WO2024140251A1 - Pharmaceutical composition for inducing immune response of mammal against norovirus, preparation method therefor, and use thereof - Google Patents

Abstract

Provided are a pharmaceutical composition for inducing an immune response of a mammal against norovirus, a preparation method therefor, and use thereof. The pharmaceutical composition is composed of the following components: i) a norovirus VLP protein, an active fragment of the protein, a variant of the protein, or a mixture of at least two thereof; ii) an aluminum adjuvant; iii) sodium chloride; and iv) water. Further provided are a vaccine and a kit comprising the pharmaceutical composition and uses thereof. The pharmaceutical composition is not only suitable for various norovirus valence types, but also has superior adsorption completeness, blocking antibody titer and specific IgG antibody titer.

Description

Pharmaceutical composition for inducing immune response against norovirus in mammals, preparation method and use thereof Technical Field

The present invention belongs to the field of biopharmaceuticals. Specifically, the present invention relates to a pharmaceutical composition for inducing an immune response against norovirus in mammals, and a preparation method and use thereof.

Background technique

Norovirus (NoV) was first discovered by American scholar Kapikian in the feces of diarrhea patients in the town of Norwalk in 1972. The virus is one of the main pathogens causing infectious gastroenteritis in humans and acute diarrhea in infants and young children.

Norovirus is a single-stranded positive-strand RNA virus belonging to the Caliciviridae family. It has a diameter of about 26-35nm, no envelope, a rough surface, a spherical shape, and a symmetrical icosahedron. It was isolated from the feces of patients with acute gastroenteritis. The full length of the Norovirus genome is about 7.7kb, containing 3 open reading frames (ORFs). Among them, ORF1 encodes non-structural proteins, including RNA polymerase (RNA-dependent RNA polymerase, RdRp); ORF2 and ORF3 encode major (VP1) and minor (VP2) capsid proteins, respectively. Based on the phylogenetics of gene sequences, Norovirus can be divided into 6 genogroups (GI-GVI), each of which is divided into multiple genotypes. Human infections are mostly caused by GI and GII genogroups, among which GII group is the main one. According to statistics, more than 75% of human NoV acute gastroenteritis is caused by GII group infection.

Norovirus has the characteristics of diverse genotypes and rapid mutation, and there is no ideal cell culture system and animal model, which to some extent limits the development of anti-norovirus drugs, resulting in the lack of effective drugs, vaccines and preventive measures. Therefore, the development of a vaccine preparation suitable for multiple norovirus valence types is an urgent problem to be solved.

Summary of the invention

In view of the above problems, the purpose of the present invention is to provide a pharmaceutical composition for inducing an immune response against norovirus in mammals, and a preparation method and use thereof. The pharmaceutical composition is not only suitable for multiple norovirus valence types, but also has better adsorption completeness, blocking antibody titer and specific IgG antibody titer.

definition:

Unless otherwise defined, all technical terms used in this article have the same meanings as understood by ordinary technicians in the field. For the definition of terms in this field, professionals can refer to Current Protocols in Molecular Biology (Ausubel).

Although the present invention shows numerical ranges and parameter approximations in a broad range, the numerical values shown in the specific embodiments are recorded as accurately as possible. However, any numerical value is bound to contain a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, the range of "2 to 40" recorded should be considered to include any and all sub-ranges between the minimum value 2 and the maximum value 40 (including endpoints), that is, all sub-ranges starting with a minimum value of 2 or greater, such as 2 to 6.1, and sub-ranges terminating with a maximum value of 40 or less, such as 5.5 to 40. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

As used herein, the term "or" is used interchangeably with the term "and/or," unless the context clearly indicates otherwise.

The term "pharmaceutical composition" as used herein is used interchangeably with "combination drug" and "drug combination" and refers 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 pharmaceutical composition includes a combination separated in time and/or space, as long as they can work together to achieve the purpose of the present invention. For example, the ingredients contained in the pharmaceutical composition can be administered to the subject as a whole, or separately. When the ingredients contained in the pharmaceutical composition are administered to the subject separately, the ingredients can be administered to the subject simultaneously or sequentially.

