CN102813920A - Vaccine adjuvant - Google Patents
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- CN102813920A CN102813920A CN2011101566022A CN201110156602A CN102813920A CN 102813920 A CN102813920 A CN 102813920A CN 2011101566022 A CN2011101566022 A CN 2011101566022A CN 201110156602 A CN201110156602 A CN 201110156602A CN 102813920 A CN102813920 A CN 102813920A
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Abstract
The invention belongs to the biological product field, relates to a vaccine adjuvant, and concretely relates to a use of the vaccine adjuvant praziquantel in the preparation of vaccine compounds, and a preparation method of the vaccine adjuvant. Praziquantel is adopted as the vaccine adjuvant in the invention; and experiments confirm that the praziquantel can be used for preparing antivirus vaccines as a vaccine adjuvant, wherein the antivirus vaccines comprise a hepatitis B vaccine, an AIDS vaccine or an influenza vaccine, and especially comprise a DNA vaccine, a nucleic acid vaccine or a subunit protein vaccine. Compared with present adjuvants, the praziquantel which is the adjuvant in the invention has the advantages of effective activation of the body fluid and cell immunity levels of bodies, substantial enhancement of the vaccine immunization effect, and improvement of the CTL response of the antigen specificity. Simultaneously the vaccine adjuvant disclosed in the invention provides a better method for the virus removal, and has the characteristics of use convenience, low cost, small side effect, and easy popularization.
Description
Technical field
The invention belongs to field of biological product, relate to a kind of vaccine adjuvant, be specifically related to praziquantel as vaccine adjuvant be used to purposes for preparing the vaccine complex and preparation method thereof.
Background technology
Hepatitis B by hepatitis B virus cause, through blood and body fluid communication, be main infectious disease with the liver injury, this illness is very big to the threat of human health, has become serious public health problem.Research shows that behind the infection hepatitis B, part patient will be developed into chronic persistent infection state, and part possibly develop into hepatitis interstitialis chronica or primary hepatoma.It is reported that China is the popular district of hepatitis b virus infected height, annual have 350,000 people to die from the disease relevant with hepatitis B (as: liver cirrhosis, hepatocarcinoma etc.) approximately; Wherein, the crowd infection rate is 60%, and hbs antigen (HBsAg) crowd carrying rate is 10%.According to estimates, the whole world has 300,000,000 HBsAg carriers, and China just account for wherein 1/3rd, hepatitis B is propagated has become the major issue that influences population quality.Haematogenous vaccine and recombinant vaccine stage have successively been experienced in the development of hepatitis B vaccine, and Hepatitis B virus vaccine plays an important role to the prevention and the control of hepatitis B.
Research shows that the effective immunization therapy means of hepatitis B should be based on the intravital immune system of challenge virus carrier.The index of the evaluation therapeutic hepatitis B vaccine of generally acknowledging in the world is the cell immune response of antigenic specificity, especially will excite the positive CTL cytoactive of high-caliber hepatitis B specificity CD8, and high-caliber γ one interferon of justacrine etc. are immune component effectively.Though there is report to utilize hepatitis B virus S or C antigen to cooperate with different immunostimulants for immunogen in the world, to improve the cellular immune level that activates body, clinical effectiveness is still in evaluation.The inoculation that prior art discloses Hepatitis B virus vaccine is the effective measures of control hepatitis B; Described Hepatitis B virus vaccine can be divided into synthetic peptide vaccine, gene (nucleic acid) vaccine and recombinant subunit vaccine according to constituent; Wherein, Synthetic peptide vaccine is the vaccine that the antigen of some peptide chain or the albumen synthetic of imitative specific antigen forms, and gene (nucleic acid) vaccine is immune with respect to gene (nucleic acid), and it is cloned on the suitable plasmid vector for containing the proteinic gene order of specific antigen of encoding; Be prepared into nucleic acid expression vector; Through methods such as intramuscular injection it is imported in the body, through the re-recording system synthetic antigen protein of host cell, the excitating organism immune system produces the specific immune response reaction to exogenous proteins.Employed nucleic acid expression vector is called as gene vaccine in the above-mentioned genetic immunization process, claims nucleic acid vaccine again.Described the 3rd type of Hepatitis B virus vaccine is recombinant subunit vaccine, is about to the virus antigen gene and in escherichia coli or Yeast system, gives expression to protein, forms with the aluminium adjuvant compatibility behind the extraction purification.Get into the eighties; The development of hepatitis B recombinant subunit vaccine is rapid; From 1981; Merck & Co., Inc. successfully develops hepatitis b gene S albumen in yeast, express back and the recombinant subunit vaccine and commercialization of aluminium adjuvant compatibility after, the prevention and the control of global hepatitis B is played an important role.
Influenza (abbreviation influenza) is the acute respiratory infectious disease that is caused by influenza virus (influenza virus).Influenza has brought huge disaster for human hygiene and health, once causes 4 flu outbreaks on the human history, and death toll reaches several ten million.Attract annual small-scale influenza pandemic in recent years in some countries and regions, there is 9% population infection influenza virus every year in the whole world.Influenza virus has the sudden change of being prone to, the characteristics of multiple serotype; Strong like highly pathogenic H5N1 hypotype and H1N1 hypotype swine flu variability in recent years, be important viral respiratory cause of disease, infection population and cause great popular very easily.Therefore, for preventing the generation of above-mentioned potential epidemic situation, need extensively promote production urgently to the effective vaccine of this new virus; But because current widely used inactivated vaccine can only cause the antibody response of antiviral protein, it fails to satisfy the demand of this respect.There is research report to point out, is reflected at control viral infection aspect to the specificity cell toxicity T lymphocyte of virus and plays an important role.Therefore, for the inactivated vaccine of control viral infection, compare and only induce the generation corresponding antibody, can it reaction of effective stimulus body generation specific CTL seem more important.
The research report, AIDS (Acquired Immunodeficiency Syndrome) is called for short AIDS (AIDS), has become a kind of serious infectious disease in the harm whole world.AIDS after getting into body by HIV virus, destroys body CD4
+The immunodeficiency diseases that the T cell is produced.The proteic gene of coding HIV retrovirus mainly contains three kinds: Env, Pol, Gag.Pol and Gag mainly the encode nuclear protein and the reverse transcriptase protein of virus, and mainly the encode albumen on its viromembrane surface of Env gene, and the epi-position of neutralizing antibody is basically all on the Env gene.Still there is not at present the HIV virus that effective vaccine and medicine are used to prevent and remove infection.Dna vaccination is as emerging vaccine; Plurality of advantages such as traditional vaccine can't replace are arranged; Also dna vaccination is regarded as one of key technology of capturing AIDS both at home and abroad; But the greatest problem of restricting current dna vaccination development is that immunogenicity is not strong; One of important channel of the evaluation control HIV viral infection of generally acknowledging in the world is how effectively to transfer cell immune response, especially will excite the positive CTL cytoactive of high-caliber HIV specificity CD8, and high-caliber γ one interferon of justacrine etc. are immune component effectively.Therefore it is extremely urgent to find effective adjuvant to strengthen the effect of dna vaccination.
