1
Ih-Jen Su, MD, Ph. D.
Chair Professor, Dept. of Pathology National Cheng Kung University;
Division of Infectious DiseasesNational Health Research Institutes;
Southern Taiwan University of Science and Technology
28 November, 2014
Vaccine Development for Enterovirus 71 Infection
Human Enteroviruses
Genus Enterovirus (family Picornaviridae)Human enteroviruses are classified into 4 species
A, B, C, D
(Bible et al., 2007)
EV71 can be classified into 11 genotypes (A, B1~B5, C1~C5).
1. Infection of EV can cause hand-foot-and-mouth disease (HFMD), aseptic meningitis, myocarditis, hepatitis, acute flaccid paralysis (AFP), brainstem encephalitis, and pulmonary edema; hundred thousands of cases and 78 mortality in 1998
2. EV71 is uniquely associated with brainstem encephalitis and pulmonary edema.
Unexpectedly, An Outbreak of EV71 in 1998 and this laboratory plays a major role in
identifying the pathogen and develop the rapid diagnosis
Unique brain stem encephalitis
and pulmonary edema
EV71 virus structureCapsid• VP1 (blue)
• VP2 (green)
• VP3 (red)
• VP4 (inner)
http://www.cgl.ucsf.edu/Research/virus/capsids/viruses.html
Picornaviridae Capsid
• EV71 VP1 DNA
• EV71 VP1, VP2, VP3 peptide
• EV71 rVP1, rVP2, rVP3 protein
• EV71 VLP
• EV71 inactivated virus particles
Working standards preparations
Enterovirus – gene structure
Picornaviridae, 7.5kb ssRNA
Enteroviruses – Species & Diseases febrile/respiratory illness,
hand-foot-and-mouth disease (HFMD), herpangina, aseptic meningitis, myo- peri- carditis, neonatal multisystem diseases, meningoencephalitis, paralysis
pulmonary edema, paralytic poliomyelitis, fatal cardiomyopathy, encephalitis
Isolation of Enterovirus 71 in Taiwan, 1998-2007
Huang et al. Virus Research 2008
Top EV serotypes, Taiwan ‘00 – ‘08
Rank
Serotype
2000 2001 2002 2003 2004 2005 2006 2007 2008
1 EV71 EV71 CA16 CA16 CA4 CB3 CA4 CA16 CA2
2 CA16 CA16 E6 EV71 CA10 CA16 CA2 CA6 EV71
3 CB3 E30 CB5 E9 CB4 EV71 CA5 CA10 CB4
4 E9 E6 EV71 E11 EV71 CA6 E18 CA4 CB1
5 CB4 Sabin CA24 CA2 CA6 CA5 CB2 E6 CA16
資料來自疾管局病毒合約實驗室
International Spread of EV71 Subgenogroup B5 in Asia
Singapore
2000
Sarawak, Malaysia
Taiwan
2003 transmission
2007 transmission
0.1
CA16N1859-TW05
1M-AUS-12-00804/NO/03
0926-OR-91KOR-EV71-0102M-AUS-3-99N6500-TW98N5811-TW98N4643-TW98N6356-TW98N9822-TW98
2779-Yamagata-02N3340-TW02N0775-TW07M229-TW10M480-TW10
N2735-TW04N2121-TW05N1375-TW05N0003-TW05N1614-TW04N2459-TW05N2411-TW04N2284-TW04N2328-TW02N2472-TW04N2490-TW04N1862-TW05
BrCr7423-MS-877673-CT-872609-AUS-74237-TW86238-TW86266-TW86EV71/SAR/SHA66EV71/SAR/SHA63SB-0635-SAR-00N1796-TW99M0177-TW00M0200-TW00962-Yamagata-00H0009-TW03S0004-TW03S0044-TW03N3496-TW02N2443-TW02N0781-TW01N3793-TW015511/SIN/002716-Yamagata-03N2776-TW03N2838-TW03SB-10712-SAR-03SB-11977-SAR-03M447-TW09M134-TW09M474-TW09M184-TW09M200-TW09M0314-TW08N0561-TW08N1324-TW08N0610-TW08M0084-TW08M0448-TW08
C5C1
C3
C2
C4
AB2
B1
B3
B4
B5
1986199819992000200120022003200420052007200820092010
99.8
99.7
1986 B1
1998 C2(major)
B4(minor)
1999 B4
2000 B4
2001 B4
2002 B4, C4 (1)
2003 B4, B5
2004 C4
2005 C4
2007 C4
2008 B5
2009
2010
B5
C4
Phylogenetic analysis of EV71 isolates in Taiwan from 1986 and 1998-2010 based on VP1 region
12
(Tee, et al. 2010)
Elevatation in genetic diversity is correlated with EV71 epidemics
13
2923(B4)
1988 (C2)
3664 (B4)7309 (C4)
7373(C4)
N0692-TW08 (B5)
N1745-TW08 (B5)
N2838-TW03 (B5)
M0448-TW08 (B5)
M0448-TW08 (B5)
BrCr (A)
S0318-TW01 (B4)
7008-TW99 (B4)
N1796-TW99 (B4)S0296-TW00 (B4)
236-TW86 (B1)
YFW
12103 (B5)1970 (C2)
12121 (B5)
3877(B4)
7265(C4)
7337(C4)
6356-TW98 (C2)
S0584-TW04 (C4)
4643-TW98 (C2)7773(C5)
2130(C4)
3872(B4)
N1859-TW05 (C5)
N3340-TW02 (C4)
N2413-TW04 (C4)
N1757-TW04 (C4)
N2121-TW04 (C4)
Antigenic map of EV71
Genotype B4/B1
Genotype B5Genotype C4
Genotype C2
(Huang, et al. J Clin Microbiol, 2009.)
