Glycosylated norovirus-like particles of mammalian cell origin and uses thereof

Abstract

This invention discloses a mammalian cell-derived glycosylated norovirus-like particle and its applications, belonging to the field of biotechnology. The amino acid sequence of the glycosylated norovirus-like particle is shown in SEQ ID NO.3; the glycosylated norovirus-like particle is obtained through recombinant expression using a mammalian cell expression system. This invention prepares GII.4 glycosylated norovirus-like particles carrying complex glycosylation modifications based on a mammalian cell system. Its serum binding titer, HBGA blocking antibody titer, and intestinal mucosal immune response are significantly superior to wild-type GII.4 norovirus-like particles. It not only achieves a secondary enhancement of isotype immunogenicity and blocking activity but also effectively broadens the spectrum of cross-reactivity against multiple circulating variants and cross-genotype strains. This provides a novel technical solution for developing safe, efficient, and broad-spectrum protective norovirus vaccines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a glycosylated norovirus-like particle of mammalian cell origin and its applications. Background Technology

[0002] Norovirus (NoV) is one of the leading pathogens causing acute gastroenteritis worldwide, posing a significant disease burden in infants, the elderly, and immunocompromised populations. Norovirus exhibits high genetic diversity and rapid evolutionary capacity, with genotype GII.4 consistently dominating global prevalence for decades and continuously generating new circulating variants through antigenic drift. Furthermore, genotypes GII.2, GII.3, GII.6, and GII.17 can also trigger regional or periodic outbreaks. However, despite the significant public health need, no norovirus vaccine has yet been approved for marketing globally.

[0003] The development of traditional inactivated or live attenuated vaccines for human norovirus is significantly constrained by the lack of stable, reliable, and readily deployable in vitro culture systems. Currently, virus-like particles (VLPs) formed by the self-assembly of the capsid protein VP1 have become the mainstream technology for norovirus vaccine development due to their ability to better mimic the surface conformation of natural virus particles, their high safety profile, and their capacity to induce humoral and cellular immune responses. Several VLP-based candidate vaccines have entered clinical trials, but publicly available results indicate that their protective efficacy, antibody breadth, and duration of immunity in different age groups remain insufficient.

[0004] Currently, most mainstream VLP candidate antigens rely on expression systems in yeast or insect cells. These non-mammalian hosts exhibit fundamental differences in post-translational modifications (especially glycosylation) compared to their natural hosts. Glycosylation is a crucial factor affecting protein folding, spatial conformation, stability, and immune recognition. For viral antigens, it can further influence key epitope exposure, receptor binding characteristics, and the quality of the immune response. Therefore, differences in glycosylation caused by variations in expression systems may be a significant factor influencing VLP immunogenicity. Existing research indicates that glycosylation modification can serve as an important strategy for vaccine antigen optimization and has been used to enhance immune responses in various viral vaccines. For norovirus, current research suggests that its capsid protein undergoes post-translational modifications, and certain specific chemical modifications can affect viral functional properties. However, for norovirus, systematic research on the glycosylation characteristics of VLPs expressed in mammalian cells and their impact on immunogenicity remains lacking.

[0005] Therefore, developing a norovirus VLP based on a mammalian cell expression system and combined with a precise glycosylation engineering optimization strategy to improve its serum antibody levels, block antibody titers, and mucosal immune responses is of great significance for the research and development and industrialization of norovirus vaccines. Summary of the Invention

[0006] The purpose of this invention is to provide a mammalian cell-derived glycosylated norovirus-like particle and its application, thereby addressing the problems existing in the prior art. This invention prepares GII.4 glycosylated norovirus-like particles carrying complex glycosylation modifications based on a mammalian cell system. These particles exhibit significantly superior serum binding titers, HBGA blocking antibody titers, and intestinal mucosal immune responses compared to wild-type GII.4 norovirus-like particles. This not only achieves a secondary enhancement in isotype immunogenicity and blocking activity but also effectively broadens the spectrum of cross-reactivity against multiple circulating variants and cross-genotype strains. This provides a novel technical solution for developing safe, efficient, and broad-spectrum protective norovirus vaccines.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing glycosylated norovirus-like particles derived from mammalian cells, comprising the following steps: (1) Insert the gene fragment with the nucleotide sequence shown in SEQ ID NO.4 into the vector to obtain the recombinant vector; (2) The recombinant vector was transfected into a mammalian cell expression system, and after culture, expression, purification, and VLP self-assembly, the glycosylated norovirus-like particles were obtained; The amino acid sequence of the glycosylated norovirus-like particle is shown in SEQ ID NO.3; N169, N172, and N307 are N-glycan sites, and T65, T309, T384, and S393 are O-glycan sites.

[0008] Furthermore, the mammalian cell expression system includes HEK 293F cells and CHO cells.

[0009] The present invention also provides glycosylated norovirus-like particles prepared by the above preparation method.

[0010] The present invention also provides a biomaterial for preparing the above-mentioned glycosylated norovirus-like particles, the biomaterial comprising a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.4 or a mammalian cell expression system containing the recombinant vector.

[0011] This invention also provides the use of the above-described glycosylated norovirus-like particles or the above-described biomaterials in any of the following: A1. Preparation of norovirus vaccine; A2. Prepare a kit for detecting norovirus or norovirus antibodies; A3. Prepare antibodies for detecting norovirus or for preventing norovirus infection; A4. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0012] The present invention also provides a norovirus vaccine comprising the above-mentioned glycosylated norovirus-like particles.

[0013] Optionally, the norovirus vaccine may also contain a pharmaceutically acceptable adjuvant.

