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1、疫苗与免疫预防Vaccine and immunoprophylaxis,Worldwide,vaccine sales are estimated to be approximately US$6.5 billion,representing only two per cent of the global pharmaceutical market,roughly equalling the sales of one successful drug(Greco,2002).,对病原的免疫力分为先天性和获得性免疫两种，免疫预防是疫病控制实践中面临的主要问题，主要通过人工被动及主动免疫式为动物提
2、供免疫保护。人工主动免疫通过接种疫苗来实现。疫苗免疫接种是控制动物疫病的主要手段，疫苗分活疫苗、灭活疫苗、亚单位疫苗及生物技术疫苗几大类，前两者最为常用，各有优缺点。,VACCINOLOGY,SUBUNITVACCINE,EPITOPEVACCINE,Vaccines induce protective immunity,an enhanced adaptive immune response to re-infection.,PATHOGEN AND HOST,如何找到疫苗株/疫苗分子？,传统方法 找到合适的疫苗株 找到合适的疫苗分子,新技术的应用：反向疫苗学免疫蛋白组学,Reverse V
3、accinology,The basic idea behind Reverse Vaccinology is that an entire pathogenic genome can be screened usingbioinformaticsapproaches to find genes.Next,those genes are filtered for desirable attributes that would make good vaccine targets such as outer membrane proteins.Those proteins then undergo
4、 normal wet lab testing for immune responses.,Genome Sequence,In silico analysis,High throughputCloning and expression,In vitro and in vivo assays forVaccine candidate identification,Global genomic approach to identify new vaccine candidates,AdvantagesFast access to virtually every antigenNon-cultiv
5、able can be approachedNon abundant antigens can be identifiedAntigens not expressed in vitro can be identified.Non-structural proteins can be usedDisadvantagesNon proteinous antigens like polysaccharides,glycolipids cannot be used.,In Silico Analysis,Gene/Protein Sequence Database,Disease related pr
6、otein DB,Candidate Epitope DB,VACCINOME,PeptideMultitope vaccines,Epitope prediction,2D-WB的免疫蛋白组学,相同的蛋白样品,进行质谱分析的是染色胶上的点这些点与转印膜上有免疫反应性的点相对应,近年发表的免疫蛋白组学文章,Zhang,W.,Liu,G.,Tang,F.,Shao,J.,Lu,Y.,Bao,Y.,Yao,H.,Lu,C.,2011a,Pre-absorbed immunoproteomics:a novel method for the detection of Streptococcus su
7、is surface proteins.PLoS One 6,e21234.Zhang,W.,Shao,J.,Liu,G.,Tang,F.,Lu,Y.,Zhai,Z.,Wang,Y.,Wu,Z.,Yao,H.,Lu,C.,2011b,Immunoproteomic analysis of bacterial proteins of Actinobacillus pleuropneumoniae serotype 1.Proteome Sci 9,32.Zhang,W.,Lu,C.P.,2007a,Immunoproteomic assay of membrane-associated prot
8、eins of Streptococcus suis type 2 China vaccine strain HA9801.Zoonoses Public Health 54,253-259.Zhang,W.,Lu,C.P.,2007b,Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2.Proteomics 7,4468-4476.Wu,Z.,Zhang,W.,Lu,C.,2008b,Immunoproteomic assay of surfac
9、e proteins of Streptococcus suis serotype 9.FEMS Immunol Med Microbiol 53,52-59.Wu,Z.,Zhang,W.,Lu,Y.,Lu,C.,2010,Transcriptome profiling of zebrafish infected with Streptococcus suis.Microb Pathog 48,178-187.Wu,Z.,Zhang,W.,Shao,J.,Wang,Y.,Lu,Y.,Lu,C.,2011,Immunoproteomic assay of secreted proteins of
10、 Streptococcus suis serotype 9 with convalescent sera from pigs.Folia Microbiol(Praha)56,423-430.Zhu,Y.Z.,Cai,C.S.,Zhang,W.,Guo,H.X.,Zhang,J.P.,Ji,Y.Y.,Ma,G.Y.,Wu,J.L.,Li,Q.T.,Lu,C.P.,Guo,X.K.,2010,Immunoproteomic analysis of human serological antibody responses to vaccination with whole-cell pertus
