CN102397538A - Application of viral vaccine prepared by using methylene blue photochemical virus inactivating method - Google Patents
Application of viral vaccine prepared by using methylene blue photochemical virus inactivating method Download PDFInfo
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- CN102397538A CN102397538A CN2011103322465A CN201110332246A CN102397538A CN 102397538 A CN102397538 A CN 102397538A CN 2011103322465 A CN2011103322465 A CN 2011103322465A CN 201110332246 A CN201110332246 A CN 201110332246A CN 102397538 A CN102397538 A CN 102397538A
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Abstract
The invention discloses application of a viral vaccine prepared by using a methylene blue photochemical virus inactivating method to preparation of a preparation for inducing the immune reaction of Th1 type cells and stimulating the cell factor secretion of lymphocytes. The invention has the beneficial effects: the viral vaccine can be used for effectively regulating the generation of cell factors with high immune regulating activities in virus resistance, and the content of tumor necrosis factors and interferon inside a patient is increased; and moreover, antigen substances for preparing the viral vaccine come from the inside of the patient, so that the viral vaccine disclosed by the invention has the advantages of specificity and pertinence specific to different patients, and is superior to the conventional vaccine consisting of an immobilized antigen and an antibody.
Description
Technical field
The present invention relates to the vaccine field, be specifically related to the application of the viral vaccine of methylene blue photochemical virus ablation method preparation.
Background technology
The disease that viral infection causes, for example hepatitis B, hepatitis C, AIDS etc. are the healthy important killers of harm humans always, the expense that is used to treat every year is up to trillion yuan.But depend on many functions of host cell during owing to viral strict cytozoicus characteristic and virus replication, make that have safe, special, the effective antiviral drugs that can suppress virus replication and not influence cell function lacks very much.Existing research shows that specificity cellular immunity response plays a significant role in removing viral infection.Therefore specific cellular immunity becomes a kind of Critical policies of treatment viral infection.
Therapeutic vaccine is through with the antigenic component of pathogen or respective components inoculation body, thereby excites, strengthens and regulate and control the former specific immune response of body disease-resistant, and then reaches the purpose of treatment.The research of exploitation therapeutic vaccine will have important in theory meaning and potential huge economy and social benefit, be subjected to worldwide extensive concern.The transformation antigen of prior art, set up new immunogen, change the antigen presentation and the course of processing, induce the research of the therapeutic vaccine of life body efficient immune, in antiviral therapy, enjoy high praise.But vaccine has just had fixed epitope from producing beginning; One of virus persistent infection mechanism is the genovariation strain that its neutrality antigenic determinant of coding occurs; Inductive body fluid of original virus or cellular immunization because variant can be escaped; Therefore the variant of virus can not be eliminated, thereby makes virus continue to exist, and causes can't effecting a radical cure of disease.
Summary of the invention
The objective of the invention is to, the viral vaccine that the preparation of a kind of methylene blue photochemical virus ablation method is provided is induced Th1 type cell immune response and is stimulated the application in the preparation of lymphocytic emiocytosis cytokine in preparation.
More excellent, said cytokine is tumor necrosis factor (TNF-α) and interferon (IFN-γ), viral vaccine of the present invention can stimulate the lymphocytic emiocytosis cytokine, improves the content of interior TNF-α of patient's body and IFN-γ.
More excellent, said viral vaccine is to obtain after adopting the deactivation of methylene blue photochemical virus ablation method to contain viral blood plasma.
More excellent, the said blood plasma that contains virus derives from the patient.
More excellent, said virus is selected from multiple RNA and DNA viruses such as hepatitis B virus, hepatitis C virus, human immunodeficiency virus type 1, HIV's 2 types, cytomegalovirus, herpesvirus or T visceral leukosis virus.
More excellent, the method for using of viral vaccine of the present invention is selected from following arbitrary:
Method one: use the blood plasma list and adopt appearance, separated plasma also carries out methylene blue photochemical virus inactivation treatment to blood plasma, the blood plasma after handling fed back to the patient, and be a course of treatment.More excellent, process of this method can be carried out deactivation to the blood plasma in whole blood circulations, handle methylene blue in the blood plasma can filtering also can not filtering.Can carry out deactivation once more in different time points according to actual needs, to consolidate, to strengthen therapeutic effect.
