Varicella-zoster virus mRNA vaccine and application thereof

Abstract

The present invention provides novel variants of varicella-zoster virus gE protein, mRNA vaccines encoding the gE variants or gE variant fusion proteins. The mRNA vaccine based on the gE variant or the gE variant fusion protein can induce high-level IgG antibody reaction, T cell immune response and T cell immune memory.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to varicella-herpes virus mRNA vaccines and their applications. Background Technology

[0002] The varicella-zoster virus (VZV) can cause two different diseases at different ages in humans: chickenpox and shingles. Chickenpox is the symptom that appears after primary VZV infection, characterized by a generalized rash or blisters, and mostly occurs in infancy and childhood. After recovery from the initial viral infection, the virus remains latent in areas such as the spinal ganglia. With age and decreased immunity, VZV is reactivated, causing clustered blisters and localized nerve pain, affecting the skin of the chest and waist, a condition known as shingles. Chickenpox in children is generally self-limiting (except in immunocompromised children) and the symptoms are relatively mild. However, chickenpox in adults is very severe; 20%–30% of cases can develop viral pneumonia, with a high mortality rate. The global incidence of shingles in the general population is 3–5 per 1,000 person-years. The incidence is 6–8 per 1,000 person-years in people over 60 years old, and can reach 8–12 per 1,000 person-years in people over 80 years old. According to data from the U.S. Centers for Disease Control and Prevention (CDC), an estimated one-third of people in the United States will have shingles at some point in their lives, with nearly one million cases of shingles occurring each year.

[0003] Shingles has two prominent characteristics: a high rate of transmission and immense suffering for patients. Statistics show that the annual incidence of shingles is approximately 2.0‰ to 5.0‰. Shingles flare-ups are extremely painful, sometimes even more so than childbirth, and often result in prolonged postherpetic neuralgia. More than half of patients experience neuralgia for about a month after recovery, and over 20% suffer from neuralgia for well over six months, earning it the nickname "the cancer that doesn't kill." With the aging population becoming increasingly prevalent globally, research on the varicella-zoster virus is being prioritized in more and more countries.

[0004] Currently, three shingles vaccines have been approved for marketing globally: Merck's Zostavax, GSK's Shingrix, and BaiKe Bio's live attenuated vaccine, Ganwei. Zostavax offers approximately 70% protection for people aged 50-59, 60-69, and over 70 years old, respectively. However, the production and storage of high-titer live attenuated shingles vaccines are technically challenging, making the manufacturing process difficult. Shingrix, a protein subunit vaccine, uses conserved viral glycoprotein gE expressed in Chinese hamster ovary cells (CHO) as an antigen. The adjuvant AS01B effectively enhances the specific cellular immune response against VZV-gE, achieving a protection rate of over 90% in all age groups over 60 years old in clinical trials. However, the key component of AS01B, QS21, cannot be artificially synthesized, presenting limitations in its availability and significant manufacturing challenges.

[0005] Therefore, how to further reduce the effectiveness of varicella vaccination, reduce the risk of shingles, and further enhance the response cycle of cellular immune response are the problems that VZV vaccines need to solve at this stage. Summary of the Invention

[0006] This invention is partly based on the inventors’ findings that designing and screening gE protein variants and mRNA vaccines based on gE variants or gE variant fusion proteins can induce high levels of IgG antibody responses, T cell immune responses and T cell immune memory.

[0007] The first aspect of the present invention provides a varicella-zoster virus (VZV) gE protein variant, wherein the gE protein variant has a mutation at one or more of the following amino acid positions 619, 437, 560, 591, 599, 266, 438, 441, 540, 546, 555, and 569 relative to the parental gE protein.

[0008] In one embodiment, the amino acid position is determined according to the amino acid sequence number shown in SEQ ID NO: 93, and the parent gE protein has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to that of SEQ ID NO: 6 or 93.

[0009] In one embodiment, the NCBI accession number of the parent gE protein is UniProtKB:Q9J3M8.1.

[0010] In one embodiment, the gE protein variant has one or more mutations at one or more of the parental gE protein, namely I619, N437, T560, E591, E599, N266, Y438, Y441, A540, A546, I555, and Y569.

[0011] In one embodiment, the gE protein variant has one or more mutations among I619S, N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A relative to the parental gE protein.

[0012] In one embodiment, the gE protein variant has the I619S mutation relative to the parental gE protein and one or more mutations selected from N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A.

[0013] In one embodiment, the gE protein variant has an N437S mutation relative to the parental gE protein and one or more mutations selected from I619S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A.

[0014] In one embodiment, the gE protein variant has the following mutation combinations relative to the parental gE protein: 1) T560S, E591S, E599S, I619S; 2) N266S, N437S, Y438S, Y441S; or 3) A540S, A546S, I555S, Y569A.

[0015] In one embodiment, the gE protein variant has an amino acid sequence shown in any one of SEQ ID NO: 8-22 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 8-22.

[0016] A second aspect of the present invention provides a fusion protein comprising a variant of the gE protein described in the first aspect of the present invention.

[0017] In one embodiment, the fusion protein further comprises a signal peptide sequence selected from IL-2 signal peptide, tPA signal peptide, IL-10 signal peptide, Ig kappa signal peptide, and gE signal peptide. In one embodiment, the signal peptide is located at the N-terminus of the fusion protein.

[0018] In one embodiment, the fusion protein has a signal peptide selected from the amino acid sequences shown in any one of SEQ ID NO:1-5 or a signal peptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with any one of SEQ ID NO:1-5.

[0019] In one embodiment, the gE protein variant in the fusion protein is fused with other antigenic peptides and / or helper epitopes of VZV.

[0020] In one embodiment, the other antigenic polypeptide is selected from one or more fusions of VZV gI, gB, gH, gK, gL, gC, gN, gM or antigenic fragments thereof.

[0021] In one embodiment, the auxiliary epitope is selected from PADRE or IL-1β epitopes.

[0022] In one embodiment, the auxiliary epitope is selected from the amino acid sequence shown in any one of SEQ ID NO:80-81 or has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:80-81.

[0023] In one embodiment, the gE protein variant is fused directly or indirectly with other antigenic peptides and / or helper epitopes.

[0024] In one embodiment, the gE protein variant is indirectly linked to other antigenic peptides and / or helper epitopes via linkers.

[0025] In one embodiment, the connector is a shearable connector, a flexible connector, or a rigid connector.

[0026] In one embodiment, the cleavable linker is selected from P2A peptide, E2A peptide, or F2A peptide.

[0027] In one embodiment, the flexible connector is (GGGGS)n, where n = 1-6, preferably 2, 3 or 4, and more preferably 3.

[0028] In one embodiment, the other antigenic peptide is located at the N-terminus or C-terminus of the gE protein variant.

[0029] In one embodiment, the gE protein variant is fused with VZV gI.

[0030] In one embodiment, the gI protein or its antigenic fragment has the amino acid sequence shown in SEQ ID NO:23 and 24 or has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and 24.

[0031] In one embodiment, the fusion protein of gE and gI has an amino acid sequence shown in any one of SEQ ID NO: 89-92 or has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO: 89-92.

[0032] A third aspect of the present invention provides a nucleic acid comprising a polynucleotide of a protein variant described in the first aspect of the present invention or a fusion protein described in the second aspect of the present invention.

[0033] In one implementation, the nucleic acid is DNA or RNA.

[0034] In one embodiment, the RNA is mRNA, circular RNA, or self-replicating RNA.

[0035] In one embodiment, the RNA further comprises a 5'UTR, a KOZAK, a 3'UTR, a polyA, and a cap structure.

[0036] In one embodiment, the RNA is modified RNA, wherein uracil, cytosine, adenine, or guanine nucleotides contain modifying groups.

[0037] In one embodiment, the modifying group is selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, and isoguanine.

[0038] In one embodiment, the mRNA molecule is a modified mRNA, the modification including the conversion of uracil nucleoside to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, or 5-methoxyuridine; and / or, the conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, or N1-methylcytosine; and / or, the conversion of adenine nucleoside to N1-methyladenosine; and / or, the conversion of adenine nucleoside to N1-methylguanine, N1-methylguanine, or isoguanine.

[0039] In one embodiment, the nucleic acid comprises a polynucleotide encoding a protein containing any of the amino acids shown in SEQ ID NO: 8-24.

[0040] In one implementation, the nucleic acid is mRNA.

[0041] In one embodiment, the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 27-43 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 27-43.

[0042] In one embodiment, the mRNA further comprises a 5'UTR as shown in any one of SEQ ID NO: 44, 46, 48, 50 or a 5'UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 44, 46, 48, 50.

[0043] In one embodiment, the mRNA further comprises a 3'UTR as shown in any one of SEQ ID NO: 45, 47, 49 or 51; or a 3'UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 45, 47, 49 or 51.

[0044] In one embodiment, the mRNA further comprises a polyA with a length of 60-150 nt, preferably 80-120 nt.

[0045] In one embodiment, the polyA is as shown in SEQ ID NO: 52 or 53.

[0046] In one embodiment, the mRNA further comprises a KOZAK sequence.

[0047] In one embodiment, the mRNA further includes a 5' cap structure.

[0048] In one embodiment, the 5' cap has the structure of a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof:

[0049]

[0050] in,

[0051] --- indicates a single key or that the key does not exist.

[0052] X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2.

