Hexavalent norovirus immune composition as well as kit and application thereof

Abstract

The invention provides a sexavalent norovirus immune composition. The sexavalent norovirus immune composition comprises virus-like particles or active fragments of GI.1 type norovirus, GII.2 type norovirus, GII.3 type norovirus, GII.4 type norovirus, GII.6 type norovirus and GII.17 type norovirus. The invention also provides a kit comprising the sexavalent norovirus immune composition. The invention also provides application of the sexavalent norovirus immune composition and the kit. The composition or the kit provided by the invention can realize effective immunization aiming at six valence types of GI.1 type, GII.2 type, GII.3 type, GII.4 type, GII.6 type and GII.17 type, and can be effective for a long time after administration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and relates to a hexavalent norovirus immune composition and a kit containing the composition. The present invention also provides uses of the composition and the kit. Background Art

[0002] Human norovirus was first discovered in 1972 and is the main pathogen causing acute viral gastroenteritis. It has high infectivity and is characterized by local outbreaks. It is mainly transmitted through the fecal-oral route. The main symptoms after infection are vomiting and diarrhea, followed by nausea, abdominal pain, headache, fever, chills, muscle soreness, etc. In severe cases, it can cause dehydration and even death.

[0003] Human norovirus is an unenveloped, single-stranded positive-sense RNA virus belonging to the family Caliciviridae. It has a diameter of about 26-35 nm, is unenveloped, has a rough surface, is spherical, and is icosahedrally symmetric. It was isolated from the feces of patients with acute gastroenteritis. The norovirus genome is about 7.7 kb in length and contains 3 open reading frames (ORFs).

[0004] Norovirus is divided into 7 genotypes according to the amino acid sequence of its VP1, and is further subdivided into more than 30 gene subtypes. Among them, G I, G II, and G VI are closely related to human diseases, and the GII genotype is the most common, followed by the G I genotype. Epidemiological studies show that in China, among the G II genotypes, G II.3, G II.4, and G II.17 are the most prevalent, and among the GI genotypes, G I.1 is relatively common.

[0005] Currently, for norovirus vaccines, most global research and development institutions are developing monovalent and bivalent norovirus vaccines. However, when more valent antigens are combined for immunization, the problem of interference between different valent types needs to be considered. For example, the trivalent polio vaccine and the quadrivalent dengue vaccine reported in previous studies have the phenomenon of interference between different valent types (Xiong Pei, Immunological evaluation of a quadrivalent norovirus vaccine and identification of neutralizing antibody epitopes of GI14 norovirus, Master's thesis, 2019: 36-37.).

[0006] Therefore, developing a multivalent norovirus vaccine with anti-interference ability and good immune effect is an urgent problem to be solved at present and also a major challenge in the multivalent vaccine industry. Summary of the Invention

[0007] Therefore, the object of the present invention is to provide, in view of the deficiencies of the prior art, an immune composition against noroviruses of multiple serotypes, which covers noroviruses of genotypes GI.1, GII.2, GII.3, GII.4, GII.6 and GII.17. The present invention also provides a kit comprising the immune composition. The present invention also provides the uses of the immune composition and the kit.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] On the one hand, the present invention provides a hexavalent norovirus immune composition, which comprises virus-like particles or active fragments thereof of noroviruses of genotypes GI.1, GII.2, GII.3, GII.4, GII.6 and GII.17.

[0010] According to the hexavalent norovirus immune composition of the present invention, the weight ratio between the virus-like particles or active fragments thereof of noroviruses of genotypes GI.1, GII.2, GII.3, GII.4, GII.6 and GII.17 is 40:20:10 to 100:40:40:40, preferably 40:20:30 to 40:40:40:40.

[0011] On the other hand, the present invention also provides an immune kit, which comprises the hexavalent norovirus immune composition according to the present invention;

[0012] Preferably, the immune kit is a kit for detecting noroviruses.

[0013] On the other hand, the present invention also provides the use of the hexavalent norovirus immune composition according to the present invention or the immune kit according to the present invention in the preparation of the following products,

[0014] (1) A drug for preventing and / or treating norovirus infection;

[0015] (2) A kit for diagnosing norovirus infection; or

[0016] (3) An immunogen for developing norovirus antibodies;

[0017] Preferably, the norovirus infection is gastroenteritis; more preferably, the norovirus infection is viral acute gastroenteritis.

[0018] As can be seen from the above technical solutions, the hexavalent norovirus immune composition, its kit and uses of the present invention have at least the following beneficial effects:

[0019] The hexavalent norovirus immunocomposition of the present invention can achieve effective immunity against 6 serotypes, namely GI.1, GII.2, GII.3, GII.4, GII.6 and GII.17. Through experimental research by the inventors, the hexavalent norovirus immunocomposition of the present invention can still maintain a high level of IgG antibodies 8 weeks after three administrations.

[0020] In addition, the inventors found through research that after combining the virus-like particles of GII.2, GII.4, GII.6 and GII.17 noroviruses with the virus-like particles of GI.1 and GII.3 noroviruses to form a hexavalent norovirus immunocomposition, the compatibility between each antigen serotype is improved, that is, the interference effect between each serotype can be significantly reduced. Brief Description of the Drawings

[0021] Figure 1 Shows the SDS-PAGE patterns of the purified virus-like particles of each serotype in step (2) of Example 1 of the present application.

