Poultry oil adjuvant and preparation method thereof

Abstract

The invention belongs to the technical field of biological products, and relates to an oil adjuvant for poultry and a preparation method thereof. The oil adjuvant for poultry comprises 5%-10% of Span, 1%-5% of Tween, 0.3%-2.0% of an AEO series surfactant, 0.1%-1.2% of a polyglycerol oleate series surfactant and the balance of white oil for injection. The poultry oil adjuvant avoids the problem of different product qualities caused by different sources of white oil, and prolongs the validity period of vaccines at the same time.

Description

Technical Field

[0001] The present application belongs to the technical field of biological products and relates to an oil adjuvant for poultry and a preparation method thereof. Background Art

[0002] White oil for injection is a commonly used adjuvant raw material for inactivated poultry vaccines. It is a mixture of various liquid hydrocarbons in petroleum. Its quality standards are included in the Chinese Pharmacopoeia of Veterinary Medicine. The quality standards only stipulate the content of harmful components such as polycyclic aromatic hydrocarbons and heavy metals, and there are no requirements for the content of various alkanes.

[0003] Due to the use of different raw materials and different processes, the composition of injection white oil from different manufacturers is quite different, mainly reflected in the ratio of normal alkanes to isoalkanes, and the inconsistency of the chain lengths of different alkanes. For example, the isoalkanes content of ESSO's Marcol 52 is 79.59% and the normal alkanes content is 17.59%; the isoalkanes content of TOTAL's E150 is 85.69% and the normal alkanes content is 14.31%; the isoalkanes content of domestic white oil is 78.21% and the normal alkanes content is 28.46%.

[0004] The poultry oil adjuvant in the prior art uses a spargan and tween system to emulsify injectable white oil and antigens, but there is a problem that vaccines prepared from injectable white oils from different sources have different stabilities, resulting in uneven quality of vaccine products, which in turn affects their immunogenicity.

[0005] Therefore, there is an urgent need for a poultry oil adjuvant with stable quality, which is of great significance to poultry vaccine products. Summary of the invention

[0006] The purpose of the present application is to provide a poultry oil adjuvant and a preparation method thereof, so as to alleviate the problem in the prior art that vaccines prepared from white oil for injection from different sources have different stability, resulting in uneven quality of vaccine products and thus affecting their immunogenicity.

[0007] In order to achieve the above purpose, this application adopts the following technical solutions:

[0008] An oil adjuvant for poultry comprises, by mass percentage, 5% to 10% of sapogenin, 1% to 5% of Tween, 0.3% to 2.0% of AEO series surfactants and 0.1% to 1.2% of polyglycerol oleate series surfactants, and the remainder is white oil for injection.

[0009] Furthermore, the AEO series surfactant is AEO5, and the polyglycerol oleate series surfactant is dipolyglycerol dioleate.

[0010] Furthermore, the Sibon is Sibon-80, and the Tween is Tween-85.

[0011] A method for preparing an oil adjuvant for poultry comprises the following steps: adding Siben and polyglycerol oleate series surfactants to white oil for injection, stirring evenly, and then adding Tween and AEO series surfactants.

[0012] A method for preparing an oil adjuvant for poultry comprises the following steps: firstly stirring Siben, polyglycerol oleate series surfactants, Tween and AEO series surfactants uniformly, and then adding the mixture into injection white oil.

[0013] The present application also relates to the use of poultry oil adjuvant in vaccines.

[0014] A vaccine composition comprises an antigen and an oil adjuvant for poultry.

[0015] Furthermore, the amount of the poultry oil adjuvant is 50% W / V to 70% W / V.

[0016] Furthermore, the antigen is an influenza virus antigen, a Newcastle disease virus antigen, an infectious bronchitis virus antigen, a parainfluenza virus antigen, a parvovirus antigen, an Actinobacillus pleuropneumoniae antigen, an infectious bursal disease virus antigen, an avian adenovirus antigen, an avian reovirus antigen, an Escherichia coli antigen, a Marek's disease virus antigen or a poxvirus antigen.

