CN101021539A - HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method - Google Patents

HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method Download PDF

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CN101021539A
CN101021539A CN 200710090957 CN200710090957A CN101021539A CN 101021539 A CN101021539 A CN 101021539A CN 200710090957 CN200710090957 CN 200710090957 CN 200710090957 A CN200710090957 A CN 200710090957A CN 101021539 A CN101021539 A CN 101021539A
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hiv
allosteric
envelope protein
env gene
gene dna
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叶新新
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Abstract

This invention is called: the experiment and method of the protein envelope which is allosteric restructuring of the HIV-Env gene DNA and its antigenic immunity responsive HIV. It relates to the domain of life sciences and medicine sciences. It is confirmed that gp120 of the HIV protein envelope or the gp160 of the antigenic vaccine combines with cell CD4+ that can induce the infirm immunogen of the alloantigen of the immune tolerant receptor CD4 and can not induce the HIV to infect the immune response of the parasitifer. It can also eliminate the multi-clone group of hill of the wild virus which is of fast copy the initial infection virus, high variability and sequence diversity. This invention can develop the non-spark human tolerant immunity HIV vaccine of the protein envelope which is allosteric restructuring of the HIV-Env gene DNA, the HIV preventive vaccine that can provide the scientific basis of the direct animal experiment and direct clinical trial of human being. It also provides the enforceable anti-HIV cell model, rodent model, non-human Primates model and the research program of human clinical trial.

Description

HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method
Technical field
A kind of HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method have been the present invention relates to.It relates to life science, reaches field of medicaments.Disclose HIV envelope protein gp120 or gp160 antigen-vaccine, be and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation; the immune response of HIV infection host be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed.Can develop and not trigger people's immune tolerance, HIV-Env gene DNA allosteric recombinant envelope protein antigen HIV treatment vaccine, HIV prevention vaccine provide enough fully direct animal model experiment scientific basis, direct clinical human trial scientific basis.Provide enforceable: anti-HIV cell model, rodent models, non-human primate's model and clinical human trial scientific research scheme.
Background technology
There are three big difficult points in modern HIV scientific research: fast, the high variability of 1.HIV reproduction speed, virus sequence diversity; Do not find---can not infect in the human body at HIV and find; can induce the immune response of initial HIV infection population; strong immunogene that effectively remove fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high, each hypotype HIV protective immunity is former---strong immunogene, the hypotype HIV protective immunity of intersecting former---.2.HIV infection duplication produces envelope protein gp120 or gp160 antigen, is and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation; the immune response of HIV infection host be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed.3. there are not in than short-term, to assess checking candidate HIV vaccine Effective Vate of Protection, the alternative terminal point of HIV vaccine clinical testing; And do not have from hundreds of candidate HIV vaccine that enters clinical research, assessment filters out a kind of ability that the HIV vaccine of actual application value is arranged.Can not develop the HIV vaccine that may effectively prevent HIV infected person types of populations.Be the inevitable science reality of outwardness; Can not be slipped 3 most important modern HIV scientific research difficult point problems that need to be resolved hurrily.
Produce security consideration, the consideration of HIV vaccine human body application security based on the HIV vaccine; modern HIV scientific research; the HIV partial immunity that can only select to use safety assurance is former; antigenic determinant is few, certain HIV protective immunity of immunogenicity difference is former, and development and production can be applied to the former synthetic vaccine of HIV protective immunity of human body.The former antigenic determinant that has of HIV protective immunity is few, is modern HIV scientific research, is difficult to use the greatest factor that it develops the synthetic HIV vaccine with higher protection ratio.HIV infects---acquired immune deficiency syndrome (AIDS) AIDS be can not self-healing human diseases; The retrovirus reproduction speed of other of the HIV and the mankind is fast, high variability, the virus sequence diversity is high, as influenza virus, Ai Bola virus ... highly pathogenic retroviral infection difference such as hepatitis C virus, all initial HIV the infected's acute stage plasma viral mass formed by blood stasis, keep that massive duplication virion degraded every day several weeks produces, various HIV immunogenes, can not induce the immune response of HIV infection host, it is fast effectively to remove initial infector virus replication speed, high variability, the wild virus polyclone group of hill that the virus sequence diversity is high, the lifelong chronic persistent infection HIV nature disease process of HIV infection host is irreversible, existing more than 6,000 ten thousand HIV infection morbidity person crowds do not have the HIV case of self-healing, there is not the HIV case that to be cured; An appropriate science inference is arranged: infect any HIV immunogene, the HIV immunogene that exist in the human body at HIV and induce the human immunity reaction mechanism, all be difficult to be used as, to develop synthetic HIV vaccine with higher protection ratio by modern HIV scientific research.The synthetic HIV vaccine of modern HIV scientific research development screens in HIV infection human body, any HIV immunogene, HIV immunogene are induced the human immunity reaction mechanism, all once when each initial HIV the infected's acute stage plasma viral mass formed by blood stasis takes place, keep and produce in a large number, existed every day several weeks, can not induce HIV the infected's immune response, effectively remove the wild HIV virus polyclone group of hill that morphs in the body; The science inference has in more than 6,000 ten thousand HIV infection morbidity person crowds' natural disease process outwardness, indubitable scientific basis.By modern HIV scientific research careless omission, epochmaking scientific research blind area; So far do not obtain the key factor of the scientific breakthrough that solves root problem.
The HIV infection duplication produces envelope protein gp120 or gp160 antigen, is and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation; can not at first produce protectiveness neutralizing antibody, blocking-up gp120 or gp160 antigen and CD4 at gp120 or gp160 antigen +The immune tolerance that cell CD4 receptors bind is induced; Make the HIV can be in the infection host immune response, before the protectiveness neutralizing antibody of generation at gp120 or gp160 antigen, when also effectively not suppressed by cellular immunity CTL, over-replicate reaches the plasma viral load peak-peak, further inducing cell immune tolerance-cellular immunity damage; Can more effectively resist host immune and suppress pressure, damage-destruction HIV infection host immune system gradually than the mankind's other the high simple immunologic escape mechanism of fast, the high variability of retroviral infection host reproduction speed, virus sequence diversity; Thereby there are not the HIV case of self-healing, the HIV case that can be cured; Be that HIV retroviral infection human body duplicates, cause the acquired immune deficiency syndrome (AIDS) crowd to continue chronic infectious disease, another main occurrence factor throughout one's life.Modern so far HIV scientific research was once developed, various HIV envelope protein gp120 or the clinical human trial of gp160 antigen vaccine, it is human to confirm to prevent HIV to infect, most important factor is, HIV envelope protein gp120 or gp160 antigen vaccine, it is immunogene a little less than the xenogenesis part antigen with CD4+ cell CD4 receptors bind, inducing immune tolerance CD4 acceptor, people's immune response be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed.Initial HIV the infected's acute stage plasma viral mass formed by blood stasis keeps that the virion degraded of massive duplication every day several weeks produces, envelope protein gp120 or gp160 antigen---with CD4 +The xenogenesis part antigen quantity of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor can surpass 1011/d, when initial HIV the infected's acute stage blood plasma poison carrying capacity reaches peak-peak, produce envelope protein gp120 or gp160 antigen by the virion of massive duplication degraded---with CD4 +The xenogenesis part antigen quantity of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor can surpass 10 12/ d can induce the immune tolerance relevant with above-mentioned immunosupress-immunologic mjury.The clinical human trial failure of numerous various HIV envelope protein gp120 or gp160 antigen vaccine has been arranged.HIV envelope protein gp120 or the clinical human body of gp160 antigen vaccine immunity inoculation, the weak immunogene humoral immune reaction of the slow generation protection antibody of performance humoral immune reaction, HIV envelope protein gp120 or gp160.Show the slow generation protection antibody of humoral immune reaction, lag behind virus variation with initial HIV infected person body surface, can not induce people's immune response at first to produce protectiveness neutralizing antibody at gp120 or gp160 antigen, weak immunogene humoral immune reaction is very similar; Can duplicate generation envelope protein gp120 or gp160 antigen for disclosing HIV, be and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor, induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation, the immune response of HIV infection host be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed; The relevant clinical human trial scientific basis of HIV vaccine is provided.Existing more than 6,000 ten thousand HIV infection morbidity person crowds, lifelong chronic persistent infection HIV nature disease process is irreversible, do not have self-healing the HIV case, do not have the HIV case that can be cured; HIV infection population immune tolerance nature disease process mechanism can be provided for this reason, and related science is according to circumstantial evidence.The lifelong chronic persistent infection HIV nature disease process of each HIV the infected, the slow generation protection antibody of humoral immune reaction, lag behind virus variation, all the time can not remove the wild HIV virus polyclone group of hill that morphs in the body, an appropriate science inference is arranged: initial HIV infection duplication produces certain important HIV immunogene, induces the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lags behind virus variation; The science inference has in more than 6,000 ten thousand HIV infection morbidity person crowds' natural disease process outwardness, indubitable scientific basis.By modern HIV scientific research careless omission, epochmaking scientific research blind area; So far do not obtain the key factor of the scientific breakthrough that solves root problem.
