Enhanced lassa fever virus immunogens

Abstract

An immunogen comprising SEQ ID NO: 4, compositions and vectors comprising the same, and methods of using the same.

Description

ENHANCED LASSA FEVER VIRUS IMMUNOGENSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 608,980, filed December 12, 2023, and U.S. Provisional Application No. 63 / 693,882, filed September 12, 2024, which are each incorporated by reference herein in their entireties.REFERENCE TO SEQUENCE LISTING

[0002] The sequence listing submitted on December 12, 2024, as an .XML file entitled “10063-093W01_ST26.xml” created on December 11, 2024, and having a file size of 15,028 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0003] Viral infections can be prevented by exposure to viral antigens via, for example, vaccination. These antigens (i.e., immunogens) are selected and designed so as to elicit a sufficient immune response for the generation of immunological memory without causing infection. Some shortcomings can include low expression of immunogens, low binding of immunogens to human neutralizing antibodies, and / or poor stability.

[0004] As such, there is a need for improved immunogens and improved immune response to the same. These needs and others are at least partially satisfied by the present disclosure.SUMMARY

[0005] Disclosed herein are variants of Lassa Fever Virus glycoprotein engineered to increase expression, stability, and ability to bind to human neutralizing antibodies. These engineered proteins are intended to be used as immunogens for vaccines. These variants can improve stabilization of Lassa glycoprotein, enhance presentation or protective epitopes, and increase expression in human cell lines. These variants can further elicit broadly neutralizing antibody response when administered as an immunogen.

[0006] In an aspect, provided is an immunogen comprising SEQ ID NO: 4, wherein SEQ ID NO: 4 comprises one or more mutations, wherein at least one of the mutations comprises A132C and I334C, A132P, A173C and T182C, A173P, A184P, A202C and T215C, A297V, A30C and F446C, A328P, A33OP, D130P, D401P, E151P, E16P, E303Q, E303Q and K304Q, E303T, E307L, G102C and T225C, G174P, G240P, G276C and N295C, G294P, H131P, H305C andN346C, H305F, H305I, H354W, H93C and A195C, I113C and S154C, I15P, I334P, I345V, I4C and L430C, K304N, K304Q, K327P, K339L, K352F, K63C and A407C, L120F, L128C and G160C, L142C and T249C, L290P, L428P, L68F, L73P, M192C and G198C, M332P, M359F, M359L, M75F, M96C and G102C, N178P, N20C and L430C, N295P, N342C and Q348C, N395P, N74C and I286C, N74D, N74P, N90C and L203C, Q189E, Q321E, Q331P, Q348P, Q406E, Q69E, R282P, R356F, R422F, S143C and R248C, S171P, S199P, S246L, S267C and I361C, S27C and S437C, S333P, S85C and G240C, S85V, T249L, T261I, T261M, T296P, T412C and G424C, T77C and W283C, T87C and G197C, V14P, V183P, V31C and Y441C, V97C and A132C, W196C and G240C, W196P, Y150P, Y200P, Y62C and A407C, Y62I, F262P, Y150C and R257C, I95A and A195F, A322V, D306W, L68F and 1403 A, H305N, L128P, E396P, L105A and A195F, Q189A and H354F, T87A and G197F, L242A and I337F, G243C and I350C, R193M, M192R, I350L, G243S, D251Q, V31I, S367T, Y369F, K339T, K339W, A343S, T261F, A177C and E329C, T87S, I334L, N346S, T281P, E329P, M19P, G197P, H179P, H230P, G294P, G206P, E287P, S216P, I213P, D229P, T215P, I201P, N99P, D175P, Q335P, A176P, N342P, G240P, G45P, N114P, A173P, T77P, G174P, N395P, M332P, WT, G27 IP, N295P, G 181P, V14P, A177P, G208P, I286A, D357A, T106A, W283A, G277A, H398A, V18A, F7A, S267A, G181A, E228A, R379A, Q406A, Q335A, P275A, T182A, T296A, G198A, S 163A, H93A, E329A, E104A, S476A, T190A, N209A, S205A, H179A, F233A, S85A, S400A, S171A, Q405A, Y200A, G206A, T397A, K481A, G45A, T274A, N295A, D268A, L203A, G208A, N158A, V65A, C53A, S27A, K33A, E76A, T249A, T412A, D402A, Q423A, M284A, T438A, P236A, H448A, M194A, D175A, D204A, G424A, E10A, L442A, T59A, T249L, H305F, T261M, R356F, L68F, R422F , A297V, T249I, T87S, Q189D, or T380H.

[0007] In some aspects, said immunogen comprises two or more mutations. In some aspects, said immunogen comprises three or more mutations. In some aspects, said immunogen comprises four or more mutations. In some aspects, said immunogen comprises five or more mutations.

[0008] In another aspect, provided is a composition comprising at least one of any of the disclosed immunogens.

[0009] In another aspect, provided is a composition comprising at least two of any of the disclosed immunogens.

[0010] In some aspects, the composition comprises at least two immunogens, wherein one immunogen comprises any of the disclosed mutations, and one additional immunogen. In some aspects, said composition further comprises an adjuvant. In some aspects, said composition further comprises a delivery vehicle. In some aspects, composition is in a vaccine form.

