (n) V of SEQ ID NO: 104 H CDR1, V of SEQ ID NO: 105 H CDR2, V of SEQ ID NO: 106 H CDR3, V of SEQ ID NO: 139 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 140 L CDR3 (from MAb 18.5C); (o) V of SEQ ID NO: 107 H CDR1, V of SEQ ID NO: 108 H CDR2, V of SEQ ID NO: 109 H CDR3, V of SEQ ID NO: 141 LCDR1, sequence Gly Ala Tyr V L CDR2 and V of SEQ ID NO: 142 L CDR3 (from MAb 8.11G); and (p) V of SEQ ID NO: 110 H CDR1, V of SEQ ID NO: 111 H CDR2, V of SEQ ID NO: 112 H CDR3, V of SEQ ID NO: 143 L CDR1, sequence Ala Ala Val V L CDR2 and V of SEQ ID NO: 144 L CDR3 (from MAb 25.10C).

[0026] Embodiment 2 is the composition of embodiment 1, wherein the composition comprises two or more of said antibodies or antigen-binding antibody fragments.

[0027] Embodiment 3 is the composition of any one of embodiments 1 and 2, wherein the composition comprises: (1) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 83 H CDR1, V of SEQ ID NO:84 H CDR2, V of SEQ ID NO:85 H CDR3, V of SEQ ID NO: 125 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 126 L CDR3 (from MAb 37.2D); (2) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 92 H CDR1, V of SEQ ID NO:93 H CDR2, V of SEQ ID NO:94 H CDR3, V of SEQ ID NO: 131 L CDR1, sequence Glu Val Ser V L CDR2 and V of SEQ ID NO: 132 L CDR3 (from MAb 8.9F); and (3) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 98 HCDR1, V of SEQ ID NO:99 H CDR2, V of SEQ ID NO: 100 H CDR3, V of SEQ ID NO: 135 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 136 L CDR3 (from MAb 12.1F).

[0028] Embodiment 4 is the composition of any one of embodiments 1 to 3, wherein the antibody is selected from the group consisting of a monoclonal antibody and a recombinantly produced antibody.

[0029] Embodiment 5 is the composition of any one of embodiments 1 to 4, wherein the antibody comprises a human monoclonal antibody.

[0030] Embodiment 6 is the composition of any one of embodiments 1 to 3, wherein the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

[0031] Embodiment 7 is a V sequence encoding an antibody or antibody fragment of the composition of embodiment 1 H A nucleic acid (e.g., cDNA) having a sequence.

[0032] Embodiment 8 is the nucleic acid of embodiment 7, wherein the nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:16.

[0033] Embodiment 9 is a V sequence encoding the antibody or antibody fragment of embodiment 1 L A nucleic acid (e.g., cDNA) having a sequence.

[0034] Embodiment 10 is the nucleic acid of embodiment 9, wherein the nucleic acid comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:17 to SEQ ID NO:32.

[0035] Embodiment 11 is an expression vector containing the nucleic acid sequence of any one of embodiments 7 to 10.

[0036] Embodiment 12 is an antigen-binding composition comprising a neutralizing antibody or a neutralizing antigen-binding antibody fragment thereof specific for glycoprotein 1 (GP1), glycoprotein 2 (GP2), glycoprotein precursor (GPC) or full-length glycoprotein (GP) of Lassa virus (LASV), wherein the antibody or antibody fragment comprises a heavy chain variable region (V H ) and light chain variable region (V L ): (a) V of SEQ ID NO: 33H and V of SEQ ID NO:49 L (from MAb 10.4B); (b) V of SEQ ID NO: 34 H and V of SEQ ID NO:50 L (from MAb 19.7E); (c) V of SEQ ID NO: 35 H and V of SEQ ID NO:51 L (from MAb 2.9D); (d) V of SEQ ID NO: 36 H and V of SEQ ID NO:52 L (from MAb 25.6A); (e) V of SEQ ID NO: 37 H and V of SEQ ID NO:53 L (from MAb 36.1F); (f) V of SEQ ID NO: 38 H and V of SEQ ID NO:54 L (from MAb 36.9F); (g) V of SEQ ID NO: 39 H and V of SEQ ID NO:55 L (from MAb 37.2D); (h) V of SEQ ID NO: 40 H and V of SEQ ID NO:56 L (from MAb 37.2G); (i) V of SEQ ID NO: 41 H and V of SEQ ID NO:57 L (from MAb 37.7F); (j) V of SEQ ID NO:42 H and V of SEQ ID NO:58 L (from MAb 8.9F); (k) V of SEQ ID NO:43 H and V of SEQ ID NO:59 L (from MAb NE13); (1) V of SEQ ID NO:44 H and V of SEQ ID NO:60 L (from MAb 12F); (m) V of SEQ ID NO:45 H and V of SEQ ID NO:61 L (from MAb 9.8A); (n) V of SEQ ID NO:46 H and V of SEQ ID NO:62 L (from MAb 18.5C); (o) V of SEQ ID NO:47 H and V of SEQ ID NO:63 L (from MAb 8.11G); and (p) V of SEQ ID NO: 48 H and V of SEQ ID NO:64 L (from MAb 25.10C).

[0037] Embodiment 13 is the composition of embodiment 12, wherein the composition comprises two or more of said antibodies or antigen-binding antibody fragments.

[0038] Embodiment 14 is the composition of any one of embodiments 12 and 13, wherein the composition comprises: (1) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 39 H and V of SEQ ID NO:55 L (from MAb 37.2D); (2) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 42 H and V of SEQ ID NO:58 L (from MAb 8.9F); and (3) an antibody or antigen-binding antibody fragment comprising V of SEQ ID NO: 44 H and V of SEQ ID NO:60 L (from MAb 12.1F).

[0039] Embodiment 15 is the composition of any one of embodiments 12 to 14, wherein the antibody is selected from the group consisting of a monoclonal antibody and a recombinantly produced antibody.

[0040] Embodiment 16 is the composition of any one of embodiments 12 to 15, wherein the antibody comprises a human monoclonal antibody.

[0041] Embodiment 17 is the composition of any one of embodiments 12 to 14, wherein the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

[0042] Embodiment 18 is a V sequence encoding the antibody or antibody fragment of embodiment 12 H A nucleic acid (e.g., cDNA) having a sequence.

[0043] Embodiment 19 is a V sequence encoding the antibody or antibody fragment of embodiment 12 L A nucleic acid (e.g., cDNA) having a sequence.

[0044] Embodiment 20 is an expression vector containing the nucleic acid sequence of any one of embodiments 18 to 19.

[0045] Embodiment 21 is a vaccine for preventing or treating infection in a patient with Lassa virus or other Arenaviridae, comprising the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17.

[0046] Embodiment 22 is the vaccine of embodiment 21 which is cross-protective against infection by other Arenaviridae.