The former antigenic determinant that has of HIV protective immunity is few; The synthetic HIV vaccine of the former development of any HIV protective immunity is used in modern HIV scientific research, all is difficult to develop the synthetic HIV vaccine with actual application value.Need to use multiple different, HIV protective immunity former with hypotype or a certain or several HIV protective immunities of different subtype, that each coordination antigenic determinant associative list amino acids sequence there are differences, coordination antigenic determinant clone Idiotype is different is former, it is former to make up various mixing HIV protective immunity; Former each coordination antigenic determinant of mixing HIV protective immunity is cloned Idiotype diversity, the coordination antigenic determinant is cloned the Idiotype diversity to increase; resist fast, the high variability of initial HIV infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high, design, the various candidates of development mix the former vaccine of HIV protective immunity.The scientific research of modern HIV vaccine, former each coordination antigenic determinant clone Idiotype of screening HIV protective immunity is different, multiple different HIV protective immunity is former, and the candidate of design, tens kinds of various various combinations of hundreds of kind of development mixes the former vaccine of HIV protective immunity; The former vaccine of mixing HIV protective immunity that therefrom screens the Effective Vate of Protection height again, has actual application value; scientific research complicacy, scientific research difficulty; exceed existing HIV scientific research theoretical thinking cognition, the big science research risk parameter that is difficult to evade of unpredictable existence the unknown.Implemented the clinical human trial of III phase of 3 kinds of candidate HIV vaccine Effective Vate of Protections of assessment checking; infect negative people at highest risk volunteer to the recruitment HIV of society and implement clinical human trial, people at highest risk volunteer's nature HIV the incidence of infection of not inoculating the HIV vaccine is low.For example, January calendar year 2001, that the HIV envelope protein gp120 antigen vaccine of Vaxgene company carries out in the North America, as to have 5400 the volunteers' participation clinical human trial of III; In February, 2003, externally announce the north America region test findings, wherein vaccine inoculation crowd nature HIV infection rate is 5.7%.Implement a kind of candidate HIV vaccine clinical human trial of Effective Vate of Protection III/IV phase of assessment checking, difficulty more than the imagination: clinical human trial of III/IV phase, respectively need be in the people at highest risk volunteer who does not inoculate the HIV vaccine, 1000/5000 natural HIV cases of infection take place, just might assess the Effective Vate of Protection that this candidate HIV vaccine of checking has; If people at highest risk volunteer's nature HIV the incidence of infection is 5%, implement a kind of candidate HIV vaccine clinical human trial of III/IV phase, actual need at least to society recruit 20000/100000 vaccinate, 20000/100000 not the HIV of vaccinate infect negative people at highest risk volunteer; Because the Effective Vate of Protection that candidate HIV vaccine may have is lower than 30%, the HIV of reception test infects negative people at highest risk volunteer's number needs will be doubled; Because, implementing the clinical human trial of candidate's HIV vaccine region, the main hypotype HIV virus infections of popular several differences of while, therefore the HIV of reception test infects negative people at highest risk volunteer's quantity also needs to add several times, and need by numerous and diverse biological detecting method screen nature HIV infect people at highest risk volunteer, whether be infect with candidate HIV vaccine with hypotype HIV virus.Therefore, implement a kind of candidate HIV vaccine clinical human trial of Effective Vate of Protection III/IV phase of assessment checking, reality needs to recruit 1,000,000 HIV to society at least and infects negative people at highest risk volunteer, raises the clinical human trial expense of over ten billion dollar; Owing to implement to finish III/IV phase in the clinical human trial cycle above 20 years, the new subtype HIV virus that the popular several main hypotype HIV virus in test region may change or popular a little speciogenesis is recombinated, make pre-stage test result and later stage test findings inconsistent, finally may can not get having the clinical human trial result of objective science researching value.Even if modern HIV scientific research has abundant scientific research resource, scientific research fund, science researcher, scientific research time, can recruit enough its to society and implement a kind of candidate HIV vaccine III/IV clinical human trial of phase, reality needs 1,000,000 HIV to infect negative people at highest risk volunteer less; Because can not evade the novel synthetic HIV vaccine of development, implement the clinical human trial of III/IV phase and have unknown big science research risk parameter, and do not have by the clinical human trial of III/IV phase, from hundreds of candidate HIV vaccine that can enter clinical research, assessment filters out a kind of ability that the HIV vaccine of actual application value is arranged.
In sum, the simple scientific knowledge thinking of experimental biology, methodology, method are studied carefully in modern HIV basic scientific research, exist obvious defects with not enough; Confine the modern HIV scientific research of constraint, can not solve HIV and infect---an acquired immune deficiency syndrome (AIDS) AIDS problem! The inventor thinks: modern HIV scientific research must be as implementing the Human Genome Project, set up big sample blood plasma, the PBMC cell bank stored of various not homology HIV viruses of initial HIV infection population Different Individual: gather various not homology HIV virus large sample blood plasma, the PBMC cell of storing initial HIV infection population Different Individual, disclose initial HIV infection population different each body, the wild HIV virus of the various not homologies of each hypotype polyclone group of hill, HIV viral gene-cDNA sequence type; The big sample blood plasma of storing of various not homology HIV viruses with initial HIV infection population Different Individual, the PBMC cell bank, various different genes sequence type allele HIV immunogenes-genetic recombination HIV immunogene, the aseptic GF mouse of immunity, it is immunogenic to obtain various different genes sequence type allele HIV, each coordination antigenic determinant specific b cells, set up by hybridoma technology, each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes: disclose initial HIV infection population different each body, the wild HIV virus of the various not homologies of each hypotype polyclone group of hill, each coordination antigenic determinant clone Idiotype of various different genes sequence type allele HIV immunogenes; Big sample blood plasma, the PBMC cell bank stored of various not homology HIV viruses with initial HIV infection population Different Individual, various different genes sequence type allele HIV immunogenes-genetic recombination HIV immunogene, immunity rodent mouse or rat or cavy or rabbit or inhuman primate monkey or the anti-HIV animal model of gorilla obtain the animal blood serum that resists various different genes sequence type allele HIV immunogene neutralizing antibodies; Application contains the animal blood serum of neutralizing antibody, implement the big sample blood plasma of storing of various not homology HIV viruses that in vitro culture is taken from initial HIV infection population Different Individual respectively, the PBMC cell bank, various not homology blood plasma, the wild HIV virus of PBMC cell polyclone group of hill infects the experiment of normal pbmc cell model, foundation suppresses the not wild HIV virus infections of homology normal pbmc cell model storehouse: the immune response that can induce initial HIV infection population is sought in screening, it is fast effectively to remove initial infector virus replication speed, high variability, the Effective Vate of Protection of the wild virus polyclone group of hill that the virus sequence diversity is high is high, and each hypotype HIV protective immunity is former---strong immunogene, the hypotype HIV protective immunity of intersecting is former---strong immunogene; With each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes, select the strong immunogene of allele HIV protectiveness, each coordination antigenic determinant clone Idiotype is different, the strong immunogene of various different genes sequence type allele HIV protectiveness, it is different more than 2 kinds or 2 kinds to instruct design to use, former with hypotype or a certain or several HIV protective immunities of different subtype, the different HIV protective immunity of each coordination antigenic determinant clone Idiotype is former, make up the novel mixing HIV of various candidate protective immunity former-vaccine; With suppressing the not wild HIV virus infections of homology normal pbmc cell model storehouse, therefrom filter out the immune response that to induce initial HIV infection population, the Effective Vate of Protection of effectively removing fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high is high, each hypotype is mixed the strong immunogene antigen-vaccine of HIV protectiveness, the hypotype of intersecting is mixed the strong immunogene antigen-vaccine of HIV protectiveness, confirmation can should be implemented clinical human experimentation as HIV treatment vaccine, HIV prevention vaccine with it; Detailed experimentation, data recording, input corresponding calculated machine overall analysis system makes up the comprehensive organism scientific research experiment porch of evading the unknown big science research risk parameter of the synthetic HIV vaccine existence of development.Instruct modern HIV scientific research, the synthetic HIV vaccine that design, development have actual application value.Rely on and enrich the big sample blood plasma of storing of various not homology HIV viruses that improves initial HIV infection population Different Individual,---each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes---suppresses the not wild HIV virus infections of homology normal pbmc cell model storehouse---computer generalization analytic system---comprehensive organism scientific research experiment porch to the PBMC cell bank, design implementation assessment checking HIV treatment vaccine, the HIV prevention vaccine, the clinical human trial of I/II/III phase--suppress the not wild HIV virus infections of homology normal pbmc cell model experiment: formulate assessment checking HIV treatment vaccine Effective Vate of Protection standard, formulate assessment checking HIV prevention vaccine Effective Vate of Protection standard; Evade the novel synthetic HIV vaccine of development, implement the clinical human trial of III/IV phase and have unknown big science research risk parameter.Implementing clinical human trial--suppress the not wild HIV virus infections of homology normal pbmc cell model experiment, experimentation, data recording are imported the computer generalization analytic system in detail; The further substantial various not homology HIV viruses of improving initial HIV infection population Different Individual are stored sample blood plasma greatly,---each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes---suppresses the not wild HIV virus infections of homology normal pbmc cell model storehouse---computer generalization analytic system---comprehensive organism scientific research experiment porch to the PBMC cell bank.Thereby can evade the novel synthetic HIV vaccine of development and have unknown big science research risk parameter, solve 3 most important difficult point problems that make modern HIV scientific research get into a difficult position, need to be resolved hurrily.