[0011] In another aspect, provided is a nucleic acid encoding at least one of any of the disclosed immunogens.

[0012] In another aspect, provided is a vector comprising any of the disclosed nucleic acids.

[0013] In another aspect, provided is a cell comprising any of the disclosed vectors.

[0014] In another aspect, provided is a method of inducing an immune response in a subject, wherein said method comprises delivering one or more of any of the disclosed immunogens into the subject in an amount sufficient to induce an immune response in the subject.

[0015] In another aspect, provided is a method of inducing an immune response in a subject, wherein said method comprises delivering one or more of any of the disclosed nucleic acids to the subject in an amount sufficient to induce an immune response in the subject.

[0016] In some aspects, the subject is a human.

[0017] In another aspect, provided is a method of inducing an immune response against an arenavirus in a subject, wherein said method comprises delivering one or more of any of the disclosed immunogens into the subject in an amount sufficient to induce an immune response in the subject.

[0018] In another aspect, provided is a method for inducing an immune response against an arenavirus in a subject, wherein said method comprises delivering one or more of any of the disclosed nucleic acids to the subject in an amount sufficient to induce an immune response in the subject.

[0019] In some aspects, the subject is a human.

[0020] In some aspects, the arenavirus comprises Lassa mammarenavirus, Argentinian mammarenavirus, Lymphocytic choriomeningitis mammarenavirus, Lujo mammarenavirus, Guanarito mammarenavirus, Flexal mammarenavirus, Machupo mammarenavirus, Oiveros mammarenavirus, and Whitewater Arroyo mammarenavirus.

[0021] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0022] FIGURE 1 depicts dose responses of 3 mAbs to the 7 LASV GPC lineages. The 3 mAbs are known to be escaped by L6.

[0023] FIGURE 2 depicts the 3 mAbs used for dose response testing shown in FIG. 1.

[0024] FIGURE 3 depicts the 3.0 A CryoEM Dataset of LASV 4P GPC in Complex with 12.1F Fab. (I15P, S171P, A33OP, Q331P mutations). The left panel displays selected 2D class averages, showcasing distinct orientations of the complex. The right panel illustrates the corresponding 3D reconstruction at atomic resolution, highlighting the structural integrity and interaction between the engineered LASV GPC and 12. IF Fab.

[0025] FIGURE 4 depicts the 3.0 A CryoEM Structure of LASV GPC (940 strain) in Complex with 12. IF Fab. Two orthogonal views are presented, with one rotated 90° for structural clarity. The LASV GPC trimer is depicted in blue, the Fab 12. IF components in yellow, and the viral membrane plane indicated below the structure. The resolution of the structure emphasizes the binding interface and spatial arrangement of the Fab relative to the viral glycoprotein.

[0026] FIGURE 5 depicts transferring mutations to Junin virus (JUNV). These variants are determined after a sequence alignment.

[0027] FIGURE 6 depicts the expression of Lassa virus (LASV) glycoprotein complex (GPC) as soluble ectodomains. The data was obtained using size-exclusion chromatography (SEC), which shows single peaks corresponding to the prefusion trimer conformation of the GPC. The SEC results were confirmed by liquid chromatography-mass spectrometry (LC-MS) to verify the molecular weight and purity of the expressed proteins, as well as by cryo-electron microscopy (cryoEM) to confirm structural integrity and the prefusion state. The data demonstrates an increase in expression levels for the 4P-stabilized GPC relative to the 5P-stabilized construct, suggesting that the 4P configuration enhances expression without compromising the prefusion trimer structure.

[0028] FIGURES 7A-7F illustrate the results of protein immunizations and enzyme-linked immunosorbent assays (ELISAs). Mice were immunized with 5 pg of each LASV GPC construct and boosted 21 days later. Depleted sera, collected 21 days post-boost, were analyzed for binding to LASV monoclonal antibodies (mAbs) by ELISA. The data shows that LASV-specific mAbs were detected in sera from immunized mice, while no antibody titers were observed in the negative control group, confirming the immunogenicity of the GPC constructs. The data highlights that the constructs successfully elicit an immune response targeting LASV.

[0029] FIGURE 8 provides a comparison of antibody titers from the protein immunization experiments described in FIGS. 7A-7F. The ELISA results indicate that sera from mice immunized with prefusion-specific GPC constructs produced higher LASV-specific antibody titers compared to other constructs. Depleted sera confirmed the specificity of the response to LASV GPC. No titers were detected in the negative control, further validating the specificity ofthe response. This data demonstrates the superior immunogenicity of the prefusion- specific GPC constructs in eliciting an immune response.

[0030] FIGURES 9A-9Y present individual mouse titers from mRNA-based immunizations and subsequent ELIS As. Each panel corresponds to a separate mouse, showing the variability and distribution of LASV-specific antibody titers across the experimental cohort. The ELISA data demonstrates that mice immunized with LASV GPC mRNA elicited detectable LASV-specific antibody titers, further supporting the immunogenicity of the prefusion-stabilized GPC delivered as an mRNA vaccine. The absence of titers in the negative control highlights the specificity of the immune response to the GPC.DETAILED DESCRIPTION

[0031] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.DEFINITIONS

[0032] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.

