Separated protein and application thereof

Abstract

The invention relates to the field of biological medicine. Specifically, the invention relates to an isolated protein, and the isolated protein is a variant protein of a VZV gE extracellular domain. The invention also relates to nucleic acid molecules encoding the isolated proteins, vectors, host cells, engineered VZV viruses comprising the isolated proteins, compositions, and their use for the prevention and / or treatment of conditions caused by VZV infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedicine. In particular, the present application relates to an isolated protein which is a variant protein of the extracellular domain of VZV gE. The present application also relates to a nucleic acid molecule encoding said isolated protein, a vector, a host cell, an engineered VZV virus comprising said isolated protein, a composition, and their use for preventing and / or treating a condition caused by VZV infection. BACKGROUND

[0002] Varicella-zoster virus (VZV) is transmitted by respiratory secretions and close contact and can cause two different clinical conditions, varicella (chickenpox) and zoster (shingles) [1,2] . Varicella is caused by the primary infection of VZV and mainly affects children who have no immunity, and has a typical spring-winter seasonality [3] . Subsequently, the VZV genome remains in the dorsal root or cranial ganglion for life [4] . When the body's immunity is reduced, the latent virus is reactivated to cause zoster [5,6] . The incidence rate increases significantly after the age of 50, with an incidence rate of about 3‰ per year at the age of 50, about 10‰ per year at the age of 80, and up to 10% to 15% in cases of low body immunity [7,8] . There is currently no cure for shingles, so vaccines are the best and necessary preventive measure.

[0003] VZV belongs to the alpha subfamily of herpesviruses and is a double-stranded DNA virus. The genome is about 125 kb and contains at least 70 open reading frames to encode protein molecules involved in biological activities related to virus replication, transcription, packaging, release, etc., as well as eight glycoproteins including gB, gC, gE, gH, gI, gK, gL, and gM; these glycoproteins play a very important role in the maturation and packaging of viruses [9] . Among them, gE is an essential glycoprotein for the generation of infectious VZV particles, and is the most antigenic and abundant on the viral envelope and infected cell membrane, induces specific humoral and cellular immunity in the body, can protect the body from viral attack, and is the main candidate antigen for preparing a subunit vaccine [10-12]The Oka strain's glycoprotein E (gE) (UniProtKB / Swiss-Prot:Q9J3M8.1) is a type I transmembrane protein containing 623 amino acids, of which 1-30 aa is a signal peptide, 1-538 aa is an extracellular domain, 539-559 aa is a transmembrane domain, and 560-623 aa is an intracellular domain. Its molecular weight is 60-100 kDa, and N-glycosylation sites are present at amino acids 266 and 437.

[0004] Currently, there are only four commercially available vaccines, three of which are from overseas: Merck's live attenuated vaccine, Zostavax, and others.

[13] GSK's subunit recombinant vaccine, Shingrix

[14] Other options include SkyZoster from SK Chemicals and Ganwei from my country's BC Biotechnology. Shingrix, approved by the FDA in 2017, is a subunit vaccine composed of gE glycoprotein (the most antigenic and abundant protein in VZV) and the novel adjuvant AS01B. Phase III clinical trial data showed that this subunit vaccine had better immunogenicity and efficacy than Zostavax in the elderly, making it suitable for people aged 50 and above.

[14] Although the vaccine was launched in China in 2019, there are currently no domestically developed recombinant subunit shingles vaccines on the market in China, indicating that subunit shingles vaccines have a huge market potential in China. Summary of the Invention

[0005] In this application, the inventors, through in-depth research, constructed an isolated protein. This isolated protein is a mutant of the extracellular domain of the wild-type VZV gE protein, exhibiting significantly enhanced gE protein expression compared to the wild-type VZV gE protein, and possessing good immunogenicity. This led to the following invention.

[0006] isolated protein

[0007] In one aspect, the present invention provides an isolated protein, wherein the isolated protein is a mutant of the extracellular domain fragment of wild-type VZV gE protein, and compared with the extracellular domain fragment of wild-type VZV gE protein, contains the following mutation: the amino acid at position 361 of the extracellular domain fragment of wild-type VZV gE protein corresponding to SEQ ID NO:1 is mutated to serine or threonine.

[0008] In some embodiments, the wild-type VZV gE protein extracellular domain fragment comprises the sequence shown in SEQ ID NO:1.

[0009] In the context of this invention, when referring to the amino acid position of the extracellular domain fragment of the wild-type VZV gE protein, the sequence shown in SEQ ID NO:1 is used for description. For example, the expression "amino acid at position 361" refers to the 361st amino acid and its corresponding position in the sequence shown in SEQ ID NO:1. The corresponding position refers to the position in the sequence to be compared that is equivalent to the specific amino acid position in SEQ ID NO:1 when the sequence to be compared is optimally aligned with SEQ ID NO:1 (i.e., to obtain the highest percentage of identity).

[0010] In some embodiments, the isolated protein comprises a sequence as shown in SEQ ID NO:2.

[0011] Fusion protein

[0012] On the other hand, the present invention provides a fusion protein comprising the isolated protein of the present invention and a signal peptide, wherein the signal peptide is located at the N-terminus of the isolated protein.