[0017] Furthermore, the antigen is an infectious bursal virus subunit antigen, an inactivated Newcastle disease virus antigen or an inactivated H9 subtype avian influenza virus antigen.

[0018] Compared with the prior art, the technical effects of this application are:

[0019] The injectable white oil in the poultry oil adjuvant of the present application can be white oil of different models from various manufacturers. The prepared vaccine has good stability, uniform product quality, and good immunogenicity, avoiding the problem of different product quality caused by white oil from different sources, and also prolonging the validity period of the vaccine. DETAILED DESCRIPTION

[0020] The technical solution of the present application will be described clearly and completely below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present application.

[0021] Experiments have shown that AEO series surfactants and polyglycerol oleate series surfactants can effectively improve the uneven quality of poultry oil adjuvants containing injectable white oil, thereby extending the shelf life of the prepared vaccine.

[0022] The poultry oil adjuvant provided in the present application comprises 5% to 10% of Siban, 1% to 5% of Tween, 0.3% to 2.0% of AEO series surfactants and 0.1% to 1.2% of polyglycerol oleate series surfactants, and the remainder is white oil for injection.

[0023] The content of Siben can be but not limited to 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%; the content of Tween can be but not limited to 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%; the content of AEO series surfactants can be but not limited to 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9% or 2%; the content of polyglycerol oleate series surfactants can be but not limited to 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1% or 1.2%.

[0024] The term "adjuvant" refers to a compound that, when administered together with an antigen, enhances a subject's immune response to the antigen. Adjuvant-mediated enhancement of the immune response can be assessed by any method known in the art, including (but not limited to) one or more of the following methods: (i) an increase in the number of antibodies produced in response to immunization with the adjuvant / antigen combination compared to the number of antibodies produced in response to immunization with the antigen alone; (ii) an increase in the number of T cells that recognize the antigen or adjuvant; (iii) an increase in the level of one or more type I cytokines; and (iv) in vivo protection after in vivo challenge. When a subject is challenged with an antigen and an adjuvant, an immune response is considered to be enhanced if any measurable parameter of antigen-specific immunoreactivity, such as antibody titer or T cell production, increases by at least 10% compared to a subject challenged with the antigen alone. In certain embodiments of the invention, an immune response is enhanced if any measurable parameter of antigen-specific immunoreactivity is increased by at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 175%, at least 200%, at least 225%, at least 250%, at least 275%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, or at least 1000%.

[0025] The term "oil adjuvant", also known as "water-in-oil" adjuvant, refers to an adjuvant whose continuous phase is an oil phase, including but not limited to adjuvants prepared with mineral oil and surfactants. For example, adjuvants prepared with mineral oil, Span (also known as Span), and Tween. The Span can be Span 60, Span 65, Span 80, Span 85 or Span 83; the Tween can be Tween 60, Tween 65, Tween 80, Tween 85, Tween 83. The amount of surfactant used can be selected by those skilled in the art according to the preparation of the stabilizer emulsion.

[0026] The term "mineral oil" refers to a mixture of various liquid hydrocarbons obtained from petroleum that meets the quality standards for white oil for injection (light mineral oil) under Appendix 42, Part III of the Veterinary Pharmacopoeia of the People's Republic of China.

[0027] The white oil for injection in the present application may be any common commercially available product, such as ESSO's Marcol 52 white oil, TOTAL's E150 white oil, Zhengxin C-ZS-004 white oil, and SEOJIN's KF50 white oil.

[0028] The term "AEO" refers to fatty alcohol polyoxyethylene ether, also known as polyoxyethylene fatty alcohol ether, which is an ether formed by the condensation of polyethylene glycol (PEG) and fatty alcohol. There are different varieties due to the different polymerization degrees of polyethylene glycol and the types of fatty alcohol. AEO5 is fatty alcohol polyoxyethylene (5) ether, which is liquid at room temperature and has good wetting and penetration effects. It is mainly used in textile printing and dyeing, papermaking and other industries as a leveling agent, penetrant, wetting agent, and auxiliary component of industrial washing.

