, people's republic of china · co., ltd. 6b-01, tian-xia industrial park, ma jia long,...

Approval No. 2010L05008 (SFDA) CONFIDENTIALCONFIDENTIALCONFIDENTIALCONFIDENTIAL Protocol Date: 20th, December 2011

Sponsor: Beijing Vigoo Biological Co., Ltd. Final protocol Version 1.0: December. 2011

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ProtocolProtocolProtocolProtocol CoverCoverCoverCover PagePagePagePage

ProtocolProtocolProtocolProtocol Title:Title:Title:Title: A multicentre, randomized, double-blind,

placebo-controlled, phase 3 trial to assess the efficacy,

safety, immunogenicity, antibody persistence, and

correlates of immunity for an inactivated enterovirus

type 71 vaccines (Vero cell) in Chinese healthy children,

aged 6 to 35 months.

ProtocolProtocolProtocolProtocol Number:Number:Number:Number: JSVCT010

ProtocolProtocolProtocolProtocol Date:Date:Date:Date: 20th December, 2011

Version:Version:Version:Version: Version 1.0 (final)

Phase:Phase:Phase:Phase: Phase 3

Sponsor:Sponsor:Sponsor:Sponsor: Beijing Vigoo Biological Co., Ltd.

No.4 Sanjianfang Nanli, Chaoyang District, Beijing

100024, People's Republic of China

ProtocolProtocolProtocolProtocol AuthorsAuthorsAuthorsAuthors Feng-Cai Zhu MSc, Zheng-Lun Liang PhD., Xin-Liang

Shen MSc

ConfidentialityConfidentialityConfidentialityConfidentiality AgreementAgreementAgreementAgreement

This document contains confidential information belonging to Beijing Vigoo Biological Co., Ltd. Except as

otherwise agreed to in writing, by accepting or reviewing this document, you agree to hold this information in

confidence and not copy or disclose it to others (except where required by applicable law) or use it for unauthorized

purposes. In the event of any actual or suspected breach of this obligation, Beijing Vigoo Biological Co., Ltd. must

be promptly notified.



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DocumentDocumentDocumentDocument HistoryHistoryHistoryHistory

DocumentDocumentDocumentDocument VersionVersionVersionVersion DateDateDateDate SummarySummarySummarySummary ofofofof ChangesChangesChangesChanges

Version1.0 20th December, 2011 N/A



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CLINICALCLINICALCLINICALCLINICAL TRIALTRIALTRIALTRIAL CONTACTCONTACTCONTACTCONTACT LISTLISTLISTLIST

SponsorSponsorSponsorSponsor Beijing Vigoo Biological Co., Ltd.

No. 4 Sanjianfang Nanli, Chaoyang District, Beijing 100024,

People's Republic of China

Tel:

Fax:

E-mail:

+86-10-65618923, 86-10-65656778

+86-10-65618923, 86-10-65656778

[email protected]，[email protected]

PrincipalPrincipalPrincipalPrincipal InvestigatorInvestigatorInvestigatorInvestigator Feng-Cai Zhu

Jiangsu Provincial Center for Diseases Control and

Prevention

No. 172, Jiangsu Road,

Nanjing 210009, Jiangsu Province,


E-mail:

Tel:

Fax:

[email protected]

+86-25-83759418

+86-25-83759409

ContractContractContractContract ResearchResearchResearchResearch

OrganizationOrganizationOrganizationOrganization

Yuan Yong

Shenzhen YingHeYuan Medical Technology Development

Co., Ltd.

6B-01, Tian-xia Industrial Park, Ma Jia Long, Yi-yuan Road,

Nan-shan District , Shenzhen 518052,


E-mail:

Tel:

Fax:

Mob. Phone:

[email protected]

+86-755-61373860

+86-755-61373008ext602

+86-13382030658

SeriousSeriousSeriousSerious AdverseAdverseAdverseAdverse EventEventEventEvent Qing-Hua Chen

mailto:[email protected]




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ContactContactContactContact SponsorSponsorSponsorSponsor:::: Beijing Vigoo Biological Co., Ltd.

No. 4 Sanjianfang Nanli, Chaoyang District, Beijing 100024,


E-mail:

Tel:

Fax:

Mob. Phone:

[email protected]

+86-10-65656778

+86-10-65656778

+86-13810946729

ClinicalClinicalClinicalClinical LaboratoryLaboratoryLaboratoryLaboratory 1:1:1:1: Zheng-Lun Liang

National Institute for Food and Drug Control

No.2,Tiantanxili, Beijing 100050,


E-mail:

Tel:

Fax:

Mob. Phone:

[email protected]

+86 -10-67095356

+86-10- 67095356

+86-13621255863

ClinicalClinicalClinicalClinical LaboratoryLaboratoryLaboratoryLaboratory 2222

(local(local(local(local laboratory)laboratory)laboratory)laboratory)

Xiao-Xiang Lv

Jiangsu Mole Bioscience Co., Ltd.

R19 Building, No.1 Yaocheng Avenue, Taizhou 225300,

Jiangsu province,


E-mail:

Tel:

Fax:

Mob. Phone:

[email protected]

+86-523-82205066

+86-523-82205066

+86-15152960668




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DSMCDSMCDSMCDSMC Xing-Wang Li

Title: Chief Physician

Ditan Hospital Attached to the Capital Medical University

Specialty: Clinical Infectious Diseases

Yuan-Wen Ze

Title: Research Fellow

Beijing Institute of Biological Products

Specialty: Epidemiology and Statistics

Zhao-Ming Wen


Beijing Union Medical College Hospital

Specialty: Allergic Reaction

Shu-Qiao Wang

Title: Research Fellow

Shanghai Institute of Biological Products

Specialty: Epidemiology and Statistics

Yao-Hua Liu


Beijing Union Medical College Hospital

Specialty: Dermatologist

Lan Hu


Children’s Hospital Attached to the Capital Institute of

Paediatrics

Specialty: Paediatrics

Hua-Qing Wang



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Center for Immunization Programme of the Chinese Center

for Disease Control and Provention

Specialty:Expanded Programme On Immunization

Gang Liu


Beijing Children’s Hospital Attached to the Capital Medical

University

Specialty: Pediatric Infectious

Zhong-Shu Zhou


Sino-Japanese Freind Hospital

Specialty: Pediatric



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PROTOCOLPROTOCOLPROTOCOLPROTOCOL SUMMARYSUMMARYSUMMARYSUMMARY

Sponsor:Sponsor:Sponsor:Sponsor: NameNameNameName ofofofof StudyStudyStudyStudy VaccineVaccineVaccineVaccine:::: ActiveActiveActiveActive Ingredient(s):Ingredient(s):Ingredient(s):Ingredient(s):

Beijing Vigoo

Biological Co., Ltd.

Inactivated Enterovirus type

71 Vaccine (Vero cell)

(EV71 vaccine )

Each dose contains 320 U antigen of the

inactivated whole EV71 virus with 0.18mg

alum-adjuvant.

Placebo:Placebo:Placebo:Placebo:

Placebo contains 0 U antigen and 0.18mg alum-adjuvant.

TitleTitleTitleTitle ofofofof thethethethe trialtrialtrialtrial::::

A multicentre, randomized, double-blind, placebo-controlled, phase 3 trial to assess the efficacy,

safety, immunogenicity, antibody persistence, and correlates of immunity type 71 vaccine (Vero cell)

in Chinese healthy children, aged 6 to 35 months.

TrialTrialTrialTrial CentCentCentCentrerereressss andandandand InvestigatorsInvestigatorsInvestigatorsInvestigators::::

Site1: Donghai County, in Jiangsu Province

Heng-Ming Ge

Tel：86-518-87775901; e-mail: [email protected]

Site2: Pizhou County, in Jiangsu Province

Lin Liu


Site3: Baoying County, in Jiangsu Province

Xin-hua Shen


Site4: Chaoyang District, in Beijing City

Nian-min Shi


TrialTrialTrialTrial Duration:Duration:Duration:Duration: TrialTrialTrialTrial Phase:Phase:Phase:Phase:

14 months Phase 3



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Objectives:Objectives:Objectives:Objectives:

PrimaryPrimaryPrimaryPrimary ObjectiveObjectiveObjectiveObjectivessss::::

To evaluate the efficacy of EV71 vaccine in healthy children (aged 6-35 months) against

EV71-associated HFMD and EV71-associated disease (defined in section 7.4.5).

SecondarySecondarySecondarySecondary ObjectiveObjectiveObjectiveObjectivessss::::


hospitalized/ severe EV71-associated diseases.

To evaluate the safety of EV71 vaccine in children (aged 6-35 months).

To evaluate the immunogenicity of EV71 vaccine in children (aged 6-35 months).

To evaluate the persistence of antibody elicited by EV71 vaccine in children (aged 6-35 months).

To evaluate the lot-to-lot consistency of EV71 vaccine.

ExploratoryExploratoryExploratoryExploratory Objective:Objective:Objective:Objective:

To explore the immunological correlate of immunity against EV71-associated diseases on day 56.

PrimaryPrimaryPrimaryPrimary Endpoints:Endpoints:Endpoints:Endpoints:

The EV71-associated HFMDs/ disease happened during the surveillance period from day 56 up to

month 14. The cases should be finally confirmed by the DSMC on the basis of lab results released by

NIFDC, epidemiological data and clinical data.

SecondarySecondarySecondarySecondary Endpoints:Endpoints:Endpoints:Endpoints:

1. Frequency of adverse events within 28 days after each vaccination.

2. Frequency of solicited adverse reactions within 7 days after each vaccination.

3. Occurrence of serious adverse events during the whole trial period.

4. Geometric mean titers (GMTs), seroconversion rates (proportion of participants with

pre-vaccination titer < 1:8 and post-vaccination titer≥1:32, or pre-vaccination titer≥1:8 and at



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least four-fold increase post-vaccination), geometric mean fold increase (GMFI) and seropositive

rates (proportion of participants with NT titer against EV71≥1:8) of EV71 neutralizing antibody

in participants measured at day 56 (28 days after two-dose vaccinations).

5. EV71 neutralizing antibody persistence as measured by GMTs, GMFIs, seropositive rates

and seroconversion rates at month 8 (six months after day 56) and month 14 (twelve months

after day 56) in subjects.

6. Lot-to-lot consistency as measured by GMTs of EV71 neutralizing antibody at day 56 in

subjects.

ExploratoryExploratoryExploratoryExploratory Endpoint:Endpoint:Endpoint:Endpoint:

EV71 neutralizing antibody titer on day 56 in the subjects who reported EV71-associated disease

during the surveillance period and the titer in their matched controls.

MethodologyMethodologyMethodologyMethodology

This clinical trial is a randomized, double-blind (subjects, guardians, investigators),

placebo-controlled phase 3 trial to assess the efficacy, safety, immunogenicity and immune

persistence and lot-to-lot consistency of a Vero cell based alum-adjuvanted inactivated EV71 vaccine

in children (aged 6-35 months). The immunological correlate of protection against EV71-associated

diseases is also explored. All subjects will receive two doses of the vaccine or placebo on day 0 and

day 28.

The planned sample size is 10,000. Three batches of EV71 vaccine and one batch of placebo will be

randomized in a ratio of 1:1:1:3. Subjects will be randomly assigned to receive EV71 vaccine or

placebo in a ratio of 1:1, i.e. 5,000 subjects in each treatment group. The injection will be given

intramuscularly in anterolateral side of thigh in the infant subjects (aged 6-11 months), and in the

deltoid muscle in children subjects (aged 12-35 months) at day 0 and day 28.

The incidence density will be calculated and compared between the vaccine group and placebo group

based on the case number in all eligible subjects against EV71-associated HFMD and

EV71-associated disease.



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The frequency of the AEs within 7 and 28 days of each vaccination and SAEs during the whole trial

duration will be calculated and compared between vaccine group and placebo group based on the

data of all eligible subjects receiving at least one injection to evaluate the safety of EV71 vaccine.

The seropositive rate, seroconversion rate, GMT and GMFI will be calculated and compared between

the vaccine group and placebo group based on the NT titers on day 0 and day 56 of the eligible

subjects in immunogenicity subgroup (1200 subjects recruited from four townships which were

selected from the three trial centres in Jiangsu Province on the basis of randomized cluster sampling)

out of the whole study cohort to evaluate the immunogenicity of EV71 vaccine.

The seropositive rate, seroconversion rate, GMT and GMFI will be calculated and compared between

the vaccine group and placebo group based on the NT titers on months 8 and 14 of the eligible

subjects in immunogenicity subgroup to evaluate the immune persistence of EV71vaccine.

The GMTs of EV71 neutralizing antibody measured at day 56 will be calculated and compared

between the different lots of EV71 vaccine based on the data of the eligible subjects in

immunogenicity subgroup to evaluate the lot-to-lot consistency of EV71 vaccine.

The sensitivity, specificity and Youden index will be calculated based on the NT titers on day 56 of

the subjects with EV71-associated diseases in both vaccine group and placebo group and their

matched controls in a ratio of 1:5 on the residence and age to evaluate the immunological correlate of

protection against EV71-associated diseases.

Subjects should attend a total of nine visits:

Visit 1 (Day 0), informed consent, eligible assessment, blood sample taking before vaccination, first

vaccination, and 30 min safety observation after injection;

Visit 2 (Day 7), assessment of safety with respect to adverse reactions/ events;

Visit 3 (Day 28), assessment of safety, eligible assessment, second dose, and 30 min safety

observation after injection;

Visit 4 (Day 35), assessment of safety with respect to adverse reactions/ events;

Visit 5 (Day 56), assessment of safety, blood sample taking for immunology analysis;



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Visit 6 (Month 5), residence of subjects (loss to follow-up, migrate out or quit) , SAEs;

Visit 7 (Month 8), residence of subjects (loss to follow-up, migrate out or quit), SAEs; blood sample

taking from participants in the immunogenicity subgroup;

Visit 8 (Month 11), residence of subjects (loss to follow-up, migrate out or quit), SAEs;

Visit 9 (Month 14), residence of subjects (loss to follow-up, migrate out or quit), SAEs, the final

assessment, blood sample taking in the immunogenicity subgroup of subjects.

TimeTimeTimeTime PointsPointsPointsPoints forforforfor BiologicalBiologicalBiologicalBiological SpecimenSpecimenSpecimenSpecimen Collection:Collection:Collection:Collection:

1. For the subjects in the immunogenicity subgroup, blood sample will be collected on day 0 (visit 1),

56 (visit 5), month 8 (visit 7) and month 14 (visit 9).

2. For all subjects, blood sample will be collected on day 0 (visit 1) for baseline antibody analysis

and day 56 (visit 5).

3. For the screening cases (defined in section 7.4.1), a throat swab and an anal swab will be collected

within 24h after the discovery of the cases for the real-time PCR in the surveillance period.

4. For the suspected enterovirus-associated cases, secondary throat and anal swabs will be collected

within 72h after the discovery of the cases in the surveillance period. A blood sample and a stool

sample will be collected at acute phase (within one week after onset) and recovery phase (3 weeks

after onset), respectively; throat and anal swabs will be collected every 3 days before the symptoms

disappear, and then every 7 days until the negative results from two consecutive sampling.

TimeTimeTimeTime PointsPointsPointsPoints forforforfor SafetySafetySafetySafety Assessment:Assessment:Assessment:Assessment:

1. Temperature and solicited adverse events will be recorded by subjects’ guardians on the diary

cards within 7 days after each vaccination.

2. Any unsolicited adverse events will be recorded by subjects’ guardians on the diary cards within

28 days following each vaccination.

3. Any serious adverse events (SAEs) happened during the whole study period (duration from day 0

to day 56 and duration from day 56 to month 14) will be reported, recorded and accessed.



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NumberNumberNumberNumber ofofofof SubjectsSubjectsSubjectsSubjects (Planned):(Planned):(Planned):(Planned):

The planned total number of subjects is 10000 with 5000 per treatment group. The subjects will

randomly receive alum-adjuvanted EV71 vaccine or placebo (alum-adjuvant).

InclusionInclusionInclusionInclusion CriteriaCriteriaCriteriaCriteria

1. Healthy subjects aged from 6 to 35 months.

2. General good health as established by medical history and physical examination.

3. The subjects' guardians are able to understand and sign the informed consent.

4. Had never received the vaccine against EV71.

5. Subjects’ guardians who allow to comply with the requirements of the protocol.

6. Able to attend all the site visits.

7. Subjects with axillary temperature ≤37.0°C upon recruitment.

ExclusionExclusionExclusionExclusion CriteriaCriteriaCriteriaCriteria forforforfor thethethethe FirstFirstFirstFirst DoseDoseDoseDose

1. Subject who has a medical history of hand, foot and mouth disease.

2. Less than 37 weeks gestation.

3. Subject that has a medical history of any of the following: allergic history of any

vaccination or drugs, or allergic to any ingredient of the inactivated Enterovirus type 71 vaccine

(Vero cell).

4. Family history of congenital or hereditary immunodeficiency, seizures or progressive

neurological disease.

5. Severe malnutrition or dysgenopathy.

6. Major congenital defects or serious chronic illness, including perinatal brain damage,

asplenia, functional asplenia, or splenic excision.

7. Autoimmune disease.

8. Bleeding disorder diagnosed by a doctor or significant bruising or bleeding difficulties

with IM injections or blood draws.



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9. Any acute infections in last 7 days.

10. Any prior administration of immunodepressant or corticosteroids in last 6 months.

11. Any prior administration of blood products in last 3 months.

12. Any prior administration of other research medicines in last 1 month.

13. Any prior administration of attenuated vaccine in last 15 days.

14. Any prior administration of inactivated vaccine in last 7 days.

15. Under the anti - TB prevention or therapy; history of asthma, angioneurotic edema,

diabetes or malignant tumour; thyroidectomy, or thyroid disease in last 12 months.

16. Any condition that in the opinion of the investigators may interfere with the evaluation of study

objectives.

EEEEliminationliminationliminationlimination CriteriaCriteriaCriteriaCriteria forforforfor thethethethe secondsecondsecondsecond dosedosedosedose

Subjects will not be eligible for the second dose if any of following happened after first dose.

However, they can continue other process of study according to the opinion of investigators.

1. Any serious adverse reactions related to vaccination within 7 days after the first dose.

2. Hypersensitivity reactions after vaccination (including urticarial/rashes that occur within 30 min

after vaccination).

3. Anaphylaxis after vaccination.

4. Any confirmed or suspected autoimmune disease or immune deficiency diseases, including human

immunodeficiency virus (HIV) infection.

5. Any condition that in the opinion of the investigator, or IRB.

DurationDurationDurationDuration ofofofof Study:Study:Study:Study:

Duration of the study is 14 months.

StatementStatementStatementStatement forforforfor furtherfurtherfurtherfurther followingfollowingfollowingfollowing studies:studies:studies:studies:

Subjects may be asked to participate in further following studies after complete this study. However,

before any further following studies initiated permission must be obtained from Beijing Vigoo

Biological Co., Ltd and institutional review board.