Age-specific incidence Rates of EV71 Infections in Taiwanese Young Children, 2008-2009
Age (Mo)
No. followed
No. of Symptomatic Infection
Virus Serology Combined
No. of Asymptom.
Infection
No. (rate*) of
infection
0-6 304 0 0 0 2 2 (1.3)
7-12 391 0 5 5 2 7 (3.6)
13-24 294 4 13 13 3 16 (5.4)
25-36 66 1 2 2 1 3 (4.6)
Total 5 20 20 8 28
* per 100 person-years
Cross-reactive Neutralizing Antibody Titers in Children Infected with EV71 B5 Genotype
(P<0.05, t test)
LN
(neu
tral
izin
g t
iter
)
B5
C4
Subgenogroups
B4
B4 B5 C4
(Lee et al. submitted)
C4
B5
B4
A
Cross-reactive Neutralizing Antibody Titers in 13 Acute Sera of Severe Cases
Kung SH et al. 2007
B4 C4B4 C4
Virulence of Different Genotypes
• CGCH in 1998 (C2), 38 CNS / 143 inpatients (27%) (Chang et al. 1999)• CGCH in 2000-02 (B4), 97 CNS / 331 inpatients (29%) (Chang et al.
2004)• CGCH in 2008 (B5), 19 CNS / 111 inpatients (17%)
Ooi et al. CID 2007
Clinical Spectrum of EV71 Infections in Taiwanese Children
Fatal Cases Severe Cases
(CNS & Cardiopulmonary)
Asymptomatic EV71 Infections
Mild Infections
Medically Attended Illness(HFMD/herpangina)
HFMD: hand-foot-mouth disease (Lin et al. EID 2003; Chang et al. JAMA 2004)
Historical Outbreaks of Enterovirus 71
(Bible et al., 2007)
EV71 Fatal Epidemics Since 2000Country Year Severe case
no.*Death
no.Source
Taiwan 2000
2001
2005
2008
152
187
82
347
25
27
7
14
Liu et al. APEC meeting 2009
Sarawak, Malaysia
2000-3 2006
185
436
4
6
Ooi et al. 2008
Ooi et al. 2009
Singapore 2000 No data 5 Chan et al. 2003
HCMC, Vietnam
2005 51 3 Tu et al. 2007
Brunei 2006 No data 3 AbuBakar et al. 2009
China 2007
2008
2009.July
No data
1165
10509
>27
126
255
Zhang et al. 2009
Fan et al. 2009; Xiao
APEC meeting 2009
* Definitions vary in different countries. Lee et al. 2010
Isolation of Enterovirus 71 in Taiwan, 2006-
Data source: Taiwan CDC, March 2010
Clinical Symptoms of Hospitalized Children With Enterovirus Infection in CGCH, 2008
EV71(n=111
)CA2
(n=202)
Other
(n=274)
Uncomplicated90
(89%)200
(99%) 258Herpangina 18 164 117HFMD 69 11 26Other illness (including fever, viral exanthema or URI) 3 25 115
Complicated21
(19%) 2 (1%) 16Aseptic meningitis 0 0 14HFMD plus aseptic meningitis 1 0 0Herpangina plus encephalitis 5 1 0HFMD plus encephalitis 6 0 0HFMD plus polio-like syndrome 3 0 0HFMD plus acute heart failure 2 0 0HFMD plus encephalitis and pulmonary complications 4* 0 0Other 0 1 2
* One fatal case and 3 other cases with physical sequelaeLee et al. Submitted
Cumulative Incidence Rates* of EV71 Infections in Taiwanese Young Children, 2008~2009
* Estimated by serology, per 100 persons
0
2
4
6
8
10
12
14
16
6 Mo 12 Mo 24 Mo 36 Mo
Age
Cum
ulat
ive
Inci
denc
e,
%
Cell-Based EV71 Vaccine Development (2007 Jan - 2011 Dec)
Pele Choi-Sing Chong 莊再成 PhD
Director
Vaccine Research and Development Center
National Health Research Institutes
E-mail: [email protected]
Co-development with Taiwan CDC
B1F Parking/Wasting treatment停車場 / 污水處理 / 消防設施
1F Production/Warehouse 製程區 / 倉儲區
2F Offices/QC/QA/QA & HVAC 辦公室 /品管分析室 / 品保文管室 / 機房
