Norovirus-like particles, immunotherapy compositions or kits, and their uses

Abstract

The present invention provides a virus-like particle or an active fragment thereof of GII.4 type norovirus and its use, the virus-like particle comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4. The present invention further provides a composition or kit comprising the virus-like particle of GII.4 type norovirus and its use. The objective of covering various types using a single antigen of the present invention is achieved, and the difficulty of the manufacturing process of the pharmaceutical composition or vaccine can be reduced and the production cost can be reduced. The present invention further provides an immune composition or kit against norovirus comprising virus-like particles or active fragments thereof of GII.2, GII.4, GII.6 and GII.17 types norovirus and its use, the composition or kit comprising the four antigens provided by the present invention can produce a cross-immune effect against the other types, and can not only cover various epidemic types of norovirus, but also has a synergistic immune effect against the same cross-type between the four antigens.

Description

Technical Field

[0001] The present invention belongs to the field of biopharmaceuticals and relates to norovirus virus-like particles and a composition or kit for inducing an immune response against norovirus. The present invention further provides uses of the norovirus virus-like particles, composition or kit.

Background Art

[0002] Norovirus was first discovered in 1972 by American scholar Kapikian from the feces of patients with diarrhea in Norwalk and named Norwalk virus (NoV). Subsequently, at the 8th International Committee on Taxonomy of Viruses, the virus was named norovirus and is one of the main pathogens causing human infectious gastroenteritis pandemics and acute diarrhea in infants and young children.

[0003] Norovirus is a single-stranded positive-strand RNA virus belonging to the family Caliciviridae, with a diameter of about 26 - 35 nm, no envelope, a rough surface, spherical shape, icosahedral symmetry, and is isolated from the feces of patients with acute gastroenteritis. The norovirus genome is approximately 7.7 kb in length and contains three open reading frames (ORFs). ORF1 encodes non-structural proteins and includes RNA-dependent RNA polymerase (RdRp), and ORF2 and ORF3 encode major (VP1) and minor (VP2) capsid proteins, respectively. According to the phylogeny of gene sequences, norovirus can be divided into six genogroups (GI - GVI), and each genogroup can be further divided into multiple genotypes. Most human infections are caused by genogroups GI and GII, among which the GII group is the main one. According to statistics, more than 75% of human NoV acute gastroenteritis is caused by infection with the GII group.

[0004] The detection rate and epidemic status of each genotype vary significantly depending on the time and region. For the past 20 years, the GII.4 genotype has been dominant in both global pandemics and sporadic cases, but genotype diversity and rapid mutation of circulating strains are important characteristics of norovirus. The detection rate and epidemic status of each genotype vary significantly depending on the time and region. In particular, GII.2, GII.3, GII.6, and GII.17 have high detection rates in genotypes other than GII.4, and in recent years, changes in epidemic trends have caused small-scale localized outbreaks.

[0005] Due to the genotypic diversity and rapid mutation characteristics of norovirus, as well as the lack of ideal cell culture systems and animal models, the development of anti-norovirus drugs has been somewhat limited, and effective drugs, vaccines, and preventive measures are still lacking. Therefore, developing effective antiviral drugs and preventive vaccines is a major challenge that must be addressed urgently.

[0006] Previous research data have shown that there is no cross-reactivity or cross-blocking activity between antibodies in the GI and GII gene groups of norovirus (Han Zibo, Zhang Xuefeng, Liu Zhaoming et al., Chinese Journal of Biological Products, 2019). Therefore, research on vaccines against various types of norovirus currently focuses on various artificially synthesized polypeptides.

[0007] In light of this, we currently need vaccines against various types of norovirus. [Overview of the project] [Problems that the invention aims to solve]

[0008] Therefore, the object of the present invention is to provide virus-like particles of various types of norovirus, immune compositions or kits against norovirus, and their applications, in order to overcome the shortcomings of the prior art.

[0009] The inventors conducted extensive research on cross-immunity between antigens of various norovirus types and unexpectedly discovered virus-like particles of norovirus type GII.4 that exhibit strong cross-immunity with GI.1. These virus-like particles of norovirus type GII.4 also exhibit cross-immunity against GII.2, GII.7, GII.12, and others. Based on this discovery, the inventors provide virus-like particles (VLPs) of norovirus type GII.4, an immune composition or kit against norovirus, and its uses.

