Truncated varicella zoster virus envelope glycoprotein gE

Abstract

The present invention relates to the fields of immunology and molecular virology, in particular the prevention and treatment of varicella zoster virus. In particular, the present invention relates to a truncated varicella zoster virus gE protein (or a mutant thereof) capable of soluble expression in an E. coli expression system, and its use in the prevention and / or treatment of varicella zoster virus infection.

Description

[Technical field]

[0001] This application relates to the fields of immunology and molecular virology, in particular to the prevention and treatment of Varicella-Zoster virus. In particular, this application relates to a truncated Varicella-Zoster virus gE protein (or a mutant thereof) expressible in a soluble form in an Escherichia coli expression system, and its use in the prevention and / or treatment of Varicella-Zoster virus infection. [Background technology]

[0002] Varicella zoster virus (VZV), also known as human herpesvirus 3 (HHV-3), belongs to the alpha subfamily of the Herpesviridae family and is a type of enveloped double-stranded DNA virus. The VZV viral genome is about 12.5 kb and contains about 70 open reading frames. VZV particles are spherical, with a diameter of about 150 nm to 200 nm, and contain four parts, viral DNA, capsid, tegument protein, and envelope protein, radially outward from the center of the sphere. The outer surface of the virus is an envelope formed by various glycoproteins and lipids, the main glycoproteins being gB, gC, gE, gH, gL, gI, etc., which play important roles in identifying host cells, stimulating immune responses, and improving the stability of the virus particles.

[0003] VZV infection can be divided into primary infection and recurrent infection. Primary infection appears as chickenpox (varicella), during which VZV mainly enters the body through respiratory droplets or skin contact of infected individuals, causing primary mucosal epithelial infection, spreads through the body via the blood or lymphatic system, causing viremia, and spreads to the skin of the whole body, appearing as maculopapular and vesicular eruptions. Chickenpox is common in children, generally heals naturally (except for immunocompromised children), and its symptoms are relatively mild, but chickenpox in adults is often accompanied by viral pneumonia and has a high mortality rate. Even after the host recovers, a small amount of VZV still remains in the sensory ganglion cells of the dorsal root ganglion of the spinal cord or in the cranial nerves. When the host becomes immunocompromised or is stimulated to a certain extent, the VZV latent in the host becomes activated, inducing shingles, causing severe pain and complications such as postherpetic neuralgia (PHN). The incidence of shingles increases with age and has a serious impact on the daily lives of the elderly.

[0004] Antiviral drugs and vaccines are the best choice for treating or preventing VZV infection. Inoculation with related vaccines is also the best treatment, which can effectively alleviate symptoms such as papules and postherpetic neuralgia. Live attenuated vaccines based on the Oka strain can be used to prevent chickenpox and shingles, but they have disadvantages such as inapplicability to immunocompromised patients, limited efficacy in preventing shingles, and risk of viral latency. Compared with live attenuated vaccines, subunit vaccines have the advantages of high safety and low production costs. Subunit vaccines using VZV envelope glycoprotein gE as an antigen have achieved significant good results in the treatment of elderly patients, and the humoral and cellular immune responses stimulated by them in vivo are stronger than those induced by live attenuated vaccines, which has become a new avenue for the development of chickenpox and shingles vaccines.

[0005] In the research and development of vaccines, safety, protective efficacy and economic efficiency must be taken into consideration. Commonly used antigen protein expression systems can be divided into eukaryotic expression systems and prokaryotic expression systems. When gE protein is expressed in a eukaryotic expression system, the damage to the natural three-dimensional structure is minimized, and the correct three-dimensional structure and epitope presentation are ensured. However, the baculovirus expression system, yeast expression system and CHO expression system used in the current eukaryotic expression systems all have defects such as high culture costs, which pose great challenges to large-scale industrial production. Currently available AS01 B Shingrix, an adjuvant-based recombinant varicella zoster vaccine, uses CHO expression system to produce gE extracellular segment protein as the vaccine antigen. The price of this product is relatively high, which affects its wide application.

[0006] Among prokaryotic expression systems, the E. coli expression system has the advantages of fast growth rate and low culture cost, and is an effective tool for producing recombinant proteins. However, viral proteins expressed in the E. coli expression system often lose the correct natural conformation and are expressed in the form of inclusion bodies in the precipitate. Currently, the renaturation of proteins expressed in inclusion bodies is still a global problem. Folding errors often occur during the renaturation process, leading to protein precipitation, or local conformational errors after folding lead to reduced protein activity and reduced immunogenicity. The complete extracellular segment of gE protein can also be expressed in a soluble form with the correct conformation in the E. coli lysis supernatant, but it is also very difficult to purify gE protein from various soluble proteins in the E. coli lysis supernatant. The use of fusion strategies and affinity chromatography is often required. The above methods often require expensive enzymes and are difficult to carry out industrial production. Currently, there are no literature reports on the highly efficient soluble expression of the extracellular segment of gE protein in E. coli and the purification of the soluble expressed protein as a VZV vaccine antigen.

[0007] Therefore, there remains a need in the art for low-cost gE proteins and vaccines thereof capable of inducing protective antibodies against VZV to enable large-scale industrial production of chickenpox and varicella-zoster vaccines. Summary of the Invention

[0008] Through multiple experiments, the inventors of the present application have selected a series of truncated VZV gE proteins that retain most of the immune epitopes, can be expressed in E. coli with higher solubility than the complete extracellular domain of gE protein, are easier to purify, and still retain the antigenicity of VZV gE protein.

[0009] Truncated gE protein or its variants Thus, in one embodiment, the present application provides a truncated Varicella-Zoster Virus (VZV) gE protein or a variant thereof, wherein the truncated VZV gE protein has a truncation of 75 to 445 amino acids at the C-terminus compared to the wild-type VZV gE protein, and the variant has at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity compared to the truncated VZV gE protein, or has one or several (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (preferably conservative substitutions), additions, or deletions, and retains the biological function of the truncated VZV gE protein (e.g., the ability to induce neutralizing antibodies against VZV and / or soluble expression in Escherichia coli).

[0010] In certain embodiments, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a truncation of at most 445 amino acids at the C-terminus, e.g. at most 442, at most 440, at most 430, at most 420, at most 410, at most 400, at most 390, at most 380, at most 370, at most 360, at most 350, at most 340, at most 330, at most 325 or at most 320 amino acids, e.g. at most 330 amino acids, and / or at least 75 amino acids at the C-terminus, e.g. at least 77, at least 80, at least 85, having a truncation of at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 193, at least 200, at least 210, at least 220, at least 222, at least 230, at least 240, at least 248, at least 250, at least 260 or at least 265 amino acids, e.g., at least 260 amino acids.

[0011] In some embodiments, compared to the wild-type VZV gE protein, the truncated VZV gE protein has 80 to 445, 190 to 445, 220 to 445, 245 to 445, 260 to 445, 75 to 330, 80 to 330, 190 to 330, 220 to 330, 245 to 330, 260 to 330, 75 to 310, 80 to 31 ... 10 pieces, 220~310 pieces, 245~310 pieces, 260~310 pieces, 75~300 pieces, 80~300 pieces, 190~300 pieces, 220~300 pieces, 245~300 pieces, 260~300 pieces, 75~270 pieces, 80~270 pieces, 190~270 pieces, 220~270 pieces, 245~270 pieces, 77 pieces~ 442 pieces, 85~442 pieces, 193~442 pieces, 222~442 pieces, 248~442 pieces, 265~442 pieces, 77~320 pieces, 85~320 pieces, 193~320 pieces, 222~320 pieces, 248~320 pieces, 265~320 pieces, 77~303 pieces, 85~303 pieces, 193~303 pieces, 222 pieces and having a truncation of 248 to 265 amino acids.

[0012] In certain embodiments, the truncated VZV gE protein has no truncation at the N-terminus compared to the wild-type VZV gE protein.

[0013] In certain embodiments, the truncated VZV gE protein has a truncation of 1-170 amino acids at the N-terminus compared to the wild-type VZV gE protein.

[0014] In certain embodiments, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a truncation of at most 170 amino acids at the N-terminus, such as at most 165, at most 160, at most 155, at most 150, at most 145, at most 140, at most 139, at most 135, at most 130 or at most 127 amino acids, such as at most 130 amino acids, and / or a truncation of at least 1 amino acid at the N-terminus, such as at least 5, at least 10, at least 15, at least 20, at least 25 or at least 30 amino acids, such as at least 30 amino acids.

[0015] In certain embodiments, the truncated VZV gE protein has a truncation of 20-170, 1-155, 20-155, 1-140, 20-140, 1-130, 20-130, 1-85, 20-85, 1-75, 20-75, 1-30, 20-30, 30-165, 30-152, 30-139, 30-127, 30-80, 30-73, 30-75, 30-85, 30-130, 30-140, 30-155, or 30-170 amino acids at the N-terminus compared to the wild-type VZV gE protein.

