CN101670102A - Method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine and application - Google Patents

Method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine and application Download PDF

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CN101670102A
CN101670102A CN200910192351A CN200910192351A CN101670102A CN 101670102 A CN101670102 A CN 101670102A CN 200910192351 A CN200910192351 A CN 200910192351A CN 200910192351 A CN200910192351 A CN 200910192351A CN 101670102 A CN101670102 A CN 101670102A
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hiv vaccine
vector hiv
poxvirus
vaccine
adenoviral vector
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陈凌
陈志伟
张林琦
孙彩军
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Guangzhou Institute of Biomedicine and Health of CAS
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Guangzhou Institute of Biomedicine and Health of CAS
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Priority to PCT/CN2010/076905 priority patent/WO2011032489A1/en
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Abstract

The invention provides a method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine, and in particular relates to a method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine to immunize organisms; the method is that: the poxvirus vector HIV vaccine is used for immunizing first and then the adenoviral vector HIV vaccine is used for immunize organisms; or the adenoviral vector HIV vaccine is used first for immunizing organism first and then the poxvirus vector HIV vaccine is used for immunizing organism. The invention also provides the applicationof the method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine on the preventive and therapeutic HIV vaccines field, so as to prevent and treat AIDS and Hepatitis B, Ebolavirus and tumor. The method can induce strong, broad-spectrum and multi-functional T leukomonocyte reaction and can be widely applied to preventing and treating AIDS and other communicable diseases and tumor.

Description

Unite method and the application thereof of using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine
Technical field
The present invention relates to biological technical field, be specifically related to unite new method and the application thereof of using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine prevention and treatment acquired immune deficiency syndrome (AIDS).
Background technology
Acquired immune deficiency syndrome (AIDS) (Acquired Immune Deficiency Syndrome AIDS) has become one of sanitarian significant challenge in the whole world, serious harm social progress and economic growth.The World Health Organization (WHO) and UNAIDS (UNAIDS) will announce that in the end of the year 2008 present global HIV the infected's total number of persons is about 3,300 ten thousand, and dead acquired immune deficiency syndrome (AIDS) patient's accumulative total has surpassed 3,000 ten thousand; Only 2007, the whole world just had 2,100,000 people to die from acquired immune deficiency syndrome (AIDS), 2,700,000 people's aids infection viruses; Asia and Eastern Europe have become HIV (human immunodeficiency virus) and have propagated area (UNAIDS report, 2008) the most rapidly.The HIV fashion trend of China is also quite serious, by in by the end of October, 2007, whole nation accumulative total is reported patients infected hiv and acquired immune deficiency syndrome (AIDS) patient 22.4 ten thousand many cases, and national epidemiological survey is estimated whole nation survival HIV the infected sum about 700,000 people, wherein about 50,000 people of new the infected in 2007.Therefore the research that prevents and treats AIDS-treating medicine and vaccine will be the most important thing of China Department of Science and Technology Eleventh Five-Year Plan plan.
In the past one, though obtaining multinomial progress aspect the Drug therapy control AIDS in 20 years, acquired immune deficiency syndrome (AIDS) case fatality rate in developed country significantly descends, some have occurred and can treat and control the novel drugs of HIV (as antiretroviral drugs, protease inhibitor etc.) and new therapy (associating cocktail), but body just can't be eradicated HIV virus in case infect the back, and because high variability, the drug resistance situation of HIV virus is on the rise, and Drug therapy has brought very big untoward reaction and patient's problems such as ability to shoulder economically to make that the prospect of these therapies is unsatisfactory for HIV the infected, therefore develops effective HIV vaccine and is still exigence.In HIV vaccine field, research that it is generally acknowledged the HIV vaccine can be divided into has experienced three phases:
Phase I: originate in AIDS vaccine research in 1984 and concentrate on to induce antibody to be main target with the prophylaxis of viral infections, not consider the effect of cellular immunization.2003 at two AIDS vaccine III clinical trial phases American-European and that Thailand finishes, i.e. the clinical verification of the first developmental stage achievement in research of the AIDS vaccine on the practical significance, and the result shows the vaccine real protection effect of tool not of inducing the antibody generation merely.
Second stage: AIDS vaccine is studied then stressed cell immunoreation effect.This stage is based on the immunoreactive recombinant viral vector vaccine of inducing cell (vaccine, adenovirus, canary pox virus etc.), because cell immune response can effectively be controlled duplicating of HIV virus and infect in theory, and mathematical model also shows, virus load reduces by 1 log value just can effectively reduce crowd's spreading rate, and this allows clinical trial see the application prospect of this vaccine.This wherein one of the most significant achievement be exactly the exploitation of Merck (Merck) company organization be the AIDS vaccine of carrier with adenovirus hominis 5 types, proved at the SHIV89.6P model and induced generation the better protect effect to be arranged at the cd8 t cell immunoreation of HIV.Merck company is pushed into clinical experiment with this vaccine thereupon, has entered the clinical IIb stage by 2004, but the clinical test results of announcing according in JIUYUE, 2007, simple stressed cell immunne response can not produce protective effect.
Comparatively popular at present viewpoint, promptly the viewpoint of phase III is thought, effectively AIDS vaccine need induce isostatic body fluid and cell immune response simultaneously.Antibody is as the first line of defence part virus that can neutralize when viral infection, this has striven for activationary time for follow-up cell-mediated memory response, and intensive cellullar immunologic response can be removed the cell of infective virus, reduces virus load, thereby can reduce the spreading rate of HIV virus the crowd.
McElrath etc. have systematically analyzed the HIV clinical experiment data of Merck; though not found that and play a protective role; but this vaccine (77%) in most of experimenter groups has produced stronger immunne response; and analyze the special CD8+T cell also find among these crowds mainly based on independent secretion of gamma-IFN cytokine (73%); some TNF secretion-a are also arranged, but the cell of little secretion IL-2 cytokine or secrete the lymphocyte of the various kinds of cell factor simultaneously.In addition, up-to-date data shows than twice India Rhesus Macacus of the independent Ad5 of using carrier immunity, behind adenovirus vector (rAd26/rAd5) combined immunization by different serotypes, has brought out stronger wider cellullar immunologic response.More secrete the various kinds of cell factor (polyfunctional CD8+ and the CD4+T lymphocyte of IFN-γ/TNF-a/IL-2) simultaneously but also produced.More meaningfully, after passing through vein mode taint with SIV mac251 virus, compare and immune twice Ad5 carrier only, rAd26/rAd5 combined immunization group has more effectively been controlled duplicating of virus and has been infected, the peak value carrying capacity of the intravital SIVmac251 virus of this group monkey has reduced by 1.4 log values, and the virus load in plateau (setpoint) has reduced by 2.4 log values especially.And all do not die from AIDS or tangible AIDS symptom takes place the monkey of this group of whole experimental session (more than 500 day), but other group monkeys all have morbidity death in various degree.
In a word; failure experience in conjunction with nearest achievement in research and Merck vaccine; the researchers in this field generally believe the developing direction based on the HIV vaccine should be can induce stronger; more wide spectrum and polyfunctional T lymphocyte reaction; bring out certain humoral immunoresponse(HI) simultaneously, so just might produce the effective immunoprotection of HIV virus.In order to search out final effectively HIV vaccine, researchers should be attempted the vaccine of number of different types and unite use in the hope of finding best of breed.
Advantages such as adenovirus vector has efficiency of infection and exogenous gene expression level height, the preparation of high titre recombinant virus is simple, capacity is big, thereby the Ad carrier enjoys favor as mammalian cell expression vector, recombiant vaccine and gene therapy vector.Have more than 342 examples as the clinical experiment of vehicle treatment infectious disease, cancer, cardiovascular disease, single-gene disorder etc. with adenovirus in the global range at present, in all kinds of carriers, account for first place (24.8%).
The Temple of Heaven strain poxvirus (VTT) was used in a large amount of crowd of the China midium or long term as the vaccine of prevention variola, had extraordinary safety, was a very ideal vaccine carrier.The poxvirus vector of using in this research is the MVTT strain of further attenuation, experimental results show that its neurotoxicity reduces greatly.Therefore both can be used as safe antismallpox vaccine, also can be used as live vector at other pathogen vaccines.And experiment shows that also this carrier can pass through the mucosa approach, nasal cavity and oral etc. for example, and the mucosa that induces general reacts.
At present, also recombinant adenoviral vector HIV/SIV vaccine and modified form poxvirus the Temple of Heaven strain (MVTT) vector HIV/SIV vaccine are not united the report of use.
Summary of the invention
In order to develop the novel vaccine of HIV safely and effectively with prevention and treatment acquired immune deficiency syndrome (AIDS), the invention provides a kind of method of using poxvirus vector and adenovirus vector of uniting, this method can be used for prevention and treatment HIV and other infectious disease and tumor.
The method of using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting of the present invention, be specially to unite and use poxvirus vector HIV vaccine and adenoviral vector HIV vaccine immunity body, bringing out stronger more wide spectrum and polyfunctional T lymphocyte reaction and humoral immunoresponse(HI), thereby the HIV viral infection is played effective prevention and therapeutic effect.
This method can be used poxvirus vector HIV vaccine immunity body earlier, re-uses adenoviral vector HIV vaccine immunity body.
This method also can be used adenoviral vector HIV vaccine immunity body earlier, re-uses poxvirus vector HIV vaccine immunity body.
Preferably, in this method, poxvirus vector HIV vaccine contains HIVgag, pol, env gene.
Preferably, poxvirus vector HIV vaccine also contains the assistant regulating and controlling protein gene, and described assistant regulating and controlling protein gene comprises nef, vpx, vpr, vif, rev and tat gene.
Preferably, in this method, adenoviral vector HIV vaccine contains HIVgag, pol, env gene.
Preferably, adenoviral vector HIV vaccine also contains the assistant regulating and controlling protein gene, and described assistant regulating and controlling protein gene comprises nef, vpx, vpr, vif, rev and tat gene.
