Recombinant glycoprotein tripolymer vaccine for preventing SFTS and application of recombinant glycoprotein tripolymer vaccine

Abstract

The invention provides a recombinant glycoprotein trimer vaccine for preventing SFTS and application thereof, the vaccine comprises a recombinant Gn or Gc glycoprotein trimer, and the recombinant Gn or Gc glycoprotein trimer is composed of a Gn or Gc protein fragment and a corresponding trimer motif; the amino acid sequences of the Gn and Gc protein fragments are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2, and the motif of the trimer is T4, DMPK, MTQ, MTI, hCorla, Langerin, T3XV, MATN1 or CAT. Results show that the neutralizing antibody level induced by the Gn or Gc trimer is superior to that of a corresponding monomer or dimer, and the Gn or Gc trimer has higher cross-neutralization activity. Meanwhile, monomers and trimers of Gn or Gc can induce high-level Th1 and Th2 cytokines and antibody-dependent cytotoxic effect (ADCC) response. According to the invention, a dominant antigen structure which can more effectively stimulate the generation of the neutralizing antibody in the SFTSV is defined, and a new target spot and a new strategy are provided for enhancing immunogenicity in the research and development of the SFTSV vaccine.

Description

Technical Field

[0001] This invention belongs to the field of vaccine preparation technology, and specifically relates to a recombinant glycoprotein trimer vaccine for the prevention of fever with thrombocytopenia syndrome (SFTS) and its application. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a tick-borne infectious disease caused by the severe fever with thrombocytopenia syndrome virus (SFTSV, also known as Dabie Bandar virus). Its clinical symptoms include high fever, thrombocytopenia, and multiple organ failure, with a mortality rate as high as 6%-30%. SFTSV has spread widely around the world, posing a serious threat to public health security. However, there are currently no approved preventive vaccines or specific treatments, so there is an urgent need to develop highly effective vaccine candidates.

[0003] The SFTSV genome contains three segments: L, M, and S. The M segment encodes the glycoproteins Gn and Gc, which are key antigens for inducing host immune responses. Existing research indicates that Gn and Gc can induce neutralizing antibodies and cellular immunity through DNA vaccines, viral vector vaccines, and mRNA vaccines, with some vaccines showing protective efficacy in animal experiments. For example, DNA vaccines based on Gn or Gc can enable immunized animals to survive viral challenge, and mRNA vaccines have also been shown to induce immune responses against SFTSV. However, these studies have significant limitations: on the one hand, existing vaccines mostly focus on monomeric forms of Gn or Gc, lacking systematic research on the immunogenicity of their naturally occurring multimeric conformations (such as dimers and trimers); on the other hand, the expression of recombinant Gn and Gc proteins faces problems such as structural instability and low expression levels, severely restricting their development and application as protein vaccines.

[0004] Studies have found that the Gn and Gc of SFTSV undergo complex conformational changes during viral invasion of host cells: Gn can form monomers or dimers, forming heterodimers with Gc and further assembling into higher-order pentamers or hexamers; Gc, under acidic conditions, transforms from monomers to trimers to mediate membrane fusion. These conformational changes may directly affect the exposure of antigenic epitopes, thereby regulating the strength and breadth of the immune response. However, current technologies have not yet determined which conformation (monomer, dimer, or trimer) of Gn and Gc has optimal immunogenicity, nor have they established a stable expression system for highly immunogenic conformations. For example, previous studies on recombinant protein vaccines against SFTSV showed that, among all groups in the study, mice challenged with combined immunization of monomeric Gc and Np had the highest survival rate (85.7%), but the evaluation of other conformations was not addressed, and further improvements in antigen immunogenicity are needed; some studies have attempted to express recombinant glycoproteins, but due to structural instability or low expression efficiency, they have failed to meet the needs of vaccine development.

[0005] Furthermore, SFTSV has multiple genotypes, and whether the neutralizing antibodies induced by existing vaccines can cross-recognize different strains is a key issue restricting their application. Currently, broad-spectrum protective vaccines against different SFTSV genotypes have not been reported, and there is a lack of in-depth analysis of the relationship between cross-neutralization ability and antigen conformation.

[0006] Therefore, in response to the technical bottlenecks in SFTSV vaccine development, such as "unclear optimal antigen conformation", "low recombinant protein expression efficiency and structural instability" and "insufficient cross-protection ability", developing glycoprotein vaccines based on highly immunogenic conformations and establishing a stable and efficient preparation system have become urgent technical problems to be solved in this field. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to construct protein vaccines with different structural conformations (monomers, dimers, and trimers of Gn or Gc), screen for antigenic forms with high immunogenicity, and evaluate the humoral and cellular immune responses induced by these forms in mice. This provides a recombinant SFTSV glycoprotein trimer vaccine, its preparation method, and its application, offering new targets and strategies for enhancing immunogenicity in SFTSV vaccine development.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] The first aspect of the present invention provides a recombinant glycoprotein trimer vaccine for the prevention of fever with thrombocytopenia syndrome, the vaccine comprising a recombinant Gn or Gc glycoprotein trimer, said recombinant Gn or Gc glycoprotein trimer being composed of a Gn or Gc protein fragment and a corresponding trimer motif;

[0010] The amino acid sequence of the Gn protein fragment is shown in SEQ ID NO:1:

[0011] DSGPIICAGPIHSNKSAGIPHLLGYSEKICQIDRLIHVSSWLRNHSQFQGYVGQR

[0012] GGRSQVSYYPAENSYSRWSGLLSPCDADWLGMLVVKKAKESDMIVPGPSYK

[0013] GKVFFERPTFDGYVGWGCGSGKSRTESGELCSSDSGTTSSGLLPSDRVLWIGDV

[0014] ACQPMTPIPEETFLELKSFSQSEFPDICKIDGIVFNQCEGESLPQPFDVAWMDV

[0015] GHSHKIIMREHKTKWVQESSSKDFVCYKEGTGPCSESEEKACKTSGSCRGDM

[0016] QFCKVAGCEHGEEASEAKCRCSLVHKPGEVVVSYGGTRVRPKCYGFSRMMA

[0017] TLEVNPPEQRIGQCTGCHLECINGGVRLITLTSELRSATVCASHFCSSASSGKKS

[0018] TEIHFHSGSLVGKTAIHVKGALVDGTEFTFEGSCMFPDGCDAVDCTFCREFLKNPQCYPAKK;

[0019] The amino acid sequence of the Gc protein fragment is shown in SEQ ID NO:2:

[0020] CDEMVHADSKLVSCRQGSGNMKECITTGRALLPAVNPGQEACLHFTAPGSPD

[0021] SKCLKIKVKRINLKCKKSSSYFVPDARSRCTSVRRCRWAGDCQSGCPPHFTSN

[0022] SFSDDWAGKMDRAGLGFSGCSDGCGGAACGCFNAAPSCIFWRKWVENPHGI

[0023] IWKVSPCAAWVPSAVIELTMPSGEVRTFHPMSGIPTQVFKGVSVTYLGSDMEV

[0024] SGLTDLCEIEELKSKKLALAPCNQAGMGVVGKVGEIQCSSEESARTIKKDGCI

[0025] WNADLVGIELRVDDAVCYSKITSVEAVANYSAIPTTIGGLRFERSHDSQGKISGSPLDITAIRGSFSVNYRGLRLSLS;

[0026] The trimer motif is T4, DMPK, MTQ, MTI, hCorla, Langerin, T3XV, MATN1, or CAT; preferably, the Gn protein corresponds to MATN1, and the Gc protein corresponds to CAT.

[0027] Furthermore, the recombinant Gn and Gc glycoprotein trimer also includes a signal peptide and a linker sequence, wherein the signal peptide is TPA, IL2Co2, or Igh epsilon-1;

[0028] The amino acid sequence of TPA is shown in SEQ ID NO:3: MDAMKRGLCCVLLLCGAVFVSP; the amino acid sequence of IL2Co2 is shown in SEQ ID NO:4: MRRMQLLLLIALSLALVTNS;

[0029] The amino acid sequence of Igh epsilon-1 is shown in SEQ ID NO:5: MDWTWILFLVAAATRVHSASS;

[0030] The amino acid sequences of the linker are shown in SEQ ID NO:6 and SEQ ID NO:7: GGGGSGGGGS; AGGGGSGGGGSGGGSA(lin3).

[0031] Preferably, the Gn protein corresponds to the signal peptide TPA, and the Gc protein corresponds to the signal peptide IL2Co2.

[0032] The second aspect of the present invention provides a gene encoding the recombinant Gn or Gc glycoprotein trimer described in the first aspect.

[0033] A third aspect of the present invention provides a method for preparing recombinant Gn or Gc glycoprotein trimers, comprising the following steps:

[0034] (1) Insert the gene described in the second aspect into the expression vector to obtain a recombinant expression vector;

[0035] (2) Transfect the recombinant expression vector into cells, and collect the supernatant by centrifugation 5-10 days after transfection;

[0036] (3) The recombinant Gn and Gc glycoprotein trimer was obtained by purifying the collected supernatant.

[0037] Furthermore, when preparing recombinant Gn glycoprotein trimers, in step (1), the expression vector is an optimized pCDNA3.1(+) or pCGS3.2 vector, the optimization including the introduction of the signal peptide TPA as shown in SEQ ID NO:3 and the linker sequence as shown in SEQ ID NO:6.

[0038] Furthermore, when preparing recombinant Gc glycoprotein trimer, in step (1), the expression vector is an optimized pCMN014 vector, the optimization including the introduction of a signal peptide and a linker sequence, the signal peptide being IL2Co2 or Igh epsilon-1, the amino acid sequences being as shown in SEQ ID NO:4 and SEQ ID NO:5 respectively; the amino acid sequence of the linker sequence being as shown in SEQ ID NO:6 or SEQ ID NO:7.

[0039] Furthermore, in step (2), the cells are HEK293FT cells, and PEI transfection reagent is used.

[0040] Furthermore, in step (3), the recombinant Gn or Gc glycoprotein trimer is purified by HisTrap HP histidine tag protein column.

[0041] The fourth aspect of the present invention provides a recombinant Gn or Gc glycoprotein trimer prepared by the method described in the third aspect.

[0042] The fifth aspect of this invention provides the use of the recombinant glycoprotein trimer vaccine of the first aspect, the gene of the second aspect, or the recombinant Gn or Gc glycoprotein trimer of the fourth aspect in the preparation of a medicament for the prevention and / or treatment of SFTSV infection.

