CN118340876A

CN118340876A - Varicella-zoster virus self-replicating mRNA vaccine pharmaceutical composition and application thereof

Info

Publication number: CN118340876A
Application number: CN202410403081.3A
Authority: CN
Inventors: 俞天奇; 马光刚; 张广远; 刘佳雯; 田家伦
Original assignee: Beijing Xiansheng Xiangrui Biological Products Co ltd
Current assignee: Beijing Xiansheng Xiangrui Biological Products Co ltd
Priority date: 2024-04-03
Filing date: 2024-04-03
Publication date: 2024-07-16

Abstract

The invention relates to a varicella-zoster virus self-replicating mRNA vaccine pharmaceutical composition and application thereof. Specifically, compared with the prior art, the preparation method has the advantages of good immunogenicity, strong immune effect, simple preparation process, suitability for clinical development and industrial production, and higher industrialization value.

Description

Varicella-zoster virus self-replicating mRNA vaccine pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a preparation method and application of a self-replicating varicella-zoster virus (VZV) mRNA vaccine preparation.

Background

Varicella-zoster virus (VZV, also known as human herpesvirus type 3) is a double stranded DNA virus with neurotropic and lymphotropic properties. Most children before age 10 will come into contact with VZV, causing fever and varicella. VZV infection is followed by latency in the dorsal sensory ganglion of the spinal cord. When immunity declines, the reactivated VZV in the elderly migrates to the skin area associated with the affected spinal cord area, causing shingles. Typical areas of skin for acute shingles include clustered blisters, causing severe pain and itching. The incidence of shingles is about 0.4% in the 50-60 year old population, while it increases significantly to 1% in the over 80 year old population. Shingles is often associated with chronic, debilitating and pain lasting for several months, known as Post Herpetic Neuralgia (PHN), especially in the elderly. About 15% of cases of post-severe PHN postherpetic neuralgia persist for at least 3 months and are not effectively alleviated by antiviral drugs.

The advantages of efficient utilization of mRNA, rapid production and development, safe administration and the like are widely paid attention to. Self-replicating mRNA (saRNA) vaccines are genetically engineered replicons derived from self-replicating single-stranded RNA viruses, and the sense alphavirus genomes commonly used in saRNA vaccine design include venezuelan equine encephalitis Virus (VEE), SINV, and Semliki Forest Virus (SFV). In the case of the alphavirus-based self-amplifying mRNA, the additional RNA contains four non-structural protein coding regions (nsP 1-4), a subgenomic promoter and a large open reading frame, and the gene encoding the viral structural protein in the viral genome is replaced by the gene of interest, so that the mRNA is not capable of producing infectious virus. The expression of conventional mRNA vaccine antigens is often proportional to the copy number of mRNA delivered into a body, so that large dosage and multiple immunity are needed, side effects are large, the problem is solved by self-replication of the saRNA, and compared with the conventional mRNA vaccine, the saRNA vaccine antigens can be delivered at a lower dosage, and the same immune protection is realized. Combining the VZV immunoprotection antigen with the self-replicating RNA is expected to be a powerful tool for preventing the VZV at low cost, quickly and efficiently.

Therefore, there is a need to develop self-replicating mRNA vaccines against VZV that are simple in process and more immunogenic.

Disclosure of Invention

It is a first object of the present invention to provide a Varicella Zoster Virus (VZV) vaccine. More specifically, an mRNA vaccine of varicella-zoster virus (VZV) is provided that can elicit an immune response against VZV and also can produce antibodies against VZV.

A second object of the present invention is to provide mRNA constructs comprised in the above vaccine, and corresponding vectors, lipid Nanoparticles (LNP).

The third object of the present invention is to provide the mRNA construct, and the corresponding vector, pharmaceutical use.

A fourth object of the present invention is to provide a method of inducing an immune response in a subject in need thereof or a method of preventing varicella-zoster in a subject in need thereof based on the vaccine described above.

In order to solve the technical problems, the invention comprises the following technical scheme:

In one aspect, the invention provides a vaccine that is a VZV mRNA vaccine comprising a VZV mRNA construct as an immunogenic component. Wherein the mRNA construct comprises mRNA encoding the gE and/or gH, gL regions of VZV.

In some specific embodiments, the mRNA encoding the gE and/or gH, gL regions of VZV is expressed alone or in fusion within the host cell.

In some specific embodiments, the mRNA encodes a protein that further comprises a mutation.

In some specific embodiments, the mRNA encodes a protein that further comprises Flt3L.

In some specific embodiments, the mRNA encodes a protein that further comprises a signal peptide sequence.

Optionally, the signal peptide is located at the N-terminus of the protein.

Preferably, the signal peptide is derived from murine H-2Kb, human IgE, HLA-B46, MICA 008, OSM, VSV-G, mouse Ig Kappa, mouse heavy chain, BM40, human chymotrypsinogen, human prothrombin-2, human IL-2, human G-CSF, human hemagglutinin IX, human albumin, gaussia luc, HAS, influenza virus, human insulin, silk LC, erenumab antibody light chain, pembrolizumab light chain, ramucirumab light chain, E SIGNAL PEPTIDE, SP1 (LZJ human IgG1, SP2, SP3 (ZLQ).

In some specific embodiments, the gE extracellular region (gE Ecto) protein binds to ferritin (Ferritin) to form a fusion protein.

The gE Ecto-ferritin fusion proteins of the invention comprise the unit subunits of ferritin which can be assembled extracellularly into nanoparticles, with 24 monomers constituting 20 panels to fully display the immunogenic portion of gE Ecto.

The ferritin subunit of the invention is a full length or any portion of ferritin, wild type or partial amino acid mutation. In a specific embodiment, the monomeric subunit is from mitochondrial ferritin or heavy chain ferritin.

In some specific embodiments, the mRNA encodes a protein comprising an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO. 13.

In some specific embodiments, the mRNA encodes a protein comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO. 13.

In some specific embodiments, the mRNA encodes a protein comprising an amino acid sequence that is at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO. 13.

In a preferred embodiment, the mRNA encodes a protein comprising the amino acid sequence set forth in SEQ ID NO. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO. 13.

In a preferred embodiment, the mRNA comprises the coding nucleotide corresponding to the amino acid sequence shown as SEQ ID NO.3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID NO. 13.

In a more preferred embodiment, the mRNA comprises the nucleotide sequence set forth in SEQ ID NO. 2,SEQ ID NO:4,SEQ ID NO:6,SEQ ID NO:8,SEQ ID NO:10 or SEQ ID NO. 12.

In some specific embodiments, the mRNA construct further comprises a 5' utr sequence.

Optionally, the 5' UTR sequence is a human alpha globulin 5' UTR or a non-native 5' UTR sequence.

