ZIKV nano vaccine taking Mi3 nano particles as core as well as construction method and application of ZIKV nano vaccine

Abstract

The invention relates to a ZIKV nano vaccine as well as a construction method and application thereof, the ZIKV nano vaccine is prepared by mixing an SC-Mi3 fusion protein and an EDIII-ST fusion protein, and carrying out self-assembly connection through a SpyTag connecting peptide in the SC-Mi3 fusion protein and a SpyCatcher connecting peptide in the EDIII-ST fusion protein, so as to obtain a nano particle vaccine with Mi3 as a core and ZIKV EDIII protein displayed on the surface. The SC-Mi3 fusion protein is formed by fusing a SpyCatcher connecting peptide into Mi3 nano particles, and the EDIII-ST fusion protein is formed by fusing a SpyTag connecting peptide into a ZIKV EDIII protein. The ZIKV nano vaccine can induce a strong specific neutralizing antibody aiming at ZIKV, can resist lethal attack of ZIKV and avoids ADE reaction.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to a method for constructing a novel nanoparticle vaccine that uses Mi3 as a carrier to display ZIKV EDIII protein on its surface. Background Technology

[0002] Zika virus (ZIKV), a mosquito-borne flavivirus, has become a major global public health threat due to its association with severe neurological complications such as microcephaly in newborns and Guillain-Barré syndrome in adults, necessitating the development of highly effective preventative vaccines. The antibody-dependent enhancement (ADE) effect between Zika and dengue is a significant challenge in vaccine development. Studies have shown that maternal ZIKV antibodies may enhance the infection efficiency of heterologous dengue virus (DENV) in newborn mouse models, leading to severe dengue fever phenotypes such as bleeding tendency and vascular leakage.

[0003] Pre-membrane protein (prM) and envelope protein (E) are the main structural components of ZIKV. They assemble into trimers on the surface of immature viral particles, and after furin cleavage of prM, infectious mature virus is formed. The E protein mediates host cell infection through its three domains (EDI, EDII, and EDIII). Currently, multiple vaccine candidates have been developed using various vaccine platforms targeting prM-E, ME, or E proteins. However, the fusion loop region of prM and E can induce cross-reactive antibodies, particularly subneutral and non-neutralizing antibodies, thereby promoting antibody-dependent enhancement (ADE). Notably, domain III (EDIII) of the E protein contains a ZIKV-specific neutralizing epitope with low sequence homology among flaviviruses. Using EDIII as an antigen can reduce or avoid the production of cross-binding antibodies while inducing neutralizing antibodies, making it a potential target for vaccine design. However, EDIII has weak immunogenicity and is difficult to elicit potent protective immunity. Self-assembled nanoparticle vaccine technology has achieved breakthroughs in the development of SARS-CoV-2 and EBV vaccines. However, self-assembled nanoparticle vaccines have high requirements for uniformity and are prone to agglomeration during preparation.

[0004] Although the number of reported ZIKV cases has decreased significantly globally, the increased transmission of DENV may indirectly enhance the potential epidemic risk of ZIKV through multiple mechanisms. Global warming is causing the suitable habitat of Aedes aegypti mosquitoes to expand northward, and it is estimated that 1 billion people will face the risk of DENV / ZIKV epidemics by 2050. The preliminary work conducted in this study to develop a ZIKV vaccine aims to provide a nanoparticle vaccine that densely displays the ZIKV EDIII protein, offering effective technical support for DENV / ZIKV prevention. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a novel nanoparticle vaccine with Mi3 as the core and high-density surface display of exogenous antigen ZIKVEDIII, as well as its construction method and application.

[0006] The first aspect of this invention is to provide a ZIKV nanovaccine, which is obtained by mixing SC-Mi3 fusion protein and EDIII-ST fusion protein, and then self-assembling and linking them through SpyCatcher linker peptide in SC-Mi3 fusion protein and SpyTag linker peptide in EDIII-ST fusion protein to obtain a nanoparticle vaccine with ZIKV EDIII protein displayed on its surface, with Mi3 nanoparticles as the core. The SC-Mi3 fusion protein is composed of Mi3 protein fused with SpyCatcher linker peptide, and the EDIII-ST fusion protein is composed of...

[0007] ZIKV EDIII protein is composed of a SpyTag linker peptide fused to it.

[0008] In some embodiments, the amino acid sequence of the SC-Mi3 fusion protein is SEQ ID NO:2.

[0009] In some embodiments, the amino acid sequence of the EDIII-ST fusion protein is SEQ ID NO:4.

[0010] In some embodiments, the amino acid sequence of the SpyCatcher linker peptide is SEQ ID NO:8; and / or the amino acid sequence of the SpyTag linker peptide is SEQ ID NO:7.

[0011] In some embodiments, the encoding nucleotide sequence corresponding to the SpyTag linker peptide is SEQ ID NO:5.

[0012] In some embodiments, the encoding nucleotide sequence of the SpyCatcher linker peptide is SEQ ID NO:6.

[0013] A second aspect of the present invention is to provide a method for constructing the ZIKV nanovaccine, comprising the following steps:

[0014] S1: Obtain SC-Mi3 fusion protein and EDIII-ST fusion protein;

[0015] S2: Mix the SC-Mi3 fusion protein and the EDIII-ST fusion protein and incubate at 3-5°C for 10-14 hours. Couple the two fusion proteins together through heteropeptide bonds between the SpyCatcher linker peptide and the SpyTag linker peptide to form the ZIKV nanovaccine Mi3-EDIII.

[0016] In some embodiments, obtaining the SC-Mi3 fusion protein and the EDIII-ST fusion protein includes the following steps: inserting the encoding nucleotide sequences of the SC-Mi3 fusion protein and the EDIII-ST fusion protein into prokaryotic expression vectors, respectively; then transforming the two prokaryotic expression vectors into prokaryotic host cells, respectively; and inducing expression with IPTG to obtain the SC-Mi3 fusion protein and the EDIII-ST fusion protein, respectively.

