AU2023387768A1 - Truncated varicella-zoster virus ge protein and use thereof - Google Patents

Abstract

The invention relates to a truncated varicella-zoster virus gE protein, a nucleic acid molecule or a vector encoding the same, a pharmaceutical composition comprising the same, and use of the pharmaceutical composition for preventing or alleviating herpes zoster and related diseases.

Description

A TRUNCATED VARICELLA-ZOSTER VIRUS gE PROTEIN AND USES THEREOF

RELATED APPLICATION The application claims priority of Chinese Patent Application No. 202310181926.4, filed on February 28, 2023, which is incorporated herein by reference in its entirety.

FIELD The present invention relates to the field of viral vaccines, more specifically, to a truncated varicella-zoster virus gE protein and a pharmaceutical composition comprising the same, uses thereof for preparing zoster vaccines, and their applications in the treatment of diseases or disorders associated with zoster virus infection.

BACKGROUND Herpes zoster (HZ) is an acutely infectious skin disease, which is mainly caused by a reactivation of the latent varicella-zoster virus (VZV) in the body. Humans are the only host of varicella zoster virus, and its incubation period is approximately 14 days. Primary infection with VZV is known as varicella or chickenpox, and the reactivation of VZV latent in sensory ganglia causes herpes zoster. Herpes zoster is mainly characterized by clustered blisters shown in zonal distribution, accompanied by obvious neuralgia. The disease is common in patients with advanced age, immunodeficiency, use of immunosuppressants etc. Herpes zoster is prone to cause complications after illness, with pain degree reaching level 7 or higher, which seriously affects the quality of life and has a certain recurrence rate. The incidence and severity of herpes zoster increase with age, and increase significantly after the age of 50, which is associated with a decline in cell-mediated immunity in the elderly. It is estimated that approximately half of the elderly at the age of 85 or older have experienced at least one episode of herpes zoster. Studies conducted in Canada, Israel, Japan, Taiwan Province of China and the United States have shown that the incidence of herpes zoster is 3.4-5.0/1,000 people/year in the overall population and 8-11/1,000 people/year in the population at the age of 65 or older. A study conducted in 27 countries in Europe has found that the incidence of herpes zoster ranges from 2.0-4.6/1,000 people/year in different countries, but does not show significant regional differences. The genome of varicella-zoster virus is a linear double-stranded DNA with a full-length of 125 kbp, containing 71 ORFs, which can encode 68 proteins, including 12 glycoproteins. Glycoproteins are particularly important for the life cycle of virus, among which gE (glycoprotein E) is the most highly expressed glycoprotein during the infectious stage of VZV and plays a vital role in mediating virus replication and spread. On the other hand, gE protein contains epitopes for neutralizing antibodies and T-cells, which can induce specific humoral and cellular immunity in the body, making it an important candidate antigen for designing zoster vaccines. To date, there hasn't been a very effective treatment for herpes zoster, and vaccination is the only effective means of prevention and control. Some vaccines against herpes zoster have been developed in the prior art, but there have been some issues, such as low protein expression efficiency, poor protein activity and unsatisfactory immune effects. Therefore, there is a need to develop improved VZV vaccines in the art.

SUMMARY The present invention provides a truncated varicella-zoster virus gE protein and its applications in the preparation of a vaccine composition for preventing or alleviating herpes zoster and related diseases. The benefits provided in this disclosure are widely applicable in the field of biomedicine. One aspect of the invention provides a glycoprotein E (gE) variant, wherein the variant comprises or consists of: (a) amino acids 1-496 or 31-496 of the full-length amino acid sequence of a native gE protein; (b) a fragment truncated by 1-50 (for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, and any subrange or individual value between that range) amino acids from the C-terminal of amino acids 31-537 of the full-length amino acid sequence of a native gE protein; or (c) an amino acid sequence having at least 90%, 95% or 99% (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 99.5%) identity with the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence as set forth in SEQ ID

NO: 2. In some embodiments, the native gE protein is from varicella-zoster virus. More specifically, the full-length amino acid sequence of the native gE protein may comprise or consist of the amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the gE protein variant is a truncated gE protein. In some embodiments, the gE protein variant consists of amino acids 1-496 or 31-496 of the full-length amino acid sequence of a native gE protein. More specifically, the gE protein variant consists of the amino acid sequence as set forth in SEQ ID NO: 2. One aspect of the invention provides a fusion protein, comprising the gE protein variant and a heterologous peptide, such as a signal peptide sequence. One aspect of the invention provides a truncated varicella-zoster virus gE protein comprising the following amino acid sequence: (a) a fragment of amino acids 1-496 or 31-496 of the full-length amino acid sequence of a native gE protein; (b) a fragment truncated by 1-50 (for example, 10, 15, 20, 25, 30, 35, 40, 45, and any subrange or individual value between that range) amino acids from the C-terminal of amino acids 31-537 of the full-length amino acid sequence of a native gE protein; or (c) an amino acid sequence having at least 90%, 95% or 99% (for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 99.5%) identity with the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the truncated varicella-zoster virus gE protein consists of the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence as set forth in SEQ ID NO: 2. One aspect of the invention provides a nucleic acid molecule encoding the gE protein variant or truncated gE protein disclosed herein. Another aspect of the invention provides a vector comprising a polynucleotide sequence encoding the gE protein variant or the truncated varicella-zoster virus gE protein. A further aspect of the invention provides a host cell comprising a vector encoding the gE protein variant or truncated varicella-zoster virus gE protein. A further aspect of the invention provides a pharmaceutical composition comprising the gE protein variant, truncated varicella-zoster virus gE protein or nucleic acid molecule encoding the same, and a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the pharmaceutical composition is a vaccine composition. The adjuvant may be any adjuvant commonly used in the art, or may be an adjuvant used in other zoster vaccines. The adjuvant can be selected from aluminum adjuvants, ASO adjuvants, squalene oil-in-water adjuvants, saponin adjuvants, a-galactosylceramide-derived adjuvants, polysaccharide adjuvants, agonists that induce immune responses in the body (for example, TLR agonists, NOD agonists, CLR agonists, RLR agonists), and any combinations thereof. In some embodiments, the adjuvant is a combination of a squalene oil-in-water adjuvant and a TLR agonist such as CpG ODN. Specifically, the squalene oil-in-water adjuvant is MF59 or an MF59 analogs, and/or the CpG ODN is CpG1018. In some embodiments, the adjuvant is a combination of an aluminum adjuvant such as aluminum hydroxide and a TLR agonist such as CpG ODN. The CpG ODN may be CpG1O18. Another aspect of the present invention provides a method of preparing a truncated varicella-zoster virus gE protein or gE protein variant disclosed herein, comprising the steps of: - expressing the truncated varicella-zoster virus gE protein or gE protein variant in a host cell; and - isolating the truncated varicella-zoster virus gE protein or gE protein variant from the host cell. Another aspect of the invention provides use of a truncated varicella-zoster virus gE protein or gE protein variant or a nucleic acid molecule encoding the same in the preparation of a vaccine composition for preventing or alleviating varicella-zoster virus infection in a subject. Another aspect of the invention provides use of a truncated varicella-zoster virus gE protein or gE protein variant or a nucleic acid molecule encoding the same in the preparation of a medicament for preventing or treating a disease or disorder related to varicella-zoster virus in a subject. The disease or disorder may be selected from the group consisting of varicella, herpes zoster and postherpetic neuralgia (PHN). Another aspect of the invention provides a method of therapeutically preventing or treating a disease or disorder related to varicella-zoster virus in a subject, which comprises administering to the subject a pharmaceutical composition, in particular a vaccine composition, comprising a truncated varicella-zoster virus gE protein or gE protein variant or a nucleic acid molecule encoding the same as disclosed herein. The disease or disorder may be selected from the group consisting of varicella, herpes zoster and postherpetic neuralgia. In some embodiments, the subject is a human or non-human mammal, preferably a human. Another aspect of the invention provides a kit comprising a container, the container comprising a truncated varicella-zoster virus gE protein or a nucleic acid molecule encoding the same disclosed herein. Another aspect of the invention provides a combination of a squalene oil-in-water adjuvant and CpG ODN for use as an adjuvant in a zoster vaccine composition. In some embodiments, the squalene oil-in-water adjuvant is MF59 or an MF59 analog, and/or the CpG ODN is CpG1018. Another aspect of the invention provides use of the combination in the preparation of a vaccine composition for preventing or alleviating varicella-zoster virus infection and a disease or disorder related to varicella-zoster virus in a subject. The vaccine composition further comprises a native gE protein, a fragment thereof (for example, an extracellular region fragment such as amino acids 1-537 or 31-537 of the full-length amino acid sequence of the native gE protein) or a gE protein variant or a nucleic acid molecule encoding the same, as an antigen. In some embodiments, the vaccine composition comprises a truncated gE protein disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS The following drawings are included to further illustrate certain aspects and features of the invention. The present invention can be better understood with reference to one or more of these drawings and in conjunction with the detailed description of specific embodiments, including examples. Figure 1A shows a map of the constructed Pee2.4-VZV-gE recombinant vector plasmid. Figure 1B shows 0.8% agarose electrophoresis for identifying the gE gene amplified by PCR, in which gE represents the expression construct of the varicella-zoster virus gE31-496 gene. The expression construct also contains part of the vector sequence and the signal peptide sequence. M represents a 5,000 bp DNA ladder.

DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, unless required otherwise in the context, all terms in singular form shall include the plural form, and terms in plural form shall include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells, and the like. In this application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "comprise" as well as other forms such as "comprising" and "comprises" is not restrictive. In addition, ranges provided in the specification and appended claims include both end points and all values between the end points. As used herein, the term "effective amount" refers to an amount of a compound, therapeutic agent, virus or drug that can achieve the intended result. As appreciated in the art, the effective amount may vary depending on the patient's medical history as well as other factors, such as the type and/or dosage of the therapeutic agent used. As used herein, the terms "subject" and "patient" may be used interchangeably and refer to any living organism, including humans and animals. As used herein, the term "about", when used in conjunction with a numerical value, is intended to encompass a numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value. A range as used herein includes both endpoint values specified by the range and any subrange or individual value therebetween. As used herein, "Percentage (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative substitution as part of the sequence identity. Sequence alignment for purposes of determining percentage amino acid sequence identity can be achieved in various ways in the art, for instance, using publicly available computer softwares such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximal alignment over the full-length of the sequences being compared. When referring to percentages of sequence identity in the present application, these percentages are calculated relative to the full-length of the longer sequence unless otherwise specifically indicated. The calculation relative to the full-length of the longer sequence is applicable to both nucleic acid sequences and polypeptide sequences. As used herein, the terms "host cell", "host cell line" and "host cell culture" are used interchangeably and refer to cells into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the primary transformed cell and progeny derived therefrom regardless of the number of passages. The progeny may not be exactly identical to the parent cell in nucleic acid content, but may contain mutations. As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that serve as self-replicating nucleic acid structures as well as vectors integrating into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of a nucleic acid to which they are operably linked. As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient, and/or salts thereof are chemically and/or physically compatible with the other ingredients in the formulation, and physiologically compatible with the recipient. As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active agent, 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, a pH adjuster, a surfactant, and an ionic strength enhancer. 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. As used herein, the term "adjuvant" refers to a non-specific immune enhancer, which can enhance the immune response to an antigen or change the type of immune response in an organism when it is delivered to the organism together with the antigen or delivered to the organism in advance. There are various adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete and Freund's incomplete adjuvants), Corynebacterium parvum, lipopolysaccharides, cytokines, etc. Freund's adjuvant is currently the most commonly used adjuvant in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials. As used herein, the term "CpG ODN" refers to a special type of DNA oligonucleotide, usually ranging from 10 to 30 bases in length, where "CpG" denotes a cytosine and a guanine linked together. These molecules play an important role in the immune system as they can mimic the DNA sequences of bacteria and viruses, thereby triggering the body's immune response. CpG ODNs are widely used in immune research and drug development, especially in the field of immunotherapy.

Varicella-zoster virus (VZV) and gE antigen Varicella-zoster virus, a virus belonging to the human herpesviridae family, is the cause of a disease that manifests as two different clinical presentations (varicella and herpes zoster). The initial infection with this virus causes varicella (blister sore), thereafter this virus latently infects ganglia, and after a number of years it is reactivated due to some inducement to provoke herpes zoster (virus particles are formed, and through nerve conduction, they reach epidermal cells, thereby forming the varicella symptom in areas where the nerves are distributed). The VZV genome is a double-stranded DNA containing approximately 125,000 bases, and it is known that there are at least 72 genes present in this genome. There are mainly four glycoproteins or complexes on the surface of VZV virus, namely gB, gC, gE/I and gH/gL. The gE protein is the most highly expressed glycoprotein and the most important surface antigen during the infectious period of VZV, which plays a vital role in mediating virus replication and spread. The native gE protein (for example, as set forth in NCBI ID Q9J3M8.1, AQT34120.1, AGY33616.1 and AEW88548.1) typically has a full-length of 623 amino acids, and is composed of a hydrophobic transmembrane region, an intracellular region, and an extracellular region. The extracellular region contains 537 amino acids (i.e., amino acids 1-537), including a signal peptide region of amino acids at the N-terminal. The antigenic epitopes of gE proteins are predominantly distributed in the extracellular region, and studies by Vafai et al. and Forghani et al. have shown that the gE protein contains at least three different antigenic epitopes, located at amino acids 109-123, 160-316, and 101-161, respectively. In some aspects, the invention provides a truncated gE protein, which is a fragment of a native gE protein (1-623), preferably a fragment of the extracellular region (1-537) of a native gE protein, and more preferably a fragment of the extracellular region (31-537) of a native gE protein with the signal peptide region removed. In some embodiments, the truncated gE protein does not contain the transmembrane region and intracellular region of a native gE protein. In some embodiments, the truncated gE protein is the extracellular region of a native gE protein. In some embodiments, the truncated gE protein is a further truncated fragment of the extracellular region of a native gE protein. In some embodiments, the truncated gE protein does not contain the signal peptide sequence at the N-terminal of a native gE protein. In some embodiments, the truncated gE protein is a fragment of a native gE protein (1-623) truncated from the C-terminal, for example, a fragment obtained by truncating 1-130 (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more) amino acids from the C-terminal, and optionally also truncated by 1-50 (for example, 40, 30, 25, , 15, 10, 9, 8, 7, 6, 5, 4 or less) amino acids from the N-terminal. In some embodiments, the truncated gE protein is a fragment obtained by truncating 1-100 (for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more) amino acids from the C-terminal of the extracellular region (31-537) of a native gE protein with the signal peptide region removed, and optionally further truncated by 1-30 (for example, 25, 20, 15, 10, 9, 8, 7, 6, , 4 or less) amino acids from the N-terminal. In some embodiments, the truncated gE protein is a fragment obtained by truncating 127 amino acids from the C-terminal of a native gE protein (1-623), that is, a fragment at positions 1-496. In some embodiments, the truncated gE protein consists of amino acids 31-496 of a native gE protein (as set forth in SEQ ID NO: 2). The inventors surprisingly found that when amino acids 31-496 of the extracellular region were used to prepare a truncated gE protein, the expression yield was significantly improved compared with that using other gE protein extracellular regions (such as amino acids 31-537), allowing for higher antigen expression level and cell yield.

