Engineered Nipah virus MRNA vaccine

Abstract

The present invention provides a Nipah virus (NiV) vaccine composition comprising (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human type I collagen alpha 1 (COL1A1) signal peptide, (ii) an mRNA comprising an ORF encoding a full length NiV-G, and (iii) a vaccine composition comprising (i) a messenger ribonucleic acid (mRNA) encoding an ORF encoding a full length NiV-G signal peptide. (iii) an mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F) or (iv) an mRNA comprising an ORF encoding a full-length NiV-G, and an mRNA comprising an ORF encoding a full-length NiV-F, and a method of inducing an immune response against Nipah virus by administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 484,339, filed February 10, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0004] This application incorporates by reference the sequence listing submitted electronically in the following file: the file named “F295207_sequence listing as filed” having a file size of 37,709 bytes, and was created on January 16, 2024. TECHNICAL FIELD

[0005] The present invention provides a Nipah virus (NiV) vaccine composition comprising: (i) a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human collagen type I alpha 1 (COL1A1) signal peptide, (ii) a mRNA comprising an ORF encoding a full-length Nipah virus glycoprotein (full-length NiV-G), (iii) a mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F), or (iv) a mRNA comprising an ORF encoding a full-length NiV-G, and a mRNA comprising an ORF encoding a full-length NiV-F, and a method of inducing an immune response against Nipah virus by administering to a subject in need thereof an effective amount of the Nipah virus vaccine composition. BACKGROUND

[0006] Nipah virus (NiV) is a zoonotic virus (transmitted from animals to humans) that can also spread through contaminated food or directly between people. In infected individuals, Nipah virus can cause a range of diseases from asymptomatic (subclinical) infection to acute respiratory illness and fatal encephalitis. There is no approved Nipah virus mRNA vaccine at present, and there is an urgent need for a Nipah virus mRNA vaccine. SUMMARY

[0007] The present disclosure provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human collagen type I alpha 1 (COL1A1) signal peptide. In one embodiment, the soluble NiV-G fused to the COL1A1 signal peptide has the amino acid sequence of SEQ ID NO: 1. In another embodiment, the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO: 2. In some embodiments, the mRNA comprising the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail, and the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In some embodiments, the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 3. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 3. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0008] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G). In one embodiment, the full-length NiV-G has the amino acid sequence of SEQ ID NO: 5. In one embodiment, the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6. In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0009] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus fusion protein (full-length NiV-F). In one embodiment, the full-length NiV-F has the amino acid sequence of SEQ ID NO: 9. In one embodiment, the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-F further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0010] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G) and a mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F). In one embodiment, the full-length NiV-G has the amino acid sequence of SEQ ID NO: 5 and the full-length NiV-F has the amino acid sequence of SEQ ID NO: 9. In one embodiment, the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6 and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6, and the mRNA comprising the ORF encoding the full-length NiV-F further comprises a 5’ UTR, a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7 and the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7 and the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0011] The present disclosure also provides a method of inducing an immune response against a Nipah virus comprising administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition according to the present disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0012] FIG. 1 In vitro transcription of NiV mRNA.

[0013] FIG. 2A and 2B NiV-G and NiV-F protein expression in 293FT cells showing mRNA infection.

[0014] FIG. 3A Mouse immunization procedures using the nipa virus vaccine compositions of the disclosure are described.

[0015] FIG. 3B Data showing mouse anti-NiV-GP titers.

[0016] FIG. 3C Data showing NiV neutralization.

[0017] Definitions

[0018] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that the present disclosure is not limited to the particular methodologies, compositions, devices, and materials described herein as these can vary and depend on the context of use and desired results. It is also to be understood that the terminology used herein is for the purpose of describing particular versions or embodiments only and is not intended to limit the scope of the embodiments described herein.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, if there is a conflict between the definitions in the specification including the definitions in the specification and the patent rules, the definition in the specification applies. Therefore, as used herein, the following terms are to be defined as follows in the context of the embodiments described herein.

[0020] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] As used herein, the term "comprising" and its grammatical variants are used inclusively of the recited features, elements, method steps, etc, and are not a limitation. In contrast, the term "consisting of and its grammatical variants are used exclusively in the recited features, elements, method steps, etc, and exclude any other feature, element, method step etc not recited. The phrase "consisting essentially of and its grammatical variants are used to indicate that the recited features, elements, method steps etc are present, but that other features, elements, method steps etc not recited can also be present, provided that those other features, elements, method steps etc do not materially affect the basic nature of the composition, system or method. Many of the embodiments herein are described using the open language "comprising". Such embodiments encompass a plurality of closed "consisting of and / or "consisting essentially of embodiments that could alternatively be described using such language.

[0022] As used herein, the term "Nipah virus vaccine composition" refers to a substance used to stimulate antibody production and provide immunity against Nipah virus.

[0023] As used herein, the term "messenger ribonucleic acid (mRNA)" refers to a single-stranded RNA molecule corresponding to the genetic sequence of a gene and is read by ribosomes during protein synthesis.

[0024] As used herein, the term "fused to" refers to a gene or gene product that, when isolated from its natural source, has the characteristics of the gene or gene product.

[0025] The term "Nipah virus glycoprotein fused to human collagen type I alpha 1 (COL1A1) signal peptide (soluble NiV-G)" refers to a recombinant fusion protein produced by genetic engineering of a fusion gene. For example, this can involve removing the stop codon from the cDNA sequence encoding soluble NiV-G and then in-frame joining the cDNA sequence of the COL1A1 signal peptide by ligation or overlap extension PCR.

[0026] Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0027] Non-natural amino acids include, but are not limited to, azetidinecarboxylic acid, 2- aminoadipic acid, 3-amino adipic acid, beta-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminohexanoic acid, 2- aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2, 7- diaminopimelic acid, t-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N- ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo- hydroxylysine, 3-hydroxyproline ("3Hyp"), 4-hydroxyproline ("4Hyp"), iso- desmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycine ("NAG") (including N-methylglycine), N-methylisoleucine, N-alkylpentylglycine ("NAPG") (including N-methylpentylglycine), N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), or ornithine ("Orn"), pentylglycine ("pG" or "PGly"), piperidinic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoglutamine ("hArg").

[0028] As used herein, the term "open reading frame (ORF)" refers to a nucleotide sequence between a start codon and a stop codon.

[0029] As used herein, the term "open reading frame (ORF) encoding" refers to a nucleotide coding sequence that encodes a polypeptide. The coding sequence can also include a start signal and a stop signal operably linked to regulatory elements, including a promoter and a polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence can also include a sequence encoding a signal peptide.

[0030] As used herein, the term "T7 promoter" refers to a promoter derived from bacteriophage T7.

[0031] As used herein, the term "5' untranslated region (UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') of a start codon (the first codon of an mRNA transcript to be translated by a ribosome) and not encoding a polypeptide.

[0032] As used herein, the term "3' untranslated region (UTR)" refers to the region of an mRNA immediately downstream (i.e., 3') of a stop codon (the codon in an mRNA transcript that signals the termination of translation) and not encoding a polypeptide.

[0033] As used herein, the term "poly(A) tail" refers to a long stretch of adenine nucleotides added to the "tail" or 3' end of an mRNA.

[0034] As used herein, the term "pharmaceutically acceptable carrier" refers to any substance or vehicle suitable for delivering an mRNA vaccine to a suitable in vivo or ex vivo site. Such carriers can include, but are not limited to, adjuvants, excipients, lipid particles, and the like.

[0035] As used herein, the term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, a lipid nanoparticle is included in a formulation that can be used to deliver an mRNA vaccine to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, an mRNA vaccine can be encapsulated in the lipid portion of a lipid nanoparticle or in the aqueous phase space surrounded by some or all of the lipid portion of a lipid nanoparticle, thereby protecting it from enzymatic degradation or other adverse effects induced by host organisms or cellular machinery, such as adverse immune responses. In some embodiments, the average diameter of a lipid nanoparticle is 50-200 nm. In some embodiments, a lipid nanoparticle includes a cationic lipid, a polyethylene glycol (PEG)-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, a lipid nanoparticle includes about 20% to 60% molar ratio of a cationic lipid, 0.5% to 15% molar ratio of a PEG-modified lipid, 25% to 55% molar ratio of a sterol, and 25% molar ratio of a non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0036] As used herein, the term "inducing an immune response against a Nipah virus" refers to providing protective immunity for prophylactic purposes and / or eliciting a desired immune response or effect against a Nipah virus in a subject in need thereof for therapeutic purposes. As used herein, the term "protective immunity" or "protective immune response" refers to a vaccinated subject being able to control an infection by a pathogen against which the subject was vaccinated. Typically, a subject who has developed a "protective immune response" only experiences mild to moderate clinical symptoms, or no symptoms at all.

