An engineered varicella-zoster virus mRNA vaccine

Abstract

Provided herein are varicella-zoster vaccine compositions comprising messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding varicella-zoster virus (VZV) glycoprotein E (gE) (which may be soluble VZV gE or full-length VZV gE), and compositions for inducing an immune response against varicella-zoster, including the varicella-zoster vaccine compositions.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 423,759, filed November 8, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] [Incorporation by reference to sequence listing] The contents of the electronically submitted sequence listing submitted herewith, with file name: F292635_sequence listing as filed, size: 25,789 bytes, and creation date: November 6, 2023, are incorporated herein by reference in their entirety.

[0003] Provided herein are varicella-zoster vaccine compositions comprising messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding varicella-zoster virus (VZV) glycoprotein E (gE), which may be soluble VZV gE or full-length VZV gE, and methods for inducing an immune response against varicella-zoster by administering an effective amount of the varicella-zoster vaccine composition to a subject in need thereof. [Background technology]

[0004] Shingles, also known as zoster or herpes zoster, is a viral disease caused by the varicella-zoster virus (VZV). Currently, there is no approved shingles mRNA vaccine, and there is a need for a shingles mRNA vaccine. Summary of the Invention

[0005] The present disclosure provides a varicella-zoster virus (VZV) vaccine composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) encoding varicella-zoster virus (VZV) glycoprotein E (gE). In one embodiment, the VZV gE has the amino acid sequence of SEQ ID NO: 1 (soluble VZV gE). In another embodiment, the VZV gE has the amino acid sequence of SEQ ID NO: 2 (full-length VZV gE). In one embodiment, the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO: 3 (ORF encoding soluble VZV gE). In another embodiment, the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO: 4 (ORF encoding full-length VZV gE). In some embodiments, the mRNA comprising an ORF encoding VZV gE further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF has a 5' UTR-ORF structure encoding VZV gE-3' UTR-poly(A) tail, and the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO: 3 (an ORF encoding soluble VZV gE). In another embodiment, the mRNA comprising an ORF encoding VZV gE further comprises a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the ORF has a 5' UTR-ORF structure encoding VZV gE-3' UTR-poly(A) tail, and the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO: 4 (an ORF encoding full-length VZV gE). In one embodiment, the poly(A) tail has a length of 50 to 250 nucleotides. In some embodiments, the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 5 (5'UTR-ORF encoding a soluble VZV gE-3'UTR-poly(A) tail). In other embodiments, the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO: 6 (5'UTR-ORF encoding a full-length VZV gE-3'UTR-poly(A) tail).In some embodiments, the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 5 (a 5'UTR-ORF encoding a soluble VZV gE-3'UTR-poly(A) tail). In another embodiment, the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has a nucleotide sequence that is at least 80% identical to SEQ ID NO: 6 (a 5'UTR-ORF encoding a full-length VZV gE-3'UTR-poly(A) tail). In one embodiment, the varicella zoster vaccine composition of the present disclosure further comprises a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

[0006] The present disclosure also provides compositions for inducing an immune response against shingles, comprising the shingles vaccine compositions of the present disclosure. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows in vitro transcription of two VZV gE mRNAs. [Figure 2] FIG. 1 shows VZV gE protein levels in gE mRNA-transfected 293FT cells and culture medium. [Figure 3] FIG. 1 shows the immunization and serum collection schedule. [Figure 4] FIG. 1 shows the results of mouse anti-VZV-gE titers. [Figure 5] FIG. 1 shows the results of VZV neutralization. DETAILED DESCRIPTION OF THE INVENTION

[0008] definition 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, certain preferred methods, compositions, devices, and materials are described herein. However, before describing the materials and methods of the present invention, it should be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described herein, as these may vary depending on routine experimentation and optimization. It should also be understood that the terminology used in the detailed description is for the purpose of describing particular forms or embodiments only, and is not intended to limit the scope of the embodiments described herein.

[0009] Unless otherwise specified, 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, in case of conflict, the present specification, including definitions, will control. Therefore, in the context of the embodiments described herein, the following definitions apply.

[0010] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] As used herein, the term "comprise" and its linguistic variants indicate the presence of the recited feature(s), element(s), method step(s), etc., but do not exclude the presence of additional feature(s), element(s), method step(s), etc. In contrast, the term "consisting of" and its linguistic variants indicate the presence of the recited feature(s), element(s), method step(s), etc., and excludes any unrecited feature(s), element(s), method step(s), etc., except for impurities ordinarily associated therewith. The phrase "consisting essentially of" refers to the recited feature(s), element(s), method step(s), etc., as well as any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic characteristics of the composition, system, or method. Many embodiments herein are described using the open-ended term "comprising." Such embodiments encompass a number of the open-ended "consisting of" and / or "consisting essentially of" embodiments, which may alternatively be claimed or described in the specification using such terms.