In some specific embodiments, the mRNA construct further comprises a 3' utr sequence.

In some specific embodiments, the mRNA construct further comprises a Poly (a) sequence.

In some specific embodiments, the mRNA construct further comprises an alphavirus nonstructural protein (nsp) gene.

Optionally, the nsp gene is shown as SEQ ID NO. 1.

In a specific embodiment, the vaccine further comprises a delivery formulation.

Preferably, the delivery formulation is a nanoparticle.

Preferably, the delivery formulation comprises lipid nanoparticles (Lipid nanoparticle, LNP), lipid multipolymers (lipopolyplex, LPP), polymer nanoparticles (Polymer nanoparticles, PNP), inorganic nanoparticles (Inorganic nanoparticles, INP), cationic nanoemulsions (Cationic nanoemulsion, CNE), exosomes, biologicals, and protamine, etc.

More preferably, the nanoparticle is a lipid nanoparticle (Lipid nanoparticle, LNP).

In some specific embodiments, the lipid comprises one or more of a cationic lipid, a neutral helper lipid, cholesterol, and a PEG-modified lipid; preferably, two or more are included.

In some specific embodiments, the lipid comprises:

a) Cationic lipids;

b) Neutral helper lipids;

c) Cholesterol; and

D) PEG modified lipids.

In some embodiments, the lipid nanoparticle has a molar ratio of each lipid component, based on 100% total molar amount of lipid, of:

a) 45% -50% of cationic lipid;

b) Neutral auxiliary lipid 5-10%;

c) 38% -48% of cholesterol;

and d) PEG modified lipid 0-3%.

In some specific embodiments, the cationic lipid is selected from the group consisting of N, N-dimethyl-2, 3-dioleoyloxypropylamine (DODMA), 1, 2-dimyristoxypropyl-3-dimethyl-hydroxyethylammonium bromide (dmrii), N-dioleoyln, N-dimethylammonium chloride (DODAC), 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (l- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTMA), 1, 2-dimethanoyloxy-N, N-dimethylaminopropane (DLin), 2-dioleylene-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin), N- (2, 3-dioleyloxy) propyl) -N, N-trimethylammonium chloride (dlma), 1- (2, 3-dioleyloxy) 2-dlm-4-dlm, dlm-2-di-N-butanoic acid (dlm), and any of more than one of these.

In some specific embodiments, the molar ratio of the cationic lipid is 45% to 48% based on 100% total molar amount of lipid.

In some specific embodiments, the neutral helper lipid is selected from any one or more of distearoylphosphatidylcholine(DSPC)、dioleoylphosphatidylcholine(DOPC)、dimyristoylphosphatidylcholine(DMPC)、dipalmitoylphosphatidylcholine(DPPC)、diarachidoylphosphatidylcholine(DAPC)、dibehenoylphosphatidylcholine(DBPC)、ditricosanoylphosphatidylcholine(DTPC)、dilignoceroylphatidylcholine(DLPC)、dioleoylphosphatidylethanolamine(DOPE)、dipalmitoyl-phosphatidylethanolamine(DPPE)、dimyristoyl-phosphatidylethanolamine(DMPE) or phosphatidylethanolamine (DLPE).

In some embodiments, the neutral helper lipid is present in a molar ratio of 6% to 10% based on 100% total molar amount of lipid.

In some embodiments, the neutral helper lipid is present in a molar ratio of 8% to 10% based on 100% total molar amount of lipid.

In some embodiments, the PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol bitetradecylamide (ALC-0159), DMG-PEG2000, DMG-PEG5000, PEG 2000.

In some embodiments, the PEG-modified lipids are present in a molar ratio of 1% to 3% based on 100% total molar amount of lipids.

In some embodiments, the PEG-modified lipids are present in a molar ratio of 1% to 2% based on 100% total molar amount of lipids.

In some embodiments, the cholesterol is in a molar ratio of 40% to 46% based on 100% total molar amount of lipid.

In some embodiments, the cholesterol is in a molar ratio of 42% to 45% based on 100% total molar amount of lipid.

In another aspect, the invention provides an mRNA construct that is any one of the mRNA constructs described herein as an immunogenic component in the vaccine, or a combination thereof.

In another aspect, the invention provides a vector comprising any one of the mRNA constructs described herein, or a combination thereof.

In another aspect, the invention provides a cell comprising any one of the mRNA constructs described herein, or a combination thereof, or a vector described herein.

In another aspect, the invention provides a nanoparticle comprising any one of the mRNA constructs described herein, or a combination thereof.

Optionally, the nanoparticles include lipid nanoparticles (Lipid nanoparticle, LNP), lipid multipolymers (lipopolyplex, LPP), polymer nanoparticles (Polymer nanoparticles, PNP), inorganic nanoparticles (Inorganic nanoparticles, INP), cationic nanoemulsions (Cationic nanoemulsion, CNE), exosomes, biological microvesicles, protamine, and the like.

Preferably, the nanoparticle is a lipid nanoparticle (Lipid nanoparticle, LNP).

In some specific embodiments, the lipid comprises:

a) Cationic lipids;

b) Neutral helper lipids;

c) Cholesterol; and

D) PEG modified lipids.

a) 45% -50% of cationic lipid;

b) Neutral auxiliary lipid 5-10%;

c) 38% -48% of cholesterol;

and d) PEG modified lipid 0-3%.

In another aspect, the invention provides a fusion protein comprising a protein encoded by any one of the mRNAs described herein and a second protein fused thereto, wherein said protein encoded by the mRNAs is an unfused protein.

In some specific embodiments, the second protein is a carrier protein.

In some specific embodiments, the second protein is ferritin (Ferritin).

Preferably, the ferritin is mitochondrial ferritin or heavy chain ferritin.

In some specific embodiments, the ferritin comprises a domain that allows the fusion protein to self-assemble into a nanoparticle.

In another aspect, the invention provides the use of any of the vaccines, mRNA constructs, vectors, or nanoparticles described herein in the manufacture of a medicament for inducing an immune response in a subject in need thereof.

In another aspect, the invention provides the use of any of the vaccines, mRNA constructs, vectors, or nanoparticles described herein in the manufacture of a medicament for treating or preventing varicella-zoster in a subject in need thereof.

In another aspect, the invention provides a method of inducing an immune response in a subject in need thereof, comprising administering to the subject any of the vaccines described herein;

preferably, the method induces an immune response against VZV by using self-replicating mRNA.

In another aspect, the invention provides a method of treating or preventing varicella-zoster in a subject in need thereof comprising administering to the subject any of the vaccines described herein;

In some specific embodiments, the vaccine composition is administered as part of a therapeutic regimen.