[0016] In certain embodiments, the truncated VZV gE protein has a truncation of 260 to 330 amino acids at the C-terminus, e.g., 260 to 310, 260 to 300, 265 to 320, 265 to 303, 265 to 293 amino acids, compared to the wild-type VZV gE protein.

[0017] In certain embodiments, compared to the wild-type VZV gE protein, the truncated VZV gE protein has no N-terminal truncation or has a truncation of 1 to 130 amino acids at the N-terminus, e.g., 20 to 130, 20 to 85, 20 to 75, 20 to 30, 30 to 127, 30 to 80, 30 to 73, 30 to 75, 30 to 85, 30 to 130 amino acids. In certain embodiments, the truncated VZV gE protein has no N-terminal truncation or has a truncation of 30 to 130 amino acids at the N-terminus.

[0018] In certain embodiments, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a truncation of 260 to 330 amino acids at the C-terminus, e.g., 260 to 310, 260 to 300, 265 to 320, 265 to 303, 265 to 293 amino acids, and no truncation at the N-terminus or a truncation of 20 to 130 amino acids at the N-terminus, e.g., 20 to 85, 20 to 75, 20 to 30, 30 to 127, 30 to 80, 30 to 73, 30 to 75, 30 to 85, 30 to 130 amino acids. In certain embodiments, the truncated VZV gE protein has no truncation at the N-terminus or a truncation of 30 to 130 amino acids at the N-terminus.

[0019] In certain embodiments, the truncated VZV gE protein has a truncation of 77, 85, 193, 222, 248, 265, 293, 303 or 320 amino acids at the C-terminus and no truncation or a truncation of 30, 73, 80, 127 or 139 amino acids at the N-terminus compared to the wild-type VZV gE protein.

[0020] In certain embodiments, the truncated VZV gE protein has a truncation of 265, 293, 303 or 320 amino acids at the C-terminus and a truncation of 30, 73, 80 or 127 amino acids at the N-terminus compared to the wild-type VZV gE protein.

[0021] In certain embodiments, the truncated VZV gE protein comprises a 265 amino acid truncation at the C-terminus and a 30 amino acid truncation at the N-terminus compared to the wild-type VZV gE protein.

[0022] In certain embodiments, the wild-type VZV gE protein has the amino acid sequence set forth in SEQ ID NO:19, or a sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0023] In certain embodiments, the truncated VZV gE protein has an amino acid sequence set forth in any one of SEQ ID NOs: 1-6, 20-30.

[0024] In certain embodiments, the truncated VZV gE protein is capable of binding to anion exchange chromatography media (eg, Q Sepharose 4 Fast Flow) in a solution having a pH between 7.5 and 8.5 (eg, pH 8.0).

[0025] In certain embodiments, the truncated VZV gE protein is capable of binding to anion exchange chromatography media (e.g., Q Sepharose 4 Fast Flow) in a solution having a pH of 7.5 to 8.5 (e.g., pH 8.0) and a salt concentration of 0 mM to 300 mM (e.g., 0 mM to 200 mM, 0 mM to 50 mM).

[0026] Isolated Nucleic Acid Molecules In another aspect, the present application provides an isolated nucleic acid molecule encoding the truncated VZV gE protein or a variant thereof described above.

[0027] In certain embodiments, the nucleotide sequence encoding a truncated VZV gE protein or variant thereof contained in the isolated nucleic acid molecule is codon-optimized or non-optimized according to the codon preference of the host cell (e.g., E. coli).

[0028] vector In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule described above.

[0029] host cell In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or vector described above.

[0030] In some embodiments, the host cell is selected from a prokaryotic cell (e.g., an E. coli cell) and a eukaryotic cell (e.g., a yeast cell, an insect cell, a plant cell, a mammalian cell). In some embodiments, the host cell is a microorganism.

[0031] In some embodiments, the host cell is an E. coli cell.

[0032] Preparation method In another aspect, the present application also provides a method for preparing the truncated VZV gE protein or a mutant thereof, comprising culturing the host cell described above under conditions allowing protein expression, and recovering the truncated VZV gE protein or a mutant thereof from the culture of the cultured host cell.

[0033] In certain embodiments, the method comprises recovering the truncated VZV gE protein or a variant thereof from the culture supernatant or lysis supernatant of the cultured host cells.

[0034] In certain embodiments, the host cell is E. coli.

[0035] In certain embodiments of the method, the recovery step comprises purification of the protein.

[0036] In certain embodiments, purifying the protein comprises performing ion exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography.

[0037] In certain embodiments, the ion exchange chromatography comprises: a) binding the truncated VZV gE protein or a variant thereof to an anion exchange chromatography medium (e.g., Q Sepharose 4 Fast Flow) in a solution having a pH of 7.5 to 8.5 (e.g., pH 8.0) and a salt concentration of 0 mM to 300 mM (e.g., 0 mM to 200 mM, 0 mM to 50 mM); b) performing gradient elution by gradually increasing the salt concentration of the solution; c) collecting elution fractions containing the truncated VZV gE protein or variants thereof when the salt concentration of the solution is in the range of 350 mM to 450 mM (e.g., 400 mM); Includes.

[0038] immunogenic composition In another aspect, the present application also provides an immunogenic composition comprising the above-mentioned truncated VZV gE protein or a mutant thereof, and optionally a pharma- ceutically acceptable carrier and / or excipient (eg, an adjuvant).

[0039] In certain embodiments, the immunogenic composition comprises the truncated VZV gE protein or variant thereof and an adjuvant, the adjuvant being a risedronate adjuvant (e.g., zinc-aluminum hybrid adjuvant containing sodium risedronate), an aluminum adjuvant (e.g., aluminum hydroxide adjuvant, aluminum phosphate adjuvant), a zinc-aluminum hybrid adjuvant (e.g., FH002C), a Freund's adjuvant, an oil emulsion adjuvant, a cytokine, a TLR agonist, a CpG adjuvant, a liposome, an AS01 adjuvant, a glycerol ... BThe adjuvant is selected from the group consisting of sodium zoledronate, monophosphoryl lipid A (MPL), cholesterol-containing liposomes, and combinations thereof. In certain embodiments, the adjuvant is a risedronate adjuvant (e.g., a zinc-aluminum hybrid adjuvant containing sodium risedronate), an aluminum adjuvant (e.g., an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant), AS01 B adjuvants, and combinations thereof.

[0040] In certain embodiments, the immunogenic composition comprises the truncated VZV gE protein or a mutant thereof, and AS01. B and an adjuvant.

[0041] In certain embodiments, the immunogenic composition is a vaccine.

[0042] The immunogenic composition 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 injections, sterile powders for injections, and concentrated solutions for injections), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The immunogenic composition of the present application must be stable in a sterile state under the conditions of manufacture and storage. The preferred dosage form is an injection. Such an injection may be a sterile injection solution. In addition, the sterile injection solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or lyophilization) for ease of storage and use. Such sterile, lyophilized powder can be dispersed in a suitable carrier before use, for example, water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., a solution containing 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0043] The immunogenic compositions described above can be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, local, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic applications, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will appreciate that the route and / or mode of administration will vary depending on the intended purpose.

[0044] The immunogenic composition 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, the severity, the age of the subject, and other personal attributes of the particular subject (e.g., the general health of the subject and the robustness of the subject's immune system). The determination of the effective amount is also guided by animal model studies, followed by human clinical trials, and dosing regimens that significantly reduce the occurrence or severity of the symptoms or conditions of the target disease in the subject.

[0045] Use in the manufacture of immunogenic compositions In another aspect, the present application also provides the use of the above-mentioned truncated VZV gE protein or a mutant thereof, or the isolated nucleic acid molecule, or the vector, or the host cell, in the manufacture of an immunogenic composition, which is used to induce an immune response against VZV in a subject and / or to prevent and / or treat VZV infection or a disease associated with VZV infection in a subject.

[0046] In certain embodiments, the immunogenic composition is a vaccine.

[0047] In certain embodiments, the VZV infection is a primary or recurrent infection with VZV.

[0048] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and complications thereof (eg, neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis).

[0049] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and post-herpetic neuralgia.

[0050] In certain embodiments, the subject is a mammal, such as a human.

[0051] Methods for preventing and / or treating diseases In another aspect, the present application also provides a method for inducing an immune response against VZV in a subject and / or preventing and / or treating VZV infection or a disease associated with VZV infection in a subject, comprising administering an effective amount of the above-mentioned truncated VZV gE protein or a variant thereof, isolated nucleic acid molecule, vector, host cell, or immunogenic composition to a subject in need thereof.