Vaccine against VZV virus There are three main types of vaccines currently in use: live attenuated vaccines, inactivated vaccines, and genetically engineered vaccines. Live attenuated vaccines induce the formation of antibodies in the body at the cost of low toxicity, which have unsatisfactory immune effects and pose potential safety risks. For inactivated vaccines, the virus is only inactivated during the in-service stage, and the infectious virus is still used in the production process, so there are risks at the production process. Genetically engineered vaccines induce the formation of antibodies in the human body through heterologous expression of viral envelope proteins, without the use of active viruses throughout the process, thus ensuring a safe process. Live attenuated VZV vaccines on the market include varicella vaccines and zoster vaccines. All attenuated varicella vaccines are currently prepared with the Oka strain, and good immunogenicity can be obtained after one or two doses of immunization. Attenuated zoster vaccines have very poor immune effects, with only 50-60% effectiveness, and they are gradually replaced by recombinant protein vaccines. Both live attenuated varicella vaccines and zoster vaccines pose risks during their use. Live attenuated zoster vaccines (Zostavax) produced by Merck & Co., Inc. are produced using the Oka attenuated strain. Shingrix produced by GSK plc. is a herpes zoster subunit vaccine containing a recombinant glycoprotein E and a adjuvant (ASOIB). Shingrix was approved by the FDA in October 2017 for the prevention of herpes zoster in adults >50 years of age.

Preparation of vaccine composition Preparation of VZV gE proteins is usually achieved by expression in cultured cells or by chemical synthesis. The host cell in the present disclosure can be any cell suitable for expressing the antigen of the present disclosure, for example, host cells that are frequently used and suitable for producing proteins, including Escherichia coli cells, yeast cells, insect cells, and mammalian cells. Mammalian host cells include Chinese hamster ovary cells (CHO cells), COS cells, SP2 cells, etc. Protein expression in eukaryotic cells will generally result in an obtained protein sequence lacking the signal peptide portion. Expression vectors and host cells are commercially available. The expression vector contains a promoter and a cloning site for a sequence encoding a protein of interest such that the promoter and sequence are operably linked. Other elements may be present, such as a signal peptide sequence (sometimes referred to as a leader sequence), a tag sequence (e.g., 6-His), a transcription termination signal, an origin of replication, and a sequence encoding a product. Methods and procedures for transfecting host cells are also well known. As mentioned above, a suitable VZV gE antigen is a VZV gE protein with a truncated C-terminal and/or N-terminal. When a recombinant expression vector encoding a protein of interest is introduced into a host cell, the protein of interest is produced by culturing the host cell for a period of time to allow expression of the protein of interest in the host cell or secretion of the protein of interest into the medium in which the host cell is cultured. The protein of interest can be recovered from the culture medium using standard protein purification methods. Preferably, the truncated gE protein antigen is combined with a specific adjuvant to prepare a vaccine composition. The vaccine composition can be in the form of aqueous solution, oil-in-water, water-in-oil, liposome, etc. A good adjuvant can promote the stimulation of producing sufficient cellular and humoral immune responses in the body to achieve a protective effect. Adjuvants currently approved for use can be roughly divided into two types: One is an adjuvant to stimulate immune response, which enhances the immunogenicity of antigens by binding to receptors on innate immune cells, such as CpG1018; the other is an adjuvant for cooperating with vaccine delivery to present a necessary amount of vaccine antigens and immunostimulants to the immune system to induce immunity, such as aluminum adjuvants and emulsions. The vaccine composition of the present invention can be prepared by mixing the truncated gE protein with various adjuvants known in the art in certain proportions. The adjuvants include, but are not limited to, TLR (Toll-like receptor) agonists, NOD agonists, CLR (C-type lectin receptor) agonists, RLR agonists, aluminum adjuvants (such as aluminum hydroxide and aluminum phosphate), squalene-containing oil-in-water emulsions (such as MF59), ASO adjuvants (such as ASO1, ASO3 and ASO4), saponin adjuvants, a-galactosylceramide-derived adjuvants, and polysaccharide adjuvants. In some embodiments, the adjuvant comprises an aluminum adjuvant, a squalene-containing oil-in-water emulsion, or a liposome-carried adjuvant. Further, the adjuvant may also comprise an agonist adjuvant, for example, a TLR agonist adjuvant such as CpG ODN, preferably CpG1O18. CpG118 belongs to the class of CpG oligodeoxyribonucleotides (CpG ODNs), which is 22-mer unmethylated CpG-B class oligonucleotide. CpG ODN, as an agonist of Toll-like receptor 9 (TLR9), can stimulate TLR9-expressing cells and activate downstream innate immune response pathways. On the one hand, it induces the expression of type I interferons and inflammatory factors, and on the other hand, it matures plasmacytoid dendritic cells, thus enhancing humoral and cellular immune responses. It can be taken up by endosomes, causing increased interferon secretion and effectively activating antigen-specific T cells by facilitating the cross-presentation of antigens. One of the clinical adjuvants early approved for use, MF59 or an MF59 analog is an oil-in-water emulsion adjuvant which is mainly composed of squalene, Tween 80 and Span85, and its oil-in-water design significantly reduces the viscosity, thus enhancing the tolerance of the human body. Squalene belongs to oil phase components, widely distributed in animals, plants and human bodies, and it is characterized by both biocompatibility and degradability. Tween 80 and Span85 are surfactants that enhance the stability of the emulsion. MF59 is an ideal adjuvant for viral vaccines, which can significantly enhance antibody titers when used in combination with influenza, HIV, etc. vaccines, and has good tolerability in different populations. It can promote antigen uptake, recruit a large number of immune cells into the injection site, and recruit monocytes and neutrophils to present antigens and transport them into lymph nodes. When used in combination with CpG, it induces higher antibody titers and ThI-type responses than one adjuvant alone. In one aspect, the invention provides a combination of MF59 and CpG for preparing a herpes virus vaccine composition. The inventors have found that when MF59 (or an MF59 analog) and CpG are used in combination, the immunogenicity of the antigen can be significantly improved, and the combination has a good synergistic effect. The simultaneous use of two adjuvants, MF59 (or an MF59 analog) and CpG ODN, can synergistically induce antigen-specific cellular immune responses, and the resulting vaccine composition has a strong ability to induce specific humoral and cellular immune responses.

Diseases or symptoms caused by herpes zoster infection Herpes zoster is caused by varicella-zoster virus (VZV) that is accquired during primary varicella infection and reactivated. Herpes zoster is mainly characterized by painful dermatomic areas and papules; the pain usually occurs a few days before the rash develops, and can persist for months after the rash subsides; the rash tends to appear on a single dermatomic area, typically subsiding within 4-5 weeks. Common complications include postherpetic neuralgia (PHN) and VZV pneumonia. In the elderly, the age-related loss of VZV-specific functional T cell-mediated immunity is consistent with an increase in the HZ incidence. VZV-specific T cell-mediated immunity is also critical for optimal recovery from herpes zoster (HZ). During the first week after the onset of the HZ rash, the magnitude of the VZV-specific T cell-mediated immune response is negatively correlated with the severity of the HZ disease and the risk of developing post-herpetic neuralgia. In one aspect, the present invention provides an use of a vaccine composition in the preparation of a medicament for preventing or ameliorating herpes zoster and/or sequelae of herpes zoster.

