Self-assembled herpes virus gE nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses self-assembled herpes virus gE nanoparticles as well as a preparation method and application thereof, and relates to the technical field of biomedicine. The self-assembled herpes virus gE nanoparticles are formed by self-assembling a plurality of monomers; the monomers include herpes virus gE and VZVgI polypeptides. The self-assembled herpesvirus gE nanoparticles can be applied to VZV and / or HSV-1 type and / or HSV-2 type vaccines, and the technical problems that the gE immunogenicity is weak and the vaccine side reaction is serious are solved. The VZV gI polypeptide and the herpes virus gE are subjected to fusion expression to prepare the self-assembled herpes virus gE nanoparticles, the preparation method is simple, and the immunogenicity of the gE is remarkably enhanced; according to the vaccine containing the nanoparticles, the immunogenicity of the vaccine can be better than that of the vaccine in the prior art, and the use of an immunopotentiator can be reduced, so that the inflammatory reaction induced by the immunopotentiator is reduced, and the clinical side reaction of the vaccine is finally reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a self-assembled herpesvirus gE nanoparticle, its preparation method, and its application. Background Technology

[0002] Herpesviruses are a class of DNA viruses that widely infect humans. Based on their pathogenic characteristics and sites of infection, they can be divided into eight main types. Among them, varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (HHV-8) cause diseases with a heavier clinical burden. Glycoprotein E is the most immunogenic of the major surface proteins of herpesviruses, but its immunogenicity is not strong. VZV glycoprotein I is a relatively abundant glycoprotein on the varicella-zoster virus envelope, which can form a heterodimer with VZV glycoprotein E. The AA82–AA101 sequence of glycoprotein I contains a confirmed Th (T helper) cell epitope and two cysteine ​​residues, which facilitate the self-assembly of the extracellular region (gE) of glycoprotein E expressed with it into nanoparticle structures.

[0003] GlaxoSmithKline's recombinant zoster vaccine Shingrix ® (Shingrix) ® This vaccine was approved by the FDA in 2017. In humans, 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 than 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 3-O-deacylated-4'-monophosphatidyllipid A (3D-MPLA), 50 μg of saponin QS-21, dioleoylphosphatidylcholine (DOPC), and cholesterol. Saponin QS-21 can activate the NLRP3 inflammasome, thereby releasing caspase-1-dependent cytokines IL-1β and IL-18, thus promoting the Th1 response; 3D-MPLA, through interaction with TLR-4, activates the MyD88 and TRIF-dependent signaling pathways, inducing the production of inflammatory cytokines and interferons, thereby enhancing the immune response. (Shingrix) ® It has a high protection rate, but a serious rate of clinical side effects.

[0004] GSK announced Shingrix in 2017.® Clinical review: Shingrix ® After injection into rabbits, it 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 main reason for the high incidence of clinical adverse reactions and serious adverse reactions (Grade 3 AEs) is that gE has poor immunogenicity and requires the use of two immune enhancers (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 herpesvirus vaccines with high immunogenicity and low side effects. Therefore, using VZVgI peptides containing Th epitopes to form herpesvirus gE into nanoparticle structures to enhance its immunogenicity, while reducing the use of immunostimulants, is an ideal technical route for developing next-generation herpesvirus vaccines. Nanoparticle antigens typically have a particle size of 10–200 nanometers, which can activate the innate immune system and induce strong humoral and cellular immune responses, with significantly higher antigen presentation efficiency than traditional vaccines.

[0007] The process for preparing VZV gE-containing nanoparticles, as described in patent CN116983403B, is particularly complex: A VZV gE fusion protein (composed of gE, linker peptide 1, and binding peptide 1) is expressed and purified in CHO cells, while a particulate protein (composed of nanoparticle protein, linker peptide 2, and binding peptide 2) is expressed and purified in E. coli. The gE fusion protein and particulate protein are then mixed to form a gE-containing nanoparticle protein mixture, which is then purified to prepare gE-containing nanoparticle protein. Although this process enhances the immunogenicity of gE-containing nanoparticle protein, it also elicits a strong non-VZV-specific (particulate protein and binding complex protein specificity) immune response. This non-VZV-specific immune response is not required for vaccines and may exacerbate clinical side effects. Summary of the Invention

[0008] The purpose of this invention is to provide self-assembled herpesvirus gE virus-like nanoparticles, their preparation method, and applications, to solve the problems of low gE immunogenicity and significant clinical side effects in existing herpesvirus vaccine preparation technologies. To achieve the above objective, this invention provides the following technical solution: A self-assembled herpesvirus gE nanoparticle is formed by the self-assembly of multiple monomers; further, the monomers include herpesvirus gE and VZVgI polypeptide; further, the herpesvirus gE includes varicella-zoster virus (VZV) gE, herpes simplex virus type 1 (HSV-1) gE, herpes simplex virus type 2 (HSV-2) gE, Epstein-Barr virus (EBV) gE, cytomegalovirus (CMV) gE, and Kaposi's sarcoma-associated herpesvirus (HHV-8) gE; further, the herpesvirus glycoside gE is preferably VZV gE.

[0009] Furthermore, the amino acid residues of the VZV gE exhibit polymorphism; further, 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). Further, the polymorphisms of glycoprotein E in some VZV strains are as follows: In OKA vaccine strain glycoprotein E (NCBI registration number: AAK19946.1; Q9J3M8.1), the amino acid residues at positions AA40 and AA536 are isoleucine (I) and leucine (L), respectively; in Dumas strain glycoprotein E (NCBI registration number: NP_040190.1), the amino acid residues at positions AA40 and AA536 are threonine (T) and leucine (L), respectively; in the 2005 German isolate glycoprotein E (NCBI registration number: AEW88764.1), the amino acid residues at positions AA40 and AA536 are isoleucine (I) and isoleucine (I), respectively.

