TW201800109A - Adjuvant for poultry Newcastle disease vaccine and preparation method thereof capable of accelerating generation of corresponding antibodies of an immunized animal and reducing vaccine manufacturing cost - Google Patents

Abstract

The present invention relates to an adjuvant for poultry Newcastle disease vaccine, which comprises: about 35.0 parts by weight to 95.0 parts by weight of oil agent; about 1.0 part by weight to 5.0 parts by weight of emulsifier; about 2.0 parts by weight to 10.0 parts by weight of gum extract; about 1.0 part by weight to 15.0 parts by weight of buffer agent; and about 1.0 part by weight to 5.0 parts by weight of surfactant. In actual application, a mixing ratio (Adg:Vat) of the adjuvant (Adj) for poultry Newcastle disease vaccine to the antigen solution (Vat) for poultry Newcastle disease is between 1:5 (v/v) to 1:1 (v/v). The antigen solution (Vat) for poultry Newcastle disease is prepared by at least one of poultry VII Newcastle disease virus strains deposited in the Food Industry Research and Development Institute with deposit numbers BCRC 970070, BCRC 970071 and BCRC 970072 and deposit date on 2016/03/24.

Description

Adjuvant for poultry Newcastle disease vaccine and preparation method thereof

The invention relates to an adjuvant for avian vaccines, in particular 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 the virus from infecting animals. However, there are still many viral or bacterial diseases that have no available vaccines or have not yet achieved sufficient immune effects. In addition, many vaccines are unsuitable because their antigenic efficacy is too low or the use of poor quality adjuvants can cause severe 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 inducing specific immunity by vaccination with live or inactivated microorganisms or their products. Effective vaccination procedures allow the formation of immune memory capacity in animals against specific antigens, thereby causing a rapid and strong immune response in the animal upon subsequent contact with the antigen. 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 upon subsequent challenge, or may require administration of other agents that help to increase immunogenicity to provide effective protection.

After mixing the antigen with a substance called an adjuvant, it can be injected into animals to enhance the immune system's immune resistance to the antigen. The substance called an adjuvant is a drug that acts directly on the immune system or modifies the antigen. Kinetic characteristics to enhance the animal's resistance to wild viruses and thereby increase the interaction time between the antigen and the immune system.

Inactivated antigen alone is not enough to produce immunogenicity, especially antigen with low immunogenicity and small molecular weight. It is therefore necessary to use different types of adjuvants to enhance the immune response induced by the antigen. The oil-based vaccines promoted by veterinary vaccine manufacturers at home and abroad contain a large amount of mineral oils. While escorting the poultry industry, they have also brought many food safety issues. For example, the problem of vaccine residues in broiler carcasses has been Cause more and more people to worry.

Water-in-oil (W / 0) type oily vaccine is prepared by mixing antigen solution, surfactant and mineral oil under the action of high shear force. Its immune mechanism is that it is stored at the inoculation site for a period of time, and locally produces granulomas and inflammatory reactions, attracting macrophages, lymphocytes and other aggregates to recognize antigens and produce antibodies. At the same time, the slow-spreading oily adjuvant prolongs the time it takes for the antigen to be delivered to the secondary lymphoid organs, constantly stimulating the body's immune system, thereby producing long-lasting, high-level immune efficacy. Although the production technology of vaccine manufacturers at home and abroad has been continuously improved and the quality of oil used has been continuously improved, due to the tendency of the continuous oil phase of this preparation to reside at the injection site, in many cases, this W / 0 type oil A large number of granulomas are produced near the vaccination site, and the production of protective antibodies is slow, which is likely to adversely affect the quality of animal carcasses, and may cause severe pain during vaccination.

The oil-in-water (0 / W) adjuvant vaccine has the advantages of showing a small local response and high stability at the injection site, however, the antigen contained or exposed to 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 immune stimulants and multiple boosts, which are expensive to prepare and are not widely used.

Therefore, people from all walks of life are looking forward to the development of an immune response that can enhance the inactivation of antigens when administered alone to immunized animals. Therefore, people from all walks of life are eager to develop a system that can increase the immune response and effect, and that can sustain the immune response. Adjuvant to the problems of traditional technology.