The term "mammal" as used herein refers to humans or other animals, such as wild animals (e.g., herons, storks, cranes, etc.), domestic animals (e.g., ducks, geese, etc.) or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

The above object of the present invention is achieved by providing the following technical solutions:

In a first aspect, the present invention provides a pharmaceutical composition for inducing an immune response against norovirus in a mammal, the pharmaceutical composition comprising the following components:

i) Norovirus virus-like particle (VLP) protein, an active fragment of the protein, a variant of the protein, or a mixture of at least two thereof;

ii) aluminum adjuvant;

iii) sodium chloride;

iv) Water.

Preferably, the concentration of component iii) in the pharmaceutical composition is 9 g/L.

Preferably, the concentration of component i) in the pharmaceutical composition is 240-480 μg/mL.

Preferably, the concentration of component ii) in the pharmaceutical composition is 0.5-1.5 mg/mL, preferably 1.5 mg/mL.

Preferably, the weight ratio between the components i), ii) and iii) is 6-12:12.5-37.5:225, preferably 6-12:37.5:225.

Preferably, the aluminum adjuvant is selected from one or more of aluminum hydroxide, aluminum phosphate or aluminum sulfate, preferably aluminum hydroxide.

Preferably, the Norovirus VLP protein is a GI group Norovirus VLP protein and/or a GII group Norovirus VLP protein.

Preferably, the GI group Norovirus VLP protein is a GI.1 Norovirus VLP protein.

Preferably, the GI.1 Norovirus VLP protein 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;

(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.

Further preferably, the GI.1 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO:1.

Preferably, the GII group Norovirus VLP protein is selected from one or more of GII.2 Norovirus VLP protein, GII.4 Norovirus VLP protein or GII.17 Norovirus VLP protein.

Preferably, the GII.2 Norovirus VLP protein 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;

(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.

Further preferably, the GII.2 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO:2.

Preferably, the GII.4 Norovirus VLP protein 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;

(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.

Further preferably, the GII.4 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO:3.

Preferably, the GII.17 Norovirus VLP protein 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;

(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.

Further preferably, the GII.17 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO:4.

Preferably, the mammal is a human.

In a second aspect, the present invention provides a method for preparing the pharmaceutical composition according to the first aspect of the present invention, comprising the following steps:

(1) preparing an aqueous solution of component iii);

(2) adding the component i) to the aqueous solution of the component iii) to obtain a diluted solution of the component i);

(3) adding the component ii) to the aqueous solution of the component iii), and then adding the diluent of the component i) to obtain the pharmaceutical composition;

Preferably, in step (3), after adding the diluent of component i), the adsorption is carried out by shaking for 0.5-20 hours to facilitate the adsorption of component i) by component ii).

Further preferably, in step (3), after adding the diluted solution of component i), the mixture is shaken and adsorbed for 2 hours.

In a specific embodiment of the present invention, the preparation method comprises the following specific steps:

1) Prepare 9g/L sodium chloride aqueous solution: Take 9g of NaCl, dissolve it in water and dilute to 1000mL;

2) Prepare aluminum hydroxide adjuvant solution: take 67.5 mL of aluminum hydroxide gel and dilute to 650 mL with 9 g/L sodium chloride aqueous solution;

3) The Norovirus VLP protein was added to a 9 g/L sodium chloride aqueous solution for dilution to obtain a dilution solution, and the dilution solution was added dropwise to a 3.0 mg/mL aluminum hydroxide adjuvant solution to dilute the aluminum hydroxide adjuvant concentration to 1.5 mg/ml, and adsorbed by shaking for 2 hours to obtain the pharmaceutical composition.

In a third aspect, the present invention provides a vaccine comprising the pharmaceutical composition according to the first aspect of the present invention.

In a fourth aspect, the present invention provides an immunological kit comprising the pharmaceutical composition according to the first aspect of the present invention.

Preferably, the kit is a kit for detecting norovirus.

