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1 National Institute of Diagnostics and Vaccine Development in Infectious Diseases & State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Xiamen University The role of quantitative anti-hbc level in chronic HBV infection 慢性乙肝感染中的核心抗体水平厦门大学, 公共卫生学院国家传染病诊断试剂与疫苗工程技术研究中心分子疫苗学与分子诊断学国家重点实验室袁权

quantitative anti-hbc level in chronic HBV infection 慢性乙肝感染中的核心抗体水平厦门大学, 公共

2 Global burden of HBV infection A B C Data from: A-B: C:

3 Natural history of chronic HBV infection Nature Immunology, 2005 (5) modified

4 Clinically-used HBV diagnostic markers

5 Clinically-used HBV diagnostic markers 乙肝两对半及 HBV DNA 商品化检测试剂发展成熟, 性能优异, 已广泛应用于临床核酸检测及测序的发展, 已能实现但尚不普及? 反映宿主免疫应答强弱的临床指标, 用于慢乙肝感染者的管理及指导临床治疗??

6 HBV 感染中的 Anti-HBc Anti-HBc 的存在指示曾经或正被 HBV 感染低流行区用于血液安全筛查定性 Anti-HBc 已经使用超过 35 年受方法学的限制,Anti- HBc 定量的意义目前所知甚少从定量角度对 Anti-HBc 进行再认识的意义 NEJM, 2005

7 Hypothesis Liver Inflammation marker: ALT AST histology CD4+ T-Cell CD8+ T-Cell B-cell Virion load marker: qhbv DNA qhbsag qhbeag cccdna HBV HBV HBV HBV HBV HBV HBV Host response marker: Anti-HBs Anti-HBe Anti-HBc T-cell activity... Qualitative to Quantitative?

qhbeag cccdna HBV HBV HBV HBV HBV HBV HBV Host response marker:

8 第一部分. Anti-HBc 定量方法学 1.1 Anti-HBc 免疫检测的三种原理 1. 竞争抑制法 HBcAb presented in specimen will block the binding between conjugated anti-hbc and coated HBcAg, so reduced reaction will indicate positive result. HRP 2. 间接法 rhbcag coated in the wells will capture the HBcAb in the specimen,then conjugated anti-human Abs will indicate the any presented human Abs. HRP 3. 夹心法 Coated r HBcAg and conjugated rhbcag will bind to the HBcAb presented in the specimen. Then the conjugated signal (such as :HRP) will indicated any presented HBcAb. HRP Ag Ag Ag Ag Ag Ag Ag Recombinant HBcAg Anti-Human Abs HRP Conjugated signal (such as HRP) HBcAb in specimen Anti-HBc Abs

间接法 rhbcag coated in the wells will capture the HBcAb in the specimen,then conjugated anti-human Abs will indicate the any presented human Abs. HRP 3.

9 1.2 Anti-HBc 免疫检测的方法学进化竞争法灵敏度较低重现性不佳不适合定量检测 1 雅培 Axysm 2 罗氏 Elecsys 间接法灵敏度提高重现性提高可用于定量检测 1 雅培 Architect ( 无定量检测模式 ) 夹心法灵敏度 / 特异性更佳特异性更高适合定量检测 1 Our shbcab SFDA/2010/

10 1.3 Anti-HBc 双抗原夹心检测方法 CpC cag 1mg/ml cag-hrp CpD 高度稳定的重组核心抗原, 标记后仍然维持良好的颗粒抗原性是实现双抗原夹心法检测 HBcAb 的基础 Clin Vac Immno, Mar. 2010, p

11 Anti-HBc 特异性单抗 Anti-HBe 特异性单抗 HBc/HBe 交叉反应单抗 Clin Vac Immno, Mar. 2010, p

12 Correlation between S-HBcAb and Abbott I2000 夹心法 Anti-HBc 试剂在不同人群中与 Abbott i2000 的符合率在 96.4~100% 两者不符合的标本经单抗中和实验结合临床背景确认, 证实夹心法 Anti-HBc 具有更强的灵敏度 / 特异性 Clin Vac Immno, Mar. 2010, p