[0014] This invention also provides the use of the above-mentioned norovirus vaccine in the following B1 or B2: B1. Prepare antibodies for detecting norovirus or for preventing norovirus infection; B2. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0015] The present invention discloses the following technical effects: This invention is the first to discover that norovirus VLPs prepared using mammalian cell-derived expression systems carry complex N-glycosylation and O-glycosylation modifications, exhibiting significantly superior immunogenicity compared to yeast-derived VLPs. Mammalian cell-derived VLPs not only induce higher levels of serum IgG antibodies and HBGA blocking antibodies but also effectively elicit a robust local mucosal immune response. Building upon the established superiority of mammalian cell-derived wild-type VLPs over yeast-derived VLPs, this invention, for the first time, applies a site-specific glycoengineering strategy to the design of a GII.4 norovirus VLP vaccine. By introducing additional N-glycosylation sites, it not only enhances isotype immunogenicity but also successfully broadens the cross-reactivity against multiple prevalent variants and non-immune genotypes (such as GII.2 and GII.17), providing a new strategy for addressing viral antigenic drift and developing broad-spectrum immunogenic vaccines. Furthermore, mammalian cells can be cultured in serum-free suspension, and the purification process is mature, which can obtain VLPs with high purity (>95%), high yield, intact structure and good uniformity. Moreover, the glycoengineering modification only involves site-directed mutation of the coding sequence, which does not increase the complexity of production and has good prospects for industrial transformation. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the structure of the GII.4-Mammal plasmid in Example 1; Figure 2 The image shows the SDS-PAGE results of the recombinant proteins GII.4-Mammal, GII.17-Mammal, and GII.4-Glyopt in Example 1. Figure 3 The images show electron micrographs of VLPs formed by the self-assembly of GII.4-Mammal, GII.17-Mammal, and GII.4-Glyopt recombinant proteins in Example 1; where A is an electron micrograph of VLPs of GII.4-Mammal, B is an electron micrograph of VLPs of GII.4-Glyopt, and C is an electron micrograph of VLPs of GII.17-Mammal. Figure 4 The graph shows the results of immune serum titers of monovalent VLPs (GII.4 and GII.17) from mammalian cell and yeast sources in Example 3. Figure 5 This is a graph showing the results of immune serum blocking activity assays for monovalent VLPs (GII.4 and GII.17) derived from mammalian cells and yeast in Example 3. Figure 6 The graph shows the binding affinity of immune serum antibodies to antigens for mammalian cell-derived and yeast-derived monovalent VLPs (GII.4 and GII.17) in Example 3. Figure 7 The image shows the results of detecting mucosal antibody levels in the feces of mice immunized with monovalent VLPs (GII.4 and GII.17) derived from mammalian cells and yeast in Example 3. Figure 8 The graph shows the serum titer and broad-spectrum cross-binding reaction results of the bivalent mixed vaccine (Biv-Mammal and Biv-Yeast) in Example 3; where A is the immune serum titer test result, B is the broad-spectrum cross-binding reaction test result with GII.4 genotype epidemic variants (GII.4 Hong Kong, GII.4 San Francisco, GII.4 Wichita), and C is the broad-spectrum cross-binding reaction test result with non-immune genotype strains (GII.2, GII.3, GII.6); Figure 9 The image shows the results of the immune serum blocking activity assay for the bivalent mixed vaccine (Biv-Mammal and Biv-Yeast) in Example 3. Figure 10This is a graph showing the results of detecting mucosal antibody levels in the feces of mice immunized with the bivalent mixed vaccine (Biv-Mammal and Biv-Yeast) in Example 3. Figure 11 The image shows the SDS-PAGE results of mammalian cell-derived VLPs (GII.4 and GII.17 types) before and after glycosidase digestion in Example 4. Figure 12 The graph shows the results of detecting the effect of glycosidase digestion on the titers of GII.4 and GII.17 immune sera in Example 4. Figure 13 The graph shows the results of detecting the effect of glycosidase digestion on the blocking activity of GII.4 and GII.17 immune sera in Example 4. Figure 14 The graph shows the immune serum titer and broad-spectrum cross-binding reaction results of GII.4-Glyopt and GII.4-Mammal in Example 5; where A is the immune serum titer test result, B is the broad-spectrum cross-binding reaction test result with GII.4 genotype epidemic variants (GII.4 Hong Kong, GII.4 San Francisco, GII.4 Wichita), and C is the broad-spectrum cross-binding reaction test result with non-immune genotype strains (GII.2, GII.3, GII.6, GII.17); Figure 15 This is a graph showing the results of detecting the blocking activity of GII.4-Glyopt and GII.4-Mammal in immune serum in Example 5; Figure 16 This is a graph showing the results of mucosal antibody level detection in the feces of mice immunized with GII.4-Glyopt and GII.4-Mammal in Example 5. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a glycosylated norovirus-like particle derived from mammalian cells, the amino acid sequence of which is shown in SEQ ID NO.3; the glycosylated norovirus-like particle is obtained by recombinant expression using a mammalian cell expression system.

[0024] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0025] In this invention, the N169, N172, and N307 positions of the glycosylated norovirus-like particles are N-glycosites, and the T65, T309, T384, and S393 positions are O-glycosites.

[0026] The present invention also provides a method for preparing the above-mentioned glycosylated norovirus-like particles, comprising the following steps: (1) Insert the gene fragment with the nucleotide sequence shown in SEQ ID NO.4 into the vector to obtain the recombinant vector; (2) The recombinant vector was transfected into a mammalian cell expression system, and after culture, expression, purification, and VLP self-assembly, the glycosylated norovirus-like particles were obtained.

[0027] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0028] The present invention also provides a biomaterial for preparing the above-mentioned glycosylated norovirus-like particles, the biomaterial comprising a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.4 or a mammalian cell expression system containing the recombinant vector.

[0029] This invention also provides the use of the above-described glycosylated norovirus-like particles or the above-described biomaterials in any of the following: A1. Preparation of norovirus vaccine; A2. Prepare a kit for detecting norovirus or norovirus antibodies; A3. Prepare antibodies for detecting norovirus or for preventing norovirus infection; A4. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0030] The present invention also provides a norovirus vaccine comprising the above-mentioned glycosylated norovirus-like particles.

[0031] In this invention, the norovirus vaccine further comprises a pharmaceutically acceptable adjuvant. In some embodiments of this invention, the adjuvant is an aluminum adjuvant.

[0032] This invention also provides the use of the above-mentioned norovirus vaccine in the following B1 or B2: B1. Prepare antibodies for detecting norovirus or for preventing norovirus infection; B2. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0033] In a second aspect, the present invention provides a norovirus-like particle of type GII.4 derived from mammalian cells, the amino acid sequence of which is shown in SEQ ID NO.1; the norovirus-like particle is obtained by recombinant expression using a mammalian cell expression system.

[0034] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0035] In this invention, the N169 and N172 positions of the GII.4 type norovirus-like particle are N-glycosites, and the T65, T384 and S393 positions are O-glycosites.

[0036] The present invention also provides a method for preparing the above-mentioned GII.4 type norovirus-like particles, comprising the following steps: (1) Insert the gene fragment with the nucleotide sequence shown in SEQ ID NO.2 into the vector to obtain the recombinant vector; (2) The recombinant vector was transfected into a mammalian cell expression system, and after culture, expression, purification, and VLP self-assembly, the GII.4 type norovirus-like particles were obtained.