Research confirms, in the preparation process of vaccine, adjuvant is as can synantigen be expelled to the internal energy enhances immunogenicity of body or change the material of immunoreation type together or in advance, receives the attention of researcher in the research and development of novel, efficient vaccine complex.Adjuvant is claimed the nonspecific immunity proliferant agent again; Itself do not have an antigenicity; Being one type acts on prior to antigen or with antigen simultaneously; Can change non-specificly or enhancing body to a kind of material of antigenic specific immune response, promptly synantigen is expelled to the internal energy enhances immunogenicity of body together or in advance or changes the immunoreation type.The main biological agent of immunological adjuvant comprises the following aspects: antigenic substance mixing adjuvant has changed antigenic physical behavior after injecting body, and antigenic substance is discharged lentamente, has prolonged antigenic action time; After adjuvant has adsorbed antigen, increased antigenic surface area, made antigen be easy to engulfed by antigen-presenting cell; Adjuvant can be the former processing of delivery cell antagonism by stimulator antigen; Adjuvant can promote the contact between the lymphocyte, strengthens the effect of helper T cell; Can stimulate the division and the plasma cell of primed lymphocyte to produce antibody.
Described adjuvant is not only the important composition composition in the inactivated vaccine production, and the immune effect that improves dna vaccination and sub-unit protein vaccine is also had effect.Especially in inducing more effective antiviral immunity reaction, the participation of T cellular immunization is absolutely necessary.At present; Improve the immunoreactive adjuvant of vaccine bebcell almost not having in clinical practice; And now use adjuvant as; Aluminium salt adjuvant; It is master's adjuvant that oil adjuvant and SF59 adjuvant are to improve antibody horizontal; So, be badly in need of clinically especially strengthening the adjuvant of CD8 t cell immune response to strengthen cell immune response.Therefore, for the screening operation of the adjuvant of the immune effect that strengthens hepatitis B, AIDS and influenza new generation vaccine, developing a kind of novel adjuvant that is used for hepatitis B and influenza vaccines has become extremely urgent research emphasis.
Praziquantel (Praziquantel PZQ) is a kind of New-type wide-spectrum antiparasitic, and the carbohydrate metabolism of worm is had the obvious suppression effect, at present, and as wide spectrum taeniacide, the anti-trematodiasis medicine of antischistosomal drug.Discover that after the praziquantel treatment, the effectively secretion of enhancing body IgE suppresses the secretion of IgG4, strengthens the Th2 cytokine expression simultaneously, can resist the generation once more of schistosomicide effectively.The article report is arranged, and after the praziquantel treatment, the ratio regular meeting of patient's regulatory T cells reduces, and the ability of the expression CD45O of natural regulatory T cells weakens greatly, makes body resist the schistosomicide ability enhancing of subinfection again.For a long time, about the research of praziquantel, mainly concentrate on people or the poultry parasitic disease field of preventing and treating.
So far, the relevant praziquantel of Shang Weijian is as the report of the vaccine adjuvant of antiviral (especially hepatitis B, AIDS and influenza).
Summary of the invention
The purpose of this invention is to provide a kind of new vaccine adjuvant, especially to strengthen cell immune response, especially strengthen the immunoreactive vaccine adjuvant of cd8 t cell, described vaccine adjuvant hepatitis B is vaccine adjuvant, AIDS vaccine adjuvant or influenza vaccines adjuvant.
The invention provides the new pharmaceutical usage of praziquantel, be specifically related to praziquantel as vaccine adjuvant be used for purposes for preparing the vaccine complex and preparation method thereof; The present invention utilizes described praziquantel can strengthen immune effect of vaccine as adjuvant, can significantly improve antibody horizontal and cellular immune level, improves the ctl response of antigenic specificity.
As vaccine adjuvant, through experiment confirm, described praziquantel can be used for preparing antiviral vaccine as vaccine adjuvant with praziquantel in the present invention, in particular for preparation anti-hepatitis B, AIDS or influenza vaccines; Wherein, described vaccine is dna vaccination, nucleic acid vaccine or sub-unit protein vaccine.
Among the present invention, the chemical name of said praziquantel: 2-cyclohexyl formoxyl-1,2,3,6,7,11b-six hydrogen-4H-pyrazine be [2,1-α] isoquinolin-4-ketone also, molecular formula: C19H24N2O2, and molecular weight: 312.41, have the structure of formula (I),
Among the present invention, described Hepatitis B virus vaccine is hepatitis B DNA vaccine, hepatitis B subunit vaccine; Wherein, described hepatitis B subunit vaccine is through technique for gene engineering the virus antigen gene to be given expression to protein in escherichia coli or Yeast system, processes with the adjuvant compatibility behind the extraction purification, especially processes with praziquantel adjuvant compatibility;
Among the present invention, described AIDS vaccine is the aids dna vaccine, can especially process with praziquantel adjuvant compatibility through processing with the adjuvant compatibility behind amplification in the escherichia coli and the extraction purification;
Among the present invention, described influenza vaccines are inactivated influenza virus vaccines, process with the adjuvant compatibility after can connecing the poison amplification and extract purification through Embryo Gallus domesticus, especially process with praziquantel adjuvant compatibility.
Another object of the present invention provides a kind of be used to prevent hepatitis B, AIDS or grippal vaccine complex; Described vaccine complex is by virus antigen or dna vaccination or sub-unit protein vaccine and praziquantel adjuvant compatibility and process.
The invention provides a kind of vaccine complex that is used to treat hepatitis B or AIDS; Described vaccine complex is by virus antigen or dna vaccination or sub-unit protein vaccine and praziquantel adjuvant compatibility and process.
Among the present invention, the vaccine of described treatment hepatitis B is hepatitis B DNA vaccine, hepatitis B subunit vaccine; Described AIDS vaccine is the aids dna vaccine; The quality of the described praziquantel adjuvant praziquantel of adjuvant (promptly as) is the 0.25-1% of vaccine complex; Wherein, The quality of preferred praziquantel adjuvant is 0.25%, 0.5%, 1%; More preferably the quality of praziquantel adjuvant is 0.5%, and described vaccine complex also can comprise pharmaceutically acceptable carrier, solvent or other auxiliary elements.
Among the present invention, the method for preparing of described vaccine complex is:
Those skilled in the art can use routine techniques that described praziquantel adjuvant and vaccine are carried out the compound vaccine complex for preparing; For example; An amount of praziquantel is dissolved in the adequate amount of ethanol; Be configured to mother solution, then mother solution added in an amount of normal saline, be configured to standard solution (can add normal saline as required again during use and adjust concentration); Vaccine with pre-prepared is dissolved in the standard solution at last, makes the vaccine complex that can directly use; Or, be prepared into dry powder formulations or injection through routine techniques (like technology such as lyophilization or cryospray dryings) behind the adding vaccine in the standard solution, in order to storage.
The invention provides described praziquantel as vaccine adjuvant be used for purposes of vaccine complex and preparation method thereof; But those skilled in the art are in the practical implementation process; Can use any known Hepatitis B virus vaccine in the prior art (like hepatitis B DNA vaccine pcD-S2, by the hepatitis B subunit vaccine of the pure antigen preparation of rHBsAg of reorganization, by influenza vaccines of inactivation of viruses or the like); In addition, also can use this area routine techniques to prepare the praziquantel adjuvant, for example; The praziquantel of 0.2 gram is dissolved in 3 milliliters the ethanol; Be configured to 6.7% mother solution, then 15 milliliters mother solution added 85 milliliters normal saline, be configured to 1.0% solution; Add normal saline at last, be configured to 0.5% and 0.25% solution.
Among the present invention; Described praziquantel can be used for preparing hepatitis B, AIDS or influenza vaccines adjuvant or vaccine complex; Described hepatitis B, AIDS or influenza vaccines are that dna vaccination, sub-unit protein or inactivation of viruses are the vaccine of antigen preparation, and described vaccine is human.
Among the present invention, the content of antigen (or vaccine) belongs to common practise or gropes and can confirm through routine in the described vaccine complex.
The present invention also confirms through test, and when the quality of the described praziquantel adjuvant praziquantel of adjuvant (promptly as) was the 0.25-1% of vaccine complex, effect was the most obvious.