13 Anti-HBc quantitative ELISA WT shbcab AxSYM Core Architect anti-hbc 1.0 IU/mL 20/20 20/20 20/ IU/mL 20/20 20/20 20/ IU/mL 20/20 20/20 0/ IU/mL 20/20 9/20 0/ IU/mL 14/20 0/20 0/20 0 IU/mL 0/20 0/20 0/20

5 IU/mL 20/20 20/20 20/20 0.25 IU/mL 20/20 20/20 0/20 0.

14 The quantifiable range of qanti-hbc ELISA at single testing tests 2 1 Measurement signal (OD value) IU/mL R 2 =0.99 Anti-HBc titer(iu/ml)= (Signal ) IU/mL D-fold Anti-HBc titer (IU/mL)

50 IU/mL R 2 =0.99 Anti-HBc titer(iu/ml)= (Signal 0.825-0.012) 2-5 2-6 0.

15 Intra- and inter-assay reproducibility of qanti-hbc T IU/mL IU/mL IU/mL IU/mL Measurement signal (OD value) IU/mL IU/mL IU/mL IU/mL 1 Con.C(IU/mL) CV(%) Measurement signal (OD value) IU/mL Con.C(IU/mL) CV(%) IU/mL IU/mL IU/mL Anti-HBc titer (IU/mL) Anti-HBc titer (IU/mL) Intraassay CV Inter-assay CV

000 2.8 2 4 8 16 Measurement signal (OD value) 1 0.5 0.25 0.125 0.0625 0.03125 0.0625 0.625 IU/mL Con.C(IU/mL) CV(%) 0.156 10.5 0.312 IU/mL 0.313 10.1 0.156 IU/mL 0.

16 The producibility of two independent qanti-hbc T Anti-HBc titer -Test1 log10(iu/ml) y=0.9904x R 2 = 份临床标本两次独立定量一致性 R2>0.98, 平均定量偏差 <10% Anti-HBc titer - Test2 (log10 IU/mL)

9904x R 2 =0.988 104 份临床标本两次独立定量一致性 R2>0.

17 第二部分. 慢性 HBV 感染自然历史过程中的 Anti-HBc 水平 Anti-HBc level in the natural history of HBV infection p=0.22 p= p<0.001 p<0.001 Anti-HBc level log10 (IU/mL) p< PBI OBI IT IC LR ENH Numbers: HBsAg: negative negative positive positive positive positive HBeAg: negative negative positive positive negative negative Viral load: negative very low very high high low high ALT: normal normal normal elevated normal elevated

0 PBI OBI IT IC LR ENH Numbers: 535 56 108 209 148 133 HBsAg: negative negative positive positive positive positive

18 The viral antigens are the essential factors to stimulate and maintain the qanti-hbc optimal cutoff: 89 IU/mL Frequency PBI N=350 CHB N=488 Sensitivity AUROC=0.991( ) p< Sensitivity: 95.8% Specificity: 98.0% Specificity

8 optimal cutoff: 89 IU/mL Frequency PBI N=350 CHB N=488 Sensitivity 0.7 0.6 0.

19 The qanti-hbc in occult HBV infection Sensitivity optimal cutoff: 6.6 IU/mL AUROC=0.723( ) p< Sensitivity: 60.7% Specificity: 75.3% Specificity

6 IU/mL AUROC=0.723(0.651-0.795) p<0.0001 Sensitivity: 60.

20 The qanti-hbc is determined by the host immune status in CHB 1.0 Sensitivity optimal cutoff: 7,400 IU/mL 84% 16%? 0.2 AUROC=0.918 p< specificity Sensitivity: 87.3% Specificity: 83.5% Anti-HBc 定量水平用于判断 CHB 患者是否处于肝炎活动 / 免疫激活状态的诊断效能

918 p<0.0001 0.0 0.0 0.2 0.4 0.6 0.8 1.0 1-specificity Sensitivity: 87.