[0017] In some embodiments, the nucleotide sequence of the Mi3 protein in the SC-Mi3 fusion protein is SEQ ID NO:1, and / or the coding sequence of the SpyCatcher linker peptide is SEQ ID NO:6.

[0018] In some embodiments, the ZIKV EDIII protein coding sequence in the EDIII-ST fusion protein is SEQ ID NO:3, and the SpyTag linker peptide coding sequence is SEQ ID NO:5.

[0019] In some embodiments, the prokaryotic expression vector is a plasmid, more preferably the pET28a plasmid.

[0020] In some embodiments, the SC-Mi3 fusion protein and the EDIII-ST fusion protein in S2 are mixed in a molar ratio of 1:3.

[0021] The prokaryotic host cell is an Escherichia coli strain, and further, the prokaryotic host cell is...

[0022] BL21(DE3) competent cells.

[0023] A third aspect of the invention is to provide the use of the ZIKV nanovaccine in the preparation of medicaments for the prevention and / or treatment of Zika virus (ZIKV) infection.

[0024] This invention utilizes the SpyTag / SpyCatcher protein self-assembly system, which has high linkage efficiency and is easy to express. It can display the surface of self-assembled nanoparticles Mi3 with icosahedral structure of ZIKV EDIII antigen at high density, successfully enhancing the immunogenicity of EDIII. At the same time, the use of EDIII effectively avoids the risk of ADE in vaccines.

[0025] In this invention, EDIII was selected as the effective immunogen, and its immunogenicity was enhanced by the multivalent display of Mi3 nanoparticles, thus preparing a Mi3-EDIII nanoparticle vaccine. This vaccine was tested in female C57BL / 6 mice and mice lacking the interferon receptor (Ifnar1). - / -The immunogenicity and protective efficacy of the vaccine were evaluated in newborn pups of C57BL / 6 mice from the ZIKV model. Results showed that, compared to the monovalent EDIII antigen, multivalent presentation of Mi3 nanoparticles not only induced a superior humoral / cellular immune response but also completely protected against lethal doses of ZIKV challenge. Importantly, the vaccine did not induce an adverse drug reaction (ADE), demonstrating its potential as a safe and effective ZIKV vaccine candidate.

[0026] Therefore, coupling the ZIKV EDIII protein to Mi3 nanoparticles forms a high-density displayed ZIKV nanovaccine. This enhances the immunogenicity of EDIII and, by selecting the EDIII antigen, avoids the risk of adverse drug reactions (ADE) in the vaccine, providing a new approach for developing highly effective novel ZIKV nanoparticle vaccines. Attached Figure Description

[0027] Figure 1 A schematic diagram of the covalent bond between EDIII-ST and SC-Mi3.

[0028] Figure 2 SDS-PAGE analysis of individual components and assembled nanoparticles, wherein lane 1: purified SC-Mi3; lane 2: purified EDIII-ST; lane 3: reaction mixture of SC-Mi3 and excess EDIII-ST (molar ratio 1:3); lane 4: purified Mi3-EDIII.

[0029] Figure 3 Transmission electron micrographs (TEM) of SC-Mi3 and Mi3-EDIII, scale bar, 100 nm.

[0030] Figure 4 The antigenic profile of Mi3-EDIII, where A. binding activities of SC-Mi3, EDIII-ST, E404, and Mi3-EDIII with each monoclonal antibody; 96-well plates were plated with each antigen at the specified dilution and then incubated with each monoclonal antibody at a concentration of 1 μg / mL. OD450 values ​​are shown in the figure. From left to right: monoclonal antibodies 7B3, 8D10, and 8-4; data points represent mean ± SD (n = 2). Statistical differences were assessed using one-way ANOVA with Dunnett's multiple comparison test; B. Binding kinetics of monoclonal antibodies 7B3 and 8D10 with Mi3-EDIII, EDIII-ST, and E404 were analyzed by BLI; equilibrium dissociation constant (KD) was indicated for each experiment, and representative results from two independent replicates are shown; **P < 0.01; ns, no significant difference. Figure 5Immunogenicity of Mi3-EDIII in C57BL / 6 mice, where: A. Immunization timing settings. Six-week-old female mice were immunized intramuscularly with three doses (weeks 0, 3, and 6) of EDIII-ST, SC-Mi3, or Mi3-EDIII, each formulated with AddaVax adjuvant; serum samples were collected at weeks 2, 5, and 8. For CMI analysis, three mice from each group were randomly selected, and spleens and ILNs were harvested at week 8; B. Quantification of endpoint IgG titers against ZIKV EDIII in immune sera after the first, second, and third immunizations by ELISA. Each data point represents the mean of three technical replicas of a single mouse. Error bars represent mean ± SD (n = 5); (C, D) Neutralizing antibody titers two weeks after the second (C) or third (D) immunization, measured by FNT. Each data point represents the mean of three technical replicas of a single mouse. Error bars represent the mean ± SD (n = 5). (E, F) Frequency of IFNγ (E) and IL-4 (F) SFCs secreted by the spleen (left) and ILN (right). One representative well (left) is shown for each group. Error bars represent the mean ± SD (n = 3). Representative results of two independent replicates are shown. Statistical comparisons were performed using one-way ANOVA and Dunnett's multiple comparison test. ***P < 0.001; ****P < 0.0001; ns, no significant difference.

