CN102813921A - 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
The invention discloses a formula of the novel adjuvant for a foot-and-mouth disease vaccine and a preparation method for the foot-and-mouth disease vaccine using the adjuvant. Two immunoenhancers PolyI:C and R848 are added to an aluminum hydroxide adjuvant to obtain a mixture, and then, the mixture is mixed with a foot-and-mouth disease antigen to immunize mice, and thereby, discovered by such a trial, the efficacy of the vaccine is similar and even superior to a traditional mineral oil imported adjuvant ISA206.
Description
Technical field
The present invention relates to biological technical field, in particular a kind of prescription of foot-and-mouth disease vaccine novel adjuvant and vaccine production method.
Background technology
(foot-and-mouth disease FMD) is harm deadly infectious disease artiodactylous to foot and mouth disease, especially to the harm especially severe of main poultry boar, cattle, sheep, can cause poultry kind of the productivity to descend and bad social influence, and social economy's loss is huge.Extremely strong because of this disease transmission property, OIE (FAO/OIE) classifies it as No.1ly must report deadly infectious disease, and strict restriction is given in international trade.China classifies this disease first of one type of zoonosis as.Many in the world countries, especially Africa, Asia and south American countries, this disease frequency is frequent, and is popular serious.The popularity outburst in the whole world appears in addition, frequently.
Vaccine is the important measures that prevention and control foot and mouth disease are taked.Foot and mouth disease immunity vaccine commonly used is an oil adjuvant killed vaccine.What the production of this Seedling was adopted is virus inoculation cell, tissue culture, and behind the virus multiplication, the results culture adds the chemical ablation agent and (like the divinyl imines, BEI) makes virus lose infectivity, process vaccine with mineral oil adjuvant mixing and emulsifying.
The natural immunity is replied and is not only body opposing virus and the first line of defence that other cause of diseases infect, and also is to start the prerequisite that acquired immunity is replied.Toll appearance receptor (Toll-like receptors; TLRs) be the cell surface receptor molecule that in the immunne response of host resisting pathogenic microbes, plays 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; 388:394-397).Up to now, 10～15 kinds of TLR and corresponding part or agonist in mammal, have been found.The TLRs wide expression is at various immunocytes, the surface of innate immunity cell especially, and like macrophage and BMDC etc., main mediated cell immunity.
TLRs mediates the secretion of host's relevant cell factor and the generation that the natural immunity is replied through identification pathogenic microorganism and special construction (the TLRs agonist generally is the analog of this structure) thereof.TLR3 identification has the viral infection of double-stranded RNA, and single strand RNA virus can produce double-stranded RNA when duplicating, also can be through TLRs identification.The corresponding identification receptor of virus single stranded RNA is TLR7 and TLR8.TLR9 is the receptor that identification has the CpG DNA viruses.Some TLRs agonist, polyinosini (polyI:C) are the agonist (Kumar H, Kawai T, Akira S.Pathogen recognition in the innate immune response, Biochem J.2009,420 (1): 1-16) of TLR3.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; Have more safety and hypoergia than oils adjuvant; Be widely used in people's epizootic disease Seedling; Mainly be to form antigen to store and slowly releasing effect; Stimulated cells immunity weak (Iyer S, 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 LPS of aluminium hydroxide absorption has been ratified as adjuvant (the Ennio De Gregorio of Hepatitis B virus vaccine in Europe in 2005; Elaine Tritto and Rino Rappuoli Alum adjuvanticity:Unraveling a century old mystery, Eur.J.Immunol.2008.38:2068-2071).Research shows that uniting of aluminium hydroxide and CpG or LPS uses immune effect to be better than its use (Vajdy, M., Selby, M. separately; 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 the 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 the present used antigen model of document is people such as hepatitis B, the hepatitis C poison of curing the desease, and makees the antigen model with animal virus and rarely reports.And; Aluminium hydroxide uses less as the adjuvant of viral vaccine for animals; This is because the effect of mineral oil adjuvant is better than aluminium hydroxide; But see that from the angle of animal welfare and meat product quality the tuberosity of the pain that mineral oil causes, side reaction, injection site etc. will be far longer than aluminium hydroxide.And aluminum hydroxide adjuvant is injected more easily.
Therefore, this research is prepared to unite and is used 2 kinds of different TLR agonist, improves the adjuvanticity of aluminium hydroxide, improves its cellular immunization effect, reduces the untoward reaction that oil adjuvant brought.
Summary of the invention
Technical problem to be solved by this invention is prescription and the vaccine production method thereof that the compound aluminum hydroxide adjuvant of a kind of foot and mouth disease is provided to the deficiency of prior art.
Technical scheme of the present invention is following:
A kind of foot-and-mouth disease vaccine NEW TYPE OF COMPOSITE aluminum hydroxide adjuvant contains the immunostimulant PolyI:C of 20mg/ml alumine hydroxide colloid adjuvant, 200 μ g/ml and the R848 of 200 μ g/ml.
