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: pelechong@nhri.org.tw

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!!