[0010] The inventors have also unexpectedly obtained a composition or kit for inducing an immune response to norovirus, comprising virus-like particles of norovirus types GII.2, GII.4, GII.6, and GII.17, and the pharmaceutical composition or kit simultaneously covers types GII.1 and GII.7 and also exhibits cross-immunity against type GII.12. Using the composition or kit of the present invention, the objective of covering seven types with four antigens can be achieved, and there is superposition of immune effects against the same cross-types among the four antigens. [Means for solving the problem]

[0011] The object of the present invention is achieved by the following technical solutions. In one embodiment, the present invention provides virus-like particles of type GII.4 norovirus or active fragments thereof for inducing an immune response in animals to various types of norovirus, wherein the virus-like particles of type GII.4 norovirus or active fragments thereof (1) The amino acid sequence shown in Sequence ID No. 4, (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in Sequence ID No. 4, (3) an amino acid sequence in which one or more amino acids are substituted, deleted or inserted at any of the amino acid sequences shown in Sequence ID No. 4, and (4) The consecutive amino acids from position X to position 480 of the amino acid sequence shown in Sequence ID No. 4 (where X is an integer from 2 to 39, preferably X is 27, 37, or 39). It contains or is composed of any amino acid sequence selected from the following.

[0012] Preferably, the animal is a mammal, more preferably a human, and / or the various types of norovirus include two or more selected from norovirus types GI.1, GII.2, GII.4, GII.7 and GII.12.

[0013] In another embodiment, the present invention provides a composition for inducing an animal immune response to norovirus, comprising virus-like particles or active fragments thereof of type GII.4 norovirus as described in the present invention.

[0014] Preferably, the animal is a mammal, and more preferably, a human.

[0015] Preferably, the norovirus is selected from one or more noroviruses of types GI.1, GII.2, GII.4, GII.7, and GII.12.

[0016] In a further embodiment, the present invention provides a composition for inducing an immune response to norovirus, comprising virus-like particles or active fragments thereof of norovirus types GII.2, GII.4, GII.6, and GII.17.

[0017] In a preferred embodiment of the present invention, the virus-like particles or active fragments of type GII.2 norovirus are, (1) The amino acid sequence shown in Sequence ID No. 2, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in Sequence ID No. 2, and (3) Amino acid sequences in which one or more amino acid substitutions, deletions, or insertions occur at one or more locations in the amino acid sequence shown in Sequence ID No. 2. It contains or is composed of any amino acid sequence selected from the following.

[0018] Preferably, the virus-like particles or active fragments of the GII.4 type norovirus are (1) The amino acid sequence shown in Sequence ID No. 4, (2) An amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in Sequence ID No. 4, (3) an amino acid sequence in which one or more amino acids are substituted, deleted or inserted at any of the amino acid sequences shown in Sequence ID No. 4, and (4) The consecutive amino acids from position X to position 480 of the amino acid sequence shown in Sequence ID No. 4 (where X is an integer from 2 to 39, preferably X is 27, 37, or 39). It contains or is composed of any amino acid sequence selected from the following.

[0019] Preferably, the virus-like particles or active fragments of the GII.6 type norovirus are (1) The amino acid sequence shown in Sequence ID No. 5, (2) Amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in Sequence ID No. 5, and (3) An amino acid sequence in which one or more amino acids are substituted, deleted, or inserted at any of the amino acid sequences shown in Sequence ID No. 5, (4) The consecutive amino acids from position X to position 547 of the amino acid sequence shown in Sequence ID No. 5 (where X is an integer from 2 to 27, preferably X is 27) It contains or is composed of any amino acid sequence selected from the following.

[0020] Preferably, the norovirus virus-like particles of the GII.17 type or active fragments thereof are (1) the amino acid sequence shown in SEQ ID NO: 8, (2) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity with the amino acid sequence shown in SEQ ID NO: 8, and (3) an amino acid sequence having an amino acid substitution, deletion or insertion at one or more positions in the amino acid sequence shown in SEQ ID NO: 8 and include or are constituted by any amino acid sequence selected from these.

[0021] More preferably, the norovirus virus-like particles of the GII.2 type or active fragments thereof include or are constituted by the amino acid sequence shown in SEQ ID NO: 2, the norovirus virus-like particles of the GII.4 type or active fragments thereof include or are constituted by the amino acid sequence shown in SEQ ID NO: 4, the norovirus virus-like particles of the GII.6 type or active fragments thereof include or are constituted by the amino acid sequence shown in SEQ ID NO: 5, and the norovirus virus-like particles of the GII.17 type or active fragments thereof include or are constituted by the amino acid sequence shown in SEQ ID NO: 8.