[0033] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

[0034] Throughout the description and claims of this specification, the word “comprise” and other forms of the word, such as “comprising” and “comprises, ” means including but not limited to, and are not intended to exclude, for example, other additives, segments, integers, or steps. Furthermore, it is to be understood that the terms comprise, comprising, and comprises as theyrelate to various aspects, elements, and features of the disclosed invention also include the more limited aspects of “consisting essentially of’ and “consisting of.”

[0035] As used herein, the singular forms “a, ” “an, ” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a “cell” includes aspects having two or more such cells unless the context clearly indicates otherwise.

[0036] Ranges can be expressed herein as from “about” one particular value and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about, ” it will be understood that the particular value forms another aspect. It should be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0037] As used herein, the terms “optional” or “optionally” mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0038] For the terms “for example” and “such as, ” and grammatical equivalences thereof, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise.

[0039] As used herein, “antibody" refers to a glycoprotein immunoglobulin which specifically binds to an antigen and comprises at least two light and two heavy chains interconnected by disulfide bonds. The antibody is composed of a variable region and a constant region where the variable region recognizes distinct antigens and the constant region is recognized by other cells of the immune system and components of the complement system.

[0040] As used herein, “monoclonal antibody" (mAb) refers to a non-naturally occurring antibody where the primary sequences are identical leading to a single binding specificity and affinity to a particular epitope. mAbs may be produced by hybridoma, recombinant, transgenic or other techniques.

[0041] As used herein, the term “antigen” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. Similarly, the term “immunogen” refers to an antigen that is able to induce an adaptive immune response (i.e., a humoral or cell-mediated immune response).

[0042] As used herein, the term “mutation” refers to the replacement, absence, or presence of additional amino acids in a peptide as compared to a control peptide.

[0043] As used herein, the term “adjuvant” refers to a drug, composition, or other substance that can increase the potency or efficacy of an administered immunogen.

[0044] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to the order or sequence of nucleotides along a strand of nucleic acids. In some cases, the order of these nucleotides may determine the order of the amino acids along a corresponding polypeptide chain. The nucleic acid sequence thus codes for the amino acid sequence. The nucleic acid sequence may be single-stranded or double-stranded, as specified, or contain portions of both double-stranded and single-stranded sequences. The nucleic acid sequence may be composed of DNA, both genomic and cDNA, RNA, or a hybrid, where the sequence comprises any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil (U), adenine (A), thymine (T), cytosine (C), guanine (G), inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc. It may include modified bases, including locked nucleic acids, peptide nucleic acids and others known to those skilled in the art.

[0045] As used herein, “amino acid” refers to a compound containing both amino ( — NH2) and carboxyl ( — COOH) groups generally separated by one carbon atom. The central carbon atom may contain a substituent which can be either charged, ionizable, hydrophilic or hydrophobic. Any of 22 basic building blocks of proteins having the formula NH2 — CHR — COOH, where R is different for each specific amino acid, and the stereochemistry is in the ‘L’ configuration. Additionally, the term “amino acid” can optionally include those with an unnatural ‘D’ stereochemistry and modified forms of the ‘D’ and ‘L’ amino acids.

[0046] As used herein, “peptide” refers to a chain of amino acids in which each amino acid is connected to the next by a formation of an amide bond. Peptides are generally considered to consist of up to 30 amino acids, or alternatively up to 25 amino acids, or alternatively up to 20 amino acids, or alternatively up to 15 amino acids, or alternatively up to 10 amino acids, or alternatively up to 5 amino acids, or alternatively between about 5-10 amino acids, or alternatively between about 10-15 amino acids, while the term “protein” is applied to compounds containing longer amino acid chains. As used herein, the term “protein domain” refers to a unit of a protein that serves a single role (e.g., functional, structural, etc.). Proteins can include a single domain or multiple domains. As used herein, the term “enzyme” refers to a protein which can catalyze or facilitate a chemical reaction or biological process.

[0047] As used herein, the term “cell” includes progeny. It is also understood that all progenies may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originallytransformed cell, are included. The “cells” referred to in the present invention generally are prokaryotic or eukaryotic hosts.

[0048] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0049] Thus, when referring to the function or activity of “modified protein, ” one of ordinary skill in the art would understand that this includes, for example, a protein that 1) performs the same activity or has the same specificity as the unmodified protein, but that may have a different level of activity; and 2) possesses an additional advantage over the unmodified protein for purposes of an assay, such as the addition of a detector. Determination of activity may be achieved using assays familiar to those of skill in the art, particularly with respect to the protein’s activity, and may include for comparison purposes, for example, the use of native and / or recombinant versions of either the modified or unmodified protein.

[0050] Modified proteins of the present invention may possess deletions and / or substitutions of amino acids; thus, a protein with a deletion, a protein with a substitution, and a protein with a deletion and a substitution are modified proteins. In some embodiments these modified proteins may further include insertions or added amino acids, such as with fusion proteins or proteins with linkers, for example. A “modified deleted protein” lacks one or more residues of the native protein, but possesses the specificity and / or activity of the native protein.

