Hexavalent norovirus immune composition, kit thereof, and use

Abstract

Provided is a hexavalent norovirus immune composition, comprising virus-like particles of norovirus genotypes GI.1, G II.2, GII.3, GII.4, GII.6, and GII.17 or active fragments thereof. Also provided is a kit comprising the hexavalent norovirus immune composition. Also provided are a use of the hexavalent norovirus immune composition and the kit. The composition or kit demonstrates effective immunization against the six norovirus genotypes GI.1, G II.2, GII.3, GII.4, GII.6, and GII.17, and can provide long-lasting efficacy after administration.

Description

Hexavalent norovirus immune composition, kit and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311729614.9 filed on December 14, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention belongs to the field of biopharmaceutical technology 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

[0004] Human norovirus was first discovered in 1972. It is the main pathogen causing acute viral gastroenteritis. It is highly contagious and 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 aches, etc. In severe cases, it can cause dehydration or even death.

[0005] Human norovirus is a non-enveloped, single-stranded, positive-sense RNA virus belonging to the Caliciviridae family. It has a diameter of approximately 26-35 nm, is non-enveloped, has a rough surface, is spherical, and has icosahedral symmetry. It was isolated from the feces of patients with acute gastroenteritis. The norovirus genome is approximately 7.7 kb long and contains three open reading frames (ORFs).

[0006] Noroviruses are classified into seven genotypes based on the amino acid sequence of their VP1, which are further subdivided into over 30 genotypes. Among these, genotypes GI, GII, and GVI are closely associated with human disease, with the GII genotype being the most common, followed by the GI genotype. Epidemiological studies have shown that in my country, among the GII genotypes, GII.3, GII.4, and GII.17 are the most prevalent, while among the GI genotypes, GI.1 is the most common.

[0007] At present, for norovirus vaccines, most research and development institutions around the world are conducting research and development of monovalent and bivalent norovirus vaccines. However, when combining multiple valent antigens for immunization, it is necessary to consider the problem of mutual interference between the valent types. For example, the trivalent polio vaccine and quadrivalent dengue vaccine reported in previous studies have the phenomenon of mutual interference between the valent types (Xiong Pei, Immunological evaluation of norovirus quadrivalent vaccine and identification of GI14 norovirus blocking antibody epitopes, Master's thesis, 2019: 36-37.).

[0008] Therefore, developing a polyvalent norovirus vaccine that has anti-interference capabilities and good immune effects is an urgent problem that needs to be solved and is also a major challenge in the polyvalent vaccine industry.

[0009] Summary of the Invention

[0010] Therefore, the present invention aims to address the deficiencies of the prior art and provide an immune composition targeting multiple valence types of norovirus, including G1.1, G1.2, G1.3, G1.4, G1.6, and G1.17. The present invention also provides a kit comprising the immune composition. The present invention also provides uses of the immune composition and kit.

[0011] The purpose of the present invention is achieved through the following technical solutions:

[0012] On the one hand, the present invention provides a hexavalent norovirus immune composition, which comprises virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus or their active fragments.

[0013] According to the hexavalent norovirus immune composition of the present invention, the weight ratio between the virus-like particles of the G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus or their active fragments is 40:20:10 to 100:40:40:40, preferably 40:20:30 to 40:40:40:40.

[0014] In another aspect, the present invention further provides an immune kit comprising the hexavalent norovirus immune composition according to the present invention;

[0015] Preferably, the immunological kit is a kit for detecting norovirus.

[0016] In another aspect, the present invention further provides use of the hexavalent norovirus immune composition according to the present invention or the immune kit according to the present invention in preparing the following products:

[0017] (1) Drugs used to prevent and / or treat norovirus infection;

[0018] (2) a test kit for diagnosing norovirus infection; or

[0019] (3) Immunogens for developing norovirus antibodies;

[0020] Preferably, the norovirus infection is gastroenteritis; more preferably, the norovirus infection is viral acute gastroenteritis.

[0021] It can be seen from the above technical solutions that the hexavalent norovirus immune composition, its kit and use of the present invention have at least the following beneficial effects:

[0022] The hexavalent norovirus immune composition of the present invention can effectively immunize against six valence types: G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17. The inventors' experimental studies have shown that the hexavalent norovirus immune composition of the present invention can still maintain high levels of IgG antibodies 8 weeks after three doses.

