GE fusion protein containing gH Th epitope and preparation method and application thereof

Abstract

The invention discloses a gE fusion protein containing a gH Th epitope and a preparation method and application thereof, and relates to the technical field of biomedicine, the fusion protein comprises a varicella-zoster virus (VZV) glycoprotein E extracellular domain (gE) and a Th epitope-containing VZV glycoprotein H extracellular domain (gH) polypeptide. The gE fusion protein can be applied to VZV vaccines, and solves the technical problems of weak immunogenicity of gE antigens and serious side reaction of vaccines in vaccines prepared in the prior art. Compared with the Xinanlietaine, the vaccine taking the gE fusion protein as the antigen has the advantages that the variety and dosage of immunopotentiators are less, the immunogenicity is better, and the clinical side reaction is lower; besides, the gE fusion protein vaccine not only can induce a gE specific CMI reaction, but also can induce a gH specific CMI reaction, so that the effectiveness of the vaccine is further improved. In short, the gE fusion protein has good clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a gE fusion protein containing the gH Th epitope, its preparation method, and its applications. Background Technology

[0002] Varicella-zoster virus (VZV) is an alpha herpesvirus that infects humans, causing diseases such as varicella, herpes zoster, and postherpetic neuralgia (PHN). The genome of VZV is approximately 125 kb, encoding 67 proteins, including envelope glycoproteins such as gB, gC, gE, gH, gI, and gL. Glycoprotein E is the most abundant and immunogenic glycoprotein on the VZV envelope and host cell membrane, containing numerous B-cell and T-cell epitopes. Glycoprotein H is also relatively abundant on the VZV envelope and possesses strong immunogenicity; its sequence contains confirmed Th (T helper) cell epitopes.

[0003] GlaxoSmithKline's recombinant zoster vaccine Shingrix ® (Shingrix) ®This vaccine was approved by the FDA in 2017 for the prevention of shingles in adults aged 50 and older and immunocompromised adults aged 18 and older. It induces a higher gE-specific CMI (Cell-Mediated Immunity) response (inducing a higher number of gE-specific IL2+ and / or IFN-γ+ CD4+ T cells) and gE-specific antibody levels in humans compared to live attenuated vaccines. The vaccine consists of the extracellular domain of glycoprotein E (gE) and the AS01B adjuvant system. The AS01B adjuvant system (compound adjuvant) contains 50 μg of the immunostimulant / adjuvant 3-O-deacylated-4'-monophospholipid A (3D-MPLA), 50 μg of the immunostimulant / adjuvant saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol. Saponin QS-21 can activate the NLRP3 (NLR family Pyrin domain protein 3) inflammasome, thereby releasing caspase-1-dependent cytokines IL-1β and IL-18. These cytokines can promote Th17 cell maturation or drive INF-γ-mediated Th1 responses. 3D-MPLA, through interaction with TLR-4, activates MyD88 (myeloid differentiation factor 88) and TRIF (TIR domain-inducing β-interferon adapter)-dependent signaling pathways, inducing the production of inflammatory cytokines and interferons, thereby enhancing the immune response. Compared to the live attenuated vaccines from Merck and Brno, GSK's Shingrix... ® It has a higher protection rate, but a higher rate of serious adverse reactions in clinical practice.

[0004] In a Phase III clinical trial, within 7 days post-vaccination, the experimental group (Shingrix) showed... ® The incidence of at least one Grade 3 adverse event (AE) in the experimental group and the placebo group was 16.5% and 3.2%, respectively; within 30 days post-vaccination, the incidence of Grade 3 AEs in the experimental group and the placebo group was 4.0% and 0.4%, respectively. In another Phase III clinical trial, within 42 days post-vaccination, the incidence of Grade 3 AEs in the Merck ZOSTAVAX live attenuated vaccine group and the placebo group was 1.4% and 1.4%, respectively. ® The incidence of Grade 3 adverse events (AEs) was significantly higher with the live attenuated vaccine than with the live attenuated vaccine. GSK announced the use of Shingrix in 2017. ® A clinical review; the review discloses: Shingrix ®Injection into rabbits elicits high levels of the inflammatory marker C-reactive protein (CRP), up to 9 times higher in males and 5 times higher in females. High CRP levels indicate the initiation of an acute phase transition response and an increased systemic inflammatory response, which may be associated with clinical side effects such as lethargy, fatigue, and nausea. A 2025 study published in *Vaccine* indicated that vaccine side effects (tolerance) have become a major concern for Americans receiving Shingrix. ® The core obstacle is that the vaccination rate for this vaccine in people aged 50 and older (17%) is significantly lower than that of the influenza vaccine (47.4% in people aged 50-64; 71.3% in people aged 65 and older) and the pneumococcal vaccine (69% in people aged 65 and older). In my country, Shingrix... ® Side effects also have a significant negative impact on vaccination rates.

[0005] Shin An Li Shi ® The high incidence of clinical adverse reactions and serious adverse reactions (Grade 3 AEs) is mainly due to the use of two immune enhancers [adjuvant saponin QS-21 and 3D-MPLA]. The use of two immune enhancers / adjuvants may overactivate the immune system, leading to a large release of inflammatory factors and enhancing local or systemic inflammatory responses, thereby causing clinical adverse reaction symptoms such as fever, fatigue, and muscle soreness.

[0006] Clinically, there is a need for a drug that can induce high levels of VZV-specific CMI and gE-specific antibodies in the human body, and is also more effective than Shingrix. ® A new generation of vaccines with fewer side effects. Therefore, using only one immune enhancer or significantly reducing the dosage of two immune enhancers while increasing the Th epitopes in the antigen to improve the levels of VZV-specific CMI and gE-specific antibodies is an ideal technical route for developing a new generation of vaccines.

[0007] Patent CN117100850A improves the immunogenicity of gE and VZV-specific CMI levels by fusing gE with multiple predicted Th epitopes of gB, gC, gH, gI, gK, gL, gM, and gN. However, most of the predicted Th epitopes have not been confirmed by research, and the immune enhancement ability is weak. Summary of the Invention

[0008] The purpose of this invention is to provide a gE fusion protein containing gH Th epitopes, its preparation method, and its application, in order to solve the problem of significant clinical side effects of vaccines in the prior art.

[0009] To achieve the above objectives, firstly, embodiments of the present invention provide a gE fusion protein containing gH Th epitopes. The gE fusion protein includes the extracellular region of varicella-zoster virus (hereinafter referred to as VZV) glycoprotein E (hereinafter referred to as gE) and the extracellular region of VZV glycoprotein H (hereinafter referred to as gH) polypeptide containing Th epitopes.

