Truncated varicella-zoster virus ge protein and use thereof

Abstract

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

Description

A truncated varicella-zoster virus gE protein and its application

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202310181926.4 filed on February 28, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to the field of viral vaccines, and more specifically, to a truncated varicella-zoster virus gE protein and a pharmaceutical composition comprising the protein, its use in preparing a herpes zoster vaccine, and its use in treating diseases or conditions associated with herpes zoster virus infection. Background Art

[0004] Herpes zoster (HZ) is an acute infectious skin disease primarily caused by the reactivation of the varicella-zoster virus (VZV), which remains dormant in the body. Humans are the sole host of the varicella-zoster virus, with an incubation period of approximately 14 days. The primary infection is chickenpox, and reactivation of VZV, dormant in the sensory ganglia, causes herpes zoster. Herpes zoster is characterized by clustered blisters distributed in a band-like pattern, accompanied by significant neuralgia. The disease is common in elderly patients, those with immunodeficiency, and those taking immunosuppressants. Complications are common after contracting herpes zoster, with pain levels reaching levels 7 or higher, severely impacting quality of life and associated with a high recurrence rate.

[0005] The incidence and severity of herpes zoster increase with age, with a significant increase after the age of 50. This is related to the decline of cell-mediated immunity in the elderly. It is estimated that approximately half of the elderly over 85 years old have experienced at least one episode of herpes zoster. Studies conducted in Canada, Israel, Japan, Taiwan, and the United States have shown that the incidence of herpes zoster in the general population is 3.4-5.0 / 1000 people / year, and in people over 65 years old it is 8-11 / 1000 people / year. A study conducted in 27 European countries found that the incidence of herpes zoster ranged from 2.0-4.6 / 1000 people / year in different countries, but did not show significant regional differences.

[0006] The varicella-zoster virus (VZV) genome is a linear double-stranded DNA sequence measuring 125 kbp in length. It contains 71 ORFs encoding 68 proteins, including 12 glycoproteins. Glycoproteins are particularly important in the viral life cycle. Glycoprotein E (gE) is the most abundant glycoprotein expressed during VZV infection and plays a crucial role in mediating viral replication and spread. Furthermore, gE contains neutralizing antibodies and T cell epitopes, which can induce specific humoral and cellular immunity, making it an important candidate antigen for the design of a shingles vaccine.

[0007] Currently, there is no specific treatment for herpes zoster (shingles), and vaccination is the only effective means of prevention and control. Several vaccines have been developed for herpes zoster, but these vaccines suffer from issues such as low protein expression efficiency, poor protein activity, and suboptimal immune responses. Therefore, there is a need for an improved VZV vaccine.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention provides a truncated varicella-zoster virus gE protein and its use in preparing a vaccine composition for preventing or alleviating herpes zoster and related diseases. The benefits provided by the present disclosure are widely applicable to the biomedical field.

[0010] One aspect of the present invention provides a glycoprotein E (gE) protein variant, the variant comprising or consisting of:

[0011] (a) amino acids 1-496 or 31-496 of the full-length amino acid sequence of native gE protein;

[0012] (b) a fragment of amino acids 31-537 of the full-length amino acid sequence of a native gE protein, truncated from the C-terminus by 1-50 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, and any subrange or individual value thereof) amino acids; or

[0013] (c) an amino acid sequence that is at least 90%, 95%, or 99% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) identical to the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2.

[0014] In some embodiments, the natural gE protein is derived from varicella-zoster virus. More specifically, the full-length amino acid sequence of the natural gE protein may comprise or consist of the amino acid sequence shown in SEQ ID NO: 3.

[0015] 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 the native gE protein. More specifically, the gE protein variant consists of the amino acid sequence shown in SEQ ID NO: 2.

[0016] One aspect of the present invention provides a fusion protein comprising the gE protein variant and a heterologous peptide such as a signal peptide sequence.

[0017] One aspect of the present invention provides a truncated varicella-zoster virus gE protein comprising the following amino acid sequence:

[0018] (a) a fragment of amino acids 1-496 or 31-496 of the full-length amino acid sequence of a native gE protein;

[0019] (b) a fragment of amino acids 31-537 of the full-length amino acid sequence of a native gE protein truncated at the C-terminus by 1-50 (e.g., 10, 15, 20, 25, 30, 35, 40, 45, and any subrange or individual value thereof) amino acids; or

[0020] (c) an amino acid sequence that is at least 90%, 95% or 99% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%) identical to the amino acid sequence encoded by SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2.