[0047] Embodiment 23 is the vaccine of any one of embodiments 21 to 22, which is cross-protective against infection with lymphocytic choriomeningitis virus.

[0048] Embodiment 24 is a pharmaceutical composition for treating or preventing infection by Lassa virus or other Arenaviridae, comprising the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 and a pharmaceutically acceptable carrier.

[0049] Embodiment 25 is the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for use in treating or preventing infection with Lassa virus or other Arenaviridae.

[0050] Embodiment 26 is the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for use in treating or preventing lymphocytic choriomeningitis virus infection.

[0051] Embodiment 27 is the use of the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for treating or preventing infection by Lassa virus or other Arenaviridae.

[0052] Embodiment 28 is the use of the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for treating or preventing lymphocytic choriomeningitis virus infection.

[0053] Embodiment 29 is the use of the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for the preparation of a medicament for treating or preventing infection with Lassa virus or other Arenaviridae.

[0054] Embodiment 30 is the use of the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 for the preparation of a medicament for treating or preventing lymphocytic choriomeningitis virus infection.

[0055] Embodiment 31 is a diagnostic kit for detecting infection by Lassa virus or other Arenaviridae hosts, comprising at least one antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 conjugated to a detectable labeling group.

[0056] Embodiment 32 is the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 conjugated to a detectable labeling group.

[0057] Embodiment 33 is a method of detecting infection with Lassa virus or other Arenaviridae, comprising contacting a biological sample from a subject with at least one antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 conjugated to a detectable labeling group; and detecting a complex between the antibody or antibody fragment and Lassa virus or other Arenaviridae present in the sample.

[0058] Embodiment 34 is a method of treating or preventing infection with Lassa virus or other Arenaviridae in a subject, comprising administering the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 to the subject.

[0059] Embodiment 35 is a method of treating or preventing lymphocytic choriomeningitis virus infection in a subject, comprising administering the antibody or antibody fragment of any one of embodiments 1 to 6 and 12 to 17 to the subject.

[0060] Other embodiments and advantages of the materials and methods described herein are set forth in part in the description which follows and, in part, may be learned by one of ordinary skill in the art from the specification or from the practice or use of the materials and methods described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 Schematic diagram depicting (A) LASV GP; (B) Arenavirus GP complex; (C) Recognition of different LASV GP species by LASV hMAbs. (A) LASV GP is synthesized as a precursor protein GPC. Signal peptidase (Spase) cleaves a small stabilizing signal peptide (SSP) that remains associated with GP1 and GP2 to form the GP complex ( Figure 1, panel B). The cellular protease SK1 / S1P cleaves GPC into GP1 and GP2. The construct rGPe corresponds to the recombinant LASV GPC extracellular domain lacking GP2 and replacing the SK1 / S1P cleavage recognition site with a non-cleavable linker. Constructs expressing recombinant SSP-GP1 (rGP1) and SSP fused to GP2 (rGP2) were also generated. (B) GP-1 forms a globular head subunit that interacts with cellular receptors, while GP2 mediates the fusion of the viral envelope with the cell membrane. SSP remains associated with GP1 and GP2 and plays a key role in the biology of the GP complex. (C) 293T cells were transfected with a plasmid expressing pCAGGS encoding LASVrGP1, rGP2 and GPC, and the reactivity of LASV hMAbs was assessed by immunofluorescence 48 hours after transfection. The distribution of LASV GP-specific hMAbs is indicated by subunit specificity, neutralization activity and reactivity to linear epitopes.

[0062] Figure 2 Depicts in vitro neutralization of LCMV ARM with 15 LASV GP-specific neutralizing hMAbs: LASV Josiah (squares) and LCMV ARM (triangles) GP-pseudotyped rLCMVΔGP / GFP viruses were incubated at 37°C with 2-fold dilutions of the indicated LASV GP-specific BnhMAbs for 90 minutes and then infected with Vero cells expressing LCMV GP (96-plate format, triplicate). Virus neutralization was determined under a fluorescent microscope and quantified using a GFP microplate reader 72 hours after infection. Results are presented as percentage inhibition normalized to the corresponding virus infection in the absence of hMAbs. Virus infection in the absence of hMAbs was used as an internal control. Mean values ​​and standard deviations are shown. Standard errors were calculated based on 2-6 replicates.

[0063] Figure 3 Depicts in vivo neutralization of 6 LCMV neutralizing antibodies (12.1F, 9.8A, 37.2D, 36.9F, 37.2G, and 18.5C) using non-cross-reactive antibodies 19.7E and 8.9F as internal controls. Mice were infected with rCl-13 (2x106 pfu; iv) and treated with the indicated hMAbs (20 mg / kg; ip) as well as isotype hMAb controls (20 mg / kg, ip) or vehicle. Post-inoculation (pi) viremia (i.e., presence of virus in the blood) was determined on days 4 and 21. Results correspond to the mean and standard deviation (SD) of four mice / group; LoD = limit of detection.

[0064] Figure 4Illustrated are viremia data from treatment and control GP plasma at days 7 and 14 PI. Viremia levels were below the limit of detection (LOD) for treatment groups 37.7H, 12.1F, and 25.6A on day 7 and 12.1F, 37.2D, 19.7E, and 10.4B on day 14. Error bars represent standard deviations from the mean. * indicates P_0.05. ** indicates P_0.001. *** indicates P<0.0001.

[0065] Figure 5 depicts clinical scores of HuMAb treated and untreated guinea pigs. GP treated with HuMAb 8.9F and 12.1 showed no difference in clinical scores from baseline (data not shown). Error bars (thin lines) represent standard deviations from the mean.

[0066] Figure 6 illustrates the effect of antibodies on rVSV-LASV GP infection and fusion. (A) Antibody-mediated neutralization of rVSV-LASV GP or (B) rVSV-VSV-G. 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 represent the standard deviation of at least six (two biological replicates, each with three or more technical replicates). (C) Antibody-mediated inhibition of rVSVLASVGP fusions on the cell surface. Error bars represent the standard error of the mean of six (except 37.7H, where N=9). (D) Fab 37.7H reduces the binding of LAMP1-Fc fusion protein to LASV GPCysR4. Error bars represent the standard deviation of six and three technical replicates.

[0067] Figure 7 Provides the use of CLUSTAL OMEGA TM(1.2.4) Sequence alignment of the heavy chain variable region amino acid sequences prepared by Multiple Sequence Alignment (from EMBL-EBI, part of the European Molecular Biology Laboratory), with the CDRs highlighted in bold font: CDR1 (marked with +), CDR2 (marked with ^) 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:47) NO:48).

[0068] Figure 8 Provides the use of CLUSTAL OMEGA TM (1.2.4) Sequence alignment of the light chain variable region amino acid sequences prepared by multiple sequence alignment, with the CDRs highlighted in bold font: CDR1 (marked with +), CDR2 (marked with ^) 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: 63) NO:64).