[0053] CH2CH2O, OCH2CH2, three-membered cycloalkyl group,

[0054] R1, R2, R3, and R4 are independently halogenated, OH-, unsubstituted, or OC-substituted, respectively. 1-3 Alkyl-substituted OC 1-3 Alkyl, unsubstituted or OC 1-3 Alkyl-substituted OC 1-3 alkyl,

[0055] B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases.

[0056] In one embodiment, the compound of formula (I) is any one of the following:

[0057]

[0058]

[0059] In one embodiment, the nucleic acid comprises the nucleotide sequence described in any one of SEQ ID NO: 54-79 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 54-79.

[0060] A fourth aspect of the present invention provides an expression vector comprising the nucleic acid described in the third aspect of the present invention.

[0061] The fifth aspect of the present invention provides a cell that includes the expression vector described in the fourth aspect of the present invention.

[0062] The sixth aspect of the present invention provides a method for preparing the gE protein variant of the first aspect of the present invention or the fusion protein of the second aspect of the present invention, the method comprising culturing the cells of the third aspect of the present invention under suitable culture conditions (e.g., in a suitable culture medium, for example, conditions that allow the gE protein variant of the first aspect of the present invention or the fusion protein of the second aspect of the present invention to be expressed).

[0063] In one embodiment, the method further includes steps of protein isolation and purification.

[0064] The seventh aspect of the present invention provides a composition comprising the gE protein variant described in the first aspect of the present invention, the fusion protein described in the second aspect, the nucleic acid described in the third aspect, and / or the vector described in the fourth aspect.

[0065] In one embodiment, the composition comprises an mRNA molecule containing the nucleotide sequences described in any one or more of SEQ ID NO: 54-79.

[0066] In one embodiment, the mRNA molecule is delivered using lipid nanoparticles (LNPs).

[0067] In one embodiment, the liposome nanoparticles comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000.

[0068] In one embodiment, the composition is a vaccine.

[0069] In one embodiment, the composition is formulated into a dosage form suitable for intramuscular or subcutaneous injection, oral administration, or inhalation.

[0070] A sixth aspect of the present invention provides the use of the gE protein variant described in the first aspect, the fusion protein described in the second aspect, the nucleic acid described in the third aspect, the vector described in the fourth aspect, and / or the composition described in the fifth aspect in the preparation of a medicament for the prevention or treatment of varicella-zoster virus infection. Preferably, the medicament is a vaccine.

[0071] The eighth aspect of the invention provides a method for preventing or treating varicella-zoster virus infection or disease caused by varicella-zoster virus infection in a subject, comprising administering to the subject the gE protein variant described in the first aspect of the invention, the fusion protein described in the second aspect, the nucleic acid described in the third aspect, the carrier described in the fourth aspect, and / or the composition described in the fifth aspect.

[0072] In one implementation, the subjects are mammals.

[0073] In one implementation, the subject is a human being.

[0074] In one implementation, it is administered via intramuscular or subcutaneous injection, oral administration, or inhalation.

[0075] A ninth aspect of the present invention provides a method for inducing an immune response in a subject, comprising administering to the subject the gE protein variant of the first aspect of the present invention, the fusion protein of the second aspect, the nucleic acid of the third aspect, the carrier of the fourth aspect, and / or the composition of the fifth aspect.

[0076] In one embodiment, the induction of an immune response includes inducing gE protein-binding antibody production and / or inducing a T-cell immune response.

[0077] In the various embodiments described herein, the disease caused by varicella-zoster virus infection may be selected from varicella and shingles.

[0078] The main advantages of this invention are: the invention designs and screens novel varicella-zoster virus gE protein variants, and the mRNA vaccine based on the gE protein variant induces IgG antibody response levels and T cell immune response levels that are superior to existing VZV protein vaccines and mRNA vaccines, and can induce effective T cell immune memory. Detailed Implementation

[0079] The following definitions are provided to enable those skilled in the art to understand the invention. Unless otherwise defined, 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 invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0080] 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. For example, terms used herein include those in Janeway CA Jr, Travers P, Walport M, et al., *Immunobiology*, 5th edition, New York: Garland Science (2001), and “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, HGW, Nagel B., and… The definition is given in H. (ed., 1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland.

[0081] The term "varicella-zoster virus" or "VZV" refers to the virus that causes chickenpox in children, remains latent in the body after recovery, and causes shingles in some patients as adults. VZV has only one serotype, and its genome contains 71 genes that encode 67 different proteins, including glycoproteins gE, gB, gH, gI, gC, and gL.

[0082] The term "protein variant" refers to a compound that has sequence homology with a wild-type / parental protein or polypeptide. Protein variants described in this application may include proteins with altered amino acid sequences obtained through insertion, deletion, modification, and / or substitution of one or more amino acid residues, while retaining at least one biological activity (e.g., immunogenicity) of the parental wild-type sequence. For example, the variant may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parental protein. Insertions, deletions, or substitutions in the variant may be conserved or non-conserved. The variant may be naturally occurring or non-naturally occurring. Non-naturally occurring variants can be generated using techniques known in the art. In this application, "gE protein variant" may refer to a protein obtained by substituting one or more amino acids based on a wild-type gE protein.

[0083] The term "mature protein" refers to a protein in its final form after translation and any post-translational modifications such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one embodiment, the mature polypeptide corresponds to the amino acid sequence having SEQ ID NO:6. In one embodiment, the mature polypeptide has the amino acid sequence having SEQ ID NO:6.

[0084] The term "fusion protein" refers to a protein formed by covalently linking two protein motifs that do not exist together under natural conditions. In this paper, "fusion protein" can be a fusion protein of gE from VZV with one or more of gI, gB, gH, gK, gL, gC, gN, and gM. For example, it could be a gE-gI fusion protein. Fusion proteins may further include auxiliary epitopes. gE can be located at the N-terminus or C-terminus of the fusion protein.

[0085] The term "signal peptide" refers to a peptide used to guide the translocation of a synthesized fusion protein into the secretory pathway. Signal peptides are generally essential for transmembrane translocation in the secretory pathway and thus universally control the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides ribosomes to the rough endoplasmic reticulum (ER) membrane and triggers the transport of the growing peptide chain across this membrane for processing. ER processing produces a mature protein, in which the signal peptide is typically cleaved from the precursor protein by the host cell's ER-resident signal peptidase, or remains unclew and acts as a membrane anchor. Signal peptides can also promote protein targeting to the cell membrane. Secretory signal peptides can be located at the N-terminus of the fusion protein. The type of secretory signal peptide is not particularly limited, as long as it can guide the secretion of the synthesized fusion protein. Secretory signal peptides can include, but are not limited to, IgG signal peptides, IL-2 signal peptides, tPA signal peptides, Ig kappa signal peptides, and SigMHC signal peptides.

[0086] The term "helper epitope" refers to all epitopes that can assist T cell activation, including PADRE and IL-1β epitopes. PADRE is a short peptide sequence of 13 amino acids that can bind to different DR molecules, be presented on the cell surface, and thereby activate CD4+ T helper cells. PADRE's ability to induce T cell responses is more than 1000 times that of natural epitopes. In vivo, PADRE can immunely activate helper T cells (Th1) to assist CTL activation and can also activate helper T cells (Th2) to assist B cells in secreting specific antibodies. The IL-1β epitope is a short linear peptide sequence of 9 amino acids. Interleukin-1 plays an important role in enhancing cellular and humoral immune responses to foreign antigens, and the IL-1β epitope can enhance immune responses without producing adverse inflammatory effects.

[0087] The terms “nucleic acid,” “polynucleotide,” and “polynucleotide sequence” are used interchangeably to refer to oligomers and polymers of any length that are essentially composed of nucleotides (such as deoxyribonucleotides and / or ribonucleotides). Nucleic acids may contain purine and / or pyrimidine bases and / or other natural (e.g., xanthine, inosine, hypoxanthine), chemically or biochemically modified (e.g., methylated), non-natural, or derived nucleotide bases. The backbone of a nucleic acid may contain sugar and phosphate groups normally present in RNA or DNA, and / or one or more modified or substituted sugars and / or one or more modified or substituted phosphate groups. Modifications to phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful properties. A “nucleic acid” can be, for example, double-stranded, partially double-stranded, or single-stranded. When single-stranded, a nucleic acid can be a sense strand or an antisense strand. A “nucleic acid” can be circular or linear. As used herein, the term “nucleic acid” encompasses DNA and RNA, including genomes, pre-mRNA, mRNA, cDNA, and recombinant or synthetic nucleic acids containing vectors. For the purposes described herein, it should be understood that polynucleotides can be modified by any method available in the art.

[0088] The terms “non-natural” or “engineered” refer to proteins, peptides, nucleic acids, or strains that are not normally present in nature, including at least one modified form.

[0089] The term "isolated nucleic acid" refers to nucleic acid fragments that are not naturally occurring as fragments and do not exist in their natural state. The term "isolated" is also used to refer to polypeptides, proteins, and / or host cells isolated from other cellular proteins, and is intended to include purified and recombinant polypeptides. In other embodiments, the term "isolated" means isolated from components, cells, and other substances that are normally naturally associated with cells, tissues, polynucleotides, peptides, polypeptides, or proteins. "Non-naturally occurring" polynucleotides, peptides, polypeptides, or proteins do not need to be "isolated" to distinguish them from their naturally occurring counterparts.

