WO2018227728A1 - 一种免疫增强剂、口蹄疫灭活疫苗及其制备方法 - Google Patents

一种免疫增强剂、口蹄疫灭活疫苗及其制备方法 Download PDF

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WO2018227728A1
WO2018227728A1 PCT/CN2017/094856 CN2017094856W WO2018227728A1 WO 2018227728 A1 WO2018227728 A1 WO 2018227728A1 CN 2017094856 W CN2017094856 W CN 2017094856W WO 2018227728 A1 WO2018227728 A1 WO 2018227728A1
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vaccine
foot
immunopotentiator
mouth disease
compound
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French (fr)
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陈瑾
于晓明
郑其升
侯立婷
王义伟
张元鹏
乔绪稳
侯继波
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江苏省农业科学院
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Priority to US16/063,209 priority patent/US20200261570A1/en
Publication of WO2018227728A1 publication Critical patent/WO2018227728A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/125—Picornaviridae, e.g. calicivirus
    • A61K39/135—Foot- and mouth-disease virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/5555—Muramyl dipeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55572—Lipopolysaccharides; Lipid A; Monophosphoryl lipid A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
    • C12N2770/32134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to the field of biopharmaceutical, in particular to an immune enhancer, a foot-and-mouth disease inactivated vaccine and a preparation method thereof.
  • Foot-and-mouth disease is an acute, heat, and highly contagious infectious disease caused by Foot-and-mouth disease virus (FMDV).
  • the foot-and-mouth disease virus belongs to the genus Aphthavirus of the genus Picornaviridae. There are seven serotypes: A, O, C, SAT1, SAT2, SAT3 and Asia type 1, each of which contains several subtypes. The virus is not only cross-immunized between the various types, but also partially cross-immunized with each subtype of the serotype.
  • the General Office of the State Council issued the “National Medium- and Long-Term Animal Disease Control Plan (2012-2020)”, which listed foot and mouth disease as one of the priority prevention and treatment diseases.
  • the foot-and-mouth disease vaccine is a mandatory immunization vaccine.
  • the inactivated vaccine is the main vaccine currently used, but it also has shortcomings such as slow antibody production, short immunization period, narrow antigen spectrum, and incomplete inactivation.
  • new inactivated vaccines such as new epidemic strains, production processes, making antigens more pure, better immune effects, more effective adjuvants, more reliable inactivation, etc., but each process Both the research cycle and the verification cycle are relatively long.
  • the routine immunization of foot-and-mouth disease vaccine in pigs is generally 2 to 3 times, but the duration of immune protection is only 3 to 4 months.
  • the protective effect after immunization is also 70-80%. Therefore, there is a great room for quality improvement in foot-and-mouth disease vaccine, and improvement of immune enhancer is one of the feasible technical approaches.
  • Astragalus polysaccharide can significantly enhance non-specific immune function and humoral immune function. Astragalus polysaccharide can induce interferon production, interfere with virus replication in the body, improve immune function of the body; and strengthen and stimulate lymphocytes and reticuloendothelial cells The production enhances the phagocytic function of reticuloendothelial cells and macrophages, and promotes and regulates humoral, mucosal and cellular immunity. As a feed additive in animal breeding, it has the effects of promoting animal growth and improving body resistance. As a natural product, it has a rich source, low price, long-term use, small side effects on tissue cells, and low residue. However, the amount added in the feed or drinking water is large, and the basic amount is at least g/day, resulting in a large waste and the immunopotentiating effect is not true or difficult to evaluate.
  • TLRs Toll-like receptors
  • TLR agonists pathogenic microbial related molecules
  • TLR agonists pathogenic microbial related molecules
  • the use of TLR agonists in veterinary vaccine research is mostly At the laboratory stage, a large number of studies have shown that TLR agonists can be used in vaccine immunopotentiators.
  • the addition of TLR agonists to the vaccine, such as CpG, polyI:C, imiquimod, etc. has significant immunopotentiating potency.
  • TLR4 agonists have been approved for use in hepatitis B and human papillomavirus vaccines in 2009.
  • the technical problem to be solved by the present invention is to provide a compound immunopotentiator.
  • the object of the present invention is to provide a compound immunopotentiator which can produce a synergistic effect by using a trace amount of TLR agonist in combination with a trace amount of a traditional Chinese medicine immune enhancer Astragalus polysaccharide, and not only reduces the cost of using a TLR agonist immunopotentiator alone, It also improves the immune efficacy, and better improves the immune effect of the foot-and-mouth disease vaccine. For piglets, it can be protected by a needle, and the window period of antibody production is shortened to 7 days. The antibody duration is extended to more than 7 months, significantly reducing the growth. Pig cost.
  • the technical problem to be solved by the present invention is to provide a preparation method of a compound immunopotentiator.
  • the technical problem to be solved by the present invention is to provide a foot-and-mouth disease inactivated vaccine containing a compound immunopotentiator.
  • the final technical problem to be solved by the present invention is to provide a method for preparing a foot-and-mouth disease inactivated vaccine containing a compound immunopotentiator.
  • the technical solution of the present invention provides a compound immunopotentiator including, but not limited to, a monophosphoryl lipid A 5-52 ⁇ g/mL, a muramyl dipeptide 10 to 520 ⁇ g/mL, ⁇ -glucan 1 to 520 ⁇ g/mL, and xanthine polysaccharide 0.05 to 5.2 mg/mL.
  • the immunopotentiator includes, but is not limited to, monophosphoryl lipid A monophosphoryl lipid A 5 to 500 ⁇ g/mL, muramyl dipeptide 10 to 500 ⁇ g/mL, and ⁇ -glucan 1 to 500 ⁇ g/ mL and Astragalus polysaccharides 0.05 to 5.0 mg/mL.
  • the immunopotentiator includes, but is not limited to, monophosphoryl lipid A 100-500 ⁇ g/mL, muramyl dipeptide 100-500 ⁇ g/mL, ⁇ -glucan 50-500 ⁇ g/mL, and astragalus polysaccharide. 1 to 5.0 mg/mL.
  • the present invention also includes a method for preparing the above-described immunopotentiator, including but not limited to the following steps:
  • the present invention also includes the use of the above-described immunopotentiator for the preparation of a vaccine.