[0021] 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 shown in SEQ ID NO: 2.

[0022] One aspect of the present invention provides a nucleic acid molecule encoding a gE protein variant or a truncated gE protein disclosed herein. Another aspect of the present invention provides a vector comprising a polynucleotide sequence encoding a truncated varicella-zoster virus gE protein.

[0023] Another aspect of the present invention provides a host cell comprising a vector encoding the gE protein variant or truncated varicella-zoster virus gE protein.

[0024] Another aspect of the present invention provides a pharmaceutical composition comprising the gE protein variant, the truncated varicella-zoster virus gE protein, or a nucleic acid molecule encoding the same, and a pharmaceutically acceptable carrier or adjuvant. In some embodiments, the pharmaceutical composition is a vaccine composition.

[0025] The adjuvant may be any adjuvant commonly used in the art, or an adjuvant used in other herpes zoster vaccine compositions. The adjuvant may be selected from aluminum adjuvants, ASO adjuvants, squalene oil-in-water adjuvants, saponin adjuvants, α-galactosylceramide derivative adjuvants, polysaccharide adjuvants, agonists that induce immune responses (e.g., TLR agonists, NOD agonists, CLR agonists, RLR agonists), and any combination thereof.

[0026] 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 analog, and / or the CpG ODN is CpG1018.

[0027] 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 CpG1018.

[0028] Another aspect of the present invention provides a method for preparing the truncated varicella-zoster virus gE protein or gE protein variant disclosed herein, comprising the following steps:

[0029] - expressing the truncated varicella-zoster virus gE protein or gE protein variant in a host cell; and

[0030] - isolating the truncated varicella-zoster virus gE protein or gE protein variant from the host cell.

[0031] Another aspect of the present invention provides use of a truncated varicella-zoster virus gE protein or gE protein variant or a nucleic acid molecule encoding the same in preparing a vaccine composition for preventing or alleviating varicella-zoster virus infection in a subject.

[0032] Another aspect of the present invention provides the 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 condition associated with varicella-zoster virus in a subject. The disease or condition may be selected from chickenpox, herpes zoster, and postherpetic neuralgia.

[0033] Another aspect of the present invention provides a method for treating or preventing a disease or condition associated with varicella-zoster virus in a subject, comprising administering to the subject a pharmaceutical composition, particularly 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 condition may be selected from chickenpox, herpes zoster, and postherpetic neuralgia.

[0034] In some embodiments, the subject is a human or non-human mammal, preferably a human.

[0035] Another aspect of the present invention provides a kit comprising a container comprising the truncated varicella-zoster virus gE protein disclosed herein or a nucleic acid molecule encoding the same.

[0036] Another aspect of the present invention provides a combination of a squalene oil-in-water adjuvant and a CpG ODN for use as an adjuvant in a herpes 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.

[0037] Another aspect of the present invention provides the use of the combination in the preparation of a vaccine composition for preventing or alleviating varicella-zoster virus infection and diseases or conditions associated with varicella-zoster virus in a subject. The vaccine composition further comprises a native gE protein, a fragment thereof (e.g., 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.

[0038] In some embodiments, the vaccine composition comprises a truncated gE protein disclosed herein.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The following figures are included to further illustrate certain aspects and features of the invention. The invention may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments (including examples).

[0041] FIG1A is a plasmid map for constructing the Pee12.4-VZV-gE recombinant vector.

[0042] FIG1B is a 0.8% agarose gel electrophoresis to identify the PCR-amplified gE gene, wherein gE represents the expression construct of the varicella-zoster virus gE31-496 gene (also comprising a partial vector sequence and a signal peptide sequence), and M represents a 5000 bp DNA molecular weight standard.

[0043] Detailed Description of the Invention

[0044] Unless otherwise defined, 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 the invention belongs. In addition, unless the context requires otherwise, terms in the singular shall include the plural form, and terms in the plural 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 indicators unless the context clearly indicates otherwise. Thus, for example, reference to "a protein" includes multiple proteins; reference to "a cell" includes a mixture of cells, etc. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. In addition, the ranges provided in the specification and the appended claims include the endpoints and all values ​​between the endpoints.

[0045] As used herein, the term "effective amount" refers to the amount of a compound, therapeutic agent, virus or drug that is capable of achieving the desired result. As understood in the art, an effective amount can vary, depending on the patient's medical history and other factors, such as the type and / or dosage of the therapeutic agent used.

