Conditions: Virus, Zika Zika Virus Disease Flavivirus Infections Healthy Participants
Keywords: Drug therapy
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 271
Healthy volunteers: yes

Brief summary:
The purpose of this study is to describe the safety, tolerability and immunogenicity of two doses of purified inactivated Zika virus vaccine (PIZV) given 28 days apart. Three different vaccine doses containing different protein concentrations (2, 5 or 10 microgram \[mcg\]) each, will be given as 2 dose schedule to flavivirus naive and primed healthy adults. Participants will be followed for 7 days post each dose for tolerability and up to 6 months post dose 2 for safety. Immunogenicity assessment will be performed at 28 days post each dose and 6 months post dose 2. In addition, the selected dose group and control group will be followed till 24 months post dose 2 for safety and persistence of immunity.

Detailed description:
The vaccine being tested in this study is called PIZV or TAK-426 adjuvanted with aluminum hydroxide. The Zika virus vaccine is being tested to provide safety and immunogenicity data to enable the vaccine to be further developed clinically.

The study will enroll approximately 240 participants. Participants will be randomly assigned (by chance, like flipping a coin) to one of the four groups-which will remain undisclosed to the study observer:

* Placebo
* PIZV: 2 microgram (mcg) Low Dose
* PIZV: 5 mcg Medium Dose
* PIZV: 10 mcg High Dose

All participants will be administered either placebo or PIZV by intramuscular (IM) injection into the middle third of the deltoid muscle, preferably in the non-dominant arm on Days 1 (Visit 1) and 29 (Visit 4).

This multi-center trial will be conducted in the United States and Puerto Rico. The overall time to participate in this study is up to 25 months. Participants will make multiple visits to the clinic on Days 1, 8, 29, 36, 57, 211, 393 and will be contacted by telephone on Day 133 (Visit 7) and Day 575 (Visit 9) and also visit the clinic on Day 757 (Visit 11) depending on the study arm, for a final follow-up assessment.

Arms:
arm 1: Placebo injection, intramuscular (IM), once on Day 1 (first dose) and Day 29 (second dose). arm 2: PIZV 0.5 mL, 2 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose). arm 3: PIZV 0.5 mL, 5 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose). arm 4: PIZV 0.5 mL, 10 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose). arm 5: Placebo injection, IM, once on Day 1 (first dose) and Day 29 (second dose). arm 6: PIZV 0.5 mL, 2 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose). arm 7: PIZV 0.5 mL, 5 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose). arm 8: PIZV 0.5 mL, 10 mcg antigen, IM injection, once on Day 1 (first dose) and Day 29 (second dose).

Interventions: intervention 1: Placebo intervention 2: PIZV
Intervention descriptions: intervention 1: Placebo (normal saline (0.9% NaCl) IM injection. intervention 2: Purified inactivated Zika virus vaccine with aluminum hydroxide adjuvant IM injection.

Protocol document:
Prot_000.pdf: 



Title: A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, and 
Dose Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in Flavivirus 
Naïve and Primed Healthy Adults Aged 18 to 49 Years 
NCT Number: NCT03343626
Protocol Approve Date: 12 September 2018 
Certain information within this protocol has been redacted (ie, specific content is masked irreversibly from 
view with a black/blue bar) to protect either personally identifiable information (PPD) or company 
confidential information (CCI). 
This may include, but is not limited to, redaction of the following: 
Named persons or organizations associated with the study.
Proprietary information, such as scales or coding systems, which are considered confidential 
information under prior agreements with license holder.
Other information as needed to protect confidentiality of Takeda or partners, personal information, or 
to otherwise protect the integrity of the clinical study.


TITLE PAGE
A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, and Dose 
Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in Flavivirus Naïve 
and Primed Healthy Adults Aged 18 to 49 Years
Safety, Immunogenicity, and Dose Ranging Study of Inactivated Zika Virus Vaccine in 
Healthy Adults
Sponsor:
Takeda Vaccines, Inc.
40 Landsdowne Street
Cambridge, MA 02139
USA
Study Identifier:
ZIK-101
IND Number:
017673
EudraCT Number:
Not applicable
Vaccine Name:

Investigational vaccine: Purified Inactivated Zika Virus Vaccine
(PIZV) candidate (TAK-426)

Placebo: saline solution
Date:
12 September 2018
Version:
Protocol Amendment 3 - Version 7.0
0002
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 4 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
INVESTIGATOR AGREEMENT
I confirm that I have read and that I understand this protocol, the Investigator’s Brochure, and any 
other product information provided by the sponsor. I agree to conduct this trial in accordance with 
the requirements of this protocol, and also protect the rights, safety, privacy, and well-being of trial 
subjects in accordance with the following:

The ethical principles that have their origin in the Declaration of Helsinki (1).
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International Council for Harmonisation of Technical Requirements for Pharmaceuticals for 
Human Use E6 Good Clinical Practice: Consolidated Guideline (2).
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All applicable laws and regulations, including, but not limited to those related to data privacy 
and clinical trial disclosure.

Regulatory requirements for reporting serious adverse events defined in Section 10.4 of this 
protocol.

Terms outlined in the Clinical Study Site Agreement.

Appendix A– Responsibilities of the Investigator.
I further authorize that my personal information may be processed and transferred in accordance 
with the uses contemplated in Appendix C of this protocol.
Signature of Investigator
Date
Investigator Name (print or type)
Investigator’s Title
Location of Facility (City, State)
Location of Facility (Country)
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 6 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
2.0
Interim Analysies:
A first interim analysis will be performed to include immunogenicity and 
safety data from all flavivirus naïve subjects up to Visit 6 (on Day 57, 
28 days post dose 2); a second interim analysis will be performed for dose 
selection, and including safety data from all flavivirus primed subjects up to 
Visit 4 (on Day 29, 28 days post dose 1). This interim analysis is not intended 
for early termination of the trial. A second interim analysis will be performed 
to include the data describing the persistence of immunity at 12 months post 
dose 2 and safety from 6 to 12 months post dose 2.
Final Analysis:
The final safety and immunogenicity analysis will be performed when all 
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development plan of PIZV. The subsequent analyses will include (1) the data 
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months post dose 2). The analyses will be performed by a separate set of 
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be involved in subsequent trial conduct. The study team at Takeda will have 
access to the group level unblinded results and will remain blinded to the 
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assignment until unblinding after trial completion (database lock for data 
through Visit 11). More details regarding the analyses will be provided in the 
Statistical Analysis Plan (SAP).
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post dose 2), and one to report the data from Visit 8 (Day 211) Visit 9 (Day 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 7 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
2.1
Visit 7 (Day 133) anchored to visit day Day 29 + 4½ 3½ mo
Visit 9 (Day 393) acceptable visit window (days before/days after) -7/+14 +97
footnote (h): Complete physical examination will be performed at Screening 
Visit(s) (Visit 1A and/or Visit 1B). All subsequent symptom-directed physical 
examinations, performed if deemed necessary or indicated by review of the 
subject’s medical history, should assess clinically significant changes from the 
baseline examination. The findings should be documented in the subject’s 
source document and; findings consistent with the definition of AEs or SAEs 
(see Section 10.0) must be transcribed into the electronic Case Report Form 
(eCRF). For any procedures at the site, the investigator shall follow his/her 
standard practice. Refer to Section 9.1.4.
9.1.4
The following should be documented in the subject’s source document and 
transcribed into the eCRF:

The findings of complete physical examinations,

the findings of symptom-directed physical examinations,

if symptom-directed physical examinations were not required
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 8 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
13.2
A first interim analysis will be performed to include immunogenicity and 
safety data from all flavivirus naïve subjects up to Visit 6 (on Day 57, 
28 days post dose 2); a second interim analysis will be performed for dose 
selection, and including safety data from all flavivirus primed subjects up to 
Visit 4 (on Day 29, 28 days post dose 1). This interim analysis is not intended 
for early termination of the trial. A second interim analysis will be performed 
to include the data describing the persistence of immunity at 12 months post 
dose 2 and safety from 6 to 12 months post dose 2.
The final safety and immunogenicity analysis will be performed when all 
(flavivirus naïve and primed) subjects have completed Visit 6 (Day 57) to 
provide data to support the planning and execution of other trials in the 
development plan of PIZV. The subsequent analyses will include (1) the data 
up to Visit 9 (12 months post dose 2) and (2) the data up to Visit 11 (24 
months post dose 2). The analyses will be performed by a separate set of 
unblinded statisticians and programmers at a Clinical Research Organization 
(CRO), who will have access to individual treatment assignments and will not 
be involved in subsequent trial conduct. The study team at Takeda will have 
access to the group level unblinded results and will remain blinded to the 
individual treatment assignment. The remaining personnel (not mentioned 
before) involved in the conduct of the trial, including those at Takeda, the 
CRO, and the trial sites, will remain blinded to the individual subject treatment 
assignment until unblinding after trial completion (database lock for data 
through Visit 11).
More details regarding the analyses will be provided in the SAP.
The final study report will be written for the data up to Visit 6 (Day 57), and 
two additional addenda will be written: one to report the data from Visit 6 (Day 
57) to Visit 8 (Day 211, 6 months post dose 2) to Visit 9 (Day 393, 12 months 
post dose 2), and one to report the data from Visit 8 (Day 211) Visit 9 (Day 
393) until the end of the study, ie, Visit 11 (Day 757, 24 months post dose 2).
0009
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 9 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
TABLE OF CONTENTS
1.0
ADMINISTRATIVE INFORMATION ..............................................................................2
1.1
Contacts..........................................................................................................................2
1.2
Approval ........................................................................................................................3
1.3
Protocol Amendment 3 Summary of Changes...............................................................5
1.3.1
Protocol Change History..........................................................................................5
1.3.2
Summary of Changes...............................................................................................5
2.0
TRIAL SUMMARY ..........................................................................................................14
2.1
Schedule of Trial Procedures.......................................................................................25
3.0
TRIAL REFERENCE INFORMATION...........................................................................29
3.1
Trial-Related Responsibilities......................................................................................29
3.2
Principal Investigator/Coordinating Investigator.........................................................29
3.3
List of Abbreviations ...................................................................................................30
3.4
Corporate Identification...............................................................................................32
4.0
INTRODUCTION .............................................................................................................33
4.1
Background..................................................................................................................33
4.1.1
Zika Virus ..............................................................................................................33
4.1.2
Epidemiology.........................................................................................................33
4.1.3
Transmission..........................................................................................................33
4.1.4
ZIKV Infection: Detection, Disease, Complications and Treatment.....................34
4.2
Rationale for the Proposed Trial..................................................................................36
4.2.1
Medical Need.........................................................................................................36
4.2.2
BARDA Funding Award .......................................................................................37
4.2.3
Vaccines Under Development ...............................................................................37
4.2.4
Summary of Available Non-clinical Data..............................................................37
4.2.5
Summary of Available Clinical Data.....................................................................37
4.2.6
Takeda Vaccine Candidate ....................................................................................37
4.2.7
Rationale for ZIK-101 Trial...................................................................................38
5.0
TRIAL OBJECTIVES AND ENDPOINTS ......................................................................40
5.1
Objectives ....................................................................................................................40
5.1.1
Primary Objectives.................................................................................................40
5.1.2
Secondary Objectives.............................................................................................40
5.2
Endpoints .....................................................................................................................40
5.2.1
Primary Endpoints .................................................................................................40
5.2.2
Secondary Endpoints .............................................................................................41
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 10 of 108
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6.0
TRIAL DESIGN AND DESCRIPTION ...........................................................................42
6.1
Trial Design .................................................................................................................42
6.2
Justification for Trial Design, Dose, and Endpoints....................................................47
6.3
Duration of Subject’s Expected Participation in the Entire Trial ................................47
6.4
Premature Termination or Suspension of Trial or Investigational Site .......................47
6.4.1
Criteria for Premature Termination or Suspension of the Trial.............................47
6.4.2
Criteria for Premature Termination or Suspension of Investigational Sites..........47
6.4.3
Procedures for Premature Termination or Suspension of the Trial or the 
Participation of Investigational Site(s)...................................................................48
7.0
SELECTION AND DISCONTINUATION/WITHDRAWAL OF SUBJECTS...............49
7.1
Inclusion Criteria .........................................................................................................49
7.2
Exclusion Criteria ........................................................................................................49
7.3
Criteria for Delay of Vaccination and/or Blood Sampling ..........................................52
7.4
Criteria for Early Study Termination of a Subject.......................................................52
7.5
Criteria for Premature Discontinuation of Trial Vaccine Application.........................53
8.0
CLINICAL TRIAL MATERIAL MANAGEMENT.........................................................55
8.1
Trial Vaccines and Materials .......................................................................................55
8.1.1
Dosage Form, Manufacturing, Packaging, and Labeling ......................................55
8.1.2
Storage ...................................................................................................................55
8.1.3
Dose and Regimen .................................................................................................55
8.2
Investigational Vaccine/Placebo Assignment and Dispensing Procedures .................56
8.3
Randomization Code Creation and Storage.................................................................57
8.4
Investigational Product Blind Maintenance.................................................................57
8.5
Unblinding Procedure..................................................................................................57
8.6
Accountability and Destruction of Sponsor-Supplied Vaccine/Placebo .....................58
9.0
TRIAL PLAN ....................................................................................................................60
9.1
Trial Procedures...........................................................................................................60
9.1.1
Informed Consent...................................................................................................60
9.1.2
Demographics, Medical History, Travel History, Prior/Concomitant 
Medications/Vaccinations, and Blood Donation ...................................................60
9.1.3
Documentation of Trial Entrance/Randomization.................................................61
9.1.4
Physical Examination.............................................................................................61
9.1.5
Vital Signs..............................................................................................................62
9.1.6
Blood Sample and Urine Sample Collection .........................................................62
9.1.7
Safety Assessments................................................................................................65
9.1.8
Contraception and Avoidance of Sexually Transmitted Disease Guidance ..........65
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 11 of 108
Protocol Amendment 3 Version 7.0 
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9.1.9
Pregnancy...............................................................................................................65
9.1.10 Documentation of Subjects who are not Randomized...........................................66
9.2
Monitoring Subject Treatment Compliance.................................................................67
9.3
Schedule of Observations and Procedures...................................................................67
9.3.1
Screening Procedures (Screening Visit 1A/ Screening Visit 1B)..........................67
9.3.2
Pre-Vaccination Procedures (Day 1 and Day 29)..................................................67
9.3.3
Vaccination Procedures (Day 1 and Day 29).........................................................68
9.3.4
Post Vaccination Procedures (Day 1 and Day 29).................................................68
9.3.5
Clinic Visits after Vaccination (Day 8, Day 36, Day 57, Day 211, and Day 393)
................................................................................................................................70
9.3.6
Phone Contacts (Day 133 and Day 575)................................................................71
9.3.7
Final Visit (Day 211 or Day 757) ..........................................................................71
9.3.8
Post-Trial Care.......................................................................................................72
9.4
Biological Sample Retention and Destruction.............................................................72
10.0
ADVERSE EVENTS.........................................................................................................73
10.1
Definitions....................................................................................................................73
10.1.1 Adverse Events (AEs)............................................................................................73
10.1.2 Solicited Adverse Events .......................................................................................73
10.1.3 Serious Adverse Events (SAEs).............................................................................76
10.2
Causality of AEs ..........................................................................................................76
10.2.1 Relationship to Trial Procedures............................................................................76
10.2.2 Outcome of AEs.....................................................................................................77
10.3
Additional Points to Consider for AEs ........................................................................77
10.4
Procedures....................................................................................................................78
10.4.1 Collection and Reporting of AEs...........................................................................78
10.4.2 Collection and Reporting of Solicited AEs............................................................79
10.4.3 Collection and Reporting of SAEs.........................................................................79
10.5
Follow-up Procedures ..................................................................................................80
10.5.1 AEs.........................................................................................................................80
10.5.2 SAEs ......................................................................................................................80
10.5.3 Safety Reporting to Investigators, IRBs or IECs, and Regulatory Authorities .....80
10.5.4 Post-Trial Events....................................................................................................80
11.0
TRIAL-SPECIFIC REQUIREMENTS .............................................................................81
11.1
Data Monitoring Committee Guidelines for Possible Study Pause.............................81
11.2
Trial-Specific Committees...........................................................................................81
11.2.1 External Data Monitoring Committee ...................................................................81
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 12 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
12.0
DATA HANDLING AND RECORDKEEPING ..............................................................82
12.1
Electronic CRFs (eCRF)..............................................................................................82
12.2
Record Retention .........................................................................................................82
13.0
STATISTICAL METHODS..............................................................................................84
13.1
Statistical and Analytical Plans....................................................................................84
13.1.1 Analysis Sets..........................................................................................................84
13.1.2 Analysis of Demographics and Other Baseline Characteristics ............................84
13.1.3 Immunogenicity Analysis......................................................................................84
13.1.4 Safety Analysis ......................................................................................................85
13.2
Interim Analysis and Sequence of Analyses................................................................86
13.3
Determination of Sample Size .....................................................................................86
14.0
QUALITY CONTROL AND QUALITY ASSURANCE.................................................87
14.1
Trial-Site Monitoring Visits.........................................................................................87
14.2
Protocol Deviations......................................................................................................87
14.3
Quality Assurance Audits and Regulatory Agency Inspections..................................87
15.0
ETHICAL ASPECTS OF THE TRIAL.............................................................................88
15.1
IRB and/or IEC Approval............................................................................................88
15.2
Subject Information, Informed Consent and Subject Authorization ...........................88
15.3
Subject Confidentiality ................................................................................................89
15.4
Publication, Disclosure, and Clinical Trial Registration Policy ..................................90
15.4.1 Publication and Disclosure ....................................................................................90
15.4.2 Clinical Trial Registration......................................................................................90
15.4.3 Clinical Trial Results Disclosure ...........................................................................90
15.5
Insurance and Compensation for Injury.......................................................................90
16.0
REFERENCES ..................................................................................................................92
0013
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 13 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
LIST OF IN-TEXT TABLES
Table 1.a
Contact Information.................................................................................................2
Table 8.a
Sponsor-Supplied Vaccine and Placebo ................................................................56
Table 9.a
Clinical Laboratory Assessments...........................................................................64
Table 10.a
Local and Systemic AEs ........................................................................................74
Table 10.b
Solicited Safety Parameters ...................................................................................75
Table 16.a
Table for Laboratory Abnormalities (Serum)......................................................106
Table 16.b
Table for Laboratory Abnormalities (Hematology).............................................107
Table 16.c
Table for Laboratory Abnormalities (Urine) .......................................................108
Table 16.d
Table for Clinical Abnormalities (Vital Signs)....................................................108
LIST OF IN-TEXT FIGURES
Figure 2.a
Schematic Representation of Trial Design ............................................................17
Figure 2.b
Staggered Enrollment.............................................................................................18
Figure 6.a
Schematic Representation of Trial Design ............................................................43
Figure 6.b
Staggered Enrollment.............................................................................................45
LIST OF APPENDICES
Appendix A
Responsibilities of the Investigator......................................................................100
Appendix B
Elements of the Subject Informed Consent Form................................................101
Appendix C
Investigator Consent to Use of Personal Information..........................................104
Appendix D
CDC Websites......................................................................................................105
Appendix E
FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials ...................................................106
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 14 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
2.0
TRIAL SUMMARY
Takeda Vaccines, Inc.
40 Landsdowne Street
Cambridge, MA 02139
USA
Product Name:
Purified Inactivated Zika Virus Vaccine (PIZV) candidate 
(TAK-426)
Trial Title: A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, and Dose 
Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in Flavivirus Naïve and Primed Healthy
Adults Aged 18 to 49 Years
IND No.: 017673
EudraCT No.: Not applicable
Study Identifier: ZIK-101
Phase: 1
Trial Blinding Schema: Observer-blind
Background and Rationale:
Zika virus (ZIKV) is a mosquito-borne single-stranded positive-sense ribonucleic acid (RNA) virus which belongs to 
the Flaviviridae family, genus Flavivirus, that presents structural similarities with other members of the Flaviviridae, 
including Spondweni virus, Yellow Fever (YF) virus, Dengue virus (DENV), West Nile virus (WNV), Japanese 
Encephalitis (JE) virus, and Tick-borne Encephalitis virus.
ZIKV was discovered in 1947 in Uganda while testing samples taken from sentinel rhesus monkeys used as bait 
during a surveillance of YF. The first human disease cases were reported in 1952 in Uganda and Tanzania. For 
55 years, the virus circulated in areas of Africa, Southeast Asia, and the Pacific Islands, and was considered a rare 
disease with mild symptoms; only 14 human cases were reported and no outbreaks were detected during that period.
The first large outbreak of ZIKV disease was reported in Yap Island (Federal state of Micronesia) in 2007, and was 
followed by an outbreak in French Polynesia in 2013-2014. The first cases of ZIKV disease in South America were 
reported in Easter Island in 2014, and the first reports of locally transmitted infection (ie, by mosquito) came from 
Brazil in May 2015, which triggered the third outbreak in 2015-2016. Since then, ZIKV disease has continued 
spreading through the South American continent and to other regions. In the current outbreak that started in 2015, 
there have been estimates of more than 750,000 ZIKV-suspected cases reported from more than 
60 countries/territories; most of these cases are from the South America continent.
The primary mode of transmission of ZIKV is through the bite of female mosquitoes, prominently Aedes aegypti and 
Ae. albopictus species that have spread globally. Vertical transmission from mother to fetus during pregnancy and the 
perinatal period, sexual contact, and blood transfusion are also confirmed modes of transmission of the disease. 
Potentially, the virus could be transmitted through laboratory exposure and by body fluid contact, eg, tears, urine, 
breast milk, or through solid organ transplants, but there is limited evidence to support this.
More than 80% of ZIKV infections remain asymptomatic, and for the symptomatic cases, the disease is in general 
mild and of short duration. Some clinical manifestations include, but are not limited to, mild fever, maculopapular 
rash, conjunctivitis and arthralgia.
Despite mild clinical symptoms in the pregnant woman, ZIKV infection during pregnancy has been associated with 
serious outcomes for the fetus and newborn. The severity of the disease is related to the consequences in the fetus and 
newborn child from women with ZIKV infection during pregnancy. The spectrum of congenital anomalies associated 
with ZIKV infection, known as Congenital Zika Syndrome (CZS), consists of severe microcephaly with partially 
collapsed skull, cerebral cortices with subcortical calcifications, macular scarring and focal pigmentary retinal 
mottling, congenital contractures, and marked early hypertonia with symptoms of extrapyramidal involvement. The 
exposure to ZIKV at any moment during pregnancy is a risk for fetal or postnatal developmental complications, 
primarily affecting brain development. Recent analysis of the U.S. Zika Pregnancy Registry (USZPR) showed that the 
prevalence of newborns with congenital defects from pregnancies with laboratory-confirmed ZIKV infection is 10% 
(95% CI: 7%-14%). This is 30 times higher than baseline prevalence before more recent Zika outbreaks.
Since the beginning of these outbreaks and up to end of September 2017, there have been 3689 confirmed CZS 
malformations potentially associated with ZIKV infection in 27 countries and territories of Latin America and the 
Caribbean.
ZIKV is a neurotropic flavivirus that can potentially cause disease within the central nervous system. Worldwide 
0015
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 15 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
concern over ZIKV causing Guillain-Barré syndrome (GBS) emerged when an increased number of GBS cases was 
temporally associated with the 2013-2014 ZIKV outbreak in French Polynesia, during which 42 GBS cases were 
reported, all of these cases had neutralizing antibodies against ZIKV, compared to 56% in the control group. Other 
neurological complications, such as encephalitis, meningoencephalitis, paresthesia, facial paralysis and myelitis, have 
also been associated with ZIKV infection. Based on these findings, the countries affected by ZIKV disease in the 
2015-2016 outbreak improved and extended the surveillance of neurological syndromes. In 2017, after the spread of 
ZIKV throughout the Pacific Islands, Latin America and the Caribbean, a total of 200 cases of GBS related to ZIKV 
infection have been reported in these regions as case series or isolated cases. The incidences reported have increased 2 
to 9.8 times compared to baseline before the epidemic in Latin America, and up to 20 times in French Polynesia. The 
association of GBS was only reported when the circulating strain was of Asian lineage. As of 01 February 2017, ZIKV 
of the Asian lineage was circulating in more than 80% of affected countries, including 48 countries in Latin America. 
The overall incidence of ZIKV-associated GBS is estimated to be 24 per 100,000 cases of ZIKV infection.
No specific antiviral treatment is available for ZIKV infections and no vaccine against ZIKV is currently available. As 
the disease is self-limiting, treatment for uncomplicated ZIKV infection is supportive and focuses on symptoms. The 
main recommendations to address outbreaks are through prevention (avoiding mosquito bites, reducing sexual 
transmission, improving the procedures for safe blood transfusion), and vector control measures. 
The risk of infection with ZIKV is increasing given that the spread of ZIKV is rapid and intense in tropical and 
subtropical countries. Vector control is difficult to establish and maintain, and new areas continue to be colonized by 
the Aedes species. Climate changes may also create favorable environments to maintain the vector. There is an 
increased urban population and mobility, and the role of sexual transmission may be an important mechanism to 
spread rapidly the virus into susceptible populations, even in regions where the vector is uncommon.
ZIKV has posed a challenging situation for health, public and economic sectors of affected countries. The World 
Health Organization (WHO) declared on 01 February 2016 the Zika outbreak as a Public Health Emergency of 
International Concern (PHEIC) and recommended to focus the research on the causal association of ZIKV infection 
during pregnancy and microcephaly and other neurological abnormalities in newborns. By August 2016, the U.S. 
Department of Health and Human Services declared Zika a public health emergency in Puerto Rico based on the 
significant threat to pregnant women and their children’s health. In November 2016, and after evaluating the evidence 
about the causal association of ZIKV infection during pregnancy and microcephaly and other neurological 
abnormalities in newborns, WHO announced that Zika no longer represents a PHEIC but stressed the need to escalate 
the disease to a sustained work program with dedicated resources to address its long-term nature and its associated 
consequences.
Considering the conclusive associations between ZIKV infections and fetal and postnatal abnormalities, the 
development of a vaccine that can provide protection is crucial for countries where the epidemic is expected to arrive 
and/or persist, as well as in countries in which the virus has not yet been introduced. In order to address the urgent 
medical need and in anticipation of possible outbreaks with rapid onsets, Takeda has initiated the development of a 
purified inactivated ZIKV vaccine candidate, PIZV, for use in endemic areas and non-endemic areas for prevention of 
ZIKV associated illness of any severity and/or infection.
The Biomedical Advanced Research and Development Authority (BARDA) from the U.S. Department of Health and 
Human Services’ Office of the Assistant Secretary for Preparedness and Response is providing funding to Takeda for 
the US-based PIZV development program. Due to the need to vaccinate women and men of reproductive age, the 
target population of PIZV will be 9 years of age and older. In this phase 1 study, study population is limited to ≥18 to 
≤49 years of age, and age expansion will be included in the next phase of development.
There are no clinical data currently available for PIZV. PIZV will be tested for nonclinical safety in a 3-dose Good 
Laboratory Practice-compliant, repeat-dose toxicity and local tolerance study in New Zealand White Rabbits. 
Toxicology data will be available prior to study start and provided in the Investigator’s Brochure (IB). 
ZIK-101 is a first-in-human study. It will enroll both flavivirus naïve and primed healthy adults aged ≥18 to ≤49 years. 
To ensure the enrollment of healthy adults, screening tests (hematology, biochemistry and urinalysis) will be 
performed prior to vaccine/placebo administration.
Flavivirus primed adults are included in this study to assess if there is any impact from pre-existing flavivirus 
antibodies. The testing of flaviviral serological status will be performed prior to randomization and will enable 
0016
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 16 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
stratification of randomization.
Flavivirus naïve subjects will be enrolled during the first phase of the study. If positive recommendations from an 
external Data Monitoring Committee (DMC) are issued following the review of safety and tolerability data in this 
population, when the last flavivirus naïve subject has completed study Visit 4 (28 days post dose 1), vaccination of 
flavivirus primed subjects will start.
Due to the ongoing threat of ZIKV disease and the urgent need of a vaccine, this phase 1 study will be accelerated to 
assess dose finding for PIZV, in addition to evaluation of safety and immunogenicity of the vaccine candidate. PIZV 
dose will be selected based on descriptive safety data in both flavivirus-naïve and primed subjects, and geometric 
mean titers (GMT) of neutralizing anti-ZIKV antibody levels at 28 days post dose 2 in flavivirus naïve subjects. 
Placebo serves as the control for the study vaccine and in the absence of effective treatment or prevention for ZIKV 
disease, the use of placebo in this trial is justified.
The rationale for the observer-blind approach is based on the different physical appearance of the investigational 
vaccine compared to the saline solution placebo that was selected and preferred to alum-containing placebo to avoid 
side effects associated with alum.
All enrolled subjects will receive two 0.5 mL doses of either PIZV or placebo intramuscularly (IM) on Day 1 and on 
Day 29. The objective of ZIK-101 is to assess the safety of PIZV and select a single vaccine dose level from three 
different antigen concentrations (2, 5 or 10 µg) for further development. 
The primary rationale for selection of the three vaccine dose levels can be summarized as follows: 