3F Offices and Lab 辦公室 / 研發實驗室
4F Mechanical Facility 機房
CRO & CMO in VRDC
Upstream ProcessingDownstream Processing
Packaging
細胞株
Cell bank
細胞庫安全性測試
Validation
製程放大
Scale up
大量細胞培養生產蛋白質藥
蛋白質回收純化
Purification
純化蛋白質原料藥
配方
Formulation
病毒庫確效
Virus seed validationl
蛋白藥物結構鑑定
Bioanalytical analysis
無菌過濾
凍晶劑型充填
Lyophilization
液劑劑型充填 Filling & Packaging
NHRI Vaccine Center
腸病毒 71 型疫苗各國研發進展 Competition
Institutes Vaccine types Status
NHRI, Taiwan Cell-base inactivated EV71 vaccine Pre-IND process rolling review
NTHU, Taiwan VLP-base recombinant EV71 vaccine R&D process
SingVac, Singapore Cell-base inactivated EV71 vaccine R&D process
SinoVac, China EV71 vaccine Submit the clinical trial application to SFDA
NIID, Japan EV71 vaccine R&D process
Dr. Ooi/Malaysia MOH Malaysia
EV71 peptide vaccine R&D process
NHRI EV71 Vaccine Product Profile
• Indication: prevent EV71-related hand-foot-mouth disease and complications
• Target population: young children • Antigen: Vero cell based inactivated whole virus• Strain: EV71 E59 (serum-free culture)• Adjuvant: alum phosphate• Administration: intramuscular injection
Cross-neutralization studies with different
EV71 strains
E59 (B4)
inactive virus
E59 (B4)
active virus
(B4)E59-06EV18#3
inactive virus
(formalin/AlPO4)
E36 (C4)
active virus
mice / i.p. rabbit /i.v. rabbit /s.c. rabbit /i.v.
1970 A BrCr <10 55 40 255
C2 E15 47 3319 1783 12915
C2 E07 224 3820 1660 13277
C2 E25 417 7130 3320 13277
C2 E32 47 5036 1783 14260
C2 E63 16 28 15 67
C4 E41 60 6638 1496 21831
1998
B1 E743 <10 569 700 3146
B4 E43 52 7130 3320 10071 2000 B4 E59 955 14260 21831 14260
B4 E73 143 33896 7130 26553
B4 E75 112 23940 6638 16948
B4 E17 158 36553 5985 28520
B4 E19 111 3557 2512 4237
B4 E20 1159 23940 12915 40286
2002
B4 E22 37 22986 4418 19953
B4 E61 209 28452 8474 30560
B4 E62 776 74161 15280 26553 2003
B4 E763 105 26553 4529 28452
C4 E36 <10 1660 782 13308
C4 E45 <10 1496 2320 25830
C4 E24 <10 16 40 285
C4 E98 <10 33 30 712
C4 E87 <10 835 391 6839
C4 E12 <10 <10 <10 54
C4 E49 14 1349 750 15280
C4 E16 28 4635 2118 14260
C4 E18 <10 879 976 8555
C4 E76 <10 <10 15 85
C4 E95 <10 28 28 188
C4 E23 <10 1259 779 5120
2004
C4 E27 <10 17 25 51
C4 E80 <10 17 201 466
C4 E79 15 891 447 5036
C4 E31 15 205 224 3090
C4 E51 15 57 531 436
2005
C4 E100 <10 20 15 36
neutralized virus
serum
Taiwan CDC
EV71 Vaccine Development
• cGMP facility– Manufacturing
clinical lots– QC testing– Pre-clinical studies
• R&D– Bioprocess development– Immunological studies – Standard reagents
preparations– Transgenic mouse model
Cell bank and virus seed validations
• Service contracts to Bioreliance (UK)• Vero cell bank (Completed)
– MCB (26/26 tests) completed
– WCB (9/9 tests) completed
– EPC (26/26 tests) completed
– Dec/2009 received final reports
• EV71 virus seed (MVS/WVS)– 97% (33/34 tests) completed
– July/2010 final report
Manufacturing Processes
• Upstream: Roller bottle (40L scale)
• Downstream: Liquid Chromatography Purification
• Inactivation: Formaldehyde (1:4000)
• Storage: Vaccine bulk stored at 4oC