  • the present invention also includes the above-mentioned foot-and-mouth disease inactivated vaccine containing the compound immunopotentiator.
  • the above-mentioned foot-and-mouth disease inactivated vaccine also includes, but is not limited to, an inactivated antigen solution.
  • the volume ratio of the inactivated antigen solution to the compound immunopotentiator in the above-mentioned foot-and-mouth disease inactivated vaccine is 9:1 to 8:1.
  • the above inactivated antigen solution is not limited to one or more of O, A, subtype I foot-and-mouth disease inactivated antigen, polypeptide or other genetically engineered expression products.
  • the invention also includes a preparation method of the above-mentioned foot-and-mouth disease inactivated vaccine containing the compound immunopotentiator, including but not limited to the following steps:
  • the present invention has the following advantages over the prior art:
  • the invention develops a compound immunopotentiator, which can effectively improve the efficacy of the vaccine by mixing it with the foot-and-mouth disease inactivated vaccine, not only can improve the antibody yield rate and the average antibody level, but also can significantly shorten the antibody production.
  • the window period is up to 7 days, while increasing the duration of the antibody to more than 7 months.
  • Astragalus polysaccharide is rich in source, low in price, long-term use has little toxic side effects on tissue cells, low residue, adding a small amount of Astragalus polysaccharide can significantly reduce the dosage of other three TOLL-like receptor agonists, reducing production cost by 90%, but The immune effect has not decreased.
  • the combined use of the compound immune enhancer and the foot-and-mouth disease inactivated vaccine of the invention can significantly improve the immune efficacy of the vaccine, and the pig farm can reduce the number of vaccine immunizations according to its own situation, thereby reducing the breeding cost and reducing the stress of the herd.
  • Figure 1 shows the average antibody level and antibody duration after immunization with compound immunopotentiator vaccine; specifically, the average liquid phase block at different time points after immunization of piglets with O-type FMD inactivated vaccine containing different compound immunopotentiator components ELISA antibody levels.
  • Example 1 Preparation of compound immune enhancer and foot-and-mouth disease vaccine
  • Monophosphoryl lipid A is abbreviated as MPL.
  • the muramyl dipeptide is abbreviated as MDP.
  • MPL MDP
  • ⁇ -glucan purchased from InvivoGen.
  • Astragalus polysaccharide was purchased from Shaanxi Zhengda Biotechnology Co., Ltd.
  • ISA206 was purchased from the company, Baiyou, Siben, and Twain.
  • porcine O-type foot-and-mouth disease virus solution (the strain is porcine O-type foot-and-mouth disease virus 98 strains in Sri Lanka), inactivated by diethyleneimine, the content of 146s is 5.87 ⁇ g/mL, which is a gift from Inner Mongolia Jinyu Group.
  • the commercialized foot-and-mouth disease O, A, and Asian I trivalent vaccines were purchased from Inner Mongolia Jinyu Group.
  • the invention adopts a foot-and-mouth disease liquid phase blocking ELISA kit (Lanzhou Veterinary Research Institute) to detect a liquid phase blocking ELISA antibody titer.
  • the main components of the immunopotentiator are: monophosphoryl lipid A (MPL), muramyl dipeptide (MDP), ⁇ -glucan and astragalus polysaccharide. It is prepared by dissolving the main components in 0.1 M Tris-HCl at pH 8.0.
  • Compound immunopotentiator 1 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharides were 5 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL and 0.05 mg/mL, respectively.
  • Compound immunopotentiator 2 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharide were 100 ⁇ g/mL, 100 ⁇ g/mL, 50 ⁇ g/mL and 1 mg/mL, respectively.
  • Compound immunopotentiator 3 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharides were 500 ⁇ g/mL, 500 ⁇ g/mL, 500 ⁇ g/mL and 5 mg/mL, respectively.
  • Compound immunopotentiator 4 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharide were 5 ⁇ g/mL, 10 ⁇ g/mL, 1 ⁇ g/mL and 20 mg/mL, respectively.
  • Compound immunopotentiator 5 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharides were 500 ⁇ g/mL, 500 ⁇ g/mL, 500 ⁇ g/mL and 20 mg/mL, respectively.
  • Immunopotentiator 6 The final concentration of the Astragalus polysaccharide was 20 mg/mL.
  • Immunopotentiator 7 The final concentration of the Astragalus polysaccharide was 5 mg/mL.
  • Immunopotentiator 8 The final concentrations of MPL, MDP, and ⁇ -glucan were 2 mg/mL, 40 mg/mL, and 0.2 mg/mL, respectively.
  • the prepared compound immunopotentiator was filtered (0.22 ⁇ m filter), sterilized, and then dispensed into a blue bottle, and stored at 4 ° C.
  • the first method preparing a compound immunopotentiator companion vaccine, that is, the compound immunopotentiator and the Tween according to a volume ratio of 96:4, fully mixed and uniformly used as an aqueous phase; the white oil and the siemen are uniformly mixed according to a volume ratio of 96:4.
  • the oil phase the vaccine is prepared according to the water phase: oil phase volume ratio of 1:2, and the prepared vaccine is the compound immunopotentiator companion vaccine.
  • the compound immune enhancer companion vaccine can be fully mixed with the commercial vaccine according to the volume ratio of 1:9 before use.
  • the second method the compound immunopotentiator and the inactivated porcine O-type foot-and-mouth disease virus solution are thoroughly mixed at a volume ratio of 1:9 to prepare an aqueous phase solution.
  • the ISA206 and aqueous phase solutions were separately placed at room temperature for approximately 30 minutes.
  • the ISA206 was placed in an emulsification tank, and the aqueous phase solution was placed in an emulsification tank at 200 rpm, and the mixture was stirred uniformly; the mixture was stirred at 2000 rpm for 10 minutes to obtain a vaccine in which the volume ratio of the aqueous phase to the ISA206 was 46: 54.
  • the vaccine prepared by the above two methods has the same immunological effects under the same conditions of the final concentration of the compound immunopotentiator, and is convenient for the users who use different requirements for production.
  • the components of the compound immunopotentiator can be flexibly proportioned within a given range, and are not enumerated here.