[0037] An“effective amount” of a Nipah virus vaccine composition (e.g., mRNA) is provided based at least in part on the target tissue, target cell type, mode of administration, physical properties of the polynucleotide (e.g., size and degree of modified nucleosides), and other components of the vaccine and other determinants. Generally, an effective amount of a Nipah virus vaccine (e.g., mRNA) can provide an induced or enhanced immune response according to antigen production in a cell, preferably more effective than a composition containing a corresponding unmodified polynucleotide or peptide antigen encoding the same antigen. Increased antigen production can be evidenced by increased cell transfection (increased percentage of cells transfected with the RNA (e.g., mRNA) vaccine), increased protein translation of the polynucleotide, decreased nucleic acid degradation (e.g., evidenced by increased duration of protein translation from the modified polynucleotide), or altered antigen-specific immune response of the host cell.

[0038] As used herein, the term“X% identity” or“sequence identity” to SEQ ID NO: Y refers to the extent to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequence of monomer subunits. The term“sequence similarity” refers to the extent to which two polymeric sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. The calculation of“percent sequence identity” (or“percent sequence similarity”) is as follows: (1) compare the two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.); (2) determine the number of positions at which the identical (or similar) monomers occur (e.g., the same amino acid occurs in both sequences, a similar amino acid occurs in both sequences), resulting in a number of matched positions; (3) divide the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); (4) multiply the result by 100 to obtain the percent sequence identity or percent sequence similarity. For example, if peptide A and peptide B are both 20 amino acids in length, and the amino acids at all but one position are identical, then peptide A and peptide B have 95% sequence identity. If the amino acids at the non-identical position have the same biophysical properties (e.g., both are acidic), then peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids in length, and peptide D is 15 amino acids in length, and 14 of the 15 amino acids of peptide D are identical to a portion of the amino acids of peptide C, then peptide C and peptide D have 70% sequence identity, but the best comparison window of peptide D to peptide C has 93.3% sequence identity. For purposes of calculating“percent sequence identity” (or“percent sequence similarity”) herein, any gaps in the aligned sequences are considered to be non-matches at those positions.

[0039] As used herein, the term "a nucleotide sequence having at least X% identity to SEQ ID NO: Y and encoding a Z protein" refers to a nucleotide sequence that satisfies both requirements of having at least X% identity to SEQ ID NO: Y and encoding a Z protein.

[0040] As used herein, the terms "about," "approximately," "around," and "substantially" mean that the amount or value in question can not be exact, but can be close to the stated or taught value. That is, it is understood that these amounts, sizes, formulas, parameters, and other quantities and characteristics are not intended to be exact, and can be approximations and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like and other factors that are well-known to those skilled in the art. In certain instances, the value, dosage, or range can be exact. In such cases, it is understood that the values are as precise as possible, given the limitations in measurement and other factors. In general, the numerical quantities mentioned in this specification are to be understood as approximated values and are indicated solely for convenience as being precise. It is further understood that where actual values are desired, the approximated values will be substituted with the desired value other than the approximated value in functionally equivalent ranges. Also, it is to be understood that the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Preferred values are those values that are within the range of values that will provide an equivalent result. Preferred values are evident to those skilled in the art.

[0041] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal, any mammalian subject, or a cell thereof, either in vitro or in situ, that can be suitable for use with the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0042] Detailed Description

[0043] 1. A Nipah virus vaccine composition

[0044] (1) A Nipah virus vaccine composition (1) comprising: an mRNA comprising an ORF encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human collagen type I alpha 1 (COL1A1) signal peptide (2) A Nipah virus vaccine composition (2) comprising: an mRNA comprising an ORF encoding a full-length Nipah virus glycoprotein (full-length NiV-G)

[0045] The present disclosure provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human collagen type I alpha 1 (COL1A1) signal peptide. In one embodiment, the soluble NiV-G fused to the COL1A1 signal peptide has the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1). In another embodiment, the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO: 2 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2). In some embodiments, the mRNA comprising the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail, and the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO: 2 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2). In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In some embodiments, the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3). In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 3. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0046] (3) A Nipah virus vaccine composition (3) comprising: an mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F) (4) A Nipah virus vaccine composition (4) comprising: an mRNA comprising an ORF encoding a full-length Nipah virus glycoprotein (full-length NiV-G) and an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F)

[0047] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G). In one embodiment, the full-length NiV-G has the amino acid sequence of SEQ ID NO: 5 (or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5). In one embodiment, the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6). In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6). In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7). In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0048] 2. A method of inducing an immune response against Nipah virus 3. Sequence information

[0049] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus fusion protein (full-length NiV-F). In one embodiment, the full-length NiV-F has the amino acid sequence of SEQ ID NO: 9 (or an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9). In one embodiment, the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10). In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-F further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10). In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 11 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11). In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding the full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11 (or a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11). In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0050] SEQ ID NO: 1 MFSFVDLRLLLLLAATALLTHG

[0051] The present disclosure also provides a Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G) and a mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F). In one embodiment, the full-length NiV-G has the amino acid sequence of SEQ ID NO: 5 and the full-length NiV-F has the amino acid sequence of SEQ ID NO: 9. In one embodiment, the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6 and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the mRNA comprising the ORF encoding the full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6; and the mRNA comprising the ORF encoding the full-length NiV-F further comprises a 5’ UTR, a 3’ UTR, and a poly(A) tail to have the structure of 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail, and the ORF encoding the full-length NiV-F has the nucleotide sequence of SEQ ID NO: 10. In one embodiment, the poly(A) tail has a length of 50-250 nucleotides. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7 and the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 11. In one embodiment, the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7 and the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 11. In one embodiment, the Nipah virus composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the mRNA encoding the full-length NiV-G and the mRNA encoding the full-length Ni-F are comprised in the composition at a ratio of about 1:1. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA is encapsulated.

[0052] In the above-described Nipah virus vaccine compositions (1) to (4), the length of the poly(A) tail can be 50-250 nucleotides. In another embodiment, the length of the poly(A) tail is 100-200 nucleotides. In another embodiment, the length of the poly(A) tail is 110-150 nucleotides. In another embodiment, the length of the poly(A) tail is 115-125 nucleotides. In another embodiment, the length of the poly(A) tail is 116-124 nucleotides. In another embodiment, the length of the poly(A) tail is 117-123 nucleotides. In another embodiment, the length of the poly(A) tail is 118-122 nucleotides. In another embodiment, the length of the poly(A) tail is 119-122 nucleotides. In another embodiment, the length of the poly(A) tail is 115 nucleotides. In another embodiment, the length of the poly(A) tail is 116 nucleotides. In another embodiment, the length of the poly(A) tail is 117 nucleotides. In another embodiment, the length of the poly(A) tail is 118 nucleotides. In another embodiment, the length of the poly(A) tail is 119 nucleotides. In another embodiment, the length of the poly(A) tail is 120 nucleotides. In another embodiment, the length of the poly(A) tail is 121 nucleotides. In another embodiment, the length of the poly(A) tail is 122 nucleotides. In another embodiment, the length of the poly(A) tail is 123 nucleotides. In another embodiment, the length of the poly(A) tail is 124 nucleotides. In another embodiment, the length of the poly(A) tail is 125 nucleotides.

[0053] In the above-described Nipah virus vaccine compositions (1) to (4), the mRNA of the present disclosure can comprise at least one chemical modification selected from the group consisting of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2’-O-methyluridine. In another embodiment, the chemical modification is at the 5 position of uracil. In another embodiment, the chemical modification is N1-methylpseudouridine. In another embodiment, the chemical modification is N1-ethylpseudouridine.

[0054] In the above Nipah virus vaccine compositions (1) to (4), the Nipah virus vaccine composition can further include a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutically acceptable carrier can include any substance or vehicle suitable for delivering the mRNA vaccine to a suitable in vivo or ex vivo site. Such a carrier can include, but is not limited to, an adjuvant, an excipient, a lipid particle, and the like. The lipid nanoparticle can be a particle having at least one dimension in the order of nanometers (e.g., 1-1,000 nm). In some embodiments, the lipid nanoparticle is included in a formulation that can be used to deliver the mRNA vaccine to a target site of interest (e.g., a cell, a tissue, an organ, a tumor, and the like). In some embodiments, the mRNA vaccine can be encapsulated in a lipid portion of the lipid nanoparticle or in an aqueous phase space surrounded by a portion or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other adverse effects induced by host organisms or cellular mechanisms, such as adverse immune reactions. In some embodiments, the lipid nanoparticle has an average diameter of 50-200 nm. In some embodiments, the lipid nanoparticle includes a cationic lipid, a polyethylene glycol (PEG)-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the lipid nanoparticle includes about 20% to 60% molar ratio of the cationic lipid, 0.5% to 15% molar ratio of the PEG-modified lipid, 25% to 55% molar ratio of the sterol, and 25% molar ratio of the non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and the sterol is cholesterol. In some embodiments, the cationic lipid is selected from 2,2-dilinoleyl-4-dimethylaminoethyl [1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyric acid ester (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0055] In one embodiment, the lipid nanoparticle includes (i) at least one lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyric acid ester (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM, (iii) a sterol, such as cholesterol, and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, at a molar ratio of about 20% to 60% cationic lipid: 5% to 25% neutral lipid: 25-55% sterol: 0.5% to 15% PEG-lipid.

