WO2005079842A1 - Adjuvant immunologique - Google Patents

Adjuvant immunologique Download PDF

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Publication number
WO2005079842A1
WO2005079842A1 PCT/CN2005/000084 CN2005000084W WO2005079842A1 WO 2005079842 A1 WO2005079842 A1 WO 2005079842A1 CN 2005000084 W CN2005000084 W CN 2005000084W WO 2005079842 A1 WO2005079842 A1 WO 2005079842A1
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Prior art keywords
levamisole
group
derivative
adjuvant
immune
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PCT/CN2005/000084
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English (en)
Chinese (zh)
Inventor
Bin Wang
Qingling Yu
Huali Jin
Youmin Kang
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China Agricultural University
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Priority to CN2005800027003A priority Critical patent/CN1909925B/zh
Publication of WO2005079842A1 publication Critical patent/WO2005079842A1/fr

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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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59

Definitions

  • the invention relates to an immune adjuvant.
  • Improving the body's immunity is the most important means of preventing infections of various pathogens, which can usually be achieved by vaccination. Therefore, vaccination is one of the effective measures to prevent infections of various pathogens.
  • Common pathogens include viruses, microorganisms, eukaryotic cells, parasites, and environmental factors.
  • Various methods have been reported to produce vaccines against infectious pathogens, such as inactivated vaccines, live attenuated vaccines, recombinant vaccines, subunit vaccines, and nucleic acid vaccines. The basic principles of these vaccines are the same, that is, with the help of The target protein bound to the pathogen stimulates the immune response, so that the immune individual is not infected by the infectious pathogen.
  • nucleic acid vaccine When an individual comes into contact with an infectious pathogen, their immune system can recognize the protein of the pathogen and produce an effective protective response against infection.
  • Many vaccines currently in use consist of non-infectious and low-infectious proteins or nucleic acid substances isolated from pathogens.
  • nucleic acid vaccine The advantage of nucleic acid vaccine is that it can activate humoral immune response and cellular immune response, and more effectively stimulate protective and clearance immune response, to achieve resistance to pathogen infection, anti-tumor, treatment of autoimmune diseases and elimination of poisoning symptoms caused by toxins. Infections and internal lesions from foreign pathogens, and nucleic acid vaccines are highly respected for their ease of preparation, low cost, and good safety.
  • nucleic acid vaccines are inefficient to activate the body to produce a complete immune response in the body, and it cannot achieve protective effects or protective instability when used alone.
  • Adjuvants that supplement, activate, and regulate the immune response of nucleic acid vaccines are the key to solving this problem.
  • each of the above adjuvants has some problems. For example, most of them have been developed and designed to increase antibody levels, but they have not promoted the T cell immune level of the body; instead, some adjuvants have immune effects on T cells. It also has a large inhibitory effect, which makes it appear weak in immunogenicity, low cell level, short protection period and other shortcomings in the application of proteins, peptides and inactivated vaccines, which makes the application of such adjuvants limited.
  • the object of the present invention is to provide an immune adjuvant which can increase the immune level of ⁇ cells.
  • the immune adjuvant provided by the present invention is a combination of levamisole, or a derivative of levamisole, or a combination of levamisole or a levamisole derivative with at least one of the following substances: white oil, glycerol, oleic acid, double ten Octamethyl dimethyl ammonium bromide or saponin.
  • composition of the immune adjuvant of the present invention is as follows:
  • the immune adjuvant is levamisole or a levamisole derivative.
  • the immune adjuvant is a combination of levamisole or a levamisole derivative and white oil.
  • the mass ratio of the white oil to the levamisole or levamisole derivative is 20: 1-1: 20, and the preferred mass ratio is 10: 1-1: 1.
  • the immune adjuvant is a combination of levamisole or a levamisole derivative and glycerol.
  • the mass ratio of glycerol to levamisole or levamisole derivative is 5: 1-1: 5, and the preferred mass ratio is 2: 1-1: 2.
  • the immune adjuvant is a combination of levamisole or a levamisole derivative and oleic acid.
  • the mass ratio of the oleic acid to the levamisole or levamisole derivative is 5: 1-1: 5, and the preferred mass ratio is 2: 1-1: 2.
  • the immune adjuvant is a combination of levamisole or a levamisole derivative and bisoctadecyldimethylammonium bromide.
  • the mass ratio of the bisoctadecyldimethylammonium bromide to the levamisole or levamisole derivative is 20: 1 to 1:20, and the preferred mass ratio is 10: 1 to 1: 5.
  • the immune adjuvant is a combination of levamisole or a levamisole derivative and saponin.