Vaccine adjuvant of the present invention compared with prior art has the following advantages:
(1) the present invention utilizes praziquantel as the Hepatitis B virus vaccine adjuvant, can strengthen the immunoreation of Hepatitis B virus vaccine effectively, for the research of vaccine adjuvant provides new research direction and focus;
(2) praziquantel adjuvant of the present invention can more effectively activate the body fluid and the cellular immune level of body, has significantly strengthened immune effect;
(3) vaccine adjuvant of the present invention is easy to use, and cost is low, and side effect is little, is easy to promote;
(4) vaccine adjuvant of the present invention, the CD8 of higher activation body
+T cell responses comprises the activation of Tc1 and Tc17, for removing virus better way is provided.
For the ease of understanding, through accompanying drawing and specific embodiment vaccine adjuvant of the present invention is carried out detailed description below.What need particularly point out is, specific embodiment and accompanying drawing only are in order to explain that obviously those skilled in the art can explain according to this paper, and the present invention is carried out various corrections or change, and these corrections and changing also will be included within the scope of the invention.
Description of drawings
Fig. 1 has shown among the present invention, detects the IgG of praziquantel as the Hepatitis B virus vaccine adjuvant through the quantitative ELISA method.
Fig. 2 has shown the testing result of the T lymphocyte amplification of adjuvant praziquantel enhancing hepatitis B DNA vaccine immunity reaction among the present invention.
Fig. 3 shown among the present invention detect IFN-γ, IL-4 in CD4 T cell, IFN-γ expression of results in CD8 T cell; Wherein, express the CD4+ cell number of IL-4 or IFN-γ,
Represent 6 normal non-immune C57BL/6 mice contrasts; Numerical value among the figure is the mean+SD of 6 mices;
*Expression P＜0.05.
Fig. 4 has shown that flow cytometer detects the reacted external ctl response of adjuvant praziquantel enhance immunity among the present invention.
Fig. 5 has shown that the adjuvant praziquantel strengthens the immunoreactive IgG antibody of hepatitis B subunit vaccine detection by quantitative result among the present invention.
Fig. 6 has shown that the adjuvant praziquantel strengthens the testing result that the hepatitis B DNA vaccine immunity reacts DTH among the present invention.
Fig. 7 has shown that the adjuvant praziquantel strengthens the testing result that the hepatitis B DNA vaccine immunity reacts Tc1 and Tc17 cytoactive among the present invention.
Fig. 8 has shown that the adjuvant praziquantel strengthens the testing result in the hepatitis B surface antigen transgenic mice that the hepatitis B DNA vaccine immunity reacts Tc1 and Tc17 cytoactive among the present invention.
Fig. 9 shown among the present invention the adjuvant praziquantel strengthen the hepatitis B DNA vaccine after the immunity of hepatitis B surface antigen transgenic mice cd8 cell to the testing result of the hepatocellular scavenging action of surperficial antigen presentation.
Figure 10 has shown the testing result of passing through the quantitative ELISA method among the present invention; Wherein, A is for detecting the total IgG content of praziquantel as the aids dna vaccine adjuvant; B is for detecting the IgG2a hypotype content of praziquantel as the aids dna vaccine adjuvant, and C is for detecting the IgG1 hypotype content of praziquantel as the AIDS vaccine adjuvant.
Figure 11 has shown that the adjuvant praziquantel strengthens detection IFN-γ expression of results in cd4 t cell in the reaction of aids dna vaccine immunity among the present invention.
Figure 12 has shown that the adjuvant praziquantel strengthens detection IL-4 expression of results in cd4 t cell in the reaction of aids dna vaccine immunity among the present invention.
Figure 13 has shown that the adjuvant praziquantel strengthens the testing result of detection IFN-γ in cd8 t cell in the reaction of aids dna vaccine immunity among the present invention.
Figure 14 has shown that flow cytometer detects ctl response in the reacted body of adjuvant praziquantel enhance immunity among the present invention.
Figure 15 has shown immunity back mice cells in vivo toxic reaction and antibody response level among the present invention; Wherein, use the H5N1 inactivated vaccine separately respectively, or solvent or praziquantel immunity C57BL/6 mice; Specific cell cracking ratio among the A by three times independently experimental summary obtain; Serum among the B is collected in the 14th day after the immunity, detects antibody horizontal through ELISA, demonstration be three independently representative datas in the experiment; *, p＜0.05.ns, p＞0.05), Inactive V represents inactivated vaccine.
Figure 16 has shown the CD8 that passes through the FACS determination and analysis among the present invention
+The expression of the T cellular antigens specific cell factor; Wherein, initial immunity sub-elected CD8 after 7 days from the C57BL/6 mouse spleen
+The T cell, and stimulate with the NP peptide and to cultivate in 6 hours; To CD8
+The IFN-γ and the IL-17 of T cell carry out cell inner dyeing; Percentage ratio among A and the B is from three independently experiments; *, p＜0.01.ns, p＞0.05.
Figure 17 has shown among the present invention the intravital cell-cytotoxic reaction level of mice behind the wild type and CD8K0 or IL-17K0 immunity; Wherein, A is behind the initial immunity 7 days, wild type and CD8K0 mice is carried out CTL in the body, and the cracked ratio of specificity is summarized; B is behind the initial immunity 7 days, wild type and IL-17K0 mice is carried out CTL in the body, and the cracked ratio of specificity is summarized; * p＜0.05.ns, p＞0.05.K0 represents gene knockout.
Figure 18 has shown the infection conditions of protection high pathogenic avian influenza virus among the present invention; Wherein, A is that immune mouse carried out counteracting toxic substances in 1 month from the 7th day to 13 days the detection of body weight change behind the infective virus behind the C57BL/6 mice initial immunity; B is that immune mouse is from the 7th day to 17 days the monitoring of death condition behind the infective virus, and data come from three independently experiments, analyze 10 at every turn.
Figure 19 has shown the symptom of CD8+T cell adoptive transfer slowing down viral infection among the present invention; Wherein, mice carries out behind the vaccine immunity taking out the CD8+T cell in 7 days, and adoptive transfer is in the normal mouse body; A is a mice from the 7th day to 13 days the detection of body weight change behind the infective virus, and each mice receives 2x10
6Cell, counteracting toxic substances immediately then; B is a mice from the 7th day to 17 days the monitoring of death condition behind the infective virus, and data come from three independently experiments, analyze 10 at every turn.
The specific embodiment
The experimental technique of being mentioned among the following embodiment is conventional method if no special instructions; The percentage composition of mentioning is the quality percentage composition if no special instructions.
Embodiment 1 preparation dna vaccination
Hepatitis B DNA vaccine pcD-S2 is a template with virus among the hepatitis B patients serum, and after reverse transcription was cDNA, again through pcr amplification preS2, PCR product enzyme action was connected on the pcDNA3.0 carrier after reclaiming, and has proved expression through the eucaryon transfection experiment.
From escherichia coli, extract DNA, in the phenol chloroformic solution, remove protein, double-stranded DNA is separated through ethanol precipitation.
Said extracted method and technology are recorded in " Molecular Cloning " (second edition 1998 of people such as Sambrook; Cold Spring Harbor Laboratory Press; New York) and li chaolong etc. compile in " Biochemistry and Molecular Biology experimental technique " (publishing house of Zhejiang University), be method known in the field and technology.
The pure antigen of hepatitis B rHBsAg through expressing cho cell, obtains rHBsAg pure antigen (purity >=99%) with HPLC system purification available from North China pharmacy group, makes DNA or nucleic acid subunit vaccine.