[0043] The sixth aspect of the present invention provides the use of component A and component B in the preparation of a vaccine for the prevention and / or treatment of SFTSV, wherein component A is the recombinant glycoprotein trimer vaccine described in the first aspect, the gene described in the second aspect, or the recombinant Gn or Gc glycoprotein trimer described in the fourth aspect; and component B is a vaccine adjuvant.

[0044] Based on the conformational characteristics and immunogenic differences of SFTSV glycoproteins Gn or Gc, this invention has achieved the following beneficial effects by constructing and screening recombinant protein vaccines with different structures, especially by optimizing the expression system of Gn and Gc trimers:

[0045] 1. Clearly define the conformation of highly immunogenic antigens and significantly increase the level of neutralizing antibodies.

[0046] This invention is the first to systematically compare the immunogenicity of Gn and Gc monomers, dimers, and trimers, finding that the neutralizing antibody induced by the Gn trimer (Gn-MATN1) was significantly higher than that induced by the Gn dimer, with an average value higher than that induced by the Gn monomer (Gn-MATN1). Figure 4 Similarly, the neutralizing antibody titers induced by Gc trimer (Gc-CAT) were significantly higher than those induced by Gc dimer, with an average higher titer than those induced by Gc monomer (Gc-CAT). Figure 4(H). When immunized with trimeric glycoprotein combined with aluminum hydroxide + CPG adjuvant, and using the HB29 strain pseudovirus homologous to the vaccine for detection of neutralizing antibodies, the neutralizing antibody titer induced by Gn trimer reached 21057, and the neutralizing antibody titer induced by Gc trimer reached 52688. Figure 6 (A). This result, to some extent, addresses the problem of insufficient immunogenicity of SFTSV vaccines in existing technologies, providing a key target for efficiently inducing protective antibodies.

[0047] 2. Achieve broad-spectrum cross-protection, covering multiple genotypes of SFTSV strains.

[0048] The neutralizing antibodies induced by the Gn trimer vaccine of this invention can cross-recognize four different genotypes of SFTSV (HB29, GangWon, HN13, SD4), while the Gc trimer vaccine can cross-recognize six different genotypes of SFTSV (HB29, GangWon, HN13, HN20, SPL030A, SD4). Figure 5 In contrast, existing vaccines are mostly limited to the protection evaluation of a single strain. This characteristic enables them to address the genetic diversity of SFTSV, significantly enhancing the practical application value of vaccines.

[0049] 3. Optimize the recombinant protein expression system to improve stability and expression efficiency.

[0050] By screening for the trimer motif (MATN1), the stability of the Gn trimer was further improved, and it also exhibited the molecular weight of the trimer in the modified gel. Figure 1 (D). Immunogenicity studies showed that the stable Gn trimer conformation (Gn-MATN1) induced a higher average neutralizing antibody titer than other unstable conformations. Figure 4 (F).

[0051] By truncating the Gc protein (retaining amino acids 563-902) and selecting vectors, Gc monomers were successfully expressed. Based on this, Gc trimers were successfully expressed by screening for trimer motifs (such as CAT), signal peptides (such as IL2Co2), and linker sequences (such as (GGGGS)3). This invention solves the problems of non-expression and low yield of full-length Gc protein. The optimized Gc monomers can be stably stored at 4°C for 12 days and show no significant structural degradation within 7 days at 25°C, providing feasibility for the large-scale production, storage, and transportation of vaccines.

[0052] 4. Induces a comprehensive immune response, achieving complete in vivo protection.

[0053] The Gn and Gc trimeric vaccine of this invention not only induces high-titer neutralizing antibodies, but also stimulates potent Th1 / Th2 cellular immunity (such as a significant increase in IFN-γ secretory T cell levels) and ADCC activity. Figure 6 (C, F). In pseudovirus challenge experiments, the Gn trimer vaccine could clear most of the SFTSV pseudovirus, while the Gc trimer vaccine could completely clear the SFTSV pseudovirus in mice. Figure 6 (I, J). Compared with the corresponding monoclonal vaccines, it improves the protective efficiency in vivo.

[0054] 5. Provides a novel strategy for SFTSV vaccine development

[0055] This invention is the first to demonstrate that the trimer conformation is the dominant antigenic structure for SFTSV, and its mechanism may be related to conserved neutralizing epitopes in stable exposure. This discovery overcomes the limitations of existing understanding of the relationship between Gn / Gc conformation and immunogenicity, and provides important reference for the design of vaccines against other Bunyaviruses. Attached Figure Description

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0057] Figure 1 The diagram illustrates plasmid design and protein expression of glycoproteins. A represents plasmid design for Gn and Gc; B shows the designed protein structures and their theoretical molecular weights; C shows purified Gn monomers (Gn), dimers (Gn-Fc), and trimers (Gn-T4, Gn-MTQ, and Gn-MATN1) detected by denaturing or non-denaturing electrophoresis; D shows Gn trimers detected using different trimer motifs (including T3XV, MTQ, MTI, CAT, hCorla, MATN1, DMPK, Langerin, and T4) detected by denaturing electrophoresis; E shows purified Gc monomers (Gc), dimers (Gc-Fc), and trimers (Gc-CAT) detected by denaturing or non-denaturing electrophoresis; and FG shows modified Gc trimers detected in cell culture supernatants by denaturing electrophoresis. Note: Sp1, Sp2, Sp3, Sp4, and Sp5 represent the signal peptides IL-2, IL2Co1, IL2Co2, IL2Co3, and pflagCMV3, respectively; Lin1, Lin2, and Lin3 represent the linker sequences (AP)8A, A(EAAAK)3A, and A(GGGGS)3A, respectively; all electrophoresis assays were performed with Coomassie Brilliant Blue staining and image acquisition. Cell culture supernatant without plasmid transfection was used as a negative control.