[0023] In addition, the inventors found through research that after combining virus-like particles of G II.2, G II.4, G II.6 and G II.17 noroviruses with virus-like particles of G I.1 and G II.3 noroviruses to form a hexavalent norovirus immune composition, the compatibility between the various antigen valences is improved, that is, the interference between the various valences can be significantly reduced.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG1 shows the SDS-PAGE profile of virus-like particles of various valencies purified in step (2) of Example 1 of the present application.

[0026] FIG2 shows the results of the test of IgG antibody levels in the serum of mice in the hexavalent preparation group and the control sample group at week 14 (wk14) in Experimental Example 1 of the present application.

[0027] FIG3 shows the results of the test of IgG antibody levels in the serum of mice in the hexavalent preparation group and the monovalent preparation group at week 14 (wk14) in Experimental Example 1 of the present application.

[0028] FIG4 shows the test results of serum GMBT50 values ​​of the hexavalent preparation group at different weight ratios at week 8 (wk08) in Experimental Example 2 of the present application.

[0029] Best Mode for Carrying Out the Invention

[0030] The present invention will be further described below with reference to the accompanying drawings and through descriptions of specific embodiments, but this is not a limitation of the present invention. Those skilled in the art may make various modifications or improvements based on the basic idea of ​​the present invention. However, as long as they do not depart from the basic idea of ​​the present invention, the modifications or improvements are within the scope of the present invention.

[0031] definition:

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0033] It should also be noted that, as used in this specification, the term "or" is used interchangeably with the term "and / or," unless the context clearly indicates otherwise.

[0034] As used herein, the terms "pharmaceutical composition," "combination drug," and "drug combination" are used interchangeably and refer to a combination of at least one drug and optional pharmaceutically acceptable excipients or adjuvants that are combined together to achieve a specific purpose. In certain embodiments, the composition includes combinations that are separated in time and / or space, as long as they can work together to achieve the purposes of the present invention. For example, the ingredients contained in the pharmaceutical composition (e.g., G I.1, G II.2, G II.3, G II.4, G II.6, G II.17, or Al(OH) 3) can be administered to a subject as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered to a subject separately, the ingredients can be administered to the subject simultaneously or sequentially.

[0035] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the individual condition and severity of the patient being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, generally taking into account the disease being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the physician.

[0036] As used herein, the term "carrier" is used to mix with the pharmaceutically active substance to form a dosage form for administration to a patient without causing significant irritation to the patient and without eliminating the activity of the pharmaceutically active ingredient. Preferred pharmaceutical carriers are, in particular, water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffered saline), 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 depends, inter alia, on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.

[0037] Hexavalent norovirus immune composition:

[0038] The present invention provides a hexavalent norovirus immune composition, which comprises virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus or their active fragments.

[0039] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of the GI.1 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

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

[0041] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 1; and

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

[0043] Preferably, the amino acid sequence of the virus-like particle of G I.1 norovirus or its active fragment is shown in SEQ ID NO: 1.

[0044] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of GII.2 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

[0045] (1) the amino acid sequence shown in SEQ ID NO: 2;

[0046] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2; and

[0047] (3) an amino acid sequence having an amino acid substitution, deletion, or insertion at one or more positions, such as position 1, 2, 3, 4, or 5, in the amino acid sequence shown in SEQ ID NO: 2;

[0048] Preferably, the amino acid sequence of the virus-like particle of GII.2 norovirus or its active fragment is shown in SEQ ID NO: 2.

[0049] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of the G II.3 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

[0050] (1) the amino acid sequence shown in SEQ ID NO: 3;

[0051] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 3; and

[0052] (3) an amino acid sequence having an amino acid substitution, deletion, or insertion at one or more positions, such as position 1, 2, 3, 4, or 5, in the amino acid sequence shown in SEQ ID NO: 3;

[0053] Preferably, the amino acid sequence of the virus-like particle of GII.3 norovirus or its active fragment is shown in SEQ ID NO: 3.

[0054] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of the GII.4 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

[0055] (1) the amino acid sequence shown in SEQ ID NO: 4;

[0056] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 4; and

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

[0058] Preferably, the amino acid sequence of the virus-like particle of GII.4 norovirus or its active fragment is shown in SEQ ID NO: 4.