[0022] Figure 2 Shows the detection results of the serum IgG antibody levels of mice in the hexavalent preparation group and the control sample group in Experimental Example 1 of the present application at the 14th week (wk14).

[0023] Figure 3 Shows the detection results of the serum IgG antibody levels of mice in the hexavalent preparation group and the monovalent preparation group in Experimental Example 1 of the present application at the 14th week (wk14).

[0024] Figure 4 Shows the detection results of the serum GMBT50 values of different weight ratios in the hexavalent preparation group at the 8th week (wk08) in Experimental Example 2 of the present application. Detailed Description of the Invention

[0025] The present invention will be further described below in conjunction with the drawings and through the description of specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the present invention. However, as long as they do not depart from the basic idea of the present invention, the said modifications or improvements are within the scope of the present invention.

[0026] Definition:

[0027] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For definitions and terms in the art, those skilled in the art may specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0028] It should also be noted that, as used in this specification, the term "or" may be used interchangeably with the term "and / or", unless the context clearly indicates otherwise.

[0029] The terms "pharmaceutical composition", "combination drug", and "drug combination" used herein may be used interchangeably, and refer to a combination of at least one drug and optionally a pharmaceutically acceptable excipient or adjuvant combined together to achieve a specific purpose. In certain embodiments, the composition includes a combination that is separated in time and / or space, as long as it can act together to achieve the purpose of the present invention. For example, the components contained in the pharmaceutical composition (such as GI.1, GII.2, GII.3, GII.4, GII.6, GII.17, or Al(OH)3) may be administered to the subject as a whole, or separately. When the components contained in the pharmaceutical composition are administered to the subject separately, the components may be administered to the subject simultaneously or sequentially.

[0030] The term "therapeutically effective amount" or "effective amount" used herein refers to a dose sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, depend on the individual circumstances and severity of the person being treated. The prescription of treatment (such as the determination of dosage, etc.) is ultimately the responsibility of the general practitioner and other physicians and depends on their decision-making, usually considering the disease being treated, the individual circumstances of the patient, the site of delivery, the method of administration, and other factors known to the physician.

[0031] The term "carrier" used herein is for mixing with a pharmaceutically active substance to form a dosage form for administration to a patient, but does not cause significant irritation to the patient and does not eliminate the activity of the pharmaceutically active ingredient. Preferred pharmaceutical carriers are especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The type of pharmaceutical carrier used especially depends on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular, or intravenous administration.

[0032] Hexavalent norovirus immunocomposition:

[0033] The present invention provides a hexavalent norovirus immunocomposition, which comprises virus-like particles or active fragments thereof of norovirus genotypes G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17.

[0034] The hexavalent norovirus immunocomposition according to the present invention, wherein the virus-like particles or active fragments thereof of norovirus genotype G I.1 comprise an amino acid sequence selected from one of the following or consist of the same:

[0035] (1) The amino acid sequence shown in SEQ ID NO: 1;

[0036] (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: 1; and

[0037] (3) An amino acid sequence having an amino acid substitution, deletion or insertion at one or more positions, such as 1, 2, 3, 4 or 5 positions, in the amino acid sequence shown in SEQ ID NO: 1;

[0038] Preferably, the amino acid sequence of the virus-like particles or active fragments thereof of norovirus genotype G I.1 is as shown in SEQ ID NO: 1.

[0039] The hexavalent norovirus immunocomposition according to the present invention, wherein the virus-like particles or active fragments thereof of norovirus genotype G II.2 comprise an amino acid sequence selected from one of the following or consist of the same:

[0040] (1) The amino acid sequence shown in SEQ ID NO: 2;

[0041] (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: 2; and

[0042] (3) An amino acid sequence having an amino acid substitution, deletion or insertion at one or more positions, such as 1, 2, 3, 4 or 5 positions, in the amino acid sequence shown in SEQ ID NO: 2;

[0043] Preferably, the amino acid sequence of the virus-like particles or active fragments thereof of norovirus genotype G II.2 is as shown in SEQ ID NO: 2.

[0044] The hexavalent norovirus immunocomposition according to the present invention, wherein the virus-like particles or active fragments thereof of norovirus genotype G II.3 comprise an amino acid sequence selected from one of the following or consist of the same:

[0045] (1) The amino acid sequence shown in SEQ ID NO: 3;

[0046] (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: 3; and

[0047] (3) An amino acid sequence having amino acid substitutions, deletions or insertions at one or more positions, such as 1, 2, 3, 4 or 5 positions, in the amino acid sequence shown in SEQ ID NO: 3;

[0048] Preferably, the amino acid sequence of the virus-like particle or its active fragment of norovirus G II.3 type is as shown in SEQ ID NO: 3.

