Stabilized modified varicella zoster virus gE protein, methods of making and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a kind of stable modified varicella-zoster virus gE protein, its preparation method and application.The stable modified varicella-zoster virus gE protein of the present application is obtained by cysteine mutation modification and structure design optimization, compared with the recombinant varicella-zoster virus gE protein with only carboxy-terminal hydrophobic transmembrane region and intracellular tail removed, has improved expression, thermal stability and immunogenicity.

Description

Technical Field

[0001] This application belongs to the field of biomedical engineering, and specifically relates to a stabilized modified varicella-zoster virus gE protein, its preparation method and application. Background Technology

[0002] Herpes zoster (HZ) is an acute infectious skin disease caused by the reactivation of the varicella-zoster virus (VZV). It is characterized by clusters of blisters distributed along nerve pathways and severe pain. Initial VZV infection manifests as chickenpox. The virus then remains latent in the dorsal root ganglia of the spinal cord or cranial nerve ganglia. When the body's immunity declines, the virus reactivates and spreads along sensory nerve axons to the skin, causing herpes zoster. Postherpetic neuralgia (PHN) is the most common complication, lasting for months or even years, severely impacting the patient's quality of life, especially in elderly patients.

[0003] Currently, the most effective way to prevent shingles is vaccination, especially recombinant subunit vaccines. GSK's recombinant shingles vaccine, Shingrix, was approved by the US FDA in 2017 and launched in China in 2020. This vaccine contains VZV gE protein and the AS01B adjuvant system, and is used to prevent HZ and PHN in adults aged 50 and older.

[0004] VZV gE protein is the most abundant glycoprotein on the viral envelope, containing 623 amino acid residues. It plays a key role in viral replication, intercellular transmission, and immune evasion, and is also the main antigen that induces a protective immune response in the host. However, wild-type gE protein has the following technical defects in recombinant expression systems (such as CHO cells): (1) The expression level of wild-type gE protein in CHO cells is usually low, which is difficult to meet the needs of large-scale vaccine production; (2) gE protein has a large structural flexibility. When used as a vaccine antigen, its extracellular domain is prone to conformational changes and proteolytic degradation during purification and storage, especially in the gI binding interface and Fc receptor functional domain, which are highly temperature sensitive, resulting in large batch-to-batch differences in immunogenicity and poor long-term stability; (3) The melting temperature (Tm) of wild-type gE protein is about 58°C, which requires strict temperature control during the production process and increases production costs. The emergence of recombinant subunit vaccines has greatly improved the stability of gE protein, but it has also sacrificed the protein expression level. The two have never achieved a perfect balance.

[0005] Disulfide bonds are crucial covalent bonds that stabilize the three-dimensional structure of proteins and are widely present in secretory and membrane proteins. Native gE proteins contain three conserved disulfide bonds (C387-C413, C396-C405, C432-C442), which are essential for maintaining their structure. However, the introduction of non-natural disulfide bonds at specific sites through protein engineering to comprehensively enhance the stability and expression levels of recombinant gE proteins has not been reported in this field. More importantly, existing techniques have not demonstrated that the introduction of non-natural disulfide bonds may simultaneously improve the immunogenicity of the protein.

[0006] Therefore, there is an urgent need in this field to develop a stable modified gE protein with high expression levels, good stability, and enhanced immunogenicity to meet the needs of vaccine production. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a stabilized modified varicella-zoster virus gE protein. By introducing four specially designed non-natural disulfide bonds, not only is the protein expression level and thermal stability significantly improved, but its immunogenicity is also unexpectedly enhanced.

[0008] The technical solution of the present invention is as follows: The present invention provides a stabilized modified varicella-zoster virus gE protein, the amino acid sequence of which is shown in SEQ ID NO: 29, wherein positions 153, 257, 165, 221, 359, 474, 367 and 456 of the protein are cysteine ​​residues.

[0009] In some embodiments, the protein is mutated at position 153 (threonine) to cysteine, position 257 (lysine) to cysteine, position 165 (arginine) to cysteine, position 221 (arginine) to cysteine, position 367 (leucine) to cysteine, position 456 (valine) to cysteine, position 359 (proline) to cysteine, and position 474 (isoleucine) to cysteine.

[0010] In some embodiments, the stabilized varicella-zoster virus gE protein forms four pairs of non-natural disulfide bonds at positions 153 and 257, 165 and 221, 359 and 474, and 367 and 456.

[0011] In some embodiments, the four pairs of non-natural disulfide bonds are C153-C257, C165-C221, C359-C474, and C367-C456, respectively.

[0012] In some embodiments, C153-C257 and C165-C221 are located in the gI domain, and C359-C474 and C367-C456 are located in the Fc domain.

[0013] The gI domain (gIBD) is located at approximately positions 116 to 305 of the amino acid residues in the wild-type gE protein, and the Fc domain (FcBD) is located at approximately positions 306 to 516 of the amino acid residues in the wild-type gE protein.