Summary of the invention
The invention discloses a kind of HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method.Disclose HIV envelope protein gp120 or gp160 antigen-vaccine, be and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation; the immune response of HIV infection host be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed.Can develop and not trigger people's immune tolerance, HIV-Env gene DNA allosteric recombinant envelope protein antigen HIV treatment vaccine, HIV prevention vaccine provide enough fully direct animal model experiment scientific basis, direct clinical human trial scientific basis.
The objective of the invention is, a kind of HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method are provided.Design, enforcement HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment disclose HIV envelope protein gp120 or gp160 antigen-vaccine, are and CD4 +The weak immunogene of the xenogenesis part antigen of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; induce the slow generation protection antibody of HIV infection host immune tolerance humoral immune reaction, lag behind virus variation; the immune response of HIV infection host be can not induce, fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high effectively removed.Can develop and not trigger people's immune tolerance, HIV-Env gene DNA allosteric recombinant envelope protein antigen HIV treatment vaccine, HIV prevention vaccine provide enough fully direct animal model experiment scientific basis, direct clinical human trial scientific basis.Provide enforceable: anti-HIV cell model, rodent models, non-human primate's model and clinical human trial scientific research scheme.
Embodiment
For achieving the above object, implement the scheme that the present invention adopts:
HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method
1. animal used as test and clinical testing human body
1.1. animal used as test: germfree animal---GF inbred strais rodent, 6 ~ 8 age in week BALB/c mouse, F344 rat; The non-human primate, rhesus macaque, gorilla.
1.2. clinical testing human body: the normal healthy people of infected by HIV not; Initial HIV the infected.
2. experiment biomaterial
2.1.HIV virus: the freezing preservation of blood plasma, liquid nitrogen cryogenics of taking from initial HIV the infected acute stage is standby; Take from initial HIV the infected human PBMC's cell of acute stage, the freezing preservation of liquid nitrogen cryogenics is standby; Obtain with normal health human PBMC cell co-incubation.
2.2. in vitro culture CD4 +Cell: take from initial HIV the infected human PBMC's cell of acute stage; Take from not infected by HIV normal health human PBMC cell.
2.3. human lymphocyte: take from initial HIV the infected acute stage; treat through the anti-HIV medicine of non-RTI NRTIs/NNRTIs drug combination; through mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; after repeatedly inoculation is treated, the human lymphocyte that blood sampling is obtained.
2.4. serum: normal BALB/c mouse serum; Mouse is through HIV-Env gene DNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen-vaccine, repeatedly after the inoculation, and the mice serum that contains anti-gp120 antibody that blood sampling is obtained.Normal F344 rat blood serum; Rat through HIV-Env gene DNA allosteric reorganization, do not induce people's immune tolerance envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, repeatedly after the inoculation, the rat blood serum that contains anti-gp120 antibody that blood sampling is obtained.Normal rhesus macaque serum; Rhesus macaque is through mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, repeatedly after the inoculation, and the rhesus macaque serum that contains anti-gp120 antibody that blood sampling is obtained.Normal gorilla serum; Gorilla is through mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, repeatedly after the inoculation, and the gorilla serum that contains anti-gp120 antibody that blood sampling is obtained.
2.5. blood plasma: infected by HIV-I normal health human plasma not, through mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV prevention vaccine, repeatedly after the inoculation, the blood plasma that contains anti-gp120 antibody that blood sampling is obtained; Initial HIV the infected acute stage do not implement blood plasma before the treatment; through mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; repeatedly after the inoculation, the blood plasma that contains anti-gp120 antibody that blood sampling is obtained.
3. experiment detection, medicine and reagent and and nutrient solution
3.1. detect the two sandwich methods or the direct sandwich method reagent of gp120 antibody.
3.2.HIV provirus detects and reagent.
3.3.HIV virus load detects HIV-1 Qiantiplex experiment and reagent, or AmplicorHIV-1Monitor experiment and reagent, or NucliSens HIV-1 experiment and reagent.
3.4.CD4 +T lymphocyte test experience and reagent.
3.5.RPMI-2 add 2% human blood nutrient solution, the nutrient solution that RPMI1640 adds 10% animal blood serum, the blood plasma of 10% initial HIV the infected acute stage contains penicillin-G100U/ml, gentamicin 50 μ g/ml; The nutrient solution that RPMI1640 adds 20% human plasma, the blood plasma of 10% initial HIV the infected acute stage contains penicillin-G100U/ml, gentamicin 50 μ g/ml; Ficoll-Hystopaque density gradient solution d=1.077g/ml Pharmacia is built in 10ml Ficoll liquid in the 50ml centrifuge tube in advance; Lymphocyte treating fluid-Nycomed, Birmingham, UK is built in 15ml lymphocyte treating fluid in the 50ml centrifuge tube in advance; The EDTA sodium salt, prepare 4% anti-freezing liquid, 1.2ml anti-freezing EDTA sodium salt solution is built in the 50ml syringe in advance, 0.6ml anti-freezing EDTA sodium salt solution is built in the 20ml syringe in advance with physiological saline.
4. prepare the Bioexperiment material
4.1. obtain initial HIV the infected human PBMC's cell of acute stage by blood sampling; With initial HIV the infected human PBMC's cell of acute stage, extract HIV provirus advantage sequence provirus gene cDNA; PCR duplicates the clone, shears preparation Env gene cDNA; With Chinese hamster ovary cell-CHO expression system, express Env gene cDNA recombinate complete glycosylated HIV envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen.Preparation when implementing experiment: with 50 μ g/ml solution of physiological saline preparation.
4.2. design, development: take from initial HIV the infected human PBMC's cell provirus advantage sequence provirus gene cDNA of acute stage, extract HIV provirus advantage sequence provirus gene cDNA; Implement various Env gene DNA allosteric recombinant expressed, change HIV envelope protein gp120 or the accurate protectiveness immunogene of gp160---with CD4 +The xenogenesis part antigen molecule of cell CD4 receptors bind, inducing immune tolerance CD4 acceptor; certain or certain a few amino acid structures with CD4 receptors bind specific bond epi-position C-end 420 ~ 463 amino acids; make its lose with the special amino acid structure epi-position of CD4 receptors bind, not with the CD4 receptors bind; the weak immunity of xenogenesis part antigen of inducing immune tolerance CD4 acceptor be can lose, people's immune tolerance envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness obtained not induce.Preparation when implementing experiment: with 100 μ g/ml of physiological saline preparation.
4.3. with human PBMC's cell of taking from more than 2 or 2 initial HIV the infected acute stage; homology is not different with the subtype virus sequence polymorphism separately; provirus Env gene cDNA more than 2 kinds or 2 kinds; implement separately that the different sequences of Env gene DNA allosteric reorganization express; different HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene of gp160 protectiveness more than 2 kinds or 2 kinds; each coordination antigenic determinant associative list amino acids sequence there are differences; coordination antigenic determinant monoclonal antibody Idiotype difference, the strong immunogene antigen-vaccine of combined hybrid HIV-Env gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness.When implementing experiment, prepare respectively: with 150 μ g/ml, the 300 μ g/ml solution of physiological saline preparation.