[0022] According to the composition described in the present invention, the norovirus may be selected from one or more types of noroviruses of the GI.1, GII.2, GII.4, GII.6, GII.7, GII.12 or GII.17 type.

[0023] Preferably, the composition described in the present invention is a pharmaceutical composition for preventing and / or treating norovirus infection, for example, a vaccine.

[0024] Preferably, the pharmaceutical composition further contains one or more adjuvants selected from aluminum adjuvants, TRL adjuvants, and saponin adjuvants. More preferably, the aluminum adjuvant is selected from one or more aluminum hydroxide, aluminum phosphate, and aluminum sulfate, and even more preferably, the aluminum adjuvant is aluminum hydroxide. More preferably, the TRL adjuvant is a TRL9 adjuvant, and even more preferably, the TRL9 adjuvant is a CPG adjuvant, and even more preferably, the nucleotide sequence of the CPG adjuvant is selected from SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. More preferably, the saponin adjuvant is QS21 or an Iscom adjuvant.

[0025] According to the composition described in the present invention, the norovirus infection may be gastroenteritis, and preferably, viral acute gastroenteritis.

[0026] In a further embodiment, the present invention provides an immunotherapy kit comprising the norovirus-like particles or active fragments thereof described in the present invention, or the composition described in the present invention. Preferably, the kit is a kit for detecting norovirus infection.

[0027] In a further embodiment, the present invention relates to the norovirus-like particles or active fragments thereof described in the present invention, the composition described in the present invention, or the kit described in the present invention. (1) Drugs for the prevention and / or treatment of norovirus infection, (2) A kit for diagnosing norovirus infection, (3) Immunogens for developing norovirus antibodies It provides applications in the manufacturing of [product name].

[0028] According to the uses described in the present invention, the norovirus infection may be gastroenteritis, and preferably viral acute gastroenteritis.

[0029] In a further embodiment, the present invention provides a method for inducing an immune response in animals to various types of norovirus, comprising administering to an animal in need a virus-like particle or active fragment thereof of type GII.4 norovirus as described in the present invention, preferably the various types of norovirus comprising one or more selected from types GI.1, GII.2, GII.4, GII.7, and GII.12 norovirus.

[0030] In another embodiment, the present invention further provides a method for inducing an animal immune response to norovirus, comprising administering the composition described in the present invention to an animal in need. [Effects of the Invention]

[0031] The inventors have discovered that virus-like particles of type GII.4 norovirus containing the amino acid sequence described in the present invention induce cross-immunity responses against four types of norovirus: GI.1, GII.2, GII.7, and GII.12. This not only achieves cross-immunity of the GII genome against the GI genome, but also exhibits significant immune effects against other types within the group, thus achieving the objective of preventing infection from various types of norovirus using a single antigen. Covering various types with a single antigen of the present invention reduces the complexity of the manufacturing process for pharmaceutical compositions or vaccines and lowers production costs, thus creating a broad market.

[0032] The inventors have also discovered that a composition containing virus-like particles of norovirus types GII.2, GII.4, GII.6, and GII.17 covers type GII.1 and exhibits cross-immunity against types GII.7 and GII.12, thus achieving the objective of covering seven types with four antigens and exhibiting superimposed immune effects against the same cross-types among the four antigens. In other words, the composition of the present invention has a synergistic immune effect against the same cross-types among the four antigens. For example, since GII.4 and GII.6 can simultaneously cover type GII.7, the antibody titer is significantly higher than that of a single type GII.4. Therefore, by using the composition of the present invention, various types of norovirus can be covered simultaneously, reducing the difficulty of the drug or vaccine manufacturing process and lowering production costs, thus opening up a broad market. [Brief explanation of the drawing]