[0037] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0038] The present invention also provides a biomaterial for preparing the above-mentioned GII.4 type norovirus-like particles, the biomaterial comprising a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.2 or a mammalian cell expression system containing the recombinant vector.

[0039] This invention also provides the use of the above-described GII.4 type norovirus-like particles or the above-described biological materials in any of the following: A1. Preparation of norovirus vaccine; A2. Prepare a kit for detecting norovirus or norovirus antibodies; A3. Prepare antibodies for detecting norovirus or for preventing norovirus infection; A4. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0040] The present invention also provides a norovirus vaccine, wherein the norovirus vaccine contains the above-mentioned GII.4 type norovirus-like particles.

[0041] In this invention, the norovirus vaccine further comprises a pharmaceutically acceptable adjuvant. In some embodiments of this invention, the adjuvant is an aluminum adjuvant.

[0042] This invention also provides the use of the above-mentioned norovirus vaccine in the following B1 or B2: B1. Prepare antibodies for detecting norovirus or for preventing norovirus infection; B2. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0043] Thirdly, the present invention provides a norovirus-like particle of type GII.17 derived from mammalian cells, the amino acid sequence of which is shown in SEQ ID NO.5; the norovirus-like particle of type GII.17 is obtained by recombinant expression using a mammalian cell expression system.

[0044] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0045] In this invention, the N308 position of the GII.17 norovirus-like particle is an N-glycosite, and the T310, S356, S374 and T384 positions are O-glycosites.

[0046] The present invention also provides a method for preparing the above-mentioned GII.17 type norovirus-like particles, comprising the following steps: (1) Insert the gene fragment with the nucleotide sequence shown in SEQ ID NO.6 into the vector to obtain the recombinant vector; (2) The recombinant vector was transfected into a mammalian cell expression system, and after culture, expression, purification, and VLP self-assembly, the GII.17 norovirus-like particles were obtained.

[0047] In this invention, the mammalian cell expression system includes HEK 293F cells and CHO cells. In some embodiments of this invention, the mammalian cell expression system is HEK 293F cells.

[0048] The present invention also provides a biomaterial for preparing the above-mentioned GII.17 type norovirus-like particles, the biomaterial comprising a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.6 or a mammalian cell expression system containing the recombinant vector.

[0049] This invention also provides the use of the above-described GII.17 norovirus-like particles or the above-described biomaterials in any of the following: A1. Preparation of norovirus vaccine; A2. Prepare a kit for detecting norovirus or norovirus antibodies; A3. Prepare antibodies for detecting norovirus or for preventing norovirus infection; A4. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0050] The present invention also provides a norovirus vaccine comprising the above-mentioned GII.17 type norovirus-like particles.

[0051] In this invention, the norovirus vaccine further comprises a pharmaceutically acceptable adjuvant. In some embodiments of this invention, the adjuvant is an aluminum adjuvant.

[0052] This invention also provides the use of the above-mentioned norovirus vaccine in the following B1 or B2: B1. Prepare antibodies for detecting norovirus or for preventing norovirus infection; B2. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

[0053] Fourthly, the present invention provides the application of the above-mentioned GII.4 type norovirus-like particles and the above-mentioned GII.17 type norovirus-like particles in the preparation of a norovirus bivalent vaccine.

[0054] The present invention also provides a norovirus bivalent vaccine, wherein the norovirus bivalent vaccine comprises the above-mentioned GII.4 type norovirus-like particles and the above-mentioned GII.17 type norovirus-like particles.

[0055] In this invention, the norovirus vaccine further comprises a pharmaceutically acceptable adjuvant. In some embodiments of this invention, the adjuvant is an aluminum adjuvant.

[0056] The present invention also provides the use of the above-mentioned norovirus bivalent vaccine in the preparation of antibodies for detecting norovirus or for preventing norovirus infection.

[0057] The present invention also provides the use of the above-mentioned norovirus bivalent vaccine in the preparation of medicaments for the prevention and / or treatment of diseases caused by norovirus.

[0058] The sequence information involved in this invention is as follows: SEQ ID NO.1: MKMASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVESRTKPFSVPVLTVEEMTNSRFPIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRNYTMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGMLTQTTRTDGSTRGHKATVYTGSADFAPKLGRVQFETDTDHDFEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTHNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMDLDCLLPQEWVQYFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAV。

[0059] SEQ ID NO.2:

[0060] SEQ ID NO.3: MKMASSDANPSDGSAANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTAGKIIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVESRTKPFSVPVLTVEEMTNSRFPIPLEKLFTGPSSAFVVQPQNGRCTTDGVLLGTTQLSPVNICTFRGDVTHITGSRNYTMNLASQNWTNYDPTEEIPAPLGTPDFVGKIQGMLTQTTRTDGSTRGHKATVYTGSADFAPKLGRVQFETDTDHDFEANQNTKFTPVGVIQDGSTTHRNEPQQWVLPSYSGRNTHNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMDLDCLLPQEWVQYFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGQHDLVIPPNGYFRFDSWVNQFYTLAPMGNGTGRRRAV。

[0061] SEQ ID NO.4:

[0062] SEQ ID NO.5: MKMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPTGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ。

[0063] SEQ ID NO.6:

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0065] Example 1 This embodiment prepared mammalian cell-derived VLPs of GII.4-Mammal, GII.17-Mammal, and GII.4-Glyopt. The specific steps are as follows: 1. Construction of VP1 protein-associated expression plasmid The gene sequences corresponding to the VP1 protein of norovirus types GII.4 and GII.17 were codon-optimized, and the optimized gene sequences are shown in SEQ ID NO.2 and SEQ ID NO.6, respectively. The whole genomes were then synthesized by a commercial sequencing / synthesis company (Qingke Biotechnology). The synthesized gene fragments were cloned into the pcDNA3.4(+) expression vector via homologous recombination, and the positive clones were verified by sequencing and named GII.4-Mammal plasmid and GII.17-Mammal plasmid, respectively.

[0066] Glycoengineering site-directed mutagenesis primers were designed (see Table 1). Using the GII.4-Mammal plasmid as a template, the N309T mutation was introduced. The gene sequence corresponding to the mutated VP1 protein is shown in SEQ ID NO.4. Positive clones were obtained through sequencing verification and named the GII.4-Glyopt plasmid. The plasmid structure map of GII.4-Mammal is shown below. Figure 1 As shown.

