CN102813921B - Novel compound aluminum hydroxide adjuvant for foot-and-mouth disease vaccine and vaccine preparation method - Google Patents
Novel compound aluminum hydroxide adjuvant for foot-and-mouth disease vaccine and vaccine preparation method Download PDFInfo
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Abstract
本发明公开了一种口蹄疫疫苗新型佐剂的配方以及用该佐剂的口蹄疫疫苗制备方法。用氢氧化铝佐剂加上2种免疫增强剂PolyI:C和R848，再和口蹄疫抗原混合后免疫小鼠，发现该疫苗的效力类似甚至优于现行的矿物油进口佐剂ISA206。The invention discloses a formula of a novel adjuvant of a foot-and-mouth disease vaccine and a preparation method of a foot-and-mouth disease vaccine using the adjuvant. After immunizing mice with aluminum hydroxide adjuvant plus two kinds of immune enhancers PolyI:C and R848, mixed with FMD antigen, it was found that the efficacy of the vaccine was similar to or even better than that of the current imported mineral oil adjuvant ISA206.
Description
Technical field
The present invention relates to biological technical field, in particular a kind of formula of foot-and-mouth disease vaccine novel adjuvant and vaccine preparation method.
Background technology
Foot and mouth disease (foot-and-mouth disease, FMD) be harm deadly infectious disease artiodactylous, especially the harm especially severe to main poultry boar, cattle, sheep, can cause poultry kind of the productivity to decline and bad social influence, and Socie-economic loss is huge.Extremely strong because of this disease transmission, OIE (FAO/OIE) is classified as No.1ly must report deadly infectious disease, and international trade strictly limits.China classifies this disease first of one class zoonosis as.Many countries in the world, especially Africa, Asia and south American countries, this disease frequency is frequent, popular serious.In addition, frequently there is global popularity outburst.
Vaccine is prevention and controls the important measures that foot and mouth disease is taked.The conventional vaccine of foot and mouth disease immunity is oil adjuvant killed vaccine.What the production of this Seedling adopted is virus inoculation cell, tissue culture, and after virus multiplication, results culture, adds chemical ablation agent (as divinyl imines, BEI) and make virus lose infectivity, makes vaccine with mineral oil adjuvant mixing and emulsifying.
Innate immunity is not only the first line of defence of body opposing virus and other Infected with Pathogenic Fungis, is also the prerequisite that starts Acquired immune response.Toll sample receptor (Toll-like receptors, TLRs) be the cell surface receptor molecule playing an important role (Medzhitov R, the Preston-Hurlburt P.Janeway CA Jr.A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature 1997 of discovered in recent years in the immunne response of host resisting pathogenic microbes; 388:394-397).Up to now, 10～15 kinds of TLR and corresponding part or agonist in mammal, have been found that there is.TLRs wide expression is at various immunocytes, the especially surface of innate immunity cell, and as macrophage and dendritic cell etc., main mediated cell immunity.
TLRs, by identification pathogenic microorganism and special construction (TLRs agonist is generally the analog of this structure) thereof, mediates the secretion of host's relevant cell factor and the generation of innate immunity.TLR3 identification has the viral infection of double-stranded RNA, and single strand RNA virus can produce double-stranded RNA when copying, and also can identify by TLRs.The corresponding identification receptor of virus single stranded RNA is TLR7 and TLR8.TLR9 is the receptor of identification with CpG DNA viruses.Some TLRs agonist, polyinosini (polyI:C) is agonist (Kumar H, Kawai T, the Akira S.Pathogen recognition in the innate immune response of TLR3, Biochem J.2009,420 (1): 1-16).Resquimod (R-848) is agonist (the Gibson SJ of TLR7 and TLR8, Lindh JM, Riter TR, Gleason RM, Rogers LM, Fuller AE, et al.Plasmacytoid dendritic cells produce cytokines and mature in response to the TLR7agonists, imiquimod and resiquimod.Cell Immunol 2002; 218 (1-2): 74-86).Historical existing more than 80 year of the use of aluminium hydroxide immunological adjuvant, than oils adjuvant, have more safety and hypoergia, be widely used in people's epizootic disease Seedling, mainly to form antigen to store and slowly releasing effect, weak (the Iyer S of the cellular immunization stimulating, HogenEsch H, Hem SL.Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production.Vaccine 2003; 21:1219-1223).The analog phosplate A (MPL) of the European LPS that has ratified aluminium hydroxide absorption in 2005 is as adjuvant (the Ennio De Gregorio of Hepatitis B virus vaccine, Elaine Tritto and Rino Rappuoli Alum adjuvanticity:Unraveling a century old mystery, Eur.J.Immunol.2008.38:2068-2071).Research shows, aluminium hydroxide is better than its use (Vajdy separately with using immune effect combining of CpG or LPS, M., Selby, M., Medina-Selby, A., Coit, D., Hall, J., Tandeske, L., Chien, D.et al., Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses.J.Gen.Virol.2006.87:2253-2262.Wack, A., Baudner, B.C., Hilbert, A.K., Manini, I., Nuti, S., Tavarini, S., Scheffczik, H.et al., Combination adjuvants for the induction of potent, long-lasting antibody and T-cell responses to influenza vaccine in mice.Vaccine 2008.26:552-561), this is because aluminium hydroxide causes Th2 effect, and TLRs causes Th1 effect (Nemazee, D., Gavin, A., Hoebe, K.and Beutler, B., Immunology:Toll-like receptors and antibody responses.Nature 2006.441:E4, discussion E4).Because current document antigen model used is the people such as hepatitis B, the hepatitis C poison of curing the desease, do the rare report of antigen model with animal virus.And, aluminium hydroxide is used less as the adjuvant of viral vaccine for animals, this is because the effect of mineral oil adjuvant is better than aluminium hydroxide, but from the angle of animal welfare and meat product quality, tuberosity of pain that mineral oil causes, side reaction, injection site etc. will be far longer than aluminium hydroxide.And aluminum hydroxide adjuvant is more easily injected.
Therefore, this research is prepared to combine and is used 2 kinds of different TLR agonist, improves the adjuvanticity of aluminium hydroxide, improves its Study On Cellular Immune, reduces the untoward reaction that oil adjuvant brings.
Summary of the invention
Technical problem to be solved by this invention is formula and the vaccine preparation method thereof that the compound aluminum hydroxide adjuvant of a kind of foot and mouth disease is provided for the deficiencies in the prior art.
Technical scheme of the present invention is as follows:
A kind of foot-and-mouth disease vaccine NEW TYPE OF COMPOSITE aluminum hydroxide adjuvant, containing 20mg/ml alumine hydroxide colloid adjuvant, the immunostimulant PolyI:C of 200 μ g/ml and the R848 of 200 μ g/ml.
The preparation method of described foot-and-mouth disease vaccine, comprises the following steps: 1, conventional method is prepared alumine hydroxide colloid adjuvant; 2, conventional method preparation deactivation foot-and-mouth disease antigen; 3, in alumine hydroxide colloid adjuvant, add respectively immunostimulant PolyI:C and the R848 of filtration sterilization, concentration is 200 μ g/ml, makes the compound aluminum hydroxide adjuvant of foot and mouth disease, and alumine hydroxide colloid adjuvant concentration is 20mg/ml.4, compound foot and mouth disease aluminum hydroxide adjuvant is mixed with volume ratio with foot-and-mouth disease antigen at 1: 1, be placed in 4 ℃ and preserve 3 days～5 days, every morning, each jolting in afternoon 2 times, each half an hour.
After the vaccine immune mouse obtaining by the inventive method, can detect that antibody and cellular level are similar or be better than oily adjuvant.
Accompanying drawing explanation
Fig. 1 PolyI:C and R848 determine as the optimal dose of immunomodulator and aluminium hydroxide combined immunization mice.The injected dose of PolyI:C is 0.2 μ g～400 μ g, and the injected dose of R848 is 0.1 μ g～200 μ g, and each dosage group has three mices.After injecting immune reinforcing agent 24 hours of mice, 72 hours, 7 days, take a blood sample and detect the content of cytokine gamma interferon (IFN-γ), interleukin 4 (IL-4) and tumor necrosis factor (TNF) for 14 days.The content of cytokine detects by commercialization ELISA test kit.The data that obtain are geometrical mean ± standard deviation.(a) content of the IL-4 in the mouse peripheral blood of the PolyI:C of different time, various dose induction; (b) content of the TNF in the mouse peripheral blood of the PolyI of different time, various dose: C induction; (c) content of the IFN-γ in the mouse peripheral blood of the PolyI:C of different time, various dose induction; (d) content of the IL-4 in the mouse peripheral blood of the R848 of different time, various dose induction; (e) content of the TNF in the mouse peripheral blood of the R848 of different time, various dose induction; (f) content of the TNF in the mouse peripheral blood of the PolyI of different time, various dose: C induction
The antibody titer that Fig. 2 produces after by different formulations immune mouse is in the dynamic change of different time.Time according to 3 days, 7 days, 14 days, 21 days, 28 days and 35 days takes a blood sample 6 times altogether, and ELISA test kit detects foot-and-mouth disease antibody and tires.Numerical value is mean+SD.