[0052] In certain embodiments, the VZV infection is a primary or recurrent VZV infection.

[0053] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and complications thereof (eg, neuropathic pain, pneumonia, encephalomyelitis, conjunctivitis).

[0054] In certain embodiments, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and post-herpetic neuralgia.

[0055] In certain embodiments, the subject is a mammal, such as a human.

[0056] Detection Method In another embodiment, the present application also provides a method for detecting the presence of VZV gE protein-specific antibodies in a sample, the method comprising using the truncated VZV gE protein or a mutant thereof as described above.

[0057] In certain embodiments, the method is an immunological assay, such as an immunoblot, an enzyme-linked immunoassay (eg, ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay.

[0058] In certain embodiments, the method comprises: (1) contacting a sample with the above-described truncated VZV gE protein or a mutant thereof; and (2) detecting the formation of a protein-antibody immune complex or detecting the amount of the immune complex, wherein the formation of the immune complex indicates the presence of VZV gE protein-specific antibodies in the sample.

[0059] In some embodiments, the method further comprises detecting the presence of VZV gE protein-specific antibodies in the sample using a second antibody having a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or ruthenium derivatives), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin).

[0060] In some embodiments, the second antibody is specific for a constant region contained in an antibody of the species (eg, human) from which the sample being tested originates.

[0061] In some embodiments, the second antibody is an anti-immunoglobulin (eg, human immunoglobulin) antibody, such as an anti-IgG antibody.

[0062] In some embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, salivary secretions, or urine) from a subject (eg, a mammal, preferably a human).

[0063] Use in the manufacture of detection reagents In another aspect, the present application also provides the use of the above-mentioned truncated VZV gE protein or mutant thereof, or the isolated nucleic acid molecule, or the vector, or the host cell, in the manufacture of a detection reagent, which is used to detect the presence of VZV gE protein-specific antibodies in a sample.

[0064] In certain embodiments, the detection reagent detects the presence of VZV gE protein specific antibodies in a sample by the detection methods described above.

[0065] In certain embodiments, the sample is a bodily fluid sample (eg, whole blood, plasma, serum, salivary secretion, or urine) from a subject (eg, a mammal, preferably a human).

[0066] kit In another aspect, the present application also provides a kit comprising the truncated VZV gE protein or a mutant thereof, or the isolated nucleic acid molecule, or the vector, or the host cell described above.

[0067] In a particular embodiment, the kit comprises a truncated VZV gE protein or a mutant thereof as described above and a second antibody, the second antibody being as defined above.

[0068] Definition of Terms In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Furthermore, the laboratory procedures of virology, biochemistry, and immunology used herein are all routine procedures widely used in the corresponding fields. At the same time, in order to better understand this application, the definitions and explanations of related terms are provided below.

[0069] When the terms "for example", "eg", "such as", "including", "comprising" or variations thereof are used herein, these terms are not to be construed as limiting terms and should be interpreted as meaning "without being limited to" or "limited to."

[0070] Unless otherwise specified herein or clearly contradicted by context, the singular forms "a," "an," and "the" and similar referents are intended to be construed as including the singular and the plural in the context of describing this application, particularly in the context of the appended claims.

[0071] As used herein, the term "VZV" is an abbreviation for "Varicella zoster virus," a DNA virus.

[0072] As used herein, the terms "gE protein", "gE" and "glycoprotein gE" refer to a type of envelope glycoprotein of VZV, have the same meaning and can be used interchangeably. A specific amino acid sequence of the wild-type gE protein can be obtained from a public database (e.g., the GenBank database), for example, the amino acid sequence shown in GenBank Accession No. CAA27951.1 (Dumas strain), AAT07825.1 (VZV-MSP strain) or AAT07749.1" (BC strain).

[0073] In the present application, the expression "truncated by X amino acids at the N-terminus" in relation to a protein means that (1) the amino acid residues at positions 1 to X at the N-terminus of the protein are deleted, or (2) the amino acid residues at positions 1 to X at the N-terminus of the protein are replaced with amino acid residues (e.g., methionine residues) encoded by a start codon (for initiating protein translation). It is known to those skilled in the art that during translation of mRNA, the amino acid at position 1 of the resulting polypeptide chain is usually the amino acid encoded by the start codon (e.g., methionine (M)) due to the influence of the start codon. Thus, the residue at position 1 of the truncated protein from which X amino acids have been truncated at the N-terminus of the present application may be the amino acid at position "X+1" of the full-length protein, or the residue at position 1 may be the amino acid encoded by the start codon additionally contained before the amino acid at position "X+1". For example, a VZV gE protein truncated by 30 amino acids at its N-terminus comprises an amino acid sequence selected from (1) an amino acid sequence in which amino acid residues 1 to 30 are deleted compared to the wild-type VZV gE protein, or (2) an amino acid sequence obtained by replacing amino acid residues 1 to 30 at the N-terminus of the wild-type VZV gE protein with amino acids encoded by the start codon (e.g., methionine).

[0074] As used herein, the phrase "X amino acid truncation at the C-terminus" in reference to a protein refers to the deletion of the X amino acid residues that are closest to the C-terminus of the protein. For example, a VZV gE protein that is C-terminally truncated by 265 amino acids refers to a VZV gE protein that has the 265 amino acid residues closest to the C-terminus deleted.

[0075] In the present application, when the amino acid sequence of the wild-type gE protein is mentioned, it is described with reference to the sequence shown in SEQ ID NO: 19. For example, the expression "amino acid residues 1 to 30 of the wild-type gE protein" refers to the amino acid residues 1 to 30 of the polypeptide shown in SEQ ID NO: 19. However, the skilled artisan understands that VZV may include multiple isolates, and there may be differences between the amino acid sequences of the gE protein of various isolates. Furthermore, the skilled artisan understands that even if there are sequence differences, the gE proteins of different VZV isolates have a very high degree of identity in amino acid sequence (usually more than 95%, for example more than 96%, more than 97%, more than 98%, or more than 99%) and have substantially the same biological function. Thus, in the present application, the term "wild-type gE protein" includes not only the protein shown in SEQ ID NO: 19, but also the gE proteins of various VZV isolates. In addition, when a sequence fragment of the wild-type gE protein is described, it includes not only the sequence fragment of SEQ ID NO: 19, but also the corresponding sequence fragment in the gE protein of various VZV isolates. For example, the phrase "amino acid residues 1 to 30 of the wild-type gE protein" includes amino acid residues 1 to 30 of SEQ ID NO: 19 and corresponding fragments in the gE proteins of various VZV isolates.

[0076] According to the present application, the expressions "corresponding sequence fragments" or "corresponding amino acid positions" refer to fragments or amino acid sites / residues that are in equivalent positions in the compared sequences when the sequences are optimally aligned, i.e. when the sequences are aligned to obtain the highest percentage identity.

[0077] According to the present application, the term "variant" refers to a protein whose amino acid sequence has at least 90%, e.g. at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity with respect to the amino acid sequence of the truncated VZV gE protein of the present application, or has one or several (e.g. 1, 2, 3, 4, 5, 6, 7, 8 or 9) amino acid substitutions (preferably conservative substitutions), additions or deletions, and retains the essential properties of the truncated VZV gE protein, where the term "essential properties" may be one or more of the following properties: ability to specifically bind to anti-gE monoclonal antibodies; ability to induce neutralizing antibodies against VZV; ability to be soluble expressed in E. coli.

[0078] As used herein, the term "identity" is used to refer to the sequence match between two polypeptides or two nucleic acids. If a position in both sequences being compared is occupied by the same nucleotide or amino acid residue (e.g., if the position in each of the two DNA molecules is occupied by an adenine nucleotide, or the position in each of the two polypeptides is occupied by a lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences are identical, then the identity of the two sequences is 60%. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions are identical). Typically, the comparison is performed when the two sequences are aligned to yield maximum identity. Such alignments can be accomplished, for example, by using the method of Needleman et al. (1970), J. Mol. Biol. 48:443-453, which can be conveniently performed by computer programs such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)), which is implemented in the GAP program of the GCG software package (available at www.gcg.com), using a Blossum62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6 or 4, and a length weight of 1, 2, 3, 4, 5 or 6.

[0079] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of an amino acid residue with an amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., similar in size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0080] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection to express the genetic material elements carried by the vector in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Vectors can contain a variety of elements that control expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may contain an origin of replication.

[0081] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as E. coli or Bacillus subtilis, a fungal cell such as a yeast cell or Aspergillus, an insect cell such as S2 Drosophila cell or Sf9, or an animal cell such as a fibroblast cell, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, a HEK 293 cell, or a human cell. In certain embodiments, the host cell is an E. coli cell.