[0049] According to the hexavalent norovirus immunocomposition of the present invention, wherein the virus-like particle or its active fragment of norovirus G II.4 type comprises an amino acid sequence selected from or consisting of one of the following:

[0050] (1) The amino acid sequence shown in SEQ ID NO: 4;

[0051] (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: 4; and

[0052] (3) An amino acid sequence having amino acid substitutions, deletions or insertions at one or more positions, such as 1, 2, 3, 4 or 5 positions, in the amino acid sequence shown in SEQ ID NO: 4;

[0053] Preferably, the amino acid sequence of the virus-like particle or its active fragment of norovirus G II.4 type is as shown in SEQ ID NO: 4.

[0054] According to the hexavalent norovirus immunocomposition of the present invention, wherein the virus-like particle or its active fragment of norovirus G II.6 type comprises an amino acid sequence selected from or consisting of one of the following:

[0055] (1) The amino acid sequence shown in SEQ ID NO: 5;

[0056] (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: 5; and

[0057] (3) An amino acid sequence having an amino acid substitution, deletion, or insertion at one or more positions, such as positions 1, 2, 3, 4, or 5, in the amino acid sequence shown in SEQ ID NO: 5;

[0058] Preferably, the amino acid sequence of the virus-like particle or its active fragment of norovirus G II.6 type is as shown in SEQ ID NO: 5.

[0059] The hexavalent norovirus immunocomposition according to the present invention, wherein the virus-like particle or its active fragment of norovirus G II.17 type comprises an amino acid sequence selected from one of the following or consists of the following:

[0060] (1) The amino acid sequence shown in SEQ ID NO: 6;

[0061] (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: 6; and

[0062] (3) An amino acid sequence having an amino acid substitution, deletion, or insertion at one or more positions, such as positions 1, 2, 3, 4, or 5, in the amino acid sequence shown in SEQ ID NO: 6;

[0063] Preferably, the amino acid sequence of the virus-like particle or its active fragment of norovirus G II.17 type is as shown in SEQ ID NO: 6.

[0064] The hexavalent norovirus immunocomposition according to the present invention, wherein the weight ratio between the virus-like particles or their active fragments of norovirus G I.1 type, G II.2 type, G II.3 type, G II.4 type, G II.6 type, and G II.17 type is 40:20:10 to 100:40:40:40, preferably 40:20:30 to 40:40:40:40.

[0065] As a preferred embodiment, in the hexavalent norovirus immunocomposition of the present invention, the amino acid sequence of the virus-like particle or its active fragment of G I.1 norovirus is as shown in SEQ ID NO: 1; the amino acid sequence of the virus-like particle or its active fragment of G II.2 norovirus is as shown in SEQ ID NO: 2; the amino acid sequence of the virus-like particle or its active fragment of G II.3 norovirus is as shown in SEQ ID NO: 3; the amino acid sequence of the virus-like particle or its active fragment of G II.4 norovirus is as shown in SEQ ID NO: 4; the amino acid sequence of the virus-like particle or its active fragment of G II.6 norovirus is as shown in SEQ ID NO: 5; the amino acid sequence of the virus-like particle or its active fragment of G II.17 norovirus is as shown in SEQ ID NO: 6; the weight ratio between the virus-like particles or their active fragments of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses is 40:20:40:40:40:40.

[0066] To enhance the immune response and enable the human body to obtain optimal protective immunity, the hexavalent norovirus immunocomposition of the present invention may further comprise an adjuvant, preferably an aluminum adjuvant, such as aluminum hydroxide, aluminum phosphate or aluminum sulfate, etc.

[0067] To maintain the balance of water and electrolytes, the hexavalent norovirus immunocomposition of the present invention may further comprise sodium chloride.

[0068] The hexavalent norovirus immunocomposition according to the present invention can be administered by any suitable route, such as orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously. Accordingly, the hexavalent norovirus immunocomposition of the present invention may further comprise a carrier, such as water. In addition, according to needs, the hexavalent norovirus immunocomposition of the present invention may further comprise additives, such as wetting agents, emulsifying agents or buffer substances, etc.

[0069] In the hexavalent norovirus immunocomposition of the present invention, the dosages of aluminum adjuvant, sodium chloride, water or additives, etc. can refer to the relevant schemes in the prior art, and the present invention does not make limitations.

[0070] Preparation method of hexavalent norovirus immunocomposition:

[0071] The preparation method of the hexavalent norovirus immunocomposition of the present invention includes: preparation of recombinant norovirus antigen strains and expression of recombinant proteins, purification of norovirus antigen proteins, mixing of norovirus antigens, and adsorption and mixing of norovirus antigen mixture with adjuvants.

[0072] As a preferred embodiment, the preparation method of the hexavalent norovirus immunocomposition of the present invention includes the following steps:

[0073] (1) Preparation of Recombinant Norovirus Antigen Strains and Expression of Recombinant Proteins

[0074] The gene sequences corresponding to the virus-like particle proteins of noroviruses of genotypes G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 were codon-optimized and then synthesized in full length.

[0075] The synthesized gene fragments were ligated into plasmid vectors and introduced into yeast by electroporation. After screening and amplification, the corresponding recombinant norovirus antigen strains were obtained. Among them, the plasmid vectors and yeast can be any commonly used plasmid vectors and yeast in the art. For example, the plasmid vector is the pMAUR(S.C)KARS1 plasmid and the yeast is Hansenula yeast.