[0012] As used herein, the term "shingles vaccine composition" refers to substances used to stimulate the production of antibodies and provide immunity against shingles.

[0013] As used herein, the term "messenger ribonucleic acid (mRNA)" refers to a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene and is read by ribosomes in the process of synthesizing proteins.

[0014] Naturally occurring 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 (Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile 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).

[0015] Unnatural amino acids include azetidine carboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, tert-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"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), N-alkylglycines ("NAG") including N-methylglycine, N-methylisoleucine, N-alkylpentylglycines ("NAPG") including N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGly"), pipecolic acid, thioproline ("ThioP" or "tPro"), homolysine ("hLys"), and homoarginine ("hArg").

[0016] As used herein, the term "open reading frame (ORF)" refers to the nucleotide sequence between the start codon and the stop codon.

[0017] As used herein, the term "open reading frame (ORF) encoding" refers to a nucleotide coding sequence that encodes a polypeptide. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered. The coding sequence may further include a sequence encoding a signal peptide.

[0018] As used herein, the term "T7 promoter" refers to the promoter derived from bacteriophage T7.

[0019] As used herein, the term "5' untranslated region (UTR)" refers to the region of an mRNA immediately upstream (i.e., 5') from the start codon (the first codon of an mRNA transcript that is translated by ribosomes) that does not encode a polypeptide.

[0020] As used herein, the term "3' untranslated region (UTR)" refers to the region of an mRNA immediately downstream (i.e., 3') from the stop codon (i.e., the codon of the mRNA transcript that signals the end of translation) that does not encode a polypeptide.

[0021] As used herein, the term "poly(A) tail" refers to the "tail" of an mRNA, i.e., the long stretch of adenine nucleotides added to the 3' end.

[0022] 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 may include, but are not limited to, adjuvants, excipients, lipid particles, etc.

[0023] As used herein, the term "lipid nanoparticle" refers to a particle having at least one dimension on the nanometer scale (e.g., 1 nm to 1000 nm). In some embodiments, lipid nanoparticles are included in formulations that can be used to deliver mRNA vaccines to desired target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the mRNA vaccine can be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, such as a harmful immune response. In some embodiments, the lipid nanoparticles have an average diameter of 50 nm to 200 nm. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of approximately 20% to 60% cationic lipid, 0.5% to 15% PEG-modified lipid, 25% to 55% sterol, and 25% 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-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0024] As used herein, the term "inducing an immune response against shingles" refers to providing protective immunity and / or vaccinating a subject against shingles for prophylactic purposes, and to generating a desired immune response or effect against shingles in a subject in need thereof for therapeutic purposes. As used herein, the term "protective immunity" or "protective immune response" means that a vaccinated subject is able to control infection by the pathogen against which the vaccine was administered. Typically, a subject who develops a "protective immune response" develops only mild to moderate clinical symptoms or no symptoms at all.

[0025] An "effective amount" of a varicella zoster vaccine composition (e.g., mRNA) is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and degree of nucleoside modification) and other components of the vaccine, as well as other determinants. Generally, an effective amount of a varicella zoster vaccine (e.g., mRNA) induces or boosts antigen production in response to cells, preferably resulting in a more efficient immune response than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (percentage of cells transfected with RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by an increased duration of protein translation from a modified polynucleotide), or a change in the antigen-specific immune response of host cells.

[0026] As used herein, the term "X% identity to SEQ ID NO: Y" or "sequence identity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the extent to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) differ only by conservative and / or semi-conservative amino acid substitutions. "Percent sequence identity" (or "percent sequence similarity") is calculated by (1) comparing 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) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid occurs in both sequences, a similar amino acid occurs in both sequences) to obtain the number of matched positions, (3) dividing the number of matched positions by the total number of positions within the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying 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 long and have identical amino acids at all positions except one, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical properties (e.g., both are acidic), peptide A and peptide B will have 100% sequence similarity. As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to those in a portion of peptide C, peptides C and D will have 70% sequence identity, but peptide D will have 93.3% sequence identity over the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0027] As used herein, the term "a nucleotide sequence having at least X% identity to sequence number Y and encoding a Z protein" means that the nucleotide sequence satisfies two distinct requirements: having at least X% identity to sequence number Y and encoding a Z protein.

[0028] As used herein, the terms "about," "approximate," "at or about," and "substantially" mean that the relevant amount or value may be the exact value set forth in the claims or taught herein, or a value that will produce an equivalent result or effect. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not, and need not be, exact, but may be approximate and / or larger or smaller, if desired, to produce an equivalent result or effect, reflecting tolerances, conversion factors, rounding, measurement errors, and the like, as well as other factors known to those skilled in the art. In some circumstances, it may not be possible to reasonably determine a value that will produce an equivalent result or effect. In such cases, generally, as used herein, "about" and "at or about" are understood to mean a ±10% variation from the nominal value indicated, unless otherwise indicated or implied. In general, amounts, sizes, formulations, parameters, or other quantities or characteristics are "about," "approximate," or "at or about," whether or not expressly stated as such. When "about," "approximate," or "at or about" is used before or after a quantitative value, it is understood that the parameter also includes the particular quantitative value itself, unless otherwise specifically stated.