In a fifth aspect, the present invention provides use of the pharmaceutical composition according to the first aspect of the present invention, the vaccine according to the third aspect of the present invention, or the immunization kit according to the fourth aspect of the present invention in the preparation of the following products;

(1) Drugs used to prevent and/or treat norovirus infection or its related diseases;

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

(3) Immunogens used to develop norovirus antibodies.

Preferably, the Norovirus infection or its related disease is gastroenteritis, preferably acute gastroenteritis.

The pharmaceutical composition, vaccine or pharmaceutical preparation according to the present invention can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

The present invention has at least the following beneficial effects:

The inventors of the present invention unexpectedly discovered that the pharmaceutical composition for inducing an immune response in a mammal against norovirus of the present invention is not only applicable to a variety of norovirus valence types, but also has better adsorption completeness, blocking antibody titer and specific IgG antibody titer than pharmaceutical compositions of other compositions for inducing an immune response in a mammal against norovirus.

In addition, sodium chloride, as a neutral salt, usually does not have a buffering effect and is not suitable for use as a buffer system. However, in the present invention, compared with conventional buffer systems such as phosphate, histidine, citric acid, etc., the vaccine composition using only sodium chloride has more stable properties.

BRIEF DESCRIPTION OF THE DRAWINGS

The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

FIG1 is an experimental result of blocking antibody titer in aluminum hydroxide adjuvant concentration screening;

FIG2 is the experimental result of blocking antibody titer in the screening of aluminum hydroxide adjuvant adsorption time;

FIG3 is an experimental result of blocking antibody titer in the screening of components of GI.1 Norovirus vaccine composition;

FIG4 is an experimental result of specific IgG antibody titer in the screening of components of GI.1 Norovirus vaccine composition;

Figure 5 shows the experimental results of blocking antibody titer in the screening of GII.2 type Norovirus vaccine composition components;

Figure 6 shows the experimental results of specific IgG antibody titers in the screening of GII.2 type Norovirus vaccine composition components;

Figure 7 shows the experimental results of blocking antibody titers in the screening of components of the GII.4 Norovirus vaccine composition;

FIG8 is the experimental results of specific IgG antibody titers in the screening of GII.4 type Norovirus vaccine composition components;

Figure 9 shows the experimental results of blocking antibody titers in the screening of components of the GII.17 Norovirus vaccine composition;

Figure 10 shows the experimental results of specific IgG antibody titers in the screening of GII.17 type Norovirus vaccine composition components.

Detailed ways

The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

The raw materials and some experimental methods used in the following examples are described as follows:

1. Norovirus VLP protein

Norovirus VLP proteins of type GII.1, GII.2, GII.4 and GII.17 were prepared respectively, and their respective amino acid sequences are shown in SEQ ID NO: 1 to 4. For specific preparation methods, please refer to patent application CN114685622A.

2. Adsorption completeness test

1) Take the sample to be tested, obtain the supernatant after centrifugation, and detect the antigen content by ELISA method, which is recorded as the antigen content of the adsorbed supernatant sample;

2) Add the sample to be tested into the centrifuge tube, add the dissociation solution and vortex to mix, invert the centrifuge tube, incubate in a 37°C incubator for 1.5 hours, and then use ELISA to detect the antigen content. is the antigen content of the sample after dissociation.

Among them, the ELISA detection steps specifically refer to the method provided in Example 4 of patent application 202111204360X.

3. Animal Immunization Experiment

3.1 Animal Dosing

Female Balb/c mice were used, Shanghai Lingchang Experimental Animal Technology Co., Ltd. The samples to be tested were injected into the left posterior thigh muscle of the mice, 100 μL/mouse, once every 3 weeks, for 3 consecutive times, i.e., injected at week 0, week 3, and week 6, respectively. Blood was collected at the endpoint two weeks after the three injections, and serum was extracted.

