基因免疫

1.基因免疫的概念和优势。,3.基因免疫中目的基因的来源,2.疫苗的主要类型,Chapter VI,IMMUNIZATION,免疫接种免疫学预防,Mortality g Highg Moderateg None or low,HIV / AIDS,Tuberculosis,Mortality g Highg Moderateg None or low,Risk Significantg Lowg None,Measles,Number of cases g More than 100g 11 - 100g 1 - 10g 0g No surveillance,Influenza,g Widespreadg Regionalg Localg Sporadicg Negligibal or no surveillance,Diarrhea Diseases,Cholera Cases g More than 1,500g 1,001 -1,500g 501 - 1,000g 1- 500g Negligibal or no surveillance,Each helper T cell expresses the CD4 antigen. HIV enters the cell by binding to CD4 and other cell surface molecules. A helper T cell is known colloquially as a CD4 cell.,Macrophages and a subset of T lymphocytes (T cells), known as helper T cells, express CD4 and so are vulnerable to HIV infection.,Edward Jenner,Louis Pasteur,Jenner and Pasteur are recognized as the immunologists who pioneered vaccination, or induction of active immunity.,脊髓灰质炎 Polio,狂犬病Rabies,风疹 Rubella,Hepatitis A,Hepatitis B,流感Influenza,白喉 diphtheria,天花Smallpox virus,轮状病毒 Rotavirus,Pertussis 百日咳,Comparison of maximum and current morbidity for vaccine-preventable diseases,Disease,Maximum cases,Year,1992,Percentage change,白喉 Diphtheria麻疹 Measles腮腺炎 Mumps百日咳 pertussis脊髓灰质炎 Polio (paralytic)风疹 RubellaCRS破伤风 Tetanus乙型流感 Heamophilusinfluenzae type B乙型肝炎 Hepatitis B,206,939694,134152,209265,269 21,269 57,686 20,000 1,560 20,000 26,611,1921194119681934195219691964, 65192319841985,4 2,237 2,572 4,083 4 160 11 45 1,41216,126,99.9999.7598.3198.4699.9899.7299.9597.1292.9439.40,患天花的小儿 （Infant with smallpox）,1979年WHO宣布在全球范围内消灭了天花病毒。,The last known person in the world to have smallpox of any kind. Variola minor in 23-year-old Ali Maow Maalin, Merka, Somalia,The last case of natural smallpox in U.K. occurred in the 1930s; the last in U.S.A. was in the 1940s. The last natural case in the world was in Somalia and occurred in October 1977.,患脊髓灰质炎的儿童 （Child with polio sequelae WHO）,WHO 宣称将在2003年在全球消灭脊髓灰质炎,In western countries polio is no longer a problem but it is still a major problem in less developed countries though some have had very major successes e.g. Cuba, Nicaragua . The wild type (non-vaccine) strain of polio has recently been declared to be wiped out from the Western Hemisphere.,Polio Virus,Polio Statistics,Wild poliovirus 1988,Know poliovirus transmission,Polio Statistics,Wild poliovirus 1998,Know poliovirus transmission,Victims of polio WHO,The control of a number of the diseases that cause significant mortality and morbidity has make outstanding progress, but there remains a crying need for vaccines against others. Every year, millions of deaths throughout the world are caused by malaria, tuberculosis, and AIDS, diseases for which there are no effective vaccines.Forty million deaths from AIDS area projected by the end of the 1990s, leaving more than 9 million orphans.,Mycobacterium tuberculosis.,HIV,Four Plasmodium species are responsible for human malaria, P. falciparum, P.vivax, P. ovale and P. malariae,IMMUNIZATION,Immunization is the means of providing specific protection