[0029] The terms "subject," "patient," "individual," and the like are used interchangeably herein and refer to any animal, any mammalian subject, or cells thereof, suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0030] Detailed Description 1. Shingles vaccine composition The present disclosure provides a varicella-zoster vaccine composition comprising an open reading frame (ORF) encoding varicella-zoster virus (VZV) glycoprotein E (gE).

[0031] In one embodiment, the VZV gE has the amino acid sequence of SEQ ID NO: 1 (soluble VZV gE). In another embodiment, the VZV gE has the amino acid sequence of SEQ ID NO: 2 (full-length VZV gE).

[0032] In one embodiment, VZV gE has an amino acid sequence at least 80% identical to SEQ ID NO:1 (soluble VZV gE). In another embodiment, VZV gE has an amino acid sequence at least 85% identical to SEQ ID NO:1 (soluble VZV gE). In some embodiments, VZV gE has an amino acid sequence at least 90% identical to SEQ ID NO:1 (soluble VZV gE). In another embodiment, VZV gE has an amino acid sequence at least 95% identical to SEQ ID NO:1 (soluble VZV gE). In one embodiment, VZV gE has an amino acid sequence at least 96% identical to SEQ ID NO:1 (soluble VZV gE). In some embodiments, VZV gE has an amino acid sequence at least 97% identical to SEQ ID NO:1 (soluble VZV gE). In another embodiment, the VZV gE has an amino acid sequence that is at least 98% identical to SEQ ID NO: 1 (soluble VZV gE). In some embodiments, the VZV gE has an amino acid sequence that is at least 99% identical to SEQ ID NO: 1 (soluble VZV gE).

[0033] In another embodiment, VZV gE has an amino acid sequence at least 80% identical to SEQ ID NO:2 (full-length VZV gE). In some embodiments, VZV gE has an amino acid sequence at least 85% identical to SEQ ID NO:2 (full-length VZV gE). In one embodiment, VZV gE has an amino acid sequence at least 90% identical to SEQ ID NO:2 (full-length VZV gE). In some embodiments, VZV gE has an amino acid sequence at least 95% identical to SEQ ID NO:2 (full-length VZV gE). In another embodiment, VZV gE has an amino acid sequence at least 96% identical to SEQ ID NO:2 (full-length VZV gE). In some embodiments, VZV gE has an amino acid sequence at least 97% identical to SEQ ID NO:2 (full-length VZV gE). In another embodiment, VZV gE has an amino acid sequence at least 98% identical to SEQ ID NO:2 (full-length VZV gE). In some embodiments, the VZV gE has an amino acid sequence that is at least 99% identical to SEQ ID NO:2 (full-length VZV gE).

[0034] The present disclosure provides two different types of varicella zoster vaccine compositions: Herpes zoster vaccine composition (1): Herpes zoster vaccine composition containing mRNA containing an ORF encoding VZV gE (soluble) Herpes zoster vaccine composition (2): Herpes zoster vaccine composition containing mRNA containing an ORF encoding VZV gE (full length)

[0035] In the two types of varicella zoster vaccine compositions described above, VZV gE (soluble) may have the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 1). In another embodiment, VZV gE (full-length) may have the amino acid sequence of SEQ ID NO: 2 (or an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 2).

[0036] In the above two types of varicella zoster vaccine compositions, the ORF encoding VZV gE (soluble) may 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). In another embodiment, the ORF encoding VZV gE (full length) may have the nucleotide sequence of SEQ ID NO: 4 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 4).

[0037] In the varicella-zoster vaccine composition (1), the mRNA containing the ORF encoding VZV gE (soluble) may further contain a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail to have a 5' UTR-ORF structure encoding VZV gE (soluble)-3' UTR-poly(A) tail, and the ORF encoding VZV gE (soluble) may 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).

[0038] In the varicella-zoster vaccine composition (2), the mRNA containing the ORF encoding VZV gE (full length) can further contain a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail to have a 5' UTR-ORF structure encoding VZV gE (full length)-3' UTR-poly(A) tail, and the ORF encoding VZV gE (full length) can have the nucleotide sequence of SEQ ID NO: 4 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 4).

[0039] In the varicella zoster vaccine composition (1), the mRNA having a 5'UTR-ORF structure encoding VZV gE (soluble)-3'UTR-poly(A) tail may have the nucleotide sequence of SEQ ID NO: 5 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 5).