[0067] Table 1. Primers for GII.4-Glyopt site-directed mutagenesis 2. Expression and purification of VP1 protein Three successfully constructed expression plasmids were transfected into HEK 293F cells using liposome transfection reagent. The transfected cells were cultured in suspension at 37°C in a shaker with 5% CO2. Cell culture supernatant was harvested 4-5 days post-transfection. The target protein was initially purified using Strep-Tactin affinity chromatography resin, followed by centrifugation to concentrate the protein in VLP assembly buffer (20 mM Tris-HCl, 150 mM NaCl, 2 mM CaCl2, 10% glycerol, pH 7.4), and incubated at 4°C for 24 h to promote VLP assembly. Finally, the purified VLPs were collected by gel filtration chromatography using Sepharose 4 Fast Flow packing material. VLPs of GII.4 and GII.17 noroviruses, as well as a glycosylated mutant GII.4 norovirus, were obtained and named GII.4-Mammal, GII.17-Mammal, and GII.4-Glyopt, respectively.

[0068] 3. SDS-PAGE identification SDS-PAGE was used to identify the purified GII.4-Mammal, GII.17-Mammal, and GII.4-Glyopt. The results showed that the molecular weight of all three recombinant proteins was approximately 65 kDa, consistent with the expected size, and the purity exceeded 95% (see [link to SDS-PAGE]). Figure 2 The amino acid sequence of the GII.4-Mammal recombinant protein is shown in SEQ ID NO.1, the amino acid sequence of the GII.17-Mammal recombinant protein is shown in SEQ ID NO.5, and the amino acid sequence of the GII.4-Glyopt recombinant protein is shown in SEQ ID NO.3.

[0069] 4. Morphological observation of VLP The VLP sample was diluted to 1 μg / μL, and 3 μL of the sample was dropped onto a copper grid. After standing for 5 min, excess liquid was blotted away with filter paper. Then, 3 μL of 2% phosphotungstic acid solution was dropped onto the copper grid for negative staining, and after standing for 5 min, excess liquid was blotted away with filter paper. The grid was then dried for at least 30 min. The morphology of the VLPs was observed under a transmission electron microscope. The electron microscopy results showed that a large number of morphologically intact and uniformly distributed VLP particles were present in the three VLP samples (see...). Figure 3 ).

[0070] 5. Preparation of yeast-derived VLP The gene sequences corresponding to the VP1 proteins of noroviruses GII.4 and GII.17 were codon-optimized, and the optimized gene sequences are shown in SEQ ID NO.2 and SEQ ID NO.6, respectively. The whole genomes were then synthesized by a commercial sequencing / synthesis company (Qingke Biotechnology). Yeast-derived norovirus VLPs of GII.4 and GII.17 were prepared using the method described in the literature “Hou W, Lv L, Wang Y, et al. 6-ValentVirus-Like Particle-Based Vaccine Induced Potent and Sustained Immunity Against Noroviruses in Mice [J]. Front Immunol, 2022, 13: 906275”, and named GII.4-Yeast and GII.17-Yeast, respectively.

[0071] Example 2 In this embodiment, glycosylation modifications in GII.4-Mammal, GII.17-Mammal, GII.4-Glyopt, GII.4-Yeast, and GII.17-Yeast prepared in Example 1 were identified using glycosylation mass spectrometry. The specific steps are as follows: 1. Enzymatic desalting (1) Take an appropriate amount of purified VLP sample and use a proteomics digestion kit to perform enzymatic desalting treatment.

[0072] (2) Incubate the sample and magnetic beads at 37°C for 30 min, then add binding solution and shake for 15 min.

[0073] (3) After washing the magnetic beads three times, add the enzyme working solution and enzymatically hydrolyze at 37°C for more than 4 hours.

[0074] (4) Add the stop solution to terminate the reaction, and freeze dry for later use.

[0075] 2. Mass spectrometry analysis (1) Prepare mobile phase A (water containing 0.1% formic acid) and mobile phase B (80% acetonitrile containing 0.1% formic acid).

[0076] (2) Take the enzyme-hydrolyzed lyophilized powder and reconstitute it with 10 μL of solution A. Centrifuge at 14000 g for 20 min at 4℃, and inject 1 μg of the supernatant. The elution conditions for liquid chromatography are shown in Table 2.

[0077] (3) Using Orbitrap Exploris TM 480 mass spectrometer, equipped with FAIMS Pro TMInterface (compensation voltage switches between -45V and -65V once per second) and Nanospray Flex TM (NSI) Ion source. Ion spray voltage 2.0 kV, ion transmission tube temperature 320℃.

[0078] (4) Mass spectrometry adopts a data-dependent acquisition mode: full scan range m / z 350-1500, first-stage resolution 120000 (200 m / z), AGC 300%, maximum injection time 50 ms; second-stage mass spectrometry adopts the "Top Speed" mode, resolution 15000 (200 m / z), AGC 75%, maximum injection time 22 ms, collision energy 33%. Raw data (.raw) is generated for subsequent analysis.

[0079] (5) The NoV VLP sequence database was compared using MaxQuant software to statistically analyze the modification sites, types and abundance.

[0080] LC-MS / MS analysis results (see Table 3) showed that GII.4-Mammal had a total of 5 glycosylation sites identified, including 2 N-glycosylation sites (N169 and N172) and 3 O-glycosylation sites (T65, T384, and S393); GII.17-Mammal had a total of 5 glycosylation sites identified, including 1 N-glycosylation site (N308) and 4 O-glycosylation sites (T310, S356, S374, and T393). 384); a total of 7 glycosylation sites were identified in GII.4-Glyopt, including 3 N-glycosylation sites (N169, N172, N307) and 4 O-glycosylation sites (T65, T309, T384, S393); in contrast, no glycosylation modification sites were detected in the GII.4 yeast VLP and GII.17 yeast VLP (GII.4-Yeast and GII.17-Yeast) with the same sequence.