[0076] The recombinant norovirus antigen strains were activated and cultured on a large scale, and the recombinant proteins were induced to express in the stationary phase.

[0077] (2) Purification of Norovirus Antigen Proteins

[0078] Each recombinant norovirus antigen strain was resuspended in a Tris buffer system, such as a 0.05M - 1.5M Tris buffer system, to prepare a bacterial suspension. The bacteria were collected by centrifugation, and the cell bodies were resuspended and lysed. The supernatant proteins were collected and subjected to gradient elution by column chromatography to collect the target proteins, thereby obtaining the virus-like particles of noroviruses of each serotype.

[0079] (3) Mixing of Norovirus Antigens

[0080] The virus-like particles of noroviruses of genotypes G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 were separately added to a sodium chloride buffer, and then the sodium chloride buffers of each virus-like particle were mixed to obtain a norovirus antigen mixture.

[0081] (4) Adsorption and Mixing of Norovirus Antigen Mixture with Adjuvant

[0082] The norovirus antigen mixture was added to aluminum adjuvant and shaken for adsorption for 0.5 - 20 hours, thereby obtaining the hexavalent norovirus immunocomposition of the present invention.

[0083] Immune kit:

[0084] The immunokit of the present invention contains the hexavalent norovirus immunocomposition of the present invention.

[0085] Preferably, the immunokit is a kit for detecting norovirus.

[0086] The immunoassay kit of the present invention may also include an instruction manual, which includes a clear description of the techniques employed when using the components of the kit to achieve the desired results, such as detection results. Optionally, the immunoassay kit of the present invention may also include other applicable components, such as measuring tools, syringes, or other applicable accessories that would be readily recognized by those skilled in the art.

[0087] Use:

[0088] The hexavalent norovirus immunocomposition or immunoassay kit of the present invention can be used for a variety of purposes. For example, it can be used for the prevention and / or treatment of norovirus infection, for the diagnosis of norovirus infection, or as an immunogen for the development of norovirus antibodies. Among them, norovirus infection is gastroenteritis, especially viral acute gastroenteritis.

[0089] Examples

[0090] The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0091] Unless otherwise specified, the experimental methods used in the following examples are all conventional experimental methods in the art. Unless otherwise specified, the experimental materials used in the following examples are all purchased from biochemical reagent sales companies.

[0092] Example 1: Preparation of Hexavalent Norovirus Immunocomposition

[0093] (1) Prepare virus-like particle (VLP) proteins of various serotypes of norovirus as follows, and their amino acid sequences are shown below:

[0094] The VLP protein of G I.1 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 1.

[0095] The VLP protein of G II.2 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 2.

[0096] The VLP protein of G II.3 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 3.

[0097] The VLP protein of G II.4 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 4.

[0098] The VLP protein of G II.6 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 5.

[0099] The VLP protein of G II.17 type norovirus, the amino acid sequence is as shown in SEQ ID NO: 6.

[0100] The specific preparation method is as follows:

[0101] The gene sequences corresponding to the virus-like particle proteins of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 were codon-optimized respectively, and entrusted to Tsingke Biotechnology Co., Ltd. for whole gene synthesis. The synthesized target gene was ligated to the pMAUR(S.C)KARS1 expression vector by Gibson assembly, and introduced into Hansenula yeast by electroporation. Positive transformants were screened using G418 resistance, and after a large amount of amplification, the recombinant Norovirus antigen strains were obtained.

[0102] The recombinant Norovirus antigen strains were inoculated into yeast extract peptone dextrose medium at an inoculation amount of 1%, cultured overnight for 24 h for small-scale activation, and the activated strains were transferred to 50 mL of yeast extract peptone dextrose medium for scale-up culture in a horizontal shaking incubator. After 16 h at 30 °C, when the strains reached the plateau phase, methanol was added to the medium for induction culture, once every 24 h for a total of two times. After the induction was completed, the cells were collected, lysed and purified to obtain the protein.

[0103] (2) Purification of Norovirus antigen protein

[0104] The cell pellet was resuspended in 0.05 M Tris buffer system to prepare a cell suspension, centrifuged for 3 h to collect the cells, the cells were redissolved and lysed using a cryogenic high-pressure cell crusher, the supernatant protein was collected, eluted by column chromatography using 0.05 M Tris buffer system containing salt, and the target protein was collected.

[0105] The purity of the protein was detected by vertical gel electrophoresis SDS-PAGE, and the results are as Figure 1 shown, where the band indicated by the arrow is the band corresponding to the virus particles of the corresponding serotype. It can be seen from Figure 1 this step that the virus particles were effectively purified.

[0106] (3) Adsorption and mixing of Norovirus antigen and adjuvant

[0107] The virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 were added to sodium chloride buffer respectively, and uniformly mixed according to the weight ratio of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 being 40:20:40:40:40:40 to obtain a Norovirus antigen mixture.

[0108] (4) Adsorption and mixing of Norovirus antigen mixture and adjuvant

[0109] Add the norovirus antigen mixture to the aluminum hydroxide solution and shake for adsorption for 2 hours.