[0058] Figure 2The images show the protein expression of glycoproteins; where A represents the expression supernatant containing Gn monomers (Gn), dimers (Gn-Fc), and trimers (Gn-T4) detected by denaturing or non-denaturing electrophoresis systems; BD represents successfully expressed (B)Gc-FC, (C)Gc, (D)Gc-CAT, and Gc-DMPK proteins; GcFrag1 represents amino acid residues 563-902, and GcFrag2 represents amino acid residues 563-800. Vec1 represents vector pCMN013, and Vec2 represents vector pCMN014. All electrophoresis assays were performed with Coomassie Brilliant Blue staining and image acquisition. Cell culture supernatant without plasmid transfection served as a negative control.

[0059] Figure 3 The results show the stability and specificity of recombinant Gn and Gc proteins. A shows the results of purified dimers (Gn-Fc and Gc-Fc) and trimers (Gn-MATN1 and Gc-CAT) after incubation at 4°C or 25°C for 0, 2, 4, 7, and 12 days, as detected by a non-denaturing electrophoresis system. B shows the results of purified monomers (Gn and Gc) after incubation at 4°C or 25°C for 0, 2, 4, 7, and 12 days, as detected by a denaturing electrophoresis system (all electrophoretic assays were performed with Coomassie brilliant blue staining and read). Image capture); C represents the specificity of Western blot detection of Gn monomer (Gn), Gn trimer (Gn-T4), Gc monomer (Gc), and Gc trimer (Gc-CAT); DF represents the specificity of ELISA detection of Gn monomer (Gn), Gn trimer (Gn-MATN1), and Gc monomer (Gc) (n≥6); CF uses serum from recovered SFTSV-infected individuals as the primary antibody, and the negative control (n=3) uses serum from uninfected SFTSV individuals as the primary antibody. Unpaired t-tests were used, and * indicates a significant difference compared to the negative group (p<0.05).

[0060] Figure 4The diagram shows the neutralizing antibodies induced by SFTSV glycoprotein; where AB represents the binding antibody titers and neutralizing antibody titers induced by Gn trimer (Gn-T4) in different mouse models detected by ELISA (A) and pseudovirus neutralization assay (B); CD represents the binding antibody titers and neutralizing antibody titers induced by Gn monomer (Gn), dimer (Gn-Fc), and trimer (Gn-T4) detected by ELISA (C) and pseudovirus neutralization assay (D); EF represents the neutralizing antibody titers induced by ELISA (E) and pseudovirus neutralization assay (F) in different mouse models. Gn or Gc trimeric motif-induced binding antibody titers and neutralizing antibody titers; GH was measured by ELISA (G) and pseudovirus neutralization assay (H) to detect Gn and Gc monomeric (Gn or Gc), dimeric (Gn-Fc or Gc-FC), or trimeric (Gn-MATN1 or Gc-CAT) binding antibody titers and neutralizing antibody titers. Balb / C mice (n=5 or n=6 per group) were immunized intramuscularly at weeks 0 and 4 with 25 μg (AF) or 10 μg (GH) of a protein vaccine containing aluminum hydroxide adjuvant. Mice injected with PBS served as the control group. Neutralizing antibody titers were detected using a universal pseudovirus containing a conserved SFTSV sequence. One-way ANOVA was used; * indicates a significant difference compared to the control group (p < 0.05); # indicates a significant difference compared to the Gn-T4 group (CD) or the Gc-CAT group (EH) (p < 0.05).

[0061] Figure 5 The results show the characteristics of neutralizing antibodies and the determination of immunization procedures; A is to evaluate the persistence of neutralizing antibodies in mice immunized with Gc protein vaccine; B is to evaluate the cross-recognition ability of neutralizing antibodies against 7 different SFTSV strains; C is to detect the titer of neutralizing antibodies in mice immunized with a mixture of Gn and Gc proteins; D is to detect the titer of neutralizing antibodies induced by Gn trimer (Gn-T4) after the third immunization using aluminum hydroxide, incomplete Freund's adjuvant, or aluminum hydroxide + CpG adjuvant; E is to investigate the results of the first and second immunizations. The effects of secondary immunization using aluminum hydroxide + CpG adjuvant were evaluated. Balb / C mice (n=5 or n=6 per group) were intramuscularly injected with 25 μg (A, D) or a total of 10 μg (B, C, and E) protein containing aluminum hydroxide adjuvant (AC) or aluminum hydroxide + CpG adjuvant (E) at weeks 0, 4 (AC, E) or 0 (A, E), 4 (A, E), or 12 (A, E, C, E, D) (A, E, A, C, or E). Mice injected with PBS served as the control group. Neutralizing antibodies in AD were detected using a pseudovirus containing a conserved SFTSV sequence, and neutralizing antibodies in E were detected using a pseudovirus of strain HB29. One-way ANOVA was used. * indicates a significant difference compared to the control group (p < 0.05); # indicates a significant difference compared to the Gc-CAT group (B) or the group shown in the figure (E) (p < 0.05).