Embodiment 3 comprises the experiment of the Hepatitis B virus vaccine complex immunity model animal of adjuvant praziquantel
Praziquantel (PZQ) crude drug is available from North China Pharmaceutical Group Company Ltd (lot number 081008);
Configuration praziquantel mother solution: the praziquantel of 0.2 gram is dissolved in 3 milliliters the ethanol; Be configured to 6.7% mother solution, then 15 milliliters mother solution added 85 milliliters normal saline, be configured to 1.0% solution; Add normal saline at last, be configured to 0.5% and 0.25% solution.
6-8 age in week, female C57BL/6 mice was divided 9 groups, and 6 every group, experiment is divided into groups as shown in table 1,
Table 1. injection is divided into groups
Every group of vaccine is dissolved in the normal saline, every injected in mice 100 microlitres, and intramuscular injection first in the 0th day, injected at interval in 14 days once more the injection back for the first time, injects altogether three times.
For the third time after the intramuscular injection 7 days the blood sampling separation of serum, the quantitative ELISA method detects IgG, IgG1 and IgG2a antibody horizontal respectively.According to anti-HBs diagnostic kit (available from Beijing Jinhao Pharmaceutical Co., Ltd., lot number S20053020) operation instruction detection by quantitative IgG, with the reference antibody 8IU/ml (IU of anti-hepatitis B; Iu) (Beijing Jinhao Pharmaceutical Co., Ltd.; Lot number 20060701) by 5 gradients of 2 times of dilutions, the back places 450nm/620nm to measure every hole OD value according to the last colour developing of explanation; Make standard curve, calculate antibody content.Detect IgG1 and IgG2a antibody horizontal respectively for the quantitative ELISA method; With 48 holes in the antigen coated 96 hole ELISA Plates of 2ug/ml rHBsAg; On same 96 hole ELISA Plate, encapsulate other 48 holes with 2ug/ml rabbit igg (Sigma), 4 ℃ are spent the night, 37 ℃ of sealings of 3% bovine serum albumin 2h; PBST (Tween 20 is dissolved in PBS, and the final concentration that makes Tween 20 is 0.05% solution that obtains) washing 4 times; Mice serum by 1: 100 times of dilution, is diluted 10 gradients from 100ng/ml by 2 times with mouse anti rabbit igg (Sigma), hatch 1h for 37 ℃; PBST washing 4 times, each 5 minutes; Add goat-anti mice horseradish peroxidase-labeled IgG1 and IgG2a antibody (Sigma) (all by dilution in 1: 4000), hatch 1h for 37 ℃.Every hole adds substrate TMB, and 37 ℃ of lucifuge colour developing 5-15min add stop buffer 2mol/L H
2SO
4, every hole 50 μ l, color development stopping places 450nm/620nm to measure every hole OD value, makes standard curve, calculates antibody content.
The result is as shown in Figure 1, and (numerical value among Fig. 1 is the mean+SD of 6 mices;
*Expression P＜0.01; MIU is a milli-international unit):
(1)
blank group, no adjuvant do not have antigenic pcDNA3 matched group, no antigenic PZQ adjuvant matched group and do not have antigenic ethanol matched group; The IgG level is minimum, shows that above combination does not possess immune effect basically;
(2) the immunizing antigen group (pcD-S2) of no adjuvant and the immunizing antigen group of ethanol adjuvant is arranged (ethanol+pcD-S2) all produces low-level IgG; This shows that immunizing antigen group (pcD-S2) has certain immune effect, and ethanol produces significantly enhancement immune effect basically;
(3) than above combination, the immunizing antigen group (pcD-S2) that contains adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce high-caliber IgG, and the effect of the antigen group of the adjuvant PZQ of 0.5% content is the most remarkable.
The above results shows that adjuvant PZQ can make Hepatitis B virus vaccine induce body to produce very high humoral immune reaction.
Embodiment 5 adjuvant praziquantel strengthen the detection of the T lymphocyte amplification of hepatitis B DNA vaccine immunity reaction
7 days execution mices after the intramuscular injection under aseptic condition, are got mouse spleen for the third time; Grind, remove erythrocyte, and cross nylon column and remove the B cell and process single cell suspension with erythrocyte cracked liquid; PBS liquid is washed 3 times, and is centrifugal and carry out cell counting, adjustment cell concentration to 1 * 10
6Individual/ml, every group of cell suspension divides 3 parts to add in 96 well culture plates; Wherein, it is 5 μ g/ml that portion adds rHBsAg antigen to final concentration, portion add ConA (concanavalin A, Con A) to final concentration be 5 μ g/ml as positive control, it is that 2 μ g/ml are as negative control to final concentration that portion adds BSA; After cultivating 48h, every hole adds the MTT (tetramethyl azo azoles salt) of 20 μ l, behind the cultivation 4h; 2,000 left the heart 5 minutes, abandoned cell conditioned medium; Add 100 μ L DMSO (dimethyl sulfoxide), 37 degree incubators were placed after 15 minutes, and ELIASA reads the OD value at 490nm place; The calculating SI (stimulated index, SI), SI=(experimental group OD-culture medium OD)/(cell OD-culture medium OD).Wherein, experimental group OD is meant the OD value that the cell of antigenic stimulus reads, and cell OD is meant the OD value that the cell without antigenic stimulus reads.
The result is as shown in Figure 2, and (among Fig. 2, ConA and BSA respectively are the mean+SD of 6 mices, and other is the mean+SD of 6 mices;
*Expression P＜0.05):
(1), considerably beyond conventional or novel antigenic stimulating effect, therefore shows the effect of the strongest stimulation T cell usually as the ConA of positive control because ConA has the effect of strong impulse T cell;
(2) negative control group of BSA,
blank group, no adjuvant do not have antigenic pcDNA3 matched group, no antigenic adjuvant PZQ matched group and do not have antigenic ethanol matched group; Basically stimulate the effect of T cell very low; Do not unite antigen if show above material as adjuvant, its effect and negative control are substantially the same;
(3) the immunizing antigen group (pcD-S2) of no adjuvant and have the immunizing antigen group (pcD-S2) of adjuvant (ethanol or PZQ) all to produce the effect of high-caliber stimulation T cell; Show that immunizing antigen group (pcD-S2) has certain immune effect, and ethanol produces significantly enhancement immune effect basically; In addition, the immunizing antigen group (pcD-S2) that contains adjuvant PZQ (0.5%, 1.0%) stimulates the effect more obvious (P＜0.05) of T cell, and the effect of the antigen group of the adjuvant PZQ of 0.5% content is the most remarkable.
Embodiment 6 flow cytometers detect the expression of adjuvant praziquantel enhance immunity reaction interior IL-4 of back cd4 cell and IFN-γ
Mice is execution in 7 days after intramuscular injection for the third time, obtains the T lymphocyte as stated above, by 10 μ g/ml rHBsAg, behind stimulation 12～14hr; The monensin effect 2hr of 2 μ L, 100 μ g/ml, PBS washes 3 times, 4 ℃ of sealings of anti-Fc gamma antibodies (eBioscience company, the U.S.) 15min; PBS washes 3 times, 4 ℃ of anti-CD4-FITC antibody (eBioscience company) dyeing 30min, and PBS washes 3 times, and 4 ℃ of 4% paraformaldehydes are 10min fixedly; PBS washes 3 times, 0.1% Saponin rupture of membranes 10min, and PBS washes 3 times; Anti-IL-4-PE or IFN-γ-FITC antibody (eBioscience company) dyeing 15min, PBS washes 3 times, and flow cytometer detects.