[0110] Obtain sample S1, wherein the concentrations of virus-like particles of norovirus genotypes GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. Specifically, as shown in Table 1.

[0111] Example 2: Preparation of a hexavalent norovirus immunocomposition

[0112] The specific preparation method of Example 2 is basically the same as that of Example 1, except that in step (3), the virus-like particles of each norovirus are uniformly mixed according to the weight ratios shown in Table 1 to obtain samples S2, S3, S4, S5, S6, S19, and S20.

[0113] Among them, in the obtained sample S2, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S3, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 40 μg / ml, 20 μg / ml, 10 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S4, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 40 μg / ml, 20 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S5, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 40 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S6, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 30 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S19, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 20 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml. In the obtained sample S20, the concentrations of virus-like particles of Norovirus GI.1, GII.2, GII.3, GII.4, GII.6, and GII.17 are successively: 10 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the concentration of aluminum hydroxide is 1.5 mg / ml.

[0114] Specifically, it is shown in Table 1 as follows.

[0115] Comparative Example 1: Preparation of monovalent preparations

[0116] Prepare monovalent preparations of GI.1 type, GII.2 type, GII.3 type, GII.4 type, GII.6 type and GII.17 type, which are samples S7, S8, S9, S10, S11, S12 in sequence.

[0117] The specific preparation method is basically the same as that of Example 1, with the only difference being that: in step (3), the mixing of virus particles of each valence type is not carried out, and in step (4), the monovalent virus particles are adsorbed and mixed with the aluminum hydroxide solution.

[0118] The concentration of aluminum hydroxide in samples S7, S8, S9, S10, S11, S12 is 1.5 mg / ml, and the protein contents are 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml in sequence. Specifically as shown in Table 1.

[0119] Comparative Example 2: Preparation of a comparative sample of a hexavalent norovirus immunocomposition

[0120] In Comparative Example 2, the preparation method of virus-like particles of GII.2 type, GII.4 type, GII.6 type and GII.17 type norovirus is the same as that of Example 1.

[0121] In Comparative Example 2, the sequence information of virus-like particles of GI.1 type and GII.3 type norovirus is as follows:

[0122] GI.1 type norovirus VLP protein comparative sequence 1 (hereinafter referred to as GI.1 comparison 1), the amino acid sequence is as shown in SEQ ID NO: 7.

[0123] GI.1 type norovirus VLP protein comparative sequence 2 (hereinafter referred to as GI.1 comparison 2), the amino acid sequence is as shown in SEQ ID NO: 8.

[0124] GII.3 type norovirus VLP protein comparative sequence 1 (hereinafter referred to as GII.3 comparison 1), the amino acid sequence is as shown in SEQ ID NO: 9, and this sequence corresponds to the amino acid sequence SEQ ID No. 6 of GII.3 in Application No. 202110861640.1.

[0125] GII.3 type norovirus VLP protein comparative sequence 2 (hereinafter referred to as GII.3 comparison 2), the amino acid sequence is as shown in SEQ ID NO: 10.

[0126] Norovirus GII.3 VLP protein comparison sequence 3 (hereinafter referred to as GII.3 comparison 3), the amino acid sequence is shown in SEQ ID NO: 11.

[0127] The preparation method of the hexavalent norovirus immunocomposition comparison sample in Comparative Example 2 was basically the same as that in Example 1, except that: according to Table 1, the corresponding virus-like particles of G I.1 type or G II.3 type norovirus were used. Samples S13, S14, S15, S16 and S17 were obtained.

[0128] Among them, in sample S13, the concentrations of virus-like particles of G I.1 comparison 1, G II.2 type, G II.3 type, G II.4 type, G II.6 type and GII.17 type norovirus were successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S14, the concentrations of virus-like particles of G I.1 comparison 2, G II.2 type, G II.3 type, GII.4 type, G II.6 type and G II.17 type norovirus were successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S15, the concentrations of virus-like particles of G I.1 type, G II.2 type, G II.3 comparison 1, G II.4 type, G II.6 type and G II.17 type norovirus were successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S16, the concentrations of virus-like particles of G I.1 type, G II.2 type, G II.3 comparison 2, G II.4 type, G II.6 type and G II.17 type norovirus were successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S17, the concentrations of virus-like particles of G I.1 type, G II.2 type, G II.3 comparison 3, G II.4 type, G II.6 type and GII.17 type norovirus were successively: 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and the aluminum hydroxide concentration was 1.5 mg / ml. Specifically as shown in Table 1.

[0129] Comparative Example 3: Preparation of the hexavalent norovirus immunocomposition comparison sample

[0130] Prepare a hexavalent norovirus vaccine according to the method described in the "Specific Embodiments" section of the Chinese invention patent application with the application number 202110861640.1 to obtain sample S18.

[0131] Table 1

[0132]

[0133]

[0134] Experimental Example 1: Screening and evaluation of hexavalent norovirus immunocompositions

[0135] (1) Experimental animals: BALB / c mice, female, 6 weeks old, 104 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number SCXK(Shanghai): 2018-0003.