[0010] Furthermore, the optional amino acid sequence length of the VZV gE includes, but is not limited to, any one of the following: AA31-AA546, AA31-AA545, AA31-AA544, AA31-AA543, AA31-AA542, AA31-AA541, AA31-AA540, AA31-AA539, ​​and AA31-AA538 of varicella-zoster virus glycoprotein E.

[0011] Further, the preferred amino acid sequence of the VZV gE is the AA31-AA544 portion of the varicella-zoster virus glycoprotein E, as shown in SEQ ID NO.1. Further, the amino acid sequence of the VZV gI is the AA21-AA272 portion of the VZV glycoprotein I, as shown in SEQ ID NO.2.

[0012] Furthermore, the VZV gE also includes a sequence that is at least 95.0% to 99.5% 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 99.5% identical.

[0013] Furthermore, the amino acid sequence of the HSV-1 type gE includes, but is not limited to, SEQ ID NO.50 (17 strains).

[0014] Furthermore, the amino acid sequence of the HSV-2 type gE includes, but is not limited to, SEQ ID NO.51 (HG52 strain).

[0015] Further, the VZV gI polypeptide sequence contains Th epitope 1 (eptope ID: 839316) and Th epitope 2 (eptope ID: 2224791); further, the amino acid sequence of Th epitope 1 is FCFRSVQVIRYDGCPRIRTS; further, the amino acid sequence of Th epitope 2 is RYDGCPRIRTSAFISCRYKH; further, both Th epitope 1 and Th epitope 2 contain two cysteine ​​residues.

[0016] Further, Th epitope 1 is located at positions AA82-AA101 in the VZV glycoprotein I sequence, and its amino acid sequence is FCFRSVQVIRYDGCPRIRTS; further, Th epitope 1 has been confirmed by Ying YingKong in 2016 as HLA-DRB1*03:01, HLA-DRB1*13:01, HLA-DRB3*03:01, and HLA-DRB3*02:02 restriction epitopes, with IDs (IEDB IDs) of 2884824, 2884827, 2884826, and 2884825, respectively; further, Th epitope 1 has been confirmed by Ying YingKong in 2017 as HLA-DRB3*01:01 and HLA-DR restriction epitopes, with IDs (IEDB IDs) of 2884824, 2884827, 2884826, and 2884825, respectively. The IDs are 3497311 and 3497310, respectively. Furthermore, Th epitope 1 was confirmed as an HLA-DRB3*03:01 restriction epitope by Kerry J. Laing in 2019, and its ID (IEDB ID) in the Immunotope Database (IEDB) is 5003302. Furthermore, Th epitope 2 is located at positions AA91–AA110 in the VZV glycoprotein I sequence, and its amino acid sequence is RYDGCPRIRTSAFISCRYKH. Furthermore, Th epitope 2 was confirmed as a Th epitope by Kerry J. Laing in 2020, and its ID (IEDB ID) in the Immunotope Database (IEDB) is 8326833.

[0017] The VZV gI polypeptide is any one of the following gI polypeptides: AA79~AA115, AA79~AA114, AA79~AA113, AA79~AA112, AA79~AA111, AA79~AA110, AA79~AA109, AA79~AA108, AA79~AA107, AA79~AA106, AA80~AA115, AA80~AA114, AA80~AA113, AA80~AA112, AA80~AA111, AA80~AA110, AA80~AA109, AA80~AA108, AA80~AA107, AA80~AA115, AA80~AA114, AA80~AA113, AA80~AA112, AA80~AA111, AA80~AA110, AA80~AA109, AA80~AA108, AA80~AA107, AA80~AA110, AA79~AA110, AA79~AA110, AA80 ... 106, AA81~AA115, AA81~AA114, AA81~AA113, AA81~AA112, AA81~AA111, AA81~AA110, AA81~AA109, AA81~AA108, AA81~AA107, AA81~AA106, AA82~AA115, AA82~AA114, AA82~AA113, AA82~AA112, AA82~AA111, AA82~AA110, AA82~AA109, AA82~AA108, AA82~AA107, or AA82~AA106, with amino acid sequences as shown in SEQ ID NO.3~SEQ ID NO.42, respectively.

[0018] Furthermore, the VZV gI polypeptide is preferably AA79~AA115 and AA82~AA115, with amino acid sequences as shown in SEQ ID NO.3 and SEQ ID NO.33, respectively.

[0019] Furthermore, the VZV gI polypeptide is most preferably AA82~AA115, with the amino acid sequence shown in SEQ ID NO.33.

[0020] Furthermore, the VZVgI polypeptide also includes a polypeptide that is at least 90.0% to 99.0% identical to any one of the above-mentioned gI polypeptides, 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] A monomer of a self-assembled herpesvirus gE nanoparticle may also contain other immune-enhancing sequences besides the VZV gI polypeptide mentioned above; further, the other immune-enhancing sequences can enhance the immunogenicity of gE; further, the other immune-enhancing sequences include, but are not limited to, any one or a combination of the following: Th epitope peptides or polypeptides containing Th epitopes, interleukins, and interferons.

[0022] A monomer of a self-assembled herpesvirus gE nanoparticle may contain a linker peptide (-); further, the possibility of containing a linker peptide (-) includes monomers that do not contain a linker peptide and monomers that do contain a linker peptide, preferably containing a linker peptide.

