WO2022000205A1

WO2022000205A1 - Avian influenza vaccine adjuvant and use thereof

Info

Publication number: WO2022000205A1
Application number: PCT/CN2020/099030
Authority: WO
Inventors: 张文炎; 刘锦容; 詹烜子; 高艳; 刘之文; 卢少华; 黎啟林
Original assignee: 肇庆大华农生物药品有限公司
Priority date: 2020-06-29
Filing date: 2020-06-29
Publication date: 2022-01-06

Abstract

A mussel polysaccharide, used in combination with an avian influenza vaccine. The two have a synergistic effect that leads to enhanced activity of the avian influenza vaccine, resulting in ideal immunization effects. An avian influenza vaccine containing the present adjuvant allows for the expression of significantly higher antibody titer levels over the entire immune period compared to avian influenza vaccines not containing said adjuvant.

Description

A kind of avian influenza vaccine adjuvant and its application

technical field

The present invention relates to an avian influenza vaccine adjuvant and its application.

Background technique

The respiratory tract is the gateway for influenza virus invasion, and the establishment of specific immune protection in the respiratory mucosa can prevent the invasion of the virus. The live attenuated vaccine is the only type of influenza vaccine that is clinically immunized via mucosa. The vaccine induces a strong SIgA antibody response at the site of virus invasion through nasal immunization, preventing influenza virus from entering the body. However, the live attenuated vaccine has its limitations, it is only recommended for people aged 5-49 and approved for use in only a few countries. Inactivated influenza vaccines include whole virus inactivated vaccines, split vaccines and subunit vaccines. They are the types of vaccines approved for use worldwide. They are administered by intramuscular injection and mainly provide specific protection by inducing systemic IgG antibodies, which cannot be induced. SIgA. In recent years, studies have shown that intranasal immunization of inactivated influenza vaccine is also effective, but mucosal adjuvants are required to enhance its immune protection effect.

The commonly used adjuvants for influenza virus inactivated vaccines include inorganic adjuvants (such as aluminum adjuvants), surfactants (such as MF59), cytokine adjuvants (such as IFN-γ) and polysaccharide adjuvants. Natural polysaccharides widely exist in nature and have a variety of biological activities. Some natural polysaccharides can regulate a variety of immune cells and immune molecules, enhance the body's resistance, and also have anti-tumor effects. In recent years, they have received extensive attention as adjuvant research objects. .

Polysaccharide Polysaccharide (POP) has an adjuvant effect and is mainly extracted from the rhizome of Polygonatum japonica. Zhang Yanan et al. immunized the BALB/c mouse model with H7N9 influenza virus split vaccine and different doses of POP as adjuvant intranasally. The results showed that POP can enhance the immune response, has mucosal adjuvant effect, specific IgG antibody and HI antibody in serum increasing trend. The results of Shan Junjie et al. showed that total polysaccharide (IIP) of blue root has adjuvant activity. Combined use of H1N1 influenza virus lysate and polysaccharide IIP-A-1 can significantly increase the titer levels of IgG1, IgG2a, IgG2b, IgG3 and IgA, and the proliferation ability of B cells is significantly higher than that of the non-adjuvant group. It has the ability to promote the differentiation of Th1 cells, and the ability of spleen lymphocytes to secrete IFNγ has been further enhanced. Xie Guoxiu et al. administered a low-dose inactivated vaccine combined with a dose of 200 μg of Poria polysaccharide, which showed that the antibody level could be increased, but it could not resist the attack of lethal influenza virus; when the dose of Poria polysaccharide increased to 1000 μg, the antibody level of the mice in the low-dose vaccine group was increased. significantly increased, and serum IgG titers were significantly higher than in the unadjuvanted group.

To sum up, although various polysaccharides have been reported in the literature as adjuvants for influenza virus vaccines, it is still of great significance to develop new natural and non-toxic polysaccharide adjuvants.

SUMMARY OF THE INVENTION

The first aspect of the present invention is to provide an avian influenza vaccine adjuvant, which can enhance the immune function of the body.

The efficient utilization and in-depth development of marine shellfish resources is an important research direction for the sustainable development of my country's economic shellfish aquaculture industry. Among them, shellfish polysaccharides are widely used in anti-tumor, anti-virus, immune regulation and liver injury protection due to their unique structure. This aspect shows a wide range of biological activities, and has a good application prospect in the research and development of functional health food and innovative drugs. The present invention selects a polysaccharide that can be used as an adjuvant for avian influenza vaccines from the mussel polysaccharides reported in the prior art.

In order to solve the technical problem of the present invention, the present invention provides an avian influenza vaccine adjuvant, which comprises mussel polysaccharide.

Preferably, the mussel polysaccharide has (1→4)-α-D-Glc as the main chain, contains (1→2)-α-D-Glc branches, and the configuration is α-glucan , there is an average (1→2) glucose branch in every 12 glucose main chains, the relative molecular mass of the mussel polysaccharide is 800-8000KDa, and its structural formula is:

The mussel polysaccharide is extracted from mussels, such as thick-shelled mussels and blue mussels. The extraction method is carried out with reference to the method reported in the document CN107056962A.

Another aspect of the present invention provides use of an avian influenza vaccine adjuvant in preparing an avian influenza vaccine formulation, the avian influenza vaccine adjuvant comprising mussel polysaccharide. Combining the mussel polysaccharide of the present invention with avian influenza vaccine can enhance the immune activity of the vaccine.

Preferably, the avian influenza vaccine is H9N2 subtype avian influenza vaccine.