21 Anti-HBc level in the natural history of HBV infection The qanti-hbc correlates with ALT when ALT<5 ULN in CHB p trend <0.001 p=0.95 Anti-HBc level log 10 (IU/mL) mean ± 95%CI ~29 folds 0.5 ULN(n=91) 0.5~1 ULN(n=171) 1~2 ULN(n=76) 2~5 ULN(n=99) 5~20 ULN(n=96) >20 ULN(n=65) ALT stratum Song LW, et al. Clin Microbiol Infect Feb;21(2):

22 ALT (U/L) HBs Ag log 10 (IU/mL) ALT (U/L) HBs Ag log 10 (IU/mL) R=0.277 p< R= p< Anti-HBc log10 (IU/mL) A C Immune tolerance phase R=0.342 p<0.001 R= p=0.206 Low-replicative phase 10 R=0.149 p= R= p< Anti-HBc log10 (IU/m L) Anti-HBc log10 (IU/mL) R=0.212 p= R= p= Anti-HBc log10 (IU/mL) AST (U/L) HBV DNA log10 (IU/mL) AST (U/L) HBV DNA log10 (IU/mL) ALT (U/L) ALT (U/L) HBs Ag log 10 (IU/mL) R=0.314 p<0.001 R=0.381 p< R= R= p<0.001 p= Anti-HBc log10 (IU/mL) Anti-HBc log10 (IU/mL) HBs Ag log 10 (IU/mL) B D R=0.300 p<0.001 Immune clearance phase HBeAg-negative hepatitis phase R=0.348 p<0.001 R=0.153 p= R= p= Anti-HBc log10 (IU/mL) Anti-HBc log10 (IU/mL) AST (U/L) HBV DNA log10 (IU/mL) AST (U/L) HBV DNA log10 (IU/mL)

23 The longitudinal cohort for CHB natural course 多因素 GEE 分析显示 qanti- HBc 与 ALT 呈纵向正相关免疫耐受期患者 ( 如 A) 的 qanti-hbc 可长期低水平在肝炎活动时,ALT 与 Anti- HBc 几乎同时升高, 前者通常比后者早 3-8 周达到峰值, 但在某些情况下,Anti- HBc 也可能先于或与 ALT 同时达到峰值在肝炎恢复期,Anti-HBc 在 ALT 复常后周方能回到基线水平

24 Is qanti-hbc level a specific marker for HBV induced liver disease? HBV infection Liver desease Hepatitis Other factor induced liver damage metabolic disorder Drug Other hepatitis virus

25 Distribution of Total Anti-HBc and Anti-HBc IgM levels in the different groups of HBV carriers Total-Anti-HBc Anti-HBc-IgM 100 p=0.86 p=0.052 p=0.055 p<0.001 Anti-HBc-IgM (S/CO) IC IT eag(+) CHB eag(-) CHB SVR EOF HDV HCC 1st d HCC ES Yuan Q, et al.plos One Jun 26;10(6):e

26 Distribution of Total Anti-HBc and Anti-HBc IgM levels in the different groups of HBV carriers Total-Anti-HBc Anti-HBc-IgM

27 Correlation between αhbc, αhbc-igm and other parameters IgMantiHBc S/CO R² = Anti HBc IU/ml Parameter Total-Anti-HBc Anti-HBc-IgM r p r p ALT < HBsAg < HBV DNA <

28 ROC analysis for distinguish HBV inactive carrier and CHB patients ROC analysis for distinguish end of treatment CHB patients with sustained response to antiviral therapy versus baseline untreated CHB AUC Cut off sensitivity specificity AUC Cut off sensitivity specificity Total- Anti-HBc % 94.4% Total- Anti-HBc % 83.3% Anti-HBc- IgM % 97.2% Anti-HBc- IgM % 80.3%

29 第三部分. qanti-hbc 预测治疗应答的研究 Derivation cohorts 队列 A: 阿德福韦酯治疗队列 49 例治疗前 ALT>2 ULN 的 HBeAg(+) 病人 ADV(10mg/day) 治疗 96 周, 停药后随访 12 周应答定义 : 随访终点 HBeAg 血清学转换合作单位 : 首都医科大学附属佑安医院队列 B: 长效干扰素治疗队列 48 例治疗前 ALT>2 ULN 的 HBeAg(+) 病人 Pegasys, 180ug/week, 治疗 24 周, 停药后随访 24 周应答定义 : 随访终点 HBeAg 血清学转换合作单位 : 台湾大学附属医院 Yuan Q, et al. Gut Jan;62(1):182-4.