[0010] Furthermore, the amino acid residues in the sequence of gE exhibit polymorphism; furthermore, the polymorphism includes, but is not limited to, the following: the amino acid residue at position AA40 is isoleucine (I) or threonine (T); the amino acid residue at position AA536 is isoleucine (I) or leucine (L).

[0011] Furthermore, the sequence length of gE includes, but is not limited to, any one of the following: AA31~AA546, AA3~AA545, AA31~AA544, AA31~AA543, AA31~AA542, AA31~AA541, AA31~AA540, AA31~AA539, ​​AA31~AA538, AA31~AA537, AA31~AA536, AA31~AA535 of VZV glycoprotein E.

[0012] Further, the preferred sequence of gE is AA31~AA544 of VZV glycoprotein E, and the amino acid sequence is shown in SEQ ID NO.1; the amino acid sequence of gH is shown in SEQ ID NO.2.

[0013] Further, the gE includes a sequence that is at least 95.0% to 99.9% identical to the sequence described in SEQ ID NO.1, such as at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or at least 99.9% identical.

[0014] Furthermore, the Th table bits include Th table bit 1, Th table bit 2, Th table bit 3, and Th table bit 4.

[0015] Furthermore, the Th epitope 1 is located at positions AA401–AA413 in the H sequence of the VZV glycoprotein, and its amino acid sequence is KFLNYILMRIGAG; ​​furthermore, the Th epitope 1 has been confirmed by Kerry J. Laing in 2018 as an HLA-DQB1*06:02 restriction epitope, with epitope ID 1596671 in the Immunotope Database (IEDB).

[0016] Furthermore, the Th epitope 2 is located at positions AA657–AA669 in the H sequence of the VZV glycoprotein, and its amino acid sequence is YVLILPAVQGHSY. Furthermore, the Th epitope 2 has been confirmed by Kerry J. Laing in 2020 as an HLA-DRB3*02:02 restriction epitope, with epitope ID 2224923 in the Immunotope Database (IEDB).

[0017] Furthermore, the Th epitope 3 is located at positions AA665–AA677 in the H sequence of the VZV glycoprotein, and its amino acid sequence is QGHSYVITRNKPQ; furthermore, the Th epitope 3 has been confirmed by Kerry J. Laing in 2019 as an HLA-DRB1*04:01 restriction epitope, with epitope ID 1597784 in the Immunotope Database (IEDB).

[0018] Furthermore, the Th epitope 4 is located at positions AA669–AA681 in the H sequence of the VZV glycoprotein, and its amino acid sequence is YVITRNKPQRGLV. Furthermore, the Th epitope 4 has been confirmed by Kerry J. Laing Ph.D. in 2019 as an HLA-DRB1*04:01 restriction epitope, with epitope ID 1597862 in the Immunotope Database (IEDB).

[0019] The gH polypeptide sequence containing Th epitopes includes at least one of gH polypeptide sequence 1 and gH polypeptide sequence 2; gH polypeptide sequence 1 contains Th epitope 1, and its amino acid sequence is shown in SEQ ID NO. 3 to SEQ ID NO. 44; gH polypeptide sequence 2 contains Th epitope 2 and / or Th epitope 3 and / or Th epitope 4, and its amino acid sequence is shown in SEQ ID NO. 45 to SEQ ID NO. 56.

[0020] Furthermore, the gH polypeptide sequence 1 also includes a sequence that is at least 90.0% to 99.0% identical to the sequences described in SEQ ID NO.3 to SEQ ID NO.44, such as at least 90.0%, at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, or at least 99.0% identical.

[0021] Furthermore, the gH polypeptide sequence 2 also includes a sequence that is at least 90.0% to 99.0% identical to the sequences described in SEQ ID NO.45 to SEQ ID NO.56, such as at least 90.0%, at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, or at least 99.0% identical.

[0022] The gE fusion protein may also contain sequences other than the gH Th epitope sequence or a polypeptide sequence containing the gH Th epitope; furthermore, the other sequences can enhance the immunogenicity of the gE fusion protein; the other sequences include, but are not limited to, one or more combinations of the following: Th epitope peptides, the Fc segment (Fc) of human immunoglobulin and its mutants, interleukins, interferons, and protein polymerization sequences; the Th epitope includes, but is not limited to, non-natural universal DR. The Th epitope padre (padre), the tetanus toxin Th epitope P2 (P2); the human immunoglobulins, including but not limited to: human immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), and immunoglobulin M (IgM); the interleukins, including but not limited to: IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-11, IL-12, IL-13, IL-15, and IL-18; the interferons, including but not limited to: α-interferon (IFN-α), β-interferon (IFN-β), and γ-interferon (IFN-γ); the protein polymerization sequence, including but not limited to: T4. Fibritin trimerization domain (GYIPEAPRDGQAYVRKDGEWVLLSTF), encapsulating protein monomer subunit polypeptide (approximately 30 kD), thiooxygenase reductase (SOR) monomer subunit polypeptide, dioxetine synthase monomer subunit polypeptide, and ferritin monomer subunit polypeptide (approximately 17-20 kD).

[0023] Further, the gE fusion protein contains a linker peptide; the linker peptide is located between gE and gH polypeptide sequence 1 or gH polypeptide sequence 2, and between gH polypeptide sequence 1 and gH polypeptide sequence 2 (if the fusion protein contains gH polypeptide sequence 1 and gH polypeptide sequence 2); further, the linker peptide is a flexible linker peptide and / or a rigid linker peptide, preferably a flexible linker peptide; further, the amino acid residues of the flexible linker peptide are glycine (G) and / or serine (S); further, the flexible linker peptide includes, but is not limited to, at least one of the following sequences: G, S, GS, SG, G G, SS, GGG, SSS, GSG, SGS, GGS, SGG, SSG, GSS, GGGG, SGGG, GGSG, GGGS, SGSG, GSGS, SGGS, GSSG, SSSG, GSSS, SGSS, SSGS, SSSS, GGGGS, GGGSG, GGSGG, GGSGG, GGSGS, GGSGS, SGGGS, SGSGG, SGGSG; Further, the flexible linker peptide is preferably SGS and / or GGSGSG.

[0024] Furthermore, the amino acid sequences of the gE fusion protein can be combined in the following ways: gE-gH polypeptide sequence 1, gE-gH polypeptide sequence 2, gH polypeptide sequence 2-gE, gH polypeptide sequence 2-gE-gH polypeptide sequence 1, gE-gH polypeptide sequence 2-gH polypeptide sequence 1, and gE-gH polypeptide sequence 1-gH polypeptide sequence 2.

