TWI603740B - Avian Newcastle disease vaccine adjuvant and its preparation method - Google Patents

Description

Adjuvant for poultry Newcastle disease vaccine and preparation method thereof

The present invention relates to an adjuvant for poultry vaccines, and more particularly to an adjuvant for avian Newcastle disease vaccine and a preparation method thereof.

Vaccination is considered to be the most effective and efficient way to prevent viral infections in animals. However, there are still many vaccines that are not available for viral or bacterial diseases, or have not yet achieved sufficient immunity. In addition, many vaccines are not suitable because of their low antigenic efficacy or the use of inferior adjuvants, which can cause serious side effects, low stability, or high cost. Therefore, there is an urgent need to develop more effective vaccines or related adjuvant products.

Many infectious disease control programs are based on the induction of specific immunity by vaccination with live microorganisms or inactivated microorganisms or their products. An effective vaccination program allows for the formation of immunological memory for a specific antigen in an animal, thereby causing a rapid and intense immune response in the animal upon contact with the antigen in the future. However, some antigens are only weakly immunogenic. These antigens may be incapable of inducing an immune response sufficient to provide effective protection to the animal in future challenge, or may require administration of other agents that enhance immunogenicity to provide effective protection.

The antigen is mixed with a substance called an adjuvant, and the injection into the animal together enhances the immune system's immune resistance to the antigen. The substance called the adjuvant is directly applied to the immune system or by modifying the antigen. Kinetic characteristics to enhance the animal's resistance to wild-type viruses and thereby increase the interaction time of the antigen with the immune system.

When the inactivated antigen is immunized to the animal alone, it is insufficient to produce immunogenicity, particularly an antigen having low immunogenicity and small molecular weight. Therefore, different types of adjuvants are needed to enhance the immune response induced by the antigen. The oily vaccines promoted and applied by domestic and foreign veterinary vaccine manufacturers contain a large amount of mineral oil, which has brought a lot of food safety problems while protecting the poultry industry. For example, the vaccine residue of broiler carcasses has been Caused more and more people's concerns.

The water-in-oil (W/0) type oily vaccine is prepared by mixing antigenic liquid, surfactant and mineral oil under high shear force. The immune mechanism is stored at the inoculation site for a period of time, local granuloma and inflammatory reaction, attracting macrophages, lymphocytes, etc. to identify antigens and produce antibodies. At the same time, the slow-diffusing oily adjuvant prolongs the delivery of antigen to the secondary lymphoid organs, constantly stimulating the body's immune system, resulting in a long-lasting, high-antibody immune system. Although the production process of vaccine manufacturers at home and abroad continues to improve, the quality of oil used is also increasing. However, due to the tendency of the continuous oil phase of this preparation to reside at the injection site, in many cases, this type of W/0 oil The cytoplasm produces a large amount of granuloma near the inoculation site, and the protective antibody is produced slowly, which is easy to adversely affect the carcass quality of the animal, and may cause severe pain during the inoculation.

The oil-in-water (0/W) type adjuvant vaccine has the advantage that the injection site shows little local reaction and high stability, but the antigen contained or exposed in the continuous phase is quickly dispersed at the injection site and decomposed by body fluid enzymes, which is difficult to provide. Long-term protective antibodies often require special immunostimulants and multiple boosts, which are costly to prepare and are not widely used.

Therefore, people from all walks of life do not expect to develop an immune response that will increase the inactivated antigen when administered to immunized animals alone. Therefore, all sectors are eager to develop an immune response and effect, and can have immune response persistence. The adjuvant of the problem of traditional technology.

In view of this, the present inventors have diligently studied various possible solutions for solving the problems of the conventional technology, and have developed a problem that not only can improve the above-mentioned conventional technology, but also provides a water-in-oil package for the deficiencies of the prior art. Water adjuvant. The adjuvant of the present invention can reduce the oil consumption of the existing inactivated vaccine, accelerate the production rate of the corresponding antibody of the immunized animal, and reduce the preparation cost of the vaccine, and thus the present invention has been completed. Summary of Invention

First, the specific terms or nouns used in the specification are described in the specification. However, the following description is merely illustrative, and is not intended to limit the scope of the invention. The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein.

As used herein, a "vaccine composition" is a composition that can be used to induce protective immunity in a recipient. Thus, after the subject has been vaccinated with the antigen, the vaccine can prevent, delay or alleviate the severity of the disease progression (relative to the unvaccinated subject) of the subject exposed to the same or related antigen. The protective immunity provided by the vaccine can be humoral (antibody mediated) immunity or cellular immunity, or both. For example, vaccination can eliminate or reduce the load on pathogens or infected cells, or produce any other measurable reduction in infection. Vaccination also reduces the tumor burden of subjects who have been immunized (vaccinated).

As used herein, the term "protective immunization" refers to the defense against antigen when the subject has been exposed to an antigen, thereby causing an immune response (active/acquired or passive/innate, or both) in the subject. Immunity, thereby inactivating antigens and/or reducing antigen loading and forming immune memory (eg, memory T cells or B cells).

The protective immunity provided by vaccination may be provided in part or only in a portion of the vaccinated subject. Thus, vaccines can induce protective immunity in a subset of immune populations, as some individuals may be unable to establish a strong immune response or a protective immune response or, in some cases, any immune response. This lack of capacity may be due to the genetic background of the individual or to an immunodeficiency condition (acquired or congenital) or immunosuppression (eg, due to chemotherapy or the use of immunosuppressive drugs to, for example, prevent organ rejection or inhibition) Physical immune condition). Vaccines that provide protection to a portion of the immune population remain applicable and considered effective.