[0056] In one embodiment, the lipid nanoparticle comprises about 25% to about 75% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3- DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), for example about 35% to about 65% by mole, about 45% to about 65% by mole, about 60% by mole, about 57.5% by mole, about 50% by mole, or about 40% by mole.

[0057] In one embodiment, the lipid nanoparticle comprises about 0.5% to about 15% by mole of a neutral lipid, for example about 3% to about 12% by mole, about 5% to about 10% by mole, or about 15%, about 10%, or about 7.5% by mole. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation comprises about 5% to about 50% by mole of a sterol, for example about 15% to about 45% by mole, about 20% to about 40% by mole, about 40% by mole, about 38.5% by mole, about 35% by mole, or about 31% by mole. An exemplary sterol is cholesterol. In some embodiments, the formulation comprises about 0.5% to about 20% by mole of a PEG or PEG-modified lipid, for example about 0.5% to about 10% by mole, about 0.5% to about 5% by mole, about 1.5% by mole, about 0.5% by mole, about 1.5% by mole, about 3.5% by mole, or about 5% by mole. In some embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of less than 2,000 Da, for example about 1,500 Da, about 1,000 Da, or about 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearyl glycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG) and PEG-cDMA.

[0058] In one embodiment, the lipid nanoparticle comprises 25% to 75% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5% to 15% by mole of a neutral lipid, 5% to 50% by mole of a sterol, and 0.5% to 20% by mole of a PEG or PEG-modified lipid.

[0059] In one embodiment, the lipid nanoparticle comprises 35% to 65% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3% to 12% of a neutral lipid, 15% to 45% of a sterol, and 0.5% to 10% of a PEG or PEG-modified lipid.

[0060] In one embodiment, the lipid nanoparticle comprises 45% to 65% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5% to 10% of a neutral lipid, 25% to 40% of a sterol, and 0.5% to 10% of a PEG or PEG-modified lipid.

[0061] In one embodiment, the lipid nanoparticle comprises about 60% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% of a neutral lipid, about 31% of a sterol, and about 1.5% of a PEG or PEG-modified lipid.

[0062] In one embodiment, the lipid nanoparticle comprises about 50% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% of a neutral lipid, about 38.5% of a sterol, and about 1.5% of a PEG or PEG-modified lipid.

[0063] In one embodiment, the lipid nanoparticle comprises about 50% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% of a neutral lipid, about 35% of a sterol, about 4.5% or about 5% of a PEG or PEG-modified lipid, and about 0.5% of a targeting lipid.

[0064] In one embodiment, the lipid nanoparticle comprises about 40% by mole of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% of a neutral lipid, about 40% of a sterol, and about 5% of a PEG or PEG-modified lipid.

[0065] In one embodiment, the Nipah virus vaccine compositions of the present disclosure can be delivered to, positioned at, and / or concentrated at a specific location by using a delivery method as described below. As a non-limiting example, empty polymeric particles can be administered to a subject prior to, concurrently with, or after the Nipah virus vaccine compositions of the present disclosure are delivered to the subject. The empty polymeric particles, once in contact with the subject, change in volume and get stuck, embedded, fixed, or entrapped at a specific location in the subject.

[0066] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated as an active substance release system. For example, the active substance release system can comprise at least one nanoparticle bound to an oligonucleotide inhibitor strand that hybridizes to a catalytically active nucleic acid, and one compound bound to at least one substrate molecule that binds to a therapeutically active substance (e.g., a polynucleotide described herein), wherein the therapeutically active substance is released by the catalytically active nucleic acid cleaving the substrate molecule.

[0067] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated as a nanoparticle comprising an inner core comprising non-cellular material and an outer surface comprising a cellular membrane. The cellular membrane can be derived from a cell, or from a membrane of a virus.

[0068] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated into porous nanoparticle supported lipid bilayers (procell).

[0069] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated into polymeric nanoparticles with high glass transition temperatures.

[0070] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated into nanoparticles for imaging. As a non-limiting example, the liposome can comprise gadolinium 2-{4,7-bis carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amidomethyl]-1,4,7,10-tetraazacyclododecan-1-yl}-acetate (III) and a neutral, fully saturated phospholipid component.

[0071] The nanoparticles of the present disclosure can also include nutrients, such as but not limited to those nutrients that are deficient in causing health hazards ranging from anemia to neural tube defects. As a non-limiting example, the nutrient can be iron in the form of ferrous, ferric salts or elemental iron, iodine, folate, vitamins, or micronutrients.

[0072] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated into swellable nanoparticles.

[0073] In another embodiment, the Nipah virus vaccine compositions of the present disclosure can be formulated into polyacid ester nanoparticles.

[0074] The nanoparticles and microparticles of the present disclosure can be geometrically engineered to modulate macrophage and / or immune responses. In some embodiments, the geometrically engineered particles can have different shapes, sizes, and / or surface charges in order to incorporate the polynucleotides of the present disclosure for targeted delivery, such as but not limited to pulmonary delivery. The geometrically engineered particles can have other physical features, including but not limited to open-pore structures, angled branches, asymmetry, surface roughness, and charge, which can alter interactions with cells and tissues.

[0075] In another embodiment, the nanoparticles of the present disclosure can be water-soluble nanoparticles. The nanoparticles can be inorganic nanoparticles with compact zwitterionic ligands to exhibit good water solubility. The nanoparticles can also have a small hydrodynamic diameter (HD), stability with respect to time, pH, and salinity, and low levels of non-specific protein binding.

[0076] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or targeted-specific stealth nanoparticles. In some embodiments, the stealth nanoparticles or targeted-specific stealth nanoparticles can include a polymeric matrix. The polymeric matrix can include two or more polymers such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropylfumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, polyorthoester, polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or combinations thereof.

[0077] In one embodiment, the nanoparticles of the present disclosure can be nanoparticle-nucleic acid hybrid structures with a high density nucleic acid layer. The nanoparticles of the present disclosure can comprise nucleic acids such as, but not limited to, polynucleotides described herein and / or known in the art.

[0078] In one embodiment, at least one nanoparticle of the present disclosure can be embedded in the core of the nanostructure or coated with a low density porous three-dimensional structure or coating capable of carrying or binding at least one payload inside or on the surface of the nanostructure.

[0079] In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle in which the mRNA of the present disclosure is encapsulated. In another embodiment, the lipid nanoparticle includes a first lipid nanoparticle encapsulating mRNA encoding soluble NiV-G fused to COL1A1 signal peptide, a second lipid nanoparticle encapsulating mRNA encoding full-length NiV-G, a third lipid nanoparticle encapsulating mRNA encoding full-length NiV-F, and a fourth lipid nanoparticle encapsulating mRNA encoding full-length NiV-G and mRNA encoding full-length NiV-F.

[0080] SEQ ID NO: 2

[0081] The present disclosure also provides a method of inducing an immune response against a Nipah virus comprising administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition of the present disclosure. In one embodiment, the effective amount of the Nipah virus vaccine composition (e.g., mRNA) is provided based at least in part on the target tissue, target cell type, mode of administration, physical properties of the polynucleotide (e.g., size and degree of modified nucleosides), and other components of the vaccine and other determinants. In general, an effective amount of the Nipah virus vaccine (e.g., mRNA) can provide an induced or enhanced immune response according to antigen production in cells, preferably more effective than a composition containing a corresponding unmodified polynucleotide or peptide antigen encoding the same antigen. Increased antigen production can be evidenced by increased cell transfection (increased percentage of cells transfected with the RNA (e.g., mRNA) vaccine), increased protein translation of the polynucleotide, decreased nucleic acid degradation (e.g., evidenced by increased duration of protein translation of the modified polynucleotide), or altered antigen-specific immune response of the host cell.

[0082] An effective amount (immunogenically effective amount) of a Nipah virus vaccine composition (e.g., Nipah virus vaccine compositions (1) to (4)) is typically administered by intramuscular injection or subcutaneous injection. Thus, the Nipah virus vaccine composition is typically formulated for intramuscular injection or subcutaneous injection, and is preferably free of any adjuvant for the purposes of the adjuvant-free formulation of the present application. However, other modes of administration are also contemplated, such as intravenous, dermal, intradermal, or nasal administration. For intravenous, dermal, or subcutaneous injection, the adenoviral vector will be in the form of a parenterally acceptable aqueous solution, having suitable values, isotonicity and stability. Likewise, the isolated envelope polypeptide will be in the form of a parenterally acceptable solution having suitable values, isotonicity and stability. One of ordinary skill in the art is well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's Injection, lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as required.

