1、疫苗研究中的的反向疫苗学疫苗研究中的的反向疫苗学 Reverse Vaccinology in Vaccine ResearchJenner策略策略:模拟自然感染过程模拟自然感染过程-病原体的致病性和保护性可以分离病原体的致病性和保护性可以分离病原体减毒病原体灭活病原体抗原多肽交叉病原体*容易模拟容易模拟*免疫致病免疫致病*免疫致敏免疫致敏基因基因临床试验临床试验反向疫苗策略反向疫苗策略技术技术Rappuoli,R.,Covacci,A.Science 2019;302(5645):602细菌细菌病毒病毒寄生虫寄生虫真菌真菌蛋白质抗原蛋白质抗原体液免疫体液免疫细胞免细胞免疫疫YYB细胞细胞YY
2、YYY抗体抗体(ThCTLT细胞细胞清除病原清除病原清除病原或病变细胞清除病原或病变细胞10101717种抗原种抗原*预防性疫苗预防性疫苗-传染病传染病*改变了人类疾病谱改变了人类疾病谱*延长了人类平均寿命延长了人类平均寿命*治疗性疫苗治疗性疫苗 -传染病和非传染病传染病和非传染病*可进一步改变人类疾病谱可进一步改变人类疾病谱*可进一步延长寿命可进一步延长寿命死亡死亡:战争死亡人数战争死亡人数健康健康:活跃人群活跃人群-平均寿命平均寿命社会经济影响社会经济影响:战争战争肿瘤肿瘤T cell clone 1012 TCR heterogenicity 1018Self-non-selfHarmf
3、ul-nonharmfulDanger signal 治疗性疫苗的研究策略-疾病过程可以用抗原疾病过程可以用抗原(表位表位)特异性免疫反应干预特异性免疫反应干预 上调或下调特异性CTL-免疫保护主要依赖细胞免疫的疾病*慢性持续性胞内感染慢性持续性胞内感染*肿瘤肿瘤*自身免疫性疾病自身免疫性疾病(负调疫苗负调疫苗)etc etc 诱生特异性抗体*抗原明确的认知性疾病抗原明确的认知性疾病 etc etc Biotech Medicines in DevelopmentProduct Category 1989 1993 2019 2019 2000 Angiogenesis Inhibitor -
4、6Antisense -5 9 9Cellular Therapy -20 16Clotting Factors 2 1 3 3 -CSFs 7 6 5 3 3Erythropoitins 4 1 1 3 3Gene Therapy -1 17 38 25Growth Factors 2 9 11 21 17hGH 3 4 7 5 5Interferons 12 11 11 12 12Interleukins 12 10 13 9 6McAb 25 50 71 74 85(59)Rec Human Proteins 3 5 6 8 14Rec Soluble Receptor 2 2 6 6
5、4Signalling -3 4Vaccines 13 20 40 77 98Others 10 23 45 53 83Total 95 143 234 350 369Immunoinformaticsthe new kid in town免疫学是一门复杂科学：免疫学是一门复杂科学：*immune system components:e.g.immunoglobulins,lymphocyte receptors,or cytokines,*the complexity of the regulatory pathways*network-type interactions目前获得的数据只是一
6、小部分，数据在指数增长目前获得的数据只是一小部分，数据在指数增长 Computational analysis has therefore become anessential element of immunology research with a main role of immunoinformatics being the management and analysis of immunological data Database In silico experiment Targeted experimentData warehousing provides a framework
7、 for this process wu,2019Immunoinformaticsthe new kid in town 进一步的数据分析，是将免疫学问题转变为计算机（模型）问题，通过解决计算问题解读出生物学意义 immunoinformatics 的基础：immunological databasessequence analysisstructure modellingmathematical modelling of the immune system simulation of laboratory experiments statistical support for immuno
8、logical experimentation and immunogenomics.候选基因候选基因预测预测抗原处理与抗原处理与MHC亲和力亲和力候选多肽候选多肽MHC结合结合实验证实实验证实抗原呈递抗原呈递自身免疫自身免疫抗原处理抗原处理预防实验预防实验癌症病人癌症病人重复重复正常志愿者正常志愿者细胞毒细胞毒T细胞库谱细胞库谱免疫反应免疫反应A2TgmiceCK 分析分析Tetramer治疗实验治疗实验确定肿瘤抗原确定肿瘤抗原临床试验临床试验*确定天然抗原的天然表位确定天然抗原的天然表位*天然表位在疾病过程中的作用天然表位在疾病过程中的作用*如何克服对天然抗原的免疫耐受如何克服对天然抗原的