As used herein, the term "adjuvant" refers to a compound that, when administered in conjunction with an antigen, enhances the subject's immune response to the antigen.

Specifically, according to one aspect of the present invention, an adjuvant for avian Newcastle disease vaccine can be provided which comprises at least: about 35.0 parts by weight to about 95.0 parts by weight of an oil agent; about 1.0 part by weight to about 5.0 parts by weight. An emulsifier; from about 2.0 parts by weight to about 10.0 parts by weight of the colloidal extract; from about 1.0 part by weight to about 15.0 parts by weight of the buffering agent; and from about 1.0 part by weight to about 5.0 parts by weight of the surfactant; The agent can increase the antigen in the avian Newcastle disease vaccine in the living body, and the stimulation time of the immune system is increased, thereby increasing the memory immunity effect of the bird.

In the adjuvant for avian Newcastle vaccine of the present invention, in order to improve the prevention of infection of Newcastle disease or increase the stability and efficacy of the vaccine to achieve the effect of epidemic prevention, the product can achieve good efficacy. According to one aspect of the present invention, the blending ratio (Adg:Vat) of the avian Newcastle disease vaccine adjuvant (Adj) to the avian Newcastle disease antigen solution (Vat) is 1:5 (v/v) at the time of actual application. ~1:1 (v/v). For example, the ratio of the avian Newcastle disease vaccine adjuvant (Adj) to the avian Newcastle disease antigen solution (Vat) of the present invention may be in the range of about 1:5 (v/v) to 1:1 (v). /v); in some embodiments, preferably in the range of about 1:6 (v/v); more preferably in the range of about 1:5 (v/v); particularly preferably in about 1 Range of 4 (v/v); preferably in the range of about 1.02:0.98 (v/v).

The above-mentioned poultry Newcastle disease antigen liquid (Vat) is stored in the Food Industry Development Research Institute of the consortium, and the storage numbers are BCRC 970070, BCRC 970071, BCRC 970072, and the poultry date of the Republic of China on March 24, 105. Prepared by a virus strain of Newcastle disease.

Specifically, according to one aspect of the present invention, an adjuvant for avian Newcastle disease vaccine can be provided, and the ratio of the oil agent, the emulsifier, the gel extract, the buffer, and the surfactant is not particularly limited, as long as it is not It can cause damage to poultry and can achieve epidemic prevention. For example, in the adjuvant for avian Newcastle disease vaccine of the present invention, the ratio of the oil agent, the emulsifier, the gel extract, the buffer, and the surfactant may be about 35 parts by weight: about 1 part by weight. : about 2 parts by weight: about 1 part by weight: about 1 part by weight to about 95 parts by weight: about 5 parts by weight: about 10 parts by weight: about 15 parts by weight: about 5 parts by weight; in some embodiments Preferably at about 25 parts by weight: about 0.25 parts by weight: about 1 part by weight: about 0.25 parts by weight: from about 0.25 parts by weight to about 99 parts by weight: about 5.75 parts by weight: about 10.75 parts by weight: about 15.75 parts by weight: A range of about 5.75 parts by weight; more preferably about 30 parts by weight: about 0.5 parts by weight: about 1 part by weight: about 0.5 parts by weight: about 0.5 parts by weight to about 96 parts by weight: about 5.5 parts by weight: about 10.5 parts by weight : about 15.5 parts by weight: a range of about 5.5 parts by weight; particularly preferably about 35 parts by weight: about 1 part by weight: about 2 parts by weight: about 1 part by weight: about 1 part by weight to about 95 parts by weight: about 5 parts by weight Parts: about 10 parts by weight: about 15 parts by weight: about 5 parts by weight.

According to one aspect of the present invention, the antigen solution to be used in combination with the adjuvant for the adjuvant for avian Newcastle disease vaccine of the present invention is not particularly limited, and for example, may be a dead antigen, a sub unit antigen, Nucleic acid antigen. According to some embodiments of the present invention, the antigenic fluid source may be, for example, at least one selected from the group consisting of a deadly antigen, a subunit antigen, a nucleic acid antigen, a derivative thereof, and a mixture thereof; The antigenic liquid of the present invention is preferably used as a deadly antigen, a subunit antigen, or a nucleic acid antigen; a more preferred source is a deadly antigen.

According to one aspect of the present invention, the oil agent for use in the adjuvant for avian Newcastle disease vaccine of the present invention is not particularly limited, and may be, for example, mineral oil or vegetable oil. According to some embodiments of the present invention, the oil source may be, for example, selected from the group consisting of pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, vitamin E, derivatives thereof, and mixtures thereof. At least one; the oil agent suitable for use in the present invention is preferably used as a pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, or vitamin E; a better source of pharmaceutical grade white oil, peanut oil, mineral oil, or vitamin E; the best source of use is pharmaceutical grade white oil, or mineral oil.

According to one aspect of the present invention, the emulsifier which can be used in the adjuvant for avian Newcastle disease vaccine of the present invention is not particularly limited. For example, it may be, for example, lecithin, soybean phospholipid, Span 85, Span 83. , Span 80, Span 65, Span 60, glyceryl monostearate, glycerol monooleate, and the like. According to some embodiments of the invention, the emulsifier may be, for example, from lecithin, soybean phospholipid, Span 85, Span 83, Span 80, Span 65, Span 60, glyceryl monostearate, At least one of glycerol monooleate; the emulsifier suitable for use in the present invention is preferably used as lecithin, soybean lecithin, Span 85, Span 83, Span 80, Span 65, Span 60, single hard Glycerylglyceride, glycerol monooleate, or; more preferably used as lecithin, soybean phospholipid, Span 85, Span 83, Span 80, Span 65, or Span 60; Phospholipids, pea phospholipids, Span 85, Span 83, Span 80, or Span 65.