Preferably, the approach of described immune body is meant by mucosal route immunity body, nasal cavity for example, oral cavity etc.; By intramuscular injection path immunity body; Use approach by intravenous injection approach and other all vaccine.
Preferably, above-mentioned adenovirus vector refers to any adenovirus hypotype well known by persons skilled in the art, the adenovirus of separated strain or other animal origins.Particularly, can be 5 types or 2 type adenovirus vectors.
Preferably, above-mentioned poxvirus vector refers to any poxvirus hypotype well known by persons skilled in the art, separated strain.Particularly, can be modified form the Temple of Heaven strain poxvirus vector.
The present invention also provides the above-mentioned method of using poxvirus vector and adenovirus vector of uniting to be applied in preventative and therapeutic HIV vaccine field, with prevention and treatment acquired immune deficiency syndrome (AIDS), and the application in prevention and control hepatitis B, Ebola virus and tumor.
Compare and the HIV vaccine that uses poxvirus vector or adenovirus vector separately; unite to use and carry HIVgag; pol; brought out stronger, more wide spectrum and polyfunctional T lymphocyte reaction behind the poxvirus vector of env gene and the adenovirus vector immunity body, and the SIVmac239 virus of rectal infection has been shown very strong protection and control action.Data shows, wide spectrum and polyfunctional T lymphocyte reaction strongly,, especially cytotoxic T lymphocyte is reflected at control various susceptible toxoinfection diseases and tumor aspect and is all bringing into play important effect, therefore the enhancing body among the present invention produces strategy stronger, more wide spectrum and polyfunctional T lymphocyte reaction and is suitable for preventing and treating various susceptible toxoinfection diseases equally, hepatitis B for example, Ebola virus etc., and various tumor.In addition because poxvirus vector and adenovirus vector have been widely used in clinical experiment, so they have good safety, make that the potential applicability in clinical practice of this strategy is very bright.Inventor's test data is that this vaccine strategy enters clinical trial and established solid foundation.The present invention can generally be used for prevention and treatment acquired immune deficiency syndrome (AIDS), and prevents and control other infectious disease and tumor.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the testing result figure that detects the recombinant adenovirus protein expression level with the western-blot method;
Fig. 2 is the initial immunity of recombinant adenovirus in mice and the immunogenicity comparison diagram behind the booster immunization; Fig. 2 A is the immunogenicity comparison diagram behind the initial immunity of recombinant adenovirus in mice; Fig. 2 B is the immunogenicity comparison diagram behind the booster immunization of recombinant adenovirus in mice;
Fig. 3 unites poxvirus vector (MVTT) SIV vaccine and immunity and the counteracting toxic substances scheme of adenovirus (Ad5) carrier S IV vaccine in Rhesus Macacus used;
Fig. 4 is that use MVTT carrier and the immunogenicity of Ad5 carrier in Rhesus Macacus are united in the ELISPOT detection; Fig. 4 A is the ELISPOT detection data apart from last immune 6 weeks, and Fig. 4 B is that the ELISPOT in last immune 21 weeks of distance detects data;
Fig. 5 is that MVTT and Ad5 vector HIV vaccine are united Rhesus Macacus is carried out 6 weeks behind the booster immunization, the testing result of the CD8+T emiocytosis cytokine that the Gag peptide is stimulated with the polychrome streaming technology;
Fig. 6 is that MVTT and Ad5 vector HIV vaccine are united Rhesus Macacus is carried out behind the booster immunization detecting the testing result of the memory t cell secrete cytokines in the peripheral blood with the polychrome streaming technology after 16 weeks; Fig. 6 A detects the responsiveness cd8 t cell to secrete the test data of various cytokines; Fig. 6 B detects the central cd8 t cell to secrete the test data of various cytokines;
Fig. 7 is that MVTT and Ad5 vector HIV vaccine are united Rhesus Macacus is carried out behind the booster immunization detecting the figure as a result of the lymphocytic multiplication capacity of T with living cells dyestuff CF 5(6)-Carboxyfluorescein acetoacetic acid succinimide ester (CFSE) dyeing after 16 weeks; Fig. 7 A is the propagation situation of cd4 t cell; Fig. 7 B is the propagation situation of cd8 t cell;
Fig. 8 be experimental monkey groups behind the SIVmac239 counteracting toxic substances at each antigenic cellullar immunologic response of SIV; Fig. 8 A is the immunne response at structural protein (Gag, Pol and Env); Fig. 8 B is the immunne response at assistant regulating and controlling albumen (Nef, Vpx, Vpr, Vif, Rev and Tat);
Fig. 9 is the special antibody response level of SIV that detects SIVmac239 infection experiment monkey front and back by elisa technique;
Figure 10 is the data result figure by the intravital virus load peak value of quantitative PCR technique test experience monkey.
The specific embodiment
For making the present invention easier to understand,, further set forth the present invention below in conjunction with specific embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in to limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Embodiment one: adenovirus and the structure of poxvirus vaccine and the immunogenicity in mice of carrying SIVgag, pol and env gene
1, the acquisition of SIVmac239 gene
Obtain testing needed each gene by full gene is synthetic, present embodiment relates to whole 3 genes of SIVmac239 virus, i.e. SIVmac239gag, pol and env.Obtaining these proteic aminoacid sequences from ncbi database, is DNA sequence guaranteeing that its aminoacid sequence does not take place under the situation of any change according to the sub-reverse translation of people's source pin then, makes these antigens efficiently express in primate cell.
1.1 the SIVmac239gag gene optimization sequence of using in the present embodiment is as follows:
cgaagcttac?catgggcgtg?aggaactctg?tgctgtctgg?caagaaggct?gatgagctgg 60
agaagatcag?gctgaggccc?aatggcaaga?agaagtacat?gctgaagcat?gtggtgtggg 120
ctgccaatga?gctggacagg?tttggcctgg?ctgagtccct?gctggagaac?aaggagggct 180
gccagaagat?cctgtctgtg?ctggcccccc?tggtgcccac?aggctctgag?aacctgaagt 240
ccctgtacaa?cacagtgtgt?gtgatctggt?gcatccatgc?tgaggagaag?gtgaagcaca 300
cagaggaggc?caagcagatt?gtgcagaggc?acctggtggt?ggagacaggc?accacagaga 360
ccatgcccaa?gacctccagg?cccacagccc?cctcctctgg?cagggggggc?aactaccctg 420
tgcagcagat?tgggggcaac?tatgtgcacc?tgcccctgtc?ccccaggacc?ctgaatgcct 480
gggtgaagct?gattgaggag?aagaagtttg?gggctgaggt?ggtgcctggc?ttccaggccc 540
tgtctgaggg?ctgcaccccc?tatgacatca?accagatgct?gaactgtgtg?ggggaccacc 600
aggctgctat?gcagatcatc?agggacatca?tcaatgagga?ggctgctgac?tgggacctgc 660
agcaccccca?gcctgccccc?cagcagggcc?agctgaggga?gccctctggc?tctgacattg 720
ctggcaccac?ctcctctgtg?gatgagcaga?tccagtggat?gtacaggcag?cagaacccca 780
tccctgtggg?caacatctac?aggaggtgga?tccagctggg?cctgcagaag?tgtgtgagga 840
tgtacaaccc?caccaacatc?ctggatgtga?agcagggccc?caaggagccc?ttccagtcct 900
acgtggacag?gttctacaag?tccctgaggg?ctgagcagac?agatgctgct?gtgaagaact 960
ggatgaccca?gaccctgctg?atccagaatg?ccaaccctga?ctgcaagctg?gtgctgaagg 1020
gcctgggggt?gaaccccacc?ctggaggaga?tgctgacagc?ctgccagggg?gtggggggcc 1080
ctggccagaa?ggccaggctg?atggctgagg?ccctgaagga?ggccctggcc?cctgtgccca 1140
tcccctttgc?tgctgcccag?cagaggggcc?ccaggaagcc?catcaagtgc?tggaactgtg 1200
gcaaggaggg?ccactctgcc?aggcagtgca?gggcccccag?gaggcagggc?tgctggaagt 1260
gtggcaagat?ggaccatgtg?atggccaagt?gccctgacag?gcaggctggc?ttcctgggcc 1320
tgggcccctg?gggcaagaag?cccaggaact?tccccatggc?ccaggtgcac?cagggcctga 1380
tgcccacagc?cccccctgag?gaccctgctg?tggacctgct?gaagaactac?atgcagctgg 1440
gcaagcagca?gagggagaag?cagagggagt?ccagggagaa?gccctacaag?gaggtgacag 1500
aggacctgct?gcacctgaac?tccctgtttg?ggggggacca?gtaaagtaaa?gcccgggtct 1560
agagg 1565
1.2 the SIVmac239pol gene optimization sequence of using in the present embodiment is as follows:
gatccaccat?ggtgctggag?ctgtgggaga?ggggcaccct?gtgcaaggcc?atgcagtccc 60
ccaagaagac?aggcatgctg?gagatgtgga?agaatggccc?atgctatggc?cagatgccca 120