[0083] In one particular embodiment, an effective amount (immunogenically effective amount) of a Nipah virus vaccine composition (e.g., Nipah virus vaccine compositions (1) to (4)) is administered by intramuscular administration. Intramuscular administration can be achieved by injecting a suspension of the adenoviral vector and / or the envelope polypeptide using a needle. Another option is to administer the composition containing the vaccine using a needle-free injection device (e.g., using a Biojector™) or a lyophilized powder.

[0084] In one embodiment, the primary immunization and / or the boost immunization, preferably both the primary immunization and the boost immunization, further comprises administering one or more adenoviral vectors encoding one or more additional Nipah virus antigens.

[0085] The timing of administration of the primary immunization and the boost immunization is not particularly limited. For example, the vaccine composition can be administered for the primary immunization, and administered again prior to administering the vaccine composition for the boost immunization. Further administration of the vaccine composition for further boost immunization is also contemplated. In certain embodiments, the boost vaccine is administered for the first time about 1-12 weeks, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, after the primary administration of the base vaccine. In other embodiments, the boost vaccine is administered for the first time about 12-52 weeks, e.g., about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 weeks, after the primary administration of the base vaccine. One of ordinary skill in the art will be able to vary the exact timing of the primary vaccine and the boost vaccine, the frequency of their administration, their dosage, etc., in light of the teachings herein and the general knowledge in the art.

[0086] In one embodiment, the Nipah virus vaccine composition can comprise the first mRNA and the second mRNA described herein, formulated into a lipid nanoparticle comprising MC3, cholesterol, DSPC, and PEG2000-DMG, trisodium citrate buffer, sucrose, and water for injection. As a non-limiting example, the composition can comprise 2.0 mg / mL of drug substance (e.g., Nipah virus vaccine compositions (1) to (4)), 21.8 mg / mL of MC3, 10.1 mg / mL of cholesterol, 5.4 mg / mL of DSPC, 2.7 mg / mL of PEG2000-DMG, 5.16 mg / mL of trisodium citrate, 71 mg / mL of sucrose, and 1.0 mL of water for injection.

[0087] In one embodiment, a method of inducing an immune response against Nipah virus comprises administering to a subject in need thereof an effective amount of the Nipah virus vaccine composition (1) of the present disclosure. In the Nipah virus vaccine composition (1), the mRNA having the structure of 5’ UTR-ORF-encoding soluble NiV-G fused to COL1A1 signal peptide-3’ UTR-poly(A) tail can have the nucleotide sequence of SEQ ID NO: 3 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 3).

[0088] In another embodiment, a method of inducing an immune response against a Nipah virus comprises administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition of the present disclosure (2). In the Nipah virus vaccine composition (2), the mRNA having the structure of 5’ UTR-ORF-3’ UTR-poly(A) tail encoding full-length NiV-G can have the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 7).

[0089] In another embodiment, a method of inducing an immune response against a Nipah virus comprises administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition of the present disclosure (3). In the Nipah virus vaccine composition (3), the mRNA having the structure of 5’ UTR-ORF-3’ UTR-poly(A) tail encoding full-length NiV-F can have the nucleotide sequence of SEQ ID NO: 11 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 11).

[0090] In another embodiment, a method of inducing an immune response against a Nipah virus comprises administering to a subject in need thereof an effective amount of a Nipah virus vaccine composition of the present disclosure (4). In the Nipah virus vaccine composition (4), the mRNA having the structure of 5’ UTR-ORF-3’ UTR-poly(A) tail encoding full-length NiV-G can have the nucleotide sequence of SEQ ID NO: 7 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 7), and the mRNA having the structure of 5’ UTR-ORF-3’ UTR-poly(A) tail encoding full-length NiV-F can have the nucleotide sequence of SEQ ID NO: 11 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 11).

[0091] SEQ ID NO: 3

[0092] 1) Protein sequence of soluble NiV-G fused with COL1A1 signal peptide (COL1A1 signal peptide sequence is underlined)

[0093] AUGUUCUCUUUCGUGGAC

[0094] CUGCGCCUGCUGCUGCUGCUCGCUGCCACCGCCCUGCUGACACACGGCCAGAACUACACCCGGAGCACCGACAACCAQNYTRSTDNQ AMIKDALQSIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYKPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNSEFYYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGMYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPVAKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDEENSKIIFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVQTVNPLVVNWRDNTVISRPGQSQCPRFNTCPEVCWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT

[0095] 2) Soluble NiV-G mRNA sequence (ORF) fused to COL1A1 signal peptide

[0096] GGCUAUGAUCAAAGACGCCCUGCAGAGCAUCCAACAGCAGAUCAAGGGCCUGGCCGACAAGAUCGGCACAGAAAUCG

[0097]

[0098] 3) Soluble NiV-G mRNA sequence fused to COL1A1 signal peptide (ORF underlined) fused to COL1A1 signal peptide (5' UTR-ORF-3' UTR-poly(A) tail)

[0099] GACCAAAGGUGUCCCUGAUCGACACGAGUUCUACAAUCACUAUCCCUGCCAACAUCGGCCUGCUGGGCAGCAAAAUC

[0100] AGGCCGGCACUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCGCCACC UCUCAGAGCACAGCCAGCAUCAACGAGAACGUGAACGAGAAAUGCAAGUUCACCCUGCCACCACUGAAGAUCCACGA AUGCAACAUCAGCUGCCCUAAUCCUCUGCCCUUCAGAGAGUACAAGCCUCAGACCGAGGGUGUGUCUAACCUGGUGG GCCUGCCGAACAACAUCUGCCUGCAAAAAACCAGCAACCAGAUCCUGAAACCUAAGCUGAUCAGCUACACACUGCCU GUGGUGGGCCAGAGCGGCACCUGUAUCACAGAUCCUCUGCUGGCCAUGGAUGAAGGCUACUUCGCCUACAGCCAUCU ​ ​ ​ ​ GGAAAAGAUCGGGAGCUGUAGCCGGGGCGUCUCCAAACAGAGAAUCAUCGGCGUGGGCGAAGUGCUGGACAGAGGCG AUGAGGUCCCCUCCCUGUUUAUGACAAAUGUGUGGACCCCUAGCAACCCUAACACAGUGUACCACUGCUCCGCCGUG UAUAAUUCUGAAUUCUACUACGUGCUGUGCGCCGUGUCCGUGGUGGGAGACCCCAUCCUGAACUCUACCUACUGGAG CGGCUCUCUGAUGAUGACCAGACUGGCUGUUAAGCCCAAGAACAACGGCGAGAGCUACAAUCAACACCAGUUCGCCC UGCGGAACAUCGAGAAGGGCAUGUACGACAAAGUGAUGCCCUACGGCCCUUCAGGAAUCAAGCAGGGCGAUACCCUG UAUUUCCCCGCUGUGGGCUUCCUGGUGCGGACCGAAUUCAAGUACAAUGACUCCAAUUGCCCCGUGGCCAAGUGUCA GUACAGCAAACCUGAAAACUGUAGACUGUCUAUGGGCAUCAGACCUAAUAGCCACUACAUCCUCAGAAGCGGACUCC UCAAGUACAACCUGUCCGACGAGGAAAACUCUAAAAUUAUCUUCAUCGAGAUCAGCGACCAGCGCCUGUCUAUCGGA UCUCCAUCUAAGAUCUACGAUAGCCUGGGCCAACCUGUGUUUUACCAGGCCAGCUUUAGCUGGGACACCAUGAUCAA GUUCGGAGAUGUGCAGACAGUGAACCCCCUAGUGGUUAACUGGAGAGAUAAUACCGUGAUUAGCAGACCCGGCCAGU CCCAGUGUCCCAGAUUCAACACCUGCCCUGAGGUGUGCUGGGAGGGCGUGUACAACGACGCCUUCCUGAUCGAUAGA AUCAACUGGAUCUCUGCCGGCGUAUUUCUGGACAGCAACCAGACCGCCGAGAAUCCUGUGUUCACCGUGUUCAAGGA UAACGAGGUGCUGUACAGAGCCCAGCUGGCCAGCGAGGACACAAACGCCCAGAAGACCAUCACAAACUGCUUCCUGC UGAAGAACAAGAUCUGGUGCAUCAGCCUGGUCGAGAUCUACGACACCGGCGACAACGUGAUCCGGCCUAAGCUGUUC GCUGUGAAGAUCCCUGAGCAGUGCACC UGAUAAAGCUGGAGCCUCGGUGGCCUUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0101] 4) Sequence of pUC57-Kan plasmid encoding soluble NiV-G fused to COL1A1 signal peptide mRNA (soluble NiV-G mRNA sequence fused to COL1A1 signal peptide underlined)