[0069] Details General Technology 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 within the ordinary skill of the art. Such techniques are fully explained in the literature, such as, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (IEC Ellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (Eds.). Cells (JM Miller and MPCabs, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology (JE Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P.Finch, 1997); Antibodies: a practical approach (D. Catty., eds., IRL Press, 1988-1989); Monoclonalantibodies: 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 JD Capra, eds., Harwood Academic Publishers, 1995).

[0070] As used herein, the singular forms "a", "an", and "the" include plural referents unless otherwise specified. For example, "a" monoclonal antibody includes one or more monoclonal antibodies.

[0071] Generally, monoclonal antibodies specific for LASV, monoclonal antibodies specific for LCMV, polynucleotides encoding the antibodies, and methods of using the 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 are provided for the development and production of diagnostic agents, vaccines, therapeutic agents, and screening tools. Generally, B cell clones that produce specific IgG for the GP of any Lassa virus isolate or strain can be used to derive the antibodies described herein.

[0072] Polynucleotide The term polynucleotide is widely used and refers to polymeric nucleotides of any length (e.g., fragments of oligonucleotides, genes, small inhibitory RNAs, protein-encoding polynucleotides, etc.). By way of example and not limitation, the polynucleotides of the present invention may comprise sequences encoding all or part of an extracellular domain and a portion of a transmembrane domain. The polynucleotides of the present invention may be, for example, linear, circular, supercoiled, single-stranded, double-stranded, branched, partially double-stranded or partially single-stranded. The nucleotides contained in the polynucleotides may be naturally occurring nucleotides or modified nucleotides.

[0073] Functional equivalents of these polynucleotides are also intended to be encompassed by the present invention. By way of example and not limitation, functionally equivalent polynucleotides are those having one or more of the following characteristics: the ability to generate antibodies (including, but not limited to, virus neutralizing antibodies) capable of recognizing LASV GP, or the ability to generate antibodies specific for LASV GP that exhibit neutralizing activity against LASV lineages I-IV and proposed new lineages (e.g., lineage V from Mali, lineage VI from Togo and Benin).

[0074] Functionally equivalent polynucleotide sequences can also be identified by methods known in the art. Various sequence alignment software programs are available to facilitate the determination of homology or identity. Non-limiting examples of these programs are the BLAST family of programs, including BLASTN, BLASTP, BLASTX, TBLASTN and TBLASTX (BLAST is available from the National Institutes of Health website), FASTA TM 、COMPARE TM , DOTPLOT TM 、BESTFIT TM 、GAP TM 、FRAMEALIGN TM CLUSTALW TM and PILEUP TM Other similar analysis and alignment programs are available from various suppliers, such as DNA Star's MEGALIGN TM , or GENEJOCKEY TM Alignment programs in . Alternatively, sequence analysis and alignment programs can be accessed through World Wide Web websites, such as the CMS Molecular Biology Resource website of the San Diego Supercomputer Center (SDSC); and the ExPASy Proteomics Server, the Swiss Institute of Bioinformatics SIB Bioinformatics Resource Portal. Any sequence database containing DNA or protein sequences corresponding to genes or fragments thereof can be used for sequence analysis. Commonly used databases include, but are not limited to, GenBank, EMBL, DDBJ, PDB, SWISS-PROT, EST, STS, GSS, and HTGS.

[0075] The parameters for determining the degree of homology described by one or more of the aforementioned alignment programs are fully established in the art. They include but are not limited to p-values, sequence identity percentages and sequence similarity percentages. The p-value is the probability of accidentally generating an alignment. 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 method, such as a method programmed in BLAST. Percent sequence identity is defined by the ratio of the number of nucleotide or amino acid matches between the two when the best alignment query sequence and the known sequence. The sequence similarity percentage is calculated in the same manner as the identity percentage, except that different but similar amino acids are scored as positive when calculating the similarity percentage. Therefore, conservative changes that often occur without changing the function, such as changes from one basic amino acid to another or changes from one hydrophobic amino acid to another, are scored as if they are the same.

[0076] The term "analog" includes any polypeptide having a substantially identical amino acid residue sequence to a polypeptide of the invention, wherein one or more residues have been conservatively substituted with a functionally similar residue, and which exhibits the functional aspects of the polypeptide as described herein. Examples of conservative substitutions include substitution of one nonpolar (hydrophobic) residue, such as isoleucine, valine, leucine or methionine, for another; substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; substitution of one basic residue, such as lysine, arginine or histidine for another; and substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

[0077] The phrase "conservative substitution" also includes the use of chemically derived residues to replace non-derivatized residues. "Chemical derivatives" refers to a subject polypeptide having one or more amino acid residues chemically derived by reaction of functional side groups. Examples of such derivatized amino acids include, for example, those amino acids in which the free amino group is derivatized to form amine hydrochlorides, p-toluenesulfonyl, benzyloxycarbonyl, tert-butyloxycarbonyl, chloroacetyl or formyl groups. Moreover, the free carboxyl groups of amino acids can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Moreover, the free hydroxyl groups of certain amino acids can be derivatized to form O-acyl or O-alkyl derivatives. Moreover, the imidazole nitrogen of histidine can be derivatized to form N-imidazole benzylhistidine. As chemical derivatives, those proteins or peptides containing one or more naturally occurring amino acid derivatives of twenty standard amino acids are also included. For example, 4-hydroxyproline can replace proline, 5-hydroxylysine can replace lysine, 3-methylhistidine can replace histidine, homoserine can replace serine, and ornithine can replace lysine. Polypeptides of the invention also include any polypeptide having one or more residue additions and / or deletions relative to the sequence of any of the polypeptides whose sequences are described herein.

[0078] Two polynucleotide or polypeptide sequences are said to be "identical" if the nucleotide or amino acid sequences in the two sequences are identical 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. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75 consecutive positions, or 40 to about 50 consecutive positions, wherein after optimally aligning the two sequences, the sequences can be compared to a reference sequence of the same number of consecutive positions.

[0079] Optimal alignment of sequences for comparison can be done using LASERGENE TM MEGALIGN in the bioinformatics software suite (DNASTAR, Inc., Madison, WI) TMThe program was performed using default parameters. The program embodies several alignment schemes described in the following references: Dayhoff, MO (1978) "A model of evolutionary change in proteins-Matrices for detecting distant relationships", in Dayhoff, MO (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, DG and Sharp, PM, 1989, CABIOS 5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson, ED, 1971, Comb. Theor. 11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ, and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0080] Preferably, "sequence percentage identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein for optimal alignment of the two sequences, the portion of the polypeptide sequence in the comparison window may contain 20% or less, typically 5 to 15% or 10 to 12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where the identical amino acid residue occurs in the two sequences to obtain 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 result by 100 to obtain the percentage of sequence identity.

