Methods to prevent e dimer mixing and heterodimer formation in multivalent dengue subunit vaccines

Abstract

Provided herein according to some embodiments are modified dengue virus E proteins, compositions comprising the modified dengue virus E proteins, and methods of use. The modified dengue virus E protein comprise three or more amino acid substitutions that enable a tetravalent mixture of DENV1, DENV2, DENV3, and DENV4 serotypes without formation of E protein heterodimers that lower immunogenic efficacy of the E proteins.

Description

METHODS TO PREVENT E DIMER MIXING AND HETERODIMER FORMATION INMULTIVALENT DENGUE SUBUNIT VACCINESSTATEMENT OF PRIORITY

[0001] This application claims the benefit, under 35 U.S.C. §119(e), of U.S. Provisional Application No. 63 / 517,424, filed August 3, 2023, the entire contents of which is incorporated by reference herein in its entirety.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing in XML format, entitled 5470-957WO_ST26.xml, 9,737 bytes in size, generated on July 31, 2024, and filed herewith, is hereby incorporated by reference into the specification for its disclosures.FIELD OF THE INVENTION

[0003] This invention relates to modified dengue virus E proteins, compositions comprising the modified dengue virus E proteins, and methods of use.GOVERNMENT SUPPORT

[0004] This invention was made with government support under Grant Number All 61025 awarded by the National Institutes of Health. The government has certain rights in this invention.BACKGROUND

[0005] Dengue virus (DENV) is a mosquito-borne flavivirus that is spreading at an unprecedented rate and has developed into a major health and economic burden in over 100 countries. The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses primarily only to the infecting serotype. Problems have arisen in previously developed Dengue vaccines; for example, in 2017, Sanofi Pasteur’s Dengvaxia raised safety concerns for some recipients because it does not protect against all 4 serotypes, and in some cases, enhanced disease. An effective vaccine against dengue needs to elicit long lastingprotective antibody responses to all four serotypes, DENV1, DENV2, DENV3, and DENV4, simultaneously.

[0006] The rise in global incidence of DENV and the resulting rise in mortality rate necessitates an effective universal vaccine against it. The E protein homodimer from dengue is the primary target of protective antibodies and is the foundation of current efforts to create a subunit vaccine for dengue virus. Improved E protein homodimers for use in universal dengue virus vaccines are needed.SUMMARY OF THE INVENTION

[0007] The present invention is based, in part, on the development of modified Dengue virus (DENV) envelope (E) polypeptides with orthogonal interfaces to enable a tetravalent mixture of E polypeptides of DENV1, DENV2, DENV 3 and DENV4 serotypes with reduced E heterodimer formation. Advantageously, the modified E proteins present epitopes recognized by neutralizing antibodies.

[0008] In an aspect, a modified DENV E polypeptide of DENV1, DENV2, DENV3, or DENV4 serotype comprising three or more amino acid mutations in domain I and / or domain II of the E polypeptide is provided, the three or more amino acid mutations inhibiting heterodimer formation between DENV E polypeptide serotypes, the modified E polypeptide retaining capability of homodimer formation.

[0009] In an aspect, a nucleic acid encoding the modified DENV E polypeptide of DENV1, DENV2, DENV3, or DENV4 serotype comprising three or more amino acid mutations in domain I and / or domain II of the E polypeptide. In some embodiments a vector comprising the nucleic acid of the invention is provided.

[0010] In another aspect, a composition comprising one or more of the modified E polypeptides or one or more of the nucleic acids or vectors is provided. In an aspect, a pharmaceutical composition comprising a composition of the invention and a pharmaceutically acceptable carrier is provided.

[0011] In an aspect, an immunogenic composition comprising an effective amount of one or more of the modified E polypeptides, one or more of the nucleic acids or vectors, the composition, or the pharmaceutical composition is provided.

[0012] In another aspect, a method of protecting a subject from the effects of dengue virus infection is provided, comprising administering to the subject an effective amount of one or more of the modified E polypeptides, one or more of the nucleic acids or vectors, the composition, the immunogenic composition and / or the pharmaceutical composition to thereby protect the subject from the effects of the dengue virus infection.

[0013] In a further aspect, a method of preventing a dengue virus infection in a subject is provided, comprising administering to the subject an effective amount of one or more of the modified E polypeptides, one or more of the nucleic acids or vectors, the composition, the immunogenic composition and / or the pharmaceutical composition, to thereby prevent the dengue virus infection in the subject.

[0014] In an aspect, a method of reducing heterodimer formation in a tetravalent dengue virus vaccine comprising DENV E polypeptides of DENV1, DENV2, DENV3, and DENV4 serotypes is provided, comprising providing three or more of the modified E polypeptides, three or more of the nucleic acids or vectors, the composition, the immunogenic composition and / or the pharmaceutical composition in the tetravalent dengue virus vaccine to thereby reduce heterodimer formation between DENV1, DENV2, DENV3, and / or DENV4 serotypes, wherein at least three of the four E proteins are modified polypeptides, or nucleic acids encoding the modified E polypeptides, and wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations.

[0015] These and other aspects of the invention are set forth in more detail in the description of the invention below.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A-1B. Mass photometry showing DENV E proteins comprising stabilizing mutations form stable homodimers when expressed on their own. (FIG. 1A) DENV serotype 1 with stabilizing mutations (DV1_SC12). (FIG. IB) DENV serotype 2 with stabilizing mutations tagged with Maltose Binding Protein (MBP) and 2x Strep-tag (DV2_SC10_MBP_2x Strep).

[0017] FIG. 2 Formation of heterodimers of E proteins from DENV1 and DENV2 serotypes each with stabilizing mutations; mass of heterodimers and homodimers (top) and mass photometry results (bottom) indicating robust formation of heterodimers with E proteins from DENV1 and DENV2 serotypes.

[0018] FIGS. 3A-3C. Example 110 modified E protein embodiment. (FIG. 3A) Ribbon diagram of example modified E protein (upper) with close up view of mutated residues (lower). (FIG. 3B) Ribbon diagram of DENY 1 serotype E protein with 110 mutations orthogonal to DV2 SCIO E protein with stabilizing mutations. (FIG. 3C) Mass photometry of example embodiment mutations shows DV1 110 example modified E protein disfavors heterodimerization with DENV2 serotype E protein comprising a stabilizing mutation set (DV2 SC10-MBP).

[0019] FIGS. 4A-4C. Example 05 modified E protein embodiment. (FIG. 4A) Ribbon diagram of example modified E protein (upper) with close up view of mutated residues (lower). (FIG. 4B) Ribbon diagram of DENV3 serotype E protein with 05 mutations orthogonal to DV2 SCIO E protein with stabilizing mutations. (FIG. 4C) Mass photometry of example embodiment mutations shows DV3 05 example modified E protein disfavors heterodimerization with DV2 SC10-MBP.

[0020] FIGS. 5A-5C. Example 06 modified E protein embodiment. (FIG. 5A) Ribbon diagram of example modified E protein (upper) with close up view of mutated residues (lower). (FIG. 5B) Ribbon diagram of DENV3 serotype E protein with 06 mutations orthogonal to DV2 SCIO E protein with stabilizing mutations. (FIG. 5C) Mass photometry of example embodiment mutations shows DV3 06 example modified E protein disfavors heterodimerization with DV2 SC10-MBP.

[0021] FIG. 6. Graph showing ELISA results of antibody experiments for orthogonal designs of example modified E proteins DV1 110, DV3 05, and DV3 06. C8, CIO, and B7 are dimer specific antibodies (Abs) that bind to all 4 DENV serotypes; 8A1 is a DENV3 Ab, and 1F4 is a DENV1 Ab.DETAILED DESCRIPTION

[0022] The present invention will now be described in more detail with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In addition, any references cited herein are incorporated by reference in their entireties.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, patent publications and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0024] Amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three-letter code, both in accordance with 37 C.F.R. §1.822 and established usage.