According to one aspect of the present invention, the gel extract of the adjuvant for avian Newcastle disease vaccine of the present invention can be used without particular limitation, and for example, may be an aloe extract, a schisandra extract, a horse tooth苋 extract, grapefruit extract, persimmon leaf extract, β-glucan, mulberry mushroom extract, Cordyceps extract. According to some embodiments of the present invention, the colloidal extract source may be, for example, from aloe extract, schisandra extract, purslane extract, grapefruit extract, persimmon leaf extract, β-glucan , the extract of Mulberry sylvestris, the extract of Cordyceps sinensis, the derivative thereof, and the mixture thereof are at least one selected from the group; the colloidal extract suitable for use in the present invention is preferably used as aloe extract, Wujiapi Extract, Portulaca oleracea extract, grapefruit extract, persimmon leaf extract, beta-glucan, mulberry mushroom extract, or cordyceps extract; better source of aloe extract, Wujiapi extract , Purslane extract, grapefruit extract, or β-glucan; the best source of use is aloe extract, schisandra extract, or β-glucan.

According to one aspect of the present invention, the buffer for the adjuvant for avian Newcastle disease vaccine of the present invention is not particularly limited, and for example, may be a sodium phosphate buffer, a potassium phosphate buffer, or a Tris-HCl. Buffer. According to some embodiments of the invention, the buffer source may be, for example, sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, sodium citrate-phosphate buffer, zwitterionic buffer, Hepes buffer. The agent, the maleate buffer, the derivative thereof, and the mixture thereof constitute at least one selected from the group; the buffer suitable for the present invention is preferably used as a sodium phosphate buffer, a potassium phosphate buffer, Tris-HCl buffer, sodium citrate-phosphate buffer, zwitterionic buffer, Hepes buffer, or maleate buffer; better use is sodium phosphate buffer, potassium phosphate buffer, Tris-HCl Buffer, sodium citrate-phosphate buffer, or zwitterionic buffer; a particularly preferred source is sodium phosphate buffer, potassium phosphate buffer, sodium citrate-phosphate buffer, or zwitterionic buffer.

According to one aspect of the present invention, the surfactant which can be used in the adjuvant for avian Newcastle disease vaccine of the present invention is not particularly limited. For example, it may be, for example, polyoxyethylene fatty alcohol or octanoic acid. Ethylene glycol glyceride (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil, Pluronic F-68, polyoxyethylene Lauryl alcohol ether and the like. According to some embodiments of the present invention, the surfactant may be, for example, self-polyoxyethylene fatty alcohol, succinic acid glycol glyceride (Labrasol), Tween 20, Tween 40, Tween 60, At least one of Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil, pluronic F-68, polyoxyethylene lauryl alcohol ether; the surfactant suitable for use in the present invention is preferably used as , polyoxyethylene fatty alcohol, octanoic acid glycol glyceride (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil , Pluronic F-68, or polyoxyethylene lauryl ether; more preferably used, polyoxyethylene fatty alcohol, octanoic acid glycol glyceride (Labrasol), Tween 20, Tween 40, spit Temperature 60, Tween 80, Tween 81, or Tween 85; the best source of use is octanoic acid glycol glyceride (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, or Tween 85.

Specifically, according to one aspect of the present invention, a method for preparing an adjuvant for avian Newcastle disease vaccine can be provided, which comprises the steps of: adding about 35.0 parts by weight to about 95.0 parts by weight of an oil agent, about 1.0 part by weight to about 5.0 parts by weight of an emulsifier, and about 1.0 part by weight to about 10.0 parts by weight of a colloidal extract, which are sufficiently emulsified and mixed under the first reaction conditions to form an emulsion; and about 1.0 is added to the emulsion obtained by the above (a) The parts by weight to about 15.0 parts by weight of the buffering agent, and from about 1.0 part by weight to about 2.5 parts by weight of the surfactant are thoroughly mixed and form an adjuvant for avian Newcastle disease vaccine under the second reaction conditions.

According to one aspect of the present invention, a method for preparing an adjuvant for avian Newcastle disease vaccine of the present invention can be used, wherein (a) the first reaction condition is included at a temperature of 65 ° C to 80 ° C, and the amplitude is 20 mm to 25 mm. After stirring for 1 to 3 hours at a stirring speed of 1200 rpm to 2400 rpm, the water oiliness (HLB) is between 10 and 16.

Further, according to one aspect of the present invention, a method for preparing an adjuvant for avian Newcastle disease vaccine of the present invention can be used, wherein (b) the second reaction condition is included at a temperature of 55 ° C to 65 ° C and the pH is adjusted to 6.8. ~7.4, the amplitude of 14 mm~18mm, stirring speed 800 rpm~1200 rpm, carry out the reaction for 2~3 hours.

Further, according to one aspect of the present invention, a method for preparing an adjuvant for avian Newcastle disease vaccine of the present invention, wherein the oil is a mineral oil; the emulsifier is a spoon 80; the colloidal extract is Aloe extract; the buffer is; the surfactant is Tween 20.

The invention will be further described below in conjunction with specific embodiments.