11、sis vaccine(WCV).PLoS One 5,e13915.,第1节 免疫的分类,天然被动免疫天然主动免疫人工被动免疫人工主动免疫,Active immunity:The production of antibodies against a specific agent by the immune system.Active immunity can be acquired in two ways:by contracting an infectious disease-such as,for example,chickenpox;or by receiving a vaccinat
12、ion usually-such as,for example,against polio.,Passive immunity is the transfer of active humoral immunity in the form of readymade antibodies,from one individual to another.Passive immunity can occur naturally,when maternal antibodies are transferred to the fetus through the placenta,and can also b
13、e induced artificially,when high levels of human(or horse)antibodies specific for a pathogen or toxin are transferred to non-immune individuals.,动物在感染某种病原微生物耐过后产生的对该病原体再次侵入的抵抗力称为天然主动免疫动物通过母体胎盘、初乳或卵黄从母体获得某种特异性抗体，从而获得对某种病原体的免疫力，称为天然被动免疫。采用人工方法向机体输入由他人或动物产生的免疫效应物，如免疫血清、淋巴因子等，使机体立即获得免疫力人工被动免疫接种抗原而获得的免疫力
14、人工主动免疫,第2节 全微生物疫苗,活疫苗（living vaccines）,毒力减弱的方法:长时间在体外连续培养传代：在高于最适生长温度条件下培养:炭疽 在含有特殊物质的培养基中培养:卡介苗 在特殊气体条件下培养:通过非易感动物:猪丹毒通过豚鼠370代后，又通过鸡42代选育而成；猪瘟兔化弱毒 通过基因工程的方法:去除毒力基因或用点突变，伪狂犬基因缺失苗,异源疫苗是用具有共同保护性抗原的不同病毒制备成的疫苗，用火鸡疱疹病毒（HVT）接种预防鸡马立克氏病用鸽痘病毒预防鸡痘牛痘预防人类天花,Edward Jenner,Discovery of small pox vaccine,Immunisat
15、ion against Smallpox WHO Programme,1978:WHO Programme completed.,Smallpox completely eradicated worldwide.,Live vaccines,Attenuated strains which replicate in hostattenuation means the virus or bacterium has been weakened to reduce virulence so it cannot cause disease in healthy peopleAct like natur
16、al infection live vaccines are the closest to actual infection and therefore elicit good,strong,long-lasting immune responses,Live vaccines,AdvantagesSingle dose often sufficient to induce long-lasting immunityStrong immune response evokedLocal and systemic immunity produced,DisadvantagesPotential t
17、o revert to virulenceContraindicated in immunosuppressed patientsInterference by viruses or vaccines and passive antibodyPoor stabilityPotential for contamination,优点：毒力弱或没有毒力，但仍然保持着原有的抗原性，可用较少的免疫剂量即或诱导产生坚实的免疫力，一般不须使用佐剂，免疫期长，不影响动物产品的品质有些弱毒病毒疫苗可刺激机体细胞产生干扰素，对抵抗其他病毒强毒的感染也是有益的,缺点许多弱毒株的致弱机制不明，有返强的危险研制周期长，
18、有一定的偶然性运输贮存不方便，一般需要冻干和低温保存,灭活疫苗,病原微生物经理化方法灭活后，仍然保持免疫原性，接种后使动物产生特异性抵抗力，这种疫苗称为灭活疫苗（killed vaccines）或死疫苗。suspensions of whole intact killed organisms e.g.whole cell pertussis,influenza,rabies,HepA目前所使用的灭活疫苗有组织灭活苗、油佐剂灭活疫苗和氢氧化铝胶灭活疫苗等。,灭活疫苗病变组织灭活苗：用患传染病病死动物的典型病变组织，经碾磨、过滤，按一定比较稀释并加入灭活剂后（一般还要加上佐剂）制备而成。鸡胚组织灭
19、活苗是用病原微生物接种鸡胚后，经一定孵育时间收获除卵黄外的所有胚组织，经碾磨、过滤，按一定比较稀释并加入灭活剂后（一般还要加上佐剂）制备而成。使用前均需要做无菌检测,灭活的方法最常用的是甲醛（0.4%）溶液，灭活机制：作用于蛋白质的氨基和酰胺基，以及核酸的嘌呤和嘧啶上的非氢键氨基团，形成交联，使其结构固定并丧失活力。其它的灭活剂还包括丙酮、乙醇、氧化乙烯等等。,killed vaccines,AdvantagesStable Constituents clearly definedUnable to cause the infection,Disadvantages Need several