the low-dose level (2 µg) was selected for purposes of potentially identify an immunological threshold, and 
was based on the limit of the analytical methods for the drug product available at the time;

the mid-dose level (5 µg) was selected to provide a mid-point on the dose response curve. In addition, this 
dose level is based on the Walter Reed Army Institute of Research (WRAIR) vaccine currently tested in 
Phase 1, which was originally based on initial studies in non-human primates (NHPs); and

the high-dose level (10 µg) was selected to follow an approximate 2-fold range and to further define the 
potential dose-response curve in both naïve and primed subjects.
The trial will be conducted in accordance with the protocol, the International Council for Harmonization of Technical 
Requirements for Pharmaceuticals (ICH) GCP Guidelines and applicable regulatory requirements.
Trial Design:
This is a phase 1, randomized, observer-blind, placebo-controlled, safety, immunogenicity, and dose ranging study of 
PIZV candidate in flavivirus naïve and primed healthy adults aged ≥18 to ≤49 years, in ZIKV endemic and 
non-endemic regions.
The trial will last approximately 7 or 25 months for each subject, following a screening period prior to Visit 2. The 
screening period will last for up to 2 weeks (14 days) prior to randomization, with the possibility that some subjects 
(Flavivirus primed subjects only) will need to repeat some screening procedures if they were not randomized right 
after the screening period (further clarification is provided in the schedule of trial procedures).
Flavivirus naïve subjects: subjects without detectable serum antibodies against a panel of flaviviruses, as measured by 
a reactive antibody based assay (Luminex). 
Flavivirus primed subjects: subjects with serum antibodies against a panel of flaviviruses, as measured by a reactive 
antibody based assay (Luminex).
Approximately 240 subjects aged ≥18 to ≤49 years will be enrolled and equally randomized into four groups (1:1:1:1) 
of approximately 60 subjects, each comprised of approximately 30 flavivirus naïve subjects and approximately 
30 flavivirus primed subjects. Randomization within each cohort (flavivirus naïve versus flavivirus primed subjects) 
will be stratified by flavivirus serostatus and age group: ≥18 to ≤29 years versus ≥30 to ≤49 years.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 17 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Figure 2.a
Schematic Representation of Trial Design
Staggered enrollment: There will be two study phases.

The first phase of the study will be aimed at enrolling flavivirus naïve subjects only.
All subjects who sign the informed consent form (ICF) and have confirmed eligibility at Screening Visit 1A 
will be tested for flavivirus serostatus.
Eligible flavivirus naïve subjects enrolled at Screening Visit 1A will be randomized at Visit 2, within 
two weeks after Screening Visit 1A.
Flavivirus primed subjects will not be enrolled during the first phase of the study (ie, when identified at 
Screening Visit 1A) and will be invited to come back for the second phase of the study. They will be 
contacted after positive recommendations from the external DMC are issued following the review of safety 
and tolerability data in flavivirus naïve subjects, when the last flavivirus naïve subject has completed study 
Visit 4 (28 days post dose 1). These flavivirus primed subjects will consequently undergo 2 screening visits: 
Visit 1A during the first phase of the study, and Visit 1B at the outset of the second phase of the study. 
Flavivirus primed subjects identified at Screening Visit 1A who do not participate in the second phase of the 
study will be declared screened failure (see Figure 2.b §).
Enrollment will be put on hold after all naïve subjects have been enrolled and will resume when positive 
recommendations from the external DMC are issued, as described above.

The second phase of the study will be aimed at enrolling flavivirus primed subjects only.
Newly identified eligible flavivirus primed subjects at Screening Visit 1B will be enrolled (randomized) at 
(second phase) Visit 2, within two weeks after Screening Visit 1B.
Previously identified flavivirus primed subjects (see Figure 2.b §§) (who were tested at Screening Visit 1A 
during the first phase and accepted to enter the second phase of the study) will retain their initial subject 
identification (ID) number and undergo an additional screening visit (Visit 1B) to be tested for eligibility 
criteria again (except for the determination of flavivirus serostatus). 
Flavivirus naïve subjects identified at Screening Visit 1B (during the second phase of the study) will be 
declared screened failure (see Figure 2.b §§§).
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 18 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Figure 2.b
Staggered Enrollment
* All subjects who were randomized to the placebo group or the PIZV dosing group that was selected for further 
development will attend Visit 9 at Day 393 (12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), 
and will also be contacted by phone on Day 575 (Visit 10) for safety follow-up.
0019
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 19 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Each subject will receive two 0.5 mL doses of either PIZV (2, 5 or 10 µg) or placebo intramuscularly (IM) – one dose 
at Visit 2 (on Day 1) and one dose at Visit 4 (on Day 29) IM into the middle third of the deltoid muscle, preferably in 
the non-dominant arm.
Each subject will be required to attend 7, 8, 9 or 10 clinical visits and will receive 1 or 2 phone calls depending on their 
flavivirus serostatus at screening and the vaccine dose level they received:

Flavivirus naïve subjects identified during the Screening Visit 1A and flavivirus primed subjects identified 
during the Screening Visit 1B will attend 7 visits and will receive 1 phone call, and possibly 2 additional 
visits and 1 phone call (thus a maximum of 9 visits and 2 phone calls) if they were randomized to the placebo 
group or the PIZV dosing group that was selected for further development 

Flavivirus primed subjects identified during the Screening Visit 1A will attend 8 visits (including Visit 1A 
and Visit 1B) and will receive 1 phone call, and possibly 2 additional visits and 1 phone call (thus a maximum 
of 10 visits and 2 phone calls) if they were randomized to the placebo group or the PIZV dosing group that 
was selected for further development
All randomized subjects will thus attend Screening Visit(s) (Visit 1A and/or Visit 1B), Visit 2 at Day 1, Visit 3 at 
Day 8 (7 days post dose 1), Visit 4 at Day 29 (28 days post dose 1), Visit 5 at Day 36 (7 days post dose 2), Visit 6 at 
Day 57 (28 days post dose 2), and Visit 8 at Day 211 (6 months post dose 2). All randomized subjects will also be 
contacted by phone on Day 133 (Visit 7) for safety follow-up. In addition, subjects who were randomized to the 
placebo group or the PIZV dosing group that was selected for further development will attend Visit 9 at Day 393 
(12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), and will also be contacted by phone on 
Day 575 (Visit 10) for safety follow-up.
Blood samples will be collected at each site visit: ie, at Screening Visit(s) (Visit 1A and/or Visit 1B) for flavivirus 
serostatus determination and eligibility screening tests (including pregnancy testing), at Visits 3 and 5 for routine 
safety laboratory testing, and at Visits 2, 4, 6, 8, and (for subjects who will be randomized to the placebo group or the 
PIZV dosing group that will be selected for further development) 9 and 11 for immunogenicity, as well as for further 
development and characterization of assays.
Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) for eligibility screening 
and at Visits 3 and 5 for routine safety laboratory testing; and for women of childbearing potential at Visits 2 and 4 
before each investigational vaccine/placebo administration for pregnancy testing. 
Each subject will receive diary cards to collect solicited adverse events (AEs) for 7 days after each dose (including the 
days of vaccine/placebo administration), and unsolicited AEs for 28 days after each dose (including the days of 
vaccine/placebo administration). Additional safety assessments will include new medical conditions (neurological and 
neuroinflammatory disorders) with onset after the first vaccination, and serious adverse events (SAEs) collection for 
the duration of the entire trial. Immunogenicity will be assessed for all subjects 28 days after each vaccine/placebo 
administration and 6 months post dose 2, and for subjects who will be randomized to the placebo group or the PIZV 
dosing group that will be selected for further development also 12 and 24 months post dose 2.
Primary Objectives

To describe the safety of two doses of PIZV given 28 days apart from three different antigen concentrations 
(2, 5 or 10 µg) in flavivirus naïve and primed healthy adults through 28 days post dose 2

To select a single vaccine dose level of PIZV for further clinical development
Secondary Objectives

To describe the safety of two doses of PIZV given 28 days apart in flavivirus naïve and primed healthy adults 
through the end of the study

To describe the immune response to PIZV in flavivirus naïve and primed healthy adults at the following 
immunogenicity time points 28 days post dose 1, 28 days post dose 2, and 6 months post dose 2

To describe the persistence of immunity to PIZV at 12 and 24 months post dose 2 in flavivirus naïve and 
primed healthy adults from the placebo group and the PIZV dosing group that will be selected for further 
development
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 20 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Subject Population:
Healthy subjects: Yes
Planned Age Range: ≥18 to ≤49 years 
Planned Number of Subjects: approximately 240
Planned Number of Arms: 4 arms (n=60 per arm: approximately 30 flavivirus naïve subjects and approximately 
30 flavivirus primed subjects)

Arm 1: Placebo saline solution

Arm 2: PIZV 2 µg (Low) dose 

Arm 3: PIZV 5 µg (Medium) dose 

Arm 4: PIZV 10 µg (High) dose 
Criteria for Inclusion:
Subject eligibility is determined according to the following criteria:

The subject is aged ≥18 to ≤49 years.

Individuals who are in good health at the time of entry into the trial as determined by medical history, 
physical examination (including vital signs) and eligibility screening tests (hematology, biochemistry and 
urinalysis) and clinical judgment of the investigator. Vital signs must be within normal limits (ie, below 
Grade 1 as specified in the FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers). 
Screening tests must be within normal limits or not be above Grade 1 as defined in the FDA Toxicity Grading 
Scale for Healthy Adult and Adolescent Volunteers.

The subject signs and dates a written, informed consent form and any required privacy authorization prior to 
the initiation of any trial procedures, after the nature of the trial has been explained according to local 
regulatory requirements.

Individuals who can comply with trial procedures and are available for the duration of follow-up.

All female participants of childbearing potential must have a negative serum beta human chorionic 
gonadotropin (-hCG) pregnancy test at screening and a negative urine -hCG pregnancy test prior to 
receiving any dose of investigational vaccine/placebo.
Criteria for Exclusion:
Any subject who meets any of the following criteria will not qualify for entry into the trial: 

Subjects and subjects’ partners with confirmed ZIKV infection by self-report.

Traveling to flavivirus endemic countries or flavivirus endemic regions of the US/US territories*, within 
4 weeks prior to screening or planned travel through to Visit 6 (applicable only to subjects to be enrolled into 
the flavivirus naïve cohort).
*CDC website defines the information about the flavivirus endemic countries and US regions and territories 
(see Appendix D for the respective website addresses).

Known hypersensitivity or allergy to any of the vaccine candidate components (including excipients of the 
investigational vaccine or placebo).

Individuals with behavioral or cognitive impairment or psychiatric disease that, in the opinion of the 
investigator, may interfere with the subject's ability to participate in the trial.

Individuals with any history of progressive or severe neurologic disorder, seizure disorder or 
neuro-inflammatory disease (eg, Guillain-Barré syndrome).

Individuals with history or any illness that, in the opinion of the investigator, might interfere with the results 
of the trial or pose additional risk to the subjects due to participation in the trial.

Known or suspected impairment/alteration of immune function, including:
0021
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 21 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
o
Chronic use of oral steroids (equivalent to 20 mg/day prednisone ≥12 weeks / ≥2 mg/kg body 
weight / day prednisone ≥2 weeks) within 60 days prior to Day 1 (use of inhaled, intranasal, or 
topical corticosteroids is allowed).
o
Receipt of parenteral steroids (equivalent to 20 mg/day prednisone ≥12 weeks / ≥2 mg/kg body 
weight / day prednisone ≥2 weeks) within 60 days prior to Day 1.
o
Receipt of immunostimulants within 60 days prior to Day 1.
o
Receipt of parenteral, epidural or intra-articular immunoglobulin preparation, blood products, 
and/or plasma derived products within 3 months prior to Day 1 or planned during the full length of 
the trial. In addition, subjects must be advised not to donate blood during the study period. 
o
Known Human Immunodeficiency Virus (HIV) infection or HIV-related disease.
o
Genetic immunodeficiency.

Individuals with known current or chronic hepatitis B and/or hepatitis C infections.

Abnormalities of splenic or thymic function.

Individuals with a known bleeding diathesis, or any condition that may be associated with a prolonged 
bleeding time.

Individuals with any serious chronic or progressive disease according to judgment of the investigator (eg, 
neoplasm, insulin-dependent diabetes, cardiac, renal, hepatic or thyroid disease, uncontrolled hypertension, 
uncontrolled asthma).

Individuals with body mass index (BMI) greater than or equal to 35 kg/m2 (= weight in kg / [height in meters 
x height in meters]).

Individuals participating in any clinical trial with another investigational product, including ZIKV vaccine 
clinical trial within 30 days prior to first trial visit or intent to participate in another clinical trial at any time 
during the conduct of this trial.

Individuals who received any other vaccines within 14 days (for inactivated vaccines) or 28 days (for live 
vaccines) prior to Day 1 in this trial or who are planning to receive any vaccine within 28 days of 
investigational vaccine/placebo administration.

Individuals involved in the trial conduct or their first degree relatives.

Individuals with history of substance or alcohol abuse within the past 2 years.

Female subjects who are pregnant or breastfeeding, or are planning to become pregnant.

Any positive or indeterminate pregnancy test.

If female subject of childbearing potential, sexually active, and who has not used any of the "acceptable 
contraceptive methods" for at least 2 months prior to trial entry:
o
“Of childbearing potential” is defined as status post onset of menarche and not meeting any of the 
following conditions: menopausal for at least 2 years without any other alternative medical cause (as 
confirmed by a healthcare professional), status after bilateral tubal ligation for at least 1 year, status 
after bilateral oophorectomy, or status after hysterectomy.
o
Acceptable birth control methods are defined as one or more of the following:

Hormonal contraceptive (such as oral, injection, transdermal patch, implant, cervical ring).

Barrier (condom with spermicide or diaphragm with spermicide) each and every time 
during intercourse.

Intrauterine device.

Monogamous relationship with vasectomized partner. Partner must have been 
vasectomized for at least six months prior to the subjects’ trial entry.

If female subject of childbearing potential and sexually active, refusal to use an "acceptable contraceptive 
method" from trial entry through 2 months after the last dose of investigational vaccine/placebo. In addition, 
female subjects of childbearing potential must be advised not to donate ova during this period.
0022
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 22 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018

To avoid sexual transmission of ZIKV from natural exposure: Refusal to use latex condoms correctly and 
consistently by sexually active subjects even if other contraceptive measures are used from signing the ICF 
through the end of the trial. Male subjects must be advised not to donate sperm during this period.
Trial Vaccines:
Investigational Vaccine:
Takeda’s PIZV is an aluminum hydroxide-adjuvanted vaccine candidate made from purified formalin-inactivated 
ZIKV. The virus seed was derived from ZIKV strain PRVABC59 propagated through infection of Vero cells (derived 
from the kidney of African green monkey) grown in culture, and further purified prior to inactivation. The inactivated 
purified virus is mixed with aluminum hydroxide to formulate the drug product, PIZV, a liquid formulation filled into 
single-use vials and stored at 2-8°C. The investigational vaccine is administered IM as a 2-dose regimen of 0.5 mL at 
2, 5, or 10 µg antigen per dose, 28 days apart. Refer to the IB for more details.
Placebo:
Sodium chloride 0.9% solution is being used as placebo. It is a sterile, clear, colorless liquid solution of sodium 
chloride without preservative designed for parenteral use only. The placebo is presented in single-use vials stored at 
2-8°C. The placebo is administered IM as a 2-dose regimen of 0.5 mL per dose, 28 days apart.
Duration of the Trial:
Approximately 7 or 25 months, following a screening period
Period of Evaluation:
7 or 25 months
Main Criteria for Evaluation and Analyses:
The primary endpoints for this trial are 
Safety and tolerability of PIZV, as determined by:

Percentage of subjects with solicited local reactions (injection site: pain, erythema, swelling, and induration), 
in each severity category, during the 7-day period after administration of each dose of PIZV or placebo.

Percentage of subjects experiencing solicited systemic adverse events (AEs) (fever, headache, fatigue, 
malaise, arthralgia, and myalgia), in each severity category, during the 7-day period after administration of 
each dose of PIZV or placebo.

Percentage of subjects experiencing non-serious unsolicited AEs during the 28-day period after 
administration of each dose of PIZV or placebo.

Percentage of subjects experiencing SAEs during the 28-day period after administration of each dose of 
PIZV or placebo.
Immunogenicity of PIZV, as determined by:

Geometric mean titers (GMT) of neutralizing anti-ZIKV antibody levels at 28 days post dose 2.
The secondary endpoints for this trial are 
Safety of PIZV, as determined by:

Percentage of subjects experiencing SAEs throughout the trial.
Immunogenicity of PIZV, as determined by:

GMT of neutralizing anti-ZIKV antibody levels at 28 days post dose 1, and 6, 12, and 24 months post dose 2 
in applicable groups.

Seropositivity rates (SPR) at each immunogenicity time point (28 days post dose 1, 28 days post dose 2, and 
6, 12, and 24 months post dose 2) in applicable groups.

Seroconversion rates (SCR) at 28 days post dose 1 and 28 days post dose 2.
0023
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 23 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Statistical Considerations:
Analysis sets:

Safety Set: The Safety Set will consist of all randomized subjects who received at least one dose of the 
investigational vaccine/placebo. 

Full Analysis Set (FAS): The FAS will include all randomized subjects who have received at least one dose of 
the investigational vaccine/placebo and provided valid baseline and at least one post-vaccination serology 
result. Subjects will be included in the FAS analysis as randomized.

Per–Protocol Set (PPS): The PPS will include all subjects in the FAS who have no major protocol violations. 
The major protocol violation criteria will be defined as part of the blinded data review prior to the unblinding 
of subject’s investigational vaccine/placebo assignment. The categories of major protocol violations include: 
1
not meeting selected entry criteria, 
2
receiving a wrong investigational vaccine/placebo, 
3
receiving prohibited therapies, and 
4
other major protocol violations that may be identified during blinded data reviews.
All summaries and analyses of safety data will be based on subjects in the Safety Set. 
The primary immunogenicity analyses will be based on the PPS, and additional immunogenicity analyses will be 
based on the FAS.
Analysis of demographics and other baseline characteristics: Summaries of age, gender, race, and other baseline 
characteristics will be presented by formulation arm.
Immunogenicity analysis: Descriptive statistics for the primary and secondary immunogenicity endpoints, including 
estimates and 95% confidence intervals (95% CI) for GMT, SPR and SCR, will be provided by time point (28 days 
post dose 1, 28 days post dose 2, and 6, 12, and 24 months post dose 2 in applicable groups) and by formulation arm. 
Point estimates and 95% CI for ratios in GMT and differences in SPR and SCR will be provided for each pair of active 
study arms to aid a single vaccine dose level selection. Immunogenicity summaries and analyses will be provided by 
dose group overall, as well as by baseline flavivirus serostatus. 

Seropositive subjects: Subjects with detectable serum antibodies (tested positive at or above limit of 
detection, LOD) as measured by the neutralization assay.

Seronegative subjects: Subjects with no detectable serum antibodies (test results are below LOD) as 
measured by the neutralization assay. 