In the following, the embodiments of the present invention will be described in more detail in the detailed description of the embodiments of the present invention so that the spirit and content of the present invention are more complete and easy to understand; however, those of ordinary skill in the art should understand The invention is of course not limited to these examples, and other similar or equivalent functions and order of steps may be utilized to achieve the invention.

In addition, the scope of the invention may be further exemplified by the following specific examples, which are not intended to limit the scope of the invention.

The various reagents used in the following examples and experiments are commercially available. For example, mineral oil is a product made by Hans H&R (for example, trade name: pharmaceutical grade white oil No. 70); poultry Newcastle disease antigen liquid is a product made by Taiwan National Science and Technology Co., Ltd., commissioned by Pingtung University of Science and Technology. , trade name: the poultry Newcastle disease antigen solution (Vat) used in the present invention is prepared by at least one of the following three strains; the three strains are deposited in the food industry development of the corporation in the Republic of China on March 24, 105. The research institute, the registration number is BCRC 970070, BCRC 970071, BCRC 970072, the virus strains of the bird type VII Newcastle disease, and the three strains of the above-mentioned bio-registered poultry type VII Newcastle disease virus were inoculated to fertilization for 11 days. In the chicken embryos, the virus was harvested 96 hours after hatching, and then the virus was deactivated at a final concentration of 0.2% formalin (Sigma, USA). The emulsifier is a spoon 80 manufactured by SIGMA Co., Ltd. (for example, trade name: S6760); and the surfactant is Tween 20 (for example, trade name: P2287) manufactured by SIGMA Corporation. "Preparation for Preparation of Avian Newcastle Disease Vaccine" "Preparation Example 1"

First, 3.5 L of pharmaceutical grade white oil, 0.5 L of lecithin and 0.5 L of Span 80 were added together in a blending bottle, followed by 1.5 L of potassium phosphate buffer (potassium dihydrogen phosphate 34 g, 1 mol/ L sodium hydroxide solution 175ml, distilled water 825ml, pH 7.2), 1.5 L zwitterionic buffer (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing 6 crystal water of magnesium chloride 0.1g dissolved in 1L ddH ₂ O), using a blender (model: UHM-10, label: NIHONSEIKI Japan) in a sterile environment, temperature 65 ° C, amplitude 20mm, stirring speed 1200 rpm, The reaction was carried out for 60 minutes, and the mixture was thoroughly mixed to obtain an emulsion M1 having a water-oiliness of 10.

Next, as shown in Table 1, 1 L of aloe vera gel extract (using 70% alcohol to extract 1000 g of aloe vera, further extracting the active ingredient of the aloe vera gel extract, and using it as an elixir, the concentration is 18.8 g/ml), 1 L of Wujipi colloidal extract (using 70% alcohol to extract 1000 g of Wujiapi, further extracting the active ingredient of Wujiapi colloidal extract, used as an elixir, its concentration Emulsion M1 was added together with 18.8 g/ml), and 0.25 L of Tween 20 and 0.25 L of Tween 80 were added at the same time, and uniformly stirred at room temperature for 2 hours under aseptic conditions; continue to use ultra-high speed homogenizer The avian Newcastle disease vaccine adjuvant (S1) of the present invention was obtained by homogenizing stirring at 800 rpm, amplitude of 14 mm, 55 ° C for 1 hour, pH adjustment to 6.8, and then cooling by condensation. Preparation Example 2

First, 6.0 L of pharmaceutical grade white oil, 0.5 L of lecithin and 0.5 L of Span 80 were added together in a blending bottle, followed by 1.5 L of phosphate buffer (potassium dihydrogen phosphate 34 g, 1 mol/ L sodium hydroxide solution 175ml, distilled water 825ml, pH 7.2), 1.5 L zwitterionic buffer (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing 6 crystal water of magnesium chloride 0.1g dissolved in 1L ddH ₂ O), using a blender (model: UHM-10, label: NIHONSEIKI Japan) in a sterile environment, temperature 70 ° C, amplitude 22 mm, stirring speed 1600 rpm The reaction was carried out for 80 minutes, and after thorough mixing, an emulsion M2 was obtained, and the oiliness was 12.

Next, as shown in Table 1, 0.5 L of aloe vera gel extract (using 70% alcohol to extract 1000 g of aloe vera, further extracting the active ingredient of the aloe vera gel extract, and using it as an elixir, the concentration is 20 g/ Ml), 0.5 L of Wujipi colloidal extract (using 70% alcohol to extract 1000 g of Wujiapi, further extracting the active ingredient of Wujiapi colloidal extract, used as an elixir, its concentration is 20g/ Ml) Add emulsion M2 together, then add 0.5 L of Tween 20, 0.5 L of Tween 80, and uniformly stir for 2.2 hours at room temperature in a sterile environment; continue to use ultra-high speed homogenizer at 900 rpm, amplitude The mixture was stirred at 160 mm and 60 ° C for 1.2 hours, and the pH was adjusted to 7.0. Then, the adjuvant for avian Newcastle disease vaccine of the present invention (S2) was obtained by cooling down by condensation. Preparation Example 3

First, add 7 L of triglyceride, 0.25 L of lecithin and 0.25 L of Span 80 to the blending bottle, then add 0.2 L of phosphate buffer (potassium dihydrogen phosphate 34 g, 1 mol/L hydrogen) 175ml of sodium oxide solution, 825ml of distilled water, pH 7.2), 0.8 L of zwitterionic buffer (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing 6 Crystallized water magnesium chloride 0.1g dissolved in 1L ddH ₂ O), using a blender (Model: UHM-10, label: NIHONSEIKI Japan) in a sterile environment, temperature 75 ° C, amplitude 23 mm, stirring speed 2000 rpm, reaction After 120 minutes, the mixture was thoroughly mixed to obtain an emulsion M3 and a water-oil ratio of 14.