9、免疫耐受*如何考虑疫苗组分如何考虑疫苗组分(含改造天然表位含改造天然表位)*如何启动表位特异性反应如何启动表位特异性反应From Reverse Biology to Reverse Vaccinology一、一、确定天然抗原的天然表位确定天然抗原的天然表位:-预测预测:免疫信息学方法免疫信息学方法/新的预测方法新的预测方法 -探测探测:新的实验技术新的实验技术二、天然表位在疾病过程中的作用二、天然表位在疾病过程中的作用-免疫病理学研究免疫病理学研究 表位特异性反应在疾病过程中的作用如表位特异性反应在疾病过程中的作用如:病毒清除病毒清除/肝损害肝损害三、如何克服对天然抗原的免疫耐受三、如何克服
10、对天然抗原的免疫耐受-“-“天然抗原加倍天然抗原加倍”失败失败“通过模拟抗原通过模拟抗原(mimogen)(mimogen)克服对天然抗原克服对天然抗原(antigen)(antigen)的耐受的耐受”四、如何考虑疫苗组分四、如何考虑疫苗组分(含改造天然表位含改造天然表位)-CTL-CTL表位的异质性与分类表位的异质性与分类-天然表位的缺陷与改造天然表位的缺陷与改造(APL)(APL)五、如何启动表位特异性反应五、如何启动表位特异性反应-mimogen-mimogen的设计的设计-Th1-Th1极化佐剂极化佐剂_ _递送系统递送系统 确定天然抗原的天然表位确定天然抗原的天然表位1.新的表位预测
11、方法Th cell epitope prediction-)1.1(/)(40ijjiijjirqqrqqE)2.1()3,2,1(/)(1kkqqnjik)5.1(.)3,2,1(/)(1kkECIECInjik)3.1(1niiqECI=(1,2,3,4,5,6,7,8,9,10)(1.6)6.1(7338.0)53.053.0()53.053.0()53.010.0()10.009.0()09.005.0()05.002.0()02.010.0()10.005.0()05.065.0()65.010.0()(/)(1011011jiijjiqqrqqSample:Ek Restricte
12、d Th epitope:LTALGAILKKK)7.1(2322.02/)53.053.0()53.010.0()53.009.0()10.005.0()09.002.0()05.010.0()02.005.0()10.065.0()05.010.0(2/)(/)(91912jiijjiqqrqq=(0.7338,0.2322,0.0546,0.0335,0.0317,0.0274,0.0620,0.0530,0.0442,0.0053)Interaction between the neighbor amino acid residuesInteraction between the ne
13、ighbor amino acid residues：L-T、T-A、A-L、L-G、G-A、A-I、I-L、L-K、K-K、K-K Interaction among amino acids with one amino acid residue intervalInteraction among amino acids with one amino acid residue interval：L-A、T-L、A-G、L-A、G-I、A-L、I-K、L-K、K-K y=0.9114-0.94572+1.43133-3.68767 -9.92708+24.179810 (1.9)Examina
14、tion:by random samplingNoNoYexpYallYpredNoNoYexpYallYpred1 115152 21616 3 317174 4 18185 519196 62020 7 721218 8 22229 9232310102424 111125251212 26261313272714142828 HLA-A2-MAGE-2112-120 complexYellow band:1domain&2 domain of HLA-A2Blue band:2microglobulinCTL epitope prediction-Molecular simulation
15、 The predicted fitting and binding sites of different Mage-2 sequences to the HLA-A*0201SequenceDistance(P2-P9)H-bondsNon-bound energySovent accessible area(2)P2P9MAGE-2（112-120）KMEELVHFL19.877-18082.11.2(0.63%)0(0%)MAGE-2(171-179)PISHLYILV17.847-18193.20.89(5.1%）0(0%)MAGE-2(220-228)KIWEELSML18.735-
16、18513.20(0%)4.8(2.8%)MAGE-2(271-279)FLWGPRALI17.926-18080.93.3(2.1%)1.6(0.9%)The predicted fitting and binding sites of different MZ2-D sequences to the HLA-A*0201Peptide H-bonds Sovent accessible area in each residue(2)Number of atomic contacts P1 P2 P3 P4 P5 P6 P7 P8 P9 P2 P9 M1 8 4.55 0.47 12.68