[0046] As used herein, the terms "subject" and "patient" are used interchangeably to refer to any living organism, including humans and animals.

[0047] As used herein, the term "about" when used in conjunction with a numerical value is intended to encompass numerical values ​​within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value. Ranges used herein include the specified endpoints of the range and any subranges or individual values ​​therebetween.

[0048] As used herein, "percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of identical amino acid residues in the candidate sequence and the reference polypeptide sequence, after the sequences are aligned (and, if necessary, introduced into gaps) to obtain maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Sequence alignment can be performed using various methods in the art to determine percent amino acid sequence identity, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to obtain maximum alignment over the full length of the compared sequences. When referring to percentages of sequence identity in this application, if not otherwise specifically noted, these percentages are calculated relative to the full length of the longer sequence. Calculations relative to the full length of the longer sequence are applicable to both nucleic acid sequences and polypeptide sequences.

[0049] As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell but may contain mutations.

[0050] As used herein, the term "vector" when used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated 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 operatively linked.

[0051] As used herein, the term "pharmaceutically acceptable" means that the vehicle, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other ingredients of the formulation and physiologically compatible with the recipient.

[0052] 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, pH adjusters, surfactants, 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; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0053] As used herein, the term "adjuvant" refers to a nonspecific immunopotentiator that can enhance the immune response to the antigen in an organism or change the type of immune response when it is delivered to the organism together with the antigen or is delivered to the organism in advance. There are multiple adjuvants, including but not limited to aluminum adjuvants (such as aluminum hydroxide), Freund's adjuvants (such as Freund's complete adjuvant and Freund's incomplete adjuvant), Corynebacterium brevis, lipopolysaccharide, cytokines, etc. Freund's adjuvant is the most commonly used adjuvant in current animal experiments. Aluminum hydroxide adjuvant is more commonly used in clinical trials.

[0054] As used herein, the term "CpG ODN" refers to a special type of DNA oligonucleotide, typically ranging from 10 to 30 bases in length. "CpG" represents a cytosine and a guanine linked together. These molecules play an important role in the immune system because they can mimic DNA sequences found in bacteria and viruses, triggering immune responses. CpG ODNs are widely used in immune research and drug development, particularly in the field of immunotherapy.

[0055] Varicella-zoster virus (VZV) and gE antigen

[0056] The varicella-zoster virus, a member of the human herpesvirus family, is the cause of two distinct clinical manifestations: chickenpox and herpes zoster. Initial infection with this virus causes chickenpox (a blistering sore). Subsequently, the virus remains latent in the nerve ganglia, and after years, reactivation by a trigger triggers the herpes zoster rash (the formation of viral particles, which travel through nerves to reach the epidermis, causing symptoms of chickenpox in the nerve supply).

[0057] The VZV genome is a double-stranded DNA containing approximately 125,000 bases, and it is known that there are at least 72 genes in the genome. There are four main glycoproteins or complexes on the surface of the VZV virus, namely gB, gC, gE / I and gH / gL. The gE protein is the most expressed glycoprotein during the VZV infection period and is also the most important surface antigen, playing an important role in mediating viral replication and spread. Natural gE protein (for example, as shown in NCBI ID Q9J3M8.1, AQT34120.1, AGY33616.1, AEW88548.1) is usually 623 amino acids in length and consists of a hydrophobic transmembrane region, an intracellular region, and an extracellular region, wherein the extracellular region contains 537 amino acids (i.e., amino acids 1-537), including a signal peptide region of 30 amino acids at the N-terminus. The antigenic epitopes of gE protein are mainly distributed in the extracellular region. Studies by Vafai et al. and Forghani et al. have shown that gE protein contains at least three different antigenic epitopes, located at amino acid positions 109-123, 160-316, and 101-161, respectively.

[0058] In some aspects, the present invention provides a truncated gE protein, which is a fragment of a natural gE protein (1-623), preferably a fragment of the extracellular region (1-537) of a natural gE protein, more preferably a fragment of the extracellular region (31-537) of a natural gE protein with a signal peptide region removed. In some embodiments, the truncated gE protein does not include the transmembrane region and the intracellular region of a natural gE protein. In some embodiments, the truncated gE protein is the extracellular region of a natural gE protein. In some embodiments, the truncated gE protein is a fragment further truncated in the extracellular region of a natural gE protein. In some embodiments, the truncated gE protein does not include the signal peptide sequence at the N-terminus of a natural gE protein.