30 3.1.1 The Baseline Characteristics Cohort A Cohort B No Treatment strategy ADV -96 weeks Pegasys -24 weeks Age, yrs, 35 (26~48) 35 (15~57) Gender, males/females 44/5 35/13 Genotype, B/C 11/38 29/19 ALT, U/L, 110 (44~402) 168 (32~626) HBV DNA, log10 copies/ml 7.58 (3.97~9.29) 7.55 (3.44~9.59) HBsAg, log10 IU/ml 4.44 (2.35~5.47) 4.06 (1.53~5.35) Anti-HBc-IgM, S/CO value 2.10(0.31~12.7) 1.78 (0.25~12.2) Anti-HBc, log10 IU/ml, 4.29 (3.08~5.11) 3.98 (2.41~5.36)

31 3.1.2 To predict SR using the baseline Characteristics ADV-cohort Characteristics SR(+) SR(-) Univariate p value Multivariate p value No Age, yrs 35±4 36± Gender, males/females 7/2 37/ Genotype, B/C 2/7 9/ ALT, U/L 170±88 137± Ns HBV DNA, log10 copies/ml 7.03± ± HBsAg, log10 IU /ml 4.32± ± Anti-HBc-IgM, S/CO value 3.13± ± Anti-HBc, log10 IU/mL 4.58± ±

32 3.1.2 To predict SR using the baseline Characteristics Pegasys-cohort Characteristics SR(+) SR(-) Univariate p value Multivariate p value No Age, yrs 34±11 36± Gender, males/females 17/6 18/ Genotype, B/C 15/8 14/ ALT, U/L 198± ± Ns HBV DNA, log10 copies/ml 7.64± ± HBsAg, log10 IU /ml 4.01± ± Anti-HBc-IgM, S/CO value 3.38± ± Anti-HBc, log10 IU/mL 4.32± ±

33 3.1.3 ROC analyses for predicting SR in derivation cohorts Sensitivity Sensitivity optimal cutoff: IU/mL Anti-HBc AUROC Anti-HBc ALT HBV DNA ALT HBsAg HBV DNA HBsAg Anti-HBc-IgM Anti-HBc-IgM optimal cutoff: 9000 IU/mL Anti-HBc AUROC Anti-HBc ALT HBV DNA ALT HBsAg HBV DNA HBsAg Anti-HBc-IgM Anti-HBc-IgM specificity ADV-cohort Pegasys-cohort p p 优化 Cutoff 值 =29000 IU/mL 灵敏度 : 77.8% 特异性 : 77.5% 优化 Cutoff 值 =9000 IU/mL 灵敏度 : 77.8% 特异性 : 77.5%

34 3.1.4 Predicting SR using baseline qanti-hbc ADV-cohort 无论 ALT 是否 >5 ULN, 基线 Anti-HBc 较高的病人接受 ADV 治疗总是具有更高 (RR=7.22) 的治疗应答率

35 3.1.4 Predicting SR using baseline qanti-hbc Pegasys-cohort 无论 ALT 是否 >5 ULN, 基线 Anti-HBc 较高的病人接受 Pegasys 治疗总是具有更高 (RR=2.10) 的治疗应答率

36 3.1.4 Predicting SR using baseline qanti-hbc

37 3.2 The Validation studies 143 例 HBeAg(+) 病人队列 1: 长效干扰素治疗队列长效干扰素治疗治疗 48 周, 停药后随访 24 周应答定义 : 随访终点 SR VR CR 合作单位 : 北京大学附属第一医院, 王贵强教授队列 2: 长效干扰素 + 替比夫定治疗队列 231 例 HBeAg(+) 病人接受 PEG-IFN,560HBeAg(+) 病人接受 NUC 长效干扰素治疗周, 停药后随访 24 周 Ldt/ADV 治疗 104 周, 停药后随访 24 周应答定义 : 随访终点 SR VR 合作单位 : 南方医科大学附属南方医院, 侯金林教授 Hou FQ, et al.theranostics 2015; 5(3): Fan R, et al. Gut 2015;0:1 8.