  • the mass ratio of the saponin to the levamisole or levamisole derivative is 20: 1-1: 20, and the preferred ratio is 10: 1-1: 5.
  • the levamisole derivative may be levamisole hydrochloride or levamisole phosphate.
  • the white oil is white oil for food or white oil for injection.
  • the adjuvant of the present invention can be combined with a vaccine to form an immune composition.
  • the vaccine may be a protein vaccine, a polypeptide vaccine, a nucleic acid vaccine or an inactivated vaccine.
  • the protein vaccine and the peptide vaccine are artificial antigens or vaccine-specific antigen substances produced by an organism.
  • the organism is Escherichia coli, Bacillus, yeast or eukaryotic cells, which can be scaled up under artificial culture conditions.
  • the inactivated vaccine is an antigenic substance used as a vaccine after the pathogen is inactivated by a known method.
  • the immune composition can be introduced into the body such as muscle, intradermal, subcutaneous, vein, mucosal tissue by injection, spray, oral, nasal, eye drop, penetration, absorption, physical or chemically mediated methods; or by other substances Into the body after mixing or wrapping.
  • the immune composition can be obtained by the following method: a protein vaccine, a nucleic acid vaccine, a polypeptide vaccine or an inactivated vaccine is dissolved in a physiological saline containing levamisole (or a levamisole derivative) at a concentration of 0.1% to 10% by mass Medium; or soluble in levamisole (or 0.1% to 10%) Levamisole derivative) and white oil in normal saline with a mass percentage of 1% to 20%; or soluble in levamisole (or levamisole derivative) containing a mass concentration of 0.1% to 10% And glycerol in normal saline with a concentration of 1% to 10%; or dissolved in levamisole (or a derivative of imidate) with a concentration of 0.1% to 10% by mass and a content of 0.1% to 5% of oleic acid in normal saline; or soluble in levamisole (or levamisole derivative) containing 0.1% to 10% by mass and 0.1% by mass % To 10% of dioc
  • the immune adjuvant of the present invention is used together with a corresponding vaccine to enhance the body's immune response ability to the following pathogenic pathogens: viruses, prokaryotic cells, and eukaryotic cells.
  • pathogenic pathogens viruses, prokaryotic cells, and eukaryotic cells.
  • eukaryotic pathogens include single cell pathogens and multicellular parasites.
  • Viral pathogens include, but are not limited to, respiratory viruses (influenza and rotavirus), sores (German measles, chicken pox, vaccinia, smallpox, shingles, etc.), central nervous system virus (prion), immune system virus (HIV), reproductive system virus (condyloma acuminatum), animal virus (foot-and-mouth disease virus, classical swine fever virus, avian influenza virus, Newcastle disease virus).
  • respiratory viruses influenza and rotavirus
  • sores German measles, chicken pox, vaccinia, smallpox, shingles, etc.
  • central nervous system virus prion
  • immune system virus HIV
  • reproductive system virus condyloma acuminatum
  • animal virus foot-and-mouth disease virus, classical swine fever virus, avian influenza virus, Newcastle disease virus.
  • Figure 1 shows the 1% agarose gel electrophoresis map of the FMDV VP1 gene amplified by PCR.
  • Figure 2 is an electrophoresis map of the recombinant expression vector SuperY / VPl for digestion and identification.
  • Figure 3 is an SDS-PAGE map of the VP1 gene expression product
  • Figure 4 shows the expression profile of VP1 protein detected by Western-blot experiments.
  • Figure 5 shows the curve of antibody titer produced by mice immunized with the immune composition formed by VP1 protein and different adjuvants.
  • Figure 6 shows the effect of T cell-specific expansion on mice immunized with an immune composition composed of 146S antigen and different adjuvants.
  • Figure 8 Comparison of T cell-specific expansion activity of immunorecombinant mice with pig reproductive and respiratory syndrome inactivated virus antigens and different adjuvants
  • Figure 9 shows the comparison of the delayed immune response of pigs reproductive and respiratory syndrome inactivated virus antigen and immune composition composed of different adjuvants.
  • Figure 10 shows the results of T cell proliferation in BABL / C mice immunized with 146S antigen
  • Figure 11 shows the results of T cell proliferation in C57BL / 6 mice immunized with 146S antigen
  • Figure 12 shows the results of T cell proliferation in BABL / C mice immunized with PRRSV antigen
  • Figure 13 shows the results of T cell proliferation of C57BL / 6 mice immunized with PRRSV antigen
  • Figure 14 is an electrophoresis diagram of cDNA concentration after quantitative regulation of HPRT housekeeping genes
  • Figure 15 is an electrophoresis image of IL-4, IL-10, IL-2 and IFN- ⁇ expressed in BABL / c mice immunized with 146S antigen
  • Figure 16 shows the results of analyzing the expression of IL-4, IL-10, IL-2 and IFN- ⁇ in C57BL / 6 mice immunized with 146S antigen by Bio-Rad Image software.