[0033] The embodiments of the present invention will be described in detail below in conjunction with the drawings, and here, [Figure 1] The cross-immunoimmune effect of the GII.4 norovirus virus-like particles (VLPs) of the present invention against each type of norovirus is demonstrated. [Figure 2] This study demonstrates the cross-immunity effect of GII.4 norovirus virus-like particles (VLPs) of other sequences against each type of norovirus. [Figure 3] This shows the immune strength of the GII.4 norovirus virus-like particles (VLPs) of the present invention against noroviruses with cross-immunity effects. [Figure 4] The present invention provides a composition containing virus-like particles of four types of norovirus, GII.2, GII.4, GII.6, and GII.17, which exhibits cross-immunity effects of each type of virus-like particle against each type of norovirus. [Figure 5] The cross-immunoimmune effect of the composition containing virus-like particles of four types of norovirus, GII.2, GII.4, GII.6, and GII.17, according to the present invention, against each type of norovirus is demonstrated. [Figure 6]This shows the immunoassay strength of compositions containing virus-like particles of four types of norovirus, GII.2, GII.4, GII.6, and GII.17, according to the present invention, against each type of norovirus with cross-immunity.

[0034] Definition: Unless otherwise defined, all technical terms used herein have the same meaning as those skilled in the art. For definitions of terms in this art, those skilled in the art can refer to Current Protocols in Molecular Biology (Ausubel). Amino acid residue abbreviations are standard three-letter and / or one-letter codes used in this art to refer to one of 20 common L-amino acids.

[0035] In this specification, the term "or" may be used interchangeably with the term "and / or" unless otherwise specified in the context.

[0036] As used herein, the terms “pharmaceutical composition,” “combination drug,” and “drug combination” are interchangeable and refer to a combination of at least one drug and any pharmaceutically acceptable excipient or additive that are combined to achieve a particular purpose. In some embodiments, the pharmaceutical composition includes combinations that are separate in time and / or space, as long as they function together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition (e.g., GII.2, GII.4, GII.6, GI.17, or Al(OH)3) may be administered to a subject as a whole or separately. If the components contained in the pharmaceutical composition are administered to a subject separately, the components may be administered to the subject simultaneously or sequentially.

[0037] As used herein, the terms “CpG oligodeoxynucleotide” or “CpG-ODN” refer to a short single-stranded synthetic DNA molecule containing one or more “CpG” units, where C represents cytosine, G represents guanine, and p represents a phosphodiester bond. In particular, the CpG oligodeoxynucleotide is unmethylated. In some embodiments, the CpG-ODN contains a phosphorothioate bond or a phosphorothioate backbone. That is, in some embodiments, the CpG-ODN is a phosphorothioate oligodeoxynucleotide (i.e., a thiooligodeoxynucleotide). Preferably, all connections between nucleotides in the CpG-ODN are phosphorothioate bonds, i.e., the CpG-ODN is a complete thiooligodeoxynucleotide. In other embodiments, the CpG-ODN contains two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif. In particular, the CpG-ODN has a sequence selected from TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 9), TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID NO: 10), or TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 11), and preferably TCG TTC GTT CGT TCG TTC GTT (SEQ ID NO: 9).

[0038] As used herein, "saponin" refers to the active ingredient present in the corresponding plant, and in particular to QS21, i.e., quillaja saponin, or Iscom adjuvant, i.e., an immunostimulatory complex adjuvant, specifically an antigen-free Iscom matrix, which is an adjuvant with a cage-like structure composed of phospholipids, saponins, and cholesterol.

[0039] As used herein, “therapeutic effective dose” or “effective dose” refers to a dose sufficient to demonstrate its benefit to the target of administration. The actual dose, rate, and duration of administration will be determined based on the patient’s own condition and severity. The prescription of treatment (e.g., dose determination) is ultimately the responsibility of the general practitioner and other physicians, and will generally take into account the disease being treated, the patient’s individual condition, the site of delivery, the method of administration, and other factors known to the physician.

[0040] As used herein, the term “mammal” refers to humans, but may also refer to other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.), or laboratory animals (e.g., orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0041] In some other embodiments, the composition of the present invention may further include, for example, a pharmaceutically acceptable carrier or additive as another additive, particularly if it exists in the form of a pharmaceutical formulation.

[0042] Preferred pharmaceutical carriers are particularly water, buffered aqueous solutions, tonic salt solutions such as PBS (phosphate-buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerin, hyaluronic acid, ethanol or polyalkylene glycol (e.g., polypropylene glycol), triglycerides, etc. The type of pharmaceutical carrier used is determined in particular by whether the composition of the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration. The composition of the present invention may also contain humectants, emulsifiers, or buffering substances as additives.