[0029] In some embodiments, the amount of AEO5 used is 1.0%.

[0030] The term "polyglycerol oleate" is also called polyglycerol fatty acid ester, which is a new type of polyhydroxy ester non-ionic surfactant with high efficiency and excellent performance, which is used in food, daily chemicals, petroleum, textiles, coatings, plastics, pesticides, rubber, medicine and other fields. Dipolyglycerol dioleate is a kind of polyglycerol oleate, a light yellow liquid with emulsification, dispersion, stabilization, defoaming and other functions. It can also be used for skin care, etc. It is widely used in cosmetics, food, feed, as well as paints, coatings, leather, textiles, papermaking, etc.

[0031] In some embodiments, the amount of diglycerol dioleate is 0.6%.

[0032] The poultry oil adjuvant provided in the present application can be prepared by the following method:

[0033] Add Siben and polyglycerol oleate series surfactants to the white oil for injection, stir evenly, then add Tween and AEO series surfactants; or, first stir Siben, polyglycerol oleate series surfactants, Tween and AEO series surfactants evenly, and then add them to the white oil for injection.

[0034] The present application also provides a vaccine composition, comprising an antigen and the above-mentioned poultry oil adjuvant.

[0035] In some embodiments, the amount of poultry oil adjuvant is 50% W / V to 70% W / V, for example, but not limited to, 50% W / V, 51% W / V, 52% W / V, 53% W / V, 54% W / V, 55% W / V, 56% W / V, 57% W / V, 58% W / V, 59% W / V, 60% W / V, 61% W / V, 62% W / V, 63% W / V, 64% W / V, 65% W / V, 66% W / V, 67% W / V, 68% W / V, 69% W / V or 70% W / V.

[0036] The term "vaccine composition" is a composition that can be used to induce protective immunity in a recipient. Thus, after a subject has been vaccinated with an antigen, a vaccine can prevent, delay or reduce the severity of disease development in a subject exposed to the same or related antigen (relative to an unvaccinated subject). The protective immunity provided by a vaccine can be humoral (antibody-mediated) immunity or cellular immunity, or both can be induced.

[0037] The term "antigen" refers to an agent that, when introduced into a subject, can be recognized by the host's immune system and is capable of inducing an immune response and producing protective immunity. Antigens include (but are not limited to) "surface antigens" naturally expressed on the surface of pathogens or the surface of infected cells or the surface of tumor cells. Non-limiting examples of disease pathogens and / or conditions for which the antigens can induce protective immunity are influenza virus, Newcastle disease virus, infectious bronchitis virus, rhinovirus, parainfluenza virus, parvovirus (e.g., duck parvovirus), adenovirus, Actinobaccilus pleuropneumoniae, avian leukosis, infectious bursal disease, reovirus, pox, laryngotracheitis, Marek's disease, tetanus, hepatitis B, malaria, pneumococcal pneumonia, rotavirus, parvovirus, caronavirus, Escherichia coli, Salmonella.

[0038] In some embodiments, the antigen is an influenza virus, such as an inactivated influenza virus, including any inactivated influenza A virus strain (e.g., an avian influenza virus strain). Inactivated influenza viruses include viruses having H1, H5, H7, or H9 hemagglutinins in combination with any of N1, N2, N3, N4, N5, N6, N7, N8, or N9. Generally, the hemagglutinin subtype of the antigen is selected based on the hemagglutinin subtype presented by circulating infectious influenza virus strains.

[0039] In some embodiments, the antigen is Newcastle disease virus, such as an inactivated Newcastle disease virus La Sota strain or a genetically modified Newcastle disease virus genotype VII strain.

[0040] In some embodiments, the antigen is an infectious bursal virus subunit antigen.

[0041] In some embodiments, the vaccine composition may also have other components added, such as other surfactants, buffers, and stabilizing compounds.