  • Example 2 Evaluation of immune efficacy of compound immunopotentiator against foot-and-mouth disease inactivated vaccine
  • the compound immunopotentiator 1 and the inactivated antigen were mixed as 1:9 as an aqueous phase solution, and the ISA206 was placed in an emulsification tank. At 200 rpm, the aqueous phase solution was placed in an emulsification tank, and the mixture was stirred evenly.
  • the foot-and-mouth disease inactivated vaccine prepared by stirring at 2000 rpm for 10 minutes is called FMD inactivated vaccine containing compound immunopotentiator 1: FMD 1 for short.
  • the vaccine preparation method of the compound immunopotentiator 2, 3, 4, 5, 6, and 7 is the same as the compound immunopotentiator 1 to prepare the FMD inactivated vaccine containing the compound immunopotentiator 2, 3, 4, 5, 6, and 7: Referred to as FMD2, 3, 4, 5, 6, and 7.
  • FMD inactivated vaccine prepared by mixing the immunopotentiator 8 and the inactivated antigen according to 1:1 is called FMD inactivated vaccine containing compound immunopotentiator 8 (referred to as FMD 8 (prepared according to patent ZL201310042983.0,
  • the immunopotentiator is mixed with the inactivated porcine foot-and-mouth disease virus solution at a volume ratio of 1:1 to obtain an aqueous phase solution.
  • the ISA206 and the aqueous phase solution are separately placed at room temperature for about 30 minutes.
  • the ISA206 is placed in an emulsification tank at 200 Transfer/min, the aqueous phase solution was placed in an emulsification tank, stirred well, and stirred at 2000 rpm for 10 minutes to obtain a vaccine).
  • Tris-HCl and inactivated antigen were mixed 1:9 as an aqueous phase solution, and ISA206 was placed in an emulsification tank. At 200 rpm, the aqueous phase solution was placed in an emulsification tank and stirred. Evenly, stirring at 2000 rpm for 10 minutes, the prepared foot-and-mouth disease inactivated vaccine is called FMD control vaccine.
  • the antibody production after immunization was monitored: on the 7th, 14th, 21st and 28th day after immunization, the serum of each group of healthy susceptible piglets was collected and tested, and the blue-beating liquid phase blocking ELISA antibody detection kit was detected. Antibody production and window period after vaccine immunization;
  • Antibody duration monitoring of immunized pigs blood samples were taken at 28 days, 60 days, 90 days, 120 days, 150 days, 180 days, and 210 days after immunization, respectively. Detection of antibody production after vaccine immunization;
  • liquid phase blocking ELISA antibody titer is greater than or equal to 26, the antibody is qualified.
  • the antibody yield after immunization is shown in Table 1, Table 2.
  • 7dpv, 14dpv, 21dpv, and 28dpv in Table 1 represent 7 days after immunization, 14 days after immunization, 21 days after immunization, and 28 days after immunization; the upper/lower numbers represent the number of pigs whose antibodies have reached the qualified line. / number of pigs immunized;
  • Group FMD 6 had a certain immune enhancement at high dose levels, but the effect was not satisfactory, which was significantly worse than FMD 1/FMD 2/FMD 3 (qualification rates 8/10, 8/10 and 9/10);
  • Immune enhancement but lower doses of Astragalus polysaccharides combined with low doses of MPL, MDP and ⁇ -glucan (FMD 1/FMD 2/FMD 3) can significantly shorten the antibody window to 7 days, 7 More than 80% of the antibodies in the day can reach the qualified line (liquid phase blocking ELISA antibody is greater than 2 6 ), and the antibody yield is not reduced (compared with FMD8).
  • FMD group 6 there was also a significant level of immune enhancement, but the antibody production period was 7 days longer than FMD 1/FMD 2/FMD3, and the antibody yield rate and compound immune enhancer 1/14 after immunization.
  • the compound immune enhancer formula 1, 2, 3, that is, FMD 1/FMD 2/FMD 3 can significantly prolong the antibody duration of the vaccine, and monitor the antibody yield rate up to 7 months after immunization. No significant decline, maintained at 10/10; and the highest qualified rate of FMD control vaccine is basically 3/10, the individual Astragalus polysaccharide group (FMD 6/FMD 7), has a certain immune enhancement, but the effect is not obvious, and FMD The difference in the control vaccine was not significant.
  • the compound immunopotentiator 1, 2, 3 (FMD 1/FMD 2/FMD 3) group and the immunopotentiator 8 (FMD 8) group can significantly improve the immune efficacy of the FMD vaccine and shorten the antibody production.
  • the compound immune booster can significantly improve the immune response of the piglets to the antigen, improve the antibody yield rate and the time of early antibody production, and further Shorten the window period of antibody production by the vaccine to 7 days after immunization, and improve the immune effect of the vaccine.
  • the components of the immunopotentiator can be flexibly proportioned within a given range, and are not enumerated here.
  • the main components of the compound immunopotentiator are: monophosphoryl lipid A, muramyl dipeptide, ⁇ -glucan and astragalus polysaccharide. It is prepared by dissolving the main components in 0.1 M Tris-HCl at pH 8.0.
  • Compound immunopotentiator 9 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharide were 5.2 ⁇ g/mL, 10.4 ⁇ g/mL, 1.04 ⁇ g/mL and 0.052 mg/mL, respectively.
  • Compound immunopotentiator 10 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharide were 104 ⁇ g/mL, 104 ⁇ g/mL, 52 ⁇ g/mL and 1.04 mg/mL, respectively.
  • Compound immunopotentiator 11 The final concentrations of MPL, MDP, ⁇ -glucan and Astragalus polysaccharide were 520 ⁇ g/mL, 520 ⁇ g/mL, 520 ⁇ g/mL and 5.2 mg/mL, respectively.
  • the prepared compound immunopotentiator was filtered (0.22 ⁇ m filter), sterilized, and then dispensed into a blue bottle, and stored at 4 ° C.
  • the compound immunopotentiator companion vaccine prepared according to this method was named as the compound immunopotentiator companion 9, 10, 11 according to the preparation of the compound immunopotentiator 9, 10, and 11, respectively.
  • the components of the compound immunopotentiator can be flexibly matched within a given range, and the volume of use can also be adjusted according to actual needs, which are not enumerated here.
  • the compound immunopotentiator companion vaccine used the three companion vaccines prepared in Example 3.