[0043] The pharmaceutical composition, vaccine, or pharmaceutical preparation of the present invention may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly, or intravenously. [Modes for carrying out the invention]

[0044] The present invention will be further described below with reference to specific embodiments using drawings, however this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements based on the spirit of the invention, and any such modifications or improvements will remain within the scope of the present invention as long as they do not depart from the spirit of the invention.

[0045] Example 1: Method for producing norovirus antigen Each of these compounds produced various types of norovirus VLP proteins, the amino acid sequences of which are shown below. This is a GII.1 type norovirus VLP protein, and its amino acid sequence is as shown in Sequence ID No. 1. This is a GII.2 type norovirus VLP protein, and its amino acid sequence is as shown in Sequence ID No. 2. This is a GII.3 type norovirus VLP protein, and its amino acid sequence is as shown in SEQ ID NO: 3. This is a GII.4 type norovirus VLP protein, and its amino acid sequence is as shown in Sequence ID No. 4. This is a GII.6 type norovirus VLP protein, and its amino acid sequence is as shown in SEQ ID NO: 5. This is a GII.7 type norovirus VLP protein, and its amino acid sequence is as shown in SEQ ID NO: 6. This is a GII.12 type norovirus VLP protein, and its amino acid sequence is as shown in Sequence ID No. 7. This is a GII.17 type norovirus VLP protein, and its amino acid sequence is as shown in Sequence ID No. 8. For comparison, the GII.4 type norovirus VLP protein has the amino acid sequence shown in SEQ ID NO: 12.

[0046] The specific manufacturing method is as follows: Based on the target protein sequence, the nucleic acid sequence was optimized so that the codons were compatible with the yeast expression system, and the target gene was synthesized. The synthesized target gene was linked to the pMAUR(SC)KARS1 plasmid by enzymatic digestion and ligation, the top 10 competent cells were transformed, positive monoclones were selected, and the positive monoclones were validated by sequencing. Monoclonal cells were amplified in large quantities, and plasmids suitable for electroporation were extracted in large quantities using a plasmid extraction kit. The plasmids were transformed into uracil-requiring Hanzenula competent cells by electroporation, and positive transformed cells were obtained by G418 resistance screening.

[0047] Recombinant strains were inoculated into yeast extract powder peptone glucose medium for activation. After activation, the strains were transferred to the yeast extract powder peptone glucose medium for expansion culture. After culturing the strains until the plateau stage, methanol was added to the medium for induction culture, which was done once every 24 hours for a total of two times. After induction was complete, the bacterial cells were collected, destroyed, and purified to obtain the protein.

[0048] Example 2: Animal Immunology Experiment 2.1 Laboratory animals Balb / c mice, female, 6 weeks old, 18 mice, provided by Shanghai Lingcheng Experimental Animal Technology Co., Ltd.

[0049] 2.2 Experimental Materials These are VLP proteins of various types of norovirus, prepared according to Example 1.

[0050] Aluminum hydroxide: Purchased from Beijing Bai'ao Laibo Technology Co., Ltd.

[0051] 2.3 Grouping of Experiments [Table 1]

[0052] 2.4 Animal immunity 1) Route of administration: Intramuscular injection.

[0053] 2) Method of administration: 100 μL / mouse was injected into the left hind thigh.

[0054] 3) Frequency and duration of administration: The drug was administered once every three weeks for three consecutive doses, specifically at week 0, week 3, and week 6. Blood samples were taken two weeks after the first immunization, three weeks after the second immunization, and two weeks after the third immunization as an endpoint.

[0055] 4) Serum recovery method: Whole blood was placed in a 37°C constant temperature incubator and left to stand for 40-50 minutes, then left to stand at 4°C for 1 hour, centrifuged at 12,000 rpm at 4°C for 10 minutes, the supernatant was aspirated and stored frozen in a medical cryogenic storage box in preparation for use.

[0056] Example 3: Cross-blocking experiment of pharmaceutical compositions 3.1 Reagent Materials 1) BSA (bovine serum albumin, Beyotime), TMB chromogenic solution (Thermo), HBGA (Glycotech), rabbit polyvalent antiserum (Kyotensei Seibutsu), goat anti-rabbit IgG-HRP (horseradish peroxidase-labeled goat anti-rabbit IgG, SIGMA), avidin plate (Thermo).