In view of this, the inventors have studied various possible solutions to solve traditional technical problems through intensive research, and then developed a method that can not only improve the problems of the conventional technology, but also provides a water-in-oil-in-oil Water-based adjuvant. The adjuvant of the present invention can reduce the amount of oil used in existing inactivated vaccines, accelerate the production rate of corresponding antibodies in the immunized animals, and reduce the cost of vaccine preparation. Thus, the present invention has been completed. "Summary of Invention"

First of all, specific terms or nouns used in this specification are described descriptively; however, the following description is merely illustrative, and is not intended to limit the scope of the present invention and patent application. Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and used by those having ordinary knowledge in the technical field to which the present invention pertains.

As used herein, a "vaccine composition" is a composition that can be used to induce protective immunity in a recipient. Therefore, after a subject has been vaccinated with an antigen, the vaccine can prevent, delay or reduce the severity of disease development in a subject exposed to the same or related antigen (as compared to an unvaccinated subject). The protective immunity provided by the vaccine can be humoral (antibody-mediated) immunity, 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 can also reduce the tumor burden in vaccinated (vaccinated) subjects.

As used herein, the term "protective immunity" refers to what is obtained against an antigen when the subject has been exposed to the antigen, thereby eliciting an immune response (active / native or passive / innate, or both) in the subject. Immune system, thereby inactivating the antigen and / or reducing the antigen load and forming immune memory (such as memory T cells or B cells).

The protective immunity provided by vaccination may be provided in part or only in a part of the vaccinated subjects. Therefore, vaccines can elicit protective immunity in a subset of the immune population, as some individuals may be unable to establish a strong or protective immune response or (in some cases) any immune response. This ability is not likely to be due to the genetic background of the individual or to an immunodeficiency condition (acquired or congenital) or immunosuppression (e.g., due to chemotherapy or the use of immunosuppressive drugs to prevent organ rejection or inhibit Body immune conditions). Vaccines that provide protection to a subset of immunized populations are still applicable and considered effective.

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

Specifically, according to an 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 parts by weight to about 5.0 parts by weight An emulsifier; about 2.0 parts by weight to about 10.0 parts by weight of a colloidal extract; about 1.0 parts by weight to about 15.0 parts by weight of a buffering agent; and about 1.0 parts by weight to about 5.0 parts by weight of a surfactant; The agent can increase the antigen in the avian Newcastle disease vaccine in the living body, increase the time for stimulating the immune system, and then increase the memory immune effect of the bird.

In the adjuvant for poultry Newcastle disease vaccine of the present invention, in order to improve the prevention of Newcastle disease infection or increase the stability and efficacy of the vaccine to achieve epidemic prevention effects, the product can achieve good efficacy. According to one aspect of the present invention, when actually applied, the admixture ratio (Adg: Vat) of the adjuvant (Adj) for avian Newcastle disease vaccine and the avian Newcastle disease vaccine (Vat) is 1: 5 (v / v) ~ 1: 1 (v / v). For example, the ratio of the admixture (Adj) of the poultry Newcastle disease vaccine to the poultry Newcastle disease vaccine (Vat) in the present invention may be about 1: 5 (v / v) to 1: 1 (v / v); In some embodiments, it is preferably in the range of about 1: 6 (v / v); more preferably in the range of about 1: 5 (v / v); particularly preferably in the range of about 1 : 4 (v / v); the best is about 1.02: 0.98 (v / v).

As mentioned above, the poultry Newcastle disease antigen solution (Vat) is deposited with the Food Industry Development Research Institute of the Consortium, and the deposit numbers are BCRC 970070, BCRC 970071, and BCRC 970072. The deposit date is Poultry VII of the Republic of China on March 24, 105. Newcastle disease virus strain.

Specifically, according to an aspect of the present invention, an adjuvant for avian Newcastle disease vaccine can be provided, which is not particularly limited by the ratio of oil agent, emulsifier, gum extract, buffer, and surfactant, as long as it is not It can cause damage to birds and achieve epidemic prevention effects. For example, for example, in the adjuvant for poultry Newcastle disease vaccine of the present invention, the ratio of the oil agent, emulsifier, colloidal extract, buffer, and 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 certain embodiments , Preferably about 25 parts by weight: about 0.25 parts by weight: about 1 part by weight: about 0.25 parts by weight: 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: about 5.5 parts by weight; particularly preferred is 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 that can be used in combination with the adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited. For example, it can be a dead virus antigen, a subunit antigen, Nucleic acid antigen. According to some specific embodiments of the present invention, the source of the antigen solution may be, for example, at least one selected from the group consisting of a dead virus antigen, a subunit antigen, a nucleic acid antigen, a derivative thereof, and a mixture thereof; The antigen solution of the present invention is preferably used from a lethal antigen, a subunit antigen, or a nucleic acid antigen; a more preferred source is a dead virus antigen.