4.4. interim the application produced human PBMC's cell: use the 50ml syringe that is built-in with 1.2ml anti-freezing EDTA sodium salt solution in advance, aseptic extraction is the normal healthy people venous blood 30ml of infected by HIV-I not, adds 2 times of RPMI-2 nutrient solution mixings; With its 3 50ml centrifuge tube upper stratas that are built-in with Ficoll liquid 10ml in advance of careful slowly adding; Not brake, 20 ℃, the centrifugal 30min of 400g; , insert new centrifuge tube and add 5 times of RPMI-2 nutrient solution mixings carefully the PBMC cell sucking-off between centrifuge tube Ficoll liquid and blood boundary layer with the sterilization suction pipe; 20 ℃, the centrifugal 10min of 300g abandon supernatant, add 5 times of RPMI-2 nutrient solution mixings; 20 ℃, the centrifugal 5min of 200g abandon supernatant.With the RPMI1640 nutrient solution that contains 10% Normal animal serum, the blood plasma of 10% initial infected by HIV person acute stage, preparation cell suspension 5 * 10 6/ ml also carries out viable count; With the RPMI1640 nutrient solution of 10% animal blood serum that contains anti-gp120 antibody, the blood plasma of 10% initial HIV the infected acute stage, preparation cell suspension 5 * 10 6/ ml also carries out viable count; With the RPMI1640 nutrient solution that contains 20% human normal plasma, the blood plasma of 10% initial HIV the infected acute stage, preparation cell suspension 5 * 10 6/ ml also carries out viable count; With the RPMI1640 nutrient solution of 20% human plasma that contains anti-gp120 antibody, the blood plasma of 10% initial HIV the infected acute stage, preparation cell suspension 5 * 10 6/ ml also carries out viable count.
4.5. interim the application produced human lymphocyte: use the 20ml syringe that is built-in with 0.6ml anti-freezing EDTA sodium salt solution in advance, aseptic extraction adds 1 times of RPMI-2 nutrient solution mixing through HIV treatment vaccine immunity treatment HIV the infected venous blood 15ml; Its careful slowly adding is built-in with in advance the 50ml centrifuge tube upper strata of lymphocyte treating fluid 15ml; Not brake, 20 ℃, the centrifugal 25min of 800g; Carefully draw the yellowish chromatograph of centrifuge tube with the sterilization suction pipe, insert new centrifuge tube and add 5 times of RPMI-2 nutrient solution mixings; 20 ℃, the centrifugal 10min of 300g abandon supernatant, add 5 times of RPMI-2 nutrient solution mixings; 20 ℃, the centrifugal 5min of 200g abandon supernatant, with the RPMI1640 nutrient solution that contains 10% people AB serum, and preparation cell suspension 2 * 10 7/ ml also carries out viable count.
4.6. interim the application produced animal blood serum: extract normal rhesus macaque or gorilla animal venous blood 30ml, immune rhesus macaque or gorilla animal venous blood 30ml; Place centrifuge tube, the centrifugal 10min of 2000g, centrifugal back 4 ℃ of stand at low temperature 60min; Push blood clot with vessel forceps then, extrude the serum that includes; Discard blood clot, place new centrifuge tube again, the centrifugal 60min of 5 ~ 10000g; Filter with a series of filtrators, after filtering with 0.1 μ m aperture sterilizing filter at last; A plurality of little aseptic high density poly propylene containers that are placed in are placed-80 ℃ of refrigerators and are preserved standby.The interim application produced mouse, rat animal serum: implement the blood sampling of etherization broken end, surplus method as above.
4.7. produce human plasma: every 10ml blood adds the healthy people's venous blood of the aseptic extraction of 0.4ml anti-freezing EDTA sodium salt solution 100ml, or the clinical human trial volunteer of HIV vaccine immunity preceding venous blood 100ml, immune posterior vein blood 100ml; Place centrifuge tube, 2000 commentaries on classics/min, centrifugal 10min.With blood plasma in the sterilization suction pipe suction pipe, give blood sampling person and feed back the residue red blood cell; Filter with a series of filtrators, after filtering with 0.1 μ m aperture sterilizing filter at last; A plurality of little aseptic high density poly propylene containers that are placed in are placed-80 ℃ of refrigerators and are preserved standby.
4.8. gather initial HIV-I the infected big storage sample of blood plasma, PBMC cell of acute stage, and store; Set up the big sample blood plasma of storing of various not homology HIV viruses of initial HIV infection population Different Individual,------suppress the not wild HIV virus infections of homology normal pbmc cell model storehouse---computer generalization analytic system---comprehensive organism scientific research experiment porch: 1. every 10ml blood adds 0.4ml anti-freezing EDTA sodium salt solution to the PBMC cell bank in various different genes sequence type allele HIV immunogenes each coordination antigenic determinant monoclonal antibody Idiotype storehouse, venous blood 200ml ~ 400ml of the initial HIV the infected of aseptic extraction acute stage.2. be placed in 4 ~ 8 100ml centrifuge tubes 2000 commentaries on classics/min, centrifugal 5min.3. with most of blood plasma in the sterilization suction pipe suction pipe; Keep about 4 ~ 6mm place, nearly red blood cell interface blood plasma, to keep top layer, red blood cell interface monokaryon buffy coat, get 2ml blood plasma and do plasma viral load, virus subtype, the detection of viral Env gene order, all the other blood plasma place 2 ~ 4 100ml centrifuge tubes.4.2400 commentaries on classics/min, centrifugal 5min removes blood platelet and other cell component, filters with a series of filtrators, after filtering with 0.15 μ m aperture sterilizing filter at last; By 1ml a plurality of little aseptic high density poly propylene containers that are placed in, mark: 1. patient's code name, 2. 3. blood plasma take a blood sample the date, 4. virus load, 5. virus subtype, viral Env gene order; Then pack in the numbered box, be stored in-80 ℃ of refrigerators standbyly, placing the seat in each initial HIV the infected's sample and the box all needs to import the computing machine detail record, so that can accurately extract in the future.5. be placed in (give blood sampling person and feed back the residue red blood cell) in 4 100ml centrifuge tubes with residual plasma+top layer, red blood cell interface monokaryon buffy coat+part red blood cell in the sterilization suction pipe suction pipe, add 2 ~ 3 times of RPMI-2 nutrient solution mixings.6. its careful slowly adding is built-in with in advance 4 100ml centrifuge tube upper stratas of Ficoll liquid; Blood-RPMI-2 nutrient solution: Ficoll liquid=3: 1.7. not brake, 20 ℃, 2000 commentaries on classics/min, centrifugal 15min.8. use the sterilization suction pipe carefully the monokaryon lymphocyte sucking-off between Ficoll liquid in the pipe and blood boundary layer.9. insert new centrifuge tube and add 5 times of RPMI-2 nutrient solution mixings.10.1500 commentaries on classics/min, centrifugal 5min; Abandon supernatant, stay cell.11. add 5 times of RPMI-2 nutrient solution mixings.12. obtained cell suspension is done viable count.13. counting simultaneously, 1500 commentaries on classics/min, centrifugal 5min; Abandon supernatant, stay cell.14. get 2 * 10 6Cell is done PBMC cell virus carrying capacity, virus subtype, the detection of viral Env gene cDNA sequence, and all the other cells add cryopreserving liquid according to total cell count, adjust cell concentration to 5 * 10 6~ 1 * 10 7Cell/ml cryopreserving liquid; Divide in the special freeze pipe of anti-low temperature of packing into every pipe 5 * 10 6Cell/ml cryopreserving liquid.15. freeze pipe mark: 1. patient's code name, 2. 3. the PBMC cell takes a blood sample the date, 4. virus load, 5. virus subtype, viral Env gene cDNA sequence, each coordination antigenic determinant associative list amino acids sequence of gp120 or gp160, coordination antigenic determinant monoclonal antibody Idiotype; And be placed on 40min in 4 ℃, and place-20 ℃ of 40min, place-80 ℃ to spend the night in the numbered box of packing into; Use CellFreezer in the frozen cell process, obtain the highest cell survival rate when being expected to obtain long storage life and cell recovery; It is standby to put into the liquid nitrogen container preservation at last, and placing the seat in each initial HIV the infected's sample and the box all needs to import the computing machine detail record, so that can accurately extract in the future.16. cryopreserving liquid: people AB serum 90ml, dimethyl sulfoxide (DMSO) 10ml.
4.9. the structure again of human lymphocyte immune defective mouse Hu-PBL-SCID: with human lymphocyte suspension 2 * 10 7/ ml, 0.2ml/ only, be expelled to 4 ~ 6 age in week the immunodeficiency mouse cavum peritoneale in.