[0040] In the varicella zoster vaccine composition (2), the mRNA having a 5'UTR-ORF structure encoding VZV gE (full-length)-3'UTR-poly(A) tail may have the nucleotide sequence of SEQ ID NO: 6 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99% identity to SEQ ID NO: 6).

[0041] In one embodiment, the poly(A) tail has a length of 50 to 250 nucleotides. In another embodiment, the poly(A) tail has a length of 100 to 200 nucleotides. In another embodiment, the poly(A) tail has a length of 110 to 150 nucleotides. In another embodiment, the poly(A) tail has a length of 115 to 125 nucleotides. In another embodiment, the poly(A) tail has a length of 116 to 124 nucleotides. In another embodiment, the poly(A) tail has a length of 117 to 123 nucleotides. In another embodiment, the poly(A) tail has a length of 118 to 122 nucleotides. In another embodiment, the poly(A) tail has a length of 119 to 122 nucleotides. In another embodiment, the poly(A) tail has a length of 115 nucleotides. In another embodiment, the poly(A) tail has a length of 116 nucleotides. In another embodiment, the poly(A) tail has a length of 117 nucleotides. In another embodiment, the poly(A) tail has a length of 118 nucleotides. In another embodiment, the poly(A) tail has a length of 119 nucleotides. In another embodiment, the poly(A) tail has a length of 120 nucleotides. In another embodiment, the poly(A) tail has a length of 121 nucleotides. In another embodiment, the poly(A) tail has a length of 122 nucleotides. In another embodiment, the poly(A) tail has a length of 123 nucleotides. In another embodiment, the poly(A) tail has a length of 124 nucleotides. In another embodiment, the poly(A) tail has a length of 125 nucleotides.

[0042] In one embodiment, the mRNA of the present disclosure may contain 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.

[0043] In one embodiment, the varicella zoster vaccine composition further comprises a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutically acceptable carrier may include any substance or vehicle suitable for delivering the mRNA vaccine to a suitable in vivo or ex vivo site. Such carriers may include, but are not limited to, adjuvants, excipients, lipid particles, etc. Lipid nanoparticles may be particles having at least one dimension on the nanometer scale (e.g., 1 nm to 1000 nm). In some embodiments, the lipid nanoparticles are included in formulations that can be used to deliver the mRNA vaccine to a desired target site (e.g., a cell, tissue, organ, tumor, etc.). In some embodiments, the mRNA vaccine can be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space surrounded by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, such as a harmful immune response. In some embodiments, the lipid nanoparticles have an average diameter of 50 nm to 200 nm. In some embodiments, the lipid nanoparticles comprise a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid. In some embodiments, the lipid nanoparticles comprise a molar ratio of about 20%-60% cationic lipid, 0.5%-15% PEG-modified lipid, 25%-55% sterol, and 25% 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-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0044] In one embodiment, the lipid nanoparticles comprise (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (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, in a molar ratio of about 20%-60% cationic lipid:5%-25% neutral lipid:25%-55% sterol:0.5%-15% PEG lipid.

[0045] In one embodiment, the lipid nanoparticles comprise about 25% to about 75% on a molar basis, e.g., about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis, of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319).

[0046] In one embodiment, the lipid nanoparticles contain about 0.5% to about 15% neutral lipid on a molar basis, e.g., about 3% to about 12%, about 5% to about 10%, or about 15%, about 10%, or about 7.5%. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation contains about 5% to about 50% sterol on a molar basis (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31%). An exemplary sterol is cholesterol. In some embodiments, the formulation comprises about 0.5% to about 20% PEG-lipid or PEG-modified lipid on a molar basis (e.g., about 0.5% to about 10%, about 0.5% to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG-lipid or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2000 Da. In other embodiments, the PEG-lipid or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2000, e.g., around 1500 Da, around 1000 Da, or around 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG) and PEG-cDMA.

[0047] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 25% to 75% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5% to 15% of a neutral lipid, 5% to 50% of a sterol, and 0.5% to 20% of a PEG lipid or a PEG-modified lipid.

[0048] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 35% to 65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-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 lipid or a PEG-modified lipid.

[0049] In one embodiment, the lipid nanoparticles comprise, on a molar basis, 45% to 65% of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-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 lipid or a PEG-modified lipid.

[0050] In one embodiment, the lipid nanoparticles comprise about 60% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol, and about 1.5% PEG lipid or PEG-modified lipid.

[0051] In one embodiment, the lipid nanoparticles comprise about 50% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 38.5% sterol, and about 1.5% PEG lipid or PEG-modified lipid.

[0052] In one embodiment, the lipid nanoparticles comprise about 50% by molar cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 35% sterol, about 4.5% or about 5% PEG lipid or PEG-modified lipid, and about 0.5% targeting lipid.

[0053] In one embodiment, the lipid nanoparticles comprise about 40% by molar of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% neutral lipid, about 40% sterol, and about 5% PEG lipid or PEG-modified lipid.