[0081] Expression vector Also described herein are expression vectors comprising at least one polynucleotide encoding an antibody or antibody fragment protein. Expression vectors are well known in the art and include, but are not limited to, viral vectors or plasmids. Virus-based vectors for delivering the desired polynucleotide and expressing it in the desired cell are well known in the art. Exemplary viral-based vectors 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 virus, baculovirus recombination systems and herpes viruses.

[0082] Non-viral vectors, such as plasmids, are also well known in the art and include, but are not limited to, yeast-based plasmids and bacterial-based plasmids.

[0083] Methods for introducing vectors into host cells and isolating and purifying expressed proteins are also well known in the art (e.g., Molecular Cloning: A Laboratory Manual, 2nd 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.

[0084] By way of example, a vector comprising a polynucleotide as described herein may further comprise a tag polynucleotide sequence to facilitate protein separation and / or purification. Examples of tags include, but are not limited to, myc-epitopes, S-tags, his-tags, HSV epitopes, V5-epitopes, FLAG and CBP (calmodulin binding protein). Such tags are commercially available or easily prepared by methods known in the art.

[0085] The vector may further comprise a polynucleotide sequence encoding a linker sequence. Typically, the linker sequence is located between the antibody polynucleotide sequence and the polynucleotide tag sequence in the vector. The linker sequence may encode random amino acids or may contain functional sites. Examples of linkers containing functional sites include, but are not limited to, sequences containing factor Xa cleavage sites, thrombin cleavage sites, or enterokinase cleavage sites.

[0086] By way of example and not limitation, antibodies specific for LASV can be produced as described herein using a mammalian expression vector in a mammalian cell culture system or a bacterial expression vector in a bacterial culture system. By way of example and not limitation, antibodies specific for LCMV can be produced as described herein using a mammalian expression vector in a mammalian cell culture system or a bacterial expression vector in a bacterial culture system.

[0087] Antibody 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, characteristics of neutralizing antibodies include the ability to block or prevent infection of host cells. Antibodies can be characterized using methods well known in the art.

[0088] Antibodies useful in the compositions and methods described herein may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain fragments (e.g., ScFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, and any other modified configurations of immunoglobulin molecules (which comprise antigen recognition sites of desired specificity), including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies).

[0089] Methods for preparing monoclonal and polyclonal antibodies are well known in the art. Polyclonal antibodies can be raised in mammals, 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 liposomes. Hemocyanin (KLH), serum albumin, bovine thyroglobulin, soybean trypsin inhibitor, complete Freund's adjuvant (CFA) and MPL-TDM adjuvant. The immunization protocol can be determined by those skilled in the art.

[0090] Alternatively, the antibody may be a monoclonal antibody.Monoclonal antibodies can be produced using the hybridoma method (see, e.g., Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, DW, et al., In Vitro, 18:377-381 (1982)).

[0091] If desired, the antibody of interest can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell, and the host cell can then be amplified and frozen for future use. In an alternative embodiment, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity or other characteristics of the antibody (e.g., genetic manipulation of the antibody sequence to obtain greater affinity for LASV and / or LCMV glycoproteins and / or greater efficacy in inhibiting LASV and / or LCMV fusion with host cells).

[0092] The antibodies may also be humanized by methods known in the art (see, e.g., 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.

[0093] In another alternative embodiment, the antibody can be recombinantly prepared and expressed using any method known in the art. By way of example, antibodies can be recombinantly prepared by phage display technology. See, for example, U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., 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 various forms; for a 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 glycoproteins as described herein can be used as antigens for the purpose of isolating recombinant antibodies by these techniques.

[0094] Antibodies can be prepared by the following recombination: first, antibodies and antibody-producing cells are separated from a host animal, a gene sequence is obtained, and the gene sequence is used to recombinantly express the antibody in a host cell (e.g., CHO cell). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or transgenic milk. Methods for recombinantly expressing antibodies 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 preparing derivatives of antibodies (e.g., humanized and single-chain antibodies, etc.) are known in the art.

[0095] The antibodies described herein can be combined with carriers by conventional methods for use, for example, in isolating or purifying LASV and / or LCMV glycoproteins or detecting LASV and / or LCMV glycoproteins, antigens or particles in biological samples or specimens. Alternatively, by way of example, the neutralizing antibodies of the present invention can be administered as a therapeutic treatment to a subject infected or suspected of being infected with LASV or LCMV. "Subject" includes, but is not limited to, humans, monkeys, farm animals, sports animals, and pets. Veterinary uses are also encompassed by the methods described herein. For diagnostic purposes, the antibodies can be labeled, for example, conjugated to a detectable labeling group, such as a fluorescent dye (e.g., ALEXA dyes), quantum dot labels (e.g. Labels), R-phycoerythrin, streptavidin, biotin, enzymes (e.g., glucose oxidase, horseradish peroxidase or alkaline phosphatase), radioactive isotopes (e.g., iodine-125, indium-111), etc. Such labeling techniques are well known in the antibody field.

[0096] Antibody DNA sequence Sixteen neutralizing antibodies against LASV were identified, which are referred to 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. The nucleotide sequences (cDNA) encoding the heavy chain (HC) and light chain (LC) portions of each antibody are shown below. The exemplified nucleotide sequences encode the variable regions (i.e., V H and V L region), together with portions of the vector sequence.

[0097] Antibody amino acid sequence The V of the antibodies are shown and discussed below. H and V L Amino acid sequence and V H and V L The complementarity determining region (CDR) of the sequence.

[0098] Figure 7 Provides the use of CLUSTAL OMEGA TM (1.2.4) Sequence alignment of the heavy chain variable region amino acid sequences prepared by Multiple Sequence Alignment (from EMBL-EBI, part of the European Molecular Biology Laboratory), with the CDRs highlighted in bold font: CDR1 (marked with +), CDR2 (marked with ^) and CDR3 (marked with #).

[0099] Figure 8 Provides the use of CLUSTAL OMEGA TM (1.2.4) Sequence alignment of the light chain variable region amino acid sequences prepared by multiple sequence alignment, with the CDRs highlighted in bold font: CDR1 (marked with +), CDR2 (marked with ^) and CDR3 (marked with #).

[0100] The following HC CDR sequence table lists the V sequences of each of the 16 neutralizing antibodies described herein. H The following LC CDR sequence table lists the V sequences of each of the 16 neutralizing antibodies described herein. L The sequences of CDR1, CDR2 and CDR3.

[0101] HC CDR sequence listing.

[0102] LC CDR sequence listing.