Method two: 1) separated plasma 200～400ml first, and carry out methylene blue photochemical virus inactivation treatment, inactivation treatment finishes after-filtration and removes methylene blue, freezing preservation; 2) separated plasma once more after 1～4 week, isolating plasma volume be last time 1～2 times of the separated plasma amount, the processing method of blood plasma feeds back the blood plasma of preserving after the viral inactivation treatment last time with isolating blood plasma method is identical first simultaneously; 3) repeating step 2), be 2800～3200ml until the displacement plasma volume, promptly whole plasma volumes are a course of treatment.
More excellent, the freezing storage temperature of method two is-20 ℃.
More excellent; Said methylene blue photochemical virus ablation method is: in 5～1000ml contains blood plasma or the solution of virus, adding methylene blue and adjust final concentration is 1.0～100 μ mol/L; Under the air-proof condition, 4～37 ℃ of concussions, 30000～35000Lux radiation of visible light is handled 30～45min.
Viral vaccine of the present invention contains the blood plasma of virus through the deactivation of methylene blue photochemical virus ablation method, and the blood plasma that contains virus will add the anticoagulant of common dose before carrying out the deactivation of aforementioned methylene blue photochemical virus, prevents solidifying of blood in the inactivation process.
The antigenic substance of viral vaccine of the present invention derives from the intravital virus of patient, and therefore vaccine of the present invention has specificity and the specific aim that is directed to different patients, and this is better than the vaccine that is made up of immobilized antigen, antibody.After vaccine is prepared by methylene blue photochemistry ablation method, have antigenicity and immunogenicity, but the effective stimulus cellullar immunologic response promotes and the generation of the closely-related Th1 cytokines of antivirus action that good antiviral application prospect is arranged.Methylene blue photochemical virus inactivation technology can all have deactivation to multiple DNA and RNA viruses.Therefore can be used for hepatitis B virus (HBV), hepatitis C virus (HCV), HIV (HIV), cytomegalovirus (HCMV), people by the vaccine of photochemistry ablation methods such as methylene blue preparation and have a liking for T cell virus several diseases viral disease patients' such as (HTLV) treatment.
Beneficial effect of the present invention is: methylene blue photochemistry inactivation technology is widely used in a lot of blood stations; This method is simple to operate, safe, do not need expensive medicine, if combine the Plasma Pheresis/Apheresis Plasma technology, can once realize the inactivation of virus in whole peripheral bloods; Can produce the virus that is inactivated in a large number; And this operation can be carried out repeatedly, constantly strengthens, consolidates immune effect, can be to different Strain in each patient's body in the clinical practice; Performance specific immunity regulating action has good antiviral application prospect.
Description of drawings
Fig. 1: the variation of hepatitis B virus surface antigen content
Fig. 2: to the influence of costimulatory molecules mRNA horizontal expression
Fig. 3: the influence of adhesion molecule mRNA horizontal expression between pair cell
Fig. 4: to the influence of lymphocyte TNF-α secretion level
Fig. 5: to the influence of lymphocyte IFN-γ secretion level
Fig. 6: to the influence of lymphocyte IL-2 secretion level
Fig. 7: to the influence of lymphocyte IL-4 secretion level
Fig. 8: to the influence of lymphocyte IL-10 secretion level
The specific embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that following examples only are used to the present invention is described and are not used in restriction protection scope of the present invention.
The preparation of embodiment 1 viral vaccine
(1) in the blood plasma 1000ml that contains hepatitis B virus, add methylene blue and to adjust final concentration be 50 μ mol/L, under the air-proof condition, 4 ℃ of concussions with 32000Lux radiation of visible light processing 45min, prepare viral vaccine.
(2) in the blood plasma 500ml that contains hepatitis B virus, add methylene blue and to adjust final concentration be 1.0 μ mol/L, under the air-proof condition, 37 ℃ of concussions with 30000Lux radiation of visible light processing 30min, prepare viral vaccine.
(3) in the blood plasma 5m that contains hepatitis B virus, add methylene blue and to adjust final concentration be 100 μ mol/L, under the air-proof condition, 20 ℃ of concussions with 35000Lux radiation of visible light processing 35min, are prepared into viral vaccine.
(4) in the blood plasma 300ml that contains hepatitis C virus, adding methylene blue adjustment final concentration is 60.0 μ mol/L, and under the air-proof condition, 4 ℃ of concussions with 31000Lux radiation of visible light 30min, prepare viral vaccine.
(5) in the viral blood plasma 600ml that contains human immunodeficiency virus type 1, adding methylene blue adjustment final concentration is 40 μ mol/L, and the following 37 ℃ of concussions of air-proof condition with 35000Lux radiation of visible light 32min, are prepared into viral vaccine.
