CN102266551A - Interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals - Google Patents
Interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals Download PDFInfo
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Abstract
The invention discloses an interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals. The interleukin-4 therapeutic vaccine is a protein vaccine or coupling protein vaccine of any form prepared by taking the natural or artificially synthesized complete protein or protein fragment of interleukin-4 as an antigen; or the interleukin-4 therapeutic vaccine is a gene vaccine or fusion gene vaccine of any form prepared by taking the complete gene or gene fragment of interleukin-4 as the antigen gene or the major antigen gene. The IL (interleukin)-4 vaccine is used for performing active immunotherapy on a host, generally, the effective time lasts for about 2-3 months by immunization for the first time, the effective therapeutic time lasts for about half a year by secondary immunization, and recovery can be achieved by 1-3 times of immunotherapy. Compared with direct application of anti-IL-4 antibody for treatment, the invention has the characteristics of few times of application, low dose and the like, thereby greatly reducing the therapeutic cost, and also greatly reducing the possibility of generating allergic reaction.
Description
Technical field
The present invention relates to can be used for treating the interleukin-4 therapeutic vaccine of human or animal's immune related diseases.
Background technology
Up to the present, the caused chronic infectious disease of the entozoic pathogen of people's pair cell (virus, antibacterial, parasite etc.) still lacks more effectively treats way, and the vaccine prevention effect is also undesirable.Theoretically, this class chronic infectious disease needs the host to produce a strong class (Th1) anti-infectious immunity reaction could remove pathogen, remains a difficult problem but how to induce body to produce strong Th1 anti-infectious immunity reaction.Discover that according to applicant's long-term exploration the clinical onset stage great majority of this class chronic infectious disease all show as very strong two classes (Th2) anti-infectious immunity reaction, promptly react based on the Th2 anti-infectious immunity; Subclinical infection then great majority all shows as very strong Th1 anti-infectious immunity reaction, promptly reacts based on the Th1 anti-infectious immunity.Studies have shown that further the generation of a class and the reaction of two para-immunities is not to take place synchronously, complementary form, but suppress mutually, the form of rising one after another.IL-4 induces to produce the immunoreactive major cytokine of Th2, if can reduce the content of IL-4 in the host, can effectively suppress the Th2 immunoreation, thereby promotes the immunoreactive generation of Th1.Experiment showed, that injecting anti-IL-4 antibody to the host can reach this purpose.But because the half-life of antibody is lacked (about 21-28 of the half-life of IgG days), need the repeated multiple times injection, not only improved cost, and easily caused allergic reaction.Be badly in need of a kind of with low cost and technological means that can not cause allergic reaction.
Summary of the invention
At above-mentioned prior art, the invention provides a class and prepare the interleukin-4 therapeutic vaccine that human or animal's immune related diseases is treated in simple, with low cost and safe and reliable can be used for.
The present invention is achieved by the following technical solutions:
Can be used for treating the interleukin-4 therapeutic vaccine of human or animal's immune related diseases, be to do any type of protein vaccine or coupling protein vaccine that antigen is made with the intact proteins of the natural or synthetic of interleukin-4 or protein fragments (polypeptide or oligopeptide need before making vaccine further and other antigen macromole coupling mutually);
Or: make any type of gene vaccine or the fusion gene vaccine that antigen gene or major antigen gene are made with the complete genome of interleukin-4 or genetic fragment;
Or: with the complete genome of any carrier cloning IL-4 or genetic fragment, with the made gene vaccine of any expression system amplification gene, or the intact proteins or the protein fragments (polypeptide or oligopeptide) of expressing IL-4 are done any type of protein vaccine or the amalgamation protein vaccine that antigen is made; If selected expression vector belongs to live vector, also can make the live vector recombinant vaccine.
Can be added with medically conventional vaccine adjuvant in the described vaccine.
Vaccine of the present invention not only can be used for treating human or animal's chronic infectious disease and parasitic disease, goes in the anaphylaxis etc. that also can further promote the use of cancer, some type and the Ia disease treatment.