[0059] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of the GII.6 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

[0060] (1) the amino acid sequence shown in SEQ ID NO: 5;

[0061] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 5; and

[0062] (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, in the amino acid sequence shown in SEQ ID NO: 5;

[0063] Preferably, the amino acid sequence of the virus-like particle of GII.6 norovirus or its active fragment is shown in SEQ ID NO: 5.

[0064] According to the hexavalent norovirus immune composition of the present invention, the virus-like particles of the G II.17 norovirus or the active fragment thereof comprise or consist of an amino acid sequence selected from one of the following:

[0065] (1) the amino acid sequence shown in SEQ ID NO: 6;

[0066] (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 6; and

[0067] (3) an amino acid sequence having an amino acid substitution, deletion, or insertion at one or more positions, such as position 1, 2, 3, 4, or 5, in the amino acid sequence of SEQ ID NO: 6;

[0068] Preferably, the amino acid sequence of the virus-like particle of GII.17 norovirus or its active fragment is shown in SEQ ID NO: 6.

[0069] According to the hexavalent norovirus immune composition of the present invention, the weight ratio between the virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus or their active fragments is 40:20:10 to 100:40:40:40, preferably 40:20:30 to 40:40:40:40.

[0070] As a preferred embodiment, in the hexavalent norovirus immune composition of the present invention, the amino acid sequence of the virus-like particles of G I.1 norovirus or its active fragment is shown as SEQ ID NO: 1; the amino acid sequence of the virus-like particles of G II.2 norovirus or its active fragment is shown as SEQ ID NO: 2; the amino acid sequence of the virus-like particles of G II.3 norovirus or its active fragment is shown as SEQ ID NO: 3; the amino acid sequence of the virus-like particles of G II.4 norovirus or its active fragment is shown as SEQ ID NO: 4; the amino acid sequence of the virus-like particles of G II.6 norovirus or its active fragment is shown as SEQ ID NO: 5; the amino acid sequence of the virus-like particles of G II.17 norovirus or its active fragment is shown as SEQ ID NO: 6; the weight ratio between the virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus or their active fragments is 40:20:40:40:40:40.

[0071] In order to enhance the immune response and enable the human body to obtain optimal protective immunity, the hexavalent norovirus immune composition of the present invention may further contain an adjuvant, preferably an aluminum adjuvant, such as aluminum hydroxide, aluminum phosphate or aluminum sulfate.

[0072] In order to maintain water and electrolyte balance, the hexavalent norovirus immune composition of the present invention may further contain sodium chloride.

[0073] The hexavalent norovirus immune composition according to the present invention can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly, or intravenously. Accordingly, the hexavalent norovirus immune composition of the present invention may further comprise a carrier, such as water. Furthermore, as desired, the hexavalent norovirus immune composition of the present invention may further comprise additives, such as wetting agents, emulsifiers, or buffer substances.

[0074] In the hexavalent norovirus immune composition of the present invention, the dosage of aluminum adjuvant, sodium chloride, water or additives can refer to the relevant schemes in the prior art, and the present invention is not limited thereto.

[0075] Preparation method of hexavalent norovirus immune composition:

[0076] The preparation method of the hexavalent norovirus immune composition of the present invention includes: preparation of recombinant norovirus antigen strain and expression of recombinant protein, purification of norovirus antigen protein, mixing of norovirus antigen, and adsorption mixing of norovirus antigen mixture and adjuvant.

[0077] As a preferred embodiment, the method for preparing the hexavalent norovirus immune composition of the present invention comprises the following steps:

[0078] (1) Preparation of recombinant norovirus antigen strain and expression of recombinant protein

[0079] The gene sequences corresponding to the virus-like particle proteins of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses were codon optimized and fully synthesized.

[0080] The synthesized gene fragment is ligated into a plasmid vector and introduced into yeast via electroporation. After screening and amplification, the corresponding recombinant norovirus antigen strain is obtained. The plasmid vector and yeast can be any commonly used plasmid vector and yeast in the art. For example, the plasmid vector is the pMAUR(SC)KARS1 plasmid, and the yeast is Hansenula.

[0081] The recombinant norovirus antigen strain was activated and expanded, and the recombinant protein was induced to express in the stationary phase.

[0082] (2) Purification of Norovirus Antigen Protein

[0083] Each recombinant norovirus antigen strain is resuspended in a Tris buffer system, for example, a concentration of 0.05M-1.5M Tris buffer system, to prepare a bacterial suspension, collect the bacteria by centrifugation, redissolve the bacteria and break the bacterial cells, collect the supernatant protein, perform gradient elution by column chromatography, collect the target protein, and thus obtain virus-like particles of each valence type of norovirus.