The antigenicity experiment of embodiment 2 viral vaccines
1. according to embodiment 1, experiment (1) method prepares the Hepatitis B virus vaccine of methylene blue photochemistry inactivation treatment.
2. detect the antigenicity of the Hepatitis B virus vaccine of preparation, experimental technique is following:
1) preparation lymphocyte: 6 parts of healthy blood donors' after the picked at random collection in the 6h ACD whole blood, the preparation White Blood Cells Concentrate is used the Ficoll-Hypaque density with reference to product operation handbook, isolated lymphocytes.With PBS (containing 0.25mM EDTA) washing 2 times, reuse RPMI1640 culture medium washing 1 time, abandon most supernatant after, cell is resuspended in the cell culture fluid (90%RPMI1640 culture medium and 10% hyclone), the adjustment cell concentration is 11 * 10
6Individual cell/ml.
2) antigenicity detects: getting concentration is 11 * 10
6The lymphocyte 2700 μ l of individual cell/ml are inoculated in 6 porocyte culture plates.Get blood plasma and each 300 μ l of the viral vaccine after the inactivation treatment of containing HBV, be inoculated in the cultured cell respectively; Wherein, hatch group altogether without the blood plasma that contains HBV of inactivation treatment and lymphocyte and be made as positive controls, the viral vaccine of methylene blue inactivation treatment and lymphocyte are hatched altogether to organize and are made as experimental group.Establish 3 multiple holes for every group.Tissue Culture Plate is placed 37 ℃, 5%CO
2In the incubator, cultivate 24h, 72h respectively.Utilize ELISA test kit (available from Beijing Wantai Biological Pharmacy Enterprise Co., Ltd.) detect before the blood plasma deactivation with deactivation afterwards 0,24, HBsAg in the 72h supernatant.
3. experiment conclusion:
HBsAg content through positive controls and experimental group respectively with untreated former blood plasma in the ratio of HBsAg content; Obtain the variation of hepatitis B virus surface antigen; Experimental result is seen Figure of description Fig. 1; The result shows there was no significant difference (P＞0.05), and vaccine carries out ELISA when detecting hatching 24h, 72h, and significant change (P＞0.05) does not take place along with the prolongation of incubation time HBsAg yet.Numerical value is the meansigma methods of triplicate testing result, proves that vaccine has good antigenicity.
The antigen presentation property experiment of embodiment 3 viral vaccines
1. according to embodiment 1, experiment (2) method prepares the Hepatitis B virus vaccine of methylene blue photochemistry inactivation treatment.
2. detect the antigenic submission property of Hepatitis B virus vaccine of preparation, experimental technique is following:
1) preparation lymphocyte: 6 parts of healthy blood donors' after the picked at random collection in the 6h ACD whole blood, the preparation White Blood Cells Concentrate is used the Ficoll-Hypaque density with reference to product operation handbook, isolated lymphocytes.With PBS (containing 0.25mM EDTA) washing 2 times, reuse RPMI1640 culture medium washing 1 time, abandon most supernatant after, cell is resuspended in the cell culture fluid (90%RPMI1640 culture medium and 10% hyclone), the adjustment cell concentration is 1.1 * 10
6Individual cell/ml.
Detect the expression of the mRNA level of the relevant costimulatory molecules CD80 of lymphocyte activation, CD86 and cell adhesion molecule ICAM2, ICAM3, LFA3: getting concentration is 1.1 * 10
6The lymphocyte 2700 μ l of individual cell/ml are inoculated in 6 porocyte culture plates.Get blood plasma and each 300 μ l of the viral vaccine after the inactivation treatment of containing HBV; Be inoculated in respectively in the cultured cell, hatch group altogether without the HBV of inactivation treatment and lymphocyte and be made as positive controls, the viral vaccine of methylene blue inactivation treatment and lymphocyte are hatched group altogether and are made as experimental group respectively; The lymphocyte that does not add any stimulation is made as negative control group; GAPDH is selected from house-keeping gene, as the confidential reference items of RT-PCR amplified reaction, establishes 3 multiple holes for every group.Tissue Culture Plate is placed 37 ℃, 5%CO
2In the incubator, cultivate 24h.2000g, the centrifugal collection lymphocyte of 5min adds 300 μ lTrizol in the lymphocyte, use the mRNA expression that the RT-PCR method detects correlation factor in the cell, and concrete steps are following:
1) extracting RNA
Centrifugal collection lymphocyte adds 300ul Trizol reagent, uses the vortex oscillation appearance, the mixing 15s that vibrates strongly, and abundant mixing, after room temperature leaves standstill 5 minutes, operation as follows:
A. add chloroform 80ml, the 15s that vibrates strongly, abundant mixing, room temperature left standstill 5 minutes.
B.4 ℃, 12000rpm, centrifugal 15min.