17、142.89 164.19 26.54 27.14 64.57 0.47 9 33 M2 9 15.08 0.47 14.20 79.46 119.29 27.73 37.94 29.78 2.10 9 33 M3 8 15.62 0.47 14.39 84.04 108.94 17.11 27.33 48.39 2.57 9 41 M4 12 40.46 2.87 32.80 40.24 107.45 31.39 36.66 55.16 4.82 9 22 CTLepitope binds to the cleft of HLA-0201CTL epitope prediction-QSAR
18、)1.2()()(992211992211050ECIcECIcECIcISAbISAbISAbapIC)2.2(09191050ECICISABaECIcISAbapICiiiiiiKIFGSLAFL with pIC50(exp)=7.478 pIC50(cal)=4.5945+(0.002 102.78)+(-0.00760154.35)+(0.4898 0.53)+(-1.2287 0.10)=7.444Example:pIC50=a0+(b1ISA1+b9 ISA9)+(c1 ECI1+c9 ECI9)NoPeptide(origin)SequenceFIapIC50scorebsc
19、orec1TRAG-3(37-45)HACWPAFTV0.615.5504-23.554.712TRAG-3(4-12)GLIQLVEGV1.027.2677-22.2812.883TRAG-3(58-66)ILLRDAGLV1.807.6175-19.85100.044TRAG-3(57-65)SILLRDAGL1.097.4092-23.258.605MAGE-2(112-120)KMVELVHFL0.896.9395-21.77115.936MAGE-2(171-179)PISHLYILV0.956.9954-21.7925.987MAGE-2(220-228)KIWEELSML1.61
20、8.0233-21.1835.458MAGE-2(271-279)FLWGPRALI1.447.8897-19.87105.889MAGE-3(108-116)ALSRKVAEL1.107.1581-22.04194.3210MAGE-3(174-182)HLYIFATCL1.087.0691-21.30128.3911MAGE-3(201-209)LLIIVLAII1.127.1739-22.521.1812MAGE-3(271-279)FLWGPRALV1.908.1336-19.47968.4313NY-BR-1(167-175)MLLQQNVDV1.6448.1177-19.58307
21、.9814NY-BR-1(1043-1051)YLLHENCML1.2837.3216-20.20100.3215NY-BR-1(1274-1282)NMWLQQQLV1.0566.9159-19.74358.11Comparison of FI and pIC50 in a series of CTL epitopes in tumor antigens 567890.511.52)9(654321050RTHMSHBMHBWHBvdwKcKcKcKcKcKcKpICCTL epitope prediction-SCORE K0c1c2c3c4c5c6statistics3.402-0.13
22、50.1000.2210.5260.988-0.129Standard coefficients0-0.6770.2830.3310.8840.911-0.564CTL epitope prediction molecular dockingRTHMSHBMHBWHBvdwKcKcKcKcKcKcKKRTG6543210logijijijijijijWHBMHBSHBhbonddWHBdMHBdSHBKKKK)()()(ijijvdwdVBK)(iiindesolvatioHMFKiiRTRTK5.0Melecular docking:CTL epitope within Tax protei
23、n of Htlv-1 to HLA-I)Pocket A(CEA691)Pocket A(CEA691Y1)疏水相互作用疏水相互作用：弱 较强氢键作用网络氢键作用网络：/N末端强大的氢键作用网络共轭共轭键键：/Trp167 与 P1P2 SAS：1.40 2 0.93 2 pIC50：6.52 7.15CEA691CEA691Y1ETotal：-112.3kcalmol-1 -138.6 kcalmol-1 与MHC I类分子的亲和力 CEA691 55 epitopes Viral antigens:HBV,HRV,Sars-Cov,RV Tumor antigens:MAGE-2,MAG
24、E-3,MAGE-1,TRAG-3,NY-BR-1,CML28,etc天然表位在疾病过程中的作用天然表位在疾病过程中的作用*病人为对象病人为对象:如急性如急性 慢性慢性 恢复期乙型肝炎病人恢复期乙型肝炎病人 肝损害肝损害 病毒载量病毒载量*表位特异性细胞免疫反应表位特异性细胞免疫反应:如如:Tetramer EliSpot,ICC,51Cr release,Facs,proliferation,differentiation,activation,polarizationThere were more perforin positive cells(mainly bystand T cells
25、)in patients with high level of ALT than in the patient with low level of ALT.Pt4ALThAlcoDNA0Pt3ALThDNAhPt5 ALThDNALPt5R ALTLDNALThe frequency of perforin positive cell out of lymphocytes in circulationThe frequency of perforin positive cell out of lymphocytes in circulation(direct ex vivo)表位存在异质性 I