[0059] In some embodiments, the truncated gE protein is a fragment of the native gE protein (1-623) truncated from the C-terminus, for example, a fragment obtained by truncating 1-130 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 or more) amino acids from the C-terminus, and optionally also truncating 1-50 (e.g., 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4 or fewer) amino acids from the N-terminus. In some embodiments, the truncated gE protein is a fragment obtained by truncating 1-100 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90 or more) amino acids from the extracellular region of the natural gE protein (31-537) with the signal peptide region removed from the C-terminus, and optionally truncating 1-30 (e.g., 25, 20, 15, 10, 9, 8, 7, 6, 5, 4 or less) amino acids from the N-terminus. In some embodiments, the truncated gE protein is a fragment obtained by truncating 127 amino acids from the C-terminus of the natural gE protein (1-623), i.e., a 1-496 fragment. In some embodiments, the truncated gE protein consists of amino acids 31-496 of the natural gE protein (as shown in SEQ ID NO: 2).

[0060] The inventors surprisingly found that when the extracellular region amino acids 31-496 were used to prepare truncated gE protein, the expression yield was significantly improved compared with using other gE protein extracellular regions (such as amino acids 31-537), and higher antigen expression and cell yield could be achieved.

[0061] Vaccines against VZV

[0062] There are three main types of vaccines currently in use: live attenuated vaccines, inactivated vaccines, and genetically engineered vaccines. Live attenuated vaccines induce antibody production at the expense of low toxicity, but their immune effectiveness is suboptimal and poses safety risks. Inactivated vaccines only inactivate the virus during use, but still use infectious viruses during production, which carries risks. Genetically engineered vaccines induce antibody production by heterologously expressing viral envelope proteins, without using active viruses, making the process safer.

[0063] Available live attenuated VZV vaccines include varicella and herpes zoster (shingles) vaccines. Currently, attenuated varicella vaccines are all prepared using the Oka strain and offer good immunogenicity after one or two doses. However, attenuated herpes zoster (shingles) vaccines have a poor immune response, with only 50-60% efficacy, and are increasingly being replaced by recombinant protein vaccines. Both live attenuated varicella and herpes zoster vaccines carry risks. Merck's live attenuated herpes zoster vaccine (Zostavax) is produced using the attenuated Oka strain. GSK's Shingrix is ​​a subunit herpes zoster vaccine containing recombinant glycoprotein E and a novel adjuvant (AS01B). Shingrix received FDA approval in October 2017 for the prevention of herpes zoster in adults 50 years of age or older.

[0064] Preparation of vaccine compositions

[0065] VZV gE protein is typically prepared by expression in cultured cells or by chemical synthesis. The host cell herein can be any cell suitable for expressing the antigens of the present disclosure. For example, commonly used and suitable host cells for protein production include Escherichia coli cells, yeast cells, insect cells, and mammalian cells. Mammalian host cells include Chinese hamster ovary (CHO cells), COS cells, and SP2 cells. Protein expression in eukaryotic cells generally results in a protein sequence lacking a signal peptide.

[0066] Expression vectors and host cells are commercially available. The expression vector comprises a promoter and a cloning site for the sequence encoding the 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 the product. Methods and procedures for transfecting host cells are also well known. As previously mentioned, a suitable VZV gE antigen is a VZV gE protein with a truncated C-terminus and / or N-terminus.

[0067] 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 the protein of interest to be expressed in the host cell or secreted into the culture medium in which the host cell is cultured. The protein of interest can be recovered from the culture medium using standard protein purification methods.

[0068] Preferably, the gE truncated protein antigen is combined with a specific adjuvant to prepare a vaccine composition. The vaccine composition can be in the form of an aqueous solution, oil-in-water, water-in-oil, liposome, etc. A good adjuvant can promote the production of sufficient cellular and humoral immune responses in the body to achieve a protective effect. Currently approved adjuvants can be roughly divided into two types: one is an adjuvant that stimulates the immune response, which enhances the immunogenicity of the antigen by connecting to receptors on innate immune cells, such as CpG1018; the other is an adjuvant that cooperates with vaccine delivery, presenting the necessary amount of vaccine antigen and immunostimulant to the immune system to induce immunity, such as aluminum adjuvants and emulsions.

[0069] The vaccine composition of the present invention can be prepared by mixing the truncated gE protein with various adjuvants known in the art in a certain proportion. 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 (e.g., aluminum hydroxide, aluminum phosphate), squalene-containing oil-in-water emulsions (e.g., MF59), ASO adjuvants (e.g., AS01, AS03, AS04), saponin adjuvants, α-galactosylceramide-derived adjuvants, and polysaccharide adjuvants.

