1、病毒感染與自身免疫病病毒感染與自身免疫病 Virus Infections and autoimmunity Exogenous viruses: EBV Molecular mechanism for autoimmunityEnogenous viruses : HERVAutoimmune disease: A role for new antiviral therapies?Outline Outline Virus Infections and autoimmunity (Background)自身免疫是機體失去對自身抗原的免疫耐受自身免疫病因與發(fā)病機制十分復雜: 遺傳背景，但環(huán)境因

2、素和生活方式等的也有重要作用。研究表明病毒和細菌等感染與自身免疫病的發(fā)生、發(fā)展密切關(guān)系。一些病毒感染可導致機體失去對自身抗原免疫耐受，因而誘發(fā)、促進或加重自身免疫病。已證明某些病毒感染與一些自身免疫病密切相關(guān)，并發(fā)現(xiàn)病毒與宿主細胞的某些基因或其蛋白分子之間存在著結(jié)構(gòu)類似性，因而可發(fā)生交叉免疫反應性。這種分子模擬假說獲動物實驗的支持。 Virus Infections and autoim少數(shù)T細胞克隆逃 避陰性選擇 進入外周識別自身抗原 通過激活誘導細胞死亡（AICD) (FasL-Fas-凋亡）部分T細胞克隆 逃避AICD識別自身抗原自身免疫病(AID)T細胞發(fā)育過程自身耐受形成及逃避機制少

3、數(shù)T細胞克隆逃識別自身抗原 通過激活誘導部分T細胞克B細胞發(fā)育過程自身耐受形成及逃避機制 克隆清除 結(jié)合 克隆無能 克隆忽視 不結(jié)合 克隆發(fā)育成熟激活自身免疫病 自身免疫病激活B細胞骨髓細胞表面抗原B細胞發(fā)育過程自身耐受形成及逃避機制 HostMHCLoss of ToleranceImmune system defectsSusceptibility GenesEndogenous VirusesAutoimmune diseaseEnvironment and autoimmunityChemical substancesMicrobial AgentsdrugsUVB lightVacc

4、inestyphoid, influenza, meningococcal, tetanus toxoid, measles, mumps, and rubellaDiet/NutritionHostEnvironment and autoimmuni新西蘭小鼠(NzB)自發(fā)產(chǎn)生抗DNA抗紅細胞抗體溶血性貧血狼瘡性腎炎自然發(fā)生新西蘭小鼠(NZW)不發(fā)生系統(tǒng)性紅斑狼瘡(SLE)NZB X NZW(F1) 雜交小鼠抗DNA和其他核抗體與人類SLE類似感染病毒感染病毒自身抗體效價更高自身免疫病的發(fā)生更早發(fā)生自身免疫病These observations lend further support th

5、at both genetic and environmental factors play a role in autoimmune disease.新西蘭小鼠(NzB)自發(fā)產(chǎn)生抗DNA溶血性貧血自然發(fā)生新西蘭病毒感染與自身免疫病課件Exogenous virusesExogenous microbial agents, such as bacteria or viruses, interact with and sometimes might override the human immune system to cause infectious diseases, cancers and

6、 autoimmunity.Exogenous virusesExogenous micExogenous viral agents have also been implicated as potential triggers or pathogenic agents of autoimmune conditions. Viruses which have been linked to the pathogenesis of SLE include EpsteinBarr Virus (EBV) Cytomegalovirus (CMV), parvovirus (細小) B19 Human

7、 Papilloma Virus (HPV), Human Herpes Virus (HHV6), HHV7, HHV8, Dengue virus HIV human T cell lymphotropic virus (HTLV,人類嗜T細胞病毒 )Exogenous viral agents have alEBV as a potential viral agent in SLEIn SLE, EBV has been highlighted as a potential pathogenic agent through serological studies of patients

8、that have shown antibodies to early antigens, capsid and latent membrane proteins in much higher frequency as compared to controlsin pediatric SLE, 99% of patients are antiEBV Ab+ compared to 70% of controlsIn people with SLE, the number of EBV infected B cells is elevated 10 to 100 times over the l