[0082] According to the present application, the term "E. coli expression system" refers to an expression system consisting of an E. coli (strain) and a vector, where the E. coli (strain) is derived from commercially available strains such as, but not limited to, ER2566, BL21(DE3), B834(DE3), BLR(DE3), etc.

[0083] One skilled in the art will understand that the design of the expression vector can depend on factors such as the choice of the host cell to be transformed, the level of expression desired, etc. The vector can be introduced into a host cell to produce a transcript, protein, or peptide, including the proteins, isolated nucleic acid molecules, etc. described herein.

[0084] According to the present application, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which are 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, and ionic strength enhancers. 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. Adjuvants include risedronate adjuvants (e.g., zinc-aluminum hybrid adjuvant containing sodium risedronate), aluminum adjuvants (e.g., aluminum hydroxide adjuvant, aluminum phosphate adjuvant), zinc-aluminum hybrid adjuvant (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokines, TLR agonists, CpG adjuvants, liposomes, AS01 B Adjuvants include, but are not limited to, sodium zoledronate, monophosphoryl lipid A (MPL), cholesterol-containing liposomes, or combinations thereof. Ionic strength enhancers include, but are not limited to, sodium chloride.

[0085] According to the present application, the term "adjuvant" refers to a non-specific immune enhancing agent that can enhance the body's immune response to an antigen or change the type of immune response when delivered to the body together with or before an antigen. There are many types of adjuvants, including risedronate adjuvants (e.g., zinc-aluminum hybrid adjuvant containing sodium risedronate), aluminum adjuvants (e.g., aluminum hydroxide adjuvant, aluminum phosphate adjuvant), zinc-aluminum hybrid adjuvant (e.g., FH002C), Freund's adjuvant, oil emulsion adjuvant, cytokine, TLR agonist, CpG adjuvant, liposome, AS01, and the like. B Adjuvants include, but are not limited to, sodium zoledronate, monophosphoryl lipid A (MPL), cholesterol-containing liposomes, or combinations thereof. In the present application, the adjuvant may be a risedronate adjuvant (e.g., a zinc-aluminum hybrid adjuvant containing sodium risedronate), an aluminum adjuvant (e.g., an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant), AS01 B Particularly preferred is an adjuvant, a medicament for treating or preventing osteoarthritis, or a combination thereof.

[0086] According to the present application, the term "effective amount" refers to an amount that can effectively achieve the intended purpose. For example, an effective amount for preventing or treating a disease (e.g., VZV infection) refers to an amount that can effectively prevent, stop or delay the onset of a disease (e.g., VZV infection), or an amount that can alleviate, reduce or treat the severity of an existing disease (e.g., a disease caused by VZV infection). It is within the ability of a person skilled in the art to determine such an effective amount. For example, the effective amount for therapeutic use depends on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other therapeutic agents administered at the same time, etc.

[0087] In this application, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. Also, in this application, amino acids are generally represented by their one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0088] As used herein, "subject" refers to an animal, such as a vertebrate. Preferably, the subject is a mammal, such as a human, cow, horse, cat, dog, rodent, or primate. Particularly preferably, the subject is a human. In this application, this term can be used interchangeably with "patient." Effect of the Invention

[0089] The truncated VZV gE protein of the present application can be expressed in E. coli with higher solubility than the complete extracellular domain of the gE protein while retaining most of the immune epitopes, and is easy to purify without the need for fusion with GST, His or other tags to aid in protein purification. The purification method employed by the truncated VZV gE protein of the present application does not require the use of expensive enzymes, is low cost, and the target protein does not undergo a harsh denaturation-renaturation process during the purification process, resulting in minimal loss and a stable protein conformation, making it suitable for large-scale industrial production.

[0090] The gE(31-358) protein provided by the present application can induce high titer neutralizing antibodies and specific cellular immune responses in serum when used as an antigen to immunize mice without the use of oil-based stimulants, and can be used to prevent and / or treat primary or recurrent VZV infection.

[0091] Furthermore, the truncated VZV gE protein of the present application has good reactivity with various gE protein-specific monoclonal antibodies and can be used as a detection antigen for diagnostic reagents, demonstrating the possibility and value of its application in VZV diagnosis.

[0092] Although the embodiments of the present application are described in detail below in conjunction with drawings and examples, those skilled in the art will understand that the following drawings and examples are used only to illustrate the present application and do not limit the scope of the present application. From the drawings and the following detailed description of the preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art. [Brief description of the drawings]

[0093] [Figure 1] This is a diagram showing the results of SDS polyacrylamide gel electrophoresis when gE proteins having different cleavage lengths were expressed in E. coli at an induction temperature of 37° C. in Example 2 of the present application, in which M: molecular weight marker, S: ultrasonic treatment supernatant sample, 2M: protein sample dissolved in 2M urea after ultrasonic precipitation and purification of inclusion bodies, 4M: protein sample dissolved in 4M urea after ultrasonic precipitation and purification of inclusion bodies, and 8M: protein sample dissolved in 8M urea after ultrasonic precipitation and purification of inclusion bodies. [Diagram 2] This figure shows the results of SDS polyacrylamide gel electrophoresis of the following samples in Example 2 of the present application: (i) lysis supernatants of E. coli expressing gE proteins with different cleavage lengths at an induction temperature of 24°C, (ii) lysis supernatants of E. coli expressing gE(337-509) at different induction temperatures (16°C, 20°C, 24°C, 37°C), and (iii) protein samples of lysis precipitates of E. coli expressing gE(182-358) dissolved in TB8.0 and solubilized by heating. In the figure, M: molecular weight marker, and the red arrow indicates the truncated gE protein band. [Diagram 3]FIG. 2 shows the results of SDS polyacrylamide gel electrophoresis of E. coli lysate supernatant after Q-FF ion chromatography in Example 2 of the present application. The E. coli lysate supernatant was obtained by ultrasonically lysing E. coli expressing gE proteins of different truncation lengths at an induction temperature of 24° C., and the ultrasonic lysis was performed using TB8.0. In the figure, M: molecular weight marker, ↑: supernatant sample after bacterial lysis, CT: sample permeated into Q-FF ion chromatography, 0.2M: sample eluted at a NaCl concentration of 200 mmol / L during Q-FF ion chromatography, 0.4M: sample eluted at a NaCl concentration of 400 mmol / L during Q-FF ion chromatography, 2M: sample eluted at a NaCl concentration of 2000 mmol / L during Q-FF ion chromatography, ↓: bacterial lysis precipitate. The red arrow indicates the truncated gE protein band. [Figure 4]1 shows the results of SDS polyacrylamide gel electrophoresis of various VZV gE truncated proteins at different purification stages in Example 2 of the present application: Panel A shows the results of electrophoresis of various gE(31-358) samples, where M is molecular weight marker, lane 1: sample eluted at a NaCl concentration of 400 mmol / L during Q-FF ion chromatography, lane 2: sample permeated during CHT chromatography with equilibration solution, lane 3: sample eluted at a NaCl concentration of 500 mmol / L during chromatography on a butyl column; Panel B shows the results of electrophoresis of various gE(31-320) samples, where M is molecular weight marker, lane 1: sample eluted at a NaCl concentration of 400 mmol / L during Q-FF ion chromatography. Lane 1: sample eluted during Q-FF ion chromatography at a NaCl concentration of 400 mmol / L; lane 2: sample eluted during CHT chromatography with equilibration solution; lane 3: sample eluted during butyl column chromatography at a NaCl concentration of 1000 mmol / L; panel C shows electrophoresis results of various gE(128-358) samples, in which M: molecular weight marker, lane 1: sample eluted during Q-FF ion chromatography at a NaCl concentration of 400 mmol / L; lane 2: sample eluted during CHT chromatography with equilibration solution; lane 3: sample eluted during butyl column chromatography at a NaCl concentration of 500 mmol / L. [Diagram 5] FIG. 13 shows the results of high-performance gel filtration chromatography (Panel A) and protein sedimentation coefficient (Panel B) analysis of purified VZV gE(31-358) protein in Example 3, as well as the results of immune reactions with gE neutralizing antibodies (11B11, 4A2, 11B12, 6H6, 10H6, 1B11, 4G4, 14G1, and 11E3) (Panel C). [Figure 6]Figure 4 shows binding antibody titers in mouse sera at different stages after immunization of mice with purified VZV gE(31-358) protein in Example 4. Panel A shows the results of Balb / C mice immunized at 0 / 2 / 4 weeks with either risedronate adjuvant or aluminum adjuvant at immunization doses of 5 μg and 1 μg. Panel B shows the results of C57 mice immunized at 0 / 4 weeks with either risedronate adjuvant or AS01B adjuvant at immunization doses of 5 μg. [Figure 7] FIG. 1 shows the serum neutralizing antibody titers of C57 mice immunized with purified VZV gE(31-358) protein in Example 4. In this experimental scheme, either risedronate adjuvant or AS01B adjuvant was used, the immunization dose was 5 μg, and a booster immunization was administered 4 weeks after the initial immunization. [Figure 8] FIG. 1 shows the results of cytokine secretion in C57 mice immunized with purified VZV gE(31-358) protein at 14 days after a single injection (Panel A) and at 30 days after two injections (Panel B) in Example 4. The immunization dose was 5 μg, and a booster immunization was administered 4 weeks after the first immunization. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Sequence information The sequence descriptions for this application are provided in the table below.