The method for preparing of described foot-and-mouth disease vaccine may further comprise the steps: 1, conventional method prepares the alumine hydroxide colloid adjuvant; 2, conventional method preparation and deactivation foot-and-mouth disease antigen; 3, the immunostimulant PolyI:C and the R848 that in the alumine hydroxide colloid adjuvant, add filtration sterilization respectively, 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, the compound aluminum hydroxide adjuvant of foot and mouth disease is mixed with volume ratio with foot-and-mouth disease antigen at 1: 1, place 4 ℃ to preserve 3 days～5 days, every morning, each jolting in afternoon 2 times, each half an hour.
Behind the vaccine immune mouse that obtains with the inventive method, it is similar or be superior to oily adjuvant to detect antibody and cellular level.
Description of drawings
Fig. 1 PolyI:C and R848 confirm as the optimal dose of immunomodulator and aluminium hydroxide combined immunization mice.The ID of PolyI:C is 0.2 μ g～400 μ g, and the ID of R848 is 0.1 μ g～200 μ g, and each dose groups has three mices.72 hours, 7 days, took a blood sample and to detect the content of cytokine gamma interferon (IFN-γ), interleukin 4 (IL-4) and tumor necrosis factor (TNF) in mice 24 hours behind the injecting immune reinforcing agent in 14 days.The content of cytokine detects through commercialization ELISA test kit.Resulting data are geometrical mean ± standard deviation.(a) content of the IL-4 in the inductive mice peripheral blood of the PolyI:C of different time, various dose; (b) content of the TNF in the inductive mice peripheral blood of the PolyI of different time, various dose: C; (c) content of the IFN-γ in the inductive mice peripheral blood of the PolyI:C of different time, various dose; (d) content of the IL-4 in the inductive mice peripheral blood of the R848 of different time, various dose; (e) content of the TNF in the inductive mice peripheral blood of the R848 of different time, various dose; (f) content of the TNF in the inductive mice peripheral blood of the PolyI of different time, various dose: C
The antibody titer that Fig. 2 produces after by the 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 the ELISA test kit detects foot-and-mouth disease antibody and tires.Numerical value is mean+SD.
Fig. 3 immunity CD8 in the mouse spleen lymphocyte after 21 days
+T lymphocyte testing result; (a) CD3CD8 in the F+206 immune group
+The lymphocytic door of T inner cell percentage ratio (10.7%); (b) CD3CD8 in the F+A immune group
+The lymphocytic door of T inner cell percentage ratio (8.4%); (c) CD3CD8 in the F+A+P immune group
+The lymphocytic door of T inner cell percentage ratio (11.5%); (d) CD3CD8 in the F+A+R immune group
+The lymphocytic door of T inner cell percentage ratio (11.5%); (e) CD3CD8 in the F+A+P+R immune group
+The lymphocytic door of T inner cell percentage ratio (12.2%).
Fig. 4 immunity CD4 in the mouse spleen lymphocyte after 21 days
+T lymphocyte testing result; (a) CD3CD4 in the F+206 immune group
+The lymphocytic door of T inner cell percentage ratio (21.6%); (b) CD3CD4 in the F+A immune group
+The lymphocytic door of T inner cell percentage ratio (17.9%); (c) CD3CD4 in the F+A+P immune group
+The lymphocytic door of T inner cell percentage ratio (19.9%); (d) CD3CD4 in the F+A+R immune group
+The lymphocytic door of T inner cell percentage ratio (19.0%); (e) CD3CD4 in the F+A+P+R immune group
+The lymphocytic door of T inner cell percentage ratio (21.2%).
35 days the mouse spleen lymphocyte of immunity of 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, utilize the erythrocyte cracked liquid splitting erythrocyte.On 25 * 16 counting chambeies, count then, process the single cell suspension of 5 * 106/mL concentration.Respectively establish three holes and repeat, every hole adds 200 microlitres, 5106 cells, adds 100 microlitre foot-and-mouth disease antigens (3 μ g/ml) then.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
Employed experimental technique is conventional method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
One, PolyI:C and R848 confirm as the optimal dose of immunostimulant and aluminium hydroxide combined immunization mice
The BALB/c mouse that selects 18-22g is 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+ R848 (0.1,1,10,20,100 or 200 μ g).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 is to confirm best immunostimulant agent dose.
In order to select optimum immuning dose, we have designed the PolyI:C and the R848 of a series of variable concentrations, to confirm ID (Fig. 1) best in the mice body.The immunoreation that the reaction of the different cells factor is different.We have chosen IL-4, and three indexs of TNF and INF-γ are weighed its immunostimulant effect.The content range that injects the intravital immunostimulant of mice is that 0.1 μ g-400 μ g does not wait.When PolyI:C content is 20 μ g/, IL-4, TNF, INF-γ are in different time, and the content of generation has reached maximum.When R848 be 1 μ g/ only or 100 μ g/ the time, the content of TNF and INF-γ is higher relatively; And when R848 was 20 μ g/, the content of cytokine IL-4 was the highest, took all factors into consideration these data and cost factor, and the immune optimal dose of confirming R-848 also is 20g/.