ggcagacagg?cggcttcttc?aggccatggt?ccatgggcaa?ggaggccccc?cagttccccc 180
atggctcctc?tgcctctggc?gctgatgcca?actgctcccc?caggggccca?tcctgtggct 240
ctgccaagga?gctgcatgct?gtgggccagg?ctgctgagag?gaaggctgag?aggaagcaga 300
gggaggccct?gcagggcggc?gacaggggct?ttgctgcccc?ccagttctcc?ctgtggagga 360
ggcctgtggt?gacagcccac?attgagggcc?agcctgtgga?ggtgctgctg?gacacaggcg 420
ctgatgactc?cattgtgaca?ggcattgagc?tgggccccca?ctacaccccc?aagattgtgg 480
gcggcattgg?cggcttcatc?aacaccaagg?agtacaagaa?tgtggagatt?gaggtgctgg 540
gcaagaggat?caagggcacc?atcatgacag?gcgacacccc?catcaacatc?tttggcagga 600
acctgctgac?agccctgggc?atgtccctga?acttccccat?tgccaaggtg?gagcctgtga 660
aggtggccct?gaagcctggc?aaggatggcc?ccaagctgaa?gcagtggccc?ctgtccaagg 720
agaagattgt?ggccctgagg?gagatctgtg?agaagatgga?gaaggatggc?cagctggagg 780
aggccccccc?caccaaccca?tacaacaccc?ccacctttgc?catcaagaag?aaggacaaga 840
acaagtggag?gatgctgatt?gacttcaggg?agctgaacag?ggtgacccag?gacttcacag 900
aggtgcagct?gggcatcccc?catcctgctg?gcctggccaa?gaggaagagg?atcacagtgc 960
tggacattgg?cgatgcctac?ttctccatcc?ccctggatga?ggagttcagg?cagtacacag 1020
ccttcaccct?gccatctgtg?aacaatgctg?agcctggcaa?gaggtacatc?tacaaggtgc 1080
tgccccaggg?ctggaagggc?tcccctgcca?tcttccagta?caccatgagg?catgtgctgg 1140
agccattcag?gaaggccaac?cctgatgtga?ccctggtgca?gtacatggat?gacatcctga 1200
ttgcctctga?caggacagac?ctggagcatg?acagggtggt?gctgcagtcc?aaggagctgc 1260
tgaactccat?tggcttctcc?acccctgagg?agaagttcca?gaaggacccc?ccattccagt 1320
ggatgggcta?tgagctgtgg?cccaccaagt?ggaagctgca?gaagattgag?ctgccccaga 1380
gggagacctg?gacagtgaat?gacatccaga?agctggtggg?cgtgctgaac?tgggctgccc 1440
agatctaccc?tggcatcaag?accaagcatc?tgtgcaggct?gatcaggggc?aagatgaccc 1500
tgacagagga?ggtgcagtgg?acagagatgg?ctgaggctga?gtatgaggag?aacaagatca 1560
tcctgtccca?agagcaggag?ggctgctact?accaggaggg?caagcccctg?gaggccacag 1620
tgatcaagtc?ccaggacaac?cagtggtcct?acaagatcca?tcaggaggac?aagatcctga 1680
aggtgggcaa?gtttgccaag?atcaagaaca?cccacaccaa?tggcgtgagg?ctgctggccc 1740
atgtgatcca?gaagattggc?aaggaggcca?ttgtgatctg?gggccaggtg?cccaagttcc 1800
atctgcctgt?ggagaaggat?gtctgggagc?agtggtggac?agactactgg?caggtgacct 1860
ggattcctga?gtgggacttc?atctccaccc?cccccctggt?gaggctggtc?ttcaacctgg 1920
tgaaggaccc?cattgagggc?gaggagacct?actacacaga?tggctcctgc?aacaagcagt 1980
ccaaggaggg?caaggctggc?tacatcacag?acaggggcaa?ggacaaggtg?aaggtgctgg 2040
agcagaccac?caaccagcag?gctgagctgg?aggccttcct?gatggccctg?acagactctg 2100
gccccaaggc?caacatcatt?gtggactccc?agtatgtgat?gggcatcatc?acaggctgcc 2160
ccacagagtc?tgagtccagg?ctggtgaacc?agatcattga?ggagatgatc?aagaagtctg 2220
agatctatgt?ggcctgggtg?cctgcccaca?agggcattgg?cggcaaccag?gagattgacc 2280
atctggtctc?ccagggcatc?aggcaggtgc?tgttcctgga?gaagattgag?cctgcccagg 2340
aggagcatga?caagtaccac?tccaatgtga?aggagctggt?cttcaagttt?ggcctgccca 2400
ggattgtggc?caggcagatt?gtggacacct?gtgacaagtg?ccatcagaag?ggcgaggcca 2460
tccatggcca?ggccaactct?gacctgggca?cctggcagat?ggactgcacc?catctggagg 2520
gcaagatcat?cattgtggct?gtgcatgtgg?cctctggctt?cattgaggct?gaggtgatcc 2580
cccaggagac?aggcaggcag?acagccctgt?tcctgctgaa?gctggctggc?aggtggccca 2640
tcacccatct?gcacacagac?aatggcgcca?actttgcctc?ccaagaggtg?aagatggtgg 2700
cctggtgggc?tggcattgag?cacacctttg?gcgtgccata?caacccccag?tcccagggcg 2760
tggtggaggc?catgaaccat?catctgaaga?accagattga?caggatcagg?gagcaggcca 2820
actctgtgga?gaccattgtg?ctgatggctg?tgcactgcat?gaacttcaag?aggaggggcg 2880
gcattggcga?catgacccct?gctgagaggc?tgatcaacat?gatcaccaca?gagcaggaga 2940
tccagttcca?gcagtccaag?aactccaagt?tcaagaactt?cagggtctac?tacagggagg 3000
gcagggacca?gctgtggaag?ggccctggcg?agctgctgtg?gaagggcgag?ggcgctgtga 3060
tcctgaaggt?gggcacagac?atcaaggtgg?tgcccaggag?gaaggccaag?atcatcaagg 3120
actatggcgg?cggcaaggag?gtggactcct?cctcccacat?ggaggacaca?ggcgaggcca 3180
gggaggtggc?tgactacaag?gatgatgatg?acaagtaaat?ctagagg 3227
1.3 the SIVmac239env gene optimization sequence of using in this research is as follows:
cgggatccac?catgggctgc?ctgggcaacc?agctgctgat?tgccatcctg?ctgctgtctg 60
tctatggcat?ctactgcacc?ctgtatgtga?cagtcttcta?tggcgtgcct?gcctggagga 120
atgccaccat?ccccctgttc?tgtgccacca?agaacaggga?cacctggggc?accacccagt 180
gcctgcctga?caatggcgac?tactctgagg?tggccctgaa?tgtgacagag?tcctttgatg 240
cctggaacaa?cacagtgaca?gagcaggcca?ttgaggatgt?ctggcagctg?tttgagacct 300
ccatcaagcc?atgtgtgaag?ctgtcccccc?tgtgcatcac?catgaggtgc?aacaagtctg 360
agacagacag?gtggggcctg?accaagtcca?tcaccaccac?agcctccacc?acctccacca 420
cagcctctgc?caaggtggac?atggtgaatg?agacctcctc?ctgcattgcc?caggacaact 480
gcacaggcct?ggagcaggag?cagatgatct?cctgcaagtt?caacatgaca?ggcctgaaga 540
gggacaagaa?gaaggagtac?aatgagacct?ggtactctgc?tgacctggtc?tgtgagcagg 600
gcaacaacac?aggcaatgag?tccaggtgct?acatgaacca?ctgcaacacc?tctgtgatcc 660
aggagtcctg?tgacaagcac?tactgggatg?ccatcaggtt?caggtactgt?gccccccctg 720
gctatgccct?gctgaggtgc?aatgacacca?actactctgg?cttcatgccc?aagtgctcca 780
aggtggtggt?ctcctcctgc?accaggatga?tggagaccca?gacctccacc?tggtttggct 840
tcaatggcac?cagggctgag?aacaggacct?acatctactg?gcatggcagg?gacaacagga 900
ccatcatctc?cctgaacaag?tactacaacc?tgaccatgaa?gtgcaggagg?cctggcaaca 960
agacagtgct?gcctgtgacc?atcatgtctg?gcctggtctt?ccactcccag?cccatcaatg 1020
acaggcccaa?gcaggcctgg?tgctggtttg?gcggcaagtg?gaaggatgcc?atcaaggagg 1080
tgaagcagac?cattgtgaag?catcccaggt?acacaggcac?caacaacaca?gacaagatca 1140
acctgacagc?ccctggcggc?ggcgaccctg?aggtgacctt?catgtggacc?aactgcaggg 1200
gcgagttcct?gtactgcaag?atgaactggt?tcctgaactg?ggtggaggac?aggaacacag 1260
ccaaccagaa?gcccaaggag?cagcacaaga?ggaactatgt?gccatgccac?atcaggcaga 1320
tcatcaacac?ctggcacaag?gtgggcaaga?atgtctacct?gccccccagg?gagggcgacc 1380
tgacctgcaa?ctccacagtg?acctccctga?ttgccaacat?tgactggatt?gatggcaacc 1440
agaccaacat?caccatgtct?gctgaggtgg?ctgagctgta?caggctggag?ctgggcgact 1500
acaagctggt?ggagatcacc?cccattggcc?tggcccccac?agatgtgaag?aggtacacca 1560
caggcggcac?ctccaggaac?aagaggggcg?tctttgtgct?gggcttcctg?ggcttcctgg 1620
ccacagctgg?ctctgccatg?ggcgctgcct?ccctgaccct?gacagcccag?tccaggaccc 1680
tgctggctgg?cattgtgcag?cagcagcagc?agctgctgga?tgtggtgaag?aggcagcagg 1740
agctgctgag?gctgacagtc?tggggcacca?agaacctgca?gaccagggtg?acagccattg 1800
agaagtacct?gaaggaccag?gcccagctga?atgcctgggg?ctgtgccttc?aggcaggtct 1860
gccacaccac?agtgccatgg?cccaatgcct?ccctgacccc?caagtggaac?aatgagacct 1920
ggcaggagtg?ggagaggaag?gtggacttcc?tggaggagaa?catcacagcc?ctgctggagg 1980
aggcccagat?ccagcaggag?aagaacatgt?atgagctgca?gaagctgaac?tcctgggatg 2040
tctttggcaa?ctggtttgac?ctggcctcct?ggatcaagta?catccagtat?ggcgtctaca 2100
ttgtggtggg?cgtgatcctg?ctgaggattg?tgatctacat?tgtgcagatg?ctggccaagc 2160
tgaggcaggg?ctacaggcct?gtcttctcct?cccccccatc?ctacttccag?cagacccaca 2220
tccagcagga?ccctgccctg?cccaccaggg?agggcaagga?gagggatggc?ggcgagggcg 2280
gcggcaactc?ctcctggcca?tggcagattg?agtacatcca?cttcctgatc?aggcagctga 2340
tcaggctgct?gacctggctg?ttctccaact?gccggaccct?gctgtccagg?gtctaccaga 2400
tcctgcagcc?catcctgcag?aggctgtctg?ccaccctgca?gaggatcagg?gaggtgctga 2460
ggacagagct?gacctacctg?cagtatggct?ggtcctactt?ccatgaggct?gtgcaggctg 2520
tctggaggtc?tgccacagag?accctggctg?gcgcctgggg?cgacctgtgg?gagaccctga 2580
ggaggggcgg?caggtggatt?ctggccatcc?ccaggaggat?caggcagggc?ctggagctga 2640
ccctgctgga?ctacaaggat?gatgatgaca?agtaaatcta?gagc 2684
2, the structure of recombinant adenovirus, amplification, purification and evaluation
Recombination adenovirus construction method according to routine is clipped to the said gene branch in the adenovirus vector, adenovirus vector wherein is the people's five type adenoviruss that lacked E1 and E3 zone, rescue obtains carrying the recombinant adenovirus of genes of interest then, express with the genome enzyme action after a small amount of amplification and identify, then amplification in a large number in the Trex293 cell, and adopt the cesium chloride gradient density centrifugal to carry out purification, the virus of purification is identified correct back its infection titer of mensuration TCID once more 50With physical particles concentration, packing frozen standby (the concrete operations step of correlation technique can be referring to the applicant's patent, and application number is 200710026295.X) then.Fig. 1 is the testing result figure that detects the recombinant adenovirus protein expression level with the western-blot method; As shown in Figure 1, the recombinant adenovirus of purification can high-caliber expression destination protein, and these albumen can be further processed into the various protein components of native form.Can express P55 and P27 as Ad5-gag, Ad5-pol can express P66/51, P31, and P10 etc., Ad5-env can express gp160, gp120, gp41.