Advantageous effects

Compared with the prior art, the technical scheme of the invention has at least one of the following beneficial effects:

1. The VZV mRNA vaccine has better immunogenicity, can effectively stimulate organisms to generate high-level immune response, and can induce high-level specific humoral immune response and cell immunity.

2. The invention provides a self-replicating VZV mRNA vaccine, which can express gH and gL proteins besides gE protein, and comprehensively verify the effect from the angles of cells and animal models, and the result proves that the VZV vaccine has stronger immune effect.

3. The mRNA vaccine provided by the invention has a simple preparation process, is more suitable for clinical development and industrial production, and has higher industrialization value.

4. The LNP delivery formulation formulations provided in the present invention were tested to exhibit excellent performance in a variety of ways.

Definition and description of terms

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meaning as understood by one of ordinary skill in the art.

Furthermore, unless otherwise indicated herein, terms in the singular herein shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.

The terms "comprising," "including," and "having" are used interchangeably herein to mean that the elements are included in an arrangement, meaning that the arrangement may exist in addition to the elements listed. It should also be understood that the use of "including," "comprising," and "having" descriptions herein also provides a "consisting of … …" scheme.

The term "and/or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term of interest".

The term "gene" as used herein refers to a nucleic acid fragment encoding a protein or RNA alone (also referred to as a "coding sequence" or "coding region") and associated regulatory regions such as promoters, operators, terminators, etc., which may be located upstream or downstream of the coding sequence.

The term "nucleic acid" herein in its broadest sense includes any compound and/or substance comprising a polymer of nucleotides. These polymers are referred to as polynucleotides.

The nucleic acid (also referred to as a polynucleotide) may be or may include, for example, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose Nucleic Acid (TNA), glycol Nucleic Acid (GNA), peptide Nucleic Acid (PNA), locked nucleic acid (LNA, including LNA having a βd ribose configuration, αlna having an αl ribose configuration (a diastereomer of LNA), 2 'amino LNA having 2' amino functionalization, and 2 'amino αlna having 2' amino functionalization), ethylene Nucleic Acid (ENA), cyclohexenyl nucleic acid (CeNA), or a chimeric or combination thereof.

The term "mRNA" herein is messenger RNA, meaning any polynucleotide that encodes (at least one) polypeptide (naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded polypeptide.

The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap, and a poly a tail. Polynucleotides of the present disclosure may serve as mrnas, but may differ from wild-type mrnas in their functional and/or structural design features that are used to overcome the existing problems of efficient polypeptide expression using nucleic acid-based therapeutics.

The mRNA as used herein refers to mRNA comprising a nucleic acid sequence encoding a VZV antigen, which may be 1) mRNA encoding and translating only a certain VZV antigen, or 2) a mixture of mRNA encoding and translating a plurality of VZV antigens, or 3) a mixture of 1) 2) mRNA encoding other non-VZV antigens.

The term 5' UTR (5 ' -untranslated region) herein refers to a specific portion of a messenger RNA (mRNA) that is located 5' of the open reading frame of the mRNA. Typically, the 5' UTR begins at the transcription initiation site and ends one nucleotide before the initiation codon of the open reading frame. The 5' utr may include elements for controlling gene expression, also known as regulatory elements. The regulatory element may be, for example, a ribosome binding site or a 5' -terminal oligopyrimidine sequence. The 5'UTR may be post-transcriptionally modified, for example by the addition of 5' CAP.

The term 3'UTR (3' -untranslated region) herein is the portion of an mRNA that is located between the protein coding region (i.e., the open reading frame) and the poly (A) sequence of the mRNA. The 3' UTR of mRNA is not translated into amino acid sequences. The 3' utr sequence is typically encoded by a gene that: the gene is transcribed into the corresponding mRNA during gene expression. The genomic sequence is first transcribed into mature pre-mRNA, which includes optional introns. The pre-mature mRNA is then further processed into mature mRNA during the maturation process. The maturation process comprises the following steps: 5' capping, splicing of the mature pre-mRNA to cleave off optional introns, and modification of the 3' end, such as polyadenylation of the 3' end of the mature pre-mRNA and optional endo-or exonuclease cleavage, etc.

The term "Poly (a)" herein refers to a stretch of (long) adenosine nucleotides added at the 3' end of an RNA of up to about 400 adenosine nucleotides, for example, about 25 to about 400, preferably about 50 to about 400, more preferably about 50 to about 300, even more preferably about 50 to about 250, most preferably about 60 to about 250 adenosine nucleotides.

The term "mutation" herein includes genetic mutations and amino acid mutations, wherein a genetic mutation refers to a deletion, insertion, inversion or substitution of a heterologous nucleic acid, which may result in an alteration of the amino acid sequence in the corresponding protein product; amino acid mutations are also known as nonsensical single nucleotide mutations, because of the change in the amino acid sequence in the protein product due to some single base changes. Amino acid changes affect protein stability, interactions and enzyme activity, resulting in disease.

The terms "protein," "polypeptide," and "peptide" are used interchangeably herein and refer to a peptide-bond-linked chain of any amino acid, whether length or co-translational or post-translational modification. Such definition of a protein polypeptide or protein not encoded on a nucleic acid construct, wherein in particular and additionally such chains are included: the chain comprises one or more unnatural amino acid or amino acid-like structural units.

The term "amino acid substitution" herein refers to those in which at least one amino acid residue in the natural or starting sequence is removed and a different amino acid is inserted into its place at the same position. Substitutions may be single, wherein only one amino acid in the molecule has been substituted, or they may be multiple, wherein two or more amino acids in the same molecule have been substituted.

The term "conservative amino acid substitution" herein refers to the substitution of an amino acid that is normally present in a sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a nonpolar (hydrophobic) residue such as isoleucine, valine and leucine for another nonpolar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, substitution of a basic residue such as lysine, arginine, or histidine for another residue, or substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue, is an additional example of a conservative substitution. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine, and/or the substitution of a polar residue for a non-polar residue.

The term "mutant" herein refers to a "variant" of the protein or peptide that may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity to the amino acid sequence of the protein or peptide.

The term "vector" as used herein refers to a DNA molecule comprising single-stranded, double-stranded, circular or supercoiled DNA. Suitable vectors include retroviruses, adenoviruses, adeno-associated viruses, poxviruses and bacterial plasmids.