[0014] The specific amino acid sequence of the wild-type herpes zoster virus gE protein described in this invention is the amino acid sequence of glycoprotein E of varicella-zoster virus strain Oka, which has GenBank accession number AAK19946.1 (SEQ ID NO:14).

[0015] The gI domain (gIBD) and Fc domain (FcBD) described in this invention can also be located in the corresponding parts of other wild-type herpes zoster virus gE proteins.

[0016] The present invention also provides an isolated nucleic acid molecule that encodes the stabilized modified varicella-zoster virus gE protein described in any of the preceding claims.

[0017] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule and an expression regulatory sequence operatively linked thereto.

[0018] The present invention also provides a host cell comprising the expression vector described above, or whose genome integrates the nucleic acid molecules described above.

[0019] In some embodiments, the host cell is selected from CHO cells, HEK293 cells, yeast cells, or insect cells.

[0020] In some embodiments, the host cell is a CHO cell.

[0021] The present invention also provides a method for preparing the stabilized modified varicella-zoster virus gE protein, comprising: S1: Synthesize the DNA sequence corresponding to the protein and clone it into an expression vector; S2: Transfect host cells with the expression vector and express the expression. S3: The culture product is purified to obtain the stabilized modified varicella-zoster virus gE protein.

[0022] In some embodiments, the expression vector described in preparation method S1 is pCHO3.1.

[0023] In some embodiments, the host cell in the preparation method is a CHO cell.

[0024] The present invention also provides an immunogenic composition comprising the stabilized modified varicella-zoster virus gE protein described above.

[0025] The present invention also provides a vaccine comprising: (i) an immunologically effective amount of the stabilized modified varicella-zoster virus gE protein; and (ii) Pharmaceutically acceptable adjuvants.

[0026] In some embodiments, the adjuvant comprises a combination of monophospholipid A and QS-21.

[0027] In some embodiments, the adjuvant comprises MPL, QS-21, DOPC, and cholesterol.

[0028] In some specific embodiments, the adjuvant per human dose (HD, 0.5 mL) comprises 50 μg MPL, 50 μg QS-21, 1 mg DOPC, 0.25 mg cholesterol, 4.385 mg NaCl, 0.54 mg KH2PO4, 0.15 mg anhydrous Na2HPO4, and water for injection.

[0029] In some implementations, the vaccine is used to prevent shingles in adults aged 50 and older.

[0030] The present invention also provides the use of the stabilized modified varicella-zoster virus gE protein in the preparation of medicaments for the prevention or treatment of shingles.

[0031] The technical solution of this invention is based on the following design principles: When wild-type gE protein is used as a vaccine antigen, its extracellular domain is prone to conformational changes and proteolytic degradation during storage, especially at the gI binding interface and the Fc receptor functional domain, exhibiting high temperature sensitivity, leading to significant batch-to-batch variations in immunogenicity and poor long-term stability. The gI domain, located in the N-terminal extracellular domain of the gE protein, is the functional region that specifically binds to the VZV gI glycoprotein to form a heterodimer. This domain contains multiple α-helices and β-sheets, forming a compact structure with a hydrophobic core. It non-covalently binds to the gI protein extracellular domain through hydrophobic interactions and hydrogen bonds, forming a stable gE-gI complex. The gE-gI interaction is crucial for viral membrane fusion activity, but this interface is prone to dissociation under physicochemical stress (such as repeated freeze-thaw cycles and oxidative environments). This domain also contains multiple flexible loop regions, which are susceptible to local conformational fluctuations in solution. Furthermore, multiple protease-sensitive sites exist within this region, leading to truncated fragments during recombinant expression. Therefore, introducing disulfide bonds into the gI domain can (1) lock the three-dimensional conformation of the gI binding interface and prevent the gE-gI complex from dissociating; (2) enhance the rigidity of the local secondary structure and reduce conformational entropy change; and (3) protect the protease-sensitive sites and improve protein integrity.

[0032] The Fc domain is located at the C-terminus of the extracellular domain of the gE protein. It is the functional region of the gE protein as an Fc receptor (FcR) that binds to the IgG Fc segment. It has the following structural features: (1) IgG binding groove: a hydrophobic pocket formed by β-sheets that specifically recognizes the CH2-CH3 hinge region of the IgG Fc segment; (2) pH-sensitive conformation switch: this domain has unique pH-dependent conformational change characteristics (binding at pH 7.4 on the cell surface and releasing at pH 6.0 in the endosomal body); (3) flexible hinge region: the linker region connecting the gI domain and the Fc domain has high flexibility.