[0023] Furthermore, when the monomer contains a linker peptide, the linker peptide is located between the herpesvirus gE and VZV gI polypeptides of the monomer; furthermore, when the self-assembled herpesvirus gE nanoparticles contain the other immune-enhancing sequences mentioned above, there are at least two linker peptides, located at gE and VZV, respectively. The linker peptide is formed between any two of the three elements: gI peptide, other immune-enhancing sequences, etc. Further, the linker peptide is a flexible linker peptide 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, any one or more combinations of the following: G, S, GS, SG, GG, SS, GGG, SSS, GSG, SGS, GGS, SGG, SSG, GSS, SGGG, GGSG, GGSGS, GGGS, SGSG, GSGS, GGGGS, GGSGG, GGSGG, SGGGG, SGGGG, SGGGG, SGGGS, SGSGG, SGGSG. Further, the flexible linker peptide is preferably SGS and / or GGSGSG. Further, the monomer contains a linker peptide, and the amino acid sequence of the monomer includes, but is not limited to, SEQ ID NO.43~SEQ ID NO.47, SEQ ID NO.52, and SEQ ID NO.53.

[0024] A combination of gE and VZV gI peptides in a monomer of a self-assembled herpesvirus gE nanoparticle includes: combination method 1 and combination method 2; further, in combination method 1, the VZV gI peptide is located at the C-terminus of the monomer (i.e., the C-terminus of gE); further, in combination method 2, the VZV gI peptide is located at the N-terminus of the monomer (i.e., the N-terminus of gE).

[0025] Furthermore, in the above-mentioned self-assembled herpesvirus gE nanoparticles, the combination of gE and VZV gI polypeptides in the monomer is preferably in combination mode 1.

[0026] Furthermore, the amino acid sequence of combination mode 1 of gE and VZV gI polypeptide in the above monomers includes, but is not limited to, SEQ ID NO.43 to SEQ ID NO.45; further, the preferred amino acid sequence is shown in SEQ ID NO.44.

[0027] Furthermore, to facilitate the purification of the self-assembled herpesvirus gE nanoparticles, various purification tags can be added to their monomers; furthermore, the purification tags include, but are not limited to, any one or more combinations of the following: GST tag, GFP tag, Fc tag, His tag, Strep-tag II tag.

[0028] Furthermore, when the purification tag is a 6X His tag located at the C-terminus of the monomer, and the VZV gI peptide is also located at the C-terminus of the monomer, the 6X His tag is located at the N-terminus or C-terminus of the gI peptide, preferably the 6X His tag is located at the C-terminus of the VZV gI peptide (the VZV gI peptide is located at the N-terminus of the 6X His tag), and the amino acid sequence of the monomer includes, but is not limited to, SEQ ID NO.46 and SEQ ID NO.47.

[0029] A nucleic acid sequence encoding a monomer of the self-assembled herpesvirus gE nanoparticles described in any of the preceding claims.

[0030] Furthermore, the aforementioned nucleic acid sequences include DNA sequences and RNA sequences.

[0031] Furthermore, the above-mentioned nucleic acid sequences can be used to construct expression vectors, viral vectors, or mRNA molecules that express any of the self-assembled herpesvirus gE nanoparticles described above.

[0032] Furthermore, the aforementioned mRNA molecules are delivered into human cells and can express the self-assembled herpesvirus gE nanoparticles described in any of the above-mentioned embodiments.

[0033] A method for preparing self-assembled herpesvirus gE nanoparticles includes 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, host cells obtained from S2 culture; S4. Collect the cell culture supernatant and / or the supernatant of cell lysates; S5. Purify the cell culture supernatant and / or the supernatant of cell lysate to obtain self-assembled herpesvirus gE nanoparticles.

[0034] Furthermore, the expression vector described in S1 is a eukaryotic expression vector.

[0035] Furthermore, the eukaryotic expression vector enables the expression of exogenous genes in eukaryotic cells, 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 (restriction sites).

[0036] Furthermore, the eukaryotic expression vector, including but not limited to empty vectors (vectors without inserted foreign genes), includes pcDNA3.4, pcDNA3.1(+), pcDNA3.1+C-His, pcDNA3.1 myc-His C, pEF1α-V5, pCMV-HA, pCMV6-AC-IRES-GFP, and pEGFP-N1; furthermore, the empty vector may contain resistance genes and / or genes of eukaryotic selection markers.

[0037] Furthermore, the construction of the expression vector or viral vector containing the above-mentioned 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 above-mentioned nucleic acid sequence (i.e., the N-terminus and / or C-terminus of the gE fusion protein).

[0038] Furthermore, the purification tag includes, but is not limited to, any one or more combinations of the following: GST tag, GFP tag, Fc tag, His tag, Strep-tag II tag.

[0039] Furthermore, the expression vector described in S1 also contains a nucleic acid sequence encoding a signal peptide that enables the secretion of self-assembled herpesvirus gE nanoparticles outside the cell.

[0040] Furthermore, the nucleic acid sequence encoding the signal peptide is located upstream of the 5' end of the aforementioned nucleic acid sequence, that is, at the N-terminus of the self-assembled herpesvirus gE nanoparticle monomer.

[0041] Furthermore, the ORF sequence in the expression vector described in S1 can be codon optimized to promote protein expression.

[0042] DNA sequences encoding herpesvirus gE virus-like nanoparticle monomers containing a 6×His tag include, but are not limited to, SEQ ID NO.48 and SEQ ID NO.49.