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TABLETABLETABLETABLEOFOFOFOF CONTENTSCONTENTSCONTENTSCONTENTS

PROTOCOLPROTOCOLPROTOCOLPROTOCOL SUMMARYSUMMARYSUMMARYSUMMARY------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7777

ABBREVIATIONSABBREVIATIONSABBREVIATIONSABBREVIATIONS---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 18181818

1.1.1.1. INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 202020201.1 Indication-------------------------------------------------------------------------------------------------- 201.2 Background and Rationale------------------------------------------------------------------------------ 201.2.1 Preclinical investigation with the EV71 Vaccine----------------------------------------------------211.2.2 Phase 1 clinical trial with the EV71 Vaccine---------------------------------------------------------211.2.3 Phase 2 clinical trial with the EV71 Vaccine---------------------------------------------------------221.2.4 Determination of dosage and regimen for phase 3 clinical trial----------------------------------- 231.2.5 Risk-benefit Assessment-------------------------------------------------------------------------------- 231.3 Investigator------------------------------------------------------------------------------------------------24

2.2.2.2. STUDYSTUDYSTUDYSTUDYOBJECTIVESOBJECTIVESOBJECTIVESOBJECTIVES ANDANDANDAND ENDPOINTSENDPOINTSENDPOINTSENDPOINTS------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 242424242.1 Primary objectives----------------------------------------------------------------------------------------242.2 Endpoint for the primary objective-------------------------------------------------------------------- 242.3 Secondary objectives------------------------------------------------------------------------------------ 242.4 Endpoints for secondary objectives--------------------------------------------------------------------252.5 Exploratory objective------------------------------------------------------------------------------------ 252.6 Endpoint for exploratory objective-------------------------------------------------------------------- 25

3.3.3.3. STUDYSTUDYSTUDYSTUDY DESIGNDESIGNDESIGNDESIGN----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------252525253.1 Description------------------------------------------------------------------------------------------------ 253.2 Duration of Study---------------------------------------------------------------------------------------- 263.3 Sample size------------------------------------------------------------------------------------------------263.4 Selection of study centres------------------------------------------------------------------------------- 263.5 Immunogenicity and immune persistence subgroup------------------------------------------------ 27

4.4.4.4. SUBJECTSUBJECTSUBJECTSUBJECT SELECTIONSELECTIONSELECTIONSELECTION------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 272727274.1 Inclusion Criteria----------------------------------------------------------------------------------------- 274.2 Exclusion Criteria for the First Dose------------------------------------------------------------------ 284.3 Elimination Criteria for the second dose--------------------------------------------------------------284.4 Contraindications to the second vaccination--------------------------------------------------------- 294.5 Withdraw from the study--------------------------------------------------------------------------------29

5.5.5.5. INVESTIGATIONALINVESTIGATIONALINVESTIGATIONALINVESTIGATIONAL PRODUCTSPRODUCTSPRODUCTSPRODUCTS ANDANDANDAND ADMINISTRATIONADMINISTRATIONADMINISTRATIONADMINISTRATION---------------------------------------------------------------------------------------------------------------------------- 303030305.1 Investigational Product Supplies----------------------------------------------------------------------- 305.1.1 Study vaccine and placebo------------------------------------------------------------------------------ 305.1.2 Dosage and administration------------------------------------------------------------------------------305.1.3 Storage and Dispensing of Study Medication--------------------------------------------------------31



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5.2 Treatment allocation and randomization--------------------------------------------------------------325.2.1 Randomization of study vaccines and placebo-------------------------------------------------------325.2.2 Treatment allocation------------------------------------------------------------------------------------- 325.3 Method of unblinding and breaking the study blind------------------------------------------------ 325.4 Packaging and Labeling--------------------------------------------------------------------------------- 335.5 Back-up vaccine doses-----------------------------------------------------------------------------------345.6 Investigational product accountability---------------------------------------------------------------- 345.7 Concomitant medication/treatment-------------------------------------------------------------------- 345.8 Compliance Monitoring--------------------------------------------------------------------------------- 36

6.6.6.6. CONDUCTCONDUCTCONDUCTCONDUCT OFOFOFOF THETHETHETHE STUDYSTUDYSTUDYSTUDY------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------373737376.1 Study Procedures Description-------------------------------------------------------------------------- 376.1.1 Outline of study procedures-----------------------------------------------------------------------------376.1.2 Informed consent----------------------------------------------------------------------------------------- 396.1.3 Eligibility assessment------------------------------------------------------------------------------------406.1.4 Safety follow-up------------------------------------------------------------------------------------------416.1.5 EV71-Associated Diseases active surveillance------------------------------------------------------ 416.1.6 Detailed description of study stages/visits------------------------------------------------------------42

7.7.7.7. SURVEILLANCESURVEILLANCESURVEILLANCESURVEILLANCE OFOFOFOF THETHETHETHE EV71-ASSOCIATEDEV71-ASSOCIATEDEV71-ASSOCIATEDEV71-ASSOCIATED DISEASESDISEASESDISEASESDISEASES------------------------------------------------------------------------------------------------------------------------------------ 444444447.1 The flow chart of the EV71-associated diseases active surveillance----------------------------- 447.2 Active surveillance---------------------------------------------------------------------------------------467.3 Duration of active surveillance Period---------------------------------------------------------------- 467.4 Case definition-------------------------------------------------------------------------------------------- 467.4.1 Screening possible cases-------------------------------------------------------------------------------- 467.4.2 Clinical classification------------------------------------------------------------------------------------ 467.4.3 Suspected enterovirus-associated diseases----------------------------------------------------------- 477.4.4 Laboratory confirmed enterovirus-associated diseases--------------------------------------------- 477.4.5 Confirmed EV71-associated diseases----------------------------------------------------------------- 487.5 Set up of the active surveillance system-------------------------------------------------------------- 497.6 Implementation of the EV71-associated disease active surveillance----------------------------- 497.6.1 Methods for discovery of screening cases------------------------------------------------------------ 497.6.2 Lab assay for suspected enterovirus-associated diseases------------------------------------------- 507.6.3 Further laboratory analysis for confirmed or suspected EV71-associated cases---------------- 517.6.4 Follow-up for laboratory confirmed EV71-associated cases---------------------------------------517.6.5 Finally confirming of EV71-associated cases--------------------------------------------------------527.7 Clinical specimens collection and lab assay methods-----------------------------------------------537.7.1 Clinical specimens collection---------------------------------------------------------------------------537.7.2 Real-time RT-PCR assay--------------------------------------------------------------------------------547.7.3 Conventional RT-PCR-----------------------------------------------------------------------------------547.7.4 Virus Isolation and Identify-----------------------------------------------------------------------------547.7.5 IgM detection--------------------------------------------------------------------------------------------- 55



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8.8.8.8. ASSESSMENTSASSESSMENTSASSESSMENTSASSESSMENTS--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------555555558.1 Safety Assessments-------------------------------------------------------------------------------------- 558.1.1 Grading for adverse events------------------------------------------------------------------------------568.2 Immunogenicity, immune persistence and lot-to-lot consistency assessments------------------608.3 Protective efficacy against EV71-associated diseases assessments-------------------------------618.4 Assessment of the immunological correlate of protection----------------------------------------- 61

9.9.9.9. ADVERSEADVERSEADVERSEADVERSE EVENT/REACTIONEVENT/REACTIONEVENT/REACTIONEVENT/REACTION REPORTINGREPORTINGREPORTINGREPORTING------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 626262629.1 Adverse event and adverse reaction definition-------------------------------------------------------629.2 Recording of Adverse Events---------------------------------------------------------------------------629.3 Serious adverse event/reaction (SAE) definition---------------------------------------------------- 639.3.1 Reporting SAEs-------------------------------------------------------------------------------------------649.3.2 Expedited reporting of SUSARs to regulatory authorities and investigators-------------------- 659.4 Follow-up of adverse events---------------------------------------------------------------------------- 659.5 Causality Assessment------------------------------------------------------------------------------------659.6 Withdrawal Due to Adverse Events (See section on Subject Withdrawal)---------------------- 669.7 Eliciting Adverse Event Information------------------------------------------------------------------ 669.8 Reporting Requirements---------------------------------------------------------------------------------669.9 Sponsor Reporting Requirements to Regulatory Authorities-------------------------------------- 66

10.10.10.10. DATADATADATADATA COLLECTIONCOLLECTIONCOLLECTIONCOLLECTION ANDANDANDANDMANAGEMENTMANAGEMENTMANAGEMENTMANAGEMENT---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 6767676710.1 Source Documents and Source Data-------------------------------------------------------------------6710.2 Data Management---------------------------------------------------------------------------------------- 6810.2.1 Clinical Data Management---------------------------------------------------------------------------6810.2.2 Surveillance Data Management--------------------------------------------------------------------- 6810.2.3 SAE Data Management-------------------------------------------------------------------------------6910.3 Clinical Investigator’s Brochure----------------------------------------------------------------------- 6910.4 Archiving-------------------------------------------------------------------------------------------------- 69

11.11.11.11. QUARLITYQUARLITYQUARLITYQUARLITY CONTROLCONTROLCONTROLCONTROLANDANDANDAND QUARLITYQUARLITYQUARLITYQUARLITY ASSURANCEASSURANCEASSURANCEASSURANCE---------------------------------------------------------------------------------------------------------------------------------------- 7070707011.1 Monitoring-------------------------------------------------------------------------------------------------7011.2 Audits------------------------------------------------------------------------------------------------------ 71

12.12.12.12. STATISTICALSTATISTICALSTATISTICALSTATISTICAL CONSIDERATIONSCONSIDERATIONSCONSIDERATIONSCONSIDERATIONS--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------7272727212.1 Sample size calculation----------------------------------------------------------------------------------7312.1.1 Overall sample size calculation--------------------------------------------------------------------- 7312.1.2 Lot-to-lot consistency analysis sample-size calculation----------------------------------------- 7412.2 Study cohorts to be evaluated-------------------------------------------------------------------------- 7412.2.1 Intention-To-Treat (ITT) cohort for safety analysis----------------------------------------------7412.2.2 Modified intention-to-treat (ITT) cohort and According-To-Protocol (ATP) cohort forefficacy 7512.2.3 According-To-Protocol (ATP) cohort for analysis of immunogenicity----------------------- 7512.2.4 According-To-Protocol (ATP) cohort for analysis of lot consistency------------------------- 76



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12.2.5 Cohort for analysis of the immunological correlate of protection------------------------------7612.3 Derived and transformed data-------------------------------------------------------------------------- 7612.4 Description of analyses---------------------------------------------------------------------------------- 77

13.13.13.13. ETHICALETHICALETHICALETHICAL ANDANDANDAND LEGALLEGALLEGALLEGAL ISSUESISSUESISSUESISSUES------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 7878787813.1 Guideline-------------------------------------------------------------------------------------------------- 7813.2 Institutional Review Board----------------------------------------------------------------------------- 7813.3 Responsibilities of the Investigator-------------------------------------------------------------------- 7913.4 Ethical Conduct of the Study--------------------------------------------------------------------------- 8013.5 Protocol Amendments and Administrative changes-------------------------------------------------8013.6 Confidentiality of Data and Access to Subject Records-------------------------------------------- 81

14.14.14.14. FINANCIALFINANCIALFINANCIALFINANCIAL CONTRACTCONTRACTCONTRACTCONTRACT ANDANDANDAND INSURANCEINSURANCEINSURANCEINSURANCE COVERAGECOVERAGECOVERAGECOVERAGE----------------------------------------------------------------------------------------------------------------------------81818181

15.15.15.15. EARLYEARLYEARLYEARLY TERMINATIONTERMINATIONTERMINATIONTERMINATIONOFOFOFOF TRIALTRIALTRIALTRIAL---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 81818181

16.16.16.16. PUBLICATIONPUBLICATIONPUBLICATIONPUBLICATIONOFOFOFOF STUDYSTUDYSTUDYSTUDY RESULTSRESULTSRESULTSRESULTS---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 82828282

17.17.17.17. REFERENCESREFERENCESREFERENCESREFERENCES------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------82828282

18.18.18.18. SIGNATURESSIGNATURESSIGNATURESSIGNATURES----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------86868686



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ABBREVIATIONSABBREVIATIONSABBREVIATIONSABBREVIATIONS

AE

AEADSS

AFP

Adverse Event

Active EV71-Associated Diseases Surveillance System

Acute flaccid paralysis

AR Adverse Reaction

ATP Accroding to Protocol

CA16 Coxsackievirus A16

CDC Center for Disease Control and Prevention

CI

CISDCP

Confidence Interval

China Information System for Diseases Control and Prevention

CNS Central nervous system

CRF Case Report Form

CRO Contract Research Organization

CTA Clinical Trial Authorization

DSMC

°C

Data Safety Monitoring Committee

Degrees Celsius

ELISA Enzyme Linked Immunosorbent Assay

EPI Expanded Program on Immunization

EV71 Human Enterovirus type 71

EV71 vaccine Inactivated Enterovirus type 71 Vaccine (Vero cell)

ITT Intention To Treat

GCP Good Clinical Practice

GLP

GM

Good laboratory practice

Geometric Mean

GMT Geometric Mean Titer



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GMTR Geometric mean of individual titer ratio

GMFI Geometric Mean Fold Increase

HA Herpangina

HFMD Hand, foot and mouth diseases

ICF Informed Consent Form

ICH International Conference on Harmonization (Technical

Requirements for Registration of Pharmaceuticals for Human Use)

IRB Institutional Review Board

ITT Intent-to-treat

IEC

MCPENT

Ethics Committee

Microtiter Cytopathogenic effect neutralization assay

NIFDC

NRCMNS

NT

ORF

National Institutes for Food and Drug Control, People’s Republic of China

New Rural Cooperative Medical Network System

Neutralization antibody

Open reading frame

PCR Polymerase chain reaction

QA Quality Assurance

P.R. China People’s Republic of China

SFDA State Food and Drug Administration, China

SAE Serious Adverse Event

SAR Serious Adverse Reaction

SOP

VLP

Standard Operation Procedure

Virus-like particle

WHO World Health Organization



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1.1.1.1. INTRODUCTIONINTRODUCTIONINTRODUCTIONINTRODUCTION

1.11.11.11.1 IndicationIndicationIndicationIndication

Hand, foot, and mouth disease (HFMD) is a common viral illness in infants and children caused by

viruses that belong to the enterovirus genus of the picornavirus family. Although most HFMD cases

do not result in serious complications, outbreaks of HFMD caused by enterovirus 71 (EV71) can

result in a high rate of neurological complications2, including meningoencephalitis, pulmonary

complications, and death. Disease caused by EV71 has become a major emerging infectious disease

in Asia and the highly pathogenic potential of EV71 clearly requires the attention of world medical

community3-7.

Recently, the phase 2 clinical trial has completed in Jiangsu Province in China. The data from the

phase 2 study suggested that the adjuvant 320 U inactivated enterovirus type 71 vaccine (Vero cell)

could be the candidate for the phase 3 trials. The 320 U inactivated enterovirus type 71 vaccine had a

clinically acceptable safety and good immunogenicity for healthy Chinese children and infants on a 0,

28 day vaccination schedule. In order to provide critical evidence for the efficacy of the EV71

vaccine, a phase 3 clinical trial is warranted.

1.21.21.21.2 BackgroundBackgroundBackgroundBackground andandandand RationaleRationaleRationaleRationale

The development of the vaccine against EV71 is active and ongoing in Asian countries or regions

now. Several Studies have examined the effectiveness of inactivated viral vaccines against EV71 in

animal model8-23. A wide range of experimental vaccine against EV71 approaches have been studied

including heat-inactivated or formaldehyde-inactivated virion16, EV71virus-like particles (VLP),

VP1 recombinant protein17, VP1 DNA vaccine18, VP1 peptide-based vaccine targeting the

neutralizing domain19, bacterial or viral vector expressing VP122, and a Vero cell-adapted live

attenuated virus23. Furthermore, neutralizing antibodies against EV71 have been suggested as one of

the most important factors in prevention of the severe EV71 infection. Passive immunization with

convalescent sera can prevent EV71 infection19.



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1.2.11.2.11.2.11.2.1 PreclinicalPreclinicalPreclinicalPreclinical investigationinvestigationinvestigationinvestigation withwithwithwith thethethethe EV71EV71EV71EV71 VaccineVaccineVaccineVaccine

Beijing Vigoo Biological (Beijing, China) developed the inactivated EV71 vaccine, using EV71

strain FY7VP5/AH/CHN/2008 (subtype C4) as the seed virus (GenBank accession number

JX025561). The strain grown in Vero cells in Dulbecco’s modified Eagle’s medium without fetal

bovine serum. The culture was clarified by filtration and concentrated by ultrafiltration. The

concentrated suspension was purified by gel filtration chromatography followed by ion exchange

chromatography, and then inactivation by formaldehyde and adsorption on aluminum hydroxide. The

antigen content was measured by double-antibody sandwich ELISA. The EV71 antigen reference for

ELISA was from NIFDC.

The immunogenicity experiment in mice with a 0, 14 day vaccination schedule, showed that the titer

of neutralization antibody began to increase at the 2nd week post vaccination, peaking at the 4th

week post vaccination; the titer of neutralization antibody after 2-dose vaccination was significantly

higher than that of 1 dose.

Besides, the titer of neutralization antibody in mice vaccinated by the vaccine with adjuvant is

significantly higher than that without adjuvant and the titer of neutralization antibody is

dose-dependent.

The protective efficacy test in suckling mice showed that the vaccine induced high levels of

neutralizing antibodies, which can protect the suckling mice from lethal infection, and the protection

is neutralizing antibody dose-dependent.

The safe evaluation in mice, guinea pig, cynomolgus showed that the vaccine is safe when the dose

is 3200U, which is 5 times of maximum clinical dosage.

1.2.21.2.21.2.21.2.2 PhasePhasePhasePhase 1111 clinicalclinicalclinicalclinical trialtrialtrialtrial withwithwithwith thethethethe EV71EV71EV71EV71 VaccineVaccineVaccineVaccine

The phase 1 study of the Inactivated Enterovirus 71 Vaccine (Vero cell) was performed in Jiangsu

Province in China, in January, 2011.

Phase 1a was an open label, non-randomized safety study in healthy young adults (aged 16-22 years)

and older children (aged 6-15 years). Total number of subjects in phase 1a was 100 with 20 per dose

group. The 40 adults aged 16 to 22 years were firstly recruited, and sequentially received vaccine in



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a manner of dose escalation, beginning with lower dosage of 320U in the first 20 adults, and then

640U in the other 20 adults. Then, 60 older children aged from 6 to 15 years were recruited, and

sequentially received vaccine of 160 U, 320 U, 640 U in the same manner. There were 20

participants in each dosage group within each age strata.

Phase 1b was a randomized, double-blind (subject, guardians, and investigators), placebo-controlled

study in children (aged 13-60 months) and infants (aged 6-12 months). Total number of subjects in

phase 1b was 360 with 45 per dose group.