The term "antigen" herein refers to a substance that can be recognized by the immune system and is capable of triggering an antigen-specific immune response. Such a response may include the formation of antibodies specific for the antigen, or the activation of T cells specific for the antigen, as part of an adaptive immune response. Typically, the antigen may be or may comprise a peptide or protein that may be presented to T cells by MHC. An antigen in the sense of the present invention may be a translation product of a provided nucleic acid molecule, preferably an mRNA as defined herein. Fragments, variants and derivatives of peptides and proteins comprising at least one epitope are also understood in this context as antigens. The term "carrier protein" as used herein refers to proteins that are non-toxic to humans, do not cause allergic reactions and enhance vaccine immune efficacy, including ferritin (Ferritin), diphtheria toxoid (DT, DT CRM 197) and Tetanus Toxoid (TT), keyhole Limpet Hemocyanin (KLH), OMPC from Neisseria meningitidis (N.menningitidis), pure protein derivatives of tuberculin (PPD), BSA and/or OVA, and the like.

The term "vaccine" herein refers to a prophylactic or therapeutic substance that provides at least one antigen or antigen function, which is capable of stimulating an immune response in the body without causing a disease. The antigen or antigen function may stimulate the adaptive immune system of the body to provide an adaptive immune response.

The terms "nanoparticle" and "nanoparticle" are used interchangeably herein.

The term "immune system" herein may protect an organism from infection. If a pathogen breaks through the physical barrier of an organism and enters the organism, the innate immune system provides an immediate but non-specific response. If the pathogen bypasses the innate response, the vertebrate has a second layer of protection, the adaptive immune system. Here, the immune system alters its response during infection to improve its recognition of pathogens. This improved response is then retained in the form of an immunological memory after the pathogen is eliminated, and allows the adaptive immune system to establish a faster and stronger attack each time the pathogen is encountered. Thus, it can be seen that the immune system comprises two main forms: innate immunity and adaptive immunity. Innate immunity recognizes pathogens through non-specific pattern recognition receptors, involving a variety of humoral and cytokines. Adaptive immunity, in turn, distinguishes pathogens through specific antigen recognition, relying on humoral immunity and cell-mediated immune responses.

The term "immune response" herein may typically be a specific response of the adaptive immune system against a specific antigen (so-called specific or adaptive immune response) or a non-specific response of the innate immune system (so-called non-specific or innate immune response). One basis of the present invention relates to the specific response of the adaptive immune system (adaptive immune response); in particular an adaptive immune response following exposure to an antigen (such as an immunogenic polypeptide). However, this specific response may be supported by an additional non-specific response (innate immune response). Thus, one basis of the present invention also relates to compounds for stimulating both the innate and adaptive immune systems simultaneously to elicit an effective adaptive immune response. In the context of the present invention, "antigen composition" refers to a compound or mixture of compounds (such as in a solution or pharmaceutical formulation): which is capable of, is used for, is capable of, or in practice can elicit, augment, generate, or elicit an immune response (preferably, an effective adaptive immune response) upon administration to or otherwise exposure to a subject.

The term "cellular immunity/cellular immune response" herein refers to an immune response that involves primarily macrophages, natural killer cells, antigen-specific T cells, and various immune cytokines. Cellular immunity does not utilize antibodies, but rather works by activating immune cells. For example, it can activate antigen-specific cytotoxic T lymphocytes, which are capable of recognizing and killing cells infected with viruses or bacteria, as well as tumor cells in vivo; it can also activate macrophages and natural killer cells to clear and kill pathogens; in addition, cellular immunity can also stimulate immune cells to secrete a variety of cytokines that can regulate the function of adaptive immunity, innate immunity, and other cells.

The term "humoral immunity/humoral immune response" herein typically refers to antibody production and possibly the ancillary processes that accompany it. For example, humoral immune responses may typically be characterized by Th2 activation and cytokine production, germinal center formation and allotypic switching, affinity maturation and memory cell production. Humoral immunity also typically may refer to effector functions of antibodies, including pathogen and toxin neutralization, classical complement activation and phagocytosis, and opsonization of pathogen elimination.

The term VZV herein, i.e., varicella-zoster virus, varicella-zoster virus, belongs to the family Herpesviridae (Herpesviridae), the subfamily alpha-Herpesviridae (alpha-hermesviridae), the genus varicella (Varicellovirus).

The term "internal aqueous phase" as used herein means that during the preparation of the lipid nanoparticle, the nucleic acid is dissolved in an aqueous medium, i.e. the internal aqueous phase, prior to mixing the nucleic acid with the lipid component to form the nanoparticle.

The term "external aqueous phase" as used herein means that, during the preparation of the lipid nanoparticle, after mixing the nucleic acid with the lipid component to form the nanoparticle, the nanoparticle is in an aqueous medium (such as a buffer), which is the external aqueous phase.

The term ALC-0315 as used herein is a structural compound with CAS number: 2036272-55-4, the structural formula is as follows:

Drawings

FIG. 1 schematic design of self-replicating repRNA-VZV constructs.

FIG. 2 is a flow chart of detection of LNP-XR003-1, LNP-XR003-2, and LNP-XR003-4 expression.

FIG. 3 shows the expression of LNP-XR003-5, LNP-XR003-6 and LNP-XR003-7 before and after the addition of protein transport inhibitor.

FIG. 4 shows the mean fluorescence intensity of LNP-XR003-5, LNP-XR003-6 and LNP-XR003-7 before and after the addition of the protein transport inhibitor.

FIG. 5 is an ELISA for detection of XR003-1 and XR003-2 group mice gE antibody levels.

FIG. 6 shows ELISPOT detection of IFN-gamma levels in mice of groups XR003-1 and XR 003-2.

FIG. 7 is a flow chart of INF-gamma, IL-2 and TNF-alpha levels released by CD4+ T cells from mice of groups XR003-1 and XR 003-2.

FIG. 8 is an ELISA for detection of XR003-5 to XR003-7 group mice gE antibody levels.

FIG. 9 shows ELISPOT detection of IFN-gamma levels in mice groups XR003-5 through XR 003-7.

FIG. 10 is a flow chart of INF-gamma, IL-2 and TNF-alpha levels released by CD4+ T cells from mice of groups XR003-5 through XR 003-7.

FIG. 11 shows ELISA detection of XR003-4 group mice gH antibody levels.

FIG. 12 shows ELISPOT detection of IFN-gamma levels in mice of group XR 003-4.

FIG. 13 is a flow chart of INF-gamma, IL-2 and TNF-alpha levels released by CD4+ T cells from mice of group XR 003-4.

FIG. 14 is a flow chart of INF-gamma, IL-2 and TNF-alpha levels released by CD8+ T cells from mice of group XR 003-4.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The present embodiments are merely examples and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.

Example 1XR003-1 to XR003-7 construct designs

6 MRNA molecules were designed using the RNA self-replication platform (NSP 1-4 nucleic acid sequence shown in SEQ ID NO: 1) as shown in FIG. 1, wherein:

1.XR003-1

The VZV-OKA strain glycoprotein gE is selected as an antigen, the C terminal 50 amino acids are deleted, and mutation Y569A is introduced, wherein the nucleotide and amino acid sequences are shown as SEQ ID NO. 2-3.