Seroconverted subjects: Seronegative subjects at baseline with detectable post-vaccination serum antibodies
(test results are at or above LOD) and seropositive subjects at baseline with different fold increases in 
post-vaccination antibodies from baseline, as measured by the neutralization assay.
Safety Analysis: Reactogenicity will be assessed for 7 days following each dose (including day of vaccine/placebo 
administration) via daily collection of solicited AEs, including local reactions (injection site: pain, erythema, swelling, 
and induration) and systemic AEs of headache, fatigue, malaise, arthralgia and myalgia. In addition, body temperature 
(preferably measured as oral temperature) as indicator of reactogenicity will be collected (with fever defined as body 
temperature ≥38.0°C/100.4°F).
For each solicited AE, including fever, the percentage of subjects will be summarized by event severity for each day 
for the 7 days after each vaccine/placebo administration and overall. For subjects with more than 1 episode of the same 
event, the maximum severity will be used for tabulations.
Unsolicited AEs and SAEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) and 
summarized by system organ class (SOC) and preferred term (PT) for each formulation arm including neurological 
and neuroinflammatory disorders and new medical conditions with onset after the first vaccination.
Safety summaries will be provided by dose group overall, as well as by baseline flavivirus serostatus.
0024
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 24 of 108
Protocol Amendment 3 Version 7.0 
12 September 2018
Sample Size Justification:
The sample size was not determined based on formal statistical power calculations.
Interim Analyses:
A first interim analysis will be performed to include immunogenicity and safety data from all flavivirus naïve subjects 
up to Visit 6 (on Day 57, 28 days post dose 2); a second interim analysis will be performed for dose selection,
including safety data from all flavivirus primed subjects up to Visit 4 (on Day 29, 28 days post dose 1). 
Final Analysis:
The final safety and immunogenicity analysis will be performed when all (flavivirus naïve and primed) subjects have 
completed Visit 6 (Day 57) to provide data to support the planning and execution of other trials in the development 
plan of PIZV. The subsequent analyses will include (1) the data up to Visit 9 (12 months post dose 2) and (2) the data 
up to Visit 11 (24 months post dose 2). The analyses will be performed by a separate set of unblinded statisticians and 
programmers at a Clinical Research Organization (CRO), who will have access to individual treatment assignments 
and will not be involved in subsequent trial conduct. The study team at Takeda will have access to the group level 
unblinded results and will remain blinded to the individual treatment assignment. The remaining personnel (not 
mentioned before) involved in the conduct of the trial, including those at Takeda, the CRO, and the trial sites, will 
remain blinded to the individual subject treatment assignment until unblinding after trial completion (database lock for 
data through Visit 11). More details regarding the analyses will be provided in the Statistical Analysis Plan (SAP).
The final study report will be written for the data up to Visit 6 (Day 57), and two additional addenda will be written: 
one to report the data from Visit 6 (Day 57) to Visit 9 (Day 393, 12 months post dose 2), and one to report the data 
from Visit 9 (Day 393) until the end of the study, ie, Visit 11 (Day 757, 24 months post dose 2). 
External Data Monitoring Committee:
An external DMC will have oversight of this trial. The external DMC functions at a program level.
The external DMC will consist of 5 independent members and an independent non-voting statistician. External DMC 
meetings will consist of open and closed face-to-face meetings or teleconference calls. The type and frequency of 
scheduled meetings will depend on the subject enrollment and safety event rates. Unscheduled ad hoc meetings will 
occur if a stopping rule occurs, or at any time consultation is requested by the Pharmacovigilance Study Team. 
The external DMC will review the safety and tolerability data of all subjects in the flavivirus naïve cohort who have 
completed Visit 4 (28 days post dose 1) for the purpose of recommendation to start vaccinating flavivirus primed 
subjects. The external DMC will review safety data on an ongoing basis and when the following set of AEs are 
reported, the external DMC will review all relevant safety data within 48 hours and consider a possible pause of the 
study, if it deems necessary:

Two or more subjects develop the same grade 3 solicited AE starting within 7 days of vaccination

Two or more subjects develop the same grade 3 unsolicited AE within 28 days of vaccination

Any SAE 

Any unexpected, serious, or unanticipated risk to the subjects enrolled.
Further information is available in the external DMC charter.
ZIK-101 Version 7.0 (12 September 2018)
0025
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 25 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
CONFIDENTIAL
2.1
Schedule of Trial Procedures
Visit Number
1A
1B (a)
2
3
4
5
6
7
8
9 (u)
10(u)
11(u)
In case of 
early 
termination
Visit Day
Day -15 
to Day 1
Day -15 
to Day 1
Day 1 Day 8
Day 29 Day 36 Day 57 Day 133 Day 211 Day 393 Day 575 Day 757
Clinical Visit
X
X
X
X
X
X
X
X
X
X
X
Phone Contact
X
X
First and 
second phase 
of the study (b)
Flavivirus naïve (1)
X
X
X
X
X
X
X
X
X
X
X
X
Flavivirus primed (2)
X
X
X
X
X
X
X
X
X
X
X
X
X
Second phase 
of the study (b) Flavivirus primed (3)
X
X
X
X
X
X
X
X
X
X
X
X
Anchored to visit day
NA
Day 1
+
7 days
Day 1
+
28 days
Day 29
+
7 days
Day 29
+
28 days
Day 29
+
3½ mo
Day 29
+
6 mo
Day 29
+
12 mo
Day 29
+
18 mo
Day 29
+
24 mo
Acceptable visit window (days before/ 
days after) (c)
NA
NA
0/+7
-1/+7
-4/+7
-1/+7
-4/+7
-7/+14
-7/+14
-7/+97
-7/+14
-14/+14
Signed informed consent
X
X (t)
Remind study procedure to flavivirus 
primed subjects identified at Visit 1A (2)
X
Assessment of eligibility criteria (d)
X
X
X
Demographics (e)
X
X (t)
Medical history (f)
X
X
X
Travel history
X
X
X
X
X
X
X
X
X
X
X
X
Concomitant medications/vaccinations (g)
X
X
X
X
X
X
X
X
X
X
Physical exam 
(h)
Complete
X
X
Symptom directed
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
Vital signs (i)
X
X
X
X
X
X
X
X
X
X
0026
Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 26 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
CONFIDENTIAL
Visit Number
1A
1B (a)
2
3
4
5
6
7
8
9 (u)
10(u)
11(u)
In case of 
early 
termination
Visit Day
Day -15 
to Day 1
Day -15 
to Day 1
Day 1 Day 8
Day 29 Day 36 Day 57 Day 133 Day 211 Day 393 Day 575 Day 757
Blood 
collection (j)
Flavivirus serostatus
X
X (t)
Eligibility screening and 
safety laboratory testing
X
X
X
X
Serum pregnancy testing
(l)
X
X
Immunogenicity and 
assay development
X
X
X
X
X
X
Urine 
collection (k)
Eligibility screening and 
safety laboratory testing 
(urinalysis)
X
X
X
X
Urine pregnancy testing
(l)
X
X
Randomization (m)
X
Check contraindications to vaccination 
and criteria for delay of vaccination
X
X
Investigational vaccine or placebo 
administration (n)
X
X
Post-vaccination and injection site 
evaluation (o)
X
X
Diary card (p)
Distribution
Days
1-7
Days
8-28
Days
29-35
Days
36-56
Review/collection
Days
1-7
Days
8-28
Days
29-35
Days
36-56
X (q)
Solicited AEs (r)
X
X
Unsolicited AEs (r)
X
X
X
X
X
SAEs and AEs leading to withdrawal or 
discontinuation (s)
X
X
X
X
X
X
X
X
X
X
X
(X) indicates procedures that are not mandatory but may be performed if deemed necessary; NA: Not applicable.
(a) Applicable only for flavivirus primed subjects
0027
keda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 27 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
CONFIDENTIAL
(b) Staggered enrollment: There will be two study phases

The first phase of the study will be aimed at enrolling flavivirus naïve subjects only (1).
All subjects who sign the ICF and have confirmed eligibility at Screening Visit 1A will be tested for flavivirus serostatus.
Eligible flavivirus naïve subjects enrolled at Screening Visit 1A (1) will be randomized at Visit 2, within two weeks after Screening Visit 1A.
Flavivirus primed subjects will not be enrolled during the first phase of the study (ie, when identified at Screening Visit 1A (2)) and will be invited to come 
back for the second phase of the study. They will be contacted after positive recommendations from the external DMC are issued following the review of 
safety and tolerability data in flavivirus naïve subjects, when the last flavivirus naïve subject has completed study Visit 4 (28 days post dose 1). These 
flavivirus primed subjects will consequently undergo 2 screening visits: Visit 1A during the first phase of the study, and Visit 1B at the outset of the second 
phase of the study. Flavivirus primed subjects identified at Screening Visit 1A who do not participate in the second phase of the study will be declared 
screened failure.
Enrollment will be put on hold after all naïve subjects (1) have been enrolled and will resume when positive recommendations from the external DMC are 
issued, as described above.

The second phase of the study will be aimed at enrolling flavivirus primed subjects only (2 and 3).
Newly identified eligible flavivirus primed subjects at Screening Visit 1B (3) will be enrolled (randomized) at (second phase) Visit 2, within two weeks after 
Screening Visit 1B.
Previously identified flavivirus primed subjects (2) (who were tested at Screening Visit 1A during the first phase and accepted to enter the second phase of the 
study) will retain their initial subject identification (ID) number and undergo an additional screening visit (Visit 1B) to be tested for eligibility criteria again 
(except for the determination of flavivirus serostatus). 
Flavivirus naïve subjects identified at Screening Visit 1B (during the second phase of the study) will be declared screened failure.
(c) If the subject has a temporary clinically significant active infection (as assessed by the investigator) or temperature ≥38.0°C/100.4°F, within 3 days of intended 
investigational vaccine/placebo administration, the investigational vaccine/placebo administration may be delayed as per the assessment of the investigator 
within the allowed visit window. The visit windows of Visit 3 and Visit 4 should be calculated from the day of the first vaccination. Following the same rule, 
the visit windows of Visit 5 and Visit 6 should be calculated from the day of the second vaccination.
(d) One single subject identification (ID) number will be assigned to each subject at Screening Visit (1A or 1B). Eligibility by review of relevant 
inclusion/exclusion criteria will be documented before enrollment.
(e) Demographic information, to be obtained at Screening Visit (1A or 1B), will include age (date of birth), sex, race, and ethnicity as provided by the subject.
(f)
Medical history will be collected at Screening Visit(s) (Visit 1A and/or Visit 1B) and at Visit 2 (Day 1) and will include any medical history that may be 
relevant to subject eligibility for trial participation such as prior vaccinations, concomitant medications, and previous and ongoing illnesses or injuries. 
Relevant medical history can also include any medical history that contributes to the understanding of an AE that occurs during trial participation, if it 
represents an exacerbation of an underlying disease/preexisting problem. Adverse occurrences before administration of the first dose of investigational 
vaccine/placebo are considered medical history.
(g) All medications, vaccines and blood products taken or received by the subjects within 3 months prior to the start of the trial are to be recorded on the source 
document (patient record) and entered on the Prior and Concomitant Medications eCRF. The use of antipyretics and/or analgesic medications within 24 hours 
prior to vaccination must be identified and the reason for their use (prophylaxis versus treatment) must be described in the source documents and the eCRF. 
Refer to Section 9.1.2.
0028
mercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 28 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
CONFIDENTIAL
(h) Complete physical examination will be performed at Screening Visit(s) (Visit 1A and/or Visit 1B). All subsequent symptom-directed physical examinations, 
performed if deemed necessary or indicated by review of the subject’s medical history, should assess clinically significant changes from the baseline 
examination. The findings should be documented in the subject’s source document; findings consistent with the definition of AEs or SAEs (see Section 10.0) 
must be transcribed into the electronic Case Report Form (eCRF). For any procedures at the site, the investigator shall follow his/her standard practice. Refer to 
Section 9.1.4.
(i)
Vital signs include (however, not limited to) systolic/diastolic blood pressure, pulse rate, respiratory rate, and temperature, and should be documented in the 
subject’s source document and transcribed into the eCRF. Follow standard of care for trial population and operational feasibility. Refer to Section 9.1.5.
(j)
Blood samples will be collected at each site visit: ie, at Screening Visit(s) (Visit 1A and/or Visit 1B) for flavivirus serostatus determination (approximately 
10 mL of blood) and eligibility screening tests (including pregnancy testing) (approximately 10 mL of blood), at Visits 3 and 5 for routine safety laboratory 
testing (approximately 10 mL of blood), and at Visits 2, 4, 6, 8, and (for subjects who will be randomized to the placebo group or the PIZV dosing group that 
will be selected for further development) 9 and 11 for immunogenicity, as well as for further development and characterization of assays (approximately 60 mL 
of blood). Should a blood sampling be performed during a vaccination visit, this sampling must occur before the administration of the investigational vaccine or 
placebo. The maximum volume of blood taken at any single visit will be between approximately 10 mL and 60 mL, and the approximate maximum total 
volume of blood for the whole trial will be 280 mL to 410 mL depending on the subject’s flavivirus serostatus at screening and the vaccine dose level the 
subject received. All samples will be collected in accordance with acceptable laboratory procedures. Blood samples will be processed and stored at the trial site 
as described in the provided Laboratory Manual. Refer to Section 9.1.6.
(k) Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) and at Visits 3 and 5; and for women of childbearing potential 
at Visits 2 and 4 before each investigational vaccine/placebo administration for pregnancy testing. Urine samples for eligibility screening and routine safety 
laboratory testing will be sent to the central laboratory. Urine pregnancy tests will be performed at the study site using kits provided by the sponsor.
(l)
Serum pregnancy tests will be conducted in the central laboratory and urine pregnancy tests will be done at the study site using kits provided by the sponsor.
Subjects must have a negative urine -hCG pregnancy test prior to receiving any dose of investigational vaccine/placebo. Refer to Section 9.1.9.
(m) If eligible, the subject will be randomized at Visit 2. Refer to Sections 9.1.3 and 9.1.10.
(n) The investigational vaccine/placebo will be administered in the deltoid muscle of the non-dominant arm (approximately at the top middle third).
(o)
After vaccine/placebo administration at Visit 2 (on Day 1) and at Visit 4 (Day 29), the subject will be observed for at least 30 minutes for observation of 
solicited AEs, unsolicited AEs, and measurement of body temperature.
(p) Diary cards will be handed out to the subjects at Visits 2, 3, 4 and 5, and reviewed with the subject and collected at the following visit. Refer to Section 9.3.4.
(q) In case of early termination during the vaccination period only.
(r)
After each dose, solicited AEs (for 7 days including the day of vaccine/placebo administration) and unsolicited AEs (for 28 days including the day of 
vaccine/placebo administration) will be collected on diary cards by each subject. The investigator will transcribe these data into eCRF. Refer to Section 9.1.7. 
(s) SAEs and AEs leading to withdrawal from the trial or discontinuation of vaccine/placebo administration will be collected for the duration of the entire trial. 
SAEs will be reported to the sponsor within 24 hours of the investigator becoming aware of the event. AEs leading to early termination will be recorded by the 
investigator.
(t)
Only for newly screened subjects at Screening Visit 1B (ie, not for flavivirus primed subjects who were tested at Screening Visit 1A and accepted to enter the 
second phase of the study).
(u) All subjects who were randomized to the placebo group or the PIZV dosing group that was selected for further development will attend Visit 9 at Day 393 
(12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), and will also be contacted by phone on Day 575 (Visit 10) for safety follow-up.
0029
on-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 29 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
3.0
TRIAL REFERENCE INFORMATION
3.1
Trial-Related Responsibilities
The sponsor will perform all trial-related activities with the exception of those identified in the 
Trial-Related Responsibilities template. The identified vendors in the template for specific 
trial-related activities will perform these activities in full or in partnership with the sponsor.
3.2
Principal Investigator/Coordinating Investigator
The sponsor will select a Signatory Principal Investigator / Coordinating Investigator from the 
investigators who participate in the trial. Selection criteria for this investigator will include 
significant knowledge of the trial protocol, the investigational vaccine, their expertise in the 
therapeutic area and the conduct of clinical research, as well as trial participation. The Signatory 
Principal Investigator / Coordinating Investigator will be required to review and sign the clinical 
study report and by doing so agrees that it accurately describes the results of the trial.
0030
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 30 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
3.3
List of Abbreviations
ADE
Antibody-Dependent Enhancement
AE
Adverse Event
BARDA
Biomedical Advanced Research and Development Authority
BMI
Body Mass Index
CI
Confidence Interval
CNS
Central Nervous System
CRO
Contract Research Organization
CZS
Congenital Zika Syndrome
DENV
Dengue Virus
DMC
Data Monitoring Committee
eCRF
electronic Case Report Form 
EDC
Electronic Data Capture system
ELISA
Enzyme-linked Immunosorbent Assay
FAS
Full Analysis Set
FDA
U.S. Food and Drug Administration
FRNT
Focus Reduction Neutralizing Test
GBS
Guillain-Barré syndrome
GCP
Good Clinical Practice
GMT
Geometric Mean Titer
-hCG
beta human Chorionic Gonadotropin
HIV
Human Immunodeficiency Virus
IB
Investigator’s Brochure
ICF
Informed Consent Form 
ICH
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use
ID
Identification
IEC
Independent Ethics Committee
Ig
Immunoglobulin
IM
Intramuscular
IRB
Institutional Review Board
IRT
Interactive Response Technology
JE
Japanese Encephalitis
LOD
Limit of Detection
MAC-ELISA
IgM-Antibody Capture Enzyme-Linked Immunosorbent assay
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Purified Inactivated Zika Virus Vaccine (PIZV)
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MedDRA
Medical Dictionary for Regulatory Activities
MN
Micro-neutralization
NHP
Non-Human Primate
PBS
Phosphate Buffered Saline solution, ie, water-based salt solution containing 
potassium dihydrogen phosphate (KH2PO4), sodium hydrogen phosphate 
(Na2HPO4), and sodium chloride
PHEIC
Public Health Emergency of International Concern
PIZV
Purified Inactivated Zika Virus Vaccine candidate
PPS
Per-Protocol Analysis Set
PRNT
Plaque Reduction Neutralization Test
PT
Preferred Term
RT-PCR
Reverse Transcription-Polymerase Chain Reaction
RVP
Reporter Virus Particle
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SCR
Seroconversion Rate
SOC
System Organ Class
SPR
Seropositivity Rate
SUSAR
Suspected Unexpected Serious Adverse Reaction
WHO
World Health Organization
WNV
West Nile Virus
WRAIR
Walter Reed Army Institute of Research
YF
Yellow Fever 
ZIKV
Zika Virus
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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3.4
Corporate Identification
Not applicable.
0033
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use


Purified Inactivated Zika Virus Vaccine (PIZV)
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4.0
INTRODUCTION
4.1
Background
4.1.1
Zika Virus
Zika virus (ZIKV) is a mosquito-borne single-stranded positive-sense ribonucleic acid (RNA) 
virus (11 kb in size) (26) which belongs to the Flaviviridae family, genus Flavivirus, that often 
causes no or only mild symptoms, including a rash and a febrile flu-like illness in the majority of 
symptomatic individuals. The ZIKV virion shows icosahedral symmetry of its nucleocapsid (27)
and presents structural similarities with other members of the Flaviviridae, including Spondweni 
virus, Yellow Fever (YF) virus, Dengue virus (DENV), West Nile virus (WNV), Japanese 
Encephalitis (JE) virus, and Tick-borne Encephalitis virus. The genome is translated into a single 
polyprotein subsequently cleaved by both viral and host cell enzymes into three structural (capsid, 
pre-membrane/membrane and envelope) proteins that form the virion and seven non-structural 
proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) (28).
ZIKV was discovered in 1947 in Uganda while testing samples taken from sentinel rhesus 
monkeys used as bait during a surveillance of YF in the Zika Forest near Entebbe in Uganda. The 
first human disease cases were reported in 1952 in Uganda (29) and Tanzania (30). For as long as 
55 years, the virus circulated in areas of Africa, Southeast Asia, and the Pacific Islands, and was 
considered a rare disease with mild symptoms (31); only 14 human cases were reported and no 
outbreaks were detected during that period (32).
4.1.2
Epidemiology
The first large outbreak of ZIKV disease was reported in Yap Island (Federal state of Micronesia) 
in 2007 (33), and was followed by an outbreak in French Polynesia in 2013-2014 (34). The first 
cases of ZIKV disease in South America were reported in Easter Island in 2014 (35), and the first 
reports of locally transmitted infection (ie, by mosquito) came from Brazil in May 2015 (36), 
which triggered the third outbreak in 2015-2016. Since then, ZIKV disease has continued 
spreading through the South American continent and to other regions. In the current outbreak that 
started in 2015, there have been estimates of more than 750,000 ZIKV-suspected cases reported 
from more than 60 countries/territories; most of these cases are from the South America continent 
(37, 38).
4.1.3
Transmission
The primary mode of transmission of ZIKV is through the bite of female mosquitoes, prominently 
Aedes aegypti and Ae. albopictus species that have spread globally (39). Other potential vectors 
for ZIKV include other Aedes species (including Ae. furcifer, Ae. vittatus, Ae. dalzieli,
Ae. metallicus, Ae. hirsutus, Ae. unilinaetus, Ae. africanus, Ae. taylori, Ae. hensilli, and 
Ae. luteocephalus) along with mosquitoes from other genera (such as Anopheles, Mansonia, and 
Culex) (40), the true extent of the vectors being still unknown. Nonhuman and human primates are 
likely the main reservoirs of the ZIKV as for most arthropod-borne viruses (arboviruses) (41), and 
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Purified Inactivated Zika Virus Vaccine (PIZV)
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anthroponotic (human-to-vector-to-human) transmission is most likely responsible for the rapid 
spreading of the virus during outbreaks.
Vertical transmission from mother to fetus during pregnancy (6, 42-44) and the perinatal period 
(45, 46), sexual contact (47-49), and blood transfusion (50, 51) are also confirmed modes of 
transmission of the disease. Potentially, the virus could be transmitted through laboratory exposure 
and by body fluid contact (52, 53), eg, tears, urine (54-56), breast milk, or through solid organ 
transplants (57, 58), but there is limited evidence to support this (59-61).
4.1.4
ZIKV Infection: Detection, Disease, Complications and Treatment
Detection
Testing for ZIKV infection is complicated by the temporal appearance and disappearance of 
biologic analytes in the infected person and thus multiple tests and sample types are often needed 
to establish a definitive laboratory diagnosis of ZIKV infection. Viral RNA is the first analyte that 
can be detected in an infected person in multiple specimen types. In blood, as the immune response 
develops (immunoglobulin M [IgM] titers rise), levels of viral RNA decline. However, viral RNA 
may be detectable in some infected persons for longer periods in certain specimen types (62, 63).
Whole blood, serum and urine are the primary diagnostic specimens for ZIKV infection. Other 
specimen types such as plasma, cerebrospinal fluid, and amniotic fluid are authorized for use with 
some tests that have received a Food and Drug Administration (FDA) Emergency Use 
Authorization (64). Other specimens that still need validation but might be appropriate for 
evaluating ZIKV disease include saliva (65, 66), semen, peripheral blood mononuclear cells, 
amniotic fluid (43), and tissue samples of, for instance, fetal brain and placenta biopsies (6, 42, 
67).
Testing methods used for ZIKV infection include molecular and serologic testing: reverse 
transcription-polymerase chain reaction (RT-PCR) for viral nucleic acid, and IgM-antibody 
capture enzyme-linked immunosorbent assay (MAC-ELISA), as well as focus reduction 
neutralizing test (FRNT), plaque reduction neutralization test (PRNT), micro-neutralization (MN) 
assay, and reporter virus particle (RVP) assay for anti-ZIKV antibodies. Although PRNT typically 
provides good specificity, serological assays are subject to cross-reactivity, especially in subjects 
with prior flavivirus infection or immunization history. In contrast, diagnosis by RT-PCR has been 
most successful within 1 week after the onset of clinical illness, though viremia is generally low 
level, which makes viral isolation from clinical samples difficult (68). Data from studies in 
non-human primates (NHP) evaluated for neutralizing antibodies by conventional FRNT, MN 
assay and RVP assay suggest that although both FRNT and MN assay strongly correlated with 
EC50 RVP values, RVP assay was more sensitive (69).
Disease
More than 80% of ZIKV infections remain asymptomatic (33), and for the symptomatic cases, the 
disease is in general mild and of short duration. Some clinical manifestations include, but are not 
limited to, mild fever, maculopapular rash, conjunctivitis and arthralgia (70).
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Purified Inactivated Zika Virus Vaccine (PIZV)
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The incubation period between infection and symptoms appears to be of 3 to 11 days (3, 4), which 
is similar to that of other mosquito-borne flavivirus diseases. The course of infection is mostly 
self-limiting. However, pre-existing cross-reactive antibodies directed against other ﬂaviviruses 
may exacerbate secondary ZIKV infection through antibody-dependent enhancement (ADE). A 
recent publication has demonstrated that human dengue virus antibodies enhance in vitro infection 
with ZIKV, suggesting that ADE is not merely a theoretical concern (71). 
Congenital Zika Syndrome
Despite mild clinical symptoms in the pregnant woman, ZIKV infection during pregnancy has 
been associated with serious outcomes for the fetus and newborn. The severity of the disease is 
related to the consequences in the fetus and newborn child from women with ZIKV infection 
during pregnancy (42, 72). The spectrum of congenital anomalies associated with ZIKV infection, 
known as Congenital Zika Syndrome (CZS), consists of severe microcephaly (5-8) with partially 
collapsed skull, cerebral cortices with subcortical calcifications, macular scarring and focal 
pigmentary retinal mottling, congenital contractures, and marked early hypertonia with symptoms 
of extrapyramidal involvement (9).
The exposure to ZIKV at any moment during pregnancy is a risk for fetal or postnatal 
developmental complications, primarily affecting brain development. Recent analysis of the U.S. 
Zika Pregnancy Registry (USZPR) showed that the prevalence of newborns with congenital 
defects from pregnancies with laboratory-confirmed ZIKV infection is 10% (95% CI: 7%-14%). 
This is 30 times higher than baseline prevalence before more recent Zika outbreaks (10).
Since the beginning of these outbreaks and up to end of September 2017, there have been 
3689 confirmed CZS malformations potentially associated with ZIKV infection in 27 countries 
and territories of Latin America and the Caribbean.
Guillain-Barré Syndrome
ZIKV is a neurotropic flavivirus that can potentially cause disease within the central nervous 
system. Worldwide concern over ZIKV causing Guillain-Barré Syndrome (GBS) emerged when 
an increased number of GBS cases was temporally associated with the 2013-2014 ZIKV outbreak 
in French Polynesia, during which 42 GBS cases were reported, all of these cases had neutralizing 
antibodies against ZIKV, compared to 56% in the control group (p<0.0001) (11-13). Other 
neurological complications, such as encephalitis, meningoencephalitis (14), paresthesia, facial 
paralysis and myelitis (15, 16), have also been associated with ZIKV infection. Based on these 
findings, the countries affected by ZIKV disease in the 2015-2016 outbreak improved and 
extended the surveillance of neurological syndromes. In 2017, after the spread of ZIKV 
throughout the Pacific Islands, Latin America and the Caribbean, a total of 200 cases of GBS 
related to ZIKV infection have been reported in these regions as case series or isolated cases. The 
incidences reported have increased 2 to 9.8 times compared to baseline before the epidemic in 
Latin America (19), and up to 20 times in French Polynesia (12). The association of GBS was only 
reported when the circulating strain was of Asian lineage (21). As of 01 February 2017, ZIKV of 
the Asian lineage was circulating in more than 80% of affected countries, including 48 countries in 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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Latin America (22). The overall incidence of ZIKV-associated GBS is estimated to be 24 per 
100,000 cases of ZIKV infection (23).
Treatment
No specific antiviral treatment is available for ZIKV infections and no vaccine against ZIKV is 
currently available (73). As the disease is self-limiting, treatment for uncomplicated ZIKV 
infection is supportive and focuses on symptoms. Treatment for more severe forms of ZIKV 
infection should address specific medical issues, and in the case of congenital ZIKV infection, 
neurodevelopmental issues for the infant’s particular needs (74).
Patient care includes proper hydration, monitoring for possible coagulopathy and multiple organ 
failure. Intensive care is recommended for the patients depicting serious conditions, such as 
showing signs of coagulopathy, tachycardia, hypotension and renal dysfunction. Patients with 
neurological complications such as GBS require hospitalization (75). Non-steroidal 
anti-inﬂammatory drugs and aspirin can be recommended for the treatment. Such medications 
should be avoided in case of concomitant infection with dengue fever virus. In case of suspected 
ZIKV infection in pregnant women, sonography is recommended to monitor the proper fetal 
growth and potential complications of infant microcephaly (76).
The main recommendations to address outbreaks are through prevention (avoiding mosquito bites, 
reducing sexual transmission, improving the procedures for safe blood transfusion), and vector 
control measures (77, 78). 
4.2
Rationale for the Proposed Trial 
4.2.1
Medical Need
ZIKV has posed a challenging situation for health, public and economic sectors of affected 
countries. The increase in microcephaly cases and other neurological disorders reported in Brazil 
following a similar cluster in the French Polynesia in 2014 (24), prompted the World Health 
Organization (WHO) to declare Zika a Public Health Emergency of International Concern 
(PHEIC) on 01 February 2016 (25) and recommended to focus research on the causal association 
of ZIKV infection during pregnancy and microcephaly and other neurological abnormalities in 
newborns. WHO recommended to standardize and enhance the surveillance for microcephaly and 
GBS particularly in areas of known or high risk of ZIKV transmission and to intensify the research 
into the etiology of new clusters of microcephaly and other neurological disorders to determine 
whether there is a causative link to ZIKV and/or other factors or co-factors.
Considering the rapid spread of ZIKV and the significant threat to public health especially to 
pregnant women and their children’s health, the U.S. Department of Health and Human Services 
declared Zika a public health emergency in Puerto Rico in August 2016 (79). In November 2016, 
after evaluating the evidence about the causal association of ZIKV infection during pregnancy and
microcephaly and other neurological abnormalities in newborns, WHO announced that Zika no 
longer represents a PHEIC (80) but stressed the need to escalate the disease to a sustained work 
program with dedicated resources to address its long-term nature and its associated consequences.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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The risk of infection with ZIKV is increasing given that the spread of ZIKV is rapid and intense in 
tropical and subtropical countries. Vector control is difficult to establish and maintain, and new 
areas continue to be colonized by the Aedes species. Climate changes may also create favorable 
environments to maintain the vector (81). There is an increased urban population and mobility, and 
the role of sexual transmission may be an important mechanism to spread rapidly the virus into 
susceptible populations, even in regions where the vector is uncommon.
Considering the conclusive associations between ZIKV infections and fetal and postnatal 
abnormalities, as well as strong associations with other medically important complications such as 
GBS, the development of a vaccine that can provide protection is crucial for countries where the 
epidemic is expected to arrive and/or persist, as well as in countries in which the virus has not yet 
been introduced. In order to address the urgent medical need and in anticipation of possible 
additional outbreaks with rapid onsets, Takeda has initiated the development of a purified 
inactivated ZIKV vaccine candidate, PIZV, for use in endemic areas and non-endemic areas for 
prevention of ZIKV associated illness of any severity and/or infection.
4.2.2
BARDA Funding Award
The Biomedical Advanced Research and Development Authority (BARDA) from the U.S. 
Department of Health and Human Services’ Office of the Assistant Secretary for Preparedness and 
Response is providing funding to Takeda for the US-based PIZV development program (82).
4.2.3
Vaccines Under Development
Currently, there is no approved vaccine against ZIKV. Various companies and academic 
organizations are using a variety of vaccine approaches, including inactivated virus, virus-like 
particles, nucleic-acid-based vaccines, live vectored vaccines, subunit vaccines, and live 
recombinant approaches. Most of these are in preclinical development and many are expected to 
enter Phase I clinical studies in 2017.
4.2.4
Summary of Available Non-clinical Data
There are no non-clinical data currently available for PIZV. 
PIZV will be tested for nonclinical safety in a 3-dose Good Laboratory Practice-compliant, 
repeat-dose toxicity and local tolerance study in New Zealand White Rabbits. Toxicology data will 
be available prior to study start and provided in the Investigator’s Brochure (IB).
4.2.5
Summary of Available Clinical Data
There are no clinical data currently available for PIZV. 
4.2.6
Takeda Vaccine Candidate
Takeda’s PIZV is an aluminum hydroxide (alum)-adjuvanted vaccine candidate made from 
purified formalin-inactivated ZIKV. The virus seed was derived from ZIKV strain PRVABC59 
propagated through infection of Vero cells (derived from the kidney of African green monkey) 
grown in culture, and further purified prior to inactivation. The choice of ZIKV strain may not be a 
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Purified Inactivated Zika Virus Vaccine (PIZV)
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Protocol Amendment 3 Version 7.0
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critical parameter as evidence suggests that infection with a single ZIKV strain can elicit broadly 
neutralizing antibodies across different strains (83).
Refer to Section 8.1 for additional details.
4.2.7
Rationale for ZIK-101 Trial
The proposed ZIK-101 clinical trial is an observer-blind placebo-controlled first-in-human study. 
The rationale for the observer-blind approach is based on the different physical appearance of the 
investigational vaccine compared to the saline solution placebo that was selected and preferred to 
alum-containing placebo to avoid side effects associated with alum. The rationale for the use of 
placebo in this trial is based on the absence of effective treatment or prevention for ZIKV disease.
Due to the need to vaccinate women and men of reproductive age, the primary target population of 
PIZV will be 9 years of age and older. In this phase 1 study, study population is limited to ≥18 to 
≤49 years, and age expansion will be included in the next phase of development.
To ensure the enrollment of healthy adults, screening tests (hematology, biochemistry and 
urinalysis) will be performed prior to vaccine administration.
Flavivirus primed adults are included in this study to assess if there is any impact from pre-existing 
flavivirus antibodies. The testing of flaviviral serological status will be performed prior to 
randomization and will enable stratification of randomization.
Due to the ongoing threat of ZIKV disease and the urgent need of a vaccine, ZIK-101 will be 
accelerated to assess dose finding for PIZV, in addition to evaluation of safety and 
immunogenicity of the vaccine candidate (PIZV) in both flavivirus seronegative (naïve) and 
seropositive (primed) subjects. 
The reactogenicity/safety of PIZV is unknown. Flavivirus naïve subjects will be enrolled during 
the first phase of the study. In the absence of clinical data on ZIKV vaccine, it is proposed to 
recruit flavivirus primed subjects in a second stage. If positive recommendations from an external
Data Monitoring Committee (DMC) are issued following the review of safety and tolerability data 
in the flavivirus naïve population, when the last flavivirus naïve subject has completed study 
Visit 4 (28 days post dose 1), vaccination of flavivirus primed subjects will start.
The primary rationale for selection of the three vaccine dose levels can be summarized as follows: 