[0084] (3) Mixture of Norovirus Antigens

[0085] Virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses were respectively added to sodium chloride buffer, and then the sodium chloride buffer solutions of the virus-like particles were mixed to obtain a norovirus antigen mixture.

[0086] (4) Adsorption mixing of norovirus antigen mixture and adjuvant

[0087] The norovirus antigen mixture is added to the aluminum adjuvant and adsorbed by shaking for 0.5-20 hours to obtain the hexavalent norovirus immune composition of the present invention.

[0088] Immunoassay kit:

[0089] The immune kit of the present invention comprises the hexavalent norovirus immune composition of the present invention.

[0090] Preferably, the immunoassay kit is a kit for detecting norovirus.

[0091] The immunoassay kits of the present invention may also include instructions for use, which include a clear description of the techniques to be employed when using the components of the kit to achieve a desired result, such as a test result. Optionally, the immunoassay kits of the present invention may also include other suitable components, such as measuring tools, syringes, or other suitable accessories that will be readily recognized by those skilled in the art.

[0092] use:

[0093] The hexavalent norovirus immune composition or immune kit of the present invention can be used for various purposes, for example, for preventing and / or treating norovirus infection, for diagnosing norovirus infection, or as an immunogen for developing norovirus antibodies. The norovirus infection is gastroenteritis, particularly viral acute gastroenteritis. Example

[0094] The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0095] The experimental methods used in the following examples, unless otherwise specified, are all conventional experimental methods in the art. The experimental materials used in the following examples, unless otherwise specified, were purchased from biochemical reagent sales companies.

[0096] Example 1: Preparation of a hexavalent norovirus immune composition

[0097] (1) The following virus-like particles (VLP) proteins of various valence types of norovirus were prepared, and their amino acid sequences are shown below:

[0098] G I.1 type Norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 1.

[0099] G II.2 type Norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 2.

[0100] G II.3 type norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 3.

[0101] G II.4 type norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 4.

[0102] G II.6 type norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 5.

[0103] G II.17 type Norovirus VLP protein, the amino acid sequence is shown in SEQ ID NO: 6.

[0104] The specific preparation method is as follows:

[0105] The gene sequences corresponding to the virus-like particle proteins of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses were codon-optimized, and Qingke Biotechnology Co., Ltd. was commissioned to perform full gene synthesis. The synthesized target genes were ligated into the pMAUR(SC)KARS1 expression vector via Gibson assembly and introduced into Hansenula by electroporation. Positive transformants were screened for G418 resistance, and recombinant norovirus antigen strains were obtained after extensive amplification.

[0106] The recombinant norovirus strain was inoculated into a yeast extract peptone-dextrose medium at a 1% inoculum size and cultured overnight for 24 hours. The activated strain was then transferred to 50 mL of yeast extract peptone-dextrose medium and expanded in a horizontal shaking incubator at 30°C for 16 hours. Once the strain reached the stationary phase, methanol was added to the medium for induction, twice every 24 hours. After induction, the cells were harvested, disrupted, and the protein was purified.

[0107] (2) Purification of Norovirus Antigen Protein

[0108] The bacterial cells were resuspended in 0.05M Tris buffer to prepare a bacterial suspension, centrifuged for 3 hours, and the bacterial cells were collected. The bacterial cells were redissolved and disrupted with a low-temperature high-pressure cell disruptor. The supernatant protein was collected and eluted with a salt-containing 0.05M Tris buffer system by column chromatography to collect the target protein.

[0109] Protein purity was determined by vertical gel electrophoresis (SDS-PAGE). The results are shown in Figure 1, where the arrows indicate the bands corresponding to the viral valences. As can be seen from Figure 1, this step effectively purified each viral particle.

[0110] (3) Adsorption and mixing of norovirus antigen and adjuvant

[0111] Virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses were respectively added to sodium chloride buffer, and evenly mixed in a weight ratio of 40:20:40:40:40:40:40 of virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 noroviruses to obtain a norovirus antigen mixture.

[0112] (4) Adsorption mixing of norovirus antigen mixture and adjuvant

[0113] The Norovirus antigen mixture was added to the aluminum hydroxide solution and adsorbed by shaking for 2 hours.