Fig. 3 immunity CD8 in mouse spleen lymphocyte after 21 days
+t lymphocyte testing result; (a) CD3CD8 in F+206 immune group
+t lymphocytic door inner cell percentage ratio (10.7%); (b) CD3CD8 in F+A immune group
+t lymphocytic door inner cell percentage ratio (8.4%); (c) CD3CD8 in F+A+P immune group
+t lymphocytic door inner cell percentage ratio (11.5%); (d) CD3CD8 in F+A+R immune group
+t lymphocytic door inner cell percentage ratio (11.5%); (e) CD3CD8 in F+A+P+R immune group
+t lymphocytic door inner cell percentage ratio (12.2%).
Fig. 4 immunity CD4 in mouse spleen lymphocyte after 21 days
+t lymphocyte testing result; (a) CD3CD4 in F+206 immune group
+t lymphocytic door inner cell percentage ratio (21.6%); (b) CD3CD4 in F+A immune group
+t lymphocytic door inner cell percentage ratio (17.9%); (c) CD3CD4 in F+A+P immune group
+t lymphocytic door inner cell percentage ratio (19.9%); (d) CD3CD4 in F+A+R immune group
+t lymphocytic door inner cell percentage ratio (19.0%); (e) CD3CD4 in F+A+P+R immune group
+t lymphocytic door inner cell percentage ratio (21.2%).
The immunity mouse spleen lymphocyte of 35 days of the each immune group of Fig. 5 carries out the content of 2 kinds of cytokines (IFN γ and IL-4) of foot-and-mouth disease antigen stimulated in vitro generation.Spleen separates grinding under aseptic condition, utilizes erythrocyte cracked liquid splitting erythrocyte.Then on 25 × 16 counting chambeies, count, make the single cell suspension of 5 × 106/mL concentration.Respectively establish three holes and repeat, every hole adds 200 microlitre 5106 cells, then adds 100 microlitre foot-and-mouth disease antigens (3 μ g/ml).Data statistics: the geometric mean ± standard deviation of getting three holes; (a) the IFN-γ content of stimulated in vitro immune mouse splenocyte; (b) the IFN-γ content of stimulated in vitro immune mouse splenocyte.
The specific embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
One, PolyI:C and R848 determine as the optimal dose of immunostimulant and aluminium hydroxide combined immunization mice
Select the BALB/c mouse of 18-22g as laboratory animal.At BALB/c mouse (n=12/ group) left hind intramuscular injection 200 μ l Al (OH)
3+ PolyI:C (0.2,2,20,40,200 or 400 μ g) or 200 μ l Al (OH)
3(0.1,1,10,20,100 or 200 μ g) for+R848.After injection 24 hours subsequently, 72 hours, 7 days, 14 days, test mice to be taken a blood sample, separation of serum, detects IL-4 with commercial kit, TNF, INF-γ, to determine best immunostimulant agent dose.
In order to select optimum immuning dose, we have designed PolyI:C and the R848 of a series of variable concentrations, to determine injected dose (Fig. 1) best in Mice Body.Different cytokines is reacted different immunoreation.We have chosen IL-4, TNF, and tri-indexs of INF-γ are weighed its immunostimulant effect.The content range that injects the immunostimulant in Mice Body be 0.1 μ g-400 μ g not etc.When PolyI:C content is 20 μ g/, IL-4, TNF, INF-γ is in different time, and the content of generation has reached maximum.When R848 be 1 μ g/ only or 100 μ g/ time, the content of TNF and INF-γ is relatively high; And when R848 is 20 μ g/, the content of cytokine IL-4 is the highest, consider these data and cost factor, the immune optimal dose of determining R-848 is also 20g/.
Two. the immune effect of more several different formulations
BALB/c mouse is divided into six groups at random, 12 every group.Foot and mouth disease inactivation antigen concentration is 3 μ g/ml.Every foot and mouth disease inactivation antigen immunizing dose is 200 μ l.According to following several group of formula muscle outside mouse hind leg, carry out intramuscular injection: (1) PBS, (2) F+206, (3) F+A, (4) F+A+P, (5), F+A+R, (F represents foot and mouth disease inactivation antigen to (6) F+A+P+R, A represents strong aluminum adjuvant, 206 represent ISA206 adjuvant, P represents PolyI:C, and R represents R848).All mices are taken a blood sample before immunity, and then carry out immunity.Blood samplings in 3,7,14,21,28,35 days after immunity.Peripheral blood leaves the heart 10 minutes through 4000, collects serum, and is stored in-20 ℃, until carry out ELISA, detects corresponding index.