[0031] Figure 6 Immunogenicity and protective effect of Mi3-EDIII in Ifnar1- / -C57BL / 6 mice, (A) Immunization and challenge schedule; 10-week-old Ifnar1- / -C57BL / 6 mice were given three intramuscular injections of a certain dose of EDIII-ST, SC-Mi3 or Mi3-EDIII; serum samples were collected 2 weeks after each immunization, and mice were subcutaneously injected with 1×10⁻⁶ EDIII at week 9. 5FFU ZIKV; Mice were sacrificed 15 days after challenge. Unchallenged mice were designated HC; (B) Quantification of endpoint IgG titers against ZIKV EDIII in immune serum after the first, second, and third immunizations by ELISA; (C) Neutralizing antibody titers two weeks after the second or third immunization, measured by FNT; (D) Changes in body weight after ZIKV challenge, monitored daily until day 15; (E) Survival curves of challenged mice; (F) Quantification of ZIKV genomic RNA copy number in mouse serum collected on days 1, 4, and 7 by qRT-PCR; Each data point represents the mean of two (F) or three (B, C) technical replicas of a mouse; Error bars represent mean ± SD (n = 5); Comparisons were made by one-way ANOVA with Dunnett's multiple comparison test (B, C, F), unpaired two-tailed Student's test, t-test (D), or log-rank test (E). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, no significant difference.

[0032] Figure 7 The ADE effect of immune serum on ZIKV or DENV infection in K562 cells was investigated. Immune serum was collected two weeks after the third immunization. Mouse serum collected two weeks after infection with ZIKV (strain GZ02) was also analyzed in parallel. Heat-inactivated serum was serially diluted (5-fold), mixed with ZIKV or DENV 1–4, and added to K562 cells. Infection rate was measured by flow cytometry after 48 hours. The percentage of infected cells is shown. Data represent two independent experiments, each repeated.

[0033] Figure 8 Transmission electron micrographs of AP205 and Mi3.

[0034] Figure 9 The coupling ratio was explored using the pattern antigen SpyTag003-sfGFP.

[0035] Figure 10 Plasmid expression profiles of .6×His-SpyCatcher-Mi3 (SC-Mi3) and SpyTag-EDIII-6×His (EDIII-ST). Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0037] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0038] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0039] To facilitate understanding of this technology, some terms and phrases are defined below.

[0040] Mi3 nanoparticles are artificially designed nanoparticles that self-assemble from 60 subunits to form an icosahedral structure. They are designed based on the KDPG aldolase model. (Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination Theodora UJBruun, §Anne-Marie C. Andersson, §Simon J. Draper, and Mark Howarth*, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX13QU, United Kingdom Jenner Institute, University of Oxford, Oxford OX3 7DQ, United Kingdom).

[0041] SpyCatcher / SpyTag molecular glue is a newly discovered and modified protein linkage system in recent years. SpyCatcher and SpyTag can form a spontaneous, specific, and irreversible covalent bond. The irreversible nature of the covalent bond endows the complex with excellent in vivo stability, making it suitable for in vivo self-assembly or long-acting drug delivery systems. SpyTag short peptide genes can be fused to the C-terminus / N-terminus of any target protein. SpyCatcher acts as a "universal adapter" to link with another functional protein (such as virus-like particles or nanocarriers), enabling controllable assembly of multiple components. Core carriers linked to SpyCatcher (such as Mi3 nanoparticles) can simultaneously bind multiple SpyTag-labeled antigens, achieving ordered, high-density antigen display and enhancing immunogenicity.

[0042] In this article, ZIKV nanovaccine, also known as Mi3-EDIII, and Mi3-EDIII nanoparticles all refer to the same substance.

[0043] SC-Mi3 protein nucleotide sequence (SEQ ID NO:1)

[0044] TCAGTAACTACACTATCTGGGTTGAGCGGCGAACAAGGTCCGAGCGGCGACATGACTACTGAGGAAGACTCGGCAACCCATATCAAATTCAGCAAACGTGATGAGGACGGCCGTGAACTCGCTGGCGCCACGATGGAACTGCGTGATTCGAGCGGCAAAACGATCAGCACCTGGATTAGCGATGGACATGTTAAGGACTTCTACCTGTATCCGGGTAAATACACCTTTGTTGAGACAGCCGCGCCAGATGGTTATGAAGTGGCGACGCCTATTGAGTTCACCGTCAACGAAGACGGTCAGGTTACTGTAGATGGTGAGGCAACCGAGGGTGACGCACATACCGGTGGTTCCGGTGGCTCTGGCGGCTCCGGCGGCTCCATGAAAATGGAAGAATTATTCAAGAAACACAAAATCGTGGCTGTACTGCGCGCGAATAGCGTGGAGGAAGCTAAGAAAAAGGCGCTGGCGGTTTTTCTTGGTGGCGTGCATCTGATCGAGATTACCTTCACCGTGCCGGATGCGGATACCGTTATTAAAGAGTTGAGTTTTTTGAAGGAGATGGGTGCAATTATCGGTGCGGGTACGGTGACCAGCGTGGAACAGGCACGTAAAGCCGTGGAGAGCGGCGCGGAGTTTATTGTGAGCCCGCATCTGGACGAAGAGATCTCTCAGTTCGCCAAGGAGAAGGGTGTTTTTTACATGCCGGGCGTCATGACCCCGACGGAGTTGGTTAAGGCTATGAAGCTGGGTCACACCATCCTGAAACTGTTCCCGGGTGAGGTGGTGGGTCCGCAATTCGTGAAGGCCATGAAGGGCCCATTTCCGAATGTCAAATTCGTTCCGACCGGTGGTGTGAACCTGGACAACGTCTGTGAATGGTTTAAAGCGGGTGTTCTGGCGGTTGGTGTGGGCAGCGCGCTGGTTAAAGGCACCCCGGTTGAAGTTGCGGAAAAGGCTAAAGCGTTTGTCGAGAAGATCCGCGGGTGCACCGAA

[0045] Amino acid sequence of SC-Mi3 protein (SEQ ID NO:2)

[0046] SVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHTGGSGGSGGSGGSMKMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQARKAVESGAEFIVSPHLDEEISQFAKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE

[0047] Nucleotide sequence of EDIII-ST protein (SEQ ID NO:3)