[0025] Except as otherwise indicated, standard methods known to those skilled in the art may be used for cloning genes, amplifying and detecting nucleic acids, and the like. Such techniques are known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Ed. (Cold Spring Harbor, NY, 2012); Ausubel et al. Current Protocols in Molecular Biology (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0026] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.

[0027] Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0028] To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0029] As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0030] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0031] The term “about,” as used herein when referring to a measurable value such as an amount of polypeptide, dose, time, temperature, enzymatic activity or other biological activityand the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

[0032] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel character! stic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0033] The term “consists essentially of’ (and grammatical variants), as applied to a polypeptide or polynucleotide sequence of this invention, means a polypeptide or polynucleotide that consists of both the recited sequence (e.g, SEQ ID NO) and a total of ten or less (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional amino acids on the N-terminal and / or C-terminal ends of the recited sequence or additional nucleotides on the 5’ and / or 3’ ends of the recited sequence such that the function of the polypeptide or polynucleotide is not materially altered. The total of ten or less additional amino acids or nucleotides includes the total number of additional amino acids or nucleotides on both ends added together. The term “materially altered,” as applied to polypeptides of the invention, refers to an increase or decrease in biological activities / properties (e.g., heterodimer formation) of at least about 50% or more as compared to the activity of a polypeptide consisting of the recited sequence.

[0034] The term “modified E polypeptide” as used herein refers to the addition, deletion, and / or substitution of one or more amino acids from the wild-type E polypeptide, wherein the modified polypeptide substantially retains at least one biological activity normally associated with that polypeptide (e.g., immunogenicity). Example wild-type serotypes of DENV1, DENV2, DENV3 and DENV4 E polypeptides are provided in Table 1 at SEQ ID NOS:l-3, and 7; however, any wild-type E polypeptide serotype can be used in accordance with the teachings of the present invention, with sequences from other strains publicly available. In some embodiments, modified E polypeptide is a modified E polypeptide from a DENV1, DENV2, DENV3 or DENV4 serotype.

[0035] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise.

[0036] As used herein, a “functional” polypeptide or “functional fragment” is one that substantially retains at least one biological activity normally associated with that polypeptide(e.g., wild-type protein or fragment thereof). In particular embodiments, the “functional” polypeptide or “functional fragment” substantially retains all of the activities possessed by the unmodified polypeptide (e.g., wild-type protein or fragment thereof). By “substantially retains” biological activity, it is meant that the polypeptide retains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native polypeptide (and can even have a higher level of activity than the native polypeptide). A “nonfunctional” polypeptide is one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only an insignificant amount, e.g., less than about 10% or even 5%). Biological activities such as recognition by neutralizing antibodies can be measured using assays that are well known in the art and as described herein.

[0037] The term “fragment,” as applied to a peptide, will be understood to mean an amino acid sequence of reduced length relative to a reference peptide (e.g., wild-type protein) or amino acid sequence and comprising, consisting essentially of, and / or consisting of an amino acid sequence of contiguous amino acids identical to the reference peptide or amino acid sequence. Such a peptide fragment according to the invention may be, where appropriate, included in a larger polypeptide of which it is a constituent. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of at least about 5, 10, 15, 20, 25, 30, 35, 46. 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more consecutive amino acids of a peptide or amino acid sequence according to the invention.

[0038] The terms “polynucleotide,” “nucleic acid,” “nucleic acid molecule,” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, genomic DNA, chimeras of RNA and DNA, isolated DNA of any sequence, isolated RNA of any sequence, synthetic DNA of any sequence (e.g., chemically synthesized), synthetic RNA of any sequence (e.g., chemically synthesized), nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Suchnucleotides can be used, for example, to prepare nucleic acid molecules that have altered basepairing abilities or increased resistance to nucleases.

[0039] If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by nonnucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and singlestranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0040] As used herein, “expression” refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0041] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 80%, 85%, 90%, or 95%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example, those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. Examples of the programs include BLASTN and BLASTP, using the following default parameters: Genetic code = standard; fdter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0042] As used herein, the term “operably linked” means that the promoter and coding sequence are joined together in a manner that allows them to carry out their normal functions, i.e., transcription of the coding sequence is under the control of the promoter and the transcript produced is correctly translated into the desired product.

[0043] Those skilled in the art will appreciate that a variety of promoters may be used depending on the level and specific expression desired. The promoter may be constitutive or regulatable, depending on the pattern of expression desired. The promoter may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0044] The promoter can be native to the target cell or subject to be treated and / or can be native to the heterologous nucleotide sequence. The promoter is generally chosen so that it will function in the target cell(s) of interest. The promoter can optionally be a mammalian promoter. The promoter may further be constitutive or regulatable (e.g, inducible).

[0045] Promoters for nucleic acid delivery can be tissue preferred and / or -specific promoters.

[0046] The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, and viruses (bacteriophage, animal viruses, and plant viruses). In some embodiments the vector is a viral vector, optionally an adeno-associated virus (AAV) vector. Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects. Viral vectors that can be used include, but are not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes virus, Epstein-Barr virus, and / or adenovirus vectors. Non-viral vectors include, but are not limited to, plasmids, liposomes, electrically charged lipids (cytofectins), lipid nanoparticles (LNPs), nucleic acid-protein complexes, and biopolymers. In addition to a nucleic acid of interest, a vector may also comprise one or more regulatory regions, and / or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.). Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al., J. Biol. Chem. 267:963(1992); Wu et al., J. Biol. Chem. 263: 14621 (1988); and Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0047] As used herein, the term "adeno-associated virus" (AAV) includes but is not limited to, AAV serotype 1 (AAV1), AAV2, AAV3 (including types 3 A and 3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS etal., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Recently, a number of putative new AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33:375-383; and Table 1). An AAV can be selected for tissue-specific delivery. AAV variants with reduced immunogenicity may also be utilized, and may comprise chimeric AAV, for example, AAV-DJ. Design strategies for AAV vectors may also be employed for the delivery of the modified E polypeptides according to the present invention. See, e.g., Lee, et al. (2018) Adeno-associated virus (AAV) vectors: rational design strategies for capsid engineering. Curr. Opin. Biomed. Eng., 7, 58-63; see also Parambi et al., 2021 Oct \5, Mol Neurobiol. 2022; 59(1): 191-233, doi: 10.1007 / sl2035-021-02555-y, incorporated herein by reference in its entirety, and specifically Table 1 for teachings of viral vectors.

[0048] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.

[0049] As used herein, the term “host cell” refers to a cell that is engineered to express the modified polypeptide or functional fragment thereof (e.g., a modified full length [protein] or a fragment thereof). “Host cell” refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0050] Host cells may be derived from prokaryotes or eukaryotes, depending upon whether the desired result is replication of the vector or expression of part or all of the vector-encoded nucleic acid sequences. Prokaryotes include gram negative or positive cells. Numerous cell lines and cultures are available for use as a host cell, and they can be obtained through the American Type Culture Collection (ATCC), which is an organization that serves as an archive for living cultures and genetic materials (atcc.org). An appropriate host can be determined by one of skill in the art based on the vector backbone and the desired result. A plasmid or cosmid, for example, can be introduced into a prokaryote host cell for replication of many vectors. Bacterial cells used as host cells for vector replication and / or expression include DH5a, JM109, and KC8, as well as a number of commercially available bacterial hosts such as SURE® Competent Cells and SOLOPACK™ Gold Cells (STRATAGENE®, La Jolla). Alternatively, bacterial cells such as E. coli LE392 could be used as host cells for phage viruses.

[0051] “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) refers to 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.

[0052] The term “modulate,” “modulates,” or “modulation” refers to enhancement (e.g., an increase) or inhibition (e.g., a decrease) in the specified level or activity.

[0053] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, twelve-fold, or even fifteen-fold and / or can be expressed in the enhancement and / or increase of a specified level and / or activity of at least about 1%, 5%, 10%, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more.