38 3.2.1 The Validation cohort 2 : Peg-IFN cohort (from Prof.Wang Guiqiang) 派格宾 90ug 0 week 12 week 24 week 48 week 72week 派格宾 135ug 派格宾 180ug 派罗欣 180ug 治疗应答定义 : 治疗随访血清学应答 (SR) 随访终点 HBeAg 血清学转换病毒学应答 (VR) 随访终点 HBV DNA<200 IU/mL 联合应答 (CR) 随访终点 HBeAg 阴转并且 HBV DNA<2000 IU/mL

39 205 patients 3 patients excluded for HBeAg negative 2 patients excluded for without serum sample 8 patients excluded for HBV DNA<10 5 IU/mL 10 patients excluded for ALT<1.5 ULN 182 patients 38 patients excluded for without follow up at week 72 4 patients excluded for inadequate data at week patients 不同治疗组应答率无显著差异, 因此将这四组治疗组合并在一起进行分析

40 3.2.1 Associations of qanti-hbc and other HBV parameters

41 3.2.1 Associations of qanti-hbc and salt A B qanti-hbc (log 10 IU/mL) qanti-hbc (log 10 IU/mL) Baseline 8 r= p< ALT (log 2 U/L) Overall 8 7 r=0.42 p< ALT (log 2 U/L) C HBV DNA (Log 10 IU/mL) HBsAg (Log 10 IU/mL) ALT (Log 2 U/L) qanti-hbc ALT HBsAg HBV DNA HBeAg 0 week 12 week 24 week 48 week 72 week Parameters correlated with qanti-hbc during treatment and follow up using GEE(GeneralizedEstimation Equations) analysis Parameter Beta p ALT <0.001 HBV DNA HBsAg HBeAg HBeAg(log 10 S/CO) qanti-hbc(log 10 IU/mL)

42 3.2.1 Associations of baseline factors and treatment response SR VR CR Baseline Univariate Multivariate * Univariate Multivariate * Univariate Multivariate * parameters OR P OR P OR P OR P OR P OR P Gender, M/F Age, yrs HBsAg, Log 10 IU/mL HBV DNA, Log 10 IU/mL HBeAg, Log 10 S/CO 0.24 < Anti-HBc, Log 10 IU/mL HBV genotype, B/C ALT, U/L ALT strata, /<5ULN AST, U/L Treatment group,

43 3.2.1 Treatment response v.s. qanti-hbc strata Rate % % (1/13) SR VR CR 18.2% (2/11) 9.1% (1/11) 18.2% (2/11) 21.2% (14/66) 15.2% (10/66) 41.9% (13/31) 32.3% (10/31) 38.7% (12/31) 52.6% (10/19) 47.4% (9/19) 42.1% (8/19) 0 < ~1 1~3 3~5 >5 Baseline qanti-hbc(10000 IU/mL)

44 3.2.1 Baseline qanti-hbc predicts EOF SR A Baseline qanti-hbc <30,000 IU/mL N=90 N=17 (18.9%) 140 patients SR(+) Baseline qanti-hbc 30,000 IU/mL N=50 N=23 (46.0%) RR(95%CI): (1.44~4.11)*** B HBc-low N=66 N=11 (16.7%) Baseline ALT <5 ULN N=94 SR(+) 140 patients HBc-highHBc-low N=28 N=11 (39.3%) N=24 N=6 (25.0%) Baseline ALT 5 ULN N=46 SR(+) HBc-high N=22 N=12 (54.5%)

45 3.2.1 Baseline qanti-hbc predicts EOF VR C Baseline qanti-hbc <30,000 IU/mL N=90 N=12 (13.3%) 140 patients VR(+) Baseline qanti-hbc 30,000 IU/mL N=50 N=19 (38.0%) RR(95%CI): (1.51~5.38)** D HBc-low N=66 N=9 (13.6%) Baseline ALT <5 ULN N=94 VR(+) 140 patients HBc-highHBc-low N=28 N=7 (25.0%) N=24 N=3 (12.5%) Baseline ALT 5 ULN N=46 VR(+) HBc-high N=22 N=12 (54.5%)