[0033] The Fc domain is the core functional area of ​​gE protein in mediating antibody-dependent enhancement of infection (ADE) and immune escape: First, it is conformation-dependent, with Fc binding activity strictly dependent on the maintenance of a specific three-dimensional conformation, which cannot be recovered after denaturation; second, it is heat-sensitive, as the domain contains multiple β-sheets, which are prone to irreversible aggregation under thermal stress (Tagg is usually 5-10°C lower than Tm); it is also oxidation-sensitive, as the domain contains multiple free thiol groups, which easily form intermolecular disulfide bonds leading to aggregation. Therefore, introducing disulfide bonds into the Fc domain can (1) stabilize the conformation of the IgG binding pocket and maintain Fc receptor activity; (2) improve the thermal stability of the domain; and (3) lock the pairing mode of the natural disulfide bonds, preventing aggregation caused by incorrect oxidation.

[0034] However, the gI and Fc domains do not exist independently in the gE protein. Instead, they work together to maintain the overall stability of the protein through long-range interactions. The formation of the gE-gI complex helps the correct folding of the Fc domain, while the conformational stability of the Fc domain in turn improves the ligand binding ability of the gI domain. The two domains form a structural stability cooperating unit through a hydrophobic core and hydrogen bond network.

[0035] Based on the above-mentioned domain characteristics, the stabilization modification of this invention specifically introduces four pairs of non-natural disulfide bonds into the gI and Fc domains of the gE protein when selecting the disulfide bond introduction sites, thereby achieving synergistic stabilization of the two domains through the following strategy: (1) Two pairs of non-natural disulfide bonds C153-C257 and C165-C221 are introduced into the gI domain. C153-C257 spans the β-sheet and C-terminal α-helix region in the middle of the gI domain, locking the core framework of the gI domain, restricting the relative movement between the β-sheet and α-helix, and preventing conformational fluctuations at the gI binding interface. C165-C221 is located between the flexible loop regions of the gI domain (aa 160-170 and aa 215-225), bridging the two flexible loop regions, enhancing the rigidity of the local secondary structure, protecting the protease-sensitive sites in the region, and avoiding direct interference with the gI protein binding interface (distance from the binding interface > 15 Å).

[0036] (2) Two pairs of non-natural disulfide bonds C359-C474 and C367-C456 are introduced into the Fc domain. C359-C474 is located outside the N-terminal β-sheet and C-terminal IgG binding groove of the Fc domain, spanning two functional subdomains of the Fc domain. It stabilizes the peripheral skeleton of the IgG binding pocket, maintains the structural integrity of the pH-sensitive conformational switch (around His334 and His431), and enhances the overall thermal stability. C367-C456 is located between the β-sheet and α-helical region in the middle and rear part of the Fc domain. The medium-long distance disulfide bond (spatial distance of about 12 Å) bridges the middle and rear parts of the Fc domain with moderate tension conformation. It works with C359-C474 to form a "front-back" dual-node support network to prevent thermally induced interdomain dissociation and conformational drift.

[0037] Four pairs of non-natural disulfide bonds form a two-node stabilization network in the gI and Fc domains. C153-C257 and C165-C221 of the gI domain synergistically lock the conformation of the N-terminal extracellular domain, ensuring the stability of the gE-gI complex assembly interface. C359-C474 and C367-C456 of the Fc domain synergistically maintain the three-dimensional structure of the C-terminal Fc receptor functional domain, preserving IgG binding activity. The two pairs of domains are connected by a natural peptide backbone, forming a rigid rod-like structure that significantly reduces overall conformational entropy.

[0038] The beneficial effects of this invention are as follows: The stabilization modification provided by this invention ensures that all non-natural disulfide bonds are located within the skeletal support regions of each domain, maintaining a safe distance (closest distance >12 Å) from key functional interfaces (gI binding site, IgG binding groove), thus guaranteeing that the stabilization modification does not interfere with the protein's natural biological function. The Cα-Cα distances of the four disulfide bonds are distributed in the range of 5.5-12 Å, conforming to the geometric characteristics of natural disulfide bonds and avoiding structural strain caused by excessive tightness (<4 Å) or conformational flexibility caused by excessive looseness (>15 Å). The introduction of two disulfide bonds into both the gI and Fc domains achieves N-terminal-C-terminal balance and stability, preventing stress concentration between domains due to excessive rigidity of a single domain. Furthermore, the selected sites are all located within the protein surface's accessible region, facilitating efficient oxidative folding during recombinant expression and improving the scalability of the preparation process.

[0039] Regarding thermal stability, UNCLE high-throughput protein stability analysis showed that the melting temperature (Tm) of the modified gE protein increased from 58.8°C in the wild type to 77.7°C, an increase of 18.9°C. This improvement allows for more relaxed temperature control in manufacturing processes, enabling the exploration of more flexible formulation conditions (such as liquid formulations, higher storage temperatures), and reducing reliance on the cold chain.