[0095] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0096] Specific Modes for Carrying Out the Invention The present application will now be described with reference to the following examples which are intended to illustrate, but not limit, the present application.

[0097] Unless otherwise specified, molecular biology experimental methods and immunoassays used in this application were essentially performed according to the methods of J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., Molecular Biology Experiment Manual, 3rd edition, John Wiley & Sons, Inc., 1995. Restriction endonucleases were used according to the conditions recommended by the product manufacturers. Those skilled in the art will understand that these examples are intended to illustrate the present application and are not intended to limit the scope of the present application. EXAMPLES

[0098] Example 1: Cloning of plasmids containing nucleotide sequences encoding truncated gE proteins Preparation of gene template for VZV gE extracellular segment A plasmid containing a nucleotide sequence encoding VZV glycoprotein E (gE) was synthesized using the DNA sequence of GenBank: AY253715.1 as a template, in which gE-1-F (SEQ ID NO: 13) was used as a forward primer and gE-546-R (SEQ ID NO: 14) was used as a reverse primer, and the PCR reaction was carried out in a PCR machine (Biometra, T3) according to the following conditions: Denaturation at 94°C for 5 min, denaturation at 94°C for 50 sec, annealing at 57°C for 50 sec, extension at 72°C for 2 min (25 cycles), extension at 72°C for 10 min.

[0099] The amplification yielded a specific product of approximately 1.6 kb in size, which was used as a template to prepare the DNA fragment encoding the truncated VZV-gE protein in this application.

[0100] Construction of a vector containing the gene for truncated VZV-gE Take the construction of a clone containing the nucleotide sequence encoding gE(31-358) (its amino acid sequence is shown in SEQ ID NO:3) as an example, B11-gE-31-F (SEQ ID NO:15) was employed as a forward primer, and a restriction endonuclease NdeI site was introduced at its 5' end, the sequence of the NdeI site was CATATG, and ATG was the start codon of the E. coli system. B11-gE-358-R (SEQ ID NO:16) was used as a reverse primer, and the 1.6 kb DNA fragment obtained in the previous step was used as a template for the second PCR reaction. The PCR reaction was carried out in a PCR thermal cycler (Biometra T3) according to the following conditions: Denaturation at 94°C for 5 min, denaturation at 94°C for 50 sec, annealing at 57°C for 50 sec, extension at 72°C for 2 min (25 cycles), extension at 72°C for 10 min.

[0101] The amplification yielded a specific DNA fragment with a size of about 1.0 kb. The PCR product was then subjected to Gibson assembly with the PCR-amplified fragment of vector pTO-T7 (Luo Wenxin et al., Journal of Biotechnology, 2000, 16:53-57), and amplified using B11-NdeI-VF of SEQ ID NO: 17 as a forward primer and B11-TGA-VR of SEQ ID NO: 18 as a reverse primer, to obtain a positive clone pTO-T7-gE(31-358) into which the truncated VZV-gE gene was inserted.

[0102] Similarly, other truncated clones were obtained.

[0103] Sequencing was commissioned to Sangon Biotech (Shanghai), and it was determined that the target nucleotide sequence inserted into the pTO-T7-gE(31~358) plasmid was SEQ ID NO:9, T7(+) / (-) primers were used as sequencing primers, and the amino acid sequence encoded thereby was SEQ ID NO:3. The protein corresponding to this sequence was a VZV-gE protein truncated at amino acid position 31 at the N-terminus and at amino acid position 358 at the C-terminus, and was named gE(31~358). The other truncated clones were named similarly: gE(31-303) (amino acid sequence shown in SEQ ID NO:1 and encoding nucleotide sequence shown in SEQ ID NO:7), gE(31-320) (amino acid sequence shown in SEQ ID NO:2 and encoding nucleotide sequence shown in SEQ ID NO:8), gE(81-358) (amino acid sequence shown in SEQ ID NO:4 and encoding nucleotide sequence shown in SEQ ID NO:10), gE(128-358) (amino acid sequence shown in SEQ ID NO:5 and encoding nucleotide sequence shown in SEQ ID NO:11), gE(140-330) (amino acid sequence shown in SEQ ID NO:6 and encoding nucleotide sequence shown in SEQ ID NO:12).

[0104] To transform 40 μL of competent E. coli ER2566 (purchased from New England Biolabs) prepared by the calcium chloride method, 1 μL of pTO-T7-gE(31-358) plasmid (0.15 mg / ml) was taken and spread on solid LB medium (ingredients: peptone 10 g / L, yeast powder 5 g / L, sodium chloride 10 g / L, same below) containing kanamycin (final concentration 25 mg / mL, same below), and cultured stationarily at 37 °C for 10 to 12 hours until a single colony was clearly recognizable. A single colony was picked up and placed in a test tube containing 4 mL of liquid LB medium containing kanamycin, and cultured at 37 °C and 220 rpm for 10 hours with shaking. 1 mL of bacterial culture was taken from it, lyophilized, and stored at -70 °C. Other truncated clones were stored in the same manner.

[0105] Example 2: Large-scale expression and purification of truncated gE protein 5 μL of the bacterial suspension of each shortened clone prepared in Example 1 was taken out of the ultra-low temperature refrigerator at -70 ° C, inoculated into 5 mL of liquid LB medium containing kanamycin, and cultured at 37 ° C and 180 rpm with shaking until OD600 reached about 0.5, then transferred to 500 mL of LB medium containing kanamycin and cultured at 37 ° C and 180 rpm with shaking for 2 to 4 hours. Low temperature induction: When OD600 reached about 0.6, the temperature of the medium was lowered to 24 ° C, IPTG was added to a final concentration of 0.4 mM, and induction was performed by shaking culture at 24 ° C for 10 hours. Induction at 37 ° C: When OD600 reached about 1.5, IPTG was added to a final concentration of 0.4 mM, and induction was performed by shaking culture at 37 ° C for 4 hours.

[0106] After induction, bacterial cells were harvested by centrifugation at 7000g for 5 min. Cells were resuspended in lysis buffer (20 mM Tris buffer, pH 8.0) at a ratio of 1 g cells per 10 mL of lysis buffer, placed on ice, and sonicated (Sonics VCX750 sonicator) (treatment conditions: 15 min working time, 2 sec pulse, 4 sec pause, 55% power). Bacterial lysate was centrifuged at 13500 rpm (30000g) for 15 min in a microcentrifuge using a JA-14 rotor, and the supernatant and precipitate were separated for further processing.

[0107] Purification of inclusion bodies of truncated gE protein The centrifuged precipitate of the lysate induced at 37°C (i.e., inclusion bodies) was washed with an equal volume of 2% Triton-100, shaken for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was then resuspended in 20 mM Tris-HCl, pH 8.0, shaken for 30 minutes, centrifuged, and the supernatant was discarded. The precipitate was then resuspended in 2 M urea, shaken for 30 minutes at 37°C, and centrifuged to obtain a supernatant and a precipitate. The supernatant was retained, and the precipitate was resuspended in an equal volume of 4 M urea, shaken for 30 minutes at 37°C, and centrifuged at 12000 rpm at 4°C for 15 minutes to obtain a supernatant and a precipitate. The supernatant (i.e., the supernatant of the 4 M urea lysate) was retained, and the precipitate was resuspended in an equal volume of 8 M urea, shaken for 30 minutes at 37°C, centrifuged, and the supernatant (i.e., the supernatant of the 8 M urea lysate) was retained.