[0136] (2) Animal grouping: Randomly divide the mice into 13 groups, with 8 mice in each group, which are used to detect sample S1, aluminum hydroxide adjuvant group (concentration is 1.5 mg / ml), samples S7 to S12, and samples S13 to S18 respectively.

[0137] (3) Animal immunization: Immunize by intramuscular injection, 100 μl per mouse in each group. Each sample is repeatedly injected 3 times at wk00, wk03, and wk06, and blood is collected at wk05, wk08, wk10, and wk14. During the experiment, detect the specific IgG antibody titer.

[0138] (4) Detection method:

[0139] Use the indirect method to detect the level of IgG specific antibodies in serum samples: That is, coat a 96-well enzyme-linked immunosorbent assay (ELISA) plate with purified VLPs to form a solid-phase antigen. After blocking treatment, serially dilute the test serum with a conventional starting dilution factor, set multiple dilution factors, add the serially diluted serum samples to the 96-well ELISA plate, and then bind them to HRP-labeled anti-IgG / IgG1 / IgG2a antibodies to form an antigen-antibody (serum)-enzyme-labeled antibody complex. Finally, add the substrate TMB for color development, and measure the absorbance (OD value) at a wavelength of 450 nm with an ELISA reader. The intensity of the developed color is positively correlated with the level of specific antibodies IgG / IgG1 / IgG2a in the test sample. Determine the antibody titer by fitting the relationship curve between the absorbance OD value and the dilution factor of the serum sample (Log).

[0140] (5) The determination method (Tukey's multiple comparison analysis after two-factor ANOVA of Log2 values) is as follows: For the antigen-antibody reaction gradient S-shaped curve, take the upper limit value of OD to be about 2.0 and the lower limit value to be about 0.2. Take the logarithmic linear part and calculate the abscissa value when the ordinate is 0, that is, the endpoint titer of the specific antibody in the serum sample. For the starting dilution gradient with a low OD value where an antigen-antibody reaction gradient S-shaped curve cannot be made, all with OD < 0.1 are determined to have a titer of 10, and all with 0.1 ≤ OD ≤ 0.2 are determined to have a titer of the starting dilution.

[0141] (6) Test results

[0142] Figure 2 Shows the levels (95% CI) of mouse serum IgG antibodies in the hexavalent preparation group (sample S1) and the control sample group (i.e., samples S13 to S18) in this experimental example at the 14th week (wk14).

[0143] It can be seen from Figure 2 that the IgG antibody titers (Lg) of each type of G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 induced by the hexavalent norovirus immunocomposition formed by sample S1 are the highest, being 5.28, 5.00, 5.38, 4.63, 4.93, and 5.44 respectively. Compared with the hexavalent compositions (i.e., samples S13 and S14) formed with different G I.1 type antigens, the IgG antibody titer of G I.1 in sample S13 is 4.70, significantly lower than that of sample S1 (5.28). The IgG antibody titer of G I.1 in sample S14 is 5.031, slightly higher than that of sample S13 but significantly lower than that of sample S1 (5.28). Therefore, the IgG antibody titer of G I.1 in sample S1 is the highest, with a significant difference, indicating that the interference between G I.1 of sample S1 and other valent types is small and the compatibility is good.

[0144] Compared with the hexavalent compositions (i.e., samples S15, S16, and S17) formed with different G II.3 type antigens, the IgG antibody titers of G II.3 in samples S15, S16, and S17 are 4.81, 4.66, and 5.02 respectively. The IgG antibody titer of G II.3 in sample S16 is the lowest, and those in samples S15 and S17 are slightly higher, but all are significantly lower than that of sample S1 (5.38). Therefore, the IgG antibody titer of G II.3 in sample S1 is the highest, with a significant difference, indicating that the interference between GII.3 of sample S1 and other valent types is small and the compatibility is good.

[0145] The G I.1 type virus-like particles with better anti-interference ability (amino acid sequence shown in SEQ ID NO: 1) and G II.3 type virus-like particles (amino acid sequence shown in SEQ ID NO: 3) were screened out and combined into a hexavalent norovirus immunocomposition (i.e., sample S1). Compared with the immunization effect of sample S18, the IgG antibody titer of G II.3 type (5.38) was significantly higher than that of sample S18 (4.83), with a significant difference. The IgG antibody titer of G I.1 type (5.28) was also significantly higher than that of sample S18 (5.08), with a significant difference. For other serotypes, namely G II.2, G II.4, G II.6, and G II.17, the immunization effect was also slightly higher than that of sample S18. Considering comprehensively, the G I.1 type and G II.3 type with better anti-interference ability were screened out and combined with G II.2, G II.4, G II.6, and G II.17 to form a hexavalent norovirus immunocomposition (sample S1), and the interference effect among various serotypes was the smallest and the compatibility was the best.

[0146] In addition, Figure 3 It shows the mouse serum IgG antibody levels (95% CI) in the hexavalent preparation group (sample S1) and the monovalent preparation groups (samples S7 to S12) in this experimental example at the 14th week (wk14). The IgG antibody titers (Lg) induced by the corresponding monovalent preparations (i.e., samples S7 to S12) of sample S1 containing G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 were 5.41, 5.00, 5.28, 4.73, 5.05, and 5.52 respectively; the IgG antibody was maintained at a high level at 8 weeks (wk14) after three administrations, and the IgG antibody titer (Lg) was above 4.50.