According to prepared vaccine form difference, the embodiment of vaccine of the present invention can have multiple: as vaccines such as aluminium glue adjuvant, oily adjuvant, liposome adjuvants, can adopt multitude of different ways such as subcutaneous injection, intradermal injection and intramuscular injection; If live vector vaccine, except above injection system, also can adopt different modes such as oral, suction, collunarium, eye drip.
The present invention be directed to the defective that exists in the prior art: " injecting anti-IL-4 antibody to the host; need the repeated multiple times injection; not only improved cost, and easily caused allergic reaction ", and the technical scheme that proposes, that is: adopt the form of IL-4 vaccine that the host is carried out active immunity treatment, generally, first immunisation can reach about 2～3 months effective time, and secondary immunity can reach effective treatment time of half a year approximately, through 1～3 time immunization therapy, can reach the purpose of rehabilitation.Compare with the anti-IL-4 Antybody therapy of direct application, have number of applications few, characteristics such as consumption is low like this, not only greatly reduce the treatment cost, and, greatly reduce and produce anaphylactoid probability.
Description of drawings
Fig. 1: give the experiment BALB/c mouse respectively at preceding 3 weeks of induced hypersensitivity reaction with inoculate prepared (face embodiment 1 as follows) the IL-4 gene recombinaton oil-adjuvant vaccine of embodiment 11 week subcutaneous (s.c.), other establishes pET-32 carrier oil Adjuvanted vaccines and the immune matched group of normal saline.In the 0th week with use low dosage (5 μ g) the pure albumen of ovum gallinaceum (OVA) aluminium glue vaccine abdominal cavity inoculation the 3rd week respectively,, splash into by the nostril in the 5th week usefulness high dose (80 μ g are dissolved among the 50 μ l PBS) OVA, to induce the generation anaphylaxis with sensitized mice.The result shows: IL-4 gene recombinaton oil-adjuvant vaccine immune group is compared with normal saline immunity matched group with pET-32 carrier oil Adjuvanted vaccines group, and IL-4 gene recombinaton oil-adjuvant vaccine can significantly reduce the generation of OVA-IgE specific antibody.
Fig. 2: inoculate prepared (face embodiment 1 as follows) the IL-4 gene recombinaton aluminium glue vaccine of embodiment 1 respectively at preceding 3 weeks of Li Shiman parasite inoculation and 1 all abdominal cavities (i.p.) for the experiment BALB/c mouse, other establishes pET-32 carrier aluminium glue vaccine and the immune matched group of normal saline.An amount of Li Shiman parasite subcutaneous (s.c.) injection inoculation is in mouse hind leg foot pad.Weekly with the kind of calliper pathological changes size at pad place enough once, continued for 8～10 weeks after the inoculation.The result shows that the IL-4 genetic vaccine can significantly reduce the parasitic infection of Li Shiman.
The specific embodiment
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1: the structure and the vaccine production of mice IL-4 genetic vaccine
According to the full gene coded sequence of mice IL-4, design a pair of suitable primer, by RT-PCR method amplification IL-4cDNA sequence.Then, through determined dna sequence, after the cDNA sequence that proof is increased is accurate, be cloned into the suitable position of pET-32 fusion expression vector again, and further change recombiant plasmid and pET-32 fusion expression vector blank plasmid over to suitable expression with in the coli strain, through the scale fermentation, extract purification IL-4 fusion rotein and the expressed sulfur hydrogen reduction albumen of vehicle Control.The sulfur hydrogen reduction albumen that purification IL-4 fusion rotein or vehicle Control is expressed mixes the back with oily adjuvant or aluminium glue adjuvant etc. by proper proportion, and [content according to purification IL-4 fusion rotein is different, to oil-adjuvant vaccine, the volume ratio of oil/protein solution can be controlled at (1: 1)～(3: 1); To the aluminium glue Adjuvanted vaccines, the volume ratio of aluminium glue/protein solution can be controlled at (1: 1)～(1: 5); Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15-20 μ g; The volume of every part vaccine is about 0.1ml; Or according to the preparation of veterinary's pharmacopeia rule of operation], further the experiment vaccine is made in homogenate emulsifying (rule of operation by used refiner is carried out).