against most common and damaging pathogens.,Immunity,Innate,Acquired,Passive,Active,Artificial,Natural,Artificial,Natural,Modes of immunization,Active Immunity,机体接受抗原，产生抗体和致敏淋巴细胞Antibody or sensitive lymphocyte produced by the body following exposure to antigens.,Naturally artificially,Active Immunity主动免疫,抗原 (Ag),与T细胞结合，激活T细胞,记忆T细胞,活化的T杀伤细胞,与B细胞结合，诱导B细胞释放抗体,记忆B细胞,活化的B细胞,抗体,免疫记忆 Immunologic memory,AB白喉vaccineDE,Anti-白喉antibody,白喉Bm cell,Anti-白喉antibody against白喉,白喉 Bm cell,days,First response to antigen白喉,白喉 vaccineimmunity,白喉Antigen B,immunity,Antibody concentration,First response to antigen B,Secondary response to antigen白喉,多价抗原,Surface antigenToxic antigen,多价抗原,Surface antigenToxic antigen,Surface antigen,vaccine,多价抗原,疫苗的发展,第一代 全菌疫苗 Inactivated, Attenuated第二代 基因工程疫苗 subunit vaccine, recombined DNA Vaccine第三代 DNA vaccine,Common currently used anti-viral vaccines,Polio,Inactivated,Attenuated,Rabies,first attenuated inactivated,Rubella Attenuated,Hepatitis A Inactivated,Hepatitis B Subunit,Varicella Attenuated,RotavirusAttenuated,InfluenzaInactivated,embryonated eggs,culture cells,many mutant viruses,primary monkey kidney cells,attenuation,Sabin polio vaccine,overgrown with a less virulent strain (for humans) that could grow well in non-nervous (kidney) tissue but not in the CNS,passage of the virus in foreign host,Attenuated Vaccines,high inocula and rapid passage,Mycobacterium bovis,On a meduim containing increasing concentration of bile,After 13 years,Bacillus Calmette-Guerin (BBC),Adapted to growth with bile and become sufficiently attenuatedand suitable as a vaccine for tuberculosis,attenuated nasal vaccine,cold-adapted vaccine,culture cells,progressively lower temperatures,dozen or more of these passages,grows well only at around 25,restricted to the upper respiratory tract.,influenza virus,用5-氟尿嘧啶（5-fluorouracil）突变并经过筛选温度敏感而获得的流感病毒A和RSV温度敏感突变株 （Temperature-sensitive mutants）. 在流感发病过程中， TS基因（TS gene）可在实验室进行重排，产生一株病毒，它具有现在流行株的外壳，和减毒株的内部蛋白。,New methods of vaccine production,* 基因重排,Attenuated influenza vaccine strain using a cold-sensitive mutant that can be reassorted with new virulent strains,NA：神经氨酸酶HA：血凝素 （中和抗体的靶位）,重组DNA技术 （ Recombinant DNA techniques）,Attenuation of virus,病毒的减毒,删除突变(Deletion mutations)是将足够大的片段除去，使其不再复毒。,保留抗原性片段,Deletion mutagenesis,Progressively deleting DNA from one end is very useful defining the importance of particular sequences. exonuclease III A strand-specific nuclease,5,3,N-,-C,cDNA,Protein,N-,- C,truncated protein,N-,- C,truncated protein,5,3,Deletion mutants,5,3,Deletion mutants,exonuclease III can remove one strand of nucleotides in a 3 to 5 direction from a recessed (凹) 3-end, but not from a 3-protruding （突出）end.,GAGCT-3 5GATCCC-5 3-G,ScaI BamHI,exonuclease III,GAGCT-3 5GATCCC-5,Nucleotides removed,Nucleotides not removed,MCS,MCS,strand-specific nuclease,S1 or mung bean nuclease remove the single protrusions at both ends,GAGCT-3 5GATCCC-5,G-3 5C-5 3,S1 or mung bean nuclease,ligate,GC,Deletion mutant,Deleted nucleotides,SacI BamHI,MCS,DNA insert,Digest with SacI and BamHI,GAGCT-3 