According to one aspect of the present invention, the oil agent that can be used in the adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited, and for example, it may be a mineral oil or a vegetable oil. According to some specific embodiments of the present invention, the oil source may be selected from the group consisting of pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, vitamin E, derivatives, and mixtures thereof. At least one; the oil source suitable for the present invention is preferably used from pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, or vitamin E; more preferably used from pharmaceutical grade white oil, peanut oil, mineral oil, or vitamin E; Extraordinarily good source is medicinal grade white oil or mineral oil.

According to one aspect of the present invention, the emulsifier that can be used as the adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited. For example, it can be lecithin, soy lecithin, Span 85, Span 83 , Span 80, Span 65, Span 60, glyceryl monostearate, glyceryl monooleate, and the like. According to some specific embodiments of the present invention, the emulsifier may be, for example, lecithin, soy lecithin, Span 85, Span 83, Span 80, Span 65, Span 60, glyceryl monostearate, At least one of glyceryl monooleate; emulsifiers suitable for use in the present invention are preferably lecithin, soy lecithin, Span 85, Span 83, Span 80, Span 65, Span 60, Single Hard Glyceryl fatty acid, glyceryl monooleate, or; better use is lecithin, soy lecithin, span 85, span 83, span 80, span 65, or span 60; particularly preferred source is egg Phospholipid, soy phospholipid, Span 85, Span 83, Span 80, or Span 65.

According to one aspect of the present invention, the colloidal extract that can be used as an adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited. For example, it can be an aloe extract, acanthopanax extract, and horse teeth Coriander extract, grapefruit extract, persimmon leaf extract, β-glucan, mulberry yellow mushroom extract, Cordyceps sinensis extract. According to some specific embodiments of the present invention, the gum extract source may be, for example, aloe vera extract, Wujiapi extract, purslane extract, grapefruit extract, persimmon leaf extract, β-glucan , Mulberry yellow mushroom extract, Cordyceps sinensis extract, its derivatives, and their mixtures constitute at least one selected from the group; the colloidal extracts suitable for the present invention are preferably used from aloe extract and Wujiapi Extract, purslane extract, grapefruit extract, persimmon leaf extract, β-glucan, mulberry yellow mushroom extract, or Cordyceps extract; better sources are aloe vera extract and Wujiapi extract , Purslane extract, grapefruit extract, or beta-glucan; a particularly good source is aloe vera extract, Wujiapi extract, or beta-glucan.

According to one aspect of the present invention, the buffer that can be used in the adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited. For example, it can be sodium phosphate buffer, potassium phosphate buffer, Tris-HCl Buffer. According to some specific embodiments of the present invention, the buffer source may be, for example, a sodium phosphate buffer, a potassium phosphate buffer, a Tris-HCl buffer, a sodium citrate-phosphate buffer, a zwitterion buffer, or a Hepes buffer. Agents, maleate buffers, their derivatives, and mixtures thereof constitute at least one selected from the group; preferred sources of buffers suitable for use in the present invention are sodium phosphate buffer, potassium phosphate buffer, Tris-HCl buffer, sodium citrate-phosphate buffer, zwitterionic buffer, Hepes buffer, or maleate buffer; more preferred sources are sodium phosphate buffer, potassium phosphate buffer, Tris-HCl Buffer, sodium citrate-phosphate buffer, or zwitterionic buffer; particularly preferred sources are sodium phosphate buffer, potassium phosphate buffer, sodium citrate-phosphate buffer, or zwitterionic buffer.