[0103] Diagnostic agents The antibodies described herein can be used in various 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 purpose of detecting antigens in biological samples. By way of example and not limitation, the antibodies of the invention can be used as reagents in ELISA assays to detect arenavirus antigens in biological samples from a subject. The antibodies can be labeled, for example, by binding to a detectable labeling group such as a fluorescent dye, a quantum dot marker, R-phycoerythrin, streptavidin, biotin, an enzyme, a radioactive isotope, or the like. Such labeling techniques are well known in the antibody field.

[0104] vaccine Vaccines for LASV, LCMV, and other arenaviruses are also described herein. In one aspect, the vaccine is a DNA-based vaccine. Those skilled in the art are familiar with administering expression vectors to obtain in vivo expression of foreign proteins. See, e.g., U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471. Virus-based vectors for delivery of desired polynucleotides and expression in desired cells are well known in the art, and non-limiting examples are described herein.

[0105] Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheterization administration, and local administration. Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery technology is described in, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (JA 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.

[0106] Non-viral delivery vehicles and methods can also be used, including but not limited to polycation-condensed DNA linked or not linked to a separate inactivated adenovirus (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 nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be utilized. 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 serve as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos. WO 95 / 13796, WO 94 / 23697, WO 91 / 14445; and EP 0524968. Additional methods are described in Philip, Mol. Cell Biol. (1994) 14:2411 and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0107] For human administration, the codons of a polynucleotide comprising an encoding one or more antibodies specific for a LASV glycoprotein and / or a LCMV glycoprotein may be optimized for human use, as is standard in the art.

[0108] In another aspect, one or more antibodies specific to LASV and / or LCMV or a combination thereof are used as vaccines. One or more antibodies or a combination thereof can be administered alone or in combination with an adjuvant. Examples of adjuvants include, but are not limited to, aluminum salts, water-in-oil emulsions, oil-in-water emulsions, saponins, QuilA and derivatives, iscoms, liposomes, cytokines (including gamma-interferon or interleukin 12), DNA (e.g., unmethylated poly-CpG), microencapsulation in solid or semisolid particles, Freund's complete and incomplete adjuvants or their active ingredients (including muramyl dipeptide and analogs), DEAE dextraril mineral oil, aluminum gel, Auspharm adjuvant and Algammulin.

[0109] Antibody vaccines comprising one or more antibodies specific for LASV and / or LCMV or a combination thereof can be administered orally or by any parenteral route, such as intravenous, subcutaneous, intraarterial, intramuscular, intracardial, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, and / or transdermal.

[0110] The dosage and schedule of administration can be determined by methods known in the art. The efficacy of one or more antibodies specific for LASV and / or LCMV, or a combination thereof, as a vaccine for Lassa virus, lymphocytic choriomeningitis virus, or a related arenavirus can also be assessed by methods known in the art.

[0111] Pharmaceutical composition The polynucleotides, polypeptides and antibodies described herein may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer known in the art (Remington: The Science and practice of Pharmacy 20th edition, 2000, Lippincott Williams and Wilkins, ed. KE Hoover), in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the doses and concentrations used, and may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethylammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol or benzyl alcohol, alkyl parabens such as methyl paraben 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, mannose or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0112] By way of example and not limitation, the compositions used in the methods described herein typically comprise an effective amount of a polynucleotide or polypeptide of the invention (e.g., an amount sufficient to induce an immune response) or an antibody of the invention (e.g., an amount of a neutralizing antibody sufficient to reduce infection, reduce symptoms of infection, and / or prevent infection).

[0113] The pharmaceutical composition may further comprise additional agents for enhancing and / or supplementing the desired effect. By way of example, in order to enhance the efficacy of one or more antibodies specific to LASV and / or LCMV or a combination thereof administered as a pharmaceutical composition, the pharmaceutical composition may further comprise an adjuvant. Examples of adjuvants are provided herein.

[0114] Also by way of example and not limitation, if one or more antibodies of the invention specific for LASV and / or LCMV or a combination thereof are administered to boost the immune response of a subject infected or suspected of being infected with LASV or LCMV and / or if the antibodies of the invention are administered as a form of passive immunotherapy, the composition may further comprise other therapeutic agents (e.g., antiviral agents).

[0115] Reagent test kit Also described are kits for use in the present methods. The kits include, by way of example and not limitation, one or more containers comprising polynucleotides encoding one or more antibodies of the present invention specific for LASV and / or LCMV, or a combination thereof, or fragments thereof, and instructions for use according to any of the methods of the invention described herein. In some embodiments of the kits, the antibodies are conjugated to a detectable marker as described above.

[0116] Typically, the instructions include a description of administration or instructions for performing an assay. The container may be a unit dose, bulk package (e.g., multi-dose package) or subunit dose. The instructions provided in the kit of the invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0117] The kit is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. Packages used in combination with specific devices such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices such as miniature pumps are also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The kit can optionally provide additional components, such as buffer and explanatory information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0118] The following non-limiting examples are provided to illustrate certain aspects and features of the materials and methods described herein. DETAILED DESCRIPTION Example

[0119] Example 1: LCMV infection of mice and recombinant arenaviruses is a powerful experimental system to evaluate the efficacy and breadth of LCMV neutralizing mMAbs in vivo.

[0120] Host and viral factors and viral infection routes and dosages affect the results of LCMV infection in mice. Therefore, intravenous (iv) inoculation of adult immunocompetent mice with LCMV Armstrong (ARM) strains leads to acute infection, which induces a protective immune response that mediates viral clearance within 10 to 14 days, a process mediated mainly by virus-specific CD8+ cytotoxic T lymphocytes (CTL). In contrast, iv inoculation with high-dose LCMV immunosuppressive clone 13 (Cl-13) strains causes a persistent infection associated with persistent viremia and extensive immunosuppression, which can last for 60 to 100 days. This model is robust and has clear results, which provides an effective and cost-saving experimental system for initial evaluation of antibody-based strategy control and removal of LCMV infection efficacy. In this regard, the use of recombinant viruses based on Cl-13 expressing target GP allows the safety and in vivo neutralization activity of GP-specific BNmMAbs to be evaluated. The method is feasible using state-of-the-art arenavirus reverse genetics, which allows the rescue of infectious recombinant LCM viruses with predetermined target mutations, as well as heterologous expression of target viral or non-viral genes. Single-cycle infectious, reporter-expressing recombinant LCMVs are generated in which the GP ORF is replaced by GFP (rLCMVΔGP / GFP). Genetic complementation with a plasmid or stable cell line expressing the target arenavirus GP generates the corresponding GP-pseudotyped rLCMVΔGP / GFP, which can be used to evaluate antibody responses against HF arenaviruses using a BSL2 platform.

[0121] Example 2: Identification of LASV (Josiah strain) GP-specific hMAbs that cross-react with the GP of the LCMV ARM strain.