[0025] Further, the amino acid sequences of the above-described gE-gH polypeptide sequence 2-gH polypeptide sequence 1 include, but are not limited to, SEQ ID NO. 58 and SEQ ID NO. 61; further, the amino acid sequences of the above-described gE-gH polypeptide sequence 1-gH polypeptide sequence 2 include, but are not limited to, SEQ ID NO. 57 and SEQ ID NO. 60; further, the amino acid sequences of the above-described gH polypeptide sequence 2-gE-gH polypeptide sequence 1 include, but are not limited to, SEQ ID NO. 59 and SEQ ID NO. 62.

[0026] Furthermore, the preferred amino acid sequence arrangement of the gE fusion protein containing the gH Th epitope is gE-gH polypeptide sequence 2-gH polypeptide sequence 1.

[0027] A nucleic acid sequence encoding a gE fusion protein containing a gH Th epitope.

[0028] Furthermore, the nucleic acid sequence includes DNA and RNA sequences.

[0029] Furthermore, the nucleic acid sequence can be used to construct an expression vector or viral vector expressing the gE fusion protein.

[0030] Furthermore, the nucleic acid sequence containing the expression vector or viral vector can express the gE fusion protein in host cells.

[0031] Furthermore, the RNA sequence can be used to construct an mRNA molecule expressing the gE fusion protein.

[0032] Further, the mRNA molecule includes the following portions (from the 5' end to the 3' end): (1) a 5' cap structure; (2) a 5' untranslated region (UTR); (3) an open reading frame (ORF) encoding the signal peptide and the gE fusion protein; (4) a 3' untranslated region; and (5) a 3' poly (A) tail.

[0033] Furthermore, the mRNA molecule is delivered into human cells, where it expresses the gE fusion protein.

[0034] Secondly, this invention also provides a method for preparing a gE fusion protein containing the gH Th epitope, comprising the following steps: S1. Construct an expression vector or viral vector containing the above nucleic acid sequence; S2. Transform the expression vector obtained in S1 into host cells or infect host cells with the viral vector obtained in S1; S3, the host cells obtained in S2; S4. Collect the cell culture supernatant; S5. Purify the cell culture supernatant to obtain the gE fusion protein.

[0035] Furthermore, the nucleic acid sequence described in S1 contains a nucleic acid sequence encoding gE and a nucleic acid sequence encoding Th epitope 1 and / or a nucleic acid sequence encoding Th epitope 2 and / or a nucleic acid sequence encoding Th epitope 3 and / or a nucleic acid sequence encoding Th epitope 4.

[0036] Furthermore, in constructing the expression vector or viral vector containing the above-mentioned nucleic acid sequence as described in S1, nucleic acid sequences encoding various purification tags that facilitate the purification of the gE fusion protein can be added upstream of the 5' end or downstream of the 3' end of the nucleic acid sequence (i.e., the N-terminus or C-terminus of the gE fusion protein).

[0037] Furthermore, the purification tags include, but are not limited to, MBP tags, GST tags, GFP tags, Fc tags, Halo tags, HA tags, Myc tags, Flag tags, His tags, and Strep-tag II tags; the amino acid sequence of the gE fusion protein with the 6X His tag is not limited to SEQ ID NO. 63 and SEQ ID NO. 64; the DNA sequence encoding the gE fusion protein with the 6X HIS tag is not limited to SEQ ID NO. 65 and SEQ ID NO. 66.

[0038] Furthermore, the expression vector described in S1 is a eukaryotic expression vector.

[0039] Furthermore, the eukaryotic expression vector can introduce exogenous genes into eukaryotic cells for expression, and its basic structure includes, but is not limited to, core elements such as origin of replication and / or selection markers and / or multiple cloning sites.

[0040] Further, the eukaryotic expression vector, its empty vector (vector without inserted foreign gene) includes, but is not limited to, pcDNA3.4, pcDNA3.1(+), pcDNA3.1+C-His, pcDNA3.1 myc-His C, pEF1α-V5, pCMV-HA, pCMV6-AC-IRES-GFP, and pEGFP-N1; further, the empty vector may contain eukaryotic selection marker resistance genes and / or genes; the eukaryotic selection marker resistance genes include, but are not limited to, any one or more combinations of the following: puromycin, neomycin, hygromycin B, blasticidin S, G418 (Geneticin), bleomycin, etc.; the eukaryotic selection marker genes include, but are not limited to, any one or more combinations of the following: dihydrofolate reductase, thymidine kinase, glutamine synthase, etc. Genes such as synthetase, asparagine synthetase, and tryptophan synthase.

[0041] Furthermore, the construction of the expression vector or viral vector containing the nucleic acid sequence described in S1 may also involve adding nucleic acid sequences encoding various purification tags upstream of the 5' end and / or downstream of the 3' end of the nucleic acid sequence (i.e., the N-terminus and / or C-terminus of the gE fusion protein).

[0042] Furthermore, the purification tags include, but are not limited to, any one or more combinations of the following: MBP tag, GST tag, GFP tag, Fc tag, Halo tag, HA tag, Myc tag, Flag tag, His tag, Strep-tagII tag.

[0043] Furthermore, the expression vector described in S1 also contains a nucleic acid sequence encoding a signal peptide that causes the gE fusion protein to be secreted / expressed outside the cell; further, the signal peptide includes, but is not limited to, any one of the following: signal peptide 1 (MHSSALLCCLVLLTGVRA); signal peptide 2 (MGTVNKPVVGVLMGFGIITGTLRITNPVRA).

[0044] Furthermore, the nucleic acid sequence encoding the signal peptide is located upstream of the 5' end of the nucleic acid sequence, i.e., at the N-terminus of the gE fusion protein.

[0045] Furthermore, the expression vector described in S1 may also contain a 5' untranslated region (UTR) sequence and / or a 3' untranslated region sequence and / or a 3' poly(A) tail sequence.

[0046] Furthermore, the ORF sequence in the expression vector described in S1 can be codon optimized to promote protein expression.

[0047] Furthermore, when the expression vector described in S1 contains a UTR sequence, the UTR sequence can be optimized.

[0048] Furthermore, the expression vector described in S1 may also contain restriction enzyme cleavage site sequences; furthermore, the function of the restriction enzyme sites is to facilitate the insertion of foreign genes into the expression vector; furthermore, the restriction enzymes include, but are not limited to, the following: Not I (recognizing restriction enzyme cleavage site sequence: GCGGCCGC), Xba I (recognizing restriction enzyme cleavage site sequence: TCTAGA), ScaI (recognizing restriction enzyme cleavage site sequence: AGTACT), and Blp I (recognizing restriction enzyme cleavage site sequence: GCTNAGC).