[0054] In one embodiment, the shingles vaccine composition of the present disclosure can be delivered, localized, and / or concentrated to a specific location using the delivery methods described below. As a non-limiting example, empty polymer particles can be administered to a subject before, simultaneously with, or after delivery of the shingles vaccine composition of the present disclosure to the subject. Upon contact with the subject, the empty polymer particles undergo a volume change and become lodged, embedded, immobilized, or trapped at a specific location within the subject.

[0055] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated in an active agent release system. For example, the active agent release system can include at least one nanoparticle bound to an oligonucleotide inhibitor chain hybridized with a catalytically active nucleic acid and a compound bound to at least one substrate molecule bound to a therapeutically active agent (e.g., a polynucleotide described herein), wherein the therapeutically active agent is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0056] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated into nanoparticles comprising an inner core comprising non-cellular material and an outer surface comprising a cellular membrane, which may be derived from a cell or a virus.

[0057] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated in porous nanoparticle-supported lipid bilayers (protocells).

[0058] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated into polymeric nanoparticles that have a high glass transition temperature.

[0059] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated into nanoparticles for use in imaging. As a non-limiting example, a liposome can comprise gadolinium(III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-acetate and a neutral, fully saturated phospholipid component.

[0060] The nanoparticles of the present disclosure may further comprise nutrients, such as, but not limited to, nutrients whose deficiency can cause adverse health effects ranging from anemia to neural tube defects. By way of non-limiting example, the nutrient can be iron in the form of ferrous salts, ferric salts, or elemental iron, iodine, folic acid, vitamins, or micronutrients.

[0061] In another embodiment, the varicella zoster vaccine composition of the present disclosure can be formulated into swellable nanoparticles.

[0062] In another embodiment, the varicella zoster vaccine composition of the present disclosure may be formulated in polyanhydride nanoparticles.

[0063] 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 various shapes, sizes, and / or surface charges to incorporate polynucleotides of the present disclosure for targeted delivery, such as, but not limited to, pulmonary delivery. Other physical features that the geometrically engineered particles can have include, but are not limited to, fenestrations, angled arms, asymmetry and surface roughness, and charge, which can alter interactions with cells and tissues.

[0064] In another embodiment, the nanoparticles of the present disclosure may be water-soluble nanoparticles. The nanoparticles may be inorganic nanoparticles that are compact and have zwitterionic ligands to exhibit good water solubility. The nanoparticles may also have a small hydrodynamic diameter (HD), stability over time, pH, and salt concentration, and low levels of nonspecific protein binding.

[0065] In some embodiments, the nanoparticles of the present disclosure are stealth nanoparticles or target-specific stealth nanoparticles. In some embodiments, the stealth nanoparticles or target-specific stealth nanoparticles can comprise a polymer matrix. The polymer matrix can comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumarate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or a combination thereof.

[0066] In one embodiment, the nanoparticles of the present disclosure may be nanoparticle-nucleic acid hybrid structures having a high-density nucleic acid layer. The nanoparticles of the present disclosure may comprise nucleic acids, such as, but not limited to, polynucleotides described herein and / or known in the art.

[0067] In one embodiment, at least one of the nanoparticles of the present disclosure may be embedded in the core of the nanostructure or coated with a low-density porous 3D structure or coating capable of carrying or associating at least one payload within or on the surface of the nanostructure.

[0068] In one embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA of the present disclosure.In another embodiment, the lipid nanoparticle comprises a first lipid nanoparticle that encapsulates the mRNA encoding VZV gE (soluble) and a second lipid nanoparticle that encapsulates the mRNA encoding VZV gE (full-length).

[0069] 2. Composition for inducing an immune response against shingles The present disclosure also provides a composition for inducing an immune response against shingles, comprising administering an effective amount of a shingles vaccine composition of the present disclosure. In one embodiment, an effective amount of a shingles vaccine composition (e.g., mRNA) is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size and degree of modified nucleosides) and other components of the vaccine, as well as other determinants. Generally, an effective amount of a shingles vaccine (e.g., mRNA) induces or boosts antigen production in cells, preferably resulting in a more efficient immune response than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (percentage of cells transfected with RNA, e.g., mRNA, vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (e.g., indicated by an increased duration of protein translation from a modified polynucleotide), or a change in the antigen-specific immune response of host cells.

[0070] Administration of an effective (immunogenically effective) amount of a varicella zoster vaccine composition (e.g., varicella zoster vaccine compositions (1) and (2)) is typically intramuscular or subcutaneous. Thus, varicella zoster vaccine compositions are typically formulated for intramuscular or subcutaneous injection, and for purposes of the present invention, are formulated without an adjuvant, preferably without any adjuvant. However, other modes of administration, such as intravenous, cutaneous, intradermal, or intranasal, are also contemplated. For intravenous, cutaneous, or subcutaneous injection, the adenoviral vector is in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Similarly, the isolated envelope polypeptide is in the form of a parenterally acceptable solution that has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using isotonic vehicles, such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required.