[0122] 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 IgG specific for LASV GP. RNA from these B cell clones was used to clone 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 originated from different germline genes and are likely independently derived. All hMAbs except one (8.9F) reacted in ELISA with GP from the Josiah strain (lineage IV) of LASV, which is closely related to the LASV strain currently circulating in Sierra Leone. LASV GP is composed of SSP and GP1 and GP2 subunits, as Figure 1 , panels A and B. To define the GP subunits recognized by the LASV hMAbs, an immunofluorescence assay was used to test the recognition of the hMAbs on human 293T cells expressing rGP1 or rGP2 alone or full-length GP, as shown in Figure 1 , as shown in panel C. 29 hMAbs, including 3 with neutralizing activity, reacted with LASV rGP1, as shown in panel Figure 1 , Panel C, left, and Table 1. 57 hMAbs recognized LASV rGP2, but none of these showed neutralizing activity, such as Figure 1 , Panel C, middle, and Table 1. Seven hMAbs reacted with peptides representing three linear epitopes in GP2, while the remaining hMAbs appeared to recognize conformational epitopes. 27 hMAbs reacted with cells expressing full-length GP, but not with rGP1 or rGP2 expressed alone. Notably, 13 of these hMAbs were neutralizing, as shown in Table 1. Figure 1 , Panel C, right and Table 1. The inhibitory concentration 50 (IC50) and 80 (IC80) neutralizing activity of LASV GP-specific hMAbs were evaluated using lentiviral particles pseudotyped with different lineages I-IV LASV GP. The results are shown in Table 1. Based on the data in Table 1, the antibodies can be classified as the most effective (hMAbs exhibiting an IC value of <1 μg / mL); effective (hMAbs exhibiting IC values ​​in the range of 1 to 2.5 μg / mL), weak (hMAbs exhibiting IC values ​​of >3 and <20 μg / mL); non-neutralizing (hMAbs exhibiting IC values ​​of >20 μg / mL).

[0123] Table 1. Neutralizing activity of LASV GP-specific hMAbs against LASV lineages I-IV.

[0124] Neutralization properties of LASV GP-specific hMAbs: The neutralization properties of LASV GP-specific hMAbs were evaluated using envelope-deficient core HIV-1 (LASVpp) pseudotyped with LASV GP (shown in Table 1) and a standard plaque reduction neutralization test (PRNT) with authentic LASV. 15 of the 120 hMAbs neutralized GP-expressing LASVpp of the Josiah strain from LASV lineage IV, as shown in Table 1. These neutralizing GP-specific hMAbs were also tested against GP-containing LASVpp of three other LASV lineages I-III (shown in Table 1). IC50 and IC80 values ​​showed that those with the greatest efficacy and breadth against all four LASV lineages 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 efficacy. The neutralizing activity of these GP-specific hMAbs against LASV Josiah strain was further confirmed using an LCMV-based pseudovirus assay. These results revealed that among the 120 tested LASV GP-specific hMAbs, 15 neutralized the LASV Josiah strain to varying degrees, and some of them exhibited broad neutralizing activity against representative strains from LASV lineages I-II.

[0125] Cross-reactivity of LASV GP-specific hMAbs with LCMV ARM: 16 LASV GP-specific hMAbs with neutralizing activity (as shown in Table 1) were characterized by immunofluorescence for their ability to recognize GP of the LCMV ARM strain expressed in human 293T cells transfected with a plasmid expressing GP. Human 293T cells transfected with LASV GP from lineages 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.

[0126] Example 3: Identification of LASV GP-specific hMAbs with broad cross-reactivity against GP from different LCMV strains.

[0127] Check for LASV GP-specific neutralizing hMAbs (eg Figure 1and Table 1) to recognize the ability of GP from 5 LCMV strains associated with human LCMV-induced disease cases. These strains correspond to the WE strain that caused zoonotic infection in New York in 1935; the Rhode Island (RI) strain that caused four human cases and three deaths due to transplant cases in 2005; the Ohio (OH) strain similar to the Michigan LCMV strain that caused human cases in 2005; the Wisconsin (WI) strain that caused 4 deaths in 2003; and the Massachusetts (MA) strain that caused 2 deaths in 2008. LASV GP-specific neutralizing hMAbs 12.1F, 37.2D, 9.8A, 18.5C, and 36.9F recognize all five LCMV GP strains. LASV GP-specific neutralizing hMAbs 37.7H, 25.6A, 37.2G, and 2.9D recognize four LCMV strains (ARM, WE, WI, and MA, but not RI or AH). The remaining hMAbs did not cross-react with any of the LCMV strains tested.

[0128] Example 4: Identification of LASV GP-specific hMAbs (BNhMAbs) with potent and broadly neutralizing activity against GPs from different LCMV strains in a cell-based assay.

[0129] 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 prime candidates that exhibit broad in vivo antiviral activity against LCMV strains previously associated with disease cases in humans. From 15 LASV GP-specific neutralizing hMAbs, 6 of them (12.1F, 37.2D, 9.8A, 18.5C, 37.2G, and 36.9F) neutralized LCMV ARM, as shown in Figure 2. Figure 2 The results are shown in Table 2, where IC50 <1 μg / mL, except that 18.5C showed a higher (>10 μg / mL) IC50. The results are shown in Table 2, which shows the neutralization activity of 15 LASV GP-specific neutralizing hMAbs against LCMV ARM, in particular, the IC50 and IC80 values ​​of 15 LASV GP-specific neutralizing hMAbs against LCMV ARM. LASV or LCMV GP-pseudotyped rLCMVΔGP / GFP viruses were used to obtain the neutralization activity of 15 LASV GP-specific neutralizing hMAbs against LCMV ARM from a cell-based microneutralization assay (shown in Figure 2 Gray indicates LASV GP-specific neutralizing hMAbs that neutralize LCMV GP ARM. Neutralization of LASV GP-pseudotyped rLCMVΔGP / GFP was similar to the neutralization results obtained using LASV GP-pseudotyped lentiviral particles shown in Table 1.

[0130] Table 2. Neutralizing activity of 15 LASV GP-specific neutralizing hMAbs against LCMV Armstrong strain (ARM).

[0131] Example 5: In vivo characterization of selected GP-specific BNhMAbs.

