Quality concern in the development of an inactivated enterovirus 71 vaccine Qihan Li Institute of medical Biology, CAMS, PUMC.
EV71 infection outside the Asia Pacific region EV71 in Norway. EV71 in Colorado, U.S. Virus Research 123 (2007) 19 29 www.eurosurveillance.org Clinical Infectious Diseases 2007; 45:950 7
EV71 infection and its risk in China The number of cases of HFMD in China, The deaths cases of HFMD in China, EV71 and HFMD Lancet Infect Dis 2010; 10:778-90
Clinical and virological features of 14 children with enterovirus 71 (EV71)-associated neurological disease. Clinical and magnetic resonance imaging data indicated that immunopathology was a major factor in the pathogenesis of neurological disease in this outbreak. McMinn P et al. Clin Infect Dis. 2001;32:236-242
Pathogenesis of EV71 infection? How does this virus infects human? What is the dynamic process of this viral infection? What is the relationship of viral infection and relative pathological change? What is the relationship of pathogenesis and immune response to viral infection?
The neonate rhesus model of EV71 infection Viremia Virus excretion
Viral antigen expression and Inflammatory response cerebrospinal fluid Peripheral blood Viral antigen expressed in various tissues
The gene profile of PBMC from infected neonate by EV71 Day 4 p.i. Day 7 p.i. Day 10 p.i. symbol foldchange symbol foldchange symbol foldchange MS4A2 12.8598 MS4A2 7.6968 MS4A2 19.6095 IL17A 3.7012 IL17A 5.8359 IL17A 16.2104 XCL1 8.4086 XCL1 7.2674 XCL1 4.2144 IL4 3.7321 IL4 7.7784 BPI 6.7284 BPI 4.0488 IL2 5.8852 IL2 4.5941 CD1A 3.2143 CD1A 23.4136 C8G 27.333 C8G 34.4187 KIR3DL1 3.7483 KIR3DL1 3.1582 CA2 4.1178 CA2 8.4027 SNCA 14.3059 SNCA 5.8654 CCL1 8.0528 CCL1 3.0783 ERAF 6.2807 ERAF 5.6899 Day 7 p.i. TRIM10 4.8866 CD300LF 10.6032 HLA-DRB3 13.283 IL6ST 10.0073 LY75 3.2395 MB 3.0113 MSR1 4.1592 PF4V1 4.0942 PLA2G1B 6.2348 CCL13 56.7043 CCL19 3.517 SELE 15.2325 BPGM 3.7486 C8A 5.4575 VPREB1 5.8522 ULBP2 6.0846 TM7SF4 5.3514 SOCS5 8.5589 Day 10 p.i. CXCL13 7.1186 CLU 3.3113 CRHR1 3.5911 CTSG 7.8076 ADSS 3.755 DEFA1 3.8451 ELANE 4.8082 ALAS2 3.0089
The gene profile of PBMC from infected neonate by EV71
Viral strains isolated from various areas in China C4 sub genotype 阜阳 昆明 合肥 中和试验鉴定 深圳
Establishment of strain for inactivated EV71 vaccine (human diploid cell) Strain cloning and its biologic characterization 8 7 6 700 血清中和抗体检测 疫苗免疫组感染对照组 5 600 4 3 2 1 0 6 7 8 9 10 11 12 13 14 15 16 17 18 FY-23K FY-23K-B FY-23K-C FY-23K-D FY-23K-G FY-23K-H 抗体效价 500 400 300 200 100 0 二免后 4 周 攻毒后 4 天 攻毒后 7 天 攻毒后 10 天 攻毒后 14 天 时间 ( 天 )
Study of immunogenicity of EV71 vaccine based upon viral structure The conformation characterization
Study of immunogenicity of EV71 vaccine based upon viral structure Two kind of virion presenting immunogenicity Page gel and Western blot: odd: the upper; even:the lower