IFN-γ expression of results is shown in the last figure of Fig. 3:
(1)
blank group, no adjuvant do not have antigenic pcDNA3 matched group, no antigenic adjuvant PZQ matched group and do not have antigenic ethanol matched group; IFN-γ expression is very low, shows that above combination does not possess immune effect basically;
(2) the immunizing antigen group (pcD-S2) of no adjuvant and have the immunizing antigen group (pcD-S2) of ethanol adjuvant all to produce low-level IFN-γ to express; This shows that immunizing antigen group (pcD-S2) has certain immune effect, and ethanol produces significantly enhancement immune effect basically;
(3) than above combination; The immunizing antigen group (pcD-S2) that contains adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce high-caliber IFN-γ; And compare with immunizing antigen group (pcD-S2) only; The level of IIFN-γ has had significant raising, and wherein the effect of the antigen group of the adjuvant PZQ of 0.5% content is the most remarkable.
The above results shows that PZQ can be good at inducing Hepatitis B virus vaccine to produce IFN-γ.
The IL-4 expression of results is shown in the middle figure of Fig. 3:
(1)
blank group, no adjuvant do not have antigenic pcDNA3 matched group, no antigenic adjuvant PZQ matched group and do not have antigenic ethanol matched group; IFN-γ expression is very low, shows that above combination does not possess immune effect basically;
(2) containing antigen pcD-S2 group (like pcD-S2, pcD-S2+ ethanol, pcD-S2+PZQ) all shows and is superior to above combined I L-4I expression; Although antigen+0.5%PZQ does not have the expression of tangible IL-4 to rise; But antigen+ethanol does not equally have the expression of tangible IL-4 to rise yet, and shows that PZQ has the immunoreactive ability that certain promotion Hepatitis B virus vaccine produces deflection Th1 direction.
Ctl response and immune IFN-γ express closely related, are a kind of important cytokine of reflection ctl response.Mice is execution in 7 days after intramuscular injection for the third time, and 2 method obtains the T lymphocyte set by step, by 1 * 10
6Individual cell adds 10
-6The hepatitis B S antigen small peptide (aa208～215) of mol (the biochemical company limited of Shanghai gill, article No. is 52633), behind stimulation 12～14hr, the monensin effect 2hr of 2 μ L, 100 μ g/ml; PBS washes 3 times, 4 ℃ of sealings of anti-Fc gamma antibodies (eBioscience company, the U.S.) 15min, and PBS washes 3 times; 4 ℃ of anti-CD8-PE antibody (eBioscience company, the U.S.) dyeing 30min, PBS washes 3 times, and 4 ℃ of 4% paraformaldehydes are 10min fixedly; PBS washes 3 times, 0.1% Saponin rupture of membranes 10min, and PBS washes 3 times, anti-IFN-γ-FITC antibody (eBioscience company; The U.S.) dyeing 15min, PBS washes 3 times, and flow cytometer detects.
(vertical coordinate is for expressing the CD8+ cell number of IFN-γ shown in figure below of Fig. 3 for the result;
Represent 6 normal non-immune C57BL/6 mice contrasts; Numerical value among the figure is the mean+SD of 6 mices;
*Expression P＜0.05):
(1)
blank group, no adjuvant do not have antigenic pcDNA3 matched group and do not have antigenic ethanol matched group; IFN-γ expression is very low and level is basic identical, shows that above combination does not possess immune effect basically;
(2) the immunizing antigen group (pcD-S2) of no antigenic adjuvant PZQ contrast, no adjuvant and the IFN-γ expression that has the immunizing antigen group (pcD-S2) of ethanol adjuvant all to produce higher level; Wherein, PZQ matched group level is the highest;, showing the immunizing antigen group (pcD-S2) that the effect of adjuvant PZQ group has been superior to not having the immunizing antigen group (pcD-S2) of adjuvant PZQ and the ethanol adjuvant is arranged, while ethanol does not produce any effect;
(3) than above combination; The immunizing antigen group (pcD-S2) that contains adjuvant PZQ (0.25%, 0.5%, 1.0%) can produce higher levels of IFN-γ; And compare with immunizing antigen group (pcD-S2) only; The level of IIFN-γ has had significant raising, and wherein, the effect of the antigen group of the adjuvant PZQ of 0.5% content is the most remarkable.
Normal non-immune 5-7 C57BL/6 mice in age in week is condemned to death, and obtains the T lymphocyte as stated above, after T lymphocyte halves that separation is obtained; Wherein, portion hatches 10
-6M hepatitis B S antigen small peptide (aa208～215) (the biochemical company limited of Shanghai gill, article No. is 52633), another part are hatched irrelevant peptide OVA (aa323～339) (the biochemical company limited of Shanghai gill of equal quantities; Article No. is P01662); The volume of every ware is 1-2mL, 37 ℃, and 5%CO
2Cultivate 4 hours (what of experimental group this step can suitably increase the target cell numbers according to); After 4 hours cell is changed in the cell pipe of 15mL; Centrifugal 5 minutes of 2000rmp; To hatch CFSE (0.5uM) dyeing of the target cell of OVA (aa 257-264) with low concentration, and hatch CFSE (5uM) dyeing of hepatitis B S antigen small peptide target cell with high concentration, 37 ℃ of lucifuge jogs dyeed 15 minutes.Dyeing back is with isopyknic hyclone cessation reaction, and centrifugal 5 minutes of 2000rmp abandons supernatant, washes 3 times with the PBS of 10mL; With low concentration and the isopyknic mixing of the painted target cell of high concentration, by every mice 2 * 10
7Individual cell carries out the reaction of cells in vivo poison by in above-mentioned each immune group mice of tail vein injection and the normal mouse body; Injected back 4 hours, and put to death experiment mice, the lucifuge separation obtains splenocyte, and copper mesh changes in the FACS dedicated pipe after filtering, and prepares to carry out instrument detecting and analysis.Kill rate (%)=[1-(percent of the specific killing specific lysis/non-specific percent that kills and wounds)] * 100.
Result's (percentage ratio that expression kills and wounds among the figure as shown in Figure 4; 6 normal non-immune C57BL/6 mice contrasts of
expression, numerical value is the meansigma methods of 6 mices):
(1)
blank group, no adjuvant do not have antigenic pcDNA3 matched group, no antigenic adjuvant PZQ matched group and do not have antigenic ethanol matched group; Ctl response is on close level low; Wherein,
blank group, no antigenic ethanol matched group approach 0;, show the effect that above combination does not possess basically stimulates ctl response;
(2) experimental group (like pcD-S2, pcD-S2+ ethanol, pcD-S2+PZQ) that contains antigen pcD-S2 all shows the level of the stimulation ctl response that significantly is superior to above combination, shows that antigen pcD-S2 plays a major role in the level that stimulates ctl response;
(3) antigen pcD-S2 plays a major role in the level that stimulates ctl response; But added in the test group of adjuvant PZQ; It stimulates the ability of ctl response level still obviously to be better than pcD-S2, pcD-S2+ ethanol group, and wherein, 0.5%PZQ can produce the ctl response of top level.
The above results shows; Adjuvant PZQ can promote immunizing antigen group (pcD-S2) to produce higher levels of ctl response; Wherein, antigen adds the ctl response that 0.5%PZQ can produce higher level, and can to excite hepatitis B nucleic acid vaccine to produce more CD8+IFN-γ consistent with PZQ.
6-8 age in week, female C57BL/6 mice was divided 6 groups, and 6 every group, experiment is divided into groups as shown in table 2,
Table 2 injection is divided into groups
Every group of vaccine is dissolved in the normal saline, every injected in mice 100 microlitres, and intramuscular injection first in the 0th day, injected at interval in 14 days once more the injection back for the first time, injects altogether twice.