[0054] “Inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 1, 5, 10, 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%). In some embodiments, the inhibition is inhibiting of dimer mixing and / or heterodimer formation between E polypeptides of different flavivirus serotypes, for example, DENV serotypes, e.g., between DENV1 and DENV2, DENV1 and DENV3, DENV1 and DENV4, DENV2 and DENV3, DENV2 and DENV4, and / or DENV3 and DENV4 serotypes.

[0055] “Retain” or “maintain” or grammatical variations thereof as used herein refers to minimal variation in a specified level or activity, for example 100%, 95%, 90%, 85% activity relative to wild-type, or resulting in little or essentially no difference in activity (at most, an insignificant amount, e.g., less than about 10% or even 5%) relative to wild-type. In some embodiments, capability of homodimer formation between modified E polypeptides of the same DENV serotype, e.g., DENV1, DENV2, DENV3, or DENV4 serotype is retained relative to unmodified E polypeptides.

[0056] The term “contact” or grammatical variations thereof as used with respect to a polypeptide or polynucleotide and a cell or aggregate, refers to bringing the polypeptide or polynucleotide and the cell or aggregate in sufficiently close proximity to each other for one to exert a biological effect on the other. In some embodiments, the term contact means binding of the polypeptide or polynucleotide to the cell or aggregate.

[0057] A “subject” may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and / or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g., a human, non-human primate, lagomorph (e.g., rabbit), or rodent (e.g., mouse, rat). In some embodiments a human subject is a neonate, child, adult or geriatric subject. In some embodiments a human subject is at least 50, 60, 70, 80, or 90 years old.

[0058] “ Treat,” “treating” and similar terms as used herein in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to asubject. Treatment is typically undertaken in an effort to alter the course of a disease, e.g., dengue virus (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and / or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be administered prophylactically, i.e., before development of any symptom or manifestation of a disease. “Prophylactic treatment” refers to providing medical and / or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur, delay the onset of the disease, or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0059] Grammatical variations of “administer,” “administration,” and “administering” to a subject include any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. “Concurrent administration,” “administration in combination,” “simultaneous administration,” or “administered simultaneously” as used herein, means that the compounds are administered at the same point in time, overlapping in time, or one following the other. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemicadministration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body (e.g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.

[0060] "Effective amount" as used herein refers to an amount of a vector, nucleic acid, epitope, polypeptide, cell, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and / or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and / or by using routine experimentation.

[0061] The term "immunogenic amount" or "effective immunizing dose," as used herein, unless otherwise indicated, means an amount or dose sufficient to induce an immune response (which can optionally be a protective response) in the treated subject that is greater than the inherent immunity of non-immunized subjects. An immunogenic amount or effective immunizing dose in any particular context can be routinely determined using methods known in the art.

[0062] The terms "vaccine," "vaccination," and "immunization" are well-understood in the art, and are used interchangeably herein. For example, the terms vaccine, vaccination or immunization can be understood to be a process or composition that increases a subject’s immune reaction to an immunogen (e.g., by providing an active immune response), and therefore its ability to resist, overcome and / or recover from infection ( / .< ., a protective immune response).

[0063] The terms "protective" immune response or "protective" immunity as used herein indicates that the immune response confers some benefit to the subject in that it prevents or reduces the incidence and / or severity and / or duration of disease or any other manifestation of infection. For example, in representative embodiments, a protective immune response or protective immunity results in reduced viremia, whether or not accompanied by clinical disease. Alternatively, a protective immune response or protective immunity may be useful in the therapeutic treatment of existing disease.

[0064] An "active immune response" or "active immunity" is characterized by "participation of host tissues and cells after an encounter with the immunogen. It involves differentiation and proliferation of immunocompetent cells in lymphoreticular tissues, which lead to synthesis of antibody or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, an active immune response is mounted by the host after exposure to immunogens by infection or by vaccination. Active immunity can be contrasted with passive immunity, which is acquired through the "transfer of preformed substances (antibody, transfer factor, thymic graft, interleukin-2) from an actively immunized host to a non-immune host." Id.

[0065] The present invention relates to modified flavivirus E polypeptides that inhibit heterodimer formation between E polypeptides, in particular, modified DENV E polypeptides that inhibit E dimer mixing and heterodimer formation between DENV E polypeptide serotypes while retaining the ability to form homodimers, and nucleic acids encoding the modified E polypeptides. Advantageously, the modified E polypeptides present epitopes recognized by neutralizing antibodies. Compositions comprising the modified E polypeptides or nucleic acids encoding the modified E polypeptides, including for example, immunogenic compositions comprising all four DENV serotypes are also provided. Methods of using the modified E polypeptides or nucleic acids encoding the modified E polypeptides are also provided, including for preventing a dengue virus infection in a subject.

[0066] In some embodiments, a modified DENV E protein of DENV1, DENV2, DENV3, or DENV4 serotype comprising three or more amino acid mutations in domain I and / or domain II of the E polypeptide, the three or more amino acid mutations inhibiting heterodimer formation between DENV E polypeptide serotypes is provided. In some embodiments, the modified DENVE protein comprises less than 20 amino acid mutations, e.g., less than 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 mutations in domain I and / or domain II of the E polypeptide. The number of mutations in domain I and / or domain II of the E polypeptide refers to the total number of mutations in both domains combined.

[0067] The Dengue virus E protein folds into three domains (DI, DII, and Dill), a membrane- proximal stem, and a transmembrane anchor. A soluble fragment of the E protein, consisting of the domains DI, DII and Dill, can comprise about 392 amino acids e.g., DENV3 serotype) or about 394 amino acids (e.g., DENV1, DENV2, or DENV4 serotype). The structure of the dengue E protein is described, for example, in Modis, et al., Proc Natl Acad Sci U S A. 2003 Jun 10; 100(12): 6986-6991, Figure 1, incorporated herein by reference in its entirety. The E protein is composed of three distinct domains in the N-terminal region, central domain I formed by eight - strand barrels, which spans residues 1-51, 132-192 and 280-295, domain II formed by 12 beta strands and two alpha helices, which spans residues 52-131, and 193-279, and domain III, an IgC-like domain formed by ten beta strands, which spans residues 296-394.

[0068] The amino acid mutations can comprise substitution of one or more amino acids each with one or more charged amino acid residues. In some embodiments, one or more amino acid mutations comprise substitution of one or more neutral or negatively charged amino acids with one or more positively charged amino acid residues. In some embodiments, one or more amino acid mutations comprise substitution of one or more neutral or positively charged amino acids with one or more negatively charged amino acid residues. In some embodiments, one or more of the amino acid mutations increase the negative charge of a first region within domain I and / or domain II and one or more of the amino acid mutations increase the positive charge of a second region within domain I and / or domain II. In some embodiments, the first region and the second region are proximal in three-dimensional space. Proximal in three-dimensional space as used herein is location in space sufficiently close to allow interaction between the first region and the second region in a homodimer. In some embodiments, the first region and the second region are sufficiently proximal in three-dimensional space, e.g., close, to allow interaction between the one or more amino acid mutations that increase the negative charge in a first region and the one or more amino acid mutations that increase the positive charge in a second region. In some embodiments, the one or more amino acid mutations comprise substitution with arginine, glutamic acid or aspartic acid.

[0069] In some embodiments, the amino acid of the wild-type E protein that is mutated is polar, but not charged (e.g., neutral), including, serine, threonine, cysteine, asparagine, glutamine and tyrosine. In some embodiments, the amino acid of the wild-type E protein that is mutated has a hydrophobic side chain, for example, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan. In some embodiments, the amino acid substituted is a charged amino acid, for example, histidine, lysine, arginine, glutamic acid, or aspartic acid.