[0081] Table 2 Separation gradients for liquid chromatography elution Table 3. Statistical results of glycosylation modification sites in norovirus VLPs from different expression systems. Example 3 In this embodiment, mice were immunized with GII.4-Mammal, GII.17-Mammal, GII.4-Glyopt, GII.4-Yeast, and GII.17-Yeast prepared in Example 1 to investigate the immunogenicity of norovirus VLP from different sources and modified types. The specific steps are as follows: 1. Animal immunization (1) Laboratory animals: All animal experiments were conducted in accordance with relevant ethical guidelines and with the approval of the institution’s Animal Care and Use Committee. Female BALB / c mice aged 6 to 8 weeks were used in the experiments (purchased from the Institute of Medical Biology, Chinese Academy of Medical Sciences).

[0082] (2) Animal grouping: Mice were randomly divided into groups of 5 each, and were designated as immunization groups for antigens of different sources and types of modifications as follows: GII.4-Yeast group (Yeast-derived GII.4 wild-type VLP control). GII.17-Yeast group (Yeast-derived GII.17 wild-type VLP control). Biv-Yeast group (a VLP control of a mixture of yeast-derived GII.4 and GII.17; the mixing ratio of GII.4-Yeast and GII.17-Yeast was 1:1). GII.4-Mammal group (mammary cell-derived GII.4 wild-type VLP); GII.17-Mammal group (mammary cell-derived GII.17 wild-type VLP); Biv-Mammal group (mixed VLP of mammalian cell-derived GII.4 and GII.17; the mixing ratio of GII.4-Mammal and GII.17-Mammal was 1:1). The GII.4-Glyopt group (glycoengineered mammalian cell-derived GII.4 mutant VLP).

[0083] (3) Animal immunization: Each dose of vaccine in the monovalent immunization group contained 10 μg of the corresponding single-genotype VLP protein and 50 μL of aluminum adjuvant; each dose of vaccine in the bivalent immunization groups (Biv-Yeast and Biv-Mammal) contained 5 μg of GII.4 VLP protein, 5 μg of GII.17 VLP protein, and 50 μL of aluminum adjuvant; each group was supplemented with physiological saline to a total volume of 100 μL and thoroughly mixed. Immunization was performed via intramuscular injection in the thigh for a total of 3 times (on days 0, 14, and 28), with an interval of 14 days between each immunization. Fourteen days after the third immunization, fecal samples were collected, and the mice were euthanized; serum was collected for subsequent testing.

[0084] 2. Indicator Testing and Evaluation (1) Detection of titer of monovalent mouse immune serum The concentration of immunogen-specific IgG antibodies in mouse serum (expressed as half-maximal effective dilution) was detected using enzyme-linked immunosorbent assay (ELISA) to assess the serum titer. The specific steps are as follows: 1) Coating: Dilute the purified VLP antigen of the corresponding genotype (GII.4 or GII.17) to 1 μg / mL with coating buffer, add 100 μL / well to a 96-well microplate, and incubate overnight (16-18 h) at 4°C.

[0085] 2) Blocking: Discard the coating solution, add 100 μL of blocking solution containing 5% bovine serum albumin (BSA) to each well, and incubate at 37°C for 1 h.

[0086] 3) Sample addition: Discard the blocking solution, wash the reaction plate, add serially diluted mouse serum, 100 μL per well, and incubate at 37°C for 1 h.

[0087] 4) Add secondary antibody: After washing the reaction plate, add 100 μL of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody diluted 1:10000 to each well and incubate at 37°C for 1 h.

[0088] 5) Color development: After thorough washing, add 100 μL of TMB substrate color development solution to each well and react at room temperature in the dark for 15 min.

[0089] 6) Termination and reading: Add 50 μL of 3 M hydrochloric acid to each well to terminate the reaction, and immediately use an ELISA reader to measure the absorbance of each well at a wavelength of 450 nm / 630 nm.

[0090] 7) Potency calculation: Fit the dose-response curve using absorbance values ​​and calculate the half-maximal effective dilution (ED). 50 () as serum titer.

[0091] Figure 4 The results of immune serum titers for monovalent VLPs (GII.4 and GII.17) from mammalian and yeast cell sources are presented. Figure 4 It can be seen that for GII.4 type, the serum titer (ED) of the GII.4-Mammal group is... 50 The value reached 22717, which is the GII.4-Yeast control group (ED). 50 The titer was 2.04 times that of the GII.17-Mammal group (11129) (P<0.001). For GII.17, the serum titer of the GII.17-Mammal group reached 55014, which was 2.04 times that of the GII.17-Yeast control group (ED). 50 The result showed that, compared with the yeast expression system, the norovirus VLP antigen prepared by the mammalian cell system could more efficiently stimulate the body to produce a strong humoral immune response and significantly increase the secretion level of antigen-specific antibodies.

[0092] (2) Detection of blocking activity of monovalent mouse immune serum The ability of immune serum to inhibit the binding of VLP to tissue blood group antigens (HBGAs) was evaluated using an in vitro simulated blocking assay (expressed as the serum sample inhibiting 50% of the maximum VLP binding dilution). The specific steps are as follows: 1) Coating HBGAs: Using porcine gastric mucus type III (PGM) as the source of HBGAs, dilute with coating buffer to 10 μg / mL, add 100 μL / well to a 96-well microplate, and incubate overnight (16-18 h) at 4°C.

[0093] 2) Blocking: Discard the coating solution, add 100 μL of blocking solution containing 5% BSA to each well, and incubate at 37℃ for 1 h.

[0094] 3) Pre-incubation of serum with VLP: The serum sample to be tested was serially diluted 2 times starting from 1:100. The diluted serum was mixed with an equal volume of 1 μg / mL VLP solution of the corresponding genotype and incubated at 37℃ for 30 min to allow the blocking antibody in the serum to bind to VLP in advance.

[0095] 4) Sample addition: Discard the blocking solution, wash the reaction plate, and transfer the mixture from step 3) to the PGM coated plate, 100 μL per well, and incubate at 37°C for 1 h.

[0096] 5) Add primary antibody: After washing the reaction plate, add 100 μL of rabbit polyclonal antibody specific to the corresponding norovirus VLP type, diluted appropriately with diluent, and incubate at 37°C for 1 h.

[0097] 6) Add secondary antibody: After washing the reaction plate, add 100 μL of HRP-labeled goat anti-rabbit IgG secondary antibody (at the recommended dilution) to each well and incubate at 37°C for 1 h.

[0098] 7) Color development and termination: After washing, add 100 μL of TMB substrate color development solution to each well and react at room temperature in the dark for 15 min; add 50 μL of 3 M hydrochloric acid to each well to terminate the reaction and read the absorbance at 450 nm / 630 nm.