[0070] In some embodiments, the adjuvant comprises an aluminum adjuvant, a squalene-containing oil-in-water emulsion, or a liposome-carried adjuvant. Furthermore, the adjuvant may also comprise an agonist adjuvant, such as a TLR agonist adjuvant such as CpG ODN, preferably CpG1018. CpG1018 belongs to the category of CpG oligodeoxyribonucleotides (CpG ODN) and is a 22-mer unmethylated CpG-B class oligonucleotide. As an agonist of Toll-like receptor 9 (TLR9), CpG ODN can stimulate cells expressing TLR9 and activate downstream innate immune response pathways, on the one hand inducing the expression of type I interferon and inflammatory factors, and on the other hand maturing plasmacytoid dendritic cells, thereby enhancing humoral immunity and cellular immune responses. It can be taken up by endosomes, causing increased secretion of interferon, and effectively activating antigen-specific T cells by promoting cross-presentation of antigens.

[0071] MF59 or its analogs are among the earliest approved clinical adjuvants. They are a water-in-oil emulsion adjuvant primarily composed of squalene, Tween 80, and Span 85. Its oil-in-water design significantly reduces viscosity and enhances human tolerability. Squalene, an oil-phase component, is widely distributed in animals, plants, and the human body, and exhibits both biocompatibility and biodegradability. Tween 80 and Span 85 are surfactants that enhance the stability of the emulsion. MF59 is an ideal adjuvant for viral vaccines. When used in combination with vaccines for influenza, HIV, and other diseases, it significantly enhances antibody titers and is well tolerated across diverse populations. It promotes antigen uptake, recruits numerous immune cells to the injection site, and recruits monocytes and neutrophils to present and transport antigens to lymph nodes. When used in combination with CpG, it induces higher antibody titers and a Th1-type response than either adjuvant alone.

[0072] In one aspect, the present invention provides a combination of MF59 and CpG for use in preparing herpesvirus vaccine compositions. The inventors have discovered that the combined use of MF59 and CpG significantly enhances the immunogenicity of antigens, demonstrating a strong synergistic effect. The simultaneous use of MF59 (or an MF59 analog) and CpG ODN adjuvants synergistically induces antigen-specific cellular immune responses, resulting in a vaccine composition with a strong ability to induce specific humoral and cellular immune responses.

[0073] Diseases or symptoms caused by herpes zoster infection

[0074] Herpes zoster is caused by reactivation of the varicella-zoster virus (VZV) during primary varicella infection and is characterized by skin pain and papules. The pain usually appears a few days before the rash and can persist for months after the rash subsides. The rash often appears in a single skin area and usually subsides within 4-5 weeks. Common complications include postherpetic neuralgia (PHN) and VZV pneumonia.

[0075] In older adults, age-related loss of functional VZV-specific T cell-mediated immunity coincides with an increased incidence of herpes zoster (HZ). VZV-specific T cell-mediated immunity is also crucial 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 inversely correlated with HZ disease severity and the risk of developing postherpetic neuralgia.

[0076] In one aspect, the present invention provides use of a vaccine composition in the preparation of a medicament for preventing or ameliorating herpes zoster and / or post-herpes zoster symptoms.

[0077] Use of compositions comprising truncated gE proteins

[0078] In one aspect, the present invention provides a method for administering or inoculating an effective amount of a truncated gE protein or a polynucleotide encoding the protein, or a pharmaceutical composition comprising the same, to a subject as a therapeutic agent or a preventive agent.

[0079] For preventive and therapeutic agents, administration should be carried out taking into account the clinical status of different patients (particularly the side effects when the preventive and therapeutic agents are used alone), the site of delivery, the method of administration, the administration plan, and other factors known to those skilled in the art. It will be understood by those skilled in the art that appropriate dosages may vary from subject to subject. Determining the optimal dosage generally involves a balance between the level of therapeutic benefit and any risks or adverse side effects. The dosage level selected will depend on a variety of factors, including but not limited to the activity of the specific administration, the route of administration, the time of administration, and the type, sex, age, weight, general health, and previous medical history of the patient. The amount of the administration and the route of administration are ultimately determined by the doctor, veterinarian, or clinician, but the dosage is generally selected to achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects.