[0114] Sample S1 was obtained, in which the concentrations of virus-like particles of norovirus types G1.1, GII.2, GII.3, GII.4, GII.6, and GII.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. The details are shown in Table 1.

[0115] Example 2: Preparation of a hexavalent norovirus immune composition

[0116] The specific preparation method of Example 2 is basically the same as that of Example 1, with the only difference being that in step (3), the virus-like particles of each norovirus are uniformly mixed according to the weight ratio shown in Table 1 to obtain samples S2, S3, S4, S5, S6, S19, and S20.

[0117] Among them, the concentrations of virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus in sample S2 were: 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the concentration of aluminum hydroxide was 1.5 mg / ml. The concentrations of virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 norovirus in sample S3 were: 40 μg / ml, 20 μg / ml, 10 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the concentration of aluminum hydroxide was 1.5 mg / ml. In sample S4, the concentrations of virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 noroviruses were 40 μg / ml, 20 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S5, the concentrations of virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 noroviruses were 40 μg / ml, 20 μg / ml, 30 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S6, the concentrations of virus-like particles of noroviruses G1.1, GII.2, GII.3, GII.4, GII.6, and GII.17 were 30 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S19, the concentrations of virus-like particles of noroviruses G1.1, GII.2, GII.3, GII.4, GII.6, and GII.17 were 20 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S20, the concentrations of virus-like particles of norovirus types G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 are 10 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the concentration of aluminum hydroxide is 1.5 mg / ml.

[0118] The details are shown in Table 1.

[0119] Comparative Example 1: Preparation of monovalent formulation

[0120] Monovalent preparations of 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 were prepared respectively, and they were samples S7, S8, S9, S10, S11 and S12 in order.

[0121] The specific preparation method is basically the same as that of Example 1, except that: in step (3), the virus particles of different valences are not mixed, and in step (4), the monovalent virus particles are adsorbed and mixed with the aluminum hydroxide solution.

[0122] The aluminum hydroxide concentration in samples S7, S8, S9, S10, S11, and S12 was 1.5 mg / ml, and the protein content was 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, as shown in Table 1.

[0123] Comparative Example 2: Preparation of a comparative sample of a hexavalent norovirus immune composition

[0124] In Comparative Example 2, the preparation method of virus-like particles of G II.2, G II.4, G II.6 and G II.17 noroviruses was the same as that in Example 1.

[0125] In Comparative Example 2, the sequence information of virus-like particles of G I.1 and G II.3 noroviruses is as follows:

[0126] G I.1 type norovirus VLP protein comparison sequence 1 (hereinafter referred to as G I.1 comparison 1), the amino acid sequence is shown in SEQ ID NO: 7.

[0127] The comparison sequence of G I.1 type norovirus VLP protein 2 (hereinafter referred to as G I.1 comparison 2) is shown in SEQ ID NO: 8.

[0128] G II.3 type norovirus VLP protein comparison sequence 1 (hereinafter referred to as G II.3 comparison 1), the amino acid sequence is shown in SEQ ID NO: 9, which corresponds to the amino acid sequence SEQ ID No. 6 of GII.3 in application number 202110861640.1.

[0129] G II.3 type norovirus VLP protein comparison sequence 2 (hereinafter referred to as G II.3 comparison 2), the amino acid sequence is shown in SEQ ID NO: 10.

[0130] G II.3 type norovirus VLP protein comparison sequence 3 (hereinafter referred to as G II.3 comparison 3), the amino acid sequence is shown in SEQ ID NO: 11.

[0131] The preparation method for the comparative sample of the hexavalent norovirus immune composition in Comparative Example 2 was essentially the same as that in Example 1, except that virus-like particles of the corresponding G1.1 or G11.3 norovirus were used according to Table 1. Samples S13, S14, S15, S16, and S17 were obtained.

[0132] In sample S13, the concentrations of virus-like particles of noroviruses G1.1 vs. 1, GII.2, GII.3, GII.4, GII.6, and GII.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S14, the concentrations of virus-like particles of noroviruses G1.1 vs. 2, GII.2, GII.3, GII.4, GII.6, and GII.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S15, the concentrations of virus-like particles of noroviruses G1.1, G1.2, G1.3 Comparison 1, G1.4, G1.6, and G1.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S16, the concentrations of virus-like particles of noroviruses G1.1, G1.2, G1.3 Comparison 2, G1.4, G1.6, and G1.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. In sample S17, the concentrations of virus-like particles of noroviruses G1.1, G1.2, G1.3 vs. 3, G1.4, G1.6, and G1.17 were 40 μg / ml, 20 μg / ml, 40 μg / ml, 40 μg / ml, 40 μg / ml, and 40 μg / ml, respectively, and the aluminum hydroxide concentration was 1.5 mg / ml. The details are shown in Table 1.