[0102] SEQ ID NO: 4

[0103] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCG GCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGTTCTCTTTCGTGGACCTGCGCCTGCTGCT GCTGCTCGCTGCCACCGCCCTGCTGACACACGGCCAGAACTACACCCGGAGCACCGACAACCAGGCTATGATCAAAG ACGCCCTGCAGAGCATCCAACAGCAGATCAAGGGCCTGGCCGACAAGATCGGCACAGAAATCGGACCAAAGGTGTCC CTGATCGACACGAGTTCTACAATCACTATCCCTGCCAACATCGGCCTGCTGGGCAGCAAAATCTCTCAGAGCACAGC CAGCATCAACGAGAACGTGAACGAGAAATGCAAGTTCACCCTGCCACCACTGAAGATCCACGAATGCAACATCAGCT GCCCTAATCCTCTGCCCTTCAGAGAGTACAAGCCTCAGACCGAGGGTGTGTCTAACCTGGTGGGCCTGCCGAACAAC ATCTGCCTGCAAAAAACCAGCAACCAGATCCTGAAACCTAAGCTGATCAGCTACACACTGCCTGTGGTGGGCCAGAG CGGCACCTGTATCACAGATCCTCTGCTGGCCATGGATGAAGGCTACTTCGCCTACAGCCATCTGGAAAAGATCGGGA GCTGTAGCCGGGGCGTCTCCAAACAGAGAATCATCGGCGTGGGCGAAGTGCTGGACAGAGGCGATGAGGTCCCCTCC CTGTTTATGACAAATGTGTGGACCCCTAGCAACCCTAACACAGTGTACCACTGCTCCGCCGTGTATAATTCTGAATT CTACTACGTGCTGTGCGCCGTGTCCGTGGTGGGAGACCCCATCCTGAACTCTACCTACTGGAGCGGCTCTCTGATGA TGACCAGACTGGCTGTTAAGCCCAAGAACAACGGCGAGAGCTACAATCAACACCAGTTCGCCCTGCGGAACATCGAG AAGGGCATGTACGACAAAGTGATGCCCTACGGCCCTTCAGGAATCAAGCAGGGCGATACCCTGTATTTCCCCGCTGT GGGCTTCCTGGTGCGGACCGAATTCAAGTACAATGACTCCAATTGCCCCGTGGCCAAGTGTCAGTACAGCAAACCTG AAAACTGTAGACTGTCTATGGGCATCAGACCTAATAGCCACTACATCCTCAGAAGCGGACTCCTCAAGTACAACCTG TCCGACGAGGAAAACTCTAAAATTATCTTCATCGAGATCAGCGACCAGCGCCTGTCTATCGGATCTCCATCTAAGAT CTACGATAGCCTGGGCCAACCTGTGTTTTACCAGGCCAGCTTTAGCTGGGACACCATGATCAAGTTCGGAGATGTGC AGACAGTGAACCCCCTAGTGGTTAACTGGAGAGATAATACCGTGATTAGCAGACCCGGCCAGTCCCAGTGTCCCAGA TTCAACACCTGCCCTGAGGTGTGCTGGGAGGGCGTGTACAACGACGCCTTCCTGATCGATAGAATCAACTGGATCTC TGCCGGCGTATTTCTGGACAGCAACCAGACCGCCGAGAATCCTGTGTTCACCGTGTTCAAGGATAACGAGGTGCTGT ACAGAGCCCAGCTGGCCAGCGAGGACACAAACGCCCAGAAGACCATCACAAACTGCTTCCTGCTGAAGAACAAGATC TGGTGCATCAGCCTGGTCGAGATCTACGACACCGGCGACAACGTGATCCGGCCTAAGCTGTTCGCTGTGAAGATCCC TGAGCAGTGCACCTGATAAAGCTGGAGCCTCGGTGGCCTTGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTC CCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAA

[0104] 5) Protein sequence of full length NiV-G

[0105] SEQ ID NO: 5

[0106] MPTESKKVRFENTASDKGKNPSKVIKSYYGTMDIKKINEGLLDSKILSAFNTVIALLGSIVIIVMNIMIIQNYTRSTDNQAMIKDALQSIQQQIKGLADKIGTEIGPKVSLIDTSSTITIPANIGLLGSKISQSTASINENVNEKCKFTLPPLKIHECNISCPNPLPFREYKPQTEGVSNLVGLPNNICLQKTSNQILKPKLISYTLPVVGQSGTCITDPLLAMDEGYFAYSHLEKIGSCSRGVSKQRIIGVGEVLDRGDEVPSLFMTNVWTPSNPNTVYHCSAVYNSEFYYVLCAVSVVGDPILNSTYWSGSLMMTRLAVKPKNNGESYNQHQFALRNIEKGMYDKVMPYGPSGIKQGDTLYFPAVGFLVRTEFKYNDSNCPVAKCQYSKPENCRLSMGIRPNSHYILRSGLLKYNLSDEENSKIIFIEISDQRLSIGSPSKIYDSLGQPVFYQASFSWDTMIKFGDVQTVNPLVVNWRDNTVISRPGQSQCPRFNTCPEVCWEGVYNDAFLIDRINWISAGVFLDSNQTAENPVFTVFKDNEVLYRAQLASEDTNAQKTITNCFLLKNKIWCISLVEIYDTGDNVIRPKLFAVKIPEQCT

[0107] 6) Full length NiV-G mRNA sequence (ORF)

[0108] SEQ ID NO: 6

[0109]

[0110] 7) Full length NiV-G mRNA sequence (5' UTR - ORF - 3' UTR - Poly(A) tail) (ORF underlined)

[0111] SEQ ID NO: 7

[0112] AGGCCGGCACUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCGCCACC AUGCCUACAGAAAGCAAA AAGGUGCGGUUCGAGAACACAGCCUCUGACAAGGGAAAGAAUCCUAGCAAGGUGAUCAAAAGCUAUUACGGCACCAU GGAUAUCAAGAAGAUUAACGAAGGCCUGCUGGACAGCAAGAUACUCAGCGCUUUUAACACCGUGAUCGCCCUGCUGG GCAGCAUCGUGAUCAUAGUUAUGAACAUCAUGAUUAUCCAGAACUACACCCGGAGCACCGACAACCAGGCUAUGAUC AAAGACGCCCUGCAGAGCAUCCAACAGCAGAUCAAGGGCCUGGCCGACAAGAUCGGCACAGAAAUCGGACCAAAGGU GUCCCUGAUCGACACGAGUUCUACAAUCACUAUCCCUGCCAACAUCGGCCUGCUGGGCAGCAAAAUCUCUCAGAGCA CAGCCAGCAUCAACGAGAACGUGAACGAGAAAUGCAAGUUCACCCUGCCACCACUGAAGAUCCACGAAUGCAACAUC AGCUGCCCUAAUCCUCUGCCCUUCAGAGAGUACAAGCCUCAGACCGAGGGUGUGUCUAACCUGGUGGGCCUGCCGAA CAACAUCUGCCUGCAAAAAACCAGCAACCAGAUCCUGAAACCUAAGCUGAUCAGCUACACACUGCCUGUGGUGGGCC AGAGCGGCACCUGUAUCACAGAUCCUCUGCUGGCCAUGGAUGAAGGCUACUUCGCCUACAGCCAUCUGGAAAAGAUC GGGAGCUGUAGCCGGGGCGUCUCCAAACAGAGAAUCAUCGGCGUGGGCGAAGUGCUGGACAGAGGCGAUGAGGUCCC CUCCCUGUUUAUGACAAAUGUGUGGACCCCUAGCAACCCUAACACAGUGUACCACUGCUCCGCCGUGUAUAAUUCUG AAUUCUACUACGUGCUGUGCGCCGUGUCCGUGGUGGGAGACCCCAUCCUGAACUCUACCUACUGGAGCGGCUCUCUG AUGAUGACCAGACUGGCUGUUAAGCCCAAGAACAACGGCGAGAGCUACAAUCAACACCAGUUCGCCCUGCGGAACAU CGAGAAGGGCAUGUACGACAAAGUGAUGCCCUACGGCCCUUCAGGAAUCAAGCAGGGCGAUACCCUGUAUUUCCCCG CUGUGGGCUUCCUGGUGCGGACCGAAUUCAAGUACAAUGACUCCAAUUGCCCCGUGGCCAAGUGUCAGUACAGCAAA CCUGAAAACUGUAGACUGUCUAUGGGCAUCAGACCUAAUAGCCACUACAUCCUCAGAAGCGGACUCCUCAAGUACAA CCUGUCCGACGAGGAAAACUCUAAAAUUAUCUUCAUCGAGAUCAGCGACCAGCGCCUGUCUAUCGGAUCUCCAUCUA AGAUCUACGAUAGCCUGGGCCAACCUGUGUUUUACCAGGCCAGCUUUAGCUGGGACACCAUGAUCAAGUUCGGAGAU GUGCAGACAGUGAACCCCCUAGUGGUUAACUGGAGAGAUAAUACCGUGAUUAGCAGACCCGGCCAGUCCCAGUGUCC CAGAUUCAACACCUGCCCUGAGGUGUGCUGGGAGGGCGUGUACAACGACGCCUUCCUGAUCGAUAGAAUCAACUGGA UCUCUGCCGGCGUAUUUCUGGACAGCAACCAGACCGCCGAGAAUCCUGUGUUCACCGUGUUCAAGGAUAACGAGGUG CUGUACAGAGCCCAGCUGGCCAGCGAGGACACAAACGCCCAGAAGACCAUCACAAACUGCUUCCUGCUGAAGAACAA GAUCUGGUGCAUCAGCCUGGUCGAGAUCUACGACACCGGCGACAACGUGAUCCGGCCUAAGCUGUUCGCUGUGAAGA UCCCUGAGCAGUGCACC UGAUAAAGCUGGAGCCUCGGUGGCCUUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0113] 8) Sequence of pUC57-Kan plasmid encoding full length NiV-G (full length NiV-G mRNA sequence underlined)