[0049] Furthermore, the viral vector in S1 includes baculovirus vectors, lentivirus vectors, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and parainfluenza virus vectors, with baculovirus vectors being preferred.

[0050] Furthermore, the expression vector described in S2 is transformed into host cells in two ways: stable transformation and transient transformation.

[0051] Furthermore, after the stable expression vector (containing a resistance gene and / or gene with a eukaryotic selection marker) is transfected into a host cell, the cell line that integrates the target gene into the host cell genome is selected using the resistance gene and / or gene with the eukaryotic selection marker.

[0052] Furthermore, the expression vector described in S2 is transformed into host cells, including CHO cells, yeast cells, and HEK293 cells, with CHO cells being preferred.

[0053] Furthermore, the CHO cells include, but are not limited to, cell lines such as CHO-S, CHO-K1, and CHO-DG44.

[0054] Furthermore, the yeast cells include, but are not limited to, yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, and Hansenula polymorpha.

[0055] Furthermore, the viral vector described in S2 infects the host cell. The viral vector includes, but is not limited to, baculovirus vectors, adenovirus vectors, adeno-associated virus vectors, and lentivirus vectors. More preferably, baculovirus vectors are used. The host cell includes, but is not limited to, insect cells and HEK293 cells. More preferably, insect cells are used.

[0056] Furthermore, the insect cells include, but are not limited to, Sf9 cells, Sf21 cells, and Hi5 cells.

[0057] Furthermore, in S3, if the host cell is a host cell transformed by the expression vector, the host cell can be either a polyclonal cell line or a monoclonal cell line, with a monoclonal cell line being preferred.

[0058] Furthermore, in S3, if the host cell is a host cell transformed by the expression vector, and the transformation is stable (the transformed host cell is screened using a resistance gene and / or gene with a eukaryotic selection marker), or transient (the transformed host cell is screened without using a resistance gene and / or gene with a eukaryotic selection marker), the host cell includes CHO cells, yeast cells, and HEK293 cells; furthermore, the host cell is preferably a CHO cell.

[0059] Furthermore, in S4, the cell culture supernatant and / or the supernatant of cell lysate contain substances such as gE fusion protein and residual components of culture medium, and may contain substances such as host cell debris, host cell protein, host cell nucleic acid, and host cell metabolites; furthermore, the supernatant of the cell lysate is the supernatant obtained by lysing host cells and centrifuging or other methods.

[0060] Furthermore, in S5, the purification method includes, but is not limited to, any one or a combination of the following methods: precipitation, chromatography, dialysis, ultrafiltration, centrifugation, nanofiltration, and microfiltration; further, the chromatography includes, but is not limited to, any one or a combination of the following chromatography methods: ion exchange chromatography, gel filtration chromatography, affinity chromatography, hydrophobic chromatography, and reversed-phase chromatography; further, the chromatography also includes composite mode chromatography, which includes a composite mode of ion exchange chromatography and / or gel filtration chromatography and / or affinity chromatography and / or hydrophobic chromatography and / or reversed-phase chromatography.

[0061] Furthermore, the purity of the gE fusion protein described in S5 (target protein; reducing SDS-PAGE method) is: not less than 30.0% to not less than 99.9%, such as not less than 30%, not less than 40%, not less than 50%, not less than 60%, not less than 70%, not less than 80%, not less than 90%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, not less than 99%, or not less than 99.9%.

[0062] Finally, this invention also provides an application of a gE fusion protein containing the gH Th epitope in a VZV vaccine for the prevention or improvement of varicella and / or shingles and / or postherpetic neuralgia.

[0063] Furthermore, the VZV vaccine contains the aforementioned gE fusion protein containing the gH Th epitope.

[0064] Furthermore, the VZV vaccine also contains an adjuvant. Further, the adjuvant is any one or a combination of any number of adjuvants listed below (compound adjuvant): aluminum hydroxide adjuvant, aluminum phosphate adjuvant, liposome adjuvant, CpG adjuvant, Poly I:C adjuvant, saponin QS-21 adjuvant, saponin QS-7 adjuvant, nanoemulsion, 3D-MPL adjuvant; more preferably, a combination of liposome adjuvant and saponin QS-21 adjuvant (compound adjuvant).

[0065] Furthermore, the method for preparing the VZV vaccine includes the following steps: S6. Prepare adjuvant or compound adjuvant solution; S7. Prepare the vaccine.

[0066] Furthermore, the VZV vaccine contains 5-200 μg / ml of gE fusion protein, and more preferably 20-150 μg / ml of gE fusion protein.

[0067] A nucleic acid sequence for use in VZV mRNA vaccines to prevent or improve varicella and / or shingles and / or postherpetic neuralgia.

[0068] Furthermore, when the aforementioned nucleic acid sequence is used in an mRNA vaccine, the nucleic acid sequence may also contain a short nucleic acid sequence (significantly shorter than the nucleic acid sequence encoding the extracellular amino acid sequence of VZV glycoprotein E) that encodes the transmembrane amino acid sequence and / or the intramembrane amino acid sequence of VZV glycoprotein E.

[0069] The VZV mRNA vaccine comprises an mRNA molecule and a system for delivering the mRNA molecule.

[0070] Further, the mRNA molecule includes the following parts (from the 5' end to the 3' end): (1) a 5' cap structure; (2) a 5' untranslated region (UTR); (3) an open reading frame (ORF) encoding the signal peptide and the gE fusion protein; (4) a 3' untranslated region; and (5) a 3' poly (A) tail.

[0071] Furthermore, the mRNA molecule also includes self-replicating mRNA.

[0072] Furthermore, the system for delivering mRNA molecules includes, but is not limited to, the following: lipid nanoparticles (LNPs), polymer nanoparticles (PNPs), protamine, cationic liposomes, cationic nanoemulsions, and preferably lipid nanoparticles (LNPs).

[0073] A viral vector vaccine based on one of the above-mentioned nucleic acid sequences, intended for the prevention or improvement of varicella and / or shingles and / or postherpetic neuralgia.

[0074] Furthermore, when the aforementioned nucleic acid sequence is applied to a viral vector vaccine, the nucleic acid sequence may also contain a short nucleic acid sequence (significantly shorter than the nucleic acid sequence encoding the extracellular amino acid sequence of VZV glycoprotein E) encoding the transmembrane amino acid sequence and / or the intramembrane amino acid sequence of VZV glycoprotein E.