[0090] The term "mRNA" refers to messenger RNA, a transcript that can be transcribed using a DNA template and may encode a peptide or protein. mRNA is typically single-stranded but may also contain its own complementary sequence. Generally, mRNA contains a 5' cap, a 5'-UTR, a coding region, a 3'-UTR, and a polyA tail. In the context of this invention, mRNA is preferably produced from a DNA template via in vitro transcription (IVT). In vitro transcription methods are known to those skilled in the art, and various in vitro transcription kits are commercially available.

[0091] The term "sequence identity" refers to the degree to which two sequences (amino acid sequences) have identical residues at the same positions when aligned. Such calculations are typically performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN (Myers and Miller, 1988), FASTA (Pearson and Lipman, 1988; Pearson, 1990), and gapped BLAST (Altschul et al., 1997), BLASTP, BLASTN, or GCG (Devereux et al., 1984). Furthermore, in determining the degree of sequence identity between two amino acid sequences, those skilled in the art may consider so-called "conserved" amino acid substitutions, which can generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, having little or no effect on the function, activity, or other biological properties of the polypeptide. Such conserved amino acid substitutions are well known in the art.

[0092] The term “identity” when used in conjunction with nucleic acids or fragments thereof means that, when an optimized alignment is performed with other nucleic acids (or their complementary strands), at least 50%, 60%, 70%, 80%, 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases have nucleotide sequence identity, as determined by any sequence identity algorithm well known in the art (such as FASTA, BLAST, or GAP) discussed below.

[0093] The term "read frame" (ORF) is a continuous extension of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). ORFs typically encode proteins. In this paper, a read frame may encode a fusion protein.

[0094] The term "5' untranslated region" (5'UTR) refers to an mRNA region located directly upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) and that does not encode a protein or peptide. When an RNA transcript is generated, the 5'UTR may contain promoter sequences. These promoter sequences are known in the art. It should be understood that these promoter sequences will not be present in the mRNA described herein.

[0095] The term "3' untranslated region" (3'UTR) refers to an mRNA region located directly downstream (i.e., 3') of a stop codon (i.e., the codon that transmits the translation termination signal in the mRNA transcript) and that does not encode a protein or peptide.

[0096] The term "Poly-A" refers to an mRNA region containing multiple consecutive adenosine monophosphates (ATPs) located downstream of the 3' UR. For example, a Poly-A tail can be located directly downstream of the 3' UR (i.e., 3'). A Poly-A tail can contain 10 to 300 ATPs, such as 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ATPs. The Poly-A tail can be used to protect mRNA, for example, from enzymatic degradation in the cytoplasm, and facilitates transcription termination and / or the export of mRNA from the nucleus for translation.

[0097] The term "KOZAK" refers to a nucleic acid sequence, usually GCCACC, located after the 5' cap structure of eukaryotic mRNA. It can bind to translation initiation factors and mediate the translation initiation of mRNA containing the 5' cap structure.

[0098] The term "ionizable lipid" refers to an amphiphilic molecule (e.g., a lipid or lipidoid, such as a synthetic lipid or lipidoid) containing a group (e.g., a head group) that can ionize, for example, dissociate under given conditions (e.g., pH) to produce one or more charged substances. In some embodiments, the ionizable lipid is SM-102.

[0099] The term "pharmaceutically acceptable" means a molecule or composition that, when administered to a recipient, is harmless to the recipient or whose benefits to the recipient outweigh any adverse effects. Regarding carriers or excipients used to formulate compositions as disclosed herein, pharmaceutically acceptable carriers or excipients must be compatible with the other components of the composition and be harmless to the recipient or whose benefits to the recipient outweigh any adverse effects.

[0100] The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, magnesium talc, calcium stearate, or zinc or stearic acid), or solvent encapsulating material, which participates in carrying or transporting a drug from one part of the body to another (e.g., from one organ to another). Each carrier must be "acceptable" in the sense that it is compatible with other components of the formulation and is not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) excipients, such as cocoa butter and suppository waxes; (8) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (9) glycols, such as propylene glycol; and (10) polyols, such as glycerol and sorbitol. (11) Mannitol and polyethylene glycol (PEG); (12) Esters, such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) Pyrothermic water; (17) Isotonic saline; (18) Ringer's solution; (19) pH buffer solution; (20) Polyesters, polycarbonates and / or polyanhydrides; (21) Fillers, such as peptides and amino acids; (22) Serum components, such as serum albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) Other non-toxic and compatible substances used in pharmaceutical formulations. In the context of this document, those skilled in the art may select a carrier suitable for acceptance on a pharmaceutically acceptable protein or nucleic acid as described herein.

[0101] The term "adjuvant" refers to a nonspecific immunoproliferator, an auxiliary substance that, when injected into the body along with or beforehand, enhances the body's immune response to the antigen or alters the type of immune response. Adjuvants may or may not be immunogenic. There are many types of adjuvants, and there is no unified classification method. The most commonly used are Freund's adjuvants and cytokine adjuvants. The immunobiological effects of adjuvants include enhancing immunogenicity, increasing antibody titers, altering the type of antibody produced, and inducing or enhancing delayed-type hypersensitivity reactions. "Adjuvants" are classified into biological adjuvants, inorganic adjuvants, synthetic adjuvants, oil-based adjuvants, and Freund's adjuvants. Biological adjuvants include bacteria or their products, which are themselves immunogenic, such as mycobacteria, Bordetella pertussis, Corynebacterium pumilus, and Gram-negative bacillus endotoxins. Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 (IL-1), interleukin-2 (IL-2), and interferon-γ (IFN-γ) also have adjuvant activity. Inorganic adjuvants include aluminum hydroxide, aluminum phosphate, and alum. Synthetic adjuvants include double-chain polyinosinic acid, cytidine monophosphate, and double-chain polyadenylate. Oil-based adjuvants include peanut oil emulsifying adjuvants, mineral oil, vegetable oil, and lanolin. Freund's adjuvants are further divided into incomplete and complete Freund's adjuvants, and are the most common adjuvants used in animal experiments. However, they are prone to forming granulomas and persistent ulcers at the injection site and are not suitable for human use.

[0102] gE variant protein

[0103] This document provides VZV gE protein variants that have one or more amino acid mutations relative to the parental gE protein. The mutation sites are selected from one or more of I619, N437, T560, E591, E599, N266, Y438, Y441, A540, A546, I555, and Y569. Those skilled in the art will understand that the parental gE protein can be any suitable gE protein. For example, the parental gE protein can be a wild-type gE protein or a gE protein variant existing in the art. For the purposes of this invention, the amino acid sequence of SEQ ID NO:93 is used to determine the corresponding amino acid residues in another VZV gE protein. The amino acid sequence of another VZV gE protein was aligned with SEQ ID NO:93, and based on this alignment, the Niederman-Onsch algorithm (Niederman and Onsch, 1970, Journal of Molecular Biology 48:443-453) implemented in the Nieder program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000) (preferably version 5.0.0 or higher) was used to determine the amino acid position number corresponding to any amino acid residue in the mature polypeptide in SEQ ID NO:93. For example, parameters that can be used are a nick opening penalty of 10, a nick extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. Those skilled in the art will also understand that another VZV gE protein can be numbered based on the mature protein (SEQ ID NO:6) in SEQ ID NO:93 (i.e., by subtracting the signal peptide length of 30 from the position determined by SEQ ID NO:93).

[0104] In some embodiments, the location of the mutation is determined according to the amino acid sequence number shown in SEQ ID NO: 93.

[0105] In some embodiments, the parental gE protein is a full-length wild-type gE protein.

[0106] In some embodiments, the wild-type gE protein accession number is UniProtKB:Q9J3M8.1.

[0107] In some embodiments, the parental gE protein is a mature wild-type gE protein that does not contain a signal peptide.

[0108] In some embodiments, the parent gE protein has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to SEQ ID NO: 6 or 93.

[0109] In some embodiments, the gE protein variant has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to SEQ ID NO: 6 or 93.

[0110] In some embodiments, the mutation is selected from one or more combinations of I619S, N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A.

[0111] In some embodiments, the gE protein variant described herein has an I619S mutation relative to the wild-type gE protein, and also includes one or more mutations selected from N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A.

[0112] In some embodiments, the gE protein variant described herein has an N437S mutation relative to the wild-type gE protein, and also includes one or more mutations selected from I619S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A.

[0113] In some embodiments, the gE protein variants described herein have mutant combinations relative to the wild-type gE protein: 1) T560S, E591S, E599S, I619S; 2) N266S, N437S, Y438S, Y441S; or 3) A540S, A546S, I555S, Y569A.

[0114] Wild-type gE protein (31-623aa)

[0115] The wild-type gE protein contains the amino acid sequence shown in SEQ ID NO: 6, while SEQ ID NO: 6 is the mature gE protein without a signal peptide.

[0116]

[0117] Wild-type gE protein signal peptide

[0118] In some embodiments, the N-terminus of the wild-type gE protein also has the natural gE signal peptide of SEQ ID NO: 5.

[0119]

[0120] Wild-type gE protein full length

[0121] In some embodiments, the wild-type gE protein has the amino acid sequence shown in SEQ ID NO: 93.

[0122]

[0123] The underlined portion in the italics represents the natural gE signal peptide.

[0124] In some embodiments, the gE protein variants described herein include gE-A (31-623AA, A540, A546, I555, Y569), gE-B (31-623AA, N266S, N437S, Y438S, Y441S) and gE-C (31-623AA, T560S, E591S, E599S, I619S).

[0125] The gE-A described herein includes the amino acid sequence shown in SEQ ID NO:20.