26、mmunol Lett 2019;92:253-258 Gastroenterol 2019，Submitted 可以象抗体识别表位类似分类:优势保护性优势保护性、非优势保护性 优势非保护性、非优势非保护性 存在优势保护性非溶破性表位优势保护性非溶破性表位 J Virol,2019，79(9):5568-5576 实验证实非溶破性CTL表位Rank position length sequence score with BIMAS score with SYFPEITHI 1 87-95 9 SYVNTNMGL 5760 26 2 117-126 10 EYLVSFGVWI 4000 23 3
27、 131-139 9 AYRPPNAPI 2880 23 4 131-140 10 AYRPPNAPIL 3456 20 Results of prediction of potential CTL epitope restricted with H-2 Kd疫苗主分的考虑（表位）预防性 治疗性优势性 优势性 保护性 保护性保守性 保守性长期记忆 长期记忆长期记忆?非溶破性非溶破性 APL APL 三、如何克服对天然抗原的免疫耐受三、如何克服对天然抗原的免疫耐受1.Strategy:“通过模拟抗原通过模拟抗原(mimogen)(mimogen)克服对天然抗原克服对天然抗原 (antigen)(
28、antigen)的耐受的耐受”Antigen EpitopeEpitope Antigen(mimogen?)2.Challenge in science:reverse immunology3.Challenge in technology:antigen engineeringEpitope-based vaccine design(EBVD)ImmunoinformaticsproteoimcscandidateAgepitopemappingimmunopathologyCandidate epitopesAPLMimeticsAntigen engineeringSynthesis&
29、purificationScreeningin vitroin vivoin patientsDelivery system Candidate vaccineClinical trialWu Y,et al.The Immunologist 2019,9(S):985-989 Wu Y,et al.Acta Academiae Medicinae Military Tertiae.2000;20:917-918 Recognition of epitope(A)epitope peptide is usually much longger in pMHCII than in pMHCI(13
30、25 vs 8-10)peptides in all MHCI have their N-and C-termini anchored into two similar pokets of MHCI,span 20longer peptide bulge out in the middlepeptide in pMHCII:smooth/cnacved away from TCR/better shape compelementarity/twice in atomic contacts/additional contacts largely accounted for by pMHCII r
31、ather than TCR-MHCII(B)MurineHuman(Molecular Immunology 38,2019:1039)TRP-2WT SVYDFFVWL -20.73 471.17 7.401TRP-2WT SVYDFFVWL -20.73 471.17 7.401TRP-2 1Y TRP-2 1Y Y YVYDFFVWL -19.80 471.17 7.704VYDFFVWL -19.80 471.17 7.704TRP-2 2L STRP-2 2L SL LYDFFVWL -19.79 8054.54 7.953YDFFVWL -19.79 8054.54 7.953T
32、RP-2 2M STRP-2 2M SM MYDFFVWL -19.48 4610.64 7.852YDFFVWL -19.48 4610.64 7.852 TRP-2 1Y2L TRP-2 1Y2L YLYLYDFFVWL -18.86 8054.54 8.256YDFFVWL -18.86 8054.54 8.256TRP-2 1Y2M TRP-2 1Y2M YMYMYDFFVWL -18.55 4610.64 8.149YDFFVWL -18.55 4610.64 8.149a:polynomial method;b:quantitative motif method c:QSARa:p
33、olynomial method;b:quantitative motif method c:QSAR APLs Sequence ScoreAPLs Sequence Scorea a Score Scoreb b Score Scorec cImmunoinformatics-APL for TCRTRP-2 WTTRP-2 WT SVYDFFVWL SVYDFFVWL0.350.351.321.32TRP-2 2LTRP-2 2L SLYDFFVWL SLYDFFVWL0.080.082.492.49TRP-2 2M TRP-2 2M SMYDFFVWL SMYDFFVWL0.130.1
