Vaccine Development for
Enterovirus 71 Infection
Ih-Jen Su, MD, Ph. D.
Chair Professor, Dept. of Pathology
National Cheng Kung University;
Division of Infectious Diseases
National Health Research Institutes;
Southern Taiwan University of Science and Technology
28 November, 2014
1
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).
Unexpectedly, An Outbreak of EV71 in 1998
and this laboratory plays a major role in
identifying the pathogen and develop the
rapid diagnosis
1. Infection of EV can cause handfoot-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.
Unique brain stem encephalitis
and pulmonary edema
EV71 virus structure
Capsid
Picornaviridae Capsid
•
•
•
VP1 (blue)
VP2 (green)
VP3 (red)
•
VP4 (inner)
Working standards preparations
• EV71 VP1 DNA
• EV71 VP1, VP2, VP3 peptide
• EV71 rVP1, rVP2, rVP3 protein
• EV71 VLP
• EV71 inactivated virus particles
http://www.cgl.ucsf.edu/Research/virus/capsids/viruses.html
Enterovirus – gene structure
Picornaviridae, 7.5kb ssRNA
Enteroviruses – Species & Diseases
 febrile/respiratory illness,
hand-foot-and-mouth
disease (HFMD), herpangina,
aseptic meningitis, myoperi- 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
Serotype
Rank
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
Taiwan
2003 transmission
Singapore
2000
Sarawak,
Malaysia
2007 transmission
CA16
N1859-TW05
1M-AUS-12-00
804/NO/03
0926-OR-91
KOR-EV71-010
2M-AUS-3-99
N6500-TW98
N5811-TW98
N4643-TW98
N6356-TW98
N9822-TW98
99.8 2779-Yamagata-02
N3340-TW02
N0775-TW07
M229-TW10
M480-TW10
N2735-TW04
N2121-TW05
N1375-TW05
N0003-TW05
N1614-TW04
N2459-TW05
N2411-TW04
N2284-TW04
N2328-TW02
N2472-TW04
N2490-TW04
N1862-TW05
BrCr
7423-MS-87
7673-CT-87
2609-AUS-74
237-TW86
238-TW86
266-TW86
EV71/SAR/SHA66
EV71/SAR/SHA63
SB-0635-SAR-00
N1796-TW99
M0177-TW00
M0200-TW00
962-Yamagata-00
H0009-TW03
S0004-TW03
S0044-TW03
N3496-TW02
N2443-TW02
N0781-TW01
N3793-TW01
5511/SIN/00
99.7 2716-Yamagata-03
N2776-TW03
N2838-TW03
SB-10712-SAR-03
SB-11977-SAR-03
M447-TW09
M134-TW09
M474-TW09
M184-TW09
M200-TW09
M0314-TW08
N0561-TW08
N1324-TW08
N0610-TW08
M0084-TW08
M0448-TW08
0.1
C5
C1
C3
C2
Phylogenetic analysis of EV71
isolates in Taiwan from 1986 and
1998-2010 based on VP1 region
C4
1986
B1
1998
C2(major)
B4(minor)
A
B2
B1
B3
B4
B5
1986
1998
1999
2000
2001
2002
2003
2004
2005
2007
2008
2009
2010
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
Elevatation in genetic diversity is
correlated with EV71 epidemics
(Tee, et al. 2010)
12
Antigenic map of EV71
3664 (B4)
7309 (C4)
Genotype B4/B1
7373(C4)
S0318-TW01 (B4)
1988 (C2)
2923(B4)
236-TW86 (B1)
7008-TW99 (B4)
S0296-TW00 (B4)
N1796-TW99 (B4)
YFW
N0692-TW08 (B5)
Genotype C4
12103 (B5)
1970 (C2)
Genotype B5
N2121-TW04 (C4)
N2838-TW03 (B5)
12121 (B5)
N1745-TW08 (B5)
M0448-TW08 (B5)
M0448-TW08 (B5)
BrCr (A)
S0584-TW04 (C4)
3877(B4)
7265(C4)
7773(C5)
2130(C4)
4643-TW98 (C2)
N1757-TW04 (C4)
7337(C4)
Genotype C2
3872(B4)
N2413-TW04 (C4)
6356-TW98 (C2)
N3340-TW02 (C4)
N1859-TW05 (C5)
13
(Huang, et al. J Clin Microbiol, 2009.)