[0070] In some embodiments, the one or more mutations, e.g., substitutions, is based on electrostatic interactions and / or charge swapping. In some embodiments, the one or more substitutions is provided to repel charge of another E protein serotype and / or repel or discourage electrostatic interactions between different E protein serotypes. In some embodiments, arginine is utilized as a positive residue for substitution of an amino acid in the wild-type E protein. In some embodiments, an amino acid in the wild-type E protein is substituted with glutamic acid, aspartic acid, or proline. In some embodiments, a lysine, histidine, glutamine, alanine, valine, or isoleucine amino acid in the wild-type E protein (DENV1, DENV2, DENV3, or DENV4 serotype) is substituted with an amino acid. In some embodiments, a glutamic acid in the wildtype E protein is substituted with an arginine. In some embodiments, the one or more mutations is at amino acid positions 27, 202, 239-243, 245-249, 267-276 of DENV3, e.g., of wild-type strain CH53489, or amino acid positions corresponding thereto in the DENV1, DENV2, or DENV4 serotype.

[0071] In some embodiments, the one or more mutations, e.g., substitutions, is based on steric properties and / or non-polar packing. In some embodiments, the one or more substitutions comprise a non-polar amino acid, e.g., alanine, valine, leucine, proline, methionine, tryptophan, glycine, cysteine, tyrosine, isoleucine, phenylalanine for an amino acid in the wild-type E protein (DENV1, DENV2, DENV3, or DENV4 serotype). In some embodiments, the one or more substitutions comprise a polar amino acid, e.g., asparagine, aspartic acid, glutamine, glutamic acid, lysine, serine, threonine, histidine, or arginine for an amino acid in the wild-type E protein (DENV1, DENV2, DENV3, or DENV4 serotype).

[0072] In some embodiments, the one or more substitutions comprise at least one substitution of a polar amino acid in the wild-type E protein, e.g., glutamine or threonine. In some embodiments, a non-polar amino acid (e.g., alanine, leucine) of the wild-type E protein is substituted with a polar amino acid (e.g., tyrosine).

[0073] In some embodiments, a polar amino acid (e.g., glutamine, threonine) of the wild-type E protein is substituted with a non-polar amino acid (e.g., leucine). In some embodiments, a nonpolar amino acid of the wild-type E protein is substituted with a positively charged amino acid e.g., arginine). In some embodiments, the one or more mutations occur at amino acid positions 253-263 of DENV 1, e.g., of wild-type strain WP74, or amino acid positions corresponding thereto in the DENV2, DENV3, or DENV4 serotype.

[0074] In some embodiments, the one or more substitutions comprise at least one substitution of a polar amino acid in the wild-type E protein, e.g., glutamine or threonine.

[0075] In some embodiments, the modified E protein comprises mutations at one or more amino acid positions corresponding to positions 27, 202, 239-243, 245-249, 267-276 of DENV3 serotype E protein or the corresponding positions of the E protein of another dengue virus (e.g., dengue virus serotypes 1 (DENV1), 2 (DENV2) or 4 (DENV4) at positions 27, 204, 241-245, 249-251, 269-278). In some embodiments, the mutations comprise mutations at one or more of amino acid positions 27, 202, 239, 241, 242, 247, 249, 267, 269, and 276 of DENV3 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide, e.g., positions 27, 204, 241, 243, 244, 249, 251, 269, 271, and 278. In some embodiments, the one or more mutations correspond to positions of DENV3 serotype E protein strain CH53489.

[0076] The modified E protein can comprise one or more mutations at amino acids selected from: H27; K202; K239; A241; H242; E247; V249; E267; Q269; and 1276 corresponding to positions of DENV3 serotype E protein. In some embodiments, the modified E protein comprises one or more mutations at amino acids selected from: K202E; K239R; H242R; E247R; V249R; Q269E; and I276D corresponding to positions of DENV3 serotype E protein strain. In some embodiments, the modified E protein has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:4. In some embodiments, the modified E protein comprises one or more mutations at amino acids selected from: H27P;K202R; K239E; A241D; H242D; V249E; E267R; Q269R; and I276R corresponding to positions of DENV3 serotype E protein strain. In some embodiments, the modified E polypeptide has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:5

[0077] In some embodiments, the modified E polypeptide can comprise amino acid mutations, e.g., substitutions, of one or more amino acids each with one or more polar or non-polar aminoacid residues and / or wherein the amino acid mutations increase steric hindrance. Steric hindrance may include intramolecular interactions or congestion caused by the physical presence of surrounding side chains in a polypeptide that may slow down or inhibit interactions or reactions between molecules at the site.

[0078] In some embodiments, one or more of the amino acid mutations comprise substitution of one or more non-polar amino acids with one or more polar amino acid residues and / or one or more of the amino acid mutations comprise substitution of one or more polar amino acids with one or more non-polar amino acid residues. In some embodiments, one or more mutations comprise substitution with tyrosine, arginine, or leucine. In some embodiments, the mutations comprise mutations at one or more of amino acid positions 253-263 of DENV1 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide. In some embodiments, the mutations comprise mutations at one or more of amino acid positions 253, 256, 259, 262 and 263 of DENV1 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide. In some embodiments, the modified E polypeptide comprises one or more mutations at amino acids selected from L253; Q256; A259; T262; and A263, corresponding to positions of DENV1 serotype E polypeptide, e.g., of strain WP74. In some embodiments, the one or more amino acid mutations are selected from: L253R; Q256L; A259Y; T262L; and A263L. In some embodiments, the modified E polypeptide has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:6

[0079] Within each of the four dengue virus serotypes, there are a number of different strains or genotypes. The dengue virus modified polypeptides of the invention can be derived from any dengue virus, including all serotypes, strains and genotypes, now known or later identified. The term “epitope” as used herein means a specific combination of amino acid residues in an amino acid sequence that, when present in the proper conformation, provides a reactive site for an antibody (e.g., B cell epitope) or T cell receptor (e.g., T cell epitope).

[0080] Regarding amino acid positions in E polypeptides of varying serotype, mutations, e.g., substitutions, at an amino acid position in one serotype can be used to provide a modified E polypeptide of another serotype. Positions in DENV1, DENV2 and DENV4 E polypeptide serotypes correspond to each other. There is a 2-residue shift after position 161 in the E polypeptide of DENV3 serotype relative to DENV1, DENV2 and DENV4 serotypes. Thus, forexample, an amino acid at position 277 in DENV3 E protein will correspond to position 279 in DENV1, DENV2, and DENV4, an amino acid at position 162 in DENV3 E protein will correspond to position 164 in DENV1, DENV2, and DENV4, an amino acid at position 161 in DENV3 E polypeptide will correspond to position 161 in DENV1, DENV2, and DENV4, and an amino acid at position 27 in DENV3 serotype, for example, SEQ ID NO:1, a wild type DENV3 E polypeptide, corresponds to position 27 in DENV1, DENV2, and DENV4 serotypes, for example, SEQ ID NOS:3, 2, and 7, wild type DENV1, DENV2 and DENV4 E polypeptides, respectively.

[0081] In some embodiments, the E protein comprises one or more stabilizing mutations at conserved regions on the E protein (see, Phan and Hvasta, et al., J. Biol. Chem. (2022) 298(7) 102079, incorporated herein by reference; see also, Kudlacek et al., Sci Adv. 2021 Oct 15; 7(42):eabg4084. doi: 10.1126 / sciadv.abg4084, incorporated herein by reference). In some embodiments, the mutations comprise one or more stabilizing mutations in the domain I core of the E protein. In some embodiments, the mutations comprise one or more mutations at positions 29, 33 and / or 35 of the wild-type E protein, (DENV1, DENV2, DENV3, or DENV4 serotype). In some embodiments, the mutation comprises the substitution 29K, 33V, and / or 35M.