[0042] The present application is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0043] Example 1. Preparation of adjuvant composition

[0044] 1. Comparison with adjuvant A

[0045] Comparative adjuvant A was prepared by adding 7.1 g of Sperm-80 and 2.3 g of Tween-85 to 90.6 g of ESSO Marcol 52 white oil and mixing them evenly.

[0046] 2. Comparison with adjuvant B

[0047] Comparative adjuvant B was prepared by adding 7.1 g of Speran-80 and 2.3 g of Tween-85 to 90.6 g of TOTAL E150 white oil and mixing them evenly.

[0048] 3. Comparison with adjuvant C

[0049] Comparative adjuvant C was prepared by adding 7.1 g of Siben-80 and 2.3 g of Tween-85 to 90.6 g of domestic white oil and mixing them evenly.

[0050] 4. Adjuvant 1 of this application

[0051] To 91.7 g of ESSO Marcol 52 white oil, 6.2 g of Sperm-80 and 0.1 g of diglycerol dioleate were added, and after stirring evenly, 1.7 g of Tween-85 and 0.3 g of AEO5 were added, and the mixture was mixed evenly to prepare adjuvant 1.

[0052] Example 2: Preparation of infectious bursal subunit vaccine composition

[0053] Take infectious bursal subunit antigen (prepared according to Chinese patent application CN112574958A) and add it to the adjuvant prepared in Example 1. Start the motor and stir at 17500r / min for 5 minutes. Before stopping stirring, add 1% thimerosal solution to make the final concentration 0.01%. See 1 for the specific ratio.

[0054] Table 1 Infectious bursal subunit vaccine ratio Components Vaccine 1 Vaccine 2 Vaccine 3 Vaccine 4 VP2 protein (AGP titer) 1:16 1:16 1:16 1:16 Comparative Adjuvant A 66％ — — — Comparative Adjuvant B — 66％ — — Comparison with adjuvant C — — 66％ — Adjuvant 1 — — — 66％

[0055] Example 3, Infectious bursal subunit vaccine stability test

[0056] The infectious bursal subunit vaccine prepared in Example 2 was subjected to a stability test and an appearance stability test at 4° C. to observe the appearance stability of the vaccine. The results are shown in Table 2.

[0057] Table 2 Results of stability test of infectious bursal subunit vaccine Group 2 months 3 months 4 months 5 months 6 months Vaccine 1 No significant changes There is obvious stratification There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 2 No significant changes No significant changes There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 3 No significant changes There is obvious stratification There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 4 No significant changes No significant changes No significant changes No significant changes No significant changes

[0058] The results showed that vaccines 1, 2 and 3 began to show obvious stratification after being stored at 4°C for 3-4 months; while vaccine 4 showed no obvious change in appearance after being stored for 6 months.

[0059] The results show that the stability of vaccines prepared with white oil for injection from different sources is different, resulting in uneven quality of vaccine products, which in turn affects their immunogenicity; while the stability of the vaccine prepared with the poultry oil adjuvant of the present application is significantly improved and remains stable after being stored for 6 months.

[0060] Example 4: Comparative test of immunogenicity of infectious bursal subunit vaccines

[0061] Take 50 SPF chickens of 21 days old and divide them into 5 groups, each group has 10 chickens. The first group and the second group are respectively injected subcutaneously through the neck with the vaccine 1 stored for 2 months and 6 months in Example 3, and the third group and the fourth group are respectively injected subcutaneously through the neck with the vaccine 4 stored for 2 months and 6 months in Example 3, 0.3 ml / chicken; the fifth group is subcutaneously injected with 0.3 ml of normal saline as a blank control. All experimental chickens are isolated and raised. 21 days after immunization, the first group to the fifth group are each inoculated with 100 times diluted chicken infectious bursal disease strong virus BC6-85 ((CVCC AV7 strain) purchased from China Veterinary Drug Supervision Institute) strain virus liquid 0.1 ml (actual toxic content ≥ 100 BID) through the eye drop route. After the attack, the clinical manifestations of the chickens are observed every day, and the number of morbidity and death chickens are recorded. After 72 to 96 hours, the surviving chickens are killed, dissected one by one, and the lesions such as bursal disease are observed. At least 8 immunized chickens should be normal without bursal lesions; at least 4 control chickens should be diseased with obvious bursal lesions (such as strip bleeding in the breast or leg muscles, enlarged or atrophic bursa, yellowing, jelly-like secretions, etc.). The results are shown in Table 3.