[0126]

[0127] The gE-B described herein includes the amino acid sequence shown in SEQ ID NO:21.

[0128]

[0129] The gE-C described in this article includes the amino acid sequence shown in SEQ ID NO:22.

[0130]

[0131] In some embodiments, the gE protein variant described herein has an alternative signal peptide sequence relative to the wild-type gE protein. The native gE signal peptide (SEQ ID NO: 5) at positions 1-30 of the gE protein is replaced with an IL-2 signal peptide (SEQ ID NO: 1), a tPA signal peptide (SEQ ID NO: 2), an IL-10 signal peptide (SEQ ID NO: 3), or an Ig kappa signal peptide (SEQ ID NO: 4).

[0132] IL-2 signal peptide: MYRMQLLSCIALSLALVTNS (SEQ ID NO: 1).

[0133] tPA signal peptide: MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 2).

[0134] IL-10 signal peptide: MHSSALLCCLVLLTGVRA (SEQ ID NO: 3).

[0135] Ig kappa signal peptide: METDTLLLWVLLLWVPGSTGD (SEQ ID NO: 4).

[0136] In some embodiments, the IL-2 signal peptide of SEQ ID NO: 1 is fused with any of the polypeptides shown in SEQ ID NO: 8-22 to generate a gE protein variant.

[0137] In some implementations, the gE protein variant described herein is further fused with an auxiliary epitope.

[0138] In some embodiments, the auxiliary epitope is PADRE (SEQ ID NO:80) or IL-1β epitope (SEQ ID NO:81).

[0139] PADRE epitope: AKFVAAWTLKAAA (SEQ ID NO:80)

[0140] IL-1β epitope: VQGEESNDK (SEQ ID NO:81).

[0141] In some embodiments, the auxiliary epitope is fused to a gE protein variant via a linker peptide. The linker peptide is selected from P2A, E2A, or F2A peptides.

[0142] Fusion protein

[0143] This article provides fusion proteins containing gE protein variants.

[0144] The fusion protein may contain a signal peptide sequence selected from IL-2 signal peptide (SEQ ID NO: 1), tPA signal peptide (SEQ ID NO: 2), IL-10 signal peptide (SEQ ID NO: 3), Ig kappa signal peptide (SEQ ID NO: 4) and gE signal peptide (SEQ ID NO: 5).

[0145] In this study, the gE protein variant was fused with other antigenic peptides of VZV.

[0146] In some embodiments, the other antigenic polypeptides are selected from one or more of VZV gI, gB, gH, gK, gL, gC, gN, gM, or antigenic fragments thereof. The antigenic fragments may be antigenic fragments of VZV gI, gB, gH, gK, gL, gC, gN, or gM known in the art.

[0147] In some embodiments, the gE protein variant is fused directly or indirectly with other antigenic peptides.

[0148] In some embodiments, the gE protein variant is indirectly linked to other antigenic peptides via linkers.

[0149] In some embodiments, the gE protein variant is fused with the VZV gI protein or an antigenic fragment thereof.

[0150] In some embodiments, the VZV gI protein has the amino acid sequence shown in SEQ ID NO:23. In some embodiments, the antigenic fragment of the VZV gI protein comprises the amino acid sequence of SEQ ID NO:24.

[0151] gIWT(1-354aa)

[0152]

[0153] gI truncated form (1-274aa)

[0154]

[0155] In some embodiments, the fusion protein has the amino acid sequence shown in any one of SEQ ID NO: 89-92.

[0156] gE-B+gI full-length fusion protein :

[0157]

[0158] gE-B+gI truncation of fusion protein

[0159] gE-C+gI full-length fusion protein

[0160] gE-C+gI cleavage of fusion protein

[0161] GSHGGSSYTVYSDKTRATNNFSLLKQAGDVEENPGPMMFLIQCLISAVIFYIQVTNALIFKG

[0162] DHVSLQVNSSLTSILIPMQNDNYTEIKGQLVFIGEQLPTGTNYSGTLELLYADTVAFCF

[0163] RSVQVIRYDGCPRIRTSAFISCRYKHSWHYGNSTDRISTEPDAGVMLKITKPGINDAGV

[0164] YVLLVRLDHSRSTDGFILGVNVYTAGSHHNIHGVIYTSPSLQNGYSTRALFQQARLCD

[0165] LPATPKGSGTSLFQHMLDLRAGKSLEDNPWLHEDVVTTETKSVVKEGIENHVYPTDMSTLPEKSLNDPPENLL(SEQ ID NO:92)

[0166] Nucleic acid

[0167] This article provides an isolated nucleic acid, which is a DNA or RNA molecule, and may be double-stranded, single-stranded, or partially double-stranded. The isolated nucleic acid encodes the fusion protein described herein. The isolated nucleic acid may contain elements that regulate the expression of the fusion protein, such as enhancer, promoter, and / or terminator sequences. These sequences may be modified or unmodified. Elements regulating the expression of the fusion protein may also not be present in the isolated nucleic acid.

[0168] The nucleic acids of particular interest in this invention are mRNA molecules, such as those containing a read frame encoding an immunogenic protein.

[0169] mRNA molecules

[0170] Messenger RNA (mRNA) is any RNA that encodes a protein and can be translated to produce the protein-encoded RNA in vitro, in vivo, in situ, or ex vivo. Unless otherwise stated, the nucleic acid sequences in this application may be described as “T” in a representative DNA sequence, but in the case of sequences representing RNA (e.g., mRNA), “T” will be replaced by “U”. Therefore, any DNA indicated herein by sequence number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each “T” in the DNA sequence is replaced by a “U”.

[0171] mRNA molecules can be synthetic and modified. mRNA can be chemically modified. mRNA molecules can be chemically synthesized or transcribed in vitro. mRNA molecules can be placed on a vector. The vector can be a viral vector, a bacterial vector, or a eukaryotic expression vector. In one embodiment, the vector is a plasmid. In some instances, mRNA molecules can be delivered to cells via transfection, electroporation, or transduction (e.g., adenovirus or lentivirus transduction).

[0172] Chemical modification

[0173] In some embodiments, the nucleic acid (e.g., mRNA) comprises RNA having a read frame encoding an immunogenic protein, wherein the nucleic acid comprises nucleotides and / or nucleosides that may be standard (unmodified) or modified as known in the art. In some embodiments, the nucleotides and nucleosides of the nucleic acid (e.g., mRNA) comprise modified nucleotides or nucleosides. These modified nucleotides and nucleosides may be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. These modifications may include modifications at the sugar, backbone, or nucleobase portions of the nucleotides and / or nucleosides as recognized in the art.

[0174] In some implementations, the nucleic acid (e.g., mRNA) may comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

[0175] In some embodiments, nucleic acids (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) comprise different types of standard and / or modified nucleotides and nucleosides. In some embodiments, specific regions of the nucleic acid contain one, two, or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0176] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms exhibit reduced degradation in cells or organisms, relative to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0177] In some implementations, modified RNA nucleic acids (e.g., modified mRNA nucleic acids) introduced into cells or organisms may exhibit reduced immunogenicity (e.g., reduced innate response) in cells or organisms, relative to unmodified nucleic acids containing standard nucleotides and nucleosides.

[0178] In some implementations, nucleic acids (e.g., mRNA) comprise non-natural modified nucleotides introduced during or after nucleic acid synthesis to achieve a desired function or property. Modifications can be present at nucleotide linkages, purine or pyrimidine bases, or sugars. Modifications can be introduced chemically or at any other location at the end of the chain or in the chain using polymerases. Any region of the nucleic acid can be chemically modified.

[0179] Nucleic acids (e.g., mRNA) can contain modified nucleosides and nucleotides. A “nucleoside” is a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate ester group. Modified nucleotides can be synthesized by any useful method, such as chemical, enzymatic, or recombinant methods, to include one or more modified or non-natural nucleosides. Nucleic acids can contain one or more linked nucleoside regions. These regions can have variable backbone linkages. The linkage can be a standard phosphodiester linkage, in which case the nucleic acid will contain the nucleotide region.

[0180] Modified nucleotide base pairings encompass not only standard adenosylthymine, adenosyluracil, or guanosine cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases. In nucleic acids, for example, those with at least one chemical modification, the arrangement of hydrogen bond donors and acceptors allows hydrogen bonding to occur between non-standard and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil.

[0181] In some embodiments, the nucleic acid (e.g., mRNA) contains uridine at one or more or all uridine sites. In some embodiments, the mRNA is uniformly modified (e.g., completely modified, modified throughout the entire sequence) for a specific modification. In some embodiments, the nucleic acid can be uniformly modified with methylpseuuridine, meaning that all uridine residues in the mRNA sequence are replaced with methylpseuuridine. Similarly, the nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacing it with modified residues (e.g., the modified residues described above).

[0182] In some embodiments, the RNA is modified RNA, wherein the uracil, cytosine, adenine, or guanine nucleotide contains a modifying group. The modifying group may be selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, and isoguanine.

[0183] In some embodiments, the mRNA molecule is a modified mRNA, the modification including the conversion of uracil nucleoside to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 5-methoxyuridine; and / or, the conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine; and / or, the conversion of adenine nucleoside to N1-methyladenosine; and / or, the conversion of adenine nucleoside to N1-methylguanine, N1-methylguanine, isoguanine.