Application of a composition comprising truncated gE protein In one aspect, the present invention provides use of an effective amount of a truncated gE protein or an encoding polynucleotide, or a pharmaceutical composition comprising the same, as a therapeutic or prophylactic agent to be administered and inoculated to a subject. For prophylactic and therapeutic agents, administration should take into account clinical condition of different patients (in particular the side effects of the prophylactic and therapeutic agents when used alone), delivery site, administration method, administration schedule and other factors known to those skilled in the art. Those skilled in the art will appreciate that the appropriate dosage may vary from subject to subject. Determination of the optimal dosage typically involves balancing the level of therapeutic benefits wtih any risks or deleterious side effects. The selected dosage level depends on a variety of factors, including, but not limited to, the activity of the particular substance to be administered, route of administration, time of administration as well as the species, gender, age, body weight, general health and prior medical history of the patient. The amount and route of administration of the substance to be administered will ultimately be at the discretion of the physician or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantially deleterious or adverse side effects. The truncated gE protein of the present invention and the vaccine composition or vaccine product prepared using the same can be administered in vivo to subjects in need thereof through various routes. The routes may be common routes of vaccine administration, including, but not limited to, subcutaneous, intramuscular, intranasal, and oral administration. Preferably, the administration is performed by subcutaneous or intramuscular injection. The truncated gE protein to be administered in the present invention is typically administered in liquid form (such as an injection), which can be diluted to the desired concentration with a buffer solution prior to use. The truncated gE protein of the present invention can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" it is meant a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can contain minor amounts of additives, such as substances with isotonicity and high chemical stability, as appropriate. These substances are not toxic to the recipient at the administration dosage and concentration employed, and such substances may include buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or salts thereof; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides such as polyarginine or tripeptide; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or derivatives thereof, glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbate, poloxamer, or PEG. Any pharmaceutical agent that can be administered therapeutically may be in a state that is free of organisms or viruses other than the active ingredient, that is, a sterile state. The sterile state can be easily achieved by filtration through a sterilized filter membrane (e.g., a 0.2 micron membrane). Generally, therapeutic/prophylactic agents are placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle. Therapeutic/prophylactic agents are typically stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of a lyophilized formulation, a 10-ml vial is filled with 5 ml of a sterile-filtered 1% (w/v) aqueous solution of the therapeutic/prophylactic agent, and the resulting mixture is lyophilized. The infusion solution may be prepared by reconstituting thelyophilized therapeutic/prophylactic agent with sterile water for injection. Therapeutic/prophylactic agents that can be administered in combination with the vaccine composition of the present invention are, such as, but not limited to: chemotherapeutic agents, antibiotics, steroidal or non-steroidal anti-inflammatory drugs, existing immunotherapeutic/prophylactic agents, other cytokines and/or proliferative factors, etc. The combination may be administered simultaneously as a composition, administered separately but simultaneously or in parallel, or administered chronologically.

Kit The present invention provides a pharmaceutical package or kit having one or more containers filled with one or more ingredients of the therapeutic/prophylactic agent of the present invention. The container may also be accompanied by a notice in the form prescribed by government agencies regulating the manufacture, use, or sale of pharmaceuticals or biological products, indicating that the relevant government agencies approves the manufacture, use, or sale for human administration.

Advantages of the invention It is surprisingly found in the present invention that when the truncated gE protein was prepared using amino acids 31-496 at the N-terminal of the extracellular region, its expression yield is significantly increased while retaining its antigenic epitopes, and when used in conjunction with adjuvants, it has a strong ability to induce specific humoral and cellular immune responses. The novel adjuvant-containing recombinant herpes zoster virus vaccine described in the present invention exhibits comparable safety and immunogenicity results at the animal level as compared to the imported recombinant herpes zoster virus vaccine (Shingrix, GSK) currently available on the market, with the following main advantages: it can help the vaccinated population effectively prevent varicella-zoster virus infection; it contains a new adjuvant system; and it is less expensive and more accessible than the imported vaccine (Shingrix).

EXAMPLES The following examples are given only for the purpose of illustrating various embodiments of the present invention, and they are not meant to limit the invention.

Example 1: stable cell line construction, protein expression and purification 1. gE31-496 protein selection and gene synthesis Through data retrieval, the amino acid sequence in the conserved region of the gE protein in the NCBI database was selected for gene sequence optimization. CHO cell preferred codons were selected for codon optimization and full gene synthesis, at the same time, in order to promote the secretion and expression of proteins and to improve the level of protein expression in mammalian cells, the IL-10 signal peptide sequence was introduced. The recognition sites of restriction endonucleases HindIll and Ecorl were introduced at the 5' and 3' ends of the target gene, respectively. The target fragments or full sequence were obtained by PCR amplification. The nucleotide sequence is shown in SEQ ID NO: 1, which contains coding sequences for the signal peptide and the truncated protein of positions 31-496 in the mature protein of the gE extracellular region. The coding sequence for the truncated protein of positions 31-537 of the gE protein linked with the IL-10 signal peptide sequence was constructed in the same manner. 2. Construction of gE protein expression vector The eukaryotic expression vector used in the present invention, PEE12.4, carries the AmpR resistance gene and the GS selection gene, which uses cytomegalovirus (CMV) promoter/enhancer sequences for the expression of the target gene. The vector PEE12.4 was double-digested using restriction endonucleases HindlIl and EcoRI, and then the recovered and purified PEE12.4 vector DNA was recombinantly ligated with the DNA fragment of the target gene. The recombinant ligation product was added to competent cells to obtain a number of single clone positive colonies, which were picked out for PCR amplification and identification (Figure 1). A large number of extracted plasmids were obtained. 3. Screening of stably transfected cell lines and expression and purification of proteins The plasmids obtained in the above section 2 were transfected into CHO-KI host cells and mini cell populations were screened. Cell populations with high expression levels were subjected to subclonal screening. The screened clones were subjected to fed-batch culture, and the supernatant was collected to detect the target protein. After a comprehensive analysis on cell growth status, protein expression level, terminal lactic acid content and related product quality, the optimal clones of 3 strains were selected. The cell strains were subjected to scale-up culture by feed culturing, and the target protein was harvested from the culture supernatant. The average content of the gE31-496 truncated protein (its amino acid sequence is set forth in SEQ ID NO: 2) was 4-5 g/L, which was much higher than the expression level of the gE31-537 protein (1.5-2 g/L) in stably transfected cell strain constructed by the same method. Table 1: Yield of truncated gE proteins Samples gE protein Average value (g/L) (g/L) gE31-496 Batch 1 4.32 4.75 Batch 2 5.28 Batch 3 4.65 gE31-537 Batch 1 1.36 1.76 Batch 2 2.31 Batch 3 1.60