3, carry the immunogenicity of adenovirus vector SIV holoantigen group vaccine in mice
Further, the immunogenicity of these vaccines has obtained checking in mice, 10 every group 6-8 age female C57BL/6 mice.Immunizing dose is 1 * 10 10The vp recombinant adenovirus/only/100 μ L, respectively inject 50 μ L at the quadriceps femoris of mice both sides respectively.Immunity is 2 times altogether, at interval 2 weeks.Fig. 2 is the initial immunity of recombinant adenovirus in mice and the immunogenicity comparison diagram behind the booster immunization; Fig. 2 A is the immunogenicity comparison diagram behind the initial immunity of recombinant adenovirus in mice; Fig. 2 B is the immunogenicity comparison diagram behind the booster immunization of recombinant adenovirus in mice; Shown in Fig. 2 A and 2B, take out the spleen of mice behind initial immunity and the booster immunization, mix by group, the preparation spleen cell carries out ELISPOT and detects, in each immune group, mice can detect stronger IFN-γ ELISPOT immunne response, wherein produces at the level of replying of SIV Gag and Pol relative higher.The free burial ground for the destitute is at the 5th group more intentionally, and promptly immune SIVgag of while can produce in the mice body of pol and env simultaneously at these 3 antigenic immunoreation of SIV.Than a kind of antigen of independent immunity, the immunne response level of this group descends to some extent, this may with a plurality of antigens of high level expression simultaneously in body, can cause between antigen vie each other relevant.Though descend to some extent at independent certain antigenic reaction, but we also can see at each antigenic W-response level and more tend to consistent, promptly more coordinate at each antigenic reaction, avoid having produced at some and antigenicly excessively reply, this may be more conducive to control duplicating of SIV virus.In control group mice, detect less than at the antigenic immunoreation of any SIV.
The poxvirus vector MVTT-gpe (strain of the modified form poxvirus the Temple of Heaven) that expresses SIVgag, pol and env is made up and is provided by the sincere medical college acquired immune deficiency syndrome (AIDS) of the Li Jia of Hong Kong University institute Chen Zhiwei professor, and its expression and antigenicity have obtained confirmation .Vaccine.2007 such as () Huang X by them.
Embodiment two: unite and use poxvirus vector SIV vaccine and the immunoreation of adenovirus vector SIV vaccine in Rhesus Macacus
1, experiment material
1.1 laboratory animal
16 non-human primate disease model study bases that Chinese Rhesus Macacus is set up cooperatively available from Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health and south China animals on the brink of extinction institute, body weight is 3-5kg, the age is 3-6 year, male and female half and half.These monkeys are carried out every detection before the experiment, determine that tubercule bacillus, shigella dysenteriae, Salmonella, STLV-1, SIV, SRV etc. are all negative.Zooperal operation meets the relevant regulations that laboratory animal is used.
1.2 virus
The poxvirus vector MVTT-gpe that expresses SIVgag, pol and env is made up and is provided by the sincere medical college acquired immune deficiency syndrome (AIDS) of the Li Jia of Hong Kong University institute Chen Zhiwei professor; Expression SIVgag, the pol of above-mentioned structure and the recombinant adenovirus of env (Ad5-SIVgag, Ad5-SIVpol and Ad5-SIVenv).
1.3 streaming antibody
Used monoclonal antibody (anti-people CD3-pacific blue (peaceful cattleya), anti-NHP CD4-FITC, anti-people CD4-AmCyan, anti-people CD8-PerCP, anti-people CD8-APC-Cy7 (SK1), anti-people CD28-FITC, anti-people CD95-PE-Cy5, anti-people TNFa-PE-CY7, anti-people IFN γ-PE, anti-people IL2-APC) available from BD company.
1.4SIV each antigenic polypeptide
All polypeptide are by the U.S.'s state-run health research institute's acquired immune deficiency syndrome (AIDS) research and reference reagent project (NIHAIDS Research ﹠amp; Reference Reagent Program) provide, most of peptide is made up of 15 aminoacid, has 11 aminoacid overlapping between any two. purity＞80%.Dissolve the storage liquid that is mixed with the 0.4mg/ml/ peptide ,-70 ℃ of preservations after the packing with dimethyl sulfoxide (DMSO).
1.5 other reagent
ELISPOT plate (brand: Millipore; Article No.: be bio tech ltd available from reaching section MSIPS4510), SIV virion pyrolysis product is oneself preparation, TMB/E substrate (article No.: ES001) available from U.S. Chemicon company.BCIP/NBT substrate (brand: Pierce; Article No.: 34042) available from the lucky safe bio tech ltd in Guangzhou.Cell dye CFSE is available from U.S.'s molecular probe (Molecule Probe) company.The link coupled alkali phosphatase of streptomycin (brand: BD PharMingen, article No.: 554065) available from the gene company limited.
2, buy the proved recipe method
2.1 animal grouping and immunization strategy
16 monkeys are divided into 4 groups, and every group of 4 monkeys are united poxvirus vector (MVTT) SIV vaccine and immunity and the counteracting toxic substances scheme of adenovirus (Ad5) carrier S IV vaccine in Rhesus Macacus used as shown in Figure 3.Detect index and comprise ELISPOT, multi-functional T cell, cell proliferation and antibody titer etc.Wherein adenovirus adopts the mode immunity of intramuscular injection, and dosage is 10 11The vp/ monkey.The consumption of poxvirus is 1 * 10 8The PFU/ml/ monkey is adopted nasal cavity immunity and Sublingual immunity.
2.2 the polychrome streaming technology detects multi-functional T cell
1) is adjusted to 2 * 10 behind the PBMC cell counting of separator well 6/ ml joins 24 well culture plates, every hole 1mL.Culture medium is R10.
2) add the specific antigen peptide in above-mentioned cell suspension, negative control hole adds the DMSO of respective concentration, positive control add Buddhist ripple ester (PMA) (20ng/ml)+ionomycin (1000ng/ml).
3) 37 ℃, 5%CO 2Cultivate 1-2h.
4) add brefeldin (BFA) (10 μ g/ml) mixing.
5) 37 ℃, 5%CO 2Cultivate 16h.
6) blow and beat even cell, be transferred to (12 * 75mm polystyrene tube) in the streaming pipe.Add 3ml streaming washing liquid.The 300g room temperature is centrifugal 10 minutes behind the mixing.
7) abandon supernatant.Add cell respectively and show traget antibody, as CD3-pacific blue (peaceful cattleya), CD4-AmCyan, CD8-APC-Cy7, CD28-FITC, CD95-PE-Cy5 vibrates 2-3 second.
8) the room temperature lucifuge was placed 25-30 minute.
9) add 3ml FACS washing liquid.The 300g room temperature is centrifugal 10 minutes behind the mixing.
10) repeated washing once.
11) the even cell that vibrates adds 250 μ l cell fixation/penetrating liquid (cytofix/cytoperm, brands: BD then; Article No.: 554722), mixing.
12) 4 ℃, lucifuge reaction 20min.
13) add 1ml1 * cell penetrating/cleaning mixture (perm/wash, brand: BD Biosciences; Article No.: 554723.Product is 10 times, with being diluted to 1 times with sterile distilled water before), 400g, centrifugal 8min.Abandon supernatant.
14) repeat once.
15) abandon supernatant, add IFN γ-PE/TNFa-PE CY7/IL2-APC (each 10ul antibody), mixing.
16) 4 ℃ lucifuge 30-60 minute.
17) add 1ml1 * cell penetrating/cleaning mixture (perm/wash), 400g, centrifugal 8min.
18) abandon supernatant, add 3ml streaming washing liquid.The 400g room temperature is centrifugal 10 minutes behind the mixing.
19) siphon away most of supernatant, stay 300 μ l approximately.Go up machine behind the vibration mixing.
20) on the FACSAria flow cytometer, detect, obtain data, use the Modfit software analysis then with Cell Quest software.