[0071] In certain embodiments, an effective amount (immunogenically effective amount) of a varicella zoster vaccine composition (e.g., varicella zoster vaccine compositions (1) and (2)) is administered intramuscularly. Intramuscular administration can be achieved by injecting a suspension of the adenoviral vector and / or envelope polypeptide using a needle. Alternatively, a needleless injection device is used to administer the composition (e.g., using a Biojector™) or a lyophilized powder containing the vaccine.

[0072] In one embodiment, the priming immunization and / or the boosting administration, preferably both the priming administration and the boosting administration, further comprises administering one or more adenoviral vectors encoding one or more additional varicella zoster antigens.

[0073] The timing of priming and boosting immunizations is not particularly limited. For example, the vaccine composition can be administered for priming immunization and then re-administered before administering the vaccine composition for boosting immunization. Further administration of the vaccine composition for further boosting immunization is also contemplated. In certain embodiments, the booster vaccine is first administered about 1 to 12 weeks after the initial administration of the primer vaccine, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks later. In another embodiment, the booster vaccine is first administered about 12 to 52 weeks after the initial administration of the primer vaccine, for example, about 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52 weeks later. Those skilled in the art can modify the exact timing, frequency of administration, dosage, etc. of the priming and boosting vaccines based on the teachings herein and general knowledge in the art.

[0074] In one embodiment, a varicella zoster vaccine composition may comprise a first mRNA and a second mRNA described herein formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC and PEG2000-DMG, trisodium citrate buffer, sucrose, and water for injection. As a non-limiting example, the composition may comprise 2.0 mg / mL of the active ingredient (e.g., varicella zoster vaccine compositions (1) and (2)), 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.

[0075] In one embodiment, a method for inducing an immune response against shingles comprises administering an effective amount of the shingles vaccine composition (1) of the present disclosure to a subject in need thereof. In the shingles vaccine composition (1), the mRNA having a 5'UTR-ORF structure encoding VZV gE (soluble)-3'UTR-poly(A) tail may have the nucleotide sequence of SEQ ID NO: 5 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 5).

[0076] In another embodiment, a method for inducing an immune response against shingles comprises administering an effective amount of the shingles vaccine composition (2) of the present disclosure to a subject in need thereof. In the shingles vaccine composition (2), the mRNA having a 5'UTR-ORF structure encoding VZV gE (full-length)-3'UTR-poly(A) tail may have the nucleotide sequence of SEQ ID NO: 6 (or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99% identity to SEQ ID NO: 6).

[0077] 3. Sequence information The specific sequence information of SEQ ID NO: 1 to SEQ ID NO: 8 cited in the present disclosure is as follows:

[0078] SEQ ID NO: 1 MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIP TLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDE LELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMN SGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAA

[0079] 2) Protein sequence of the ORF encoded by full-length VZV gE SEQ ID NO: 2 MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTNSVYEPYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYG DVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGGTST YATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVD TPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRWDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR

[0080] 3) soluble VZV gE mRNA sequence (ORF) sequence number 3

[0081] 4) Full-length VZV gE mRNA sequence (ORF) SEQ ID NO:4

[0082] 5) Soluble VZV gE mRNA sequence (5'UTR-ORF-3'UTR-poly(A) tail) SEQ ID NO:5

[0083] 6) Full-length VZV gE mRNA sequence (5'UTR-ORF-3'UTR-poly(A) tail) SEQ ID NO:6

[0084] 7) pUC57-Kan plasmid encoding soluble VZV gE mRNA (soluble VZV gE mRNA sequence is underlined) SEQ ID NO:7 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGGGCACCGTGAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAGGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCATGATAAAGCTGGAGCCTCGGTGGCCTTGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0085] 8) pUC57-Kan plasmid encoding full-length VZV gE mRNA (full-length VZV gE mRNA sequence is underlined) SEQ ID NO:8 TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGAAATACCGCACAGATGCGTAAGGAGAAAA TACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGAGAATTCGAGCTCGGTACCTCGCGAATACATCTAGATTAATACGACTCACTATA AGGCCGGCACTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCGCCACCATGGGCACCGTGAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAGGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTAAACGAATGAGGGTTAAAGCCTATAGGGTAGACAAGTCCCCGTATAACCAAAGCATGTATTACGCTGGCCTTCCAGTGGACGATTTCGAGGACTCGGAATCTACGGATACGGAAGAAGAGTTTGGTAACGCGATTGGAGGGAGTCACGGGGGTTCGAGTTACACGGTGTATATAGATAAGACCCGGTGATAAAGCTGGAGCCTCGGTGGCCTTGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA [Example]