Example 2: immunogenicity evaluation of gE protein-containing vaccine compositions The truncated gE31-496 protein produced in Example 1 was purified and mixed evenly with MF 59+CpG adjuvant (MF59: supplied by Invivogen, Cat. No. vac-adx-10; CpG1018: supplied by Novus, Cat. No. NBP2-31142) so as to prepare a recombinant zoster vaccine containing the novel adjuvant. Animal experiments were conducted. A vaccine of the truncated gE31-537 protein mixed with MF 59+CpG adjuvant was prepared similarly as a control. The steps for animal experiments were as follows: C57BL/6 mice aged 6-8 weeks were selected and randomly divided into groups, with 6 mice in each group. C57BL/6 mice were pre-immunized with the attenuated varicella vaccine (Shanghai Institute of Biological Products Co., Ltd.) and blood was collected. Mice in each group were immunized with the first shot after 35 days and the second immunization was given after 28 days, respectively, and the specific immunization procedures were shown in Table 2. Among them, the amount of MF59 used was 25 pl per mouse. On days 56-58, blood was collected from C57BL/6 mice in each group, and then the ELISA method was used to detect the titer of total IgG antibodies produced by specific gE protein induction. At the same time, the spleen was collected, splenic lymphocytes were isolated, and the expression of INF-y and IL-2 was detected by the ELISPOT method. The detailed results were shown in Table 3. As could be seen from the results, the truncated gE protein (31-496) and vaccine composition thereof of the present invention had good immunogenicity, and the immunogenicity was at least comparable to that of the mature gE31-537 extracellular region protein. MF59+CpG had a very good synergistic effect and could be used as an adjuvant candidate for zoster vaccines. Table 2. Procedure of animal experiments and the composition of the vaccine Immunization procedures Pre- First immunization (dayO) Second immunization Spleen removal immunization (day28) (day56-58) (day-35)

Group 1 Live attenuated Normal saline

Group 2 varicella gE31-496 (5 g) + MF59 (25 l)

Group 3 vaccine gE31-496 (5 g) + CpG1018 (10 [g)

Group 4 gE31-496 (5 g) + MF59 + CpG1018 (10 g)

Group 5 gE31-537 (5 g) + MF59 + CpG1018 (10 g)

Table 3. Summary of immunogenicity evaluation results Vaccines IgG (GMT) IFN-y (number of IL-2 (number of

positive cells per positive cells per 100,000 splenocytes) 100,000 splenocytes)

Normal saline Less than 3.3 3.2

1:100

gE31-496 (5 g) + MF59 1:87241 192.8 176.5 (25 [l)

gE31-496 (5 g) + 1:43620 94.3 70.6

CpG1018 (10 g)

gE31-496 (5 g) + MF59 1:1280000 440.1* 328.2 (25 l) + CpG1018 (10

[g) gE31-537 (5 g) + MF59 1:485029 376.5 314.3 (25 l) + CpG1018 (10

[g) * Significant difference compared to gE31-537 (5 pg) + MF59 + CpG1018 (10 pg) (p<

0.05) Under the same conditions, immunogenicity results of the vaccine compositions of the truncated gE protein (31-496) using aluminum hydroxide (supplied by Invivogen, Cat. No. vac-alu-250) instead of MF59 was tested. Table 4. Summary of immunogenicity evaluation results Vaccines IgG (GMT) IFN-y (number of IL-2 (number of

positive cells per positive cells per

100,000 splenocytes) 100,000 splenocytes)

Normal saline Less than 3.3 3.2

1:100

gE31-496 (5 g) + 1:144815 21.0 18.2

Aluminum hydroxide (50

pg) gE31-496(5 g)+ 1:43620 94.3 70.6

CpG1018 (10 [g)

gE31-496 (5 g) + 1:650199 172.4* 162.0*

Aluminum hydroxide (50

rig) + CpG1018 (10 ug) gE31-537 (5 g) + 1:409600 134.5 127.2

Aluminum hydroxide (50

rig) + CpG1018 (10 _g) * Significant difference compared to gE31-537 (5 pg) + Aluminum hydroxide (50 pg)

+ CpG1018 (10 pg) (p<0.05) The results showed that the immunogenicity effects of gE31-496 vaccines were significantly superior to that of gE31-537 truncated protein vaccine compositions, regardless of whether MF59+CpG1018 or aluminum hydroxide+CpG1018 was used as adjuvant. In the case of using MF59+CpG1018 as adjuvant, the vaccine compositions with both truncated proteins showed a significant increase in IgG production, IFN-y and IL-2 production, as compared to aluminum hydroxide+CpG1018 as adjuvant, demonstrating the advantages of MF59+CpG1018 over aluminum hydroxide+CpG1018. When MF59 or aluminum hydroxide was used alone as adjuvant, MF59 was inferior to aluminum hydroxide in producing IgG, therefore the combination of MF59+CpG1018 produced a synergistic effect.

The sequences involved in the present invention are as follows: SEQ ID No: 1 encodes the nucleotide sequence of the recombinant varicella-zoster virus gE31-496 truncated protein, where the box indicates the start codon, the underlined portion indicates the signal peptide sequence, and the remaining portion is the gE31-496 sequence.

CACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCCAGCGTGC TGAGATACGACGACTTCCACATCGACGAGGACAAGCTGGACACAAACAGCGTGTACGAAC CCTACTACCACAGCGACCACGCCGAATCATCATGGGTGAACAGAGGCGAGAGCTCCAGAA AGGCCTACGACCACAACAGCCCCTACATCTGGCCCAGAAACGACTACGACGGCTTTCTGG AGAACGCCCACGAGCACCACGGCGTGTACAACCAGGGCAGAGGCATCGACAGCGGAGAG AGACTGATGCAGCCCACCCAGATGAGCGCCCAGGAGGACCTGGGCGACGACACCGGAAT CCACGTGATCCCCACACTGAACGGAGACGACAGACACAAGATCGTGAACGTGGACCAGA GACAGTACGGCGACGTGTTCAAGGGCGACCTGAATCCCAAACCCCAGGGACAGAGACTG ATCGAGGTGTCCGTGGAAGAGAACCACCCCTTCACCCTGCGCGCCCCCATTCAGAGAATC TACGGCGTGAGATACACCGAGACATGGAGCTTCCTGCCCAGCCTGACATGCACCGGCGAC GCTGCCCCCGCTATTCAGCACATCTGCCTGAAGCACACCACCTGCTTCCAGGACGTGGTGG TGGATGTGGACTGCGCCGAAAACACCAAGGAAGACCAGCTGGCCGAGATCTCTTATAGAT TTCAGGGAAAGAAGGAGGCCGACCAGCCATGGATCGTGGTGAACACCTCAACACTGTTTG ACGAGCTGGAGCTGGACCCCCCAGAGATCGAGCCCGGCGTGCTGAAGGTGCTGAGAACCG AAAAGCAGTACCTGGGAGTGTATATTTGGAACATGAGAGGCAGCGACGGCACCTCTACTT ATGCCACCTTCCTGGTGACCTGGAAGGGAGACGAGAAGACCAGAAATCCCACACCCGCCG TGACCCCCCAGCCCAGGGGAGCTGAATTTCACATGTGGAACTATCATAGCCACGTGTTCA GTGTGGGCGACACCTTCAGCCTGGCTATGCACCTGCAGTACAAGATCCACGAAGCCCCTTT CGATCTGCTGCTGGAGTGGCTGTACGTGCCCATCGACCCCACATGCCAGCCCATGAGACTG TATAGCACCTGCCTGTACCATCCCAACGCCCCCCAGTGCCTGAGCCACATGAACAGCGGCT GCACCTTCACCTCCCCCCACCTGGCCCAGAGAGTGGCCAGCACCGTGTACCAGAACTGCG AGCACGCCGACAACTACACCGCCTACTGCCTGGGCATCTCCCACATGGAACCATCCTTTGG CCTGATCCTGCACGACGGCGGCACCACACTGAAATTCGTGGACACCCCCGAAAGCCTGAG CGGACTGTACGTGTTCGTGGTGTACTTCAACGGCCACGTGGAGGCCGTGGCTTACACAGTG GTGTCCACC SEQ ID No: 2, amino acid sequence of the recombinant varicella-zoster virus gE31-496 truncated protein (without signal peptide): SVLRYDDFHIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDG

FLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVD QRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTG DAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTL FDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTP AVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPM RLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEP SFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVST

SEQ ID NO: 3, full-length gE protein mgtvnkpvvg vlmgfgiitg tlritnpvra svlryddfhi dedkldtnsv yepyyhsdha

esswvnrges srkaydhnsp yiwprndydg flenahehhg vynqgrgids gerlmqptqm

saqedlgddt gihviptlng ddrhkivnvd qrqygdvfkg dlnpkpqgqr lievsveenh

pftlrapiqr iygvrytetw sflpsltctg daapaiqhic lkhttcfqdv vvdvdcaent

kedqlaeisy rfqgkkeadq pwivvntstl fdeleldppe iepgvlkvlr tekqygvyi

wnmrgsdgts tyatflvtwk gdektrnptp avtpqprgae fhmwnyhshv fsvgdtfsla

mhlqykihea pfdlllewly vpidptcqpm rlystelyhp napqclshmn sgetftsphl

aqrvastvyq neehadnyta yclgishmep sfglilhdgg ttlkfvdtpe slsglyvfvv

yfnghveava ytvvstvdhf vnaieergfp ptagqppatt kpkeitpvnp gtspllryaa

wtgglaavvl lefviflict akrmrvkayr vdkspynqsm yyaglpvddf edsestdtee

efgnaiggsh ggssytvyid ktr

Those skilled in the art will further realize that the invention can be implemented in other specific forms without departing from the spirit or central features thereof. In that the foregoing description of the present invention discloses only exemplary embodiments thereof, it is to be understood that other variations are recognized as being within the scope of the present invention. Accordingly, the present invention is not limited to the particular embodiments which have been described in detail herein. Rather, reference should be made to the appended claims as indicative of the scope and content of the present invention.

Sequence Listing Sequence Listing 1 1 Sequence Sequence Listing Listing Information Information 1-1 1-1 File Name File Name Seq.xml Seq.xml 1-2 1-2 DTD Version DTD Version V1_3 V1_3 1-3 1-3 Software Name Software Name WIPOSequence WIPO Sequence 1-4 1-4 Software Version Software Version 2.3.0 2.3.0

1-5 1-5 Production Date Production Date 2024-04-28 2024-04-28 1-6 1-6 Originalfree Original freetext textlanguage language code code 1-7 1-7 NonEnglish Non English freefree texttext

languagecode language code 2 2 GeneralInformation General Information 2-1 2-1 Currentapplication: Current application: IP IP

Office Office

2-2 2-2 Currentapplication: Current application: Application number Application number 2-3 2-3 Currentapplication: Current application: Filing Filing

date date 2-4 2-4 Currentapplication: Current application: IEC230148PCT IEC230148PCT Applicantfile Applicant filereference reference 2-5 2-5 Earliest priority Earliest priority application: application: CN CN IP Office IP Office

2-6 2-6 Earliestpriority Earliest priority application: application: 202310181926.4 202310181926.4 Application number Application number 2-7 2-7 Earliestpriority Earliest priority application: application: 2023-02-28 2023-02-28 Filing date Filing date

2-8en 2-8en Applicant name Applicant name 2-8 2-8 Applicant name: Applicant name: NameName YitherBiotech Yither Biotech Co.,Ltd Co., Ltd

Latin Latin

2-9en 2-9en Inventor name Inventor name 2-9 2-9 Inventor name: Inventor name: NameName Latin Latin 2-10en 2-10en Inventiontitle Invention title 2-11 2-11 SequenceTotal Sequence TotalQuantity Quantity 33

3-1 3-1 Sequences Sequences 3-1-1 3-1-1 Sequence Number Sequence Number [ID]

[ID] 1 1

3-1-2 3-1-2 MoleculeType Molecule Type DNA DNA 3-1-3 3-1-3 Length Length 1452 1452 3-1-4 3-1-4 Features Features source1..1452 source 1..1452 Location/Qualifiers Location/Qualifiers mol_type=otherDNA mol_type=other DNA organism=syntheticconstruct organism=synthetic construct NonEnglishQualifier Value NonEnglishQualifier Value 3-1-5 3-1-5 Residues Residues atgcacagctcagcactgct atgcacagct cagcactgct ctgttgcctg ctgttgcctg gtcctcctga gtcctcctga ctggggtgag ctggggtgag ggccagcgtg ggccagcgtg 60 60 ctgagatacg acgacttcca ctgagatacg acgacttcca catcgacgag catcgacgag gacaagctgg gacaagctgg acacaaacag acacaaacag cgtgtacgaa cgtgtacgaa 120 120 ccctactacc acagcgacca ccctactacc acagcgacca cgccgaatca cgccgaatca tcatgggtga tcatgggtga acagaggcga acagaggcga gagctccaga gagctccaga 180 180 aaggcctacgaccacaacag aaggcctacg accacaacag cccctacatc cccctacatc tggcccagaa tggcccagaa acgactacga acgactacga cggctttctg cggctttctg 240 240 gagaacgcccacgagcacca gagaacgccc acgagcacca cggcgtgtac cggcgtgtac aaccagggca aaccagggca gaggcatcga gaggcatcga cagcggagag cagcggagag 300 300 agactgatgcagcccaccca agactgatga agcccaccca gatgagcgcc gatgagcgcc caggaggacc caggaggace tgggcgacga tgggcgacga caccggaatc caccggaatc 360 360 cacgtgatcc ccacactgaa cacgtgatcc ccacactgaa cggagacgac cggagacgac agacacaaga agacacaaga tcgtgaacgt tcgtgaacgt ggaccagaga ggaccagaga 420 420 cagtacggcg acgtgttcaa cagtacggcg acgtgttcaa gggcgacctg gggcgacctg aatcccaaac aatcccaaac cccagggaca cccagggaca gagactgatc gagactgatc 480 480 gaggtgtccgtggaagagaa gaggtgtccg tggaagagaa ccaccccttc ccaccccttc accctgcgcg accctgcgcg cccccattca cccccattca gagaatctac gagaatctac 540 540 ggcgtgagatacaccgagac ggcgtgagat acaccgagac atggagcttc atggagcttc ctgcccagcc ctgcccagcc tgacatgcac tgacatgcac cggcgacgct cggcgacgct 600 600 gcccccgcta ttcagcacat gcccccgcta ttcagcacat ctgcctgaag ctgcctgaag cacaccacct cacaccacct gcttccagga gcttccagga cgtggtggtg cgtggtggtg 660 660 gatgtggactgcgccgaaaa gatgtggact gcgccgaaaa caccaaggaa caccaaggaa gaccagctgg gaccagctgg ccgagatctc ccgagatctc ttatagattt ttatagattt 720 720 cagggaaaga aggaggccga cagggaaaga aggaggccga ccagccatgg ccagccatgg atcgtggtga atcgtggtga acacctcaac acacctcaac actgtttgac actgtttgac 780 780 gagctggagctggacccccc gagctggaga tggacccccc agagatcgag agagatcgag cccggcgtgc cccggcgtgc tgaaggtgct tgaaggtgct gagaaccgaa gagaaccgaa 840 840 aagcagtacctgggagtgta aagcagtacc tgggagtgta tatttggaac tatttggaac atgagaggca atgagaggca gcgacggcac gcgacggcac ctctacttat ctctacttat 900 900 gccaccttcctggtgacctg gccaccttcc tggtgacctg gaagggagac gaagggagac gagaagacca gagaagacca gaaatcccac gaaatcccac acccgccgtg acccgccgtg 960 960 accccccagcccaggggage accccccage ccaggggagc tgaatttcac tgaatttcac atgtggaact atgtggaact atcatagcca atcatagcca cgtgttcagt cgtgttcagt 1020 1020 gtgggcgacaccttcageet gtgggcgaca ccttcagcct ggctatgcac ggctatgcac ctgcagtaca ctgcagtaca agatccacga agatccacga agcccctttc agcccctttc 1080 1080 gatctgctgctggagtggct gatctgctgc tggagtggct gtacgtgccc gtacgtgccc atcgacccca atcgacccca catgccagcc catgccagcc catgagactg catgagactg 1140 1140 tatagcacct gcctgtacca tatagcacct gcctgtacca tcccaacgcc tcccaacgcc ccccagtgcc ccccagtgcc tgagccacat tgagccacat gaacagcggc gaacageggc 1200 1200 tgcaccttca cctcccccca tgcaccttca cctcccccca cctggcccag cctggcccag agagtggcca agagtggcca gcaccgtgta gcaccgtgta ccagaactgc ccagaactgc 1260 1260 gagcacgccgacaactacac gagcacgccg acaactacac cgcctactgc cgcctactgc ctgggcatct ctgggcatct cccacatgga cccacatgga accatccttt accatccttt 1320 1320 ggcctgatcc tgcacgacgg ggcctgatcc tgcacgacgg cggcaccaca cggcaccaca ctgaaattcg ctgaaattcg tggacacccc tggacacccc cgaaagcctg cgaaagcctg 1380 1380 agcggactgtacgtgttcgt agcggactgt acgtgttcgt ggtgtacttc ggtgtacttc aacggccacg aacggccacg tggaggccgt tggaggccgt ggcttacaca ggcttacaca 1440 1440 gtggtgtccaCCcc gtggtgtcca 1452 1452 3-2 3-2 Sequences Sequences 3-2-1 3-2-1 SequenceNumber Sequence Number [ID]