9、evels in controls, and the amount of free virus in serum is also elevated.molecular analysis of complementary EBV transcripts appears to substantiate these observationIncreased virus levels are associated with disease flares LMP1 mRNA is the most frequently detected latent EBV product in the blood o

10、f SLE patientsa metaanalysis of serological studies revealed that antibodies to capsid and early antigen proteins were particularly prominent.EBV is recognized as a strong polyclonal B cell activator and therefore could stimulate a substantial number of potentially autoreactive B cells.EBV as a pote

11、ntial viral agentEBV 結(jié)構(gòu)和蛋白（Epstein-Barr virus，EBV）Epstein和Barr：1964年首次成功地在Burkitt非洲兒童淋巴瘤細胞中發(fā)現(xiàn)靶細胞： B細胞、鼻咽上皮細胞、胃上皮感染類型： 潛伏感染 核抗原（EBNA），潛伏膜蛋白（LMP） 增殖感染 早期蛋白（EA）， 病毒衣殼抗原（VCA） 膜抗原（MA）受體：CD21分子EBV 結(jié)構(gòu)和蛋白（Epstein-Barr virus，E增殖性感染表達的抗原 當EBV進入宿主細胞后，首先表達反式激活蛋白ZERBA，進而激活EBV早期基因，產(chǎn)生增殖性感染。 EBV早期抗原(early antigen,

12、EA) 增殖早期誘導的非結(jié)構(gòu)蛋白，分為 EAR存在于細胞漿中 EAD存在于細胞漿和細胞核內(nèi)，并具有EBV特異的DNA多聚酶活性。 EA表示EBV增殖活躍，是感染細胞進入溶解性周期的標志。 非洲兒童惡性淋巴瘤患者抗EAR抗體陽性，鼻咽癌患者抗EAD抗體陽性。EBV衣殼抗原(viral capsid antigen, VCA) : 病毒增殖晚期合成的結(jié)構(gòu)蛋白，存在于細胞漿和細胞核內(nèi)。 VCA: 與病毒DNA組成EBV的核衣殼，在核膜出芽時獲得包膜裝配成完整病毒體。 VCAIgM: 出現(xiàn)早，消失快，VCAIgG:出現(xiàn)晚，持續(xù)時間長。EBV膜抗原(membrance antigen, MA) 存在于細

13、胞表面，為包膜糖蛋白，其中 糖蛋白gp320/220能誘導產(chǎn)生中和抗體。 MAIgM: 用于早期診斷，MAIgG: 可體內(nèi)持續(xù)存在。增殖性感染表達的抗原 當EBV進入宿主細胞后，首先表潛伏性感染表達的抗原 EBV在記憶B細胞及某些上皮細胞中可表現(xiàn)為潛伏感染，僅部分表達基因發(fā)生轉(zhuǎn)錄， 選擇性表達EBV潛伏感染期蛋白。 EBV核抗原（EB nuclear antigen, EBNA）: DNA結(jié)合蛋白，所有EBV感染 和轉(zhuǎn)化的B細胞核內(nèi)均可檢出該抗原。 (EBNA1, 2, 3A, 3B, and 3C) EBNA1: 與EBV基因組以環(huán)狀附加體(episome)形式持續(xù)存在，對細 胞處理和抗原提

14、呈功能具有抑制作用，從而逃避宿主細胞的 CTL殺傷作用，因此與維持EBV基因組在感染細胞內(nèi)潛伏有關(guān)； EBNA2和EBNA3為轉(zhuǎn)錄因子: 可調(diào)控多種病毒蛋白和宿主細胞蛋白的表 達，與誘導B細胞轉(zhuǎn)化有關(guān)。潛伏感染膜蛋白(latent membrance protein, LMP) : 存在于潛伏感染B細胞表面，有LMP1和LMP2 (LMP2A,LMP2B)兩種。 LMP1: 功能類似活化的生長因子受體，能與細胞抑癌蛋白（腫瘤壞死因子受體相關(guān) 因子，TRAF）相互作用，抑制細胞凋亡，誘導B細胞轉(zhuǎn)化，是一種致癌蛋白； LMP2: 細胞酪氨酸激酶的底物，具有阻止?jié)摲《炯せ畹淖饔谩摲愿腥颈磉_的抗