[0147] Compared with the corresponding monovalent preparations, the IgG antibody level produced by the hexavalent norovirus immunocomposition (sample S1) was consistent with that of the corresponding monovalent preparations, reaching substantially the same IgG antibody titer induced by the monovalent preparations, with no significant difference (P < 0.05), indicating good compatibility among the hexavalent norovirus antigens. That is to say, there was almost no interference among the antigens of the hexavalent norovirus immunocomposition. After combining G II.2, G II.4, G II.6, and G II.17 with G I.1 type and G II.3 type to form a hexavalent norovirus immunocomposition, the compatibility among its antigens was greatly improved, the interference among its antigens was reduced, and an unexpected synergistic effect was presented, with a significant immunization effect.

[0148] Experimental Example 2: Screening of the antigen ratio of hexavalent norovirus

[0149] (1) Experimental animals: BALB / c mice, female, 6 weeks old, 48 in number, from Shanghai Lingchang Biotechnology Co., Ltd., animal license number SCXK(Shanghai): 2018-0003.

[0150] (2) Animal grouping: The mice were randomly divided into 8 groups, 6 in each group, and were used to detect samples S1 to S6, S19 and S20 respectively.

[0151] (3) Animal immunization: Immunization by intramuscular injection, 100 μl per mouse in each group. Each sample was repeatedly injected three times at wk00, wk03, and wk06, and blood was collected at wk05, wk06 or wk08. During the experiment, the blocking antibody titer was detected.

[0152] (4) Detection method for blocking antibody titer in serum

[0153] The serum to be tested was serially diluted with a diluent at a conventional starting dilution, and multiple dilutions were set. Biotin-labeled human histo-blood group antigen (HBGA) was fully bound to the streptavidin microplate. At the same time, the diluted serum sample was mixed and incubated with an equal volume of purified VLPs protein working solution. The avidin microplate bound with HBGA was washed. The incubated VLPs-serum mixture was added to the microplate. After incubation, the plate was washed. Rabbit polyclonal antibody was added. After incubation, the plate was washed. HRP-labeled goat anti-rabbit IgG was added to form an antigen-antibody (serum)-enzyme-labeled antibody complex. Finally, the substrate TMB was added for color development, and the absorbance (OD value) at a wavelength of 450 nm was measured with an enzyme-linked immunosorbent assay reader.

[0154] (5) Evaluation index: Blocking index = (1 - OD value of serum group / OD value of positive control) × 100%, and the BT50 value was calculated, that is, the highest dilution of serum that could block 50% of the binding of VLPs to HBGA.

[0155] (6) Detection results

[0156] The detection results (95% CI) of serum GMBT50 value two weeks after the third immunization (wk08) are as Figure 4 shown. In sample S1 (4 / 2 / 4 / 4 / 4 / 4), the GMBT50 values of serum against the hexavalent composition containing G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 were 720, 1524, 640, 679, 539 and 1210 respectively, and the immune effect was the best;

[0157] In the hexavalent composition of sample S2 (4 / 4 / 4 / 4 / 4 / 4), for the immunization effect against G II.2, the GMBT50 value of G II.2 is 1440, slightly lower than that of sample S1 (1524), and the difference is not significant. Increasing the dosage ratio of G II.2 instead reduces the immunization effect of G II.2. Therefore, the immunization effect of the hexavalent composition of sample S2 (4 / 4 / 4 / 4 / 4 / 4) is poor.

[0158] In the screening of the weight ratio of G II.3, different weight ratios have different effects on the immunization effect. The GMBT50 values of G II.3 for sample S3 (4 / 2 / 1 / 4 / 4 / 4), sample S4 (4 / 2 / 2 / 4 / 4 / 4), and sample S5 (4 / 2 / 3 / 4 / 4 / 4) are 400, 480, and 600 respectively. Compared with the GMBT50 value of sample S1 (640), the immunization effect of sample S3 is the worst, followed by sample S4, and the difference is significant. The GMBT50 value of sample S5 is slightly lower than that of sample S1, and the difference is not significant. Moreover, the screening of the weight ratio of G II.3 for samples S3, S4, and S5 also affects the immunization effect against G II.2, and correspondingly reduces the GMBT50 value level of G II.2. In addition, the screening of the weight ratio of G II.3 for samples S3, S4, and S5 has little effect on the immunization effects against G I.1, G II.4, G II.6, and G II.17. Compared with sample S1, the GMBT50 value levels are basically the same.

[0159] In the screening of the weight ratio of G I.1, different weight ratios have different effects on the immunization effect. The GMBT50 values of G I.1 for sample S6 (3 / 2 / 4 / 4 / 4 / 4), sample S19 (2 / 2 / 4 / 4 / 4 / 4), and sample S20 (1 / 2 / 4 / 4 / 4 / 4) are 560, 440, and 400 respectively. Compared with the GMBT50 value of sample S1 (720), the immunization effect of sample S20 is the worst, followed by sample S19, sample S6 is slightly better, slightly lower than sample S1, but all differences are significant. In addition, the screening of the weight ratio of G I.1 for samples S6, S19, and S20 has little effect on the immunization effects against GII.2, G II.3, G II.4, G II.6, and G II.17, slightly lower than sample S1, and the difference is not significant.