Age-specific incidence Rates of EV71 Infections
in Taiwanese Young Children, 2008-2009
Age
No.
(Mo) followed
No. of Symptomatic
No. of
No. (rate*)
Infection
Asymptom.
of
Virus Serology Combined Infection
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)
5
20
20
8
28
Total
* per 100 person-years
Cross-reactive Neutralizing Antibody Titers in
Children Infected with EV71 B5 Genotype
B5
LN(neutralizing titer)
B4
A
C4
B5
B4
C4
(P<0.05, t test)
B4
B5
C4
Subgenogroups
(Lee et al. submitted)
Cross-reactive Neutralizing Antibody Titers
in 13 Acute Sera of Severe Cases
B4
B4
Kung SH et al. 2007
C4
C4
Virulence of Different Genotypes
Ooi et al. CID 2007
• 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%)
Clinical Spectrum of EV71 Infections in
Taiwanese Children
Fatal Cases
Severe Cases
(CNS & Cardiopulmonary)
Medically Attended Illness
(HFMD/herpangina)
Mild Infections
Asymptomatic EV71 Infections
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 2000
Country
Year
Severe case
no.*
Death
no.
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.
Source
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
Uncomplicated
Herpangina
HFMD
Other illness (including fever, viral exanthema or URI)
Complicated
Aseptic meningitis
HFMD plus aseptic meningitis
Herpangina plus encephalitis
HFMD plus encephalitis
HFMD plus polio-like syndrome
HFMD plus acute heart failure
HFMD plus encephalitis and pulmonary complications
Other
EV71
CA2
Other
(n=111) (n=202) (n=274)
90 (89%) 200 (99%) 258
18
164
117
69
11
26
3
25
115
21 (19%) 2 (1%)
16
0
0
14
1
0
0
5
1
0
6
0
0
3
0
0
2
0
0
4*
0
0
0
1
2
* One fatal case and 3 other cases with physical sequelae
Lee et al. Submitted
Cumulative Incidence, %
Cumulative Incidence Rates* of EV71 Infections
in Taiwanese Young Children, 2008~2009
16
14
12
10
8
6
4
2
0
6 Mo
12 Mo
Age
* Estimated by serology, per 100 persons
24 Mo
36 Mo
Cell-Based EV71 Vaccine Development
(2007 Jan - 2011 Dec)
Co-development with Taiwan CDC
Pele Choi-Sing Chong 莊再成 PhD
Director
Vaccine Research and Development Center
National Health Research Institutes
E-mail: pelechong@nhri.org.tw
4F Mechanical Facility 機房
3F Offices and Lab 辦公室/研發實驗室
2F Offices/QC/QA/QA & HVAC 辦公室/品
管分析室/品保文管室/機房
1F Production/Warehouse 製程區/倉儲區
B1F Parking/Wasting treatment
停車場/污水處理/消防設施
CRO & CMO in VRDC
Upstream Processing
細胞株
Cell
bank
製程放
大
Scale
up
大量細胞
培養生產
蛋白質藥
Downstream
Processing
蛋白質回收
純化
Packaging
純化蛋白
質原料藥
配方
凍晶劑型充填
Formulation
Lyophilization
Purification
細胞庫安全
性測試
病毒庫確效
蛋白藥物結構鑑
定
Validation
Virus seed
validationl
Bioanalytical
analysis
NHRI Vaccine Center
無菌
過濾
液劑劑型充填
Filling &
Packaging
腸病毒71型疫苗各國研發進展
Institutes
Competition
Vaccine types
Status
NHRI, Taiwan
Cell-base inactivated EV71 vaccine
NTHU, Taiwan
VLP-base recombinant EV71 vaccine
R&D process
Cell-base inactivated EV71 vaccine
R&D process
SingVac, Singapore
Pre-IND process rolling review
SinoVac, China
EV71 vaccine
Submit the clinical trial
application to SFDA
NIID, Japan
EV71 vaccine
R&D process
EV71 peptide vaccine
R&D process
Dr. Ooi/Malaysia
MOH Malaysia
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
serum
neutralized
virus
1970
1998
2000
2002
Cross-neutralization
studies with different
EV71 strains
2003
2004
Taiwan CDC
2005
A
C2
C2
C2
C2
C2
C4
B1
BrCr
E15
E07
E25
E32
E63
E41
E743
B4
B4
B4
B4
B4
B4
B4
B4
B4
B4
B4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
C4
E43
E59
E73
E75
E17
E19
E20
E22
E61
E62
E763
E36
E45
E24
E98
E87
E12
E49
E16
E18
E76
E95
E23
E27
E80
E79
E31
E51
E100
E59 (B4)
inactive virus
E59 (B4)
active virus
(B4)E59-06EV18#3
inactive virus
(formalin/AlPO4)
E36 (C4)
active virus
mice / i.p.