15、原 EBV在記憶B細胞及某些上皮細Gp350-CD21BMRF2-1整合素gp110-?Gp350-CD21BMRF2-1整合素gp110-?EBV specific CD8+ Tcells are enriched in or near the diseased organs of patients with RA and MS .膜抗原（MA）Bystander Activation（旁路激活）MSRV 體內(nèi)外均可激活Tcells 并誘導產(chǎn)生 cytokines.EBV as a potential viral agent in SLEEBV 誘導內(nèi)源性逆轉(zhuǎn)錄病毒產(chǎn)生 HERVK18HER

16、VK18編碼超抗原激活 T細胞HERV數(shù)量雖多，但大部分由于突變、缺失等的積累，已經(jīng)沒有編碼能力。in serum is also elevated.domains, termed CTAR1, CTAR2 and CTAR3, which recruit TNFR-associated signalling adapter proteinsautoimmune disease.抗DNA和其他核抗體與人類SLE類似Serum antibodies from SLE patients have also demonstrated to an envelope derived peptide of

17、 HERV K10typhoid, influenza, meningococcal, tetanus toxoid, measles, mumps, and rubellaERV3，HERVE 41HERV抗體在睪丸腫瘤、乳癌和黑素瘤中很常見。(HTLV,人類嗜T細胞病毒 )molecular analysis of complementary EBV transcripts appears to substantiate these observation結(jié)合 克隆無能Immunological Mechanisms for AutoimmunityMolecular MimicryBys

18、tander Activation and Epitope Spreading Polyspecific Bcell Activation Accumulation of EBVspecific CD8+ Tcells in Sites of Inflammation Transactivation of Human Endogenous Retroviruses （反式激活）EBV specific CD8+ Tcells are eMolecular mimicrySequence or structural similarities between microbial and selfa

19、ntigens are believed to cause crossreactivity of Tcells, Bcells and antibodies Molecular mimicrySequence or sEBNA1In SLE, autoantibodies against epitopes on SmBB and SmD have been shown to crossreact with different domains of EBNA1 EBNA1 motif PPPGRRP (aa 398404) Rabbits immunized lupuslike autoimmu

20、ne disease EBNA1 full length protein mice immunized antidsDNA and antiSm antibodiesRo (aa 169180) autoantibodies SLE EBNA1 (aa 5872) cross-reactionEBNA1In SLE, autoantibodies ag李玲玲，朱珊麗，李文姝，薛向陽，張麗芳* EBV核蛋白-1B細胞表位的預測及其同源性分析 生物醫(yī)學工程學雜志 2011，28（2）：371-375李玲玲，朱珊麗，李文姝，薛向陽，張麗芳* EBV核蛋白-1LMP1LMP1 expression h

21、as been implicated in making important contributions to a variety of human malignancies, as well as to autoimmune diseases.LMP1 alters Bcell biology and the molecular mechanisms by which it exerts these effects by LMP1mediated signaling pathwaysLMP1LMP1 expression has been iFig. 1. Activation of cel

22、l signalling pathways by LMP1. The carboxyl terminus of LMP1 contains three signalling domains, termed CTAR1, CTAR2 and CTAR3, which recruit TNFR-associated signalling adapter proteins (TRAF, TRADD, RIP), BS69 and Janus kinase (JAK)-3 proteins. These activate the NF-B, JNK/SAPK, PI3-K/Akt, ERK-MAPK,

23、 PLC/PKC and JAK/STAT signalling pathways, which collectively induce the expression of numerous downstream effectors that impact on a variety of cellular processes such as proliferation, survival, motility and invasion. 187-386: 200個氨基酸殘基為C端胞漿區(qū)，含活性區(qū)域1-3（CTAR1，2，3） IFNIL-6IL-10IFN- play an important