2.3IFN-γ elispot assay
First day:
Get a pipe coated antibody (anti-monkey IFN-gamma antibodies is to wherein including this antibody, brand: U-Cytech, the clone number be MD-1) and dilute at 1: 100 with aseptic PBS (pH7.2-7.4), 100 μ l/ holes join 96 hole pvdf membrane plates, and 4 ℃ of bags are spent the night.
Second day:
1. get rid of liquid, with aseptic PBS washing 3 times, every hole adds 200 μ l R10 complete mediums, and 37 ℃ were sealed 2 hours.
2. closed period separates PBMC.Adopt lymphocyte separation medium (commodity the are called opti-prep) isolated lymphocytes of 95% dilution.
3. discard confining liquid, every hole adds 100 μ l PBMC.
4. establish positive controls (ConA group), special peptide, blank (DMSO) in the experiment.In 37 ℃ of 5%CO2 incubators, hatch.
The 3rd day:
5. behind the 24h, discard cell suspension, with aseptic PBST 220 μ l/well washing 6 times.
6. get rid of washing liquid, on aseptic dry paper, buckle and do.
7. detect antibody (biotin labeled anti-monkey IFN-gamma antibodies) with the PBST that contains 5%FBS by dilution in 1: 400,100 μ l/well, 4 ℃ are spent the night.
The 4th day:
1. with aseptic PBST 220 μ l/ holes washing 4 times.
2. get rid of washing liquid, on aseptic dry paper, buckle and do.
3. dispose the link coupled alkali phosphatase of streptomycin: use the PBST that contains 5%FBS by 1: 2500 link coupled alkali phosphatase of dilution streptomycin, 100 μ l/ holes, 37 ℃, 2 hours.
4. (Pierce, Cat:34042), 37 ℃ of temperature are bathed 30min to treatments B CIP/NBT substrate.
5. with aseptic PBST 220 μ l/ holes washing 5 times, get rid of washing liquid, on aseptic dry paper, buckle and do.
6. add the BCIP/NBT substrate, 100 μ l/ holes, lucifuge reaction 7min.
7. discard substrate, wash with water 2 times.The air-dry plate of reading.
2.4CFSE detect the lymphopoiesis situation
1) the PBMC cell of separation Rhesus Macacus.
2) centrifugal 10 minutes of 300g behind the cell counting.Abandon supernatant, adding 0.1%FBS/PBS liquid is resuspended, washs once, and the 0.1%FBS/PBS of reuse suitable volumes number is resuspended, obtains 4 * 10 6The cell suspension of/ml.
3) with 0.1%FBS/PBS dilution CFSE storage liquid to 2 μ mol/L.Below operation lucifuge operation as far as possible.
4) respectively get above-mentioned cell suspension of 0.25ml and CFSE diluent, soft mixing.(this moment cell fragility, action will be tried one's best softly! )
5) place 10min for 37 ℃.
6) add ice-cold RPMI 1640 complete culture solutions, ice bath 5 minutes is to end dyeing.
7) the 300g room temperature is centrifugal 5 minutes.Abandon supernatant, add the ice-cold complete RPMI-1640 that contains 10%FBS and wash 2 times.Use 1ml R10 culture medium resuspended at last.
8) above-mentioned cell suspension is added 24 well culture plates, every hole 1ml uses SEB (final concentration 1ug/ml) and specific polypeptide storehouse (2 μ g/ml) to stimulate respectively.
9) 37 ℃, the 5%CO2 lucifuge was cultivated 5-6 days.
10) blow and beat even cell, be transferred to (12 * 75mm polystyrene tube) in the streaming pipe.Add the 4mlFACS washing liquid again.The 250g room temperature is centrifugal 10 minutes behind the mixing.
11) vacuum pump aspirates supernatant.Vibrate on the vortex oscillation instrument 2-3 second.
12) add 20ul CD4-PE, CD8-Percp, CD3-APC respectively.Vibrate 2-3 second.
13) the room temperature lucifuge was placed 25-30 minute.
14) add 4ml FACS washing liquid.The 300g room temperature is centrifugal 10 minutes behind the mixing.
15) siphon away most of supernatant, stay 300ul approximately.The vibration mixing.Directly go up machine.Or continued for the 15th step.
16) add 30ul 10% formalin (brand: Polysciences; Article No.: 04018), vibrate 10-20 second.
17) 4 ℃ keep in Dark Place, and go up machine testing in 24 hours.
18) on the FACScalibur flow cytometer, detect, obtain data, use the Modfit software analysis then with Cell Quest software.
2.5 data analysis
Use FlowJo software analysis polychrome fluidic cell data.Carry out data statistic analysis and mapping with JMP version 6.0.3 software, relatively the difference between each group adopts nonparametric Wilcoxon rank check.
3, experimental result
3.1MVTT/Ad5 the combined immunization strategy brings out stronger more persistent cellullar immunologic response in Chinese Rhesus Macacus
We have compared independent use or have united and use MVTT carrier and the immunogenicity of Ad5 carrier in monkey, the result shows that the immunoreation that an independent immune Ad5 carrier brings out will be higher than the MVTT carrier far away, is better than any carrier of independent use far away and unite the immune effect that uses these two kinds of carriers.Fig. 4 is that use MVTT carrier and the immunogenicity of Ad5 carrier in Rhesus Macacus are united in the ELISPOT detection; Fig. 4 A is the ELISPOT data apart from last immune 6 weeks, and Fig. 4 B is the ELISPOT data apart from last immune 21 weeks.As shown in Figure 4, we observe an independent immune modified form poxvirus the Temple of Heaven strain (MVTT) carrier, in the monkey body, brought out the cellullar immunologic response of certain intensity, at the antigenic stimulation average generation of its purpose of carrying 237 speckles (1,000,000 PBMC cells); Separately immunity our 5 type adenovirus vectors of making up have once then produced stronger cellullar immunologic response in the monkey body, at the antigenic stimulation average generation of its purpose of carrying 2643 speckles (1,000,000 PBMC cells).And just exempt from the MVTT carrier, at interval 6 week back reuse Ad5 carriers are strengthened, and have then brought out the cellullar immunologic response of high level in the monkey body, at the antigenic stimulation average generation of its purpose of carrying 7383 speckles (1,000,000 PBMC cells).Therefore this strategy on average is eager to excel 31 times more than than the level of replying of the cellular immunization that uses MVTT carrier to bring out separately, and also is eager to excel 2.8 times than the level of replying of using an Ad5 carrier separately.This strategy has not only brought out the cellullar immunologic response of high level soon in the monkey body, the more important thing is that this duration of the reaction is very long.Still can detect the ELISPOT reaction of high level the monkey of the last immunity 21 all backs combined immunization groups of distance.
3.2MVTT/Ad5 the combined immunization strategy has brought out more polyfunctional memory lymphocyte in Chinese Rhesus Macacus
Though IFN-γ ELISPOT technology has become the index of estimating the more common of HIV vaccine and extensively being approved; but current more approved viewpoint is immunoprotection and the lymphocyte quantity with multiple function dependency is arranged more; so-called multi-functional lymphocyte is meant that secretion simultaneously comprises the lymphocyte of the various kinds of cell factor of IFN-γ; these cytokines comprise IL-2; TNFa, MIP1 β etc.Estimating multi-functional T lymphocyte will become the important indicator of estimating the HIV vaccine effect gradually, and our development and perfect polychrome streaming technology are measured the polyfunctional T lymphocyte reaction situation that this strategy produces for this reason.The result as shown in Figure 5, Fig. 5 is that MVTT and Ad5 carrier are united Rhesus Macacus is carried out 6 weeks behind the booster immunization, the testing result of the CD8+T emiocytosis cytokine that the Gag peptide is stimulated with the polychrome streaming technology; At MVTT and after 6 weeks of Ad5 carrier combined immunization, the ability of not only secreting certain cytokine separately in the monkey of combined immunization group is strengthened greatly, and what is more important has detected the multi-functional CD8+T lymphocyte of more while secretion of gamma-IFN/TNF-a/IL-2 cytokine.Particularly, CD8+T lymphocyte while these 3 kinds of cytokines of secretion of gamma-IFN/TNF-α/IL-2 of 0.2464 ± 0.2770% are arranged in the monkey of MVTT and Ad5 carrier combined immunization, and 1.4668 ± 1.1845% CD8+T lymphocyte is these 2 kinds of cytokines of secretion of gamma-IFN/TNF-α simultaneously; And having only CD8+T lymphocyte while these 3 kinds of cytokines of secretion of gamma-IFN/TNF-α/IL-2 of 0.0320 ± 0.0472% in the monkey of independent immune Ad5 carrier, 0.1587 ± 0.2035% CD8+T lymphocyte is these 2 kinds of cytokines of secretion of gamma-IFN/TNF-α simultaneously.Promptly compare the immunity of independent Ad5 carrier, produced the multi-functional CD8+T lymphocyte of secretion of gamma-IFN/these the 3 kinds of cytokines of TNF-α/IL-2 simultaneously more than 7.7 times and the multi-functional CD8+T lymphocyte of secretion of gamma-IFN/these 2 kinds of cytokines of TNF-α simultaneously more than 9 times behind MVTT and the Ad5 carrier combined immunization.
We have further detected the generation of the specificity memory cell behind MVTT and the Ad5 carrier combined immunization and the ability of secrete cytokines.The result as shown in Figure 6, Fig. 6 is that MVTT and Ad5 carrier are united Rhesus Macacus is carried out behind the booster immunization detecting the testing result of the memory t cell secrete cytokines in the peripheral blood with the polychrome streaming technology after 16 weeks; Fig. 6 A detects the responsiveness cd8 t cell to secrete the test data of various cytokines; Fig. 6 B detects the central cd8 t cell to secrete the test data of various cytokines.The ability of the generation of SIV antigenic specificity memory CD4+T cell and CD8+T cell and secrete cytokines is all obviously stronger in these monkey bodies.Than other groups, the ability of the responsiveness memory CD8+ emiocytosis cytokine in the monkey peripheral blood of MVTT and Ad5 combined immunization group is obviously stronger, and these cells are based on two kinds of cytokines of while secretion of gamma-IFN/TNF-a.Though it is little respectively to organize the difference of ability of central memory CD8+ emiocytosis cytokine of experimental monkey groups, compares and the responsiveness memory T cell, the ability of these emiocytosis IL-2 is obviously stronger.In a word, we have detected MVTT and the Ad5 combined immunization can bring out more multipurpose memory T lymphocyte in the Rhesus Macacus body.The analysis showed that further that this method is induced has simultaneously produced central memory and responsiveness memory T cell, wherein remembers the CD8+T cell based on responsiveness.