34、32.152.15TRP-2 1Y2L TRP-2 1Y2L YLYDFFVWL YLYDFFVWL0.040.041.451.45TRP-2 1Y2M TRP-2 1Y2M YMYDFFVWL YMYDFFVWL0.190.191.821.82HBV(18-27)HBV(18-27)FLPSDFFPSV FLPSDFFPSV0.190.192.002.00APLsAPLsSequenceSequenceFIFIa a/H-2K/H-2Kb bFI/HLA-A2.1FI/HLA-A2.1OVA(257-264)OVA(257-264)SIINFEKLSIINFEKL 3.08 3.08 0.1
35、0 0.10Affinity of peptides to HLA-A2.1 or H-2Kb molecules 506070809010002468Time post treatment of brefeldin(hour)%of Complexes Remaining2M1Y2M1Y2LWT2L Comparison of the stability of the complex of TRP-2Comparison of the stability of the complex of TRP-2(180-188)(180-188)wild peptide or APLs with HL
36、A-A2wild peptide or APLs with HLA-A2APL could induce more IFN-and granzyme B in PBMC 如何在体诱导如何在体诱导Th1/CTLTh1/CTL反应反应?抗原颗粒化-表位噬菌体展示 HBV的Phage-displayed particle -Vaccine 2019;19:2918 肿瘤的Phage-displayed particle-Inter J Cancer 2019;98:748 HBV的ISCOMs-World Journal astroenterology 2019;8:294抗原颗粒化-模拟病毒研究
37、HBV的 mimovirusAB3.抗原颗粒化-转基因植物口服疫苗 轮状病毒转基因口服疫苗 Li J,et al.Virology 2019；313:337-345 012345123456789 10 11 12 13 14 15 16 17 18 19 20 21抗原颗粒化抗原颗粒化-Semiliki forest virus-based tumor vaccine Ni B,et al.Induction of specific human primary immune response to a Semiliki forest virus-based tumor vaccine in
38、a trimera mouse model.Cancer Immunol Immunother 2019;54(5):489-98Ni B,et al.Induction of P815 Tumor Immunity by DNA-Based Recombinant Semliki Forest Virus orReplicon DNA Expressing the P1A Gene.Cancer DetectPrev 2019;28(6):418-425 Ni B,et al.Induction of P815 Tumor Immunity by DNA-Based Recombinant
39、Semliki Forest Virus orReplicon DNA Expressing the P1A Gene.Cancer DetectPrev 2019;28(6):418-425 Immunoinformatics-mimogen for HBV1.Kinds of epitope2.Refined epitope3.Number of epitope4.Permutation and combination of epitopesUnder phase-II-III clinical trial国际上第一个模拟抗原疫苗国内第一个CTL疫苗国内第一个多肽疫苗国内第一个脂质体疫苗颗
40、粒颗粒(抗原抗原)疫苗的呈递机理疫苗的呈递机理 -ER-ER靶向抗原交叉呈递特征和机制靶向抗原交叉呈递特征和机制 0204060801000246810Time of incubation(h)Mean fluorescence：OVA257-264-KDEL：OVA257-264：OVA257-264-TEWTEur J Immunol 2019；34(12):3582-95颗粒颗粒(抗原抗原)疫苗的呈递机理疫苗的呈递机理These data suggest that endocytosed phage particles may be processed and cross-present
41、ed in organelles positive for phagosome and endoplasmic reticulum(ER)markers via a classical ER MHCclass I loading mechanism.Eur J Immunol 2019;35(7):2041-50Vaccine+免疫调节免疫调节 Song J,et al.Sustained Survivin Expression from OX40 Costimulatory Signals Drives T Cell Clonal Expansion.Immunity 2019;22:621