[0080] The truncated gE protein of the present invention and the vaccine composition or vaccine product prepared using the same can be administered to a subject in need thereof in vivo via various routes, which are common routes of vaccine administration, including but not limited to subcutaneous, intramuscular, intranasal, and oral administration. Preferably, administration is performed by subcutaneous or intramuscular injection.

[0081] The truncated gE protein to be administered in the present invention is usually administered in a liquid form (eg, injection) and can be diluted with a buffer to a desired concentration before use.

[0082] The truncated gE protein of the present invention can be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier. The so-called "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, coating material or any form of formulation adjuvant.

[0083] The carrier may contain minor additives as appropriate, such as substances with high isotonicity and chemical stability. These substances are non-toxic to the recipient at the dosages and concentrations used, such as: buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than 10 residues) polypeptides, such as polyarginine or tripeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, or dextrins; chelating agents such as ethylenediaminetetraacetic acid; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and / or nonionic surfactants such as polysorbates, poloxamers, or PEG.

[0084] Any medicament that can be used therapeutically can be in a state that does not contain organisms or viruses other than viruses as active ingredients, i.e., a sterile state. Sterile conditions can be easily achieved by filtering through a sterile filter membrane (e.g., a 0.2 micron membrane). Generally speaking, therapeutic agents / prophylactic agents are placed in containers with sterile access ports, for example, intravenous solution bags with stoppers that can be pierced with a hypodermic needle, or vials with stoppers.

[0085] Therapeutic / prophylactic agents are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or freeze-dried formulations that require reconstitution. As an example of a freeze-dried 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 freeze-dried. The freeze-dried therapeutic / prophylactic agent is reconstituted with sterile water for injection to prepare an infusion solution.

[0086] Therapeutic / prophylactic agents that can be administered in combination with the vaccine composition of the present invention include, but are not limited to, chemotherapeutic agents, antibiotics, steroidal or non-steroidal anti-inflammatory drugs, existing immunotherapeutic / prophylactic agents, other cytokines and / or growth factors, etc. The so-called combination includes, for example, simultaneous administration as a composition, separate but simultaneous or concurrent administration, or chronological administration.

[0087] Reagent test kit

[0088] The present invention provides a pharmaceutical packaging or kit comprising one or more containers filled with one or more ingredients of the therapeutic / prophylactic agents of the present invention. The containers are also accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, indicating that the governmental agency approves the manufacture, use, or sale for human administration.

[0089] Beneficial effects of the present invention

[0090] The present invention surprisingly found that when the truncated gE protein is prepared using the N-terminal 31-496 amino acids of the extracellular region, its expression yield is significantly improved while retaining its antigenic epitope. When used in combination with an adjuvant, it has a strong ability to induce specific humoral immunity and cellular immune responses.

[0091] Compared with the imported recombinant herpes zoster virus vaccine (Shingrix, GSK) currently available on the market, the recombinant herpes zoster virus vaccine containing the novel adjuvant of the present invention has shown comparable safety and immunogenicity results at the animal level. Its main advantages are:

[0092] It can help vaccinated people effectively prevent varicella-zoster virus infection;

[0093] This novel adjuvanted recombinant herpes zoster virus vaccine contains a new adjuvant system;

[0094] This new adjuvanted recombinant herpes zoster virus vaccine is cheaper and more accessible than imported vaccines (Shingrix). Example

[0095] The following examples are given only for the purpose of illustrating various embodiments of the present invention and are not meant to limit the invention.

[0096] Example 1: Construction of stable cell lines, protein expression and purification

[0097] 1. gE31-496 protein selection and gene synthesis

[0098] Through data retrieval, the conserved amino acid sequence of the gE protein from the NCBI database was selected for gene sequence optimization. Codons preferred by CHO cells were selected for codon optimization and whole-gene synthesis. To promote secretory expression and improve protein expression in mammalian cells, the IL10 signal peptide sequence was introduced. Restriction endonuclease recognition sites for HindIII and EcoRI were introduced at the 5' and 3' ends of the target gene, respectively. PCR amplification was used to amplify the synthesized single-stranded DNA primers and connect them to obtain the target fragments. PCR amplification was then used to splice the target fragments into the full-length sequence. The nucleotide sequence is shown in SEQ ID NO:1, which contains the coding sequence for the signal peptide and the truncated protein from positions 31-496 of the mature gE extracellular domain.

[0099] The coding sequence of the truncated protein at position 31-537 of the gE protein connected to the IL-10 signal peptide sequence was constructed in the same way.