[0048] ATGGGCCTAAGGTTAAAAGGAGTAAGTTATTCACTTTGCACCGCGGCATTTACCTTCACGAAAATCCCGGCTGAAACCCTGCACGGCACTGTCACCGTTGAGGTACAGTATGCCGGTACGGATGGTCCGTGTAAAGTTCCGGCGCAAATGGCGGTTGACATGCAGACCTTGACCCCGGTGGGTCGCCTGATTACTGCGAATCCGGTCATCACGGAAAGCACCGAGAACAGCAAAATGATGCTGGAACTCGATCCGCCTTTTGGTGATAGCTACATCGTGATCGGCGTGGGTGAGAAGAAGATTACCCATCATTGGCATCGTAGCGGCAGCACCATTGGTAAAGGTGGCTCCGGCGGCTCCGGCGGTTCTGGCCGCGGGGTGCCGCATATTGTGATGGTTGACGCTTACAAGCGTTATAAG

[0049] Amino acid sequence of EDIII-ST protein (SEQ ID NO:4)

[0050] MGLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKGGSGGSGGSGRGVPHIVMVDAYKRYK

[0051] SpyTag nucleotide sequence: (SEQ ID NO:5)

[0052] CGCGGGGTGCCGCATATTGTGATGGTTGACGCTTACAAGCGTTATAAG SpyCatcher nucleotide sequence: (SEQ ID NO:6)

[0053] ATGAAACTGGGCGAGATTGAATTTATTAAAGTGGACAAAACCGATAAAAAGCCGCTGCGTGGTGCCGTGTTTAGCCTGCAGAAACAGCATCCCGACTATCCCGATATCTATGGCGCGATTGATCAGAATGGGACCTATCAAGATGTGCGTACCGGCGAAGATGGTAAACTGACCTTTACGAATCTGAGCGATGGCAAATATCGCCTGATTGAAAATAGCGAACCCCCGGGCTATAAACCGGTGCAGAATAAGCCGATTGTGAGCTTTCGTATTGTGGATGGCGAAGTGCGTGATGTGACCAGTATTGTGCCGCAG

[0054] SpyTag amino acid sequence: (SEQ ID NO:7)

[0055] RGVPHIVMVDAYKRYK

[0056] SpyCatcher amino acid sequence: (SEQ ID NO:8)

[0057] MKLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGKLTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ

[0058] 6×His gene sequence: (SEQ ID NO:9)

[0059] CATCACCACCACCACCAC

[0060] 6×His amino acid sequence:(SEQ ID NO:10)

[0061] HHHHHH

[0062] The present invention will be further described in detail below with reference to specific embodiments.

[0063] The HEK293 cells used in the following procedures were purchased from the ATCC cell bank; pET28a and pET28a-MBP plasmids were purchased from Dalian Takara Bio Inc.; competent cells BL21(ED3) and DH5α were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0064] Example 1: Protein Production in Escherichia coli

[0065] The coding sequences (GenScript) for 6×His-SpyCatcher-Mi3 (SC-Mi3) or SpyTag-EDIII-6×His (EDIII-ST) were synthesized (the SpyCatcher linker peptide was inserted into the N-terminus of the Mi3 protein coding sequence and the SpyTag sequence was inserted into the C-terminus of the EDIII sequence), and then inserted into the pET28a plasmid. Figure 9SC-Mi3 was expressed as a soluble protein in *Escherichia coli* strain BL21. Expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) (420 μM) at an optical density (OD) of 0.6–0.8 and incubated at 22 °C and 220 rpm for 16 h. Cell particles were collected by centrifugation and resuspended in a buffer of pH 8.0 containing 25 mM Tris-HCl, 300 mM NaCl, and 1 mM benzoyl fluoride (PMSF), a protease inhibitor. Cells were then lysed using a high-pressure cell homogenizer and centrifuged at 4 °C and 12,000 rpm for 30 min. The supernatant was loaded onto a Ni affinity column, HisTrap HP 5 mL (Cytiva). The column was washed with a buffer of pH 8.0 containing 25 mM Tris-HCl, 300 mM NaCl, and 20 mM imidazole. The target protein was then eluted with a buffer containing pH 8.0, 20 mM Tris-HCl, 300 mM NaCl, and 300 mM imidazole. The eluted protein was purified by gel filtration (Superose 6-increase 10 / 300GL, Cytiva) and then equilibrated with a buffer containing pH 8.0, 20 mM Tris, and 150 mM NaCl. EDIII-ST was expressed as inclusion bodies in *E. coli* strains. Expression was induced with 1 mM IPTG at an OD value of 0.8–1.0 and incubated at 37°C and 220 rpm for 3–4 h. Cells were lysed and centrifuged, and the particles were dissolved in a buffer containing 8 M urea for denaturation and then centrifuged at high speed. The supernatant was diluted dropwise at 4°C to a stirred refolding buffer (100 mM Tris, pH 8.0, 400 mM L-Arg-HCl, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, and 5% glycerol) and incubated overnight. After refolding, the refolded protein was loaded onto a nickel affinity column. The eluted protein was purified by gel filtration (Superdex™ 75Increase 10 / 300GL, Cytiva) and then equilibrated with a buffer containing pH 8.0, 20 mM Tris, 150 mM NaCl, and 5% glycerol.

[0066] The plasmid expression maps of 6×His-SpyCatcher-Mi3 (SC-Mi3) and SpyTag-EDIII-6×His (EDIII-ST) are shown below. Figure 10 .

[0067] Example 2: Preparation and characterization of Mi3-EDIII nanoparticle vaccine.