[0160] Therefore, compared with samples S2, S3, S4, S5, S6, S19, and S20, sample S1 containing six serotypes, namely G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17, has the highest GMBT50. The immune effect of sample S5 is slightly lower than that of sample S1, indicating that when the weight ratio of each serotype is 4 / 2 / 4 / 4 / 4 / 4, the interference between serotypes is minimized, the compatibility is the best, the interference between serotypes is greatly reduced, the compatibility between serotypes is further improved, and the immune effect is enhanced.

[0161] The above are only several exemplary embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed above with preferred embodiments, it is not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by making some changes or modifications to the disclosed technical content by those skilled in the art without departing from the technical solution of the present invention all fall within the scope of the present invention.

Claims

1. A hexavalent norovirus immune composition, characterized in that, It includes virus-like particles of Norovirus GI.1 type, GII.2 type, GII.3 type, GII.4 type, GII.6 type and GII.17 type or active fragments thereof.

2. The hexavalent norovirus immune composition according to claim 1, characterized in that, The virus-like particles of Norovirus GI.1 type or active fragments thereof include an amino acid sequence selected from one of the following or consist of the following: (1) The amino acid sequence shown in SEQ ID NO: 1; (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: 1; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 1; Preferably, the amino acid sequence of the virus-like particles of Norovirus GI.1 type or active fragments thereof is as shown in SEQ ID NO:

1.

3. The hexavalent norovirus immune composition according to claim 1 or 2, characterized in that, The virus-like particles of Norovirus GII.2 type or active fragments thereof include an amino acid sequence selected from one of the following or consist of the following: (1) The amino acid sequence shown in SEQ 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 SEQ ID NO: 2; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 2; Preferably, the amino acid sequence of the virus-like particles of Norovirus GII.2 type or active fragments thereof is as shown in SEQ ID NO:

2.

4. The hexavalent norovirus immune composition according to any one of claims 1 to 3, characterized in that, The virus-like particles of Norovirus GII.3 type or active fragments thereof include an amino acid sequence selected from one of the following or consist of the following: (1) The amino acid sequence shown in SEQ ID NO: 3; (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: 3; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 3; Preferably, the amino acid sequence of the virus-like particles of Norovirus GII.3 type or active fragments thereof is as shown in SEQ ID NO:

3.

5. The hexavalent norovirus immune composition according to any one of claims 1 to 4, characterized in that, The virus-like particles of Norovirus GII.4 type or active fragments thereof include an amino acid sequence selected from one of the following or consist of the following: (1) The amino acid sequence shown in SEQ 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 SEQ ID NO: 4; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 4; Preferably, the amino acid sequence of the virus-like particle of norovirus G II.4 or its active fragment is as shown in SEQ ID NO:

4.

6. The hexavalent norovirus immune composition according to any one of claims 1 to 5, characterized in that, The virus-like particle of norovirus GII.6 or its active fragment comprises or consists of an amino acid sequence selected from one of the following: (1) The amino acid sequence shown in SEQ ID NO: 5; (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: 5; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 5; Preferably, the amino acid sequence of the virus-like particle of norovirus G II.6 or its active fragment is as shown in SEQ ID NO:

5.

7. The hexavalent norovirus immune composition according to any one of claims 1 to 6, characterized in that, The virus-like particle of norovirus GII.17 or its active fragment comprises or consists of an amino acid sequence selected from one of the following: (1) The amino acid sequence shown in SEQ ID NO: 6; (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: 6; and (3) An amino acid sequence having one or more amino acid substitutions, deletions or insertions in the amino acid sequence shown in SEQ ID NO: 6; Preferably, the amino acid sequence of the virus-like particle of norovirus G II.17 or its active fragment is as shown in SEQ ID NO:

6.

8. The hexavalent norovirus immunocomposition according to any one of claims 1 to 7, characterized in that, The weight ratio between the virus-like particles of noroviruses G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 or their active fragments is 40∶20∶10~100∶40∶40∶40, preferably 40∶20∶30~40∶40∶40∶40.

9. The hexavalent norovirus immunocomposition according to any one of claims 1 to 8, characterized in that, It further comprises an adjuvant, preferably an aluminum adjuvant; Optionally, it further comprises sodium chloride; Optionally, it further comprises water.

10. An immune kit, characterized in that, It comprises the hexavalent norovirus immunocomposition according to any one of claims 1 to 9; Preferably, the immunokit is a kit for detecting norovirus.

11. Use of the hexavalent norovirus immunocomposition according to any one of claims 1 to 9 or the immune kit according to claim 10 in the preparation of the following products, (1) A drug for preventing and / or treating norovirus infection; (2) A kit for diagnosing norovirus infection; or (3) An immunogen for developing norovirus antibodies; Preferably, the norovirus infection is gastroenteritis; more preferably, the norovirus infection is viral acute gastroenteritis.

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