[0051] Substitutional or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide, particularly to reduce its immunogenicity / antigenicity, reduce any side effects in a subject, or increase its efficacy. Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. An antigenic region of a polypeptide may be substituted for a lessantigenic region; the less antigenic region may contain residues that are identical to the corresponding residues in the native protein, yet also contain some conservative substitutions and / or nonconservative substitutions.

[0052] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids or 5’ or 3’ sequences, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various non-coding sequences flanking either of the 5’ or 3’ portions of the coding region or may include various internal sequences, i.e., introns, which are known to occur within genes.

[0053] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, modified molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, binding sites to substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below.

[0054] A proteinaceous molecule has “homology” or is considered “homologous” to a second proteinaceous molecule if one of the following “homology criteria” is met: 1) at least 30% of the proteinaceous molecule has sequence identity at the same positions with the second proteinaceous molecule; 2) there is some sequence identity at the same positions with the second proteinaceous molecule and at the nonidentical residues, at least 30% of them are conservative differences, as described herein, with respect to the second proteinaceous molecule; or 3) at least 30% of the proteinaceous molecule has sequence identity with the second proteinaceous molecule, but with possible gaps of nonidentical residues between identical residues. As used herein, the term “homologous” may equally apply to a region of a proteinaceous molecule, instead of the entire molecule. If the term “homology” or “homologous” is qualified by a number, for example, “50% homology” or “50% homologous, ” then the homology criteria, with respect to 1), 2), and 3), is adjusted from “at least 30%” to “at least 50%. ”

[0055] “Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dos may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0056] A "pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the disclosure and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0057] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0058] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0059] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a non-immunogenic cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0060] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g. a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of type I diabetes. In some embodiments, a desired therapeutic result is the control of obesity. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.

[0061] As used herein, by “combination therapy” is meant that a first agent is administered in conjunction with another agent. “In conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during, or after delivery of the other treatment modality to the individual. Such combinations are considered to be part of a single treatment regimen or regime.

[0062] As used herein, the term “concurrent administration” means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.

[0063] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0064] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0065] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0066] The term “immunoglobulin” or “Ig, ” as used herein, is defined as a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0067] As used herein, the term “immune response” includes T-cell mediated and / or B-cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokineproduction and cellular cytotoxicity, and B cell responses, e.g., antibody production. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages. Immune cells involved in the immune response include lymphocytes, such as B cells and T cells (CD4+, CD8+, Thl and Th2 cells); antigen presenting cells (e.g., professional antigen presenting cells such as dendritic cells, macrophages, B lymphocytes, Langerhans cells, and non-professional antigen presenting cells such as keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes); natural killer cells; myeloid cells, such as macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0068] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated, ” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0069] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0070] The term “simultaneous administration, ” as used herein, means that a first therapy and second therapy in a combination therapy are administered with a time separation of no more than about 15 minutes, such as no more than about any of 10, 5, or 1 minutes. When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition comprising both a first and second therapy) or in separate compositions (e.g., a first therapy in one composition and a second therapy is contained in another composition).

[0071] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. Tn some instances, the terms “specific binding” or “specifically binding, ” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to meanthat the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A, ” the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0072] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.METHODS AND COMPOSITIONS

[0073] The present invention provides immunogens that, when administered to a subject in need thereof, elicit an immune response directed against an arenavirus. In some embodiments, the composition includes polypeptides, nucleotides, vectors, or vaccines. Further, when the compositions are administered to a subject, they elicit an immune response that serves to protect the inoculated mammal against conditions associated with an arenavirus.

[0074] In one embodiment, the present invention provides compositions that are useful as immunomodulatory agents, for example, in stimulating immune responses and in preventing arenavirus related pathology.

[0075] Also disclosed are engineered viruses which have been modified with one or more of the modifications disclosed herein.

[0076] The present invention should be construed to encompass any other arenavirus in which codon deoptimization may be useful to generate an immune response. In one embodiment, the arenavirus is lymphocytic choriomeningitis virus (LCMV). Additional non-limiting examples of arenaviruses include Lassa virus (LASV), Lujo virus (LUJV), Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabia virus (SABV), Chapare virus (CHPV), Ocozocoautla de Espinosa virus (OCEV), and Whitewater Arroyo virus (WWAV).

[0077] Also disclosed are nucleic acids encoding the protein sequences disclosed herein. The nucleic acid can be RNA or DNA. In one embodiment, the composition comprises an RNA or DNA vaccine.

[0078] The skilled artisan would understand that the nucleic acids of the invention encompass a RNA or a DNA sequence encoding a polypeptide of the invention, and any modified formsthereof, including chemical modifications of the DNA or RNA which render the nucleotide sequence more stable when it is cell-free or when it is associated with a cell. Chemical modifications of nucleotides may also be used to enhance the efficiency with which a nucleotide sequence is taken up by a cell or the efficiency with which it is expressed in a cell. Any and all combinations of modifications of the nucleotide sequences are contemplated in the present invention.