[0184] mRNA molecule preparation methods

[0185] Methods for preparing and purifying mRNA molecules are known and disclosed in the art. mRNA molecules can be prepared using only in vitro transcription (IVT) enzymes. Methods for preparing IVT polynucleotides are known in the art and described in WO 2013 / 151666, WO 2013 / 151668, etc. Purification methods include purifying RNA transcripts including polyA tails by contacting a sample with a surface linked to multiple thymidines or their derivatives and / or multiple uracils or their derivatives (polyT / U) under conditions that allow RNA transcripts to bind to said surface, and eluting the purified RNA transcripts from said surface (WO2014 / 152031); using ion (e.g., anion) exchange chromatography, which allows for the separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014 / 144767); and subjecting the modified mRNA sample to DNase treatment (WO 2014 / 152030).

[0186] In some embodiments, a method for preparing mRNA molecules is provided, comprising (1) transcribing a downstream DNA sequence of a promoter using an RNA polymerase to synthesize mRNA, using linear double-stranded DNA containing a promoter sequence as a template and ATP, GTP, CTP, or N1-Me-pUTP as substrates; and (2) capping the synthesized mRNA using a one-step chemical method with a capping analogue CAP m7Gppp(2'OMeA)pG or CAP5 m7G(5')vppp(5')(2'OMeA)pG. In some embodiments, the promoter is a T7 promoter. In some embodiments, the RNA polymerase is a T7 RNA polymerase.

[0187] During mRNA processing, characteristic structural features of mature mRNA, such as the 5' cap and Poly-A tail, are typically added to the transcribed (immature) mRNA.

[0188] During the in vitro synthesis of mRNA molecules, 5' capping of polynucleotides can be performed simultaneously using chemical RNA cap analogs to produce 5' guanosine cap structures: 5'-guanosine cap structures: 3'-O-Me-m7G(5')ppp(5')G[ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G. In some embodiments described herein, capping is performed using the following kits: CAP m7Gppp(2'OMeA)pG or CAP5m7G(5')vppp(5')(2'OMeA)pG. 5' capping of mRNA can also be performed post-transcriptionally using a vaccinia virus capping enzyme to produce the Cap 0 structure. The Cap 1 structure can be generated using both vaccinia virus capping enzyme and 2'-O methyltransferase to produce m7G(5')ppp(5')G 2'O-methyl. The Cap 2 structure can be generated from the Cap 1 structure, followed by 2'-O methylation of the 5' penultimate nucleotide using 2'-O methyltransferase. The Cap 3 structure can be generated from the Cap 2 structure, followed by 2'-O methylation of the 5' penultimate nucleotide using 2'-O methyltransferase. The 3' Poly-A tail is typically an extension of an adenine nucleotide added to the 3' end of the transcribed mRNA. In some embodiments, it can include up to approximately 400 adenine nucleotides.

[0189] Elements of mRNA molecules

[0190] In some implementations, in addition to the read frame encoding the immunogenic protein, the mRNA molecule also contains a 5'UTR and a 3'UTR, as well as a 5' cap structure or a 3' Poly-A tail. The 5'UTR and 3'UTR are typically transcribed from genomic DNA and are elements of immature mRNA.

[0191] When mRNA is engineered to encode immunogenic proteins, it may contain one or more of these untranslated regions (UTRs). Wild-type untranslated regions of nucleic acids are transcribed but not translated. In mRNA, the 5' UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3' UTR begins immediately after the stop codon and continues until the transcription termination signal. UTRs may play a regulatory role in the stability of nucleic acid molecules and translation. A variety of 5' UTR and 3' UTR sequences are known and available in the art. The 5' UTR is the mRNA region 5' directly upstream of the start codon (the first codon of the mRNA transcript translated by ribosomes). The 5' UTR does not encode proteins (it is non-coding). Native 5' UTRs have features that play a role in translation initiation. They possess features such as the Kozak sequence, which are well known to be involved in the ribosome-initiated translation of many genes. It is also known that 5' UTRs form secondary structures involved in elongation factor binding.

[0192] The 3'UTR is the mRNA region directly downstream (3') of a stop codon (the codon that transmits the translation termination signal in the mRNA transcript). The 3'UTR does not encode proteins (it is non-coding). Strains containing adenosine and uridine are known to be embedded in natural or wild-type 3'UTRs. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories (Chen et al., 1995): Class I AREs contain several scattered copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonmers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less clearly defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this category. Most proteins that bind to AREs are known to disrupt messenger stability, while members of the ELAV family, particularly HuR, have been shown to increase mRNA stability. HuR binds to all three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule will result in HuR binding, thereby stabilizing the messenger in vivo. The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to modulate the stability of polynucleotides (e.g., mRNA). When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to make the nucleic acid of this disclosure less stable, thereby reducing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to increase intracellular stability, thereby increasing the translation and production of the resulting protein. Those skilled in the art will understand that the 5'UTR can be used with any desired 3'UTR sequence.

[0193] A poly-A tail is an mRNA region containing multiple consecutive adenosine monophosphates (ATPs) located downstream of the 3' UTR, for example, directly downstream (i.e., 3'). A poly-A tail can contain 10 to 300 ATPs. In some embodiments, the poly-A tail contains 10 to 400 ATPs (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400). Poly-A tails can be used to protect mRNA from enzymatic degradation, for example, in the cytoplasm, and can facilitate transcription termination and / or the export of mRNA from the nucleus for translation.

[0194] In some implementations, the Poly-A tail contains a polynucleotide sequence of SEQ ID NO:52 or 53.

[0195] In some embodiments, the 5'UTR contains the polynucleotide sequence of SEQ ID NO:44, 46, 48, 50 or a variant polynucleotide sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:44, 46, 48, 50.

[0196] In some embodiments, the 3'UTR contains the polynucleotide sequence of SEQ ID NO:45, 47, 49, 51 or a variant polynucleotide sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO:45, 47, 49, 51.

[0197] In some embodiments, the 5'UTR and 3'UTR are selected from combinations of the following sequences or variant polynucleotide sequences: SEQ ID NO:44 and 45; SEQ ID NO:46 and 47; SEQ ID NO:48 and 49; SEQ ID NO:50 and 51.

[0198] Typical mRNA sequence

[0199] The mRNA of the present invention comprises an open reading frame containing the nucleotide sequence shown in SEQ ID NO: 27-43 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 27-43.

[0200] In some embodiments, the open reading frame of the mRNA of the present invention further comprises a nucleotide sequence encoding a signal peptide; in some embodiments, the nucleotide sequence encoding the signal peptide comprises a nucleotide sequence as described in any one of SEQ ID NO: 84-88 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 84-88.

[0201] In some embodiments, the mRNA of the present invention further comprises a 5'UTR, which comprises the nucleotide sequence shown in SEQ ID NO: 44, 46, 48, 50 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 44, 46, 48, 50.

[0202] In some embodiments, the mRNA of the present invention further comprises a 3'UTR, which comprises a 3'UTR as shown in SEQ ID NO: 45, 47, 49 or 51; or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 45, 47, 49 or 51.

[0203] In some embodiments, the mRNA of the present invention further comprises a polyA tail, which contains nucleotide sequences as shown in SEQ ID NO: 52, 53.

[0204] In some embodiments, the mRNA of the present invention further comprises a KOZAK sequence located between the 5'UTR and the promoter.

[0205] In some embodiments, the mRNA of the present invention comprises the following structure: 5'-UTR-signal peptide-gE protein-optionally gI protein or a fragment thereof-3'-UTR-PolyA. The individual elements in this structure (5'-UTR, signal peptide, gE protein, gI protein or a fragment thereof, 3'-UTR, and PolyA) can be any corresponding element herein.

[0206] In some embodiments, the mRNA of the present invention comprises the nucleotide sequence described in any one of SEQ ID NO: 54-79 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with SEQ ID NO: 54-79.

[0207] In some embodiments, the mRNA of the present invention further comprises the nucleotide sequence shown in SEQ ID NO: 66.

[0208]

[0209]

[0210] In some embodiments, the mRNA of the present invention further comprises the nucleotide sequence shown in SEQ ID NO: 72.

[0211]

[0212]

[0213] In some embodiments, the mRNA of the present invention further comprises the nucleotide sequence shown in SEQ ID NO: 74.

[0214]

[0215]

[0216] In some embodiments, the mRNA of the present invention further comprises the nucleotide sequence shown in SEQ ID NO: 75.

[0217]

[0218]

[0219] In some embodiments, the mRNA of the present invention further comprises a 5' cap structure.

[0220] Formulations or compositions

[0221] Formulations or compositions containing nucleic acids (e.g., mRNA molecules) are known in the art and are described, for example, in WO2013 / 090648. For example, compositions or formulations may be, but are not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microspheres, lipids, lipid complexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin binders, fibrinogen, thrombin, rapidly eliminating lipid nanoparticles (reLNPs), and combinations thereof.

[0222] vaccine

[0223] This document also provides information on nucleic acid vaccines. Nucleic acid vaccines can be mRNA vaccines. In some embodiments, an mRNA vaccine may contain the same mRNA molecule or multiple different mRNA molecules. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein, and the mRNA molecule may be the same. In some embodiments, the mRNA in an mRNA vaccine may contain the same read frame of an immunogenic protein, and the mRNA molecules may be different. That is, an mRNA vaccine contains multiple different mRNA molecules that encode the same immunogenic protein. In some embodiments, the mRNA in an mRNA vaccine may contain different read frames of immunogenic proteins. Nucleic acid vaccines may contain mRNA molecules containing any one or more of SEQ ID NO: 54-79 (e.g., 2-35, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35).