5. set up initial HIV the infected HIV virus infected cell vitro culture system of acute stage
5.1. with the RPMI1640 nutrient solution that contains 10% Normal animal serum, the blood plasma of 10% initial HIV the infected acute stage, adjusting the normal pbmc cell concentration is 5 * 10 6/ ml cultured cell suspension, inoculation 1 * 10 6/ m takes from the liquid nitrogen cryogenics of initial HIV the infected acute stage and preserves human PBMC's cell of standby recovery, simulate initial HIV the infected host tissue cell pathology/physiology/immune microenvironment of acute stage, plasma viral sequence polymorphism, the multifarious virus of PBMC virus sequence and CD4 +Cyto-dynamics; With 24 orifice plates implement 60 or the more initial HIV-I the infected of multidigit acute stage, HIV virus infections normal pbmc cells in vitro cultivates.Each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole, every hole perfusion 1ml; Be placed on certain humidity, 37 ℃, 5%CO 2Incubator in cultivate.Every cultivation 3d draws supernatant 0.5ml, replenishes fresh medium.Observe cultured cell pathology at inverted microscope every day after cultivating 4d.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.
5.2. with the RPMI1640 nutrient solution of 10% animal blood serum that contains anti-gp120 antibody, the blood plasma of 10% initial HIV the infected acute stage, adjusting the normal pbmc cell concentration is 5 * 10 6/ ml cultured cell suspension, inoculation 1 * 10 6/ m gets the liquid nitrogen cryogenics of initial HIV the infected acute stage and preserves human PBMC's cell of standby recovery, simulation HIV treatment vaccine immunity is treated initial HIV the infected host tissue cell pathology/physiology/immune microenvironment of acute stage, plasma viral sequence polymorphism, the multifarious virus of PBMC virus sequence and CD4 +Cyto-dynamics; With 24 orifice plates implement 60 or the more initial HIV the infected of multidigit acute stage, HIV virus infections normal pbmc cells in vitro cultivates.Each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole, every hole perfusion 1ml; Be placed on certain humidity, 37 ℃, 5%CO 2Incubator in cultivate.Every cultivation 3d draws supernatant 0.5ml, replenishes fresh medium.Observe cultured cell pathology at inverted microscope every day after cultivating 4d.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.
5.3. with the RPMI1640 nutrient solution that contains 20% human normal plasma, the blood plasma of 10% initial HIV the infected acute stage, adjusting the normal pbmc cell concentration is 5 * 10 6/ ml cultured cell suspension, inoculation 1 * 10 6/ ml takes from the liquid nitrogen cryogenics of initial HIV the infected acute stage and preserves human PBMC's cell of standby recovery, simulate initial HIV the infected host tissue cell pathology/physiology/immune microenvironment of acute stage, plasma viral sequence polymorphism, the multifarious virus of PBMC virus sequence and CD4 +Cyto-dynamics; With 24 orifice plates or 96 orifice plates implement 100 or the more initial HIV the infected of multidigit acute stage, HIV virus infections normal pbmc cells in vitro cultivates.Each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole, every hole perfusion 1ml or 0.2ml; Be placed on certain humidity, 37 ℃, 5%CO 2Incubator in cultivate.Every cultivation 3d draws supernatant 0.5ml or 0.1ml, replenishes fresh medium.Observe cultured cell pathology at inverted microscope every day after cultivating 4d.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.
5.4. with the RPMI1640 nutrient solution that contains 20% human plasma that contains anti-gp120 antibody, the blood plasma of 10% initial HIV the infected acute stage, adjusting the normal pbmc cell concentration is 5 * 10 6/ ml cultured cell suspension, inoculation 1 * 10 6/ m takes from the liquid nitrogen cryogenics of initial HIV the infected acute stage and preserves human PBMC's cell of standby recovery, after simulation HIV vaccine immunity is treated clinical human body, host tissue cell pathology/physiology/immune microenvironment of initial HIV the infected acute stage, plasma viral sequence polymorphism, the multifarious virus of PBMC virus sequence and CD4 +Cyto-dynamics; With 24 orifice plates or 96 orifice plates implement 100 or the more initial HIV the infected of multidigit acute stage, HIV virus infections normal pbmc cells in vitro cultivates.Each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole, every hole perfusion 1ml or 0.2ml; Be placed on certain humidity, 37 ℃, 5%CO 2Incubator in cultivate.Every cultivation 3d draws supernatant 0.5ml or 0.1ml, replenishes fresh medium.Observe cultured cell pathology at inverted microscope every day after cultivating 4d.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.
6. experiment: method, biological detection, data recording, result evaluation
6.1. take from the HIV provirus advantage sequence provirus gene cDNA of human PBMC's cell of initial HIV the infected acute stage; express HIV-Env gene cDNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen---not with mouse CD4 receptors bind, the strong immunogene antigen of inducing immune tolerance not; immunity BALB/c mouse anti-HIV animal model-suppress the not wild HIV virus infections of homology normal pbmc cell model, experimental technique, biological detection, data recording, result evaluation:
6.1.1. respectively to 30 BALB/c mouse, implement the blood sampling of etherization broken end, produce Normal animal serum.
6.1.2.HIV-Env the accurate protectiveness immunogene of gene cDNA recombinant envelope protein gp120 or gp160---not with mouse CD4 receptors bind, the strong immunogene antigen of inducing immune tolerance not; the immunity BALB/c mouse; 30 BALB/c mouse of intramuscular injection immunity: 50 μ g/ml, 0.2ml/, 0d, 14d, 28d, 42d are respectively once.At immunity inoculation 14d, 21d, 28d, 35d, 42d, the afterbody trace blood detects the titre of the reaction of every mouse humoral immune, antibody.49d or 60d, 30 mouse are put to death in the blood sampling of etherization broken end separately respectively, produce the titre that contains anti-gp120 antibody animal serum, detects antibody; Set up each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes.
6.1.3. respectively with the mixing Normal animal serum of 6.1.1.30 mouse, 6.1.2.30 only mouse contains anti-gp120 antibody mixing animal blood serum, the experiment of 5.1.HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.2.HIV virus infected cell vitro culture system experiment: sum is no less than 60 initial HIV the infecteds and reserves the liquid nitrogen preservation acute stage, blood plasma and PBMC cell not homology are cultivated with hypotype HIV virus infections normal pbmc cells in vitro, and each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detection is used with hypotype HIV envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen; the anti-HIV model of immune mouse; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.1.4. experimentation, data recording: 1. detail record, 6.1.1./6.1.2./6.1.3. tests overall process.Detailed experimentation, data recording, input computer generalization analytic system.2. record; implement the experiment of HIV virus infected cell vitro culture system; with HIV-Env gene cDNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen-vaccine; the immunity BALB/c mouse; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage; multiple not homology infects the normal pbmc cell ability with the wild HIV of hypotype virus polyclone group of hill---promptly: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity=? figure place: total bit * 100%.Mouse anti HIV model, the neutralizing antibody that immune response produces, having neutralization and suppressing the multiple not homology of blood plasma, PBMC cell greater than 6 initial HIV the infected acute stages with hypotype HIV virus infections normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 10%; Can be considered to: have actual application value HIV-Env gene cDNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen.
6.1. the not wild HIV virus infections of homology normal pbmc cell model experiment of mouse anti HIV animal model-suppress: 1. can be applied to the initial HIV infection population of biological detection Different Individual, various each coordination antigenic determinant associative list amino acids sequences of different genes sequence type allele HIV immunogene, coordination antigenic determinant monoclonal antibody Idiotype; The various not homology HIV viruses of setting up initial HIV infection population Different Individual are stored sample blood plasma greatly,---each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes---suppresses the not wild HIV virus infections of homology normal pbmc cell model storehouse---computer generalization analytic system---comprehensive organism scientific research experiment porch to the PBMC cell bank; Therefrom filter out; neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 10%, have actual application value HIV-Env gene cDNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen.2. confirm; HIV provirus advantage sequence provirus Env gene cDNA with human PBMC's cell of taking from initial HIV the infected acute stage; express HIV-Env gene DNA recombinant envelope protein gp120 or the accurate protectiveness immunogene of gp160 antigen; be the immune response that to induce initial HIV infection population, effectively remove the accurate protectiveness immunogene of the same hypotype antigen of fast, the high variability of initial infector virus replication speed, wild virus polyclone group of hill that the virus sequence diversity is high.3. further confirm; development and design; implement the HIV-Env gene DNA allosteric and recombinate, can lose the weak immunogenicity of xenogenesis part antigen of inducing immune tolerance CD4 acceptor; obtain not induce people's immune tolerance envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; might aspect development screening HIV treatment vaccine, the scientific research of HIV prevention vaccine, obtain scientific breakthrough.The not wild HIV virus infections of homology normal pbmc cell model experimental science foundation of further anti-HIV animal model-suppress is provided.
6.2.HIV-Env the strong immunogene antigen-vaccine of gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness; immunity F344 rat anti HIV animal model-suppress the not wild HIV virus infections of homology normal pbmc cell model, experimental technique, biological detection, data recording, result evaluation:
6.2.1. respectively to 10 F344 rats, implement the blood sampling of etherization broken end, produce Normal animal serum.