Results of the phase 1 study suggested that the Inactivated Enterovirus 71 Vaccine (Vero cell) had a

clinically acceptable safety for healthy young adults, children and infants aged from 6 month to 22

years. A single dose induced typical immune response in healthy young adults aged 16-22 years and

children aged 6-15 years. In children (aged 13-60 months) and infants (aged 6-12 months), two doses

could elicit sufficient immune response. This phenomenon may be associated with their low baseline

neutralizing antibody level. However, all the investigational vaccines used in Phase 1 were

alum-adjuvant, the comparison between the alum-adjuvant vaccines and adjuvant-free vaccines had

not been evaluated. Also, the cross-immunity against poliovirus of the EV71 vaccines had not been

studied in the Phase 1 trial 24.

1.2.31.2.31.2.31.2.3 PhasePhasePhasePhase 2222 clinicalclinicalclinicalclinical trialtrialtrialtrial withwithwithwith thethethethe EV71EV71EV71EV71 VaccineVaccineVaccineVaccine

The phase 2 clinical trial was completed in 2011. Data from phase 2 trial showed that the EV71

vaccine was well tolerated with no treatment-related serious adverse events. Adverse events were

mainly mild and transient. Infants who received the 640 U adjuvant vaccine had the highest GMTs

on day 56 (742.2 [95% CI 567.3–954.3]), followed by those who received the 320 U formulation

(497.9 [383.1–647.0]). For children, those who received the 320 U formulation had the highest

GMTs on day 56 (1383.2 [1037.3–1844.5]). Participants who received the vaccine had significantly

higher GMTs than did who received placebo (p<0.0001). For the subgroup of participants who were

seronegative at baseline, both infants and children who received the 640 U adjuvant vaccine had the

highest GMTs on day 56 (522.8 [403.9–676.6] in infants and 708.4 [524.1–956.6] in children),

followed by those who received the 320 U adjuvant vaccine (358.2 [280.5–456.5] in infants and



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498.0 [383.4–646.9] in children). 549 (45.8%) of 1200 participants (95 CI 42.9–48.6%) reported at

least one injection-site or systemic adverse reaction, but the incidence of adverse reactions did not

differ significantly between groups (p=0.36). The 640 U alum-adjuvant vaccine group had a

significantly higher incidence of induration than did the 640 U adjuvant-free group (p=0.001).

Taking immunogenicity, safety, and production capacity into account, the 320 U alum-adjuvant

formulation of the EV71 vaccine is probably the best possible formulation for phase 3 trials 25.

1.2.41.2.41.2.41.2.4 DeterminationDeterminationDeterminationDetermination ofofofof dosagedosagedosagedosage andandandand regimenregimenregimenregimen forforforfor phasephasephasephase 3333 clinicalclinicalclinicalclinical trialtrialtrialtrial

Based on the results from phase 1 and 2 studies, although the 640U adjuvanted formulation was

more immunogenic than the 320U formulation, taking safety, immunogenicity and product capacity

together into account, the 320U with two-dose regimen will be more suitable for being used in the

phase 3 study to evaluate the prophylactic efficacy of the EV71 vaccine in a larger population of

infant and children (aged 6-35 months).

1.2.51.2.51.2.51.2.5 Risk-benefitRisk-benefitRisk-benefitRisk-benefit AssessmentAssessmentAssessmentAssessment

Benefits:Benefits:Benefits:Benefits:

Subjects may be benefit from following three aspects:

― immunization against EV71-associated disease;

― pathogenic assay for free during the surveillance period, privilege medical care and covered

treatment fee for EV71-associated diseases cases;

― free vaccination of the EV71 vaccine for the subjects who will receive placebo by randomization

in this study after the approval for marketing of the EV71 vaccine.

Risks:Risks:Risks:Risks:

The EV71 vaccine is supposed to have a satisfying safety profile, according to the safety data from

phase 1 and phase 2 clinical trials. However, subjects may suffer from adverse reactions after

vaccination including pain at injection-site, fever, diarrhea, nausea and vomiting. Other common

reactions might include pyrexia, headache, dizziness, chills, malaise, fatigue, arthralgia, urticaria,

and exanthema.



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1.31.31.31.3 InvestigatorInvestigatorInvestigatorInvestigator

The study will be performed by:

Jiangsu Center for Disease Control and Prevention (CDC)

No. 172, Jiangsu Road, Nanjing 210009, Jiangsu Province

The Principal investigator is Professor Zhu, the vice director of Jiangsu Province Center for Disease

Control and Prevention.Professor Zhu is very experienced with vaccine clinical trials. At the Jiangsu

CDC, a considerable amount of vaccine clinical studies have been performed in the recent years.

Professor Zhu is also a recognized expert in China regarding HFMD and enterovirus infectious

diseases.

2.2.2.2. STUDYSTUDYSTUDYSTUDYOBJECTIVESOBJECTIVESOBJECTIVESOBJECTIVES ANDANDANDAND ENDPOINTSENDPOINTSENDPOINTSENDPOINTS

2.12.12.12.1 PrimaryPrimaryPrimaryPrimary objectivesobjectivesobjectivesobjectives


EV71-associated HFMD and EV71-associated disease.

2.22.22.22.2 EEEEndpointndpointndpointndpoint forforforfor thethethethe primaryprimaryprimaryprimary objectiveobjectiveobjectiveobjective

The EV71-associated HFMDs / disease happened during the surveillance period from day 56 up to

month 14. The cases should be finally confirmed by the DSMC on the basis of lab results (i.e. one

viral isolations positive and/or two successive positive in EV71-specific RNA by real time PCR

assay), epidemiological data and clinical data.

2.32.32.32.3 SecondarySecondarySecondarySecondary objectivesobjectivesobjectivesobjectives

To evaluate the efficacy of EV71 vaccine in healthy children (aged 6-35 months) against hospitalized

/severe EV71-associated diseases.

To evaluate the safety of EV71 vaccine in children (aged 6-35 months).

To evaluate the immunogenicity of EV71 vaccine in children (aged 6-35 months).



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To evaluate the persistence of antibody elicited by EV71 vaccine in children (aged 6-35 months).

To evaluate the lot-to-lot consistency of EV71 vaccine.

2.42.42.42.4 EndpointsEndpointsEndpointsEndpoints forforforfor secondarysecondarysecondarysecondary objectivesobjectivesobjectivesobjectives

1) Frequency of adverse reactions within 28 days after each vaccination.

2) Frequency of solicited adverse reactions within 7 days after each vaccination.

3) Occurrence of SAE during the whole trial period.

4) Geometric mean titers (GMTs), seroconversion rates, geometric mean fold increase

(GMFI) and seropositive rates of EV71 neutralizing antibody in participants measured at day 56.

5) EV71 neutralizing antibody persistence as measured by seropositive rate, GMTs, GMFIs

and seroconversion rates at month 8 (six months after day 56) and month 14 (twelve months

after day 56) in subjects.

6) Lot-to-lot consistency as measured by GMTs of EV71 neutralizing antibody at day 56 in

subjects.

2.52.52.52.5 ExploratoryExploratoryExploratoryExploratory objectiveobjectiveobjectiveobjective

To explore the immunological correlate of protection against EV71-associated diseases based on the

antibody titer on day 56.

2.62.62.62.6 EndpointEndpointEndpointEndpoint forforforfor exploratoryexploratoryexploratoryexploratory objectiveobjectiveobjectiveobjective

EV71 neutralizing antibody (NT) titer on day 56 in the subjects who reported EV71-associated

disease during the surveillance period and the titer in their matched controls.

3.3.3.3. STUDYSTUDYSTUDYSTUDY DESIGNDESIGNDESIGNDESIGN

3.13.13.13.1 DescriptionDescriptionDescriptionDescription

This clinical trial is a multicentre, randomized, double-blind, placebo-controlled, phase 3 trial to

assess the efficacy, safety, immunogenicity, antibody persistence, and correlates of immunity for an



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inactivated enterovirus type 71 vaccines (Vero cell) in 10,000 Chinese healthy children, aged 6 to 35.

Subjects were randomized in a 1:1 ratio to receive two dose of the EV71 vaccine (including three

different batches) or the placebo on day 0 and day 28.

The active surveillance on EV71-associated disease will commence since 56 days following the first

dose vaccination.

.

3.23.23.23.2 DurationDurationDurationDuration ofofofof StudyStudyStudyStudy

The study will be completed in 14 months after the start of the study. The participants’ completion of

12-month follow-up and at least 31 finally confirmed EV71-associated HFMDs will lead to the

closure of the study fields. At that time, sites will be closed and the institutional review

board/independent ethics committee (IRB/IEC) will be informed.

3.33.33.33.3 SampleSampleSampleSample sizesizesizesize

The overall sample size calculation is detailed described in section 12.1.1 of this protocol. A total of

10,000 subjects will be randomized in a 1:1 ratio to receive two doses of the EV71 vaccine (N=5,000)

or the placebo (N=5,000) on day 0 and day 28 (detailed information on sample size calculation see

section 12.1).

3.43.43.43.4 SelectionSelectionSelectionSelection ofofofof studystudystudystudy centcentcentcentrerereressss

The following factors were taken into account when selecting the study centres :1) having relative

high incidence of EV71-associated diseases; 2) sufficient subjects available for the recruitment; 3)

sufficient professionals available; 4) medical and health insurance available for subjects, Baoying

county, Pizhou county and Donghai County in Jiangsu Province were chosen as the study centres

according to epidemiological and pathogenic data of Jiangsu province collected from national

laboratory network and notifiable disease surveillance system from 2009 to 2011, and also Chaoyang

district in Beijing city is chosen as another study centre in considering of the higher population

movement in cities comparing to counties. The planned recruitment numbers of participants from

Donghai, Pizhou, Baoying and Chaoyang are 2800, 4400, 2000 and 800, respectively.



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A high vaccination coverage in the study centers may contribute to an obvious indirect effect through

reducing unvaccinated people’s exposure to EV71 virus which as a result, the efficacy obtained in

this study may underestimate the total effect of the vaccine in realistic setting. To reduce the indirect

effect as much as possible, a predefined proportion of vaccinated persons in every township should

not exceed 40 percent of the total number of the target children. Therefore, not more than 20% of the

target children or a much lower proportion of the general population will receive the EV71 vaccine,

which makes the indirect effect less significant and as a result, the direct effect as close to the total

effect as possible. (detailed information see section 12.1).

3.53.53.53.5 IIIImmunogenicitymmunogenicitymmunogenicitymmunogenicity andandandand immuneimmuneimmuneimmune persistencepersistencepersistencepersistence subgroupsubgroupsubgroupsubgroup

1,200 subjects will be recruited from four townships (Quyang township in Donghai county,

Zhancheng township in Pizhou county, Xi’anfeng township and Jinghe township in Baoying county).

The four townships were selected on the basis of randomized cluster sampling in townships from

three centres in Jiangsu Province with 400 subjects in each study centre to form the immunogenicity

subgroup. Blood samples will be collected from these subjects at day 0, day 56, month 8 and

month 14.

4.4.4.4. SUBJECTSUBJECTSUBJECTSUBJECT SELECTIONSELECTIONSELECTIONSELECTION

The following inclusion and exclusion criteria will be used to select the subjects for this study.

4.14.14.14.1 InclusionInclusionInclusionInclusion CriteriaCriteriaCriteriaCriteria

1. Healthy subjects aged from 6 to 35 months

2. General good health as established by medical history and physical examination

3. The subjects' guardians are able to understand and sign the informed consent

4. Had never received the vaccine against EV71

5. Subjects’ guardians who allow to comply with the requirements of the protocol

6. Able to attend all the site visits

7. Subjects with axillary temperature ≤37.0°C upon recruitment



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4.24.24.24.2 ExclusionExclusionExclusionExclusion CriteriaCriteriaCriteriaCriteria forforforfor thethethethe FirstFirstFirstFirst DoseDoseDoseDose

1. Subject who has a medical history of hand, foot and mouth disease.

2. Less than 37 weeks gestation.

3. Subject that has a medical history of any of the following: allergic history of any vaccination or

drugs, or allergic to any ingredient of the inactivated Enterovirus type 71 vaccine (Vero cell).

4. Family history of congenital or hereditary immunodeficiency, seizures or progressive

neurological disease.

5. Severe malnutrition or dysgenopathy.

6. Major congenital defects or serious chronic illness, including perinatal brain damage, asplenia,

functional asplenia, or splenic excision.

7. Autoimmune disease.

8. Bleeding disorder diagnosed by a doctor or significant bruising or bleeding difficulties with IM

injections or blood draws.

9. Any acute infections in last 7 days.

10. Any prior administration of immunodepressant or corticosteroids in last 6 months.

11. Any prior administration of blood products in last 3 months.

12. Any prior administration of other research medicines in last 1 month.

13. Any prior administration of attenuated vaccine in last 15 days.

14. Any prior administration of inactivated vaccine in last 7 days.

15. Under the anti - TB prevention or therapy; history of asthma, angioneurotic edema, diabetes or

malignant tumour; thyroidectomy, or thyroid disease in last 12 months.

16. Any condition that in the opinion of the investigators may interfere with the evaluation of study

objectives.

4.34.34.34.3 EliminationEliminationEliminationElimination CriteriaCriteriaCriteriaCriteria forforforfor thethethethe secondsecondsecondsecond dosedosedosedose

Subjects will not be eligible for the second dose if any of following happened after first dose.

However, they can continue other process of study according to the opinion of investigators.



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1. Any SAE related to the vaccination of the study vaccine.

2. Hypersensitivity reactions after vaccination (including urticarial/rashes that occur within 30 min

after vaccination).

3. Anaphylaxis after vaccination.

4. Any confirmed or suspected autoimmune disease or immune deficiency diseases,

including human immunodeficiency virus (HIV) infection.

5. Any condition that in the opinion of the investigator, or IRB.

4.44.44.44.4 ContraindicationsContraindicationsContraindicationsContraindications totototo thethethethe secondsecondsecondsecond vaccinationvaccinationvaccinationvaccination

The following AEs constitute contraindications to administration of EV71 vaccine at that point in

time; if any one of these AEs occurs at the time scheduled for vaccination, the subject may be

vaccinated at a later date, within the time window specified in the protocol, or withdrawn at the

discretion of the investigator. The subject must be followed until resolution of the event, as with any

AE.

1. Acute disease at the time of vaccination. (Acute disease is defined as the presence of a moderate

or severe illness with or without fever.)

2. Axillary temperature >37.0°C at time of vaccination.

4.54.54.54.5 WithdrawWithdrawWithdrawWithdraw fromfromfromfrom thethethethe studystudystudystudy

1. Severe violation of the protocol.

2. Have some food and/or medicine that can interfere with the immune response during the

observation period of the primary objectives (day 0 to day 56).

3. Unwilling to continue the study and request to withdraw.

4. Any intolerable adverse events (related to vaccination or not).

5. New diagnosed disorder which makes the subjects not suitable to continue the study.

6. The presence of any condition needs to be withdrawn determined by investigators.



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5.5.5.5. INVESTIGATIONALINVESTIGATIONALINVESTIGATIONALINVESTIGATIONAL PRODUCTSPRODUCTSPRODUCTSPRODUCTS ANDANDANDAND ADMINISTRATIONADMINISTRATIONADMINISTRATIONADMINISTRATION

5.15.15.15.1 InvestigationalInvestigationalInvestigationalInvestigational ProductProductProductProduct SuppliesSuppliesSuppliesSupplies

5.1.15.1.15.1.15.1.1 StudyStudyStudyStudy vaccinevaccinevaccinevaccine andandandand placeboplaceboplaceboplacebo

The experimental vaccines are EV71 inactivated vaccines with aluminium hydroxide adjuvant and

are developed and produced by Beijing Vigoo Biotech Co., LTD. The EV71 inactivated vaccines are

produced by first inoculating EV71 strain (FY7VP5/AH/CHN/2008 (subtype C4)) onto Vero cells,

followed by culture, harvest, formalin inactivation, purification, and aluminum hydroxide absorption.

The composition of the vaccine includes EV71 virus antigen, aluminum hydroxide (adjuvant),

disodium hydrogen phosphate, sodium chloride, and water for injection. The EV71 vaccines are

injectable formulation (0.5 ml/vial). The EV71 antigen contents of the vaccines used in this study is

320 U/0.5 ml, (batch numbers: 201108002, 201108003, and 201108004). The EV71 antigen contents

were measured according EV71 antigen standard (National biological reference: 20100023) which

provided by NIFDC.

The placebo contains aluminum hydroxide diluents without EV71 (0.5 ml/vial, batch No.:

201108001). The aluminum hydroxide content of both the placebo and the studied vaccines was 0.18

mg /0.5 ml (Table 1). To allow 10% deviation in the participants number in practical operation, a

total of 22020 doses of vaccine and placebo are prepared for 11010 persons.

Table 1 Information of investigational vaccine and placebo.

name specification batch Expiry date number

vaccine 320U/0.5ml (with adjuvant) 201108002 Aug, 31,2014 1835×2



placebo 0U/0.5ml (adjuvant only) 201012001 Aug, 31,2014 5505×2

Total 22020

5.1.25.1.25.1.25.1.2 DosageDosageDosageDosage andandandand administrationadministrationadministrationadministration

The specification of the EV71 vaccines is 320 U/0.5 ml /vial.



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The injection will be given intramuscularly in anterolateral side of thigh in the infant subjects (aged

6-11 months), and in the deltoid muscle in children subjects (aged 12-36 months) at day 0 and day

28.

Only the investigator or medically qualified designee will give the injection. For the purpose of

vaccine/placebo administration, a medically qualified member of the study staff is a study physician

or a study nurse, as applicable to the investigator’s local practice. (see Table 2).

Table 2: Dosage and Administration

visit vaccination dose vaccine routesite

aged 6-11 months aged 12-36 months

1 D 0 1st EV71 vaccine / placebo IManterolateral side ofthigh

deltoid muscle

3 D 28 2nd EV71 vaccine / placebo IManterolateral side ofthigh

deltoid muscle

5.1.35.1.35.1.35.1.3 StorageStorageStorageStorage andandandand DispensingDispensingDispensingDispensing ofofofof StudyStudyStudyStudy MedicationMedicationMedicationMedication

A site assessment will be performed during the pre-study activities to ensure that the study vaccines/

placebo will be stored in appropriate conditions during the study.

All vaccines will be shipped at +2°C to +8°C to the study site upon request. Upon receipt at the study

site, the vaccines should be immediately transferred to a +2°C to +8°C temperature-monitored

refrigerator for storage. Vaccines must not be frozen. Storage conditions stated in the investigator

brochure may be superseded by the label storage.

The refrigerator in which vaccines are stored must be secure and have limited access. The storage

temperature of study vaccines/ placebo will be monitored daily while using validated temperature

monitoring devices and the temperature measurements will be recorded during working days,

preferably at the same time of the day (e.g. at the beginning of the day). Freezing indication will be

continuously controlled by an appropriate device placed close to the study vaccines.

Any temperature deviation, meaning temperature outside the defined range (i.e. +2 to +8°C), must be

reported within 2 working days to the sponsor and investigator Unit. Following exposure to a



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temperature deviation, the study vaccines/ placebo should be placed separately to avoid the confused

with normal vaccines, and will not be used until written approval is given by the sponsor.