  • Trivalent vaccine is O, A, sub-I trivalent inactivated vaccine (fine seedlings) batch number 5235039, 20151224
  • Each group of vaccines was immunized with a group of healthy susceptible piglets.
  • the antibody production after immunization was monitored: on the 7th, 14th, 21st and 28th day after immunization, the serum of each group of healthy susceptible piglets was collected and tested, and the blue-beating liquid phase blocking ELISA antibody detection kit was detected. Antibody production and window period after vaccine immunization;
  • Antibody duration monitoring was performed on the compound immunopotentiator group with better immune enhancement after immunization: blood was collected at 28 days, 60 days, 90 days, 120 days, 150 days, 180 days, and 210 days after immunization, respectively.
  • the liquid phase continuous ELISA antibody detection kit detects the antibody production after the vaccine is immunized;
  • the antibody yield after immunization is shown in Table 4 and Table 5.
  • the piglets immunized with the trivalent vaccine were only 20%, 30% and 10% qualified for the three serotypes 7 days after immunization; and the compound immunostimulant companion vaccine was immunized 9/10.
  • the piglets of the /11+ trivalent vaccine can reach a qualified rate of 70-90% 7 days after immunization, and the window period is significantly shortened; the pass rate of the liquid phase blocking ELISA antibody is also significantly increased.
  • the antibodies of piglets immunized with the trivalent vaccine began to decline slowly from the 90th day after immunization; and the piglets immunized with the compound immunopotentiator companion vaccine 9/10/11+ trivalent vaccine, after immunization After 28 days, the antibody level was basically stable, and there was no obvious downward trend until 7 months.
  • the immunization group supplemented with the compound immunopotentiator partner vaccine significantly prolonged the antibody duration of the vaccine.
  • the immune efficacy of the compound immune enhancer companion vaccine against O, A, and sub-I trivalent foot-and-mouth disease inactivated vaccines
  • the antibodies against the three serotypes of O, A and sub-I have obvious immunopotentiating effects, and significantly shorten the window period of vaccine antibody production and increase the antibody duration of the vaccine.
  • the compound immunopotentiator companion vaccine used the three companion vaccines prepared in Example 3.
  • Each group of vaccines was immunized with a group of healthy susceptible piglets.
  • Antibody duration monitoring was performed on the compound immunopotentiator group with better immune enhancement after immunization: blood was collected at 28 days, 60 days, 90 days, 120 days, 150 days, 180 days, and 210 days after immunization, respectively.
  • the liquid phase blocking ELISA antibody detection kit detects the production of serum antibodies after vaccine immunization;