[0114] SEQ ID NO: 8

[0115] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCG GCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGCCTACAGAAAGCAAAAAGGTGCGGTTCGA GAACACAGCCTCTGACAAGGGAAAGAATCCTAGCAAGGTGATCAAAAGCTATTACGGCACCATGGATATCAAGAAGA TTAACGAAGGCCTGCTGGACAGCAAGATACTCAGCGCTTTTAACACCGTGATCGCCCTGCTGGGCAGCATCGTGATC ATAGTTATGAACATCATGATTATCCAGAACTACACCCGGAGCACCGACAACCAGGCTATGATCAAAGACGCCCTGCA GAGCATCCAACAGCAGATCAAGGGCCTGGCCGACAAGATCGGCACAGAAATCGGACCAAAGGTGTCCCTGATCGACA CGAGTTCTACAATCACTATCCCTGCCAACATCGGCCTGCTGGGCAGCAAAATCTCTCAGAGCACAGCCAGCATCAAC GAGAACGTGAACGAGAAATGCAAGTTCACCCTGCCACCACTGAAGATCCACGAATGCAACATCAGCTGCCCTAATCC TCTGCCCTTCAGAGAGTACAAGCCTCAGACCGAGGGTGTGTCTAACCTGGTGGGCCTGCCGAACAACATCTGCCTGC AAAAAACCAGCAACCAGATCCTGAAACCTAAGCTGATCAGCTACACACTGCCTGTGGTGGGCCAGAGCGGCACCTGT ATCACAGATCCTCTGCTGGCCATGGATGAAGGCTACTTCGCCTACAGCCATCTGGAAAAGATCGGGAGCTGTAGCCG GGGCGTCTCCAAACAGAGAATCATCGGCGTGGGCGAAGTGCTGGACAGAGGCGATGAGGTCCCCTCCCTGTTTATGA CAAATGTGTGGACCCCTAGCAACCCTAACACAGTGTACCACTGCTCCGCCGTGTATAATTCTGAATTCTACTACGTG CTGTGCGCCGTGTCCGTGGTGGGAGACCCCATCCTGAACTCTACCTACTGGAGCGGCTCTCTGATGATGACCAGACT GGCTGTTAAGCCCAAGAACAACGGCGAGAGCTACAATCAACACCAGTTCGCCCTGCGGAACATCGAGAAGGGCATGT ACGACAAAGTGATGCCCTACGGCCCTTCAGGAATCAAGCAGGGCGATACCCTGTATTTCCCCGCTGTGGGCTTCCTG GTGCGGACCGAATTCAAGTACAATGACTCCAATTGCCCCGTGGCCAAGTGTCAGTACAGCAAACCTGAAAACTGTAG ACTGTCTATGGGCATCAGACCTAATAGCCACTACATCCTCAGAAGCGGACTCCTCAAGTACAACCTGTCCGACGAGG AAAACTCTAAAATTATCTTCATCGAGATCAGCGACCAGCGCCTGTCTATCGGATCTCCATCTAAGATCTACGATAGC CTGGGCCAACCTGTGTTTTACCAGGCCAGCTTTAGCTGGGACACCATGATCAAGTTCGGAGATGTGCAGACAGTGAA CCCCCTAGTGGTTAACTGGAGAGATAATACCGTGATTAGCAGACCCGGCCAGTCCCAGTGTCCCAGATTCAACACCT GCCCTGAGGTGTGCTGGGAGGGCGTGTACAACGACGCCTTCCTGATCGATAGAATCAACTGGATCTCTGCCGGCGTA TTTCTGGACAGCAACCAGACCGCCGAGAATCCTGTGTTCACCGTGTTCAAGGATAACGAGGTGCTGTACAGAGCCCA GCTGGCCAGCGAGGACACAAACGCCCAGAAGACCATCACAAACTGCTTCCTGCTGAAGAACAAGATCTGGTGCATCA GCCTGGTCGAGATCTACGACACCGGCGACAACGTGATCCGGCCTAAGCTGTTCGCTGTGAAGATCCCTGAGCAGTGC ACCTGATAAAGCTGGAGCCTCGGTGGCCTTGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGC ACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAA

[0116] 9) Protein sequence of full-length NiV-F

[0117] SEQ ID NO: 9

[0118] MAVILNKRYYSNLLLLILMISECSVGILHYEKLSKIGLVKGITRKYKIKSNPLTKDIVIKMIPNVSNMSQCTGSVMENYKTRLNGILTPIKGALEIYKNNTHDLVGDVRLAGVIMAGVAIGIATAAQITAGVALYEAMKNADNINKLKSSIESTNEAVVKLQETAEKTVYVLTALQDYINTNLVPTIDKISCKQTELSLDLALSKYLSDLLFVFGPNLQDPVSNSMTIQAISQAFGGNYETLLRTLGYATEDFDDLLESDSITGQIIYVDLSGYYIIVRVYFPILTEIQQAYIQELLPVSFNNDNSEWISIVPNFILVRNTLISNIEIGFCLITKRSVICNQDYATPMTNNMRECLTGSTEKCPRELVVSSHVPRFALSNGVLFANCISVTCQCQTTGRAISQSGEQTLLMIDNTTCPTAVLGNVIISLGKYLGSVNYNSEGIAIGPPVFTDKVDISSQISSMNQSLQQSKDYIKEAQRLLDTVNPSLISMLSMIILYVLSIASLCIGLITFISFIIVEKKRNTYSRLEDRRVRPTSSGDLYYIGT

[0119] 10) Full-length NiV-F mRNA sequence (ORF)

[0120] SEQ ID NO: 10

[0121]

[0122] 11) Full length NiV-F mRNA sequence (5' UTR - ORF - 3' UTR - Poly(A) tail) (ORF underlined)

[0123] SEQ ID NO: 11

[0124] AGGCCGGCACUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCGCCACC AUGGCCGUGAUCCUGAAC AAGCGGUACUACUCUAAUCUGCUGCUGCUGAUCCUGAUGAUCAGCGAGUGCAGCGUGGGCAUUCUCCACUACGAGAA GCUGUCCAAAAUCGGCCUGGUGAAAGGCAUCACAAGAAAGUACAAGAUCAAAAGCAACCCUCUGACCAAGGACAUCG UGAUUAAGAUGAUCCCUAAUGUGAGCAAUAUGAGCCAGUGCACCGGUAGCGUGAUGGAAAACUACAAGACCAGACUG AACGGCAUCCUGACCCCUAUCAAGGGCGCCCUGGAAAUCUAUAAGAACAACACACAUGACCUGGUGGGAGAUGUGCG GCUGGCUGGCGUGAUUAUGGCCGGCGUGGCCAUCGGAAUCGCCACAGCCGCCCAGAUCACCGCCGGCGUCGCCCUGU ACGAGGCCAUGAAAAACGCCGAUAAUAUCAACAAGCUGAAGUCUAGCAUCGAGUCUACAAACGAGGCCGUGGUGAAG CUGCAGGAGACAGCAGAGAAGACCGUGUACGUGCUGACCGCCCUGCAGGACUACAUUAACACCAAUCUGGUGCCUAC CUACAGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGAUGA ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ GAUCAUCCUGUACGUGCUGAGCAUCGCCUCCCUUUGUAUCGGCCUGAUCACCUUCAUCAGCUUUAUCAUCGUGGAAA AAAAGAGAAACACCUACAGUAGACUGGAAGAUAGAAGGGUGCGCCCCACAAGCAGCGGCGACCUGUACUACAUAGGC ACC UGACUCGAGUAAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0125] 12) Sequence of pUC57-Kan plasmid encoding full length NiV-F (full length NiV-F mRNA sequence underlined)