[0013] Typically, a signal peptide is a polypeptide sequence to which a linked sequence is targeted to a desired site in the cell. In some embodiments, the signal peptide is selected from: HAS, Modified HAS, tissue-type plasminogen activator (t-PA), Modified t-PA-1, Modified t-PA-2, Modified t-PA-3, or Modified t-PA-4.

[0014] In some embodiments, the signal peptide comprises a sequence as shown in any one of SEQ ID NO:12-20.

[0015] In some embodiments, the signal peptide comprises a sequence as shown in SEQ ID NO:12 or SEQ ID NO:13.

[0016] In some embodiments, the fusion protein comprises a sequence as shown in SEQ ID NO:3.

[0017] In some embodiments, the fusion protein further includes, at its C-terminus, a transmembrane domain and an intracellular domain of the wild-type VZV gE protein. In the context of this invention, "transmembrane domain," "transmembrane domain," and "membrane transdomain" have the same definition and can be used interchangeably.

[0018] In some embodiments, the isolated protein comprises a sequence as shown in SEQ ID NO:11.

[0019] Preparation of isolated proteins

[0020] The isolated proteins of this invention can be prepared by various methods known in the art, such as by genetic engineering methods (recombinant technology) or by chemical synthesis methods (e.g., the Fmoc solid-phase method). The isolated proteins of this invention are not limited by their method of preparation.

[0021] On the other hand, the present invention provides an isolated nucleic acid molecule encoding the isolated protein or fusion protein of the present invention. In some embodiments, the isolated nucleic acid molecule comprises a sequence as shown in any one of SEQ ID NO:4-9.

[0022] On the other hand, the present invention provides a vector (e.g., a cloning vector or an expression vector) containing the isolated nucleic acid molecules of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.

[0023] On the other hand, the present invention provides a host cell comprising the isolated nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell is a mammalian cell, such as a human cell.

[0024] On the other hand, a method for preparing the isolated protein or fusion protein of the present invention is provided, comprising the following steps:

[0025] Under conditions that allow protein expression, host cells are cultured as described above, and the isolated protein or fusion protein is recovered from the cultured host cell culture.

[0026] Modified VZV virus

[0027] On the other hand, the present invention provides a modified VZV virus that expresses the isolated protein or fusion protein described in the present invention.

[0028] The modified VZV virus of this invention will lose its ability to infect cells and animals. As an immunogen for vaccines, it can simulate the immune response process of VZV infection in humans to the greatest extent, potentially generating protective immunity while also having good safety.

[0029] In some embodiments, the genome of the modified VZV virus includes the following modifications: the wild-type gE gene is replaced with a nucleotide sequence encoding either the isolated protein of the present invention or the nucleotide sequence encoding the fusion protein of the present invention.

[0030] On the other hand, the present invention provides an isolated nucleic acid molecule or vector containing a nucleotide sequence encoding the genome of the modified VZV virus described above.

[0031] Composition

[0032] On the other hand, the present invention provides a composition comprising the isolated protein, fusion protein, isolated nucleic acid molecule, vector, host cell, or modified VZV virus described in the present invention; and one or more pharmaceutically acceptable excipients.

[0033] In some embodiments, the composition is an immunogenic composition or a vaccine. In such embodiments, the isolated protein, fusion protein, isolated nucleic acid molecule, vector, host cell, or modified VZV virus described in this invention serves as an immunogen.

[0034] In some embodiments, the composition is a subunit vaccine comprising the isolated or fusion protein described in this invention as an immunogen.

[0035] In some embodiments, the composition is a live attenuated vaccine comprising the modified VZV virus described in this invention as an immunogen.

[0036] In some embodiments, the composition is a nucleic acid vaccine comprising the isolated nucleic acid molecule or carrier described in this invention as an immunogen.

[0037] In some embodiments, the nucleic acid vaccine comprises DNA or RNA. In some embodiments, the DNA or RNA may be naked or encapsulated within a shell having delivery and / or protective functions. In some embodiments, the shell may be the shell of an adenovirus, adeno-associated virus, lentivirus, retrovirus, etc., or other materials synthesized chemically that perform similar functions.

[0038] In some embodiments, the composition comprises an adjuvant. The adjuvant used for co-administration or included in the composition according to the invention should preferably be a potentially safe, well-tolerated, and effective adjuvant in humans. Such adjuvants are well known to those skilled in the art, and non-limiting examples include HA201, QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminum salts such as aluminum phosphate (e.g., AdjuPhos) or aluminum hydroxide, and MF59.

[0039] The immunogenic compositions or vaccines of the present invention can also be combined with other drugs known in the art for the treatment and / or prevention of VZV infection. Therefore, in some embodiments, the compositions of the present invention may also contain antiviral drugs. In some embodiments, the antiviral drug is an antiherpesvirus drug. In some embodiments, the antiviral drug includes acyclovir, valacyclovir, and famciclovir.

[0040] In some embodiments, the immunogenic composition or vaccine of the present invention and the antiviral drug are present as individual components or as a single formulation. In some embodiments, the immunogenic composition or vaccine of the present invention and the antiviral drug are present as a mixed component or as a mixed formulation. In some embodiments, the immunogenic composition or vaccine of the present invention and the antiviral drug may be administered simultaneously, separately, or sequentially.