[0108] The results of SDS-PAGE electrophoresis of the various fractions obtained are shown in Figure 1. The results showed that (1) when the N-terminus of gE protein was cut at a fixed amino acid position 31 and the C-terminus was cut at amino acid positions 181 / 303 / 320 / 358 / 375 / 401 / 430 / 538 / 546 in sequence, the total amount of inclusion body protein gradually increased, the amount of inclusion bodies dissolved in 4M urea gradually decreased, while the amount of inclusion bodies dissolved in 8M urea gradually increased. (2) it was also found that when the C-terminus of gE protein was cleaved at a fixed amino acid position 358 and the N-terminus was cleaved sequentially at amino acid positions 81 / 128 / 182, the amount of gE protein in the ultrasonic supernatant gradually decreased, the ratio of the amount of protein in the ultrasonic supernatant to the total amount of inclusion body protein decreased, and the inclusion body protein gradually changed from being soluble in 4 M urea to being soluble in 8 M urea; and (3) it was also found that when the C-terminus of gE protein was cleaved at a fixed amino acid position 537 and the N-terminus was cleaved sequentially at amino acid positions 74 / 153, the amount of gE protein in the ultrasonic supernatant gradually decreased, the ratio of the amount of protein in the ultrasonic supernatant to the total amount of inclusion body protein decreased, and the inclusion body protein gradually changed from being soluble in 4 M urea to being soluble in 8 M urea. In summary, the above results indicate that under induction at 37°C, the N-terminally truncated gE protein tends to form inclusion bodies when exogenously expressed in E. coli, and the inclusion bodies require a relatively high concentration of urea to dissolve, whereas the C-terminally truncated gE protein tends to be soluble when exogenously expressed in E. coli, and the amount of inclusion bodies formed decreases with increasing degree of truncation, making the inclusion bodies more soluble at low concentrations of urea.

[0109] Purification of truncated gE protein by anion exchange chromatography Supernatant samples of the lysates under induction conditions at 24° C. were filtered using a 0.22 μm pore size filter membrane and the samples were used for the next step of anion exchange chromatography.

[0110] Instrument system: AKTA Explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia company). Chromatography media: Q Sepharose 4 Fast Flow Column volume: 15mm x 20cm Buffer: 20mM Tris buffer pH 8.0 20mM Tris buffer pH8.0, 0.2M NaCl Flow rate: 8mL / min Detector wavelength: 280nm

[0111] The samples were lysed supernatants of bacterial cells containing truncated gE proteins of different lengths.

[0112] The elution procedure involved eluting impurities using 200 mM NaCl, eluting the target protein using 400 mM NaCl, and collecting the fractions eluted at 400 mM NaCl.

[0113] The results of SDS-PAGE analysis of various fractions obtained are shown in Figures 2 and 3. The results in Figure 2 show that, similar to the expression induced at 37°C, when the gE protein was truncated at the C-terminus at amino acid position 358 (330) and truncated at the N-terminus at amino acid positions 81 / 128 / 140 / 182, the amount of gE protein in the sonication supernatant gradually decreased, and gE(182-358) was mainly expressed in the form of inclusion bodies. When the gE protein was truncated at the C-terminus at amino acid position 537 and truncated at the N-terminus at amino acid positions 74 / 153, the amount of gE protein in the sonication supernatant gradually decreased, and gE(337-509) protein did not show obvious soluble expression at different induction temperatures. In summary, the above results indicate that, similar to expression under induction at 37°C, a certain degree of C-terminal truncation promotes soluble expression of gE protein in E. coli under induction at 24°C, whereas excessive N-terminal truncation changes the protein from soluble expression to inclusion body expression.

[0114] The results in Figure 3 show that the truncation of the C-terminus brings certain advantages to the purification of the protein, and that under these purification conditions the protein can achieve the column purification effect of low salt binding and high salt elution. Specifically, the uncleaved or slightly cleaved gE extracellular segments (31-375 / 401 / 430 / 538 / 546) in the lysis supernatant of E. coli could not bind to the Q-FF column under these purification conditions, and most of them permeated through, whereas the gE protein with further C-terminus truncation (31-358 / 320 / 303 / 181) bound to the column and could be eluted at a salt concentration of 0.4M. Both N- and C-terminally truncated gE(81-358) and gE(128-358) also bound to the column and could be eluted at a salt concentration of 0.4 M, whereas N-terminally truncated gE(74-537) could not bind to the chromatography column, and N-terminally truncated gE(153-537) and gE(182-358) showed no expression in the supernatant. In summary, the above results indicate that a certain degree of C-terminal truncation promotes the binding of gE protein in the ultrasonication supernatant to the anion column under TB8.0 buffer conditions, achieving the purification effect.

[0115] Next, the subsequent purification process of the lysate supernatant was described using the gE(31–358) protein as an example.

[0116] Hydroxyapatite (CHT) purification of gE(31-358) Instrument system: AKTA Explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia company). Chromatography medium: CHT-II Column volume: 15mm x 20cm Buffer: 5mM phosphate buffer pH 8.0, 0.4M NaCl Equilibrium solution: 20mM Tris buffer pH8.0, 0.4M NaCl Eluent: 200mM phosphate buffer pH 8.0, 0.4M NaCl Flow rate: 8mL / min Detector wavelength: 280nm Sample: Product eluted at 400mM NaCl during Q Sepharose 4 Fast Flow chromatography Elution procedure: Permeate was collected after loading, and fractions were collected continuously during column equilibration with equilibration solution after loading was completed.

[0117] The permeate was collected at equilibrium.

[0118] HIC (hydrophobic interaction chromatography) purification of gE(31-358) Instrument system: AKTA Explorer 100 preparative liquid chromatography system manufactured by GE Healthcare (formerly Amershan Pharmacia company). Chromatography media: Butyl Sepharose 4 Fast Flow Column volume: 15mm x 20cm Buffer: 20 mM Tris buffer pH 8.0, 1.5 M NaCl Eluent: 20mM Tris buffer pH8.0 Flow rate: 8mL / min Detector wavelength: 280nm

[0119] The sample was a CHT permeation product, and was treated with an appropriate amount of salt to give a salt concentration of 1.5M.

[0120] The elution procedure consisted of using 500 mM NaCl to elute the target protein and 0 mM NaCl to elute impurity proteins.

[0121] The elution product was collected at a concentration of 500 mM NaCl to obtain the purified gE(31-358) sample.

[0122] The SDS-PAGE analysis electrophoresis results of the samples of gE(31–358), gE(31–320), and gE(128–358) after purification at each stage are shown in Figure 4. The results showed that VZV gE truncated protein with purity of more than 90% could be obtained after anion chromatography, hydroxyapatite chromatography, and hydrophobic interaction chromatography.

[0123] In the following examples, the protein characteristics were detected using gE(31-358) protein as an example.

[0124] Example 3: Analysis of the properties of gE(31-358) protein Analysis of gE(31-358) protein by high-performance gel filtration chromatography. The instrument used was a Waters analytical high performance liquid chromatograph, using a TSK Gel G5000PW column.

[0125] The results of high-performance gel filtration chromatography of the VZV gE(31-358) protein purified in Example 2 are shown in panel A of Figure 5, which demonstrated that the gE(31-358) protein obtained in Example 2 had good purity and homogeneity.

[0126] Sedimentation velocity analysis of gE(31–358) protein The instrument was a Beckman-XL-A analytical ultracentrifuge with an An60-Ti rotor at a rotation speed of 30,000 rpm, and the collected data was fitted and analyzed using SEDFIT software.

[0127] The results of the analysis of the sedimentation coefficient of the VZV gE(31-358) protein purified in Example 2 are shown in panel B of Figure 5. The sedimentation coefficient C(s) of the VZV gE(31-358) protein obtained in Example 2 was approximately 2.3S, and the molecular weight was approximately 39.4 kDa, which was consistent with the results of SDS-PAGE, indicating that the purity and homogeneity of the protein were good.

[0128] Activity analysis of gE(31-358) protein The gE(31-358) protein obtained in Example 2 was coated (100ng / well) and incubated at 37℃ for 2 hours. The plate was washed and then blocked with 1×EDTA at room temperature for 2 hours. A gE protein-specific neutralizing monoclonal antibody (screened via gE protein expressed in our laboratory by a baculovirus expression system; see Liu, J., Zhu, R., Ye, X., et al. (2015). A monoclonal antibody-based VZV glycoprotein E quantitative assay and its application on antigen quantitation in VZV vaccine. Applied microbiology and biotechnology, 99(11), 4845-4853. https: / / doi.org / 10.1007 / s00253-015-6602-5) was used at a concentration of 0.1μg / mL or 1μg / mL in the first well, and then serially diluted 2-fold and incubated at 37℃ for 0.5 hours. The plate was washed five times, and the secondary antibody GAM-HRP (1:5000) was added and incubated at 37°C for 0.5 h, washed five times, developed, and terminated after 10 min. The plate was detected at a wavelength of 450 nm using an ELISA reader, and GraphPad Prism 5 (GraphPad, USA) software was used for data analysis. The results are shown in panel C of Figure 5, which indicates that the gE(31-358) protein obtained in Example 2 maintains good reactivity with gE-specific monoclonal antibodies.