20、dosesLocal reactions common Adjuvant neededkeeps vaccine at injection siteactivates antigen presenting cellsShorter lasting immunity,优点：研制周期短、使用安全和易于保存。缺点：接种剂量大，免疫期较短，需加入适当的佐剂以增强免疫效果，接种时反应较大，对肉品品质有影响。,多价苗与联苗,多价苗：指将同一种微生物的不同血清型混合制成的疫苗。如巴氏杆菌多价苗、大肠杆菌多价苗等。多联苗：是指由两种以上的细菌（或病毒）联合制成的疫苗。,三 提纯的大分子疫苗,多糖蛋白结合疫苗（
21、polysaccharide conjugate vaccines）是将多糖与蛋白载体（如白喉、破伤风或霍乱类毒素）结合制成疫苗，这样多糖抗原转变成为T细胞信赖性抗原，能诱导产生IgG和记忆淋巴细胞。如人的肺炎链球菌的多种血清型荚膜多糖蛋白载体疫苗。,CHARACTERISTICS OF IMMUNE RESPONSES TO T-CELL INDEPENDENT VS.T-CELL DEPENDENT ANTIGENS,Polysaccharide antigen,T cell independent,Production dysproportionate IgMLack of immuno
22、logical memoryShort-lived immune responseFailure to produce high affinity antibody,Protein-Polysachharide conjugate antigen,T cell dependent,Stimulate T-helper cell responseStrong booster response upon re-exposureiii.High affinity antibody,Carrierprotein,Polysaccharide linked to carrier protein,Conj
23、ugate vaccine,Bacteria,Polysaccharide(sugar)coating,Conjugation,Conjugation is the process of attaching(linking)the polysaccharide antigen to a protein carrier(e.g.diphtheria or tetanus)that the infants immune system already recognises in order to provoke an immune response,毒素疫苗(toxoid vaccines)是将细菌
24、外毒素经甲醛脱毒，使其失去致病性而保留免疫原性的制剂，如破伤风类毒素、白喉类毒素、肉毒类毒素等等。,Modification of Toxin to Toxoid,toxin moiety,antigenic determinants,Toxin,Toxoid,亚单位疫苗（subunit vaccine）利用微生物的某种表面结构成分（抗原）制成不含有核酸、能诱发机体产生抗体的疫苗，称为亚单位疫苗。微生物的免疫原性结构成分包括细菌的荚膜、鞭毛和病毒的囊膜、膜粒、衣壳蛋白等。,第3节 基因工程重组亚单位疫苗,用基因工程技术将具有免疫保护力的蛋白质基因克隆，并进行表达，以获得亚单位疫苗的方法常用的表
25、达表达系统有大肠杆菌、酵母和病毒等等。,重组载体的构建,重组质粒的表达（如大肠杆菌中）,第4节 基因工程重组活载体疫苗,概念：基因工程重组活载体疫苗（recombinant living vectored vaccines）是用基因工程技术将保护性抗原基因转移到载体中使之表达的活疫苗。有多种可用于活载体的疫苗：如痘病毒、腺病毒和疱疹病毒。,基因工程重组活载体疫苗的种类,重组载体病毒活疫苗痘病毒，疱疹病毒，腺病毒重组载体细菌活疫苗沙门氏菌，卡介苗等,重组载体细菌死疫苗菌蜕,菌蜕系统是一个自身具有佐剂性质的新型疫苗体系,不含细胞质内容物但具有细菌的完整表面抗原结构,可诱导机体的体液、细胞免疫应答及
26、增强黏膜免疫反应。Bacterial ghosts are a novel nonliving vaccination technology platform,which is based on the conditional expression of the lethal lysis gene E from bacteriophage PhiX174 in Gram negatives,迟缓爱德华菌菌蜕疫苗的制备,迟缓爱德华菌菌蜕扫描电镜照片孔径大约200 to 500 nm（箭头处）,迟缓爱德华菌扫描电镜照片表面光滑、无孔,迟缓爱德华菌菌蜕疫苗的制备,迟缓爱德华菌菌蜕透射电镜照片无内含物
27、（箭头处）,第5节 基因缺失疫苗,基因缺失疫苗（gene deleted vaccines）是用基因工程技术将强毒株毒力相关基因切除构建的活疫苗 该类疫苗安全性好、不易返祖；其免疫接种与强毒感染相似，机体可对病毒的多种抗原产生免疫应答；免疫力坚实，免疫期长，尤其是适于局部接种，诱导产生黏膜免疫力，因而是较理想的疫苗。,牛传染性气管炎病毒伪狂犬病毒的tK基因缺失疫苗,第6节 核酸疫苗,核酸疫苗(nucleic acid vaccines)包括 DNA疫苗和RNA疫苗，由编码能引起保护性免疫反应的病原体抗原基因片段和载体构建而成。导入机体的方式主要是肌肉注射，或用基因枪将带有基因的金粒子注入。,H
28、OW DNA VACCINE IS MADE,Viral gene,Expression plasmid,Plasmid with foreign gene,Recombinant DNA Technology,Bacterial cell,Transform into bacterial cell,Plasmid DNA,Plasmid DNA get Amplified,Plasmid DNA Purified,Ready to use,Gene gun delivery:-,Adsorbed plasmid DNA into gold particlesBallastically acc
29、elerated into body with gene gun.,第7节 合成肽疫苗与表位疫苗,合成肽疫苗（synthetic peptide vaccines)是用化学合成法人工合成病原微生物生物的保护性多肽，并将其连接到大分子载体上，再加入佐剂制成的疫苗。最早报道（1980）成功的是口蹄疫多肽疫苗,Synthetic Peptide,The synthetic peptide approach to vaccine development arose in response to rapid DNA cloning and sequencing technology.This made