[0041] The compositions of the present invention (e.g., immunogenic compositions) can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The compositions of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. Furthermore, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), antibacterial water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0042] The compositions of the present invention (e.g., immunogenic compositions) can be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / method of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or method of administration will vary depending on the intended purpose.

[0043] The immunogenic compositions of the present invention should be administered in an amount sufficient to induce an immune response against VZV. The appropriate amount of immunogen can be determined based on the specific disease or condition to be treated or prevented, its severity, the age of the subject, and other individual attributes of the subject (e.g., the general health status of the subject and the robustness of the subject's immune system). The determination of effective dosage is also guided by animal model studies, followed by human clinical trials, and by administration regimens that significantly reduce the occurrence or severity of the target disease symptoms or condition in the subjects.

[0044] application

[0045] The compositions of the present invention (e.g., immunogenic compositions) can be readily used in a variety of therapeutic or preventative applications to treat VZV infection or to elicit an immune response to VZV in subjects. For example, the compositions can be administered to subjects to induce an immune response to VZV, such as inducing the production of broadly neutralizing antibodies against VZV. For subjects at risk of developing VZV infection, the immunogenic compositions of the present invention can be administered to provide prophylactic protection against viral infection.

[0046] Therefore, on the other hand, the present invention provides a method for inducing an immune response against VZV in a subject or for preventing and / or treating symptoms caused by VZV infection in a subject, comprising administering to a subject in need an immunologically effective amount of the isolated protein, isolated nucleic acid molecule, vector, host cell, modified VZV virus, or composition of the present invention.

[0047] In some embodiments, the method may further include administering to the subject other drug combinations for the treatment or prevention of symptoms caused by VZV infection. In some embodiments, the method includes administering antiviral drugs, such as acyclovir, valacyclovir, famciclovir, etc.

[0048] In some implementations, the symptoms caused by the VZV infection are chickenpox and / or shingles.

[0049] For prophylactic application, the isolated proteins, fusion proteins, isolated nucleic acid molecules, vectors, host cells, modified VZV viruses, or compositions (e.g., immunogenic compositions) of the present invention are provided prior to any symptoms, such as before infection. Prophylactic application of the immunogenic composition is intended to prevent or mitigate any subsequent infection, to reduce the expected severity, duration, or extent of symptoms of infection and / or related disease after exposure to or suspected exposure to the virus, or after actual onset of infection. Therefore, in some embodiments, the subject to be treated is a subject with VZV infection or at risk of developing VZV infection, for example, due to exposure to or potential exposure to VZV. Following administration of a therapeutically effective amount of the disclosed therapeutic composition, the subject may be monitored for VZV infection, or symptoms associated with VZV infection.

[0050] For therapeutic applications, the isolated proteins, fusion proteins, isolated nucleic acid molecules, vectors, host cells, modified VZV viruses, or compositions (e.g., immunogenic compositions) of the present invention are provided at or after the onset of symptoms of disease or infection, such as after the onset of symptoms of VZV infection or after a diagnosis of VZV infection.

[0051] On the other hand, the present invention also relates to the use of the isolated proteins, fusion proteins, isolated nucleic acid molecules, vectors, host cells, modified VZV viruses or compositions of the present invention in the preparation of pharmaceutical agents for inducing an immune response against VZV in subjects and / or for preventing and / or treating conditions caused by VZV infection in subjects.

[0052] In some implementations, the symptoms caused by the VZV infection are chickenpox and / or shingles.

[0053] In some implementations, the agent is a vaccine.

[0054] In some implementations, the subject is a mammal, such as a human.

[0055] In some embodiments, the isolated protein, fusion protein, isolated nucleic acid molecule, vector, host cell, modified VZV virus, or composition may be used alone or in combination with other antiviral agents (e.g., acyclovir, valacyclovir, famciclovir, etc.).

[0056] On the other hand, the present invention also relates to the use of the isolated or fusion protein of the present invention for the preparation of a subunit vaccine, said subunit vaccine for inducing an immune response against VZV in a subject and / or for preventing and / or treating symptoms caused by VZV infection in a subject.

[0057] On the other hand, the present invention also relates to the use of the modified VZV virus of the present invention for the preparation of a live attenuated vaccine, said live attenuated vaccine for inducing an immune response against VZV in subjects and / or for preventing and / or treating symptoms caused by VZV infection in subjects.

[0058] On the other hand, the present invention also relates to the use of the isolated nucleic acid molecules or vectors of the present invention for the preparation of nucleic acid vaccines, said nucleic acid vaccines being used to induce an immune response against VZV in subjects and / or to prevent and / or treat symptoms caused by VZV infection in subjects.

[0059] In any of the above embodiments, the symptoms caused by the VZV infection are chickenpox and / or herpes zoster.

[0060] On the other hand, the present invention provides a method for preparing a vaccine for the prevention and / or treatment of symptoms caused by VZV infection, the method comprising the following steps:

[0061] The amino acid at position 391 of SEQ ID NO:10 in the VZV gE protein was replaced with a serine, and the modified protein was expressed in cells.

[0062] In some embodiments, the method further includes replacing the signal peptide of the VZV gE protein with the IL6SP signal peptide.

[0063] In some embodiments, the VZV gE protein comprises the sequence shown in SEQ ID NO:10.

[0064] In some embodiments, the modified protein comprises a sequence as shown in SEQ ID NO:11.