[0086] Example 1 - DNA templates used for in vitro transcription and protein expression The DNA template sequence for mRNA in vitro transcription (IVT) consisted of a T7 promoter, 5' untranslated region (UTR), a modified glycoprotein E (gE) open reading frame (ORF) of VZV ORF68 from Oka strain DNA (GenBank: AH010548.2), 3' UTR, and 120 bases of polyadenine (polyA). The 5' UTR and 3' UTR were from human hemoglobin subunit α1 (HBA1) mRNA (GenBank: NM_000558.5). For the soluble form of glycoprotein E (gE sol), the transmembrane domain and carboxy terminus were removed from the full-length gE sequence to obtain the secreted form (ΔTrp541-Arg623). All DNA fragments were synthesized by GenScript (Piscataway, NJ) and subcloned into the pUC57-Kan vector. The sequence of the pUC57-Kan plasmid encoding the soluble VZV gE mRNA is shown in SEQ ID NO: 7. The sequence of the pUC57-Kan plasmid encoding the full-length VZV gE mRNA is shown in SEQ ID NO: 8.

[0087] Example 2 - In vitro transcription (IVT) For gE and its soluble forms, the plasmid vectors were linearized with the restriction enzyme BspQI (New England Biolabs). N1-methylpseudouridine (m1Ψ) was purchased from BOC Sciences (Shirley, NY). IVT conditions followed the manufacturer's recommendations (TranscriptAid T7 High Yield Transcription Kit, ThermoFisher) as follows: ATP / CTP / GTP / m1ψTP: 5mM each SmartCap (SC101, ST Pharm): 4mM Linear template DNA: 1 μg of plasmid T7 RNA polymerase enzyme mix: 2 μl

[0088] IVT was performed in 20 μl reactions by incubation for 2 h at 37°C. Template DNA was removed by treatment with 2 units of DNase I (Invitrogen) for 15 min at 37°C, followed by column purification (Monarch RNA Cleanup Kit, New England Biolabs).

[0089] After IVT from a DNA template of VZV gE mRNA, 100 ng of mRNA was run on 1% E-GEL EX agarose in an E-Gel Power Snap Electrophoresis Device (ThermoFisher) (one of three independent IVT products). Analysis of the IVT products of the two VZV gE constructs by agarose gel revealed approximately 2 knt mRNAs for these two mRNAs. See Figure 1. IVT was performed in triplicate.

[0090] Example 3 - Transfection 1 μg of mRNA (synthesized in triplicate) was individually transfected into 293FT cells (Invitrogen) in 12-well plates using 2 μl of Lipofectamine MesseangerMax (Invitrogen) at a 1:2 ratio according to the manufacturer's protocol. After 24 h of transfection, samples were collected from both the medium and cells. Cell lysates were prepared in NP-40 lysis buffer (150 mM sodium chloride / 1% NP-40 / 50 mM Tris (pH 8.0)). As a transfection control, 0.1 μg of EGFP mRNA (L-7601, TriLink) was cotransfected.

[0091] Example 4 - Western Blot Mouse anti-VZV gE monoclonal antibody (#9) was purchased from antibody.com. Protein detection was performed using an HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) and SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Scientific). EGFP was detected with an HRP-conjugated mouse monoclonal antibody (sc-9996, Santa Cruz Biotechnology). Target protein levels were quantified from triplicates by analyzing protein band intensity.

[0092] As shown in Figure 2, gE protein levels were determined by Western blot. 293FT cells were individually transfected with 1 μg of the two gE mRNAs. 24 hours after transfection, both cell lysates and culture medium were collected and subjected to Western blot analysis using a gE-specific antibody. GFP mRNA was cotransfected for normalization, and untransfected 293FT cells served as a negative control (lanes 1 and 3).

[0093] Western blots were performed (three independent replicates) on samples collected from both the medium to detect secreted "soluble" gE protein and from cells to detect intracellular / unsecreted gE protein. As shown in lane 2 of Figure 2, full-length gE protein was detected in the cell lysate but not in the medium (not shown). Conversely, soluble gE protein was detected only in the medium (lane 4) but not in the cell lysate (not shown).

[0094] Example 5 - Immunogenicity Studies This study was designed to test the immunogenicity in mice of the varicella zoster vaccine compositions of the present disclosure (eg, varicella zoster vaccine compositions (1) and (2)).

[0095] Mice were immunized intramuscularly (IM) with 1 μg, 5 μg, or 10 μg of the mRNA formulation of varicella zoster vaccine composition (1) or (2) of the present disclosure (five mice per dose). The vaccine composition of the present disclosure may be chemically modified or unmodified. A total of two immunizations were administered at 3-week intervals (i.e., at weeks 0 and 3), and serum was collected after each immunization on days 20, 41, and 56 until week 8, as shown in Figure 3. Serum antibody titers against VZV gE (soluble) and VZV gE (full-length) were determined by ELISA (AcroBiosystems, RAS-T103). As shown in Figure 4, after the booster immunization, all immunized mice showed high IgG titers against VZV gE (sera at D41 and D56). VZV gE-specific IgG titers of varicella zoster vaccine composition (1) (full-length gE) were higher than those of varicella zoster vaccine composition (2) (soluble gE) at low doses (1 μg), but at higher doses (5 μg and 10 μg), both compositions showed similar titers.