6.2.2. with HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, immune F344 rat, 10 F344 rats of intramuscular injection immunity: 100 μ g/ml, 0.2ml/, 0d, 14d, 28d, 42d are respectively once.At immunity inoculation 14d, 21d, 28d, 35d, 42d, the afterbody trace blood detects the titre of every rat humoral immune reaction, antibody.49d or 60d implement the blood sampling of etherization broken end respectively separately and put to death 10 rats, produce the animal blood serum that contains anti-gp120 antibody, the titre that detects antibody.
6.2.3. respectively with the mixing Normal animal serum of 6.2.1.10 rat, 6.2.2.10 only rat contains anti-gp120 antibody mixing animal blood serum, the experiment of 5.1.HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.2.HIV virus infected cell vitro culture system experiment: sum is no less than 60 initial HIV the infecteds and reserves the liquid nitrogen preservation acute stage, blood plasma and PBMC cell not homology are cultivated with hypotype HIV virus infections normal pbmc cells in vitro, and each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates each 3 hole.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detection is used with hypotype HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; the anti-HIV model of immune rat; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.2.4. experimentation, data recording: 1. detail record, 6.2.1./6.2.2./6.2.3. tests overall process.Detailed experimentation, data recording, input computer generalization analytic system.2. record; implement the experiment of HIV virus infected cell vitro culture system; with HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; immunity F344 rat; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage; multiple not homology infects the normal pbmc cell ability with the wild HIV of hypotype virus polyclone group of hill---promptly: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity=? figure place: total bit * 100%.Rat anti HIV-I animal model, the neutralizing antibody that immune response produces, having neutralization and suppressing the multiple not homology of blood plasma, PBMC cell greater than 6 initial HIV the infected acute stages with hypotype HIV virus infections normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 10%; Can be considered to: have the strong immunogene antigen of actual application value HIV-Env gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness.
6.2. rat anti HIV animal model-suppress the not wild HIV virus infections of homology normal pbmc cell model experiment: 1. can recombinate from implementing various different HIV-Env gene DNA allosterics; can lose the weak immunogenicity of xenogenesis part antigen of inducing immune tolerance CD4 acceptor; obtain not induce in people's immune tolerance envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; filter out; neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 10%, have the strong immunogene antigen of actual application value HIV-Env gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness.2. further confirm; HIV provirus advantage sequence provirus Env gene cDNA with human PBMC's cell of taking from initial HIV the infected acute stage; implement the reorganization of HIV-Env gene DNA allosteric; can lose the weak immunogenicity of xenogenesis part antigen of inducing immune tolerance CD4 acceptor; obtain not induce people's immune tolerance envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; be the immune response that can induce initial HIV infection population, it is fast effectively to remove initial infector virus replication speed; high variability; the strong immunogene antigen of same hypotype protectiveness of the wild virus polyclone group of hill that the virus sequence diversity is high.3. further confirm again; development and design; implement the HIV-Env gene DNA allosteric and recombinate, can lose the weak immunogenicity of xenogenesis part antigen of inducing immune tolerance CD4 acceptor; obtain not induce people's immune tolerance envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; might aspect the anti-HIV treatment of development screening vaccine, the scientific research of anti-HIV prevention vaccine, obtain scientific breakthrough.The not wild HIV virus infections of homology normal pbmc cell model experimental science foundation of further anti-HIV animal model-suppress is provided.
6.3. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; immunity rhesus macaque anti-HIV animal model-suppress the not wild HIV virus infections of homology normal pbmc cell model, experimental technique, biological detection, data recording, result evaluation:
6.3.1. to 8 ~ 10 rhesus macaquies venous blood samples 30ml separately, produce Normal animal serum respectively.
6.3.2. with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, 8 ~ 10 rhesus macaquies of intramuscular injection immunity, 150 μ g/ml, 1ml/, 0d, 21d, 42d are respectively once.At immunity inoculation 14d, 21d, 28d, 35d, 42d, 49d, blood sampling detects the titre of every rhesus macaque humoral immune reaction, antibody.60d, difference is venous blood samples 30ml separately, produces the titre that contains anti-gp120 antibody animal serum, detects antibody.
6.3.3. respectively with the Normal animal serum of every rhesus macaque, contain anti-gp120 antibody animal serum, the experiment of 5.1.HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.2.HIV virus infected cell vitro culture system experiment: sum be no less than 60 initial HIV the infecteds reserves acute stage that liquid nitrogen is preserved, blood plasma and PBMC cell not homology cultivate with hypotype HIV virus infections normal pbmc cells in vitro, each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates respectively 3 holes.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detect with using and mix HIV-I-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; the anti-HIV animal model of immunity rhesus macaque; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.3.4. experimentation, data recording: 1. detail record, 6.3.1./6.3.2./6.3.3. tests overall process.Detailed experimentation, data recording, input computer generalization analytic system.2. record; implement the experiment of HIV virus infected cell vitro culture system; with mixing HIV-I-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; the immunity rhesus macaque; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage; multiple not homology infects the normal pbmc cell ability with the wild HIV of hypotype virus polyclone group of hill---promptly: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity=? figure place: total bit * 100%.The anti-HIV animal model of rhesus macaque, the neutralizing antibody that immune response produces, having neutralization and suppressing the multiple not homology of blood plasma, PBMC cell greater than 18 initial HIV the infected acute stages with the wild HIV virus infections of hypotype normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 30%; Can be considered to: have actual application value and mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness.
6.3. the anti-HIV animal model of rhesus macaque-suppress the not wild HIV virus infections of homology normal pbmc cell model experiment: can mix from the candidate of various various combinations HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, filter out, neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 30%, have actual application value and mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; Promptly confirm: infect any HIV immunogene that exists in the human body at HIV, all be difficult to be used as, to develop synthetic HIV vaccine with higher protection ratio by modern HIV scientific research; The former antigenic determinant that has of HIV protective immunity is few; modern HIV scientific research; use the synthetic HIV vaccine of the former development of any HIV protective immunity; all be difficult to develop the immune response that to induce initial HIV infection population; fast, the high variability of effective initial infector virus replication speed of removing, the wild virus polyclone group of hill that the virus sequence diversity is high, the synthetic HIV vaccine with actual application value.Need to use multiple differently, former with hypotype or a certain or several HIV protective immunities of different subtype HIV protective immunity former, that each coordination antigenic determinant associative list amino acids sequence there are differences, coordination antigenic determinant clone Idiotype is different, the mixing HIV protective immunity of design, the various various combinations of development is former; Former each coordination antigenic determinant of mixing HIV protective immunity is cloned Idiotype diversity, the coordination antigenic determinant is cloned the Idiotype diversity to increase, the high wild virus polyclone group of hill of fast, the high variability of provirus reproduction speed, virus sequence diversity that resists initial infected by HIV crowd continues variation, and design, the various candidates of development mix the former vaccine of HIV protective immunity; It is the only way that to develop synthetic HIV vaccine future with actual application value.Might aspect development screening HIV treatment vaccine, the scientific research of HIV prevention vaccine, obtain scientific breakthrough.Provide to have to human similar life mechanism the not wild HIV virus infections of homology normal pbmc cell model experimental science foundation of the anti-HIV animal model of rhesus macaque-suppress.
6.4. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; immunity gorilla anti-HIV animal model-suppress the not wild HIV virus infections of homology normal pbmc cell model, experimental technique, biological detection, data recording, result evaluation:
6.4.1. to 4 ~ 6 gorillas venous blood samples 30ml separately, produce Normal animal serum respectively.
6.4.2. with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, 4 ~ 6 gorillas of intramuscular injection immunity, 300 μ g/ml, 2ml/, 0d, 21d, 42d are respectively once.At immunity inoculation 14d, 21d, 28d, 35d, 42d, 49d, blood sampling detects the titre of every gorilla humoral immune reaction, antibody.60d, difference is venous blood samples 30ml separately, produces the titre that contains anti-gp120 antibody animal serum, detects antibody.