[0068] Preparation of Mi3-EDIII nanoparticle vaccine: The EDIII-ST and SC-Mi3 fusion proteins obtained from prokaryotic expression were incubated overnight at 4°C. EDIII-ST and SC-Mi3 were effectively coupled through isopeptide bonds between SC and ST to form an icosahedron composed of 60 identical subunits, Mi3-EDIII ( Figure 1 The EDIII-ST fusion protein was expressed as inclusion bodies in *E. coli* strain BL21(DE3). After denaturation of the inclusion bodies, it successfully renatured in vitro. SC-Mi3 was expressed in a soluble form in the same *E. coli* strain. EDIII-ST and SC-Mi3 were further purified by nickel-nitrotriacetic acid (Ni-NTA) affinity chromatography and size exclusion chromatography (SEC). To achieve full loading of the EDIII-ST antigen onto SC-Mi3 nanoparticles, SC-Mi3 was conjugated to EDIII-ST at a molar ratio of 1:3 at 4 °C (incubated for 12 h). Then, Mi3-EDIII nanoparticles were purified and separated from the excess EDIII-ST by SEC. SDS-PAGE analysis showed a high conjugation efficiency between EDIII-ST and SC-Mi3. Figure 2 ).

[0069] Negative staining electron microscopy: A 300-mesh carbon-coated copper mesh treated with glow discharge was incubated for 30 s in a droplet containing purified nanoparticles (0.05 mg / mL). After washing twice with ultrapure water, the mesh was negatively stained with freshly prepared 2% (w / v) uranyl acetate for 1 min and then air-dried. Finally, the mesh was imaged using a 120 kV negative staining electron microscope on a FEI TEM Imaging and Analysis (TIA) system. Under the negative staining electron microscope, it was observed that the surface of the Mi3-EDIII nanoparticles was fully loaded with EDIII-ST antigen, and the particle uniformity was good. Figure 3 This indicates that Mi3-EDIII nanoparticles, namely the ZIKV nanovaccine, have been successfully constructed, hereinafter referred to as Mi3-EDIII.

[0070] In selecting nanoparticle carriers, this invention compared AP205 and Mi3, and observed the morphology of the formed nanoparticles using negative staining transmission electron microscopy. It was found that under the same conditions, Mi3 protein exhibited better uniformity. Figure 8 Therefore, Mi3 was chosen as the core particle for the nanovaccine.

[0071] In addition, we also used the model antigen SpyTag003-sfGFP to obtain the coupling ratio of EDIII-ST and EDIII antigens ( Figure 9By controlling the molar ratio of SC-Mi3 (i.e., the SC-Mi3 fusion protein) and ST-sfGFP (the fusion protein of fluorescent tag proteins GFP and ST), the mixture was incubated at 4°C for 12 hours. Initially, when SC-Mi3 was in excess, the excess ST-sfGFP protein was removed by gel filtration chromatography. When the molar ratio of SC-Mi3 to ST-sfGFP was 1:0.75, ST-sfGFP was completely displayed on the nanoparticle surface, with some SC-Mi3 remaining. As the molar amount of ST-sfGFP increased, when the SC-Mi3:ST-sfGFP ratio reached 1:1.5, ST-sfGFP became excess, and the excess antigen ST-sfGFP was removed by gel filtration chromatography. The optimal coupling ratio of SC-Mi3:ST-sfGFP was finally determined to be 1:3. Based on this condition, Mi3-EDIII nanoparticles were further prepared (the SC-Mi3 fusion protein and the EDIII-ST fusion protein were mixed in a molar ratio of 1:3).

[0072] Example 3: Antigen profile detection of Mi3-EDIII

[0073] To determine whether Mi3-EDIII retains the epitope recognized by nAbs, this invention tested the binding activity of several monoclonal antibodies (mAbs) to Mi3-EDIIII. Three monoclonal antibodies were used: 7B3, 8D10 (Niu X, et al., 2019), and 8-4 (from the Eighth Affiliated Hospital of Guangzhou Medical University). These antibodies were isolated from convalescent ZIKV-infected patients. 7B3 and 8D10 targeted EDIII and exhibited neutralizing activity against ZIKV (IC50 values: 7B3 and 8B10, respectively, 0.024 and 0.555 μg / ml), while 8-4 targeted EDII and showed only mild neutralizing activity (IC50 value: 67.970 μg / ml). E404 protein (a recombinant protein corresponding to amino acids 1 to 404 of the ZIKVE protein) was expressed in Epxi293F cells and purified by Ni-NTA affinity chromatography.

[0074] The results showed that Mi3-EDIII, EDIII-ST, and E404, but not SC-Mi3, were recognized by mAb 7B3 in a dose-dependent manner. Figure 4A). Notably, mAb 7B3 exhibited significantly enhanced binding to Mi3-EDIII, with the minimum required mAb 7B3 concentration being approximately 10 ng / ml, about three times lower than the concentrations required for binding to EDIII-ST and E404. A similar pattern was observed with mAb 8D10 binding to these proteins. mAb 8D10 recognized Mi3 EDIII, EDIII-ST, and E404, but not SC-Mi3. The minimum required mAb 8D10 concentration for significant binding to Mi3-EDIII was also lower than the minimum required for binding to EDIII-ST and E404. Figure 4 A). The results showed that mAb 8-4 could only recognize E404, but not Mi3EDIII, EDIII-ST, or SC-Mi3. Figure 4 A). This indicates that EDIII polymerization does not impair or even enhances the recognition rate of nAbs. The binding kinetics of monoclonal antibodies 7B3 and 8D10 with each form of EDIII were studied using biolayer interferometry (BLI). Figure 4 B). The equilibrium dissociation constants (KD) of mAb 7B3 for Mi3-EDIII, EDIII-ST, and E404 are <1.0 pM, 45.1 nM, and 2.17 nM, respectively, indicating that mAb 7B3 has a much higher binding activity for Mi3-EDIII than for EDIII-ST and E404. Similarly, the KD values ​​of 8D10 for Mi3-EDIII, EDIII-ST, and E404 are <1.0 pM, 173 pM, and 132 pM, respectively, indicating that mAb 8D10 also has a higher binding activity for Mi3-EDIII than for EDIII-ST and E404. In summary, compared with the monomeric EDIII-ST, Mi3-EDIII nanoparticles retain the original epitopes of EDIII.