[0132] A well-characterized mouse model of LCMV infection was used to test LASV GP-specific neutralizing hMAbs with broad neutralizing activity against LCMV (shown in Figure 2 and Table 2) also showed in vivo neutralizing activity. The immunosuppressive clone 13 (Cl-13) strain of LCMV was used. 6 Infection of B6 WT mice with Cl-13 at 100 PFU (pfu) of Cl-13 (iv) resulted in transient systemic immunosuppression and establishment of a persistent infection with well-established parameters. Viral clearance occurred between days 60 and 100 post-inoculation (pi). However, treatment of Cl-13-infected mice, which resulted in a reduction in viral load, accelerated Cl-13 clearance. Therefore, it was predicted that LASV GP-specific neutralizing hMAbs exhibiting in vivo neutralizing activity could prevent the establishment of Cl-13 persistence or accelerate its clearance. Mice were treated intraperitoneally (ip) with the indicated hMAbs at 20 mg / Kg and infected with 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 cytoplasmic domain of GP, which enhance persistence in mice. The in vivo results are shown in Figure 3 in the literature and correlate with those previously documented in cultured cells (shown in Figure 2 and Table 2). Mice treated with hMAbs 12.1F, 37.2D, 9.8A, and 36.9F were protected from the persistence of rCl-13 / WT. Unexpectedly, hMAbs 37.2G and 18.5C did not protect against the persistence of Cl-13 in vivo. As expected, based on cross-reactivity and neutralization in cultured cells, hMAbs 19.7E and 8.9F were not able to protect against the persistence of rCl-13.

[0133] Table 3 presents a summary of the in vitro and in vivo cross-reactivity and neutralization activities of LASV GP-specific hMAbs against the six LCMV strains tested (ARM, WE, RI, OH, WI, and MA).

[0134] Table 3. Summary of in vitro and in vivo cross-reactivity and neutralization activity of LASV GP-specific hMAbs against LCMV.

[0135] Example 6: Assay development.

[0136] A panel of murine antibodies to Fab or F(ab')2 fragments of lead candidate therapeutic BNhMAbs was obtained for the isolation of highly specific anti-idiotypic reagents for assay development. In order to develop a highly protective therapeutic BNhMAb mixture containing 2 to 4 antibodies that together confer maximum pre-exposure and post-exposure protection against LCMV infection while minimizing the appearance of escape mutants, it is important to characterize the PK of each antibody when administered as a mixture. In order to distinguish all BNhMAbs included in the mixture after administration, highly specific anti-idiotypic antibodies are the best tools available for rapid determination of the concentration and clearance of individual hMAbs in the blood. A panel of anti-idiotypic antibodies against 37.2D and 12.1F has been developed. The anti-idiotypic mMAb against 37.2D specifically detects the BNhMAb when incorporated into human serum. The anti-idiotypic antibodies do not capture or detect any other tested arenavirus BNhMAbs or any other IgG specificity present in human serum in studies based on ELISA and SPR, and can therefore be used to determine 37.2D.

[0137] Example 7: Therapeutic efficacy of first-in-human LASV-specific antibodies in the guinea pig (GP) and cynomolgus monkey (CM) models of Lassa fever.

[0138] These studies were performed under BSL-4 biocontainment at the Galveston National Laboratory. Outbred Hartley strain GP were challenged ip with 1,000 pfu of GP-adapted (GPA) LASV Josiah strain (N=5 / group). This model has recently been described for testing therapeutic agents against LASV. The advantage of using outbred animals to simulate human infection is inferred from the higher variability of the inherent immune response in outbred populations. Kruskal-Wallis test supported by Dunn's multiple comparison post-test (PRISM 5 available from GraphPad Software, La Jolla, CA) TM Software) to detect differences from the control group from time points associated with onset of disease (day 7) or peak viremia (day 14), as determined from historical data.

[0139] The 11 LASV hMAbs tested in the Hartley GP model of LF fell 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 scores in GP. (2) 37.2D, 19.7E, and 37.2G protected 80 to 90% of the animals. (3) 10.4B, 25.10C, and 36.1F conferred 40%, 30%, and 20% protection, respectively. An irrelevant recombinant human isotype control (IgG1) Ab conferred no protection (0% survival).

[0140] Regarding viremia, untreated control animals had an average of 3.5 and 4.5 LogPFU / mL on days 7 and 14, respectively. Figure 4 As shown in. Although there is 100% protection at the end of the study, some animals from treatment groups 8.9F or 37.7H, 2.9D and 25.6A have low-level viremia on the 7th day or the 14th day, respectively. The treatment groups (37.2D and 19.7E) providing 90% protection have reduced average viremia titers and minimal clinical score values. The treatment groups with 80% or lower survival rates have average viremia titers comparable to control animals on the 7th day, but by the 14th day, average viremia is significantly lower than that of control animals. The group with 80 to 90% survival rate shows a relatively low average clinical score (Fig. 5), and all remaining treatment groups show an accompanying increase in average clinical score as each group's survival rate decreases. Since survival rate is the main objective metric, the endpoint viremia of these studies is not determined, although all surviving animals do not show clinical signs.

[0141] Results from guinea pig studies informed studies in the cynomolgus monkey (CM) model of LF. These studies showed that several of the antibodies with high efficacy in the GP model also protected 100% of CM when administered on the day of challenge. 19.7E protected 75% of CM. It is noteworthy that therapeutic doses as low as 6 mg / kg hMAb 37.2D provided 100% protection in CM, while 19.7E protected 75% of CM. A mixture of three human MAbs (37.2D, 12.1F and 8.9F, each 15 mg / kg) rescued 100% of CM even after the start of treatment was delayed to 3, 6 or 8 days after infection (therapeutic agent withdrawal (walk-out) study). 8 days after infection, untreated CM had developed high viral loads and were extremely ill. CMs were also protected from lethal LF induced by challenge with strain Josiah (lineage IV) or a contemporary lineage II strain from a fatal case of LF originating from Nigeria, both with the first treatment administered 8 days after infection.

[0142] Example 8: Structural definition of the anti-LASV 37.7H epitope.

[0143] Monomeric GPCysR4 was incubated with excess Fab 37.7H and subjected to SEC-MALS analysis. SEC-MALS indicated that trimeric GP-Fab complexes were formed in addition to monomeric GP-Fab complexes. Crystals of monomeric and trimeric fractions of the GPCysR4-Fab 37.7H complex formed in space group P6122 and diffracted to The GP trimer was bound to three Fabs in the asymmetric unit. The phases were determined by an iterative approach using molecular replacement with related Fab structures and the LCMV GP crystal structure.

[0144] The antibody 37.7H against LASV neutralizes viruses representing all four known LASV lineages in vitro and provides protection against lethal LASV challenge in guinea pigs and non-human primates. The antibody simultaneously binds to two GP monomers at the base of the GP trimer, where it engages four discrete 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 387-408 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 approximately The GP at site A is about and the GP at site B Although almost the entire surface buried on GP belongs to GP2, the presence of both GP1 and GP2 is critical for 37.7H recognition, probably because GP1 is required to maintain the proper prefusion conformation of GP2 for 37.7H binding.

[0145] Antibody 37.7H also recognizes the GPC of LCMV, but not that of the more distantly related Old World Arenavirus LUJV, nor that of the New World Arenaviruses. Sequence comparisons between these arenaviruses show almost complete sequence conservation throughout the 37.7H epitope for all LASV lineages and LCMV. However, the sequences of the LUJO, JUNV, and MACV GPCs are much different, particularly in HR2 of GP2, which is heavily involved in binding to 37.7H. The 37.7H antibody neutralizes by stabilizing the prefusion GP.