5-GATCCC-5 3-G,ScaI BamHI,GAGCT-3 5-GATCCC-5,Exonuclease III,GAGCT-3 5-GATCCC-5,GAGCT-3 5-GATCCC-5,MCS,5,3,N,C,GAGCT-3 5-GATCCC-5,GAGCT-3 5-GATCCC-5,GAGCT-3 5-GATCCC-5,G-3 5C-5 3,S1 or mung bean nuclease,ligate,G-3 5C-5 3,G-3 5C-5 3,GC,GC,GC,MCS,DNA insert,MCS,DNA insert,MCS,DNA insert,MCS,DNA insert,Deletion mutants,MCS,3,C,MCS,3,C,MCS,3,C,MCS,3,C,5,N,5,N,5,N,5,N,5,3,N-,-C,cDNA,Protein,N-,- C,truncated protein,N-,- C,5,3,5,3,3,5,Deletion mutants,N-,- C,Approximately one in three of the deletions will be in frame and could be used to express truncated protein.,Gene encoding immunogenic protein ( from viral bacterial, and protozoan pathogens),Be cloned,Bacterial, yeast, inset, mammalian expression systems,expressed,immunogenic protein,vaccine,单基因法 Single gene approach,Be cloned,Yeast,In large fermenters,HBsAg protein,vaccine,First recombinant antigen vaccine Hepatitis B vaccine,HBsAg,Purified,induce,Protective antibodies,Cloning of protein into another virus.Cloning a protective antigen into another harmless virus,HIV vaccine,multivalent vaccine (多价疫苗)virus strain in this way as Vaccinia will accept several foreign genes.,Vaccinia(牛痘) ( the smallpox vaccine virus ),DNA Vaccines,The Third Vaccine Revolution,Plasmidvaccine,DNA Vaccines,基因免疫（gene immunization）DNA免疫（DNA immunization）核酸疫苗（polynucleotide vaccine）DNA疫苗 （DNA Vaccine）体细胞转基因免疫（somatic transgene immunization）裸DNA免疫（naked DNA immunization） 系指将靶抗原编码基因置于真核表达调控元件的调控下，将该质粒DNA直接进行动物体内接种，并以与自然感染类似的方式呈递抗原，诱生特异性体液和细胞免疫应答的新理论和技术。,一、概述,DNA表达载体,一个强的真核表达调控元件（通常为HCMV即刻早期启动子IE和增强子）、几个单一酶切点的多克隆位点（MCS）、一个多聚腺苷poly（A）加尾终止信号、一个原核复制启始位点启动质粒DNA在大肠杆菌中增殖及抗生素抗性选择标志。,CMV,MCS,原核复制启始位点启动,Plasmid vaccine,Gene for antigenic protein,Plasmid yield antigenic protein,antigenic peptides,Memory T cell,Activated cytotoxic T cell,Memory B cell,Antibodies,cellular immunity humoral immunity,（1）体内合成的蛋白抗原与自然感染状态下相同；（2） MHC-I类分子可有效呈递内源合成的蛋白，诱生CTL； （3） MHC-II分子呈递表达的产物诱生体液和细胞免疫应答； （4）质粒DNA不在体内复制；（5）DNA的制备、贮存极为方便和廉价，易操作和改造； （6）基因免疫可应用在一些特殊领域.,基因免疫的优点,表 2 基因免疫的优点,疫 苗 类 型,减毒活疫苗 复制型 死疫苗或 DNA疫苗 重组疫苗 亚单位疫苗,1、内源合成的抗原与MHC I 是 是 否 是 分子结合诱生CD8+CTL2、与MHC II类分子结合诱 是 是 是 是 生CD4+Th细胞免疫3、是否可覆盖所有的病原体 是 少数 是（亚单位疫苗否） 是（可能）4、低水平抗载体应答的存在是 / ？ / 是 否允许同一载体被反复使用 5、在孕妇和免疫功能低下 否 否 可能是 可能是 者中是否安全 6、回复毒力的危险性 是 是 否 否7、被有害成分污染的危险性 是 是 否 否8、制备和纯化是否简便 否 否 否 是9、热稳定 否 否 否 是 10、是否价廉 否 否 否 是,二 、基因免疫中目的基因的来源,1、完整抗原(complete antigen)编码基因的基因免疫2 、以抗原表位(Epitope)为基础的基因免疫3 、表达文库（expression library）基因免疫。,1、完整抗原(complete antigen)编码基因的基因免疫,抗原编码基因: 已知的病原微生物中抗原性较强的蛋白抗原,PCR,encoded,Th,Transfection,Expression,Secretion,Ingestion,Presentation,Specific humoral immunity and cellular immunity,2、以抗原表位(Epitope)为基础的基因免疫,Epitope or Antigenic Determinant,antigen,antigen,antigen,Antigenic determinants or epitopes are the immunologically active regions of an immunogen that bind to antigen-specific membrane receptors on lymphocytes (TCR/BCR) or to secreted antibodies.,T cell Epitope and B cell Epitope,In immune response, the epitopes that TCR and BCR recognize is different, and are called T cell epitope and B cell epitope, separately.,（1）human pancreatic glucagon (人胰高血