3.2 Blocking antibody titer experiment

Blocking antibody titer experiment: Avidin plate was washed with 200 μL/well washing solution (0.1M PB buffer) was used to wash the plate once; HBGA (Glycotech) was diluted to 4 μg/mL, added to the avidin plate at 100 μL/well, and incubated at 25°C; after the serum was diluted in multiple ratios in a 96-well plate, the same volume of the same valence type of Norovirus VLP protein was added to each well to form a VLP-serum mixture, and a positive control (no serum) and a blank control (diluent) were set up at the same time, and incubated at 4°C; the avidin plate coated with HBGA was washed twice, the VLP-serum mixture was aspirated from the 96-well plate and added to the avidin plate, and incubated at 4°C; the avidin plate was washed 3 times, rabbit antiserum (1:10000) was added, and incubated at 4°C; the avidin plate was washed 3 times, goat anti-rabbit IgG-HRP (1:10000) was added, and incubated at 37°C; the avidin plate was washed 3 times, TMB color development solution was added, and color was developed at 25°C; stop solution (2M H ₂ SO ₄ ) to terminate the reaction; measure the OD450nm value (corrected by OD630nm).

Detection standard: BT50 was calculated as blocking index % = (1-serum group A value/positive control A value) × 100%, that is, the highest serum dilution that can block 50% of the binding between VLP and HBGA.

3.3 Specific IgG antibody titer experiment

Specific IgG antibody titer experiment: dilute the same type of Norovirus VLP protein to 1 μg/ml with coating solution (0.5% carbonate solution), coat the ELISA enzyme-linked plate, and incubate at 4°C overnight; wash the plate 3 times with washing buffer PBST; add blocking solution 5% milk, block at 37°C; wash the plate 3 times; add serum diluted in multiples, set up two blank control wells, incubate at 37°C; wash the plate 3 times; add HRP-sheep anti-mouse IgG antibody (1:10000), incubate at 37°C; wash the plate 3 times; add TMB colorimetric solution After color development, the reaction was terminated by adding a stop solution (2M H ₂ SO ₄ ); and the OD450nm value (calibrated with OD630nm) was measured by an enzyme-linked analyzer.

Example 1 Screening of aluminum hydroxide adjuvant concentration

1.1 Solution preparation

① 3.0 mg/mL aluminum hydroxide adjuvant solution: measure 67.5 mL of aluminum hydroxide gel and dilute it to 650 mL with 9 g/L sodium chloride aqueous solution;

②9g/L sodium chloride aqueous solution: Take 9g of NaCl, dissolve it in water and dilute it to 1000mL.

1.2 Experimental procedures

Take 3 portions of prepared GII.17 Norovirus VLP proteins, dilute them all with 9 g/L sodium chloride aqueous solution, add the diluted solutions dropwise into 3 portions of 3.0 mg/mL aluminum hydroxide adjuvant solutions, dilute the concentration of aluminum hydroxide adjuvant to 0.5 mg/mL, 1.0 mg/mL and 1.5 mg/mL respectively, and shake and adsorb for 2 hours to obtain Group C, Group B and Group A GII.17 Norovirus vaccine compositions, respectively.

Among them, in the GII.17 Norovirus vaccine compositions of Group C, Group B and Group A, the concentration of GII.17 Norovirus VLP protein was 480 μg/mL, and the weight ratios of GII.17 Norovirus VLP protein, aluminum hydroxide adjuvant and sodium chloride were 12:12.5:225, 12:25:225 and 12:37.5:225, respectively.

The GII.17 norovirus vaccine composition was selected for screening of aluminum hydroxide adjuvant concentration. Table 1.1 shows the aluminum hydroxide concentration screening scheme in the GII.17 norovirus VLP protein adsorption stock solution formulation screening process. Groups A-C compare the effects of different aluminum hydroxide adjuvant concentrations on the adsorbed stock solution. Six mice were included in each group, and the adsorption completeness and blocking antibody titer were measured respectively.

Table 1.2 and Figure 1 are the test data of the aluminum hydroxide adjuvant concentration screening experiment. The results show that the effect is optimal when the aluminum hydroxide adjuvant concentration is 1.5 mg/mL.

Table 1.1 Aluminum hydroxide adjuvant concentration screening experimental plan

Table 1.2 Aluminum hydroxide adjuvant concentration screening test results

Example 2 Screening of aluminum hydroxide adjuvant adsorption time

2.1 Solution preparation

The preparation of 3.0 mg/mL aluminum hydroxide adjuvant solution and 9 g/L sodium chloride aqueous solution is the same as in Example 1.