[ID] 2 2 3-2-2 3-2-2 MoleculeType Molecule Type AA AA 3-2-3 3-2-3 Length Length 466 466 3-2-4 3-2-4 Features Features source 1..466 source 1..466 Location/Qualifiers Location/Qualifiers mol_type=protein mol_type=protein organism=syntheticconstruct organism=synthetic construct NonEnglishQualifier Value NonEnglishQualifier Value 3-2-5 3-2-5 Residues Residues SVLRYDDFHI DEDKLDTNSV SVLRYDDFHI DEDKLDTNSV YEPYYHSDHA YEPYYHSDHA ESSWVNRGES ESSWVNRGES SRKAYDHNSP SRKAYDHNSP YIWPRNDYDG YIWPRNDYDG 60 60 FLENAHEHHGVYNQGRGIDS FLENAHEHHG VYNQGRGIDS GERLMQPTQM GERLMQPTQM SAQEDLGDDT SAQEDLGDDT GIHVIPTLNG GIHVIPTLNG DDRHKIVNVD DDRHKIVNVD 120 120 QRQYGDVFKGDLNPKPQGQR QRQYGDVFKG DLNPKPQGQR LIEVSVEENH LIEVSVEENH PFTLRAPIQR PFTLRAPIQR IYGVRYTETW IYGVRYTETW SFLPSLTCTG SFLPSLTCTG 180 180 DAAPAIQHICLKHTTCFQDV DAAPAIQHIC LKHTTCFQDV VVDVDCAENT VVDVDCAENT KEDQLAEISY KEDOLAEISY RFQGKKEADQ RFQGKKEADQ PWIVVNTSTL PWIVVNTSTL 240 240 FDELELDPPEIEPGVLKVLR FDELELDPPE IEPGVLKVLR TEKQYLGVYI TEKQYLGVYI WNMRGSDGTS WNMRGSDGTS TYATFLVTWK TYATFLVTWK GDEKTRNPTP GDEKTRNPTP 300 300 AVTPQPRGAEFHMWNYHSHV AVTPQPRGAE FHMWNYHSHV FSVGDTFSLA FSVGDTFSLA MHLQYKIHEA MHLQYKIHEA PFDLLLEWLY PFDLLLEWLY VPIDPTCQPM VPIDPTCQPM 360 360 RLYSTCLYHPNAPQCLSHMN RLYSTCLYHP NAPQCLSHMN SGCTFTSPHL SGCTFTSPHL AQRVASTVYQ AQRVASTVYQ NCEHADNYTA NCEHADNYTA YCLGISHMEP YCLGISHMEP 420 420 SFGLILHDGG SFGLILHDGG TTLKFVDTPE TTLKFVDTPE SLSGLYVFVV YFNGHVEAVAYTVVST SLSGLYVFW YFNGHVEAVA YTVVST 466 466 3-3 3-3 Sequences Sequences 3-3-1 3-3-1 SequenceNumber Sequence Number [ID]

[ID] 3 3 3-3-2 3-3-2 MoleculeType Molecule Type AA AA 3-3-3 3-3-3 Length Length 623 623 3-3-4 3-3-4 Features Features source1..623 source 1..623 Location/Qualifiers Location/Qualifiers mol_type=protein mol_type=protein organism=syntheticconstruct organism=synthetic construct NonEnglishQualifier Value NonEnglishQualifier Value 3-3-5 3-3-5 Residues Residues MGTVNKPVVGVLMGFGIITG MGTVNKPVVG VLMGFGIITG TLRITNPVRA TLRITNPVRA SVLRYDDFHI SVLRYDDFHI DEDKLDTNSV DEDKLDTNSV YEPYYHSDHA YEPYYHSDHA 60 60 ESSWVNRGES SRKAYDHNSP ESSWVNRGES SRKAYDHNSP YIWPRNDYDG YIWPRNDYDG FLENAHEHHG FLENAHEHHG VYNQGRGIDS VYNQGRGIDS GERLMQPTQM GERLMQPTQM 120 120 SAQEDLGDDT GIHVIPTLNG SAQEDLGDDT GIHVIPTLIG DDRHKIVNVD DDRHKIVNVD QRQYGDVFKG QRQYGDVFKG DLNPKPQGQR DLNPKPQGQR LIEVSVEENH LIEVSVEENH 180 180 PFTLRAPIQRIYGVRYTETW PFTLRAPIQR IYGVRYTETW SFLPSLTCTG SFLPSLTCTG DAAPAIQHIC DAAPAIQHIC LKHTTCFQDV LKHTTCFQDV VVDVDCAENT VVDVDCAENT 240 240 KEDQLAEISYRFQGKKEADQ KEDOLAEISY RFQGKKEADQ PWIVVNTSTL PWIVVNTSTL FDELELDPPE FDELELDPPE IEPGVLKVLR IEPGVLKVLR TEKQYLGVYI TEKQYLGVYI 300 300 WNMRGSDGTSTYATFLVTWK WNMRGSDGTS TYATFLVTWK GDEKTRNPTP GDEKTRNPTP AVTPQPRGAE AVTPQPRGAE FHMWNYHSHV FHMWNYHSHV FSVGDTFSLA FSVGDTFSLA 360 360 MHLQYKIHEAPFDLLLEWLY MHLQYKIHEA PFDLLLEWLY VPIDPTCQPM VPIDPTCQPM RLYSTCLYHP RLYSTCLYHP NAPQCLSHMN NAPQCLSHMN SGCTFTSPHL SGCTFTSPHL 420 420 AQRVASTVYQ NCEHADNYTA AQRVASTVYQ NCEHADNYTA YCLGISHMEP YCLGISHMEP SFGLILHDGG SFGLILHDGG TTLKFVDTPE TTLKFVDTPE SLSGLYVFW SLSGLYVFVV480 480 YFNGHVEAVAYTVVSTVDHF YFNGHVEAVA YTVVSTVDHF VNAIEERGFP VNAIEERGFP PTAGQPPATT PTAGQPPATT KPKEITPVNP KPKEITPVNP GTSPLLRYAA GTSPLLRYAA 540 540 WTGGLAAVVLLCFVIFLICT WTGGLAAVVL LCFVIFLICT AKRMRVKAYR AKRMRVKAYR VDKSPYNQSM VDKSPYNQSM YYAGLPVDDF YYAGLPVDDF EDSESTDTEE EDSESTDTEE 600 600 EFGNAIGGSH GGSSYTVYID EFGNAIGGSH GGSSYTVYID KTR KTR 623