  • Figure 17 shows the results of analyzing the expression of IL-4, IL-10, IL-2 and IFN- ⁇ in BABL / c mice immunized with porcine reproductive and respiratory syndrome virus antigen by Bio-Rad Image software.
  • Figure 18 shows the analysis of immune system of porcine reproductive and respiratory syndrome virus antigen by Bio-Rad Image software.
  • Fig. 19 is an electrophoresis diagram of MHC and costimulatory molecules expressed by BABL / c mice immunized with 146S antigen
  • Fig. 20 is a result of analysis of MHC and costimulatory molecules expression of C57BL / 6 mice immunized with 146S antigen by Bio-Rad Image software
  • Figure 21 shows the analysis of the immune response of porcine reproductive and respiratory syndrome virus antigen by Bio-Rad Image software.
  • Figure 22 shows the results of analysis of MH (:) and co-stimulatory molecules in C57BL / 6 mice immunized with PRSV antigen by Bio-Rad Image software.
  • Figure 23 shows the analysis results of SOCS1 and SOCS3 expressions of BABL / c mice immunized with 146S antigen by Bio-Rad Image software.
  • Figure 24 shows the results of analyzing the expression of SOCS1 and SOCS3 of C57BL / 6 mice immunized with 146S antigen by Bio-Rad Image software.
  • Figure 25 shows the analysis results of SOCS1 and SOCS3 expressions of BABL / c mice immunized with porcine reproductive and respiratory syndrome virus antigen by Bio-Rad Image software.
  • Figure 26 shows the analysis of the immune system of porcine reproductive and respiratory syndrome virus antigen by Bio-Rad Image software.
  • Figure 27 shows the results of T cell proliferation of BABL / C mice immunized with chicken Newcastle disease virus antigen.
  • RNA extraction kit purchased from Shanghai Biological Engineering Co., Ltd.
  • RNA extraction kit purchased from Shanghai Biological Engineering Co., Ltd.
  • RNA extraction kit obtained from Shanghai Biological Engineering Co., Ltd.
  • the first-strand cDNA was synthesized under the following reaction conditions: 2 ⁇ g RNA of Foot and Mouth Disease Virus, 50 mmol / L Tris-HCl (pH8.3), 75 ⁇ l / LKCl, 10 mmol / L DTT, 3 mmol / L MgCl 2 , 500 ⁇ mol / L dNTPs, 100 ⁇ g random hexamer primer, 500 units of LV reverse transcriptase, total volume 20 L, incubated at 37 ° C for 1 h.
  • primer 1 5 '-AAG_
  • the reaction conditions were: 94 ⁇ denaturation for 30 seconds, 54 ° C renaturation. 30 seconds, 72 ° C extension for 1 minute, a total of 30 cycles.
  • the results of the 1% agarose gel electrophoresis of the FMDV VP1 gene amplified by PCR are shown in Figure 1 (lane M is the DNA Marker; lane 1 is the PCR product).
  • the position of the target band indicated by the arrow in the figure indicates the purpose
  • the size of the fragment was 639bp, which was consistent with the size of the VP1 gene fragment.
  • the amplified fragment was recovered using a low melting point gel.
  • Foot-and-mouth disease VP1 cDNA fragment obtained by restriction enzyme ⁇ and 3 ⁇ 4al digestion step 1 was electrophoresed and recovered.
  • the VP1 gene fragment was cloned into the shuttle plasmid SuperY (in the Sphl and HPal sites of plasmid pGAPZa purchased from Invitrogen, USA). Kanamycin resistance gene (Kan r ) was added to get the EcoRl and al restriction sites of SuperY, and then the recombinant vector was digested with EcoRl and 3 ⁇ 4al restriction enzymes to identify the recombinant vector.
  • the recombinant expression vector SuperY / VPl constructed in step 2 was transformed into yeast SMD1168 by electric shock method, and positive clones were selected and identified. Single colonies were picked and cultured at 30 ° C for 48-96 hours (also set yeast SMD1168 and yeast SMD1168 transformed with SuperY).
  • lane 1 showed specific color bands near 66kD and 43kD, but no bands appeared in lanes 2 and 3, indicating that the expressed protein can react with anti-FMDV serum to generate specific reaction bands.
  • the protein product has the immunogenicity of FMDV.
