CN111544587B - Avian influenza vaccine adjuvant and application thereof - Google Patents
Avian influenza vaccine adjuvant and application thereof Download PDFInfo
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- CN111544587B CN111544587B CN202010606523.6A CN202010606523A CN111544587B CN 111544587 B CN111544587 B CN 111544587B CN 202010606523 A CN202010606523 A CN 202010606523A CN 111544587 B CN111544587 B CN 111544587B
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Abstract
An avian influenza vaccine adjuvant and its application are provided. The invention discloses mussel polysaccharide which is matched with an avian influenza vaccine for use, plays a synergistic effect in effect, can enhance the activity of the avian influenza vaccine and obtains an ideal immune effect. Compared with the avian influenza vaccine without the adjuvant, the avian influenza vaccine with the adjuvant has the advantage that the antibody titer of immune expression can be obviously improved in the whole immune period.
Description
Technical Field
The invention relates to an avian influenza vaccine adjuvant and application thereof.
Background
The respiratory tract is a portal for invasion of influenza viruses, and specific immunity protection is established on the mucous membrane of the respiratory tract, so that the invasion of the viruses can be prevented. Attenuated live vaccines are the only influenza vaccine species clinically used at present and are immunized through mucous membrane, and the vaccine induces strong SIgA antibody response at the invasion site of viruses through nasal cavity immunization to prevent influenza viruses from entering the body. However, live attenuated vaccines have their limitations, recommending vaccination for humans between 5 and 49 years of age, and are approved for use in only a few countries. Influenza inactivated vaccines, including whole virus inactivated vaccines, split vaccines and subunit vaccines, are types of vaccines approved for use worldwide, vaccinated by intramuscular injection, provide specific protection mainly by induction of systemic IgG antibodies, and are incapable of inducing production of SIgA. In recent years, research shows that influenza inactivated vaccines are also effective in nasal immunization, but mucosal adjuvants are required to promote their immunoprotection effect.
Adjuvants for inactivated influenza virus vaccines are commonly used at present, such as inorganic adjuvants (e.g. aluminum adjuvants), surfactants (e.g. MF 59), cytokine adjuvants (e.g. IFN-gamma) and polysaccharide adjuvants. Natural polysaccharide is widely existed in nature, has various biological activities, and some natural polysaccharide can regulate various immune cells and immune molecules, enhance the body resistance, has the anti-tumor effect, and has been widely paid attention as an adjuvant research object in recent years.
Polygonatum Odoratum Polysaccharide (POP) has adjuvant effect component, and is mainly extracted from rhizoma Polygonati Odorati of Liliaceae. Zhang Yanan A nasal drop immunization BALB/c mouse model using H7N9 influenza virus split vaccine and POP with different doses as adjuvants shows that POP can enhance immune response, has mucosa adjuvant effect, and has increasing trend of specific IgG antibody and HI antibody in serum. Shan Junjie, etc., show that the total polysaccharide (IIP) of the radix isatidis has adjuvant activity. The H1N1 influenza virus lysate is combined with polysaccharide IIP-A-1, so that the titer level of IgG1, igG2a, igG2B, igG3 and IgA can be obviously improved, the proliferation capacity of B cells is obviously higher than that of a non-adjuvant group, the IIP-A adjuvant has the capacity of promoting Th1 cell differentiation, and the capacity of spleen lymphocytes for secreting IFNgamma is further enhanced. Xie Guoxiu and the like, which combine low-dose inactivated vaccine with pachymaran with 200 mu g dose, show that the antibody level can be improved, but the attack of lethal influenza virus cannot be resisted; when the pachyman dose was raised to 1000 μg, the low dose vaccine group mice had significantly increased antibody levels, with serum IgG titers significantly higher than the adjuvant-free group.
In summary, although a variety of polysaccharide adjuvants for influenza virus vaccines have been reported in the literature, the development of new natural non-toxic polysaccharide adjuvants is still of great importance.
Disclosure of Invention
The first aspect of the invention: an avian influenza vaccine adjuvant is provided which is capable of enhancing the immune function of the body.