[0079] Disclosed herein are polypeptides or combination of polypeptides which are capable of generating an arenavirus-specific immune response. In another embodiment, the composition of the invention, comprising the polypeptide or combination of polypeptides of the present invention, is capable of generating arenavirus-specific antibodies.

[0080] The mutations disclosed herein can be engineered into an arenavirus such as lymphocytic choriomeningitis virus (LCMV), Lassa virus (LASV), Lujo virus (LUJV), Junin virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabia virus (SABV), Chapare virus (CHPV), Ocozocoautla de Espinosa virus (OCEV), and Whitewater Arroyo virus (WWAV). For example, these mutations can be engineered into vaccine strains.

[0081] These mutations can include, but are not limited to, any of the following: an immunogen comprising SEQ ID NO: 4, wherein SEQ ID NO: 4 comprises one or more mutations, wherein at least one of the mutations comprises A132C and I334C, A132P, A173C and T182C, A173P, A184P, A202C and T215C, A297V, A30C and F446C, A328P, A33OP, D130P, D401P, E151P, E16P, E3O3Q, E303Q and K304Q, E3O3T, E307L, G102C and T225C, G174P, G240P, G276C and N295C, G294P, H131P, H305C and N346C, H305F, H305I, H354W, H93C and A195C, I113C and S154C, I15P, I334P, I345V, I4C and L430C, K304N, K304Q, K327P, K339L, K352F, K63C and A407C, L120F, L128C and G160C, L142C and T249C, L290P, L428P, L68F, L73P, M192C and G198C, M332P, M359F, M359L, M75F, M96C and G102C, N178P, N20C and L430C, N295P, N342C and Q348C, N395P, N74C and I286C, N74D, N74P, N90C and L203C, Q189E, Q321E, Q331P, Q348P, Q406E, Q69E, R282P, R356F, R422F, S 143C and R248C, S171P, S199P, S246L, S267C and I361C, S27C and S437C, S333P, S85C and G240C, S85V, T249L, T261I, T261M, T296P, T412C and G424C, T77C and W283C, T87C and G197C, V14P, VI 83P, V31C and Y441C, V97C and A132C, W196C and G240C, W196P, Y150P, Y200P, Y62C and A407C, Y62I, F262P, Y150C and R257C, I95A and A195F, A322V, D306W, L68F and I403A, H305N, L128P, E396P, L105A and A195F, Q189A and H354F, T87A and G197F, L242A and I337F, G243C and I350C, R193M, M192R, I350L, G243S, D251Q, V31I, S367T, Y369F, K339T, K339W, A343S, T261F, A177C and E329C, T87S, I334L, N346S, T281P,E329P, M19P, G197P, H179P, H230P, G294P, G206P, E287P, S216P, I213P, D229P, T215P, I201P, N99P, D175P, Q335P, A176P, N342P, G240P, G45P, N114P, A173P, T77P, G174P, N395P, M332P, WT, G271P, N295P, G181P, V14P, A177P, G208P, I286A, D357A, T106A, W283A, G277A, H398A, V18A, F7A, S267A, G181A, E228A, R379A, Q406A, Q335A, P275A, T182A, T296A, G198A, S163A, H93A, E329A, E104A, S476A, T190A, N209A, S205A, H179A, F233A, S85A, S400A, S 171A, Q405A, Y200A, G206A, T397A, K481A, G45A, T274A, N295A, D268A, L203A, G208A, N158A, V65A, C53A, S27A, K33A, E76A, T249A, T412A, D402A, Q423A, M284A, T438A, P236A, H448A, M194A, D175A, D204A, G424A, E10A, L442A, T59A, T249L, H3O5F, T261M, R356F, L68F, R422F , A297V, T249I, T87S, Q189D, or T380H.

[0082] It is noted that in the above list, where “and” is present between mutations, it is meant that particular mutation includes BOTH of those residues. Otherwise, those in the list are considered alternatives, although two, three, four, five, six, seven, eight, nine, ten, or more mutation of the list can be used in the same immunogen.

[0083] It is noted that the mutations described above with reference to SEQ ID NO: 4 can likewise be made in any other strain, such as SEQ ID NO: 1-3 or 5-7, or other strains not specifically disclosed herein. SEQ ID NO: 4 is simply used as a point of reference and one of skill in the art can use this sequence as a guide to produce identical mutations in closely related homologs of SEQ ID NO: 4.

[0084] Furthermore, the immunogen can comprise a fragment of SEQ ID NO: 4. An immunogen is designed to elicit an immune response, so any length immunogen is contemplated, as long as that immunogen is able to elicit an immune response. This is described herein as a “functional fragment” or an “immunogenic fragment.” For example, disclosed is a peptide sequence which is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179,180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191 , 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255,256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312,313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350,351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407,408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426,427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445,446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464,465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483,484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 amino acids in length, and which includes 1, 2, 3, 4, 5, or more of the mutations specified herein.