According to one aspect of the present invention, the surfactant that can be used in the adjuvant for the poultry Newcastle disease vaccine of the present invention is not particularly limited. For example, it may be, polyoxyethylene fatty alcohol, caprylic acid and caprylic acid. Ethylene glycol glyceride (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, Polyoxyethylene hydrogenated castor oil, Planonic F-68, Polyoxyethylene Lauryl alcohol ether and so on. According to some specific embodiments of the present invention, the surfactant may be, for example, self-, polyoxyethylene fatty alcohol, caprylic caprylic acid polyethylene glycol glyceryl ester (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil, Planic F-68, polyoxyethylene lauryl alcohol ether; the surfactants suitable for the present invention are preferably used as , Polyoxyethylene Fatty Alcohol, Polyethylene Glycerol Caprylate (Labrasol), Tween 20, Tween 40, Tween 60, Tween 80, Tween 81, Tween 85, Polyoxyethylene Hydrogenated Castor Oil , Planic F-68, or polyoxyethylene lauryl alcohol ether; more preferably, polyoxyethylene fatty alcohols, capraic acid caprylic acid polyethylene glycol glyceride (Labrasol), Tween 20, Tween 40, Tween Tween 60, Tween 80, Tween 81, or Tween 85; particularly good sources of use are Labrasol Caprylate Caprylate (Trapon 20), 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 following steps: about 35.0 parts by weight to about 95.0 parts by weight of an oil agent, about 1.0 parts by weight to about 5.0 parts by weight of an emulsifier and about 1.0 parts by weight to about 10.0 parts by weight of a colloidal extract are sufficiently emulsified and mixed under a first reaction condition to form an emulsion; about 1.0 is added to the emulsion obtained from the above (a) A part of the buffering agent to about 15.0 parts by weight, and a surfactant of about 1.0 to about 2.5 parts by weight are sufficiently mixed and formed an adjuvant for avian newcastle disease vaccine under the second reaction condition.

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

In addition, according to one aspect of the present invention, a method for preparing an adjuvant for a poultry Newcastle disease vaccine of the present invention can be used, wherein (b) the second reaction condition includes a temperature of 55 ° C to 65 ° C and an adjusted pH value of 6.8 ~ 7.4, vibration amplitude 14 mm ~ 18 mm, stirring speed 800 rpm ~ 1200 rpm, and reaction was performed for 2 ~ 3 hours.

In addition, according to one aspect of the present invention, a method for preparing an adjuvant for a poultry Newcastle disease vaccine of the present invention can be used, wherein the oil agent is mineral oil; the emulsifier is Span 80; the colloidal extract is Aloe vera extract; the buffering agent is; the surfactant is Tween 20.

The present invention is further described below with reference to specific embodiments.

In the following, different specific embodiments are listed and described in more detail for the implementation aspects of the present invention in order to make the spirit and content of the present invention more complete and easy to understand; however, those with ordinary knowledge in this technology should understand The invention is of course not limited to these examples, and other identical or equal functions and sequence of steps can be used to achieve the invention.

In addition, through the following specific examples, the scope of the present invention can be further proved, but it is not intended to limit the scope of the present invention in any form.

Various reagents used in the following examples and experiments are commercially available. For example, mineral oil is a product made by H & R (for example, trade name: pharmaceutical grade white oil No. 70); poultry Newcastle disease antigen solution is produced by Pingtung University of Science and Technology commissioned by Taiwan Guonian Industrial Co., Ltd. , Trade name: The poultry Newcastle disease antigen solution (Vat) used in the present invention is prepared by at least one of the next three virus strains; these three virus strains were deposited in the food industry development of the consortium on March 24, 105 Institute, the deposit numbers are BCRC 970070, BCRC 970071, and BCRC 970072, all of which are poultry type VII Newcastle disease virus strains. The three poultry type VII Newcastle disease virus strains that have completed the above biological deposit are inoculated to 11 days of fertilization. In chicken embryos, the virus was harvested after 96 hours of incubation, and then the virus was deactivated with formalin (Sigma, USA) at a final concentration of 0.2%. ; The emulsifier is Span 80 (for example, trade name: S6760) produced by SIGMA company; the surfactant is Tween 20 (for example, trade name: P2287) produced by SIGMA company. "Adjuvant for preparing poultry Newcastle disease vaccine" "Preparation Example 1"

First, add 3.5 L of pharmaceutical-grade white oil, 0.5 L of lecithin, and 0.5 L of Span 80 to a blending bottle, and then add 1.5 L of potassium phosphate buffer (34 g of potassium dihydrogen phosphate, 1 mol / 175 ml of sodium hydroxide solution, 825 ml of distilled water, pH 7.2), 1.5 L of amphoteric buffer solution (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing Six grams of crystal water 0.1g of magnesium chloride was dissolved in 1L ddH ₂ O), using a mixer (model: UHM-10, brand: NIHONSEIKI Japan) in a sterile environment, at a temperature of 65 ° C, an amplitude of 20mm, and a stirring speed of 1200 rpm, The reaction was carried out for 60 minutes, and the emulsified liquid M1 was obtained after thorough mixing.