[0040] Regarding protein expression levels, the modified gE protein of this invention overcomes the technical deficiency of wild-type gE protein, which is characterized by "high expression but extreme instability." By introducing four pairs of non-natural disulfide bonds, the residual rate at 55°C is increased from 16% (WT) to 80%, achieving a qualitative leap in process stability and product uniformity at the cost of a moderate reduction in basal expression (4°C). Moreover, under extreme heat stress conditions (75°C), the expression stability of the modified gE protein of this invention surpasses that of the existing optimal construct Des-13 disclosed in GSK's patent, achieving a breakthrough improvement in thermal stability. This indicates that the disulfide bond design of this technical solution is more advantageous than the modification strategy of Des-13 in protecting the core protein structure. The basal expression level-first strategy (45.19 mg / L) used by Des-13 is effective under mild conditions, but loses 79% at 75°C; this technical solution adopts a stability-first strategy, losing only 73% at 75°C, retaining more functional protein.

[0041] Regarding protein availability and product quality, under the same purification process, BCA detection results showed that the gE protein modified by this invention had a high purification yield, and SEC-HPLC detection results showed that the purity of the gE protein modified by this invention was comparable to that of Des-13. This result indicates that the gE protein modified by this invention maintains structural integrity during purification (especially in chromatography steps involving room temperature operation), reduces activity loss, and is beneficial for achieving scalability of the preparation process and improving the quality of vaccine products.

[0042] Regarding immunogenicity, animal immunization experiments showed that, under the same antigen dose and adjuvant conditions, the immune response induced by the modified gE protein of this invention was stronger than that of the construct Des-13 (containing cysteine ​​substitutions A216C, I251C, I158C, G254C, H427C, K434C, T365C, and R477C) disclosed by GSK in WO2023223255A1, which has the best thermostability and in vivo immunogenicity, as well as GSK's marketed recombinant shingles vaccine Shingrix. This is an unexpected positive technical effect that could not have been anticipated during the design and validation of the technical solution. Attached Figure Description

[0043] Figure 1 This is a schematic diagram of the linear structure of the extracellular region of the wild-type gE protein, with the gI and Fc domains highlighted.

[0044] Figure 2 The results are SDS-PAGE of the purified protein in Example 4 of this invention. 7# represents gI1220F24, 11# represents gI920F24, 13# represents Des-13, and 14# represents wild-type WT.

[0045] Figure 3 The results show the antigen-specific IgG antibody titers induced by each group of protein samples in Example 6 of this invention.

[0046] Figure 4 The results show the expression of gE-specific CD4⁺ T cell cytokines induced by each group of protein samples in Example 6 of this invention. Detailed Implementation

[0047] The present invention will be further illustrated below by means of non-limiting embodiments. Those skilled in the art will recognize that many modifications can be made to the present invention without departing from its spirit, and such modifications also fall within the scope of the present invention. The following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention, as implementations are inevitably diverse. Whether or not a purification tag or N-terminal signal peptide is present in the embodiments does not affect the scope of protection. Non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the content of this application still fall within the scope of protection of this application. The terminology used in this specification is only for illustrating specific implementations and is not intended as limitation; the scope of the present invention is defined in the appended claims.

[0048] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Preferred methods and materials of the invention are described below; however, any methods and materials similar to or equivalent to those described in this specification may be used to practice or test the invention. Unless otherwise specified, the experimental methods described below are conventional methods or methods described in product manuals, and the experimental materials used are readily available from commercial companies.

[0049] Example 1: Design and Gene Synthesis of gE Proteins with Single-Domain Monostabilization Modification Based on the gE sequence of VZV Oka strain (GenBank accession number AAK19946.1), its C-terminus was truncated, and the hydrophobic transmembrane region and intracellular tail (starting from amino acid 547) at the C-terminus were removed to obtain the herpes zoster virus gE protein sequence without any stabilization modifications (Shingrix control group, amino acid sequence as shown in SEQ ID NO:31). Then, according to Table 1, different cysteine ​​substitutions were introduced into the gI domain (gI in the table) or Fc domain (gF in the table) through site-directed mutagenesis to obtain the amino acid sequences of each construct with a pair of non-natural disulfide bonds added to a single domain. In addition to the introduced mutations, these proteins also contain an IgG signal peptide (MGWSCIILFLVATATGVHS) added to the N-terminus, and purified tags His Tag (HHHHHHHH) and Strep-tag®II (WSHPQFEK) added to the C-terminus. In addition, the construct Des-13 (containing cysteine ​​substitutions for A216C, I251C, I158C, G254C, H427C, K434C, T365C, and R477C, amino acid sequence as shown in SEQ ID NO:30), constructed according to GSK's WO2023223255A1, which exhibits the best thermostability and in vivo immunogenicity, served as a positive control. The amino acid sequence was determined by codon optimization according to host CHO cells for gene synthesis, which was performed by a commercial gene synthesis service company. The sequence was verified to be correct by Sanger sequencing.

[0050] Table 1 Single-domain single disulfide bond design

[0051] The synthesized target gene was inserted into the eukaryotic plasmid pCHO3.1 vector for transfection and expression. Each recombinant plasmid was transformed into *E. coli* DH5α competent cells. Glycerol-containing bacteria were then transferred to 150 mL LB liquid medium (Amp final concentration 100 μg / mL) and cultured overnight at 37°C and 2000 rpm. Plasmids were extracted using an endotoxin-free plasmid extraction kit. The plasmid concentration of the extract was determined, and enzyme digestion was performed for identification. The specific reaction system is shown in Table 2.