• Produced 3 test Lot and 3 clinical Lot
cGMP facility
Vaccine Research and Development Center
Roller Bottle Process Flow
Batch
Batch
00
55
99
1515
2121
2727
3333
3636
4141
4747
6868
7575
Cell Production BankCell Production Bank
850RB x 10850RB x 10
1700RB x 100 (40L)1700RB x 100 (40L)
Virus InfectionVirus Infection
Harvest & ConcentrationHarvest & Concentration
Purification Purification (3 working Days(3 working Days))
In Process AssayIn Process Assay(5 working Days(5 working Days))
InactivationInactivation ( 4-6 Days( 4-6 Days))
ImmunizationImmunization(21 Days,(21 Days, 11st st 3 3 rdrd week IP) week IP)
NeutralizationNeutralization (5-7 Days)(5-7 Days)
Seed Seed (Day (Day 00))
Bulk Bulk (Day 75)(Day 75)
150T-flask x 4150T-flask x 4
75T-flask75T-flask
850RB x 2850RB x 2
Taiwan CDC transfer
Roller bottle process
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
0 1 2 3 4 5 6
DPI
Vir
us
tite
r (T
CID
50/m
L)
R1(200mL)
R2(200mL)
R3(400mL)
Roller bottle #1Roller bottle #2Roller bottle #3
Days post-infection
Liquid Chromatography Purification System
Liquid Chromatography
M F1 F2 F3 F4 F5 F6 F7 F8 PC
50kD36kD
64kD
-SDS-PAGE(silver stain)
-Western blot
-ELISA titer
EV71 virus inactivation
Virus titer 37oC 25oC 4oC
10-8 1.71 d 6.45 d 24.90 d
10-12 2.16 d 8.27 d 31.64 d
Inactivation time prediction
1.E+00
1.E+01
1.E+02
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
1.E+08
0 10 20 30 40 50 60 70 80Hours
Vir
us
tite
r (P
FU
/mL
)
)
37oC
25oC
4oC
Formaldehyde solution (1:4000)37oC
25oC
4oC
Tests Seed In-Process Bulk
Mycoplasma test (PCR) V
Sequence analysis (PCR) V
Q-ELISA VP2 assay V V
Residual Host cell assay V
Neutralization assay (TCID50) V
Plaque Assay V V
Virus titration (TCID50) V V
SDS-PAGE V V
Protein content assay V V
Virus Inactivation assay V
Residual DNA content V
QC Tests Development
Immunogenicity study (Mouse)
Adjuvant 2 dosages
Test year SampleTotal protein
(ug)type ug
Specific antibody titer
Neutralization antibody (TCID50)
2008 07EV03 0.5 PBS 0 116 105
2008 07EV03 2.5 PBS 0 266 214
2008 07EV03 0.5 AlPO4 300 166 322
2008 07EV03 2.5 AlPO4 300 2066 644
2009 r2F304 0.2 AlPO4 300 3733 604
2009 r2F304 1 AlPO4 300 6133 199
2009 r2F304 2 AlPO4 300 4800 707
2009 r2F337 0.2 AlPO4 300 2967 100
2009 r2F337 1 AlPO4 300 1920 854
2009 r2F337 2 AlPO4 300 3467 911
靜脈 (iv) 免疫中和效價 neutralization titer
免疫劑量
Total Protein week2 week4 Week6
07EV01 4.36ug 28 224 1496
07EV02 5.56ug 72 750 X
07EV03 5.39ug 20 158 982
肌肉 ( im) 免疫中和效價 neutralization titer
免疫劑量
Total Protein week2 week4 Week6
07EV01 4.36ug 105 417 1135
07EV02 5.56ug 112 1259 1496
07EV03 5.39ug 94 417 631
皮下 ( sc) 免疫中和效價 neutralization titer
免疫劑量
Total Protein week2 week4 Week6
07EV01 4.36ug 20 224 531
07EV02 5.56ug 376 809 3548
07EV03 5.39ug 203 1059 1959
Immunogenicity studies (Rabbit)
Virus Neutralization titers of animals immunized with EV71vac using either Vero or RD cell assay systems
Results from RD cell were provided by Taiwan CDC
MAb or
Antisera
Virus Neutralization Titer (GMT)
NHRI VRDC (Vero) Taiwan CDC (RD)