ELISA quantitative technique for vaccine antigen Using checkerboard assay to determine the optimum concentration of capture antibodies, detecting antibodies and enzyme labeled antibody. Using the standard strain as reference antigen to draw standard curves. OD 值 0.9 1 0.8 0.7 0.6 0.5 0.4 0.3 0.2 含量 0.1 0 0.0 200.0 400.0 600.0 800.0 1000.0 EV71 蛋白含量与 OD 值的反应曲线 biotin AB Labeled EV71 Ab EV71 antigen Coated EV71 Ab
The immune response induced by EV71 inactivated vaccine in rhesus Immunized animal Infectious control animal
Protective effect of EV inactivated vaccine in monkey 婴猴保护性实验体温分析 41 40.5 40 39.5 39 38.5 38 37.5 37 36.5 36 体温 ( ) 疫苗免疫组 1# 疫苗免疫组 2# 疫苗免疫组 16# 疫苗免疫组 6# 阴性对照组 1 2 3 4 5 6 7 8 9 10 11 12 13 14 时间 ( 天 ) 婴猴保护性实验体温分析 体温 ( ) 41 40.5 40 39.5 39 38.5 38 37.5 37 36.5 36 感染对照组 37# 感染对照组 3# 感染对照组 10# 感染对照组 13# 阴性对照组 1 2 3 4 5 6 7 8 9 10 11 12 13 14 时间 ( 天 ) 婴猴保护性实验淋巴结病毒载量检测 ( 感染对照组 ) 病毒拷贝数 cop ies/ 100mg 1.60E+05 1.40E+05 1.20E+05 1.00E+05 8.00E+04 6.00E+04 4.00E+04 2.00E+04 0.00E+00 婴猴保护性实验主要器官病毒载量检测 ( 感染对照组 ) 病毒拷贝数 c o p i e s / 1 0 0 m g 5.00E+03 4.00E+03 3.00E+03 2.00E+03 1.00E+03 0.00E+00 颌下淋巴结肺门淋巴结腹股沟淋巴结 4 7 10 14 时间 ( 天 ) 4 7 10 14 时间 ( 天 ) 腋窝淋巴结肠系膜淋巴结 婴猴保护性实验淋巴结病毒载量检测 ( 疫苗免疫组 ) 病毒拷贝数 copies/100m g 心脏 肾脏 脾脏 肺脏 胰脏 膀胱 气管 支气管 胸肌 生殖器 肝脏 胆囊 胃 食管 空肠 回肠 升结肠 横结肠 降结肠 乙状结肠 直肠 十二指肠 1.60E+05 1.40E+05 1.20E+05 1.00E+05 8.00E+04 6.00E+04 4.00E+04 2.00E+04 0.00E+00 婴猴保护性实验主要器官病毒载量检测 ( 疫苗免疫组 ) 病毒拷贝数 c o p i e s / 1 0 0 m g 5.00E+03 4.00E+03 3.00E+03 2.00E+03 颌下淋巴结肺门淋巴结 腹股沟淋巴结 4 7 10 14 时间 ( 天 ) 腋窝淋巴结肠系膜淋巴结 心脏 肾脏 脾脏 肺脏 胰脏 膀胱 气管 支气管 胸肌 生殖器 肝脏 胆囊 胃 食管 空肠 回肠 升结肠 横结肠 降结肠 乙状结肠 直肠 十二指肠 病毒拷贝数 c o p i e s / 1 0 0 m g 病毒拷贝数 copies/100mg 婴猴保护性实验中枢神经系统病毒载量检测 ( 感染对照组 ) 6.00E+03 5.00E+03 4.00E+03 3.00E+03 2.00E+03 1.00E+03 0.00E+00 婴猴保护性实验中枢神经系统病毒载量检测 ( 疫苗免疫组 ) 6.00E+03 5.00E+03 4.00E+03 3.00E+03 2.00E+03 1.00E+03 0.00E+00 大脑 小脑 丘脑 延脑 中脑 桥脑 脊髓 4 7 10 14 时间 ( 天 ) 大脑 小脑 丘脑 延脑 中脑 桥脑 脊髓 4 7 10 14 时间 ( 天 ) 1.00E+03 0.00E+00 4 7 10 14 时间 ( 天 ) Viral loads detection of various organs of immunized animal at viral challenge 血清中和抗体检测 疫苗免疫组 感染对照组 700 600 500 抗体效价 400 300 200 100 0 二免后 4 周 攻毒后 4 天 攻毒后 7 天 攻毒后 10 天 攻毒后 14 天 时间 ( 天 )
Comparison of the pathological observation in immunized and control animals Immunized Control CNS tissue Immunized Control Lung tissue Immunized Control Trachea
Cytokines modulation in rhesus immunized by vaccine followed by virus challenge
The immune response induced by EV71 vaccine is not interfered with CA16 infection
The immune response induced by EV71 vaccine is not interfered with CA16 infection
Clinical trial of EV71 inactivated vaccine (human diploid cell) Effect: immunity; clinical protective efficacy Safety: advent; SAE. Clinicaltrials.gov, NCT01569581 Phase I:safety; Phase II:safety; immunogenicity; effective dose and immunization program Phase III:safety; effect and clinical protective efficacy.