[0082] In some embodiments, the mutations comprise one or more stabilizing mutations in the Domain I-II Hinge of the E protein. In some embodiments, the mutations comprise one or more stabilizing mutations at positions 279 and / or 280 of DENV1, DENV2 or DENV4, or positions 277 and / or 278 of DENV3 serotype. In some embodiments, the substitution comprises 279W and / or 280P in DENV1, DENV2 or DENV4 or 277W and / or 278P in DENV3 serotype relative to the wild-type E protein.

[0083] In some embodiments, the mutations comprise one or more stabilizing mutations in the Central aB helix of the E protein. In some embodiments, the mutations comprise one or more stabilizing mutations at positions 259 and / or 262 of DENV1, DENV2 or DENV4, or positions 257 and / or 260 of DENV3 serotype. In some embodiments, the substitution comprises 259W and / or 262R in DENV1, DENV2 or DENV4 or 257W and / or 260R in DENV3 serotype relative to the wild-type E protein.

[0084] In some embodiments, the modified E polypeptide comprises one or more mutations at amino acid positions 29, 33, 24, 259, 279 and 280 in DENV1 serotype, or amino acid positions 29,33, 35, 257, 260, 277, and 278 in DENV3 serotype, or the corresponding amino acid positionsin another DENV serotype E polypeptide. The modified E proteins of the present invention can further comprise one or more amino acid mutations selected from: G29K; T33V; A35M;A257W; T260R; F277W; and A278P of DENV3 or the corresponding positions of the E protein of other dengue viruses (e.g., dengue virus serotypes 1 (DENV1), 2 (DENV2) or 4 (DENV4)). In some embodiments, the modified E protein comprises one or more mutations selected from: S29K; T33V; A35M; A259Y; F279W; and A280P ofDENVl or the corresponding positions of the E protein of other dengue viruses (e.g., dengue virus serotypes 2 (DENV2), 3 (DENV3), or 4 (DENV4)).

[0085] It is also contemplated that embodiments of this invention can include any functional fragment of the amino acid sequence of the modified E polypeptide sequences provided herein, in particular, retaining domain I and / or domain II, the mutations described herein, and immunogenic epitopes. Immunogenic epitopes of DENV E protein are described, for example, in Kudlacek et al., SciAdv, 7:42 (2021); Nadugala, et al., BMC Immunol 22, 71 (2021); Lai etal., J of Virology, 82: 13 (2008), Fahimi, et al ., Applied Microbiol. andBiotech. 102, 2977-2996 (2018), each of which is incorporated herein in their entirety. For example, some embodiments of the invention can include a fragment of any 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 or 350 contiguous amino acids, including any values not explicitly recited herein. These fragments can be at either end and / or at any internal location in the amino acid sequence (e.g., residues 1-50 and / or residues 75-100).

[0086] In addition, the present invention provides an isolated nucleic acid encoding the modified E proteins of the present invention; in some embodiments, a plurality of nucleic acids, for example, encoding two, three, or four different DENV serotypes. The present invention provides an isolated nucleic acid encoding the modified E proteins of a DENV1 serotype, a DENV2 serotype, a DENV3 serotype and a DENV4 serotype. In some embodiments, the nucleic acid comprises RNA, optionally mRNA.

[0087] In some embodiments, a vector comprising a nucleic acid molecule encoding a modified E polypeptide is provided. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a non-viral vector. Example non-viral vectors include a nanoparticle, optionally a lipid nanoparticle or virus-like particle. A plurality of vectors can be provided, the vectors each comprising a nucleic acid encoding an E polypeptide from each of the DENV1, DENV2, DENV3 and DENV4 serotypes. In some embodiments, a modified E polypeptide of atleast three of the four serotypes, or of all four serotypes is provided. The compositions of the present invention can optionally be formulated for administration as a pharmaceutical composition, for example, an immunogenic composition e.g., a vaccine, as described in further detail herein.

[0088] In some embodiments, a composition comprising one or more of the modified E polypeptides, one or more nucleic acids encoding the modified E polypeptides, or one or more vectors comprising the nucleic acids encoding the modified E polypeptides is provided. In some embodiments, the composition comprises two, three, or four modified E polypeptides, nucleic acids encoding the modified E polypeptides, or one or more vectors comprising the nucleic acids encoding the modified E polypeptides. In some embodiments, the modified E polypeptides or nucleic acids encoding the modified E polypeptides of each serotype has different amino acid mutations.

[0089] In some embodiments, compositions of the present invention, such as a vaccine, can comprise a combination of modified DENV E polypeptides or nucleic acids encoding the E polypeptides with one or more other flavivirus vaccines. Example flaviviruses include yellow fever virus (YFV), West Nile fever virus, hepatitis C virus, Japanese encephalitis virus (JEV), Kyasanur Forest Disease (KFD), Alkhurma disease, Zika virus (ZIKV), St. Louis encephalitis virus, and Omsk hemorrhagic fever. The combination compositions can be tailored according to the dominant viruses in a region; for example YFV is prevalent in Africa and Latin America with JEV prevalent in Asia, ZFV prevalent in Africa, Asia, and South America, West Nile and St Louis Encephalitis virus present in the U.S. Accordingly, in some embodiments, compositions of the present invention, such as a vaccine, can comprise a combination of modified DENV E polypeptides or nucleic acids encoding the E polypeptides with one or more other flavivirus E polypeptide(s) or nucleic acids encoding the E polypeptides, such ZIKV. In some embodiments, a combination vaccine comprising modified DENV E polypeptides or nucleic acids encoding the modified E polypeptides with a ZIKV vaccine is provided, optionally wherein the ZIKV vaccine comprises E polypeptides or nucleic acids encoding E polypeptides. In some embodiments, a combination vaccine comprising modified DENV E polypeptides or nucleic acids encoding the modified E polypeptides with a ZIKV vaccine and / or JEV vaccine. In some embodiments, a combination vaccine comprising modified DENV E polypeptides or nucleic acids encoding the modified E polypeptides with a ZIKV vaccine and / or YFV vaccine.In some embodiments, a combination vaccine comprising modified DENV E polypeptides or nucleic acids encoding the modified E polypeptides with West Nile virus vaccine and / or St. Louis Encephalitis vaccine. Without being bound by theory, the modified E polypeptides of the present invention reduce E dimer mixing and heterodimer formation and may reduce cross- reactive antibody-dependent enhancement-facilitating antibodies between one or more other flavivirus compositions, e.g., ZIKV, and DENV.Methods

[0090] A method of protecting a subject from the effects of dengue virus infection is provided, comprising administering to the subject an effective amount of one or more of the modified E polypeptides, one or more of the nucleic acids, one or more of the vectors, the composition, the pharmaceutical composition, and / or the immunogenic composition of the present invention, to thereby protect the subject from the effects of the dengue virus infection.

[0091] A method of preventing a dengue virus infection in a subject is provided, the method comprising administering to the subject an effective amount of one or more of the modified E polypeptides, one or more of the nucleic acids, one or more of the vectors, the composition, the pharmaceutical composition, and / or the immunogenic composition of the present invention, to thereby prevent the dengue virus infection in the subject.

[0092] A method of producing neutralizing antibodies in a human subject against DENV1. DENV2, DENV3 and DENV4 serotypes is provided, comprising administering to the subject an effective amount of three or more of the modified E polypeptides, three or more of the nucleic acids, three or more of the vectors, the composition, the pharmaceutical composition, and / or the immunogenic composition of the present invention, to thereby induce the production of neutralizing antibodies in the human subject against the serotypes, optionally wherein a DENV1, DENV2, DENV3 and a DENV4 serotype are administered.

[0093] The methods of administration comprise E polypeptides, or nucleic acids encoding the E polypeptides, of DENV1, DENV2, DENV3 and DENV4 serotypes administered to the subject, optionally in a composition, wherein at least three of the four E proteins can be modified polypeptides, or nucleic acids encoding the modified E polypeptides, and wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations.