[0075] Furthermore, the viral vector vaccine includes adenovirus vectors, attenuated influenza virus vectors, adeno-associated virus vectors, herpes simplex virus vectors, and parainfluenza virus vectors, with adenovirus vectors being preferred.

[0076] Compared with the prior art, the embodiments of the present invention have the following advantages and beneficial effects: This invention provides a gE fusion protein comprising VZV gE and VZV gH peptides. This gE fusion protein can be used in VZV vaccines, solving the technical problems of weak gE immunogenicity and severe vaccine side effects in existing vaccine preparation techniques.

[0077] Mice immunized twice with this vaccine had higher levels of gE-specific antibodies in their serum than mice immunized with Shingrix. ® Furthermore, the induced gE-specific CMI response (splenic cells) is similar to that of Shingrix. ® Comparable; the immunogenicity of the gE fusion protein is higher than that of gE, thus reducing the type and dosage of immunostimulants in the vaccine (this vaccine contains 50 μg of the immunostimulant saponin QS-21; Shingrix). ® Containing 50 μg of the immune enhancer saponin QS-21 and 50 μg of the immune enhancer 3D-MPLA, this vaccine can reduce the inflammatory response induced by the immune enhancer, thereby reducing the clinical side effects of the vaccine. Furthermore, this vaccine can induce a gH-specific CMI response, which is beneficial for further improving vaccine efficacy.

[0078] In short, with Xin An Li Shi ® Compared to other vaccines, this VZV vaccine has better potential for clinical application! Therefore, this gE fusion protein has good potential for clinical application. Attached Figure Description

[0079] To more clearly illustrate the technical solutions in this invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art are briefly introduced below. Obviously, the drawings described below are only some embodiments of this invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0080] Figure 1 These are SDS-PAGE detection results of the gE fusion protein in Examples 1, 2, and 3 of this invention; In the figure, M / M1: protein standard; R: reducing SDS-PAGE; NR: non-reducing SDS-PAGE; FP-3: gE fusion protein FP-3; FP-13: gE fusion protein FP-13; FP-18: gE fusion protein FP-18; FP-25: gE fusion protein FP-25; FP-22: gE fusion protein FP-22; Figure 2 This is a graph showing the level of gE-specific antibody GMT in the serum of mice in each vaccine group after secondary immunization in Example 5 of this invention; Figure 3 This is a graph showing the percentage of gE-specific CD4+ T cells in the spleen of mice in each vaccine group after a second immunization in Example 5 of this invention. Figure 4 This is a graph showing the percentage of gH-specific CD4+ T cells in the spleen of mice in each vaccine group after a second immunization in Example 5 of this invention. Figure 5 This is a graph showing the percentage of VZV(gE+gH) specific CD4+ T cells in the spleen of mice in each vaccine group after secondary immunization in Example 5 of this invention. Detailed Implementation

[0081] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0082] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, regarding numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the numerical range is also specifically disclosed. Every smaller range between any stated numerical value or an intermediate value within a stated numerical range, and any other stated numerical value or an intermediate value within said numerical range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0083] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and / or materials have been described herein, any methods and / or materials similar or equivalent to those described herein may be used in the implementation and / or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe the methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the description herein shall prevail.

[0084] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope and / or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. The specification and embodiments of this invention are merely exemplary.

[0085] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0086] Example 1: Screening for gE fusion proteins containing gHPP1 (1) Design gE fusion proteins containing gH Th epitopes I. Extracellular sequences of VZV glycoprotein gE and gH, detailed information is shown in Table 1 below: Table 1. Sequence information of the extracellular regions of VZV glycoproteins gE and gH.

[0087] II. The selected gH Th table position (IEDB table position database), the relevant information of the gH Th table position is shown in Table 2 below: Table 2. Information related to gH Th epitopes

[0088] III. Design information for gH polypeptides (PP) containing gH Th epitopes (gHPP1), as shown in Table 3: Table 3 Information on gH polypeptides (PP) containing gH Th epitopes

[0089] S1. Construct an expression vector containing a nucleic acid sequence encoding the target protein (a gE fusion protein containing gHPP1). IV. Design of gE fusion proteins containing gHPP1, detailed information is shown in Table 4: Table 4. Information on gE fusion proteins containing gHPP1

[0090] (2) Codon optimization and whole-genome synthesis of nucleic acids At the 3' end of the DNA sequence encoding the gE fusion protein (3' end of the DNA positive strand; C-terminus of the gE fusion protein), a DNA sequence encoding a 6X His tag (HHHHHH) was added; at the 5' end of the DNA sequence encoding the gE fusion protein (5' end of the DNA positive strand; N-terminus of the gE fusion protein), a DNA sequence encoding a signal peptide (MHSSALLCCLVLLTGVRA) was added. Then, the DNA sequence was codon-optimized; next, a UTR sequence was added to the 5' end of the codon-optimized DNA sequence, and a stop codon (TGA) was added to its 3' end; then, a Not I restriction enzyme cleavage site sequence was added to the 5' end of the 5' UTR sequence, and an Xba I restriction enzyme cleavage site sequence and protective bases were added to the 3' end of the stop codon (TGA). Finally, the entire DNA sequence was synthesized.

[0091] (3) Construction of expression plasmids A synthetic DNA sequence containing the complete genome was ligated into the pCDNA3.4 vector. The cloning vector was transformed into competent bacteria, and the bacteria were then amplified in large quantities. The amplified bacteria were then spread onto solid culture medium in petri dishes and incubated for approximately 8 hours. Eight single colonies were then selected for cloning screening (PCR).

[0092] (4) Amplification of expression plasmids After the positive clones were verified to be correct by sequencing, they were cultured and amplified in bacterial culture, and plasmids were extracted.

[0093] S2. Transform the expression vector obtained in S1 into host cells. (5) Transfection of CHO cells with expression vector (plasmid) Expression plasmids were transfected into CHO cells.

[0094] S3. Cultivate the host cells described in S2. (6) Cell culture and expression of gE fusion protein CHO cells with the transfection complex added were placed in an incubator and cultured for 3-7 days on a shaker at 36.5°C (±0.5°C) + 5% CO2, with appropriate feed added as needed.

[0095] S4. Collect cell culture supernatant. (7) Collect cell culture supernatant When cell viability is below 50%, harvest the cell culture supernatant: centrifuge the harvested culture medium at 4500 rpm for 30 min, collect the cell culture supernatant, and filter it through a 0.22 μm filter.