[0133] Comparative Example 3: Preparation of a comparative sample of a hexavalent norovirus immune composition

[0134] The hexavalent norovirus vaccine was prepared according to the method described in the "Specific Implementation Method" section of the Chinese invention patent application with application number 202110861640.1 to obtain sample S18.

[0135] Table 1

[0136] Experimental Example 1: Screening and efficacy evaluation of hexavalent norovirus immune composition

[0137] (1) Experimental animals: BALB / c female mice, 6 weeks old, 104 mice, Shanghai Lingchang Biotechnology Co., Ltd., animal license number SCXK (Shanghai): 2018-0003.

[0138] (2) Animal grouping: The mice were randomly divided into 13 groups, with 8 mice in each group, which were used to detect sample S1, aluminum hydroxide adjuvant group (concentration was 1.5 mg / ml), samples S7 to S12, and samples S13 to S18.

[0139] (3) Animal Immunization: Immunization was performed by intramuscular injection, with 100 μl injected per animal per group. Each sample was administered three times at wk00, wk03, and wk06. Blood was collected at wk05, wk08, wk10, and wk14. Specific IgG antibody titers were measured during the experiment.

[0140] (4) Detection method:

[0141] The indirect method is used to detect the level of IgG-specific antibodies in serum samples: purified VLPs are coated on a 96-well ELISA plate to form a solid-phase antigen. After blocking treatment, the serum to be tested is diluted in multiple ratios at the conventional starting dilution. Multiple dilutions are set and the serially diluted serum samples are added to the 96-well ELISA plate. The serum samples are then combined with HRP-labeled anti-IgG / IgG1 / IgG2a antibodies to form an antigen-antibody (serum)-enzyme-labeled antibody complex. Finally, the substrate TMB is added for color development and the absorbance (OD value) at a wavelength of 450 nm is measured using a microplate reader. The depth of the color is positively correlated with the level of specific antibodies IgG / IgG1 / IgG2a in the test sample. The antibody titer is determined by fitting the relationship curve between the absorbance OD value and the serum sample dilution multiple (Log).

[0142] (5) The determination method (Tukey's multiple comparison analysis after two-way ANOVA of Log2 values) is as follows: for the antigen-antibody reaction gradient S-shaped curve, the OD upper limit is about 2.0 and the lower limit is about 0.2. The logarithmic linear part is used to calculate the value of the horizontal axis when the vertical axis is 0, which is the endpoint titer of the specific antibody in the serum sample; for the initial dilution gradient OD value is too low to make the antigen-antibody reaction gradient S-shaped curve, all OD values ​​less than 0.1 are determined to have a titer of 10, and all 0.1≤OD≤0.2 are determined to have a titer of the starting dilution.

[0143] (6) Test results

[0144] FIG2 shows the serum IgG antibody levels (95% CI) of mice in the hexavalent preparation group (sample S1) and the control sample group (ie, samples S13 to S18) at week 14 (wk14) of this experimental example.

[0145] The hexavalent norovirus immune composition formed by sample S1 induced the highest IgG antibody titers (Lg) of G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17, which were 5.28, 5.00, 5.38, 4.63, 4.93, and 5.44, respectively; compared with the hexavalent compositions formed by different G I.1 type antigens (i.e., samples S13 and S14), the IgG antibody titer of G I.1 in sample S13) was 4.70, which was significantly lower than that of sample S1 (5.28), and the IgG antibody titer of G I.1 in sample S14 was 5.031, which was slightly higher than sample S13, but significantly lower than sample S1 (5.28). Therefore, the IgG antibody titer of G I.1 of sample S1 was the highest, and the difference was significant, indicating that the interference between G I.1 of sample S1 and other valence types was small and the compatibility was good.

[0146] Compared with the hexavalent compositions formed by different G II.3 antigens (i.e., samples S15, S16, and S17), the IgG antibody titers of G II.3 in samples S15, S16, and S17 were 4.81, 4.66, and 5.02, respectively, with sample S16 being the lowest, and samples S15 and S17 being slightly higher, but all significantly lower than sample S1 (5.38). Therefore, the IgG antibody titer of G II.3 in sample S1 was the highest, and the difference was significant, indicating that the interference between G II.3 in sample S1 and other valence types was small, and the compatibility was better.