C. after the centrifugal end, upper strata water 200ul is carefully drawn in the solution layering, adds the equal-volume isopropyl alcohol.
D. after putting upside down mixing 10 times, room temperature 10 minutes.
E.4 ℃, 14000rpm, centrifugal 15min.
F. abandon supernatant, 75% pre-cooling ethanol, the 400ul washing is put upside down 1 time.
G.4 ℃, 14000rpm, centrifugal 5min.
H. abandon supernatant, 4 ℃, 14000rpm, centrifugal 5min abandons a small amount of supernatant of residue, natural volatile dry with careful suction of suction pipe.
2) reverse transcription
A. remove genomic DNA
B. reverse transcription program:
25℃，10min?50℃，1h→85℃，5min
The quantitative fluorescent PCR of C.cDNA
The PCR system:
Amplification program:
Fluorescence signal is collected and is located at 72 ℃, the 45s place, and reaction system is made as 25ul.
D. the design of primers of quantitative PCR is with synthetic as follows:
3. experimental result:
The T cellular immunization plays a significant role in removing viral infection, and effective activation of T cell, except that the needs antigenic stimulus, the antigen-presenting cell surface costimulatory molecules expression relevant with the T cell also plays a significant role therein.The present invention adopts the Real-Time PCR method to detect the mRNA horizontal expression of CD80 and CD86 in the cell; The ratio of the mRNA expression through positive controls or experimental group costimulatory molecules and the mRNA expression of negative control group costimulatory molecules; Detect the variation multiple of mRNA expression, experimental result is seen Figure of description Fig. 2, and the result shows; Compare with negative control group, positive controls and experimental group all can significantly raise the mRNA expression (P＜0.05) of CD80 and CD86.Above results suggest vaccine has the ability that raises costimulatory molecules CD80 and CD86 expression.
The activation of T cell is except that the influence of costimulatory moleculeses such as CD80 and CD86, and cell adhesion molecule also plays a significant role therein.Use the Real-Time PCR method,, detect the situation of change of CAF mRNA levels such as ICAM2, ICAM3, LFA3 in the cell through the ratio of positive controls or experimental group and negative control group.Experimental result is seen Fig. 3, and the result shows that the mRNA expression of the ICAM2 of experimental group, ICAM3, LFA3 is compared with negative control group, expresses and significantly raises (P＜0.05).Above results suggest viral vaccine of the present invention has the ability that raises the relevant cell adhesion molecule.Numerical value is the meansigma methods of triplicate testing result.
The above results proof viral vaccine can be discerned by antigen presenting cell, submission, and viral vaccine has the ability of activation antigen presenting cell, has immunogenicity.The situation of change of the mRNA horizontal expression of CD80, CD86,
1. according to embodiment 1, experiment (3) method prepares the Hepatitis B virus vaccine of methylene blue photochemistry inactivation treatment.
2. the Hepatitis B virus vaccine that detects preparation is to T lymphocyte and the excretory influence of Th1 type antiviral relevant cell factor, and experimental technique is following:
1) preparation lymphocyte: 6 parts of healthy blood donors' after the picked at random collection in the 6h ACD whole blood, the preparation White Blood Cells Concentrate is used the Ficoll-Hypaque density with reference to product operation handbook, isolated lymphocytes.With PBS (containing 0.25mM EDTA) washing 2 times, reuse RPMI1640 culture medium washing 1 time, abandon most supernatant after, cell is resuspended in the cell culture fluid (90%RPMI1640 culture medium and 10% hyclone), the adjustment cell concentration is 1.1 * 10
6Individual cell/ml.
2) detect cytokine levels: getting concentration is 1.1 * 10
6The lymphocyte 2700 μ l of individual cell/ml are inoculated in 6 porocyte culture plates.Get blood plasma and each 300 μ l of the viral vaccine after the inactivation treatment of containing HBV; Be inoculated in the cultured cell respectively; Hatch group altogether without the HBV of inactivation treatment and lymphocyte and be made as positive controls; The viral vaccine of methylene blue inactivation treatment and lymphocyte are hatched group altogether and are made as experimental group respectively, and the lymphocyte that does not add any stimulation is made as negative control group, establish 3 multiple holes for every group.Tissue Culture Plate is placed 37 ℃, 5%CO
2In the incubator, cultivate 72h.2000g, the centrifugal collection lymphocyte of 5min adds 300 μ lTrizol in the lymphocyte, use ELISA test kit (available from R&D company) and detect cytokine levels such as TNF-α, IFN-γ, IL-2, IL-4, IL-10 in the supernatant.