[0129] Example 4: Immunogenicity analysis of gE(31-358) Detection of binding antibody titers induced by gE(31-358) The mice used in this experiment were 6-week-old female BALB / C or C57 mice. The gE(31-358) protein prepared by the method of Example 2 was administered in a 10-mL dose in a 10-mL ... B Mice were immunized intramuscularly with adjuvant (purchased from GSK) at an injection volume of 0.05 mL and a dose of 5 μg or 1 μg. The primary immunization was performed at week 0, and booster immunization was performed at week 2 and / or 4. ELISA was used to detect the binding antibody level in serum after antigen immunization. gE (Bac) protein (amino acid sequence shown in SEQ ID NO: 31) obtained by baculovirus-insect cell expression system and GAM-HRP served as the capture antigen and detection antibody, respectively. The binding titer was defined as the highest serum dilution ratio at which the absorbance value exceeded the critical value, and the critical value was calculated as the average value of the negative control OD450 value plus 3 times the standard deviation (sd). The detection results of the binding antibody titer in the immune serum are shown in Figure 6. The results showed that mice immunized with gE (31-358) protein in combination with different adjuvants could induce gE protein-specific binding antibodies in the serum. In this case, the binding antibody titer increased significantly after the first immunization, and after one or two booster immunizations, the antibody titer increased to 10 4 Times to 10 5 It can be more than double.

[0130] Determination of neutralizing antibody titers in serum after immunization The serum neutralizing antibody titers of the above immunized mice at weeks 2 and 6 (i.e., 2 weeks after primary / boosting immunization) were detected by serum antibody-mediated virus neutralization experiments. On the previous day, ARPE-19 cells (stored in our laboratory) were plated in 24-well cell culture plates, and the cells were used for infection when the cell density reached 70%-80% per well. A virus protection solution (25 mM histidine, 150 mM NaCl, 9% sucrose, pH 7.4) was used to prepare a virus standard solution containing vOka virus (500 pfu / mL-1000 pfu / mL, prepared by harvesting vOka virus passaged in ARPE-19 cells) and 10% (v / v) guinea pig serum (purchased by Beijing Bersee Science and Technology Co., Ltd., catalog number: BM361Y). The serum samples to be tested were inactivated at 56°C for 30 minutes, and then serially diluted with virus standard solution. The diluted serum mixture was incubated at 37°C for 1 hour. The culture medium of the 24-well plate pre-plated with ARPE-19 cells was discarded, and the serum mixture was added respectively and incubated at 37°C for 1 hour, after which the supernatant was discarded. DMEM / F12 medium was added, and the culture was continued, and the pathological changes of the cells were observed after 48 hours. If obvious lesion cells were observed, the cells in the 24-well plate were fixed and permeabilized in the conventional manner, and incubated with gE-specific enzyme-labeled antibody (1B11-HPR, 1 / 2000 dilution, prepared by our laboratory) for immunoadsorption. After rinsing three times with PBST, ELISPOT color development was performed, and the cell plate was photographed using a fluorescent spot analyzer. The lesion spots in the photograph were counted, and the neutralization titer of the serum was calculated. In the experiment, the serum samples to be tested were not added to the control wells, and the number of lesion spots in the wells was taken as the number of unneutralized viruses. The neutralizing titer of serum antibodies was defined as the maximum dilution at which serum could neutralize 50% of the virus. The results of the neutralizing titer are shown in Figure 7. From the results, gE(31-358) protein was adjuvanted with risedronate or AS01. BIt was shown that mice immunized with the adjuvant were able to induce the production of neutralizing antibodies in the serum. In this case, the titer of neutralizing antibodies was 10 3 It may reach such a high level.

[0131] Median effective dose (ED) of gE(31-358) 50 ) In this study, the median effective dose (ED 50 The immunogenicity of gE(31-358) protein was examined by determining the immunogenicity of gE(31-358) protein. The experimental animals were 3-4 week old female BALB / c mice. The gE(31-358) protein prepared in Example 2 was adsorbed to aluminum adjuvant, and the protein concentrations were 1.00μg / mL, 0.50μg / mL, 0.25μg / mL, 0.125μg / mL, 0.0625μg / mL, and 0.03125μg / mL, respectively, for a total of six dose groups. In each group, six BALB / c mice were intraperitoneally injected once with 1mL of the above concentration. In addition, a blank group containing six BALB / c mice was set. Serum was collected 4 weeks after injection, and varicella-zoster virus IgG was detected using a varicella-zoster virus IgG detection kit (enzyme-linked immunosorbent assay, EIA) (National Medical Device Registration No.: 20173403325) according to the instructions.

[0132] For the EIA detection results, the mean × 50% of the A value of the negative control wells (NC) (one negative control well with A value less than 0.80 should be discarded, and two negative control wells with A value less than 0.80 should be repeated) was calculated and used as the cutoff value. All BALB / c mice were negative for VZV antibodies before injection. The detection results are shown in Table 2.

[0133] [Table 2]

[0134] ED 50The ED was calculated according to the Reed-Muench method. After 4 weeks of observation, blood samples were taken and the ED 50 The results showed that the ED of gE(31-358) protein 50 It was shown that the dose was 0.063 μg, indicating that this dose was capable of producing a high level of immune antibodies.

[0135] Assessment of gE-specific cellular immune responses (flow cytometry) The mice used in this experiment were 6-week-old female C57 mice. The gE(31-358) protein prepared by the method of Example 2 was injected into AS01 B Mice were immunized with adjuvant by injection into the tibialis muscle at an injection volume of 0.05 mL and a dose of 5 μg. The control group was immunized with the same dose of Shingrix vaccine (GSK), and the blank group was immunized with saline. The primary immunization was performed at week 0, and the booster immunization was performed at week 4. Four and eight mice were sacrificed at week 2 (14 days after the single injection) and week 8 (30 days after the second injection), respectively. Spleens were removed under aseptic conditions, and spleen cell suspensions were prepared after crushing, filtering and lysing red blood cells, and 2 × 10 cells were placed in a 96-well U-bottom plate. 6 The cells were plated at a density of 1000 cells / well. A gE peptide mixture library (1.25 μg / mL; an overlapping peptide library covering aa 22-537 of gE protein, with a length of 15 aa, overlapping 11 aa, and synthesized by Sangon Biotech (Shanghai) Co., Ltd.) was added to the culture medium as a stimulus. After 18 h of culture, Golgi inhibitors were added and cultured for another 6 h. The stimulated cells were washed, fixed, permeabilized, and incubated with specific antibodies labeled with fluorescent dyes, followed by flow cytometry using a BD LSRFortessa™ X-20 cell analyzer to measure CD4 + and CD8 + The expression levels of cytokines such as IFN-γ and IL-2 in cell subsets were analyzed. The results are shown in Figure 8. gE(31-358) protein and AS01 BIt was found that the combination with an adjuvant was able to stimulate mice to produce a specific cellular immune response equivalent to that of the Shingrix vaccine.

[0136] The results of this example showed that the gE(31-358) protein obtained in Example 2 can be mixed with an adjuvant to formulate a vaccine with good immunogenicity, and can induce high titer binding antibodies, neutralizing antibodies, and specific cellular immune responses in animals, and can be used as a vaccine to prevent primary and recurrent VZV infection.

[0137] Although the specific embodiments of the present application have been described in detail, it will be understood that those skilled in the art can make various modifications and changes to the details based on all the teachings disclosed, and these modifications are within the scope of protection of the present application. The entirety of the present application is intended to be provided by the appended claims and their equivalents.

Claims

1. A truncated varicella-zoster virus (VZV) gE protein or a mutant thereof, wherein the truncated VZV gE protein has a truncation of 75 to 445 amino acids at the C-terminus compared to a wild-type VZV gE protein, and the mutant has at least 90%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity compared to the truncated VZV gE protein, or has a substitution, addition, or deletion of one or several amino acids, and retains the biological function of the truncated VZV gE protein.

2. The truncated VZV gE protein or variant thereof according to claim 1, wherein the truncated VZV gE protein has a truncation of at most 445 amino acids at the C-terminus and / or has a truncation of at least 75 amino acids at the C-terminus compared to the wild-type VZV gE protein.

3. The truncated VZV gE protein or variant thereof according to claim 1, wherein the truncated VZV gE protein has a truncation of at most 330 amino acids at the C-terminus and / or has a truncation of at least 260 amino acids at the C-terminus compared to the wild-type VZV gE protein.

4. the truncated VZV gE protein has no N-terminal truncation or an N-terminal truncation of 1 to 170 amino acids compared to the wild-type VZV gE protein; Preferably, the truncated VZV gE protein has an N-terminal truncation of at most 170 amino acids, such as at most 165, at most 160, at most 155, at most 150, at most 145, at most 140, at most 139, at most 135, at most 130 or at most 127 amino acids, such as at most 130 amino acids, compared to the wild-type VZV gE protein; and / or has an N-terminal truncation of at least 1 amino acid, such as at least 5, at least 10, at least 15, at least 20, at least 25 or at least 30 amino acids, such as at least 30 amino acids; Preferably, the truncated VZV gE protein has a lower nucleotide sequence than the wild-type VZV gE protein.