For the third time after the intramuscular injection 7 days the blood sampling separation of serum, the quantitative ELISA method detects the IgG antibody horizontal respectively.According to anti-HBs diagnostic kit (Beijing Jinhao Pharmaceutical Co., Ltd., lot number S20053020) operation instruction detection by quantitative IgG, with the reference antibody 8IU/ml (IU of anti-hepatitis B; Iu) (Beijing Jinhao Pharmaceutical Co., Ltd.; Lot number 20060701) by 5 gradients of 2 times of dilutions, the back places 450nm/620nm to measure every hole OD value according to the last colour developing of explanation; Make standard curve, calculate antibody content.
The result is as shown in Figure 5, and (numerical value is the mean+SD of 6 mices;
*Expression P＜0.05):
(1)
blank group, no antigenic ethanol matched group, no antigenic adjuvant PZQ matched group; The IgG antibody response is on close level low, shows the effect that single adjuvant (ethanol or PZQ) does not possess basically stimulates the IgG antibody response;
(2) experimental group (like rHBsAG, rHBsAG+ ethanol, rHBsAG+PZQ) that contains antigen rHBsAG all shows the level of the stimulation ctl response that significantly is superior to combinations thereof, shows that antigen rHBsAG plays a major role in the level that stimulates the IgG reaction;
(3) antigen rHBsAG plays a major role in the level that stimulates the IgG reaction, but has added in the test group of adjuvant PZQ, and it stimulates the ability of IgG reaction level obviously to be better than rHBsAG, rHBsAG+ ethanol group, shows that ethanol does not have the effect of any promotion immunity.
The above results shows that immunizing antigen group (rHBsAg) produces low-level IgG, and ethanol does not have the effect of any promotion immunity, and antigen adds IgG (P＜0.05=that 0.5%PZQ can produce higher level.
The adjuvant praziquantel is prepared in laboratory and purification; 100ug interleukin-17 A is dissolved in the PBS buffer of pH7.0 of 10ml 10mM, obtains the solution of 10ug/ml.AIDS Env protein-specific neutralizing antibody epitope peptide mB1 (ENFDMWKNDM), mB2 (QKVYALFYRLD), mB3 (PNNNTRKSIRIGPGQTFYAT), specific CTL epitope peptide mCTL1 (WYIKIFIMI), mCTL2 (RYLKDQQLL), mCTL3/Th (TSAITQACPKVSFDPIPIHYCAPAG) epitope peptide are given birth to the biochemical company limited of worker by Shanghai and are synthesized.
AIDS nucleic acid vaccine pGX-Envc source and preparation:
The preparation of dna vaccination and DNA: dna vaccination pGX-Env plasmid is presented by Dr.David professor Weiner of Univ Pennsylvania USA; Change DNA among DH5 α fermentation culture, adopt extensive alkaline lysis method of extracting, Qiagen company purification column plasmid DNA purification, and with normal saline quality of regulation concentration to 1mg/ml in-20 ℃ of preservations.Animal divides into groups and immunization method: the Balb/c mice is divided into 6 groups at random; Every group 10, the 1st group
(blank group); The 2nd group of immunity 30 μ g pGX-Envc; The 3rd group of immunity 30 μ g pGX-Env+EP (electric shock appearance method); The 4th group of immunity 30ug pGX-Env+PZQ+EP; The 5th group of immunity 30ug pGX-Env+CIM adjuvant+EP; The 6th group of immunity 30ugpGX-Env+LMS+EP.Immunity employing intramuscular injection adds 70 volts of methods in mice back leg quadriceps femoris of electric shock appearance and carries out immunity; Each organizes mice all immune 3 times, and each 14 days at interval, 7 days after the immunity were put to death mice and detected each item amynologic index for the third time,
(1)?
(2)pGX-ENVC；
(3)pGX-ENVC+E.P；
(4)pGX-ENVC+PZQ+E.P；
(5)pGX-ENVC+CIM+E.P；
(6)pGX-ENVC+LMS+E.P。
Embodiment 12 adjuvant praziquantel strengthen IgG, IgG1 and the IgG2a antibody detection by quantitative of aids dna vaccine immunity reaction
7 days blood sampling separation of serum after the intramuscular injection by 3 gradients of 5 times of dilutions, detect IgG, IgG1 and IgG2a antibody horizontal respectively in the ELISA method for the third time; According to last colour developing, place 450nm/620nm to measure every hole OD value then, calculate the antibody titer curve, be specially with 2ug/ml HIV-1ENV polypeptide be in the antigen coated 96 hole ELISA Plates 4 ℃ spend the night 37 ℃ of sealings of 3% bovine serum albumin 2h; PBST (Tween 20 is dissolved in PBS, and the final concentration that makes Tween 20 is 0.05% solution that obtains) washing 4 times; By 1: 10,1: 50,1: 250 times of dilution was hatched 1h for 37 ℃ with mice serum; PBST washing 4 times, each 5 minutes; Add goat-anti mice horseradish peroxidase-labeled total IgG, IgG1 and IgG2a antibody (Sigma) (all by dilution in 1: 4000) are hatched 1h for 37 ℃; Every hole adds substrate TMB, and 37 ℃ of lucifuge colour developing 5-15min add stop buffer 2mol/L H
2SO
4, every hole 50 μ l, color development stopping places 450nm/620nm to measure every hole OD value, makes standard curve, calculates the antibody titer curve.
Result's (numerical value among the figure is the meansigma methods of 6 mices) shown in figure 10:
(1)
blank group antibody horizontal is minimum, shows that combinations thereof does not possess immune effect basically;
(2) the dna vaccination immunity of the dna vaccination immune group (pGX-ENVC) of no adjuvant and no adjuvant adds electric shock appearance method group (pGX-ENVC+EP); And dna vaccination has immunizing antigen group (pGX-ENVC+CIM+EP) that the CIM adjuvant adds electric shock appearance method and dna vaccination to have the LMS adjuvant to add to shock by electricity the immunizing antigen group (pGX-ENVC+LMS+EP) of appearance method all to produce low-level IgG; IgG2a and IgG1 show that immune dna vaccination has certain immune effect, but other adjuvant group produces significantly enhancement immune effect basically;
(3) than combinations thereof, the immunizing antigen group (pGX-ENVC+LMS+EP) that contains adjuvant PZQ can produce high-caliber IgG, and the effect of the antigen group of the adjuvant PZQ of 0.5% content is the most remarkable.
The above results shows that adjuvant PZQ can make the vaccine-induced body of aids dna produce very high humoral immune reaction.
Flow cytometer detects the cytokine-expressing level
Back 7 days of the 3rd immunity, separating Morr. cell under the aseptic condition is removed the B cell through nylon column and is processed single cell suspension, adjustment cell concentration to 5 * 10
6Individual/mL, every hole adds 100 μ L cell suspension to 96 porocyte culture plates, and adding the every hole of HIV-1 type epitope peptide final concentration is 5 μ g/mL, and every group of cell established 3 repeating holes.37 ℃, 5%CO
2, cultivate 8h, after the adding monensin is cultivated 2h again, centrifugal collecting cell; Anti-Fc gamma antibodies sealing, 4% paraformaldehyde is 12min fixedly, 0.1% Saponin rupture of membranes 7min; PBS washes 2 times, with 4 ℃ of dark place dyeing of 10 μ L fluorescent monoclonal antibodies 30min, gets 300 μ L flow cytometers and detects.FlowJo software is analyzed.
Shown in Figure 11～13, CD4
+And CD8
+The result of T emiocytosis IL-4 and IFN-γ shows, PZQ reaches as adjuvant group and other adjuvant group and do not add adjuvant group comparing difference statistical significance (P＜0.05) is arranged, and explains that PZQ can obviously promote CD4 and CD8 as the adjuvant of dna vaccination
+The activation of T emiocytosis IL-4 and IFN-γ and promotion T cell, thus cellular immune level strengthened.