[0094] In some embodiments, a method of reducing heterodimer formation in a tetraval ent dengue virus vaccine comprising DENV E polypeptides of DENV1, DENV2, DENV3, and DENV4 serotypes is provided. The method comprises providing three or more of the modified E polypeptides, three or more of the nucleic acids, three or more of the vectors, the composition, the pharmaceutical composition, and / or the immunogenic composition of the present invention, in the tetravalent dengue virus vaccine to thereby reduce heterodimer formation between DENV1, DENV2, DENV3, and / or DENV4 serotypes, wherein at least three of the four E polypeptides are modified polypeptides, or nucleic acids encoding the modified E polypeptides, and wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations. In some embodiments, the differing amino acid mutations on at least three of the four E polypeptides reduces, inhibits, and / or disfavors heterodimer formation in the vaccine. In some embodiments, the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprising different amino acid mutations improve the efficacy of a composition, e.g., vaccine, relative to a vaccine comprising E polypeptides without the amino acid mutations, for example, eliciting a stronger immune response. Without being bound by theory, the reduction in heterodimer formation may reduce eliciting antibodies that are poorly neutralizing against any of the serotypes, and / or improve the safety profile of administered compositions, e.g., vaccines, comprising the modified E polypeptides or nucleic acids encoding the modified E polypeptide.

[0095] Administration to a subject can be by any route known in the art. As non-limiting examples, the route of administration can be by inhalation (e.g., oral and / or nasal inhalation), oral, buccal (e.g., sublingual), rectal, vaginal, topical (including administration to the airways), intraocular, transdermal, parenteral (e.g., intramuscular [e.g., administration to skeletal muscle], intravenous, intra-arterial, intraperitoneal and the like), subcutaneous (including administration into the footpad), intradermal, intrapleural, intracerebral, and / or intrathecal routes.

[0096] In some embodiments, a pharmaceutical composition comprising a composition as disclosed herein and a pharmaceutically acceptable carrier is provided. Pharmaceutical formulations (e.g., immunogenic formulation) comprising the dengue virus polypeptides, nucleic acids, vectors, or compositions of the invention and a pharmaceutically acceptable carrier can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition). In the manufactureof a pharmaceutical composition according to embodiments of the present invention, the composition of the invention is typically admixed with, inter alia, a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is meant a carrier that is compatible with other ingredients in the pharmaceutical composition and that is not harmful or deleterious to the subject. The carrier may be a solid or a liquid, or both, and is preferably formulated with the composition of the invention as a unit-dose formulation, for example, a tablet, which may contain from about 0.01% or 0.5% to about 95% or 99% by weight of the composition. The pharmaceutical compositions are prepared by any of the well-known techniques of pharmacy including, but not limited to, admixing the components, optionally including one or more accessory ingredients. In certain embodiments, the pharmaceutically acceptable carrier is sterile and would be deemed suitable for administration into human subjects according to regulatory guidelines for pharmaceutical compositions comprising the carrier.

[0097] A pharmaceutically acceptable component such as a salt, carrier, excipient or diluent of a composition according to the present invention is a component that (i) is compatible with the other ingredients of the composition in that it can be combined with the compositions of the present invention without rendering the composition unsuitable for its intended purpose, and (ii) is suitable for use with subjects as provided herein without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are "undue" when their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable components include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil / water emulsion, microemulsions and various types of wetting agents.

[0098] In some embodiments, the compositions of the invention can further comprise one or more than one adjuvant. The adjuvants of the present invention can be in the form of an amino acid sequence, and / or in the form or a nucleic acid. In some embodiments, the nucleic acid encodes an adjuvant. When in the form of a nucleic acid, the adjuvant can be a component of a nucleic acid encoding the polypeptide(s) or fragment(s) and / or a separate component of the composition comprising the nucleic acid encoding the polypeptide(s) or fragment(s) of the invention. According to the present invention, the adjuvant can also be an amino acid sequence that is a peptide, a protein fragment or a whole protein that functions as an adjuvant, and / or the adjuvant can be a nucleic acid encoding a peptide, protein fragment or whole protein thatfunctions as an adjuvant. As used herein, "adjuvant" describes a substance, which can be any immunomodulating substance capable of being combined with a composition of the invention to enhance, improve or otherwise modulate an immune response in a subject.

[0099] The pharmaceutical formulations of the invention can optionally comprise other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, diluents, salts, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0100] For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and is typically in a solid or liquid particulate form.

[0101] The compositions of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques. See, e.g., Remington, The Science And Practice of Pharmacy (latest edition). In the manufacture of a pharmaceutical composition according to the invention, the polypeptides, nucleic acids, or vectors of the invention are typically admixed with, inter alia, an acceptable carrier. The carrier can be a solid or a liquid, or both, and is optionally formulated with the compound as a unit-dose formulation, for example, a tablet. A variety of pharmaceutically acceptable aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, pyrogen-free water, pyrogen-free phosphate-buffered saline solution, bacteriostatic water, or Cremophor EL[R] (BASF, Parsippany, N.J.), and the like. These compositions can be sterilized by conventional techniques. The formulations of the invention can be prepared by any of the well-known techniques of pharmacy.

[0102] The pharmaceutical formulations can be packaged for use as is, or lyophilized, the lyophilized preparation generally being combined with a sterile aqueous solution prior to administration. The compositions can further be packaged in unit / dose or multi-dose containers, for example, in sealed ampoules and vials.

[0103] The pharmaceutical formulations can be formulated for administration by any method known in the art according to conventional techniques of pharmacy. For example, the compositions can be formulated to be administered intranasally, by inhalation (e.g., oral inhalation), orally, buccally (e.g., sublingually), rectally, vaginally, topically, intrathecally,intraocularly, transdermally, by parenteral administration (e.g, intramuscular [e.g, skeletal muscle], intravenous, subcutaneous, intradermal, intrapleural, intracerebral and intra-arterial, intrathecal), or topically (c.g, to both skin and mucosal surfaces, including airway surfaces).

[0104] For intranasal or inhalation administration, the pharmaceutical formulation can be formulated as an aerosol (this term including both liquid and dry powder aerosols). For example, the pharmaceutical formulation can be provided in a finely divided form along with a surfactant and propellant. Typical percentages of the composition are 0.01-20% by weight, preferably 1- 10% by weight. The surfactant is generally nontoxic and soluble in the propellant.Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1-20% by weight of the composition, preferably 0.25-5% by weight. The balance of the composition is ordinarily propellant. A carrier can also be included, if desired, as with lecithin for intranasal delivery. Aerosols of liquid particles can be produced by any suitable means, such as with a pressure- driven aerosol nebulizer or an ultrasonic nebulizer, as is known to those of skill in the art. See, e.g., U.S. Patent No. 4,501,729, which is hereby incorporated by reference in its entirety. Aerosols of solid particles can likewise be produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art. Intranasal administration can also be by droplet administration to a nasal surface.

[0105] Injectable formulations can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the pharmaceutical formulations in a local rather than systemic manner, for example, in a depot or sustained-release formulation.

[0106] Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. For example, an injectable, stable, sterile formulation of the invention in a unit dosage form in a sealed container can be provided. The formulation can be provided in the form of a lyophilizate, which can be reconstituted with a suitable pharmaceutically acceptable carrier to form a liquid composition suitable for injection into a subject. The unit dosage form can be from about 1 pg to about 10 grams of the formulation. When the formulation is substantially water-insoluble, a sufficientamount of emulsifying agent, which is pharmaceutically acceptable, can be included in sufficient quantity to emulsify the formulation in an aqueous carrier. One such useful emulsifying agent is phosphatidyl choline.