[0075] Example 4: Immunological and immunological detection in Mi3-EDIII mice.

[0076] The specific experimental procedure is as follows:

[0077] 1) ELISA detection of binding antibodies produced by immunized mice. Specific procedure: Coat 96-well plates overnight at 4°C with purified ZIKV EDIII protein (1 μg / mL). Alternatively, serially dilute EDIII-ST, E404, and Mi3-EDIII proteins starting at 1 μg / mL (3-fold) and coat them onto 96-well plates overnight at 4°C. Wash plates with PBST (0.05% Tween 20 in 1×PBS) and block with 5% skim milk in PBS at room temperature for 1 h. After washing three times with PBST, add heat-inactivated, serially diluted (5-fold) mouse serum to the plates and incubate at 37°C for 1 h. Finally, develop the plates with 100 μL of TMB substrate (Merck Millipore) and stop with 50 μL of 2M sulfuric acid. Measure the OD value at 450 nm (OD450). IgG titer is calculated as the reciprocal of the maximum dilution with an OD450 value equal to or higher than the critical value (2.1 times that of the negative control well).

[0078] 2) FACS-based neutralization test (FNT). 2 × 10 4 10 Vero cells were seeded in 96-well plates. Heat-inactivated mouse serum or monoclonal antibodies 7B3, 8D10, or 8-4 were serially diluted with an equal volume of 2 × 10⁻⁴ cells per mL. 3 The FFU was mixed with ZIKV and incubated at 37°C for 1 hour. The mixture was then added to the wells and incubated for 2 hours. Cells were subsequently washed twice with PBS, and the medium was replaced with DMEM containing 2% FBS. After 3 days of culture, cells were fixed and infiltrated using the BD Cytofix / Cytoperm™ Fixation / Infiltration Kit (BD Biosciences) according to the commercial manufacturer's protocol and stained with mAb 8D10. After washing, cells were incubated with Alexa Fluor 647-conjugated goat anti-human IgG antibody (SouthernBiotech) and analyzed using the BD Accuri™ C6 Plus personal flow cytometer (BD Biosciences). For mouse serum, the neutralizing titer (NT50) was calculated as a 50% reduction in infection compared to the control wells containing only virus. For each monoclonal antibody, the IC50 value was calculated as the concentration of the monoclonal antibody that resulted in a 50% reduction in E-positive cells relative to the negative control.

[0079] 3) ELISApot assay. Sterile 96-well microtiter plates were coated with mouse IFN-γ or IL-4 capture antibodies (Mabtech) and incubated overnight at 4°C. Freshly isolated spleen lymphocytes were then inoculated at 3 × 10⁶ cells / well. 5Each well was incubated with an EDIII peptide pool (1 μg / mL per peptide; Genscript, China) for 24 h. After stimulation, a biotinylated detection antibody (U-CyTech) was added, followed by incubation with streptavidin conjugated with alkaline phosphatase (U-CyTech). Finally, the plates were scanned, and SFCs were quantified using an ELISpot reader (Bioreader4000, BIOSYS).

[0080] To test the immunogenicity of Mi3-EDIII nanoparticles, this invention used AddaVax adjuvant and immunized 6-week-old female C57BL / 6 mice by intramuscular injection (IM) with 5 μg of EDIII-ST and Mi3-EDIII nanoparticles coupled with an equimolar amount of EDIII antigen, respectively. Immunization was performed three times, with each immunization spaced 3 weeks apart. Figure 5 A). Mice inoculated with the unconjugated SC-Mi3 fusion protein served as controls. Two weeks after immunization, serum samples were collected from mice, and the titers of EDIII-specific IgG antibodies were measured.

[0081] In the Mi3-EDIII group, EDIII-specific IgG antibodies were detectable in all mice 2 weeks after the first immunization, increased 70-fold after the second immunization, and remained unchanged after the third immunization. Figure 5 (B) In the EDIII-ST group, EDIII-specific IgG antibodies were not detectable until 2 weeks after the second immunization, with a slight increase of 2.6-fold after the third immunization. At the final test time point, mice immunized with Mi3-EDIII nanoparticles had approximately 20-fold higher IgG titers than mice immunized with EDIII-ST. Mice immunized with unconjugated SC-Mi3 did not elicit an antibody response against EDIII. Therefore, Mi3-EDIII nanoparticles are superior to soluble EDIII-ST in inducing EDIII-specific IgG antibodies.

[0082] To determine the effectiveness of the immune-induced antibody response, this invention used a fluorescence-activated cell sorting (FACS)-based neutralization assay (FNT) to assess the neutralizing titer of antibodies in the immune serum. Following the first immunization, nAbs were not detected in any group of mice. Following the second immunization, neutralizing antibodies were detected in 2 out of 5 mice in the EDIII-ST group and in all mice in the Mi3-EDIII group, but not in the SC-Mi3 group. Figure 5 C). Notably, the neutralizing antibody titer in the Mi3-EDIII group was significantly higher than that in the EDIII-ST group ( Figure 5 C). After the third immunization, the nAb titer in the Mi3-EDIII group increased approximately fourfold. Figure 5D), while only residual nAb (if present) was detected in the EDIII-ST group. In summary, Mi3-EDIII nanoparticles were significantly more effective than monomeric EDIII-ST protein in inducing ZIKV-specific antibody responses.