Next, as shown in Table 1, 1 g of aloe vera extract (using 70% alcohol to extract 1000 g of aloe vera) can further extract the active ingredients of the aloe vera extract and use it in the form of tincture. Its concentration is 18.8 g / ml), 1 L of Wujiapi Glia extract (using 70% alcohol to extract 1000 g Wujiapi, can further extract the active ingredients of Wujiapi Glia extract, use it in the form of tincture, its concentration (18.8 g / ml), add emulsion M1 together, and then add 0.25 L Tween 20 and 0.25 L Tween 80 at the same time, and first stir for 2 hours at room temperature in a sterile environment; continue to use the ultra-high-speed homogenizer to The adjuvant (S1) for the poultry Newcastle disease vaccine of the present invention was obtained by homogeneously stirring at 800 rpm, an amplitude of 14 mm, and 55 ° C. for 1 hour, adjusting the pH value to 6.8, and lowering the temperature by condensation. << Preparation Example 2 >>

First, add 6.0 L of pharmaceutical-grade white oil, 0.5 L of lecithin, and 0.5 L of Span 80 to the blending bottle, and then add 1.5 L of phosphate buffer solution (34 g of potassium dihydrogen phosphate, 1 mol / 175 ml of sodium hydroxide solution, 825 ml of distilled water, pH 7.2), 1.5 L of amphoteric buffer solution (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing Six grams of crystal water 0.1g of magnesium chloride are dissolved in 1L ddH ₂ O), using a mixer (model: UHM-10, brand: NIHONSEIKI Japan) under a sterile environment, the temperature is 70 ℃, the amplitude is 22 mm, and the stirring speed is 1600 rpm The reaction was carried out for 80 minutes. After being thoroughly mixed, an emulsion M2 was obtained with a water and oil content of 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, the active ingredients of the aloe vera gel extract can be further extracted and used in the form of tincture, with a concentration of 20 g / ml), 0.5 L of Wujiapi Glia extract (using 70% alcohol to extract 1000 g Wujiapi, further extract the active ingredients of Wujiapi Glia extract and use it in the form of tincture. Its concentration is 20g / ml) Emulsion M2 was added together, and then 0.5 L of Tween 20 and 0.5 L of Tween 80 were added at the same time, and the mixture was stirred uniformly for 2.2 hours at room temperature under a sterile environment; the ultra-high-speed homogenizer was used at 900 rpm, and the amplitude was increased. It was homogeneously stirred at 16 mm and 60 ° C. for 1.2 hours, the pH was adjusted to 7.0, and then the adjuvant (S2) for poultry Newcastle disease vaccine of the present invention was obtained through cooling and cooling. << Preparation Example 3 >>

First, add 7 L of triglyceride, 0.25 L of lecithin, and 0.25 L of Span 80 to the preparation bottle, and then add 0.2 L of phosphate buffer solution (34 g of potassium dihydrogen phosphate, 1 mol / L hydrogen). 175 ml of sodium oxide solution, 825 ml of distilled water, pH 7.2), 0.8 L of amphoteric buffer solution (NaCl 8g, KCl 0.2g, disodium hydrogen phosphate 1.15g, potassium dihydrogen phosphate 0.2g, CaCl ₂ 0.1g, containing 6 0.1 g of magnesium chloride in crystal water was dissolved in 1 L of ddH ₂ O), using a stirrer (model: UHM-10, brand: NIHONSEIKI Japan) in a sterile environment, at a temperature of 75 ℃, an amplitude of 23 mm, and a stirring speed of 2000 rpm, the reaction After 120 minutes of intensive mixing, an emulsion M3 was obtained with a water-oil content 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, can further extract the active ingredients of aloe vera gel extract, and use it in the form of tincture, its concentration is 22.1 g / ml), 0.5 L of Wujiapi Glia extract (using 70% alcohol to extract 1000 g Wujiapi, further extract the active ingredients of Wujiapi Glia extract, use it in the form of tincture, its concentration is 22.1 g / ml) together with emulsion M3, and then add 0.25 L Tween 20 and 0.25 L Tween 80 at the same time, and first stir uniformly for 2 hours at room temperature in a sterile environment; continue to use the ultra-high speed homogenizer at 1000 rpm The mixture was homogeneously stirred at an amplitude of 17 mm and 62 ° C. for 1 hour, and the pH was adjusted to 7.2. Then, the adjuvant (S3) for the poultry Newcastle disease vaccine of the present invention was obtained through cooling and cooling. << Preparation Example 4 >>