[0062] Figure 6 The images show the T cell response and protection stimulated by Gn and Gc glycoproteins; where A represents the neutralizing antibody titer induced by monomers (Gn or Gc) or trimers (Gn-MATN1 or Gc-CAT); B represents the neutralizing antibody titer induced by Gn-MATN1 or Gc-CAT against SARS-CoV-2 or influenza virus pseudoviruses; C represents the ADCC activity assay after serum serial dilution; DF represents the T cells secreting IFN-γ, IL-2, or IL-4 after protein vaccine immunization as detected by ELISpot; GH represents the establishment of a bioluminescent imaging mouse challenge model based on the SFTSV pseudovirus system, with images acquired at 4, 6, 8, 10, 12, and 14 hours after pseudovirus challenge; IJ represents SFTSV pseudovirus challenge performed 2 weeks after secondary immunization, with bioluminescent imaging performed 6 hours after challenge. Balb / C mice (n=5 or n=8 per group) were intramuscularly injected with 10 μg of a protein vaccine containing aluminum hydroxide + CpG adjuvant at weeks 0 and 4. Mice injected with PBS served as the control group. Neutralizing antibody titers and pseudovirus challenge experiments were performed using SFTSV HB29 pseudovirus. One-way ANOVA was used. * indicates a significant difference compared to the control group (p < 0.05); # indicates a significant difference compared to the Gc-CAT group (A) or the groups shown in the figure (F and J) (p < 0.05). Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0065] Example 1: Construction, expression and identification of recombinant SFTSV Gn trimer protein

[0066] 1. Target gene and vector design

[0067] Using the M fragment of SFTSVHB29 strain (GenBank accession number: NC_018138) as a template, the coding sequence of the Gn protein (nucleotides 76-1374) was synthesized after CHO codon optimization, as shown in SEQ ID NO:8:

[0068] GACAGCGGCCCTATCATCTGCGCCGGCCCTATCCACAGCAACAAGAGCGCC

[0069] GGCATCCCTCACCTGCTGGGCTACAGCGAGAAGATCTGTCAGATCGACAGA

[0070] CTGATCCACGTGAGCAGCTGGCTGAGAAACCACAGCCAATTCCAAGGCTA

[0071] CGTCGGACAGAGAGGCGGCAGAAGCCAAGTGAGCTACTACCCTGCCGAG

[0072] AACAGCTACAGCAGATGGAGCGGACTGCTCAGCCCCTGCGACGCCGACTG

[0073] GCTGGGCATGCTGGTGGTGAAGAAGGCCAAGGAGAGCGACATGATCGTGC

[0074] CTGGCCCTAGCTACAAGGGCAAGGTGTTCTTCGAGAGACCTACCTTCGACG

[0075] GCTACGTGGGCTGGGGCTGCGGCTCCGGCAAGAGCAGAACCGAGAGCGG

[0076] CGAGCTGTGCAGCAGCGACAGCGGCACAAGCAGCGGCCTGCTGCCTTCCG

[0077] ACAGAGTGCTGTGGATCGGCGATGTCGCCTGTCAGCCTATGACCCCTATCC

[0078] CTGAGGAGACCTTCCTGGAGCTGAAGAGCTTCAGCCAAAGCGAGTTCCCT

[0079] GACATCTGCAAGATCGACGGCATCGTGTTCAATCAGTGCGAGGGCGAGAG

[0080] CCTGCCTCAGCCTTTCGACGTGGCCTGGATGGACGTGGGCCACAGCCACA

[0081] AGATCATCATGAGAGAGCACAAGACCAAGTGGGTGCAAGAGAGCAGCAG

[0082] CAAGGACTTCGTGTGCTACAAGGAGGGCACCGGCCCTTGCAGCGAGAGCG

[0083] AGGAGAAGGCCTGCAAGACAAGCGGCAGCTGCAGAGGCGACATGCAGTT

[0084] CTGCAAGGTGGCGGCTGCGAGCACGGCGAGGAGGCTAGCGAGGCCAAG

[0085] TGCAGATGCAGCCTGGTGCACAAGCCTGGCGAGGTGGTCGTGAGCTACGG

[0086] CGGCACAAGAGTGAGACCTAAGTGCTACGGCTTCAGCAGAATGATGGCCA

[0087] CCCTGGAGGTGAACCCTCCTGAGCAGAGAATCGGACAGTGCACCGGCTGC

[0088] CACCTGGAGTGCATCAACGGCGGCGTGAGACTGATCACCCTGACAAGCGA

[0089] GCTGAGAAGCGCCACCGTGTGCGCTAGCCACTTCTGCAGCAGCGCTAGCA

[0090] GCGGCAAGAAATCCACCGAGATCCACTTCCACAGCGGCAGCCTGGTGGGC

[0091] AAGACCGCCATCCACGTGAAGGGCGCCCTGGTGGACGGCACCGAGTTCAC

[0092] CTTCGAGGGCAGCTGCATGTTCCCTGACGGCGCCGACGCCGTGGACGCCACTTCGCTAGAGAGTTCCTGAAGAACCCTCAAGCCTACCCTGCCAAGAAG。

[0093] Using pCGS3.2 as the basic vector, the tPA signal peptide (SP: SEQ ID NO:3), His tag, and trimer motif (MATN1) were introduced to construct the recombinant expression plasmid Gn-MATN1 (see schematic diagram). Figure 1 (A). Among them, the nucleotide sequence of the trimer motif MATN1 was optimized based on known databases and tandemly linked with the Gn sequence via the linker sequence (GGGGS)2 (SEQ ID NO:6).