5.25.25.25.2 TreatmentTreatmentTreatmentTreatment allocationallocationallocationallocation andandandand randomizationrandomizationrandomizationrandomization

5.2.15.2.15.2.15.2.1 RandomizationRandomizationRandomizationRandomization ofofofof studystudystudystudy vaccinesvaccinesvaccinesvaccines andandandand placeboplaceboplaceboplacebo

All the vaccine and placebo doses will be subjected to randomization. Three different batches of

vaccines and one batch of placebo will be randomized at a ratio of 1:1:1:3.

The investigational vaccines and placebo will be randomized and coded by independent statistician

from the School of Public Health, Southeast University, People's Republic of China, using SAS 9.1

software based on the predefined block size of 30.

All the randomized vaccine and placebo doses will be re-labeled with sequential numbers which are

the sole identifier for each dose. Therefore, all the vaccine and placebo doses are blindly coded. The

sequential numbers to label each dose consist of 5 digits, ranging from 00001 to 11010, containing

367 blocks.

This study will be performed in a double-blinded manner. Blinding will be maintained for all

subjects and investigators and their study staff participating in this study. Beijing Vigoo personnel

directly involved in the conduct of this study (e.g. site monitors, medical monitors, laboratory

personnel, etc.) will also be blinded to the subject’s treatment assignments.

5.2.25.2.25.2.25.2.2 TreatmentTreatmentTreatmentTreatment allocationallocationallocationallocation

A sequential number will be assigned to each participant based on study centre, the order of

enrollment to identify them. The sequential number (as ID number for each subject) must be

recorded by the investigator in the CRF (Randomization/Treatment Allocation Section). The

participants will receive doses labeled with the same sequential numbers. Therefore, the participants

will be randomly assigned to receive either EV71 vaccine or placebo in a 1:1 ratio.

5.35.35.35.3 MethodMethodMethodMethod ofofofof unblindingunblindingunblindingunblinding andandandand breakingbreakingbreakingbreaking thethethethe studystudystudystudy blindblindblindblind

The treatment allocation corresponding to each sequential number is provided to the investigator in

separate sealed envelopes labeled with the sequential numbers. Additionally, each envelope is

labeled “To Be Opened Only in Case of Emergency”.



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The breaking of the treatment code is forbidden, except in the event of a medical emergency where

the investigator believes it is necessary to determine the treatment code in order to initiate

appropriate treatment. If knowledge of the treatment code is required, the investigator will open only

the specific subject’s code envelope. A signature, date, time and reason will be written on the opened

code envelope and the participant with this code has to be withdrawn from this study.

The Investigator will assess the relationship of the adverse event to the study vaccine before the

treatment code is unblinded. The investigator will immediately notify Beijing Vigoo Biological Co.,

Ltd. at the 24 hours emergency call number (see contact list) when the treatment code is broken on

any subject for any reason during the study. The reason for the treatment code being broken must be

documented in the subject’s medical records and in the CRF. If a code envelope is opened for any

reason other than a medical emergency, the subject must be withdrawn.

At the end of the study, all code envelopes (intact and opened) must be accounted for and are to be

collected by the Monitor to be destroyed.

In case of an emergency, when knowledge of the investigational product assignment for subjects

allocated to which treatment group for the medical management of an individual subject, the

investigational product may be unblinded. The investigator must notify the sponsor within 24 hours

after determining that it is necessary to unblind the vaccine assignment. The investigator must also

indicate in source documents and in the CRF that the blind was broken and provide the date, time,

and reason for breaking the blind. And after the unblinding, the subject has to be withdrawn from

this trial.

5.45.45.45.4 PackagingPackagingPackagingPackaging andandandand LabelingLabelingLabelingLabeling

All the experimental vaccines and placebo will be supplied in coded, identical-appearing single-dose

vials by Beijing Vigoo Biological Co., Ltd. (Beijing, China). Any flaws in the investigational

products will be reported to Beijing Vigoo Biological Co., Ltd.

The vaccines will be packed in labeled boxes. Each label will contain the following information:

name of vaccine, manufacturing enterprises, vaccine code, expiry date, storage conditions and “only

used for investigation”.



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5.55.55.55.5 Back-upBack-upBack-upBack-up vaccinevaccinevaccinevaccine dosesdosesdosesdoses

In cases of the breakage of the investigational vaccine/placebo, 600 additional vaccine/placebo will

be supplied as back-up in a 1:1:1:3 ratio of the three lots of vaccine and placebo.

5.65.65.65.6 InvestigationalInvestigationalInvestigationalInvestigational productproductproductproduct accountabilityaccountabilityaccountabilityaccountability

The investigator will maintain accurate records of the disposition of all study vaccine supplies

received during the study. These records will include the amounts and dates that clinical supplies

were received from Beijing Vigoo Biological Co., Ltd, dispensed to the subject and returned by the

subject. If errors or damages in the investigational vaccine supply shipments occur, the investigator

will contact Beijing Vigoo Biological Co., Ltd through the CRO YingHeYuan immediately.

Unused investigational product must be destroyed according to procedures and local environment

regulations once all investigational product accountability documentation has been completed unless

there are regulatory requirements for this to be returned. Empty investigational product containers

may be destroyed after the sponsor has performed accountability.

All investigational vaccine supplies will be accounted for at the termination of the study and written

explanation provided for discrepancies. All unused investigational vaccine supplies and packaging

materials will be inventoried and returned to Beijing Vigoo Biological Co., Ltd, by the monitor. The

investigator is not permitted to return or destroy unused investigational vaccine supplies or

packaging materials unless authorized by Beijing Vigoo Biological Co., Ltd.

5.75.75.75.7 ConcomitantConcomitantConcomitantConcomitant medication/treatmentmedication/treatmentmedication/treatmentmedication/treatment

All concomitant medication, with the exception of vitamins and/or dietary supplements, administered

at ANY time during the period starting with administration of each dose of study vaccine and ending

one month (minimum 28 days) after each dose of study vaccine will be recorded with generic name

of the medication (trade names are allowed for combination drugs, i.e. multi-component drugs),

medical indication, total daily dose, route of administration, start and end dates of treatment.

Any treatments and/or medications specifically contraindicated, e.g. any immunoglobulins and other

blood products administered three months preceding the first dose of the study vaccine or at any time

during the study period and any immune modifying drugs administered within six months preceding



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the first dose of the study vaccine or at any time during the study period will be recorded with

generic name of the medication (trade names are allowed for combination drugs only), medical

indication, total daily dose, route of administration, start and end dates of treatment.

Any vaccine not foreseen in the study protocol administered in the period beginning 28 days

preceding each dose of study vaccine and ending one month (minimum 28 days) after each dose of

study vaccine is to be recorded with trade name, route of administration and date(s) of

administration.

During the study, generally other vaccines will not be permissible to be administrated, except some

vaccines needed in some emergency conditions such rabies vaccine, tetanus vaccine, and some EPI

vaccines such as hepatitis B vaccine, encephalitis B vaccine, measles vaccine, mumps vaccine,

rubella vaccine, or MMR vaccine, DPT vaccine, hepatitis A vaccine and OPV vaccine. Any foreseen

vaccinations need to be recorded in detail.

A prophylactic medication is a medication administered in the absence of ANY symptom and in

anticipation of a reaction to the vaccination (e.g. an anti-pyretic is considered to be prophylactic

when it is given in the absence of fever [Axillary temperature ≤37.0°C] and any other symptom, to

prevent fever from occurring).

Any concomitant medication administered prophylactically in anticipation of reaction to the

vaccination must be recorded in the CRF with generic name of the medication (trade names are

allowed for combination drugs only), total daily dose, route of administration, start and end dates of

treatment and coded as ‘Prophylactic’.

During the period starting with administration of each dose of study vaccine and ending one month

(minimum 28 days) after each dose of study vaccine, concomitant medication administered for the

treatment of an AE must be recorded in the CRF with generic name of the medication (trade names

are allowed for combination drugs only), medical indication (including which AE), total daily dose,

route of administration, start and end dates of treatment. Similarly, concomitant medication

administered for the treatment of an SAE, at any time, must be recorded on the SAE Report Form/

SAE page in the CRF, as applicable.



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The guardians of subjects should record the concomitant medication 28 days after each vaccination

on the diary cards. At each study visit/contact, the investigator should question the guardians of

subject about any medication(s) taken.

Systemic medication of Corticosteroid drug will be forbidden, but the local medication of this kind

of drug will be permissible, such as cream, eye drops, the inhaler or nasal spray, the taking dose

should not exceed the recommended dose by the leaflet or lead to any systemic exposure signs.

The necessary medication and treatment will be permissible and should be recorded in time, once

any AE occurs during the study.

5.85.85.85.8 ComplianceComplianceComplianceCompliance MonitoringMonitoringMonitoringMonitoring

The following measures will ensure that the study vaccine administered complies with the treatments

planned, or that any non-compliance is documented so that it can be accounted for in the data

analyses. All vaccines and placebo will be administered by trial personnel. The Investigator or the

person in charge of product management will maintain records of product delivery to the trial site,

product inventory at the study centre, number assigned to each subject, and the return of unused

doses to the Sponsor. Labels with bar code will be affixed in the source document, in the CRF and on

the product dispensing list if needed after administration of each dose of vaccine.



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6.6.6.6. CONDUCTCONDUCTCONDUCTCONDUCT OFOFOFOF THETHETHETHE STUDYSTUDYSTUDYSTUDY

6.16.16.16.1 StudyStudyStudyStudy ProceduresProceduresProceduresProcedures DescriptionDescriptionDescriptionDescription

6.1.16.1.16.1.16.1.1 OutlineOutlineOutlineOutline ofofofof studystudystudystudy proceduresproceduresproceduresprocedures

An overview of study procedures of to be followed by the investigator is summarized in Figure 1,

Table 3 and 4.

FigureFigureFigureFigure 1111:::: PPPPlannedlannedlannedlanned visitvisitvisitvisitssss inininin thisthisthisthis trialtrialtrialtrial

Table 3: Phase 3 Trial, 9 Visits, 2 Vaccinations, 12 Months Duration/Subject

Visit timing

Sampling time point

Visit

1

Visit

2

Visit

3

Visit

4

Visit

5

Visit

6

Visit

7

Visit

8

Visit

9

Day

0

Day

7

Day

28

Day

35

Day

56

Month

5

Month

8

Month

11

Month

14

Informed consent ●

Collecting demographic information ●

Pre-vaccination body temperature ● ●

Physical examination ●



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Visit timing

Sampling time point

Visit

1

Visit

2

Visit

3

Visit

4

Visit

5

Visit

6

Visit

7

Visit

8

Visit

9

Day

0

Day

7

Day

28

Day

35

Day

56

Month

5

Month

8

Month

11

Month

14

Check inclusion criteria ●

Check exclusion criteria ●

Check elimination criteria ● ●

Check contraindications to 2nd vaccination ● ●

Blood sampling (antibody detection)a ● ● ● ●

Blood sampling (storage for antibody detection) ● ●

vaccination ● ●

Observation for 30 minutes post-vaccination ● ●

Post-vaccination recording of solicited symptoms

(Days 0-7) by guardians● ● ● ●

Post-vaccination recording of unsolicited symptoms

(Days 0-28) by guardians● ● ● ● ●

Distribution of new diary cards ● ●

Return of diary cards ● ●

Record any concomitant medication/vaccination ● ● ● ● ●

cases surveillance for EV71 associated diseases ● ● ● ● ●

Final analysis ●

Study Conclusion ●a: is applicable for immunogenicity subgroup only

Table 4: Intervals between study visits*

IntervalIntervalIntervalInterval LengthLengthLengthLength ofofofof intervalintervalintervalinterval (days)(days)(days)(days)RecommendedRecommendedRecommendedRecommended intervalintervalintervalinterval betweenbetweenbetweenbetween

scheduledscheduledscheduledscheduled visitsvisitsvisitsvisits (days)(days)(days)(days)

Visit 1 (Day 0)→ Visit 2 (Day 7) 4-10 7

Visit1 (Day 0)→ Visit 3 (Day 28) 21-42 28

Visit1 (Day 0)→ Visit 4 (Day 35) 32-38 35

Visit1 (Day 0)→Visit 5 (Day 56) 56-70 56

Visit1 (Day 0)→Visit 6 (Month 5) 150-164 150




* If the investigator determines that the subject’s health on the day of vaccination temporarily precludes vaccination, the visit will berescheduled within the interval for this visit.



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6.1.26.1.26.1.26.1.2 InformedInformedInformedInformed consentconsentconsentconsent

In obtaining and documenting informed consent, the investigator should comply with the applicable

regulatory requirement(s), and should adhere to GCP and to the ethical principles that have their

origin in the Declaration of Helsinki. Prior to the beginning of the trial, the investigator should have

the IRB/IEC’s written approval/favorable opinion of the written informed consent form and any

other written information to be provided to the guardians of subjects.

Before any study-related procedures are performed, voluntary written

study-specific informed consent must be obtained from the parent/legal guardian of the subject.

A copy of the signed and dated ICF must be given to the parent/legal guardian. The source data must

reflect that the informed consent was obtained before participation in the study.

Both the informed consent discussion and the written Informed Consent Form and any other written

information to be provided to the subjects should include explanations of the following:

1. The nature of the study.

2. The purpose of the trial.

3. The trial treatment(s) and the probability for random assignment to each treatment.

4. The trial procedures to be followed, including all invasive procedures.

5. The subject’s responsibilities.

6. All contents related to the nature of the study.

7. The reasonably foreseeable risks or inconveniences to the subjects.

8. The reasonable expected benefits. When there is no intended clinical benefit to subjects,

the subjects should be made aware of this.

9. Replacement therapy/prophylaxis available for selection and its reasonably foreseeable

benefits and risks.

10. The compensation and/or treatment available to subjects in the event of trial-related

injury.

11. That the subjects’ participation in the trial is voluntary and subjects may refuse to

participate or withdraw from the trial, at any time, without penalty or loss of benefits to which

subjects are otherwise entitled.



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12. That the monitor(s), the auditor(s), the IRB/IEC, and the regulatory authority will be

granted direct access to the subject’s original medical records for verification of clinical trial

procedures and/or data, without violating the confidentiality of subjects, to the extent permitted

by the applicable laws and regulations and that, by signing a written informed consent, the

subject is authorizing such access.

13. The records for identifying subjects will be kept confidential and, to the extent permitted

by the applicable laws and/or regulations, will not be made publicly available. If the results of

the trial are published, subjects’ identity will remain confidential.

14. Offer the contact information of the person(s) to contact for further information regarding

the trial and the rights of trial subjects, and who to contact in the event of trial-related injury.

15. The expected duration of the trial.

16. The number of subjects involved in the trial.

Beijing Vigoo will prepare a model Informed Consent Form which will embody all the elements

described above. While it is strongly recommended that this model document be followed as closely

as possible, the informed consent requirements given in this document are not intended to pre-empt

any local regulations which require additional information to be disclosed for informed consent to be

legally effective. Clinical judgment, local regulations and requirements should guide the final

structure and content of the document.

The investigator has the final responsibility for the final presentation of Informed Consent Form,

respecting the mandatory requirements of local regulations. The consent form generated by the

investigator with the assistance of the sponsor’s representative, must be approved (along with the

protocol, and any other necessary documentation) by the IRB/IEC and be acceptable to Beijing

Vigoo.

6.1.36.1.36.1.36.1.3 EligibilityEligibilityEligibilityEligibility assessmentassessmentassessmentassessment

Eligible assessment will take place on day 0, before vaccination. Prior to inclusion into the study, all

subjects will be assessed for their eligibility to participate in the study.

The following procedures/assessments will be performed:



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- Compliance with inclusion/exclusion criteria.

- Recording of demographic data.

- Recording of concomitant medication.

- A physical examination including vital signs (height, weight, temperature).

6.1.46.1.46.1.46.1.4 SSSSafetyafetyafetyafety follow-upfollow-upfollow-upfollow-up

Safety follow-ups are divided into two phases: the vaccination phase (from Day 0 to 56) and the

active surveillance phase (from Day 56 to Month 14).

After an on-site safety observation of 30 minutes’ duration following vaccination, subjects or their

guardians will be asked to record axillary temperature and data on solicited and unsolicited adverse

events (AEs) for seven consecutive days, in a diary card provided by the investigators. Subjects will

return on Day 7 (Visit 2) or Day 35 (Visit 4) for review of the diary card, concomitant medications

and medical history, and examination of the vaccination site. During 8-28 days after each vaccination,

subjects or their guardians continue to record any unsolicited AEs and concomitant medications into

the diary card. Subjects also will return approximately 28 days after vaccination for collection of

safety data and next vaccination. In the whole study period from day 0 to month 14, serious adverse

event (SAEs) will be reported and recorded (detailed see 9.3.1).

6.1.56.1.56.1.56.1.5 EV71-AssociatedEV71-AssociatedEV71-AssociatedEV71-Associated DiseasesDiseasesDiseasesDiseases activeactiveactiveactive surveillancesurveillancesurveillancesurveillance

An active surveillance system including all the clinics in villages, hospitals in townships and seven

biggest hospitals in cities will be set to capture the cases, since Day 56 (56 days after the first

vaccination) onward. The expected total duration for case surveillance is 12 months. All the eligible

subjects will be included in the surveillance. Within the surveillance period, when a subject has any

possible illness or unwell been reported, investigators will collect clinical data, take throat and anal

swabs samples for the detection of EV71 specific RNA. The ad hoc local laboratories in each study

centre will conduct the detection of enterovirus including CA16, EV71 and other enterovirus using

real-time PCR. The possible cases with EV71-specific RNA positive will be visited by investigators

to collect acute blood sample, serial swabs/stool samples. Then, all the specimens (acute blood, serial

swabs/stool samples) obtained from the suspected cases were shipped to NIFDC for laboratory



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confirmation. All the clinical, epidemiologic and lab data of suspected cases were submitted to

DSMC to confirm the final EV71-associated cases.

The detailed description of the surveillance is seen in the section 7.

6.1.66.1.66.1.66.1.6 DetailedDetailedDetailedDetailed descriptiondescriptiondescriptiondescription ofofofof studystudystudystudy stages/visitsstages/visitsstages/visitsstages/visits

6.1.7.16.1.7.16.1.7.16.1.7.1 VisitVisitVisitVisit 1:1:1:1: DayDayDayDay 0000

� Obtain written informed consent. (All subjects’ guardians who agree to participate in the trial

must sign an original written informed consent prior to any study procedures.)

� Collect demographic data (birth date, sex, height and weight).

� Record pre-vaccination body temperature.

� Collect medical history data.

� Check inclusion and exclusion criteria.

� Take 3 ml whole blood sample.

� Allocate vaccine code to each subject.

� Administration of the first dose.

� The vaccinee will be observed closely for any AE for at least 30 minutes, with appropriate

medical treatment readily available in case of a rare anaphylactic reaction following the

administration of vaccine.

� Distribute diary cards for daily post-vaccination recording of safety observation (day 0-28 after

vaccination) and instruct subjects’ guardians to bring the diary card to investigators next visit (i.e.

Visit 2).


� Check elimination criteria and contraindications.

� Check the safety observation data in the diary card and record.

� Check concomitant medication/vaccination and record.