[0065] In some embodiments, the amino acid substitution is achieved by modifying the nucleotide sequence encoding the VZV gE protein through site-directed mutagenesis and expressing the modified nucleotide sequence.

[0066] In some implementations, the symptoms caused by the VZV infection are chickenpox and / or shingles.

[0067] In some embodiments, the method further includes adding an adjuvant to the vaccine. The adjuvant used to prepare the vaccine is preferably an adjuvant that is potentially safe, well-tolerated, and effective in humans. Such adjuvants are well known to those skilled in the art, and non-limiting examples include HA201, QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, aluminum salts such as aluminum phosphate (e.g., AdjuPhos) or aluminum hydroxide, and MF59.

[0068] Terminology Definition

[0069] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, and immunology, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0070] When the terms “for example,” “such as,” “like,” “including,” “contains,” or variations thereof are used herein, these terms will not be considered restrictive terms but will be interpreted as meaning “but not limited to” or “not limited to.”

[0071] Unless otherwise specified herein or clearly contradicted by the context, the terms “an” and “a kind” as well as “the” and similar designations shall be interpreted to cover both the singular and plural in the context of describing the invention.

[0072] As used herein, the term "VZV gE protein" refers to one of the glycoproteins of the varicella-zoster virus (VZV) envelope. VZV glycoproteins are associated with host infectivity and are target antigens for herpesvirus vaccine development. Among them, gE is an important target in VZV vaccine development, and is one of the most abundant glycoproteins in VZV, playing a crucial role in viral replication and transmission between ganglion cells. The sequence of the VZV gE protein is well known to those skilled in the art (see, for example, UniProtKB / Swiss-Prot:Q9J3M8.1).

[0073] As used herein, when referring to the amino acid sequence of the wild-type VZV gE protein extracellular domain fragment, the sequence shown in SEQ ID NO:1 is used. When referring to the amino acid sequence of the VZV gE protein, the sequence shown in SEQ ID NO:10 is used. For example, the statement "the amino acid at position 361 of the wild-type VZV gE protein extracellular domain fragment" refers to the 361st amino acid and its corresponding position in the sequence shown in SEQ ID NO:1. However, those skilled in the art will understand that mutations or variations can be naturally generated or artificially introduced into the amino acid sequence of the wild-type VZV gE protein extracellular domain fragment or the VZV gE protein without affecting its biological function. Therefore, in this invention, the term "wild-type VZV gE protein extracellular domain fragment" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO:1 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity), and when describing a sequence fragment of the wild-type VZV gE protein extracellular domain fragment, it includes not only the sequence fragment of SEQ ID NO:1 but also the corresponding sequence fragment in its natural or artificial variants; the term "VZV gE protein" and similar expressions should include all such sequences, including, for example, the sequence shown in SEQ ID NO:10 and its natural or artificial variants (e.g., variants having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity), and when describing a sequence fragment of the VZV gE protein, it includes not only the sequence fragment of SEQ ID NO:10 but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid at position 361 of the extracellular domain fragment of wild-type VZV gE protein" includes the 361st amino acid residue of SEQ ID NO:1, and the corresponding fragment in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" refers to a fragment located at an equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0074] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0075] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0076] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoIBiol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0077] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0078] As used herein, the term "pharmaceuticalally acceptable excipient" means an excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (such as buffered saline), alcohols and polyols (such as glycerol).

[0079] As used in this article, the term "subunit vaccine" refers to a vaccine made by extracting specific protein structures from bacteria or viruses through chemical decomposition or controlled protein hydrolysis, and then screening out the immunologically active fragments.

[0080] As used herein, the term “nucleic acid vaccine” refers to a vaccine based on DNA or RNA (e.g., plasmids, such as expression plasmids), which optionally also contains an adjuvant.

[0081] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a disease caused by VZV infection) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of disease, stabilizing (i.e., no longer worsening) the state of disease, delaying or slowing the progression of disease, improving or alleviating the state of disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0082] Beneficial effects of the invention

[0083] This invention provides an isolated protein, which is a mutant of the extracellular domain of wild-type VZV gE protein. Compared with wild-type VZV gE protein, it has significantly enhanced gE protein expression and the same good immunogenicity. It can be used for the development of VZV subunit vaccines to prevent VZV infection and / or diseases caused by said infection, and has important clinical value. Attached Figure Description

[0084] Figure 1 Comparison of VZV gE and gE(R361S) protein expression levels. The X-axis represents the group, and the Y-axis represents the protein expression level, expressed as Mean ± SEM.

[0085] Figure 2 Comparison of VZV gE and gE(R361S) specific IgG antibody titers. The X-axis represents the group, the Y-axis represents the anti-gE IgG titer, and the IgG antibody titer for each group is expressed as Mean±SEM.

[0086] Figures 3A-3B Detection of IFN-γ levels in mouse cells 5 weeks after immunization. Figure 3A It uses gE protein as a stimulant. Figure 3B The gE(R361S) protein was used as a stimulant. In the figure, the X-axis represents the group and the Y-axis represents the cytokine level. The cytokine intensity of each group is expressed as Median ± 95% CI.