2.XR003-2

XR003-2 is connected with Flt3L gene through P2A at N end of XR003-1, and nucleotide and amino acid sequences are shown in SEQ ID NO. 4-5.

3.XR003-4

XR003-4 lacks 8AA at the C end of gH protein, the N end is connected through the C end of P2A gL, his tag is added at gLC end for protein expression detection, and the nucleotide and amino acid sequences are shown in SEQ ID NO. 6-7.

4.XR003-5

XR003-5 inserts signal peptide into N end of gE protein, and lacks gE transmembrane domain and intracellular region, and its nucleotide and amino acid sequences are shown in SEQ ID NO. 8-9.

5.XR003-6

XR003-6 is connected with human mitochondrial ferritin through (G4S) 4 at the C end of XR003-5 protein sequence, and the nucleotide and amino acid sequences are shown in SEQ ID NO 10-11.

6.XR003-7

XR003-7 is connected with human heavy chain ferritin through (G4S) 4 at the C end of XR003-5 protein sequence, and the nucleotide and amino acid sequences are shown in SEQ ID NO. 12-13.

TABLE 1 sequence information Table

Note that: the nucleotide sequences in the tables are indicated by ATGC. Those skilled in the art know that ATGC when the nucleotide is DNA; AUGC when transcribed into mRNA.

EXAMPLE 2 preparation of XR003-1 to XR003-7 stock solutions

The 6 construct plasmids designed in example 1 were subjected to enzymatic tangentially and mRNA was prepared by In Vitro Transcription (IVT), capping and purification. The method comprises the following specific steps:

2.1 plasmid linearization cleavage

The six XR003 plasmids were subjected to enzymatic cleavage and purification according to conventional plasmid linearization cleavage and purification methods in the art, and subjected to capping and purification by in vitro co-transcription to prepare mRNA.

Plasmid linearized cleavage was performed using NotI (NEB, R3189L) or BspQ I (Nanjinovazan, DD 4302-03) under conventional systems. After the enzyme digestion, 1% agarose gel electrophoresis and a gel imager are carried out to observe whether the enzyme digestion of the sample is complete.

2.2 In vitro transcription and purification

An in vitro transcription system was prepared using linearized plasmid templates according to methods conventional in the art, incubated at 37℃for 2h, subjected to in vitro transcription, then digested at 37℃for 15min with DNase I, and the DNA template removed, followed by purification of mRNA by ion exchange chromatography media. Gel electrophoresis was performed on the purified RNA, resulting in: the size of all RNA molecules after electrophoresis was consistent with that expected, confirming that RNA was successfully prepared.

2.3MRNA integrity detection

2.3.1 Sample treatment: mRNA dilution with 1 XTE buffer

2.3.2 Adding the RNA diluent standard into a 96-well sample plate, adding diluted sample solution and RNA LADDER solution, sealing by using a sealing plate film, and centrifuging for 2 minutes at 3000 rpm/min. Mix well for 2 minutes with shaking at 2000 rpm. RNA integrity was obtained using a 5200Fragment Analyzer assay.

2.4 Detection of RNA concentration

Ribogreen dyes (ThermoFisher, R11490) were mixed with 1×te buffer for use. RNA standards were diluted with 1 XTE. 100. Mu.L of mRNA sample to be detected was added to a 96-well plate, followed by addition of 100. Mu. L Ribogreen dye solution and placed on a plate shaker for 5 minutes. And detecting by using a SpectraMax iD3 multifunctional enzyme-labeled instrument. Total RNA concentration was calculated using a standard curve.

Concentration and integrity measurements were performed on the transcribed and purified XR003 mRNA. The integrality of six mRNA in XR003 exceeds 85%, the concentration is between 2.4mg/ml and 2.5mg/ml, and the subsequent preparation requirements of LNP can be met.

EXAMPLE 3 preparation of XR003-1 to 7 constructs LNP

TABLE 2 materials and reagents

Sample name	Sample name
		ALC0315	Quant-it^TMRiboGreen RNA Assay Kit
ALC0159	1 XTE buffer
		Cholesterol	Triton X-100
DSPC	Spike protein liquid

3.1LNP/mRNA preparation

The lipid nanoparticle is prepared by a microfluidic method.

Preparing an aqueous phase: the RNA stock solutions (XR 003-1, XR003-2, XR003-3, XR003-4, XR003-5, XR003-6, XR 003-7) were treated with 50mM potassium dihydrogen phosphate solution pH4.5;

Organic phase preparation: the lipid was diluted with absolute ethanol (ALC 0315: ALC0159: DSPC: cholesterol, molar ratio 47.4%:1.8%:9.9%: 40.8%).

The aqueous phase and the organic phase are subjected to micro-fluidic, the prepared LNP is diluted by PBS diluent, an ultrafiltration centrifuge tube is used for concentration, liquid exchange, constant volume, split charging by a microporous filter membrane with the size of 0.22 mu m in a biosafety cabinet, and after frozen for 24 hours at the temperature of minus 60 ℃, particle size, polydispersity index, zeta potential, total concentration of mRNA, encapsulation rate and mRNA integrity detection are carried out.

3.2LNP/mRNA preparation particle size, PDI and Zeta potential detection

LNP/mRNA preparation particle size and potential were measured using Malvern Zetasizer ultra. mu.L of the solution containing LNP particles was diluted to 1mL with water for injection, and placed in a detection cell to detect the particle size, PDI and Zeta potential of LNP/mRNA preparation particles.

3.3 Detection of Total RNA concentration and encapsulation efficiency in LNP/mRNA preparation

Total RNA concentration and encapsulation efficiency in LNP/mRNA preparations were measured by the RiboGreen method. The LNP/mRNA preparation solution was demulsified with triton X-100, 100. Mu.L of the demulsified solution was added to a 96-well plate, and then 100. Mu.L of the riboGreen dye solution was added, and the solution was placed on a plate shaker and shaken for 5 minutes at 600rpm. And detecting by using a SpectraMax iD3 multifunctional enzyme-labeled instrument. Total RNA concentration was calculated using a standard curve.

10. Mu.l of LNP/mRNA preparation solution was taken, 990. Mu.l of buffer was added, mixed well, 100. Mu.l of riboGreen dye solution was added, and placed on a plate shaker, and the shaking was carried out for 5 minutes at 600rpm. And detecting by using a SpectraMax iD3 multifunctional enzyme-labeled instrument. The free RNA concentration was calculated using a standard curve. The encapsulation efficiency was then calculated according to the following formula.