[0083] Given that cell-mediated immune (CMI) responses also play an important role in the prevention of ZIKV infection, this invention compares the CMI responses induced by Mi3-EDIII and EDIII-ST. Figure 5 A). Two weeks after the last immunization, lymphocytes were isolated from the spleen and inguinal lymph nodes (ILN), and IFN-γ and IL-4 secreting cells were detected by enzyme-linked immunospot assay (ELISpot). Figure 5 E, Figure 5 F). Strong IFN-γ+ spotted cells (SFCs) were detected in the spleen and ILN of mice stimulated with the ZIKV EDIII peptide library and then immunized with Mi3-EDIII. Figure 5 E) indicates that Mi3-EDIII may induce a CMI response that secretes IFN-γ. In contrast, residual IFN-γ+ SFCs were detected in the spleen of mice immunized with EDIII-ST, but not in the inguinal lymph nodes (ILN), meaning that soluble EDIII-ST protein, even with Addavax adjuvant, only elicits a mild CMI response that secretes IFN-γ. Notably, Mi3-EDIII immunization induced robust IL-4+ SFCs (779 ± 106 SFCs per million spleen lymphocytes) in the spleen, while the EDIII-ST group only showed IL-4+ SFCs in the spleen (106 ± 74 SFCs per million spleen lymphocytes). Figure 5 F). These results indicate that Mi3-EDIII nanoparticles are significantly more effective than monomeric EDIII-ST proteins in inducing the CMI response.

[0084] Example 5: Mi3-EDIII nanoparticles can protect adults from Ifnar1 antibodies. - / - C57BL / 6 mice were protected from ZIKV infection.

[0085] The specific experimental procedure is as follows:

[0086] 1) Challenge Experiment: ZIKV strain GZ02 (GenBank ID KX056898.1) was isolated from a Chinese patient returning from Venezuela in 2016. DENV-1 strain (Hawaii strain, GenBank ID EU848545.1), DENV-2 strain 16681 (GenBank ID U87411.1), DENV-3 strain D191267 (GenBank ID OQ948473.1), and DENV-4 strain H241 (GenBank ID AY947539.1) were also tested (Liu X et al., 2024). All viruses multiplied in Vero cells. - / - C57BL / 6 mice were constructed using CRISPR-Cas9 technology from Cyagen Biosciences. Mice were housed in an animal center. Challenge experiments were conducted under biosafety level 2 (ABSL-2) conditions. The mice were 10-week-old male Ifnar1 mice. - / - C57BL / 6 mice underwent three immunizations. Three weeks after the last immunization, the mice were subcutaneously injected with 1.0 × 10⁻⁶ mmol / L. 5 FFU ZIKV GZ02 strain. Animal body weight and survival rate were monitored daily for 15 consecutive days. Serum samples were collected at 1, 4, and 7 days post-challenge.

[0087] 2) RT-qPCR: using Total RNA was extracted from the brain or serum of mouse pups using the Viral DNA / RNA Miniprep Kit (Vazyme). One-step RT-qPCR was performed using the HiScript II One-Step qRT-PCR SYBR Green Kit (Vazyme). The forward primer (NS5-F): 5′-TG-GAGGCTGAGGAAGTTCTAG-3′ (SEQ ID NO:11); the reverse primer (NS5-R): 5′-CTTCACACGCAATCATCTCCACTG-3′ (SEQ ID NO:12). The program was set as follows: 50℃ for 10 min; 95℃ for 30 s; 40 cycles of 10 s at 95℃, followed by 30 s at 55℃, followed by melting curves at 65℃ to 95℃, increasing by 0.5℃ for 5 s per cycle. A standard curve was constructed using RNA fragments covering the NS5 region generated by in vitro transcription. The limit of detection (LOD) was determined by serially diluting the ZIKV RNA fragment from the RNA extract of healthy control mouse pups; the LOD was ~1×10⁻¹¹ per mg brain tissue. 3 Copy number or 1×10 per serum sample 3 Copy number.

[0088] To determine the protective effect of Mi3-EDIII nanoparticles on adult mice, 10-week-old Ifnar1- / -C57BL / 6 mice were immunized with 5 μg of EDIII-ST or an equimolar amount of Mi3-EDIIII or SC-Mi3 via the IM route, with immunizations repeated every 3 weeks for a total of three immunizations. Figure 6 A). Two weeks after each immunization, mouse serum was collected and antibody levels were measured. After the first immunization, only Mi3-EDIII nanoparticles elicited EDIII-specific IgG antibodies (A). Figure 6 B). The second immunization significantly increased the IgG antibody titer, and Mi3-EDIII nanoparticles showed a greater immune response than EDIII-ST. After the third immunization, the EDIII-specific IgG titer in mice immunized with Mi3-EDIII nanoparticles was still higher than that in mice immunized with EDIII-ST. Figure 6 B). Although the first immunization did not induce nAbs in each group, the second immunization with Mi3-EDIII nanoparticles and EDIII-ST improved neutralizing antibody titers, with the Mi3-EDIII group showing significantly higher titers than the EDIII-ST group. Figure 5 C). The third immunization further increased the titer of nAbs, and the nAbs titer in the Mi3-EDIII group was significantly higher than that in the EDIII-ST group ( Figure 6 C). These results indicate that in Ifnar1 - / - In C57BL / 6 mice, Mi3-EDIII nanoparticles exhibited better immunogenicity than soluble EDIII-ST protein, similar to that observed in wild-type C57BL / 6 mice.

[0089] Three weeks after the last immunization, at a dose of 1.0 × 10⁻⁶ 5 FFU-ZIKV was used to subcutaneously challenge immunized mice. Compared with HC mice, mice immunized with SC-Mi3 showed severe weight loss, with 80% dying within 15 days after challenge. Figure 6 D, E). In contrast, mice immunized with Mi3-EDIII showed only slight weight loss and all survived. Mice immunized with EDIII-ST also showed weight loss, but less severe than those immunized with SC-Mi3, with 80% surviving. Figure 6 D, E). Viral load in mouse serum collected at 1, 4, and 7 days post-challenge was further measured by RT-qPCR. The results showed that a large viral genome was detected in SC-Mi3 immunized mice 1 day after challenge, increasing approximately 10-fold by day 4, while viral load began to decline by day 7. Figure 6F). In contrast, only small amounts of residual viral genome were detected in mice immunized with Mi3-EDIII at each test time point. However, in mice immunized with EDIII-ST, the viral load was comparable to that in mice immunized with SC-Mi3 (F). Figure 6 F). Therefore, immunization with Mi3-EDIII can effectively protect Ifnar1. - / - C57BL / 6 mice were protected from fatal ZIKV infection, while immunization with EDIII-ST only moderately reduced the mortality rate caused by ZIKV, but did not reduce viral replication.