[0107] Pharmaceutical formulations suitable for oral administration can be presented in discrete units, such as capsules, cachets, lozenges, or tables, as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in- oil emulsion. Oral delivery can be performed by complexing a compound(s) of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the gut of an animal. Examples of such carriers include plastic capsules or tablets, as known in the art. Such formulations are prepared by any suitable method of pharmacy, which includes the step of bringing into association the protein(s) and a suitable carrier (which may contain one or more accessory ingredients as noted above). In general, the pharmaceutical formulations are prepared by uniformly and intimately admixing the compound(s) with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture. For example, a tablet can be prepared by compressing or molding a powder or granules, optionally with one or more accessory ingredients. Compressed tablets are prepared by compressing, in a suitable machine, the formulation in a free-flowing form, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and / or surface active / dispersing agent(s). Molded tablets are made by molding, in a suitable machine, the powdered protein moistened with an inert liquid binder.

[0108] Pharmaceutical formulations suitable for buccal (sub-lingual) administration include lozenges comprising the compound(s) in a flavored base, usually sucrose and acacia or tragacanth; and pastilles in an inert base such as gelatin and glycerin or sucrose and acacia.

[0109] Pharmaceutical formulations suitable for parenteral administration can comprise sterile aqueous and non-aqueous injection solutions, which preparations are preferably isotonic with the blood of the intended recipient. These preparations can contain antioxidants, buffers, bacteriostats and solutes, which render the composition isotonic with the blood of the intended recipient. Aqueous and non-aqueous sterile suspensions, solutions and emulsions can include suspending agents and thickening agents. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0110] Pharmaceutical formulations suitable for rectal administration are optionally presented as unit dose suppositories. These can be prepared by admixing the active agent with one or more conventional solid carriers, such as for example, cocoa butter and then shaping the resulting mixture.[0U1] Pharmaceutical formulations suitable for topical application to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers that can be used include, but are not limited to, petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof. In some embodiments, for example, topical delivery can be performed by mixing a pharmaceutical formulation of the present invention with a lipophilic reagent (e.g., DMSO) that is capable of passing into the skin.

[0112] Pharmaceutical formulations suitable for transdermal administration can be in the form of discrete patches adapted to remain in intimate contact with the epidermis of the subject for a prolonged period of time. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3:318 (1986)) and typically take the form of a buffered aqueous solution of the compound(s). Suitable formulations can comprise citrate or bis\tris buffer (pH 6) or ethanol / water and can contain from 0.1M to 0.2M active ingredient.

[0113] An immunogenic composition comprising an effective amount of one or more of the modified E polypeptides, one or more nucleic acids encoding the modified E polypeptides, one or more vectors comprising the nucleic acids encoding the modified E polypeptides, the composition and / or the pharmaceutical composition is provided. The modified proteins, nucleic acids, vectors, and compositions can further be administered to elicit an immunogenic response e.g., as a vaccine). Typically, immunogenic compositions of the present invention comprise an immunogenically effective amount of proteins or nucleic acids in combination with a pharmaceutically acceptable carrier. Optionally, the dosage is sufficient to produce a protectiveimmune response (as defined above). The degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.

[0114] In an embodiment, the modified E polypeptide or a functional fragment thereof or a nucleic acid encoding the polypeptide is administered via a nucleic acid delivery vector. In embodiments, the modified E polypeptide or functional fragment thereof or nucleic acid encoding the protein is delivered via a viral vector, nanoparticle or hydrogel. In an embodiment, the modified E polypeptide or a functional fragment thereof is administered via a nucleic acid delivery vector comprising a nucleic acid encoding the modified E polypeptide. In an embodiment, the delivery vehicle may comprise a nanoparticle comprising modified dendrimers for the enclosure of a nucleic acid, e.g., a nucleic acid encoding a modified E polypeptide. Exemplary dendrimers include polyester dendrimers, which may be modified with amine linkers and / or fatty acid derivatives. Exemplary molecules and methods of making nanoparticle compositions may be found at International Publication WO 2020 / 132196. Lipid particles, for example, lipid nanoparticles and liposomes may also be used. In an example embodiment, the lipid particles comprise one or more polynucleotides encoding a modified E polypeptide according to the present invention. Example lipid nanoparticles can be found in the art, for example, in U.S. Pat. Nos. 9,868,692, 10,266,485, 10,442,756, and 10,272,150. Liposomes and stable nucleic acid lipid particles (SNALPs) can also be used for delivery, including commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega, Madison, WI), as well as other liposomes developed according to procedures standard in the art.

[0115] The present invention further provides a kit comprising one or more compositions of this invention. It would be well understood by one of skill in the art that the kit of this invention can comprise one or more containers and / or receptacles to hold the reagents (e.g., modified polypeptides, nucleic acids) of the kit, along with appropriate buffers and / or diluents and / or other solutions and directions for using the kit, as would be well known in the art. Such kits can further comprise adjuvants and / or other immunostimulatory or immunomodulating agents, as are well known in the art. The compositions and kits of the present invention can also include other medicinal agents, pharmaceutical agents, carriers, diluents, immunostimulatory cytokines, etc.Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art.

[0116] Having described the present invention, the same will be explained in greater detail in the following examples, which are included herein for illustration purposes only, and which are not intended to be limiting to the invention.EXAMPLES

[0117] Universally safe vaccines for dengue virus should provide protection against all four serotypes of the virus (DENV1, DENV2, DENV3, DENV4). The E protein homodimer from dengue is the primary target of protective antibodies and is the foundation of current efforts to create a subunit vaccine for dengue virus (protein-based or mRNA based). Applicant recently discovered that E dimers from different serotypes readily mix in solution to form heterodimers. While DENV E proteins comprising stabilizing mutations form stable homodimers when expressed on their own (FIGS. 1A-1B), robust formation of heterodimers with E proteins from DENV1 and DENV2 serotypes with stabilizing mutations occurs when the serotypes are mixed (FIG. 2)

[0118] Applicant designed mutations in E polypeptides using a combination of ProteinMPNN, Rosetta, and Rationale Design. Designs targeted at primary interface of DENV3 amino acid positions 7, 98, 110, 154, 244, and 314 were unsuccessful.

[0119] Modified E proteins 05 and 06, Table 1 mutations are based on electrostatic interactions and charge swapping. Modifications to E protein 110 in Table 1 are based on steric and non-polar packing. Advantageously, the designed E proteins provide orthogonality towards each other, allowing the selective assembly of serotype-specific homodimer.

[0120] Ribbon diagrams of example modified E proteins 110, 05 and 06 with close-up views of mutated residues are shown in FIGS. 3A, 4A and 5A, respectively. A ribbon diagram of the example modified proteins orthogonal to DV2-SC10 E protein with stabilizing mutations is shown in FIGS. 3B, 4B, and 5B. The SCIO stabilizing mutation set is described in Phan, et al., “A conserved set of mutations for stabilizing soluble envelope protein dimers from dengue and Zika viruses to advance the development of subunit vaccines” J. Biol. Chem. 2022 Jul; 298(7): 102079; doi: 10.1016 / j .jbc.2022.102079, in particular, at FIGS. 1 and 2, incorporated herein by reference. Mass photometry of the mutations show the example modified E proteins disfavorheterodimerization with DENV2 serotype E protein comprising a stabilizing mutation set (DV2 SC10-MBP)(FIGS. 3C, 4C, 5C).

[0121] The orthogonal designs of example modified E proteins DV1 110, DV3 05, and DV3 06 still present epitopes recognized by neutralizing antibodies, as shown in the ELISA results of FIG. 6. Example modified E polypeptides, DV1 110, DV3 05, and DV3 06 which also comprise stabilizing mutations PM4, Intfc2, and UndPk6 as described in Kudlacek et al., Sci Adv. 2021 Oct; 7(42):eabg4084, are included in Table 1 below.Table 1Table 2. Mutation Sets

[0122] The foregoing examples are illustrative of the present invention and are not to be construed as limiting thereof. Although the invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention as described and defined in the following claims

Claims

CLAIMS1. A modified Dengue virus (DENV) envelope (E) polypeptide of DENV 1 , DENV2, DENV3, or DENV4 serotype comprising three or more amino acid mutations in domain I and / or domain II of the E polypeptide, the three or more amino acid mutations inhibiting heterodimer formation between DENV E polypeptide serotypes, the modified E polypeptide retaining capability of homodimer formation.