[0126] SEQ ID NO: 12

[0127] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCG GCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGGCCGTGATCCTGAACAAGCGGTACTACTC TAATCTGCTGCTGATCCTGATGATCAGCGAGTGCAGCGTGGGCATTCTCCACTACGAGAAGCTGTCCAAAATCG GCCTGGTGAAAGGCATCACAAGAAAGTACAAGATCAAAAGCAACCCTCTGACCAAGGACATCGTGATTAAGATGATC CCTAATGTGAGCAATATGAGCCAGTGCACCGGTAGCGTGATGGAAAACTACAAGACCAGACTGAACGGCATCCTGAC CCCTATCAAGGGCGCCCTGGAAATCTATAAGAACAACACACATGACCTGGTGGGAGATGTGCGGCTGGCTGGCGTGA TTATGGCCGGCGTGGCCATCGGAATCGCCACAGCCGCCCAGATCACCGCCGGCGTCGCCCTGTACGAGGCCATGAAA AACGCCGATAATATCAACAAGCTGAAGTCTAGCATCGAGTCTACAAACGAGGCCGTGGTGAAGCTGCAGGAGACAGC AGAGAAGACCGTGTACGTGCTGACCGCCCTGCAGGACTACATTAACACCAATCTGGTGCCTACCATCGACAAGATCT CATGTAAGCAGACCGAGCTGTCTCTGGATCTGGCCCTGAGCAAATATCTGTCTGATCTGCTGTTCGTGTTCGGCCCT AACCTGCAAGACCCTGTTTCCAATTCCATGACAATCCAAGCCATAAGCCAGGCCTTCGGCGGCAATTACGAAACCCT GCTTAGAACCCTGGGCTACGCCACAGAGGACTTCGACGACCTGCTGGAGAGCGACAGCATCACCGGACAGATCATCT ACGTTGATCTGTCCGGCTACTATATCATCGTGAGAGTGTACTTCCCCATCCTGACTGAGATCCAGCAGGCTTACATA CAGGAGCTGCTGCCAGTGAGCTTCAACAACGACAATTCTGAATGGATCAGCATCGTGCCCAACTTCATCCTGGTGCG GAACACCCTCATCAGCAACATCGAGATCGGATTTTGCCTGATCACCAAGCGGAGCGTGATCTGCAACCAGGATTACG CCACACCTATGACCAACAATATGCGGGAATGCCTGACAGGATCTACCGAGAAGTGCCCCAGAGAACTGGTCGTGTCC AGCCACGTGCCAAGATTCGCCCTGTCTAACGGCGTGCTGTTTGCCAACTGCATCTCTGTGACCTGTCAGTGTCAGAC AACCGGCAGAGCCATCAGCCAGAGCGGCGAGCAGACCCTGCTGATGATCGACAACACCACATGCCCTACAGCTGTTC TGGGCAACGTGATCATTTCCCTGGGAAAGTACCTGGGCTCTGTGAACTATAACAGCGAAGGCATCGCGATTGGACCT CCTGTGTTCACCGACAAGGTGGACATCAGCAGCCAAATCAGTAGCATGAACCAGAGCCTGCAGCAGAGCAAGGACTA CATTAAGGAAGCTCAGAGACTGCTGGACACCGTGAACCCCAGCCTGATCTCTATGCTGTCTATGATCATCCTGTACG TGCTGAGCATCGCCTCCCTTTGTATCGGCCTGATCACCTTCATCAGCTTTATCATCGTGGAAAAAAAGAGAAACACC TACAGTAGACTGGAAGATAGAAGGGTGCGCCCCACAAGCAGCGGCGACCTGTACTACATAGGCACCTGACTCGAGTA AGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACC CCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA Examples

[0128] Example 1 - DNA templates for in vitro transcription and protein expression

[0129] The DNA template sequence for mRNA in vitro transcription (IVT) consists of a T7 promoter, a 5’ untranslated region (UTR), open reading frames (ORF) for the Nipah virus glycoprotein (NiV-G) and fusion protein (NiV-F) modified from MCL-18-H-1088 strain (GenBank: MH523642.1), a 3’ UTR, and 120 polyadenine bases (polyA). The 5’ UTR and 3’ UTR are from human hemoglobin alpha 1 subunit (HBA1) mRNA (GenBank: NM_000558.5). For the soluble form of glycoprotein (NiV-G_sol), the amino-terminal and transmembrane domain were removed from the full-length glycoprotein sequence, resulting in a potential secreted form (AMet1-Ile70). A signal peptide from collagen alpha 1 (COL1A1) (ColSP, MFSFVDLRLLLLLAATALLTHG, GenBank: Z74615.1) was added to the N-terminus of the full-length and soluble glycoprotein ORF to facilitate its secretion into the culture medium. The initial NiV-G DNA was synthesized by GenScript (Piscataway, NJ) and subcloned into the pUC57-Kan vector. Constructs for NiV-G_sol, ColSP-NiV-G, and ColSP-NiV-G_sol were subsequently made by PCR and subcloned into the NiV-G construct and the sequences were verified by Sanger sequencing (Azenta). NiV-F DNA was synthesized by Twist Bioscience and subsequently subcloned into the pUC57-Kan vector. NiV-F constructs with ColSP (ColSP-NiV-F) and without ColSP (NiV-F) were made by PCR, as well as NiV-F constructs with a FLAG tag (ColSP-NiV-F-FLAG and NiV-F-FLAG) to ensure protein expression and detection.

[0130] The sequence of the pUC57-Kan plasmid encoding soluble NiV-G fused to the COL1A1 signal peptide mRNA is set forth in SEQ ID NO: 4. The sequence of the pUC57-Kan plasmid encoding full-length NiV-G mRNA is set forth in SEQ ID NO: 8. The sequence of the pUC57-Kan plasmid encoding full-length NiV-F mRNA is set forth in SEQ ID NO: 12.

[0131] Example 2 - In vitro transcription (IVT)

[0132] For each NiV form, plasmid vectors were linearized using the restriction enzyme BspQI (New England Biolabs). N1-methylpseudouridine (m1y) was purchased from BOC Sciences (Shirley, NY). IVT conditions followed the manufacturer’s recommendations (TranscriptAid T7 High Yield Transcription Kit, ThermoFisher) and are shown below:

[0133] • ATP / CTP / GTP / m1yTP: 5 mM each

[0134] • SmartCap (SC101, ST Pharm): 4 mM

[0135] • Linear template DNA: 1 pg plasmid or 0.5 pg PCR product

[0136] • T7 RNA polymerase mix: 2 pl

[0137] IVT was performed in 20 pl reactions and incubated at 37 °C for 2 hours. Template DNA was removed with 2 units of DNase I (Invitrogen) at 37 °C for 15 min followed by column purification (Monarch RNA Cleanup Kit, New England Biolabs).

[0138] IVT products of the four NiV-G constructs (A) and four NiV-F constructs (B) were analyzed by agarose gel, detecting the four mRNAs at a length of ~2 knt. After IVT from DNA templates of NiV-G mRNA (A) and NiV-F mRNA (B), 100 ng of mRNA was analyzed by electrophoresis using 1% E-GEL EX agarose in an E-Gel Power Snap Electrophoresis Device (ThermoFisher) (one of three independent IVT products). See Figure 1 .

[0139] Example 3 - Transfection

[0140] One μg of mRNA (synthesized in triplicate) was transfected into 293FT cells (Invitrogen) in 12-well plates using Lipofectamine MesseangerMax (Invitrogen) at a 1 :2 ratio of reagent to mRNA according to the manufacturer's protocol. Samples were collected from the media and cells 24 hours post-transfection. Cell lysates were prepared in NP-40 lysis buffer (150 mM NaCl / 1% NP-40 / 50 mM Tris pH 8.0). As a transfection control, 0.1 μg of EGFP mRNA (L-7601, TriLink) was co-transfected.

[0141] Example 4 - Western blotting

[0142] Rabbit anti-Nipah glycoprotein antibody (#NIV11-S) was purchased from Alpha Diagnostic (San Antonio, TX). Protein detection used HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) and SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher Scientific). GAPDH was detected with HRP-conjugated mouse monoclonal antibody (sc-47724, Santa Cruz Biotechnology) as a loading control. EGFP as a transfection control was detected with mouse monoclonal antibody (sc-9996, Santa Cruz Biotechnology). To detect NiV-F protein, anti-FLAG tag monoclonal antibody (Sigma-Aldrich) and anti-Nipah F Fl rabbit antibody (Absolute Antibody, Wilton, UK) were purchased.

[0143] NiV-G protein levels were determined by Western blotting as shown in Figure 2A Four different NiV-G mRNAs were each transfected into 293FT cells at 1 μg. Twenty-four hours post-transfection, cell lysates and media were collected and subjected to Western blot analysis using NiV-G specific antibodies. GFP mRNA was co-transfected as an mRNA transfection control, and untransfected 293FT cells were used as a negative control. GAPDH was used as a loading control. As shown in Figure 2BNiV-F proteins with or without ColSP were detected in cell lysates using FLAG tag antibody and NiV-F specific antibody, showing full-length NiV-F protein of 60 kDa (arrow) and smaller cleavage products. Untransfected cell lysate or media were used as negative controls ((-) control).