  • the expressed supernatant was desalted and stored at 20 ° C, which can be used as a vaccine for foot-and-mouth disease VP1 protein.
  • VP1 protein The purified VP1 protein is dissolved in 0.9% physiological saline and stored at 4 ° C.
  • Inactivated avian influenza virus H5N1 vaccine (purchased from Harbin Veterinary Research Institute) Remove the emulsion and store at 4 ° C.
  • Saponin was purchased from Sigma, USA.
  • the above antigen and the adjuvant of the present invention are soluble in 0.9 ° /. NaCl in water (saline).
  • the levamisole hydrochloride was dissolved in a 0.9% NaCl solution, configured as an adjuvant with the following concentration: 0.5% salt Levamisole acid, 1% levamisole hydrochloride, 1% white oil for injection + 1% levamisole hydrochloride, 5% white oil for injection + 1% levamisole hydrochloride, 1% glycerol + 1% levamisole hydrochloride, 5% glycerol + 1% levamisole hydrochloride, 0.5% oleic acid + 1% levamisole hydrochloride, 0.5% bisoctadecyldimethylammonium bromide + 1% levamisole hydrochloride, 1% bisoctadecyldimethylammonium bromide + 1% levamisole hydrochloride, 0.5% saponin + 1% levamisole hydrochloride, 1% saponin + 1% levamisole hydrochloride.
  • the detection method is: 96-well microtiter plate Coat with 8ug / ml antigen, overnight at 4 ° C, block with 3% calf serum at 37 ° C for 1 h; wash with PBST (0.05% Tween20 in PBS) 3 times for 5 minutes each time; add not less than 1: 100 Diluted serum from immunized animals (mouse), use unimmunized mouse serum as a control, and incubate at 37 ° C for 2 hours. After washing the plate three times with PBST, horseradish peroxidase-labeled goat anti-mouse IgG ( Secondary antibody, Sigma, St.
  • mice Twenty-four 6-8 week-old BALB / c (H-2 d ) female mice were divided into two groups. One group was intramuscularly injected with 1% levamisole hydrochloride and the VP1 protein (20 micrograms) of foot-and-mouth disease obtained in Example 1. The composition was 100 microliters, and another group was intramuscularly injected with 100 microliters of a composition containing 1% white oil for injection and 20 micrograms of foot-and-mouth disease VP1 protein for immunization, and the immunization was boosted again at the same dose on the 14th day after the first immunization. Serum was taken at 14, 28, 42 and 56 days after the second immunization to determine the antibody titer by ELISA.
  • the detection method is the same as that in step 1.
  • the results are shown in Figure 5 ( ⁇ : Foot-and-mouth disease VP1 protein + 1% White oil for injection, ⁇ : VP1 protein + 1% levamisole hydrochloride), indicating that levamisole hydrochloride or a combination vaccine of oil adjuvant and VP1 protein can stimulate the production of specific antibodies, but vaccine antibodies using levamisole hydrochloride as an adjuvant The titer is high.
  • mice 60 6-8 week-old BALB / c (H-2d) female mice were divided into two groups.
  • the first group was intramuscularly injected with 100 microliters of a vaccine composition containing 50% white oil for injection and 20 micrograms of 146S antigen.
  • Two groups were intramuscularly injected with 100 microliters of a composition containing 20 micrograms of antigen 146S and 1% levamisole hydrochloride.
  • boost the immunization again at the same dose and take the spleen T cells on the 14th day after the second immunization to determine its T cell expansion activity.
  • the specific method is: under sterile conditions, take the spleen Make a single cell suspension, remove the red blood cells with red blood cell lysate, then wash three times with PBS solution, centrifuge and count the cells, adjust the cell concentration to 1 X 10 6 cells / ml, and add 96 cells of each group in three portions.
  • Well plate. One of them added 100 ⁇ l concanavalin (Con-A) to a final concentration of 5 g / ml, one added the corresponding specific antigen (146S antigen) as a stimulant to a final concentration of 2 g / ml, and the other did not Add the stimulus. After 24 hours, add 100 ⁇ MTTT to each well to a final concentration of 5mg / ml.
  • the adjuvant is: 50% white oil for injection, 0.5% levamisole hydrochloride, 1% levamisole hydrochloride, 2% levamisole hydrochloride or 1% levamisole hydrochloride + 0.5% glycerol
  • the serum and measure its antibody titer by ELISA take the serum and measure its antibody titer by ELISA. The result is the same as that in step 1.
  • FIG. 7 it is shown that using different concentrations of levamisole hydrochloride and levamisole hydrochloride + glycerol as adjuvants, the antibody titer is significantly improved in time compared with the traditional white oil adjuvant for injection.