the low-dose level (2 µg) was selected for purposes of potentially identify an immunological 
threshold, and based on the limit of the analytical methods for the drug product available at the 
time;

the mid-dose level (5 µg) was selected to provide a mid-point on the dose response curve. In 
addition, this dose level is based on the Walter Reed Army Institute of Research (WRAIR) 
vaccine currently tested in Phase 1 (84), which was originally based on initial studies in NHPs 
(85); and
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the high-dose level (10 µg) was selected to follow an approximate 2-fold range and to further 
define the potential dose-response curve in both naïve and primed subjects.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 39 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
The initial immunogenicity and efficacy studies in mice support the rationale for formulating 
PIZV with aluminum hydroxide adjuvant in order to elicit a robust immune response and survival 
following wild-type challenge.
PIZV dose will be selected based on descriptive safety data and geometric mean titers (GMT) of 
neutralizing anti-ZIKV antibody levels at 28 days post dose 2 in both flavivirus naïve and primed 
subjects. 
The trial will be conducted in accordance with the protocol, the International Council for 
Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) GCP 
Guidelines and applicable regulatory requirements.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 40 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
5.0
TRIAL OBJECTIVES AND ENDPOINTS
5.1
Objectives
5.1.1
Primary Objectives

To describe the safety of two doses of PIZV given 28 days apart from three different antigen 
concentrations (2, 5 or 10 µg) in flavivirus naïve and primed healthy adults through 28 days 
post dose 2

To select a single vaccine dose level of PIZV for further clinical development
5.1.2
Secondary Objectives

To describe the safety of two doses of PIZV given 28 days apart in flavivirus naïve and primed 
healthy adults through the end of the study

To describe the immune response to PIZV in flavivirus naïve and primed healthy adults at the 
following immunogenicity time points 28 days post dose 1, 28 days post dose 2, and 6 months 
post dose 2

To describe the persistence of immunity to PIZV at 12 and 24 months post dose 2 in flavivirus 
naïve and primed healthy adults from the placebo group and the PIZV dosing group that will be 
selected for further development
5.2
Endpoints
5.2.1
Primary Endpoints
Safety and Tolerability of PIZV, as determined by:

Percentage of subjects with solicited local reactions (injection site: pain, erythema, swelling, 
and induration), in each severity category, during the 7-day period after administration of each 
dose of PIZV or placebo.

Percentage of subjects experiencing solicited systemic adverse events (AEs) (fever, headache, 
fatigue, malaise, arthralgia, and myalgia), in each severity category, during the 7-day period 
after administration of each dose of PIZV or placebo.

Percentage of subjects experiencing non-serious unsolicited AEs during the 28-day period 
after administration of each dose of PIZV or placebo.

Percentage of subjects experiencing serious AEs (SAEs) during the 28-day period after 
administration of each dose of PIZV or placebo.
Immunogenicity of PIZV, as determined by:

Geometric mean titers (GMT) of neutralizing anti-ZIKV antibody levels at 28 days post 
dose 2.
0041
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 41 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
5.2.2
Secondary Endpoints
Safety of PIZV, as determined by:

Percentage of subjects experiencing SAEs throughout the trial.
Immunogenicity of PIZV, as determined by:

GMT of neutralizing anti-ZIKV antibody levels at 28 days post dose 1, and 6, 12, and 
24 months post dose 2 in applicable groups.

Seropositivity rates (SPR) at each immunogenicity time point (28 days post dose 1, 28 days 
post dose 2, and 6, 12, and 24 months post dose 2) in applicable groups.

Seroconversion rates (SCR) at 28 days post dose 1 and 28 days post dose 2.
0042
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 42 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
6.0
TRIAL DESIGN AND DESCRIPTION
6.1
Trial Design
This is a phase 1, randomized, observer-blind, placebo-controlled, safety, immunogenicity, and 
dose ranging study of PIZV candidate in flavivirus naïve and primed healthy adults aged ≥18 to 
≤49 years, in ZIKV endemic and non-endemic regions.
The trial will last approximately 7 or 25 months for each subject, following a screening period 
prior to Visit 2. The screening period will last for up to 2 weeks (14 days) prior to randomization, 
with the possibility that some subjects (Flavivirus primed subjects only) will need to repeat some 
screening procedures if they were not randomized right after the screening period (further 
clarification is provided in the schedule of trial procedures).

Flavivirus naïve subjects: subjects without detectable serum antibodies against a panel of 
flaviviruses as measured by a reactive antibody-based assay (Luminex) (refer to the Serology 
Plan, available on file).

Flavivirus primed subjects: subjects with serum antibodies against a panel of flaviviruses as 
measured by a reactive antibody-based assay (Luminex) (refer to the Serology Plan, available 
on file). 
Approximately 240 subjects aged ≥18 to ≤49 years will be enrolled and equally randomized into 
four groups (arms) (1:1:1:1) of approximately 60 subjects, each comprised of approximately 
30 flavivirus naïve subjects and approximately 30 flavivirus primed subjects. Randomization 
within each cohort (flavivirus naïve versus flavivirus primed subjects) will be stratified by 
flavivirus serostatus and age group: ≥18 to ≤29 years versus ≥30 to ≤49 years.
A schematic representation of the trial design is included as Figure 6.a. A schedule of trial 
procedures is provided in Section 2.1.
0043
mercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 43 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Figure 6.a
Schematic Representation of Trial Design
Staggered enrollment (Figure 6.b): There will be two study phases. 

The first phase of the study will be aimed at enrolling flavivirus naïve subjects only. All 
subjects who sign the informed consent form (ICF) and have confirmed eligibility at Screening 
Visit 1A will be tested for flavivirus serostatus.
Eligible flavivirus naïve subjects enrolled at Screening Visit 1A will be randomized at Visit 2, 
within two weeks after Screening Visit 1A.
Flavivirus primed subjects will not be enrolled during the first phase of the study (ie, when 
identified at Screening Visit 1A) and will be invited to come back for the second phase of the 
study. They will be contacted after positive recommendations from the external DMC are 
issued following the review of safety and tolerability data in flavivirus naïve subjects, when the 
last flavivirus naïve subject has completed study Visit 4 (28 days post dose 1). These flavivirus 
primed subjects will consequently undergo 2 screening visits: Visit 1A during the first phase of 
the study, and Visit 1B at the outset of the second phase of the study. Therefore, it is 
recommended that the key aspects of the ICF already signed during the Screening Visit 1A are
reinforced with the subject. Flavivirus primed subjects identified at Screening Visit 1A who do 
not participate in the second phase of the study will be declared screened failure (see 
Figure 6.b §).
Enrollment will be put on hold after all naïve subjects have been enrolled and will resume 
when positive recommendations from the external DMC are issued, as described above.
0044
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 44 of 108
Protocol Amendment 3 Version 7.0
12 September 2018

The second phase of the study will be aimed at enrolling flavivirus primed subjects only.
Newly identified eligible flavivirus primed subjects at Screening Visit 1B will be enrolled 
(randomized) at (second phase) Visit 2, within two weeks after Screening Visit 1B.
Previously identified flavivirus primed subjects (see Figure 6.b §§) (who were tested at 
Visit 1A during the first phase and accepted to enter the second phase of the study) will retain 
their initial subject identification (ID) number and undergo an additional screening visit 
(Visit 1B) to be tested for eligibility criteria again, (except for the determination of flavivirus 
serostatus). 
Flavivirus naïve subjects identified at Screening Visit 1B (during the second phase of the 
study) will be declared screened failure (see Figure 6.b §§§).
0045
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 45 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Figure 6.b
Staggered Enrollment
* All subjects who were randomized to the placebo group or the PIZV dosing group that was selected for further 
development will attend Visit 9 at Day 393 (12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), 
and will also be contacted by phone on Day 575 (Visit 10) for safety follow-up.
Each subject will receive two 0.5 mL doses of either PIZV (2, 5 or 10 µg) or placebo 
intramuscularly (IM) – one dose at Visit 2 (on Day 1) and one dose at Visit 4 (on Day 29) IM into 
the middle third of the deltoid muscle, preferably in the non-dominant arm.
0046
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 46 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Each subject will be required to attend 7, 8, 9 or 10 clinical visits and will receive 1 or 2 phone 
calls depending on their flavivirus serostatus at screening and the vaccine dose level they received:

Flavivirus naïve subjects identified during the Screening Visit 1A and flavivirus primed 
subjects identified during the Screening Visit 1B will attend 7 visits and will receive 1 phone 
call, and possibly 2 additional visits and 1 phone call (thus a maximum of 9 visits and 2 phone 
calls) if they were randomized to the placebo group or the PIZV dosing group that was selected 
for further development

Flavivirus primed subjects identified during the Screening Visit 1A will attend 8 visits 
(including Visit 1A and Visit 1B) and will receive 1 phone call, and possibly 2 additional visits 
and 1 phone call (thus a maximum of 10 visits and 2 phone calls) if they were randomized to 
the placebo group or the PIZV dosing group that was selected for further development
All randomized subjects will thus attend Screening Visit(s) (Visit 1A and/or Visit 1B), Visit 2 at 
Day 1, Visit 3 at Day 8 (7 days post dose 1), Visit 4 at Day 29 (28 days post dose 1), Visit 5 at 
Day 36 (7 days post dose 2), Visit 6 at Day 57 (28 days post dose 2), and Visit 8 at Day 211 
(6 months post dose 2). All randomized subjects will also be contacted by phone on Day 133 
(Visit 7) for safety follow-up. In addition, all subjects who were randomized to the placebo group 
or the PIZV dosing group that was selected for further development will attend Visit 9 at Day 393 
(12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), and will also be 
contacted by phone on Day 575 (Visit 10) for safety follow-up.
Blood samples will be collected at each site visit: ie, at Screening Visit(s) (Visit 1A and/or 
Visit 1B) for flavivirus serostatus determination and eligibility screening tests (including
pregnancy testing); at Visits 3 and 5 for routine safety laboratory testing; and at Visits 2, 4, 6, 8, 
and (for subjects who will be randomized to the placebo group or the PIZV dosing group that will 
be selected for further development) 9 and 11 for immunogenicity, as well as for further 
development and characterization of assays. Refer to Section 9.1.6 and the Serology Plan
(available on file). 
Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) for 
eligibility screening, and at Visits 3 and 5 for routine safety laboratory testing; and for women of 
childbearing potential at Visits 2 and 4 before each investigational vaccine/placebo administration 
for pregnancy testing. Refer to Section 9.1.6.
Each subject will receive diary cards to collect solicited AEs for 7 days after each dose (including 
the days of vaccine/placebo administration), and unsolicited AEs for 28 days after each dose 
(including the days of vaccine/placebo administration). Additional safety assessments will include 
new medical conditions (neurological and neuroinflammatory disorders) with onset after the first 
vaccination, and SAEs collection for the duration of the entire trial. Immunogenicity will be 
assessed for all subjects 28 days after each vaccine/placebo administration and 6 months post 
dose 2, and for subjects who will be randomized to the placebo group or the PIZV dosing group 
that will be selected for further development also 12 and 24 months post dose 2.
Refer to Section 13.2 for details about sequence of analyses.
0047
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 47 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
6.2
Justification for Trial Design, Dose, and Endpoints
The trial design and the collection of solicited and unsolicited AEs following vaccination are 
consistent with guidelines on vaccine evaluation trials, including dose-finding studies.
The design of the study answers to the principles outlined in the ICH Considerations on Clinical 
Trials E8 (86) and the WHO Guidelines for Evaluation of Vaccines (87), and more specifically for 
phase 1 trials. As such, the study design, objectives and endpoints were defined so as to assess the 
initial safety and reactogenicity of the vaccine candidate, obtain preliminary information on its 
immunogenicity, and provide data useful for the design of further clinical studies. The 
dose-ranging design adopted is in line with the ICH and FDA guidance (88, 89). The use of 
placebo as control for the investigational vaccine is justified in the absence of a proven 
intervention (90), ie, as no prophylactic vaccine against ZIKV infection is available to date. 
As this is a first in human trial, a relatively high serum volume (60 mL) will be needed for assay 
development and other pre-clinical studies to support possible alternative license pathways which 
may include bridging animal data to human data.
The rationale for the proposed trial is given in Section 4.2.7.
Please also refer to the IB.
6.3
Duration of Subject’s Expected Participation in the Entire Trial
Each enrolled subject’s participation in the trial will last approximately 7 or 25 months, following 
a screening period prior to Visit 2 of two-weeks for flavivirus naïve subjects and of more than two 
weeks for flavivirus primed subjects.
6.4
Premature Termination or Suspension of Trial or Investigational Site
6.4.1
Criteria for Premature Termination or Suspension of the Trial
The trial will be completed as planned unless one or more of the following criteria are satisfied that 
require temporary suspension or early termination of the trial.

New information or other evaluation regarding the safety or efficacy of the investigational 
vaccine that indicates a change in the known risk/benefit profile, such that the risk/benefit is no 
longer acceptable for subjects participating in the trial.

The external DMC recommends that the trial should be suspended or terminated.

Significant deviation from Good Clinical Practice (GCP) that compromises the ability to 
achieve the primary trial objectives or compromises subject safety.
6.4.2
Criteria for Premature Termination or Suspension of Investigational Sites
A trial site may be terminated prematurely or suspended if the site (including the investigator) is 
found in significant deviation from GCP, protocol, or contractual agreement, is unable to ensure 
adequate performance of the trial, or as otherwise permitted by the contractual agreement.
0048
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 48 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
6.4.3
Procedures for Premature Termination or Suspension of the Trial or the 
Participation of Investigational Site(s)
In the event that the sponsor, an institutional review board (IRB)/independent ethics committee 
(IEC) or regulatory authority elects to terminate or suspend the trial or the participation of an 
investigational site, a trial-specific procedure for early termination or suspension will be provided 
by the sponsor; the procedure will be followed by applicable investigational sites during the course 
of termination or trial suspension.
0049
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 49 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
7.0
SELECTION AND DISCONTINUATION/WITHDRAWAL OF SUBJECTS
All entry criteria, including test results, need to be confirmed prior to randomization.
7.1
Inclusion Criteria
Subject eligibility is determined according to the following criteria:
1. The subject is aged ≥18 to ≤49 years.
2. Individuals who are in good health at the time of entry into the trial as determined by medical 
history, physical examination (including vital signs) and eligibility screening tests 
(hematology, biochemistry and urinalysis) and clinical judgment of the investigator. Vital 
signs must be within normal limits (ie, below Grade 1 as specified in the FDA Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers, see Appendix E). Screening tests 
must be within normal limits or not be above Grade 1 as defined in the FDA Toxicity Grading 
Scale for Healthy Adult and Adolescent Volunteers (see Appendix E).
3. The subject signs and dates a written, informed consent form and any required privacy 
authorization prior to the initiation of any trial procedures, after the nature of the trial has been 
explained according to local regulatory requirements (Appendix B).
4. Individuals who can comply with trial procedures and are available for the duration of 
follow-up.
5. All female participants of childbearing potential must have a negative serum beta human 
chorionic gonadotropin (-hCG) pregnancy test at screening and a negative urine -hCG 
pregnancy test prior to receiving any dose of investigational vaccine/placebo.
7.2
Exclusion Criteria
Any subject who meets any of the following criteria will not qualify for entry into the trial:
1. Subjects and subjects’ partners with confirmed ZIKV infection by self-report.
2. Traveling to flavivirus endemic countries or flavivirus endemic regions of the US/US 
territories*, within 4 weeks prior to screening or planned travel through to Visit 6 (applicable 
only to subjects to be enrolled into the flavivirus naïve cohort).
*CDC website defines the information about the flavivirus endemic countries and US regions 
and territories (see Appendix D for the respective website addresses).
3. Known hypersensitivity or allergy to any of the vaccine candidate components (including 
excipients of the investigational vaccines, control or placebo as summarized in Section 8.1).
4. Individuals with behavioral or cognitive impairment or psychiatric disease that, in the opinion 
of the investigator, may interfere with the subject's ability to participate in the trial.
5. Individuals with any history of progressive or severe neurologic disorder, seizure disorder or 
neuro-inflammatory disease (eg, Guillain-Barré syndrome).
0050
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 50 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
6. Individuals with history or any illness that, in the opinion of the investigator, might interfere 
with the results of the trial or pose additional risk to the subjects due to participation in the trial.
7. Known or suspected impairment/alteration of immune function, including:
o
Chronic use of oral steroids (equivalent to 20 mg/day prednisone ≥12 weeks / 
≥2 mg/kg body weight / day prednisone ≥2 weeks) within 60 days prior to Day 1 (use 
of inhaled, intranasal, or topical corticosteroids is allowed).
o
Receipt of parenteral steroids (equivalent to 20 mg/day prednisone ≥12 weeks / 
≥2 mg/kg body weight / day prednisone ≥2 weeks) within 60 days prior to Day 1.
o
Receipt of immunostimulants within 60 days prior to Day 1.
o
Receipt of parenteral, epidural or intra-articular immunoglobulin preparation, blood 
products, and/or plasma derived products within 3 months prior to Day 1 or planned 
during the full length of the trial. In addition, subjects must be advised not to donate 
blood during the study period.
o
Known Human Immunodeficiency Virus (HIV) infection or HIV-related disease.
o
Genetic immunodeficiency.
8. Individuals with known current or chronic hepatitis B and/or hepatitis C infections.
9. Abnormalities of splenic or thymic function.
10. Individuals with a known bleeding diathesis, or any condition that may be associated with a 
prolonged bleeding time.
11. Individuals with any serious chronic or progressive disease according to judgment of the 
investigator (eg, neoplasm, insulin-dependent diabetes, cardiac, renal, hepatic or thyroid 
disease, uncontrolled hypertension, uncontrolled asthma).
12. Individuals with body mass index (BMI) greater than or equal to 35 kg/m2 (= weight in kg / 
[height in meters x height in meters]).
13. Individuals participating in any clinical trial with another investigational product, including 
ZIKV vaccine trial, within 30 days prior to first trial visit or intent to participate in another 
clinical trial at any time during the conduct of this trial.
14. Individuals who received any other vaccines within 14 days (for inactivated vaccines) or 
28 days (for live vaccines) prior to Day 1 in this trial or who are planning to receive any 
vaccine within 28 days of investigational vaccine/placebo administration.
15. Individuals involved in the trial conduct or their first degree relatives.
16. Individuals with history of substance or alcohol abuse within the past 2 years.
17. Female subjects who are pregnant or breastfeeding, or are planning to become pregnant.
18. Any positive or indeterminate pregnancy test.
0051
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 51 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
19. If female subject of childbearing potential, sexually active, and who has not used any of the 
"acceptable contraceptive methods" for at least 2 months prior to trial entry:
o
“Of childbearing potential” is defined as status post onset of menarche and not meeting 
any of the following conditions: menopausal for at least 2 years without any other 
alternative medical cause (as confirmed by a healthcare professional), status after 
bilateral tubal ligation for at least 1 year, status after bilateral oophorectomy, or status 
after hysterectomy.
o
Acceptable birth control methods are defined as one or more of the following:
–
Hormonal contraceptive (such as oral, injection, transdermal patch, implant, cervical 
ring).
–
Barrier (condom with spermicide or diaphragm with spermicide) each and every time 
during intercourse.
–
Intrauterine device.
–
Monogamous relationship (only have one sexual partner) with vasectomized partner. 
Partner must have been vasectomized for at least six months prior to the subjects' trial 
entry.
20. If female subject of childbearing potential and sexually active, refusal to use an "acceptable 
contraceptive method" from trial entry through 2 months after the last dose of investigational 
vaccine/placebo. In addition, female subjects of childbearing potential must be advised not to 
donate ova during this period.
21. To avoid sexual transmission of ZIKV from natural exposure: Refusal to use latex condoms 
correctly and consistently by sexually active subjects even if other contraceptive measures are 
used from signing the ICF through the end of the trial. Male subjects must be advised not to 
donate sperm during this period. 
Temporary screening failure
A subject may be a temporary screen failure under the following circumstances: due to temporary 
conditions such as receipt of immunosuppressive or immunomodulatory therapies, blood products, 
immunoglobulins, or vaccines, in the respective prohibited periods (see exclusion criteria 7, 13 or 
14), or in the event that a subject has an indeterminate pregnancy test result or has not used 
acceptable contraceptive methods in the period defined in the protocol, prior to the start of the 
study (exclusion criteria 19). Subjects screen failed for the above-mentioned reasons may be 
rescreened once they cease to meet the respective exclusion criteria. Should the respective 
repeated test results be available within the initial screening period, no new subject ID numbers 
will be required; the repeated screening will be recorded in the appropriate CRF (unscheduled lab 
form). Should the respective repeated test results not be available within the initial screening 
period, the subject would need to be screen failed and all screening procedures and tests would 
need to be repeated with a new subject ID number.
0052
cial Use Only and Subject to the Applicable Terms of Use
ing
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 52 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
7.3
Criteria for Delay of Vaccination and/or Blood Sampling
After enrollment, subjects may encounter clinical circumstances that warrant a delay in the 
administration of the subsequent dose of investigational vaccine/placebo. These situations are 
listed below:

Individuals with a clinically significant active infection (as assessed by the investigator) or 
temperature ≥38.0°C/100.4°F, within 3 days of intended investigational vaccine/placebo 
administration.