  • liquid phase blocking ELISA antibody titer is greater than or equal to 26
  • the antibody is qualified, and the polypeptide antibody detection is determined by the kit criteria to determine the positive yin.
  • the level of the liquid phase blocking ELISA antibody has a certain correlation with the protection of the drug, especially the higher the antibody level, the better the protective effect.
  • the compound immunopotentiator companion vaccine can significantly shorten the window period of the polypeptide seedling antibody production and improve the pass rate of the liquid phase blocking ELISA antibody.
  • the antibodies of the piglets of the immunopolypeptide vaccine began to decrease slowly from the 90th day after immunization; and the piglets immunized with the compound exemptionist companion vaccine 9/10/11+ polypeptide vaccine, 21 days after immunization
  • the antibody level was basically stable and could not be maintained until 7 months without a significant downward trend; the immunization group supplemented with the compound immunopotentiator companion vaccine significantly prolonged the antibody duration of the vaccine.
  • the compound immune booster companion vaccine has a significant effect on the immune efficacy of the foot-and-mouth disease polypeptide vaccine. And obviously can improve the pass rate of the liquid phase blocking ELISA antibody, and significantly shorten the window period of the vaccine antibody production, and improve the antibody duration of the vaccine.

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Abstract

本发明提供了一种复方免疫增强剂，含有单磷酰脂A 5-520μg/mL、胞壁酰二肽10-520μg /mL、β-葡聚糖1-520μg/mL和黄芪多糖0.05-5.2mg/mL。本发明还提供了含有该复方免疫增强剂的口蹄疫灭活疫苗，以及该复方免疫增强剂和口蹄疫灭活疫苗的制备方法。

Description

一种免疫增强剂、口蹄疫灭活疫苗及其制备方法 技术领域
本发明涉及生物制药领域，具体涉及一种免疫增强剂、口蹄疫灭活疫苗及其制备方法。
背景技术
口蹄疫(Foot-and-mouth disease,FMD)是由口蹄疫病毒(Foot-and-mouth disease virus,FMDV)引起的一种急性、热性、高度接触传染性的传染病。口蹄疫病毒属小RNA病毒科(Picornaviridae)口蹄疫病毒属(Aphthavirus)，有7个血清型：A、O、C、SAT1、SAT2、SAT3以及Asia 1型，每个血清型又包含若干个亚型。该病毒不仅在各型间没有交叉免疫性，同血清型的各亚型之间也仅有部分交叉免疫性。2012年，国务院办公厅印发了《国家中长期动物疫病防治规划(2012—2020年)》，将口蹄疫列为优先防治病种之一。
在我国，口蹄疫疫苗属于强制免疫疫苗，灭活疫苗是目前使用的疫苗主体，但其也有如抗体产生慢、免疫期短、抗原谱窄，灭活不彻底等不足之处。现在，许多研究人员正在改进和研究新的灭活疫苗，如新的流行毒株，生产工艺，使抗原更纯，免疫效果更好，佐剂更有效，灭活更加可靠等，但每项工艺的研究周期和验证周期都比较长。而猪群常规的口蹄疫疫苗免疫次数一般要免疫2～3次，但免疫保护持续期仅3～4个月，加强免疫后保护效力也最高仅为70～80％。因此，口蹄疫疫苗存在很大的质量提升空间，免疫增强剂改进是可行技术途径之一。
黄芪多糖可显著增强非特异性免疫功能和体液免疫功能，黄芪多糖能诱导机体产生干扰素，干扰病毒在机体内的复制，提高机体的免疫功能；并能强化和刺激淋巴细胞和网状内皮层细胞的生成，增强网状内皮层细胞和巨噬细胞的吞噬功能，并对体液、粘膜和细胞免疫有很好的促进和调节作用。作为饲料添加剂在动物养殖上的应用，具有促进动物生长，提高机体抵抗力等效力。其作为天然产品具有来源丰富、价格低廉、长期使用对组织细胞毒副作用小、残留低。然而在饲料或饮水中添加量较大，基本用量至少为克/天，造成较大的浪费且免疫增强效力不确实或者较难评价。
Toll样受体(TLR)是存在于哺乳动物免疫细胞的一类跨膜蛋白，其主要免疫学功能是监视与识别各种不同的病原微生物相关分子(TLR激动剂)，快速诱发先天性免疫反应，为抗原特异性获得性免疫反应打下基础。TLR激动剂应用于兽用疫苗研究多处于实 验室阶段，大量研究结果表明TLR激动剂可以用于疫苗免疫增强剂。在疫苗中添加TLR激动剂，例如CpG、polyI:C、咪喹莫特等，均有明显的免疫增强效力。其中TLR4激动剂已于2009年批准应用于乙型肝炎和人乳头瘤病毒疫苗。
现存的主要应用瓶颈是多数TLR激动剂制造成本太高问题。
发明内容
发明目的：本发明所要解决的技术问题是提供了一种复方免疫增强剂。本发明的目的在于提供一种复方免疫增强剂，使用微量TLR激动剂，配合使用微量中药免疫增强剂黄芪多糖可以产生协同增效作用，不仅降低了单独使用TLR激动剂类免疫增强剂的成本，也提高了免疫效力，更好的提高了口蹄疫疫苗的免疫效果，对于仔猪可以一针保护到出栏，同时缩短抗体产生的窗口期至7天，抗体持续期延长至7个月以上，显著降低养猪成本。