1. anti-foot-and-mouth disease antibody is tired.
By ELISA method, detect.As can be seen from Figure 3, F+A group and F+206 group difference on antibody titer numerical value are little, the highest antibody titer of F+206 group is 1:128, and F+A+P+R group has produced higher in the 14/21st day, (be respectively 1:256,1:512) antibody titer, illustrate that this combination antibody titer that relatively other groups produce is higher, more early, 2 kinds of immunostimulant PolyI:C and R848 have played very important effect in enhancing antibody produces.
2. with flow cytometer, detect CD8
+t cell and CD4
+t lymphocyte.
Spleen aseptic separation, is made single cell suspension (10 in the Mice Body of putting to death
6～10
7/ mL).In 2ml centrifuge tube, add 100 microlitre lymphocyte suspensions, add double labelling monoclonal antibody CD3 (ALEXA
488)/CD4 (ALEXA
647) or CD3 (ALEXA
488)/CD8 (ALEXA
647), room temperature lucifuge 20 minutes, then 3000 revs/min of centrifugal 5min, discard supernatant.Every pipe adds the PBS buffer of 2ml to wash cell, and 3000 revs/min, centrifugal 5min, discards supernatant, repeats twice.Last every pipe adds the PBS suspension cell of 0.5ml, upper machine testing in four hours.
Flow cytometry analysis CD3CD8
+proportion of composing result (Fig. 3 and Fig. 4) demonstration of T lymphocyte in spleen lymphocyte, in F+A+P+R group, its proportion of composing is the highest, reaches 12.2%, and the lymphocytic proportion of composing of CD3CD8+T of mineral oil 206 is 10.7%; The ratio of F+A+P and F+A+R is respectively 11.5%.And in F+A group, ratio is minimum, only have 8.4%.In latter 21 days mouse spleens of immunity, CD3CD4+T lymphocyte testing result shows, CD4 in F+206 group
+t lymphocyte proportion is the highest, reach 21.6%, and F+A+R+P is 21.2%, and the two approaches, and illustrates that the effect of this combination and mineral oil adjuvant approaches, and high compared with ratio in F+A+R group or F+A+P group.Illustrate that PolyI:C and R848 synergy are more effective than independent role in promotion T cell differentiation.
3. stimulated in vitro splenocyte is surveyed cytokine production
Separate the mouse spleen lymphocyte of all immune 35 days, make single cell suspension.Splenocyte (5 × 10
6/ ml) on 96 orifice plates, cultivate every hole 0.1mL.Then use the splenocyte of the foot-and-mouth disease antigen 100 μ L immune stimulatory mices of deactivation, then 37 ℃, containing hatching 48h in 5% CO2 gas incubator, finally by centrifugal 96 well culture plates, cell aggregation is in bottom, hole, collect supernatant, with ELISA method detection cytokine (IL-4, IFN-γ) production.
Immune mouse splenocyte stimulated in vitro result of the test (Fig. 5) shows, the IFN-γ secreting for specific antigen, and the numerical value of F+206 group is the highest, is that the numerical value of 489.16, F+A+R+P group is 462.62, and the two numerical value approaches.And the numerical value of F+A group is 98.67, the numerical value of F+A+P is 144.15, and the numerical value of F+A+R is 232.5.Illustrate that compound aluminum hydroxide adjuvant is better than the effect of a certain immunostimulant of simple use.IL-4 generation is respectively organized difference not obvious (P > 0.05).Wherein the numerical value of F+206 group is the highest, and the numerical value that is 75.61, F+A+R+P group is that the numerical value that numerical value that the numerical value of 65.83, F+A group is 65.02, F+A+P is 60.95, F+A+R is 63.93.Comprehensive above-mentioned data, illustrate and use New Hydrogen aluminium oxide combination adjuvant (A+R+P) and the effect of mineral oil 206 adjuvants to approach.
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| CN110624100B (en) * | 2019-09-06 | 2021-03-16 | 中国农业科学院兰州兽医研究所 | Application of foot-and-mouth disease virus 3D protein as activator of cellular TLR3 pathway |
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