[0043] Furthermore, the viral vector in S1 includes baculovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and parainfluenza virus vectors, with baculovirus vectors being preferred.

[0044] Furthermore, the expression vector described in S2 is transformed into host cells in two ways: stable transformation and transient transformation.

[0045] Furthermore, after the stable transformation 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; the resistance gene with the eukaryotic selection marker includes, but is not limited to, any one or more combinations of the following: neomycin, hygromycin B, blastcin S, bleomycin, etc.; the gene with the eukaryotic selection marker includes, but is not limited to, any one or more combinations of the following: dihydrofolate reductase, glutamine synthetase, tryptophan synthase, etc.

[0046] 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.

[0047] 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.

[0048] Furthermore, the insect cells include, but are not limited to, Sf9 cells, Sf21 cells, and Hi5 cells.

[0049] Furthermore, the cell culture supernatant and / or cell lysate supernatant mentioned in S4 contains gE nanoparticles, residual components of the culture medium, and may contain host cell debris, host cell proteins, host cell nucleic acids, host cell metabolites, and other substances; furthermore, the cell lysate supernatant is a supernatant obtained by lysing host cells and then centrifuging or other methods.

[0050] Further, the purification of cell culture supernatant and / or cell lysate supernatant described in S5 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 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.

[0051] Furthermore, the self-assembled herpesvirus gE nanoparticles described in S5 have a purity (nanoparticles) of not less than 10.0% to not less than 99.9%, such as not less than 10.0%, not less than 20.0%, 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%.

[0052] Application of a self-assembled herpesvirus gE nanoparticle for the prevention and / or improvement of herpesvirus-induced diseases in a herpesvirus vaccine; further, the herpesvirus includes VZV, HSV-1, HSV-2, EBV, CMV, and HHV-8.

[0053] Furthermore, the herpesvirus vaccine contains self-assembled herpesvirus gE nanoparticles as described in any of the above-mentioned embodiments; furthermore, the herpesvirus vaccine also contains an adjuvant.

[0054] Further, the adjuvant is any one or any combination of adjuvants (compound adjuvant / adjuvant system) listed below: aluminum adjuvant, liposome adjuvant, CpG (non-methylated oligodeoxynucleotide) adjuvant, Poly I:C (polyinosinic acid: polycytidylic acid) adjuvant, saponin QS-21 adjuvant, saponin QS-7 adjuvant, nanoemulsion, MPLA (monophosphoryl lipid A) adjuvant, manganese adjuvant; further, a combination of liposome adjuvant and saponin QS-21 adjuvant is preferred.

[0055] Furthermore, the herpesvirus vaccine, the method for preparing the vaccine includes the following steps: S6. Prepare adjuvant or compound adjuvant solution; S7. Prepare the vaccine.

[0056] Further, in the herpesvirus vaccine, the concentration of self-assembled herpesvirus gE nanoparticles is 10-1000 μg / ml; more preferably, the concentration of self-assembled herpesvirus gE nanoparticles is 40-400 μg / ml.

[0057] Based on the above-mentioned nucleic acid sequence, it was applied to a herpesvirus mRNA vaccine.

[0058] Furthermore, when the aforementioned nucleic acid sequence is applied to an mRNA vaccine, the nucleic acid sequence may also contain a short nucleic acid sequence encoding the transmembrane region and / or intramembrane region of herpesvirus glycoprotein E.

[0059] The herpesvirus mRNA vaccine includes an mRNA molecule and a system for delivering the mRNA molecule.

[0060] Based on the above-mentioned nucleic acid sequence, it was applied to a viral vector herpesvirus vaccine.

[0061] Furthermore, when the aforementioned nucleic acid sequence is used in a viral vector vaccine, the nucleic acid sequence may also contain a short nucleic acid sequence encoding the transmembrane region and / or intramembrane region of herpesvirus glycoprotein E.

[0062] Furthermore, the viral vector herpesvirus vaccine uses a viral vector including adenovirus vector, attenuated influenza virus vector, and parainfluenza virus vector; more preferably, an adenovirus vector is used.

[0063] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects: This invention provides a self-assembled herpesvirus gE nanoparticle, which is self-assembled from multiple monomers; the monomers include herpesvirus gE and VZV gI polypeptide sequences. This self-assembled herpesvirus gE nanoparticle can be used in herpesvirus vaccines, solving the technical problems of weak gE immunogenicity and severe vaccine side effects in existing vaccine preparation techniques.

[0064] Mice immunized twice with this herpesvirus vaccine had higher serum gE-specific antibody levels and gE-specific CMI response (splenic cells) than mice immunized with Shingrix. ® (Prior Art) Group; Self-assembled herpesvirus gE nanoparticles have higher immunogenicity than gE, thus reducing the type and dosage of immunostimulants in vaccines (this vaccine contains only 50 μg of the immunostimulant saponin QS-21; Shingrix). ® It contains 50 μg of the immune enhancer saponin QS-21 and 50 μg of the immune enhancer 3D-MPLA, thereby reducing the inflammatory response induced by the immune enhancer and thus reducing the clinical side effects of the vaccine.

[0065] Compared with the nanoparticles and preparation method provided in patent CN116983403B, the self-assembled herpesvirus gE nanoparticles do not contain exogenous nanoparticle sequences (non-herpesvirus sequences), use VZV gI peptides as the elements driving the nanoparticleization of herpesvirus gE, and have a simpler preparation method, and only induce herpesvirus-related immune responses; in addition, the VZV gI peptides contain confirmed gI Th epitopes, therefore, vaccines containing self-assembled herpesvirus gE nanoparticles can also induce VZVgI-specific CMI responses.