[0224] In some embodiments, two or more different mRNAs may be formulated into the same lipid nanoparticle. In some embodiments, two or more different RNAs encoding antigens may be formulated into separate lipid nanoparticles. The lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., containing multiple RNAs encoding multiple antigens), or they may be administered alone.

[0225] Vaccine administration

[0226] In some embodiments, the compositions, formulations, or vaccines described herein may be administered to a subject (e.g., a mammalian subject, such as a human subject). The dosage is an effective amount to enable the nucleic acid to be translated in vivo to produce an immunogenic protein. An "effective amount" is based at least in part on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and / or degree of nucleoside modification), other components of the vaccine, and other determinants such as the subject's age, weight, height, sex, and general health condition. Typically, an effective amount of vaccine provides an induced or enhanced immune response based on the antigen produced in the subject's cells.

[0227] The term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or in vitro. A "pharmaceutically acceptable carrier" will not cause undesirable physiological effects when administered to or to a subject. A carrier in a pharmaceutical composition must also be "acceptable" in the sense that it is compatible with and can stabilize the active ingredient. One or more solubilizers may be used as drug carriers to deliver the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible mediators, adjuvants, additives, and diluents to achieve compositions usable as dosage forms. Other examples of carriers include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate. Further suitable drug carriers and diluents, as well as the pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0228] In some embodiments, the vaccine described herein can be used to treat or prevent VZV infection. The vaccine can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization program, or during the incubation period or during active infection after the onset of symptoms. In some embodiments, the vaccine can treat subjects already infected with VZV. In some embodiments, the amount of RNA provided to cells, tissues, or subjects can be an amount effective for immunoprophylaxis or treatment.

[0229] Vaccines may be administered in combination with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition such as a vaccine, the term "booster" refers to an additional administration of the prophylactic (vaccine) composition. In exemplary embodiments, the time interval between the initial administration of the prophylactic composition and the administration of the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year. In some embodiments, the vaccine may be administered intramuscularly, intranasally, or intradermally.

[0230] In some implementations, the mRNA molecules or vaccines described herein are administered at doses of 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or more. In some implementations, the mRNA molecules or vaccines described herein are in unit dose form, each unit dose may contain 1 μg, 2 μg, 5 μg, 10 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1 μg, etc. The mRNA molecule or vaccine described herein may be in doses of g, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or more.

[0231] Example

[0232] The invention will be more readily understood by referring to the following examples, which are only used to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0233] Material

[0234] Unless otherwise stated, all reagents used in this embodiment are commercially available or conventional materials.

[0235] Example 1: VZV gE and gI variant design

[0236] Based on the amino acid sequence of VZV gE protein (UniProtKB:Q9J3M8.1) in the NCBI (National Center for Biotechnology Information) database, affinity and endoplasmic reticulum anchoring were predicted for the extracellular region, transmembrane region, and intracellular tail of the gE protein by increasing the affinity for the mature target antigen and the flow affinity from the endoplasmic reticulum to the trans Golgi apparatus. Mutants were designed at sites I619, N437, T560, E591, E599, N266, Y438, Y441, A540, A546, I555, and Y569 in the antigenic region, transmembrane region, and carboxyl hydrophobic region. A truncated variant was designed for the extracellular region of VZV gI protein (UniProtKB:Q77NN4.1) in the NCBI database, as shown in Table 1.

[0237] Table 1. Wild-type and variants of VZV gE and gI proteins

[0238]

[0239]

[0240] Example 2: Design of VZV mRNA vaccine construct

[0241] Using human hosts, mammalian codon preference optimization was employed to design wild-type sequences and variants of VZV gE and gI antigens, constructing plasmids (Tables 5-7) carrying nucleic acid sequences (SEQ ID NO: 54-79) encoding the corresponding gE protein amino acid sequence (SEQ ID NO: 6-22) or gE-gI fusion protein (SEQ ID NO: 89-92). Conventional molecular biology methods were used to construct the relevant plasmids. In short, the preferred nucleotide sequences encoding the varicella-zoster virus glycoprotein gE or gE-gI fusion protein containing the signal peptide (Table 2), along with the 5' untranslated region sequence (Table 3), 3' untranslated region sequence (Table 3), and 3' polyadenylate sequence (Table 4), were synthesized by Genscript Biotech and constructed on the plasmid pVAX1-BsaI-SpeI-SapI-BspMI-free-Terminator-T7 deleted.

[0242] Table 2: Signal peptide sequences

[0243] element amino acid sequence Nucleic acid sequence IL-2 signal peptide SEQ ID NO:1 SEQ ID NO:84 tPA signal peptide SEQ ID NO:2 SEQ ID NO:85 IL-10 signal peptide SEQ ID NO:3 SEQ ID NO:86 Ig kappa signal peptide SEQ ID NO:4 SEQ ID NO:87 gE signal peptide SEQ ID NO:5 SEQ ID NO:88

[0244] Table 3: UTR sequences

[0245] element Nucleic acid sequence 5'-UTR SEQ ID NO:44 5'-UTR SEQ ID NO:46 5'-UTR SEQ ID NO:48 5'-UTR SEQ ID NO:50 3'-UTR SEQ ID NO:45 3'-UTR SEQ ID NO:47 3'-UTR SEQ ID NO:49 3'-UTR SEQ ID NO:51

[0246] Table 4: polyA sequences

[0247] element Nucleic acid sequence PolyA nucleotide sequence SEQ ID NO:52 PolyA nucleotide sequence SEQ ID NO:53

[0248] Table 5: mRNA constructs of gE-A fusion proteins with different signal peptides

[0249] Construct Nucleic acid sequence 5'-UTR-IL2(SP)-gE-WT-3'-UTR-PolyA SEQ ID NO:54 5'-UTR-gE(SP)-gE-WT-3'-UTR-PolyA SEQ ID NO:55 5'-UTR-IL-10(SP)-gE-WT-3'-UTR-PolyA SEQ ID NO:56 5'-UTR-tPA(SP)-gE-WT-3'-UTR-PolyA SEQ ID NO:57 5'-UTR-Ig kappa(SP)-gE-WT-3'-UTR-PolyA SEQ ID NO:58

[0250] Table 6: mRNA constructs of IL-2 signal peptide fused with different gE proteins

[0251]

[0252]

[0253] Table 7: mRNA constructs of gE mutant and gI fusion protein

[0254]

[0255] Table 8: Auxiliary epitopes for immune enhancement

[0256]

[0257]

[0258] Example 3: Validation of in vitro transcription and different cap analogues

[0259] Using linear double-stranded DNA containing the T7 promoter sequence (as described in Example 2, SEQ ID NO: 55) as a template, and ATP, GTP, CTP, and N1-Me-pUTP as substrates, the DNA sequence downstream of the promoter was transcribed using T7 RNA polymerase and the cap analog CAP m7GpppAmG or CAP5m7G(5')vppp(5')(2'OMeA)pG. The transcribed DNA was then capped using T7 RNA polymerase and the cap analog CAP m7GpppAmG or CAP5m7G(5')vppp(5')(2'OMeA)pG in a 20 μL system (Table 9), incubated at 37°C for 2.5 hours. After incubation, 2 μL of DNase I was added and incubated for 15 minutes to synthesize mRNA. The results are shown in Table 9. The cap analog CAP m7GpppAmG (structural formula: ...) ... ) or CAP5m7G(5')vppp(5')(2'OMeA)pG (structural formula is All of these methods can achieve an mRNA template capping rate of over 95%. Therefore, in subsequent examples, CAP5 m7G(5')vppp(5')(2'OMeA)pG was selected for mRNA template preparation.

[0260] Table 9: In vitro transcription system and capping rate of different capping analogues

[0261]

[0262]

[0263] Example 4: Preparation of VZV mRNA-LNP formulation

[0264] Encapsulation was performed using a fixed lipid formulation. SM-102, DSPC, cholesterol, and DMG-PEG2000 were completely dissolved in ethanol at a molar percentage of (50:10:38.5:1.5) to obtain a lipid mixture. The lipids and mRNA were mixed in a nanomedicine preparation system at a volume ratio of 1:3 (lipids:mRNA) in 20mM sodium citrate buffer (pH 4.0) at a flow rate of 12 ml / min. The collected sample solution was diluted 10-fold in Tris-0.1% NaCl buffer, passed through a 100 kDa Pall ultrafiltration tube, centrifuged, and concentrated to remove ethanol. Finally, the solution was adjusted to a suitable concentration with DPBS buffer for use in the subsequent mRNA vaccine formulation preparation.