本发明还要解决的技术问题是提供了复方免疫增强剂的制备方法。
本发明还要解决的技术问题是提供了含复方免疫增强剂的口蹄疫灭活疫苗。
本发明最后要解决的技术问题是提供了含复方免疫增强剂的口蹄疫灭活疫苗的制备方法。
技术方案：为了解决上述问题，本发明的技术方案是提供了一种复方免疫增强剂，所述复方免疫增强剂包括但不仅限于含有单磷酰脂A 5～520μg/mL、胞壁酰二肽10～520μg/mL、β-葡聚糖1～520μg/mL和黄芪多糖0.05～5.2mg/mL。
其中，作为优选，上述免疫增强剂包括但不仅限于含有单磷酰脂A单磷酰脂A 5～500μg/mL、胞壁酰二肽10～500μg/mL、β-葡聚糖1～500μg/mL和黄芪多糖0.05～5.0mg/mL。
其中，作为优选，所述免疫增强剂包括但不仅限于含有单磷酰脂A 100～500μg/mL、胞壁酰二肽100～500μg/mL、β-葡聚糖50～500μg/mL和黄芪多糖1～5.0mg/mL。
本发明内容还包括上述的免疫增强剂的制备方法，包括但不仅限于以下步骤：
1).配制含有单磷酰脂质A、胞壁酰二肽、β-葡聚糖和黄芪多糖的溶液，与吐温-80混合，得到成水相溶液；
2).将白油和司盘-80混合，得到油相溶液；
3).将所述水相溶液与油相溶液混合乳化后得到含复方免疫增强剂的伴侣疫苗。
本发明内容还包括上述的免疫增强剂在制备疫苗方面的应用。
本发明内容还包括上述的一种含有所述的含复方免疫增强剂的口蹄疫灭活疫苗。
其中，上述口蹄疫灭活疫苗还包括但不仅限于灭活的抗原溶液。
其中，上述口蹄疫灭活疫苗中的灭活的抗原溶液与复方免疫增强剂的体积比为9:1～8:1。
其中，上述灭活的抗原溶液但不仅限于含O，A，亚I型口蹄疫灭活抗原、多肽或其他基因工程表达产物中的一种或几种。
本发明内容还包括上述含复方免疫增强剂的口蹄疫灭活疫苗的制备方法，包括但不仅限于以下步骤：
1)将复方免疫增强剂与灭活的抗原溶液混合，再与吐温-80混合得到水相溶液；
2)将白油和司盘-80混合，得到油相溶液；
3)将所述水相溶液与油相溶液充分混合即得含复方免疫增强剂的口蹄疫灭活疫苗。
有益效果：本发明相对于现有技术，具有以下优点：
1、本发明研制了一种复方免疫增强剂，将其与口蹄疫灭活疫苗混合后使用可以有效的提高疫苗的效力，不但可以提高抗体的合格率和平均抗体水平，还可以明显的缩短抗体产生的窗口期至7天，同时提高抗体的持续期至7个月以上。
2、黄芪多糖来源丰富、价格低廉、长期使用对组织细胞毒副作用小、残留低，添加少量黄芪多糖可以明显降低其他三种TOLL样受体激动剂的用量，降低了90％的生产成本，但免疫效力却没有下降。
3、本发明的复方免疫增强剂和口蹄疫灭活疫苗联合使用，可以明显提高疫苗的免疫效力，猪场可以根据自身情况减少疫苗免疫次数，从而减少养殖成本，减少猪群应激。
附图说明
图1含复方免疫增强剂疫苗免疫后的平均抗体水平及抗体持续期；具体为各组含不同复方免疫增强剂组分的O型FMD灭活疫苗免疫仔猪后不同时间点的平均液相阻断ELISA抗体水平。
具体实施方式
下面结合附图对本发明作更进一步的说明。
实施例1：复方免疫增强剂、口蹄疫疫苗的制备
1、实验材料
单磷酰脂质A缩写为MPL。
胞壁酰二肽缩写为MDP。
MPL、MDP和β-葡聚糖均购自InvivoGen公司。
黄芪多糖购自陕西正大生物科技有限公司。
ISA206购自赛百克公司，白油，司本，吐温为本实验室自行采购。
灭活的猪O型口蹄疫病毒液(毒株为猪O型口蹄疫病毒缅甸98株)，经二乙烯亚胺灭活，146s含量为5.87μg/mL，内蒙古金宇集团惠赠。
商品化的口蹄疫O、A、亚I三价疫苗购自内蒙古金宇集团。
6-7周龄健康易感仔猪，液相阻断ELISA抗体滴度≤1:8。
本发明采用口蹄疫液相阻断ELISA试剂盒(兰州兽医研究所)检测液相阻断ELISA抗体滴度。
1、免疫增强剂的配置
免疫增强剂的主要成份为：单磷酰脂质A(MPL)，胞壁酰二肽(MDP)，β-葡聚糖和黄芪多糖。其制备方法为将各主要成份溶解于pH8.0的0.1MTris-HCl中。
复方免疫增强剂1：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为5μg/mL、10μg/mL、1μg/mL和0.05mg/mL。
复方免疫增强剂2：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为100μg/mL、100μg/mL、50μg/mL和1mg/mL。
复方免疫增强剂3：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为500μg/mL、500μg/mL、500μg/mL和5mg/mL。
复方免疫增强剂4：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为5μg/mL、10μg/mL、1μg/mL和20mg/mL。
复方免疫增强剂5：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为500μg/mL、500μg/mL、500μg/mL和20mg/mL。
免疫增强剂6：配置黄芪多糖终浓度为20mg/mL。
免疫增强剂7：配置黄芪多糖终浓度为5mg/mL。
免疫增强剂8：配置MPL、MDP、β-葡聚糖终浓度分别为2mg/mL、40mg/mL、0.2mg/mL。
将配制好的复方免疫增强剂过滤(0.22μm滤器)除菌后分装至青瓶中，4℃保存。
口蹄疫疫苗制备方法
第一种方法：制备复方免疫增强剂伴侣疫苗，即复方免疫增强剂与吐温按照体积比为96:4，充分混合均匀做为水相；白油与司本按照体积比96:4混合均匀作为油相；按照水相：油相体积比为1:2进行疫苗制备，制备成的疫苗即为复方免疫增强剂伴侣疫苗。复方免疫增强剂伴侣疫苗使用前按照体积比1:9与商品化疫苗充分混匀即可。
第二种方法：将复方免疫增强剂与灭活的猪O型口蹄疫病毒液按照体积比为1:9充分混合，制备为水相溶液。先将ISA206和水相溶液分别放置于室温，大约30分钟。将ISA206放入乳化罐中，在200转/分，将水相溶液放入乳化罐中，搅拌均匀；在2000转/分搅拌10分钟，得到疫苗，其中水相与ISA206的体积比为46:54。
采用上述两种方法制备的疫苗，在复方免疫增强剂终浓度相同的条件下，免疫效果上基本一致，只是方便生产上不同需求的用户使用。
本实施例中，复方免疫增强剂的各组分可以在给出范围内灵活配比，在此不一一列举。
实施例2：复方免疫增强剂对口蹄疫灭活疫苗的免疫效力评价
1、疫苗制备
本实施例按照实施例1的第二种方法制备口蹄疫疫苗：
将复方免疫增强剂1与灭活抗原按照1:9混合后做为水相溶液，将ISA206放入乳化罐中，在200转/分，将水相溶液放入乳化罐中，搅拌均匀，在2000转/分搅拌10分钟，制备成的口蹄疫灭活疫苗称为含复方免疫增强剂的FMD灭活疫苗1：简称FMD 1。