Encapsulation (%) = 100% - (free mRNA concentration/total RNA concentration)%

3.4 Detection of mRNA integrity in LNP/mRNA preparation

MRNA integrity detection: 10% of triton X-100 is added into LNP/mRNA sample to carry out demulsification, thus obtaining broken emulsion. Diluting to a concentration of about 8 ng/. Mu.l by using a1 XTE buffer solution, uniformly mixing, denaturing at 70 ℃ for 2min, rapidly transferring to ice for preservation, then adding a 96-well sample plate, sealing by using a sealing plate film, and centrifuging to remove bubbles. And detecting by using an Agilent 5200 fragment analyzer to obtain the mRNA integrity.

3.5 Detection results

The mass measurements of the seven LNP formulations LNP/XR003-1, LNP/XR003-2, LNP/XR003-3, LNP/XR003-4, LNP/XR003-5, LNP/XR003-6, LNP/XR003-7 are shown in the following table. The indexes of LNP/XR003-1, LNP/XR003-2, LNP/XR003-3, LNP/XR003-4, LNP/XR003-5, LNP/XR003-6 and LNP/XR003-7 prepared by the representative process meet the standard specification. The particle size of the 7 preparations is 64-69 nm, PDI is 0.084-0.153, electric potential is-5 mV-7 mV, total RNA concentration is 0.32-0.45 mg/ml, encapsulation rate is above 92%, and mRNA integrity is 65% -70%.

TABLE 3 detection results

Name of the name

Particle size

PDI

Potential of

Total RNA concentration

Encapsulation efficiency

MRNA integrity

LNP/XR003-1

66nm

0.084

-5mV

0.45mg/ml

93％

68％

LNP/XR003-2

69nm

0.153

7mV

0.42mg/ml

92％

65％

LNP/XR003-4

64nm

0.098

7mV

0.32mg/ml

92％

67％

LNP/XR003-5

64nm

0.111

5mV

0.41mg/ml

92％

70％

LNP/XR003-6

66nm

0.123

6mV

0.38mg/ml

92％

68％

LNP/XR003-7

65nm

0.101

6mV

0.37mg/ml

92％

66％

Example 4 detection of expression of LNP-XR003-1 to 7 in 293T cells

293T cells were aliquoted into 24 well plates, cultured overnight, 100. Mu.l/well was aspirated when the cell fusion should be greater than or equal to 80%, opti-MEM medium (gibco, cat# 31985-062) was placed into sterile centrifuge tubes, 1. Mu. gLNP-XR003-1, 2, 4, 5, 6, 7 molecules were added to each well, mixed well, 293T cells were added, XR003-5 to XR003-7 transfected 2 parts, 1. Mu.l of the eBioscience ^TM protein transport inhibitor mixture (Thermo fisher, cat# 00-4980-03) was added in one part, and sample treatment and expression measurement were performed after 48 h.

As shown in FIG. 2, the gE expression can be detected by all the XR003-1 to XR003-3 molecules, the XR003-4 gL protein can be expressed on a membrane, and the XR003-5 to XR003-7 molecules can be secreted outside the cell after the transmembrane region sequence is removed; as shown in fig. 3 and 4, the protein transport inhibitor mixture prevents transport of proteins outside the cell, and is effective to increase intracellular gE protein accumulation upon addition of the inhibitor.

Example 5XR003-1 and 2 molecular mouse immunogenicity detection

The animals groups referred to in example 5

SPF-class female 6-8 week-old C57BL/6 mice were randomly divided into 3 groups of 6 animals each, grouped as follows:

a first group: LNP-XR003-1 1. Mu.g/dose

Second group: LNP-XR003-2 1. Mu.g/dose

Third group: 50 μl PBS

Each group was vaccinated with 1 μg (50 μl) of vaccine, and 2 immunizations were performed at 4 weeks intervals, and ELISA detection of gE antibody levels was performed by taking blood on days D14, D28, and D47, respectively, and spleen of mice was collected on day D47 for IFN-. Gamma.ELISPOT detection and cytokine detection.

5.1ELISA method for detecting gE antibody level in mouse serum

96-Well ELISA plates were coated with VZV-gE (0.5. Mu.g/ml) overnight at 4 ℃. Plates were washed 5 times with 260 μl/well PBST and blocked with 3% BSA in PBST for 2 hours at 37deg.C. Immunized mouse serum was serially diluted twice and added to each well, incubated at 37 ℃ for 1 hour, plated 5 times with PBST, and dried by pipetting with HRP-labeled goat anti-mouse IgG. TMB was added to develop the color for 10min, ELISA stop solution was added to stop the reaction. The absorbance at 450nm of each well is read by an enzyme-labeled instrument, and the sample OD450 is equal to or greater than the maximum dilution corresponding to the average value of all negative control samples multiplied by 2.1.

As shown in fig. 5, the differences in the levels of gE antibodies were not significant in the XR003-1 and XR003-2 groups D14, D28, and the levels of gE antibodies to D47 were significantly increased after D28 days of booster immunization; d47 Both XR003-1 and XR003-2 groups had higher antibody levels, with the XR003-2 group being slightly higher than the XR003-1 group, but the difference was not significant.

5.2ELISPot detection of IFN-gamma levels secreted by mouse spleen cells

Incubation of spleen cell suspensions (2X 10 ⁵ cells) with PRIM 1640 medium (negative control), gE peptide pool (6.67. Mu.g/ml), or ConA (6.67. Mu.g/ml, positive control) for 16-20h was performed using the mouse IFN-. Gamma.ELISPOT kit (Abcam ab 64029) according to the manufacturer's instructions. The number of spots was determined using an enzyme-linked immunosorbent assay (Cellular Technology Limited, S6 Entry). The prevention of shingles requires intense VZV-specific cellular immunity, which is characterized by IFN- γ secretion. T cells are responsible for producing IFN- γ in adaptive immunity, which means that the level of IFN- γ produced by a herpes zoster vaccine may reflect its ability to induce specific cellular immunity. To study VZV-specific cellular immunity, the enzyme-linked immunosorbent spot assay stimulated the number of IFN- γ secreting splenocytes with the gE protein peptide pool on day 47.

As shown in FIG. 6, both XR003-1 and XR003-2 had higher levels of IFN-gamma secretion, and in addition XR003-2 induced higher specific cellular immunity when splenocytes were stimulated with the gE peptide pool than XR003-1, but the difference was not significant.

5.3 Intracellular cytokine detection

1.5X10 ⁶ spleen cells were incubated with 0.2. Mu.g of gE protein peptide library or 1 XeBioscience ^TM cell stimulating mixture (Thermo, 00-4970-03) at 37℃for 1h followed by addition of 1 XeBioscience ^TM protein transport inhibitor mixture (Thermo, 00-4980-93) and incubation in a 5% carbon dioxide incubator for 5h to block cytokine release.