[0096] Example 6 - In vitro neutralization studies This study was designed to test the neutralizing ability of the disclosed varicella zoster vaccine compositions (e.g., varicella zoster vaccine compositions (1) and (2)) against VZV infection in cultured human cell lines. A patient-isolated VZV strain (human herpesvirus 3; ATCC, VR-1367; Ellen strain) was propagated in the human epithelial cell line ARPE-19 (ATCC, CRL-2302). Infected cells were resuspended in PBS-sucrose-glutamate-serum (PSGC; 5% sucrose, 0.1% L-glutamate, and 10% FBS) buffer and sonicated three times for 2 minutes with 15-second intervals (PC3 Ultrasonic Unit, L&R) to release cell-free virus particles. Three volumes of PSGC supernatant containing cell-free VZV were combined with one volume of a Lenti-X concentrator (Takara Bio Inc.) for concentration, followed by centrifugation at 1500 × g for 45 minutes at 4°C. The VZV pellet was resuspended in 1 / 50 of the initial volume. VZV viral titers were determined by infecting ARPE-19 cells with serial dilutions of VZV and measuring gE production using ELISA (AcroBiosystems, RAS-A135).

[0097] To evaluate mouse sera in neutralizing VZV infection, ARPE-19 cells were infected with VZV along with serially diluted mouse sera from zoster compositions (1) and (2). Five days after infection, the amount of gE protein produced by infected cells was measured as a neutralizing titer (NT50) using an ELISA assay (AcroBiosystems, RAS-A135). Figure 5 shows that full-length gE serum (VER-009) elicited higher neutralizing antibodies than soluble gE (VER-008) at all three dose levels. Even at the lowest dose (1 μg), full-length serum was able to elicit potent neutralizing antibodies, while soluble gE required a higher dose for neutralization.

Claims

1. A varicella-zoster vaccine composition comprising a messenger ribonucleic acid (mRNA) containing an open reading frame (ORF) encoding varicella-zoster virus (VZV) glycoprotein E (gE).

2. 2. The varicella-zoster vaccine composition of claim 1, wherein the VZV gE has the amino acid sequence of SEQ ID NO:

1.

3. 2. The varicella-zoster vaccine composition of claim 1, wherein the VZV gE has the amino acid sequence of SEQ ID NO:

2.

4. 3. The varicella-zoster vaccine composition of claim 2, wherein the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO:

3.

5. 4. The varicella-zoster vaccine composition of claim 3, wherein the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO:

4.

6. The mRNA comprising an ORF encoding VZV gE has the following structure: VZV gE-3'UTR-5'UTR-ORF encoding poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the 3. The varicella-zoster vaccine composition of claim 2, wherein the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO:

3.

7. The mRNA comprising an ORF encoding VZV gE has the following structure: VZV gE-3'UTR-5'UTR-ORF encoding poly(A) tail and further comprising a 5' untranslated region (UTR), a 3' UTR, and a poly(A) tail, such that the 4. The varicella-zoster vaccine composition of claim 3, wherein the ORF encoding VZV gE has the nucleotide sequence of SEQ ID NO:

4.

8. 7. The varicella-zoster vaccine composition of claim 6, wherein the poly(A) tail has a length of 50 to 250 nucleotides.

9. 8. The varicella-zoster vaccine composition of claim 7, wherein the poly(A) tail has a length of 50 to 250 nucleotides.

10. The varicella-zoster vaccine composition according to claim 6, wherein the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

5.

11. The varicella-zoster vaccine composition according to claim 7, wherein the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has the nucleotide sequence of SEQ ID NO:

6.

12. The varicella-zoster vaccine composition according to claim 6, wherein the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:

5.

13. The varicella-zoster vaccine composition according to claim 7, wherein the mRNA having a 5'UTR-ORF structure encoding VZV gE-3'UTR-poly(A) tail has a nucleotide sequence having at least 80% identity with SEQ ID NO:

6.

14. The varicella zoster vaccine composition of claim 1, further comprising a pharmaceutically acceptable carrier.

15. 15. The varicella zoster vaccine composition of claim 14, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle that encapsulates the mRNA.

16. A composition for inducing an immune response against shingles, comprising the shingles vaccine composition of claim 1.

17. A composition for inducing an immune response against shingles, comprising the shingles vaccine composition of claim 12.

18. A composition for inducing an immune response against shingles, comprising the shingles vaccine composition of claim 13.

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