First, add 9.5 L of mineral oil and 0.1 L of lecithin to the preparation bottle, and then add 0.1 L of phosphate buffer solution (34 g of potassium dihydrogen phosphate, 175 ml of 1 mol / L sodium hydroxide solution, 825 ml of distilled water, pH Value 7.2), using a blender (model: UHM-10, brand: NIHONSEIKI Japan) under a sterile environment, temperature 80 ℃, amplitude 25 mm, stirring speed 2400 rpm, reaction for 180 minutes, fully mixed to obtain emulsion M4 , Water and oil degree 16.

Next, as shown in Table 1, 0.1 g of aloe vera gel extract (using 70% alcohol to extract 1000 g of aloe vera, the active ingredients of the aloe vera gel extract can be further extracted and used in the form of tincture, the concentration of which is 25.8 g / ml), 0.1 L of Wujiapi Glia extract (using 70% alcohol to extract 1000 g Wujiapi, further extract the active ingredients of Wujiapi Glia extract, use it in the form of tincture, its concentration is 25.8 g / ml) together with emulsion M4 and 0.1 L of Tween 20 at the same time. Stir for 1 hour at room temperature in a sterile environment. Continue to use an ultra-high speed homogenizer at 1200 rpm, 18 mm, 65 ° C. The mixture was homogeneously stirred for 2 hours, the pH was adjusted to 7.4, and then the temperature was reduced by condensation to obtain the adjuvant (S4) for poultry Newcastle disease vaccine of the present invention.

According to the above-mentioned "Preparation Examples 1 to 4", the proportion and data are described in "Table 1" as follows:

"Table 1"

Next, put 450 ml to 600 ml of Preparation Examples S1 to S4 in a 2000 ml beaker, and then put the emulsifying homogenizer into the beaker, and let the poultry new town disease of the present invention in Preparation Examples S1 to S4. The emulsifying homogenizer covers the stirring rod of the emulsification homogenizer for 2/3 (about 15 cm). Turn the emulsification homogenizer (model: TT-SM-S, brand: Taiwan Dading Machinery) to a high speed of 3000 rpm and slowly 350 Add ml to 600 ml of antigen to Preparation Example S1 to Preparation S4 in rotation (buffer to 350 ml when antigen is less than 350 ml); Finally, when 350 ml of antigen is added, the speed can be adjusted to 5000 rpm During the final stirring time, five to ten minutes. The detailed proportioning method is recorded in "Table 2" below.

"Table 2"

ND Experiment 1 In order to test the efficacy of vaccines V1 to V4 containing the adjuvant for poultry Newcastle disease vaccine of the present invention in preventing poultry type VII Newcastle disease, 30 1-day-old SPF chickens were divided into 5 groups, respectively for control Example: 2.5 μL of phosphate buffered saline (PBS) was injected subcutaneously. Example 1 was a vaccination method V1 containing the adjuvant for the poultry Newcastle disease vaccine of the present invention in a nasal manner of 2.0 μL. Example 2 was a 2.5 μL subcutaneous injection of the Invented vaccine V2 for poultry Newcastle disease vaccine, Example 3 is an intramuscular injection of 3.0 μL of vaccine V3 containing an adjuvant for poultry Newcastle disease vaccine, and Example 4 is a subcutaneous injection of 3.2 μL of a vaccine containing the invention Adjuvant vaccine V4 for poultry Newcastle disease vaccine.

Three weeks after vaccination, the vaccine was boosted again. The vaccine was the same as the first time. The chickens were sacrificed two weeks after the booster vaccination. 1wk), 2 weeks after the inoculation (PI-2wk), and 3 weeks after the inoculation (PI-3wk), blood was collected and the cell proliferation test in blood was detected. The spleen cells of the chickens were sacrificed and the percentages of CD4 ⁺ and CD8 ⁺ cells in the spleen cells were detected.