复方免疫增强剂2、3、4、5、6、7的疫苗制备方法同复方免疫增强剂1，制备获得含复方免疫增强剂的FMD灭活疫苗2、3、4、5、6、7：简称FMD2、3、4、5、6、7。
将免疫增强剂8与灭活抗原按照1:1混合后，制备成的口蹄疫灭活疫苗称为含复方免疫增强剂的FMD灭活疫苗8：简称FMD 8(按照专利ZL201310042983.0进行制备疫苗，将免疫增强剂与灭活的猪口蹄疫病毒液按照体积比1:1混合，得到水相溶液。将ISA206和水相溶液分别放置于室温，大约30分钟。将ISA206放入乳化罐中，在200转/分，将水相溶液放入乳化罐中，搅拌均匀，在2000转/分搅拌10分钟，得到疫苗)。
将0.1M pH 8.0的Tris-HCl与灭活抗原按照1:9混合后做为水相溶液，将ISA206放入乳化罐中，在200转/分，将水相溶液放入乳化罐中，搅拌均匀，在2000转/分搅拌10分钟，制备成的口蹄疫灭活疫苗称为FMD对照疫苗。
2、分组、免疫和抗体检测
实验分组及免疫：将健康易感仔猪随机分组，每组10头，共6组。每组疫苗各免疫一组健康易感仔猪，免疫剂量为2mL。
免疫后采血：
对免疫后的抗体产生情况进行监测：免疫后第7天、14天、21天和第28天，对各组健康易感仔猪采血分离血清，兰兽研液相阻断ELISA抗体检测试剂盒检测疫苗免疫后的抗体产生情况和窗口期；
对免疫后猪只进行抗体持续期监测：分别在免疫后28天、60天、90天、120天、150天、180天和210天分别采血，兰兽研液相续断ELISA抗体检测试剂盒检测疫苗免疫后的抗体产生情况；
(当液相阻断ELISA抗体滴度大于等于26为抗体合格。)
免疫后抗体合格率如表1，表2所示。
免疫后的平均抗体水平如图1所示。
表1各组仔猪免疫后抗体合格率
Figure PCTCN2017094856-appb-000001
备注：表1中7dpv、14dpv、21dpv、28dpv分别代表免疫后7天、免疫后14天、免疫后21天、免疫后28天；上/下数字分别代表：抗体达到合格线的猪的头数/免疫猪的头数；
从表1可以看出，免疫FMD对照疫苗的仔猪，在免疫后7天抗体10头免疫猪的抗体仅有2头达到合格线，合格率为2/10；单独添加黄芪多糖为免疫增强剂的组别FMD 6，在高剂量水平有一定的免疫增强作用，但是效果也不理想，明显较FMD 1/FMD 2/FMD  3组差(合格率8/10，8/10及9/10)；单独的低剂量黄芪多糖组(FMD 7)的免疫增强效果更差；高剂量的黄芪多糖与不同剂量的MPL、MDP和β-葡聚糖复配后(FMD 4/FMD 5)，没有明显的免疫增强作用，但更低剂量的黄芪多糖与低剂量的MPL、MDP和β-葡聚糖复配后(FMD 1/FMD 2/FMD 3)，可以明显缩短抗体的窗口期至7天，7天即可达到80％以上的抗体达到合格线以上(液相阻断ELISA抗体大于26)，且不降低抗体的合格率(与FMD8比较)。FMD 6组，也有明显的免疫增强水平，但是在抗体产生的窗口期上要比FMD 1/FMD 2/FMD3相对延后7天，在免疫后14天的抗体合格率与复方免疫增强剂1/2/3(FMD 1/FMD 2/FMD3)相当，在免疫后14天的抗体合格率与对应的复方免疫增强剂1，2，3(FMD 1/FMD 2/FMD 3)组免疫后7天合格率相当。
表2各组仔猪免疫后抗体持续期内的抗体合格率
Figure PCTCN2017094856-appb-000002
从表2可以看出，复方免疫增强剂配方1，2，3即FMD 1/FMD 2/FMD 3三组免疫后均可明显延长疫苗的抗体持续期，监测至免疫后7个月抗体合格率无明显下降，维持在10/10；而FMD对照疫苗最高合格率基本在3/10，单独的黄芪多糖组(FMD 6/FMD 7)，有一定的免疫增强作用，但效果不明显，与FMD对照疫苗差异不显著。
从图1可以看出，复方免疫增强剂1，2，3(FMD 1/FMD 2/FMD 3)组及免疫增强剂8(FMD 8)组可以明显提高FMD疫苗的免疫效力，缩短抗体产生的窗口期；其中复方免疫增强剂1，2，3(FMD 1/FMD 2/FMD 3)组在免疫后7天，平均液相阻断ELISA抗体水平已高于26，远远高于对照疫苗组的23，显著提高平均抗体水平和抗体持续期。 单独的黄芪多糖(FMD 6/FMD 7)和高剂量黄多糖与MPL、MDP和β-葡聚糖配伍组(FMD 4/FMD 5)没有明显的免疫增强效果。
因此，由于复方免疫增强剂中添加了一定剂量的黄芪多糖与MPL、MDP和β-葡聚糖的协同效应，能够显著提高仔猪对抗原的免疫应答，提高抗体合格率和提早抗体产生时间，进而缩短疫苗产生抗体的窗口期至免疫后7天，提高疫苗的免疫效果。
本实施例中，免疫增强剂的各组分可以在给出范围内灵活配比，在此不一一列举。
实施例3：配制复方免疫增强剂伴侣疫苗
1、复方免疫增强剂的配制：
复方免疫增强剂的主要成份为：单磷酰脂质A，胞壁酰二肽，β-葡聚糖和黄芪多糖。其制备方法为将各主要成份溶解于pH8.0的0.1MTris-HCl。
复方免疫增强剂9：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为5.2μg/mL、10.4μg/mL、1.04μg/mL和0.052mg/mL。
复方免疫增强剂10：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为104μg/mL、104μg/mL、52μg/mL和1.04mg/mL。
复方免疫增强剂11：配置MPL、MDP、β-葡聚糖和黄芪多糖终浓度分别为520μg/mL、520μg/mL、520μg/mL和5.2mg/mL。
将配制好的复方免疫增强剂过滤(0.22μm滤器)除菌后分装至青瓶中，4℃保存。
2、复方免疫增强剂的伴侣疫苗制备：
(1)、将复方免疫增强剂与吐温按照96:4进行混匀，制备为水相。
(2)、将白油与司本按照96:4进行充分混合。
(3)、将水相与油相按照体积比1:2充分混合制备为含复发免疫增强剂的伴侣疫苗。
按照此方法制备好复方免疫增强剂伴侣疫苗分别根据有复方免疫增强剂9、10、11制备而来命名为复方免疫增强剂伴侣9、10、11。
3、使用方法
取300μL含复方免疫增强剂的伴侣疫苗与1头份疫苗充分混合后免疫。
本实施例中，复方免疫增强剂的各组分可以在给出范围内灵活配比，使用体积也可依据实际需要调整，在此不一一列举。
实施例4复方免疫增强剂伴侣疫苗对商品化O、A、亚I三价苗的免疫效力评价
1、疫苗制备：
复方免疫增强剂伴侣疫苗采用实施例3中制备的三种伴侣疫苗。
三价疫苗为O，A，亚I三价灭活疫苗(精品苗)批号5235039，20151224
2、分组、免疫和抗体检测
实验分组及免疫：将健康易感仔猪随机分组，每组10头，共4组。
每组疫苗各免疫一组健康易感仔猪。
表3疫苗免疫及分组
Figure PCTCN2017094856-appb-000003
免疫后采血：