[0094] 2. Cell transfection and protein purification

[0095] The recombinant plasmid was transfected into HEK293FT cells using PEI transfection reagent (Polysciences). Cells were cultured at 37°C and 5% CO2 for 7 days post-transfection. On day 1 post-transfection, 5% Pro-feed was added, followed by 0.2% and 1% glucose on days 3 and 5, respectively. Cell supernatant was collected and transfected using a HisTrap HP nickel column (GE Healthcare) combined with AKTAstart. TM The protein was purified systematically, and the elution buffer was PBS containing 500 mM imidazole, finally yielding purified Gn-MATN1 trimer protein.

[0096] 3. Protein structure identification

[0097] (1) Denaturing PAGE analysis: 5 μg of purified protein was subjected to 10% SDS-PAGE. Coomassie brilliant blue staining showed a single band at approximately 165 kDa, consistent with the theoretical molecular weight (including glycosylation modification). Figure 1 (D).

[0098] (2) Non-denaturing PAGE analysis: The protein forms a higher-order hexamer in its native state, with a molecular weight of approximately 440 kDa. Figure 1 (C)

[0099] (3) Stability analysis: The Gn monomer conformation is stable. Figure 3 (B) showed no significant degradation at 4℃ for 7 days, but the trimer conformation degraded on the second day at 4℃. Figure 3 Therefore, the purified Gn-MATN1 trimer protein needs to be stored at -80℃.

[0100] Example 2: Construction, expression optimization, and identification of recombinant SFTSV Gc trimer protein

[0101] 1. Target gene truncation and vector optimization

[0102] Since the full-length Gc protein is not expressed, its head domain was truncated, retaining amino acids 563-902 (GcFrag1: SEQ ID NO:1), which encodes nucleotides 1705-2727 of the M fragment. Using the modified pCMN014 vector, the signal peptide IL2Co2 (Sp3: SEQ ID NO:4), the linker sequence A (GGGGS)3A (lin3: SEQ ID NO:7), and the trimer motif CAT were introduced to construct the recombinant plasmid Gc-CAT (see schematic diagram). Figure 1 (A)

[0103] 2. Optimization of expression conditions

[0104] Comparing the effects of different signal peptides (Sp1-Sp5) and linkers (Lin1-Lin3) on expression levels, the results showed that the Sp3+lin3 combination could increase Gc-CAT expression by approximately 2-fold. Figure 1 (F, G).

[0105] The transfection and purification methods were the same as in Example 1, and the Gc-CAT trimer protein with a purity of >90% was finally obtained.

[0106] In this sequence, Sp1 represents the signal peptide IL-2, with the sequence MYRMQLLSCIALSLALVTNS; Sp2 represents the signal peptide IL2Co1, with the sequence MRMQLLLLIALSLALVTNS; Sp3 represents the signal peptide IL2Co2, with the sequence MRRMQLLLLIALSLALVTNS; Sp4 represents the signal peptide IL2Co3, with the sequence MRRKKMQLLLLIALSLALVTNS; and Sp5 represents the signal peptide pflagCMV3, with the sequence MSALLILALVGAAVA.

[0107] Lin1, Lin2, and Lin3 represent the connection sequences (AP)8A, A(EAAAK)3A, and A(GGGGS)3A, respectively.

[0108] 3. Structural verification

[0109] (1) Non-denaturing PAGE showed that Gc-CAT formed higher-order oligomers. Figure 1 (E);

[0110] (2) Antigen-specific detection

[0111] Gc monomer or trimer (Gc-CAT) proteins were collected and their specific reaction with serum from SFTSV convalescent patients was detected by Western blot and ELISA.

[0112] Western blot results ( Figure 3(C): Gc and Gc-CAT showed specific bands at 41kDa and 440kDa, respectively, indicating that they are indeed Gc glycoproteins of SFTSV.

[0113] ELISA test ( Figure 3 (E): When the Gc sequence was bound to the patient's serum, its OD value (450nm) was positive, which was significantly higher than that of the negative control (p<0.05), confirming the specificity of its antigen sequence.

[0114] The above results demonstrate the antigen specificity of the Gc-CAT trimer protein.

[0115] Example 3: Expression optimization and fragment screening of recombinant SFTSV Gn / Gc protein

[0116] To address the issue of non-expression of full-length Gc proteins, this embodiment focuses on fragment truncation and expression system screening for Gc proteins.