  • mice 72 6-8 week-old BALB / c (H-2d) female mice were divided into seven groups, and intramuscularly injected with 20 micrograms of reproductive and respiratory syndrome virus (PRRSV) antigen and one of the following adjuvants. 100 microliters of the composition, the adjuvant is: no adjuvant, 50% white oil for injection, 0.25% levamisole hydrochloride, 0.5% levamisole hydrochloride, 1% levamisole hydrochloride, 2% levamisole hydrochloride, 1% hydrochloric acid Levamisole + 0.5% glycerol.
  • PRRSV reproductive and respiratory syndrome virus
  • mice Sixty 6-8 week-old female BALB / c (H-2d) mice were divided into five groups. Each group was injected with 20 micrograms of porcine reproductive and respiratory syndrome virus (PRRSV) antigen and one of the following groups. 100 microliters of adjuvant composition was used for immunization, the adjuvant was: 50% white oil for injection, 0.5% levamisole hydrochloride, 1% levamisole hydrochloride, 2% levamisole hydrochloride, 1% levamisole hydrochloride + 0.5% glycerin.
  • PRRSV porcine reproductive and respiratory syndrome virus
  • mice The mice were divided into two groups: BALB / c mice (126) and C57BL / 6 mice (126).
  • the mouse immune antigens were inactivated foot-and-mouth disease (FMD) virus antigen 146S and porcine reproductive and respiratory syndrome virus (PRRSV) antigens.
  • FMD foot-and-mouth disease
  • PRRSV porcine reproductive and respiratory syndrome virus
  • Each large group was further divided into 1, 2, 3, 4, 5, 6, and 7 groups with seven treatment groups, with 18 mice in each group, of which 9 were immunized with 146S antigen and 9 were immunized with porcine reproductive and respiratory syndrome ( PRRS) virus antigen, 1 group was the control group, and only injected 100 microliters of 0.9% NaCl solution containing 20 micrograms of mouse immune antigen; 2 groups were injected with 20 micrograms of mouse immune antigen and 50% white oil composition for injection 100 microliters; three groups of intramuscular injections containing 20 micrograms of mouse immune antigen and 0.25% levamisole hydrochloride composition 100 microliters; four groups of intramuscular injections containing 20 micrograms of mouse immune antigen and 0.5% levamisole hydrochloride composition 100 microliters; 5 groups of intramuscular injections containing 20 micrograms of mouse immune antigen and 1% levamisole hydrochloride composition 100 microliters; 6 groups of intramuscular injections containing 20 micrograms of mouse immune antigen and
  • mice On the 14th day after the first immunization, boost the immunization with the same dose again, and take 3 mice from each group for the anti-foot-and-mouth disease antibody and porcine reproductive and respiratory syndrome inactivated virus antibody titer; The effects of the above composition on the specific expansion of T cells were measured in mice; the remaining 3 mice in each group were tested for the expression of cytokines, MHC, costimulatory molecules and SOCS by RT-PCR, as follows:
  • mice group 28 days 42 days 56 days 70 days 84 days
  • Table 2 shows that the antibody titers of the 2% levamisole hydrochloride group and the 1% levamisole hydrochloride + 1% glycerol composition group were significantly higher than those of the white oil adjuvant group for injection, while the 1% levamisole hydrochloride + 1% glycerol composition Group antibodies extended the longest.
  • the antibody titers of the other concentrations of the levamisole hydrochloride group were comparable to those of the white oil injection group adjuvant group.
  • mice immunized with 146S antigen and 3 immunized with PRRS virus antigen were taken from each group, and the spleen T cells were measured for T cell expansion activity.
  • the stimulus was the same as that in step 3 except Porcine Reproductive and Respiratory Syndrome Virus or 6S antigen.
  • the results are shown in Figures 10-13, indicating that different concentrations of levamisole hydrochloride as an adjuvant group and traditional white oil for injection Compared with the adjuvant group, the T cell expansion activity was significantly improved. * In Figs. 10-13 indicates that p ⁇ 0.05.
  • RNA TriZ0L, Dingguo Biological Co., Ltd.
  • Reverse transcription was performed according to the Dalian RT-PCR RNA RT-PCR operating guide.
  • RNA was placed in a 250 ⁇ centrifuge tube, and related reagents were added in this order: 4 ⁇ 1 MgCl 2 , 2 ⁇ 1 10X buffer, 8.5 ⁇ 1 DEPC water, 2 ⁇ 1 dNTP mixture, 0.5 ⁇ 1 RNase inhibitor, 0.5 ⁇ 1 M-MLV reverse transcriptase (Promage), 0.5 ⁇ Oligo (dT) ⁇ 2 primer; reaction conditions were 42 ° C 30 min, 99 ° C 5 min, 5 ° C 5 min.