3. experiment conclusion:
Use the ELISA method and detect TNF-α, IFN-γ, IL-2, IL-4, IL-10 level in the supernatant; Ratio through positive controls or experimental group and negative control group; Detect the situation of change of the mRNA level of TNF-α, IFN-γ, IL-2, IL-4, IL-10 etc. in the cell, experimental result is seen Fig. 4～8, and the result shows: compare with negative control group; Experimental group can make TNF-α secretory volume raise 14 ± 10 times, makes IFN-γ secretory volume raise 12 ± 6 times (Fig. 4, Fig. 5); But the secretory volume of IL-2, IL-4 and IL-10 there is not remarkable rise effect (Fig. 6-8, numerical value are the meansigma methods of triplicate testing result).This shows that vaccine can stimulate the lymphocytic emiocytosis cytokine, and the Th1 cytokines of being correlated with antiviral such as TNF-α, IFN-γ is main.
Claims (10)
1. the viral vaccine of methylene blue photochemical virus ablation method preparation is induced Th1 type cell immune response and is stimulated the application in the preparation of lymphocytic emiocytosis cytokine in preparation.
2. application as claimed in claim 1 is characterized in that, said cytokine is tumor necrosis factor and interferon.
3. application as claimed in claim 1 is characterized in that, said viral vaccine is to obtain after adopting the deactivation of methylene blue photochemical virus ablation method to contain viral blood plasma.
4. application as claimed in claim 3 is characterized in that, the said blood plasma that contains virus derives from the patient.
5. application as claimed in claim 3; It is characterized in that said virus is selected from hepatitis B virus, hepatitis C virus, human immunodeficiency virus type 1, HIV's 2 types, cytomegalovirus, herpesvirus or T visceral leukosis virus.
6. application as claimed in claim 1 is characterized in that, it is arbitrary that the method for using of said viral vaccine is selected from following two kinds of methods:
Method one: use the blood plasma list and adopt appearance, separated plasma also carries out methylene blue photochemical virus inactivation treatment to blood plasma, the blood plasma after handling fed back to the patient, and be a course of treatment;
Method two: 1) separated plasma 200～400ml first, and carry out methylene blue photochemical virus inactivation treatment, inactivation treatment finishes after-filtration and removes methylene blue, freezing preservation; 2) separated plasma once more after 1～4 week, isolating plasma volume be last time 1～2 times of the separated plasma amount, the processing method of blood plasma feeds back the blood plasma of preserving after the viral inactivation treatment last time with isolating blood plasma method is identical first simultaneously; 3) repeating step 2), be 2800～3200ml until the displacement plasma volume, be a course of treatment.
7. application as claimed in claim 6 is characterized in that, carry out deactivation to the blood plasma in whole blood circulations a course of treatment of method one.
8. application as claimed in claim 6 is characterized in that, is feeding back to filtering before the patient or not filtering through the methylene blue in the blood plasma of methylene blue photochemical virus inactivation treatment in the method one.
9. application as claimed in claim 6 is characterized in that, the freezing storage temperature described in the method two is-20 ℃.
10. like the described application of the arbitrary claim of claim 1-9; It is characterized in that; Said methylene blue photochemical virus ablation method is: in 5～1000ml contains blood plasma or the solution of virus, adding methylene blue and adjust final concentration is 1.0～100 μ mol/L; Under the air-proof condition, 4～37 ℃ of concussions, 30000～35000Lux radiation of visible light is handled 30～45min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498795A (en) * | 2018-03-22 | 2018-09-07 | 耿艳春 | The one-step preparation process of therapeutic type drug resistance AIDS self-plasma virus deactivation vaccine |
| CN110870912A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Application of methylene blue dye as vaccine adjuvant, vaccine containing adjuvant and application of vaccine |
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| CN1211277A (en) * | 1996-10-28 | 1999-03-17 | 巴克斯特国际有限公司 | Systems and methods for removing viral agents from blood |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108498795A (en) * | 2018-03-22 | 2018-09-07 | 耿艳春 | The one-step preparation process of therapeutic type drug resistance AIDS self-plasma virus deactivation vaccine |
| CN110870912A (en) * | 2018-08-31 | 2020-03-10 | 成都夸常奥普医疗科技有限公司 | Application of methylene blue dye as vaccine adjuvant, vaccine containing adjuvant and application of vaccine |
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Application publication date: 20120404 |