The efficient utilization and deep development of the marine shellfish resources are important research directions of sustainable development of the economic shellfish culture industry in China, wherein shellfish polysaccharide has wide biological activity in various aspects of anti-tumor, antivirus, immunoregulation, liver injury protection and the like due to the unique structure of the shellfish polysaccharide, and has good application prospect in the aspects of research and development of functional health-care foods and innovative medicaments. The invention screens out polysaccharide which can be used as an avian influenza vaccine adjuvant from mussel polysaccharide reported in the prior art.
In order to solve the technical problem, the invention provides an avian influenza vaccine adjuvant which comprises mussel polysaccharide.
Preferably, the mussel polysaccharide takes (1-4) -alpha-D-Glc as a main chain, contains (1-2) -alpha-D-Glc branches, is configured as alpha-pyrane glucan, and has 1 (1-2) glucose branches in every 12 glucose main chains on average, and the mussel polysaccharide has a relative molecular mass of 800-8000kDa and a structural formula:
the mussel polysaccharide is extracted from animals of mussel genus such as Mytilus coruscus and Mytilus edulis. The extraction process was carried out with reference to the process reported in document CN107056962 a.
Another aspect of the invention provides the use of an avian influenza vaccine adjuvant comprising mussel polysaccharide in the manufacture of an avian influenza vaccine formulation. The mussel polysaccharide and the avian influenza vaccine are combined to enhance the immune activity of the vaccine.
Preferably, the avian influenza vaccine is an H9N2 subtype avian influenza vaccine.
Another aspect of the present invention provides a mussel polysaccharide that is capable of enhancing the immune response of an avian influenza vaccine, useful as an avian influenza vaccine adjuvant.
The invention has the beneficial effects that:
the invention adopts the mussel polysaccharide and the avian influenza vaccine to be matched for use, plays a synergistic effect in effect, can enhance the activity of the avian influenza vaccine and obtains ideal immune effect.
Specifically, the avian influenza vaccine using the adjuvant of the present invention has significantly improved antibody titers in immune expression throughout the immune cycle compared to avian influenza vaccines without immunopotentiator added.
Detailed Description
The following is a detailed description of the present invention by way of examples. In the present invention, the following examples are provided for better illustration of the present invention and are not intended to limit the scope of the present invention. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1
H9N2 subtype avian influenza vaccine immunopotentiator:
step one: preparation of Bei Yi polysaccharide aqueous solution: adding 15 g mussel polysaccharide into 85 ml water, and stirring to dissolve to obtain mussel polysaccharide water solution (i.e. mussel polysaccharide content is 15%).
Step two: preparing an oil phase: respectively taking 56 ml white oil and 4 ml span-80, and uniformly mixing the two.
Step three: preparing an aqueous phase: 35 mL of the Bei Yi polysaccharide aqueous solution prepared in the first step of H9N2 embryo liquid inactivation antigen (107 EID50/0.1mL, zhaoqing Dahua agricultural and biological medicine Co., ltd.), 1.7 mL Tween-80 and 3.3 mL are respectively taken and uniformly mixed.
Step four: preparation of immunopotentiator: the oil phase and the water phase were mixed to prepare a formulation (200 parts by weight).
Example 2
H9N2 subtype avian influenza vaccine immunopotentiator: it is substantially the same as in example 1, wherein the mussel polysaccharide content in step 1 is 10 wt%.
Example 3
H9N2 subtype avian influenza vaccine immunopotentiator: it is substantially the same as in example 1, wherein the mussel polysaccharide content in step 1 is 20 wt%.
Comparative example 1
H9N2 subtype avian influenza vaccine: it is substantially the same as in example 1, wherein no mussel polysaccharide is added in step 1.
The mussel polysaccharide takes (1-4) -alpha-D-Glc as a main chain, contains (1-2) -alpha-D-Glc branches, is configured as alpha-pyrane glucan, and has 1 (1-2) glucose branches in every 12 glucose main chains on average, and the mussel polysaccharide has a relative molecular mass of 800-8000kDa and has a structural formula as follows:
example 4 in vitro Activity test
1. H9N2 subtype avian influenza vaccine immunoassay
1.1 animal Experimental groups
40 SPF chickens of 7 days of age were randomly divided into four groups of 10: a: the H9N2 subtype inactivated avian influenza vaccine prepared in the example 1 is subcutaneously injected into the nape of the neck, and the concentration is 0.5 ml/feather; b: the H9N2 subtype inactivated avian influenza vaccine prepared in the example 2 is subcutaneously injected into the nape of the neck, and the concentration is 0.5 ml/feather; c: the H9N2 subtype inactivated avian influenza vaccine prepared in the example 3 is subcutaneously injected into the nape of the neck, and the concentration is 0.5 ml/feather; the H9N2 subtype inactivated avian influenza vaccine prepared in example 4 is subcutaneously injected into the nape of the neck, and 0.5 ml/feather.