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

Three weeks after vaccination, the vaccine was boosted again. The vaccine was the same as the first time. The chickens were sacrificed two weeks after the booster vaccination. 1wk), 2 weeks after the inoculation (PI-2wk), and 3 weeks after the inoculation (PI-3wk), blood was collected and the cell proliferation test in blood was detected. The spleen cells of the chickens were sacrificed and the percentages of CD4 ⁺ and CD8 ⁺ cells in the spleen cells were detected. 《Detection method of cell proliferation test》

For cell proliferation test, chicken blood was used. Histopaque ^® 1077 density gradient medium was used to isolate PBMC cells. Count the cells and adjust the cell number to 2 x 10 ⁵ cells / 100 μL / well. Dispense into 96-well plates and add 1 g / 50 μL / well of Con A (Sigma, USA) and the same volume of culture solution, each three times, put in a 5% CO ₂ , 37 ° C incubator for 48 hours, add 10 μL / well BrdU reagent to mark the cells, and then Incubate for 24 hours, centrifuge at 1000 rpm for 10 minutes, remove the supernatant, add 200 μL / well FixDenat solution, place at room temperature for 30 minutes, remove FixDenat solution and add 100 μL / well anti-Brd U-pod working solution, set Remove the anti-Brd U-pod working solution at room temperature for 90 minutes, add 300 μL / well wash solution, repeat the washing three times, remove the wash solution, add 100 μL / well substrate solution, and let it stand at room temperature for 5 minutes. Read at 370 nm. The average values of the percentage of cell proliferation in the test results of 6 chickens per group in each example are recorded in "Table 3", respectively.

《CD4⁺ /CD8⁺ 細胞百分比之檢測方法》"table 3"

"Detection method of CD4 ⁺ / CD8 ⁺ cell percentage"

The detection of CD4 ⁺ / CD8 ⁺ cell surface antigens adjusted the cytosol concentration to 2 × 10 ⁵ cells, and the cytosols stained with avian monoclonal antibodies IgG1- FITC and IgG1-PE were used as negative control groups (isotype control). Detect chicken cell surface antigens CD4 ⁺ and CD8 ⁺ , add anti-CD4 monoclonal antibody and anti-CD8 monoclonal antibody (Beckman Coulter, Inc. USA) to the cell fluid to be tested, mix well, and place on ice After 30 minutes of protection from light, centrifuge at 300 xg for 5 minutes at 4 ° C. After removing the supernatant, add PBS to suspend the cells, wash the cells by centrifugation at 300 xg for 5 minutes at 4 ° C, and repeat the washing twice. Finally, 100 μL of PBS suspension cells containing 1% paraformaldehyde was added, and their cell surface antigens were analyzed with a Coulter Epics Altra Flow Cytometry (Beckman Coulter, CA). The cells were then analyzed with Expo 32 v1.0 MultiCOMP Software (Applied Cytometry Systems, Sheffield, UK) analyzed its percentage of CD4 ⁺ and CD8 ⁺ cells. The average values of the virus power values of the test results of 6 chickens per group in each example are recorded in "Table 4".

"Table 4"

As shown in Table 4 above, in the control group, the immune cells in the blood of the chicken not administered with the adjuvant for the poultry Newcastle disease vaccine of the present invention only increased by 15-18%; relatively, in the examples, In 1-4, chickens administered with the adjuvant vaccine for the Newcastle disease vaccine for avians of the present invention showed an increase in immune cells in the blood of at least more than 30%, and after a long observation period, the new birds of the present invention Adjuvants for disease vaccines can increase immune protection, because immune cells in the blood of chickens are tested after three weeks and are 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 Examples 1 to In 4, chickens treated with the adjuvant vaccine for poultry Newcastle disease vaccine of the present invention had a CD4 ⁺ / CD8 ⁺ cell percentage of about 25% / 35% or more, showing that inoculation with the vaccine using the adjuvant of the present invention can improve The ratio of CD4 ⁺ / CD8 ⁺ cells in chickens has further demonstrated the effect of improving immunity and combating Newcastle disease.

Thus, it was confirmed that chickens administered with the vaccine using the adjuvant for avian Newcastle disease vaccine can increase the immune protection ability, and further have an excellent effect against Newcastle disease infection. Therefore, the adjuvant for the poultry Newcastle disease vaccine of the present invention can significantly improve the immunity and immune cell proliferation of chickens, and enhance the vaccine to induce protective immune responses to chickens in Newcastle disease.