[0057] 2) Washing solution: 0.1M PB buffer Diluent: 0.1M PB buffer + 0.25% BSA Stop liquid: 2M H2SO4 All were prepared by Nanjing Yuanda Sai Weixin Biotechnology Co., Ltd.

[0058] 3) Norovirus VLP proteins: types GI.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, and GII.17, each prepared according to Example 1, with a concentration of 8 μg / mL.

[0059] 3.2 Detection Step 1) Wash the avidin plate once with 200 μL of washing solution / well. 2) Dilute HBGA to 4 μg / mL and add it to the avidin plate from step 1) in 100 μL / well, then incubate at 25°C for 1 hour. 3) In a 96-well plate, serum (from Example 2) was diluted 20-fold to 2-fold, resulting in a 1280-fold dilution, with a final volume of 60 μL. 60 μL of a single type of norovirus VLP protein was added to each well and mixed to form a VLP-serum mixture. Simultaneously, a positive control (no serum) and a blank control (diluted solution) were placed, and the mixture was incubated at 4°C for 1 hour. 4) Wash the avidin plate coated with HBGA in step 2) twice with 200 μL of washing solution / well, aspirate 100 μL of VLP-serum mixture from the 96-well plate in step 3) and add it to the avidin plate, then incubate at 4°C for 2 hours. 5) Wash the avidin plate three times with 200 μL of washing solution / well, add rabbit antiserum (1:10000), and incubate at 4°C for 1 hour. 6) Wash the avidin plate three times with 200 μL of washing solution / well, add goat anti-rabbit IgG-HRP (1:10000), and incubate at 37°C for 40 minutes. 7) Wash the avidin plate three times with 200 μL of washing solution / well, add 100 μL of TMB chromogenic solution / well, and allow to develop color at 25°C for 5-15 minutes. 8) Stop: Add 100 μL / well of stop solution to stop the reaction. 9) Reading: OD450nm value was measured (corrected for OD630nm).

[0060] Detection criteria: The blocking index % = (1 - serogroup A value / positive control A value) × 100% is used to calculate BT50, i.e., the highest serum dilution that can block the binding of VLP and HBGA by 50%.

[0061] Cut-off value setting: The cut-off value was set to twice the BT50 of the Al(OH)3 group to determine whether or not there was a cross-immunoactive effect.

[0062] 3.3 Experimental Results The experimental results are shown in Figure 1. The GII.4 norovirus VLP protein provided by the present invention exhibits remarkable cross-immunity against GI.1, and its BT50 can reach over 250. It also shows some cross-immunity against GII.2, GII.7, and GII.12. A different sequence of GII.4 protein used for comparison showed no cross-immunity against GI.1, GII.7, or GII.12 (Figure 2).

[0063] Example 4: Detection of IgG antibody levels in a pharmaceutical composition 4.1 Reagent Materials 1) Skim milk powder (BD), PBS (Nakasugi Kanehashi), carbonate (SIGMA), polysorbate 20 (BIO-LINK), TMB colorant (Thermo), horseradish peroxidase-labeled goat anti-rabbit IgG (hereinafter abbreviated as goat anti-rabbit IgG-HRP, SIGMA) 2) Coating solution: 0.5% (g / 100mL) carbonate solution Washing buffer PBST: 10 g / L PBS + 0.5 mL / L Polysorbate 20 Dilution: 2% Skim Milk PBST Stop liquid: 2M H2SO4 All of the above were prepared by Nanjing Yuandasai Weixin Biopharmaceutical Co., Ltd.

[0064] 4.2 Detection Step 1) Coating: Norovirus VLP proteins of types GI.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.12, and GII.17 were each diluted to 1 μg / mL with the coating solution, and the ELISA enzyme-conjugated plates were coated with these solutions. The plates were incubated overnight at 4°C in 50 μL / wells. 2) Washing: Wash the plate three times with washing buffer PBST, for 2-3 minutes each time. 3) Blocking: Add 5% milk as a blocking solution to a concentration of 200 μL / well, and block at 37°C for 1 hour. 4) Washing: Wash the plate three times with washing buffer PBST, for 2-3 minutes each time. 5) Primary antibody: Add the serum after digit dilution (from Example 2), dilute 90-fold, then 3-fold to 196,830-fold, resulting in 50 μL / well. Place two blank control wells and incubate at 37°C for 1 hour. 6) Washing: Wash the plate three times with washing buffer PBST, for 2-3 minutes each time. 7) Secondary antibody: HRP-goat anti-mouse IgG antibody (1:10000), incubated at 37°C for 40 minutes. 8) Washing: Wash the plate three times with washing buffer PBST, for 2-3 minutes each time. 9) Color development: Add TMB color developer to a well of 50 μL and allow to develop color for 10 minutes. 10) Stop: The stop solution is 50 μL / well, and the reaction is stopped. 11) Reading: The OD450nm value was measured using a microplate reader (corrected for OD630nm).