2.2 Experimental procedures

Five GII.17 Norovirus vaccine compositions with an aluminum hydroxide adjuvant concentration of 1.5 mg/mL were prepared according to the method of Example 1, except that the oscillation adsorption times were 0.5 h, 1.0 h, 2.0 h, 4.0 h and 20.0 h, respectively, to obtain GII.17 Norovirus vaccine compositions of groups A-E, respectively.

The GII.17 type Norovirus vaccine composition was selected for the screening of adsorption time. Table 2.1 shows the screening scheme for adsorption time in the screening process of the GII.17 Norovirus VLP protein adsorbed stock solution. The effects of 5 groups of different adsorption times on the adsorbed stock solution were compared. Each group had 6 mice, and the adsorption completeness and blocking antibody titer were measured respectively.

Table 2.2 and Figure 2 show the test data of the GII.17 adsorption time screening experiment. The results show that the best effect is achieved when the adsorption time is 2 hours.

Table 2.1 Screening scheme for aluminum hydroxide adjuvant adsorption time

Table 2.2 Screening test results of aluminum hydroxide adjuvant adsorption time

Example 3 Component screening experiment of GI.1 type Norovirus vaccine composition

3.1 Solution preparation

The preparation of 3.0 mg/mL aluminum hydroxide adjuvant solution and 9 g/L sodium chloride aqueous solution is the same as in Example 1;

20 mM phosphate solution: Na ₂ HPO ₄ ·12H ₂ O 1.61 g, NaH ₂ PO ₄ ·H ₂ O 2.14 g, NaCL 9 g, dissolve in water and dilute to 1000 mL, adjust pH to 6.3 with hydrochloric acid or sodium hydroxide.

3.2 Experimental procedures

Take 2 portions of the prepared GI.1 Norovirus VLP protein, dilute them with 9 g/L sodium chloride aqueous solution and phosphate solution, respectively, and add the diluted solutions dropwise to 2 portions of 3.0 mg/mL aluminum hydroxide adjuvant solution, so that the concentration of the aluminum hydroxide adjuvant is diluted to 1.5 mg/mL, and adsorb by shaking for 2 hours to obtain Group B and Group C GI.1 Norovirus vaccine compositions, respectively, and Group A (antigen group) is diluted with 20 mM phosphate solution. Among them, in each composition prepared in this embodiment, the concentration of GI.1 Norovirus VLP protein is 480 μg/mL.

The component screening experimental scheme is shown in Table 3.1. There were 6 mice in each group. The adsorption completeness, blocking antibody titer, and specific IgG antibody titer were measured respectively. The test results are shown in Table 3.2, Figure 3 and Figure 4.

A comprehensive comparison of the results showed that the best effect was achieved when 9 g/L sodium chloride aqueous solution was used after adding aluminum hydroxide adjuvant.

Table 3.1 Component screening scheme for GI.1 Norovirus vaccine composition

Table 3.2 Component screening test results of GI.1 Norovirus vaccine composition

Example 4 Component screening experiment of GII.2 Norovirus vaccine composition

4.1 Solution preparation

The preparation of 3.0 mg/mL aluminum hydroxide adjuvant solution, 20 mM phosphate solution, and 9 g/L sodium chloride aqueous solution is the same as in Example 3.

4.2 Experimental procedures

Take 2 portions of prepared GII.2 Norovirus VLP protein, dilute with 9g/L sodium chloride aqueous solution and 20mM phosphate solution, respectively, and add the diluted solution dropwise to 2 portions of 3.0mg/mL aluminum hydroxide adjuvant solution, so that the concentration of aluminum hydroxide adjuvant is diluted to 1.5mg/mL, and adsorb by shock for 2h to obtain Group B and Group C GII.2 Norovirus vaccine compositions, respectively, and Group A (antigen group) is diluted with 20mM phosphate solution. Among them, in each composition prepared in this embodiment, the concentration of GII.2 Norovirus VLP protein is 240μg/mL, the concentration of aluminum hydroxide adjuvant is 1.5mg/mL, and the weight ratio of GII.2 Norovirus VLP protein, aluminum hydroxide adjuvant and sodium chloride is 6:37.5:225, respectively.