[0052] Table 2 Enzyme digestion reaction system

[0053] After plasmid amplification, the concentration was measured, and each plasmid achieved a high amplification concentration, approximately 600-900 μg / ml, with a total amount of about 1.0 μg. Enzyme digestion results were detected by 1% agarose gel electrophoresis, showing clear bands at the corresponding positions. Sequencing of all plasmids confirmed that the target gene sequences of all plasmids were completely correct. The recombinant plasmid vector carrying the target gene has been successfully transferred into host cells and successfully amplified.

[0054] Example 2: Analysis of expression level and thermal stability of gE protein with single-domain monostable modification The obtained plasmid was introduced into CHO cells via transient transfection, and the protein supernatant was collected after culture. ExpiCHO-S cells (Thermo) were cultured in EmCD CHO-S 203 (Eminence / L20301) medium supplemented with 6 mM L-glutamine (sigma / G5146) one day in advance, seeded in 96-well plates at a density of 0.25E5 cells / well (supplemented with 10% FBS), and incubated statically at 37°C and 8% CO2. On the day of transfection, the cell-seeded 96-well plates were replaced with fresh EmCD CHO-S 203 medium containing L-glutamine. A transfection incubation system (50% of the transfection volume) was prepared using EmCD CHO-S 203 medium containing L-glutamine, with DNA at a concentration of 1 μg / ml and a PEI (Polysciences / 24765-1) to DNA ratio of 3. After mixing the PEI and DNA and incubating for 5 min, the mixture was added to 96-well plates containing cells. The culture was then incubated at 37°C with 8% CO2. 24 h after transfection, the temperature was lowered to 32°C, and AdvancedFeed1 (sigma / 24368-1L) was added at a 5% (v / v) feed ratio. The supernatant was collected on day 7 post-transfection after incubation.

[0055] After incubating the protein supernatant at 4℃ and 55℃ for 1 h, respectively, a double-antibody sandwich ELISA method was used. Rabbit polyclonal antibody 1701-36B3 was used as the coating antibody, and 9H10-HRP was used as the labeling antibody for detection pairing. The expression levels of each protein sample were measured, and the thermostability was analyzed. Expression levels were expressed as protein concentration, and thermostability was expressed as the ratio of activity concentration at 55℃ / 4℃. To evaluate the comprehensive effect of monostabilization modification on the expression and stability of recombinant proteins, this study established a quantitative scoring model consisting of three weighted components, as shown in the formula: S = 2 × E 4℃ +E 55℃ +R thermo,55℃ , E 4℃ The soluble expression level, representing the expression level under low temperature (4°C) conditions, was assigned a weighting factor of 2 to serve as the primary screening indicator. E 55℃ Represents the residual expression level after high-temperature (55°C) treatment; R thermo,55℃ To characterize the thermal stability under 55℃ treatment, the activity concentration ratio was determined. Mutation designs for gI1, gI5, and gI7, referencing WO2023223255A1, were used as a positive control group. The final S value was obtained by adding the S values ​​of the two batches. 总 Based on the scoring results in Table 3-1, three groups, gI20, gI12, and gI9, were selected for further research.

[0056] Table 3-1 Results of protein expression levels and thermostability assays for gI domain mutant designs

[0057] The expression levels and thermostability of the Fc domain mutant protein are shown in Table 3-2. Similarly, the S values ​​of the two batches were calculated using the quantitative scoring model formula. 总 Of the values, gF5 has the lowest rating.

[0058] Table 3-2 Results of protein expression levels and thermostability assays for Fc domain mutant designs

[0059] Example 3: Design, gene synthesis, expression level, and stability analysis of gE proteins with single-domain multi-stabilization modifications. Based on the screening results of single-domain stabilization modifications in Example 2, different cysteine ​​substitution combinations were introduced into the gI or Fc domains through site-directed mutagenesis according to Table 4, resulting in amino acid sequences for each construct with two pairs of non-natural disulfide bonds added to a single domain. In addition to the introduced mutations, these proteins also contain an IgG signal peptide (MGWSCIILFLVATATGVHS) added to the N-terminus, and purified tags His Tag (HHHHHHHH) and Strep-tag®II (WSHPQFEK) added to the C-terminus. The amino acid sequences were determined by codon optimization in host CHO cells for gene synthesis, which was performed by a commercial gene synthesis service company. The sequences were verified as correct by Sanger sequencing.

[0060] Table 4. Single-domain multi-disulfide bond design

[0061] A recombinant plasmid vector carrying the target gene was constructed according to Example 1. The obtained plasmid was introduced into CHO cells by transient transfection according to Example 2. After culture, the protein supernatant was collected. The collected supernatant was incubated at 4℃, 55℃, 65℃ and 75℃ for 1 h and then used for expression level detection, verification of the proportion of functionally correct folded proteins and thermal stability analysis.