Cell surface marker staining: cells were blocked with 50. Mu.l of 4% TruStain FcX ^TM anti-mouse CD16/32 (Biolegend, 101320) in PBS and incubated at 4℃for 8-10min, 50. Mu.l of 1:500LIVE/DEAD ^TM A mixture of pale green DEAD cell stain/L34957 (Thermo, L34957) with 2% CD45 (bioleged, 103116), CD8 (bioleged, 100734), CD4 (bioleged, 100422) direct fluorescent antibody in PBS was incubated at 4℃for 20min.

Cytokine staining: the mixture was fixed at IC Fixation Buffer (thermo Fisher, 00-8222-49) for 20min at 4 ℃. Membrane-through staining was performed with 100. Mu.l (containing 1% IL-2 (Biolegend, 503808), IFN-. Gamma. (Biolegend, 505830), TNF-. Alpha. (Biolegend, 506304) direct-labeled fluorescent antibody) 1X Permeabilization Buffer (ThermoFisher, 00-8333-56) and incubated at 4℃for 20min in the absence of light. After staining, the cells were subjected to loop gating (forward and side scatter, FSC/SSC) and samples were analyzed using Attune NxT acoustic focusing flow cytometry (Thermo, 2AFC 236901121).

As a result, as shown in FIG. 7, CD4 ⁺ cells, after stimulation with gE peptide library, can specifically secrete Th1 type cytokines IFN-gamma, IL-2 and TNF-alpha, and XR003-2 is superior to XR003-1.

Based on the experimental data, XR003-1 and XR003-2 can both induce corresponding antibodies to generate better, and have better immunogenicity, and the addition of FIT3L ligand has a certain degree of improvement on the immunogenicity.

EXAMPLE 6XR003-5 to 7 molecular mouse immunogenicity

The animals group referred to in example 6

SPF-class female 6-8 week-old C57BL/6 mice were randomly divided into 4 groups of 6 animals each, each group being as follows:

A first group: LNP-XR003-5 1. Mu.g/dose

Second group: LNP-XR003-6 1. Mu.g/dose

Third group: LNP-XR003-7 1. Mu.g/dose

Fourth group: 50 μl PBS

6.1ELISA method for detecting gE antibody level in mouse serum the experimental method is the same as that of 5.1, and the results are shown in FIG. 8, D14 and D28 immune groups show that gE antibody level is not obviously different, gE antibody level to D47 is obviously increased after D28 days of booster immunization, and XR003-5 to XR003-7 groups show that the XR003-5 group has the highest antibody level.

6.2ELISPot the same experimental procedure as 5.2 for detecting INF-gamma secretion from spleen cells of mice, and the results are shown in FIG. 9, XR003-5, XR003-6, XR003-7, in which gE peptide library-specific cellular immunity was generated and the number of IFN-gamma spots in XR003-6 group was the greatest.

6.3 Intracellular cytokine detection experiments were performed in the same manner as 5.3, and as shown in FIG. 10, after stimulation with gE peptide library, the vaccine group and the control group, CD4+ T cells can specifically secrete Th1 type cytokines IFN-gamma, IL-2 and TNF-alpha, wherein the cell proportion of IFN-gamma, IL-2 and TNF-alpha secreted by the XR003-6 group CD4 ⁺ T cells is the highest.

Based on the experimental data, XR003-5 to XR003-7 can induce corresponding antibodies to generate better, have better immunogenicity, and the added mitochondrial ferritin has a certain degree of improvement on the immunogenicity.

EXAMPLE 7XR003-4 molecular mouse immunogenicity

Animal grouping in example 7

SPF-class female 6-8 week-old C57BL/6 mice were randomly divided into 2 groups of 6 animals each, grouped as follows:

a first group: LNP-XR003-4 1. Mu.g/dose

Second group: 50 μl PBS

The first group was vaccinated with 1 μg (50 μl) of vaccine, and immunized 2 times at 4 weeks intervals, ELISA was performed to detect the gH antibody level by taking blood on days D14, D28, and D47, and spleen of the mice was collected on day D47 for IFN-. Gamma.ELISPOT detection and intracellular cytokine detection, respectively.

7.1ELISA method for detecting gH antibody level in mouse serum

96-Well ELISA plates were coated overnight with VZV-gH protein (4. Mu.g/ml) at 4℃and tested in the same manner as 5.1. As a result, as shown in FIG. 11, XR003-4 group produced gH-specific antibodies, which had slightly higher antibody titer at D28 than at D14, and significantly increased gH antibody levels in XR003-4 group after booster immunization;

7.2ELISPot detection of INF-gamma levels secreted by mouse spleen cells

Experimental methods were similar to 5.2, and as shown in FIG. 12, XR003-4 immunized mice produced a high level of gH-specific Th1 type cellular immune response;

7.3 intracellular cytokine detection

The experimental method is the same as 5.3, and the results are shown in FIG. 13 and FIG. 14, and CD4 ⁺ and CD8 ⁺ T cells (cell surface CD8 antibody-derived Biolegend, 100734) can specifically secrete Th1 type cytokines IFN-gamma, IL-2 and TNF-alpha after being stimulated by the gH peptide library;

Based on the experimental data, XR003-4 can induce humoral immunity and cellular immune response well, and the induced cellular immune response comprises two T cell types of CD4 ⁺ and CD8 ⁺, so that the cell type has good immunogenicity.

Claims

1. A pharmaceutical composition comprising an mRNA molecule construct and a delivery formulation; preferably, the mRNA construct comprises mRNA encoding the gE and/or gH, gL regions of VZV; more preferably, the mRNAs encoding the gE and/or gH, gL regions of VZV are expressed alone or in fusion in a host cell.

2. The pharmaceutical composition of claim 1, wherein the mRNA encodes a protein further comprising a mutation; optionally, the mRNA encodes a protein that further comprises Flt3L; optionally, the mRNA encodes a protein further comprising a signal peptide sequence; optionally, the signal peptide is located at the N-terminus of the protein; preferably, the signal peptide is derived from murine H-2Kb, human IgE, HLA-B46, MICA 008, OSM, VSV-G, mouse Ig Kappa, mouse heavy chain, BM40, human chymotrypsinogen, human prothrombin-2, human IL-2, human G-CSF, human hemagglutinin IX, human albumin, gaussia luc, HAS, influenza virus, human insulin, silk LC, erenumab antibody light chain, pembrolizumab light chain, ramucirumab light chain, E SIGNAL PEPTIDE, SP1 (LZJ human IgG1, SP2, SP3 (ZLQ).