3.3MVTT/Ad5 the combined immunization strategy has brought out stronger lymphopoiesis ability in Chinese Rhesus Macacus
Studies show that HIV does not make progress patient's CD4+T cell and CD8+T cell for a long time and has very by force at the antigenic in-vitro multiplication ability of HIV, and HIV morbidity patient's CD4+T cell and CD8+T cell are very weak at the antigenic in-vitro multiplication ability of HIV.And the viral copy number in this multiplication capacity and the patient's blood plasma exists negative correlation to a certain degree, and becomes positive correlation with the quantity of CD4+T cell.Therefore detecting the lymphocytic multiplication capacity of T can reflect the control degree of body to the HIV virus replication preferably.
Fig. 7 is that MVTT and Ad5 carrier are united Rhesus Macacus is carried out behind the booster immunization detecting the figure as a result of the lymphocytic multiplication capacity of T with CFSE dyeing after 16 weeks; Fig. 7 A is the propagation situation of cd4 t cell; Fig. 7 B is the propagation situation of cd8 t cell.Shown in Fig. 7 A, the PBMC of MVTT and Ad5 carrier combined immunization monkey has the CD4+ cell of (4.2725 ± 4.5516) % after external use SIV Gag stimulates be CFSElow, i.e. the daughter cell of division growth; And after the PBMC of Ad5 carrier immune monkey stimulates with SIV Gag separately the CD4+T cell of (3.2350 ± 1.7008) % is arranged is CFSElow.Therefore than independent Ad5 carrier, the CD4+T lymphopoiesis ability at antigenic stimulus behind MVTT and the Ad5 carrier combined immunization slightly strengthens.And shown in Fig. 7 B, after stimulating with SIV Gag the CD8+T cell of (7.4825 ± 5.8550) % being arranged is CFSElow, i.e. the daughter cell of division growth; And have only the CD8+T cell of (2.9475 ± 1.6951) % among the PBMC of independent Ad5 carrier immune monkey is CFSElow.Therefore than independent Ad5 carrier, the CD8+T lymphopoiesis ability at antigenic stimulus behind MVTT and the Ad5 carrier combined immunization has strengthened more than 2.5 times.
The MVTT carrier is just exempted from a word, and the immunization method that the Ad5 carrier is strengthened has produced stronger in Chinese Rhesus Macacus, the t cell responses of wide spectrum and greater functionality more, and this meets the index of nearest evaluation effective vaccine very much.This strategy is worth further research.
Embodiment three: unite and use the immunoprotection test in Rhesus Macacus of poxvirus vector and adenovirus vector SIV vaccine
1 experiment material:
1.1 laboratory animal is with embodiment 2.
1.2 virus
SIVmac239 virus obtains for inventor's rescue, and used SIVmac239phage molecular cloning is provided by the U.S.'s state-run health research institute's acquired immune deficiency syndrome (AIDS) research and reference reagent project.
1.3 antibody
Used monoclonal antibody (anti-people CD3-pacific blue, anti-NHP CD4-FITC, anti-people CD4-AmCyan, anti-people CD8-PerCP, anti-people CD8-APC-Cy7 (SK1), anti-people CD28-FITC, anti-people CD95-PE-Cy5, anti-people TNFa-PE-CY7, anti-people IFN γ-PE, anti-people IL2-APC) available from BD company.The anti-monkey IgG of HRP-is available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing.
1.4 other reagent
Fluorescence quantitative RT-PCR detecting kit (QuantiTect SYBR Green RT-PCR Kit, article No.:: 204243) available from Qiagen company, erythrocyte cracked liquid (FACS lysis solution), absolute counting pipe (BDTruCOUNT Tubes, article No. 340334) is available from BD Biosciences company.Other reagent are referring to embodiment 2.
2 experimental techniques
2.1 animal grouping and counteracting toxic substances experiment
16 monkeys are divided into 4 groups, every group of 4 monkeys, each the group immune programme for children as shown in Figure 3, the last time the immunity 22 all rectum taint with SIV mac239 viruses, dosage is 10 5TCID 50Concrete grammar is monkey fasting 24h, keeps a buttocks high position, inserts anus 4-7cm with children stomach vessel, squeezes into virus, and squeezes into little air.Keep this position 20min at least.
2.2 quantitative PCR is measured the SIV virus load
1) conventional method separated plasma.
2) the SIV viral RNA in the extraction blood plasma.
3) carry out one-step method fluorescent quantitation RT-CR, standard curve is all done in each test.
4) the configuration reaction system (use the SYBR Green RT-PCR test kit of QuantiTect, article No.: 204243):
2x QuantiTect SYBR Green RT-PCR primary response liquid 25 μ l
QuantiTect?RT?mix 0.5μl
Test kit carries primer 1 (10 μ M) 0.5 μ l
Test kit carries primer 2 (10 μ M) 0.5 μ l
RNA template 2 μ l
RNase-free water 21.5 μ l
Cumulative volume 50 μ l
5) layout is tested in the reasonable quantity arrangement per sample.Carry out the RT-PCR reaction condition according to following condition then:
a)50℃ 30min
b)95℃ 15min
c)94℃ 15s
d)60℃ 30s
e)72℃ 30s
F) read plate;
G) 3)-6) 45 weeks of circulation;
H) the 65-95 degree is done melt curve analysis, every 0.5 ℃ of reading, keeps for 1 second;
I) remain on 24 ℃;
6) according to standard curve, utilize Opticon Monitor 3 softwares to obtain the viral copy number of each sample, reduction obtains viral copy number in every milliliter of blood according to extension rate then.
2.3SIV virion lysate ELISA method detects binding antibody
1) envelope antigen: the every hole of 96 orifice plates drips 100 μ l antigens (1 μ g/ml dilutes with the PBS of pH7.4 or the carbonate buffer solution of pH9.6), and 4 ℃ of bags are spent the night.
2) wash plate: outwell antigen next day, dry, PBS washing 3 times.
3) sealing: add the PBST that contains 5% defatted milk powder, every hole 250 μ l, 37 ℃ of sealing 1h.
4) wash plate: every hole adds 200 μ l PBST, and 3min/ time, wash 3 times, preserving moisture, it is standby to preserve.
5) add specimen to be measured: every hole adds 100 μ l, and each sample is done diplopore and measured, 37 ℃ of reaction 2h.(confining liquid is used in dilution if desired)
6) wash plate: every hole adds 200 μ l PBST, 3min/ time, washes 6 times.
7) in conjunction with second antibody: every hole adds anti-Mus IgG (1: 8000) antibody of the suitable dilution factor HRP-of 100 μ l or anti-human IgG (1: 4000), hatches 1h for 37 ℃.(diluting) with confining liquid
8) wash plate: every hole adds 200 μ l PBST, 3min/ time, washes 6 times.
9) colour developing: every hole adds 100 μ l enzyme reaction substrate TMB, and room temperature was placed 5-10 minute.
10) H of the isopyknic 1M of adding 2SO 4Cessation reaction.
11) result measures: measure OD with the ELISA Plate spectrophotometer at the 450nm wavelength 450Value
2.4 additive method is referring to embodiment 2.
3, experimental result
3.1 behind the Rhesus Macacus counteracting toxic substances at each antigenic cellullar immunologic response situation
Fig. 8 be experimental monkey groups behind the SIVmac239 counteracting toxic substances at each antigenic cellullar immunologic response of SIV; Fig. 8 A is the immunne response at structural protein (Gag, Pol and Env); Fig. 8 B is the immunne response at assistant regulating and controlling albumen (Nef, Vpx, Vpr, Vif, Rev and Tat).Shown in Fig. 8 A, after being subjected to the SIVmac239 virus attack, risings that be swift in response of the immunogen (gag/pol/env) of carrying at vaccine in the monkey body of combined immunization vaccine group, the reaction of matched group are then relatively slower, and response strength also relatively a little less than.This shows that immunization method of the present invention has brought out memory T cell effectively, and these cells are activated rapidly after running into the same antigen stimulation and produce corresponding immunne response.The significant reaction of the immunogen (nef/vpx/vpr/vif/rev/tat) of more ironically in the monkey body of combined immunization group, not carrying at vaccine than matched group a little less than, we infer that this is because SIV virus is controlled at lower level effectively or does not have infected, just lower at the viral antigenic reaction level that carries itself like this in these monkey bodies.This conjecture has obtained confirmation in ensuing virus load determination experiment.
3.2 the antibody mediated immunity at SIV behind the Rhesus Macacus counteracting toxic substances is replied situation
We also detect the antibody in these experiment monkey different times serum, as shown in Figure 9, detect the special antibody response level of SIV of SIVmac239 infection experiment monkey front and back by elisa technique, no matter be MVTT carrier or Ad5 carrier, the antibody horizontal of most of monkey is all very low behind the initial immunity, but the antibody horizontal of most of monkey raises rapidly behind booster immunization.The free burial ground for the destitute is that the monkey of immune group antibody horizontal after being subjected to virus attack has been elevated to high level rapidly more intentionally.And the experimental monkey groups of matched group detects before counteracting toxic substances less than antibody, and the time that antibody produces behind the counteracting toxic substances is slow relatively, and titre is lower.