[0066] In short, with Xin An Li Shi ® Compared with the nanoparticle vaccine provided by patent CN116983403B, this herpes virus vaccine has better potential for clinical application. Attached Figure Description

[0067] 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.

[0068] Figure 1 This is a graph showing the SDS-PAGE detection results of VZV gE or VZV gE fusion protein in Example 1 of this invention. In the graph, M / M1: protein marker; R: reducing SDS-PAGE; NR / NR: non-reducing SDS-PAGE; gE(VZV)-gI2: gE(VZV)-gI2 fusion protein; gE(VZV)-gI(VZV): gE(VZV)-gI(VZV) fusion protein; NP-3: self-assembled gE(VZV) nanoparticles NP-3.

[0069] Figure 2 This is the SEC-HPLC detection chromatogram of the self-assembled gE (VZV) nanoparticles NP-3 in Example 1 of this invention.

[0070] Figure 3 This is the particle size distribution (light intensity distribution) of the self-assembled gE (VZV) nanoparticles NP-3 in Example 1 of this invention.

[0071] Figure 4 This is an electron microscope image (40,000x magnification) of the self-assembled gE (VZV) nanoparticles NP-3 in Example 1 of this invention.

[0072] Figure 5 This is an electron microscope image (80,000x magnification) of the self-assembled gE (VZV) nanoparticles NP-3 in Example 1 of this invention.

[0073] Figure 6 This is a graph showing the level of gE (VZV) specific antibody GMT in the serum of mice in each VZV vaccine group after secondary immunization in Example 3 of this invention.

[0074] Figure 7 This is a graph showing the percentage of gE (VZV) specific IL2+ and / or IFN-γ+ CD4+ T cells in the spleen CD4+ T cells of mice in each VZV vaccine group after secondary immunization in Example 3 of this invention. Detailed Implementation

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] Example 1: Study on the effect of different VZV gI polypeptide sequences on the preparation of self-assembled VZV gE nanoparticles (1) Design of self-assembled VZV gE nanoparticles containing different VZV gI polypeptide sequences The extracellular sequence information of VZV glycoprotein gE and gI is shown in Table 1 below: Table 1 Sequence information of VZV glycoproteins gE and gI

[0081] The amino acid sequence information for the selected VZV gI Th epitopes (IEDB epitope database) is shown in Table 2 below: Table 2. Amino acid sequence information of the selected gI Th epitopes.

[0082] Information on VZV gI polypeptide (gIpolypeptide, gIPP) The amino acid sequence information of gIPP is shown in Tables 3 and 4 below. gI2 has been disclosed in CN117100850A to enhance the immunogenicity of gE.

[0083] Table 3 contains different gI peptides with Th epitope 1.

[0084] S1. Construct an expression vector containing a nucleic acid sequence encoding a self-assembled VZV gE nanoparticle monomer. Design self-assembled VZV gE nanoparticles containing different gIPPs Table 4 Information on self-assembled VZV gE nanoparticle monomers or VZV gE fusion proteins

[0085] (2) Nucleic acid codon optimization and whole gene synthesis First, a DNA sequence encoding the signal peptide (MHSSALLCCLVLLTGVRA) was added to the 5' end (5' end of the positive strand) of the protein-coding DNA sequence, and a DNA sequence encoding the 6×His tag was added to the 3' end (3' end of the positive strand). Then, the DNA sequence was codon-optimized to improve translation efficiency. Next, a 5' UTR sequence and a stop codon (TGA) were added to the 5' and 3' ends (3' end of the positive strand), respectively, of the codon-optimized DNA sequence. Then, a Not I restriction enzyme cleavage site was added to the 5' end of the 5' UTR sequence, and an Xba I restriction enzyme cleavage site and protective bases were added to the 3' end of the stop codon (TGA). Finally, the entire DNA sequence was synthesized. The amino acid sequences of the NP-2 and NP-3 monomers containing the 6×His tag are shown in SEQ ID NO.46 and SEQ ID NO.47, and the nucleic acid sequences are shown in SEQ ID NO.48 and SEQ ID NO.49, respectively.

[0086] (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, which were then amplified and plated onto solid culture medium in petri dishes. The dishes were then incubated in an incubator for approximately 8 hours. Eight single colonies were then selected for cloning screening (PCR).

[0087] (4) Expression plasmid amplification After the positive clones were verified to be correct by sequencing, they were cultured and amplified in bacterial culture, and plasmids were extracted.

[0088] 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.

[0089] S3. Cultivate the host cells described in S2. (6) Cell culture and protein expression 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.

[0090] 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.

[0091] S5. Purify the cell culture supernatant to obtain gE virus-like nanoparticles. (8) Protein purification Incubate the cell culture supernatant with AmMag™ Ni Magnetic Beads for at least 120 min, remove the supernatant, then equilibrate with 25 mM Tris-HCl + 300 mM NaCl (pH 7.0) buffer, and elute and collect with 25 mM Tris-HCl + 300 mM NaCl + 500 mM Imidazole (pH 7.0) buffer. Then, dialyze the collected protein three times using histidine buffer (10 mM histidine + 10% sucrose + 0.05% Tween 80; pH 6.5 ± 0.1) through a SNAKESKIN™ DIALYSIS TUBING™ dialysis bag, with each dialysis session lasting at least 2 hours.