3. The pharmaceutical composition of claim 1 or 2, wherein the gE extracellular region (gE Ecto) protein binds to ferritin (Ferritin) to form a fusion protein; preferably, gE Ecto-ferritin fusion proteins comprise the unit subunit of ferritin; alternatively, the ferritin subunit is a full length or any portion of ferritin, wild type or partial amino acid mutation; alternatively, the monomeric subunit is from mitochondrial ferritin or heavy chain ferritin.

4. A pharmaceutical composition according to any one of claims 1 to 3, wherein the protein encoded by the mRNA comprises an amino acid sequence having at least 90%, 95%, 99%, 100% identity to the amino acid sequence as set forth in SEQ ID No. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID No. 13.

5. The pharmaceutical composition of any one of claims 1-4, wherein the mRNA comprises a coding nucleotide corresponding to the amino acid sequence set forth in SEQ ID No. 3,SEQ ID NO:5,SEQ ID NO:7,SEQ ID NO:9,SEQ ID NO:11 or SEQ ID No. 13.

6. The pharmaceutical composition of any one of claims 1-5, wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID No. 2,SEQ ID NO:4,SEQ ID NO:6,SEQ ID NO:8,SEQ ID NO:10 or SEQ ID No. 12.

7. The pharmaceutical composition of any one of claims 1-6, wherein the mRNA construct further comprises a 5'utr and/or a 3' utr sequence; optionally, the 5' UTR sequence is a human alpha globulin 5' UTR or a non-native 5' UTR sequence; optionally, the mRNA construct further comprises a Poly (a) sequence.

8. The pharmaceutical composition of any one of claims 1-7, wherein the mRNA construct further comprises an alphavirus nonstructural protein (nsp) gene; optionally, the nsp gene is shown as SEQ ID NO. 1.

9. The pharmaceutical composition of any one of claims 1-8, wherein the delivery formulation is a nanoparticle; preferably, the delivery formulation comprises lipid nanoparticles (Lipid nanoparticle, LNP), lipid multipolymer (lipopolyplex, LPP), polymer nanoparticles (Polymer nanoparticles, PNP), inorganic nanoparticles (Inorganic nanoparticles, INP) cationic nanoemulsions (Cationic nanoemulsion, CNE), exosomes, biologicals microvesicles, protamine, etc.; more preferably, the nanoparticle is a lipid nanoparticle (Lipid nanoparticle, LNP); more preferably, the lipid comprises one or more of a cationic lipid, a neutral helper lipid, cholesterol, and a PEG-modified lipid; more preferably, two or more are included.

10. The pharmaceutical composition of any one of claims 1-9, wherein the lipid nanoparticle comprises: a) a cationic lipid, b) a neutral helper lipid, c) cholesterol, and d) a PEG-modified lipid; preferably, in the lipid nanoparticle, the molar ratio of each lipid component is as follows, based on 100% of the total molar amount of lipid: 45% -50% of a cationic lipid, 5% -10% of b) a neutral auxiliary lipid, 38% -48% of c) cholesterol and 0% -3% of d) PEG modified lipid.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the molar ratio of the cationic lipid is 45% to 48% based on 100% of the total molar amount of lipid; preferably, the cationic lipid is selected from the group consisting of N, N-dimethyl-2, 3-dioleoyloxypropylamine (DODMA), 1, 2-dimyristoxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N, N-dioleoyln, N-dimethylammonium chloride (DODAC), 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N- (l- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA), 1, 2-dimethanooxy-N, N-dimethylaminopropane (DLinDMA), 2-dioleylene-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLIn), N- (2, N-dimethyl-MC-4- (DLMC-2-DMA), or any one of the methyl-2- (3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP), N- (1- (2, 3-dioleyloxy) propyl) -N, N-trimethyl ammonium chloride (DLMA).

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the molar ratio of neutral helper lipids is 6% to 10% based on 100% total molar amount of lipids; preferably, the molar ratio of the neutral auxiliary lipid is 8% -10%; optionally, the neutral helper lipid is selected from any one or more of distearoylphosphatidylcholine(DSPC)、dioleoylphosphatidylcholine(DOPC)、dimyristoylphosphatidylcholine(DMPC)、dipalmitoylphosphatidylcholine(DPPC)、diarachidoylphosphatidylcholine(DAPC)、dibehenoylphosphatidylcholine(DBPC)、ditricosanoylphosphatidylcholine(DTPC)、dilignoceroylphatidylcholine(DLPC)、dioleoylphosphatidylethanolamine(DOPE)、dipalmitoyl-phosphatidylethanolamine(DPPE)、dimyristoyl-phosphatidylethanolamine(DMPE) or phosphatidylethanolamine (DLPE).

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the PEG-modified lipid is present in a molar ratio of 1% to 3% based on 100% total molar amount of lipid; preferably, the molar ratio of the PEG modified lipid is 1% -2%; optionally, the PEG modified lipid is selected from any one or more of methoxy polyethylene glycol bitetradecylacetamide (ALC-0159), MG-PEG2000, DMG-PEG5000 and PEG 2000.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the molar ratio of cholesterol is 40% to 46% based on 100% of the total molar amount of lipid; preferably, the molar ratio of the cholesterol is 42% -45%.

15. A vaccine comprising the pharmaceutical composition of any one of claims 1-14; preferably, the vaccine is a VZV vaccine.

16. An mRNA construct as an immunogenic component in the medicament of any one of claims 1-14 or the vaccine of claim 15, or a combination thereof.

17. A vector comprising the mRNA construct of claim 16.

18. A nanoparticle in a pharmaceutical composition as claimed in any one of claims 9 to 14.

19. A fusion protein comprising a protein encoded by the mRNA construct of claim 16 and a second protein fused thereto; optionally, the protein encoded by the mRNA is an unfused protein; preferably, the second protein is a carrier protein; more preferably, the second protein is ferritin (Ferritin); more preferably, the ferritin is mitochondrial ferritin or heavy chain ferritin; more preferably, the ferritin comprises a domain that allows the fusion protein to self-assemble into a nanoparticle.

20. Use of the pharmaceutical composition of any one of claims 1-14, the vaccine of claim 15, the mRNA construct of claim 16, the vector of claim 17, the nanoparticle of claim 18 in the preparation of a medicament for inducing an immune response in a subject in need thereof.

21. Use of the pharmaceutical composition of any one of claims 1-14, the vaccine of claim 15, the mRNA construct of claim 16, the vector of claim 17, the nanoparticle of claim 18 in the manufacture of a medicament for the treatment or prevention of a VZV-related disease in a subject in need thereof.

22. A method of inducing an immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of any one of claims 1-14 or the vaccine of claim 15.

23. A method of treating or preventing a VZV-related disease in a subject in need thereof, wherein the pharmaceutical composition of any one of claims 1-14 or the vaccine of claim 15 is administered to the subject.