[0117] 1. Gc protein fragment design and vector screening

[0118] Based on the Gc protein sequence of SFTSVHB29 strain, two truncated fragments were designed: GcFrag1 (amino acids 563-902) and GcFrag2 (amino acid sequence 563-800 as shown in SEQ ID NO:9: CDEMVHADSKLVSCRQGSGNMKECITTGRALLPAVNPGQEACLHFTAPGSPDSKCLKIKVKRINLKCKKSSSYFVPDARSRCTSVRRCRWAGDCQSGCPPHFTSNSFSDDWAGKMDRAGLGFSGCSDGCGGAACGCFNAAPSCIFWRKWVENPHGIIWKVSPCAAWVPSAVIELTMPSGEVRTFHPMSGIPTQVFKGVSVTYLGSDMEVSGLTDLCEIEELKSKKLALAPCNQAGMGV), corresponding to nucleotides 1705-2727 and 1705-2418 of the M fragment, respectively.

[0119] Two modified vectors, pCW1151 and Vec1 (pCMN013), and Vec2 (pCMN014), were used to construct monoclonal, dimeric (Fc tag), and trimer (CAT / DMPK motif) expression plasmids for GcFrag1 and GcFrag2, respectively. Figure 2 (BD).

[0120] 2. Expression Validation and Result Analysis

[0121] After transfecting HEK293FT cells, the cell supernatant was analyzed by denaturing PAGE. Figure 2 (Chinese BD):

[0122] GcFrag1 expression efficiency was significantly higher in Vec2 (pCMN014) than in Vec1. Based on this, by adding a trimer motif and screening nine trimer motifs, it was found that using the CAT or DMPK trimer motif successfully expressed the GcFrag1 fragment trimer. Figure 2 (C, D)

[0123] Comparison of Gn protein expression supernatant ( Figure 2 (A) Gn-T4 trimer is prone to depolymerization into monomers under denaturing conditions, while Gn-Fc dimer can be stably expressed.

[0124] Example 4: Comparison of immunogenicity of Gn / Gc proteins with different conformations

[0125] This embodiment compares the immunogenicity of proteins with different conformations, specifically including:

[0126] 1. Experimental grouping and immunization procedure:

[0127] Five 6-8 week old Balb / C mice per group were selected and administered the following vaccines intramuscularly (with aluminum hydroxide as adjuvant):

[0128] Control group: PBS;

[0129] Gn monomer group: 10 μg Gn protein;

[0130] Gn dimer group: 10 μg Gn-Fc (Fc tag-mediated dimerization);

[0131] Gn trimer group: 10 μg Gn-MATN1;

[0132] Gc monomer group: 10 μg Gc protein;

[0133] Gc dimer group: 10 μg Gc-Fc;

[0134] Gc trimer group: 10 μg Gc-CAT.

[0135] The patients were immunized once at week 0 and once at week 4, and serum was collected two weeks after the second immunization.

[0136] 2. Antibody level detection

[0137] (1) ELISA detection of binding antibodies: After the second immunization, the antibody titers in each vaccine-immunized group were significantly increased, all higher than those in the control group (p<0.05). Figure 4 (G);

[0138] (2) Pseudovirus neutralization test: The neutralizing antibody titer (against the universal strain) in the Gc-CAT group reached 1:7200, which is 3.2 times that of the Gn-MATN1 group. Figure 4 (H).

[0139] (3) Cross-neutralization capacity assessment

[0140] The cross-neutralizing activity of Gn or Gc-induced antibodies against different SFTSV strains (HB29, C3 genotype; GangWon, C2 genotype; HN13, C4 genotype; HN20, J genotype; SPL030A, J genotype; SD4, C1 genotype) was tested. Neutralizing antibodies induced by all individuals in the Gn group showed positive cross-neutralizing effects against HB29 and GangWon strains, while neutralizing antibodies induced by Gn trimer (Gn-MATN1) were positive against HB29, GangWon, SD4, and HN13 strains. In contrast, Gc trimer (Gc-CAT) immunization significantly enhanced the neutralizing activity against all six wild-type viruses studied. Figure 5 (B)

[0141] Furthermore, four combined antigens were used to determine the immunization procedure for the glycoproteins. Regardless of whether monomeric or trimeric forms were used, the neutralizing antibody titer of mixed immunization with Gn and Gc was not significantly increased compared to immunization with Gc trimer alone. Figure 5 (C). A third immunization was administered to the Gn-T4 group, and the results showed that even with incomplete Freund's adjuvant or aluminum hydroxide + CpG adjuvant for the third immunization, two immunizations were sufficient to achieve a neutralizing antibody titer >7200 for universal pseudovirus detection containing the conserved SFTSV sequence. Figure 5 (D).

[0142] To enhance immunogenicity, both the initial and secondary immunizations used 10 μg of Gn-MATN1 protein combined with aluminum hydroxide and CpG adjuvant. The induced neutralizing antibodies were detected using HB29 pseudovirus. Results showed that the aluminum hydroxide + CpG group exhibited a significant increase in neutralizing antibodies immediately after the initial immunization, and this increase was higher than that in the aluminum hydroxide adjuvant group. Figure 5 (E).