[0265] Example 5: Validation of different signal peptides

[0266] Babl / c mice were used in this experiment. On day 1, mice were injected intramuscularly with a single dose of different vaccines (as shown in Table 10), with each mouse receiving 5 μg of mRNA. Blood was collected on day 14 post-immunization, and PBMC cells were isolated to detect vaccine-induced cellular immunity IFNγ. The specific method was as follows: 70 μL of orbital blood was collected from mice, and PBMC cells were separated by lysis using RBC Lysis Buffer (10X, Lot-420301, BioLegend). gE protein (sequence SEQ ID NO: 6, expressed and purified by Jiangsu Shenji Biotechnology) was added to a final concentration of 10 μg / mL as a stimulant. Stimulate with 100 μl of the solution, incubate at 37°C and 5% CO2 for 2 hours, then add brefeldin A inhibitor (5 μg / ml, Lot-420601, BioLegend), incubate at 37°C and 5% CO2 for 5 hours. Transfer cells to BD Falcon flow cytometry tubes, add CD16 / 32Fc-receptor (clone2.4G2, Lot-101302, BioLegend) blocking solution, and stain the cells with the following staining solutions: anti-CD4-FITC (1:100 in PBS with 2% FBS, Lot-100406, BioLegend); anti-CD8-PE (1:100 in PBS with 2% FBS, Lot-100708, BioLegend); and anti-CD3-PerCP-Cy5.5 (1:100 in PBS). Cells were incubated with 2% FBS (Lot-317336, BioLegend) at 4°C for 30 minutes. Intracellular cytokine staining was then performed: 4% paraformaldehyde was added at a standard concentration of 100 μL / sample for fixation, and the cells were incubated at room temperature for 15 minutes. Freshly prepared 1× Permeabilization Buffer (10×, Lot-421002, BioLegend) was added at a concentration of 200 μL / sample, and the cells were permeabilized at room temperature for 15 minutes. After permeabilization, the cells were centrifuged at 500×g for 3 minutes, and the supernatant was discarded. Anti-IFN-γ-APC (1:100 in Permeabilization Buffer) was added, and the sample was added at a concentration of 100 μL / sample. The mixture was gently mixed, and the cells were incubated at 4°C for 25 minutes. Data were obtained using flow cytometry, and the flow cytometry results were analyzed using FlowJo software.

[0267] The results are shown in Table 10. On day 14, all VZV-gE-mRNA vaccine immunization groups with different signal peptides produced a certain amount of gE-specific IFNγ. The signal peptides IL-2 and IL-10 increased the T-cell immune response to the VZV-gE-mRNA vaccine to 9.28% and 8.11%, respectively. After T lymphocytes were activated by the immune system, CD4+ T lymphocytes differentiated into TH1 and TH2 cells. IFN-γ is a marker cytokine for TH1 cells. As VZV is a lifelong latent virus, the T-cell response capacity determines the strength of the vaccine's protective efficacy.

[0268] As shown in Table 10, compared with the gE signal peptide itself, tPA, and Ig Kappa signal peptide, IL-2 or IL-10 signal peptides have a better effect on enhancing the T-cell immune response of VZV-gE-mRNA vaccine. Therefore, in subsequent examples, IL-2 signal peptide (SEQ ID NO:1) was selected as the secretion signal peptide of mRNA.

[0269] Table 10: Validation of different signal peptides

[0270]

[0271] Example 6: Validation of antibody binding to different gE variant mRNA vaccines

[0272] Balb / c mice were used in this experiment. Two doses of the vaccine were administered intramuscularly to mice on days 1 and 22, with each mouse receiving a dose of 5 μg (comparative vaccine 1 – Shingrix subunit shingles vaccine, 5 μg / mouse, 1 / 10 human dose, intramuscular injection, purchased from GlaxoSmithKline; comparative vaccine 2 – full-length gE sequence (SEQ ID NO: 59); comparative vaccine 3 – gE truncated sequence (Y569A mutation, SEQ ID NO: 60). Blood was collected on day 7 after each vaccination, and serum was separated to detect the vaccine-induced binding antibody immune response. The coating antigen was the gE antigen protein (sequence of which is SEQ ID NO: 60). NO:6 (Expression and purification by Jiangsu Shenji Biotechnology), incubated overnight at 4℃; blocked the ELISA plate with PBST solution containing 5% skim milk powder, and incubated at 37℃ for 2 hours; serially diluted the inactivated serum three times, added the diluted serum to the ELISA plate, and incubated at 37℃ for 1 hour; washed the plate three times with 1×PBST, added HRP-conjugated anti-mouse IgG antibody, and incubated at 37℃ for 1 hour; discarded the supernatant, washed the plate three times with 1×PBST, added TMB substrate for color development for 5 minutes, and terminated the color development reaction with 2M HCl. The OD450 value of each well was read using a microplate reader, and the statistical data and endpoint dilution of each serum were calculated.

[0273] The results are shown in Table 11. Antibodies targeting the gE protein were detected in the serum of each experimental group on days 8 and 29. The levels of IgG antibodies induced by the gE-N437S vaccine, the gE-I619S vaccine, and the gE-mutant combined gE-B-mRNA and gE-C-mRNA vaccine groups were higher than those induced by the control vaccine Shingrix, the control vaccine 2-gE full-length sequence mRNA, and the control vaccine 3-gE truncated sequence mRNA. The gE-mutant combined gE-C-mRNA vaccine induced the highest level of IgG antibody response.

[0274] Table 11 Validation of antibody binding to different gE variant mRNA vaccines

[0275]

[0276]

[0277]

[0278] Example 7: Cellular Immunoassay Validation of Different gE Variant mRNA Vaccines

[0279] Balb / c mice were administered two doses of the vaccine via intramuscular injection on days 1 and 22, with each mouse receiving a dose of 5 μg (comparative vaccine 1 – Shingrix subunit shingles vaccine, 5 μg / mouse, 1 / 10 human dose, intramuscular injection, purchased from GlaxoSmithKline; comparative vaccine 2 – full-length gE sequence (SEQ ID NO: 59); comparative vaccine 3 – truncated gE sequence (Y569A mutation, SEQ ID NO: 60)). On day 14 post-vaccination, the spleens of the mice were harvested, and spleen lymphocyte suspensions were prepared using Ficoll separation medium (purchased from Cytiva) to detect vaccine-induced cellular immunity to IFNγ. The specific method was the same as in Exercise 4.

[0280] The results are shown in Table 12. Significant differences were observed in the production of gE-specific IFNγ among the experimental groups on day 36. Specifically, the T-cell immune response of the control vaccine 1 (Shingrix) was 13.07%, the T-cell immune response of the control vaccine 2 (full-length gE mRNA) was 10.26%, and the T-cell immune response of the control vaccine 3 (cut-out gE mRNA) was 15.15%. Compared to the control group, the gE-mutant derived gE-N437S and gE-I619S vaccines, as well as the gE-mutant combined gE-B-mRNA and gE-C-mRNA vaccine groups, induced significantly higher levels of T-cell immune responses, at 17.24%, 19.33%, 22.43%, and 25.77%, respectively.

[0281] Table 12 Validation of Specific Cellular Immunity for Different gE Variant mRNA Vaccines

[0282]

[0283] Example 8: Validation of T-cell immune memory from gE mutant mRNA vaccine

[0284] Balb / c mice were used in this experiment. Two doses of the vaccine were administered intramuscularly on days 1 and 22, with each mouse receiving a dose of 5 μg (comparative vaccine 1 – Shingrix subunit shingles vaccine, 5 μg / mouse, 1 / 10 human dose, intramuscular injection, purchased from GlaxoSmithKline; comparative vaccine 2 – full-length gE sequence (SEQ ID NO: 59); comparative vaccine 3 – gE truncated sequence (Y569A mutation, SEQ ID NO: 60). On day 14 after the second vaccination, mouse spleens were harvested, and spleen lymphocyte suspensions were prepared using Ficoll separation medium (purchased from Cytiva) to detect vaccine-induced T-cell immune memory (primarily detecting the proportion of CD44+CD62L+ cells in the central memory T cells Tcm). Specifically, gE protein (sequence SEQ ID NO: 59) was added to mouse spleen lymphocytes at a final concentration of 10 μg / mL. NO:1 (Jiangsu Shenji Biotechnology Expression and Purification) was used as a stimulant. 100 μL of the stimulant was added, and the cells were cultured at 37°C and 5% CO2 for 2 hours. Then, brefeldin A blocking agent (5 μg / ml, Lot-420601, BioLegend) was added, and the cells were incubated at 37°C and 5% CO2 for 5 hours. Cells were then transferred to BD Falcon flow cytometry tubes, and CD16 / 32Fc-receptor (clone 2.4G2, Lot-101302, BioLegend) blocking solution was added. Surface staining was performed: cells with 2%... Anti-CD44-FITC (Lot-103016, BioLegend), anti-CD62L-APC (Lot-104412, BioLegend), and anti-CD3-PerCP-Cy5.5 (Lot-317336, BioLegend) were diluted 1:100 in PBS with FBS and incubated at 4°C for 30 minutes. Data were obtained by flow cytometry and the flow cytometry results were analyzed using FlowJo software.

[0285] The results are shown in Table 13. The proportion of CD44+CD62L+ T cells in the control vaccine 1 (Shingrix) was relatively low at 6.26%, while the proportions of CD44+CD62L+ T cells in the mRNA vaccines (control vaccines 2 and 3) were 7.28% and 7.09%, respectively, indirectly suggesting that the long-term cellular immunity level of protein vaccines is weaker than that of mRNA vaccines. The CD44+CD62L+ T cell proportions of the gE-mutant derived gE-N437S and gE-I619S vaccines increased to 16.25% and 17.08%, respectively. Meanwhile, the CD44+CD62L+ T cell proportions of the gE-mutant derived combinations gE-B and gE-C increased to 20.19% and 25.04%, respectively.

[0286] Table 13. Detection of CD44+CD62L+ central memory T cell ratio in gE mutant mRNA vaccine at different time points.