Next, as shown in Table 1, 0.5 L of aloe vera gel extract (using 70% alcohol to extract 1000 g of aloe vera, further extracting the active ingredient of the aloe vera gel extract, and using it as an elixir, the concentration is 22.1 g. /ml), 0.5 L of Wujipi colloidal extract (using 70% alcohol to extract 1000 g of Wujiapi, further extracting the active ingredient of Wujiapi colloidal extract, used as an elixir, its concentration is 22.1 g/ml) together with emulsion M3, then add 0.25 L of Tween 20, 0.25 L of Tween 80, and uniformly stir for 2 hours at room temperature in a sterile environment; continue to use ultra-high speed homogenizer at 1000 rpm The sample was homogenized for 17 hours at an amplitude of 17 mm and 62 ° C, and the pH was adjusted to 7.2. Then, the adjuvant for avian Newcastle disease vaccine of the present invention (S3) was obtained by cooling down by condensation. Preparation 4

First, add 9.5 L of mineral oil and 0.1 L of lecithin to the mixing bottle, then add 0.1 L of phosphate buffer (potassium dihydrogen phosphate 34 g, 1 mol/L sodium hydroxide solution 175 ml, distilled water 825 ml, pH) Value 7.2), using a blender (Model: UHM-10, label: NIHONSEIKI Japan) In a sterile environment, the temperature is 80 °C, the amplitude is 25 mm, the stirring speed is 2400 rpm, the reaction is carried out for 180 minutes, and the emulsion M4 is fully mixed. , water oil level 16.

Next, as shown in Table 1, 0.1 L of aloe vera gel extract (using 70% alcohol to extract 1000 g of aloe vera, further extracting the active ingredient of the aloe vera gel extract, and using it as an elixir, the concentration is 25.8 g /ml), 0.1 L of Wujipi colloidal extract (using 70% alcohol to extract 1000 g of Wujiapi, further extracting the active ingredient of Wujiapi colloidal extract, used as an elixir, its concentration is 25.8 g/ml) Add emulsion M4 together, then add 0.1 L of Tween 20, and uniformly stir for 1 hour at room temperature in a sterile environment; continue to use ultra-high speed homogenizer at 1200 rpm, amplitude 18 mm, 65 °C The mixture was homogenized for 2 hours, the pH was adjusted to 7.4, and then the adjuvant for avian Newcastle disease vaccine of the present invention (S4) was obtained by cooling down by condensation.

According to the above "Preparation Examples 1 to 4", the proportions and data are listed in "Table 1" as follows:

"Table 1"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> Adjuvant for Newcastle Disease Vaccine</td><td> Preparation 1 </td> <td> Preparation Example 2 </td><td> Preparation Example 3 </td><td> Preparation Example 4 </td></tr><tr><td> Oil Agent</td><td> White Oil</td><td> 3.5 </td><td> 6 </td><td> 0 </td><td> 0 </td></tr><tr><td> Grease</td><td> 0 </td><td> 0 </td><td> 7 </td><td> 0 </td></tr><tr><td> mineral oil< /td><td> 0 </td><td> 0 </td><td> 0 </td><td> 9.5 </td></tr><tr><td> emulsifier</td ><td> Lecithin</td><td> 0.5 </td><td> 0.5 </td><td> 0.25 </td><td> 0.1 </td></tr><tr>< Td> Span 80 </td><td> 0.5 </td><td> 0.5 </td><td> 0.25 </td><td> 0 </td></tr><tr><td > Gum Extract </td><td> Aloe </td><td> 1 </td><td> 0.5 </td><td> 0.5 </td><td> 0.1 </td></ Tr><tr><td> Wujiapi</td><td> 1 </td><td> 0.5 </td><td> 0.5 </td><td> 0.1 </td></tr ><tr><td> Buffering agent</td><td> Phosphate buffer solution</td><td> 1.5 </td><td> 0.5 </td><td> 0.2 </td><td > 0.1 </td></tr><tr><td> zwitterionic buffer</td><td> 1.5 </td><td> 0.5 </td><td> 0.8 </td><td> 0 </td></tr><tr><td> Surfactant </td><td> Tween 20 </td><td> 0.25 </td><td> 0.5 </td><td> 0.25 </td><td> 0.1 </td></tr><tr><td> Tween 80 </td><td> 0.25 </td><td> 0.5 </td><td> 0.25 </td><td> 0 </td></tr><tr><td> Adjuvant for Newcastle Disease Vaccine</td>< Td> S1 </td><td> S2 </td><td> S3 </td><td> S4 </td></tr><tr><td> traits</td><td> transparent Smoothing emulsion</td></tr><tr><td> Viscosity</td><td> 40°C/(mm²/sec) </td><td> 5.3 </td><td> 7.4 </td><td> 8.2 </td><td> 9.1 </td></tr><tr><td> 100°C/(mm<sup>2</sup >/sec) </td><td> 1.2 </td><td> 2.2 </td><td> 2.8 </td><td> 3.6 </td></tr><tr><td> Oil droplet size</td><td> (nm) </td><td> 2.3 </td><td> 2.6 </td><td> 3.2 </td><td> 2.6 </td>< /tr></TBODY></TABLE>