[0143] The persistence and cross-reactivity of glycoprotein-induced neutralizing antibodies were investigated. Neutralizing antibodies induced by Gn or Gc trimers persisted for 4 months after secondary immunization without a significant decrease. Figure 4 This result is consistent with the finding that high antibody levels persisted for 5 months after Gc monomeric immunization. Figure 5 (A)

[0144] Example 5: Validation of the protective efficacy of Gn-MATN1 and Gc-CAT trimeric vaccines

[0145] 1. Immunization and Virus Challenge Program

[0146] The mouse immunization regimen was the same as in Example 4 (the dose was adjusted to 10 μg, and the adjuvant was aluminum hydroxide + CpG). Two weeks after the second immunization, SFTSV HB29 pseudovirus was injected intraperitoneally (400 μL of stock solution; the pseudovirus was used to titrate Huh7 cells; after 48 hours of undiluted titration, the fluorescence value detected by PerkinElmer microplate reader was 1 × 10⁻⁶). 7 RLU).

[0147] 2. Immune response detection

[0148] Cellular immunity: ELISpot showed that, compared with the control group, the number of T cells secreting IFN-γ, IL-2, and IL-4 induced by Gn-MATN1 and Gc-CAT groups was significantly increased after secondary immunization. Figure 6 (DF).

[0149] ADCC activity: Serum was serially diluted 5-fold starting at 1:70. After the first and second immunizations, ADCC activity induced by both Gn-MATN1 and Gc-CAT groups was significantly increased compared with the control group. Figure 6 (C)

[0150] 3. Results of virus attack protection

[0151] Bioluminescence imaging showed that 6 hours after challenge, all mice in the Gn monomer group had strong signals, while in the Gn-MATN1 group, only one mouse showed a small amount of fluorescence signal, and the other mice did not show any fluorescence signal. Mice in the Gc-CAT group showed no pseudovirus fluorescence signal (photon flux <1×10⁻⁶). 4 ()( Figure 6 (I, J). This indicates that the immunogenicity induced by Gn or Gc trimer vaccines is stronger than that induced by the corresponding monomers. In pseudovirus challenge experiments, the Gc trimer provides complete protection.

[0152] 4. Conclusion

[0153] Gn (amino acids 20-452) efficiently and stably expresses monomeric, dimeric, and trimer proteins in the eukaryotic cell expression vector pCGS3.2. GcFrag1 (amino acids 563-902) efficiently expresses stable trimer (Gc-CAT) and monomer (Gc) proteins in Vec2 (pCMN014), and stably expresses Gc dimer (Gc-Fc) in the vector pCW1151. This provides optimized protein fragments and vector systems for subsequent vaccine development.

[0154] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A recombinant glycoprotein trimer vaccine for preventing sepsis with thrombocytopenia syndrome, characterized by, The vaccine comprises a recombinant Gn or Gc glycoprotein trimer, which consists of a Gn or Gc protein fragment and a corresponding trimer motif; The amino acid sequences of the Gn and Gc protein fragments are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the trimer motif is T4, DMPK, MTQ, MTI, hCorla, Langerin, T3XV, MATN1 or CAT.

2. The recombinant glycoprotein trimer vaccine of claim 1, characterized in that, The recombinant Gn or Gc glycoprotein trimer further comprises a signal peptide and a linker sequence, the signal peptide is TPA, IL2Co2 or Igh epsilon-1, the amino acid sequences of the respective signal peptide are shown in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively; and the amino acid sequence of the linker sequence is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

3. A gene encoding the recombinant Gn or Gc glycoprotein trimer of claim 1 or 2.

4. A method of preparing a recombinant Gn or Gc glycoprotein trimer, characterized in that, The method comprises the following steps: (1) inserting the gene of claim 3 into an expression vector to obtain a recombinant expression vector; (2) transfecting the recombinant expression vector into cells, and collecting the supernatant by centrifugation after 5-10 days of transfection; (3) purifying the recombinant Gn or Gc glycoprotein trimer from the collected supernatant.

5. The method of claim 4, wherein, When preparing the recombinant Gn glycoprotein trimer, in step (1), the expression vector is an optimized pCDNA3.1(+) or pCGS3.2 vector, and the optimization comprises introducing the signal peptide TPA shown in SEQ ID NO: 3 and the linker sequence shown in SEQ ID NO:

6.

6. The method of claim 4, wherein, When preparing the recombinant Gc glycoprotein trimer, in step (1), the expression vector is an optimized pCMN014 vector, and the optimization comprises introducing a signal peptide and a linker sequence, the signal peptide is IL2Co2 or Igh epsilon-1, and the amino acid sequences are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively; and the amino acid sequence of the linker sequence is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

7. The method of claim 4, wherein, In step (3), the recombinant Gn or Gc glycoprotein trimer is purified by a HisTrap HP histidine tag protein column.

8. The recombinant Gn or Gc glycoprotein trimer prepared by the method of any one of claims 4-7.

9. Use of the recombinant glycoprotein trimer vaccine of claim 1 or 2, the gene of claim 3, or the recombinant Gn or Gc glycoprotein trimer of claim 8 in the preparation of a medicament for preventing and / or treating SFTSV infection.

10. Use of component A and component B for the preparation of a vaccine for the prevention and / or treatment of SFTSV, characterized in that, The component A is the recombinant glycoprotein trimer vaccine of claim 1 or 2, the gene of claim 3, or the recombinant Gn or Gc glycoprotein trimer of claim 8; and the component B is a vaccine adjuvant.

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