2. The modified E polypeptide of claim 1, wherein the amino acid mutations comprise substitution of one or more amino acids each with one or more charged amino acid residues.

3. The modified E polypeptide of claim 1, wherein one or more of the amino acid mutations comprise substitution of one or more neutral or negatively charged amino acids with one or more positively charged amino acid residues.

4. The modified E polypeptide of claim 1 or 2, wherein one or more amino acid mutations comprise substitution of one or more neutral or positively charged amino acids with one or more negatively charged amino acid residues.

5. The modified E polypeptide of any one of the previous claims, wherein one or more of the amino acid mutations increase the negative charge of a first region within domain I and / or domain II and one or more of the amino acid mutations increase the positive charge of a second region within domain I and / or domain II, wherein the first and the second region are proximal in three-dimensional space and interact in a homodimer.

6. The modified E polypeptide of any one the previous claims, wherein one or more amino acid mutations comprise substitution with arginine, glutamic acid, or aspartic acid.

7. The modified E polypeptide of any one of the previous claims, wherein the mutations comprise mutations at one or more of amino acid positions 27, 202, 239-243, 245-249, 267-276 of DENV3 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide.

8. The modified E polypeptide of any one of the previous claims, wherein the mutations comprise mutations at one or more of amino acid positions 27, 202, 239, 241, 242, 247, 249, 267, 269, and 276 of DENV3 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide.

9. The modified E polypeptide of claim 8, comprising one or more mutations at amino acids selected from:H27;K202;K239;A241;H242;E247;V249;E267;Q269; and1276 or the corresponding amino acid positions in another DENV serotype E polypeptide.

10. The modified E polypeptide of claim 9, comprising one or more mutations at amino acids selected from:K202E;K239R;H242R;E247R;V249R;Q269E; andI276D or the corresponding amino acid positions in another DENV serotype E polypeptide.11 . The modified E polypeptide of claim 10, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:4.

12. The modified E polypeptide of claim 11, comprising one or more mutations at amino acids selected from:H27P;K202R;K239E;A241D;H242D;V249E;E267R;Q269R; andI276R or the corresponding amino acid positions in another DENV serotype E polypeptide.

13. The modified E polypeptide of claim 12, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:5.

14. The modified E polypeptide of claim 1, wherein the amino acid mutations comprise substitution of one or more amino acids each with one or more polar or non-polar amino acid residues and / or wherein the amino acid mutations increase steric hindrance.

15. The modified E polypeptide of claim 14, wherein one or more of the amino acid mutations comprise substitution of one or more non-polar amino acids with one or more polar amino acid residues.

16. The modified E polypeptide of claim 14, wherein one or more of the amino acid mutations comprise substitution of one or more polar amino acids with one or more non-polar amino acid residues.

17. The modified E polypeptide of any one of claims 14-16, wherein one or more mutations comprises substitution with tyrosine, arginine, or leucine.

18. The modified E polypeptide of any one of claims 14-17, wherein the mutations comprise mutations at one or more of amino acid positions 253-263 of DENV1 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide.

19. The modified E polypeptide of any one of claims 14-18, wherein the mutations comprise mutations at one or more of amino acid positions 253, 256, 259, 262 and 263 of DENV1 serotype E polypeptide or the corresponding amino acid positions in another DENV serotype E polypeptide.

20. The modified E polypeptide of claim 19, comprising one or more mutations selected from:L253;Q256;A259;T262; andA263 or the corresponding amino acid positions in another DENV serotype E polypeptide.

21. The E polypeptide of claim 20, comprising one or more mutations selected from: L253R;Q256L;A259Y;T262L; andA263L or the corresponding amino acid positions in another DENV serotype E polypeptide.

22. The modified E polypeptide of claim 21, that has at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:6.

23. The modified E polypeptide of any one of claims 1-22, further comprising one or more mutations at amino acid positions 29, 33, 24, 259, 279 and 280 in DENV1 serotype, or amino acid positions 29,33, 35, 257, 260, 277, and 278 in DENV3 serotype, or the corresponding amino acid positions in another DENV serotype E polypeptide.

24. The modified E polypeptide of claim 23, comprising one or more mutations selected from:S29K;T33V;A35M;A259Y;F279W; andA280P or the corresponding amino acid positions in another DENV serotype E polypeptide.

25. The modified E polypeptide of claim 23, comprising one or more mutations selected from:G29K;T33V;A35M;A257W;T260R;F277W; andA278P or the corresponding amino acid positions in another DENV serotype E polypeptide.

26. A nucleic acid encoding the polypeptide of any one of the previous claims.

27. The nucleic acid of claim 26, comprising RNA, optionally mRNA.

28. A vector comprising the nucleic acid of claim 26 or 27.

29. The vector of claim 29, wherein the vector is a nanoparticle, optionally a lipid nanoparticle.

30. A plurality of vectors according to claim 29 or 30, the vectors each comprising a nucleic acid encoding an E polypeptide from each of the DENV1, DENV2, DENV3 and DENV4 serotypes.

31. A composition comprising one or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, or the vector of any one of claims 28-30.

32. The composition of claim 31, comprising an E polypeptide from each of the DENV1, DENV2, DENV3 and DENV4 serotypes or a nucleic acid encoding an E polypeptide from each of the DENV1, DENV2, DENV3 and DENV4 serotypes, wherein at least three of the four serotypes are modified E polypeptides, or nucleic acids encoding the modified E polypeptides, wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations.

33. A pharmaceutical composition comprising the composition of claim 31 or 32, and a pharmaceutically acceptable carrier.

34. An immunogenic composition comprising an effective amount of one or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, the vector of any one of claims 28-30, the composition of claim 31 or 32, and / or the pharmaceutical composition of claim 33.

35. A method of protecting a subject from the effects of dengue virus infection, comprising administering to the subject an effective amount of one or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, the vector of anyone of claims 28-30, the composition of claim 31 or 32, and / or the pharmaceutical composition of claim 33, thereby protecting the subject from the effects of the dengue virus infection.

36. A method of preventing a dengue virus infection in a subject, the method comprising administering to the subject an effective amount of a composition comprising one or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, the vector of any one of claims 28-30, the composition of claim 31 or 32, and / or the pharmaceutical composition of claim 33, thereby preventing the dengue virus infection in the subject.

37. A method of producing neutralizing antibodies in a human subject against DENV1, DENV2, DENV3 and DENV4 serotypes, comprising administering to the subject an effective amount of three or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, the vector of any one of claims 28-30, the composition of claim 31 or 32, and / or the pharmaceutical composition of claim 33, thereby inducing the production of neutralizing antibodies in the human subject against the serotypes.

38. The method of any one of claims 35-37, wherein E polypeptides, or nucleic acids encoding the E polypeptides, of DENV1, DENV2, DENV3 and DENV4 serotypes are administered to the subject, and wherein at least three of the four E proteins are modified polypeptides, or nucleic acids encoding the modified E polypeptides, and wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations.

39. A method of reducing heterodimer formation in a tetravalent dengue virus vaccine comprising Dengue virus (DENV) envelope (E) polypeptides of DENV1, DENV2, DENV3, and DENV4 serotypes, the method comprising providing three or more of the modified E polypeptides of any one of claims 1-25, one or more of the nucleic acids of claim 26 or 27, the vector of any one of claims 28-30, the composition of claim 31 or 32, and / or the pharmaceutical composition of claim 33 in the tetraval ent dengue virus vaccine to thereby reduce heterodimer formation between DENV1, DENV2, DENV3, and / or DENV4 serotypes, wherein at least threeof the four E proteins are modified polypeptides, or nucleic acids encoding the modified E polypeptides, and wherein each of the modified E polypeptides or nucleic acids encoding the modified E polypeptides comprise different amino acid mutations.

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