[0087] Figures 4A-4B Detection of IL-4 levels in mice immunized for 5 weeks. Figure 4A It uses gE protein as a stimulant. Figure 4B The gE(R361S) protein was used as a stimulant. In the figure, the X-axis represents the group and the Y-axis represents the cytokine level. The cytokine intensity of each group is expressed as Median ± 95% CI.

[0088] Sequence information

[0089] Information about the sequences involved in this invention is described in the table below:

[0090] Table 1: Sequence Description

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Detailed Implementation

[0099] The invention will now be described in the following non-limiting embodiments.

[0100] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0101] Example 1: Comparison of VZV gE and gE(R361S) protein expression levels

[0102] 1.1 Construction of VZV gE and gE(R361S) protein expression vectors

[0103] The extracellular domain amino acid sequence (1-538 aa, SEQ ID NO:23) of VZV Oka strain gE protein (UniProtKB / Swiss-Prot:Q9J3M8.1) was retrieved and downloaded from NCBI. Eight additional histidine residues were added to the 3' end. The sequence was synthesized at Jiangsu Saisofe Biotechnology Co., Ltd. and inserted into the pCDNA3.1 vector (provided by Jiangsu Saisofe Biotechnology Co., Ltd.). Then, the signal peptide (1-30 aa, SEQ ID NO:12) in the original sequence was replaced with the IL6SP signal peptide (1-29 aa, SEQ ID NO:13). The 361st amino acid of the extracellular domain amino acid sequence of the gE protein (SEQ ID NO:1), excluding the signal peptide, was mutated from R to S, yielding the gE(R361S) mutant protein (SEQ ID NO:2). The eukaryotic expression vector used in the construction of the VZV gE and gE(R361S) protein expression vectors was the pCGS3.2 vector from Merck. The VZV gE gene and the gE(R361S) mutant gene were constructed into the pCGS3.2 vector, named pCGS3.2-gE and pCGS3.2-gE(R361S), respectively. The PCR primers for the VZV gE gene were analyzed and designed as shown in Table 2. The PCR reaction system was configured according to Table 3, and the target sequence was amplified according to the PCR reaction conditions in Table 4. The pCGS3.2 vector was double-digested with EcoI (NEB, USA) and HindIII (NEB, USA), and the large fragment was recovered using an agarose gel DNA recovery kit (enhanced version) (Tiangen Biotech (Beijing) Co., Ltd., DP219). The PCR-amplified fragments were ligated to the digested vector according to the configuration in Table 5, and then... HiFi DNA AssemblyMaster Mix (NEB, USA) completes the connection process.

[0104] Remove trans5α competent cells (Beijing TransGen Biotech Co., Ltd., China) and place them on ice. Add 10 μL of the ligation product to the freshly thawed competent cells, mix gently, and incubate on ice for 30 minutes. Then, heat shock the cells in a 42°C water bath for 45 seconds, quickly transfer the EP tube to ice, and incubate on ice for 2 minutes, avoiding vigorous shaking. Add 500 μL of antibiotic-free LB medium to the centrifuge tube, mix well, and incubate at 200 rpm for 1 hour at 37°C on a shaker to revive the bacteria. Centrifuge at 5000 rpm for 1 minute to collect the bacterial cells, discard 300-400 μL of supernatant, resuspend the cells, and spread them on solid LB agar plates containing Amp. Invert the plates and incubate overnight at 37°C for 12-16 hours. Extract plasmids using a plasmid mini-extraction kit (Zhuangmeng Biotechnology, ZP101) and send them to Beijing Nuosai Genome Research Center Co., Ltd. for sequencing. Compare the sequencing results with the target sequence; correct plasmids can be used for large-scale preparation. A large number of correctly sequenced plasmids were extracted using a plasmid extraction kit (Kangwei Century, CW2107M) for subsequent experiments.

[0105] Table 2: PCR primers for VZV gE

[0106]

[0107] Table 3: PCR reaction system

[0108]

[0109]

[0110] Table 4: PCR Amplification Conditions

[0111]

[0112] Table 5: Target Fragment-Vector Ligation System

[0113]

[0114] 1.2 Expression of gE and gE(R361S) proteins

[0115] Expi293F cells (Gibco A14527) were cultured and passaged at 80% relative humidity, 8% CO2, 37℃, and 120 rpm. The transfected viable cell density and viability should reach 4.5-5.5 × 10⁻⁶ cells / year. 6 Live cells / mL and 95%, plasmid usage was 2 μg / 10 6Cells were cultured at a PEI / DNA ratio of 3:1. The plasmid DNA and PEI transfection reagent (1 mg / mL, PEI Max 40k, Polysciences) complex was incubated at room temperature. Then, while shaking the flask, the PEI-DNA complex was added to the transfection flask, and the cells were cultured for 5-7 days, with glucose supplementation as needed. The cells were centrifuged at 300×g for 10 min at 4°C, and the supernatant was collected. Then, the cells were centrifuged again at 5000×g for 30 min at 4°C, and the expressed protein in the supernatant was collected for subsequent protein quantification.