  • Target gene primer reaction conditions HPRT 5 'GTTGGATACAGGCCAGACTTTGTTG 94 ° C 30 sec, 60 ° C 30 sec and
  • the 2% levamisole hydrochloride adjuvant group produced high levels of IL-4 and IL-10 after immunization, but low levels of IL-2 and IFN- Y .
  • the adjuvant group in the white oil injection group produced low levels of Thl and Th2 cytokines.
  • high levels of IL-10 were produced. This shows that the injection of the white oil group adjuvant group not only failed to activate T cells, but also produced IL-10 that inhibited T cell levels.
  • the 2 ⁇ 1 cDNA synthesized in the previous paragraph was used as a template for PCR amplification of the MHCI, MHCII, CD40, CD80, CD86, SOCS1 and SOCS3 genes.
  • the primer sequences and PCR reaction conditions are shown in Table 4.
  • the electrophoretic detection results are shown in Figure 19 (lanes 1-7 are 1-7 treatment groups, respectively).
  • the electrophoresis images were analyzed with Bio-Rad Image software (Quantity One 4. 2. 0) Analysis and mapping.
  • the results are shown in Figs. 19-26, which show that the immunization with levamisole hydrochloride adjuvant group can produce high levels of MHC-I and MHC-II.
  • MHC-1 and MHC-II expression were lowest in the adjuvant group of the white oil group for injection.
  • MHC molecules play an important role in the activation of immune T cells
  • the white oil group adjuvant also fails to play a role in this process.
  • Another class of co-stimulatory molecules, CD40, CD80, and CD86 produced high levels of expression after immunization with the 0.5% levamisole hydrochloride composition group, indicating that 0.5% levamisole hydrochloride has a better effect in enhancing T cell immunity. .
  • the white oil group had the worst adjuvant group.
  • the 6-month-old bulls were divided into five groups, two in each group.
  • the first group was a control group without any injection.
  • the second group was injected with a composition containing 50 micrograms of 146S antigen and 50% white oil for injection. 100 microliters;
  • the third group was intramuscularly injected with 100 microliters of a composition containing 50 micrograms of 146S antigen and 5% levamisole;
  • the fourth group was intramuscularly injected with 100 microliters of a composition containing 50 micrograms of VP1 protein and 50% white oil for injection;
  • the fifth group was intramuscularly injected with 100 microliters of a composition containing 50 micrograms of VP1 protein and 5% levamisole.
  • the immunization was boosted again at the same dose.
  • the serum was taken to determine the antibody titer. The results are shown in Table 5:
  • Table 5 shows that the use of levamisole as an adjuvant for cattle anti-foot-and-mouth disease vaccine has significantly improved antibody titer compared to traditional mineral oil as an adjuvant.
  • the SPF chickens were divided into six groups, six in each group.
  • the first group was a blank control group, which was injected with 100 microliters of a 0.9% NaCl aqueous solution.
  • the second group was intramuscularly injected with 20 micrograms of Lasota Newcastle disease virus antigen.
  • the third group was intramuscularly injected with 20 micrograms of Lasota Newcastle disease virus antigen, 2% levamisole hydrochloride and 0.5 100 microliters of a composition of bisoctadecyldimethylammonium bromide; the fourth group was injected intramuscularly with 20 micrograms of Lasota Newcastle disease virus antigen, 2% levamisole hydrochloride and 1% bisoctadecyldimethyl bromide 100 microliters of ammonium chloride composition; 100 microliters of a composition containing 20 micrograms of Lasota Newcastle disease virus antigen, 2% levamisole hydrochloride, and 2% saponin in the fifth group; intramuscular injection of chickens against Newcastle disease in the sixth group Commercial seedlings of white oil adjuvant for injection (containing 50% white oil for injection, purchased by
  • Table 6 shows that compared with traditional mineral oil adjuvants, levamisole hydrochloride, levamisole hydrochloride + dioctadecyldimethylammonium bromide, levamisole hydrochloride + saponin were used as adjuvants for chicken against Newcastle disease vaccine Antibody titers were similar.
  • the chicken antibody hemagglutination inhibition titer (HI) detection method is as follows: In the two rows of wells on the microaggregation plate, from the 1st to 12th or according to the number of antiserum titers, each well is added to 0. 025ml physiological saline; unknown sera 0.025ml were added to the first row and the first well, respectively, and diluted to the last well in order, each well was filled with a known virus solution containing 4 agglutination units. Add 0. 025ml of PBS to the first well in the second row, and then dilute to the last well in multiples, and add a known virus solution containing 4 agglutination units to each well. After shaking and mixing, 30 minutes at room temperature, it can be suppressed by unknown serum, and it is judged as the HI titer in serum.