Next, 450 ml to 600 ml of Preparation Example S1 to Preparation S4 were placed in a 2000 ml beaker, and the emulsification homogenizer was placed in a beaker to prepare the poultry Newcastle disease of the present invention of Preparation Examples S1 to S4. The agent is covered with an emulsifier homogenizer with a stirring bar of 2/3 (about 15 cm), and the high-speed shearing force of the rotary emulsifier homogenizer (model: TT-SM-S, brand: Taiwan Dading Machinery) to 3000 rpm slowly will be 350 ML ~ 600 ml of antigen is added to the rotating preparations S1 to S4 (the buffer can be added to 350 ml when the antigen is less than 350 ml); finally, when the 350 ml antigen is added, the rotation speed can be adjusted to 5000 rpm. , at the final stirring time of five to ten minutes. The detailed matching method is recorded in Table 2 below.

"Table 2"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Mixing Example 1 </td><td> Mixing Example 2 < /td><td> Mixing Example 3 </td><td> Mixing Example 4 </td></tr><tr><td> Source of infection (registered number) </td><td> TW-CB- ND02 (BCRC 970070) </td><td> 350 ml </td><td> 0 </td><td> 0 </td><td> 200 ml </td></tr><tr> <td> TW-CB-ND04 (BCRC 970071) </td><td> 0 </td><td> 400 ml </td><td> 0 </td><td> 200 ml </td> </tr><tr><td> TW-CB-ND06 (BCRC 970072) </td><td> 0 </td><td> 0 </td><td> 500 ml </td><td > 200 ml </td></tr><tr><td> Adjuvant</td><td> S1(ml) </td><td> 650 </td><td> 0 </td> <td> 0 </td><td> 0 </td></tr><tr><td> S2(ml) </td><td> 0 </td><td> 600 </td> <td> 0 </td><td> 0 </td></tr><tr><td> S3(ml) </td><td> 0 </td><td> 0 </td> <td> 500 </td><td> 0 </td></tr><tr><td> S4(ml) </td><td> 0 </td><td> 0 </td> <td> 0 </td><td> 400 </td></tr><tr><td> Vaccine containing adjuvant for Newcastle disease vaccine of the present invention</td><td> V1 </td ><td> V2 </td><td> V3 </td><td> V4 </td></tr></TBODY></TABLE>

ND experiment 1 is to test the effect of the vaccine V1~V4 containing the adjuvant for the Newcastle disease vaccine of the present invention for the epidemic-type bird type VII Newcastle disease, and divide the 30-day-old SPF chicken into 5 groups, respectively, for control For example, 2.5 μL of phosphate buffer solution (PBS) was injected subcutaneously, and Example 1 was 2.0 μL of a vaccine V1 containing the adjuvant for avian Newcastle disease vaccine of the present invention, and Example 2 was a subcutaneous injection of 2.5 μL. The vaccine V2 of the adjuvant for avian Newcastle disease vaccine of the invention, the third embodiment is an intramuscular injection of 3.0 μL of the vaccine V3 containing the adjuvant for avian Newcastle disease vaccine of the present invention, and the fourth embodiment is a subcutaneous injection of 3.2 μL of the present invention. Vaccine V4 for adjuvants for avian Newcastle disease vaccines.

Three weeks after the inoculation, the immunization was boosted once again. The vaccine was injected the same as the first time. The chickens were sacrificed two weeks after the immunization, and all the test chickens were separately before the inoculation (PI-0wk) and one week after the inoculation (PI- 1 wk), 2 weeks after inoculation (PI-2wk) and 3 weeks after inoculation (PI-3wk), blood was collected, and a cell proliferation test in blood was examined. The chickens were sacrificed and their spleen cells were taken for detection of the percentage of CD4 ⁺ and CD8 ⁺ cells in the spleen cells.

The method for detecting cell proliferation in blood and the percentage of CD4 ⁺ /CD8 ⁺ cells in spleen cells are described in the latter section.

Three weeks after the inoculation, the immunization was boosted once again. The vaccine was injected the same as the first time. The chickens were sacrificed two weeks after the immunization, and all the test chickens were separately before the inoculation (PI-0wk) and one week after the inoculation (PI- 1 wk), 2 weeks after inoculation (PI-2wk) and 3 weeks after inoculation (PI-3wk), blood was collected, and a cell proliferation test in blood was examined. The chickens were sacrificed and their spleen cells were taken for detection of the percentage of CD4 ⁺ and CD8 ⁺ cells in the spleen cells. "Detection method of cell proliferation test"

Chickens take blood cell proliferation assay, to Histopaque ^® 1077 density gradient medium which PBMC isolated cells, cells were counted, the number of cells adjusted to ^{2 x 10 5 cells / 100 μL} / well dispensed into 96-well plates, were added to 1 g / 50 μL/well of Con A (Sigma, USA) and the same volume of medium, three replicates, placed in a 5% CO ₂ , 37 ° C incubator for 48 hours, add 10 μL / well BrdU reagent labeled cells, and then Incubate for 24 hours, centrifuge at 1000 rpm for 10 minutes, remove the supernatant, add 200 μL/well FixDenat solution, leave it at room temperature for 30 minutes, remove FixDenat solution and add 100 μL/well anti-Brd U-pod working solution. Remove anti-Brd U-pod working solution at room temperature for 90 minutes, add 300 μL/well wash solution, repeat three washes, remove wash solution, add 100 μL /well substrate solution, place at room temperature for 5 minutes, color to ELISA reader The value is read at 370 nm wavelength. The average percentage of cell proliferation of the test results of each group of 6 chickens in each example was recorded in Table 3, respectively.