[0147] The G I.1 type virus-like particles (amino acid sequence shown in sequence SEQ ID NO: 1) and G II.3 type virus-like particles (amino acid sequence shown in sequence SEQ ID NO: 3) with good anti-interference ability were screened out and combined into a hexavalent norovirus immune composition (i.e., sample S1). Compared with the immune 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. The immune effects of other valence types, i.e., G II.2, G II.4, G II.6, and G II.17, were also slightly higher than that of sample S18. After comprehensive consideration, G I.1 and G II.3 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 immune composition (sample S1). The interference between each valence type is minimal and the compatibility is best.

[0148] In addition, Figure 3 shows the serum IgG antibody levels (95% CI) of mice in the hexavalent formulation group (sample S1) and the monovalent formulation group (samples S7 to S12) at week 14 (wk14) of this experimental example. The IgG antibody titers (Lg) induced by the monovalent formulations corresponding to sample S1 containing G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 (i.e., samples S7 to S12) were 5.41, 5.00, 5.28, 4.73, 5.05, and 5.52, respectively; high levels of IgG antibodies were maintained for 8 weeks (wk14) after three doses, with IgG antibody titers (Lg) above 4.50.

[0149] Compared with the corresponding monovalent preparations, the IgG antibody level produced by the hexavalent norovirus immune composition (sample S1) is consistent with the corresponding monovalent preparations, reaching substantially the same IgG antibody titer as that induced by each monovalent preparation, and the difference is not significant (P < 0.05), indicating that the hexavalent norovirus antigens are compatible with each other. In other words, there is almost no interference between the antigens in the hexavalent norovirus immune composition. After G II.2, G II.4, G II.6, G II.17 are combined with G I.1 and G II.3 to form a hexavalent norovirus immune composition, the compatibility between the antigens is greatly improved, the interference between the antigens is reduced, and an unexpected synergistic effect is presented, with a significant immune effect.

[0150] Experimental Example 2: Screening of Hexavalent Norovirus Antigen Ratios

[0151] (1) Experimental animals: BALB / c female mice, 6 weeks old, 48 mice, Shanghai Lingchang Biotechnology Co., Ltd., animal license number SCXK (Shanghai): 2018-0003.

[0152] (2) Animal grouping: The mice were randomly divided into 8 groups, with 6 mice in each group, and used to detect samples S1 to S6, S19 and S20, respectively.

[0153] (3) Animal Immunization: Immunization was performed by intramuscular injection, with 100 μl injected per animal per group. Each sample was administered three times at wk00, wk03, and wk06. Blood was collected at wk05, wk06, or wk08. During the experiment, blocking antibody titers were measured.

[0154] (4) Method for detecting blocking antibody titer in serum

[0155] The serum to be tested is serially diluted with a conventional starting dilution using a diluent, and multiple dilutions are set. The biotin-labeled human blood group antigen (HBGA) is fully combined with the streptavidin microplate. At the same time, the diluted serum sample is mixed with an equal volume of purified VLPs protein working solution and incubated. The avidin microplate bound to HBGA is washed, and the incubated VLPs-serum mixture is added to the microplate. After incubation, the plate is washed, rabbit polyclonal antibody is added, the plate is washed after incubation, and HRP-labeled goat anti-rabbit IgG is added to form an antigen-antibody (serum)-enzyme-labeled antibody complex. Finally, the substrate TMB is added for color development, and the absorbance (OD value) at a wavelength of 450nm is measured using a microplate reader.

[0156] (5) Evaluation index: blocking index = (1-serum group OD / positive control OD) × 100%, and calculate the BT50 value, that is, the highest dilution of serum that can block 50% of the binding of VLPs to HBGA.

[0157] (6) Test results

[0158] The results (95% CI) of serum GMBT50 values ​​two weeks after the triple immunization (wk08) were as follows: in sample S1 (4 / 2 / 4 / 4 / 4 / 4), the GMBT50 values ​​of the serum against the hexavalent combination 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, indicating the best immune effect;

[0159] In the hexavalent composition of sample S2 (4 / 4 / 4 / 4 / 4 / 4), the GMBT50 value of G II.2 was 1440 for the immune effect of G II.2, which was slightly lower than that of sample S1 (1524). The difference was not significant. Increasing the dosage ratio of G II.2 actually reduced the immune effect of G II.2. Therefore, the immune effect of the hexavalent composition of sample S2 (4 / 4 / 4 / 4 / 4 / 4) was poor.