24、rolein SLE pathogenesis. (development of the inflammatory)in theFig. 1. Activation of cell sigBystander Activation（旁路激活）Bystander activation lymphocytes are stimulated by cytokines or superantigens, or antigendependent in the setting of tissue destruction and presentation of selfantigens by APC to a

25、utoreactive T or Bcells抗微生物 抗體（T細胞表位不同，無Th活化信號）Bystander Activation（旁路激活）Byst抗微生物 抗體抗微生物Epitope Spreading（表位擴展）抗原刺激免疫系統(tǒng)，首先針對免疫優(yōu)勢表位產(chǎn)生免疫應答，如果抗原沒有及時清除，免疫應答持續(xù)時，機體可相繼針對隱蔽表位產(chǎn)生應答表位擴展。Epitope Spreading（表位擴展）抗原刺激免疫系Polyspecific Bcell Activation（非特異性激活）Polyspecific Bcell Activation（Accumulation of EBVspecific

26、 CD8+ Tcells in Sites of InflammationEBV specific CD8+ Tcells are enriched in or near the diseased organs of patients with RA and MS . EBVspecific CD8+ Tcells have also been reported to accumulate in synovial fluid (滑液)from patients with psoriatic arthritis, osteoarthritis and Reiters syndrome局部炎癥部位

27、EBV特異性CTL細胞的集聚，加劇了局部的炎癥反應Accumulation of EBVspecific CDTransactivation of Human Endogenous Retroviruses EBV 誘導內(nèi)源性逆轉(zhuǎn)錄病毒產(chǎn)生 HERVK18HERVK18編碼超抗原激活 T細胞 HERVK18表達產(chǎn)物在RA的外周血和關(guān)節(jié)液中出現(xiàn) ，但SLE尚未報道。 已經(jīng)證明EBV 在體外可激活HERVW（MS相關(guān)逆轉(zhuǎn)錄病毒（MSRV），在MS患者的星形膠質(zhì)細胞，B細胞和單核細胞中存在MSRVMSRV 體內(nèi)外均可激活Tcells 并誘導產(chǎn)生 cytokines. Transactivation

28、of Human EndogEnogenous viruses : Human Endogenous Retroviruses （HERV） HERV是幾百萬年前整合到人類基因組中，并以孟德爾方式遺傳至今的逆轉(zhuǎn)錄病毒的殘余物，約占了人類基因組DNA的8HERV可以通過轉(zhuǎn)座作用而增加其在基因組中的拷貝數(shù)，因此有些家族的成員數(shù)達上千個，現(xiàn)已發(fā)現(xiàn)的HERV家族至少有31個。HERV數(shù)量雖多，但大部分由于突變、缺失等的積累，已經(jīng)沒有編碼能力。HERV各家族基因結(jié)構(gòu)基本相同，但許多功能都不明確，過去大多數(shù)研究人員將內(nèi)源性逆轉(zhuǎn)錄病毒視為垃圾DNA。Enogenous viruses : Human End

29、HERV 編碼的超抗原、HERV 病毒顆粒及 B細胞表達的蛋白 激活和巨噬細胞1997年，發(fā)現(xiàn)多發(fā)性硬化癥(MS)患者細胞培養(yǎng)過程中產(chǎn)生了EAR存在于細胞漿中Environment and autoimmunityBystander Activation and Epitope SpreadingLMP1 alters Bcell biology and the molecular mechanisms by which it exerts these effects by LMP1mediated signaling pathwaysEAD存在于細胞漿和細胞核內(nèi)，并具有EBV特異的DNA多聚

30、酶活性。B細胞發(fā)育過程自身耐受形成及逃避機制EBNA2和EBNA3為轉(zhuǎn)錄因子: 可調(diào)控多種病毒蛋白和宿主細胞蛋白的表和轉(zhuǎn)化的B細胞核內(nèi)均可檢出該抗原。Cytomegalovirus (CMV),HERVK18表達產(chǎn)物在RA的外周血和關(guān)節(jié)液中出現(xiàn) ，但SLE尚未報道。EBNA2和EBNA3為轉(zhuǎn)錄因子: 可調(diào)控多種病毒蛋白和宿主細胞蛋白的表28kD nuclear autoantigen (p28) HRES1/p28HERV與系統(tǒng)性紅斑狼瘡的關(guān)系EBNA1 motif PPPGRRP (aa 398404)Schematic representation of HERV-K10.具有高度的病毒特