� Instruct subjects’ guardians to record unsolicited symptoms (Day 8-28), and to bring the diary

card to the next visit (i.e. Visit 3).



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� Check elimination criteria and contraindications for the 2nd vaccination.



� Collection of diary cards for the first dose post-vaccination safety observation.

� Record pre-vaccination body temperature.

� Administration of the second dose.

� The vaccinee will be observed closely for any AE for at least 30 minutes, with appropriate

medical treatment readily available in case of a rare anaphylactic reaction following the

administration of vaccine.

� Distribute diary cards for daily post-vaccination recording of safety observation (Day 0-28 after

the 2nd vaccination) and instruct subjects’ guardians to bring the diary card to the next visit (i.e.

Visit 4).


� Check elimination criteria and contraindications.



� Instruct subjects’ guardians to record unsolicited symptoms (Day 7-28), and to bring the diary

card to the next visit (i.e. Visit 5).




� Collection of diary cards.

� Take 3 ml whole blood (for all subjects).

� Confirm the beginning of active surveillance for EV71-associated cases.

� Distribute emergency contact card and instruct guardians to immediately report to investigator

once SAE occurs.



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6.1.7.66.1.7.66.1.7.66.1.7.6 VisitVisitVisitVisit 6:6:6:6: MonthMonthMonthMonth 5555

� Check the status changes of subjects (migrated out, absent, and withdrawal).

� Check the overall physical conditions of the participants.


once SAE occur.




� Take 3 ml whole blood (only for immunogenicity subgroup).


once SAE occurs.





once SAE occurs.




� Take 3 ml whole blood (only for immunogenicity subgroup).


once SAE occurs.

� Conclusion of study.

7.7.7.7. SURVEILLANCESURVEILLANCESURVEILLANCESURVEILLANCE OFOFOFOF THETHETHETHE EV71-ASSOCIATEDEV71-ASSOCIATEDEV71-ASSOCIATEDEV71-ASSOCIATED DISEASESDISEASESDISEASESDISEASES

7.17.17.17.1 TheTheTheThe flowflowflowflow chartchartchartchart ofofofof thethethethe EV71-associatedEV71-associatedEV71-associatedEV71-associated diseasesdiseasesdiseasesdiseases activeactiveactiveactive surveillancesurveillancesurveillancesurveillance



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FigureFigureFigureFigure 2222.... TheTheTheThe procedureprocedureprocedureprocedure ofofofof thethethethe EV71-associatedEV71-associatedEV71-associatedEV71-associated diseasesdiseasesdiseasesdiseases surveillancesurveillancesurveillancesurveillance

Case reported by subjects’ guardians; Irregularly follow-up by village doctorsCase reported by medical institutions at all levels;

NRCMNS; CIDSP

EV-G (-),

EV71 (-),

CA16 (-),

Screening all the possible cases

EV71 (-), CA16 (-),EV-G (+),

Terminate surveillance visits

EV71 (-), CA16 (+),EV-G (+),

EV71EV71EV71EV71 (+),(+),(+),(+), CA16 (+/-),EV-G (+),

Epidemiological data and series samplescollection for both casesSetting matchers for cases

Data submitted to DSMC

Finally confirmedby DSMC

LabLabLabLab assayassayassayassay bybybyby Real-timeReal-timeReal-timeReal-time PCRPCRPCRPCR

Collecting clinical data;Collecting throat and anal swabs samples

Suspected enterovirus-associated diseases

Collecting throat and anal swabsTheTheTheThe secondsecondsecondsecond LabLabLabLab assayassayassayassay bybybyby RRRReal-timeeal-timeeal-timeeal-time PCRPCRPCRPCR

EV71EV71EV71EV71 ((((++++))))

Isolation and identification of EV71Seroepidemiological assays

IgM in acute blood

Surveillance target subjects



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7.27.27.27.2 ActiveActiveActiveActive ssssurveillanceurveillanceurveillanceurveillance

All subjects with at least one dose of vaccination who are available at day 56 will be the target

population of the EV71-associated diseases surveillance system.

7.37.37.37.3 DurationDurationDurationDuration ofofofof activeactiveactiveactive ssssurveillanceurveillanceurveillanceurveillance PeriodPeriodPeriodPeriod

The surveillance period will last 12 months (from day 56 to month 14).

7.47.47.47.4 CaseCaseCaseCase definitiondefinitiondefinitiondefinition

7.4.17.4.17.4.17.4.1 SSSScreeningcreeningcreeningcreening possiblepossiblepossiblepossible casescasescasescases

Considering the complexity of the clinical manifestation of the enterovirus-associated diseases, 24-26

the “preclusive diagnosis” method will be used to ensure the sensitivity of the local surveillance

system in this study. Excepting the subjects showing confirmed etiological evidence not involving

EV71 [including surgical diseases (i.e. hernia, fracture, empyrosis, electric shock, drowning, etc.),

congenital diseases, neoplasms, mental/behavioral disorders, etc.], all subjects (receiving at least one

dose of the vaccine or placebo) with other symptoms, signs and diseases without confirmed

diagnosis or pathogenesis, mainly including HFMD, HA, respiratory disease, gastrointestinal disease,

with or without other systemic complications, such as central nervous system (CNS) complications

and cardiopulmonary failure, etc., will be considered as screening cases for the

enterovirus-associated diseases surveillance.

Illness separated by a symptom-free interval of 14 days or longer will be regarded as separate

incident.

7.4.27.4.27.4.27.4.2 ClinicalClinicalClinicalClinical classificationclassificationclassificationclassification

According to the Guidance by WHO24 and (HFMD diagnosis and treatment guide (2011 version)) and (Expert

consensus of clinical treatment on severe cases by EV71 infection) by Chinese Ministry of Health and other

related references, the diagnostic criteria of clinical enterovirus associated diseases are as the following.

Table 5: Clinical case definition



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CCCClinicallinicallinicallinical diagnoseddiagnoseddiagnoseddiagnosed

diseasediseasediseasediseaseCaseCaseCaseCase definitionsdefinitionsdefinitionsdefinitions

HFMD

1. Papulovesicular rash on one or more of the hands, feet, mouth, and buttocks,

sometimes associated with fever.

2. With or without oral ulcers.

3. Severe CNS complications, including one or more of the following signs: Fever ≥

39°C or ≥ 48 hours, Vomiting, Lethargy, Agitation/irritability, Myoclonic jerks,

Limb weakness, Truncal ataxia, “Wandering eyes”, Dyspnea/tachypnea.

4. Simple rash in trunk, limbs, face and other parts cannot be involved into HFMD.

5. Sever HFMD means that HFMD accompanied with other systemic complications,

such as CNS complications, = cardiopulmonary failure and so on.

HA

Fever and multiple oral ulcers on the posterior parts of the oral cavity, sometimes

associated with mouth rash (but no papulovesicular rash on one or more of the hands,

feet, mouth, and buttocks).

Respiratory diseases

1. Signs including two and more of the following respiratory symptoms: Cough,

runny nose, sore throat, lung sputum, lung wet rales; or

2. One of the above symptoms and fever.

3. If a patient present both respiratory symptoms and gastrointestinal symptom, he or

she is diagnosed as the respiratory diseases case.

Gastrointestinal diseases

1. Signs including two and more of the following gastrointestinal symptom: Nausea,

vomiting, abdominal pain, diarrhea; or

2. One of the above gastrointestinal symptoms and fever.

Other diseases Simple fever or other signs not involved into any of the above diseases.

7.4.37.4.37.4.37.4.3 SSSSuspecteduspecteduspecteduspected enterovirus-associatedenterovirus-associatedenterovirus-associatedenterovirus-associated diseasesdiseasesdiseasesdiseases

If at least one of the first biological samples (i.e. throat and anal swabs) from screening cases shows

positive for general enterovirus (EVG)-specific RNA and/ or Coxsackievirus group A type 16

(CA16)-specific RNA or EV71-specific RNA, the screening case will then be considered as

suspected enterovirus-associated cases.

7.4.47.4.47.4.47.4.4 LLLLaboratoryaboratoryaboratoryaboratory confirmedconfirmedconfirmedconfirmed enterovirus-associatedenterovirus-associatedenterovirus-associatedenterovirus-associated diseasesdiseasesdiseasesdiseases

If a suspected case is confirmed again by laboratory test with throat and anal swabs collected second

time from suspected enterovirus-associated case within 24 h after the first lab results released, the

case will be considered as laboratory confirmed enterovirus-associated diseases.



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Table 6: Definitions of laboratory confirmed EV71-associated cases

ClinicalClinicalClinicalClinical diagnosisdiagnosisdiagnosisdiagnosis LaboratoryLaboratoryLaboratoryLaboratory assayassayassayassay CaseCaseCaseCase definitionsdefinitionsdefinitionsdefinitions

HFMD

Plus

1)1)1)1) TwoTwoTwoTwo consecutiveconsecutiveconsecutiveconsecutive positivpositivpositivpositiveeee ofofofof

EV71-specificEV71-specificEV71-specificEV71-specific RNARNARNARNA bybybyby Real-timeReal-timeReal-timeReal-time

PCRPCRPCRPCR assay;assay;assay;assay;

2)2)2)2) VirusVirusVirusVirus isolation:isolation:isolation:isolation: EV71EV71EV71EV71 positivepositivepositivepositive;;;;

Laboratory confirmedEV71-associated HFMD

HALaboratory confirmedEV71-associated HA

Respiratory diseasesLaboratory confirmedEV71-associated other disease


Other diseases

Table 7: Definitions of laboratory confirmed CA16-associated cases

ClinicalClinicalClinicalClinical diagnosisdiagnosisdiagnosisdiagnosis LaboratoryLaboratoryLaboratoryLaboratory assayassayassayassay**** CaseCaseCaseCase definitionsdefinitionsdefinitionsdefinitions

HFMD

Plus

Two consecutive CA16-specific RNA

positive by Real-time PCR assay;

Laboratory confirmedCA16-associated HFMD

HALaboratory confirmedCA16-associated HA

Respiratory diseasesLaboratory confirmedCA16-associated other disease


Other diseases

* Meet at least one of the criteria.

Table 8: Definitions of laboratory confirmed EVG-associated cases

ClinicalClinicalClinicalClinical diagnosisdiagnosisdiagnosisdiagnosis LaboratoryLaboratoryLaboratoryLaboratory assayassayassayassay CaseCaseCaseCase definitionsdefinitionsdefinitionsdefinitions

HFMD

Plus

Two consecutive positive of EVG

-specific RNA and negative of

EV71-specific RNA and

CA16-specific RNA by Real-time

PCR assay

Laboratory confirmedEVG-associated HFMD

HALaboratory confirmedEVG-associated HA

Respiratory diseases

Laboratory confirmedEVG-associated other disease


Other diseases

7.4.57.4.57.4.57.4.5 ConfirmedConfirmedConfirmedConfirmed EV71EV71EV71EV71-associated-associated-associated-associated diseasesdiseasesdiseasesdiseases

Based on the clinical data, lab results released by NIFDC, epidemiological information of laboratory

confirmed EV71-associated cases, Data Safety and Monitoring Committee (DSMC) will review all



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laboratory confirmed EV71-associated disease and then makes the decision on the finally confirmed

EV71-associated disease according to the case definition listed in section 7.4.4. And then DSMC will

classify these confirmed cases into three categories according to the clinical classification (defined in

section 7.4.2).

7.57.57.57.5 SetSetSetSet upupupup ofofofof thethethethe activeactiveactiveactive surveillancesurveillancesurveillancesurveillance systemsystemsystemsystem

The EV71-associated diseases active surveillance system will be set up based on the existing medical

infrastructure in the 4 study centres, including 485 village clinics, 32 township hospitals, 7 county/

district/ city hospitals, 4 county/ district/ city centers for diseases control and prevention(CDC), New

Rural Cooperative Medical Network System (NRCMNS), China Information System for Diseases

Control and Prevention (CISDCP) and 1 provincial CDC, in addition, 4 ad hoc local labs for

real-time fluorescence quantitative PCR assay will be set up by the Mole Bioscience CO., Ltd. and

the National Institutes for Food and Drug Control (NIFDC) will consist the lab assay system to

provide lab assays and evidence for case diagnosis for the screening cases.

7.67.67.67.6 ImplementationImplementationImplementationImplementation ofofofof thethethethe EV71-associatedEV71-associatedEV71-associatedEV71-associated diseasediseasediseasedisease activeactiveactiveactive surveillancesurveillancesurveillancesurveillance

7.6.17.6.17.6.17.6.1 MethodsMethodsMethodsMethods forforforfor discoverydiscoverydiscoverydiscovery ofofofof screeningscreeningscreeningscreening casescasescasescases

7.6.1.17.6.1.17.6.1.17.6.1.1 AnAnAnAn activeactiveactiveactive CaseCaseCaseCase reportedreportedreportedreported bybybyby medicalmedicalmedicalmedical institutionsinstitutionsinstitutionsinstitutions atatatat allallallall levelslevelslevelslevels

Doctors from medical institutions at all levels included in the surveillance system (i.e. village clinics,

township hospitals, county/district/city hospitals) will make effort to discover and report screening

cases from surveillance subjects who visit for medical-care-seeking. And then, for screening cases,

corresponding investigators will collect clinical data and throat and anal samples within 24 hours

from screening cases for EV71-specific RNA detection by real-time fluorescence quantitative PCR.

7.6.1.27.6.1.27.6.1.27.6.1.2 CaseCaseCaseCase reportedreportedreportedreported bybybyby subjectssubjectssubjectssubjects’’’’ guardiansguardiansguardiansguardians

Doctors at all levels actively and frequently provide health education around the hazards caused by

EV71 infection on infants and children and the diagnosis and treatment methods to the subjects’



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parents or guardians, as well as the screening criteria, screening test items, case reporting and

consulting methods to encourage the subjects’ guardians and parents to contact corresponding

investigators actively once their children’s health status change. If there is a case reported from

subjects’ guardians, the investigators will firstly make judgment that whether the sick subjects fit the

criteria of screening cases, if so, and then collect the clinical data and throat and anal swabs samples

within 24 hours for EV71-specific RNA detection by real-time fluorescence quantitative PCR.

7.6.1.37.6.1.37.6.1.37.6.1.3 IrregularlyIrregularlyIrregularlyIrregularly scheduledscheduledscheduledscheduled follow-upfollow-upfollow-upfollow-up bybybyby villagevillagevillagevillage doctorsdoctorsdoctorsdoctors

The village doctors will perform irregularly scheduled and multiform (including face to face visit,

telephone visit etc.) follow-up visits to subjects within the jurisdiction in order to keep abreast of the

subjects' health status and medical-care-seeking behavior and so will be able to perform face to face

visit to sick subjects. The village doctors will firstly make judgment that whether the sick subjects fit

the criteria of screening cases, if so, and then collect the clinical data and throat and anal swabs

samples within 24 hours for EV71-specific RNA detection by real-time fluorescence quantitative

PCR.

7.6.1.47.6.1.47.6.1.47.6.1.4 NewNewNewNew RuralRuralRuralRural CooperativeCooperativeCooperativeCooperative MedicalMedicalMedicalMedical NetworkNetworkNetworkNetwork SystemSystemSystemSystem (NRCMNS)(NRCMNS)(NRCMNS)(NRCMNS) andandandand ChinaChinaChinaChina

InformationInformationInformationInformation SystemSystemSystemSystem forforforfor DiseasesDiseasesDiseasesDiseases ControlControlControlControl andandandand PreventionPreventionPreventionPrevention (CISDCP)(CISDCP)(CISDCP)(CISDCP)

Investigators from county/city CDC will perform daily monitoring of the medical-care-seeking

behavior and morbidity situation of surveillance subjects through the New Rural Cooperative

Medical Network System (NRCMNS) and China Information System for Diseases Control and

Prevention (CISDCP). Once sick subjects fit the criteria for screening cases, corresponding

investigators will be informed to collect clinical data and throat and anal samples within 24 hours

from them for EV71-specific RNA detection by real-time fluorescence quantitative PCR.

7.6.27.6.27.6.27.6.2 LabLabLabLab assayassayassayassay forforforfor suspectedsuspectedsuspectedsuspected enterovirus-associatedenterovirus-associatedenterovirus-associatedenterovirus-associated diseasesdiseasesdiseasesdiseases

The lab assay results should be released within 24 hours after the collection of the first throat and

anal swabs. According to the lab assay results released within 24 hours, the suspected

enterovirus-associated diseases cases will be divided into following three categories:



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Table 9: Three categories for suspected enterovirus-associated disease cases according to the resultsof real-time fluorescence quantitative PCR assay

Categories: EV71 CA16 EV-G

Suspected CA16-associated disease Neg (-) Pos (+) Pos (+)

Suspected EVG-associated disease Neg (-) Neg (-) Pos (+)

Suspected EV71-associated disease Pos (+) Neg (-) Pos (+)

For all suspected enterovirus-associated cases, repeated sampling including throat and anal swabs

will be collected within 24 hours after the first lab results released for laboratory confirmed diagnosis

of enterovirus-associated diseases.

For the laboratorylaboratorylaboratorylaboratory confirmedconfirmedconfirmedconfirmed CA16/CA16/CA16/CA16/ EVG-associatedEVG-associatedEVG-associatedEVG-associated diseasesdiseasesdiseasesdiseases casescasescasescases, the surveillance visit and

samples collection will be terminated after the lab confirmation again.

7.6.37.6.37.6.37.6.3 FurtherFurtherFurtherFurther laboratorylaboratorylaboratorylaboratory analysisanalysisanalysisanalysis forforforfor confirmedconfirmedconfirmedconfirmed orororor suspectedsuspectedsuspectedsuspected EV71-associatedEV71-associatedEV71-associatedEV71-associated casecasecasecasessss

For laboratory suspected EV71-associated diseases cases, one aliquot of blood samples, stool

samples and serial throat and anal swabs will be shipped to the NIFDC. The blood samples will be

used for seroepidemiologic assay (blood samples collected at acute phase for detection of IgM

against EV71). Stool samples will be used for isolation of EV71. Serial throat and anal swabs will be

used for EV71-specific RNA detection again and for EV71 VP1 region sequencing.

7.6.47.6.47.6.47.6.4 Follow-upFollow-upFollow-upFollow-up forforforfor laboratorylaboratorylaboratorylaboratory confirmedconfirmedconfirmedconfirmed EV71EV71EV71EV71-associated-associated-associated-associated casescasescasescases

For the laboratorylaboratorylaboratorylaboratory confirmedconfirmedconfirmedconfirmed EV71-associatedEV71-associatedEV71-associatedEV71-associated casescasescasescases, the investigators from the county CDCs will

select 5 matched controls (confirmed by detection of EV71-specific RNA using throat and anal

swabs). The matched factors include age (difference within three months) and place of residence

(living in the same village or nearby villages). For both cases and their matched controls, the

investigators will:

1) perform surveillance visits to collect clinical and epidemiological data during the observation

period;

2) collect serum samples at acute phase (within 1 week after the onset of the case) and recovery

phase (three weeks after the onset of the case) for seroepidemiological evaluation, a stool sample



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at acute phase for the isolation and identifying of EV71, a series of throat and anal swabs

samples every 3 days before the symptoms disappear, and then every 7 days until the assay

results from two consecutive sampling indicate negative for the EV71-specific RNA detection

and the lab re-checking by the NIFDC to study dynamic change of the virus shedding.