[0062] A double-antibody sandwich ELISA method was used to quantify the function of each sample based on conformation-sensitive antibodies (identifying only correctly folded proteins). The total protein content of each sample (including correctly folded, partially folded, and aggregated proteins) was determined using the BCA (Bicinchoninic Acid) method. The specific activity of proteins was characterized by the ratio of ELISA concentration (mg / L) to BCA concentration (mg / L), reflecting the proportion of functionally correctly folded proteins in the protein sample.

[0063] Elevated temperature can cause protein denaturation / aggregation, leading to disruption of protein conformational epitopes, which manifests as decreased ELISA signal and reduced specific activity. Therefore, specific activity retention rate (specific activity retention rate = high-temperature specific activity / 4℃ control specific activity × 100%) can be used to characterize the thermal stability of protein samples. To assess the comprehensive impact of mutations on recombinant protein expression and stability, this study established a quantitative scoring model, the formula of which is: S = 2 × E 4℃ +E 55℃ +E 65℃ +E 75℃ +R thermo,55℃ + R thermo,65℃+R thermo,75℃ For an explanation of each term in the formula, please refer to Example 2.

[0064] In the study of Fc domain mutation design, gF5, which had the lowest score in Example 2, was used as the negative control group, and F3, which had the best stability reproducibility among the remaining groups, was also considered. Table 5 shows that gF3, gF23, and gF24 all had high scores and warrant further investigation.

[0065] Table 5. Results of protein expression levels and thermostability assays for Fc domain mutation designs.

[0066] In the study of the gI domain mutation design, gI720 and gI19 were used as control groups (obtained by combining gI1 and gI7, which were disclosed in WO2023223255A1, with gI9 and gI20, which were selected from Example 2 and had higher scores). In this section, gI12, which had the best thermal stability reproducibility, was further investigated. Table 6 shows that gI920, gI12, gI720, and gI1220 had high overall scores and are suitable for further research.

[0067] Table 6. Results of thermostability assay for protein expression levels designed with gI domain mutations.

[0068] The above results lead to the following conclusions: Regarding protein expression levels, introducing two pairs of non-natural disulfide bonds into a single domain significantly increases protein expression compared to introducing only one pair in a single domain as in Example 2. Furthermore, the stabilizing modification of the gI domain contributes more positively to expression levels than the stabilizing modification of the gF domain. Among the gF series proteins, gF24 shows particularly strong expression levels. Combining the results of Examples 2 and 3, it was found that although introducing one pair of disulfide bonds into a single domain significantly enhances protein thermostability compared to the wild type, protein expression levels are not significantly increased. Introducing two pairs of disulfide bonds into a single domain, while significantly increasing expression levels, only maintains thermostability at a low to medium level. These results suggest that combining multiple stabilizing modifications across multiple domains may be a promising direction for improving the expression levels and thermostability of gE protein.

[0069] Example 4: Design, expression level, and stability analysis of gE proteins with multiple structural domains and multiple stabilization modifications. For example, different cysteine ​​substitution combinations were introduced into the gI and Fc domains through site-directed mutagenesis, as shown in Table 7, to obtain amino acid sequences for each construct with at least two pairs of non-natural disulfide bonds added to multiple domains. In addition to the introduced mutations, these proteins also include an IgG signal peptide (MGWSCIILFLVATATGVHS) added to the N-terminus, and purified tags His Tag (HHHHHHHH) and Strep-tag®II (WSHPQFEK) added to the C-terminus. The amino acid sequences were determined by codon optimization in host CHO cells for gene synthesis, which was performed by a commercial gene synthesis service company, and the sequences were verified to be correct by Sanger sequencing.

[0070] Table 7 Multi-domain multi-disulfide bond design

[0071] A recombinant plasmid vector carrying the target gene was constructed according to Example 1. The obtained plasmid was introduced into CHO cells by transient transfection according to Example 2. After culturing, the protein supernatant was collected and incubated at 4℃, 55℃, 65℃ and 75℃ for 1 h, respectively, for protein expression determination, verification of the proportion of functionally correct folded proteins and thermal stability analysis.

[0072] A double-sandwich antibody ELISA method was used to perform functional quantification of each sample based on conformation-sensitive antibodies (identifying only correctly folded proteins). The thermostability of the protein samples was characterized by (correct conformation retention rate = high-temperature protein concentration / 4℃ protein concentration × 100%). To assess the comprehensive impact of mutations on the expression and stability of recombinant proteins, a quantitative scoring model was established, with the formula: S = 2 × E 4℃ +E 55℃ +E 65℃ +E 75℃ +R thermo,55℃ +R thermo,65℃ +R thermo,75℃ The definitions of each term in the formula are provided in Example 2. For ease of analysis and evaluation, the expression levels and thermostability detection results are presented in the following table, grouped into F23, F24, and F3 with reordered names. The candidate proteins with higher scores all come from group F24, especially the F24gI920 design in this group, which has the highest score. The results are shown in Table 8.