The component screening experimental scheme is shown in Table 4.1. There were 6 mice in each group. The adsorption completeness, blocking antibody titer, and specific IgG antibody titer were measured respectively. The test results are shown in Table 4.2, Figures 5 and 6.

A comprehensive comparison of the results showed that the best effect was achieved when 9 g/L sodium chloride aqueous solution was used after adding aluminum hydroxide adjuvant.

Table 4.1 Component screening scheme for GII.2 Norovirus vaccine composition

Table 4.2 Component screening test results of GII.2 Norovirus vaccine composition

Example 5 GII.4 Component screening experiment of Norovirus vaccine composition

5.1 Solution preparation

The preparation of 3.0 mg/mL aluminum hydroxide adjuvant solution, 20 mM phosphate solution, and 9 g/L sodium chloride aqueous solution is the same as in Example 3.

① Histidine solution: 1.55g histidine, 2.095g histidine hydrochloride, 9g NaCl, dissolve in water and dilute to 1000mL.

②Citric acid solution: C ₆ H ₈ O ₇ ·H ₂ O 2.1g, Na ₃ C ₆ H ₅ O ₇ ·2H ₂ O 2.4g, NaCl 9g, dissolve in water and dilute to 1000mL;

5.2 Experimental procedures

Take 4 portions of prepared GII.4 Norovirus VLP protein, dilute with histidine solution, citric acid solution, 9g/L sodium chloride aqueous solution and 20mM phosphate solution, respectively, and add the diluted solution dropwise to 4 portions of 3.0mg/mL aluminum hydroxide adjuvant solution, so that the concentration of aluminum hydroxide adjuvant is diluted to 1.5mg/mL, and adsorb by shock for 2h to obtain B-E group GII.4 Norovirus vaccine compositions, and group A (antigen group) is diluted with 20mM phosphate solution. Among them, in each composition prepared in this embodiment, the concentration of GII.4 Norovirus VLP protein is 480μg/mL.

The experimental scheme for component screening is shown in Table 5.1. Each group had 6 mice, and the adsorption completeness, The blocking antibody titer and specific IgG antibody titer are shown in Table 5.2, Figure 7 and Figure 8.

A comprehensive comparison of the results showed that the best effect was achieved when 9 g/L sodium chloride aqueous solution was used after adding aluminum hydroxide adjuvant.

Table 5.1 Component screening scheme for GII.4 Norovirus vaccine composition

Table 5.2 Component screening scheme for GII.4 Norovirus vaccine composition

Example 6 GII.17 Norovirus vaccine composition component screening experiment

6.1 Solution preparation

The preparation of 3.0 mg/mL aluminum hydroxide adjuvant solution, 20 mM phosphate solution, and 9 g/L sodium chloride aqueous solution was the same as in Example 3, and the preparation of histidine solution was the same as in Example 5.

5 mM phosphate buffer solution: Na ₂ HPO ₄ ·12H ₂ O 0.40 g, NaH ₂ PO ₄ ·H ₂ O 0.54 g, NaCl 9 g, dissolve in water and dilute to 1000 ml, adjust pH to 6.3 with hydrochloric acid or sodium hydroxide.

6.2 Experimental procedures

Take 4 portions of prepared GII.17 Norovirus VLP protein, dilute with histidine solution, 20mM phosphate solution, 5mM phosphate solution and 9g/L sodium chloride aqueous solution, respectively, and add the diluted solution dropwise to 4 portions of 3.0mg/mL aluminum hydroxide adjuvant solution, so that the concentration of aluminum hydroxide adjuvant is diluted to 1.5mg/mL, and adsorb by shock for 2h to obtain B-D group GII.17 Norovirus vaccine composition, and group A (antigen group) is diluted with 20mM phosphate solution. Among them, in the composition prepared in this embodiment, the concentration of GII.17 Norovirus VLP protein is 480μg/mL.