6.4.3. respectively with the Normal animal serum of every gorilla, contain anti-gp120 antibody animal serum, the experiment of 5.1 HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.2.HIV virus infected cell vitro culture system experiment: sum be no less than 60 initial HIV the infecteds reserves acute stage that liquid nitrogen is preserved, blood plasma and PBMC cell not homology cultivate with hypotype HIV virus infections normal pbmc cells in vitro, each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates respectively 3 holes.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detect with mixing HIV-I-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness; the anti-HIV animal model of immunity gorilla; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.4.4. experimentation, data recording: 1. detail record, 6.4.1./6.4.2./6.4.3. tests overall process.Detailed experimentation, data recording, input computer generalization analytic system.2. record is implemented the experiment of HIV virus infected cell vitro culture system, with mixing the HIV-Env gene DNA AllostericRecombinant envelope protein gp120 or gp160 The strong immunogene of protectivenessAntigen; the immunity gorilla; the neutralizing antibody that immune response produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity=? figure place: total bit * 100%.The anti-HIV animal model of gorilla, the neutralizing antibody that immune response produces, having neutralization and suppressing the multiple not homology of blood plasma, PBMC cell greater than 18 initial HIV-I the infected acute stages with the wild HIV virus infections of hypotype normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 30%; Can be considered to: have actual application value and mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness.
6.4. the not wild HIV virus infections of homology normal pbmc cell model experiment of the anti-HIV animal model of gorilla-suppress; confirm; can HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness will be mixed; as HIV treatment vaccine, HIV prevention vaccine, implement clinical human trial.Provide have with human life's mechanism the most approaching, the not wild HIV virus infections of homology normal pbmc cell model experimental science foundation of the anti-HIV animal model of gorilla-suppress.
6.5. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; as HIV treatment vaccine; with the initial HIV the infected of anti-HIV medicine drug combination immunization therapy acute stage; the anti-HIV immunization therapy test one of clinical human body of I phase suppresses not homology HIV wild virus infection normal pbmc cell model experiment, experimental technique, biological detection, data recording, the result evaluation of relevant vaccinology:
6.5.1. the big storage sample of blood plasma, PBMC cell of the initial HIV the infected of collection acute stage before implementing treatment; See: 4.8..
6.5.2. be chosen in initial HIV the infected acute stage, through the treatment of the anti-HIV medicine of non-RTI NRTIs/NNRTIs drug combination, can drop in 14d inner virus carrying capacity can not detect, CD4 in the 28d +The T lymphocyte detects counting and can recover 〉=800/ μ l, 60 infection are with hypotype HIV patient.Divide 2 groups; with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; with the initial HIV-I the infected of anti-HIV medicine drug combination immunization therapy: 1 group of 150 μ g/ml/ people, 2 group of 300 μ g/ml/ people, the immune separately subcutaneous multiple spot vaccinal injection of 0d, 14d, 28d, 60d is once.At immunity inoculation 14d, 21d, 28d, 35d, 49d, blood sampling detects every and implements HIV treatment vaccine immunity curer's humoral immune reaction, the titre of antibody.60d, difference is venous blood samples 30ml separately: the titre that detects antibody; Set up the human monoclonal antibody Idiotype storehouse of former each coordination antigenic determinant of different genes sequence type allele HIV protective immunity; Produce lymphocyte.
6.5.3. implement the treatment of the anti-HIV medicine of non-RTI NRTIs/NNRTIs drug combination with every; mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness with novel with hypotype---HIV treatment vaccine immunity curer's lymphocyte; implement 3 ~ 5 SCID mouse, the structure again of human lymphocyte immune defective mouse Hu-PBL-SCID separately respectively.21d, the Hu-PBL-SCID mouse is put to death in blood sampling respectively, does provirus detection, plasma viral load detection respectively.
6.5.4.HIV it is can not the tester that treatment vaccine immunity curer's lymphocyte is rebuild provirus, the plasma viral load of Hu-PBL-SCID mouse, after stopping the anti-HIV medicine of non-RTI NRTIs/NNRTIs drug combination treatment 7d, venous blood samples 100ml produces blood plasma.The blood plasma that contains anti-gp120 antibody respectively with human normal plasma, HIV treatment vaccine immunity curer, the experiment of 5.3.HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.4.HIV virus infected cell vitro culture system experiment: 100 or the more initial HIV the infected of multidigit reserves acute stage that liquid nitrogen is preserved, blood plasma and PBMC cell not homology cultivate with hypotype HIV virus infections normal pbmc cells in vitro, each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates respectively 3 holes.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detection is with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; the initial HIV the infected of immunization therapy; the neutralizing antibody that humoral immunoresponse(HI) produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.5.5. experimentation, data recording: 1. detail record 6.5.1./6.5.2./6.5.3./6.5.4. tests overall process.Detailed experimentation, data recording, input computer generalization analytic system.2. record; implement the experiment of HIV-I virus infected cell vitro culture system; with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; the clinical human body of anti-HIV immunization therapy; the neutralizing antibody that humoral immune reaction produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage; multiple not homology infects the normal pbmc cell ability with the wild HIV of hypotype virus polyclone group of hill---promptly: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity=? figure place: total bit * 100%.The clinical human body of anti-HIV immunization therapy, the neutralizing antibody that humoral immune reaction produces, having to neutralize suppresses the multiple not homology of blood plasma, PBMC cell greater than 30 initial HIV the infected acute stages with the wild HIV virus infections of hypotype normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 30%; Can be considered to: have actual application value and mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness.Can implement the clinical human trial of I phase HIV prevention vaccine as HIV treatment vaccine, HIV prevention vaccine, implement II phase HIV treatment vaccine, the clinical human trial of HIV prevention vaccine.
6.5.6.HIV treatment vaccine immunity curer's the anti-gp120 antibody of blood plasma, have and suppress me and implement to reserve before the treatment that liquid nitrogen is preserved, blood plasma and PBMC cell HIV virus infected cell ability, solicit immunization therapy person and sign and agree to stop the therapeutic alliance of the anti-HIV medicine of non-RTI NRTIs/NNRTIs.Implementing regularly to immunization therapy person, blood drawing detects provirus, plasma viral load 3 years; observe provirus, plasma viral load knock-on recovery can take place? if HIV treatment vaccine immunity treatment patient's provirus, plasma viral load are still for detecting; can be considered to: the strong immunogene antigen of application mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness---HIV treats vaccine, initial HIV the infected of several examples of clinical cure.
6.5. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; as HIV treatment vaccine; with the initial HIV the infected's of anti-HIV medicine drug combination immunization therapy; clinical human immunity therapeutic test--suppress the not wild HIV virus infections of homology normal pbmc cell model experiment, can the initial HIV the infected of several examples of clinical cure.Be that assessment checking HIV treatment vaccine, HIV prevention vaccine have actual application value, golden standard.
6.6. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; as HIV treatment vaccine; with the initial HIV the infected of anti-HIV medicine drug combination immunization therapy acute stage; the anti-HIV immunization therapy test of clinical human body of II phase--suppress not homology HIV wild virus infection normal pbmc cell model experiment, experimental technique, biological detection, data recording, the result evaluation of relevant vaccinology:
6.6.1. the big storage sample of blood plasma, PBMC cell of the initial HIV the infected of collection acute stage before implementing treatment; See: 4.8..
6.6.2.800 homology is infected not at first with hypotype HIV patients during acute stage in the position.Divide 3 immunization therapy groups, 1 control group, every group of each 200 people; With mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine, treat 3 initial HIV the infecteds of immunization therapy group with anti-HIV medicine drug combination: behind anti-HIV medicine drug combination treatment 28d, 1 group of 100 μ g/ml/ people, 2 group of 200 μ g/ml/ people, 3 group of 300 μ g/ml/ people, the immune separately subcutaneous multiple spot vaccinal injection of 0d, 14d, 28d, 60d is once; Initial HIV the infected is organized in 1 contrast of anti-HIV medicine drug combination treatment treatment: behind anti-HIV medicine therapeutic alliance 28d, and physiological saline 1ml/ people, the subcutaneous separately multiple spot vaccinal injection of 0d, 14d, 28d, 60d is once.At immunity inoculation 14d, 21d, 28d, 35d, 49d, 60d, blood sampling detects the titre of every immunization therapy person's humoral immunoresponse(HI), antibody.60d stops whole 1 group, 2 groups, 3 groups, 4 groups anti-HIV medicine drug combination treatment.Behind the 7d, 1 group of immunization therapy, 2 groups, 3 groups, extract the medium 10 minimum people of 10 people, antibody titer of humoral immune reaction antibody titer the highest 10 people, antibody titer separately respectively out, venous blood samples 100ml separately produces blood plasma respectively.