[0073] Table 8. Protein expression levels and thermostability at different temperatures.

[0074] Meanwhile, the results showed that not all gE proteins with multiple stabilization modifications introduced into multiple domains could simultaneously improve expression levels and thermostability. gI1220F24 and gI920F24 performed well in both expression levels and thermostability. Specifically, gI1220F24, after treatment at 4℃ or 55℃, showed comparable expression levels and stability to Des-13, but after treatment at 65℃, it significantly outperformed Des-13. Particularly unexpectedly, after treatment at 75℃, its expression level (20.26 mg / L) and stability were significantly better than Des-13. gI920F24, on the other hand, maintained superior expression levels and thermostability compared to Des-13 at all treatment temperatures.

[0075] Based on the ELISA detection results, the inventors further prepared large quantities of gI1220F24 and gI920F24 proteins, purified them using nickel affinity chromatography and size exclusion chromatography (SEC), and determined the purity using the BCA method. The results are shown in Table 9. SDS-PAGE results are also shown. Figure 2 As shown, the electrophoresis results indicate that the molecular weight of the stabilized modified herpes zoster virus gE protein gI920F24 monomer provided by this invention is similar to that of Des-13 and WT, while the molecular weight of gI1220F24 is smaller.

[0076] Table 9. Results of protein purification yield and purity testing

[0077] Table 9 shows that the stabilized modified herpes zoster virus gE protein gI920F24 provided by this invention has a higher purification yield, with purity comparable to Des-13, and elution time similar to Des-13 and WT. However, gI1220F24 does not appear to be the optimal choice for large-scale preparation.

[0078] Example 5: UNCLE High-Throughput Protein Stability Analysis UNCLE (Unchained Labs) is an integrated protein stability analysis platform that combines three detection technologies: full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS), enabling the simultaneous acquisition of multiple stability parameters in a single experiment. The inventors performed joint detection of melting temperature (Tm) and aggregation initiation temperature (Tagg) according to the following steps: The purified protein was diluted to 0.5–2 mg / mL with formulation buffer, and 9 μL / sample was added to a dedicated Uni quartz cuvette. The instrument was set to the "Tm&Tagg" application, with the temperature set to 25–95 °C, the heating rate 0.3–1 °C / min, and the equilibration time 0–180 s. Full-spectrum fluorescence and static light scattering (SLS) signals were acquired simultaneously, and the complete fluorescence spectrum (290–400 nm) was recorded at each temperature point. The fluorescence data were analyzed using the Barycentric Mean (BCM) method, and the Tm value (reflecting protein conformational stability; a higher Tm indicates greater stability) was calculated. The detection results are shown in Table 10. The Tm of the wild-type gE protein was 58.8 °C. The stabilized herpes zoster virus gE protein of this invention provided superior thermal stability compared to Des-13 (Tm of 77.7 °C), resulting in an 18.9 °C increase in Tm compared to the wild type.

[0079] Table 10 Results of UNCLE High-Throughput Protein Stability Assay

[0080] Example 6 Immunological Effect Test Using 6-8 week old C57BL / 6N mice as an animal model, stable modified herpes zoster virus gE proteins gI920F24, gI12F24, Des-13, and Shingrix recombinant gE protein (i.e., gE-PPQ group) purified in Example 5 were used as antigens. Each human dose contained 50 μg of recombinant gE protein, combined with liposome adjuvant for immunization. The adjuvant formulation was: 0.5 mL per human dose containing 50 μg MPL, 50 μg QS-21, 4.385 mg sodium chloride, 1 mg DOPC, 0.54 mg potassium dihydrogen phosphate, 0.25 mg cholesterol, and 0.15 mg anhydrous disodium phosphate. Five mice were immunized with the recombinant herpes zoster vaccine (1 / 10 human dose) via intramuscular injection on days 0 and 28, and samples were collected on day 42 for immunological marker detection.

[0081] The titer of gE-specific IgG antibodies in the serum of immunized mice was detected by ELISA, and the results are as follows: Figure 3 As shown, after two immunizations, high geometric mean titers (GMT) of gE-specific IgG antibodies were detected in the serum of mice in all groups. The levels in the gI920F24 group and the Des-13 group were comparable, and both were higher than those in the Shingrix group. This indicates that the stabilized and modified herpes zoster virus gE protein provided by this invention can effectively enhance antigen-specific humoral immune responses and is superior to GSK's marketed recombinant herpes zoster vaccine, Shingrix.