＜10
47
224
417
47
16
60
＜10
rabbit /i.v.
55
3319
3820
7130
5036
28
6638
569
rabbit /s.c.
40
1783
1660
3320
1783
15
1496
700
rabbit /i.v.
255
12915
13277
13277
14260
67
21831
3146
52
955
143
112
158
111
1159
37
209
776
105
7130
14260
33896
23940
36553
3557
23940
22986
28452
74161
26553
3320
21831
7130
6638
5985
2512
12915
4418
8474
15280
4529
10071
14260
26553
16948
28520
4237
40286
19953
30560
26553
28452
＜10
＜10
1660
782
13308
1496
2320
25830
＜10
＜10
16
40
285
33
30
712
＜10
＜10
14
835
391
6839
＜10
1349
＜10
750
54
15280
28
4635
2118
14260
＜10
＜10
879
976
8555
＜10
28
15
85
28
188
1259
779
5120
17
17
891
205
57
20
25
201
447
224
531
15
51
466
5036
3090
436
36
＜10
＜10
＜10
＜10
15
15
15
＜10
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
Vaccine Research and Development Center
cGMP facility
Seed (Day 0) Cell Production Bank
0
Roller Bottle Process Flow
75T-flask
150T-flask x 4
5
9
850RB x 2
15
850RB x 10
1700RB x 100 (40L)
Batch
21
27
Virus Infection
33
Harvest & Concentration
Purification (3 working Days)
36
41
In Process Assay(5 working Days)
47
Inactivation ( 4-6 Days)
Immunization(21 Days,
68
Bulk (Day 75)
1st 3 rd week IP)
75
Taiwan CDC transfer
Neutralization
(5-7 Days)
Roller bottle process
1.E+08
1.E+07
Virus titer (TCID50/mL)
1.E+06
1.E+05
1.E+04
1.E+03
R1(200mL)
Roller bottle
#1
R2(200mL)
Roller bottle #2
Roller bottle #3
R3(400mL)
1.E+02
1.E+01
1.E+00
0
1
2
3
4
DPI
Days post-infection
5
6
Liquid Chromatography Purification System
Liquid Chromatography
-ELISA titer
-SDS-PAGE
(silver stain)
M F1 F2 F3 F4 F5 F6 F7 F8 PC
64kD
50kD
36kD
-Western blot
EV71 virus inactivation
Formaldehyde solution (1:4000)
1.E+07
4oC
4 oC
)
1.E+08
37oC
37oC
25oC
25oC
Virus titer (PFU/mL)
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
0
10
20
30
40
Hours
50
60
70
Inactivation time prediction
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
80
QC Tests Development
Tests
Seed
Mycoplasma test (PCR)
V
Sequence analysis (PCR)
V
Q-ELISA VP2 assay
In-Process
Bulk
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
Immunogenicity study (Mouse)
Adjuvant
2 dosages
ug
Specific
antibody titer
Neutralization
antibody
(TCID50)
Test year
Sample
Total protein
(ug)
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
type
Immunogenicity studies (Rabbit)
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
Virus Neutralization titers of animals immunized with
EV71vac using either Vero or RD cell assay systems
Virus Neutralization Titer (GMT)
MAb or
Antisera
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
ND
ND
ND
>1659
3169
5623
Monkey
NHRI VRDC (Vero)
Taiwan CDC (RD)
Results from RD cell were provided by Taiwan CDC
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
Lot.