Individuals who received any other vaccines within 28 days of planned vaccination.
In the event that a subject meets a criterion for delay of vaccination, the subject may receive trial 
vaccination once the window for delay has passed as long as the subject is otherwise eligible for 
trial participation. The decision to vaccinate in those situations will be taken by the investigator.
There are also circumstances under which receipt of further vaccines is a contraindication in this 
trial. These circumstances include anaphylaxis or severe hypersensitivity reactions following the 
initial vaccine/placebo administration. If these reactions occur, the subject must not receive 
additional vaccinations; however, the subject is encouraged to continue in trial participation for 
safety reasons.
7.4
Criteria for Early Study Termination of a Subject
Under some circumstances, a subject’s trial participation may be terminated early. This means that 
no further trial procedures (including data collection) will be performed on that subject beyond the 
specific date of early termination. The primary reason for early termination of the subject from the 
trial should be recorded in the electronic Case Report Form (eCRF “end of study visit” page) using 
the following categories. For screen failure subjects, refer to Section 9.1.10.
1. Adverse Event: The subject has experienced an AE (irrespective of being related / unrelated to 
the Trial Vaccine or trial-related procedures) that requires early termination because continued 
participation imposes an unacceptable risk to the subject’s health and / or the subject is 
unwilling to continue participation because of the AE. If the subject is unwilling to continue 
because of the AE the primary reason for early termination in this case will be ‘withdrawal due 
to AE’ and not ‘withdrawal of consent’, see below. Any ongoing AEs leading to early 
termination will be followed by the investigator until resolution or stabilization.
2. Lost to follow-up: The subject did not return to the clinic and at least three attempts to contact 
the subject were unsuccessful. Attempts to contact the subject must be documented.
3. Withdrawal of consent: The subject wishes to withdraw from the trial. The primary reason for 
early termination will be ‘withdrawal of consent” if the subject withdraws from participation 
due to a non-medical reason (ie, reason other than AE). The reason for withdrawal, if provided, 
should be recorded in the eCRF.
Note: All attempts should be made to determine the underlying reason for the withdrawal and, 
where possible, the primary underlying reason should be recorded. 
0053
on-Commercial Use Only and Subject to the Applicable Terms of Use
ator) or 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 53 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
4. Premature study termination by sponsor, a regulatory agency, the IEC/IRB, or any other 
authority.
If the clinical study is prematurely terminated by the sponsor, the investigator is to promptly 
inform the study subjects and local EC/IRB and should assure appropriate follow up for the 
subjects. The primary reason for early termination in this case will be ‘trial termination”.
5. Subject’s death during trial participation.
6. Other.
Note: The specific reasons should be recorded in the “specify” field of the eCRF.
7.5
Criteria for Premature Discontinuation of Trial Vaccine Application 
Early (premature) study termination of a subject will by default prevent the subject from continued 
Trial Vaccine administration, as the subject will no longer be participating in the study.
In addition to early termination (see Section 7.4) criteria, other situations may apply in which 
subjects may continue participating in the trial (eg, contributing safety data according to protocol) 
but Trial Vaccine application is discontinued selectively. Regardless of the reasons for 
discontinuation of Trial Vaccine application, this must be documented as protocol deviation. Even 
if the subject is deemed ineligible to continue to receive Trial Vaccine, all efforts should be made 
to continue the collection of safety data according to protocol. In addition, the one primary reason 
for premature discontinuation of Trial Vaccine application should be recorded in the eCRF, “end 
of Trial Vaccine application” page using the following categories.
1. Adverse Event: The subject has experienced an AE (irrespective of being related / unrelated to 
the Trial Vaccine or trial-related procedures) for which subsequent Trial Vaccine applications 
impose an unacceptable risk to the subject’s health, but the subject will continue trial 
participation for safety, or a subset of other study procedures. 
2. Lost to follow-up: The subject did not return to the clinic and at least three attempts to contact 
the subject were unsuccessful. Attempts to contact the subject must be documented
3. Withdrawal of consent: The subject wishes to withdraw from the trial. The primary reason for 
early termination will be ‘withdrawal of consent” if the subject withdraws from participation 
due to a non-medical reason (ie, reason other than AE). The reason for withdrawal, if provided, 
should be recorded in the eCRF.
4. Premature study termination by sponsor, a regulatory agency, the IEC/IRB, or any other 
authority.
If the clinical study is prematurely terminated by the sponsor, the investigator is to promptly 
inform the study subjects and local EC/IRB and should assure appropriate follow up for the 
subjects. The primary reason for early termination in this case will be ‘trial termination”.
5. Subject´s death during trial participation.
0054
keda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
he eCRF
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auth


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 54 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
6. Protocol deviation: A protocol deviation is any change, divergence, or departure from the 
study design or procedures of a study protocol. The subject may remain in the trial unless 
continuation in the trial jeopardizes the subject’s health, safety or rights (see Section 7.4).
7. Pregnancy: Any subject who, despite the requirement for adequate contraception, becomes 
pregnant during the trial will not receive further Trial Vaccine applications. Pregnant subjects 
should, however, be asked to continue participating in the trial contributing data to the safety 
follow-up according to protocol. In addition, the site should maintain contact with the pregnant 
subject and complete a “Clinical Trial Pregnancy Form” as soon as possible. The subject 
should be followed-up until the birth of the child, or spontaneous or voluntary termination; 
when pregnancy outcome information becomes available, the information should be captured 
using the same form. Data obtained from the “Clinical Trial Pregnancy Form” will be captured 
in the safety database.
8. Other.
Note: The specific reasons should be recorded in the “specify” field of the eCRF.
0055
pplicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 55 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
8.0
CLINICAL TRIAL MATERIAL MANAGEMENT
This section contains information regarding all vaccines and materials provided directly by the 
sponsor, and/or sourced by other means, that are required by the trial protocol, including important 
sections describing the management of clinical trial material.
8.1
Trial Vaccines and Materials
8.1.1
Dosage Form, Manufacturing, Packaging, and Labeling
In this protocol, the investigational vaccine (PIZV) refers to Zika purified formalin-inactivated 
virus formulated with 200 µg aluminum hydroxide, Al(OH)3, as adjuvant, in phosphate buffered 
saline solution (PBS). The final liquid formulated product is filled into single-use vials and sealed 
with tamper-evident seals at Takeda’s manufacturing plant in Hikari, Japan. The vaccine is 
packaged, labeled, and shipped to unblinded study site pharmacist/designee. The investigational 
vaccine is administered IM as a 2-dose regimen of 0.5 mL at 2, 5, or 10 µg antigen per dose, 
28 days apart. Refer to the IB and the Pharmacy Manual for more details.
Sodium chloride (NaCl) 0.9% solution for injection is being used as placebo. It is supplied in 
single-use vials. It is a sterile, clear, colorless liquid solution of sodium chloride without 
preservative designed for parenteral use only. The placebo is packaged, labelled and shipped to 
unblinded study site pharmacist/designee. The placebo is administered IM as a 2-dose regimen of 
0.5 mL per dose, 28 days apart. Refer to the Pharmacy Manual for more details.
8.1.2
Storage
PIZV/placebo will be shipped in refrigerated and temperature-monitored containers. From receipt 
and prior to use, the investigational vaccine and the placebo must be protected from light and 
stored at 2°C to 8°C in a temperature monitored refrigerator with controlled access available only 
to authorized trial personnel. The investigational vaccine/placebo must not be frozen. In case of 
accidental freezing, frozen investigational vaccine/placebo should never been administered to 
subjects. The investigational vaccine/placebo is single use only.
All clinical trial material must be kept in an appropriate, limited-access, secure place until it is used 
or returned to the sponsor or designee for destruction. All sponsor-supplied vaccines/placebos 
must be stored under the conditions specified on the label, and remain in the original container 
until dispensed. A daily temperature log of the vaccine/placebo storage area must be maintained 
every working day. Temperature excursions must be reported to the sponsor as soon as possible 
and use of these vaccines/placebos requires sponsor approval. Refer to the Pharmacy Manual.
8.1.3
Dose and Regimen
Three formulations of the vaccine containing different antigen concentrations will be used, where 
low dose contains 2 µg, medium dose contains 5 µg, and high dose contains 10 µg of the vaccine. 
Each dose will be packaged and appropriately labeled in compliance with regional regulatory 
requirements.
0056
mmercial Use Only and Subject to the Applicable Terms of Use
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8.1.


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 56 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Table 8.a describes the doses that will be provided to each group.
Table 8.a
Sponsor-Supplied Vaccine and Placebo
Treatment Group
Dose
Treatment Description
Timing (Dose 1/Dose 2)
Arm 1
2 doses
Placebo
Day 1/Day 29
Arm 2
2 doses
PIZV 2 µg
Day 1/Day 29
Arm 3
2 doses
PIZV 5 µg
Day 1/Day 29
Arm 4
2 doses
PIZV 10 µg
Day 1/Day 29
8.2
Investigational Vaccine/Placebo Assignment and Dispensing Procedures
The investigator or investigator’s designee will access the Interactive Response Technology (IRT) 
at Screening Visit (1A or 1B) to obtain the subject ID number. 
The investigator or investigator’s designee will utilize the IRT to randomize the subject at Visit 2. 
The investigational vaccine/placebo identification numbers to be administered will be assigned by 
the IRT. Details of randomization and the IRT will be covered in the IRT User Manual.
The investigational vaccine/placebo will be prepared and administered by the unblinded designee 
who is qualified to perform that function under applicable laws and regulations for that specific 
trial, according to the instructions in the Pharmacy Manual. All investigational vaccine/placebo 
preparation will be documented.
All doses of the investigational vaccine/placebo will be administered IM into the middle third of 
the deltoid muscle of the subject’s arm, preferably the non-dominant arm.
The investigator or designee will be responsible for overseeing the administration of 
investigational vaccine/placebo to subjects enrolled in the trial according to the procedures 
stipulated in this trial protocol.
If vials containing the investigational vaccine/placebo are lost or damaged, the site can request a 
replacement from the IRT (refer to the IRT manual supplied separately). Expired trial vaccines 
must not be administered.
PRECAUTIONS TO BE OBSERVED IN ADMINISTERING THE TRIAL VACCINE:
Prior to vaccination, a subject must be determined to be eligible for trial vaccination and it must be 
clinically appropriate in the judgment of the investigator to vaccinate. Eligibility for vaccination 
prior to first trial vaccine administration is determined by evaluating the entry criteria outlined in 
this protocol (Sections 7.1 and 7.2).
Eligibility for subsequent trial vaccination is determined by following the criteria outlined in 
Section 7.0.
Trial vaccines should not be administered to individuals with known hypersensitivity to any 
component of the vaccines.
0057
Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 57 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Standard immunization practices are to be observed and care should be taken to administer the 
injection intramuscularly. In addition, WHO recommendations to reduce anxiety and pain at the 
time of vaccination should be followed (91). Before administering the vaccine, the vaccination site 
is to be disinfected with a skin disinfectant (eg, 70% alcohol). Allow the skin to dry. DO NOT 
inject intravascularly.
As with all injectable vaccines, trained medical personnel and appropriate medical treatment 
should be readily available in case of anaphylactic reactions following vaccination. For example, 
epinephrine 1:1000, diphenhydramine, and/or other medications for treating anaphylaxis should 
be available.
8.3
Randomization Code Creation and Storage
Randomization personnel of the IRT provider or designee will generate the randomization 
schedule(s). Randomization information will be stored in a secured area, accessible only by 
authorized personnel.
8.4
Investigational Product Blind Maintenance
This trial is an observer-blind study. The subjects, data collectors (eg, investigator), and data 
evaluators (eg, trial statisticians) are blinded to the material administered. The investigational 
product assignment will be maintained by the unblinded site staff designee.
All care must be taken to ensure that the unblinded reports and documents are shared only with 
unblinded personnel and properly stored in a secured area, accessible only by authorized 
personnel.
8.5
Unblinding Procedure
The investigational vaccine/placebo blind shall not be broken by the investigator unless 
information concerning the investigational vaccine/placebo is necessary for the medical treatment 
of the subject. In the event of a medical emergency, if possible, the medical monitor should be 
contacted before the investigational vaccine/placebo blind is broken to discuss the need for 
unblinding.
For unblinding a subject, the investigational vaccine/placebo blind can be obtained by the 
investigator, by accessing the IRT.
The sponsor’s Pharmacovigilance Department must be notified as soon as possible if the 
investigational vaccine/placebo blind is broken by the investigator; and if the unblinding was for 
medical reasons (SAE), the completed SAE form must be sent within 24 hours (ie, the site will 
follow the standard process for reporting of SAEs). The date, time, and reason the blind is broken 
must be recorded in the source document and the same information (except the time) must be 
recorded on the eCRF.
If any subject is unblinded, no further doses of investigational vaccine/placebo are to be 
administered. However, the subject will be followed up for safety and safety data will be collected 
according to protocol. 
0058
of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 58 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
8.6
Accountability and Destruction of Sponsor-Supplied Vaccine/Placebo
Investigational vaccine/placebo supplies will be counted and reconciled at the site before being 
returned to the sponsor or designee as noted below. Sites will maintain source documents in 
addition to entering data in the IRT.
The investigator or designee must ensure that the sponsor-supplied vaccine/placebo is used in 
accordance with the approved protocol and is administered only to subjects enrolled in the trial. To 
document appropriate use of sponsor-supplied vaccines/placebos, the investigator must maintain 
records of all sponsor-supplied vaccine/placebo delivery to the site, site inventory, administration 
and use by each subject, and return to the sponsor or designee.
Upon receipt of sponsor-supplied vaccines/placebos, the investigator or designee must verify the 
contents of the shipments against the packing list. The verifier should ensure that the quantity is 
correct, the investigational vaccine/placebo is received within the labeled storage conditions (ie, 
no cold chain break has occurred during transit), and is in good condition. If quantity and 
conditions are acceptable, investigator or designee will acknowledge receipt of the shipment by 
recording in IRT.
If there are any discrepancies between the packing list versus the actual product received, the 
sponsor or designee must be contacted to resolve the issue. The packing list should be filed in the
investigator’s essential document file.
The investigator must maintain 100% accountability for all sponsor-supplied vaccines/placebos 
received and administered during his or her entire participation in the trial. Proper vaccine/placebo 
accountability includes, but is not limited to:

Verifying that actual inventory matches documented inventory;

Verifying that the log is completed for the vaccine/placebo used to prepare each dose;

Verifying that all containers used are documented accurately on the log;

Verifying that required fields are completed accurately and legibly.
If any dispensing errors or discrepancies are discovered, the sponsor must be notified immediately.
The investigator must record the current inventory of all sponsor-supplied vaccines/placebos on a 
sponsor-approved vaccine/placebo accountability log. The following information will be recorded 
at a minimum: protocol number and title, name of investigator, site identifier and number, 
description of sponsor-supplied vaccines/placebos, date and amount. The log should include all 
required information as a separate entry for each subject to whom sponsor-supplied 
vaccine/placebo is administered.
The investigator will be notified of any expiry date or retest date extension of clinical trial material 
during the trial conduct. On expiry date notification from the sponsor or designee, the site must 
complete all instructions outlined in the notification, including segregation of expired clinical trial 
material for return to the sponsor or its designee for destruction.
0059
da: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 59 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Prior to site closure or at appropriate intervals throughout the trial, before any clinical trial 
materials are returned to the sponsor or its designee for destruction, a representative from the 
sponsor or its designee will perform clinical trial material accountability and reconciliation. The 
investigator will retain a copy of the documentation regarding clinical trial material accountability, 
return, and/or destruction, and originals will be sent to the sponsor or designee.
The pharmacist (or designated individual) at each site will be responsible for vaccine/placebo 
accountability and will document receipt, use, return, or destruction of PIZV. Vaccine/placebo 
accountability documentation will be reviewed by the monitor during clinical monitoring visits.
0060
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 60 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
9.0
TRIAL PLAN
9.1
Trial Procedures
The following sections describe the trial procedures and data to be collected. For each procedure, 
subjects are to be assessed by the same investigator or site personnel whenever possible. The 
Schedule of Trial Procedures is located in Section 2.1.
9.1.1
Informed Consent
The requirements of the informed consent are described in Section 15.2.
Informed consent must be obtained at Screening Visit (1A or 1B), prior to the subject entering into 
the trial and before any protocol-directed procedures are performed.
A unique subject ID number will be assigned at Screening Visit (1A or 1B) to each subject after 
informed consent is obtained from the appropriate coding eg, IRT. If all eligibility criteria are 
fulfilled, this subject ID number will be used throughout the trial. Subject ID numbers assigned to 
subjects who fail screening should not be reused (Section 9.1.10).
Flavivirus primed subjects who were tested at Screening Visit 1A and accepted to enter the second 
phase of the study (at Screening Visit 1B) do not need to sign the ICF again. Refer to Section 6.1
for more details about the staggered enrollment.
9.1.2
Demographics, Medical History, Travel History, Prior/Concomitant 
Medications/Vaccinations, and Blood Donation
Demographic information, to be obtained at Screening Visit (1A or 1B), will include age (date of 
birth), sex, race, and ethnicity as provided by the subject. Refer to Section 6.1 for more details 
about the staggered enrollment.
Medical history will also be collected at Screening Visit(s) (Visit 1A and/or Visit 1B) and at 
Visit 2 (Day 1) and will include any medical history that may be relevant to subject eligibility for 
trial participation such as prior vaccinations, concomitant medications, and previous and ongoing 
illnesses or injuries. Relevant medical history can also include any medical history that contributes 
to the understanding of an AE that occurs during trial participation, if it represents an exacerbation 
of an underlying disease/preexisting problem.
Medical occurrences before administration of the first dose of investigational vaccine/placebo are 
considered medical history.
Travel history to flavivirus endemic countries and flavivirus endemic regions of the US/US 
territories within 4 weeks prior to screening will be collected at Screening Visit 1A. Travel history 
to flavivirus endemic countries and flavivirus endemic regions of the US/US territories will be 
collected at each visit.
All medications, vaccines and blood products taken or received by the subjects within 3 months 
prior to the start of the trial are to be recorded on the source document (patient record) and entered 
on the Prior and Concomitant Medications eCRF. The use of antipyretics and/or analgesic 
0061
Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 61 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
medications within 24 hours prior to vaccination must be identified and the reason for their use 
(prophylaxis versus treatment) must be described in the source documents and the eCRF.
Medications taken for prophylaxis are those intended to prevent the onset of AEs following 
vaccination. Medications taken for treatment are intended to reduce or eliminate the presence of 
symptoms that are present. These data must be recorded in the source documents.
Prohibited Therapies (See Section 7.2)

Parenteral, epidural or intra-articular immunoglobulin preparation, blood products, and/or 
plasma derivatives within 3 months of trial vaccination. 

Immunosuppressive therapy within 3 months or systemic (eg, oral or parenteral) corticosteroid 
treatment within 60 days prior to trial vaccine administration.

Other vaccines within 14 days (for inactivated vaccines) or 28 days (for live vaccines) prior to 
Day 1 in this trial. Any planned vaccinations during the trial should be discussed with the 
investigator and if given recorded on the trial eCRFs.

Immunostimulants within 60 days prior to trial vaccine administration.
Subjects should not donate blood during the study period. 
Medical history (including corresponding medication) to be obtained will include any significant 
conditions or diseases that have disappeared or resolved at or prior to signing of informed consent.
All subjects will be tested for flaviviral serological status at Screening Visit (1A or 1B).
9.1.3
Documentation of Trial Entrance/Randomization
Only subjects who have signed an ICF at Screening Visit (1A or 1B), meet all of the inclusion 
criteria and none of the exclusion criteria, are eligible for entrance/randomization into the 
vaccination phase.
One single subject ID number will be assigned to each subject at Screening Visit (1A or 1B). 
Eligible subjects will be assigned randomly to one of the four study arms in a (1:1:1:1) ratio by a 
computer.
The randomization schedule will be created and controlled by the IRT provider. The 
randomization specification will be approved by the sponsor’s trial statistician or designee. 
If the subject is found to be ineligible for randomization/trial phase, the investigator should record 
the primary reason for failure on the subject enrollment screening log.
9.1.4
Physical Examination
Physical examinations must be performed by a qualified health professional in accordance with 
local regulations and licensing requirements designated within the Site Responsibility Delegation 
Log. Complete physical examination, including height and weight, will be performed at Screening 
Visit(s) (Visit 1A and/or Visit 1B) according to the investigator’s standard practice. Measurement 
0062
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 62 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
of height and weight is only required at Screening Visit(s) (Visit 1A and/or Visit 1B). BMI will be 
calculated using standard BMI calculator.
Additional symptom-directed physical examinations may be performed at following visits if 
deemed necessary or indicated by review of the subject’s medical history, and should assess 
clinically significant changes from the baseline examination.
The following should be documented in the subject’s source document:

The findings of complete physical examinations,

the findings of symptom-directed physical examinations,

if symptom-directed physical examinations were not required.
Findings consistent with the definition of AEs or SAEs (see Section 10.0) must be transcribed into 
the eCRF.
9.1.5
Vital Signs
Vital signs include (however, not limited to) systolic/diastolic blood pressure, pulse rate, 
respiratory rate, and temperature. Follow standard of care for trial population and operational 
feasibility.
Vital signs must be within normal limits (ie, below Grade 1 as specified in the FDA Toxicity 
Grading Scale for Healthy Adult and Adolescent Volunteers, see Appendix E). 
In the event of abnormal heart rate or blood pressure due to physiological variation or activity, the 
subject may rest for 10 minutes in a quiet room, and then blood pressure and/or heart rate may be 
re-measured. Repeated vital signs may be used to determine eligibility. 
9.1.6
Blood Sample and Urine Sample Collection
Blood samples will be collected at each site visit: ie, at Screening Visit(s) (Visit 1A and/or Visit 
1B) for flavivirus serostatus determination (see Section 9.1.6.1) and eligibility screening tests 
(including pregnancy testing) (see Section 9.1.6.2), at Visits 3 and 5 for routine safety laboratory 
testing (see Section 9.1.6.3), and at Visits 2, 4, 6, 8, and (for subjects who will be randomized to 
the placebo group or the PIZV dosing group that will be selected for further development) 9 and 
11 for immunogenicity, as well as for further development and characterization of assays (see 
Section 9.1.6.4). Should a blood sampling be performed during a vaccination visit, this sampling 
must occur before the administration of the investigational vaccine or placebo. The maximum 
volume of blood taken at any single visit will be between approximately 10 mL and 60 mL, and 
the approximate maximum total volume of blood for the whole trial will be 280 mL to 410 mL 
depending on the subject’s flavivirus serostatus at screening and the vaccine dose level the subject 
received. Blood should be taken from the subject using an aseptic venipuncture technique. Blood 
samples will be sent to the central laboratory.
Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) for 
eligibility screening (see Section 9.1.6.2), and at Visits 3 and 5 for routine safety laboratory testing 
0063
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Trial No. ZIK-101 
Page 63 of 108
Protocol Amendment 3 Version 7.0
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(see Section 9.1.6.3); and for women of childbearing potential at Visit 2 and 4 before each 
investigational vaccine/placebo administration for pregnancy testing (see Section 9.1.9). Urine 
samples for eligibility screening and routine safety laboratory testing will be sent to the central 
laboratory. Urine pregnancy tests will be performed at the study site using kits provided by the 
sponsor.
All samples will be collected in accordance with acceptable laboratory procedures. All samples 
will be processed and stored at the trial site as described in the provided Laboratory Manual.
9.1.6.1
Flavivirus Serostatus Determination
All subjects who sign the ICF will be tested for flavivirus serostatus determination at Screening 
Visit (1A or 1B). For Screening, the subject’s serostatus relevant to major flavivirus(es) will be 
evaluated (including but not limited to Dengue, ZIKA, West Nile, Japanese Encephalitis).
Blood samples for flavivirus serostatus determination will be approximately 10 mL. Refer to the 
Serology Plan, available on file.
Each subject will be tested only once for flavivirus serostatus determination.
The first phase of the study will be aimed at enrolling flavivirus naïve subjects only, while the 
second phase of the study will be aimed at enrolling flavivirus primed subjects only. Flavivirus 
primed subjects will not be enrolled (randomized) during the first phase of the study (ie, when 
identified at Screening Visit 1A) and will be invited to come back for the second phase of the 
study. Refer to Section 6.1 for more details about the staggered enrollment.
9.1.6.2
Eligibility Screening Tests
Screening laboratory tests that will be performed on blood and urine samples at Screening Visit(s) 
(Visit 1A and/or Visit 1B) are outlined in Table 9.a. 
In order for the subjects to be enrolled, they must have laboratory values within normal limits or 
not be above Grade 1 as defined in the FDA Toxicity Grading Scale for Healthy Adult and 
Adolescent Volunteers (see Appendix E) within 15 days before enrollment. If laboratory screening 
tests are out of acceptable range, repeat of screening tests is permitted once, provided there is an 
alternative explanation for the out of range value.
Should the respective repeated test results be available within the initial screening period, no new 
subject ID numbers will be required; the repeated screening tests will be recorded in the 
appropriate CRF (unscheduled lab form). Should the respective repeated test results not be 
available within the initial screening period, the subject would need to be screen failed and all 
screening procedures and tests would need to be repeated with a new subject ID number.
Blood samples for screening laboratory testing will be approximately 10 mL. 
Subjects who are tested flavivirus primed at Screening Visit 1A (during the first phase of the 
study) and accept to enter the second phase of the study will undergo an additional Screening Visit 
(Visit 1B) during which screening laboratory testing will be tested again. Refer to Section 6.1 for 
more details about the staggered enrollment.
0064
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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Table 9.a
Clinical Laboratory Assessments
Sample
Test
Blood
Hematology
Hemoglobin
Platelet count
White blood cell count
Total neutrophils
Eosinophils
Lymphocytes
PT (prothrombin time)
APTT (activated partial thromboplastin time)
Fibrinogen
Biochemistry
Blood urea nitrogen
Creatinine
AST (aspartate transaminase)
ALT (alanine transaminase)
Alkaline phosphatase
Sodium 
Potassium
Chloride
Bicarbonate
Pregnancy test
-hCG blood test (only at Screening Visit(s) 
Visit 1A and/or Visit 1 B)
Urine
Urinalysis
Protein
Glucose
Blood (microscopic)
Red blood cells per high power field
9.1.6.3
Safety Laboratory Testing 
Routine safety laboratory parameters that will be performed on blood and urine samples at Visit 3 
(Day 8) and Visit 5 (Day 36) are outlined in Table 9.a.
Each blood sample will be approximately 10 mL.
9.1.6.4
Immunogenicity Assessments
Subjects in all study arms will undergo blood sampling for serological immunogenicity testing at 
Visit 2 (Day 1), Visit 4 (Day 29), Visit 6 (Day 57), Visit 8 (Day 211), and (for subjects who will 
be randomized to the placebo group or the PIZV dosing group that will be selected for further 
development) Visit 9 (Day 393) and Visit 11 (Day 757). Should a blood sampling be performed 
during a vaccination visit, this sampling must occur before the administration of the 
investigational vaccine or placebo. Refer to the Serology Plan, available on file.
Each blood sample for immunogenicity assessments will be approximately 60 mL. Some of it will 
be used for further development and characterization of assays if subjects agree and sign the ICF.
0065
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 65 of 108
Protocol Amendment 3 Version 7.0
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9.1.7
Safety Assessments
During the trial, safety assessments will include collection and recording of solicited local 
(injection site) and systemic AEs (including fever), unsolicited AEs (serious and non-serious), and 
new medical conditions (neurological and neuroinflammatory disorders) with onset after the first 
vaccination. Refer to Section 10.1 for safety definitions. Details on collection and reporting of AEs 
are in Section 10.5. Refer to Section 9.3.4 for details about the diary cards distribution, review and 
collection processes.
9.1.8
Contraception and Avoidance of Sexually Transmitted Disease Guidance
Subjects will be provided with information on acceptable methods of contraception and of 
protection against sexually transmitted diseases.
Female subject of childbearing potential and sexually active will have to use an "acceptable 
contraceptive method" from trial entry through 2 months after the last dose of trial vaccine, and 
will be advised not to donate ova during this period.
Furthermore, to avoid sexual transmission of ZIKV from natural exposure, all enrolled sexually 
active subjects (both male and female) will have to use latex condoms correctly and consistently 
even if other contraceptive measures are used from signing the ICF through the end of the trial.
Male subjects must be advised not to donate sperm during this period.
Refer to Section 7.2 for more details.
9.1.9
Pregnancy
In women of childbearing potential, a serum pregnancy testing will be performed at Screening 
Visit(s) (Visit 1A and/or Visit 1B), and a urine pregnancy testing will be performed at Visit 2 
(Day 1) before randomization (and thereby before investigational vaccine/placebo dose 1 
administration) for confirmation of eligibility, as well as at Visit 4 (Day 29) before investigational 
vaccine/placebo dose 2 administration. 
Serum pregnancy tests will be conducted in the central laboratory (and will be part of the eligibility 
screening tests) and urine pregnancy tests will be performed at the study site using kits provided by 
the sponsor. 
Subjects must have a negative serum -hCG pregnancy test at screening and a negative urine 
-hCG pregnancy test prior to receiving any dose of investigational vaccine/placebo.
To ensure subject safety and the safety of the unborn child, each pregnancy in a subject having 
received an investigational vaccine/placebo must be reported to the sponsor within 24 hours of the 
site learning of its occurrence. If the subject becomes pregnant during the trial, she will not receive 
any further doses of any sponsor-supplied investigational vaccine/placebo. The pregnancy must be 
followed to determine outcome, including spontaneous or voluntary termination, details of birth, 
and the presence or absence of any birth defects, congenital abnormalities, or maternal and/or 
newborn complications. This follow-up should occur even if the intended duration of safety 
follow-up for the trial has ended.
0066
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 66 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Any pregnancy occurring following investigational vaccine/placebo administration should be 
reported immediately, using a pregnancy notification form, to the contact listed in the Investigator 
Site File.
Should the pregnancy occur after administration of a blinded investigational vaccine/placebo, the 
investigator must inform the subject of their right to receive information on the study 
vaccine/placebo administered. If the subject chooses to receive unblinded information, the 
individual blind should be broken by the investigator and procedures must be followed, as 
described in Section 8.5.
9.1.10 Documentation of Subjects who are not Randomized
Investigators must account for all subjects who sign an ICF. If the subject is found to be not 
eligible at this visit, the investigator should complete the eCRF. The IRT supplier should be 
contacted as a notification of non-randomization.
The primary reason for non-randomization is recorded in the eCRF using the following categories:

Screen failure
o
did not meet one or more inclusion criteria
o
did meet one or more exclusion criteria
o
tested flavivirus naïve at Screening Visit 1B (during the second phase of the study)
o
tested flavivirus primed at Screening Visit 1A (during the first phase of the study) and 
did not come back for the second phase of the study
o
with a non-determinant screening test result

Withdrawal by subject,

Trial terminated by sponsor.
A subject may be a temporary screen failure under the following circumstances: due to temporary 
conditions such as receipt of immunosuppressive or immunomodulatory therapies, blood products, 
immunoglobulins, or vaccines, in the respective prohibited periods (see exclusion criteria 7, 13 or 
14), or in the event that a subject has an indeterminate pregnancy test result or has not used 
acceptable contraceptive methods in the period defined in the protocol, prior to the start of the 
study (exclusion criteria 19). Subjects screen failed for the above-mentioned reasons may be 
rescreened once they cease to meet the respective exclusion criteria. Should the respective 
repeated test results be available within the initial screening period, no new subject ID numbers 
will be required; the repeated screening tests will be recorded in the appropriate CRF (unscheduled 
lab form). Should the respective repeated test results not be available within the initial screening 
period, the subject would need to be screen failed and all screening procedures and tests would 
need to be repeated with a new subject ID number.
Subject ID numbers assigned to subjects who fail screening should not be reused.
0067
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 67 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
9.2
Monitoring Subject Treatment Compliance
The investigator records all injections of investigational vaccine/placebo given to the subject in the 
eCRF.
9.3
Schedule of Observations and Procedures
The schedule for all trial-related procedures for all evaluations is shown in Section 2.1. 
Assessments should be completed at the designated visit(s)/time point(s).
9.3.1
Screening Procedures (Screening Visit 1A/ Screening Visit 1B)
Refer to Section 6.1 for more details about the staggered enrollment.
The following screening procedures will be performed at Screening Visit(s) (Visit 1A and/or Visit 
1B):
1. Confirm informed consent, and complete and collect ICF (see Section 9.1.1). Before 
performing any other trial procedure, the signed ICF needs to be obtained. Only applicable for 
newly screened subjects.
2. Assign subject ID number (see Section 6.1 for more details about the staggered enrollment).
3. Assess eligibility criteria (see Sections 7.1 and 7.2).
4. Collect demographics (see Section 9.1.2). Only applicable for newly screened subjects.
5. Collect medical history, travel history (to flavivirus endemic countries and flavivirus endemic 
regions of the US/US territories), and prior/concomitant medications/vaccinations (see 
Section 9.1.2).
6. Perform “complete” physical examination (see Section 9.1.4).
7. Check vital signs (see Section 9.1.5).
8. Collect blood sample for flavivirus serostatus (only applicable for newly screened subjects, see 
Section 9.1.6.1) and eligibility screening tests (hematology, biochemistry and pregnancy 
testing, see Section 9.1.6.2). 
9. Collect urine sample for eligibility screening urinalysis (for all subjects, see Section 9.1.6.2).
10. Provide contraception and avoidance of sexually transmitted disease guidance (see 
Section 9.1.8).
9.3.2
Pre-Vaccination Procedures (Day 1 and Day 29)
The following procedures will be performed at Visit 2 (Day 1) and Visit 4 (Day 29):
1. Collect medical history, travel history (to flavivirus endemic countries and flavivirus endemic 
regions of the US/US territories), and concomitant medications/vaccinations (see 
Section 9.1.2).
2. Perform symptom-directed physical examination (if deemed necessary, see Section 9.1.4).
0068
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 68 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
3. Assess eligibility criteria (see Sections 7.1 and 7.2) at Visit 2 (Day 1).
4. Check vital signs (see Section 9.1.5).
5. Perform a urine pregnancy test (if the subject is a woman of childbearing potential, see 
Section 9.1.9).
6. Randomize subject (only at Visit 2 on Day 1) (see Section 9.1.3).
7. Collect blood sample for immunogenicity assessment and assay development (see 
Section 9.1.6.4).
9.3.3
Vaccination Procedures (Day 1 and Day 29)
The following procedures will be performed at Visit 2 (Day 1) and Visit 4 (Day 29):
1. Check contraindications to vaccination and criteria for delay of vaccination (see Sections 7.2
and 7.3).
2. Prepare the investigational vaccine/placebo according to the Pharmacy Manual (see 
Section 8.2).
3. Administer the investigational vaccine or placebo (see Section 8.2). 
9.3.4
Post Vaccination Procedures (Day 1 and Day 29)
The following post-vaccination procedures will be performed at Visit 2 (Day 1) and Visit 4 
(Day 29):
1. After vaccination, the subject will be observed for at least 30 minutes including observation for 
immediate reactions and body temperature measurement. Information should be recorded in 
the eCRF as immediate reactions. The investigator or delegate will take the opportunity to 
remind the subject how to measure solicited AEs and body temperature as part of this 
observation period. All safety data will be collected in the subject’s source documents.
2. Provide the subject with a diary card, and train the subject on how to use it.
Careful training of the subject on how to record concomitant medications, how to measure 
solicited AEs and body temperature, how to complete the diary card and how often to complete 
the diary card. Training should be directed at the individual(s) who will perform the 
measurements of solicited AEs and those who will enter the information into the diary card. 
This individual may or may not be the subject, but if a person other than the subject enters 
information into the diary card, this person’s identity must be documented in the trial file and 
this person must receive training on the diary card. Training of the subject on how to measure 
an injection site AE should be performed while the subject is under observation after 
vaccination.
Diary card instructions must include the following: The subject must understand that timely 
completion of the diary card on a daily basis is a critical component of trial participation. The 
subject should also be instructed to write clearly and to complete the diary card in pen. Any 
corrections to the diary card that are performed by the person completing the diary card should 
0069
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 69 of 108
Protocol Amendment 3 Version 7.0
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include a single strikethrough line with a brief explanation for any change and be initialed and 
dated.
Please note:
Diary cards will be the only source document allowed for remote collection of solicited local 
and systemic AEs (including body temperature measurements). The following additional rules 
apply to the documentation of safety information collected by diary card:
o
Diary cards should be reviewed with the subject.
o
No corrections or additions to the diary card will be allowed after it is reviewed with 
the investigator/designee.
o
Any data that are identified as implausible or incorrect, and confirmed by the subject to 
be a transcription error should be corrected by the subject on the diary card (the 
correction should include a single strikethrough line and should be initialed and dated 
by the subject).
o
Any blank or illegible fields on the diary card not otherwise corrected as above will be 
missing in the eCRF.
o
The site must enter all readable entries on the diary card into the eCRF.
o
Any newly described solicited safety information should be added to the diary card by 
the subject and initialed and dated. Any new unsolicited safety information would be 
recorded in the subject source document as a verbally reported event and therefore 
captured as an AE and recorded in the AE eCRF.
o
Starting on the day of vaccine/placebo administration, the subject will check for 
specific types of events at the injection site (solicited local AEs), any specific 
generalized symptoms (solicited systemic AEs), body temperature (preferably oral), 
any other symptoms or change in the subject’s health status, and any medications taken 
(excluding vitamins and minerals). These solicited AEs and body temperature will be 
recorded in the diary. Assessments should preferably take place in the evening at day’s 
end using the same route of measurement every day.
o
Temperature measurement is to be performed using the thermometer provided by the 
site. If the subject feels unusually hot or cold during the day, the subject should check 
his/her temperature. The highest body temperature observed that day should be 
recorded on the diary card.
o
The measurement of solicited local AEs (erythema, swelling, and induration) is to be 
performed using the ruler provided by the site. If multiple measurements are taken 
during the day, the highest measured value should be recorded on the diary card.
o
The collection on the diary card of body temperature, solicited local AEs, and solicited 
systemic AEs will continue for a total of 7 days following each vaccine/placebo 
administration. The collection on the diary card of unsolicited AEs and medications 
will continue for 28 days following each vaccine/placebo administration.
0070
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 70 of 108
Protocol Amendment 3 Version 7.0
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o
The healthcare professional reviewing the data on the diary cards will discuss the AEs 
(if any) reported by the subject and will determine if any additional diagnoses and/or 
AEs are present and/or concomitant medications have been used.
3. At Visit 4 (Day 29), review the previous diary card with the subject and collect it (see 
Section 9.3.4).
4. Provide contraception and avoidance of sexually transmitted disease guidance (see 
Section 9.1.8).
5. Provide the subject with a written reminder of the next planned trial activity. The subject will 
be reminded to complete the diary card daily and to contact the site if there are any questions 
and to contact the site immediately (or as soon as the subject is medically stable) if the subject 
has a medical condition that leads to a hospitalization or an emergency room visit or is 
otherwise perceived as serious. All contact details will be provided to the subject. 
6. Schedule the next trial clinic visit with the subject.
9.3.5
Clinic Visits after Vaccination (Day 8, Day 36, Day 57, Day 211, and Day 393)
The following procedures will be performed at Visit 3 (Day 8), Visit 5 (Day 36), Visit 6 (Day 57), 
and for subjects randomized to the placebo group or the PIZV dosing group that was selected for 
further development, Visit 8 (Day 211) and Visit 9 (Day 393):
1. Collect travel history (to flavivirus endemic countries and flavivirus endemic regions of the 
US/US territories) and concomitant medications/vaccinations (see Section 9.1.2).
2. Perform symptom-directed physical examination (if deemed necessary, see Section 9.1.4).
3. Check vital signs (see Section 9.1.5).
4. Collect blood sample for routine safety laboratory testing (only at Visit 3 on Day 8 and Visit 5 
on Day 36, see Section 9.1.6.3) and immunogenicity assessment and assay development (only 
at Visit 6 on Day 57, Visit 8 (Day 211), and Visit 9 (Day 393); see Section 9.1.6.4).
5. Collect urine sample for routine safety laboratory testing (urinalysis; only at Visit 3 on Day 8 
and Visit 5 on Day 36; see Section 9.1.6.3).
6. Review the previous diary card with the subject and collect it (see Section 9.3.4).
7. At Visit 3 (Day 8) and Visit 5 (Day 36), provide the subject with a new diary card, and remind 
the subject on how to use it (see Section 9.3.4).
8. Provide contraception and avoidance of sexually transmitted disease guidance (see 
Section 9.1.8).
9. Provide the subject with a written reminder of the next planned trial activity. The subject will 
be reminded (only at Visit 3 on Day 8 and Visit 5 on Day 36) to complete the diary card daily. 
The subject will be reminded to contact the site if there are any questions and to contact the site 
immediately (or as soon as the subject is medically stable) if the subject has a medical 
0071
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 71 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
condition that leads to a hospitalization or an emergency room visit or is otherwise perceived 
as serious. All contact details will be provided to the subject.
10. Schedule the next trial clinic visit with the subject.
9.3.6
Phone Contacts (Day 133 and Day 575)
A phone contact will be made on Day 133 (Visit 7) and Day 575 (Visit 10) with each subject to 
collect SAEs that may have occurred since Visit 6 (Day 57) and Visit 9 (Day 393), respectively, 
and to remind the subject of the next planned (end of) trial activity. The subject will also be 
reminded to contact the site if there are any questions, and to contact the site immediately (or as 
soon as the subject is medically stable) if the subject has a medical condition that leads to a 
hospitalization or an emergency room visit or is otherwise perceived as serious. All contact details 
will be provided to the subject. The subject should be reminded to contact the site via the telephone 
number provided in the ICF to discuss medical questions. 
Travel history (to flavivirus endemic countries and flavivirus endemic regions of the US/US 
territories) will be collected.
If the subject wishes to describe safety information, this information should only be collected by a 
trained healthcare professional at the site, and the safety data described must be written down in 
source documents.
The subject will be interviewed according to a script, and information relating to SAEs and/or AEs 
leading to trial or vaccine withdrawal and concomitant medications or vaccinations associated 
with those events will be collected. All safety information described by the subject must be written 
down in a designated location within the source documents and not written on the script used for 
the telephone call.
The site should schedule the next clinic visit with the subject, as appropriate. 
9.3.7
Final Visit (Day 211 or Day 757)
The Final Visit will be performed at Visit 8 (Day 211) or, for subjects randomized to the placebo 
group or the PIZV dosing group that was selected for further development, Visit 11 at Day 757 
(24 months post dose 2). If a subject terminates earlier, Final Visit procedures should be 
performed, if possible.
The following procedures will be performed at Visit 8 (Day 211) or Visit 11 (Day 757):
1. Collect travel history (to flavivirus endemic countries and flavivirus endemic regions of the 
US/US territories) and concomitant medications/vaccinations (see Section 9.1.2).
2. Perform symptom-directed physical examination (if deemed necessary, see Section 9.1.4).
3. Check vital signs (see Section 9.1.5).
4. Collect blood sample for immunogenicity assessment and assay development (see 
Section 9.1.6.4).
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 72 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
5. For all subjects having received the investigational vaccine or the placebo, the investigator 
must complete the End of Trial eCRF page.
9.3.8
Post-Trial Care
No post-trial care will be provided.
9.4
Biological Sample Retention and Destruction
In this trial, specimens for immune response testing will be collected as described in 
Section 9.1.6.4. After blood draw and serum processing, the serum samples will be preserved and 
retained at a central storage location that was contracted by the sponsor for this purpose for up to 
but not longer than 20 years or as required by applicable law. The sponsor has put into place a 
system to protect the subjects’ personal information to ensure optimal confidentiality and defined 
standard processes for sample and data collection, storage, analysis, and destruction.
Serum samples will be used for the analyses defined in this protocol, but can also, with permission 
from the subject, be used to assess, improve or develop tests related to the disease(s) or the vaccine 
under trial that will allow more reliable measurement of the response to the vaccine.
0073
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 73 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
10.0
ADVERSE EVENTS
10.1
Definitions
10.1.1 Adverse Events (AEs)
An AE is defined as any untoward medical occurrence in a clinical investigation subject 
administered a trial vaccine; it does not necessarily have to have a causal relationship with trial 
vaccine administration.
An AE can therefore be any unfavorable and unintended sign (eg, a clinically significant abnormal 
laboratory finding), symptom, or disease temporally associated with the administration of a trial 
vaccine whether or not it is considered related to the trial vaccine.
AEs will be graded by the investigator in the following manner:
Mild
Grade 1

Awareness of symptoms that are easily tolerated, causing minimal 
discomfort and not interfering with everyday activities. Relieved with 
or without symptomatic treatment.
Moderate
Grade 2

Sufficient discomfort is present to cause interference with normal 
activity. Only partially relieved with symptomatic treatment.
Severe
Grade 3

Extreme distress, causing significant impairment of functioning or 
incapacitation. Prevents normal everyday activities. Not relieved with 
symptomatic treatment
10.1.2 Solicited Adverse Events
The occurrence of selected indicators of safety (Table 10.a) will be measured/collected for 7 days 
after each dose (including the days of vaccine administration) and will be recorded on the “Local 
and Systemic AEs” eCRF as applicable. These will be summarized in the final report under the 
category “solicited adverse events” to differentiate them from other AEs which were not solicited. 
Any solicited local or systemic AE observed as continuing beyond 7 days after vaccination will be 
recorded as an unsolicited AE on the Adverse Event eCRF. Any solicited local (injection site) or 
systemic AE observed as continuing beyond 7 days following each trial vaccination will be 
additionally recorded on the Adverse Event eCRF for follow-up. For these persistent/prolonged 
solicited AEs, the end date will be captured on the Adverse Event eCRF to permit a separate 
analysis from the unsolicited AEs.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 74 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Table 10.a
Local and Systemic AEs
Local AEs (injection site):
Pain
Erythema
Swelling
Induration
Systemic AEs:
Fever (a)
Headache
Fatigue
Malaise
Arthralgia
Myalgia
(a) Based on recorded body temperature, fever is defined as body temperature ≥38.0°C/100.4°F regardless of method 
taken (Brighton Case Definition (92)).
The intensity of solicited safety parameters will be assessed as described in Table 10.b.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 75 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Table 10.b
Solicited Safety Parameters
Adverse Event
Intensity grade
Severity/Intensity
Pain at injection site
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity with or without treatment
3
Severe: Prevents daily activity with or without treatment
Erythema at injection 
site (a)
0
<25 mm
1
Mild: >25 – ≤50 mm
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Induration at injection 
site (a)
0
<25 mm
1
Mild: >25 – ≤50 mm
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Swelling at injection 
site (a)
0
<25 mm
1
Mild: >25 – ≤50 mm 
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Fever (b)
Record body temperature in °C/°F
Headache
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity with or without treatment
3
Severe: Prevents normal activity with or without treatment
Fatigue
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Malaise 
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Arthralgia
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Myalgia
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
(a) Subjects are to record greatest surface diameter in mm in the Diary.
(b) Based on recorded body temperature, fever is defined as body temperature ≥38.0°C/100.4°F regardless of method taken 
(Brighton Case Definition (92)).
0076
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 76 of 108
Protocol Amendment 3 Version 7.0
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10.1.3 Serious Adverse Events (SAEs)
An SAE is defined as any untoward medical occurrence that:
1. Results in DEATH.
2. Is LIFE THREATENING.

The term “life threatening” refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might have caused death 
if it were more severe.
3. Requires inpatient HOSPITALIZATION or prolongation of existing hospitalization.
4. Results in persistent or significant DISABILITY/INCAPACITY.
5. Leads to a CONGENITAL ANOMALY/BIRTH DEFECT in the offspring of a subject.
6. Is an IMPORTANT MEDICAL EVENT that satisfies any of the following:

May require intervention to prevent items 1 through 5 above.