[0062] Table 3 Comparative test results of immunogenicity of infectious bursal subunit vaccines

[0063] The results showed that vaccine 1 stored for 2 months without stratification and vaccine 4 stored for 2 months and 6 months without stratification could completely protect against the attack of virulent infectious bursal disease virus 21 days after immunization; while vaccine 1 stored for 6 months (stratification occurred at 3 months) could not completely protect against the attack of virulent infectious bursal disease virus 21 days after immunization. This indicates that the vaccine prepared with the poultry oil adjuvant provided by the present application has good stability and can provide complete protection to the chickens after immunization.

[0064] Example 5: Preparation of adjuvant composition using white oil of different types

[0065] 1. Adjuvant 2 of this application

[0066] To 90.5 g of TOTAL E150 white oil, 6.2 g of Sperm-80 and 0.6 g of diglycerol dioleate were added, and after stirring evenly, 1.7 g of Tween-85 and 1 g of AEO5 were added, and the mixture was mixed evenly to prepare adjuvant 2.

[0067] 2. Adjuvant 3 of this application

[0068] To 88.9 g of domestic white oil, 6.2 g of Siben-80 and 1.2 g of diglycerol dioleate were added, and after stirring evenly, 1.7 g of Tween-85 and 2 g of AEO5 were added, and the mixture was mixed evenly to prepare adjuvant 3.

[0069] Example 6. Preparation of Newcastle disease vaccine composition

[0070] Newcastle disease antigen (prepared according to Chinese patent application CN112574958A) was added to the adjuvants prepared in Example 1 and Example 5, and the motor was started at 17500 r / min for 5 minutes. Before stopping the stirring, 1% thimerosal solution was added to make the final concentration 0.01%. The specific ratios are shown in Table 4.

[0071] Table 4 Newcastle disease vaccine ratio Components Vaccine 5 Vaccine 6 Vaccine 7 Vaccine 8 <![CDATA[Newcastle disease antigen (EID 50 / 0.1 ml)]]> <![CDATA[10 8.0 ]]> <![CDATA[10 8.0 ]]> <![CDATA[10 8.0 ]]> <![CDATA[10 8.0 ]]> Comparative Adjuvant B 66％ — — — Comparative Adjuvant C — 66％ — — Adjuvant 2 — — 66％ — Adjuvant 3 — — — 66％

[0072] Example 7, Newcastle disease vaccine stability test

[0073] The Newcastle disease vaccine prepared in Example 6 was subjected to a stability test and an appearance stability test at 4° C. to observe the appearance stability of the vaccine. The results are shown in Table 5.

[0074] Table 5 Newcastle disease vaccine stability test results Group 2 months 3 months 4 months 6 months 12 months Vaccine 5 No significant changes There is obvious stratification There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 6 No significant changes No significant changes There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 7 No significant changes No significant changes No significant changes No significant changes No significant changes Vaccine 8 No significant changes No significant changes No significant changes No significant changes No significant changes

[0075] The results showed that vaccines 5 and 6 began to show obvious stratification after being stored at 4°C for 3-4 months; vaccines 7 and 8 showed no obvious changes in appearance after being stored for 12 months.

[0076] The results show that the poultry oil adjuvant prepared with white oil for injection from different sources has uniform quality, and the stability of the vaccine prepared with it is significantly improved, and it is still stable after being stored for 12 months; it further shows that the poultry oil adjuvant of the present application can better solve the problem of different stability of vaccines prepared with white oil for injection from different sources.