  • the SPF chickens were divided into six groups, six in each group.
  • the first group was a negative control group and was injected only with 100 microliters of a 0.9% NaCl aqueous solution.
  • the second group was intramuscularly injected with an aqueous solution containing 20 micrograms of Lasota Newcastle disease virus antigen.
  • the third group was injected intramuscularly with 20 ⁇ g Lasota Newcastle disease virus antigen and 2% levami microphone 100 ⁇ l of the immune composition of azole; fourth group of 100 ⁇ l of the immune composition containing 20 micrograms of Lasota Newcastle disease virus antigen and 4% levamisole; intramuscular injection of chicken against Newcastle white oil Commercial vaccine (containing 50% white oil for injection, purchased by Beijing Biological Products Factory) 100 microliters, intramuscular injection 4 ° / in the sixth group. 100 microliters of levamisole.
  • the immunization was boosted again with the same dose, and the serum was taken to determine the antibody hemagglutination value on the 28th and 60th days after the second immunization.
  • Table 7 The results are shown in Table 7:
  • Table 7 shows that the use of levamisole as an adjuvant for chicken anti-Newcastle disease vaccine, compared with the traditional mineral oil adjuvant, the antibody titer does not decrease significantly in time.
  • the SPF chickens were divided into six groups of six animals.
  • the first group was injected intramuscularly with 20 micrograms of Lasota Newcastle disease virus antigen, 2% levamisole hydrochloride, and 0.5% bisoctadecyldimethylammonium bromide.
  • the composition 100 microliters of the composition; the second group was intramuscularly injected with 20 micrograms of Lasota Newcastle disease virus antigen, 100 microliters of the composition with 2% levamisole hydrochloride and 2% saponin; the third group was intramuscularly injected with 20 micrograms of Lasota Newcastle disease virus antigen.
  • the fourth group was intramuscularly injected with 100% of a composition containing 2% levamisole hydrochloride and 0.5% bisoctadecyldimethylammonium bromide; the fifth group was intramuscularly injected with 2% levamisole hydrochloride and 2% soap 100 microliters of the composition; the sixth group was intramuscularly injected chicken with Newcastle disease-resistant white oil adjuvant commercial vaccine (containing 50% white oil for injection, purchased from Beijing Biological Products Factory) 100 microliters.
  • Newcastle disease-resistant white oil adjuvant commercial vaccine containing 50% white oil for injection, purchased from Beijing Biological Products Factory
  • the specific method is: under sterile conditions, take the spleen Make a single cell suspension, remove the red blood cells with red blood cell lysate, then wash three times with PBS solution, centrifuge and count the cells, adjust the cell concentration to IX 10 6 cells / ml, and add the cell suspension of each group into 4 wells into 96 wells In a culture plate.
  • a ⁇ Con A was added to a final concentration of 5 ⁇ ⁇ / ⁇ 1, corresponding to a specific antigen was added (inactivated virus antigen of Lasota) as a stimulus to a final concentration of 2 g / ml, lOO l BSA was added to a The final concentration was 2 g / ml as the irrelevant antigen, and another portion was added without stimulus.
  • 100 ⁇ l of MTT was added to a final concentration of 5 mg / ml.
  • 100 ⁇ l of SDS-DMS0 (20% SDS) was added to each well. Dissolved in 50% DMSO, pH 2.0) to completely dissolve.
  • 1 indicates that the ConA stimulation group is a positive control
  • 2 indicates the T cell expansion activity of the first group after immunization
  • 3 indicates the T cell expansion activity of the second group after immunization.
  • 4 indicates the T cell expansion activity after the third group of immunization
  • 5 indicates the T cell expansion activity after the fourth group of immunization
  • 6 indicates the T cell expansion activity after the fifth group of immunization
  • 7 indicates the sixth group after immunization T cell expansion activity.
  • the SPF chickens were divided into six groups of four, the first group was a negative control group, and only 100 microliters of 0.9% NaCl aqueous solution was injected; the second group was intramuscularly injected with 20 micrograms of Lasota Newcastle disease virus antigen and 1 100 microliters of immune composition of levamisole levamisole; 100 microliters of immune composition of 20 micrograms of Lasota Newcastle disease virus antigen and 2% levamisole phosphate intramuscularly in the third group; 20 micrograms of Lasota Newtown intramuscular injection 100 microliters of immune composition of epidemic virus antigen and 4% levamisole phosphate; fifth group of intramuscularly injected chicken vaccines against Newcastle disease white oil adjuvant (containing 50% white oil for injection, purchased by Beijing Biological Products Factory) 100 In the sixth group, 100 microliters of 4% levamisole phosphate was injected intramuscularly.