[0160] In the weight ratio screening of G II.3, different weight ratios have different effects on the immune effect. The GMBT50 values ​​of 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) on G II.3 are 400, 480, and 600, respectively. Compared with the GMBT50 value of sample S1 (640), sample S3 has the worst immune effect, followed by sample S4, with significant differences. The GMBT50 value of sample S5 is slightly lower than that of sample S1, with no significant difference. Furthermore, the weight ratio screening of samples S3, S4, and S5 for G II.3 also affects the immune effect of G II.2, and correspondingly lowers the GMBT50 value of G II.2. In addition, the weight ratio screening of samples S3, S4, and S5 for G II.3 had little effect on the immune effects of G I.1, G II.4, G II.6, and G II.17, and the GMBT50 value levels were basically the same as those of sample S1.

[0161] In the weight ratio screening of G I.1, different weight ratios have different effects on the immune effect. The GMBT50 values ​​of 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) on G I.1 are 560, 440, and 400, respectively. Compared with the GMBT50 value of sample S1 (720), sample S20 has the worst immune effect, followed by sample S19, and sample S6 is slightly better, slightly lower than sample S1, but the differences are significant. In addition, the weight ratio screening of samples S6, S19, and S20 for G I.1 has little effect on the immune effect of G II.2, G II.3, G II.4, G II.6, and G II.17, which are slightly lower than sample S1, and the differences are not significant.

[0162] Therefore, compared with samples S2, S3, S4, S5, S6, S19, and S20, sample S1 containing six valence types G I.1, G II.2, G II.3, G II.4, G II.6, and G II.17 has the highest GMBT50, and the immune effect of sample S5 is slightly lower than that of sample S1, indicating that when the weight ratio of each valence type is 4 / 2 / 4 / 4 / 4 / 4, the interference between the valence types is minimal and the compatibility is best, which greatly reduces the interference between the valence types, further improves the compatibility between the valence types, and improves the immune effect.

[0163] The above descriptions are merely exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention is disclosed above with reference to preferred embodiments, they are not intended to limit the present invention. Any equivalent or equivalent embodiments obtained by any person skilled in the art, without departing from the scope of the present invention, by making slight changes or modifications to the above-disclosed technical contents, fall within the scope of the present invention.

Claims

1. A hexavalent norovirus immune composition, characterized in that: It contains virus-like particles of G I.1, G II.2, G II.3, G II.4, G II.6 and G II.17 Norovirus or their active fragments.

2. The hexavalent norovirus immune composition according to claim 1, characterized in that: The virus-like particle of the G1.1 type Norovirus 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: 1; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 1; 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: 1; Preferably, the amino acid sequence of the virus-like particle of the G I.1 type Norovirus or its active fragment 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 particle of the GII.2 Norovirus 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: 2; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO: 2; 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: 2; Preferably, the amino acid sequence of the virus-like particle of the G II.2 type Norovirus or its active fragment 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 particle of the GII.3 type Norovirus 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: 3; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO:3; 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: 3; Preferably, the amino acid sequence of the virus-like particle of the G II.3 type Norovirus or its active fragment 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 particle of the GII.4 type Norovirus 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: 4; (2) an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO:4; 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: 4; Preferably, the amino acid sequence of the virus-like particle of the G II.4 type Norovirus 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 the GII.6 type Norovirus 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO:5; 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: 5; Preferably, the amino acid sequence of the virus-like particle of the G II.6 type Norovirus 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 the GII.17 type Norovirus 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 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence set forth in SEQ ID NO:6; 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: 6; Preferably, the amino acid sequence of the virus-like particle of the G II.17 type Norovirus or its active fragment is as shown in SEQ ID NO:

6.

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

40.

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

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

11. Use of the hexavalent norovirus immune composition 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) Drugs used to prevent and / or treat norovirus infection; (2) a test kit for diagnosing norovirus infection; or (3) Immunogens for the development of norovirus antibodies; Preferably, the Norovirus infection is gastroenteritis; more preferably, the Norovirus infection is viral acute gastroenteritis.

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