[0146] The quaternary nature and involvement of the fusion peptide in the 37.7H epitope suggest that the antibody neutralizes the virus by stabilizing GPC in a prefusion conformation, thereby preventing conformational changes required for infection. This was validated by analyzing the ability of LASV GP-pseudotyped recombinant vesicular stomatitis virus (rVSV-LASV GP) to mediate fusion with cell membranes.

[0147] First, the ability of 37.7H to neutralize rVSVLASV GP was determined. Figure 6 shows the effects of antibodies on rVSV-LASV GP infection and fusion. Antibody-mediated neutralization of rVSV-LASV GP is shown in Figure 6, panel A. Antibody-mediated neutralization of rVSV-VSV-G is shown in Figure 6, panel B. Antibody 9.7A is a non-neutralizing antibody and is 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 represent the standard deviation of at least six (two biological replicates, each with three or more technical replicates). Figure 6, panel C shows antibody-mediated inhibition of rVSVLASV GP fusions on the cell surface. Error bars represent the standard error of the mean of six (except 37.7H, where N=9). Figure 6, Panel D shows that Fab 37.7H reduces the binding of LAMP1-Fc fusion protein to LASV GPCysR4. Error bars represent the standard deviation of six and three technical replicates.

[0148] 37.7H effectively blocked cell infection of rVSV-LASV GP, as did antibody 12.1F, which binds to the upper β-sheet surface of LASVGP1 and presumably blocks cell attachment. In contrast, antibody 13.4E, which binds to a linear epitope in the T-loop, and 9.7A, a non-neutralizing GPC-B antibody, did not prevent virus infection (Fig. 6, panels A and B).

[0149] Next, the ability of 37.7H to prevent rVSV-LASV GP from fusing with cell membranes when exposed to low pH was examined. Unlike non-neutralizing antibodies 9.7A and 13.4E, which were not effective in preventing fusion, 37.7H reduced fusion by nearly 80% compared to 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, indicating that the effect of 37.7H was strictly due to the disruption of GPC fusion rather than attachment to cells.

[0150] LASV GP1 engages LAMP1 before exposing the GP2 fusion peptide and loop and subsequently fusing the viral and host cell membranes. It is believed that the engagement of this receptor requires a conformational change in GP1, which is triggered by exposure to low pH in the endosome. Tomography of the LASV spike in the presence of low pH and LAMP1 shows the opening of the trimer compared to its neutral pH conformation. To determine whether 37.7H can prevent these conformational changes, the ability of GPCysR4 to bind to a separate soluble LAMP1-Fc fusion and to bind to a soluble LAMP1-Fc fusion when bound to Fab 37.7H was analyzed. In the absence of Fab 37.7H, GPCysR4 effectively binds to LAMP1 when exposed to low pH. However, in the presence of Fab37.7H, the interaction between GPCysR4 and LAMP1 was significantly reduced (Figure 6, panel D).

[0151] Based on the crystallographic data, the footprint of 37.7H and that of LAMP1 are separated by approximately 50 Å, and the angle adopted by the bound Fab fragment of 37.7H suggests that it is unlikely to interfere sterically with LAMP1. Therefore, there may be conformational changes in GP1 required for LAMP1 binding that are prevented by this human survival antibody. Taken together, these results suggest that the likely mechanism of action of 37.7H and perhaps other antibodies in its potent GPC-B competition group is to stabilize the prefusion GPC trimer and prevent conformational changes required for binding of LAMP1 and triggering the GP2 fusion peptide and fusion loop in the endosome.

[0152] By considering the specification and implementation of the invention disclosed herein, other embodiments and uses of the present invention will be apparent to those skilled in the art. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and integrally incorporated herein by reference. It is intended that this specification and examples be considered to be exemplary only in the context of following the true scope and spirit of the invention as indicated by the claims.

[0153] The following references are incorporated herein by reference.

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Claims

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

2. The composition of claim 1, wherein the composition comprises: A recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising: V of SEQ ID NO: 39 H and V of SEQ ID NO:55 L ; A recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising: V of SEQ ID NO: 42 H and V of SEQ ID NO:58 L ;and A recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprising: V of SEQ ID NO: 44 H and V of SEQ ID NO: 60 L .

3. The composition of claim 1 or claim 2, wherein each antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

4. A pharmaceutical composition for treating infection by Lassa virus or lymphocytic choriomeningitis virus, comprising the composition of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

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

6. The composition of claim 5, wherein the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

7. The composition of claim 5 or claim 6, wherein the composition comprises a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof, wherein the recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprises V of SEQ ID NO: 39 H and V of SEQ ID NO:55 L .

8. A pharmaceutical composition for treating infection by Lassa virus or lymphocytic choriomeningitis virus, comprising the composition of any one of claims 5 to 7 and a pharmaceutically acceptable carrier.

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

10. The composition of claim 9, wherein the antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

11. The composition of claim 9 or claim 10, wherein the composition comprises a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof, wherein the recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprises V of SEQ ID NO:

42. H and V of SEQ ID NO:58 L .

12. A pharmaceutical composition for treating infection by Lassa virus or lymphocytic choriomeningitis virus, comprising the composition of any one of claims 9 to 11 and a pharmaceutically acceptable carrier.

13. An antigen binding composition comprising a recombinant human monoclonal neutralizing antibody or a neutralizing antigen binding antibody fragment thereof specific for Lassa virus glycoprotein; the antibody or antibody fragment thereof comprising: V of SEQ ID NO: 98 H CDR1, V of SEQ ID NO:99 H CDR2, V of SEQ ID NO: 100 H CDR3, V of SEQ ID NO: 135 L CDR1, sequence Gly Ala Ser V L CDR2 and V of SEQ ID NO: 136 L CDR3.

14. The composition of claim 13, wherein each antigen-binding antibody fragment is selected from the group consisting of Fab, Fab' and F(ab')2 fragments.

15. The composition of claim 13 or claim 14, wherein the composition comprises a recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof, wherein the recombinant human monoclonal antibody or an antigen-binding antibody fragment thereof comprises V of SEQ ID NO: 44 H and V of SEQ ID NO:60 L .

16. A pharmaceutical composition for treating infection by Lassa virus or lymphocytic choriomeningitis virus, comprising the composition of claims 13 to 15 and a pharmaceutically acceptable carrier.

17. Use of a composition according to any one of claims 1 to 16 for the preparation of a medicament for treating Lassa virus infection.

18. Use of the composition of any one of claims 1 to 16 for the preparation of a medicament for treating lymphocytic choriomeningitis virus infection.

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