[0077] Example 8: Newcastle disease vaccine immunogenicity test

[0078] SPF chickens were purchased from Spafas, and the efficacy test was performed using a serological method. 85 21-day-old SPF chickens were used, including 5 unimmunized control groups and 10 vaccine test groups. Groups 6 and 7 were immunized with vaccine 5 stored for 2 months and 12 months in Example 7 by subcutaneous injection in the neck, respectively; Groups 8 and 9 were immunized with vaccine 7 stored for 2 months and 12 months in Example 7 by subcutaneous injection in the neck; Groups 10 and 11 were immunized with vaccine 6 stored for 2 months and 12 months in Example 7 by subcutaneous injection in the neck, respectively; Groups 12 and 13 were immunized with vaccine 8 stored for 2 months and 12 months in Example 7 by subcutaneous injection in the neck; each chicken in the test group was immunized with 20μ1 of vaccine; Group 14 was subcutaneously injected with 20μ1 of saline as a blank control. All experimental chickens were isolated and raised. 21 days after immunization, blood was collected from the immunized chickens in Groups 6 to 13, together with the control chickens in Group 14, and serum was separated to detect Newcastle disease virus HI antibodies. The results are shown in Table 6.

[0079] Table 6 Results of the immunogenicity test of chicken Newcastle disease vaccine

[0080] Note: The HI antibody titer in the table is the geometric mean

[0081] The results showed that vaccines 5 and 6 stored for 2 months without stratification and vaccines 7 and 8 stored for 2 months and 12 months without stratification could produce high Newcastle disease antibodies 21 days after immunization; while vaccines 5 and 6 stored for 12 months and stratified showed a significant decrease in Newcastle disease antibodies 21 days after immunization.

[0082] This indicates that the Newcastle disease vaccine prepared with the poultry oil adjuvant provided in this application has good stability and can provide complete protection to the chickens after immunization.

[0083] Example 9: Preparation of H9 subtype avian influenza vaccine composition

[0084] Take H9 subtype avian influenza antigen (prepared according to Chinese patent application CN112574958A) and add it to the adjuvant prepared in Example 1 and Example 5, start the motor at 17500r / min and stir for 5 minutes, and add 1% thimerosal solution before stopping stirring to make the final concentration 0.01%. See 7 for specific ratios.

[0085] Table 7 H9 subtype avian influenza vaccine ratio Components Vaccine 9 Vaccine 10 <![CDATA[Avian influenza antigen (EID 50 / 0.1 ml)]]> <![CDATA[10 8.0 ]]> <![CDATA[10 8.0 ]]> Comparative Adjuvant B 66％ — Adjuvant 2 — 66％

[0086] Example 10: Stability test of H9 subtype avian influenza vaccine

[0087] The H9 subtype avian influenza vaccine prepared in Example 9 was subjected to a stability test and an appearance stability test at 4° C. to observe the appearance stability of the vaccine. The results are shown in Table 8.

[0088] Table 8 Results of stability test of H9 subtype avian influenza vaccine Group 2 months 3 months 4 months 6 months 12 months Vaccine 9 No significant changes There is obvious stratification There is obvious stratification There is obvious stratification There is obvious stratification Vaccine 10 No significant changes No significant changes No significant changes No significant changes No significant changes

[0089] The results showed that vaccine 9 began to show obvious stratification after being stored at 4°C for 3 months, while vaccine 10 showed no obvious change in appearance after being stored for 12 months.

[0090] The results show that the poultry oil adjuvant prepared in the present application has uniform quality, and the stability of the H9 subtype avian influenza vaccine prepared therefrom is significantly improved, and it remains stable after being stored for 12 months; it further illustrates that the poultry oil adjuvant of the present application can better solve the problem of different stability of vaccines prepared from white oil for injection from different sources.