3.3 the intravital virus load of Rhesus Macacus is measured
Behind the counteracting toxic substances, the virus load of all monkeys reached peak value at 10-14 days.As can be seen from Figure 10, the monkey body inner virus carrying capacity peakedness ratio matched group of combined immunization group has obvious minimizing.Have 1 in 4 monkeys of combined immunization group and also do not detect SIV virus so far, other 3 virus load is compared than matched group and has been reduced the 1.37log value.This federation policies that shows us has been controlled the SIV viral infection effectively and has been duplicated at acute infection period, and the permanent immunity protection effect of this method needs longer observation time to confirm.Have any to need to specify, we are with more difficult neutral SIVmac239 strain high dose mucosal infections, and before others' research major part with virulence more weak strain relatively.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention has been done detailed description with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from the essence and the scope of technical solution of the present invention.
Sequence table
＜120〉unite method and the application thereof of using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine
<160>3
<210>1
<211>1565
<212>DNA
<213>SIVmac239gag
<222>(1)...(1565)
<400>1
cgaagcttac?catgggcgtg?aggaactctg?tgctgtctgg?caagaaggct?gatgagctgg 60
agaagatcag?gctgaggccc?aatggcaaga?agaagtacat?gctgaagcat?gtggtgtggg 120
ctgccaatga?gctggacagg?tttggcctgg?ctgagtccct?gctggagaac?aaggagggct 180
gccagaagat?cctgtctgtg?ctggcccccc?tggtgcccac?aggctctgag?aacctgaagt 240
ccctgtacaa?cacagtgtgt?gtgatctggt?gcatccatgc?tgaggagaag?gtgaagcaca 300
cagaggaggc?caagcagatt?gtgcagaggc?acctggtggt?ggagacaggc?accacagaga 360
ccatgcccaa?gacctccagg?cccacagccc?cctcctctgg?cagggggggc?aactaccctg 420
tgcagcagat?tgggggcaac?tatgtgcacc?tgcccctgtc?ccccaggacc?ctgaatgcct 480
gggtgaagct?gattgaggag?aagaagtttg?gggctgaggt?ggtgcctggc?ttccaggccc 540
tgtctgaggg?ctgcaccccc?tatgacatca?accagatgct?gaactgtgtg?ggggaccacc 600
aggctgctat?gcagatcatc?agggacatca?tcaatgagga?ggctgctgac?tgggacctgc 660
agcaccccca?gcctgccccc?cagcagggcc?agctgaggga?gccctctggc?tctgacattg 720
ctggcaccac?ctcctctgtg?gatgagcaga?tccagtggat?gtacaggcag?cagaacccca 780
tccctgtggg?caacatctac?aggaggtgga?tccagctggg?cctgcagaag?tgtgtgagga 840
tgtacaaccc?caccaacatc?ctggatgtga?agcagggccc?caaggagccc?ttccagtcct 900
acgtggacag?gttctacaag?tccctgaggg?ctgagcagac?agatgctgct?gtgaagaact 960
ggatgaccca?gaccctgctg?atccagaatg?ccaaccctga?ctgcaagctg?gtgctgaagg 1020
gcctgggggt?gaaccccacc?ctggaggaga?tgctgacagc?ctgccagggg?gtggggggcc 1080
ctggccagaa?ggccaggctg?atggctgagg?ccctgaagga?ggccctggcc?cctgtgccca 1140
tcccctttgc?tgctgcccag?cagaggggcc?ccaggaagcc?catcaagtgc?tggaactgtg 1200
gcaaggaggg?ccactctgcc?aggcagtgca?gggcccccag?gaggcagggc?tgctggaagt 1260
gtggcaagat?ggaccatgtg?atggccaagt?gccctgacag?gcaggctggc?ttcctgggcc 1320
tgggcccctg?gggcaagaag?cccaggaact?tccccatggc?ccaggtgcac?cagggcctga 1380
tgcccacagc?cccccctgag?gaccctgctg?tggacctgct?gaagaactac?atgcagctgg 1440
gcaagcagca?gagggagaag?cagagggagt?ccagggagaa?gccctacaag?gaggtgacag 1500
aggacctgct?gcacctgaac?tccctgtttg?ggggggacca?gtaaagtaaa?gcccgggtct 1560
agagg 1565
<210>1
<211>3227
<212>DNA
<213>SIVmac239pol
<222>(1)...(3227)
<400>2
gatccaccat?ggtgctggag?ctgtgggaga?ggggcaccct?gtgcaaggcc?atgcagtccc 60
ccaagaagac?aggcatgctg?gagatgtgga?agaatggccc?atgctatggc?cagatgccca 120
ggcagacagg?cggcttcttc?aggccatggt?ccatgggcaa?ggaggccccc?cagttccccc 180
atggctcctc?tgcctctggc?gctgatgcca?actgctcccc?caggggccca?tcctgtggct 240
ctgccaagga?gctgcatgct?gtgggccagg?ctgctgagag?gaaggctgag?aggaagcaga 300
gggaggccct?gcagggcggc?gacaggggct?ttgctgcccc?ccagttctcc?ctgtggagga 360
ggcctgtggt?gacagcccac?attgagggcc?agcctgtgga?ggtgctgctg?gacacaggcg 420
ctgatgactc?cattgtgaca?ggcattgagc?tgggccccca?ctacaccccc?aagattgtgg 480
gcggcattgg?cggcttcatc?aacaccaagg?agtacaagaa?tgtggagatt?gaggtgctgg 540
gcaagaggat?caagggcacc?atcatgacag?gcgacacccc?catcaacatc?tttggcagga 600
acctgctgac?agccctgggc?atgtccctga?acttccccat?tgccaaggtg?gagcctgtga 660
aggtggccct?gaagcctggc?aaggatggcc?ccaagctgaa?gcagtggccc?ctgtccaagg 720
agaagattgt?ggccctgagg?gagatctgtg?agaagatgga?gaaggatggc?cagctggagg 780
aggccccccc?caccaaccca?tacaacaccc?ccacctttgc?catcaagaag?aaggacaaga 840
acaagtggag?gatgctgatt?gacttcaggg?agctgaacag?ggtgacccag?gacttcacag 900
aggtgcagct?gggcatcccc?catcctgctg?gcctggccaa?gaggaagagg?atcacagtgc 960
tggacattgg?cgatgcctac?ttctccatcc?ccctggatga?ggagttcagg?cagtacacag 1020
ccttcaccct?gccatctgtg?aacaatgctg?agcctggcaa?gaggtacatc?tacaaggtgc 1080
tgccccaggg?ctggaagggc?tcccctgcca?tcttccagta?caccatgagg?catgtgctgg 1140
agccattcag?gaaggccaac?cctgatgtga?ccctggtgca?gtacatggat?gacatcctga 1200
ttgcctctga?caggacagac?ctggagcatg?acagggtggt?gctgcagtcc?aaggagctgc 1260
tgaactccat?tggcttctcc?acccctgagg?agaagttcca?gaaggacccc?ccattccagt 1320
ggatgggcta?tgagctgtgg?cccaccaagt?ggaagctgca?gaagattgag?ctgccccaga 1380
gggagacctg?gacagtgaat?gacatccaga?agctggtggg?cgtgctgaac?tgggctgccc 1440
agatctaccc?tggcatcaag?accaagcatc?tgtgcaggct?gatcaggggc?aagatgaccc 1500
tgacagagga?ggtgcagtgg?acagagatgg?ctgaggctga?gtatgaggag?aacaagatca 1560
tcctgtccca?agagcaggag?ggctgctact?accaggaggg?caagcccctg?gaggccacag 1620
tgatcaagtc?ccaggacaac?cagtggtcct?acaagatcca?tcaggaggac?aagatcctga 1680
aggtgggcaa?gtttgccaag?atcaagaaca?cccacaccaa?tggcgtgagg?ctgctggccc 1740
atgtgatcca?gaagattggc?aaggaggcca?ttgtgatctg?gggccaggtg?cccaagttcc 1800
atctgcctgt?ggagaaggat?gtctgggagc?agtggtggac?agactactgg?caggtgacct 1860
ggattcctga?gtgggacttc?atctccaccc?cccccctggt?gaggctggtc?ttcaacctgg 1920
tgaaggaccc?cattgagggc?gaggagacct?actacacaga?tggctcctgc?aacaagcagt 1980
ccaaggaggg?caaggctggc?tacatcacag?acaggggcaa?ggacaaggtg?aaggtgctgg 2040
agcagaccac?caaccagcag?gctgagctgg?aggccttcct?gatggccctg?acagactctg 2100
gccccaaggc?caacatcatt?gtggactccc?agtatgtgat?gggcatcatc?acaggctgcc 2160
ccacagagtc?tgagtccagg?ctggtgaacc?agatcattga?ggagatgatc?aagaagtctg 2220
agatctatgt?ggcctgggtg?cctgcccaca?agggcattgg?cggcaaccag?gagattgacc 2280
atctggtctc?ccagggcatc?aggcaggtgc?tgttcctgga?gaagattgag?cctgcccagg 2340
aggagcatga?caagtaccac?tccaatgtga?aggagctggt?cttcaagttt?ggcctgccca 2400
ggattgtggc?caggcagatt?gtggacacct?gtgacaagtg?ccatcagaag?ggcgaggcca 2460
tccatggcca?ggccaactct?gacctgggca?cctggcagat?ggactgcacc?catctggagg 2520
gcaagatcat?cattgtggct?gtgcatgtgg?cctctggctt?cattgaggct?gaggtgatcc 2580
cccaggagac?aggcaggcag?acagccctgt?tcctgctgaa?gctggctggc?aggtggccca 2640
tcacccatct?gcacacagac?aatggcgcca?actttgcctc?ccaagaggtg?aagatggtgg 2700
cctggtgggc?tggcattgag?cacacctttg?gcgtgccata?caacccccag?tcccagggcg 2760
tggtggaggc?catgaaccat?catctgaaga?accagattga?caggatcagg?gagcaggcca 2820
actctgtgga?gaccattgtg?ctgatggctg?tgcactgcat?gaacttcaag?aggaggggcg 2880
gcattggcga?catgacccct?gctgagaggc?tgatcaacat?gatcaccaca?gagcaggaga 2940
tccagttcca?gcagtccaag?aactccaagt?tcaagaactt?cagggtctac?tacagggagg 3000
gcagggacca?gctgtggaag?ggccctggcg?agctgctgtg?gaagggcgag?ggcgctgtga 3060
tcctgaaggt?gggcacagac?atcaaggtgg?tgcccaggag?gaaggccaag?atcatcaagg 3120
actatggcgg?cggcaaggag?gtggactcct?cctcccacat?ggaggacaca?ggcgaggcca 3180
gggaggtggc tgactacaag?gatgatgatg?acaagtaaat ctagagg 3227
<210>1
<211>2684
<212>DNA
<213>SIVmac239env
<222>(1)...(2684)
<400>3
cgggatccac?catgggctgc?ctgggcaacc?agctgctgat?tgccatcctg?ctgctgtctg 60
tctatggcat?ctactgcacc?ctgtatgtga?cagtcttcta?tggcgtgcct?gcctggagga 120
atgccaccat?ccccctgttc?tgtgccacca?agaacaggga?cacctggggc?accacccagt 180