[0090] Example 6: The Mi3-EDIII nanovaccine can induce potent and specific neutralizing antibodies against ZIKV, circumventing ADE reactions.

[0091] The specific experimental procedure is as follows:

[0092] To assess whether immunization with Mi3-EDIII nanoparticles would induce antibodies with an antibody-dependent enhancement (ADE) risk, we examined the infectivity of ZIKV and DENV in K562 cells in the presence of immune serum. This cell line does not inherently support infection by either virus in the absence of antibodies. In our experiments, we co-incubated ZIKV or dengue virus types 1-4 (DENV1-4) with serum from C57BL / 6 mice immunized with Mi3-EDIII, EDIII-ST, or SC-Mi3, respectively, using serum from Zika virus-infected convalescent mice as a control. Specifically, mouse serum was serially diluted 5-fold from 1:100 using RPMI 1640 medium and mixed with ZIKV or DENV3 (0.5 FFU / cell) or DENV1, DENV2, or DENV4 (0.2 FFU / cell), then incubated at 37°C for 1 h. C57BL / 6 mice were then subcutaneously injected with ZIKV (1×10⁻⁶). 5 FFU (Fluorescent Fumes), mouse serum was collected two weeks after infection as a control. The mixture was then added to K562 cells and incubated at 37°C for 3 consecutive days. Subsequently, cells were fixed with Cytofix / Cytoperm buffer (BD Biosciences) to make them permeable and stained with mAb 8D10 or mAb 8-4, the latter of which is cross-reactive to DENVE protein. Cells were then labeled with goat anti-human IgG antibody conjugated to Alexa Fluor 647 (SouthernBiotech) and analyzed on an Accuri C6 flow cytometer (BD Biosciences).

[0093] Results showed that Mi3-EDIII immune sera enhanced ZIKV infection of K562 cells within the dilution range of 1:100 to 1:2500, but the enhancing effect at the 1:100 dilution was weaker than that at 1:500( Figure 7 ), suggesting that partial neutralizing activity might exist at this dilution. Although EDIII-ST immune sera could also enhance ZIKV infection within the same dilution range, no significant neutralizing activity was detected. Notably, at all dilutions tested, neither Mi3-EDIII nor EDIII-ST immune sera showed enhanced dengue virus infection; while Zika recovered sera significantly promoted dengue virus infection within the dilution range of 1:100 to 1:2500( Figure 7 ).

[0094] In summary, under the existing experimental conditions, the ADE-related risk antibodies induced by immunization with Mi3-EDIII nanoparticles are extremely few (if any).

[0095] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A ZIKV nanovaccine, characterized in that, The ZIKV nanovaccine is prepared by mixing SC-Mi3 fusion protein and EDIII-ST fusion protein, and then self-assembling them using SpyCatcher linker peptide in SC-Mi3 fusion protein and SpyTag linker peptide in EDIII-ST fusion protein to obtain a nanoparticle vaccine with ZIKV EDIII protein displayed on its surface, with Mi3 nanoparticles as the core. The SC-Mi3 fusion protein is composed of Mi3 protein fused with SpyCatcher linker peptide, and the EDIII-ST fusion protein is composed of ZIKV EDIII protein fused with SpyTag linker peptide.

2. The ZIKV nanovaccine according to claim 1, characterized in that, The amino acid sequence of the SpyCatcher linker peptide is SEQ ID NO:8; and / or the amino acid sequence of the SpyTag linker peptide is SEQ ID NO:

7.

3. The ZIKV nanovaccine according to claim 1, characterized in that, The amino acid sequence of the SC-Mi3 fusion protein is shown in SEQ ID NO:2; and / or the amino acid sequence of the EDIII-ST fusion protein is shown in SEQ ID NO:

4.

4. A method for constructing a ZIKV nanovaccine according to any one of claims 1-3, characterized in that, Includes the following steps: S1: Obtain SC-Mi3 fusion protein and EDIII-ST fusion protein; S2: The SC-Mi3 fusion protein and the EDIII-ST fusion protein are mixed and incubated at 3-5°C for 10-14 hours. The two fusion proteins are coupled together by heteropeptide bonds between the SpyCatcher linker peptide and the SpyTag linker peptide to form the ZIKV nanovaccine.

5. The construction method according to claim 4, characterized in that, The process of obtaining the SC-Mi3 fusion protein and the EDIII-ST fusion protein includes the following steps: inserting the encoding nucleotide sequences of the SC-Mi3 fusion protein and the EDIII-ST fusion protein into prokaryotic expression vectors, respectively; then transforming the two prokaryotic expression vectors into prokaryotic host cells, respectively; and inducing expression with IPTG to obtain the SC-Mi3 fusion protein and the EDIII-ST fusion protein, respectively.

6. The construction method according to claim 4, characterized in that, The nucleotide sequence of the SC-Mi3 fusion protein is SEQ ID NO:1, and / or the coding sequence of the SpyCatcher linker peptide is SEQ ID NO:

6.

7. The construction method according to claim 4, characterized in that, The nucleotide sequence of the EDIII-ST fusion protein is SEQ ID NO:

3.

8. The construction method according to claim 4, characterized in that, The prokaryotic expression vector is a plasmid, preferably the pET28a plasmid. The prokaryotic host cell is an Escherichia coli strain, preferably a BL21(DE3) competent cell.

9. The construction method according to any one of claims 4-8, characterized in that, In step S2, the SC-Mi3 fusion protein and the EDIII-ST fusion protein are mixed in a molar ratio of 1:

3.

10. The use of the ZIKV nanovaccine according to any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of Zika virus (ZIKV) infection.

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