[0096] S5. Purify the cell culture supernatant to obtain the gE fusion protein. (8) Purification of gE fusion protein Mix cell culture supernatant with AmMag TM Incubate with Ni Magnetic Beads for at least 120 min, remove the supernatant, then equilibrate with 10 CV of 25 mM Tris-HCl + 300 mM NaCl (pH 7.0) buffer, and elute with 25 mM Tris-HCl + 300 mM NaCl + 500 mM Imidazole (pH 7.0) buffer and collect. Then, shred the collected protein using histidine buffer (10 mM histidine + 10% sucrose + 0.05% Tween 80; pH 6.5 ± 0.1) with SNAKESKIN. TM DIALYSIS TUBING TM The dialysis bag is used for dialysis and fluid replacement 3 times, with each dialysis and fluid replacement lasting no less than 2 hours.

[0097] (9) Purity detection of gE fusion protein Detection method (SDS-PAGE): Voltage 140~160V, electrophoresis for 45~60 minutes, stop electrophoresis after bromophenol blue reaches the bottom of the separating gel, and remove the gel; use eStain TMThe L1 protein staining system was used for staining and destaining. The difference between reduction electrophoresis and non-reduction electrophoresis is that a reducing agent (such as dithiothreitol) needs to be added to the electrophoresis system for reduction electrophoresis; no reducing agent is added for non-reduction electrophoresis. The gel was scanned and the gel pattern was saved. The detection results are shown in Table 5: Table 5 Purity detection results of gE fusion protein containing gHPP1

[0098] FP-3 containing gHPP1-6 has the highest purity, such as... Figure 1 As shown in the table above.

[0099] (10) Detection of gE fusion protein expression level Fusion protein (vaccine stock solution) concentration detection: The protein concentration was detected by Nanodrop using the UV-Vis spectrophotometric method according to the general rules of the Chinese Pharmacopoeia; then, the protein expression level was obtained by multiplying the protein concentration by the protein volume. The specific results are shown in Table 6 below.

[0100] Table 6. Concentration detection of gE fusion protein containing gHPP1

[0101] The expression level of FP-3 containing gHPP1-6 was the highest, as shown in the table above.

[0102] Example 2: Expression and screening of gE fusion proteins containing gHPP2 (1) Design gE fusion proteins containing gH Th epitopes I. Extracellular sequences of VZV glycoprotein gE and gH, detailed information is shown in Table 7: Table 7. Extracellular sequence information of VZV glycoprotein gE and gH regions

[0103] II. Selected gH Th tabletop (IEDB tabletop database), details are shown in Table 8: Table 8. Information related to gH Th epitopes

[0104] III. Information on the design of gH polypeptides (PP) containing gH Th epitopes (gHPP2) is shown in Table 9. Table 9 Information on gH polypeptides (PP) containing gH Th epitopes

[0105] S1. Construct an expression vector containing a nucleic acid sequence encoding the target protein (gE fusion protein containing gHPP2). Information on the gE fusion protein containing gHPP2 is shown in Table 10 below: Table 10 Information on gE fusion proteins containing gHPP2

[0106] (2) Codon optimization and whole-genome synthesis of nucleic acids The method of codon optimization and whole gene synthesis is consistent with "(2) Codon optimization and whole gene synthesis of nucleic acid" in "Example 1".

[0107] (3) Construction of expression plasmids This is consistent with “(3) Construction of gE fusion protein expression plasmid” in “Example 1”.

[0108] (4) Amplification of expression plasmids Consistent with “(4) Amplification of expression plasmid” in “Example 1”.

[0109] S2. Transform the expression vector obtained in S1 into host cells. (5) Transfection of CHO cells with expression vector (plasmid) Consistent with “(5) Expression vector (plasmid) transfection of CHO cells” in “Example 1”.

[0110] S3. Cultivate the host cells described in S2. (6) Cell culture and expression of gE fusion protein Consistent with “(6) Cell culture and expression of gE fusion protein” in “Example 1”.

[0111] S4. Collect cell culture supernatant. (7) Collect cell culture supernatant Consistent with “(7) Collect cell culture supernatant” in “Example 1”.

[0112] S5. Purify the cell culture supernatant to obtain the gE fusion protein. (8) Purification of gE fusion protein Consistent with “(8) Purification of gE fusion protein” in “Example 1”.

[0113] (9) Purity detection of gE fusion protein Consistent with "(9) Purity detection of gE fusion protein" in "Example 1", the purity detection results of gE fusion protein containing gHPP2 are shown in Table 11 below: Table 11 Purity test results of gE fusion proteins containing gHPP1 and gHPP2

[0114] FP-13 (containing gHPP2-4), FP-15, FP-16, and FP-18 (containing gHPP2-8) had the highest purity. Results for FP-13 and FP-18 are as follows... Figure 1 As shown in Table 11.

[0115] (10) Detection of gE fusion protein expression level Consistent with "(10) Detection of expression level of gE fusion protein" in "Example 1", the detection results of gE fusion protein containing gHPP2 are shown in Table 12 below: Table 12 Detection results of gE fusion protein containing gHPP2

[0116] The expression levels of FP-10, FP-11, FP-13 (containing gHPP2-4) and FP-18 (containing gHPP2-8) were relatively high.

[0117] Based on a comprehensive analysis of purity and expression levels, the preferred gE fusion proteins containing gHPP2 are FP-13 (containing gHPP2-4) and FP-18 (containing gHPP2-8).

[0118] Example 3: Expression and screening of gE fusion proteins containing gHPP1 and gHPP2 S1. Construct an expression vector containing a nucleic acid sequence encoding the target protein (a gE fusion protein containing gHPP1 and gHPP2). (1) Design gE fusion proteins containing gHPP1 and gHPP2. Specific information is shown in Table 13: Table 13 Information on gE fusion proteins containing gHPP1 and gHPP2

[0119] (2) Codon optimization and whole-genome synthesis of nucleic acids Consistent with “(2) Codon optimization and whole gene synthesis of nucleic acid” in “Example 1”.

[0120] (3) Construction of expression plasmids This is consistent with “(3) Construction of gE fusion protein expression plasmid” in “Example 1”.

[0121] (4) Amplification of expression plasmids Consistent with “(4) Amplification of expression plasmid” in “Example 1”.

[0122] S2. Transform the expression vector obtained in S1 into host cells. (5) Transfection of CHO cells with expression vector (plasmid) Consistent with “(5) Expression vector (plasmid) transfection of CHO cells” in “Example 1”.