31、異性和有效性即可針對病毒復制又可針對病毒的長期持續(xù)感染。Class II HERVs:(HTLV,人類嗜T細胞病毒 )Class III :Human Endogenous Retroviruses （HERV）隨著研究的深人，人們發(fā)現(xiàn)少部分HERV保留了產(chǎn)生逆轉(zhuǎn)錄病毒產(chǎn)物和病毒顆粒的能力HERV與人類的進化關(guān)系密切，是哺乳動物生殖所必需的，并且影響哺乳動物胎盤發(fā)育，是妊娠所不可或缺的基因。同時和胎盤共同構(gòu)建了一個防止微生物感染胎盤的屏障。HERV還參與人體多種自身免疫性疾病和腫瘤的發(fā)生和發(fā)展過程。 HERV抗體在睪丸腫瘤、乳癌和黑素瘤中很常見。HERV 編碼的超抗原、HERV 病毒顆粒及 B

32、細胞表達的蛋幾百萬年前，HERV感染人類的生殖細胞，從而成為宿主遺傳基因組的一部分。遺傳給宿主的子孫后代。整合在人類基因組中的內(nèi)源性逆轉(zhuǎn)錄病毒無法從宿主的DNA上轉(zhuǎn)錄。幾百萬年前，HERV感染人類的生殖細胞，從而成為宿主遺傳基因病毒感染與自身免疫病課件 Schematic representation of HERV-K10. Insert: electron micrograph (magnification: 15,000) of HERV-K particles budding from a teratocarcinoma（畸胎瘤） cell line TERA-1. Schematic

33、 repreHERVsClass I HERVs: HRES1， (HERVR) ERV3 HERVE 41Class II HERVs: HERVK10， HERVK18Class III : HERVsClass I HERVs:HRES1two Gagrelated products : 28kD nuclear autoantigen (p28) HRES1/p28 24kD small GTPase (termed HRES1/Rab4)higher titres of autoantibodies to HRES1/p28 nuclear protein or synthetic

34、peptides in patients with SLEMolecular mimicry may be responsible in generating crossreactive HRES1/p28 and the 70kD spliceome protein U1 snRNP.In lupus patientsCD4 + T cells appear to overexpress HRES1/Rab4， which regulates the CD4.Thus, Gagrelated products could play a significant role in modulati

35、ng B and T cell reactivity in SLEHRES1two Gagrelated products :Figure 3 (a) Molecular models of a homologous epitopes located on SmD1 and HRES-1. (b) Amino acid sequence homology between HRES-1 gag and SLE autoantigens.Figure 3 (a) Molecular models ERV3，HERVE 41ERV3 Env and several SLE autoantigens,

36、 RibosomalP,Ro/SSAribo nuclear protein and Beta2glycoprotein共同抗原Beta2glycoprotein is associated with antiphospholipid syndrome（抗磷脂綜合征）, whilst RibosomalP （核糖體磷脂）antibodies are strongly associated with SLE, as demonstrated in a mouse model.Peripheral blood mononuclear cells derived from SLE patients

37、generate mRNA gag transcripts of HERVE 41ERV3，HERVE 41ERV3 Env and seveHERV K10Serum antibodies from SLE patients have also demonstrated to an envelope derived peptide of HERV K10Herpes viruses such as EBV and CMV have been shown to influence HERVK10 activity and upregulate HERV transcriptionEBV gen