[0116] 1.3 Protein quantification: ELISA detection

[0117] The mouse anti-gE hybridoma monoclonal antibody (obtained by screening hybridoma cells after immunizing mice with gE antigen standard (sequence same as SEQ ID NO:23)) was diluted to 2 μg / ml with carbonate buffer, seeded at 100 μL / well, washed with PBS, blocked with 1% BSA blocking buffer, washed with PBST, and then the gE antigen standard (sequence same as SEQ ID NO:23) diluted with PBST and the protein sample to be tested were added. After washing, 4 μg / mL of Biotin-conjugated rabbit anti-gE polyclonal antibody (antiserum obtained by immunizing rabbits with gE antigen standard, purified by Cytiva Protein A column chromatography, and then purified according to Thermo Fisher Scientific's instructions and Thermo EZ-Link) was added. TM Prepared by Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) coupling, 100 μL / well, wash plate, add 1:1000 diluted Streptavidin-HRP dye, incubate for 30 min, wash plate, develop with TMB, stop with 2M H2SO4, measure absorbance of each well at 450 nm, perform Logistic four-parameter curve fitting using ELISACalc software or similar software, and calculate the antigen content of the sample based on the obtained standard curve. Figure 1 The results showed that the protein expression levels in pCGS3.2-gE and pCGS3.2-gE(R361S) were 25.5 μg / mL and 992.7 μg / mL, respectively. In the pCGS3.2 vector, the expression level of gE protein containing the R361S mutation was increased by 33-fold, therefore, the R361S mutation in the gE gene can significantly increase protein expression.

[0118] Example 2: Immunogenicity evaluation of VZV gE and gE(R361S) proteins

[0119] 2.1 Animal Experiments

[0120] To further evaluate whether the R361S mutation affects the immunogenicity of the gE protein, the gE and gE(R361S) proteins were purified. The gE and gE(R361S) proteins collected in Example 1.2 were loaded onto a Cytiva HisTrap HP chromatography column. The chromatography column was equilibrated with Solution A (50 mM PBS + 10 mM imidazole, pH 7.4), and then eluted with a linear gradient of Solution A + 0.5 M imidazole to obtain the purified gE and gE(R361S) proteins. Then, the purified proteins were used to immunize Balb / C mice alone or in combination with the HA201 adjuvant (Beijing Huonotai Biomedical Technology Co., Ltd.). Female Balb / C mice aged 6 - 8 weeks (Small Animal Experiment Department of the Institute of Medical Biology, mouse license number: SCXK(Yunnan) K2019 - 0002, experimental animal welfare ethics approval number: DWLL202202 0008) were immunized by intramuscular injection into the hind legs at week 0 and week 4. The gE protein group and the gE(R361S) protein group were immunized at 5 μg / mouse, and the gE protein + HA201 adjuvant group and the gE(R361S) protein + HA201 adjuvant group were formulated and immunized at 5 μg of protein + 50 μL of HA201 / mouse. A PBS blank control group was set; blood was collected at week 6 to measure serum anti-gE IgG, and spleen cells were isolated at week 5 for cytokine detection.

[0121] 2.2 Detection of serum - gE IgG antibody titer

[0122] The indirect ELISA method was used to detect the IgG antibody titer against the VZV gE protein in mouse serum. The serum of Balb / C mice immunized in the sixth week as described above was collected. The test serum was diluted 200 - fold on a 96 - well plate and then serially diluted 2 - fold. The negative control serum was mixed equally and diluted 200 - fold. The diluted serum was added to the enzyme - coated plate coated with VZV gE protein (2 μg / mL) at a volume of 100 μL / well and incubated at room temperature for 1 h, and then the plate was washed 4 times; 100 μL / well of goat anti - mouse IgG - HRP secondary antibody (Invitrogen, 31430) diluted 1:10000 was added and incubated at room temperature for 1 h, and then the plate was washed 4 times; 100 μL / well of TMB chromogenic solution was added and developed at room temperature for 5 - 10 min; 50 μL / well of the termination solution was added to terminate the reaction, and the plate was read on an enzyme - label instrument (OD450 - OD630). 2.1 times the OD value of the negative control group was used as the cutoff value, and the dilution multiple ≥ cutoff value was the antibody titer. The geometric mean titer (GMT) of the serum titers of each group of mice was used as the antibody titer.

[0123] The results are as Figure 2As shown, the gE group, gE(R361S) group, gE+HA201 adjuvant group, and gE(R361S)+HA201 adjuvant group all produced high titers of IgG antibodies, with average IgG antibody titers of 1.28 × 10⁻⁶ and 1.28 × 10⁻⁶, respectively. 5 2.45×10 4 1.47×10 6 and 1.02×10 6 Statistical analysis showed no statistically significant difference in IgG antibody titers between the gE group and the gE(R361S) group (P > 0.05). Similarly, there was no statistically significant difference in IgG antibody titers between the gE+HA201 adjuvant group and the gE(R361S)+HA201 adjuvant group (P > 0.05).

[0124] 2.3 Detection of IL-4 and IFN-γ

[0125] Splenic lymphocytes from mice immunized for 5 weeks were isolated using mouse lymphocyte separation medium (Dakow, China). Mouse splenic lymphocytes were analyzed using the ELISpot PLUS (ALP) kit (Mabtech, Sweden) for mouse IFN-γ, IL-2, and IL-4 detection, following the manufacturer's instructions: Pre-coated ELISpot 96-well plates were washed with sterile PBS and serum-free culture medium. The stimulants were diluted with serum-free culture medium to a final gE protein concentration of 20 μg / mL, 50 μL / well. A negative control was set up, and 50 μL of cell suspension was added to each well. Cell volume: 3 × 10⁶ cells / well for IFN-γ detection plates. 5 Number of wells / holes, IL-4 detection plate is 7×10 5 Cells / well. Incubate at 37°C in a 5% CO2 incubator for 40 hours.