Another aspect of the present invention provides a mussel polysaccharide, which can enhance the immune response of avian influenza vaccine and can be used as an avian influenza vaccine adjuvant.

The beneficial effects of the present invention are:

The invention adopts the mussel polysaccharide and the avian influenza vaccine in combination, and has a synergistic effect in effect, can enhance the activity of the avian influenza vaccine, and obtain an ideal immune effect.

Specifically, in the whole immunization cycle of the avian influenza vaccine using the adjuvant of the present invention, compared with the avian influenza vaccine without the addition of the immune enhancer, the antibody titer of the immune expression will be significantly improved.

detailed description

The content of the present invention will be specifically described by the following examples. In the present invention, the following examples are intended to better illustrate the present invention and are not intended to limit the scope of the present invention. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1

H9N2 subtype avian influenza vaccine immune booster:

Step 1: Prepare an aqueous solution of mussel polysaccharide: add 15 g of mussel polysaccharide to 85 ml of water, and stir until dissolved to obtain an aqueous solution of mussel polysaccharide (that is, the content of mussel polysaccharide is 15%).

Step 2: Prepare the oil phase: take 56ml of white oil and 4ml of Siben-80, respectively, and mix them evenly.

Step 3: Configure the water phase: respectively take 35ml H9N2 embryo fluid inactivated antigen (107EID50/0.1mL, Zhaoqing Dahuanong Bio-Pharmaceutical Co., Ltd.), 1.7ml Tween-80 and 3.3ml Beiyi polysaccharide aqueous solution prepared in step 1, well mixed.

Step 4: Preparation of immune enhancer: the oil phase and the water phase are mixed to prepare a preparation (200 feathers).

Example 2

H9N2 subtype avian influenza vaccine immune enhancer: it is generally the same as in Example 1, wherein the content of mussel polysaccharide in step 1 is 10wt%.

Example 3

H9N2 subtype avian influenza vaccine immune enhancer: it is generally the same as in Example 1, wherein the content of mussel polysaccharide in step 1 is 20wt%.

Comparative Example 1

H9N2 subtype avian influenza vaccine: it is basically the same as in Example 1, wherein in step 1, mussel polysaccharide is not added.

Among them, the above-mentioned mussel polysaccharides have (1→4)-α-D-Glc as the main chain and (1→2)-α-D-Glc branches, and the configuration is α-glucan. There is one (1→2) glucose branch in every 12 glucose main chains, the relative molecular mass of the mussel polysaccharide is 800-8000KDa, and its structural formula is:

Example 4 In vitro activity test

1. H9N2 subtype avian influenza vaccine immunization test

1.1 Animal experiment grouping

40 7-day-old SPF chickens were randomly divided into four groups, 10 in each group: A: H9N2 subtype inactivated avian influenza vaccine prepared in Example 1, subcutaneously injected on the back of the neck, 0.5ml/feather; B: Example 2 The prepared H9N2 subtype inactivated avian influenza vaccine was subcutaneously injected on the nape of the neck, 0.5ml/feather; C: the H9N2 subtype inactivated avian influenza vaccine prepared in Example 3 was subcutaneously injected on the back of the neck, 0.5ml/feather; The H9N2 subtype inactivated avian influenza vaccine prepared in Example 4 was subcutaneously injected on the back of the neck, 0.5 ml/feather.

1.2 Sample Collection

Before immunization and from 1 to 9 weeks after immunization, 1 mL of blood was collected from the lower vein of each chicken wing every week, centrifuged at 3000 rpm for 10 min to separate serum, and frozen at -20 °C.

1.3 Results

Antibody level determination. The level of specific antibodies was detected by hemagglutination inhibition test (HI test), as shown in Table 1 (where the first week refers to the 7th day after the injection of the vaccine), 3 weeks after immunization, the mussel polysaccharide added vaccine group The level of avian influenza virus (H9) antibodies started to be significantly higher than that of the conventional vaccine group (Comparative Example 1, no mussel polysaccharide was added), and continued until the end of the experiment.

Table 1 Antibody level table of vaccine immunization experiment (HI mean log2)

This embodiment is intended to illustrate that the above embodiments are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description of the present invention, or directly or indirectly used in Other related technical fields are similarly included in the scope of patent protection of the present invention.

Claims

An avian influenza vaccine adjuvant comprising mussel polysaccharide.
The avian influenza vaccine adjuvant according to claim 1, wherein the mussel polysaccharide has (1→4)-α-D-Glc as the main chain and contains (1→2)-α-D- Glc branches, the configuration is α-glucan, there is an average (1→2) glucose branch in every 12 glucose main chains, and the relative molecular mass of the mussel polysaccharide is 800-8000KDa, Its structural formula is:
The avian influenza vaccine adjuvant according to claim 1, wherein the avian influenza vaccine is an H9N2 subtype avian influenza vaccine.
A use of an avian influenza vaccine adjuvant in preparing an avian influenza vaccine formulation, the avian influenza vaccine adjuvant comprising mussel polysaccharide.
The use according to claim 4, characterized in that: the mussel polysaccharide has (1→4)-α-D-Glc as the main chain and contains (1→2)-α-D-Glc branches, The configuration is α-glucan, and there is an average (1→2) glucose branch in every 12 glucose main chains. The relative molecular mass of the mussel polysaccharide is 800-8000KDa, and its structural formula is:
The use according to claim 4, wherein the avian influenza vaccine is an H9N2 subtype avian influenza vaccine.
The mussel polysaccharide as claimed in claim 2, which can enhance the immune response of avian influenza vaccine and can be used as an avian influenza vaccine adjuvant.