[0082] The expression of cytokines in gE-specific CD4⁺ T cells was detected using intracellular cytokine staining. The results are as follows: Figure 4 As shown, the stabilized and modified herpes zoster virus gE protein provided by the present invention can effectively induce antigen-specific cellular immune responses, promote the secretion of Th1 cytokines, and generate a protective immune response that combines humoral and cellular immunity. Its overall effect is superior to Des-13 and GSK's marketed recombinant herpes zoster vaccine Shingrix.

[0083] Through analysis of the results of the embodiments, the inventors found that the stabilized modified herpes zoster virus gE protein provided by the present invention and the prior art Des-13 (GSK WO2023223255A1) both adopt a stabilization strategy of 4 pairs of non-natural disulfide bonds, but there are essential differences between the two in the selection of disulfide bond sites and spatial distribution, which leads to different temperature-dependent characteristics of thermal stability.

[0084] In the gI domain (gIBD) region, Des-13 employs a "C-terminal anchoring + central crosslinking" strategy: its cross-boundary disulfide bonds I158C-G254C anchor the N-terminal leader region to the gIBD C-terminus (position 254), while the internal disulfide bonds A216C-I251C are located in the rear center of the gIBD. This invention, however, employs an "N-terminal anchoring + core crosslinking" strategy: the anchoring starting point of the cross-boundary disulfide bonds C153-C257 is moved forward (153 vs 158), and the internal disulfide bonds C165-C221 are located in the N-terminal core of the gIBD, adjacent to the gIBD initiation boundary (116), directly covering the functional core region of the gE-gI interaction (positions 178-206).

[0085] In the Fc structural domain (FcBD) region, Des-13 employs a "uniform coverage + rear-end local reinforcement" strategy: short-range disulfide bonds C427-C434 are located in the middle and rear part of the FcBD. This technical solution, however, adopts a "front-back dual-node support" strategy: medium- and long-range disulfide bonds C367-C456 are located in the middle and rear part of the FcBD, shifted forward by approximately 60 aa compared to C427-C434 in Des-13, and closer to the functional core region of the FcBD (positions 322-469). This medium bond, together with the long-range disulfide bonds C359-C474, forms a "front-back" dual-node support network, collaboratively stabilizing the full-length conformation from the N-terminus to the C-terminus of the FcBD, preventing thermally induced interdomain dissociation and conformational drift.

[0086] The aforementioned site differences result in different stabilization centers: Des-13 exhibits "backward-tilting" stabilization (gIBDC end + FcBD back end), while this invention employs "dual-domain core-enhanced" stabilization (gIBD N-terminal core + FcBD full-length coverage). This difference is directly reflected in stability performance under heating conditions: Des-13 retains 75% (33.83 mg / L) and 47% (21.38 mg / L) at 55℃ and 65℃, respectively, while this technical solution retains 80% (48.63 mg / L) and 48% (29.37 mg / L) at 55℃ and 65℃, respectively, achieving improved stability under heating processes.

[0087] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A stabilized and modified varicella-zoster virus gE protein, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO: 29, wherein positions 153, 257, 165, 221, 359, 474, 367 and 456 of the protein are cysteine ​​residues.

2. The stabilized and modified varicella-zoster virus gE protein as described in claim 1, characterized in that, The protein forms four pairs of non-natural disulfide bonds at positions 153 and 257, 165 and 221, 359 and 474, and 367 and 456.

3. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the stabilized modified varicella-zoster virus gE protein as described in claim 1 or 2.

4. An expression carrier, characterized in that, It comprises the nucleic acid molecule of claim 3, and a guide expression regulatory sequence operatively linked thereto.

5. A host cell, characterized in that, It includes the expression vector of claim 4, or the nucleic acid molecule of claim 3 integrated into the genome.

6. The host cell as described in claim 5, characterized in that, The host cells are selected from CHO cells, HEK293 cells, yeast cells, or insect cells.

7. A method for preparing the stabilized modified varicella-zoster virus gE protein according to claim 1 or 2, comprising: S1: Synthesize the DNA sequence corresponding to the protein and clone it into an expression vector; S2: Transfect host cells with the expression vector and express the expression. S3: The culture product is purified to obtain the stabilized modified varicella-zoster virus gE protein.

8. The preparation method according to claim 7, characterized in that, The expression vector described in S1 is pCHO3.

1.

9. The preparation method according to claim 7, characterized in that, The host cell is a CHO cell.

10. An immunogenic composition, characterized in that, Includes the stabilized modified varicella-zoster virus gE protein as described in claim 1 or 2.

11. A vaccine, characterized in that, Include: (i) an immunologically effective amount of the stabilized modified varicella-zoster virus gE protein as described in claim 1 or 2; and (ii) Pharmaceutically acceptable adjuvants.

12. The vaccine as described in claim 11, characterized in that, The adjuvant comprises a combination of monophospholipid A and QS-21.

13. The vaccine as described in claim 11, characterized in that, The vaccine is intended to prevent shingles in adults aged 50 and older.

14. Use of the stabilized modified varicella-zoster virus gE protein according to claim 1 or 2 in the preparation of a medicament for the prevention or treatment of shingles.

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