[0092] (9) Protein purity detection Detection method (SDS-PAGE): Voltage 140~160V, electrophoresis for 45~60 minutes; use the eStain™ L1 protein staining system for gel staining and destaining, then scan the gel and save the gel pattern. For reduction electrophoresis, a reducing agent (e.g., dithiothreitol) must be added to the electrophoresis system; no reducing agent is added for non-reduction electrophoresis.

[0093] Detection method (SEC-HPLC): The sample must be filtered through a 0.2 μm filter membrane before being injected into the chromatographic column. The chromatographic column is a TSKgel G3000SWxl, and the injection volume is approximately 20 μg.

[0094] Test results: See Table 5 below. Figure 1 and Figure 2 As shown.

[0095] Table 5. Purity test results of self-assembled gE nanoparticles or gE fusion proteins

[0096] Note: 1. " / " means "No protein expression detected".

[0097] The reason why the nanoparticle bands in the SDS-PAGE (non-reduction) purity (nanoparticle) spectrum are located in the sample wells is that the molecular weight is extremely large, and the molecules have not moved by the end of electrophoresis, so the bands are still located in the sample wells.

[0098] Analysis of purity test results: 1. When gE is fused with gI2 (AA141-AA155) (gI2 is located at the C-terminus of the fusion protein), although the purity of the target protein (gE fusion protein) is 95%, no nanoparticle bands are detected by SDS-PAGE (non-reduction). When gE is fused with gIPP or gI (AA21-AA272) containing Th epitopes 1 (AA82-AA101) and Th epitopes 2 (AA91-AA110) (gIPP or gI is located at the C-terminus of the fusion protein), the purity of the target protein (gE fusion protein) is also 95%, but nanoparticle bands are detected by SDS-PAGE (non-reduction). Therefore, Th epitopes 1 (AA82-AA101) and Th epitopes 2 (AA91-AA110) are essential for the formation of nanoparticles from the gE fusion protein monomer.

[0099] When gE is fused with gI (AA21-AA272) (gI is located at the C-terminus of the fusion protein), the purity of the target protein (gE fusion protein) is 95%, but the purity of the nanoparticles is very low: the purity detected by SEC-HPLC is 31%. When gE is fused with gIPP (gIPP is located at the C-terminus of the fusion protein) in NP-2 and NP-3, the purity of the target protein (gE fusion protein) is also 95%, but the purity of the nanoparticles is higher: the purity detected by SEC-HPLC is 77% and 80%, respectively, that is, most of the gE fusion protein monomers formed nanoparticles. Therefore, in order to allow monomers to form nanoparticles better, gIPP is not necessarily longer and taller.

[0100] No protein expression was detected at NP-1, which is located at the N-terminus of the monomer; significant protein expression (above 1.5 mg) was detected at NP-2 and NP-3, which are located at the C-terminus of the monomer, and the purity (target protein) of both was 95%. Nanoparticle bands were detected by non-reducing SDS-PAGE, and the purity (nanoparticles; SEC-HPLC) of both was not less than 77%. Therefore, in order for the monomer to better form nanoparticles, gIPP should be located at the C-terminus of the monomer.

[0101] Located at the C-terminus of monomers, gIPP was used to prepare NP-2 and NP-3 by fusion expression of gIPP and gE, and nanoparticle bands were detected (non-reducing SDS-PAGE). However, when gE was fused with gI2 (gI2 is located at the C-terminus of the fusion protein), nanoparticle bands were not detected by SDS-PAGE (non-reducing). The main difference in amino acid residue composition between gIPP and gI2 sequences is that gIPP contains three cysteine ​​residues (C83, C95, and C106). Therefore, gIPP may facilitate the formation of disulfide bonds between monomers, which is beneficial for the formation of nanoparticles.

[0102] In summary, it is speculated that within the range of gIAA79 to AA115, sequences containing three cysteine residues other than gIPP1 and gIPP2, such as: AA79 to AA114 (SEQ ID NO.4), AA79 to AA113 (SEQ ID NO.5), AA79 to AA112 (SEQ ID NO.6), AA79 to AA111 (SEQ ID NO.7), AA79 to AA110 (SEQ ID NO.8), AA79 to AA109 (SEQ ID NO.9), AA79 to AA108 (SEQ ID NO.10), AA79 to AA107 (SEQ ID NO.11), AA79 to AA106 (SEQ ID NO.12), AA80 to AA115 (SEQ ID NO.13), AA80 to AA114 (SEQ ID NO.14), AA80 to AA113 (SEQ ID NO.15), AA80 to AA112 (SEQ ID NO.16), AA80 to AA111 (SEQ ID NO.17), AA80 to AA110 (SEQ ID NO.18), AA80 to AA109 (SEQ ID NO.19), AA80 to AA108 (SEQ ID NO.20), AA80 to AA107 (SEQ ID NO.21), AA80 to AA106 (SEQ ID NO.22), AA81 to AA115 (SEQ ID NO.23), AA81 to AA114 (SEQ ID NO.24), AA81 to AA113 (SEQ ID NO.25), AA81 to AA112 (SEQ ID NO.26), AA81 to AA111 (SEQ ID NO.27), AA81 to AA110 (SEQ ID NO.28), AA81 to AA109 (SEQ ID NO.29), AA81 to AA108 (SEQ ID NO.30), AA81 to AA107 (SEQ ID NO.31), AA81 to AA106 (SEQ ID NO.32), AA82 to AA114 (SEQ ID NO.34), AA82 to AA113 (SEQ ID NO.35), AA82 to AA112 (SEQ ID NO.36), AA82 to AA111 (SEQ ID NO.37), AA82 to AA110 (SEQ ID NO.38), AA82 to AA109 (SEQ ID NO.39), AA82 to AA108 (SEQ ID NO.40), AA82 to AA107 (SEQ ID NO.41), AA82 to AA106 (SEQ ID NO.42) The presence of gI polypeptide sequences also promotes the formation of gE nanoparticles.