CTL detects in the body
Back 8 days of the 3rd immunity, put to death the blank mice of end through immunity, separate obtaining splenocyte; Process single splenocyte suspension, be divided into halves as being target cell, portion adds 10mol/L Env specific CTL epitope polypeptide pond (mCTL1+mCTL2+mCTL3) to stimulate; Portion does not stimulate, and 37 ℃, 5%CO
2, cultivate 4h, the centrifugal supernatant of abandoning; PBS washes 1 time, and the target cell that the end stimulates is with 0.5 μ mol/L CFSE dark place dyeing 10min, and the target cell of stimulation is with 5 μ mol/LCFSE dark places dyeing 10min; Add the equal-volume calf serum and stop dyeing 2min, the centrifugal supernatant of abandoning, PBS are washed 5 times; At last with two kinds of isopyknic mixing of target cell, with 1 * 10
7Individual target cell tail vein injection carries out cells in vivo poisoning traumatic response in the immune mouse body, put to death mice after killing and wounding 4h, and the dark place is separated and obtained splenocyte, gets the check and analysis of 1mL flow cytometer.
Result of the test is shown in figure 14, and PZQ is as adjuvant group and other adjuvant and not add the adjuvant group more variant, explain that the intensive CTL of generation kills and wounds reaction to PZQ in the body for the aids dna vaccine adjuvant can be induced.
1, materials and methods
(1) reagent and laboratory animal
Praziquantel (North China pharmacy, Hebei, China) is made into 6.7% solution with dissolve with ethanol, is diluted to 0.5% with normal saline subsequently.Group of solvents is that 7.5% ethanol is dissolved in the normal saline.The fluorescently-labeled monoclonal antibody of all anti-mices that is used for the fluidic cell detection is all available from BD Pharmingen company (Santiago, California, the U.S.).NP366-374 peptide as the influenza virus nucleoprotein (NP) of CD8+T cell epitope (ASNENMETM) representative is synthetic by the biochemical company limited of gill.The C57BL/6 female mice, age in 8-10 week is available from Institute of Experimental Animals, Chinese Academy of Medical Sciences (Beijing, China).All laboratory animals all under 12 hours periodicity of illumination conditions, the feedstuff and the drinking water raising of polluting with no cause of disease.
(2) vaccine
(HPAIV, H5N1 A/Chicken/Henan/1/04) deposit in 3 grades of laboratorys of biosafety level (China Agricultural University, Beijing, China) to high pathogenic avian influenza virus; The Embryo Gallus domesticus virus of proliferation, and carry out the whole virus vaccine production of formalin-inactivated; The protein content of this vaccine is measured by Pierre Si BCA protein detection kit (Rockford, Illinois, the U.S.), and the H1N1 vaccine is provided by the big Hua Nong in Guangdong.
(3) immunization method
The C57BL/6 mice is divided into 5 groups at random, 10 every group.With the independent immune mouse of 0.1 μ g inactivated vaccine, dosage inactivated vaccine and adjuvant combined immunization group, intramuscular injection such as set up simultaneously; Every dosage volume for injections is 100 μ l.
(4) counteracting toxic substances
Single dose drips infecting mouse H5N1 influenza virus with nose after 4 weeks of immunity, and the counteracting toxic substances amount is the lethal dose (10LD50) of mice adapted strain A/Chicken/Henan/1/04 (H5N1), and dosage is the viral suspension of 20 μ l; All of observing that this infection causes are the death condition of immune mouse in 7-12 days not.
(5) CTL detects in the body
At immune back 7 days for the third time, put to death the end through the blank mice of the C57BL/6 of immunity, separate obtaining splenocyte, process single splenocyte suspension, be divided into halves, portion adds 10
-6M NP antigenic peptides stimulates and with CFSE (15 μ M, the CFSE of high concentration
HighCells) dark place dyeing; Another part stimulates without antigenic peptides, with CFSE (0.5 μ M, the CFSE of low concentration
LowCells) dyeing is as non-specific target cell matched group.With two kinds of isopyknic mixing of target cell, by every mice 2 * 10
7Individual cell by tail vein injection in the experiment mice body; Put to death mice behind the 4H, separate to obtain lymph node and splenocyte, different through target cell and cellular control unit CFSE fluorescence intensity can use flow cytometer FACSCalibur (BD company, the U.S.) to analyze.
The computing formula of specificity cracking intensity level is following: ratio=CFSE
Low%/CFSE
High%;
Specificity cracking percentage ratio=[1-(not by antigenic peptides stimulating group ratio/antigenic peptides stimulating group ratio) * 100].
(6) detection of anti-H5N1 avian influenza virus antibody level
Detect the antibody horizontal of anti-H5N1 bird flu virus in the serum through ELISA.
Behind initial immunity 14 days, gather serum; Elisa is carried out on one 96 hole polystyrene microwell plate, and reagent comprises the inactivated vaccine of H5N1 virus etc.; With the sealing of 3% bovine serum albumin after 1 hour, the mice serum that every hole adds dilution on this plate is hatched; Then, 1000 times sheep anti-mouse igg antibody (Sigma company, St. Louis, the Missouri State) has been diluted in adding; Behind 0.025M phosphoric acid-citrate buffer solution 10 milligrams of TMB of dissolving (Sigma company, St. Louis, the Missouri State), add in each hole and develop the color; H with 2M
2SO
4Chromogenic reaction is at 450nm/620nm place photometry density value; The OD value of experimental port is thought the positive during for the twice of control wells.
(7) analyzing and testing of flow cytometer
After the immunity the 7th day sub-elects the CD8+T cell that mouse boosting cell is originated with separating magnetic bead (R&D company, Heng Tingdeng Valley, Pennsylvania, the U.S.) for the third time; On 96 orifice plates, every hole concentration is 0.5 * 10
6The CD8+T cell of individual/20 μ l stimulates with NP antigenic peptides (5 μ g/ml) and anti--CD28 (5 μ g/ml) monoclonal antibody cultivates 6H, 37 ℃, 5%CO
2Last 4 hours, add monensin (2 μ g/ml) and handle, wash cell three times with PBS/10%FCS then.In PBS, 4 ℃, with Fc receptor antibody (BD company; Santiago; The U.S.) closing cell is 30 minutes, adds 4% paraformaldehyde then and changes cell thoroughly with Saponin, uses immunohistochemical assay to carry out the homotype contrast; Or carry out two dying---as with anti-CD8-FITC and anti-IFN-γ-PE, or anti-CD8-FITC and anti-IL-17-PE dyeed 1 hour at 4 ℃.
Detect cell with flow cytometer FACScalibur, and use software CellQuest Pro Software (BD bioscience) to analyze.
(8) statistical analysis
The result adopts the t method of inspection to carry out data analysis with ± S.E.M statistical representation; There is statistical significance P＜0.05 for difference.
2, experimental result
(1) praziquantel is induced inactivated vaccine and is produced high-level cell-cytotoxic reaction
Because the ctl response of virus-specific is absolutely necessary for the infection of control virus, whether can induce the specific cytotoxicity lethal effect of the high-caliber nucleoprotein of generation with the praziquantel combined immunization so at first detect inactivated vaccine.
Mice after first immunisation 7 days carries out immunity and cells in vivo toxicity test once more.Than independent with inactivated vaccine immunity and group of solvents; Experimental group with inactivated vaccine and praziquantel combined immunization shows higher levels of antigenic specificity lysis reaction (shown in figure 15); The result shows that praziquantel can strengthen the cytotoxicity that inactivated vaccine is induced generation.