[0085] Attenuated viruses generated by the reverse genetics approach can be used in a vaccine and pharmaceutical formulations described herein. Reverse genetics techniques can also be used to engineer additional mutations to other viral genes important for vaccine production — i.e., the epitopes of useful vaccine strain variants can be engineered into the attenuated virus. Alternatively, completely foreign epitopes, including antigens derived from other viral or non- viral pathogens can be engineered into the attenuated strain.

[0086] In an alternate embodiment, a combination of reverse genetics techniques and reassortant techniques can be used to engineer attenuated viruses having the desired epitopes in arenaviruses. For example, an attenuated virus (generated by natural selection, mutagenesis or by reverse genetics techniques) and a strain carrying the desired vaccine epitope (generated by natural selection, mutagenesis or by reverse genetics techniques) can be co-infected in hosts that permit reassortment of the segmented genomes. Reassortants that display both the attenuated phenotype and the desired epitope can then be selected.

[0087] The immunogens described herein can itself be used as the active ingredient in vaccine or pharmaceutical formulations. These immunogens can be capable of inducing a robust anti-arenavirus response in the host — a feature which contributes to the generation of a strong immune response when used as a vaccine.

[0088] The immunogens, which induce an arenavirus -specific immune response in hosts, may also be used in pharmaceutical formulations for the prophylaxis or treatment of other viral infections, or arenavirus related diseases. For an immunological composition to be useful as a vaccine, the antigenic composition must induce an immune response to the antigen in a cell, tissue or mammal (e.g., a human). Preferably, the vaccine induces a protective immune response in the mammal. As used herein, an “immunological composition” may comprise, by way of examples, an antigen (e.g., immunogen, such as a polypeptide), a nucleic acid encoding an antigen (e.g., an antigen expression vector), or a cell expressing or presenting an antigen or cellular component. In particular embodiments the immunological composition comprises or encodes all or part of any polypeptide antigen described herein, or an immunologically functional equivalent thereof. In other embodiments, the immunological composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination.

[0089] In the context of the present invention, the term “vaccine” refers to a substance that induces anti-arenavirus immunity or suppresses an arenavirus upon inoculation into an animal.

[0090] Either a live recombinant viral vaccine or an inactivated recombinant viral vaccine can be formulated using the polypeptides disclosed herein. Many methods may be used to introduce the vaccine formulations described above, these include but are not limited to introduction intranasally, intratracheally, orally, intradermally, intramuscularly, intraperitoneally, intravenously, and subcutaneously. It may be preferable to introduce the virus vaccine formulation via the natural route of infection of the pathogen for which the vaccine is designed, or via the natural route of infection of the parental attenuated virus. Where a live arenavirus vaccine preparation is used, it may be preferable to introduce the formulation via the natural route of infection for arenavirus virus.

[0091] The invention provides a method for treating or preventing arenavirus infection or an arenavirus related disease or disorder. In one embodiment, the method comprises administering an immunogen as described herein.

[0092] The therapeutic compositions of the invention may be administered prophylactically or therapeutically to subjects suffering from, or at risk of, or susceptible to, developing the disease or condition. Such subjects may be identified using standard clinical methods. In the context of the present invention, prophylactic administration occurs prior to the manifestation of overt clinicalsymptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease-related complications.

[0093] The composition may be combined with an adjuvant. An adjuvant refers to a compound that enhances the immune response when administered together (or successively) with the immunological composition. Examples of suitable adjuvants include cholera toxin, salmonella toxin, alum and such, but are not limited thereto. Furthermore, a vaccine of this invention may be combined appropriately with a pharmaceutically acceptable carrier. Examples of such carriers are sterilized water, physiological saline, phosphate buffer, culture fluid and such. Furthermore, the vaccine may contain as necessary, stabilizers, suspensions, preservatives, surfactants and such. The vaccine is administered systemically or locally. Vaccine administration may be performed by single administration or boosted by multiple administrations.

[0094] In one embodiment, the methods of the present invention comprise administering an immunological composition of the invention directly to a subject in need thereof. Administration of the composition can comprise, for example, intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

[0095] Furthermore, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. One skilled in the art can easily make any necessary adjustments in accordance with the exigencies of the particular situation.

[0096] The present invention envisions treating or preventing a disease or condition associated with an arenavirus in a mammal by the administration of a therapeutic composition of the invention to a mammal in need thereof. Administration of the composition in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient’s physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the compositions of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. The amount administeredwill vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art.

[0097] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of the immunogen, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0098] The amount of the pharmaceutical composition of the invention which will be effective in the treatment of a particular disease or disorder will depend on the nature of the disease or disorder, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.EXAMPLES

[0099] Protein Design and Mutagenesis: LASV glycoprotein constructs were engineered using site-directed mutagenesis to introduce mutations intended to enhance protein expression, stability, and immunogenicity. Mutations included proline substitutions, disulfide bonds, and other modifications. Codon optimization was performed as needed. Constructs were synthesized and constructed with Golden Gate assembly. All constructs were verified with DNA sequencing.