[0099] 8) Control and Potency Calculation: VLP samples without mouse serum were used as positive controls (maximum binding). The dilution that inhibited 50% of maximum VLP binding in each serum sample was calculated, which is the blocking potency (BT). 50 ).

[0100] Figure 5 The results of immunosera blocking activity assays for monovalent VLPs (GII.4 and GII.17) from mammalian and yeast cell sources are presented. Figure 5 It can be seen that for GII.4 type, the average blocking titer (BT) of serum in the GII.4-Mammal group is...50 The value reached 1657, which is higher than the control group GII.4-Yeast (BT). 50 3.23 times that of 513 P <0.01). For GII.17, the mean blocking titer in the GII.17-Mammal group reached 6893, which is higher than that in the control GII.17-Yeast group (BT). 50 4.17 times that of 1653 P <0.01). The results indicate that mammalian cell-derived VLPs not only increase serum binding antibody titers but also effectively induce specific blocking antibodies with neutralizing potential.

[0101] (3) Detection of binding affinity between monovalent mouse immune serum antibodies and antigens 1) The binding affinity between immune serum antibodies and antigens was assessed using the sodium thiocyanate dissociation method (NaSCN). The antigen coating, blocking, and serum loading were performed according to the ELISA method described above. After the serum bound to the antigen and was washed, different concentrations of NaSCN (0, 1, 2, 3, 4, 5 M) were added and incubated at 37°C for 30 min. 0 M NaSCN (i.e., PBS) served as the undissociated control. After washing, secondary antibody was added, and subsequent color development and reading procedures were the same as above.

[0102] 2) Affinity Calculation: A dissociation curve was plotted with NaSCN concentration on the x-axis and the percentage of absorbance values ​​at each concentration to the absorbance value of 0 M NaSCN on the y-axis. The NaSCN concentration at which the absorbance value decreased to 50% of the untreated control was calculated and recorded as the Affinity Index (half-maximum dissociation concentration). The higher the Affinity Index, the stronger the affinity between the serum antibody and the antigen.

[0103] Figure 6 The results of antibody-antigen binding affinity assays for mammalian cell-derived and yeast-derived VLPs (GII.4 and GII.17) are presented. Figure 6 It can be seen that for type GII.4, the half-maximum dissociation concentration (WMC) of the GII.4-Mammal group is 3.59, and that of the GII.4-Yeast group is 2.00. P <0.0001). For GII.17, the half-maximum dissociation concentration (MCD) of the GII.17-Mammal group was 3.49, which was also significantly better than the 2.62 of the GII.17-Yeast control group. P <0.01). The results indicate that mammalian cell-derived VLPs can produce high-affinity antibodies that bind more tightly to antigens.

[0104] (4) Detection of mucosal immune antibody levels in feces of monovalently immunized mice Collect mouse fecal samples and detect the levels of VLP antigen-specific mucosal immune antibodies (IgG and IgA). The specific steps are as follows: 1) Fecal sample processing: Collect fresh fecal samples from mice, add 90% (W / V) of resuspension containing protease and phosphatase inhibitors, homogenize thoroughly, centrifuge, collect the supernatant to obtain fecal suspension, and store at -80℃ for later use.

[0105] 2) Perform antigen coating and blocking according to the above ELISA method, replacing the serum with serially diluted fecal suspension, and replacing the secondary antibody with HRP-labeled goat anti-mouse IgG or IgA secondary antibody, and the remaining steps are the same.

[0106] 3) Titer calculation: The antibody endpoint titer of each sample was calculated by using 2.5 times the OD value of the control group of the same batch of PBS as the positive judgment threshold (cut-off value).

[0107] Figure 7 The results of fecal mucosal antibody level detection in mice immunized with monovalent VLPs (GII.4 and GII.17) derived from mammalian cells and yeast were presented. Figure 7 It was found that the endpoint titer of specific antibodies (IgG + IgA) in the feces of mice in the GII.4-Mammal group reached 256.0, while that in the GII.4-Yeast control group was 60.8. The level of local mucosal antibodies induced in the GII.4-Mammal group was 4.21 times that in the yeast group. P <0.01). For GII.17, the average endpoint titer of specific antibodies (IgG + IgA) in the feces of GII.17-Mammal group mice reached 838.4, which was significantly higher than that of the GII.17-Yeast control group (titer of 76.8) by 10.92 times. P <0.05). The results indicate that mammalian cell-derived VLPs not only enhance systemic humoral immunity but also more effectively promote the secretion of specific antibodies into the intestinal mucosa.

[0108] (5) Detection of titer and broad-spectrum cross-reactivity of bivalent mouse immune serum The same ELISA method as described above was used, except that the serum samples to be tested were replaced with mouse serum from the Biv-Mammal and Biv-Yeast groups. The specific IgG titers of the serum from the two groups of bivalent mixed-immune mice were detected using the VLPs of the immunogens (GII.4, GII.17), the VLPs of the GII.4 genotype epidemic variants (GII.4 Hong Kong, GII.4 San Francisco, GII.4 Wichita), and the VLPs of the non-immune genotype strains (GII.2, GII.3, GII.6) as coating antigens.

[0109] The methods for preparing VLPs of the prevalent variant strain of GII.4 genotype and VLPs of non-immune genotype strains are as follows: The VP1 gene sequences of the above-mentioned strains (corresponding amino acid sequence GenBank accession numbers: GII.4HongKong-QEL43936.1, GII.4San Francisco-WKD84242.1, GII.4Wichita-XER91914.1, GII.2-ARQ84857.1, GII.3-AFK75854.1 (STM), and GII.6-CRL46967.1 (STM)) were codon-optimized, and the whole genome was synthesized by a commercial sequencing / synthesis company (Qingke Biotechnology). The genome was then cloned into a pcDNA3.4(+) expression vector carrying a Strep-tag II tag. Following the method in Example 1, VLPs of the prevalent GII.4 genotype variant strain and the non-immune genotype strain from HEK293F cell lines were prepared through cell transfection, expression, and purification.