The experimental scheme for component screening is shown in Table 6.1. There were 6 mice in each group. The completeness of adsorption, blocking antibody titer, and specific IgG antibody titer were measured respectively. The test results are shown in Table 6.2, Figures 9 and 10.

A comprehensive comparison of the results showed that the best effect was achieved when 9 g/L sodium chloride aqueous solution was used after adding aluminum hydroxide adjuvant.

Table 6.1 GII.17 buffer system screening experimental plan

Table 6.2 GII.17 buffer system screening test results

The above descriptions are only some exemplary embodiments of the present invention and do not constitute any form of interpretation of the present invention. Although the present invention is disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make slight changes or modifications using the above disclosed technical content to obtain equivalent or equivalent embodiments, which belong to the scope of the present invention.

Claims (10)

A pharmaceutical composition for inducing an immune response against norovirus in a mammal, the pharmaceutical composition comprising the following components: i) Norovirus VLP protein, an active fragment of the protein, a variant of the protein, or a mixture of at least two thereof; ii) aluminum adjuvant; iii) sodium chloride; iv) Water. The pharmaceutical composition according to claim 1, wherein the concentration of component iii) in the pharmaceutical composition is 9 g/L. The pharmaceutical composition according to claim 1 or 2, wherein the concentration of component i) in the pharmaceutical composition is 240-480 μg/mL; Preferably, the concentration of component ii) in the pharmaceutical composition is 0.5-1.5 mg/mL, preferably 1.5 mg/mL. The pharmaceutical composition according to any one of claims 1 to 3, wherein the weight ratio between the components i), ii) and iii) is 6-12:12.5-37.5:225, preferably 6-12:37.5:225. The pharmaceutical composition according to any one of claims 1 to 4, wherein the aluminum adjuvant is selected from one or more of aluminum hydroxide, aluminum phosphate or aluminum sulfate, preferably aluminum hydroxide. The pharmaceutical composition according to any one of claims 1 to 5, wherein the norovirus VLP protein is a GI group norovirus VLP protein and/or a GII group norovirus VLP protein; Preferably, the GI group Norovirus VLP protein is a GI.1 Norovirus VLP protein; Preferably, the GI.1 Norovirus VLP protein 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; (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; More preferably, the GI.1 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 1; Preferably, the GII group Norovirus VLP protein is selected from GII.2 Norovirus VLP protein, One or more of GII.4 Norovirus VLP protein or GII.17 Norovirus VLP protein; Preferably, the GII.2 Norovirus VLP protein 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; (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; More preferably, the GII.2 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; Preferably, the GII.4 Norovirus VLP protein 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; (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; More preferably, the GII.4 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO: 3; Preferably, the GII.17 Norovirus VLP protein 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; (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; More preferably, the GII.17 Norovirus VLP protein comprises or consists of the amino acid sequence shown in SEQ ID NO:4. A method for preparing a pharmaceutical composition according to any one of claims 1 to 6, comprising the following steps: (1) preparing an aqueous solution of component iii); (2) adding the component i) to the aqueous solution of the component iii) to obtain the component i) diluent; (3) adding the component ii) to the aqueous solution of the component iii), and then adding the diluent of the component i) to obtain the pharmaceutical composition; Preferably, in step (3), after adding the diluted solution of component i), the adsorption is carried out by shaking for 0.5-20 hours, preferably for 2 hours. A vaccine comprising the pharmaceutical composition according to any one of claims 1 to 6. An immunological kit comprising the pharmaceutical composition according to any one of claims 1 to 6; Preferably, the kit is a kit for detecting norovirus. Use of the pharmaceutical composition according to any one of claims 1 to 6, the vaccine according to claim 8 or the immunization kit according to claim 9 in the preparation of the following products: (1) Drugs used to prevent and/or treat norovirus infection or its related diseases; (2) a test kit for diagnosing norovirus infection; or (3) Immunogens for the development of norovirus antibodies; Preferably, the Norovirus infection or its related disease is gastroenteritis, preferably acute gastroenteritis.