7.6.57.6.57.6.57.6.5 FinalFinalFinalFinallylylyly confirmingconfirmingconfirmingconfirming ofofofof EV71-associatedEV71-associatedEV71-associatedEV71-associated casescasescasescases

7.6.5.17.6.5.17.6.5.17.6.5.1 DataDataDataData SafetySafetySafetySafety andandandand MonitoringMonitoringMonitoringMonitoring CommitteeCommitteeCommitteeCommittee (DSM(DSM(DSM(DSMCCCC))))

An independent committee consisting of experts in epidemiology, pediatrics, statistics and other

appropriate disciplines will be constituted by Beijing Vigoo to oversee ethical and safety aspects of

the study conduct and determine the efficacy endpoints. A quorum of 3 members is required at

scheduled meetings. There are 9 members of the DSMC in this study and the composition of the

DSMC is outlined in the contact list.

7.6.5.27.6.5.27.6.5.27.6.5.2 FinallyFinallyFinallyFinally confirmingconfirmingconfirmingconfirming bybybyby DSMCDSMCDSMCDSMC

After finishing the final surveillance visit of a laboratory confirmed EV71-associated case, the

investigators will integrate the clinical data, lab test results released by NIFDC, epidemiological

information and then write the case final report. The relevant source documents will be submitted to

DSMC for review prior to study unblinding, to finally confirm the EV71-associated diseases.

The independent DSMC will review overall subject data, as well as endpoint-specific data, applying

complex case definitions to provide standardized adjudicated outcomes. DSMCs should be blinded

to treatment when performing centralized adjudication, regardless of whether the trial is conducted in

a blinded manner. Also, the centralized adjudication process should be designed to both preserve the

independence of the DSMC and prevent any undue bias that could impact its decision-making

processes.

DSMC-adjudicated outcomes typically either validate, negate, upgrade, downgrade, or otherwise

modify initial classifications of the suspected endpoints (i.e., those assigned by investigators).

Therefore, the occurrence of differences between initial classifications and final DSMC-adjudicated

outcomes is the expected result of any centralized adjudication process. In addition, for validated



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endpoints, the DSMC may provide further endpoint sub-classifications. DSMC adjudicators can also

identify new, previously un-reported suspected endpoints for investigation and follow-up. Final

DSMC-adjudicated outcomes are not provided to investigators, as they have the potential to unduly

bias investigator reporting of suspected endpoints, and their intended use is for the purposes of

performing uniform analysis of key clinical efficacy and safety variables: DSMC-adjudicated

outcomes are not intended or suited for medical treatment decisions. Final DSMC-adjudicated

outcomes do not replace or overwrite initial classifications; however, it is the DSMC-adjudicated

outcomes that are used to assess key efficacy and safety variables in primary and secondary endpoint

analyses, rather than the initial classifications.

7.77.77.77.7 ClinicalClinicalClinicalClinical specimensspecimensspecimensspecimens collectioncollectioncollectioncollection andandandand lablablablab assayassayassayassay methodsmethodsmethodsmethods

7.7.17.7.17.7.17.7.1 ClinicalClinicalClinicalClinical specimensspecimensspecimensspecimens collectioncollectioncollectioncollection

Samples will be collected in hospital and kept anonymous with a coding number. The stool samples

will be collected in sterile tubes and detected within 24 hours. Throat and vesicle swab samples will

be collected in 5ml saline solution contained 5% FBS and detected within 24 hour. Then all the

samples will be divided into aliquots and stored at -20°C until further tested. All the operation will

be performed according to the guild to prevent and control for Hand, Foot and Mouth Disease (2009

Version), approved by the Ministry of Health of People’s Republic of China.

Blood samples should be sent to NIFDC after all subjects finish each required visit for each blood

collection. Other biological samples collected during the surveillance period should be sent to

NIFDC after laboratory confirmed EV71-associated cases’ biological samples show consecutive

negative EV71-specific RNA released by ad hoc local labs. Biological samples should always be

sent by air, road or train and must be placed with cold chain during shipment. Biological Specimen

listing form should always accompany the shipment.



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7.7.27.7.27.7.27.7.2 Real-timeReal-timeReal-timeReal-time RT-PCRRT-PCRRT-PCRRT-PCR assayassayassayassay

The real-time PCR kit will be used is manufactured by Jiangsu Mole Bioscience Co., LTD (SFDA,

Approval No: 34011300). Extracting and amplifying of the nucleic acid will be performed according

to the kit’s instruction.

In order to ensure the accuracy of the results, eight negative samples, nucleotide free water will be

added to monitor the process of nucleic acid extraction and amplification, avoiding false positive

result. In addition, one strong positive sample and one weak positive sample in the kit will be used to

monitor the amplification efficiency, avoiding false negative result. Only if the 8 negative and 2

positive are right at same time, the results of the test (96 samples) will be accept, otherwise it must

be re-tested.

7.7.37.7.37.7.37.7.3 ConventionalConventionalConventionalConventional RT-PCRRT-PCRRT-PCRRT-PCR

VP1 was amplified to determine the EV71 and subtype. The EV71 positive samples confirmed by

real-time RT-PCR will be identified by conventional RT-PCR. The MagMAX™-96 extraction system

and Viral RNA Isolation Kit (Cat No, AM1836) from ABI will be used to extract RNA, according to

the kit’s instruction. The DNA will be generated using SuperScript III RT/ Platinum Taq High

Fidelity one step RT-PCR kit (Invitrogen). The primers will be designed according to the conserved

regions of C4 subtype. The forward and reverse primers for detection of the EV71 is F1

(5’-ATAATAGCACTAGCGGCAGCC-3’) and F2 (5’-CAAGATGTCGGTTGACCACTC-3’).

Amplifications of conventional RT-PCR will be purified with an E.Z.N.A Gel Extraction kit

(OMEGA) and sequenced with F1 and F2. All sequencing will be performed by invitrogen using the

ABI-PRISM3730XL DNA sequencer. The alignments of these EV71 isolates and reference

sequences will be achieved with MEGA4 program27. Phylogenetic analysis will be conducted using

the neighbor-joining method and Kimura 2-parameter model with 1000 bootstrap psesudoreplicated

with MEGA4. The full length of VP1 (891bp) will be used for the analysis.

7.7.47.7.47.7.47.7.4 VirusVirusVirusVirus IsolationIsolationIsolationIsolation andandandand IdentifyIdentifyIdentifyIdentify

Vero and RD cells will be used for isolation of enteroviruses. Confluent cell cultures will be seeded

in microplate wells and inoculated with 100μl of mantenance medium and 50μl of pharyngeal



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swab samples. The cell culture will then be incubated at 37°C in 5% CO2 and observed for 7 days to

check for cytopathic effect. A blind passage will be performed twice if no cytopathic effect is

observed by the end of the observation period.

Sera contained specific neutralizing antibody for EV71 will be used to identify the serotype. The

isolated virus will identify EV71 if it is neutralized by the sera in Vero and RD cell.

7.7.57.7.57.7.57.7.5 IgMIgMIgMIgM detectiondetectiondetectiondetection

The detection of IgM using blood samples collected at acute phase from EV71-associated cases will

be performed by NIFDC according to corresponding specification for operating28.

8.8.8.8. ASSESSMENTSASSESSMENTSASSESSMENTSASSESSMENTS

Every effort should be made to ensure that the protocol required tests and procedures are completed

as described. However it is anticipated that from time to time there may be circumstances, outside of

the control of the investigator, may make it unfeasible to perform the test. In these cases the

investigator will take all steps necessary to ensure the safety and wellbeing of the subject. When a

protocol required test cannot be performed, the investigator will document the reason for this and any

corrective and preventive actions which he/she has taken to ensure that normal processes are adhered

to as soon as possible. The study team will be informed of these incidents in a timely fashion.

8.18.18.18.1 SafetySafetySafetySafetyAssessmentsAssessmentsAssessmentsAssessments

For safety assessments, the outcome measure is to evaluate the solicited adverse reactions within 7

days, all the adverse events within 28 days after each vaccination, and the serious adverse events

during the whole study period. Participants will remain in the clinic to observe for the occurrence of

any adverse events for 30 minutes after receipt of each dose. For the next 28 days following each

vaccination, any injection-site adverse events and systematic adverse events will be recorded by their

guardians on the diary cards. The diameters of any erythema, swelling, induration and exanthema at

the injection site will be reviewed by members of the study staff, and daily axillary temperature will



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be measured by their guardians. And the adverse events recorded by their guardians will be reviewed

by study staffs.

Adverse events will be coded according to the requirements of the China state Food and Drug

Administration (SFDA) 28. The presence of solicited and unsolicited adverse events and any serious

adverse events will be described in terms of the incidence, intensity and relation to vaccination. The

incidence of adverse events will be based on the most severe response, and expressed in terms of the

number and proportion of individuals who had adverse events in each group.

8.1.18.1.18.1.18.1.1 GradingGradingGradingGrading forforforfor adverseadverseadverseadverse eventseventseventsevents

Injection-siteInjection-siteInjection-siteInjection-site reactionsreactionsreactionsreactions forforforfor thethethethe subjectssubjectssubjectssubjects agedagedagedaged fromfromfromfrom 6-356-356-356-35 monthsmonthsmonthsmonths：

- Pain

- none = lack of symptoms

- Grade 1(mild) = mild reactions such as withdrawal when the injection

site was touched

- Grade 2(moderate) = moderate reactions such as withdrawal when the

injection site was touched

- Grade 3(severe) = crying or refuse to be touched when the injection

site was touched severe

- Grade 4(potentially life threatening) = emergency or hospitalization

- Mucocutaneous


- Grade 1(mild) = redness

- Grade 2(moderate) = disperse, rash-like tetter, dry, desquamation,

- Grade 3(severe) = blister-like eruptions, moist, desquamation or

ulceration

- -Grade 4( potentially life threatening) = Peeling dermatitis, or Erythema

multiforme , or like Stevens-Johnsons-syndrom

- Induration



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- Grade 1(mild) = diameter < 10 mm

- Grade 2(moderate) = diameter ≥ 10 mm - ≤ 25 mm

- Grade 3(severe) = diameter ≥26 mm- ≤ 50 mm

- Grade 4(potentially life threatening) = diameter ＞50 mm

- Erythema






- Swelling


- Grade 1(mild) = diameter < 10 mm and no impairment of movements

- Grade 2(moderate) = diameter ≥ 10 mm - ≤ 25mm or impairment of

movements


- Grade 4(potentially life threatening) = ＞50 mm

- Exanthema (at the injection site)






SystemicSystemicSystemicSystemic reactionsreactionsreactionsreactions forforforfor thethethethe subjectssubjectssubjectssubjects agedagedagedaged fromfromfromfrom 6-356-356-356-35 months:months:months:months:

- Fever (axillary)

- none = ≤ 37.0°C



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- Grade 1(mild) = ≥ 37.1°C - ≤ 37.5°C

- Grade 2(moderate) = ≥ 37.6°C - ≤ 39.0°C

- Grade 3(severe) =＞39.0°C

- Allergic reactions


- Grade 1(mild) = pruritus without rashes

- Grade 2(moderate) = localized urticaria

- Grade 3(severe) = generalized urticaria, angioedema

- Grade 4(potentially life threatening) = severe allergic reactions

- Fatigue


- Grade 1(mild) = presence of mild symptoms that do not interfere with

normal daily activities, last <=48 hours

- Grade 2(moderate) = symptoms that have an impact on normal daily

activities, last >48 hours

- Grade 3 (severe) = symptoms that have an impact on normal daily

activities, last >72 hours

- Grade 4 (potentially life threatening) =emergency or hospitalization

- Activity


- Grade 2(moderate) = a little irritable or silent last over 42 hours

- Grade 3(severe) = irritable or lethargy

- Grade 4(potentially life threatening) = constant crying or unconscious

- Vomiting:


- Grade 1(mild) = once per 24 hours, normal food intake and no

impairment of activities



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- Grade 2(moderate) = twice to three times per 24 hours, food intake

significantly reduced or limitations of activities

- Grade 3(severe) = > four to six times per 24 hours, little food intake,

necessity of intravenous infusion

- Grade 4(potentially life threatening) = > not eating or liquid food for more than 24hs

- Diarrhea:


- Grade 1(mild) = slightly or transient, twice to three times of watery

stools per day, or continuously slight diarrhea within

one week

- Grade 2(moderate) = moderate or continuously, four to five times per day

or diarrhea > one week

- Grade 3(severe) = > six times of watery stool per day, or bloody stool,

postural hypotension, electrolyte imbalance and

necessity of intravenous infusion > 2 liter

- Grade 4(potentially life threatening) = hospitalization due to hypotensive shock

- Nausea:


- Grade 1(mild) = mild

- Grade 2(moderate) = moderate, food intake reduced severe

- Grade 3(serve) = food intake significantly reduced

- Grade 4(potentially life threatening) = > six times per 24 hours

- Convulsion:


- Grade 2(moderate) = once or twice per 24 hours, unnecessity of drugs

- Grade 3(severe) = more than twice per 24 hours, necessity of drugs

- Grade 4(potentially life threatening) =shock, hospitalization

- Crying



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- Grade 1(mild) = a slight increase than usual

- Grade 2(moderate) = double the usual,

- Grade 3(severe) = more than double the usual, duration for more than 3

days

- Grade 4(potentially life threatening)= hospitalization

- Decreased feeding


- Grade 1(mild) = feeding times or food intake less than usual

- Grade 2(moderate) = double the usual reduction of the feeding times or

food intake

- Grade 3(severe) = more than double the usual reduction of the feeding

times or food intake, duration for more than 1 day

- Grade 4(potentially life threatening)= hospitalization

- Cough


- Grade 1(mild) = transient, treatment unnecessary

- Grade 2(moderate) = continuous coughs, response to treatment

- Grade 3(severe) = paroxysmal coughs, treatment uncontrolled

- Grade 4(potentially life threatening) = emergency of hospitalization

8.28.28.28.2 ImmunogenicityImmunogenicityImmunogenicityImmunogenicity,,,, immuneimmuneimmuneimmune persistencepersistencepersistencepersistence andandandand lot-to-lotlot-to-lotlot-to-lotlot-to-lot consistencyconsistencyconsistencyconsistency aaaassessmentsssessmentsssessmentsssessments

Blood will be obtained at Day 0, Day 56, Month 8, and Month 14 from subjects in immunogenicity

subgroup for immunogenicity, antibody persistence and lot-to-lot consistency assessment.

Neutralizing antibody titers will be analyzed by the NIFDC.

Immunogenicity, immune persistence and lot-to-lot consistency will be assessed by measuring serum

neutralization antibody (NT) titer against EV71 by Microtiter Cytopathogenic effect neutralization



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assay (MCPENT) 30, which was modified from the standard method used for the determination of the

antibody against Polio and developed by the World Health Organization 31.

The cutoff value for seropositivity will be 1:832-34. All samples will be blindly assayed in duplicate

and double-checked.

8.38.38.38.3 ProtectiveProtectiveProtectiveProtective efficacyefficacyefficacyefficacy againstagainstagainstagainst EV71-associatedEV71-associatedEV71-associatedEV71-associated diseasesdiseasesdiseasesdiseases assessmentsassessmentsassessmentsassessments

Based on the fixed-period follow-up every 3 months, the status changes (loose to follow-up, migrate

out or quite) during the surveillance period (day 56 to month 14 from the first vaccination) in

subjects that received at least one dose of EV71 vaccine or placebo will be recorded for calculating

effective observational person-year. Protective efficacy against EV71-associated diseases will be

assessed by measuring the accumulated numbers of the EV71-associated cases in each group and the

effective observational person-years to calculate the incidence density. The EV71-associated diseases

will be captured through the Active EV71-Associated Diseases Surveillance System (AEADSS)

described in section 7.

8.48.48.48.4 AAAAssessmentssessmentssessmentssessment ofofofof thethethethe immunologicalimmunologicalimmunologicalimmunological correlatecorrelatecorrelatecorrelate ofofofof protectionprotectionprotectionprotection

The EV71-associated cases (finally confirmed by DMSC) and their corresponding event-free

matched controls will be included in the exploratory analysis for correlate of protection. Based on

their NT titers on day 56, sensitivity, specificity and Youden index will be calculated at each antibody

titer. The antibody titer on day 56 with maximum Youden index will be considered as the

immunological correlate of protection against EV71-associated diseases.



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9.9.9.9. ADVERSEADVERSEADVERSEADVERSE EVENT/REACTIONEVENT/REACTIONEVENT/REACTIONEVENT/REACTION REPORTINGREPORTINGREPORTINGREPORTING

9.19.19.19.1 AdverseAdverseAdverseAdverse eventeventeventevent andandandand adverseadverseadverseadverse reactionreactionreactionreaction definitiondefinitiondefinitiondefinition

An adverse event (AE) is any untoward medical occurrence in a subject administered an

investigational product and which does not necessarily have a causal relationship with this treatment.

An adverse event can therefore be any unfavorable and unintended sign (including an abnormal

laboratory finding), symptom or disease temporally associated with the use of the investigational

products, whether or not considered related to it.

An adverse reaction (AR) is all untoward and unintended responses to a medical product related to

any dose administered.

An unexpected adverse reaction is an adverse reaction, the nature or severity of which is not

consistent with the applicable product information:

− investigator's brochure for an unauthorized experimental product

− Summary of product characteristics for an authorized product.

9.29.29.29.2 RecordingRecordingRecordingRecording ofofofofAdverseAdverseAdverseAdverse EventsEventsEventsEvents

Any adverse event will be recorded in the CRF and source document. In order to avoid vague,

ambiguous or colloquial expressions, the adverse event will be recorded in standard medical

terminology rather than the subject’s own words. Whenever possible, the investigator will group

together into a single term, signs and symptoms which constitute a single diagnosis.

The existence of or change in an adverse event may be concluded due to the necessity to administer a

concomitant medication, from a spontaneous report of the subject, from the physical examination or

from special tests like ECG, EEGs, laboratory assessments or other study specified tests (source of

AE).

Adverse events may occur in the specified follow-up period and, regardless of the interval since the

last administration of the study medication, will be treated as any other adverse event occurring

during the treatment with study medication.



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Each adverse event is to be evaluated for duration, severity, seriousness and causal relationship to the

vaccinations. The action taken with study medication, the possible concomitant treatment/therapy

introduced, and the outcome as well as whether the event led to study termination must also be

recorded.

Severity

The severity of the adverse event will be characterized as mild (Grade 1), moderate (Grade 2), or

severe (Grade 3), life-threatening (Grade 4), according to the following definitions:

− Mild events are usually transient and do not interfere with the subject’s daily activities.

− Moderate events introduce a low level of inconvenience or concern to the subject and may

interfere with daily activities.

− Severe events interrupt the subject’s usual daily activity.

− Life-threatening events forbid subject’s usual daily activity and introduce needs of

hospitalization.