46 3.2.1 Baseline qanti-hbc predicts EOF CR Baseline qanti-hbc <30,000 IU/mL N=90 N=13 (14.4%) 140 patients CR( BR(+) +) Baseline qanti-hbc 30,000 IU/mL N=50 N=20 (40.0%) RR(95%CI): (1.51~5.08)** HBc-low N=10 (15.2%) Baseline ALT <5 ULN N=94 N=66 N=28 CR( BR(+) +) 140 patients HBc-highHBc-low N=8 (28.6%) N=3 (12.5%) Baseline ALT 5 ULN N=46 HBc-high N=24 N=22 CR( BR(+) +) N=12 (54.5%)

47 不同基线 qanti-hbc 水平病人病毒学参数动力学变化

48 不同基线 qanti-hbc 水平病人 ALT 动力学变化

49 根据基因型和 SR 分组后的病毒学参数动力学变化

50 根据基因型和 VR 分组后的病毒学参数动力学变化

51 3.2.2 The predictive value of baseline qanti-hbc level in PEG-IFN and NUC therapy

52 3.2.2 The kinetics of qanti-hbc during therapy PEG-IFN LdT

53 3.2.2 Baseline qanti-hbc predict PEG-IFN treatment response Factors Univariate Multivariate Age Gender Genotype Baseline ALT level Baseline HBV DNA level Baseline HBsAg level Baseline HBeAg level Baseline Anti-HBc level Odd-ratio 95% CI P-Value Oddratio 95% CI P- Value <

54 3.2.2 Baseline qanti-hbc predict LdT treatment response Univariate Multivariate Factors Odd-ratio 95% CI P-Value Oddratio 95% CI P- Value Age Gender Genotype Baseline ALT level Baseline HBV DNA level Baseline HBsAg level Baseline HBeAg level Baseline Anti-HBc level <

55 3.2.2 Treatment response among subgroups of patients stratified by parameters alone or combined

57 Profiles of serum marker levels during the natural history of chronic hepatitis B virus infection Viral production Host response

58 Anti-HBc titer is determined by both viral Hypothesis: loads and host immune response Low anti-hbc Virus surge Infected hepatocyte Low ALT ADCC/CDC Effectors AP Release virus C and HBc Ag Uptake of viral Ag by APC T cell independent activation of B cells Release intrahepatic enzymes killing Activation Of cytotoxic T cells Activation Of Th cells Activation Of B cells ALT qanti HBc Core Ag ALT MHC I HBV TCR MHC II Cytokines complement ADCC: Ab-dependent cellular cytotoxicity; CDC: complement dependent cytotoxicity qanti HBc

59 Published paper derived this study Yuan Q, et al. Gut Jan;62(1): Yuan Q, et al. PLoS One Jun 26;10(6):e Song LW, et al. Clin Microbiol Infect Feb;21(2): Hou FQ, et al. Theranostics Jan 1;5(3): Jia W, et al. Medicine (Baltimore) Dec;93(29):e322. Fan R, et al. Gut Jan 13. pii: gutjnl

60 Acknowledgments National Taiwan University Prof. Per-Jer Chen Prof. Chien-Jen Chen Nanfang hospital of FIMMU Prof. Jin-Lin Hou Prof. Jian Sun First hospital of PKU Prof. Gui-Qiang Wang Xiamen Blood Service Director Chang-Rong Chen Dr. Shan-Hai Ou Capital Medical University Prof. Zhuo Li Zhongshan Hospital of XMU Prof. Ping-Guo Liu

61 谢谢大家!

人工抗原的鉴定

人工抗原的鉴定生物技术通报张海棠王自良刘耀东传统的理化分析方法在食品污染检测方面发挥了重要的作用但均存在一定的缺陷急需建立快速高效的检测方法食品污染免疫检测是一种新型的分析方法与理化分析方法相比具有省时省力费用低廉便