[0110] Figure 8 The results of serum titers and broad-spectrum cross-binding reactivity assays for the bivalent combination vaccine (Biv-Mammal and Biv-Yeast) are presented. Figure 8 It can be seen that, in terms of isotype serum titers, the Biv-Mammal group showed better efficacy against GII.4 and GII.17 ED. 50 The results were 37,950 and 67,931 respectively, which were significantly better than the Biv-Yeast control group (10,059 and 16,566 respectively). P All <0.001). Regarding cross-recognition of GII.4 variants, the cross-recognition efficiency of Biv-Mammal serum against the GII.4 Hong Kong, GII.4 San Francisco, and GII.4 Wichita variants was <0.001. 50 The titers reached 7921, 9800, and 20823, respectively, significantly higher than the Biv-Yeast control group (titers of 991, 1045, and 2578, respectively), and all showed statistically significant differences (respectively). P <0.01, P <0.001, P <0.01). Regarding cross-genotype cross-reactivity, the ED50 of Biv-Mammal serum against non-immune types GII.2, GII.3, and GII.6 was [not specified]. 50 The titers reached 21125, 28401, and 7673 respectively, also showing a significant advantage over the Biv-Yeast control group (titers of 5547, 5196, and 527 respectively).P <0.0001, P <0.05, P <0.01). The results showed that, compared with the monovalent immunization data, the isotype titer of the bivalent mixed immunization group did not decrease significantly, and the mammalian cell-derived VLP antigen did not cause immune interference during mixed immunization. Furthermore, it was more effective in responding to multiple variants and non-immune types of the GII.4 genotype, demonstrating broad cross-reactivity.

[0111] (6) Detection of blocking activity of bivalent mouse immune serum The same in vitro simulated blocking experiment as described above was used to evaluate the ability of the two groups of bivalent mixed immune serums to inhibit the binding of homotype wild-type VLPs (GII.4 and GII.17) to tissue blood group antigens (PGMs). The difference was that the test serum samples pre-incubated with VLPs were mouse immune serums from the Biv-Mammal and Biv-Yeast groups.

[0112] Figure 9 The results of immunoseroprotective activity assays for the bivalent combination vaccine (Biv-Mammal and Biv-Yeast) are presented. Figure 9 It can be seen that the serum of the Biv-Mammal group has BT levels against GII.4 and GII.17. 50 The BT values ​​reached 1978 and 6378 respectively, significantly higher than those of the traditional bivalent yeast control group (Biv-Yeast group). 50 The numbers were 571 and 1030 respectively, and both showed statistically significant differences (respectively). P <0.01, P <0.0001). The results indicate that the mammalian cell-derived bivalent vaccine can simultaneously induce high levels of neutralizing blocking antibodies against two core circulating strains.

[0113] (7) Detection of mucosal immune antibodies in bivalent immunized mice The same method as described above was used to detect the levels of antigen-specific antibodies (IgG + IgA) in fecal samples from two groups of bivalent mixed-immune mice. The difference was that the fecal samples used for the test were derived from experimental mice immunized with Biv-Mammal and Biv-Yeast.

[0114] Figure 10 The results of fecal mucosal antibody level detection in mice immunized with a bivalent combined vaccine (Biv-Mammal and Biv-Yeast) are shown. Figure 10 It can be seen that when GII.4 and GII.17 VLP were used as coating antigens, the specific antibody endpoint titers in the feces of Biv-Mammal group mice reached 275.0 and 563.0, respectively, which were 2.21 times that of the yeast bivalent control group. P<0.01) and 1.33 times ( P <0.001).

[0115] Example 4 This embodiment verifies the direct effect of glycosylation modification on enhancing immunogenicity by digesting and removing the glycan modifications on the surface of mammalian cell-derived norovirus VLPs using glycosidases. The specific steps are as follows: 1. Glycosidase digestion and SDS-PAGE identification of mammalian VLPs In vitro deglycosylation of purified GII.4-Mammal and GII.17-Mammal VLPs was performed using a mixed enzyme system. This mixed enzyme system contained PNGase F, O-glycosidase, α2-3,6,8,9 neuraminidase A, β1-4 galactosidase S, and β-N-acetaminohexosidase, enabling simultaneous and efficient cleavage of N-linked and O-linked glycan modifications on the antigen surface. The specific operational steps are as follows: (1) System preparation: Take a sample containing no more than 100 μg of target protein (GII.4-Mammal or GII.17-Mammal) and place it in a centrifuge tube. Make up the volume to 40 μL with sterile deionized water.

[0116] (2) Add buffer and enzyme: Add 5 μL of 10× Deglycosylation Mix Buffer 1 and 5 μL of protein deglycosylation mixed enzyme to the protein solution in sequence, and gently mix with a pipette to prepare a final reaction system of 50 μL.

[0117] (3) Temperature incubation: The reaction system was first incubated at 25°C for 30 min to allow the enzyme to bind initially and start the reaction; then it was transferred to 37°C for 16 h to ensure that the complex glycosylation modification on the surface of the VLP antigen was completely digested.

[0118] (4) Electrophoretic identification: After the reaction, the same batch of mammalian cell-derived VLPs that were not digested by enzymes were used as a control, and an appropriate amount of digestion products were taken for SDS-PAGE electrophoresis analysis.

[0119] Figure 11 The SDS-PAGE spectra of GII.4-Mammal and GII.17-Mammal before and after complete digestion with glycosidase are shown. Figure 11It was observed that after complete digestion with the aforementioned glycosidase, the protein bands of GII.4-Mammal and GII.17-Mammal showed significant downward migration. This result confirms that the mammalian cell-derived norovirus VLP antigen prepared in this invention carries a high level of complex glycan modification, and that the in vitro enzymatic digestion process effectively removes the glycans.

[0120] 2. Effects of deglycosylation on mouse immune serum titers The same ELISA method as in Example 3 was used, except that the serum samples to be tested were replaced with mouse serum that had been immunized with GII.4-Mammal and GII.17-Mammal glycosidases before and after digestion of VLPs, respectively. The immunogen was used as the coating antigen to detect the specific IgG concentration in the serum of the two groups of mice.

[0121] Figure 12 The results show the effect of glycosidase digestion on serum titers of GII.4 and GII.17 immunized mice. Figure 12 It can be seen that for type GII.4, the undigested wild-type group ED 50 The serum titer was 22402, while the VLP immunization group, after deglycosylation by glycosidase digestion, showed a significant decrease to 13572. P <0.01); for GII.17, the undigested wild-type group ED 50 The value is 49434, and the deglycosylated ED is... 50 Decreased to 24422 ( P <0.001). The results showed that after the complex glycan chains on the surface were removed, the ability of mammalian cell-derived VLPs to stimulate humoral immune responses was significantly reduced, directly confirming the importance of glycosylation modification for the high immunogenicity of norovirus VLPs.