[0103] The purity (nanoparticles) of NP-3 containing gIPP2 (AA82~AA115) is higher than that of NP-2 containing gIPP1 (AA79~AA115); therefore, gIPP2 is more conducive to the formation of monomer nanoparticles.

[0104] (10) Protein expression level detection Detection method: Protein concentration was detected using Nanodrop according to the UV-Vis spectrophotometry method in the general chapter of the Chinese Pharmacopoeia; then, the protein expression level was obtained by multiplying the protein concentration by the protein volume.

[0105] Test results are shown in Table 6 below.

[0106] Table 6 shows the concentration detection of the designed self-assembled gE nanoparticles or gE fusion proteins.

[0107] Note: " / " indicates that no protein expression was detected.

[0108] Analysis of protein expression levels: No protein expression was detected in the designed NP-1; the protein expression levels of the designed NP-2 and NP-3 were both high (above 1.5 mg), with NP-3 showing the highest level; the results indicate that the expression level of self-assembled VZV gE nanoparticles is higher when VZV gIPP is located at the C-terminus of the monomer compared to when it is located at the N-terminus of the monomer.

[0109] (11) Particle size detection Detection method: NP-3 with high protein expression levels was selected for particle size analysis. The samples were diluted to a protein concentration of 0.1~0.3 mg / ml and mixed thoroughly; particle size and PDI distribution were detected using a Malvern particle size analyzer (Zetasizer Advance).

[0110] Test results: See Table 7 below. Figure 3 As shown in (NP-3).

[0111] Table 7. Particle size determination of self-assembled gE nanoparticles

[0112] Particle size analysis: The average particle size of NP-3 is 33 nm.

[0113] (12) Morphological (electron microscopy) detection Detection method: NP-3 with high protein expression levels was selected for microscopic morphology detection. The sample was diluted to a concentration of 0.1~0.3 mg / ml; 10 μL of the diluent was dropped onto the surface of a 300-mesh carbon copper mesh and left to adhere for 10 min; then rinsed with distilled water, followed by rapid staining twice with 2% sodium phosphotungstenate, 10 s each time; then the stained copper mesh was completely dried; finally, imaging and observation were performed using a Hitachi HT7800 transmission electron microscope at an accelerating voltage of 80 kV.

[0114] Test results: such as Figure 4 and Figure 5 As shown.

[0115] Electron microscopy results analysis: Nanoparticles could be observed in the NP-3 sample under magnification conditions of 40,000 and 80,000 times.

[0116] Example 2: Preparation of VZV vaccine using saponin QS-21 as an immune enhancer 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 pore size membrane to remove residual ethanol. Finally, the liposomes were sterilized by filtration using a 0.22 μm sterilizing filter to obtain the final liposome product.

[0117] (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.

[0118] (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.

[0119] S7, Vaccine preparation (4) Vaccine preparation [5ml volume; target concentration of VZV gE is 100μg / ml; 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 VZV gE nanoparticles, VZV gE fusion protein, or VZV gE solution to the adjuvant, then supplement the total volume to 5 ml with histidine solution (10 mM; pH 6.5 ± 0.1; 10% sucrose) and stir well. This is the gE nanoparticle, gE fusion protein, or gE adjuvant vaccine. The formula for calculating the target volume (ml) of added gE nanoparticles, gE fusion protein, or gE solution is: target mass (μg) of added gE nanoparticles, gE fusion protein, or gE / concentration (μg / ml) of gE nanoparticles, gE fusion protein, or gE solution. See Table 8 below for vaccine prescription information.

[0120] Table 8. Vaccine Prescription Information (Vaccine volume: 50 μl)

[0121] Note: Xin An Li Shi ® This is a commercial vaccine, no preparation is required, batch number F7P54; " / " indicates not applicable.

[0122] Example 3: Immunogenicity study of VZV vaccine using saponin QS-21 as an immune enhancer I. Animals immunized with vaccines Thirty-six female C57BL / 6 mice aged 6–8 weeks were randomly divided into 6 groups of 6 mice each. On day 35, each mouse was pre-immunized subcutaneously in the neck with one dose of attenuated varicella vaccine. On day 1 and day 29, the mice were injected intramuscularly in the leg with the vaccine described in Example 2 (50 μl / mouse). On day 0 and day 57, blood was collected and serum was separated. On day 57, the animals were sacrificed, and the spleen was removed and spleen cells were separated.

[0123] II. Methods for Detecting Vaccine Immunogenicity (1) Detection method for VZV gE specific antibody in serum The level of gE antibody in mouse serum 28 days after secondary immunization was detected using an indirect ELISA method. The method was as follows: First, gE was coated into 96-well plates at a rate of 3 μg / well and incubated at 37°C for 60 min. Then, the plates were blocked with TPBS solution containing BSA and washed four times with TPBS. Next, serum was diluted at different dilutions (two-fold serial dilutions; with an initial dilution of 62,500 times, for a total of 8 dilutions), and 100 μl was added to each well. After incubation at 37°C for 1 hour, the plates were washed four times with TPBS. Then, the plates were incubated with HRP-labeled goat anti-mouse secondary antibody at 37°C for 1 hour and washed four times with TPBS. Finally, the plates were developed with TMB in the dark for 15 min, and the reaction was terminated by adding 0.2 M sulfuric acid. The OD value at 450 was read using a microplate reader. The cut-off value was set at four times the average OD value of the mixed serum samples collected on day 0, and the serum gE antibody level was determined. The antibody GMT was then calculated.