9.39.39.39.3 SeriousSeriousSeriousSerious adverseadverseadverseadverse event/reactionevent/reactionevent/reactionevent/reaction (SAE)(SAE)(SAE)(SAE) definitiondefinitiondefinitiondefinition

A serious adverse event/reaction is occurrence of any untoward medical during the whole studyperiod that:

− Results in death.

− Is life-threatening (an event in which the subject is at risk of death at the time of the event; it

does not refer to an event which hypothetically might have caused death if it was more

severe).

− Results in persistent or significant disability/incapacity.

− Requires hospitalization or prolongation of an existing hospitalization.

− Is a congenital anomaly/birth defect.

In addition, medical and scientific judgment will be exercised in deciding whether other conditions

will also be considered serious, such as important medical events that may not be immediately

life-threatening or result in death or hospitalization but may jeopardize the subject’s safety or may

require intervention to prevent one of the other outcomes listed in the definition above. These will



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also be considered serious. Examples of such events are intensive treatment in an emergency room or

at home for allergic bronchospasm; blood dyscrasias or convulsions that do not result in

hospitalization; or development of drug dependency or drug abuse.

9.3.19.3.19.3.19.3.1 ReportingReportingReportingReporting SAEsSAEsSAEsSAEs

Any serious adverse event, including death due to any cause, which occurs during this study, whether

or not related to the investigational products, must be reported immediately (within 24 hours of the

study site’s knowledge of the event) by telephone or fax to the China SFDA and the Jiangsu

provincial SFDA, Beijing Vigoo Biological Co., Ltd., Jiangsu Center for Diseases Control and

Prevention, CRO at the following number:

Principal Investigator: Fengcai Zhu, Tel +86-25-8375 9418, Fax: +86-25-8375 9409

Representative of sponsor: Qing-Hua Chen, Tel/Fax: +86-10-65656778

The unblinding of single cases by investigators in the course of the clinical trial will only be

performed if relevant for the safety of the subject.

The report for CRO will contain as much available information concerning the serious adverse event

to enable CRO to file a report which satisfies regulatory reporting requirements. This will be done on

the SFDA SAE-form. This will be a preliminary report. The final report will be provided after

evaluation by Beijing Vigoo Biological Co., Ltd. according to ICH standard timelines. In addition to

the initial 24-hour report, a completed, separate SAE report is to be sent to CRO via fax or mail

within 48 hours of the event.

All serious AEs will be recorded on the case report form and source documents. Criteria for

documenting the relationship to study medication and severity will be the same as those previously

described.

9.3.29.3.29.3.29.3.2 ExpeditedExpeditedExpeditedExpedited reportingreportingreportingreporting ofofofof SUSARsSUSARsSUSARsSUSARs totototo regulatoryregulatoryregulatoryregulatory authoritiesauthoritiesauthoritiesauthorities andandandand investigatorsinvestigatorsinvestigatorsinvestigators

All unexpected and serious (SUSARs) events are subject to expedited reporting. Also post-study

SUSARs that occur after the patient has completed a clinical trial and are reported by an investigator

to the sponsor, qualify for expedited reporting.



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Beijing Vigoo Biological Co., Ltd. is responsible for the prompt notification to all concerned

investigators, the JSCDC Ethics Committees and competent authority of all relevant safety

information previously described.

Fatal or life-threatening SUSARs will be notified by Beijing Vigoo Biological Co., Ltd. to the

competent authority and the JSCDC Ethics Committee as soon as possible but no later than seven

calendar days after the sponsor has first knowledge of the minimum criteria for expedited reporting.

In each case relevant follow-up information will be sought and a report completed as soon as

possible. It will be communicated to the competent authority and the JSCDC Ethics Committee

within an additional eight calendar days.

All other SUSARs and safety issues deserving expedited reporting must be reported to the competent

authority and -dependent on national provisions - to the JSCDC Ethics Committee in the concerned

countries as soon as possible but no later than 15 calendar days after the sponsor has first knowledge

of the minimum criteria for expedited reporting.

Expedited reporting is not usually required for reactions which are serious but expected, or for

non-serious adverse reactions whether expected or not.

9.49.49.49.4 Follow-upFollow-upFollow-upFollow-up ofofofof adverseadverseadverseadverse eventseventseventsevents

All adverse events occurring during the study are to be followed up in accordance with good medical

practice until resolved or judged no longer clinically significant, or if a chronic condition, until fully

characterized. All follow-up results are to be reported to Beijing Vigoo Biological Co., Ltd.

9.59.59.59.5 CausalityCausalityCausalityCausalityAssessmentAssessmentAssessmentAssessment

The assessment of causality must be provided for all AEs (serious and non-serious): All the

information related to SAEs will be reported to DSMC, and the DSMC are responsible to give a final

decision of causality for SAEs.



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9.69.69.69.6 WithdrawalWithdrawalWithdrawalWithdrawal DueDueDueDue totototoAdverseAdverseAdverseAdverse EventsEventsEventsEvents (See(See(See(See sectionsectionsectionsection onononon SubjectSubjectSubjectSubjectWithdrawal)Withdrawal)Withdrawal)Withdrawal)

Withdrawal due to AE should be distinguished from withdrawal due to insufficient response,

according to the definition of AE noted earlier and recorded on the appropriate AE CRF page.

When a subject withdraws due to an SAE, the SAE must be reported in accordance with the

reporting requirements

9.79.79.79.7 ElicitingElicitingElicitingElicitingAdverseAdverseAdverseAdverse EventEventEventEvent InformationInformationInformationInformation

The investigator is to report all directly observed AEs and all AEs spontaneously reported by the

study subject’s guardians as required by the protocol. In addition, each study subject’s guardians will

be questioned about AEs.

9.89.89.89.8 ReportingReportingReportingReporting RequirementsRequirementsRequirementsRequirements

Each AE is to be assessed to determine if it meets the criteria for SAEs. If an SAE occurs, expedited

reporting will follow local and international regulations, as appropriate.

9.99.99.99.9 SponsorSponsorSponsorSponsor ReportingReportingReportingReporting RequirementsRequirementsRequirementsRequirements totototo RegulatoryRegulatoryRegulatoryRegulatoryAuthoritiesAuthoritiesAuthoritiesAuthorities

Adverse event reporting, including suspected serious unexpected adverse reactions, will be carried

out in accordance with applicable local regulations.

10.10.10.10. DATADATADATADATA COLLECTIONCOLLECTIONCOLLECTIONCOLLECTION ANDANDANDANDMANAGEMENTMANAGEMENTMANAGEMENTMANAGEMENT

10.110.110.110.1 SourceSourceSourceSource DocumentsDocumentsDocumentsDocuments andandandand SourceSourceSourceSource DataDataDataData

The purpose of source documents is to document the existence of the subject and substantiate the

integrity of the trial data collected. The Investigator must maintain the trial source documents

accurate, complete, legible and up to date.

Examples of source documents are: subject eligible assessment, and enrolment log, subject’s diary

cards, hospital records, subject’s surveillance investigation forms (including initial, follow-up and



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final forms), informed consent forms, investigational dispensing and reconciliation forms, subject’s

file and records kept at the pharmacy or at the laboratories, mail, certified letters.

Source data are the data contained in source documents (originals or certified copies).

Source documentation include two major parts, Case Report Forms (CRFs) which will be used to

record data on the planned visit foreseen in the protocol, and Disease Surveillance Forms (DSFs).

Surveillance data reported in the DSFs will be integrated into the surveillance database (i.e. data

management software EV71VCTSM (version 1.30), which will be used to record the clinical and

epidemiological data collected from the suspected EV71-associated disease cases as well as the lab

confirmation reports. In addition, supplementary documents (lab test results, supplementary hospital

or medical records, etc.) may form part of the source documentation for a study subject. The

Investigator is responsible for the accuracy and completeness of the data reported in CRFs, DSFs and

other source documents. Data reported in the CRFs and DSFs that are derived from source

documents should be consistent with source documents and any discrepancies should be explained.

All CRFs and DSFs must be signed by the Investigator. Incorrect data must be crossed-out with a

single line, then initialed and dated. Correction fluid or similar corrective methods that mask the

original data will not to be used. These rules also apply to the completion of SAE Reporting Forms,

Data Correction Forms, and ICFs.

10.210.210.210.2 DataDataDataDataManagementManagementManagementManagement

Data generated during the trial will be managed following three different processes, one related to

visit data, defined as all data reported in the CRFs, one related to the surveillance data from the

suspected EV71-associated diseases cases, defined as all data reported in the DSFs, and the third one

related to data pertaining to SAEs (i.e. data reported by the Investigator on the SAE Reporting

Forms).

10.2.110.2.110.2.110.2.1 ClinicalClinicalClinicalClinical DataDataDataDataManagementManagementManagementManagement

During the trial, through regular data collection and monitoring, clinical data reported in the CRFs

will be integrated into the clinical database under the responsibility of Beijing Vigoo Clinical Data



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Management team. For each batch of data, double entry, quality control and triggers to computerized

logic and/or consistency checks will be systematically applied in order to detect errors or omissions.

Medical safety data reviews may be performed several times during the course of the trial. Queries

will be generated and submitted through Data Clarification Forms to the Investigator for resolution.

Each step of this process will be monitored through the implementation of individual passwords and

regular backups to maintain appropriate database access and to ensure database integrity.

10.2.210.2.210.2.210.2.2 SurveillanceSurveillanceSurveillanceSurveillance DataDataDataData ManagementManagementManagementManagement

During the surveillance period, through active data collection and monitoring, surveillance data

reported in the DSFs will be integrated into the surveillance database (i.e. data management software

EV71VCTSM (version 1.30). For each batch of data, double entry, quality control and triggers to

computerized logic and/or consistency checks will be systematically applied in order to detect errors

or omissions. Clinical and epidemiological data reviews may be performed several times during the

course of the trial. Queries will be generated and submitted through Data Clarification Forms to the

Investigator for resolution. Each step of this process will be monitored through the implementation

of individual passwords and regular backups to maintain appropriate database access and to ensure

database integrity.

The validation of the central lab data will be performed at the laboratory level following the

laboratory’s procedures. Information from the laboratories such as subject identifiers and dates or

sample numbers will be checked for consistency before the integration into the surveillance database.

After integration of all corrections in the complete set of data, and after the SAE information has

been reconciled, the whole database will be locked and saved before being released for statistical

analysis.

10.2.310.2.310.2.310.2.3 SAESAESAESAE DataDataDataDataManagementManagementManagementManagement

During the trial, data pertaining to SAEs reported on SAE Reporting Forms required by SFDA will

be integrated into the Sponsor’s SAE database. Upon receipt of an SAE Reporting Form, the data

will be entered into the database after a duplicate check. Each SAE is assigned a case identification

number. Entered data will be independently verified against the original SAE forms. All SAEs are



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then reviewed by the Product Safety Officer. Each SAE is reviewed, locked and approved in the

database. Any follow-up information concerning a locked and approved SAE will be incorporated

and a new version of the SAE will be created.

10.310.310.310.3 ClinicalClinicalClinicalClinical InvestigatorInvestigatorInvestigatorInvestigator’’’’ssss BrochureBrochureBrochureBrochure

Investigators will receive the current version of the Clinical Investigator’s Brochure, which

comprehensively describes all the available preclinical and human experience with the experimental

vaccine. If relevant new information becomes available during the course of the trial, the

investigators will receive a revised Investigator’s Brochure or an amendment to the current version.

10.410.410.410.4 ArchivingArchivingArchivingArchiving

Following closure of the study, the investigator must maintain all site study records in a safe and

secure location. The records must be maintained to allow easy and timely retrieval, when needed

(e.g., audit or inspection), and, whenever feasible, to allow any subsequent review of data in

conjunction with assessment of the facility, supporting systems, and staff. Where permitted by

applicable laws/regulations or institutional policy, some or all of these records can be maintained in a

validated format other than hard copy; however, caution needs to be exercised before such action is

taken. The investigator must assure that all reproductions are legible and are a true and accurate copy

of the original ones, and meet accessibility and retrieval standards, including re-generating a hard

copy, if required. Furthermore, the investigator must ensure there is an acceptable back-up of these

reproductions and that an acceptable quality control process exists for making these reproductions.

Trial-related documents will be maintained for a period of 10 years after final marketing approval of

the vaccine, or 10 years after the formal discontinuation of clinical development of the product per

the requirements by SFDA and IRB/IEC. The site investigators must be aware of all requirements

and retain protocol records in accordance with the longest requirement that pertains to the study. No

study document should be destroyed without prior written agreement between the Beijing Vigoo, the

Principal Investigator and the site investigators. Storage of all trial-related documents will be such



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that confidentiality will be strictly maintained. Should the site investigators wish to assign the study

records to another party or move them to another location, Beijing Vigoo must be notified in writing

of the new responsible person and/or the new location.

11.11.11.11. QUARLITYQUARLITYQUARLITYQUARLITY CONTROLCONTROLCONTROLCONTROLANDANDANDAND QUARLITYQUARLITYQUARLITYQUARLITY ASSURANCEASSURANCEASSURANCEASSURANCE

11.111.111.111.1 MonitoringMonitoringMonitoringMonitoring

Before the start of the trial (i.e. before the inclusion of the first subject by the first centre), the

Investigators and the Sponsor’s monitoring staff will meet at the "Site-initiation visit" to discuss the

trial protocol and the detailed trial procedures, with emphasis on inclusion and exclusion criteria,

visit timing, safety procedures, informed consent procedures, SAE reporting procedures, CRF

completion, and sample and product handling.

The Sponsor’s monitoring staff will ensure and document that all material to be used during the trial

has been received and that the investigational team and local monitoring staff have been properly

informed about the trial, GCP and regulatory requirements, and the Sponsor’s procedures. Specific

training sessions for the investigational team and CRAs on these topics may be performed, as

necessary.

Specific instruction manuals will be provided for the completion of the CRF and for the detailed trial

procedures such as the laboratory and sample handling procedures (Operating Guidelines).

After the start of the trial, the Sponsor’s monitoring staff will be in regular contact with the

investigational team through telephone calls and regular follow-up visits to the trial centres. The

Investigator must be available for these visits and will allow the monitoring staff direct access to

subject medical files and CRFs. During monitoring visits, the monitoring staff will:

• Control the quality of the trial progress (e.g., with respect to the protocol and operating guidelines,

quality of data collection and document completion, signature of consent forms, appearance of SAEs,

sample and product management, cold chain monitoring, and archiving).

• Collect completed CRFs and DSFs and any corresponding queries (Data Correction Forms).

• Evaluate the number of complete or ongoing observations.



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Any identified problems will be discussed with the Investigator and corrective or preventive actions

will be determined, as appropriate.

Once the CRF pages corresponding to the last visit have been returned duly completed and signed,

the Investigator must be available to complete any queries (Data Correction Forms) forwarded by the

Sponsor until database lock.

At the end of the trial, a close-out visit will be performed to ensure that:

• The centre has all the documents necessary for archiving.

• All samples have been shipped to the appropriate laboratories.

• All unused material and products have been returned to the Sponsor.

11.211.211.211.2 AuditsAuditsAuditsAudits

For the purpose of compliance with Good Clinical Practice (GCP) and Regulatory Agency

Guidelines it may be necessary for Beijing Vigoo or a Drug Regulatory Agency to conduct a site

audit. This may occur at any time from start to after conclusion of the study.

When an investigator signs the protocol, he agrees to permit drug regulatory agencies and Beijing

Vigoo audits, providing direct access to source data/ documents. Furthermore, if an investigator

refuses an inspection, his data will not be accepted in support of a New Drug Registration and/or

Application, Biologics Licensing Application.

A quality assurance (QA) audit may be performed by the Sponsor’s QA Department or by

independent auditors to verify that the trial has been conducted according to the protocol, GCP, ICH

requirements and the applicable regulations. These audits usually take 1 to 2 days. Beijing Vigoo’s

audits entail review of source documents supporting the adequacy and accuracy of CRFs, review of

documentation required to be maintained, and checks on vaccine accountability. Beijing Vigoo’s

audit therefore helps prepare an investigator for a possible regulatory agency inspection as well as

assuring Beijing Vigoo of the validity of the database across investigational sites. The Inspector will

be especially interested in the following items:

- Log of visits from the sponsor’s representatives



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- Study personnel

- Study file

- Safety reporting

- IRB/IEC and regulatory authority approvals

- Facilities

- monitoring

- Vaccine accountability

- Approved study protocol and amendments and investigator brochure

- Informed consent of the subjects

- Medical records and other source documents supportive of CRF data

- Reports to the IRB/IEC and the sponsor

- Record retention.

12.12.12.12. STATISTICALSTATISTICALSTATISTICALSTATISTICAL CONSIDERATIONSCONSIDERATIONSCONSIDERATIONSCONSIDERATIONS

Detailed methodology for summary and statistical analyses of the data collected in this study will be

documented in a Statistical Analysis Plan (SAP), which will be maintained by the sponsor.

Before analysis, the blinded data will be inspected for inconsistencies by performing several checks.

Any inconsistencies found will be resolved by the monitor after contacting the investigator. When

the data in the database is clean, the database will be locked to prevent unauthorized access. The

database will be made available as SAS®files for statistical analysis. The statistical analysis of the

data will be performed by The School of Public Health, Southeast University, People's Republic of

China. All statistical analyses will be performed by means of SAS software (version 9.1).

12.112.112.112.1 SampleSampleSampleSample sizesizesizesize calculationcalculationcalculationcalculation

12.1.112.1.112.1.112.1.1 OverallOverallOverallOverall samplesamplesamplesample sizesizesizesize calculationcalculationcalculationcalculation

Sample size calculation was done by SAS software (version 9.1) for this phase 3 trial. According to

the results of phase I and II clinical trials, the dynamic changes of the neutralizing antibody titers of

the EV71 vaccine were similar to the NT titers of the inactivated poliovirus vaccines (mainly NT



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against polioviruses type II and type III)35. Assuming a vaccine protective efficacy of 80% with the

lower limit of 95% CI exceed 20%. An EV71-associated HFMD incident rate of 8 per 1000

population for participants received placebo was assumed according to HFMD epidemiological

survey data in Jiangsu province in 2010.

1 0= 1- =0.2 0.008=0.0016λ µ λ ×（）

1λ : Incident rate of EV71-associated disease in vaccine group;

0λ : Incident rate of EV71-associated disease in placebo group;

µ : Vaccine protective efficacy;

The sample size was calculated on the basis of type I error rateα =0.05 (two-sided test), and with a

1:1 ratio of the allocation between placebo group and vaccine group. The minimum sample size of

3253 per group was required. And at least 31 EV71 associated HFMDs would be captured during the

follow-up period.

Considering a drop-out rate of 20% in the follow-up, and other potential confounding factors may

affect the case capturing, a sample-size of 5000 per group was determined. Thus, the overall sample

size of this trial should be 10000.

According to the previous seroepidemiologic study carried out in Jiangsu Province, a baseline EV71

seropositive rate of 15%-35% was assumed in children aged from 6 to 35 months, in the study areas.