May expose the subject to danger, even though the event is not immediately life 
threatening or fatal or does not result in hospitalization.
10.2
Causality of AEs
Relatedness (causality) to vaccine will also be assessed by the investigator. The relationship of 
each AE, including solicited systemic AEs (solicited local AEs are considered as related) to trial 
vaccine(s) will be assessed using the following categories:
Related:
There is suspicion that there is a relationship between the trial vaccine and the 
AE (without determining the extent of probability); there is a reasonable 
possibility that the trial vaccine contributed to the AE.
Not Related:
There is no suspicion that there is a relationship between the trial vaccine and the 
AE; there are other more likely causes and administration of the trial vaccine is 
not suspected to have contributed to the AE.
10.2.1 Relationship to Trial Procedures
Relationship (causality) to trial procedures should be determined for all AEs.
The relationship should be assessed as “Yes” if the investigator considers that there is reasonable 
possibility that an event is due to a trial procedure. Otherwise, the relationship should be assessed 
as “No”.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 77 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
10.2.2 Outcome of AEs
Resolved:
The subject has fully recovered from the event or the condition has 
returned to the level observed at baseline
Resolving:
The event is improving but the subject is still not fully recovered
Not resolved:
The event is ongoing at the time of reporting and the subject has still not 
recovered
Resolved with 
sequelae:
As a result of the AE, the subject suffered persistent and significant 
disability/incapacity (eg, became blind, deaf or paralyzed)
Fatal:
The subject died due to the event. If the subject died due to other 
circumstances than the event, the outcome of the event per se should be 
stated otherwise (eg, Not Resolved or Resolving)
Unknown:
If outcome is not known or not reported.
10.3
Additional Points to Consider for AEs
An untoward occurrence generally may:

Indicate a new diagnosis or unexpected worsening of a pre-existing condition. Intermittent 
events for pre-existing conditions or underlying disease should not be considered as AEs.

Necessitate therapeutic intervention.

Require an invasive diagnostic procedure.

Require vaccine discontinuation or a change in concomitant medication.

Be considered unfavorable by the investigator for any reason.
Diagnoses vs signs and symptoms:

Each event should be recorded to represent a single diagnosis. Accompanying signs (including 
abnormal laboratory values) or symptoms should NOT be recorded as additional AEs. If a 
diagnosis is unknown, signs or symptoms should be recorded appropriately as AEs.
Worsening of AEs:

If the subject experiences a worsening or complication of an AE after starting administration of 
the trial vaccine, the worsening or complication should be recorded as a new AE. Investigators 
should ensure that the AE term recorded captures the change in the condition (eg, “worsening 
of…”).

If the subject experiences a worsening or complication of an AE after any change in trial 
vaccine, the worsening or complication should be recorded as a new AE. Investigators should 
ensure that the AE term recorded captures the change in the condition (eg, “worsening of…”).
0078
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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Changes in severity of AEs:

If the subject experiences changes in severity of an AE, the event should be captured once with 
the maximum severity recorded.
Preplanned surgeries or procedures:

Preplanned procedures (surgeries or therapies) that were scheduled prior to signing of 
informed consent are not considered AEs. Complications resulting from any planned surgery 
should be reported as AEs.
Elective surgeries or procedures:

Elective procedures performed where there is no change in the subject’s medical condition 
should not be recorded as AEs; however, these procedures should be documented in the 
subject’s source documents. Complications resulting from an elective surgery should be 
reported as AEs.
Medical occurrences emerging during the time between signing of informed consent and the first 
administration of investigational vaccine/placebo will be recorded on the medical history eCRF 
page. However, if such medical occurrence is assessed as ’related to a study procedure’ this should 
be reported as an AE ‘related to study procedure’ in the eCRF.
10.4
Procedures
10.4.1 Collection and Reporting of AEs
All AEs, whether considered related with the use of the investigational vaccine/placebo or not, 
must be monitored until symptoms subside and any abnormal laboratory values have returned to 
baseline, or until there is a satisfactory explanation for the changes observed, or until death, in 
which case a full pathologist’s report should be supplied, if possible. All findings must be reported 
on an AE eCRF and on the SAE form, if necessary (see Section 10.4.2). All findings in subjects 
experiencing AEs must also be reported in the subject’s source documents. Any unsolicited AEs 
will be collected for 28 days after each vaccine/placebo administration (ie, through trial Day 28 
and trial Day 56) via diary cards. AEs leading to withdrawal or discontinuation will be collected 
throughout the trial.
The following information will be documented for each event:

Reported term for the Adverse Event,

Start and end date,

Serious (Y/N),

Severity,

Investigator’s opinion of the causality (relationship) between the event and administration of 
trial vaccine(s) (“related” or “not related”),
0079
da: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 79 of 108
Protocol Amendment 3 Version 7.0
12 September 2018

Investigator’s opinion of the causality (relationship) to trial procedure(s), including the details 
of the suspected procedure,

Action taken with the trial treatment (trial vaccine),

Outcome of event.
10.4.2 Collection and Reporting of Solicited AEs
The occurrence of selected indicators of safety will be collected on diary cards by the subjects for 
7 days following each investigational vaccine/placebo administration (ie, the day of vaccination 
+6 subsequent days) and will be recorded on the “Local and Systemic AE” eCRF, as applicable. 
These will be summarized in the final report under the category “solicited adverse events” to 
differentiate them from unsolicited AEs. Any solicited local (injection site) or systemic AE 
observed as continuing beyond 7 days following each trial vaccination will be additionally 
recorded on the Adverse Event eCRF for follow-up. For these persistent/prolonged solicited AEs, 
the end date will be captured on the Adverse Event eCRF to permit a separate analysis from the 
unsolicited AEs. 
Any solicited AE that meets any of the following criteria must be entered as an AE on the Adverse 
Event eCRF.

Solicited local or systemic AEs that lead the subject to withdraw from the trial.

Solicited local or systemic AEs that lead to the subject being withdrawn from the trial or to 
discontinuation of vaccination by the investigator.

Solicited local and systemic AEs that otherwise meet the definition of an SAE (see 
Section 10.1.2).
10.4.3 Collection and Reporting of SAEs
Collection of SAEs will commence from the time that the subject is first administered the 
investigational vaccine/placebo (Day 1). Routine collection of SAEs will continue up to the end of 
the trial at Visit 8 (Day 211) or Visit 11 (Day 757) for subjects who were randomized to the 
placebo group or the PIZV dosing group that was selected for further development.
SAEs should be reported according to the following procedure:
A sponsor SAE form must be completed, in English, and signed by the investigator immediately or 
within 24 hours of first onset or notification of the event. The information should be completed as 
fully as possible but contain, at a minimum:

A short description of the event and the reason why the event is categorized as serious.

Subject identification number.

Investigator’s name.

Name of the trial vaccine(s) – in a blinded way at all times, unless there is a valid reason to 
unblind.
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of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 80 of 108
Protocol Amendment 3 Version 7.0
12 September 2018

Causality assessment.
The SAE form should be transmitted within 24 hours to the attention of the contact(s) in the list 
provided to each site. 
Note: For this study, SAE reporting will be performed via eCRF. Only if the Electronic Data 
Capture system (EDC) is unavailable, a paper sponsor SAE form should be completed, signed by 
the investigator and transmitted within 24 hours. The SAE must be entered into the eCRF once 
access to the EDC is restored.
10.5
Follow-up Procedures
10.5.1 AEs
All AEs will be monitored until resolution or a stable status is reached or until a formal diagnosis 
can be made or until the end of the trial, whichever occurs first. 
10.5.2 SAEs
If information not available at the time of the first report becomes available at a later date, the 
investigator should complete a follow-up SAE form or provide other written documentation and 
fax it immediately within 24 hours of receipt. Copies of any relevant data from the hospital notes 
(eg, laboratory tests, discharge summary, postmortem results) should be sent to the sponsor.
All SAEs should be followed up until resolution or permanent outcome of the event(s) or until they 
are otherwise explained (outside of the trial, after Visit 11 at Day 757, whichever applicable). The 
timelines and procedure for follow-up reports are the same as those for the initial report.
10.5.3 Safety Reporting to Investigators, IRBs or IECs, and Regulatory Authorities
The sponsor or designee will be responsible for the reporting of all suspected unexpected serious
adverse reactions (SUSARs) and any other applicable SAEs to regulatory authorities, investigators 
and IRBs or IECs, as applicable, in accordance with national regulations in the countries where the 
trial is conducted. Relative to the first awareness of the event by/or further provision to the sponsor 
or sponsor’s designee, SUSARs will be submitted within 7 days for fatal and life-threatening 
events and 15 days for other SUSARs, unless otherwise required by national regulations. The 
sponsor will also prepare an expedited report for other safety issues where these might materially 
alter the current benefit-risk assessment of an investigational medicinal product or that would be 
sufficient to consider changes in the trial vaccine administration or in the overall conduct of the 
trial. The investigational site also will forward a copy of all expedited reports to their IRB or IEC 
in accordance with national regulations.
10.5.4 Post-Trial Events
Any SAE that occurs outside of the protocol-specified observation period or after the end of the 
trial but considered to be caused by the trial vaccine(s) must be reported to the sponsor. These 
SAEs will be processed by the sponsor’s Pharmacovigilance Department. Instructions for how to 
submit these SAEs will be provided in a handout in the Investigator Site File.
0081
ommercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 81 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
11.0
TRIAL-SPECIFIC REQUIREMENTS
11.1
Data Monitoring Committee Guidelines for Possible Study Pause
The external DMC will review safety data on an ongoing basis (refer to Section 11.2.1). More 
specifically, when the following set of AEs are reported, the external DMC will review all relevant 
safety data within 48 hours and consider a possible pause of the study, if it deems necessary:

Two or more subjects develop the same grade 3 solicited AE starting within 7 days of 
vaccination

Two or more subjects develop the same grade 3 unsolicited AE within 28 days of vaccination

Any SAE 

Any unexpected, serious, or unanticipated risk to the subjects enrolled.
11.2
Trial-Specific Committees
11.2.1 External Data Monitoring Committee
An external DMC will have oversight of this trial. The external DMC functions at a program level 
and further information is available in the external DMC Charter. The external DMC may review 
the data in an unblinded manner; however, the study team will remain blinded. The external DMC 
will receive predefined standard tables and listings of exposure and demographic data, solicited 
and unsolicited AEs, SAEs, new medical conditions (neurological and neuroinflammatory 
disorders) with onset after the first vaccination, and laboratory data obtained at specific data lock 
points for review. Any additional data requested by the external DMC will also be provided as 
needed. 
The external DMC will consist of 5 independent members and an independent non-voting 
statistician. External DMC meetings will consist of open and closed face-to-face meetings or 
teleconference calls. The type and frequency of scheduled meetings will depend on the subject 
enrollment and safety event rates. Unscheduled ad hoc meetings will occur at any time 
consultation is requested by the Pharmacovigilance Study Team. The recommendations will be 
communicated by the Chair of the external DMC to the Head of Pharmacovigilance in a timely 
manner. Takeda Vaccines will notify investigators and the appropriate regulatory authorities if the 
study is modified, placed on hold, completed, or closed.
0082
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 82 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
12.0
DATA HANDLING AND RECORDKEEPING
The full details of procedures for data handling will be documented in the Data Management Plan. 
AEs, medical history, and concurrent conditions will be coded using the Medical Dictionary for 
Regulatory Activities (MedDRA – System Organ Class [SOC], High Level Group Term [HLGT], 
High Level Term [HLT], Lowest Level Term [LLT], Preferred Term [PT], and their 
corresponding descriptive terms). Drugs will be coded using the WHO Drug Dictionary.
12.1
Electronic CRFs (eCRF)
Completed eCRFs are required for each subject who provides a signed ICF.
The sponsor or its designee will supply investigative sites with access to eCRFs. The sponsor will 
make arrangements to train appropriate site staff in the use of the eCRF. These forms are used to 
transmit the information collected in the performance of this trial to the sponsor and regulatory 
authorities. eCRFs must be completed in English.
After completion of the entry process, computer logic checks will be run to identify items, such as 
inconsistent dates, missing data, and questionable values. Queries may be issued by sponsor 
personnel (or designees) and will be answered by the site.
Corrections to eCRFs are recorded in an audit trail that captures the old information, the new 
information, identification of the person making the correction, the date the correction was made, 
and the reason for change.
The principal investigator or designee must review the eCRFs for completeness and accuracy and 
must sign and date the appropriate eCRFs as indicated. Furthermore, the investigator must retain 
full responsibility for the accuracy and authenticity of all data entered on the eCRFs.
eCRFs will be reviewed for completeness and acceptability at the trial site during periodic visits by 
trial monitors. The sponsor or its designee will be permitted to review the subject’s medical and 
hospital records pertinent to the trial to ensure accuracy of the eCRFs. The completed eCRFs are 
the sole property of the sponsor and should not be made available in any form to third parties, 
except for authorized representatives of appropriate governmental health or regulatory authorities, 
without written permission of the sponsor.
12.2
Record Retention
The investigator agrees to keep the records of the eCRF and those documents that include (but are 
not limited to) the trial-specific documents, the identification log of all participating subjects, 
source documents. Temporary media such as thermal sensitive paper should be copied and 
certified, source worksheets, all original signed and dated ICFs, subject authorization forms 
regarding the use of personal health information (if separate from the ICFs), electronic copy of 
eCRFs, including the audit trail, and detailed records of vaccine disposition to enable evaluations 
or audits from regulatory authorities, the sponsor or its designees. Furthermore, ICH E6 (2)
Section 4.9.5 requires the investigator to retain essential documents specified in ICH E6 (2)
Section 8 until at least 2 years after the last approval of a marketing application for a specified 
vaccine indication being investigated or, if an application is not approved, until at least 2 years 
0083
keda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 83 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
after the investigation is discontinued and regulatory authorities are notified. In addition, ICH E6 
(2) Section 4.9.5 states that the trial records should be retained until an amount of time specified by 
applicable regulatory requirements or for a time specified in the Clinical Study Site Agreement 
between the investigator and sponsor.
Refer to the Clinical Study Site Agreement for the sponsor’s requirements on record retention. The 
investigator should contact and receive written approval from the sponsor before disposing of any 
such documents.
0084
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 84 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
13.0
STATISTICAL METHODS
13.1
Statistical and Analytical Plans
A statistical analysis plan (SAP) will be prepared and finalized prior to unblinding of subject’s 
treatment assignment. This document will provide further details regarding the definition of 
analysis variables and analysis methodology to address all trial objectives.
A blinded data review will be conducted prior to unblinding of subject’s vaccination assignment. 
This review will assess the accuracy and completeness of the trial database, subject evaluability, 
and appropriateness of the planned statistical methods.
13.1.1 Analysis Sets

Safety Set: The Safety Set will consist of all randomized subjects who received at least one 
dose of the investigational vaccine/placebo.

Full Analysis Set (FAS): The FAS will include all randomized subjects who have received at 
least one dose of the investigational vaccine/placebo and provided valid baseline and at least 
one post-vaccination serology result. Subjects will be included in the FAS analysis ‘as 
randomized’.

Per–Protocol Set (PPS): The PPS will include all subjects in the FAS who have no major 
protocol violations. The major protocol violation criteria will be defined as part of the blinded 
data review prior to the unblinding of subject’s investigational vaccine/placebo assignment. 
The categories of major protocol violations include: 
a) not meeting selected entry criteria, 
b) receiving a wrong investigational vaccine/placebo, 
c) receiving prohibited therapies, and 
d) other major protocol violations that may be identified during blinded data reviews.
All summaries and analyses of safety data will be based on subjects in the Safety Set. 
The primary immunogenicity analyses will be based on the PPS, and additional immunogenicity 
analyses will be based on the FAS.
13.1.2 Analysis of Demographics and Other Baseline Characteristics
Summaries of age, gender, race, and other baseline characteristics will be presented by 
formulation arm.
13.1.3 Immunogenicity Analysis

Seropositive subjects: Subjects with detectable serum antibodies (tested positive at or above 
limit of detection, LOD) as measured by the neutralization assay.
0085
of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 85 of 108
Protocol Amendment 3 Version 7.0
12 September 2018

Seronegative subjects: Subjects with no detectable serum antibodies (test results are below 
LOD) as measured by the neutralization assay.

Seroconverted subjects: Seronegative subjects at baseline with detectable post-vaccination 
serum antibodies (test results are at or above LOD) and seropositive subjects at baseline with 
different fold increases in post-vaccination antibodies from baseline, as measured by the 
neutralization assay.
Descriptive statistics for the primary and secondary immunogenicity endpoints, including 
estimates and 95% confidence intervals (95% CI) for GMT, SPR and SCR, will be provided by 
time point (28 days post dose 1, 28 days post dose 2, and 6, 12, and 24 months post dose 2 in 
applicable groups) and by formulation arm.
Point estimates and 95% CI for ratios in GMT and differences in SPR and SCR will be provided 
for each pair of active study arms (ie, low, medium and high doses) to aid a single vaccine dose 
level selection.
Immunogenicity summaries and analyses will be provided by dose group overall, as well as by 
baseline flavivirus serostatus.
13.1.4 Safety Analysis
Reactogenicity will be assessed for 7 days following each dose (including day of vaccine/placebo 
administration) via daily collection of solicited AEs, including local reactions (injection site: pain, 
erythema, swelling, and induration) and systemic AEs of headache, fatigue, malaise, arthralgia and 
myalgia. In addition, body temperature (preferably measured as oral temperature) as indicator of 
reactogenicity will be collected (with fever defined as body temperature ≥38.0°C/100.4°F).
For each solicited AE and fever, the percentage of subjects will be summarized by event severity 
for each day for the 7 days after each vaccine/placebo administration and overall. For subjects with 
more than 1 episode of the same event, the maximum severity will be used for tabulations.
Unsolicited AEs and SAEs will be coded using MedDRA and summarized by SOC and PT for 
each formulation arm including new medical conditions (neurological and neuroinflammatory 
disorders) with onset after the first vaccination.
In general, unsolicited AEs will be tabulated at each of the following levels: overall summary 
(subject with at least 1 AE), and by SOC and PT. Unsolicited AEs will be summarized as follows: 
by PT; by SOC and PT; by SOC, PT, and severity; by SOC, PT, and relationship (causality) to the 
investigational vaccine/placebo; and by SOC and PT, including events with frequency greater than 
a pre-defined frequency. 
Safety summaries will be provided by dose group overall, as well as by baseline flavivirus 
serostatus.
0086
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 86 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
13.2
Interim Analysis and Sequence of Analyses
A first interim analysis will be performed to include immunogenicity and safety data from all 
flavivirus naïve subjects up to Visit 6 (on Day 57, 28 days post dose 2); a second interim analysis 
will be performed for dose selection, including safety data from all flavivirus primed subjects up to 
Visit 4 (on Day 29, 28 days post dose 1).
The final safety and immunogenicity analysis will be performed when all (flavivirus naïve and 
primed) subjects have completed Visit 6 (Day 57) to provide data to support the planning and 
execution of other trials in the development plan of PIZV. The subsequent analyses will include 
(1) the data up to Visit 9 (12 months post dose 2) and (2) the data up to Visit 11 (24 months post 
dose 2). The analyses will be performed by a separate set of unblinded statisticians and 
programmers at a Clinical Research Organization (CRO), who will have access to individual 
treatment assignments and will not be involved in subsequent trial conduct. The study team at 
Takeda will have access to the group level unblinded results and will remain blinded to the 
individual treatment assignment. The remaining personnel (not mentioned before) involved in the 
conduct of the trial, including those at Takeda, the CRO, and the trial sites, will remain blinded to 
the individual subject treatment assignment until unblinding after trial completion (database lock 
for data through Visit 11).
More details regarding the analyses will be provided in the SAP.
The final study report will be written for the data up to Visit 6 (Day 57), and two additional 
addenda will be written: one to report the data from Visit 6 (Day 57) to Visit 9 (Day 393, 12 
months post dose 2), and one to report the data from Visit 9 (Day 393) until the end of the study, ie, 
Visit 11 (Day 757, 24 months post dose 2).
13.3
Determination of Sample Size
The sample size was not determined based on formal statistical power calculations. Stochastic 
simulations suggest that the proposed sample size is deemed adequate under a variety of 
decision-making scenarios.
0087
Commercial Use Only and Subject to the Applicable Terms of Use
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14.0
QUALITY CONTROL AND QUALITY ASSURANCE
14.1
Trial-Site Monitoring Visits
Monitoring visits to the trial site will be made periodically during the trial to ensure that all aspects 
of the protocol are followed. Source documents will be reviewed for verification of data recorded 
on the eCRFs. Source documents are defined as original documents, data, and records. The 
investigator and institution guarantee access to source documents by the sponsor or its designee 
(CRO) and by the IRB or IEC.
All aspects of the trial and its documentation will be subject to review by the sponsor or designee 
(as long as blinding is not jeopardized), including but not limited to the Investigator’s Binder, 
investigational vaccine/placebo, subject medical records, ICF documentation, documentation of 
subject authorization to use personal health information (if separate from the ICF), and review of 
eCRFs and associated source documents. It is important that the investigator and other trial 
personnel are available during the monitoring visits and that sufficient time is devoted to the 
process.
14.2
Protocol Deviations
The investigator should not deviate from the protocol, except where necessary to eliminate an 
immediate hazard to trial subjects. Should other unexpected circumstances arise that will require 
deviation from protocol-specified procedures, the investigator should consult with the medical 
monitor (and IRB or IEC, as required) to determine the appropriate course of action. There will be 
no exemptions (a prospective approved deviation) from the inclusion or exclusion criteria.
14.3
Quality Assurance Audits and Regulatory Agency Inspections
The trial site also may be subject to quality assurance audits by the sponsor or designees. In this 
circumstance, the sponsor-designated auditor will contact the site in advance to arrange an 
auditing visit. The auditor may ask to visit the facilities where laboratory samples are collected, 
where the vaccine/placebo is stored and prepared, and any other facility used during the trial. In 
addition, there is the possibility that this trial may be inspected by regulatory agencies, including 
those of foreign governments (eg, the FDA, the United Kingdom Medicines and Healthcare 
products Regulatory Agency, the Pharmaceuticals and Medical Devices Agency of Japan). If the 
trial site is contacted for an inspection by a regulatory body, the sponsor should be notified 
immediately. The investigator and institution guarantee access for quality assurance auditors to all 
trial documents as described in Section 14.1.
0088
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Trial No. ZIK-101 
Page 88 of 108
Protocol Amendment 3 Version 7.0
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15.0
ETHICAL ASPECTS OF THE TRIAL
This trial will be conducted with the highest respect for the individual participants (ie, subjects) 
according to the protocol, the ethical principles that have their origin in the Declaration of 
Helsinki (1), and ICH E6 (2). Each investigator will conduct the trial according to applicable local 
or regional regulatory requirements and align his or her conduct in accordance with the 
“Responsibilities of the Investigator” that are listed in Appendix A.
. The principles of Helsinki are addressed through the protocol and through appendices containing 
requirements for informed consent and investigator responsibilities.
15.1
IRB and/or IEC Approval
IRBs and IECs must be constituted according to the applicable requirements of each participating 
region. The sponsor or designee will require documentation noting all names and titles of members 
who make up the respective IRB or IEC. If any member of the IRB or IEC has direct participation 
in this trial, written notification regarding his or her abstinence from voting must also be obtained.
The sponsor or designee will supply relevant documents for submission to the respective IRB or 
IEC for the protocol’s review and approval. This protocol, the IB, a copy of the ICF, and, if 
applicable, subject recruitment materials and/or advertisements and other documents required by 
all applicable laws and regulations, must be submitted to a central or local IRB or IEC for 
approval. The IRB’s or IEC’s written approval of the protocol and subject ICF must be obtained 
and submitted to the sponsor or designee before commencement of the trial (ie, before shipment of 
the sponsor-supplied Vaccine or trial specific screening activity). The IRB or IEC approval must 
refer to the trial by exact protocol title, number, and version date; identify versions of other 
documents (eg, ICF) reviewed; and state the approval date. The sponsor will notify the site once 
the sponsor has confirmed the adequacy of site regulatory documentation and, when applicable, 
the sponsor has received permission from the competent authority to begin the trial. Until the site 
receives notification, no protocol activities, including screening may occur.
Sites must adhere to all requirements stipulated by their respective IRB or IEC. This may include 
notification to the IRB or IEC regarding protocol amendments, updates to the ICF, recruitment 
materials intended for viewing by subjects, local safety reporting requirements, reports and 
updates regarding the ongoing review of the trial at intervals specified by the respective IRB or 
IEC, and submission of the investigator’s final status report to IRB or IEC. All IRB and IEC 
approvals and relevant documentation for these items must be provided to the sponsor or its 
designee.
Subject incentives should not exert undue influence for participation. Payments to subjects must 
be approved by the IRB or IEC and sponsor.
15.2
Subject Information, Informed Consent and Subject Authorization
Written consent documents will embody the elements of informed consent as described in the 
Declaration of Helsinki (1) and ICH E6 (2) and will be in accordance with all applicable laws and 
regulations. The ICF, subject authorization form (if applicable), and subject information sheet (if 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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applicable) describe the planned and permitted uses, transfers, and disclosures of the subject’s 
personal and personal health information for purposes of conducting the trial. The ICF and the 
subject information sheet (if applicable) further explain the nature of the trial, its objectives, and 
potential risks and benefits, as well as the date informed consent / is given. The ICF will detail the 
requirements of the participant and the fact that he or she is free to withdraw at any time without 
giving a reason and without prejudice to his or her further medical care.
The investigator is responsible for the preparation, content, and IRB or IEC approval of the ICF 
and if applicable, the subject authorization form. The ICF, subject authorization form (if 
applicable), and subject information sheet (if applicable) must be approved by both the IRB or IEC 
and the sponsor prior to use.
The ICF, subject authorization form (if applicable), and subject information sheet (if applicable) 
must be written in a language fully comprehensible to the prospective subject. It is the 
responsibility of the investigator to explain the detailed elements of the ICF, subject authorization 
form (if applicable), and subject information sheet (if applicable) to the subject. Information 
should be given in both oral and written form whenever possible and in the manner deemed 
appropriate by the IRB or IEC.
The subject must be given ample opportunity to: (1) inquire about details of the trial and (2) decide 
whether or not to participate in the trial. If the subject determines he or she will participate in the 
trial, then the informed consent and subject authorization form (if applicable) must be signed and 
dated by the subject, at the time of consent and prior to the subject entering into the trial. The 
subject should be instructed to sign using their legal names, not nicknames, using blue or black 
ballpoint ink. The investigator must also sign and date the informed consent and subject 
authorization (if applicable) at the time of consent and prior to subject entering into the trial; 
however, the sponsor may allow a designee of the investigator to sign to the extent permitted by 
applicable law.
Once signed, the original ICF, subject authorization form (if applicable), and subject information 
sheet (if applicable) will be stored in the investigator’s site file. The investigator must document 
the date the subject signs the ICF in the subject’s medical record and eCRF. Copies of the signed 
ICF, the signed subject authorization form (if applicable), and subject information sheet (if 
applicable) shall be given to the subject.
All revised ICFs must be reviewed and signed by the relevant subject in the same manner as the 
original ICF. The date the revised consent was obtained should be recorded in the subject’s 
medical record and eCRF, and the subject should receive a copy of the revised ICF.
15.3
Subject Confidentiality
The sponsor and designees affirm and uphold the principle of the subject’s right to protection 
against invasion of privacy. Throughout this trial, a subject’s source data will only be linked to the 
sponsor’s clinical trial database or documentation via a unique identification number. As permitted 
by all applicable laws and regulations, limited subject attributes, such as sex, age, or date of birth, 
and subject initials may be used to verify the subject and accuracy of the subject’s unique 
identification number.
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Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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To comply with ICH E6 (2) and to verify compliance with this protocol, the sponsor requires the 
investigator to permit its monitor or designee’s monitor, representatives from any regulatory 
authority (eg, FDA, Medicines and Healthcare products Regulatory Agency, Pharmaceuticals and 
Medical Devices Agency), the sponsor’s designated auditors, and the appropriate IRBs and IECs 
to review the subject’s original medical records (source data or documents), including, but not 
limited to, laboratory test result reports, electrocardiography reports, admission and discharge 
summaries for hospital admissions occurring during a subject’s trial participation, and autopsy 
reports. Access to a subject’s original medical records requires the specific authorization of the 
subject as part of the ICF process (see Section 15.2).
Copies of any subject source documents that are provided to the sponsor must have certain 
personally identifiable information removed (ie, subject name, address, and other identifier fields 
not collected on the subject’s eCRF).
15.4
Publication, Disclosure, and Clinical Trial Registration Policy
15.4.1 Publication and Disclosure
The results of this trial are expected to be published in a scientific journal. It is anticipated that 
clinical and laboratory co-investigators will participate in authorship. The order of authorship and 
choice of journal will be proposed by the sponsor to the principal investigator(s), to be eventually 
agreed upon by all authors. The data analysis center for this trial will provide the analyses needed 
for publication. Information regarding this trial will be posted on ClinicalTrials.gov.
15.4.2 Clinical Trial Registration
In order to ensure that information on clinical trials reaches the public in a timely manner and to 
comply with applicable law, regulation and guidance, the sponsor will, at a minimum register all 
clinical trials conducted in subjects that it sponsors anywhere in the world on ClinicalTrials.gov or 
other publicly accessible websites before trial initiation. The sponsor contact information, along 
with investigator’s city, country, and recruiting status will be registered and available for public 
viewing.
15.4.3 Clinical Trial Results Disclosure
The sponsor will post the results of this clinical trial, regardless of outcome, on ClinicalTrials.gov 
or other publicly accessible websites, as required by applicable laws and/or regulations.
Trial completion corresponds to the date on which the final subject was examined or received an 
intervention for the purposes of final collection of data for the primary outcome, whether the 
clinical trial concluded according to the pre-specified protocol or was terminated.
15.5
Insurance and Compensation for Injury
Each subject in the trial must be insured in accordance with the regulations applicable to the site 
where the subject is participating. If a local underwriter is required, then the sponsor or sponsor’s 
designee will obtain clinical trial insurance against the risk of injury to clinical trial subjects. Refer 
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Trial No. ZIK-101 
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Protocol Amendment 3 Version 7.0
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to the Clinical Study Site Agreement regarding the sponsor’s policy on subject compensation and 
treatment for injury. If the investigator has questions regarding this policy, he or she should contact 
the sponsor or sponsor’s designee.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 92 of 108
Protocol Amendment 3 Version 7.0
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16.0
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For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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29. Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. 
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Epub 2014/10/28.
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outbreak on Yap Island, Federated States of Micronesia. The New England journal of 
medicine. 2009;360(24):2536-43. Epub 2009/06/12.
34. Cao-Lormeau VM, Roche C, Teissier A, Robin E, Berry AL, Mallet HP, et al. Zika virus, 
French polynesia, South pacific, 2013. Emerging infectious diseases. 2014;20(6):1085-6. 
Epub 2014/05/27.
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December 2016]; Press release 1 September 2016]. Available from: 
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Trial No. ZIK-101 
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12 September 2018
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 100 of 108
Protocol Amendment 3 Version 7.0
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Appendix A
Responsibilities of the Investigator
Clinical research studies sponsored by the sponsor are subject to ICH GCP and all the applicable 
local laws and regulations.
The investigator agrees to assume the following responsibilities:
1. Conduct the trial in accordance with the protocol.
2. Personally conduct or supervise the staff who will assist in the protocol.
3. Ensure that trial related procedures, including trial specific (non-routine/non-standard panel) 
screening assessments are NOT performed on potential subjects, prior to the receipt of written 
approval from relevant governing bodies/authorities.
4. Ensure that all colleagues and employees assisting in the conduct of the trial are informed of 
these obligations.
5. Secure prior approval of the trial and any changes by an appropriate IRB/IEC that conform to 
21 CFR Part 56, ICH, and local regulatory requirements.
6. Ensure that the IRB/IEC will be responsible for initial review, continuing review, and approval 
of the protocol. Promptly report to the IRB/IEC all changes in research activity and all 
anticipated risks to subjects. Make at least yearly reports on the progress of the trial to the 
IRB/IEC, and issue a final report within 3 months of trial completion.
7. Ensure that requirements for informed consent, as outlined in 21 CFR Part 50, ICH, and local 
regulations, are met.
8. Obtain valid informed consent from each subject who participates in the trial, and document 
the date of consent in the subject’s medical chart. Valid ICF is the most current version 
approved by the IRB/IEC. Each ICF should contain a subject authorization section that 
describes the uses and disclosures of a subject’s personal information (including personal 
health information) that will take place in connection with the trial. If an ICF does not include 
such a subject authorization, then the investigator must obtain a separate subject authorization 
form from each subject.
9. Prepare and maintain adequate case histories of all persons entered into the trial, including 
eCRFs, hospital records, laboratory results, etc., and maintain these data for a minimum of 
2 years following notification by the sponsor that all investigations have been discontinued or 
that the regulatory authority has approved the marketing application. The investigator should 
contact and receive written approval from the sponsor before disposing of any such 
documents.
10. Allow possible inspection and copying by the regulatory authority of GCP-specified essential 
documents.
11. Maintain current records of the receipt, administration, and disposition of sponsor-supplied 
vaccines, and return all unused sponsor-supplied vaccines to the sponsor.
12. Report AEs to the sponsor promptly. In the event of an SAE, notify the sponsor within 
24 hours.
13. Review and provide a signature as approval of the content of the clinical study report.
0101
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M