Two. the immune effect of more several kinds of 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.Muscle carries out intramuscular injection: (1) PBS, (2) F+206, (3) F+A, (4) F+A+P, (5) in the mouse hind leg outside according to following a few assembly sides; F+A+R, (F represents the foot and mouth disease inactivation antigen to (6) F+A+P+R; A represents strong aluminum adjuvant; 206 represent the ISA206 adjuvant, and 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 left the heart 10 minutes through 4000, collected serum, and was stored in-20 ℃, detected corresponding index up to carrying out ELISA.
1. anti-foot-and-mouth disease antibody is tired.
Detect through the ELISA method.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 that the F+A+P+R group produced in the 14/21st day is higher (be respectively 1:256,1:512) antibody titer; This combination is described, and relatively antibody titer of producing of other groups is higher, more early, and 2 kinds of immunostimulant PolyI:C have played very important effect with R848 in the enhancing antibody generation.
2. detect CD8 with flow cytometer
+T cell and CD4
+The T lymphocyte.
Spleen after the aseptic separation, is processed single cell suspension (10 in the mice sacrificed body
6～10
7/ mL).In the 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; 3000 rev/mins of centrifugal 5min discard supernatant then.Every pipe adds the PBS buffer of 2ml and washes cell, and 3000 rev/mins, centrifugal 5min discards supernatant, repeats twice.Last every pipe adds the PBS suspension cell of 0.5ml, goes up machine testing in four hours.
Flow cytometry analysis CD3CD8
+The T lymphocyte is the proportion of composing result (Fig. 3 and Fig. 4) in spleen lymphocyte show, its proportion of composing is the highest in the F+A+P+R group, reaches 12.2%, and the lymphocytic proportion of composing of the 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 the F+A group, ratio is minimum, has only 8.4%.CD3CD4+T lymphocyte testing result shows in the immune back 21 days mouse spleens, CD4 in the F+206 group
+T lymphocyte proportion is the highest, reach 21.6%, and F+A+R+P is 21.2%, and the two is approaching, explains that the effect of this combination and mineral oil adjuvant is approaching, and high than ratio in F+A+R group or the F+A+P group.Explain that PolyI:C and R848 synergy are better than independent action effect in promoting the T cell differentiation.
3. the stimulated in vitro splenocyte is surveyed the cytokine production
Separate 35 days mouse spleen lymphocyte of all immunity, process single cell suspension.Splenocyte (5 * 10
6/ ml) on 96 orifice plates, cultivate every hole 0.1mL.Use the splenocyte of the foot-and-mouth disease antigen 100 μ L immune stimulatory mices of deactivation then; Then 37 ℃, contain in 5% CO2 gas incubator and hatch 48h; At last through centrifugal 96 well culture plates; Cell aggregation is collected supernatant in the bottom, hole, detects cytokine (IL-4, IFN-γ) production with the ELISA method.
Immune mouse splenocyte stimulated in vitro result of the test (Fig. 5) shows that to the excretory IFN-γ of specific antigen, the numerical value of F+206 group is the highest, is 489.16, and the numerical value of F+A+R+P group is 462.62, and the two numerical value is approaching.And the numerical value of F+A group is 98.67, and the numerical value of F+A+P is 144.15, and the numerical value of F+A+R is 232.5.Explain that compound aluminum hydroxide adjuvant is better than the effect of a certain immunostimulant of simple use.The IL-4 generation is respectively organized difference not obvious (P＞0.05).Wherein the numerical value of F+206 group is the highest, is 75.61, and the numerical value of F+A+R+P group is 65.83, and the numerical value of F+A group is 65.02, and the numerical value of F+A+P is 60.95, and the numerical value of F+A+R is 63.93.Comprehensive above-mentioned data explain that the novel aluminium hydroxide combination adjuvant of use (A+R+P) is approaching with the effect of mineral oil 206 adjuvants.
Claims (2)
1. a foot-and-mouth disease vaccine NEW TYPE OF COMPOSITE aluminum hydroxide adjuvant is characterized in that, contains the immunostimulant PolyI:C of 20mg/ml alumine hydroxide colloid adjuvant, 200 μ g/ml and the R848 of 200 μ g/ml.
2. the method for preparing foot-and-mouth disease vaccine according to the said foot-and-mouth disease vaccine NEW TYPE OF COMPOSITE of claim 1 aluminum hydroxide adjuvant; May further comprise the steps: the immunostimulant PolyI:C and the R848 that in the alumine hydroxide colloid adjuvant, add filtration sterilization respectively; Concentration is 200 μ g/ml; Make the compound aluminum hydroxide adjuvant of foot and mouth disease, alumine hydroxide colloid adjuvant concentration is 20mg/ml.The compound aluminum hydroxide adjuvant of foot and mouth disease is mixed with volume ratio with foot-and-mouth disease antigen at 1: 1, place 4 ℃ to preserve 3 days～5 days, every morning, each jolting in afternoon 2 times, each half an hour.
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