[0091] Example 11: Immunogenicity test of H9 subtype avian influenza vaccine

[0092] Serological methods were used to test the efficacy of H9 subtype avian influenza. 45 21-day-old SPF chickens were used, 5 non-immunized control groups, and 10 vaccine test groups in each group. Groups 15 and 16 were subcutaneously injected with vaccine 9 stored for 2 months and 12 months in Example 10, respectively, and groups 17 and 18 were subcutaneously injected with vaccine 10 stored for 2 months and 12 months in Example 10, respectively; each chicken in the test group was injected with 0.3ml of vaccine; Group 19 was subcutaneously injected with 0.3ml of normal saline as a blank control. All test chickens were isolated and raised. 21 days after immunization, blood was collected from the immunized chickens in Groups 15 to 18, together with the control chickens in Group 19, and serum was separated to detect the titer of H9 subtype avian influenza HI antibodies. The results are shown in Table 9.

[0093] Table 9 Results of immunogenicity test of avian influenza vaccine in chicken

[0094] Note: The HI antibody titer in the table is the geometric mean

[0095] The results showed that vaccine 9 that was stored for 2 months without stratification and vaccine 10 that was stored for 2 months and 12 months without stratification could produce high levels of H9 subtype avian influenza antibodies 21 days after immunization; while vaccine 9 that was stored for 12 months and stratified showed a significant decrease in avian influenza antibodies 21 days after immunization.

[0096] This indicates that the H9 subtype avian influenza vaccine prepared with the poultry oil adjuvant provided in the present application has good stability and can provide complete protection to the chickens after immunization.

[0097] Note that the above are only preferred embodiments of the present application and the technical principles used. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, readjustments and substitutions can be made by those skilled in the art without departing from the protection scope of the present application. Therefore, although the present application is described in more detail through the above embodiments, the present application is not limited to the above embodiments, and may also include more other equivalent embodiments without departing from the technical concept of the present application, all of which belong to the protection scope of the present application.

Claims

1. A poultry oil adjuvant, characterized in that: Calculated by mass percentage, it includes 5% to 10% of Siban, 1% to 5% of Tween, 0.3% to 2.0% of AEO series surfactants and 0.1% to 1.2% of polyglycerol oleate series surfactants, and the remainder is white oil for injection.

2. The poultry oil adjuvant according to claim 1, characterized in that The AEO series surfactant is AEO5, and the polyglycerol oleate series surfactant is dipolyglycerol dioleate.

3. The poultry oil adjuvant according to claim 1 or 2, characterized in that The Sibon is Sibon-80, and the Tween is Tween-85.

4. A method for preparing the poultry oil adjuvant according to any one of claims 1 to 3, characterized in that: Add Siben and polyglycerol oleate series surfactants to the white oil for injection, stir evenly, then add Tween and AEO series surfactants.

5. A method for preparing the poultry oil adjuvant according to any one of claims 1 to 3, characterized in that: First, stir the Siben, polyglycerol oleate series surfactants, Tween and AEO series surfactants evenly, and then add them into the injection white oil.

6. Use of the poultry oil adjuvant according to any one of claims 1 to 3 in vaccines.

7. A vaccine composition, characterized in that The invention comprises an antigen and the poultry oil adjuvant according to any one of claims 1 to 3.

8. The vaccine composition according to claim 7, characterized in that The amount of poultry oil adjuvant is 50% W / V to 70% W / V.

9. The vaccine composition according to claim 7, characterized in that The antigen is an influenza virus antigen, a Newcastle disease virus antigen, an infectious bronchitis virus antigen, a parainfluenza virus antigen, a parvovirus antigen, an Actinobacillus pleuropneumoniae antigen, an infectious bursal disease virus antigen, an avian adenovirus antigen, an avian reovirus antigen, an Escherichia coli antigen, a Marek's virus antigen or a poxvirus antigen.

10. The vaccine composition according to claim 9, characterized in that The antigen is an infectious bursal virus subunit antigen, an inactivated Newcastle disease virus antigen or an inactivated H9 subtype avian influenza virus antigen.

Images