[0100] 2. Construction of gE protein expression vector

[0101] The eukaryotic expression vector PEE12.4 used in the present invention carries the AmpR resistance gene and the GS screening gene. The vector uses a cytomegalovirus (CMV) promoter / enhancer sequence to express the target gene.

[0102] The vector PEE12.4 was double-digested with restriction endonucleases HindIII and EcoRI. Finally, the recovered and purified PEE12.4 vector DNA was recombined and ligated with the target gene DNA fragment. The recombinant ligation product was added to competent cells to obtain several monoclonal positive colonies. Parts of these colonies were selected for PCR amplification and verification (Figure 1), thereby obtaining a large amount of extracted plasmid.

[0103] 3. Screening of Stable Transfected Cell Lines and Protein Expression and Purification

[0104] The plasmid obtained in 2 above was transfected into the host cell CHO-K1, the mini cell group was screened, and the cell group with higher expression level was subcloned. The screened clones were fed-batch cultured, and the supernatant was collected to detect the target protein. The three best clones were selected based on the cell growth, protein expression, terminal lactic acid content and related product quality. The cell line was expanded by fed-batch culture, and the target protein in the culture supernatant was harvested. The average content of the gE31-496 truncated protein (whose amino acid sequence is shown in SEQ ID NO: 2) was 4-5 g / L, which was much higher than the target protein expression level (1.5-2 g / L) of the gE31-537 stably transfected cell line constructed by the same method.

[0105] Table 1: Yields of gE truncated proteins

[0106] Example 2: Evaluation of the immunogenicity of a vaccine composition containing gE protein

[0107] The truncated gE31-496 protein produced in Example 1 was purified and mixed uniformly with MF59+CpG adjuvant (MF59: Invivogen, Catalog No. vac-adx-10; CpG1018: Novus, Catalog No. NBP2-31142) to prepare a recombinant herpes zoster vaccine containing the novel adjuvant. Animal experiments were conducted. Similarly, a vaccine containing the truncated gE31-537 protein mixed with MF59+CpG adjuvant was prepared as a control.

[0108] The animal experiment procedures are as follows:

[0109] C57BL / 6 mice aged 6-8 weeks were randomly divided into groups of 6. The C57BL / 6 mice were pre-immunized with an attenuated varicella vaccine (Shanghai Institute of Biological Products Co., Ltd.), and blood was collected. Each group of mice received the first dose 35 days later, and the second dose 28 days later. The specific immunization schedule is shown in Table 2. The amount of MF59 used per mouse was 25 μl. Blood was collected from each group of C57BL / 6 mice on days 56-58, and the titer of induced total IgG antibodies to the specific gE protein was measured by ELISA. Spleens were collected, and splenic lymphocytes were isolated and analyzed for INF-γ and IL-2 expression using ELISPOT. The results are shown in Table 3.

[0110] The results indicate that the truncated gE protein (31-496) and its vaccine composition have good immunogenicity, comparable to that of the mature gE 31-537 extracellular region protein. MF59+CpG exhibit excellent synergistic effects and can be used as a candidate for a herpes zoster vaccine.

[0111] Table 2. Animal experimental vaccine numbers and composition information

[0112] Table 3. Summary of immunogenicity evaluation results

[0113] *Significant difference compared with gE31-537 (5μg) + MF59 + CpG1018 (10μg) (p < 0.05)

[0114] Under the same conditions, the immunogenicity of a truncated gE protein (31-496) vaccine composition prepared using aluminum hydroxide (supplier: Invivogen, catalog number: vac-alu-250) instead of MF59 was tested.

[0115] Table 4. Summary of immunogenicity evaluation results

[0116] *Compared with gE31-537 (5 μg) + aluminum hydroxide (50 μg) + CpG1018 (10 μg), there was a significant difference (p < 0.05)

[0117] The results showed that the immunogenicity of the gE31-496 vaccine composition was significantly superior to that of the gE31-537 truncated protein vaccine composition, regardless of whether MF59+CpG1018 or aluminum hydroxide+CpG1018 was used as an adjuvant. When MF59+CpG1018 was used as an adjuvant, both truncated protein vaccine compositions significantly improved IgG production, IFN-γ, and IL-2 compared to aluminum hydroxide+CpG1018, demonstrating the superiority of MF59+CpG1018 over aluminum hydroxide+CpG1018. When MF59 or aluminum hydroxide was used alone as an adjuvant, MF59 was inferior to aluminum hydroxide in producing IgG, so the combination of MF59+CpG1018 produced a synergistic effect.