B4 B5 C4 B4 B5 C5
MAb E1 64 <8 8 <8 <8 <8
MAb979 128 128 32 64 128 32
Mice antisera 1 1774 8 16 8 31 <8
Mice antisera 2 1581 64 32 8 <8 <8
Rabbit antisera 1 32768 16384 8192 ND 16384 13520
Rabbit antisera 2 16384 ND ND ND 16384 16384
Monkey ND ND ND >1659 3169 5623
Additional Product characterizations• Working Standards Preparations • Neutralization epitope(s) mapping• Q-ELISA for Quantify EV71 VP2 Antigen• Transmission electron microscopy• Mass spectrometry analysis• Immuno-plaque assay for validating formalin virus
inactivation process• Real-Time PCR for validating formalin virus inactivation
process
Identification of Neutralization epitopes
ELISA for Quantify EV71 VP2 Antigen
y = 0.7389Ln(x) + 1.3906
R2 = 0.9922
0
0.5
1
1.5
2
2.5
3
3.5
0.1 1 10
OD
450
nm
VP2 concentration (ug/ml)
Inactivated EV71(dilution fold)
Concentrationof VP2(ug/ml)
Mean(ug/ml)
Total protein(ug/ml)
Lot. 07EV01
4X 5.84
6.01 18.598X 5.71
16X6.48
Lot. 07EV02
4X4.23
4.46 24.048X4.07
16X 5.08
Lot. 07EV03
4X10.08
9.81 23.868X 9.51
16X9.82
腸病毒 71 型疫苗 (EV71vac) 發展現況
• 細胞庫確效執行進度 : 100%• 病毒庫確效執行進度 : 97% (Q2/2010 完成 )• 疫苗生產 : 完成 3 批次試產與 3 批臨床批次生產• 完成臨床前毒理動物實驗 (Rat and Rabbit models)• 已送審 CMC 文件並準備新藥臨床試驗 (IND) 文件• 預定於 Sept/2010 執行第一期人體臨床試驗• 與台北榮總、台大醫院臨床試驗中心共同合作• 目前 : 持續 (Scale up Bioreactor) 量產臨床批次
EV71 Bulk assayTest Item Method/Criteria Result
Free formaldehyde Test for free formaldehyde / <0.2 mg/mL 0.106 mg/mL
Total Protein content Bicinchoninic acid (BCA) method 53.04 ug/mL
VP2 antigen content Q-ELISA 198.4 Unit/mL
Sterility Sterility test No microbial growth
Inactivation Plaque assay No plaque formed
Bacterial endotoxin Bacterial endotoxin ELISA method/ <5 EU/dose
0.1 EU/mL
Residual host cell protein Immunochemical method 2.72 ug/mL
Residual Benzonase content
Detected by ELISA / <0.156 ng/mL <0.156 ng/mL
Residual host cell DNA content
Detected by Thershold system / <10ng/dose
0.01667 ng/mL
※ NuPAGE Bis-Tris Mini Gels 4-12% (1.5mm), loading 0.5ug total protein per well
188
98
62
3
6
14
17
28
38
49
Bulk M P
SDS-PAGE (silver stain)
P: positive control
188
98
62
3
6
14
17
28
38
49
Bulk M P
Western blot
1° Ab: Mab979 (1:1000)
2° Ab: Goat- anti-mouse
(1:5000)
Table 1. Summary of predicted and observed molecular weights (MW) of EV71/E59 viral proteins and incompletely processed viral
polypeptides
EV71 antigens Amino acid sequences Residues Predicted MWa (kDa) Observed MWb (kDa)
Viral antigen in virion
VP4 1-69 69 7.49 8
VP2 70-323 254 27.78 28
VP3 324-565 242 26.52 27
VP1 566-862 297 32.73 36
Incompletely processed viral polypeptides
VP0 (VP4-VP2) 1-323 323 35.27 38
VP3-VP1 324-862 519 59.25 60
Viral antigens cross-linked by formalin inactivation based on VP2 recognized by MAB979
VP4-VP2 1-323 323 35.27 38
VP4-VP2-VP4 42.76 45
VP2-VP2 55.56 58
VP2-VP4-VP2 63.05 64
VP4-VP2-VP3-VP1 1-862 862 94.52 98
a The predicted MW is based on the corresponding amino acid sequence derived from the EV71/E59 strain genome. b The observed MW is based on the SDS-PAGE and western blot analyses of the EV71/E59 virus bulk.