30、it possible to quickly obtain primary sequences and construct various peptides.It is important to consider the dynamics of humoral versus cell mediated immunity when developing a synthetic peptide vaccine.In terms of humoral responses the focus is primarily B cells.Since B cells recognize antigens i
31、n their native form vaccinologists must identify the spatially accessible epitopes of the naturally occuring antigen and sequence these key peptides.Whether humoral or cell mediated immunity is desired T cells are vital to the generation of effective memory.Synthetic peptide vaccines are particularl
32、y useful in inducing the generation of memory helper T cells.,In contrast to B-cell epitopes,T-cell epitopes are linear peptides of a relatively short length of 9 to 11 amino acids which are presented within MHC molecules.However,it is not understood in detail which peptides are immunodominant T cel
33、l epitopes.Furthermore,immunodominance of T cell epitopes is associated with MHC haplotype,so certain peptides are only effective for those with certain haplotypes.Currently,computer-driven algorithms are used to predict T-cell epitopes based on data of already known epitopes for specific haplotypes
34、.,The synthetic peptide vaccine has had an impact on three major fronts:,Bacteria-synthetic peptide vaccines for bacterial diseases have focused primarily on trying to neutralize toxins.For example,synthetic peptide vaccines have been developed for both the diphtheria and cholera toxins.Viruses-curr
35、ently,invariant regions(amino acid sequence highly conserved)are the focus of synthetic peptide vaccine design for viruses.Synthetic peptide vaccines for HIVproteins and glycoproteins are currently under development.Parasites-scientists hope to achieve stage specific anti-malarial immunity by constr
36、ucting synthetic peptide vaccines for key malarial sporozoite epitopes.,表位疫苗（epitope vaccines）是通过确定抗原蛋白上的B细胞、TH细胞以及CTL细胞识别的表位，经人工合成，并与大分子载体连接，加入佐剂制成；也可通过基因工程技术表达抗原蛋白表位多肽，或表达与大分子蛋白的融合蛋白制成。,其它形式或功能的疫苗,转基因植物疫苗、(transgenic plant vaccines)负疫苗:中止免疫应答计划生育疫苗抗独特型疫苗,疫苗的接种方法,皮内皮下肌肉静脉腹腔,皮内,皮下,1.注射法：将疫苗直接注射到肌肉、腹
37、腔或心脏的免疫接种方法。优点：免疫效果好；缺点：操作困难，易损伤接种鱼类，幼鱼无法使用。2.口服法：将疫苗通过投喂的方法接种到鱼类机体 优点：操作简单；缺点：免疫效果不稳定（因消化液对疫苗的消化分解）3.浸泡法：将鱼类浸泡于疫苗溶液中以达到免疫接种目的 优点：操作简单；缺点：免疫效果不稳定（因浸泡进入机体的疫苗数量有限）,鱼用疫苗的接种方法,A certain dose of vaccine(usually 0.1ml to 0.2ml)is injected in the abdominal area of each fish held with the ventral side up an
38、d the head away from the operators body.,4.喷雾法：将疫苗以高压喷射到接种生物体表以进行免疫的方法。优点：操作简单；缺点：免疫效果差。5.其他方法：如超声免疫法等，该法是在浸泡法基础上外加超声作用而实施的接种方法，目前尚在探索中。,第10节 细菌组织灭活疫苗的制备举例,取患有典型症状病畜的肝、脾、肾等病变组织，用清水冲洗后称重，用研钵磨碎，加510倍的生理盐水，成匀浆后用两层纱布过滤，取滤液。将滤液经60-65水浴灭活2小时后，加入福尔马林使其最终浓度为1%，封口后，置4并向中保存。做安全及效力试验后即可使用。,免疫失败原因,遗传因素营养状况环境因素疫苗方面病原的血清型疾病对免疫的影响母源抗体病原微生物之间的相互干扰,谢 谢,