To avoiding the forming of herd immunity by mass vaccination of EV71 vaccines in the population

at the study areas, the seropositive rate after vaccinations should be controlled < 50%. On condition

that 100% of children receiving EV71 vaccines would be seropositive post-vaccinations, the

enrollment of volunteers should not exceed 40% of the total number of children at that age in each

township, according to the following results (Table 10).

Table 10: The overall post-seropositive rate calculated based on the different pre-seropositive rate

pre-seropositive

total no.of children6-35 m*

enrollmentrate (%)

no. ofchildrenenrolled

vaccinegroup

placebogroup

seropositivechildren

post-seropositive

15% 800 0.4 320 160 160 256 32%20% 800 0.4 320 160 160 288 36%



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25% 800 0.4 320 160 160 320 40%30% 800 0.4 320 160 160 352 44%

35% 800 0.4 320 160 160 384 48%15% 800 0.5 400 200 200 290 36%

20% 800 0.5 400 200 200 320 40%25% 800 0.5 400 200 200 350 44%30% 800 0.5 400 200 200 380 48%35% 800 0.5 400 200 200 410 51%

* assuming around 800 children at age of 6-35 months in a township.

12.1.212.1.212.1.212.1.2 Lot-to-lotLot-to-lotLot-to-lotLot-to-lot consistencyconsistencyconsistencyconsistency analysisanalysisanalysisanalysis sample-sizesample-sizesample-sizesample-size calculationcalculationcalculationcalculation

According to the immunologic data in previous trials, the GMTs elicited by two-dose vaccination of

the EV71 vaccine at day 56 would be ranging from 497.9 (383.1–647.0). An equivalence range of

[0.5, 2.0] for GMT ratios is assumed. 200 per vaccine lot could provide at least 80% power to detect

the consistency with respect to the vaccine-induced GMTs at day 56.

12.212.212.212.2 StudyStudyStudyStudy cohortscohortscohortscohorts totototo bebebebe evaluatedevaluatedevaluatedevaluated

12.2.112.2.112.2.112.2.1 Intention-To-TreatIntention-To-TreatIntention-To-TreatIntention-To-Treat (ITT)(ITT)(ITT)(ITT) cohortcohortcohortcohort forforforfor safetysafetysafetysafety analysisanalysisanalysisanalysis

The ITT cohort for analysis of safety will include all subjects with at least one dose.

The ITT cohort analysis will be performed per treatment assignment.

Safety analysis will be performed on the basis of ITT cohort. Descriptive statistics will be used to

calculate the rates of adverse reactions and adverse events, and Chi-square test or Fisher Exact test

for the comparison between groups, as appropriate.

Events will be reported on per-subject basis, i.e. counting subjects rather than events. This means

that even if a subject suffered the same event (i.e. with the same assigned Preferred Term) repeatedly

during the follow-up, the event will be counted only once. Repeated adverse events per subject will

be summarized according to the following rule: if a subject suffered the same adverse event more

than once, the event will be assigned the worst severity, the closest relationship to the vaccination

and the earliest starting date. In the listings, however, all occurrences of the adverse events will be

shown.



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12.2.212.2.212.2.212.2.2 ModifiedModifiedModifiedModified iiiintentionntentionntentionntention-to-treat-to-treat-to-treat-to-treat (ITT)(ITT)(ITT)(ITT) cohortcohortcohortcohort andandandand According-To-ProtocolAccording-To-ProtocolAccording-To-ProtocolAccording-To-Protocol (ATP)(ATP)(ATP)(ATP) cohortcohortcohortcohort forforforforefficacyefficacyefficacyefficacy

The modified intention-to-treat (ITT) cohort for efficacy analysis will include all eligible subjects

(receive at least one dose and enter the surveillance period at day 56) for whom data concerning

efficacy endpoint measures are available.

For this cohort, the follow-up time for a subject will start at Day 56 (28 days after the first dose).

The ATP cohort for analysis of efficacy will include all evaluable subjects (i.e. those meeting all

eligibility criteria, complying with the procedures defined in the protocol, with no elimination

violations) for whom data concerning efficacy endpoint measures are available. The primary efficacy

will be estimated on the basis of ATP cohort.

For this cohort, the follow-up time for a subject will start at Day 56 (56 days after the first

vaccination). The efficacy analysis will be performed using ATP population and will be repeated

using the ITT population as supplementary results.

12.2.312.2.312.2.312.2.3 According-To-ProtocolAccording-To-ProtocolAccording-To-ProtocolAccording-To-Protocol (ATP)(ATP)(ATP)(ATP) cohortcohortcohortcohort forforforfor analysisanalysisanalysisanalysis ofofofof immunogenicityimmunogenicityimmunogenicityimmunogenicity

The ATP cohort for analysis of immunogenicity and immune persistence will include all evaluable

subjects in the immunogenicity subset (i.e. those meeting all eligibility criteria, complying with the

procedures defined in the protocol, with no elimination criteria during the study) for whom data

concerning immunogenicity and immune persistence endpoints measures are available. This will

include subjects for whom assay results are available for antibodies against after vaccination.

Subjects who acquire EV71-associated diseases during the trial will be excluded from the ATP

cohort for immunogenicity and immune persistence.

12.2.412.2.412.2.412.2.4 According-To-ProtocolAccording-To-ProtocolAccording-To-ProtocolAccording-To-Protocol (ATP)(ATP)(ATP)(ATP) cohortcohortcohortcohort forforforfor analysisanalysisanalysisanalysis ofofofof lotlotlotlot consistencyconsistencyconsistencyconsistency

The ATP cohort for analysis of lot consistency will include all evaluable subjects (i.e. those meeting

all eligibility criteria, complying with the procedures defined in the protocol, with no elimination

criteria during the study) for whom data concerning lot consistency endpoint measures are available.



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This will include subjects for whom assay results are available for antibodies against EV71 infection

after vaccination.

12.2.512.2.512.2.512.2.5 CohortCohortCohortCohort forforforfor analysisanalysisanalysisanalysis ofofofof thethethethe immunologicalimmunologicalimmunologicalimmunological correlatecorrelatecorrelatecorrelate ofofofof protectionprotectionprotectionprotection

The cohort for analysis of the immunological correlate of protection will be consisted of all the

confirmed EV71-associated cases and their event-free matchers at a ratio of 1:5 who meet all

eligibility criteria, comply with the procedures and intervals defined in the protocol, with no

elimination criteria during the study, with at least one dose vaccination, with their antibody titre at

day 56 available.

12.312.312.312.3 DerivedDerivedDerivedDerived andandandand transformedtransformedtransformedtransformed datadatadatadata

• The cut-off value is defined by the laboratory before the analysis.

• A seronegative subject is a subject whose titer is below the cut-off value (with titer below1:8).

• A seropositive subject is a subject whose titer is greater than or equal to the cut-off value (with titer

greater than or equal to 1:8).

• A seroconversion subject is a subject whose titer is below 1:8 before vaccination and becomes

greater than or equal to 1:32 after vaccination, or whose titer is greater than or equal to 1:8 before

vaccination and gets a post-vaccination relative increase of 4 folds.

• The Geometric Mean Titers (GMTs) calculations are performed by taking the antilog of the mean

of the log titer transformations. Antibody titers below the cut-off of the assay will be given an

arbitrary value of half the cut-off for the purpose of GMT calculation.

• The Geometric Mean Fold Increase (GMFIs) calculations are performed by taking the antilog of the

mean of the log titer increase folds transformations. Antibody titers below the cut-off of the assay

will be given an arbitrary value of half the cut-off for the purpose of GMT calculation.

• For a given subject and a given immunogenicity measurement, missing or non-evaluable

measurements will not be replaced. Therefore, an analysis will exclude subjects with missing or

non-evaluable measurements.



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• For the analysis of solicited symptoms, missing or non-evaluable measurements will not be

replaced. Therefore the analysis of the solicited symptoms based on the ITT cohort will include only

subjects/doses with documented safety data (i.e. symptom eligible assessment/sheet completed).

• For the analysis of unsolicited adverse events/serious adverse event/concomitant medication, all

vaccinated subjects will be included. Subjects who did not report an event will be considered as

subjects without an event.

12.412.412.412.4 DescriptionDescriptionDescriptionDescription ofofofof analysesanalysesanalysesanalyses

The analyses will evaluate efficacy, safety, immunogenicity, immune persistence, lot consistency

and exploratory endpoints. The analysis will be performed when the twelve months surveillance for

all participants from day 56 to month 14 have been completed as well as at least 31 cases of

EV71-associated HFMD confirmed by DSMC have been accrued in the ATP cohort for efficacy.

Vaccine efficacy will be estimated according to Cox regression. If the estimated risk in the vaccine

group is zero, the vaccine efficacy will be evaluated using an exact conditional procedure depended

on Poisson distribution.

Analyses for safety and immunogenicity endpoints will be descriptive, using 95%CI, Student t test

for log-transformed antibody titers, and Fisher’ exact or Chi-square for categorical data. Equivalence

test will be used for analyses of the lot consistency.

To assess the correlation between neutralizing antibody titer and protective efficacy, the sensitivity

(defined as the proportion of EV71 cases having a titre < the cutoff value at day 56 in total EV71

cases), specificity (defined as the proportion of participants without EV71-associated cases having a

titre ≥ the cutoff values at day 56 in total participants without EV71-associated cases) and

corresponding Youden indexes (the sum of sensitivity and specificity minus one) will be computed.

The best cutoff value with maximum Youden index will be considered as the correlate of protection.

Antibody titers below 1:8 will be assigned values of 1:4 for calculation.

The analyses will be detailed described in detail in the Statistical Analysis Plan (SAP).



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13.13.13.13. ETHICALETHICALETHICALETHICAL ANDANDANDAND LEGALLEGALLEGALLEGAL ISSUESISSUESISSUESISSUES

13.113.113.113.1 GuidelineGuidelineGuidelineGuideline

The study will be conducted according to Good Clinical Practice (GCP), the Declaration of Helsinki,

and local rules and regulations of China.

Submission of the protocol and any protocol amendments to regulatory agencies will occur in

accordance with local regulatory requirements. When submission to the local regulatory authority is

required, the timing of the submission relative to IEC/IRB submission or approval and whether or

not the authority will provide their approval of or favorable opinion on the protocol or amendment

before it can be implemented will depend on local regulatory requirements.

13.213.213.213.2 InstitutionalInstitutionalInstitutionalInstitutional ReviewReviewReviewReview BoardBoardBoardBoard

The investigator is responsible for obtaining written approval for the clinical study protocol

(including all substantial protocol amendments), the written subject informed consent form, informed

consent updates, subject recruitment procedures (e.g. advertisements) and any other written

information to be provided to subjects’ guardians from an Institutional Review Board which

complies with local regulatory requirements. Any amendments will require approval by the

Institutional Review Board.

Written approval of the study will be obtained from the JSCDC Institutional Review Board prior to

the study being implemented. The investigator is responsible for maintaining a copy of the approval

document in the study documentation files and for providing a copy to CRO.

The only circumstance in which an amendment may be initiated prior to JSCDC Institutional Review

Board approval is where the change is necessary to eliminate apparent immediate hazards to the

subjects. In that event, the investigator must notify the Institutional Review Board and Beijing Vigoo

in writing immediately after the implementation.

A final study notification will be forwarded by the investigator to the JSCDC Institutional Review

Board within 90 days after the study has been completed or in the event of premature termination of

the study within 15 days. Copies of all clinical study status reports (including termination) will be

provided by an investigator to CRO.



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13.313.313.313.3 ResponsibilitiesResponsibilitiesResponsibilitiesResponsibilities ofofofof thethethethe InvestigatorInvestigatorInvestigatorInvestigator

- To ensure that he/she has sufficient time to conduct and complete the study and has adequate staff

and appropriate facilities and equipment which are available for the duration of the study and to

ensure that other studies do not divert essential subjects or facilities away from the study at hand.

- To submit an up-to-date curriculum vitae or Investigator Biography and other credentials to Beijing

Vigoo and—where required—to relevant authorities. It is recommended that this documentation

indicates any previous clinical research experience and history of training in GCP.

- To acquire the reference ranges for laboratory tests performed locally and, if required by local

regulations, obtain the laboratory’s current certification or Quality Assurance procedure manual.

- To ensure that all back-up clinical samples (including serum samples) are retained onsite according

to the approval of Beijing Vigoo.

- To perform no other biological assays on the clinical samples except those described in the protocol

or its amendment(s).

- To prepare and maintain adequate subject source data or raw data designed to record observations,

and other data pertinent to the study.

- To conduct the study in compliance with the protocol any amendment and “Good Clinical Practice”

(GCP) and all applicable regulatory requirements.

- To co-operate with a representative of Beijing Vigoo in the monitoring process of the study and in

resolution of queries about the data.

- To permit drug regulatory agencies and Beijing Vigoo audits.

13.413.413.413.4 EthicalEthicalEthicalEthical ConductConductConductConduct ofofofof thethethethe StudyStudyStudyStudy

The study will be conducted in accordance with the principles of the Declaration of Helsinki1, the

standards of Good Clinical Practice (as defined by the International Conference on Harmonization),

and Chinese regulatory requirements, as stipulated by the Chinese Food and Drug Administration.

The policies and procedures of ShenzhenYingHeYuan Medical Technology Development Co., Ltd,

the Contract Research Organization (CRO) responsible for the monitoring of the trial, will also be

followed. These policies and procedures ensure that personnel are adequately trained to perform the



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study complying with these guidelines mentioned above. Written informed consent was obtained

from each subject’s guardians.

13.513.513.513.5 ProtocolProtocolProtocolProtocol AmendmentsAmendmentsAmendmentsAmendments andandandand AdministrativeAdministrativeAdministrativeAdministrative changeschangeschangeschanges

- No changes to the study protocol will be allowed unless discussed in detail with the Beijing Vigoo'

Clinical Research Manager/Medical Monitor and filed as an amendment/administrative change to

this protocol. This does not apply to changes made to reduce discomfort or avert risk to study

volunteers. Furthermore, in the event of a medical emergency, the investigators shall perform any

medical procedures that are deemed medically appropriate. The PI must notify the sponsor of all

such occurrences.

- Any amendment/administrative change to the protocol will be adhered to by the participating

centre(s) and will apply to all subjects. Written IRB/IEC approval of protocol amendments is

required prior to implementation, except where permitted by all applicable regulatory requirements;

administrative changes and amendments not submitted for approval are submitted to IRBs/IECs for

information only. Submission of protocol amendments to regulatory agencies will occur in

accordance with local regulatory requirements. When submission to the local regulatory authority is

required, the timing of the submission relative to IEC/IRB submission or approval and whether or

not the authority will provide their approval of or favorable opinion on the amendment before it can

be implemented will depend on local regulatory requirements.

13.613.613.613.6 ConfidentialityConfidentialityConfidentialityConfidentiality ofofofof DataDataDataData andandandandAccessAccessAccessAccess totototo SubjectSubjectSubjectSubject RecordsRecordsRecordsRecords

Prior to initiation of the trial, the Investigator will sign a fully executed confidentiality agreement

with the Sponsor. All study-related information will be stored securely at the study sites. All

participant information will be stored in locked file cabinets in areas with access limited to study

staff. All laboratory specimens, reports, study data collection, process, and administrative forms will

be identified by coded number only to maintain participant confidentiality. All computer entries will

be done by coded numbers only, and all local databases will be secured with password-protected

access systems. Forms, lists, logbooks, appointment books, and any other listings that link participant



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ID numbers to other identifying information will be stored in a separate, locked file in an area with

limited access.

Sponsor personnel, the IRB/IEC and the regulatory authorities will have direct access to source

data/documents.

14.14.14.14. FINANCIALFINANCIALFINANCIALFINANCIAL CONTRACTCONTRACTCONTRACTCONTRACT ANDANDANDAND INSURANCEINSURANCEINSURANCEINSURANCE COVERAGECOVERAGECOVERAGECOVERAGE

An agreement will be signed by all the parties involved in the trial’s performance, if relevant.

Adequate insurance coverage for all subjects to be included in the trial is supplied by the Sponsor.

15.15.15.15. EEEEARLYARLYARLYARLY TERMINATIONTERMINATIONTERMINATIONTERMINATIONOFOFOFOF TRIALTRIALTRIALTRIAL

If any of the following situation appear, the study may come to thethethethe earlyearlyearlyearly terminationterminationterminationtermination, under the

joint decision of sponsor, investigator and Data and Safety Monitoring Committees (DSMC).

― If occurrence of vaccination-associated adverse event grading 4 or serious adverse event,

sponsor, investigator and DSMC will have a panel meeting to determine whether the study

should be terminated;

― If occurrence of adverse events grading 3 or more in 15% of participants or more, sponsor,

investigator and DSMC will have a panel meeting to determine whether the study should be

terminated;

― If occurrence of adverse events grading 2 in 70% participants or more. Sponsor, investigator

and DSMC will have a panel meeting to determine whether the study should be terminated;

― Required by sponsor;

― Required by regulatory authority.

16.16.16.16. PUBLICATIONPUBLICATIONPUBLICATIONPUBLICATIONOFOFOFOF STUDYSTUDYSTUDYSTUDY RESULTSRESULTSRESULTSRESULTS

Beijing Vigoo has no objection to publication by Investigator of any information collected or

generated by Investigator, whether or not the results are favorable to the Investigational Product.



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However, to ensure against inadvertent disclosure of Confidential Information or unprotected

Inventions, Investigator will provide Beijing Vigoo an opportunity to review any proposed

publication or other type of disclosure before it is submitted or otherwise disclosed.

17.17.17.17. REFERENCESREFERENCESREFERENCESREFERENCES

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newborn mice by passive immunization with subunit VP1 vaccines and inactivated virus. Vaccine

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model of enterovirus 71 encephalomyelitis against disease. J Virol 2010;84(1):661-5.

18. Tung WS, Bakar SA, Sekawi Z, Rosli R. DNA vaccine constructs against enterovirus 71 elicit immune

response in mice. Genet Vaccines Ther 2007;5:6.

19. Foo DG, Alonso S, Chow VT, Poh CL. Passive protection against lethal enterovirus 71 infection in newborn

mice by neutralizing antibodies elicited by a synthetic peptide. Microbes Infect 2007;9(11):1299-6.

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22. Sivasamugham LA, Cardosa MJ, Tan WS, Yusoff K. Recombinant Newcastle Disease virus capsids

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18.18.18.18. SIGNATURESSIGNATURESSIGNATURESSIGNATURES

SPONSOR: Beijing Vigoo Biological Co.,LTD.

This Study Protocol was subject to critical review and has been approved.

Clinical Development Manager: [Signature]

Xin-Liang Sheng, PhD

Beijing Vigoo Biological Co.,LTD.

Date

LABORATORY TEST: National Institute for Food and Drug Control

Responsible Officer: [Signature]

Zheng-Lun Liang

National Institute for Food and Drug Control

Date

INVESTIGATOR:

I have reviewed this Study Protocol, including Appendices. I will conduct the clinical study as

described and will adhere to GCP and all the ethical and regulatory requirements stated.

I have read and understood the contents of the Investigator's Brochure.

Principal Investigator: [Signature]

Professor Fen-Cai Zhu

Center for Disease Control and Prevention, Jiangsu Province

Date