  • the SPF chickens were divided into three groups of 6 each.
  • the first group was a negative control group and was injected with only 100 microliters of normal saline.
  • the second group was intramuscularly injected with an inactivated avian influenza virus H5N1 vaccine (containing 50% white for injection). oil, (Purchased from Harbin Veterinary Research Institute) 100 microliters;
  • the third group was intramuscularly injected with 100 microliters of a composition containing 5 micrograms of the emulsion-inactivated avian influenza virus H5N1 vaccine and 5% levamisole hydrochloride.
  • the immunization was boosted again at the same dose. Serum was taken on day 7 after the first and second immunizations to determine the antibody titer.
  • Table 9 The results are shown in Table 9:
  • the bird flu virus H5N1 is inactivated by formaldehyde, it is divided into two parts. One is emulsified with 5% white oil for injection to make a traditional mineral oil vaccine. The other is mixed with 5% levamisole hydrochloride as an adjuvant. Into a vaccine. SPF chickens were divided into three groups (10 chickens per group).
  • the first group was intramuscularly injected with 100 ⁇ l of the above-mentioned avian influenza virus H5N1 mineral oil vaccine; the second group was intramuscularly injected with 5 ⁇ g of inactivated avian influenza virus H5N1 antigen and 5 100 microliters of the composition of levamisole hydrochloride%; the third group was a control group, and 100 microliters of 5% levamisole hydrochloride was injected.
  • virus 100-fold lethal dose of the avian influenza virus i.e. 0.1 ml 2 X 10- 9 chickens were diluted virus challenge, the results as shown in Table 10:
  • Table 10 shows that the use of levamisole hydrochloride as an adjuvant for chicken anti-avian influenza H5N1 vaccine has a higher protection rate than the traditional mineral oil adjuvant.
  • the immune adjuvant of the present invention can effectively improve the ability of protein vaccines, nucleic acid vaccines, peptide vaccines and inactivated vaccines to stimulate the body's complete immune response, enhance the immune effect of T cells of the body, and prolong the protection period; Simple does not require complicated equipment and operating steps, is easy to implement, has excellent application prospects in the medical field, and will produce huge social and economic benefits.

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Abstract

L'invention concerne un adjuvant immunologique qui est le lévomisole ou un dérivé de celui-ci ou une composition constituée de lévomisole ou d'un dérivé de celui-ci et d'au moins l'un des composés suivants : huile, glycérol, acide oléique, DDA ou saponine. L'adjuvant immunologique selon l'invention permet de stimuler efficacement la capacité de réaction immunologique d'un vaccin à base de protéines, à base d'acide nucléique, à base de polypeptides et d'un vaccin inactivé. Cet adjuvant immunologique permet également d'améliorer l'effet immunologique des lymphocytes T et de prolonger la période de protection. La méthode de préparation de cet adjuvant immunologique est facile à mettre en oeuvre. Cet adjuvant immunologique peut trouver un grand nombre d'applications et apporter de nombreux avantages.
PCT/CN2005/000084 2004-02-20 2005-01-19 Adjuvant immunologique WO2005079842A1 (fr)

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CN103961306A (zh) * 2013-02-05 2014-08-06 日东电工株式会社 经皮给予用疫苗组合物
CN104667273A (zh) * 2015-02-06 2015-06-03 吉林正业生物制品股份有限公司 疫苗佐剂及其在制备新城疫灭活疫苗中的应用

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CN102416177A (zh) * 2011-12-12 2012-04-18 天津瑞普高科生物药业有限公司 鸡新城疫-h9亚型禽流感二联双佐剂灭活疫苗及其制备方法
CN104248760B (zh) * 2013-12-16 2021-07-23 普莱柯生物工程股份有限公司 一种疫苗组合物及其制备方法和应用
CN111467488B (zh) * 2020-04-17 2023-09-01 内蒙古必威安泰生物科技有限公司 一种水溶性复合免疫佐剂及其应用

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103961306A (zh) * 2013-02-05 2014-08-06 日东电工株式会社 经皮给予用疫苗组合物
CN104667273A (zh) * 2015-02-06 2015-06-03 吉林正业生物制品股份有限公司 疫苗佐剂及其在制备新城疫灭活疫苗中的应用

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