[0126] Discard the cell suspension, wash with PBS, dilute the primary antibody 1:1000 with PBS containing 0.5% fetal bovine serum, add to the ELISpot plate, and incubate at room temperature for 2 hours. Wash the plate with PBS. Dilute the ALP-labeled streptavidin secondary antibody 1:1000 with PBS containing 0.5% fetal bovine serum, add to the ELISpot plate, and incubate at room temperature for 1 hour. Wash the plate with PBS. Add 100 μL of BCIP / NPT-plus to each well for color development, incubate at 37°C in the dark for about 2–5 minutes, or until obvious spots appear in the positive wells, then discard the color development solution. Rinse the membrane with deionized water, air dry, and count the spots using an ELISpot plate reader (CTL, USA).

[0127] IFN-γ detection results are as follows Figure 3A , 3B As shown. When gE protein is used as a stimulant ( Figure 3A Or use gE(R361S) protein as a stimulant.Figure 3B At the same time, there was no statistically significant difference in the secretion level of cytokine IFN-γ between the gE group and the gE(R361S) group (P>0.05), and there was also no statistically significant difference in the secretion level of cytokine IFN-γ between the gE+HA201 adjuvant group and the gE(R361S)+HA201 adjuvant group (P>0.05); however, compared with the PBS group (blank control group), the combination of gE+HA201 adjuvant and the combination of gE(R361S)+HA201 adjuvant significantly increased the secretion of cytokine IFN-γ in immunized animals (P<0.05).

[0128] IL-4 test results as follows Figure 4A , 4B As shown. When gE protein is used as a stimulant ( Figure 4A ) or gE(R361S) protein as a stimulant ( Figure 4B At the same time, there was no statistically significant difference in the ability of the gE group and the gE(R361S) group to promote IL-4 secretion (P>0.05); there was no statistically significant difference in the ability of the gE+HA201 adjuvant group and the gE(R361S)+HA201 adjuvant group to promote IL-4 secretion (P>0.05); however, compared with the PBS group (blank control group), both the gE+HA201 adjuvant and gE(R361S)+HA201 adjuvant immunization methods had a stronger ability to promote IL-4 secretion (P<0.05).

[0129] Antibody titer and cytokine assays demonstrated that the R361S mutation does not affect the immunogenicity of the gE protein.

[0130] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

[0131] References

[0132] [1]FUSCO D, KRAWITZ P, LARUSSA P, et al. VZV meningitis following varicella vaccine [J]. Journal of Clinical Virology, 2010.

[0133] [2]GERSHON A A,GERSHON M D,BREUER J,et al.Advances in theunderstanding of the pathogenesis and epidemiology of herpes zoster[J].Journal of Clinical Virology,2010.

[0134] [3]MILLER E,MARSHALL R,VURDIEN J.Epidemiology,outcome and control ofvaricella-zoster infection[J].Reviews in Medical Microbiology,1993,4(4):222-30.

[0135] [4]HOPE-SIMPSON R E.The Nature of Herpes Zoster:A Long-Term Study anda New Hypothesis[J].Proceedings of the Royal Society of Medicine,1965,58(1):9-20.

[0136] [5]ISABEL L R,GEERT L R, C,et al.A Phase 1 / 2ClinicalTrial Evaluating Safety and Immunogenicity of a Varicella Zoster GlycoproteinE Subunit Vaccine Candidate in Young and Older Adults[J].Journal ofInfectious Diseases,2012,(8):1280-90.

[0137] [6]OLIVER,STEFAN L,SOMMER,et al.Mutagenesis of Varicella-Zoster VirusGlycoprotein I(gI)Identifies a Cysteine Residue Critical for gE / gIHeterodimer Formation,gI Structure,and Virulence in Skin Cells[J].

[0138] [7]EDMUNDS W J,BRISSON M,ROSE J D.The epidemiology of herpes zosterand potential cost-effectiveness of vaccination in England and Wales[J].Vaccine,2001,19(23-24):3076-90.

[0139] [8]GAUTHIER A,BREUER J,CARRINGTON D,et al.

[0140] Epidemiology and cost of herpes zoster and post-herpetic neuralgia inthe United Kingdom[J].Epidemiology&Infection,2009,137(1):38-47.

[0141] [9]DAVISON A J,SCOTT J E.The complete DNA sequence of Varicella-Zoster Virus[J].Journal of General Virology,1986,67(Pt

[0142] 9)(9):1759-816.

[0143]

[10] MERCEDES,GARCIA-VALCARCEL,AND,et al.Induction of neutralizingantibody and T-cell responses to Varicella-zoster virus(VZV)using Ty-virus-like particles carrying fragments of glycoprotein E(gE)[J].Vaccine,1997.

[0144]

[11] HASAN U A,ABAI A M,HARPER D R,et al.Nucleic acid immunization:concepts and techniques associated with third generation vaccines[J].Journalof Immunological Methods,1999,(1 / 2):229.

[0145]

[12] GILLER,ROGER H,WINISTORFER,et al.Cellular and Humoral Immunity toVaricella Zoster Virus Glycoproteins in Immune and Susceptible Human Subjects[J].Journal of Infectious Diseases.