In summary, the content of the present invention has been illustrated by the above examples, but the present invention is not limited to these embodiments. Those with ordinary knowledge in the technical field to which the present invention pertains may make various changes and modifications without departing from the spirit and scope of the present invention; for example, combining or changing the technical contents illustrated in the foregoing embodiments As new embodiments, these embodiments are of course regarded as the content of the present invention. Therefore, the scope of protection in this case also includes the scope of patent application and its defined scope.

Claims (10)

An adjuvant for avian Newcastle disease vaccine, comprising at least: about 35.0 parts by weight to about 95.0 parts by weight of an oil agent; about 1.0 parts by weight to about 5.0 parts by weight of an emulsifier; about 2.0 parts by weight to about 10.0 parts by weight A colloidal extract; from about 1.0 part by weight to about 15.0 parts by weight of a buffer; and from about 1.0 part by weight to about 5.0 parts by weight of a surfactant; and when actually applied, the adjuvant for the avian Newcastle disease vaccine ( Adj) and avian Newcastle disease antigen solution (Vat) have a blending ratio (Adg: Vat) of 1: 5 (v / v) to 1: 1 (v / v), and the avian newcastle disease antigen solution (Vat) is Prepared by at least one of the following three virus strains; these three virus strains were deposited with the Food Industry Development Research Institute of the Consortium on March 24, 105 under the registration numbers of BCRC 970070, BCRC 970071, and BCRC 970072. Avian strain of Newcastle disease type VII. The adjuvant for a poultry Newcastle disease vaccine according to claim 1, wherein the oil agent is at least one selected from pharmaceutical grade white oil, peanut oil, mineral oil, vegetable oil, or vitamin E. The adjuvant for poultry Newcastle disease vaccine according to claim 1, wherein the emulsifier is selected from the group consisting of lecithin, soy lecithin, Span 85, Span 83, Span 80, Span 65, Span 60, Single Hard At least one of glyceryl fatty acid or glyceryl monooleate. The adjuvant for poultry Newcastle disease vaccine according to claim 1, wherein the colloidal extract is selected from the group consisting of aloe extract, Wujiapi extract, purslane extract, grapefruit extract, persimmon leaf extract, β -At least one of dextran, mulberry extract, or cordyceps extract. The adjuvant for poultry 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, zwitterionic buffer, At least one of Hepes buffer, or maleate buffer. The adjuvant for poultry Newcastle disease vaccine according to claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene fatty alcohol, caprylic caprylic acid polyethylene glycol glyceride (Labrasol), Tween 20, Tween 40, At least one of Tween 60, Tween 80, Tween 81, Tween 85, polyoxyethylene hydrogenated castor oil, Planic F-68, and polyoxyethylene lauryl alcohol ether. A method for preparing an adjuvant for a poultry Newcastle disease vaccine comprises the following steps: (a) about 35.0 parts by weight to about 95.0 parts by weight of an oil agent, about 1.0 parts by weight to about 5.0 parts by weight of an emulsifier, and about 1.0 parts by weight to about 10.0 parts by weight of the colloidal extract is sufficiently emulsified and mixed under the first reaction condition to form an emulsion; (b) about 1.0 part by weight to about 15.0 parts by weight is added to the emulsion obtained in the above (a) Parts of the buffer, and about 1.0 parts 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. The method for preparing an adjuvant for a poultry Newcastle disease vaccine as described in claim 7, wherein (a) the first reaction conditions include a temperature range of 65 to 80 ° C, a vibration amplitude of 20 mm to 25 mm, and a stirring speed of 1200 rpm to 2400. After the reaction was performed at rpm for 1 to 3 hours, the water-oil degree (HLB) was between 10 and 16. The method for preparing an adjuvant for a poultry Newcastle disease vaccine as described in claim 7, wherein (b) the second reaction condition includes adjusting the pH to 6.8 to 7.4 at a temperature of 55 ° C to 65 ° C and a vibration amplitude of 14 mm to 18mm, stirring speed 800 rpm ~ 1200 rpm, and reaction was performed for 2 ~ 3 hours. The method for preparing an adjuvant for a poultry Newcastle disease vaccine according to claim 7, wherein the oil agent is mineral oil; the emulsifier is Span 80; the colloidal extract is an aloe extract; the buffer agent is Is a phosphate buffer; the surfactant is Tween 20.