Phase III Subjects: Children (6 71 month old) 12000 subjects; who distributed in 7 counties, Guilin, Guangxi, China, received vaccine and placebo Principle: random, double blind and placebo control Dose: 100U (320EU) Program: 0, 28 days
Process of phase III
Phase III protective efficacy
Safety observation servation Placebo Group N=6000 Number of cases Occurrence (%) Number of cases Occurrence (%) Solicited systemic adverse events within 7 days after each injection Total 2916 48.60 2574 42.90 <0.0001 Fever 2498 41.63 2111 35.18 <0.0001 Fever 3 grade 147 2.45 149 2.48 0.95 Diarrhoea: overall 498 8.30 535 8.92 0.24 Diarrhoea 3 grade 8 0.13 12 0.20 0.50 Nausea/vomiting/anorexia 530 8.83 475 7.92 0.08 Nausea/vomiting/anorexia 3 grade 6 0.10 7 0.12 1.00 Irritability/Drowsiness/acratia 360 6.00 303 5.05 0.03 Irritability/Drowsiness/acratia 3 grade 3 0.05 7 0.12 0.34 Allergy 166 2.77 156 2.60 0.61 Allergy 3 grade 2 0.03 0 0.00 0.50 Solicited local adverse events within 7 days after each injection Total 356 5.93 138 2.30 <0.0001 Pain 211 3.52 80 1.33 <0.0001 Pain 3 grade 1 0.02 0 0.00 1.00 Redness 130 2.17 34 0.57 <0.0001 Redness 3 grade 1 0.02 0 0.00 1.00 Itching 59 0.98 31 0.52 <0.004 Itching 3 grade 0 0.00 0 0.00 - Swelling 106 1.77 22 0.37 <0.0001 Swelling 3 grade 0 0.00 0 0.00 - Serious adverse events* Vaccination associated 2 0.03 2 0.03 1.0 HFMD associated hospitalization 41 0.68 88 1.47 <0.0001 Overall 68 1.13 125 2.08 <0.0001 Unsolicited events within 28 days after each injection 2841 47.35 2985 49.75 0.009 Unsolicited events within 28 days after each injection 3 grade 136 2.27 136 2.27 1.00
Levels of serum pro inflammatory cytokines were maintained normal in vaccinee
0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 0 56 180 360 540 720 1024 256 64 16 4 1024 256 64 16 4 The dynamic profile of antibody levels in participant for 2 years 6-11 12-23 24-35 36-71 1024 1024 1024 256 64 16 256 64 16 256 64 16 4 Day post 1st immunization Day post 1st immunization Day post 1st immunization Day post 1st immunization Day post 1st immunization Day post 1st immunization Day post 1st immunization Day post 1st immunization Neutralization antibody N eu traliz ation antib od y N eu traliz ation antib od y 4 1024 256 64 16 Neutralization antibody Neutralization antibody 4 1024 256 64 16 Neutralization antibody Neutralization antibody 1024 256 64 16 Neutralization antibody 4 4 4 Placebo Vaccine
Cross neutralization of serum from immunized individuals to different viral strains
Cost of treatment on HFMD rural residents urban residents mild case severe case mild case severe case 2887 5274 Average Annual Income of rural residents 13302 13106 Good news: 20,000 doses EV71 vaccine had been used in areas with population of 12.43 million, incidences of HFMD and its severe case decreased 41% and 68% respectively. 0 2000 4000 6000 8000 100001200014000 Zheng-kai Gan, Hui Jin, Jing-xin Li,(2015) Disease burden of enterovirus 71 in rural central China: A community-based survey, Human Vaccines & Immunotherapeutics, 11:10, 2400-2405
Analysis of the protective effect among large populations Post-licensure clinical trials Establishment of the database system Safety analysis Post-licensure clinical trials of EV71 inactivated vaccine The protective effect of EV71 vaccine combined with other vaccines The consistency of three batches of vaccine Analysis of the protective effect on severe cases
This work was coordinated by many people, who were come from SFDC, NIFDC, Chinese CDC, Guanxi CDC, CAMS and Fudan university and etc.. I hope to express much thanks to WHO for its supporting during this work. I do want to express my thanks to my lab team!
Much thanks for your attention!