2. The truncated VZV gE protein or variant thereof of claim 1, wherein the gE protein has an N-terminal truncation of 20 to 170, 1 to 155, 20 to 155, 1 to 140, 20 to 140, 1 to 130, 20 to 130, 1 to 85, 20 to 85, 1 to 75, 20 to 75, 1 to 30, 20 to 30, 30 to 165, 30 to 152, 30 to 139, 30 to 127, 30 to 80, 30 to 73, 30 to 75, 30 to 85, 30 to 130, 30 to 140, 30 to 155, or 30 to 170 amino acids.

5. the truncated VZV gE protein has a C-terminal truncation of 260 to 330 amino acids compared to the wild-type VZV gE protein; 2. The truncated VZV gE protein or variant thereof according to claim 1, wherein the truncated VZV gE protein has no N-terminal truncation or a truncation of 1 to 130 amino acids at the N-terminus compared to the wild-type VZV gE protein.

6. The truncated VZV gE protein or its variant according to claim 1, wherein, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a truncation of 260 to 330 amino acids at the C-terminus and either no truncation or a truncation of 20 to 130 amino acids at the N-terminus.

7. The truncated VZV gE protein or variant thereof according to claim 1, wherein, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a C-terminal truncation of 77, 85, 193, 222, 248, 265, 293, 303, or 320 amino acids and either no N-terminal truncation or an N-terminal truncation of 30, 73, 80, 127, or 139 amino acids.

8. The truncated VZV gE protein or its variant according to claim 1, wherein, compared to the wild-type VZV gE protein, the truncated VZV gE protein has a truncation of 265, 293, 303, or 320 amino acids at the C-terminus and a truncation of 30, 73, 80, or 127 amino acids at the N-terminus.

9. The truncated VZV gE protein or variant thereof according to claim 1, wherein the truncated VZV gE protein has a truncation of 265 amino acids at the C-terminus and a truncation of 30 amino acids at the N-terminus compared to the wild-type VZV gE protein.

10. 2. The truncated VZV gE protein or variant thereof of claim 1, wherein said wild-type VZV gE protein has the amino acid sequence set forth in SEQ ID NO:

19.

11. 2. The truncated VZV gE protein or a mutant thereof according to claim 1, wherein the truncated VZV gE protein has an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6, 20 to 30.

12. An isolated nucleic acid molecule encoding the truncated VZV gE protein or a variant thereof according to any one of claims 1 to 11.

13. A vector comprising the isolated nucleic acid molecule of claim 12.

14. 13. A host cell comprising the isolated nucleic acid molecule of claim 12 or a vector comprising the isolated nucleic acid molecule, Preferably, the host cell is selected from the group consisting of a prokaryotic cell (e.g., an E. coli cell) and a eukaryotic cell (e.g., a yeast cell, an insect cell, a plant cell, a mammalian cell); Preferably, the host cell is an E. coli cell.

15. A method for preparing the truncated VZV gE protein or variant thereof according to any one of claims 1 to 11, comprising culturing a host cell comprising a nucleotide sequence encoding the truncated VZV gE protein or variant thereof under conditions allowing protein expression, and recovering the truncated VZV gE protein or variant thereof from a culture of the cultured host cell, Preferably, the method comprises recovering the truncated VZV gE protein or a variant thereof from a culture supernatant or a lysis supernatant of cultured host cells; Preferably, the host cell is Escherichia coli.

16. the recovering step comprises purifying the protein; Preferably, purifying the protein comprises performing ion exchange chromatography, hydroxyapatite chromatography, and / or hydrophobic interaction chromatography; Preferably, the ion exchange chromatography comprises: a) binding the truncated VZV gE protein or variant thereof to an anion exchange chromatography medium (e.g., Q Sepharose 4 Fast Flow) in a solution having a pH of 7.5 to 8.5 (e.g., pH 8.0) and a salt concentration of 0 mM to 200 mM (e.g., 0 mM to 50 mM); b) performing gradient elution by gradually increasing the salt concentration of said solution; c) collecting elution fractions containing the truncated VZV gE protein or variants thereof when the salt concentration of the solution is in the range of 350 mM to 450 mM (e.g., 400 mM); 16. The method of claim 15, comprising:

17. An immunogenic composition comprising the truncated VZV gE protein or variant thereof according to any one of claims 1 to 11, and optionally a pharmaceutically acceptable carrier and / or excipient (e.g., adjuvant), Preferably, the immunogenic composition comprises a truncated VZV gE protein or a mutant thereof and an adjuvant, and the adjuvant is selected from the group consisting of a risedronate adjuvant (e.g., a zinc-aluminum hybrid adjuvant containing sodium risedronate), an aluminum adjuvant (e.g., an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant), an oil emulsion adjuvant, a cytokine, a TLR agonist, a CpG adjuvant, a liposome, and ASO1. Ｂ The adjuvant is selected from the group consisting of sodium zoledronate, monophosphoryl lipid A (MPL), cholesterol-containing liposomes, and combinations thereof, and preferably, the adjuvant is selected from the group consisting of a risedronate adjuvant (e.g., a zinc-aluminum hybrid adjuvant containing sodium risedronate), an aluminum adjuvant (e.g., an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant), ASO1 Ｂ adjuvants, and combinations thereof; Preferably, the immunogenic composition comprises a truncated VZV gE protein or a mutant thereof and AS01. Ｂ and an adjuvant, Preferably, the immunogenic composition is a vaccine.

18. A pharmaceutical composition comprising: (i) a truncated VZV gE protein or variant thereof according to any one of claims 1 to 11; or (ii) an isolated nucleic acid molecule, or vector, or a host cell comprising a nucleotide sequence encoding the truncated VZV gE protein or variant thereof; or (iii) an immunogenic composition comprising a nucleotide sequence encoding the truncated VZV gE protein or variant thereof for inducing an immune response against VZV in a subject and / or for preventing and / or treating VZV infection or a disease associated with VZV infection in a subject; Preferably, said immunogenic composition is a vaccine, Preferably, the VZV infection is a primary or recurrent VZV infection; Preferably, the disease associated with VZV infection is selected from the group consisting of shingles, chickenpox, and postherpetic neuralgia; Preferably, the subject is a mammal such as a human.

19. A method for detecting the presence of VZV gE protein-specific antibodies in a sample, comprising using a truncated VZV gE protein or a variant thereof according to any one of claims 1 to 11, Preferably, the method is an immunological detection, such as an immunoblot, an enzyme-linked immunosorbent assay (e.g., ELISA), a chemiluminescent immunoassay, a fluorescent immunoassay, or a radioimmunoassay; Preferably, the method comprises: (1) contacting the sample with a truncated VZV gE protein or a mutant thereof; and (2) detecting the formation of a protein-antibody immune complex or detecting the amount of said immune complex, wherein the formation of said immune complex indicates the presence of VZV gE protein-specific antibodies in the sample; Preferably, the method further comprises detecting the presence of VZV gE protein-specific antibodies in the sample using a second antibody having a detectable label (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent agent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., fluorescein or a fluorescent protein), a radionuclide, or biotin); Preferably, the second antibody is specific for a constant region contained in an antibody of the species (e.g., human) from which the sample to be tested originates; Preferably, the second antibody is an anti-immunoglobulin (e.g., human immunoglobulin) antibody, such as an anti-IgG antibody; Preferably, the method, wherein the sample is a body fluid sample (eg, whole blood, plasma, serum, salivary secretions, or urine) from a subject (eg, a mammal, preferably a human).

20. 12. Use of a truncated VZV gE protein or a variant thereof, or an isolated nucleic acid molecule, or a vector, or a host cell comprising a nucleotide sequence encoding a truncated VZV gE protein or a variant thereof according to any one of claims 1 to 11, in the manufacture of a detection reagent, wherein said detection reagent is used to detect the presence of VZV gE protein-specific antibodies in a sample, Preferably, the use, wherein said sample is a body fluid sample (eg whole blood, plasma, serum, salivary secretion, or urine) from a subject (eg a mammal, preferably a human).

21. A kit comprising the truncated VZV gE protein or variant thereof according to any one of claims 1 to 11, or an isolated nucleic acid molecule, or a vector, or a host cell comprising a nucleotide sequence encoding the truncated VZV gE protein or variant thereof, Preferably, the kit comprises a truncated VZV gE protein or a mutant thereof and a second antibody, said second antibody carrying a detectable label.

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