<Table 3><TABLEborder="1"borderColor="#000000"width="85%"><TBODY><tr><td></td><td> Control example</td><td> Implementation Example 1 </td><td> Example 2 </td><td> Example 3 </td><td> Example 4 </td></tr><tr><td></td><td> CB-ND02 </td><td> V1 </td><td> V2 </td><td> V3 </td><td> V4 </td></tr><tr><td> How to apply</td><td> Subcutaneous injection</td><td> Point nose</td><td> Subcutaneous injection</td><td> Intramuscular injection</td><Td> subcutaneous injection</td></tr><tr><td> dose (μl) </td><td> 25 </td><td> 20 </td><td> 25 </ Td><td> 30 </td><td> 32 </td></tr><tr><td> dose (ml) </td><td> 2.5 </td><td> 2.0 </td><td> 2.5 </td><td> 3.0 </td><td> 3.2 </td></tr><tr><td> number of hits (only) </td><Td> 6 </td><td> 6 </td><td> 6 </td><td> 6 </td><td> 6 </td></tr><tr><td> Percentage of proliferation (%) </td><td> PI-1wk </td><td> 15 ± 9.3 </td><td> 30 ± 1.3 </td><td> 35 ± 0.3 </td><td> 38 ± 6.3 </td><td> 30 ± 1.3 </td></tr><tr><td> PI-2wk </td><td> 17 ± 9.3 </td><td> 40 ± 1.3 </td><td> 49 ± 0.3 </td><td> 42 ± 6.3 </td><t d> 40 ± 1.3 </td></tr><tr><td> PI-3wk </td><td> 18 ± 9.3 </td><td> 52 ± 1.3 </td><td> 53 ± 0.3 </td><td> 55 ± 6.3 </td><td> 52 ± 1.3 </td></tr></TBODY></TABLE>"Method for detecting percentage of CD4 ⁺ /CD8 ⁺ cells"

CD4 ⁺ /CD8 ⁺ cell surface antigen detection The cell fluid concentration was adjusted to 2 × 10 ⁵ cells, and the cell cultures stained with avian monoclonal antibodies IgG1- FITC and IgG1-PE were used as a negative control. The chicken cell surface antigens CD4 ⁺ and CD8 ^{+ were} detected, and the anti-CD4 monoclonal antibody and the anti-CD8 monoclonal antibody (Beckman Coulter, Inc. USA) were separately added to the cell liquid to be tested, mixed uniformly, and placed on ice. After 30 minutes in the dark, centrifuge at 300 xg for 5 minutes at 4 °C. After removing the supernatant, the cells were suspended in PBS, and the cells were washed by centrifugation at 300 xg for 5 minutes at 4 ° C, and the washing was repeated twice. Finally, 100 μL of PBS suspension cells containing 1% paraformaldehyde were added, and the cell surface antigen was analyzed by flow cytometry (Coulter Epics Altra Flow Cytometry, Beckman Coulter, CA), and then Expo 32 v1.0 MultiCOMP Software (Applied Cytometry Systems, Sheffield, UK) analyzed the percentage of CD4 ⁺ and CD 8 ⁺ cells. The average values of the viral power values of the test results of each group of 6 chickens in each example were recorded in Table 4, respectively.

"Table 4"         <TABLE border="1" borderColor="#000000" width="85%"><TBODY><tr><td> </td><td> Control example</td><td> Example 1 </ Td><td> Example 2 </td><td> Example 3 </td><td> Example 4 </td></tr><tr><td> Shit Group </td> <td> CB-ND02 </td><td> V1 </td><td> V2 </td><td> V3 </td><td> V4 </td></tr><tr>< Td> Application method</td><td> Subcutaneous injection</td><td> Point nose</td><td> Subcutaneous injection</td><td> Intramuscular injection</td><td> Subcutaneous injection </td></tr><tr><td> dose (μl) </td><td> 25 </td><td> 20 </td><td> 25 </td><td > 30 </td><td> 32 </td></tr><tr><td> hit time (week) </td><td> 2 </td><td> 2 </td> <td> 2 </td><td> 2 </td><td> 2 </td></tr><tr><td> Number of hits (only) </td><td> 6 < /td><td> 6 </td><td> 6 </td><td> 6 </td><td> 6 </td></tr><tr><td> CD4⁺(%) </td><td> 10 ± 9.3 </td><td> 30 ± 1.3 </td><td> 35 ± 0.3 </td><td> 38 ± 6.3 </td ><td> 30 ± 1.3 </td></tr><tr><td> CD8⁺(%) </td><td> 17 ± 9.3 </td><td> 40 ± 1.3 </td><td> 49 ± 0.3 </td><td> 42 ± 6.3 </td><td> 40 ± 1.3 </td></tr> </TBODY></TABLE>

According to the above Table 4, in the control group, the immune cells in the blood of the chicken which did not apply the vaccine for the adjuvant of the Newcastle disease vaccine of the present invention increased by only 15-18%; in contrast, in the examples In 1 to 4, the chickens which have been administered the vaccine for the adjuvant of the Newcastle disease vaccine of the present invention show that the increase of the immune cells in the blood is at least 30%, and the observation of the time is elongated, the poultry new city of the present invention. Adjuvants for disease vaccines can increase immune protection, because the immune cells in the blood of the chickens are tested three weeks later, which is 50% higher than before the original control group.