[0100] Native Display of Viral Glycoproteins and Detection by Conformational Antibodies: To assess stabilizing mutations in viral glycoproteins, a native display platform was employed using transfected cells. Plasmids encoding wild-type (WT) or mutant viral glycoproteins were transfected into cells, with a P2A-mScarlet cassette to monitor transfection efficiency. After 48 hours of expression, cells were labeled with a panel of conformational antibodies that target distinct epitope groups. Primary antibody labeling was performed at room temperature for 1 hour to ensure optimal binding under native conditions. Afterward, cells were washed three times with PBSA to remove unbound antibodies. Secondary labeling was conducted at 4°C to minimize nonspecific binding, using Alexa Fluor 647-conjugated anti-human IgG (AF-647) for 30 minutes in the dark.

[0101] To evaluate the stability of mutant glycoproteins, cells were subjected to a heat shock treatment at 50°C for 10 minutes prior to the labeling process, followed by a 30-minute recovery at 37°C. Antibody binding was quantified using flow cytometry, with measurements performed in duplicate for each condition. Mean fluorescence intensities (MFIs) were averaged, and binding of mutant glycoproteins was normalized to WT glycoproteins for comparison. Statistical analyses were conducted to determine the significance of binding differences between WT and mutant groups. Enhanced antibody binding post-heat shock was used as an indicator of stabilizing mutations. mScarlet fluorescence was monitored to validate transfection efficiency across all samples.

[0102] Expression and Purification of Proteins: Initial screening of glycoprotein variants was performed by transient transfection in HEK293T cells to assess expression and antigenicity (see below). Constructs identified as promising were subsequently scaled up and expressed as soluble ectodomains in Expi293 cells to achieve higher yields and quality. Proteins were purified by affinity chromatography (e.g., Strep-tagll) and further polished by size-exclusion chromatography (SEC) in a buffer containing 20 mM Tris, pH 7.5, and 150 mM NaCl.

[0103] Structural Integrity and Biophysical Characterization: The conformational integrity and prefusion state of the purified glycoprotein ectodomains were examined by cryo-electron microscopy (cryo-EM). Samples were vitrified on copper grids and imaged using a direct electron detector. Three-dimensional density maps were reconstructed to confirm that the proteins adopted the desired prefusion trimer conformation. Bottom-up LC-MS was performed to verify protein preps.

[0104] Antigenicity and Immunogenicity Assays: To determine antigenicity, an enzyme- linked immunosorbent assay (ELISA) was performed. Purified glycoprotein constructs were immobilized on ELISA plates and incubated with serial dilutions of LASV-specific monoclonal antibodies. Bound antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, and absorbance at 450 nm was measured. Constructs were assessed relative to wild-type controls and monocolonal antibodies (8.9F, 12. IF, and isotype controls) , enabling evaluation of how introduced mutations affected antibody binding.

[0105] Immunogenicity Studies in Mice: Immunogenicity was tested using female CD1 mice, aged 6-8 weeks. Mice were immunized intramuscularly with 5 pg of the purified glycoprotein ectodomains formulated in a squalene-based adjuvant. Mice received a prime immunization on day 0 and a booster immunization on day 21. Blood was collected 21 days postboost, and serum samples were prepared by depleting nonspecific antibodies using protein G beads. ELISA assays with coated wild-type or mutant glycoproteins were conducted to measure the LASV-specific antibody titers induced by each construct.

[0106] Animal Welfare: All animal work was performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC). Mice were housed in pathogen-free facilities with ad libitum access to food and water, and every effort was made to minimize animal distress during the study.TABLE 1. Viral sequences.TABLE 2. Singleton mutations to LASV lineage L4 (SEQ ID NO: 4).TABLE 3. Key for TABLE 4 - TABLE 19. Antibodies marked with * can be found in U.S.Patent No. 11,198,723, which is incorporated by reference herein in its entirety. Antibodies marked with ** can be found in Li, H., et al. A cocktail of protective antibodies subverts the dense glycan shield of Lassa virus. Science Translational Medicine. 2022;14(668):eabq0991, which is incorporated by reference herein in its entirety.TABLE 4. Room temperature antibody panel (controls).TABLE 5. Room temperature antibody panel (prolines).TABLE 6. Room temperature antibody panel (prolines).TABLE 7. Room temperature antibody panel (disulfides).TABLE 8. Room temperature antibody panel (disulfides).TABLE 9. 50°C antibody panel (controls).TABLE 10.50°C antibody panel (prolines).TABLE 11. 50°C antibody panel (prolines).TABLE 12. 50°C antibody panel (disulfides).TABLE 13. 50°C antibody panel (disulfides).TABLE 14. Stacks of mutations to LASV lineage L4 (SEQ ID NO: 4).TABLE 15. L4 normalized binding.TABLE 16. L4 normalized binding.TABLE 17. Normalized by L4.TABLE 18. Normalized by L4.TABLE 19. LASV soluble GPC epitope screening by BLI.TABLE 20. Transfer of a few mutations to Junin Virus (JUNV).TABLE 21. Transfer of a few mutations to JUNV

Claims

CLAIMSWhat is claimed is:

1. An immunogen comprising SEQ ID NO: 4, wherein SEQ ID NO: 4 comprises one or more mutations, wherein at least one of the mutations comprises A132C and I334C, A132P, A173C and T182C, A173P, A184P, A202C and T215C, A297V, A30C and F446C, A328P, A330P, D130P, D401P, E151P, E16P, E303Q, E303Q and K304Q, E303T, E307L, G102C and T225C, G174P, G240P, G276C and N295C, G294P, H131P, H305C and N346C, H305F, H3O5I, H354W, H93C and A195C, Il 13C and S154C, Il 5P, I334P, I345V, I4C and L430C, K304N, K304Q, K327P, K339L, K352F, K63C and A407C, L120F, L128C and G160C, L142C and T249C, L290P, L428P, L68F, L73P, M192C and G198C, M332P, M359F, M359L, M75F, M96C and G102C, N178P, N20C and L430C, N295P, N342C and Q348C, N395P, N74C and I286C, N74D, N74P, N90C and L203C, Q189E, Q321E, Q331P, Q348P, Q406E, Q69E, R282P, R356F, R422F, S143C and R248C, S171P, S199P, S246L, S267C and I361C, S27C and S437C, S333P, S85C and G240C, S85V, T249L, T261I, T261M, T296P, T412C and G424C, T77C and W283C, T87C and G197C, V14P, V183P, V31C and Y441C, V97C and A132C, W196C and G240C, W196P, Y150P, Y200P, Y62C and A407C, Y62I, F262P, Y150C and R257C, I95A and A195F, A322V, D306W, L68F and 1403 A, H3O5N, L128P, E396P, L105A and A195F, Q189A and H354F, T87A and G197F, L242A and I337F, G243C and I350C, R193M, M192R, I35OL, G243S, D251Q, V31I, S367T, Y369F, K339T, K339W, A343S, T261F, A177C and E329C, T87S, I334L, N346S, T281P, E329P, M19P, G197P, H179P, H230P, G294P, G206P, E287P, S216P, 1213?, D229P, T215P, I201P, N99P, D175P, Q335P, A176P, N342P, G240P, G45P, N114P, A173P, T77P, G174P, N395P, M332P, WT, G271P, N295P, G181P, V14P, A177P, G208P, I286A, D357A, T106A, W283A, G277A, H398A, VI 8A, F7A, S267A, G181A, E228A, R379A, Q406A, Q335A, P275A, T182A, T296A, G198A, S163A, H93A, E329A, E104A, S476A, T190A, N209A, S205A, H179A, F233A, S85A, S400A, S171A, Q405A, Y200A, G206A, T397A, K481A, G45A, T274A, N295A, D268A, L203A, G208A, N158A, V65A, C53A, S27A, K33A, E76A, T249A, T412A, D402A, Q423A, M284A, T438A, P236A, H448A, M194A, D175A, D204A, G424A, E10A, L442A, T59A, T249L, H305F, T261M, R356F, L68F, R422F , A297V, T249I, T87S, QI89D, or T38OH.

2. The immunogen of claim 1, wherein said immunogen comprises two or more mutations.

3. The immunogen of claim 1, wherein said immunogen comprises three or more mutations.

4. The immunogen of claim 1, wherein said immunogen comprises four or more mutations.

5. The immunogen of claim 1, wherein said immunogen comprises five or more mutations.

6. A composition comprising at least one immunogen of claim 1.

7. A composition comprising at least two immunogens of claim 1.

8. The composition of claim 6, wherein the composition comprises at least two immunogens, wherein one immunogen comprises a mutation as listed in claim 1, and one additional immunogen.

9. The composition of any one of claims 6-8, wherein said composition further comprises an adjuvant.

10. The composition of any one of claims 6-9, wherein said composition further comprises a delivery vehicle.

11. The composition of any one of claims 6-10, wherein said composition is in a vaccine form.

12. A nucleic acid encoding at least one immunogen of claim 1.

13. A vector comprising the nucleic acid of claim 12.

14. A cell comprising the vector of claim 13.

15. A method of inducing an immune response in a subject, wherein said method comprises delivering one or more of the immunogens of claim 1 into the subject in an amount sufficient to induce an immune response in the subject.

16. A method of inducing an immune response in a subject, wherein said method comprises delivering one or more of the nucleic acids of claim 12 to the subject in an amount sufficient to induce an immune response in the subject.

17. The method of claim 16, wherein the subject is a human.

18. A method of inducing an immune response against an arenavirus in a subject, wherein said method comprises delivering one or more of the immunogens of claim 1 into the subject in an amount sufficient to induce an immune response in the subject.

19. A method for inducing an immune response against an arenavirus in a subject, wherein said method comprises delivering one or more of the nucleic acids of claim 12 to the subject in an amount sufficient to induce an immune response in the subject.

20. The method of claim 18 or 19, wherein the subject is a human.

21. The method of claim 18 or 19, wherein the arenavirus comprises Lassa mammarenavirus, Argentinian mammarenavirus, Lymphocytic choriomeningitis mammarenavirus, Lujo mammarenavirus, Guanarito mammarenavirus, Flexal mammarenavirus, Machupo mammarenavirus, Oiveros mammarenavirus, and Whitewater Arroyo mammarenavirus.

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