[0065] 3.6 Experimental Results The results are shown in Figure 3. The GII.4 type virus-like particle VLP provided by the present invention elicits an immune response to GI.1, GII.2, GII.7, and GII.12, with the antibody titer against GI.1 reaching levels of 10,000 or higher.

[0066] As described above, the present invention provides applications for the production of compositions and kits for the prevention, treatment, and / or diagnosis of infection with one or more types of norovirus (GI.1, GII.2, GII.7, and GII.12) using GII.4 norovirus-like particles (VLPs). It not only achieves cross-immunity with the GII genome against the GI genome, but also with GII.2, GII.7, and GII.12, achieving the objective of covering various types with a single antigen, reducing process complexity and production costs, and is expected to have a broad market.

[0067] Example 5: Animal Immunology Experiment 2.1 Laboratory animals Balb / c mice, female, 6 weeks old, 36 mice, provided by Shanghai Lingcheng Experimental Animal Technology Co., Ltd.

[0068] 2.2 Experimental Materials These are VLP proteins of various types of norovirus, prepared according to Example 1.

[0069] Aluminum hydroxide: Purchased from Beijing Bai'ao Laibo Technology Co., Ltd.

[0070] 2.3 Grouping of Experiments [Table 2]

[0071] 2.4 Animal immunity 1) Route of administration: Intramuscular injection.

[0072] 2) Method of administration: 100 μL / mouse was injected into the left hind thigh.

[0073] 3) Frequency and duration of administration: The drug was administered once every three weeks for three consecutive doses, specifically at week 0, week 3, and week 6. Blood samples were taken two weeks after the first immunization, three weeks after the second immunization, and two weeks after the third immunization as an endpoint.

[0074] 4) Serum recovery method: Whole blood was placed in a 37°C constant temperature incubator and left to stand for 40-50 minutes, then left to stand at 4°C for 1 hour, centrifuged at 12,000 rpm at 4°C for 10 minutes, the supernatant was aspirated and stored frozen in a medical cryogenic storage box in preparation for use.

[0075] Example 6: Cross-blocking experiment of pharmaceutical compositions The reagent materials and detection steps were the same as those described in Example 3. The serum sample used in the experiment was the same as that used in Example 5.

[0076] The experimental results are shown in Figures 4 and 5. Figure 4 shows the cross-immunity of each single antigen. It was found that GII.2 has a cross-immunity effect against GII.12, GII.4 has a cross-immunity effect against GI.1, GII.2, GII.7, and GII.12, GII.6 has a cross-immunity effect against GII.7, and GII.17 has a cross-immunity effect against GII.6, GII.7, and GII.12.

[0077] Figure 5 shows the composition provided by the present invention, which contains four types of norovirus VLP proteins, GII.2, GII.4, GII.6, and GII.17, and can cover seven types of norovirus. GI.1, GII.7, and GII.12 are types that are further covered by cross-immunity, and among them, the cross-immunity effect against type GI.1 is the strongest, with a BT50 of 150 or more.

[0078] Example 7: Detection of IgG antibody levels in a pharmaceutical composition The reagent materials and detection steps were the same as those described in Example 4. The serum sample used in the experiment was the same as that used in Example 5.

[0079] The experimental results are shown in Figure 6. Pharmaceutical compositions containing virus-like particles of four types of norovirus—GII.2, GII.4, GII.6, and GII.17—induced an immune response against GII.1, GII.7, and GII.12, with antibody titers reaching levels of 10,000 or higher. When comparing the IgG antibody levels of the single antigen of type GII.4 against each type, the levels for both GII.7 and GII.12 were increased by more than 10 times, demonstrating a synergistic effect of antigen cross-reactivity.