1.2 sample collection
Blood was collected 1mL per chicken wing vein every week at 1-9 weeks before and after immunization, and serum was separated by centrifugation at 3000 rpm for 10min and frozen at-20 ℃.
1.3 results
Antibody level determination. Specific antibody levels were tested by the hemagglutination inhibition assay (HI assay) as shown in table 1 (where the first week refers to day 7 after vaccine injection), and the avian influenza virus (H9) antibody levels of the vaccine group with mussel polysaccharide added began to be significantly higher than the conventional vaccine group (comparative example 1, no mussel polysaccharide added) and continued until the end of the assay, 3 weeks after immunization.
Table 1 antibody level Table for vaccine immunization experiments (HI mean log 2)
| Week 1 | Week 2 | Week 3 | Week 4 | Week 5 | Week 6 | Week 7 | Week 8 | Week 9 | |
| Example 1 | 5.5 | 6.2 | 7.0 | 8.3 | 8.0 | 7.9 | 7.4 | 7.0 | 6.4 |
| Example 2 | 5.8 | 6.5 | 7.9 | 9.2 | 8.5 | 8.0 | 7.9 | 7.5 | 6.9 |
| Example 3 | 5.8 | 6.6 | 7.9 | 9.1 | 8.7 | 8.2 | 7.8 | 7.4 | 6.6 |
| Comparative example 1 | 4.0 | 5.7 | 6.3 | 7.5 | 7.8 | 7.4 | 7.1 | 6.5 | 6.0 |
The present embodiment is intended to be illustrative of the preferred embodiments of the present invention, and not to limit the scope of the present invention, and all equivalent structures or equivalent processes using the content of the present invention, or direct or indirect application in other related technical fields, are included in the scope of the present invention.
Claims (1)
1. Use of an avian influenza vaccine adjuvant comprising mussel polysaccharide in the preparation of an avian influenza vaccine formulation; the avian influenza vaccine is an H9N2 subtype avian influenza vaccine; the mussel polysaccharide takes (1-4) -alpha-D-Glc as a main chain, contains (1-2) -alpha-D-Glc branches, is configured as alpha-pyrane glucan, and has 1 (1-2) glucose branches in every 12 glucose main chains on average, and has the relative molecular mass of 800-8000kDa, and has the structural formula:
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103223164A (en) * | 2013-04-15 | 2013-07-31 | 华南农业大学 | Water-in-oil-in-water adjuvant vaccine and preparation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273496C (en) * | 2004-06-07 | 2006-09-06 | 中国人民解放军第二军医大学 | Polysaccharose MF4 of mussel with enhancing immunity and anti-tumour activity |
| CN107056962B (en) * | 2017-05-18 | 2019-06-11 | 山东省药学科学院 | A kind of mussel polysaccharide and its preparation method and application |
| CN109966484B (en) * | 2019-04-23 | 2023-08-01 | 肇庆大华农生物药品有限公司 | Immunopotentiator, preparation method, avian influenza vaccine and application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103223164A (en) * | 2013-04-15 | 2013-07-31 | 华南农业大学 | Water-in-oil-in-water adjuvant vaccine and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Bioactive compounds from marine mussels and their effects on human health;Ulrike Grienke等;FOOD CHEMISTRY;第142卷;第48-60页 * |
| Endemic Variation of H9N2 Avian Influenza Virus in China;Yu-Fu Liu等;Avian Diseases;第60卷(第4期);第817-825页 * |
| 多糖佐剂在疫苗中的应用;姜威;朱婷;王玉霞;张振学;单俊杰;;中国新药杂志(13);第1470-1478页 * |
| 禽流感病毒BEI灭活疫苗制备与免疫效果;施维松等;中国兽医杂志;第48卷(第08期);第21-23页 * |
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