On the other hand, to induce the antibody horizontal of generation that mice is resisted virus also most important for inactivated vaccine.Back 14 days of immunity, whether the antibody titer of detection serum also can influence the inductive HI of inactivated vaccine to judge praziquantel.(shown in Figure 15 B), than independent with inactivated vaccine immunity and solvent experimental group, the antibody horizontal no significant difference of inactivated vaccine and praziquantel combined immunization group.
(2) praziquantel and H5N1 combined immunization can strengthen the Tc17 cell activity
Because the CD8 of secretion of gamma-IFN
+The CD8 of T cell (Tc1) and generation IL-17
+T cell (Tc17) all has cytotoxic activity, so need to confirm that which T cell subsets plays significant feature therein.Behind initial immunity 7 days, sub-elect the CD8+T cell, the laggard hand-manipulating of needle of stimulated in vitro is to the cell inner dyeing of IFN-γ and IL-17.
Shown in figure 16, in praziquantel and the inactivated vaccine combined immunization group by the CD8 of antigen induction
+The ability of T emiocytosis IL-17 significantly increases, and its secretion of gamma-IFN level is not significantly improved, and shows that Tc17 induces body to produce the key of high-level cytotoxic effect.
(3) knock out the enhanced level that CD8 or IL-17 can weaken cell-cytotoxic reaction
Further checking Tc17 is most important in the inducing cytotoxic mechanism; At first use inactivated vaccine and praziquantel combined immunization CD8 gene knockout (KO) mice and wild type C57BL/6 mice, detect the specific cytotoxicity dissolution of nucleoprotein.Shown in Figure 17 A, the forfeiture of CD8 gene can weaken cytotoxicity, shows that the cytotoxicity in this system is mainly cell-mediated by CD8+T.
Then, with inactivated vaccine and praziquantel combined immunization interleukin-17 gene knockout (KO) mice and wild type C57BL/6 mice, carry out CTL detection in the body; Compare with wild-type mice, after the immunity, the intravital cytotoxicity ability of IL-17KO mice (like Figure 17 B) obviously descends, and the effective expression that shows IL-17 in the CD8+T cell is that the enhancing body cytotoxicity is necessary.
(4) protection of infecting to high pathogenic avian influenza virus
Confirm to use inactivated vaccine and praziquantel combined immunization whether can enhancing body to the cause death resistance of infection of high pathogenic avian influenza.In four week behind the initial immunity, mice is carried out counteracting toxic substances; In behind the counteracting toxic substances 7 days, observe the mice body weight change, the result is shown in Figure 18 B, and in negative control group and the praziquantel experimental group, the body weight of all mices nearly all descends 30%; Than independent immunity of inactivated vaccine or solvent experimental group, the minimizing degree of mice body weight is obviously controlled in inactivated vaccine and the praziquantel combined immunization group.
Shown in Figure 18 A, the survival of the experiment mice of inactivated vaccine and praziquantel simultaneous inoculation is obviously longer, shows that high-caliber cytotoxicity has effectively stoped the infection of H5N1 virus.
Mice in negative control group and the praziquantel group is just all dead in the 12d behind counteracting toxic substances.
(5) the Tc17 cell is participated in the prevention of body to H5N1 type viral infection
With inactivated vaccine and praziquantel combined immunization IL-17KO and wild type C57BL/6 mice, behind initial immunity 7 days, the CD8+T cell that sub-elects.Cell purification is 96-98% to purity, before counteracting toxic substances, by whenever receiving only mice 2 * 10
6Individual cell is through the vein adoptive transfer.
Shown in Figure 19 A, if the experimental group adoptive transfer comes the CD8+T cell for immune wild-type mice, the weight loss degree that then wherein receives mice is obviously low; What shift be immune IL-17KO or the CD8+T cell of IFN-γ KO mice, weakens the reduction of mice body weight to a certain extent, but with respect to the CD8+T cell of wild-type mice, the degree that weakens is limited.
Shown in Figure 19 B; Behind the counteracting toxic substances; Adoptive transfer comes in the experimental group of immune wild-type mice CD8+T cell, and four one-tenth reception mices survive at last, receives all finally death of experiment mice of the IL-17KO mice CD8+T cell of immunity; And IFN-γ KO mice CD8+T cell has the certain protection effect, shows that the prevention and control of the cytotoxic response reaction pair H5N1 type viral infection that Tc17 mediates are most important.
Above result of the test shows; Praziquantel of the present invention can strengthen the immunoreation of Hepatitis B virus vaccine effectively as the Hepatitis B virus vaccine adjuvant, and it is compared with existing adjuvant; Can more effectively activate the body fluid and the cellular immune level of body, significantly strengthen immune effect; Simultaneously, vaccine adjuvant of the present invention, the CD8 of activation body that can be higher
+T cell responses comprises the activation of Tc1 and Tc17, for removing virus better way is provided, and easy to use, cost is low, side effect is little, be easy to promote.
Claims (11)
1. a vaccine adjuvant is characterized in that, described vaccine adjuvant is Hepatitis B virus vaccine adjuvant, AIDS vaccine adjuvant or influenza vaccines adjuvant, and described vaccine adjuvant adopts the praziquantel preparation of formula (I) structure; Its chemical name: 2-cyclohexyl formoxyl-1,2,3,6; 7,11b-six hydrogen-4H-pyrazine is [2,1-α] isoquinolin-4-ketone also; Molecular formula: C19H24N2O2, molecular weight: 312.41
2. the vaccine adjuvant of claim 1 is in the purposes of preparation in the antiviral vaccine.
3. by the described purposes of claim 2, it is characterized in that described antiviral vaccine is Hepatitis B virus vaccine, AIDS vaccine or influenza vaccines.
4. by the described purposes of claim 2, it is characterized in that described vaccine is dna vaccination, nucleic acid vaccine or sub-unit protein vaccine.
5. by the described purposes of claim 3, it is characterized in that described Hepatitis B virus vaccine is hepatitis B DNA vaccine, hepatitis B subunit vaccine; Described AIDS vaccine is the aids dna vaccine; Described influenza vaccines are inactivated influenza virus vaccines.
6. by arbitrary described purposes among the claim 3-5, it is characterized in that described vaccine is the human vaccine.
7. the vaccine adjuvant of claim 1 prevents the purposes in hepatitis B, AIDS or the grippal vaccine complex in preparation.
8. the purposes of the vaccine adjuvant of claim 1 in the vaccine complex of preparation treatment hepatitis B or AIDS.
9. by claim 7 or 8 described purposes, it is characterized in that in the described vaccine complex, the quality of adjuvant praziquantel is the 0.25-1% of vaccine complex.
10. by claim 7 or 8 described purposes, it is characterized in that in the described vaccine complex, the quality of adjuvant praziquantel is 0.5% of a vaccine complex.
11. the method for preparing of the vaccine adjuvant of claim 1; It is characterized in that it comprises step: the praziquantel of 0.2 gram is dissolved in 3 milliliters the ethanol, is configured to 6.7% mother solution; The normal saline that then 15 milliliters mother solution is added 85 milliliters; Be configured to 1.0% solution, add normal saline at last, be configured to 0.5% and 0.25% solution.
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| CN104984334B (en) * | 2015-06-26 | 2018-06-26 | 金宇保灵生物药品有限公司 | A kind of rabies Attenuate vaccine-praziquantel complexing agent and preparation method and application |
| CN111494620A (en) * | 2020-05-05 | 2020-08-07 | 华中科技大学同济医学院附属协和医院 | Application of trametinib in preparation of vaccine |
| CN116283976A (en) * | 2023-04-04 | 2023-06-23 | 佛山职业技术学院 | Rapid detection device for praziquantel in animal tissue sample, preparation and application thereof |
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