对免疫后的抗体产生情况进行监测：免疫后第7天、14天、21天和第28天，对各组健康易感仔猪采血分离血清，兰兽研液相阻断ELISA抗体检测试剂盒检测疫苗免疫后的抗体产生情况和窗口期；
对免疫后免疫增强效果较好的复方免疫增强剂组进行抗体持续期监测：分别在免疫后28天、60天、90天、120天、150天、180天和210天分别采血，兰兽研液相续断ELISA抗体检测试剂盒检测疫苗免疫后的抗体产生情况；
(O型液相阻断ELISA抗体滴度大于等于26为抗体合格，A和亚I型液相阻断ELISA抗体滴度大于等于27为抗体合格。)
免疫后抗体合格率如表4，表5所示。
表4各组仔猪免疫后抗体合格率
Figure PCTCN2017094856-appb-000004
Figure PCTCN2017094856-appb-000005
从表4可以看出，免疫三价疫苗的仔猪，在免疫后7天三个血清型的抗体合格率仅为20％，30％和10％；而免疫了复方免疫增强剂伴侣疫苗9/10/11+三价疫苗的仔猪，在免疫后7天即可达到合格率为70～90％，窗口期明显缩短；液相阻断ELISA抗体合格率也明显升高。
表5各组仔猪免疫后抗体持续期内的抗体合格率
Figure PCTCN2017094856-appb-000006
从表5可以看出，免疫三价疫苗的仔猪的抗体从免疫后90日龄开始出现缓慢下降；而免疫了复方免疫增强剂伴侣疫苗9/10/11+三价疫苗的仔猪，在免疫后28天后抗体水平基本稳定，持续至7个月没有明显的下降趋势；添加了复方免疫增强剂伴侣疫苗的免疫组，明显延长了疫苗的抗体持续期。
综上，复方免疫增强剂伴侣疫苗对O，A，亚I三价口蹄疫灭活疫苗的免疫效力有 明显的提高作用。对O，A，亚I三种血清型的抗体均有明显的免疫增强作用，且明显缩短疫苗抗体产生的窗口期，提高疫苗的抗体持续期。
实施例5复方免疫增强剂伴侣疫苗对商品化多肽疫苗的免疫效力评价
1、疫苗制备：
复方免疫增强剂伴侣疫苗采用实施例3中制备的三种伴侣疫苗。
多肽疫苗批号：(2014)090297522
2、分组、免疫和抗体检测
实验分组及免疫：将健康易感仔猪随机分组，每组10头，共4组。
每组疫苗各免疫一组健康易感仔猪。
表6疫苗免疫及分组
Figure PCTCN2017094856-appb-000007
免疫后采血：
对免疫后的抗体产生情况进行监测：免疫后第7天、14天、21天和第28天，对各组健康易感仔猪采血分离血清，兰兽研液相阻断ELISA抗体检测试剂盒及猪口蹄疫病毒VP1结构蛋白抗体酶联免疫吸附试验诊断试剂盒(多肽抗体检测试剂盒，购自上海申联公司)，分别检测疫苗免疫后的抗体产生情况；
对免疫后免疫增强效果较好的复方免疫增强剂组进行抗体持续期监测：分别在免疫后28天、60天、90天、120天、150天、180天和210天分别采血，兰兽研液相阻断ELISA抗体检测试剂盒检测疫苗免疫后的血清抗体产生情况；
(当液相阻断ELISA抗体滴度大于等于26为抗体合格，多肽抗体检测采用试剂盒判定标准判定阴阳性。)
免疫后两种试剂盒检测的抗体合格率如表7，表8所示。
表7各组仔猪免疫后抗体合格率
Figure PCTCN2017094856-appb-000008
从表7可以看出，免疫多肽疫苗的仔猪，在免疫后7天后多肽抗体合格率为4/10，但液相阻断ELISA抗体合格率为0；而免疫了复方免疫增强剂伴侣疫苗9/10/11+多肽苗的仔猪，在免疫后7天多肽疫苗的抗体合格率为8/10或者9/10，液相阻断ELISA抗体抗体的合格率也可以提高至5/10或6/10左右；在兰兽研的液相阻断ELISA抗体检测试剂盒中可见，液相阻断ELISA抗体水平与攻毒保护具有一定的相关性，特别是抗体水平越高，保护效力越好。复方免疫增强剂伴侣疫苗可以明显缩短多肽苗抗体产生的窗口期，提高液相阻断ELISA抗体的合格率。
表8各组仔猪免疫后抗体持续期内的抗体合格率
Figure PCTCN2017094856-appb-000009
Figure PCTCN2017094856-appb-000010
从表8可以看出，免疫多肽苗的仔猪的抗体从免疫后90日龄开始出现缓慢下降；而免疫了复方免增强剂伴侣疫苗9/10/11+多肽疫苗的仔猪，在免疫后21天后抗体水平基本稳定，可维持至7个月无明显的下降趋势；添加了复方免疫增强剂伴侣疫苗的免疫组，明显延长了疫苗的抗体持续期。
综上，复方免疫增强剂伴侣疫苗对口蹄疫多肽疫苗的免疫效力有明显的提高作用。且明显可以提高液相阻断ELISA抗体的合格率，且明显缩短疫苗抗体产生的窗口期，提高疫苗的抗体持续期。
以上所述仅是本发明的优选实施方式，应当指出：对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (10)

  1. 一种复方免疫增强剂，其特征在于，所述复方免疫增强剂含有单磷酰脂A 5～520μg/mL、胞壁酰二肽10～520μg/mL、β-葡聚糖1～520μg/mL和黄芪多糖0.05～5.2mg/mL。
  2. 根据权利要求1所述的复方免疫增强剂，其特征在于，所述复方免疫增强剂含有单磷酰脂A 5～500μg/mL、胞壁酰二肽10～500μg/mL、β-葡聚糖1～500μg/mL和黄芪多糖0.05～5.0mg/mL。
  3. 根据权利要求1所述的复方免疫增强剂，其特征在于，所述复方免疫增强剂含有单磷酰脂A 100～500μg/mL、胞壁酰二肽100～500μg/mL、β-葡聚糖50～500μg/mL和黄芪多糖1～5.0mg/mL。
  4. 权利要求1～3任一项所述的复方免疫增强剂的制备方法，其特征在于，包括以下步骤：
    1)配制含有单磷酰脂质A、胞壁酰二肽、β-葡聚糖和黄芪多糖的溶液，与吐温-80混合，得到成水相溶液；
    2)将白油和司盘-80混合，得到油相溶液；
    3)将所述水相溶液与油相溶液混合乳化后得到含复方免疫增强剂的伴侣疫苗。
  5. 权利要求1～3任一项所述的免疫增强剂在制备疫苗方面的应用。
  6. 一种含有权利要求1～3任一项所述的含复方免疫增强剂的口蹄疫灭活疫苗。
  7. 根据权利要求6所述的口蹄疫灭活疫苗，其特征在于，所述口蹄疫灭活疫苗还包括灭活的抗原溶液。
  8. 根据权利要求7所述的口蹄疫灭活疫苗，其特征在于，所述口蹄疫灭活疫苗中的灭活的抗原溶液与复方免疫增强剂的体积比为9:1～8:1。
  9. 根据权利要求7所述的口蹄疫灭活疫苗，其特征在于，所述灭活的抗原溶液为含O，A，亚I型口蹄疫灭活抗原、多肽或其他基因工程表达产物中的一种或几种。
  10. 权利要求6所述的含复方免疫增强剂的口蹄疫灭活疫苗的制备方法，其特征在于，包括以下步骤：
    1)将复方免疫增强剂与灭活抗原溶液混合，再与吐温-80充分混合得到水相溶液；
    2)将白油和和司盘-80混合，得到油相溶液；
    3)将所述水相溶液与油相溶液充分混合即得含复方免疫增强剂的口蹄疫灭活疫苗。
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