[0287]

[0288]

[0289] Example 9: Validation of the gE mutant-gI fusion mRNA vaccine

[0290] Balb / c mice were used in this experiment. Two doses of the vaccine were administered intramuscularly to mice on days 1 and 22, with each mouse receiving a dose of 5 μg (comparative vaccine 1 – Shingrix subunit shingles vaccine, 5 μg / mouse, 1 / 10 human dose, intramuscular injection, purchased from GlaxoSmithKline; comparative vaccine 2 – full-length gE sequence (SEQ ID NO: 59); comparative vaccine 3 – gE truncated sequence (Y569A mutation, SEQ ID NO: 60). Blood was collected on day 7 after each vaccination, and serum was separated to detect the vaccine-induced binding antibody immune response. The coating antigen was the gE antigen protein (sequence of which is SEQ ID NO: 60). NO:6 (Expression and purification by Jiangsu Shenji Biotechnology), incubated overnight at 4℃; blocked the ELISA plate with PBST solution containing 5% skim milk powder, and incubated at 37℃ for 2 hours; serially diluted the inactivated serum three times, added the diluted serum to the ELISA plate, and incubated at 37℃ for 1 hour; washed the plate three times with 1×PBST, added HRP-conjugated anti-mouse IgG antibody, and incubated at 37℃ for 1 hour; discarded the supernatant, washed the plate three times with 1×PBST, added TMB substrate for color development for 5 minutes, and terminated the color development reaction with 2M HCl. The OD450 value of each well was read using a microplate reader, and the statistical data and endpoint dilution of each serum were calculated.

[0291] The results are shown in Table 14. Antibodies targeting the gE protein were detectable in the serum of all experimental groups on days 8 and 29. Compared with the gE-mutant gE-B-mRNA vaccine, there was no significant difference in the IgG antibody levels induced by the gE-B+gI full-length-mRNA vaccine group and the gE-B+gI truncated-mRNA vaccine group. Compared with the gE-mutant gE-C-mRNA vaccine, the IgG antibody level induced by the gE-C+gI truncated-mRNA vaccine group increased from 10.28 to 14.19 on day 29.

[0292] Table 14. Detection of gE mutation and gI dual-antigen mRNA vaccine binding antibodies at different time points.

[0293]

[0294]

[0295] It should be understood that although the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A varicella-zoster virus (VZV) gE protein variant, characterized in that, The gE protein variant has a mutation at one or more of the following amino acid positions relative to the parental gE protein: 619, 437, 560, 591, 599, 266, 438, 441, 540, 546, 555, 569, wherein the amino acid positions are numbered according to the amino acid sequence shown in SEQ ID NO: 93, and the parental gE protein has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical or 100% identical to SEQ ID NO: 6 or 93. Preferably, the gE protein variant has one or more mutations in one or more of the parental gE protein, namely I619, N437, T560, E591, E599, N266, Y438, Y441, A540, A546, I555, and Y569. Preferably, the gE protein variant has one or more mutations among I619S, N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S, and Y569A relative to the parental gE protein. Preferably, the gE protein variant has an I619S mutation relative to the parental gE protein and one or more mutations selected from N437S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S and Y569A. Preferably, the gE protein variant has an N437S mutation relative to the parental gE protein and one or more mutations selected from I619S, T560S, E591S, E599S, N266S, Y438S, Y441S, A540S, A546S, I555S and Y569A. Preferably, the gE protein variant has the following mutation combinations relative to the parental gE protein: 1) T560S, E591S, E599S and I619S; 2) N266S, N437S, Y438S and Y441S; or 3) A540S, A546S, I555S and Y569A; Preferably, the gE protein variant has an amino acid sequence shown in any one of SEQ ID NO: 8-22 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 8-22.

2. A fusion protein comprising the gE protein variant of claim 1; Preferably, the fusion protein comprises a signal peptide sequence selected from IL-2 signal peptide, tPA signal peptide, IL-10 signal peptide, Ig kappa signal peptide and gE signal peptide; Preferably, the fusion protein has a signal peptide selected from the amino acid sequences shown in any one of SEQ ID NO:1-5 or a signal peptide having an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NO:1-5; Preferably, the gE protein variant in the fusion protein is fused with other antigenic peptides and / or helper epitopes of VZV; Preferably, the other antigenic polypeptides are selected from one or more of VZV gI, gB, gH, gK, gL, gC, gN, gM and their antigenic fragments; Preferably, the auxiliary epitope is selected from PADRE or IL-1β epitopes; Preferably, the auxiliary epitope comprises an amino acid sequence selected from any one of SEQ ID NO:80-81 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO:80-81; Preferably, the gE protein variant is directly or indirectly fused with other antigenic peptides and / or helper epitopes; Preferably, the gE protein variant is indirectly linked to other antigenic peptides and / or helper epitopes via linkers; Preferably, the connector is a shearable connector, a flexible connector, or a rigid connector; Preferably, the cleavable linker is selected from P2A peptide, E2A peptide, or F2A peptide; Preferably, the other antigenic polypeptides are located at the N-terminus or C-terminus of the gE protein variant; Preferably, the gE protein variant is fused with VZV gI; Preferably, the gI protein or its immune fragment has the amino acid sequences shown in SEQ ID NO:23 and 24, or has an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and 24. Preferably, the fusion protein of gE and gI has an amino acid sequence shown in any one of SEQ ID NO: 89-92 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 89-92.

3. Nucleic acid, characterized in that, Contains a polynucleotide encoding the protein variant of claim 1 or the fusion protein of claim 2; Preferably, the nucleic acid is DNA or RNA; Preferably, the RNA is mRNA, circular RNA, or self-replicating RNA; Preferably, the RNA further comprises a 5'UTR, KOZAK, 3'UTR, polyA, and / or a cap structure; Preferably, the RNA is modified RNA, wherein uracil, cytosine, adenine or guanine nucleotides contain modifying groups; Preferably, the modifying group is selected from at least one of pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine, 2-thiouridine, 5-methoxyuridine, or N1-methyladenosine, N1-methylguanine, N1-methylguanine, and isoguanine. Preferably, the mRNA molecule is a modified mRNA, the modification including the conversion of uracil nucleoside to pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 5-methoxyuridine; and / or, the conversion of cytosine nucleoside to 5-methylcytosine, 5-methoxycytosine, N1-methylcytosine; and / or, the conversion of adenine nucleoside to N1-methyladenosine; and / or, the conversion of adenine nucleoside to N1-methylguanine, N1-methylguanine, isoguanine.

4. The nucleic acid according to claim 3, comprising a polynucleotide encoding a protein comprising any of the amino acid sequences shown in SEQ ID NO: 8-24; Preferably, the nucleic acid is mRNA; Preferably, the mRNA comprises the nucleotide sequence shown in any one of SEQ ID NO: 27-43 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 27-43; Preferably, the mRNA further comprises a 5'UTR as shown in any one of SEQ ID NO: 44, 46, 48, 50 or a 5'UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 44, 46, 48, 50; Preferably, the mRNA further comprises a 3'UTR as shown in any one of SEQ ID NO: 45, 47, 49 or 51; or a 3'UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 45, 47, 49 or 51. Preferably, the mRNA further comprises a polyA with a length of 60-150 nt, more preferably 80-120 nt; Preferably, the polyA is as shown in SEQ ID NO: 52 or 53; Preferably, the mRNA further comprises a KOZAK sequence; Preferably, the mRNA further includes a 5' cap structure; Preferably, the 5' cap is a compound of formula (I), or a pharmaceutically acceptable salt, stereoisomer, tautomer, or isotopic variant thereof: in, --- indicates a single key or that the key does not exist. X1 is selected from O, S, CH2, CH2CH2, CH=CH, CH=CHO, CH2O, OCH2, CH2CH2O, OCH2CH2, and tricyclic alkyl groups. R1, R2, R3, and R4 are independently halogenated, OH-, unsubstituted, or OC-substituted, respectively. 1-3 Alkyl-substituted OC 1-3 Alkyl, unsubstituted or OC 1-3 Alkyl-substituted OC 1-3 alkyl, B1 and B2 are each independently selected from natural, modified, or non-natural nucleoside bases, preferably, the compound of formula (I) is any one of the following:

5. The nucleic acid according to claim 3 or 4, comprising the nucleotide sequence of any one of SEQ ID NO: 54-79 or a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 54-79.

6. An expression vector comprising the nucleic acid according to any one of claims 2-5.

7. A cell comprising the expression vector of claim 6, preferably a eukaryotic cell or a prokaryotic cell.

8. A method for preparing the gE protein variant of claim 1 or the fusion protein of claim 2, the method comprising culturing the cells of claim 7 in a culture medium; preferably, the method further comprises the steps of protein isolation and purification.

9. A composition comprising the gE protein variant of claim 1, the fusion protein of claim 2, the nucleic acid of any one of claims 3-5, and / or the vector of claim 6; Preferably, the composition comprises an mRNA molecule containing the nucleotide sequences described in any one or more of SEQ ID NO: 54-79; Preferably, the mRNA molecule is a lipid nanoparticle (LNP); Preferably, the liposome nanoparticles comprise ionizable lipids, DSPC, cholesterol, and DMG-PEG2000; Preferably, the composition is a vaccine.

10. The composition of claim 9, wherein it is formulated into a formulation suitable for intramuscular or subcutaneous injection, oral administration, or inhalation.

11. The use of the gE protein variant of claim 1, the fusion protein of claim 2, the nucleic acid of any one of claims 3-5, the vector of claim 6, and / or the composition of claim 9 in the preparation of a medicament for the prevention or treatment of varicella-zoster virus infection or diseases caused by varicella-zoster virus infection; preferably, the medicament is a vaccine; preferably, the disease is selected from varicella and shingles.

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