[0146]

[13] OXMAN M N,LEVIN M J,JOHNSON G R,et al.A Vaccine to Prevent HerpesZoster and Postherpetic Neuralgia in Older Adults[J].New England Journal ofMedicine,2005,352(22):2271-84.

[0147]

[14] CUNNINGHAM A L,LAL H,KOVAC M,et al.Efficacy of the Herpes ZosterSubunit Vaccine in Adults 70 Years of Age or Older[J].New England Journal ofMedicine,2016,375(11):1019.

Claims

1. An isolated protein which is a mutant of a wild-type VZV gE protein ectodomain fragment and which comprises the following mutation as compared to the wild-type VZV gE protein ectodomain fragment: the arginine at a position corresponding to position 361 of SEQ ID NO: 1 in the wild-type VZV gE protein ectodomain fragment is mutated to a serine or a threonine.

2. The isolated protein of claim 1, wherein, the wild-type VZV gE protein ectodomain fragment comprises a sequence as set forth in SEQ ID NO:

1.

3. A fusion protein comprising the isolated protein of claim 1 or 2, and a signal peptide, wherein, the signal peptide is located at the N-terminus of the isolated protein; preferably, the signal peptide comprises a sequence as set forth in any one of SEQ ID NOs: 12-20; preferably, the signal peptide comprises a sequence as set forth in SEQ ID NO: 12 or SEQ ID NO:

13.

4. The fusion protein of claim 3, which comprises a sequence as set forth in SEQ ID NO:

3.

5. The fusion protein of claim 3 or 4, which further comprises the transdomain and the endodomain of a wild-type VZV gE protein at the C-terminus; preferably, the isolated protein comprises a sequence as set forth in SEQ ID NO:

11.

6. An isolated nucleic acid molecule encoding the isolated protein of claim 1 or 2, or the fusion protein of any one of claims 3-5; preferably, the isolated nucleic acid molecule comprises a sequence as set forth in any one of SEQ ID NOs: 4-9.

7. A vector comprising the isolated nucleic acid molecule of claim 6; preferably, the vector is a cloning vector or an expression vector.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A method of preparing the isolated protein of claim 1 or 2, or the fusion protein of any one of claims 3-5, comprising the steps of: culturing the host cell of claim 8 under conditions permitting expression of the protein, and recovering the isolated protein or fusion protein from the cultured host cell culture.

10. An engineered VZV virus expressing the isolated protein of claim 1 or 2, or the fusion protein of any one of claims 3-5; preferably, the genome of the engineered VZV virus comprises the following engineering: the wild-type gE gene is replaced with a nucleotide sequence encoding the isolated protein of claim 1 or 2, or a nucleotide sequence encoding the fusion protein of any one of claims 3-5.

11. A composition comprising the isolated protein of claim 1 or 2, the fusion protein of any one of claims 3-5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, or the engineered VZV virus of claim 10; and one or more pharmaceutically acceptable excipients; preferably, the composition further comprises an additional antiviral drug.

12. The composition of claim 11, wherein, the composition is an immunogenic composition or a vaccine; preferably, the composition comprises an adjuvant; Preferably, the composition is a subunit vaccine comprising the isolated protein of claim 1 or 2, or the fusion protein of any one of claims 3-5; Preferably, the composition is a live attenuated vaccine comprising the engineered VZV virus of claim 10; Preferably, the composition is a nucleic acid vaccine comprising the isolated nucleic acid molecule of claim 6, or the vector of claim 7; preferably, the nucleic acid vaccine comprises DNA or RNA; preferably, the DNA or RNA can be naked or can be encapsulated in a coat with delivery or / and protection functions.

13. Use of the isolated protein of claim 1 or 2, the fusion protein of any one of claims 3-5, the isolated nucleic acid molecule of claim 6, the vector of claim 7, the host cell of claim 8, the engineered VZV virus of claim 10, or the composition of claim 11 or 12, in the manufacture of a medicament for inducing an immune response against VZV in a subject and / or for preventing and / or treating a condition caused by VZV infection in a subject; Preferably, the condition caused by VZV infection is chickenpox and / or shingles; Preferably, the medicament is a vaccine; Preferably, the subject is a mammal, such as a human; Preferably, the isolated protein, fusion protein, isolated nucleic acid molecule, vector, host cell, engineered VZV virus or composition is used alone or in combination with another antiviral drug.

14. A method of preparing a vaccine for preventing and / or treating a condition caused by VZV infection, the method comprising the steps of: replacing the amino acid at a position corresponding to position 391 of SEQ ID NO: 10 in a VZV gE protein with a serine, and expressing the engineered protein in a cell; Preferably, the method further comprises replacing the signal peptide of the VZV gE protein with an IL6SP signal peptide; Preferably, the VZV gE protein comprises a sequence as set forth in SEQ ID NO: 10; Preferably, the engineered protein comprises a sequence as set forth in SEQ ID NO: 11; Preferably, the amino acid replacement is achieved by engineering the nucleotide sequence encoding the VZV gE protein through a gene site-directed mutagenesis technique, and expressing the engineered nucleotide sequence; Preferably, the condition caused by VZV infection is chickenpox and / or shingles.

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