[0123] S3. Cultivate the host cells described in S2. (6) Cell culture and expression of gE fusion protein Consistent with “(6) Cell culture and expression of gE fusion protein” in “Example 1”.

[0124] S4. Collect cell culture supernatant. (7) Collect cell culture supernatant Consistent with “(7) Collect cell culture supernatant” in “Example 1”.

[0125] S5. Purify the cell culture supernatant to obtain the gE fusion protein. (8) Purification of gE fusion protein Consistent with “(8) Purification of gE fusion protein” in “Example 1”.

[0126] (9) Purity detection of gE fusion protein Detection method: consistent with "(9) purity detection of gE fusion protein" in "Example 1".

[0127] Test results: FP-22 and FP-25 had the highest purity, as shown in Table 14 below: Table 14 Purity test results of gE fusion proteins containing gHPP1 and gHPP2

[0128] FP-22 and FP-25 had the highest purity, as shown in Table 14. Figure 1 As shown.

[0129] (10) Detection of gE fusion protein expression level Detection method: consistent with "(10) gE fusion protein expression level detection" in "Example 1".

[0130] Detection results: FP-22 and FP-25 showed the highest expression levels, as shown in Table 15 below.

[0131] Table 15. Concentration detection results of gE fusion proteins containing gHPP1 and gHPP2

[0132] FP-22 and FP-25 were expressed at high levels.

[0133] (11) Information on preferred gE fusion proteins containing gHPP1 and gHPP2 The amino acid sequence of FP-22 with the 6X His tag is shown in SEQ ID NO.63, and the DNA sequence is shown in SEQ ID NO.65; the amino acid sequence of FP-25 with the 6X His tag is shown in SEQ ID NO.64, and the DNA sequence is shown in SEQ ID NO.66.

[0134] Example 4: Formulation of gE fusion protein vaccine containing gH Th epitopes S6. Preparation of adjuvant or compound adjuvant solution (1) Preparation of liposomes (theoretical concentrations of dioleoylphosphatidylcholine and cholesterol are 5 mg / ml and 1.25 mg / ml, respectively) 1000 mg of dioleoylphosphatidylcholine (DOPC; Nippon Fine Chemicals Co., Ltd.) and 250 mg of cholesterol (Nippon Fine Chemicals Co., Ltd.) were accurately weighed into 20 ml volumetric flasks. DOPC and cholesterol were then dissolved in 10 ml of anhydrous ethanol, and the volume was adjusted to 20 ml. The mixture was thoroughly mixed to obtain the organic phase. 20 ml of the organic phase was injected into 180 ml of 10 mM histidine buffer solution (pH 6.5 ± 0.1; sucrose concentration 10%) to prepare the colostrum. The colostrum was then granulated using a liposome extruder to achieve a particle size of approximately 100 nm. The granulated liposomes were then ultrafiltered using a 30 kDa membrane to remove residual ethanol. Finally, the liposomes were filtered through a 0.22 μm sterile filter to obtain the final liposome product.

[0135] (2) Preparation of saponin QS-21 solution (theoretical concentration 2 mg / ml) Accurately weigh 20 mg of saponin QS-21 (Desert King), then dissolve it completely in 5 ml of 5 mM histidine buffer solution (pH 6.5 ± 0.1), and then bring the volume up to 10 ml with the buffer solution and mix well to obtain the saponin QS-21 solution.

[0136] (3) Preparation of adjuvants Take 2 ml of liposomes, add 0.25 ml of saponin QS-21 solution, and stir well to make an adjuvant.

[0137] S7, Vaccine preparation (4) Vaccine preparation [5ml volume; target concentration of saponin QS-21 is 100μg / ml; liposome components: target concentrations of dioleoylphosphatidylcholine and cholesterol are 2mg / ml and 0.5mg / ml, respectively] Add the calculated volume of gE fusion protein solution to the adjuvant, then use histidine solution (10mM; pH 6.5±0.1; 10% sucrose) to make up the total volume to 5ml, and stir well to obtain the gE fusion protein adjuvant vaccine. The formula for calculating the target volume (ml) of gE fusion protein solution added is: target mass of gE fusion protein added (μg) / concentration of gE fusion protein solution (μg / ml).

[0138] Vaccine prescription information is shown in Table 16 below: Table 16 Vaccine Prescription Information (Vaccine volume: 50 μl)

[0139] Note: Xin An Li Shi ® This is a commercial vaccine, no preparation is required, batch number F7P54; " / " indicates not applicable.

[0140] Example 5: Immunogenicity Study of gE Fusion Protein Vaccine Containing gH Th Epitope I. Animals immunized with vaccines Forty-eight female C57BL / 6 mice were randomly divided into eight groups of six each. The animals were housed for approximately 100 days. On day -35, they were subcutaneously immunized with a live attenuated varicella vaccine in the neck. On days 1 and 29, they were intramuscularly injected with the vaccine described in Example 2 in the legs. Blood was collected and serum separated on days 0 and 57. On day 57, after blood collection, the animals were sacrificed, and the spleen was harvested and spleen cells separated. The following parameters were measured: serum gE binding antibody; number of gE-specific IL2+ and IFN-γ+ CD4+ T cells; and number of gH-specific IL2+ and IFN-γ+ CD4+ T cells.

[0141] II. Methods for Detecting Vaccine Immunogenicity (1) Detection of gE-specific binding antibodies in serum Total gE-specific antibodies in serum samples from all mice 28 days after secondary immunization were detected using an indirect ELISA method. The procedure was as follows: gE protein was coated onto 96-well plates with carbonate buffer at a concentration of 3 μg / well, incubated at 37°C for 60 min, blocked with TPBS containing BSA, and washed four times with TPBS. Serum from all mice was then diluted at different dilutions (two-fold serial dilutions; with an initial dilution of 62,500, for a total of 8 dilutions) and added to each well at 100 μl. Samples, negative controls, and positive controls were all replicated. The plate was incubated at 37°C for 1 hour, washed 4 times with TPBS solution, and then incubated with HRP-labeled goat anti-mouse secondary antibody at 37°C for 1 hour. After washing 4 times with TPBS solution, the plate was developed with TMB light-protected chromogenic solution for 15 minutes. The reaction was terminated by adding 0.2M sulfuric acid, and the OD450 value was read by an ELISA reader. The cut-off value was determined by using 4 times the reading of the serum mixed sample prepared from blood collected on day 0. The serum gE-specific antibody level was then determined.