EV71vac 原液 電子顯微鏡檢查
150K X Cooperation with NHRI Nanomedicine center
EV71vac 無菌充填包裝10 ug protein/300ug Al/dose
Bioreactor Process Development
• Upstream: Bioreactor (Microcarrier)
• Downstream: Sucrose gradient ultracentrifuge
• Inactivation: Formaldehyde (1:4000)
• Storage: Virus samples stored at 4oC
Bioreactor System in NHRI Vaccine Center
2.2 L 7.5 L 30 L 150 L
Sucrose gradient ultracentrifuge
1.E+03
1.E+04
1.E+05
1.E+06
1.E+07
0 10 20 30 40 50 60
Sucrose gradient (%)
Vir
us ti
ter
(TC
ID50
/mL
)
EV71 virus
Transmission electron microscopy
Full virus particle
150K X
Serum-Free culture, Formaldehyde solution (1:4000)
Empty virus particle
Cooperation with NHRI Nanomedicine center
Mass spectrometry analysis
m/z800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
%
0
100
EV71_A1_2 11 (0.306) 1: TOF LD+ 1.24e41072.6973
1058.6251
1042.6378
926.5388
2603.4385
1074.6440
2078.2000
2077.1914
1075.64071570.7391
1076.6379
1283.8030 1532.8632
1571.7256
1811.0808
1572.7396
1778.0740
1573.7268
2063.0342
2062.02912018.7096
1813.0527
2602.4504
2079.1770
2173.1653
2174.1650
2175.1643
2187.1782
2287.35522288.3801
2604.4617
2605.4500
2606.47362979.5542
2936.0032
polyprotein [Human enterovirus 71] major capsid protein VP1
1 GDRVADVIES SIGDSVRRAL TQALPAPTGQ NTQVSSHRLD TGEVPALQAA51 EIGASSNASD ESMIETRCVL NSHSTAETTL DSFFSRAGLV GEIDLPLEGT 101 TNPNGYANWD IDITGYAQMR RKVELFTYMR FDAEFTFVAC TPTGGVVPQL 151 LQYMFVPPGA PKPESRESLA WQTATNPSVF VKLTDPPAQV SVPFMSPASA 201 YQWFYDGYPT FGEHKQEKDL EYGACPNNMM GTFSVRNVGS SKSKYPLVVR 251 IYMRMKHVRA WIPRPMRNQN YLFKANPNYA GNSIKPTGTS RTAITTL
Sequence coverage = 43%
Neutralization assay (3rd week)
0
200
400
600
800
1000
1200
1400
1600
0.5ug/dose Empty virus particle
2.5ug/dose Empty virus particle
0.5ug/doseFull virus particle
2.5ug/doseFull virus particle
AlPO4
Serum-Free culture, Formaldehyde solution (1:4000)
Neu
tral
izat
ion
titer
VP2 is responsible for SCARB2 binding & X-neutralization
X-Y SCARB2EV71 & CoxA16 receptor
Receptors identified for EV71 virus, Nat. Med. 15 (2009): 728-729, 794-797, 798-801.
Vero RD 3T3
s ss
ss
ss
ss s ss
s
s ss
X-Y
X-Y
X-Y
X-Y
X-Y
X-Y
s
Current Concerns of EV71 Vaccine Licensing
• Given the disease burden of EV71, how can efficacy be demonstrated in Taiwan?
• Are we using WHO-qualification approach?• Would serologic endpoints like in IPV be feasible
for licensure for EV71?• Would be EV71 sufficient or a combination with
CV16?• Target population: infants (2, 4 & 6) Children 1 to 5 year as
booster• Is adjuvant acceptable?
謝 謝 Thank you for Your Attention!!
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