6.6.3. contain the blood plasma of anti-gp120 antibody respectively with human normal plasma, HIV treatment vaccine immunity curer, the experiment of 5.3.HIV virus infected cell vitro culture system is implemented in contrast respectively, 5.4.HIV virus infected cell vitro culture system experiment: 500 or the more initial HIV the infected of multidigit reserves acute stage that liquid nitrogen is preserved, blood plasma and PBMC cell not homology cultivate with hypotype HIV virus infections normal pbmc cells in vitro, each is numbered the HIV virus infections normal pbmc cells in vitro of initial HIV the infected acute stage and cultivates respectively 3 holes.After cultivating 12d, to not infected the viable count that causes death in each cellular incubation hole by HIV.Detection is with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine; the initial HIV the infected of immunization therapy; the neutralizing antibody that humoral immunoresponse(HI) produces, how many positions does having effectively to neutralize suppress? initial infected by HIV person acute stage, multiple not homology infects the normal pbmc cell ability with the wild HIV virus of hypotype polyclone group of hill---promptly: neutralizing antibody suppresses the Effective Vate of Protection of multiple not homology with the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity.
6.6.4. experimentation, data recording: detailed same 6.5.1./6.5.2./6.5.3. is with mixing HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen-vaccine of gp160 protectiveness, the clinical human body of anti-HIV immunization therapy, the neutralizing antibody that humoral immune reaction produces, having to neutralize suppresses blood plasma greater than 180 initial HIV the infected acute stages, the multiple not homology of PBMC cell is with the wild HIV virus infections of hypotype normal pbmc cell ability, that is: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 30%; Can be considered to: have actual application value and mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness.Can implement III phase HIV-I treatment vaccine, the clinical human trial of I/II/III phase HIV-I prevention vaccine as HIV treatment vaccine, HIV prevention vaccine.Detailed experimentation, data recording, the input Computerized analysis system.
6.6. to 3 immunization therapy groups, 1 contrast treatment group; implementing regularly, blood drawing detects provirus, plasma viral load 3 years; observe provirus, plasma viral load knock-on? if immunization therapy person's provirus, plasma viral load are for detecting; can be considered to: the strong immunogene antigen of application mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or gp160 protectiveness---HIV treats vaccine, initial HIV the infected of clinical cure.Be assessment checking candidate HIV treatment vaccine, HIV prevention vaccine, have the golden standard of actual application value.Formulate assessment checking HIV treatment vaccine Effective Vate of Protection standard: in 3 immunization therapy groups: have 30 or initial HIV the infected of multidigit clinical cure more; Plasma viral load detects to be counted/discontinues medication 1 year ÷ plasma viral load of treatment and detect and count/discontinue medication 3 months ratio of treatment, plasma viral load and detect and count/discontinue medication 3 years ÷ plasma viral loads of treatment and detect and count/discontinue medication treatment ratio, the CD4 in 1 year +The T lymphocyte detects counts/discontinues medication 1 year ÷ CD4 of treatment +The T lymphocyte detects counts/discontinues medication treatment 3 months ratio, CD4 +The T lymphocyte detects counts/discontinues medication 3 years ÷ CD4 of treatment +The T lymphocyte detects counts/discontinues medication the ratio in 1 year of treatment, can compare that to organize ratio of the same period according to treatment low more than 30%.Can think: have with the initial HIV the infected's of anti-HIV medicine drug combination immunization therapy; the clinical treatment practical value, can go on the market, mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV treats vaccine.
6.7. mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness; as the HIV-I prevention vaccine; enforcement I/II (/III) clinical human trial of phase--suppress not homology HIV wild virus infection normal pbmc cell model experiment; experimental technique, biological detection, data recording, the result evaluation of relevant vaccinology are implemented with reference to 6.5./6.6.HIV treatment vaccine.With the blood plasma that contains anti-gp120 antibody after reservation blood plasma, the immunity of HIV prevention vaccine before the test, the experiment of 5.3/5.4.HIV virus infected cell vitro culture system is implemented in contrast respectively respectively.Formulate assessment checking HIV prevention vaccine Effective Vate of Protection standard: neutralizing antibody suppress multiple not homology with the Effective Vate of Protection of the high polyclone group of hill of the wild HIV virus sequence of hypotype diversity greater than 35%.Can think: have prevention with hypotype HIV infected person types of populations actual application value, can go on the market, mix HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---HIV prevention vaccine.Detailed experimentation, data recording, the input Computerized analysis system.
Modern HIV scientific research development is synthesized the HIV vaccine, is had unknown big science research risk parameter with the clinical human trial of enforcement III/IV phase.HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment: the anti-HIV animal model of HIV-Env gene DNA allosteric recombinant envelope protein antigen-cell model experiment, the clinical human trial of HIV-Env gene DNA allosteric recombinant envelope protein antigen HIV vaccine-cell model experiment is set up the various not homology HIV viruses of initial HIV infection population Different Individual and is stored sample blood plasma greatly,---each coordination antigenic determinant monoclonal antibody Idiotype storehouse of various different genes sequence type allele HIV immunogenes---suppresses the not wild HIV virus infections of homology normal pbmc cell model storehouse---computer generalization analytic system---comprehensive organism scientific research experiment porch to the PBMC cell bank; 3 methods that make the most important difficult point problem that modern HIV scientific research gets into a difficult position, needs to be resolved hurrily that solve are provided, evade the synthetic HIV vaccine of development, have unknown big science research risk parameter with the clinical human trial of enforcement III/IV phase, instruct modern HIV scientific research, the synthetic HIV vaccine that design, development have actual application value.Can in 5 ~ 6 years, implement to finish; the assessment checking mixes HIV-Env gene DNA allosteric recombinant envelope protein gp120 or the strong immunogene antigen of gp160 protectiveness---and HIV treatment vaccine, HIV prevention vaccine have the clinical human trial of Effective Vate of Protection I/II/III phase--suppress the not wild HIV virus infections of homology normal pbmc cell model experiment.

Claims (10)

1. HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method is characterized in that: use HIV-Env gene DNA allosteric recombinant envelope protein antigen (1) immunity inoculation animal used as test (2), blood sampling detects the titre (5) of immunization experiment animal (2) at blood constituent (3) antibody (4) of HIV-Env gene DNA allosteric recombinant envelope protein antigen (1).
2. HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method, it is characterized in that: use HIV-Env gene DNA allosteric recombinant envelope protein antigen (1) immunity inoculation animal used as test (2), blood sampling detects the titre (5) of immunization experiment animal (2) at blood constituent (3) antibody (4) of HIV-Env gene DNA allosteric recombinant envelope protein antigen (1), uses blood constituent (3), HIV virus (6), nutrient solution (7) the in vitro culture CD4 of immunization experiment animal (2) +Cell (8), blood constituent (3) antibody (4) that detects immunization experiment animal (2) suppresses HIV virus (6) and infects cultivation CD4 +The ability (9) of cell (8).
3. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1,2, it is characterized in that: animal used as test (2) is rodent (10).
4. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1,2, it is characterized in that: animal used as test (2) is the primate (11) of non-human.
5. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1,2, it is characterized in that: animal used as test (2) is clinical testing human body (12).
6. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 2 to 5, it is characterized in that: cultivate CD4 +Cell (8) is human PBMC's cell (13).
7. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 2 to 6, it is characterized in that: HIV virus (6) is to take from the HIV virus (15) of initial HIV the infected's acute stage (14) blood constituents (3).
8. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1 to 7, it is characterized in that: HIV-Env gene DNA allosteric recombinant envelope protein antigen (1) is an Env gene DNA allosteric recombinant envelope protein antigen (1) of taking from the HIV virus (15) of initial HIV the infected's acute stage (14) blood constituents (3).
9. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1 to 8, it is characterized in that: HIV-Env gene DNA allosteric recombinant envelope protein antigen (1), be the HIV-Env gene DNA allosteric recombinant envelope protein antigen (1) that there are differences by 2 kinds of antigenic determinants clone Idiotypes, the mixing HIV-Env gene DNA allosteric recombinant envelope protein antigen (16) of combination.
10. according to HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and the method described in the claim 1 to 9, it is characterized in that: HIV-Env gene DNA allosteric recombinant envelope protein antigen (1), be the HIV-Env gene DNA allosteric recombinant envelope protein antigen (1) that there are differences by antigenic determinant clone Idiotype more than 2 kinds, the mixing HIV-Env gene DNA allosteric recombinant envelope protein antigen (17) of combination.
CN 200710090957 2007-03-29 2007-03-29 HIV-Env gene DNA allosteric recombinant envelope protein antigen immune response anti HIV experiment and method Pending CN101021539A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106924726A (en) * 2015-12-29 2017-07-07 普莱柯生物工程股份有限公司 A kind of vaccine combination for preventing porcine reproductive and respiratory syndrome and its preparation method and application

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106924726A (en) * 2015-12-29 2017-07-07 普莱柯生物工程股份有限公司 A kind of vaccine combination for preventing porcine reproductive and respiratory syndrome and its preparation method and application
CN106924726B (en) * 2015-12-29 2021-02-09 普莱柯生物工程股份有限公司 Vaccine composition for preventing porcine reproductive and respiratory syndrome and preparation method and application thereof

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