38、e products, EBNA1, Latent Membrane Protein (LMP1) and LMP2A, significantly enhance HERVK10 transcripts.HERV K10Serum antibodies from HERV與系統(tǒng)性紅斑狼瘡的關(guān)系在系統(tǒng)性紅斑狼瘡患者的器官和血清中發(fā)現(xiàn)了HERV成分的抗原及相應 的抗體， 電鏡觀察系統(tǒng)性紅斑狼瘡患者的組織，發(fā)現(xiàn)了逆轉(zhuǎn)錄病毒顆粒HRES1, ERV3, HERVE 41, HERVK10 and HERVK18 have also been implicated in SLE.在體外實驗中HERV逆

39、轉(zhuǎn)錄病毒成分可以誘導出系統(tǒng)性紅斑狼瘡樣的免疫異常合成的HERV E 41來源的多肽P15E (env序列編碼的跨膜蛋白)，能 夠誘導許多免疫異常，如CD4+ T細胞的活化和失能，細胞因子的產(chǎn)生(如IL6、IL16)和細胞因子相關(guān)的多克隆B細胞活化。近來有研究者報道，在系統(tǒng)性紅斑狼瘡患者中HERV E 41序列比正 常對照組轉(zhuǎn)錄明顯增加，大約有50 的患者血清中發(fā)現(xiàn)能與其轉(zhuǎn)錄產(chǎn)物結(jié)合的抗體，而對照組是零 。HERV與系統(tǒng)性紅斑狼瘡的關(guān)系在系統(tǒng)性紅斑狼瘡患者的器官和血HERV與類風濕性關(guān)節(jié)炎的關(guān)系與健康對照人群或者骨關(guān)節(jié)炎患者相比，類風濕性關(guān)節(jié)炎患者的HERVK10 mRNA表達增加，提示HERV

40、K10可能與這種自身免疫性疾病的致病性有關(guān)研究結(jié)果顯示，68 的類風濕性關(guān)節(jié)炎患者的外周血中單核細胞中HERVK10 mRNA表達水平增加，骨關(guān)節(jié)炎患者為l7 ，對照組為185。在類風濕性關(guān)節(jié)炎患者中HERV增加可能與宿主蛋白的分子模擬相關(guān)，或由環(huán)境因素觸發(fā)激活了HERVs。含活化HERV的宿主細胞移行到滑膜并產(chǎn)生促炎細胞因子，誘導RA疾病的發(fā)生。HERV與類風濕性關(guān)節(jié)炎的關(guān)系與健康對照人群或者骨關(guān)節(jié)炎患者Ro (aa 169180) autoantibodies SLE抗DNA和其他核抗體與人類SLE類似在類風濕性關(guān)節(jié)炎患者中HERV增加可能與宿主蛋白的分子模擬相關(guān)，或由環(huán)境因素觸發(fā)激活了H

41、ERVs。Class I HERVs:EBNA1 motif PPPGRRP (aa 398404)in pediatric SLE, 99% of patients are antiEBV Ab+ compared to 70% of controlsEpsteinBarr Virus (EBV)EpsteinBarr Virus (EBV)BMRF2-1整合素Immune system defectsRetroviral integrase inhibitors as a novel form of antiviral therapy for autoimmune disease28kD

42、nuclear autoantigen (p28) HRES1/p28局部炎癥部位EBV特異性CTL細胞的集聚，加劇了局部的炎癥反應用免疫組化法對HERVW家族檢測在健康人腦的神經(jīng)細胞中有HERVW 的Gag蛋白的生理表達，而在MS患者的脫髓鞘腦白質(zhì)的軸突結(jié)構(gòu)中Gag蛋白有顯著的積累，在病理組織的內(nèi)皮細胞中也觀察到了Gag蛋白的高表達。HERV 編碼的超抗原、HERV 病毒顆粒及 B細胞表達的蛋白 激活和巨噬細胞(FasL-Fas-凋亡）EAD存在于細胞漿和細胞核內(nèi)，并具有EBV特異的DNA多聚酶活性。in pediatric SLE, 99% of patients are antiEBV Ab+ compared to 70% of controlsHERV與多發(fā)性硬化癥的關(guān)系1997年，發(fā)現(xiàn)多發(fā)性硬化癥(MS)患