[0144] Collected media samples were subjected to Western blot analysis to detect any secreted NiV-G protein, and collected cell lysates were subjected to Western blot analysis to detect intracellular / non-secreted NiV-G protein. As shown in Figure 2A Wild-type NiV-G protein without COL1A1 signal peptide (lane 2) and soluble NiV-G protein with COL1A1 signal peptide (lane 5) were detected in cell lysates. However, only soluble NiV-G protein with COL1A1 signal peptide was detected in media. Since NiV-G and ColSP-NiV-G_sol were well expressed compared to ColSP-NiV-G and NiV-G_sol, NiV-G and ColSP-NiV-G_sol were selected as mRNA vaccine candidates. For NiV-F protein, cell lysates were probed with FLAG antibody and further confirmed with NiV-F specific antibody to detect NiV-F protein in Figure 2B Similar to NiV-G, full-length form of wild-type NiV-F was better expressed than ColSP-NiV-F, thus wild-type full-length NiV-F was selected as another mRNA vaccine candidate.

[0145] Example 5 - Immunogenicity studies

[0146] The present study aimed to test the immunogenicity of the disclosed Nipah virus vaccine compositions (e.g., Nipah virus vaccine compositions (1) and (4)) in mice.

[0147] Mice were immunized intramuscularly (IM) with the disclosed Nipah virus vaccine compositions. The vaccine compositions of the disclosure were chemically modified or unmodified. A total of two immunizations were performed, with a 3-week interval (i.e., at week 0 and week 3), and serum was collected after each immunization. See Figure 3ASerum antibody titers against soluble NiV-G fused to the COL1A1 signal peptide and full-length NiV-G were determined using a mouse anti-Nipah virus glycoprotein IgG ELISA kit (NIV-025, Alpha Diagnostics International). Overall, both mRNA vaccine formulations (VER-012 containing soluble NiV-G (Nipah virus vaccine composition (1)) and VER-015 containing full-length NiV-G and NiV-F (Nipah virus vaccine composition (4))) showed high titers of IgG antibodies against NiV-G in a dose-dependent manner. Figure 3B As shown. Serum collected from each mouse was also used for, for example... Figure 3C The in vitro NiV neutralization assay against Nipah virus is shown to evaluate its in vitro protective effect. Both vaccine formulations showed strong neutralizing activity against Nipah virus in a dose-dependent manner. Overall, the full-length formulation of NiV-G and NiV-F (VER-015) performed better in neutralizing Nipah virus than the soluble NiV-G (VER-012).

[0148] Example 6 - Additional immunogenicity studies

[0149] This study aims to test the immunogenicity of the Nipah virus vaccine compositions disclosed herein (e.g., Nipah virus vaccine compositions (2) and (3)) in mice.

[0150] Mice were immunized intramuscularly (IM) with the Nipah virus vaccine composition disclosed herein. The vaccine composition disclosed herein may be chemically modified or unmodified. A total of two immunizations were performed, three weeks apart (i.e., week 0 and week 3), with serum collected after each immunization.

[0151] Both mRNA vaccine formulations (VER-013 containing full-length NiV-G (Nipah virus vaccine composition (2)) and VER-014 containing full-length NiV-F (Nipah virus vaccine composition (3)) are expected to exhibit high titers of IgG antibodies against Nipah virus in a dose-dependent manner. Serum collected from each mouse was also used in an in vitro NiV neutralization assay against Nipah virus to assess its in vitro protective effect. Both vaccine formulations are expected to demonstrate strong neutralizing activity against Nipah virus.

Claims

1. A Nipah virus vaccine composition comprising: A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a soluble Nipah virus glycoprotein (soluble NiV-G) fused to a human collagen type I alpha 1 (COL1A1) signal peptide.

2. The Nipah virus vaccine composition of claim 1, wherein the soluble NiV-G fused to the COL1A1 signal peptide has the amino acid sequence of SEQ ID NO:

1.

3. The Nipah virus vaccine composition of claim 1, wherein the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO:

2.

4. The Nipah virus vaccine composition of claim 1, wherein the mRNA comprising the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail, to have the structure: 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail, and wherein the ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide has the nucleotide sequence of SEQ ID NO:

2.

5. The Nipah virus vaccine composition of claim 4, wherein the poly(A) tail is 50-250 nucleotides in length.

6. The Nipah virus vaccine composition of claim 4, wherein the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

3.

7. The Nipah virus vaccine composition of claim 4, wherein the mRNA having the structure of 5’ UTR-ORF encoding the soluble NiV-G fused to the COL1A1 signal peptide-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

3.

8. A method of inducing an immune response against a Nipah virus comprising:

8. A method of treating a Nipah virus infection in a subject in need thereof, comprising administering to the subject an effective amount of the Nipah virus vaccine composition of claim 1.

9. A Nipah virus vaccine composition comprising:

9. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G).

10. The Nipah virus vaccine composition of claim 9, wherein the full-length NiV-G has the amino acid sequence of SEQ ID NO:

5.

11. The Nipah virus vaccine composition of claim 9, wherein the ORF encoding the full-length NiV-G has the nucleotide sequence of SEQ ID NO:

6.

12. The Nipah virus vaccine composition of claim 9, wherein the mRNA comprising an ORF encoding a full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure: 5’ UTR-ORF encoding a full-length NiV-G-3’ UTR-poly(A) tail, and wherein the ORF encoding a full-length NiV-G has the nucleotide sequence of SEQ ID NO:

6.

13. The Nipah virus vaccine composition of claim 12, wherein the poly(A) tail is 50-250 nucleotides in length.

14. The Nipah virus vaccine composition of claim 12, wherein the mRNA having the structure of 5’ UTR-ORF encoding a full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

7.

15. The Nipah virus vaccine composition of claim 12, wherein the mRNA having the structure of 5’ UTR-ORF encoding a full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

7.

16. A method of inducing an immune response against a Nipah virus comprising:

16. A method of vaccinating a subject in need thereof, comprising administering to the subject an effective amount of the Nipah virus vaccine composition of claim 9.

17. A Nipah virus vaccine composition comprising:

17. A messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus fusion protein (full-length NiV-F).

18. The Nipah virus vaccine composition of claim 17, wherein the full-length NiV-F has the amino acid sequence of SEQ ID NO:

9.

19. The Nipah virus vaccine composition of claim 17, wherein the ORF encoding a full-length NiV-F has the nucleotide sequence of SEQ ID NO:

10.

20. The Nipah virus vaccine composition of claim 17, wherein the mRNA comprising an ORF encoding a full-length NiV-F further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure: 5’ UTR-ORF encoding a full-length NiV-F-3’ UTR-poly(A) tail, and wherein the ORF encoding a full-length NiV-F has the nucleotide sequence of SEQ ID NO:

10.

21. The Nipah virus vaccine composition of claim 20, wherein the poly(A) tail is 50-250 nucleotides in length.

22. The Nipah virus vaccine composition of claim 20, wherein the mRNA having the structure of 5’ UTR-ORF encoding a full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

11.

23. The Nipah virus vaccine composition of claim 20, wherein the mRNA having the structure of 5’ UTR-ORF encoding a full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

11.

24. A method of inducing an immune response against a Nipah virus comprising: administering to a subject in need thereof an effective amount of the Nipah virus vaccine composition of claim 17.

25. A Nipah virus vaccine composition comprising: a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding a full-length Nipah virus glycoprotein (full-length NiV-G), and a mRNA comprising an ORF encoding a full-length Nipah virus fusion protein (full-length NiV-F).

26. The Nipah virus vaccine composition of claim 25, wherein the full-length NiV-G has the amino acid sequence of SEQ ID NO: 5, and wherein the full-length NiV-F has the amino acid sequence of SEQ ID NO:

9.

27. The Nipah virus vaccine composition of claim 25, wherein the ORF encoding full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6, and wherein the ORF encoding full-length NiV-F has the nucleotide sequence of SEQ ID NO:

10.

28. The Nipah virus vaccine composition of claim 25, wherein the mRNA comprising the ORF encoding full-length NiV-G further comprises a 5’ untranslated region (UTR), a 3’ UTR, and a poly(A) tail to have the structure: 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail, and the ORF encoding full-length NiV-G has the nucleotide sequence of SEQ ID NO: 6, and wherein the mRNA comprising the ORF encoding full-length NiV-F further comprises a 5’ UTR, a 3’ UTR, and a poly(A) tail to have the structure: 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail, and the ORF encoding full-length NiV-F has the nucleotide sequence of SEQ ID NO:

10.

29. The Nipah virus vaccine composition of claim 28, wherein the poly(A) tail is 50-250 nucleotides in length.

30. The Nipah virus vaccine composition of claim 28, wherein the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 7, and wherein the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

11.

31. The Nipah virus vaccine composition of claim 28, wherein the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-G-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 7, and wherein the mRNA having the structure of 5’ UTR-ORF encoding full-length NiV-F-3’ UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO:

11.

32. A method of inducing an immune response against a Nipah virus comprising: administering to a subject in need thereof an effective amount of the Nipah virus vaccine composition of claim 25.

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