[0118] The sequence involved in the present invention is as follows:

[0119] SEQ ID No: 1 Nucleotide sequence encoding recombinant varicella-zoster virus gE31-496 truncated protein, wherein the box is the start codon, the underlined portion is the signal peptide sequence, and the rest is the gE31-496 sequence

[0120] SEQ ID No: 2 Amino acid sequence of recombinant varicella-zoster virus gE31-496 truncated protein (excluding signal peptide):

[0121] SEQ ID NO: 3 Full-length gE protein

[0122] Those skilled in the art will further appreciate that the present invention may be embodied in other specific forms without departing from its spirit or central features. Since the foregoing description of the present invention discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims for an indication of the scope and content of the present invention.

Claims

1. A glycoprotein E (gE) protein variant, consisting of the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 2.

2. A fusion protein, comprising the gE protein variant of claim 1 and a heterologous peptide, optionally wherein the heterologous peptide is a signal peptide.

3. A nucleic acid molecule encoding the gE protein variant of claim 1.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A pharmaceutical composition comprising the gE protein variant of claim 1 or the nucleic acid molecule of claim 3, and a pharmaceutically acceptable carrier and / or adjuvant, optionally wherein the pharmaceutical composition is a vaccine composition.

7. The pharmaceutical composition of claim 6, wherein the adjuvant is selected from the group consisting of aluminum adjuvants, AS0 adjuvants, squalene oil-in-water adjuvants, saponin adjuvants, a-galactosylceramide-derived adjuvants, polysaccharide adjuvants, agonists that induce immune responses in the body, and any combination(s) thereof,optionally wherein the agonists that induce immune response in the body are selected from the group consisting of TLR agonists, NOD agonists, CLR agonists, and RLR agonists.

8. The pharmaceutical composition of claim 7, wherein the adjuvant is a2023387768   16 Sep 2026combination of a squalene oil-in-water adjuvant and a TLR agonist, optionally wherein the TLR agonist is a CpG ODN.

9. The pharmaceutical composition of claim 8, wherein the squalene oil-in-water adjuvant is MF59 or an MF59 analog, and / or the CpG ODN is CpG1018.

10. The pharmaceutical composition of claim 7, wherein the adjuvant is a combination of an aluminum adjuvant and a TLR agonist, optionally wherein the aluminum adjuvant is an aluminum hydroxide and / or the TLR agonist is a CpG ODN.

11. A method for preparing the gE protein variant of claim 1, comprising the steps of:- culturing a host cell transfected with the vector of claim 4 under appropriate conditions; and- isolating the gE protein variant from the supernatant of the host cell.

12. Use of the gE protein variant of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, the host cell of claim 5 or the pharmaceutical composition of any one of claims 6-10 in the manufacture of a vaccine composition for preventing or alleviating varicella-zoster virus infection in a subject.

13. Use of the gE protein variant of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, the host cell of claim 5 or the pharmaceutical composition of any one of claims 6-10 in the manufacture of a medicament for preventing or treating a disease or disorder related to varicella-zoster virus in a subject.

14. The use of claim 12 or claim 13, wherein the subject is a human or a nonhuman animal, optionally wherein the non-human animal is a non-human mammal.

15. The use according to claim 14, wherein the disease related to varicella-zoster2023387768   16 Sep 2026virus is selected from the group consisting of varicella, herpes zoster and postherpetic neuralgia.

16. A method of preventing or alleviating varicella-zoster virus infection in a subject, comprising administering to the subject the pharmaceutical composition of any one of claims 6-10.

17. A kit comprising a container containing the gE protein variant of claim 1, the nucleic acid molecule of claim 3, or the pharmaceutical composition of any one of claims 6-10.

18. Use of a squalene oil-in-water adjuvant and CpG ODN in combination as an adjuvant in a zoster vaccine composition comprising the gE protein variant of claim 1.

19. Use of a squalene oil-in-water adjuvant and / or CpG ODN in the preparation of a medicament for preventing or alleviating varicella-zoster virus infection and diseases or disorders related to varicella-zoster virus in a subject, wherein the medicament is a vaccine composition comprising the gE protein variant of claim 1, the squalene oil-in-water adjuvant and the CpG ODN.

20. The use of claim 18 or claim 19, wherein the squalene oil-in-water adjuvant is MF59 or an MF59 analog, and / or the CpG ODN is CpG1018.

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