[0080] As described above, the composition containing virus-like particles of four types of norovirus, GII.2, GII.4, GII.6, and GII.17, as described in the present invention, not only achieves the objective of preventing infection with seven types of norovirus through the cross-immunity effect of the four antigens, but also exhibits a synergistic immune effect against the same cross-types among the four antigens, increasing the antibody levels of the covered types and reducing the number of antigen types, thereby lowering the complexity of the process. Therefore, it has high clinical value and is expected to have a broad market.

[0081] Although the present invention has been described in detail above, it will be obvious to those skilled in the art that various modifications and changes can be made to the present invention as long as they do not depart from the spirit and scope of the invention. The claims of the present invention are not limited to the detailed description above, and such modifications and changes fall within the scope of the claims. Although specific embodiments of the present invention have been described above as examples, these are merely illustrative examples, and it will be obvious to those skilled in the art that the claims of the present invention are limited by the attached claims. Those skilled in the art can make various changes or modifications to these embodiments as long as they do not depart from the principles and spirit of the present invention, provided that such changes or modifications all fall within the scope of the claims of the present invention.

Claims

1. A composition for inducing an animal immune response to noroviruses of types GI. 1, GII. 2, GII. 4, GII. 7 and GII. 12, comprising a virus-like particle of norovirus type GII. 4 or an active fragment thereof comprising or composed of the amino acid sequence shown in Sequence ID No.

4.

2. The composition according to claim 1, wherein the animal is a mammal.

3. The composition according to claim 1, wherein the animal is a human.

4. A composition for inducing an immune response to norovirus types GI. 1, GII. 2, GII. 4, GII. 6, GII. 7, GII. 12, and GII. 17, It contains virus-like particles or active fragments thereof of norovirus types GII.2, GII.4, GII.6, and GII.

17. GII. Virus-like particles or active fragments of norovirus type 4 contain or are composed of the amino acid sequence shown in Sequence ID No.

4. The aforementioned GII.2 type norovirus virus-like particles or their active fragments contain or are composed of the amino acid sequence shown in Sequence ID No.

2. The aforementioned GII.6 norovirus virus-like particles or active fragments thereof contain or are composed of the amino acid sequence shown in Sequence ID No.

5. A composition comprising the virus-like particles or active fragments of norovirus type GII.17, which include or are composed of the amino acid sequence shown in Sequence ID No.

8.

5. The composition according to any one of claims 1 to 4, wherein the composition is a pharmaceutical composition for preventing and / or treating norovirus infection.

6. The composition according to claim 5, wherein the pharmaceutical composition is a vaccine.

7. The composition according to claim 5 or 6, further comprising one or more adjuvants selected from aluminum adjuvants, TRL adjuvants, or saponin adjuvants.

8. The composition according to claim 7, wherein the aluminum adjuvant is selected from one or more of aluminum hydroxide, aluminum phosphate, and aluminum sulfate.

9. The composition according to claim 7, wherein the aluminum adjuvant is aluminum hydroxide.

10. The composition according to claim 7, wherein the TRL adjuvant is a TRL9 adjuvant.

11. The composition according to claim 10, wherein the TRL9 adjuvant is a CPG adjuvant.

12. The composition according to claim 11, wherein the nucleotide sequence of the CPG adjuvant is selected from SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

13. The composition according to claim 7, wherein the saponin adjuvant is QS21 or an ISCOm adjuvant.

14. The composition according to any one of claims 5 to 13, wherein the norovirus infection is gastroenteritis.

15. The composition according to any one of claims 5 to 14, wherein the norovirus infection is viral acute gastroenteritis.

16. An immunotherapy kit for detecting norovirus types GI. 1, GII. 2, GII. 4, GII. 7, and GII. 12, comprising the composition according to any one of claims 1 to 15.

17. A composition according to any one of claims 1 to 15, (1) Drugs for the prevention and / or treatment of norovirus infections of types GI. 1, GII. 2, GII. 4, GII. 7, and GII.

12. (2) A kit for diagnosing norovirus infection of types GI. 1, GII. 2, GII. 4, GII. 7, and GII. 12, or (3) Use in the manufacture of immunogens for developing norovirus antibodies of types GI. 1, GII. 2, GII. 4, GII. 7, and GII.

12.

18. The use according to claim 17, wherein norovirus infection of types GI. 1, GII. 2, GII. 4, GII. 7, and GII. 12 is gastroenteritis.

19. The use according to claim 18, wherein the gastroenteritis is viral acute gastroenteritis.

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