[0124] (2) VZV gE-specific cell-mediated immunoassay (CMI) detection method Twenty-eight days after the second immunization, the spleen of mice was harvested to prepare a single-cell suspension; the cells were diluted with culture medium to 1×10⁶. 7 Cells / ml; then add 100 μl to each well of a U-shaped 96-well plate, and seal the remaining four wells with 250 μl of PBS each; stimulate cells with gE peptide pool to induce cytokine secretion; then add Containing Brefeldin A to block secretion; after cell live / dead staining, FcR blocking, CD3, CD45, and CD4 surface staining, fixation and perforation, and IL-2 and IFN-γ intracellular staining, the number of gE-specific IL2+ and / or IFN-γ+ CD4+ T cells in CD4+ T cells is detected by flow cytometry.

[0125] III. Results of Vaccine Immunogenicity Testing (1) The results of the detection of VZV gE-specific binding antibody in serum are shown in Table 9 below. Figure 6 As shown: Table 9 Serum gE-specific antibody levels

[0126] Note: # represents the initial dilution of serum samples in group 1 (buffer). Both wells were negative, and the antibody level was marked as "0" or "<62500".

[0127] (2) The detection results of VZV gE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+) are shown in Table 10 below. Figure 7 As shown Table 10. GE-specific CD4+ T cell levels (IL2+ and / or IFN-γ+)

[0128] 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 self-assembled herpesvirus gE nanoparticle, characterized in that, It is self-assembled from multiple monomers; the monomers include herpesvirus gE and VZVgI polypeptide; the herpesvirus gE includes at least one of VZVgE, HSV-1 gE, and HSV-2 gE.

2. The self-assembled herpesvirus gE nanoparticles according to claim 1, characterized in that, The amino acid sequence of the VZV gI polypeptide in the monomer is any one of SEQ ID NO.3 to SEQ ID NO.

42.

3. The self-assembled herpesvirus gE nanoparticle according to claim 2, characterized in that, Contains a linker peptide; the linker peptide is located between the monomeric herpesvirus gE and VZV gI polypeptides; the linker peptide is a flexible linker peptide or a rigid linker peptide; the amino acid residues of the flexible linker peptide are glycine (G) and / or serine (S); the flexible linker peptide includes any one or more combinations of the following: G, S, GS, SG, GG, SS, GGG, SSS, GSG, SGS, GGS, SGG, SSG, GSS, SGGG, GGSG, GGGS, SGSG, GSGS, GGGGS, GGGSG, GGSGG, GGSGG, SGGGG, GGSGS, SGGGS, SGSGG, SGGSG.

4. The self-assembled herpesvirus gE nanoparticles according to claim 3, characterized in that, The combination of herpesvirus gE and VZVgI polypeptide includes combination mode 1 or combination mode 2; in combination mode 1, the VZVgI polypeptide sequence is located at the C-terminus of the monomer; in combination mode 2, the VZVgI polypeptide sequence is located at the N-terminus of the monomer.

5. A nucleic acid sequence, characterized in that, It encodes the monomer of the self-assembled herpesvirus gE nanoparticles as described in any one of claims 1 to 4.

6. A method for preparing self-assembled herpesvirus gE nanoparticles, characterized in that, Includes the following steps: S1. Construct an expression vector or viral vector containing the nucleic acid sequence described in claim 5; S2. Transform the expression vector obtained in S1 into host cells or infect host cells with the viral vector obtained in S1; S3, host cells obtained from S2 culture; S4. Collect the cell culture supernatant and / or the supernatant of cell lysates; S5. Purify the cell culture supernatant and / or the supernatant of cell lysate to obtain self-assembled herpesvirus gE nanoparticles.

7. The method for preparing self-assembled herpesvirus gE nanoparticles according to claim 6, characterized in that, The expression vector described in S1 is a eukaryotic expression vector; the host cells into which the expression vector is transformed in S2 include at least one of CHO cells, yeast cells, and HEK293 cells; the viral vector described in S1 includes at least one of baculovirus vector, adenovirus vector, adeno-associated virus vector, and lentivirus vector; the host cells into which the viral vector infects the host cells in S2 include at least one of insect cells and HEK293 cells.

8. An application of the self-assembled herpesvirus gE nanoparticles as described in any one of claims 1 to 4, characterized in that, For use in herpesvirus vaccines; the herpesvirus includes at least one of VZV, HSV-1 and HSV-2.

9. The application of the self-assembled herpesvirus gE nanoparticles according to claim 8, characterized in that, The herpesvirus vaccine contains the self-assembled herpesvirus gE nanoparticles; the concentration of the self-assembled herpesvirus gE nanoparticles in the herpesvirus vaccine is 10–1000 μg / ml; the herpesvirus vaccine also contains an adjuvant; the adjuvant includes any one or any combination of the following adjuvants: aluminum adjuvant, liposome adjuvant, CpG adjuvant, Poly I:C adjuvant, saponin QS-21 adjuvant, saponin QS-7 adjuvant, nanoemulsion, MPLA adjuvant, and manganese adjuvant.

10. A nucleic acid sequence according to claim 5, characterized in that, It is applied to herpesvirus mRNA vaccines, DNA vaccines, or viral vector vaccines; the herpesvirus includes at least one of VZV, HSV-1, and HSV-2.

Images