[0142] (2) Intracellular cytokine detection Twenty-eight days after the second immunization, spleens were harvested from mice, and spleen single-cell suspensions were prepared. After adjusting the cell concentration, erythrocytes in the single-cell suspension were lysed using erythrolysis buffer. 10 μl of cell suspension from each sample was added to 30 μl of trypan blue solution mixed with culture medium at a 1:2 ratio. After thorough mixing, cell counts were performed. Based on the technical results, the T-cell culture medium was diluted to 1×10⁻⁶. 7 Add 100 μl of cells / ml to the corresponding wells of a U-shaped 96-well plate (i.e., 1 × 10⁶ cells / ml) to each well. 6 (cells / well), with 250 μl PBS added to each of the remaining four wells for sealing. Splenic cells were stimulated with peptide pools (gE peptide pool or gH peptide pool) to induce cytokine secretion. Secretion was then blocked by adding ContainingBrefeldin A secretion inhibitor. After cell live / dead staining, surface receptor FcR blocking, CD3, CD45, and CD4 surface staining, fixation and perforation, and intracellular staining for IL-2 and IFN-γ, the number of CD4+ T cells secreting IL-2 and IFN-γ was detected by flow cytometry.

[0143] III. Results of Vaccine Immunogenicity Testing (1) gE-specific binding antibody like Figure 2 As shown, FP-22+QS-21 induces the strongest gE-specific antibody response.

[0144] (2) Intracellular cytokines (gE specific) like Figure 3As shown, QS-21 adjuvant significantly enhances the secretion of gE-specific CD4+ T cytokines.

[0145] (3) Intracellular cytokines (gH specific) like Figure 4 As shown, FP-25+QS-21 induced the highest level of gH-specific CD4+ T cell response, which was superior to the control vaccine.

[0146] (4) Intracellular cytokines (VZV specific) like Figure 5 As shown, FP-25+QS-21 induced the highest level of VZV(gE+gH) specific CD4+ T cell response, which was superior to the control vaccine.

[0147] Finally, it should be noted that: The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A gE fusion protein containing gH Th epitopes, characterized in that, The gE fusion protein comprises a varicella-zoster virus gE and a varicella-zoster virus gH polypeptide sequence containing Th epitopes; The amino acid sequence of the gE is shown as SEQ ID NO. 1; The Th epitopes comprise at least one of Th epitope 1, Th epitope 2, Th epitope 3, and Th epitope 4; The amino acid sequence of the Th epitope 1 is KFLNYILMRIGAG, the amino acid sequence of the Th epitope 2 is YVLILPAVQGHSY, the amino acid sequence of the Th epitope 3 is QGHSYVITRNKPQ, and the amino acid sequence of the Th epitope 4 is YVITRNKPQRGLV; The gH polypeptide sequence comprises at least one of gH polypeptide sequence 1 and gH polypeptide sequence 2; The amino acid sequence of the gH polypeptide sequence 1 is shown as SEQ ID NO. 3~SEQ ID NO. 44, and the amino acid sequence of the gH polypeptide sequence 2 is shown as SEQ ID NO. 45~SEQ ID NO.

56.

2. The gE fusion protein containing gH Th epitopes according to claim 1, characterized in that, The gE fusion protein further comprises a connecting peptide; the connecting peptide is located between the gE and the gH polypeptide sequence 1 or the gH polypeptide sequence 2, and between the gH polypeptide sequence 1 and the gH polypeptide sequence 2; the connecting peptide is a flexible connecting peptide; the amino acid residues of the flexible connecting peptide are glycine G and / or serine S; the flexible connecting peptide comprises at least one of the following sequences: G, S, GS, SG, GG, SS, GGG, SSS, GSG, SGS, GGS, SGG, SSG, GSS, GGGG, SGGG, GSGG, GGSG, GGGS, SGSG, GSGS, SGGS, GSSG, SSSG, GSSS, SGSS, SSSG, SSSS, GGGGS, GGGSG, GGSGG, GSGGG, SGGGG, GSGGS, GSGSG, GGSGS, SGGGS, SGSGG, SGGSG.

3. The gE fusion protein containing gH Th epitopes according to claim 1, characterized in that, The amino acid sequence combination of the gE fusion protein comprises the following combinations: gE-gH polypeptide sequence 1, gE-gH polypeptide sequence 2, gH polypeptide sequence 2-gE, gH polypeptide sequence 2-gE-gH polypeptide sequence 1, gE-gH polypeptide sequence 2-gH polypeptide sequence 1, gE-gH polypeptide sequence 1-gH polypeptide sequence 2.

4. A nucleic acid sequence encoding the gE fusion protein of any one of claims 1-3.

5. A method of producing a gE fusion protein comprising a gH Th epitope according to any one of claims 1 to 3, characterized in that, The nucleic acid sequence comprises the following steps: S1, constructing an expression vector or a viral vector; S2, transforming the expression vector into a host cell or infecting the host cell with the viral vector; S3, culturing the host cell treated in S2; S4, collecting the cell culture supernatant; S5, purifying the cell culture supernatant to obtain the gE fusion protein.

6. The method of claim 5, wherein the gE fusion protein comprising a gH Th epitope is prepared by, The expression vector comprises a eukaryotic expression vector, and the host cell used in S2 for transforming the expression vector into the host cell comprises at least one of a CHO cell, a yeast cell, and a HEK293 cell.

7. The method of claim 5, wherein the gE fusion protein comprising a gH Th epitope is prepared by, The viral vector comprises at least one of a baculovirus vector, an adenovirus vector, an adeno-associated virus vector, and a lentivirus vector. The host cells in the step S2 of infecting the host cells with the virus vector include at least one of insect cells, HEK293 cells.

8. Use of a gE fusion protein containing a gH Th epitope, which is prepared by the preparation method according to any one of claims 5 to 7, characterized in that, The gE fusion protein is applied to a varicella-zoster virus vaccine.

9. The use of a gE fusion protein containing a gH Th epitope according to claim 8, characterized in that, The varicella-zoster virus vaccine includes an adjuvant; the adjuvant includes at least one of an aluminum adjuvant, a liposome adjuvant, a CpG adjuvant, a Poly I:C adjuvant, a Quillaja saponins QS-21 adjuvant, a Quillaja saponins QS-7 adjuvant, a nanoemulsion, an MPLA adjuvant, a manganese adjuvant; the content of the gE fusion protein in the varicella-zoster virus vaccine is 5-200 μg / ml.

10. A nucleic acid sequence according to claim 4, applied to a varicella-zoster virus mRNA vaccine and a virus vector vaccine for preventing chickenpox and / or shingles and / or postherpetic neuralgia.

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