42、31 Jia Z,et al.Effective induction of anti-tumor immunity by immunization with plasmid DNA encoding TRP-2 plus meutralizing of TGF-betta,Cancer Immunol Immunother 2019;54(5):446-52 Chen Y,et al.Cross-talk between complement system and T cells through B7-H1 on human renal tubular epithelial cells in
43、inflammation.JCI 2019(submitted)在SCI收录期刊发表主要论著目录 Protection of mammalian cells from severe acute respiratory syndrome coronavirus infection by equine neutralizing antibody.Antiviral Therapy,2019,10(4):671-80.Xiliang Wang,Bing Ni,Xinan Du,Guangyu Zhao,Wenda Gao,Xinfu Shi,Songle Zhang,Liangyan Zhang,D
44、ong Wang,Deyan Luo,Li Xing,Haiyan Jiang,Wanling Li,Man Jiang,Liwei Mao,Yangdong He,Yu Xiao and Yuzhang Wu.Inhibition of replication and infection of severe acute respiratory syndrome-associated coronavirus with plasmid-mediated interence RNA;Antiviral Therapy 2019,10(1):65-71;Ni B,Shi XF,Yang Li,Gao
45、 WD,Wang XL,Wu YZ*.Sustained Survivin Expression from OX40 Costimulatory Signals Drives T Cell Clonal Expansion.Immunity 2019;22:62131.J Song,Takanori So,Mary Cheng,Xiaohong Tang,and Michael Croft.Effective induction of anti-tumor immunity by immunization with plasmid DNA encoding TRP-2 plus meutral
46、izing of TGF-betta;Cancer Immunol Immunother 2019;54(5):446-52.Jia ZC,Zou LY,NI B,Wan Y,Zhou W,LV YB,Geng M,Wu YZ*Induction of specific human primary immune response to a Semiliki forest virus-based tumor vaccine in a trimera mouse model.Cancer Immunol Immunother 2019;54(5):489-98.Ni B,Gao W,Zhu B,L
47、in ZH,Jia ZC,Zhou W,Zhao JP,Wang L,Wu YZ*.A H-2 kd restricted hepatitis B virus-derived epitope induced production of interferon-gamma without cytotoxicity;J Virol 2019;79(9):5568-76;Chen A,Wang L,Zhang JB,Jia ZC,Zou LY,Zhou W,Wu YZ*Cross-presentation of phage particle antigen in MHC class II and en
48、doplasmic reticulum marker-positive compartments;Eur J Immunol 2019;35(7):2041-50.Wan Y,Wu YZ*,Zhou JR,Zou LY,Liang YF,Zhao JP,Jia ZC,Enberg J,Bian J,Zhou W.Comprehensive antibody epitope mapping of the nucleocapsid protein of severe acute respiratory syndrome-coronavirus(SARS-CoV):insight into the
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50、M,Zhao TT,Wu JH,Ni B,Mou ZR,Han JF,Chen YW,Zhi Y.Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells.Int Immunopharmacol 2019;5(9):1446-57.Chen Y,Li J,Zhang J,Zhao T,Zou L,Tang Y,Zhang X,Wu Y.Triptolide inhibits B7-H1 expression on proinflammatory factor activated