[0122] 3. Effects of deglycosylation on the blocking activity of immune serum The ability of immune serum to inhibit the binding of VLP to tissue blood group antigens (HBGAs) was evaluated using an in vitro simulated blocking experiment. The specific implementation steps were the same as in Example 3, except that the test serum samples pre-incubated with VLP were mouse serum immunized with GII.4-Mammal and GII.17-Mammal before and after glycosidase digestion of VLP.

[0123] Figure 13 The results of the assay show the effect of glycosidase digestion on the blocking activity of GII.4 and GII.17 immune sera. Figure 13 It can be seen that for GII.4 type, after deglycosylation, the BT targeting GII.4-Mammal 50 The value decreased significantly from 1625 to 736. P<0.01); BT for GII.17-Mammal 50 It also decreased significantly from 5517 to 1653. P <0.001).

[0124] Example 5 This example compares the immunogenicity differences between GII.4-Glyopt and GII.4-Mammal. Evaluation indicators include serum titer, cross-reactivity breadth, serum blocking activity, and mucosal immune antibody levels.

[0125] 1. Detection of mouse immune serum titer and broad-spectrum cross-reactivity The same ELISA method as in Example 3 was used, except that the serum samples to be tested were replaced with mouse serum from the GII.4-Glyopt group and the GII.4-Mammal group. The specific IgG concentrations in the serum of the two groups of mice were detected using the immunogen, the VLP of the GII.4 genotype epidemic variants (GII.4 Hong Kong, GII.4 San Francisco, GII.4 Wichita), and the VLP of the non-immune genotype strains (GII.2, GII.3, GII.6, GII.17) as coating antigens.

[0126] Figure 14 The results of serum titers and broad-spectrum cross-reactivity assays for GII.4-Glyopt and GII.4-Mammal are presented. Figure 14 It can be seen that, against the GII.4 antigen, the ED in the GII.4-Glyopt group is... 50 The concentration reached 47,950, a significant increase of 1.85 times compared to the unmodified GII.4-Mammal group (25,971). P <0.001). Regarding cross-recognition of GII.4 variants, GII.4-Glyopt targets the ED of three GII.4 variants (Hong Kong, San Francisco, and Wichita). 50 The values ​​were 2261, 5523, and 10544, respectively, all higher than the unmodified GII.4-Mammal group (1594, 3476, and 5646, respectively). The improvement in GII.4 Wichita strain was statistically significant. P <0.05%. Regarding cross-genotype cross-reactivity, for the four non-immune types (GII.2, GII.3, GII.6, and GII.17), the ED50 of GII.4-Glyopt was <0.05. 50 Increased to 8143 respectively P <0.01), 1432, 1134 and 4386 ( P<0.001), significantly higher than the unmodified GII.4-Mammal group (3080, 743, 770, and 1032, respectively). The results indicate that site-specific sugar engineering significantly enhances homotype immunogenicity while broadening the spectrum of cross-reactivity against mutant strains and across genotypes.

[0127] 2. Detection of blocking activity in mouse serum The ability of immune serum to inhibit the binding of VLP to tissue blood group antigens (HBGAs) was evaluated using an in vitro simulated blocking experiment. The specific implementation steps were the same as in Example 3, except that the test serum samples pre-incubated with VLP were mouse serum from the GII.4-Glyopt group and the GII.4-Mammal group.

[0128] Figure 15 The results of immunosera blocking activity assays for GII.4-Glyopt and GII.4-Mammal are shown. Figure 15 It can be seen that the GII.4-Glyopt group is affected by the binding of GII.4-Mammal to HBGAs in BT. 50 The value reached 2183, a significant increase of 1.81 times compared to the unmodified GII.4-Mammal group (1204). P <0.0001). The results showed that the engineered modification of the site-directed sugar not only increased the serum antibody concentration, but also induced the body to produce higher titers of blocking antibodies.

[0129] 3. Detection of mucosal immune antibodies in immunized mice Collect mouse fecal samples and detect the levels of antigen-specific IgG and IgA antibodies. The specific implementation steps are the same as in Example 3, except that the fecal samples used for the test are from experimental mice immunized with GII.4-Glyopt and GII.4-Mammal.

[0130] Figure 16 The results of mucosal antibody level detection in feces of GII.4-Glyopt and GII.4-Mammal immunized mice are shown. Figure 16 It was found that the average endpoint titer of specific antibodies (IgG+ IgA) in the feces of mice in the GII.4-Glyopt group reached 550.0, which was significantly increased by 2.15 times compared with the unmodified GII.4-Mammal group (256.0). P <0.05).

[0131] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing glycosylated norovirus-like particles derived from mammalian cells, characterized in that, Includes the following steps: (1) Insert the gene fragment with the nucleotide sequence shown in SEQ ID NO.4 into the vector to obtain the recombinant vector; (2) The recombinant vector was transfected into a mammalian cell expression system, and after culture, expression, purification, and VLP self-assembly, the glycosylated norovirus-like particles were obtained; The amino acid sequence of the glycosylated norovirus-like particle is shown in SEQ ID NO.3; N169, N172, and N307 are N-glycan sites, and T65, T309, T384, and S393 are O-glycan sites.

2. The preparation method according to claim 1, characterized in that, The mammalian cell expression system includes HEK293F cells and CHO cells.

3. Glycosylated norovirus-like particles prepared by the preparation method according to claim 1 or 2.

4. A biomaterial for preparing the glycosylated norovirus-like particles of claim 3, characterized in that, The biomaterials include a recombinant vector containing the nucleotide sequence shown in SEQ ID NO.4 or a mammalian cell expression system containing the recombinant vector.

5. The use of the glycosylated norovirus-like particles of claim 3 or the biomaterial of claim 4 in any of the following: A1. Preparation of norovirus vaccine; A2. Prepare a kit for detecting norovirus or norovirus antibodies; A3. Prepare antibodies for detecting norovirus or for preventing norovirus infection; A4. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

6. A norovirus vaccine, characterized in that, The norovirus vaccine comprises the glycosylated norovirus-like particles as described in claim 3.

7. The norovirus vaccine according to claim 6, characterized in that, The norovirus vaccine also contains a pharmaceutically acceptable adjuvant.

8. The use of the norovirus vaccine according to claim 6 or 7 in B1 or B2 below: B1. Prepare antibodies for detecting norovirus or for preventing norovirus infection; B2. To prepare drugs for the prevention and / or treatment of diseases caused by norovirus.

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