Claims (1)

1. CLAIMS 1. A glycoprotein E (gE) protein variant consisting of: (a) Amino acids 31-496 of the full-length amino acid sequence of a native gE protein; (b) A fragment truncated by 1-50 amino acids from the C-terminal of amino acids 31-537 of the full-length amino acid sequence of a native gE protein; or (c) An amino acid sequence having at least 90%, 95% or 99% identity with the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence as set forth in SEQ ID NO: 2.

   2. The gE protein variant of claim 1, consisting of the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence as set forth in SEQ ID NO: 2.

   3. A fusion protein, comprising the gE protein variant of claim 1 or 2 and a heterologous peptide, such as a signal peptide.

   4. A nucleic acid molecule encoding the gE protein variant of claim 1 or 2.

   5. A vector comprising the nucleic acid molecule of claim 4.

   6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

   7. A pharmaceutical composition (such as a vaccine composition) comprising the gE protein variant of claim 1 or 2 or the nucleic acid molecule of claim 4, and a pharmaceutically acceptable carrier and/or adjuvant.

   8. The pharmaceutical composition of claim 7, wherein the adjuvant is selected from aluminum adjuvants, ASO adjuvants, squalene oil-in-water adjuvants, saponin adjuvants, a-galactosylceramide-derived adjuvants, polysaccharide adjuvants, agonists that induce immune responses in the body (for example, TLR agonists, NOD agonists, CLR agonists, RLR agonists), and any combination(s) thereof.

   9. The pharmaceutical composition of claim 8, wherein the adjuvant is a combination of a squalene oil-in-water adjuvant and a TLR agonist such as CpG ODN.

   10. The pharmaceutical composition of claim 9, wherein the squalene oil-in-water adjuvant is MF59 or an MF59 analog, and/or the CpG ODN is CpG1018.

   11. The pharmaceutical composition of claim 8, wherein the adjuvant is a combination of an aluminum adjuvant such as aluminum hydroxide and a TLR agonist such as CpG ODN.

   12. A method for preparing the gE protein variant of claim 1 or 2, comprising the steps of: - culturing a host cell transfected with the vector of claim 5 under appropriate conditions; and - isolating the gE protein variant from the supernatant of the host cell.

   13. Use of the gE protein variant of claim 1 or 2 or the nucleic acid molecule of claim 4 in the manufacture of a vaccine composition for preventing or alleviating varicella-zoster virus infection in a subject.

   14. Use of the gE protein variant of claim 1 or 2 or the nucleic acid molecule of claim 4 in the manufacture of a medicament for preventing or treating a disease or disorder related to varicella-zoster virus in a subject.

   15. The use according to claim 13 or 14, wherein the subject is a human or an animal such as a non-human mammal, preferably the subject is a human.

   16. The use according to claim 15, wherein the disease related to varicella-zoster virus is selected from varicella, herpes zoster and postherpetic neuralgia.

   17. A method of preventing or alleviating varicella-zoster virus infection in a subject, comprising administering to the subject the pharmaceutical composition according to any one of claims 7-11.

   18. A kit comprising a container containing the gE protein variant as claimed in claim 1 or the nucleic acid molecule of claim 4.

   19. A combination of a squalene oil-in-water adjuvant and CpG ODN for use as an adjuvant in a zoster vaccine composition.

   20. The combination of claim 19, wherein the squalene oil-in-water adjuvant is MF59 or an MF59 analog, and/or the CpG ODN is CpG1018.

   21. Use of the combination according to claim 19 or 20 in the preparation of a vaccine composition for preventing or alleviating varicella-zoster virus infection and diseases or disorders related to varicella-zoster virus in a subject.

   22. The use according to claim 21, wherein the vaccine composition comprises a native gE protein, a fragment thereof (e.g., a fragment of the extracellular region such as amino acids 31-537 of the full-length amino acid sequence of a native gE protein) or a variant thereof.

   23. The use according to claim 22, wherein the vaccine composition comprises the gE protein variant according to claim 1 or 2.

   1 / 1 1/1

   (8906) MauBI (8906) MauBI HindIII HindiII (8991) (8991) (8777) Affil (877?) AflII DraIII Oralll(270) (270) (8561) BspQI SapI (8561) BipQI Sapl (8543) Bspfl (8543) BspEI PfoI* (650) PIoI* (650)

   (8098) Sac (8098) 11 SacII BstEII BstEII (918) (918)

   (7783) SnaBI (7783) Snaf (7777) Btq/I (7777) BtgZI (7677) NdrI Ndel ECORI EcoRI(1463) (1463) Bdl** (1471) Bell* (1471) (7442) S|)«*I (7442) Spel

   i r.s, NotI (1710)

   ¥ SbfI (1724)

   (6919) Mlul (6919) f I Bl Xbal Xbal (2151) BamH (2157) BamHI r&n i Salt (2433) Sail (2433)

   J DrdI DrdI (2508) (2508)

   S *\ (5680) Kpnl (5680) KpnI SV40 ori «V40 on / (5676) (5676) ACC65I Acc651 X (5673) Agcl (5673) Agel T7Te terminator (5301) Ap.ll* (5301) Apal *K*0 SV40 promoter (5297) PspOMI" (5297) PspOMI (5241) BstZI/I (5241) BstZ171 Fspl (3515) FspI (3515) 5000 (5089) Kfll (5089) Kill Pvul Pvul(3663) (3663)

   (4800) BmtI (4800) BillII XmnI(3892) XmnI (3892) (4796) Nhcl (4796) NheI (87 78) Awrll (4778) AvrIl (4731) Sfil (4731) sm SexAI*(4545) SexAI* (4545)

   Figure Figure 1A 1A

   gE M M bp bp

   5000 5000 3000 3000 2000 2000 1500 1500

   1000 1000

   750 750

   500 500

   250 250

   Figure 1B Figure 1B