gcctgcctga?caatggcgac?tactctgagg?tggccctgaa?tgtgacagag?tcctttgatg 240
cctggaacaa?cacagtgaca?gagcaggcca?ttgaggatgt?ctggcagctg?tttgagacct 300
ccatcaagcc?atgtgtgaag?ctgtcccccc?tgtgcatcac?catgaggtgc?aacaagtctg 360
agacagacag?gtggggcctg?accaagtcca?tcaccaccac?agcctccacc?acctccacca 420
cagcctctgc?caaggtggac?atggtgaatg?agacctcctc?ctgcattgcc?caggacaact 480
gcacaggcct?ggagcaggag?cagatgatct?cctgcaagtt?caacatgaca?ggcctgaaga 540
gggacaagaa?gaaggagtac?aatgagacct?ggtactctgc?tgacctggtc?tgtgagcagg 600
gcaacaacac?aggcaatgag?tccaggtgct?acatgaacca?ctgcaacacc?tctgtgatcc 660
aggagtcctg?tgacaagcac?tactgggatg?ccatcaggtt?caggtactgt?gccccccctg 720
gctatgccct?gctgaggtgc?aatgacacca?actactctgg?cttcatgccc?aagtgctcca 780
aggtggtggt?ctcctcctgc?accaggatga?tggagaccca?gacctccacc?tggtttggct 840
tcaatggcac?cagggctgag?aacaggacct?acatctactg?gcatggcagg?gacaacagga 900
ccatcatctc?cctgaacaag?tactacaacc?tgaccatgaa?gtgcaggagg?cctggcaaca 960
agacagtgct?gcctgtgacc?atcatgtctg?gcctggtctt?ccactcccag?cccatcaatg 1020
acaggcccaa?gcaggcctgg?tgctggtttg?gcggcaagtg?gaaggatgcc?atcaaggagg 1080
tgaagcagac?cattgtgaag?catcccaggt?acacaggcac?caacaacaca?gacaagatca 1140
acctgacagc?ccctggcggc?ggcgaccctg?aggtgacctt?catgtggacc?aactgcaggg 1200
gcgagttcct?gtactgcaag?atgaactggt?tcctgaactg?ggtggaggac?aggaacacag 1260
ccaaccagaa?gcccaaggag?cagcacaaga?ggaactatgt?gccatgccac?atcaggcaga 1320
tcatcaacac?ctggcacaag?gtgggcaaga?atgtctacct?gccccccagg?gagggcgacc 1380
tgacctgcaa?ctccacagtg?acctccctga?ttgccaacat?tgactggatt?gatggcaacc 1440
agaccaacat?caccatgtct?gctgaggtgg?ctgagctgta?caggctggag?ctgggcgact 1500
acaagctggt?ggagatcacc?cccattggcc?tggcccccac?agatgtgaag?aggtacacca 1560
caggcggcac?ctccaggaac?aagaggggcg?tctttgtgct?gggcttcctg?ggcttcctgg 1620
ccacagctgg?ctctgccatg?ggcgctgcct?ccctgaccct?gacagcccag?tccaggaccc 1680
tgctggctgg?cattgtgcag?cagcagcagc?agctgctgga?tgtggtgaag?aggcagcagg 1740
agctgctgag?gctgacagtc?tggggcacca?agaacctgca?gaccagggtg?acagccattg 1800
agaagtacct?gaaggaccag?gcccagctga?atgcctgggg?ctgtgccttc?aggcaggtct 1860
gccacaccac?agtgccatgg?cccaatgcct?ccctgacccc?caagtggaac?aatgagacct 1920
ggcaggagtg?ggagaggaag?gtggacttcc?tggaggagaa?catcacagcc?ctgctggagg 1980
aggcccagat?ccagcaggag?aagaacatgt?atgagctgca?gaagctgaac?tcctgggatg 2040
tctttggcaa?ctggtttgac?ctggcctcct?ggatcaagta?catccagtat?ggcgtctaca 2100
ttgtggtggg?cgtgatcctg?ctgaggattg?tgatctacat?tgtgcagatg?ctggccaagc 2160
tgaggcaggg?ctacaggcct?gtcttctcct?cccccccatc?ctacttccag?cagacccaca 2220
tccagcagga?ccctgccctg?cccaccaggg?agggcaagga?gagggatggc?ggcgagggcg 2280
gcggcaactc?ctcctggcca?tggcagattg?agtacatcca?cttcctgatc?aggcagctga 2340
tcaggctgct?gacctggctg?ttctccaact?gccggaccct?gctgtccagg?gtctaccaga 2400
tcctgcagcc?catcctgcag?aggctgtctg?ccaccctgca?gaggatcagg?gaggtgctga 2460
ggacagagct?gacctacctg?cagtatggct?ggtcctactt?ccatgaggct?gtgcaggctg 2520
tctggaggtc?tgccacagag?accctggctg?gcgcctgggg?cgacctgtgg?gagaccctga 2580
ggaggggcgg?caggtggatt?ctggccatcc?ccaggaggat?caggcagggc?ctggagctga 2640
ccctgctgga?ctacaaggat?gatgatgaca?agtaaatcta?gagc 2684

Claims (12)

1, unites the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine, it is characterized in that, unite and use poxvirus vector HIV vaccine and adenoviral vector HIV vaccine immunity body.
2, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that, uses poxvirus vector HIV vaccine immunity body earlier, re-uses adenoviral vector HIV vaccine immunity body.
3, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that, uses adenoviral vector HIV vaccine immunity body earlier, re-uses poxvirus vector HIV vaccine immunity body.
4, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that described poxvirus vector HIV vaccine contains HIVgag, pol and env gene.
5, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 4, it is characterized in that, described poxvirus vector HIV vaccine also can contain the assistant regulating and controlling protein gene, and described assistant regulating and controlling protein gene comprises nef, vpx, vpr, vif, rev and tat gene.
6, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that described adenoviral vector HIV vaccine contains HIVgag, pol and env gene.
7, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 6, it is characterized in that, described adenoviral vector HIV vaccine also can contain the assistant regulating and controlling protein gene, and described assistant regulating and controlling protein gene comprises nef, vpx, vpr, vif, rev and tat gene.
8, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that the approach of described immune body comprises mucosal route, intramuscular injection path and intravenous injection approach.
9, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that described adenovirus vector is 5 type adenovirus vectors.
10, the method for using poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of uniting according to claim 1 is characterized in that described poxvirus vector is a modified form the Temple of Heaven strain poxvirus vector.
11, the described application of method in preventing and treating acquired immune deficiency syndrome (AIDS) of uniting use poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of one of claim 1-10.
12, according to the described application of method in prevention and control hepatitis B, Ebola virus and tumor of uniting use poxvirus vector HIV vaccine and adenoviral vector HIV vaccine of one of claim 1-10.
CN200910192351A 2009-09-15 2009-09-15 Method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine and application Pending CN101670102A (en)

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Cited By (4)

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WO2011032489A1 (en) * 2009-09-15 2011-03-24 中国科学院广州生物医院与健康研究院 Method of using poxvirus vector hiv vaccine combined with adenoviral vector hiv vaccine and the uses thereof
WO2013010300A1 (en) * 2011-07-18 2013-01-24 Zeng Yi Sequential and repeated application of four or more hiv vector gene vaccines
CN107106673A (en) * 2014-09-03 2017-08-29 巴法里安诺迪克有限公司 Method and composition for strengthening immune response
CN110624106A (en) * 2018-06-22 2019-12-31 嘉和生物药业有限公司 Combined application of PD-1 signal channel antagonist and AIDS vaccine

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CN1321694C (en) * 2005-05-20 2007-06-20 吉林大学 Freezedrying type vaccine of AIDS recombined poxvirus with Ankara gene stock carrier, and preparation method
CN101394868B (en) * 2006-03-07 2011-08-10 浙江海正药业股份有限公司 Strong immune induction by using combination of adenovirus type-5/type-35 vector and vaccinia virus mva vector
CN101219221B (en) * 2007-01-12 2011-06-15 中国科学院广州生物医药与健康研究院 Adenovirus carrier vaccine carrying HIV gene
CN101670102A (en) * 2009-09-15 2010-03-17 中国科学院广州生物医药与健康研究院 Method for combining poxvirus vector HIV vaccine and adenoviral vector HIV vaccine and application

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011032489A1 (en) * 2009-09-15 2011-03-24 中国科学院广州生物医院与健康研究院 Method of using poxvirus vector hiv vaccine combined with adenoviral vector hiv vaccine and the uses thereof
WO2013010300A1 (en) * 2011-07-18 2013-01-24 Zeng Yi Sequential and repeated application of four or more hiv vector gene vaccines
CN107106673A (en) * 2014-09-03 2017-08-29 巴法里安诺迪克有限公司 Method and composition for strengthening immune response
CN110624106A (en) * 2018-06-22 2019-12-31 嘉和生物药业有限公司 Combined application of PD-1 signal channel antagonist and AIDS vaccine

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