The pale yellow oily liquid body of described oily adjuvant for forming after Span-80,2% (g/V) the aluminium stearate heating for dissolving by 94% (V/V) mineral oil (No. 10 white oils), 6% (V/V); The oil adjuvant is the heavy-gravity milky water-in-oil type vaccine that vaccine protein solution and oily adjuvant form through emulsifying.The general injection inoculation that adopts is a product commonly used in the prior art, is to use animal at present to use wider adjuvant in the inactivated vaccine.
Described aluminium glue adjuvant is also used for animal and is used wider adjuvant in the inactivated vaccine.
Embodiment 2: the safety testing of mice IL-4 genetic vaccine and anaphylaxis test
Press the method for embodiment 1, the sulfur hydrogen reduction albumen that purification IL-4 fusion rotein or vehicle Control is expressed suitably dilutes back and oily adjuvant or aluminium glue adjuvant with normal saline and is mixed with into IL-4 amalgamation protein vaccine or vehicle Control sulfur hydrogen reduction protein vaccine by 1: 1 volume ratio.The IL-4 amalgamation protein vaccine for preparing divides low (0.1ml, about 1 part), (0.25ml in, about 2.5 parts), high (0.5ml, about 5 parts) three immune BALB/c mouse of various dose group difference, other establishes pET-32 carrier bacterin and normal saline matched group, every group of 5 mices, subcutaneous multi-point injection.At interval 3 weeks, carry out the second time and inoculation for the third time respectively.Observe phenomenons such as mice has no adverse reaction, allergy, death after the vaccine injection respectively.The result shows: physically well develop behind the mouse inoculation vaccine, the mental status and appetite are all normal, and inoculation position has untoward reaction such as the property a crossed swelling, heating, and is especially more obvious in the high dose group performance.For oily adjuvant Seedling, the high dose group mice because the vaccine absorption is slower, has slight granuloma reaction.
Embodiment 3: mice IL-4 genetic vaccine strengthens the test of anti-tumor activity
Press the method for embodiment 1, the IL-4 fusion rotein of purification or the expressed sulfur hydrogen reduction albumen of vehicle Control are suitably diluted back and mixed preparation experiment or the control vaccine of aluminium glue adjuvant by 1: 1 with normal saline.Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15～20 μ g; The volume of every part vaccine is about 0.1ml.Attack preceding 3 weeks and 1 all abdominal cavities (i.p.) respectively at tumor cell for experiment mice and inoculate the prepared IL-4 gene recombinaton aluminium glue vaccine of embodiment 1, other establishes pET-32 carrier aluminium glue vaccine and the immune matched group of normal saline.Check a tumor growth situation every 3 days behind the tumor inoculation, test end in 60 days.The result shows that the IL-4 genetic vaccine can significantly reduce the incidence rate (seeing Table 1) of tumor.
Table 1: mice IL-4 genetic vaccine strengthens the result of the test (10 every group, data are survival rate %) of anti-tumor activity
Embodiment 4: the test of relapse rate behind the mice IL-4 genetic vaccine reduction tumor operation
Press the method for embodiment 1, the IL-4 fusion rotein of purification or the expressed sulfur hydrogen reduction albumen of vehicle Control are suitably diluted back and mixed preparation experiment or the control vaccine of aluminium glue adjuvant by 1: 1 with normal saline.Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15-20 μ g; The volume of every part vaccine is about 0.1ml.The prepared IL-4 gene recombinaton aluminium glue vaccine of abdominal cavity inoculation embodiment 1 when the same day and the 14th day after giving experiment mice respectively at tumor operation, other establishes pET-32 carrier aluminium glue vaccine and normal saline immunity matched group.Checked a tumor growth situation every 3 days behind the tumor operation, test after 100 days and finish.The result shows that the IL-4 genetic vaccine can significantly reduce the relapse rate (seeing Table 2) of tumor.