In addition, the results of the percentage of CD4 ⁺ /CD8 ⁺ cells also showed that the vaccine of the present invention was not used in the control group, and the percentage of CD4 ⁺ /CD8 ⁺ cells was about 10% / 18%; on the other hand, in Example 1~ In the case of 4, the percentage of CD4 ⁺ /CD8 ⁺ cells in the chickens administered with the vaccine for the adjuvant of the Newcastle disease vaccine of the present invention is about 25%/35% or more, indicating that the vaccine vaccinated with the adjuvant of the present invention can be improved. The proportion of CD4 ⁺ /CD8 ⁺ cells in chickens further demonstrates the effect of improving immunity and fighting Newcastle disease.

Therefore, it was confirmed that the chicken which was administered with the vaccine for the adjuvant for the Newcastle disease vaccine of the poultry can increase the immunoprotective ability, and thus has an excellent effect against the infection of Newcastle disease. Therefore, the adjuvant for avian Newcastle disease vaccine of the present invention can significantly improve the immunity of the chicken and the proliferation of the immune cells, and enhance the vaccine to induce a protective immune response to the chicken in Newcastle disease.

In summary, the content of the present invention has been exemplified by the above embodiments, but the present invention is not limited to the embodiments. A person skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention; for example, combining or modifying the technical contents exemplified in the foregoing embodiments. As a new embodiment, these embodiments are of course considered as belonging to the present invention. Therefore, the scope of protection to be covered in this case also includes the scope of the patent application described below and the scope defined by it.

Claims (7)

A method for preparing an adjuvant for avian Newcastle disease vaccine, comprising the steps of: (a) about 35.0 parts by weight to about 95.0 parts by weight of an oil agent, about 1.0 part by weight to about 5.0 parts by weight of an emulsifier, and about 1.0 part by weight to about 10.0 parts by weight of the colloidal extract, which is sufficiently emulsified and mixed under the first reaction conditions to form an emulsion; (b) from the emulsion obtained in the above (a), about 1.0 part by weight to about 15.0 by weight. a buffer, and from about 1.0 part by weight to about 2.5 parts by weight of the surfactant are thoroughly mixed and form an adjuvant for avian Newcastle disease vaccine under the second reaction condition; wherein the first reaction condition in step (a) is included At a temperature of 65 ° C ~ 80 ° C, the amplitude is 20mm ~ 25mm, the stirring speed is 1200rpm ~ 2400rpm, after the reaction for 1~3 hours, the water oil degree (HLB) is between 10~16; the second in step (b) The reaction conditions are included at a temperature of 55 ° C to 65 ° C and the pH is adjusted to 6.8 to 7.4, the amplitude is 14 mm to 18 mm, the stirring speed is 800 rpm to 1200 rpm, and the reaction is carried out for 2 to 3 hours; and the colloidal extract is selected from the aloe extract. , Wujiapi extract, purslane extract, grapefruit extract, persimmon leaves Extract, [beta] glucan, Phellinus mushroom extract, or at least one extract of Cordyceps sinensis. The method for preparing an adjuvant for avian Newcastle disease vaccine according to claim 1, wherein the oil agent is at least one selected from the group consisting of pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, or vitamin E. The preparation method of the adjuvant for avian Newcastle disease vaccine according to claim 1, wherein the emulsifier is selected from the group consisting of lecithin, soybean phospholipid, Span 85, Span 83, Span 80, Span 65, Span 60 At least one of glyceryl monostearate or glycerol monooleate. The method for preparing an adjuvant for avian Newcastle disease vaccine according to claim 1, wherein the buffer is selected from the group consisting of phosphate buffer (PBS), Tris-HCl buffer, sodium citrate-phosphate buffer, zwitterion At least one of a buffer, a Hepes buffer, or a maleate buffer. The method for preparing an adjuvant for avian Newcastle disease vaccine according to claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene fatty alcohol, succinic acid glycol glyceride (Labrasol), Tween 20, and spit. At least one of temperature 40, Tween 60, Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil, pluronic F-68, polyoxyethylene lauryl alcohol ether. An adjuvant for avian Newcastle disease vaccine, which is prepared by the method of any one of the above claims 1 to 5, wherein the adjuvant for the avian Newcastle vaccine comprises at least: about 35.0 parts by weight to about 95.0. Parts by weight of the oil; from about 1.0 part by weight to about 5.0 parts by weight of the emulsifier; from about 2.0 parts by weight to about 10.0 parts by weight of the gum extract; from about 1.0 part by weight to about 15.0 parts by weight of the buffer; and about 1.0% by weight a mixture of about 5.0 parts by weight of a surfactant; and at the time of actual application, the blending ratio of the avian Newcastle disease vaccine adjuvant (Adj) to the avian Newcastle disease antigen solution (Vat) (Adg:Vat) is 1 : 5 (v / v) ~ 1:1 (v / v), the poultry Newcastle disease antigen liquid (Vat) is prepared for at least one of the following three strains; the three strains of the virus in the Republic of China 105 years On March 24th, it was deposited in the Food Industry Development Research Institute of the Foundation. The registration numbers are BCRC 970070, BCRC 970071, BCRC 970072, and the names are all strains of avian type VII Newcastle disease. The adjuvant for avian Newcastle disease vaccine according to claim 6, wherein the oil agent is mineral oil; the emulsifier is a spoon 80; the colloidal extract is an aloe extract; the buffer is phosphate Buffer; the surfactant is Tween 20.