07EV01
4X
5.84
8X
5.71
16X
4X
Lot.
07EV02
8X
16X
4X
Lot.
07EV03
Concentration
of VP2(ug/ml)
Mean
(ug/ml)
2.5
6.01
18.59
6.48
4.23
4.07
3
OD 450nm
Inactivated EV71
(dilution fold)
3.5
Total
protein
(ug/ml)
2
1.5
1
4.46
24.04
y = 0.7389Ln(x) + 1.3906
R2 = 0.9922
0.5
0
5.08
0.1
1
10.08
VP2 concentration (ug/ml)
8X
16X
9.51
9.82
9.81
23.86
10
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 assay
Test 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
SDS-PAGE (silver stain)
Bulk
M
Western blot
P
Bulk
M
P
188
188
98
98
62
62
49
49
38
38
28
28
1° Ab: Mab979 (1:1000)
17
14
17
14
2° Ab: Goat- anti-mouse
6
3
(1:5000)
6
3
P: positive control
※ NuPAGE Bis-Tris Mini Gels 4-12% (1.5mm), loading 0.5ug total protein per well
Table 1. Summary of predicted and observed molecular weights (MW) of EV71/E59 viral proteins and incompletely processed viral
polypeptides
Residues
Predicted MW a (kDa)
1-69
70-323
324-565
69
254
242
7.49
27.78
26.52
8
28
27
566-862
297
32.73
36
Incompletely processed viral polypeptides
VP0 (VP4-VP2)
1-323
323
35.27
38
VP3-VP1
519
59.25
60
EV71 antigens
Amino acid sequences
Viral antigen in virion
VP4
VP2
VP3
VP1
324-862
Observed MW b (kDa)
Viral antigens cross-linked by formalin inactivation based on VP2 recognized by MAB979
VP4-VP2
1-323
323
35.27
VP4-VP2-VP4
42.76
VP2-VP2
55.56
38
45
58
VP2-VP4-VP2
VP4-VP2-VP3-VP1
64
98
1-862
862
63.05
94.52
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
EV71 virus
Virus titer (TCID50/mL)
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
0
10
20
30
Sucrose gradient (%)
40
50
60
Transmission electron microscopy
Serum-Free culture, Formaldehyde solution (1:4000)
150K X
Empty virus particle
Full virus particle
Cooperation with NHRI Nanomedicine center
Mass spectrometry analysis
polyprotein [Human enterovirus 71] major capsid protein VP1
EV71_A1_2 11 (0.306)
100
1: TOF LD+
1.24e4
1072.6973
1 GDRVADVIES SIGDSVRRAL TQALPAPTGQ NTQVSSHRLD TGEVPALQAA
51 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%
2603.4385
1074.6440
%
2604.4617
2078.2000
2602.4504
2077.1914 2079.1770
1075.6407
1570.7391
2605.4500
2173.1653
1571.7256
2174.1650
1811.0808
2606.4736
1058.6251
1042.6378
926.5388
0
800
2063.0342
1572.7396
1076.6379
2062.0291
2018.7096
1778.0740
1283.8030 1532.8632
1573.7268
1813.0527
2979.5542
2175.1643
2187.1782
2936.0032
2287.3552
2288.3801
m/z
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
Neutralization assay (3rd week)
Serum-Free culture, Formaldehyde solution (1:4000)
1600
Neutralization titer
1400
1200
1000
800
0.5ug/dose
Empty virus particle
2.5ug/dose
Empty virus particle
0.5ug/dose
Full virus particle
600
400
2.5ug/dose
Full virus particle
200
AlPO4
0
VP2 is responsible for SCARB2 binding &
X-neutralization
s
s
X-Y X-Y
X-Y
X-Y
Vero
X-Y
s
EV71 & CoxA16 receptor
s s s
s
s
s
s s RD
X-Y
SCARB2
X-Y
s
Receptors identified for EV71 virus,
Nat. Med. 15 (2009): 728-729, 794-797, 798-801.
s
s
s
3T3
s
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!!