Table 2: mice IL-4 genetic vaccine strengthens the result of the test (10 every group, data are survival rate %) of anti-tumor activity
Embodiment 5: mice IL-4 genetic vaccine suppresses the anaphylactoid test of respiratory tract type
Press the method for embodiment 1, the IL-4 fusion rotein of purification or the expressed sulfur hydrogen reduction albumen of vehicle Control are suitably diluted back and mixed preparation experiment or the control vaccine of oily adjuvant by 1: 1 with normal saline.Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15-20 μ g; The volume of every part vaccine is about 0.1ml.Give the experiment BALB/c mouse respectively at preceding 3 weeks of induced hypersensitivity reaction with inoculate the prepared IL-4 gene recombinaton oil-adjuvant vaccine of embodiment 11 week subcutaneous (s.c.), other establishes pET-32 carrier oil Adjuvanted vaccines and the immune matched group of normal saline.In the 0th week with use low dosage (5 μ g) the pure albumen of ovum gallinaceum (OVA) aluminium glue vaccine abdominal cavity inoculation the 3rd week respectively,, splash into by the nostril in the 5th week usefulness high dose (80 μ g are dissolved among the 50 μ l PBS) OVA, to induce the generation anaphylaxis with sensitized mice.The result shows: IL-4 gene recombinaton oil-adjuvant vaccine immune group is compared with normal saline immunity matched group with pET-32 carrier oil Adjuvanted vaccines group, IL-4 gene recombinaton oil-adjuvant vaccine can significantly reduce the generation (seeing accompanying drawing 1) of OVA-IgE specific antibody and the quantity (seeing Table 3) of the oxyphil cell in the pulmonary branches trachea folliculus flushing liquor, and significantly suppresses the loose and histiocyte degree of inflammation of hypertrophy of goblet cell.
Table 3: mice IL-4 genetic vaccine suppresses the secretion of oxyphil cell in the pulmonary branches trachea folliculus
Embodiment 6: the test of the chronic leishmaniasis of mice IL-4 genetic vaccine treatment mice
Press the method for embodiment 1, the IL-4 fusion rotein of purification or the expressed sulfur hydrogen reduction albumen of vehicle Control are suitably diluted back and mixed preparation experiment or the control vaccine of aluminium glue adjuvant by 1: 1 with normal saline.Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15-20 μ g; The volume of every part vaccine is about 0.1ml.Inoculate the prepared IL-4 gene recombinaton aluminium glue vaccine of embodiment 1 respectively at preceding 3 weeks of Li Shiman parasite inoculation and 1 all abdominal cavities (i.p.) for the experiment BALB/c mouse, other establishes pET-32 carrier aluminium glue vaccine and the immune matched group of normal saline.An amount of Li Shiman parasite subcutaneous (s.c.) injection inoculation is in mouse hind leg foot pad.Weekly with the kind of calliper pathological changes size at pad place enough once, continued for 8～10 weeks after the inoculation.The result shows that the IL-4 genetic vaccine can significantly reduce the parasitic infection of Li Shiman (seeing accompanying drawing 2).
Embodiment 7: mice IL-4 genetic vaccine strengthens the test of mice prevention chronic tuberculosis disease
Press the method for embodiment 1, the IL-4 fusion rotein of purification or the expressed sulfur hydrogen reduction albumen of vehicle Control are suitably diluted back and mixed preparation experiment or the control vaccine of aluminium glue adjuvant by 1: 1 with normal saline.Require in every part vaccine IL-4 fusion rotein or the proteic amount of vehicle Control sulfur hydrogen reduction to be about 15-20 μ g; The volume of every part vaccine is about 0.1ml.Inoculate the prepared IL-4 gene recombinaton aluminium glue vaccine of embodiment 1 respectively at preceding 3 weeks of bacillus calmette-guerin vaccine (BCG) inoculation and 1 all abdominal cavities (i.p.) for experiment mice, other establishes pET-32 carrier aluminium glue vaccine and the immune matched group of normal saline.An amount of bacillus calmette-guerin vaccine is given mice subcutaneous (s.c.) or vein (i.v.) injection inoculation.12～16 weeks of inoculation back are attacked (i.v. inoculation) with the high dose recombinant BCG, and then, 10～12 weeks were got mouse spleen, grinding, filtration, centrifugal, get an amount of single-cell suspension liquid and be taped against on the agar version, insert in 37 ℃ of incubators and cultivate about 3 weeks, count each dull and stereotyped colony counts that goes up.The result shows that the IL-4 genetic vaccine can significantly reduce the infection (seeing Table 4) of BCG.
Table 4: mice IL-4 genetic vaccine strengthens the result of the test (10 every group, data are each dull and stereotyped bacterium colony average of going up) of mice prevention chronic tuberculosis disease
Claims (4)
1. can be used for treating the interleukin-4 therapeutic vaccine of human or animal's immune related diseases, it is characterized in that: it is to make any type of protein vaccine or the coupling protein vaccine that antigen is made with the intact proteins of the natural or synthetic of interleukin-4 or protein fragments.
2. can be used for treating the interleukin-4 therapeutic vaccine of human or animal's immune related diseases, it is characterized in that: it is to make any type of gene vaccine or the fusion gene vaccine that antigen gene or major antigen gene are made with the complete genome of interleukin-4 or genetic fragment.
3. can be used for treating the interleukin-4 therapeutic vaccine of human or animal's immune related diseases, it is characterized in that: it is with the complete genome of any carrier cloning IL-4 or genetic fragment, with the made gene vaccine of any expression system amplification gene, or the intact proteins or the protein fragments of expressing IL-4 are done any type of protein vaccine or the amalgamation protein vaccine that antigen is made.
4. according to claim 1 or the 2 or 3 described interleukin-4 therapeutic vaccines that can be used for treating human or animal's immune related diseases, it is characterized in that: be added with medically conventional vaccine adjuvant in the described vaccine.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510010040.9A CN104623688B (en) | 2011-06-27 | 2011-06-27 | It can be used for treating the interleukin-4 therapeutic vaccine of human or animal's tumor disease |
| CN201110174918.4A CN102266551B (en) | 2011-06-27 | 2011-06-27 | Interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110174918.4A CN102266551B (en) | 2011-06-27 | 2011-06-27 | Interleukin-4 therapeutic vaccine for treating immune related diseases of humans or animals |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510009584.3A Division CN104623647A (en) | 2011-06-27 | 2011-06-27 | Therapeutic interleukin-4 vaccine capable of treating human or animal chronic tuberculosis |
| CN201510010040.9A Division CN104623688B (en) | 2011-06-27 | 2011-06-27 | It can be used for treating the interleukin-4 therapeutic vaccine of human or animal's tumor disease |
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| CN102266551A true CN102266551A (en) | 2011-12-07 |
| CN102266551B CN102266551B (en) | 2015-05-13 |
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Cited By (3)
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| CN104826093A (en) * | 2015-04-16 | 2015-08-12 | 刘永庆 | Th2 immune response antagonist for treating chronic infectious hepatopathy, and yolk antibody thereof |
| CN104922659A (en) * | 2015-07-03 | 2015-09-23 | 刘永庆 | Th2 immunoreaction inhibitor for preventing and treating tumors and/or chronic tuberculosis and application thereof |
| CN108324948A (en) * | 2018-03-28 | 2018-07-27 | 潍坊康奥思生物技术有限公司 | Application of the Th2 immune responses inhibitor in preparing treatment and/or preventing cancer drug |
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| CN1703239A (en) * | 2002-10-09 | 2005-11-30 | 遗传工程与生物技术中心 | Vaccine composition comprising interleukin-15 (IL-15) |
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| CN1703239A (en) * | 2002-10-09 | 2005-11-30 | 遗传工程与生物技术中心 | Vaccine composition comprising interleukin-15 (IL-15) |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104826093A (en) * | 2015-04-16 | 2015-08-12 | 刘永庆 | Th2 immune response antagonist for treating chronic infectious hepatopathy, and yolk antibody thereof |
| CN104922659A (en) * | 2015-07-03 | 2015-09-23 | 刘永庆 | Th2 immunoreaction inhibitor for preventing and treating tumors and/or chronic tuberculosis and application thereof |
| CN104922659B (en) * | 2015-07-03 | 2018-04-20 | 刘永庆 | The Th2 immune responses inhibitor of anti-curing oncoma and/or chronic tuberculosis disease and its application |
| CN108324948A (en) * | 2018-03-28 | 2018-07-27 | 潍坊康奥思生物技术有限公司 | Application of the Th2 immune responses inhibitor in preparing treatment and/or preventing cancer drug |
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