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 101 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Appendix B
Elements of the Subject Informed Consent Form
In seeking informed consent, the following information shall be provided to each subject:
1. A statement that the trial involves research.
2. An explanation of the purposes of the research.
3. The expected duration of the subject’s participation.
4. A description of the procedures to be followed, including invasive procedures.
5. The identification of any procedures that are experimental.
6. The estimated number of subjects involved in the trial.
7. A description of the subject’s responsibilities.
8. A description of the conduct of the trial.
9. A statement describing the vaccination(s) and the probability for random assignment to each 
treatment.
10. A description of the possible side effects following vaccine administration that the subject may 
receive.
11. A description of any reasonably foreseeable risks or discomforts to the subject and, when 
applicable, to an embryo, fetus, or nursing infant.
12. A description of any benefits to the subject or to others that reasonably may be expected from 
the research. When there is no intended clinical benefit to the subject, the subject should be 
made aware of this.
13. Disclosures of appropriate alternative procedures or courses of treatment, if any, that might be 
advantageous to the subject and their important potential risks and benefits.
14. A statement describing the extent to which confidentiality of records identifying the subject 
will be maintained, and a note of the possibility that regulatory agencies, auditor(s), IRB/IEC, 
and the monitor may inspect the records. By signing a written informed consent, the subject is 
authorizing such access.
15. For research involving more than minimal risk, an explanation as to whether any compensation 
and an explanation as to whether any medical treatments are available if injury occurs and, if 
so, what they consist of or where further information may be obtained.
16. The anticipated prorated payment(s), if any, to the subject for participating in the trial.
17. The anticipated expenses, if any, to the subject for participating in the trial.
18. An explanation of whom to contact for answers to pertinent questions about the research 
(investigator), subject’s rights, and IRB/IEC and whom to contact in the event of a 
research-related injury to the subject.
19. A statement that participation is voluntary, that refusal to participate will involve no penalty or 
loss of benefits to which the subject otherwise is entitled, and that the subject may discontinue 
participation at any time without penalty or loss of benefits to which the subject is otherwise 
entitled.
20. The consequences of a subject’s decision to withdraw from the research and procedures for 
orderly termination of participation by the subject.
21. A statement that the subject will be informed in a timely manner if information becomes 
available that may be relevant to the subject’s willingness to continue participation in the trial.
0102
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 102 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
22. A statement that results of pharmacogenomic analysis will not be disclosed to an individual, 
unless prevailing laws require the sponsor to do so.
23. The foreseeable circumstances or reasons under which the subject’s participation in the trial 
may be terminated.
24. A written subject authorization (either contained within the informed consent or provided as a 
separate document) describing to the subject the contemplated and permissible uses and 
disclosures of the subject’s personal information (including personal health information) for 
purposes of conducting the trial. The subject authorization must contain the following 
statements regarding the uses and disclosures of the subject’s personal information:
a) that personal information (including personal health information) may be processed by or 
transferred to other parties in other countries for clinical research and safety reporting 
purposes, including, without limitation, to the following: (1) Takeda, its affiliates, and 
licensing partners; (2) business partners assisting Takeda, its affiliates, and licensing 
partners; (3) regulatory agencies and other health authorities; and (4) IRBs/IECs;
b) it is possible that personal information (including personal health information) may be 
processed and transferred to countries that do not have data protection laws that offer 
subjects the same level of protection as the data protection laws within this country; 
however, Takeda will make every effort to keep your personal information confidential, 
and your name will not be disclosed outside the clinic unless required by law;
c) that personal information (including personal health information) may be added to 
Takeda’s research databases for purposes of developing a better understanding of the 
safety and effectiveness of the investigational vaccine(s), studying other therapies for 
patients, developing a better understanding of disease, and improving the efficiency of 
future clinical studies;
d) that subjects agree not to restrict the use and disclosure of their personal information 
(including personal health information) upon withdrawal from the trial to the extent that 
the restricted use or disclosure of such information may impact the scientific integrity of 
the research; and
e) that the subject’s identity will remain confidential in the event that trial results are 
published.
25. The vaccination may involve risks to the subject (or to the embryo or fetus, if the subject is or 
may become pregnant) that are currently unforeseeable. Female subjects of childbearing 
potential, sexually active, must use "acceptable contraceptive methods" for at least 2 months 
prior to trial entry through to 2 months after the last dose of investigational vaccine/placebo. 
Regular pregnancy tests will be performed throughout the trial for all female subjects of 
childbearing potential. If a subject is found to be pregnant during trial, no further vaccine doses 
will be administered and the investigator will offer the subject the choice to receive unblinded 
treatment information. In addition, female subject of childbearing potential must be advised 
not to donate ova during the study period.
26. To avoid sexual transmission of ZIKV from natural exposure: Sexually active subjects (both 
male and female) should use latex condoms correctly and consistently even if other 
contraceptive measures are used from signing the ICF through the end of the trial. Male 
subjects must be advised not to donate sperm during this period.
0103
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 103 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
27. A statement that clinical trial information from this trial will be publicly disclosed in a publicly 
accessible website, such as ClinicalTrials.gov.
28. Subject intending to donate blood/blood product for transfusion might have positive ZIKV 
serological test result due to the participation in this study. Subject will be required to inform 
the health professional about the participation in the ZIKV vaccine study when intending to 
donate blood/blood product.
0104
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
form
ms o
form
ing to
erms
ing to


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 104 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Appendix C
Investigator Consent to Use of Personal Information
Takeda will collect and retain personal information of investigator, including his or her name, 
address, and other personally identifiable information. In addition, investigator’s personal 
information may be transferred to other parties located in countries throughout the world (eg, the 
United Kingdom, United States, and Japan), including the following:

Takeda, its affiliates, and licensing partners.

Business partners assisting Takeda, its affiliates, and licensing partners.

Regulatory agencies and other health authorities.

IRBs and IECs.
Investigator’s personal information may be retained, processed, and transferred by Takeda and 
these other parties for research purposes including the following:

Assessment of the suitability of investigator for the trial and/or other clinical studies.

Management, monitoring, inspection, and audit of the trial.

Analysis, review, and verification of the trial results.

Safety reporting and pharmacovigilance relating to the trial.

Preparation and submission of regulatory filings, correspondence, and communications to 
regulatory agencies relating to the trial.

Preparation and submission of regulatory filings, correspondence, and communications to 
regulatory agencies relating to other vaccines used in other clinical studies that may contain the 
same chemical compound present in the investigational vaccine.

Inspections and investigations by regulatory authorities relating to the trial.

Self-inspection and internal audit within Takeda, its affiliates, and licensing partners.

Archiving and audit of trial records.

Posting investigator site contact information, trial details and results on publicly accessible 
clinical trial registries, databases, and websites.
Investigator’s personal information may be transferred to other countries that do not have data 
protection laws that offer the same level of protection as data protection laws in investigator’s own 
country. Investigator acknowledges and consents to the use of his or her personal information by 
Takeda and other parties for the purposes described above.
0105
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
,
d (eg, th
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 105 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Appendix D
CDC Websites

World Map of Areas with Risk of Zika: 
https://wwwnc.cdc.gov/travel/page/world-map-areas-with-zika

Travel & Dengue Outbreaks: https://www.cdc.gov/dengue/traveloutbreaks/index.html

Yellow Fever Maps: https://www.cdc.gov/yellowfever/maps/index.html

Geographic Distribution of Japanese Encephalitis Virus: 
https://www.cdc.gov/japaneseencephalitis/maps/index.html
0106
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
T


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 106 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Appendix E
FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials 
Toxicity grading scales for laboratory abnormalities should consider the local laboratory reference 
values when the parameter limit values are defined. The characterization of laboratory parameters 
among some populations of healthy adults and adolescents may require the exercise of clinical 
judgment. Source: U.S. Department of Health and Human Services, Food and Drug 
Administration, and Center for Biologics Evaluation and Research, September 2007 (93)
Table 16.a
Table for Laboratory Abnormalities (Serum)
Serum(a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life 
Threatening
(Grade 4)(b)
Sodium – Hyponatremia mEq/L
132 – 134
130 – 131
125 – 129
< 125
Sodium – Hypernatremia mEq/L
144 – 145
146 – 147
148 – 150
> 150
Potassium – Hyperkalemia mEq/L
5.1 – 5.2
5.3 – 5.4
5.5 – 5.6
> 5.6
Potassium – Hypokalemia mEq/L
3.5 – 3.6
3.3 – 3.4
3.1 – 3.2
< 3.1
Glucose – Hypoglycemia mg/dL
65 – 69
55 – 64
45 – 54
< 45
Glucose – Hyperglycemia
Fasting – mg/dL
Random – mg/dL
100 – 110
110 – 125
111 – 125
126 – 200
>125
>200
Insulin requirements or 
hyperosmolar coma
Blood Urea Nitrogen
BUN mg/dL
23-26
27 – 31
> 31
Requires dialysis
Creatinine – mg/dL
1.5 – 1.7
1.8 – 2.0
2.1 – 2.5
> 2.5 or requires dialysis
Calcium – hypocalcemia mg/dL
8.0 – 8.4
7.5 – 7.9
7.0 – 7.4
< 7.0
Calcium – hypercalcemia mg/dL
10.5 – 11.0
11.1 – 11.5
11.6 – 12.0
> 12.0
Magnesium – hypomagnesemia mg/dL
1.3 – 1.5
1.1 – 1.2
0.9 – 1.0
< 0.9
Phosphorous – hypophosphatemia mg/dL
2.3 – 2.5
2.0 – 2.2
1.6 – 1.9
< 1.6
CPK – mg/dL
1.25 – 1.5 x 
ULN(c)
1.6 – 3.0 x 
ULN
3.1 –10 x 
ULN
> 10 x ULN
Albumin – Hypoalbuminemia g/dL
2.8 – 3.1
2.5 – 2.7
< 2.5
--
Total Protein – Hypoproteinemia g/dL
5.5 – 6.0
5.0 – 5.4
< 5.0
--
Alkaline phosphate – increase by factor
1.1 – 2.0 x 
ULN
2.1 – 3.0 x 
ULN
3.1 – 10 x 
ULN
> 10 x ULN
Liver Function Tests –ALT, AST increase by factor
1.1 – 2.5 x 
ULN
2.6 – 5.0 x 
ULN
5.1 – 10 x 
ULN
> 10 x ULN
Bilirubin – when accompanied by any increase in Liver 
Function Test increase by factor
1.1 – 1.25 x 
ULN
1.26 – 1.5 x 
ULN
1.51 – 1.75 x 
ULN
> 1.75 x ULN
Bilirubin – when Liver Function Test is normal; 
increase by factor
1.1 – 1.5 x 
ULN
1.6 – 2.0 x 
ULN
2.0 – 3.0 x 
ULN
> 3.0 x ULN
Cholesterol
201 – 210
211 – 225
> 226
---
Pancreatic enzymes – amylase, lipase
1.1 – 1.5 x 
ULN
1.6 – 2.0 x 
ULN
2.1 – 5.0 x 
ULN
> 5.0 x ULN
0107
 Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 107 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
(a)
The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. 
Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
(b)
The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory 
abnormalities as Potentially Life Threatening (Grade 4). For example. a low sodium value that falls within a grade 3 parameter 
(125-129 mEq/L) should be recorded as a grade 4 hyponatremia event if the subject had a new seizure associated with the low 
sodium value.
(c)
“ULN” is the upper limit of the normal range.
Table 16.b
Table for Laboratory Abnormalities (Hematology)
Hematology(a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Hemoglobin (Female) – gm/dL
11.0 – 12.0
9.5 – 10.9
8.0 – 9.4
< 8.0
Hemoglobin (Female) change from 
baseline value – gm/dL
Any 
decrease –
1.5
1.6 – 2.0
2.1 – 5.0
> 5.0
Hemoglobin (Male) – gm/dL
12.5 – 13.5
10.5 – 12.4
8.5 – 10.4
< 8.5
Hemoglobin (Male) change from 
baseline value – gm/dL
Any 
decrease –
1.5
1.6 – 2.0
2.1 – 5.0
> 5.0
WBC Increase – cell/mm3
10,800 –
15,000
15,001 –
20,000
20,001 – 25, 
000
> 25,000
WBC Decrease – cell/mm3
2,500 –
3,500
1,500 – 2,499
1,000 –
1,499
< 1,000
Lymphocytes Decrease – cell/mm3 750 – 1,000
500 – 749
250 – 499
< 250
Neutrophils Decrease – cell/mm3
1,500 –
2,000
1,000 – 1,499
500 – 999
< 500
Eosinophils – cell/mm3
650 – 1500
1501 - 5000
> 5000
Hypereosinophilic
Platelets Decreased – cell/mm3
125,000 –
140,000
100,000 –
124,000
25,000 –
99,000
< 25,000
PT – increase by factor 
(prothrombin time)
1.0 – 1.10 x 
ULN(b)
ï€ 1.11 – 1.20 x 
ULN
1.21 – 1.25 
x ULN
> 1.25 ULN
PTT – increase by factor (partial 
thromboplastin time)
1.0 – 1.2 x 
ULN
1.21 – 1.4 x 
ULN
1.41 – 1.5 x 
ULN
> 1.5 x ULN
Fibrinogen increase – mg/dL
400 – 500
501 – 600
> 600
--
Fibrinogen decrease – mg/dL
150 – 200
125 – 149
100 – 124
< 100 or associated with gross bleeding or 
disseminated intravascular coagulation (DIC)
(a)
The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. 
Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
(b)
“ULN” is the upper limit of the normal range.
0108
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101 
Page 108 of 108
Protocol Amendment 3 Version 7.0
12 September 2018
Table 16.c
Table for Laboratory Abnormalities (Urine)
Urine(a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Protein
Trace
1+
2+
Hospitalization or dialysis
Glucose
Trace
1+
2+
Hospitalization for hyperglycemia
Blood (microscopic) – red blood cells 
per high power field
1 - 10
11 - 50
> 50 and/or 
gross blood
Hospitalization or packed red blood 
cells (PRBC) transfusion
(a) The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
Table 16.d
Table for Clinical Abnormalities (Vital Signs)
Vital Signs(a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Fever (°C)(b)
(°F)(b)
38.0 – 38.4
100.4 –
101.1
38.5 – 38.9
101.2 –
102.0
39.0 – 40
102.1 –
104
> 40
> 104
Tachycardia – beats per minute
101 – 115
116 – 130
> 130
ER visit or hospitalization for arrhythmia
Bradycardia – beats per minute(c) 50 – 54
45 – 49
< 45
ER visit or hospitalization for arrhythmia
Hypertension (systolic) – mm 
Hg
141 – 150
151 – 155
> 155
ER visit or hospitalization for malignant 
hypertension
Hypertension (diastolic) – mm 
Hg
91 – 95
96 – 100
> 100
ER visit or hospitalization for malignant 
hypertension
Hypotension (systolic) – mm Hg 85 – 89
80 – 84
< 80
ER visit or hospitalization for hypotensive 
shock
Respiratory Rate – breaths per 
minute
17 – 20
21 – 25
> 25
Intubation
(a) Subject should be at rest for all vital sign measurements.
(b) Oral temperature; no recent hot or cold beverages or smoking.
(c) When resting heart rate is between 60 – 100 beats per minute. Use clinical judgement when characterizing 
bradycardia among some healthy subject populations, for example, conditioned athletes.
0109
t to the Applicable Terms of Use
of
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SAP_001.pdf: 
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
Other information as needed to protect confidentiality of Takeda or partners, personal
information, or to otherwise protect the integrity of the clinical study.
Title: A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, 
and Dose Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in 
Flavivirus Naïve and Primed Healthy Adults Aged 18 to 49 Years 
NCT Number: NCT03343626
SAP Approve Date: 26 April 2019 
Certain information within this Statistical Analysis Plan has been redacted (ie, specific content is masked 
irreversibly from view with a black/blue bar) to protect either personally identifiable (PPD) information 
or company confidential information (CCI). 
This may include, but is not limited to, redaction of the following: 
Named persons or organizations associated with the study.
Proprietary information, such as scales or coding systems, which are considered confidential 
information under prior agreements with license holder.


1.0
TITLE PAGE
STATISTICAL ANALYSIS PLAN
STUDY NUMBER: ZIK-101
A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity, and 
Dose Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in 
Flavivirus Naïve and Primed Healthy Adults Aged 18 to 49 Years
Safety, Immunogenicity and Dose Ranging Study of Inactivated Zika Virus Vaccine in 
Healthy Adults 
PHASE 1
Version: 2.0
Date: 26 April 2019
Prepared by:
Based on:
Protocol Amendment 3 Version: 7.0 
Protocol Date: 12 September 2018
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 3 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
2.0
TABLE OF CONTENTS
1.0
TITLE PAGE.......................................................................................................................1
1.1
Approval Signatures.......................................................................................................2
2.0
TABLE OF CONTENTS.....................................................................................................3
List of In-Text Tables ................................................................................................................5
List of In-Text Figures...............................................................................................................5
List of Appendices .....................................................................................................................5
3.0
LIST OF ABBREVIATIONS..............................................................................................6
4.0
OBJECTIVES......................................................................................................................8
4.1
Primary Objectives.........................................................................................................8
4.2
Secondary Objectives.....................................................................................................8
4.3
Study Design..................................................................................................................8
4.4
Investigational Study Vaccine .....................................................................................13
4.5
Placebo.........................................................................................................................13
5.0
ANALYSIS ENDPOINTS ................................................................................................14
5.1
Primary Endpoints .......................................................................................................14
5.2
Secondary Endpoints ...................................................................................................14
6.0
DETERMINATION OF SAMPLE SIZE..........................................................................15
7.0
METHODS OF ANALYSIS AND PRESENTATION.....................................................16
7.1
General Principles........................................................................................................16
7.1.1
Definition of Study Days .......................................................................................16
7.1.2
Definition of Study Visit Windows .......................................................................17
7.1.3
Conventions for Missing Adverse Event Data.......................................................17
7.1.4
Conventions for Missing Concomitant Medication Dates.....................................18
7.2
Analysis Sets................................................................................................................18
7.2.1
Major Protocol Violations and Evaluability Criteria.............................................19
7.3
Disposition of Subjects ................................................................................................21
7.4
Demographic and Other Baseline Characteristics .......................................................22
7.5
Medical History and Concurrent Medical Conditions.................................................22
7.6
Prior and Concomitant Medications/Vaccinations ......................................................22
7.7
Study Vaccine Exposure and Compliance...................................................................23
7.8
Traveling During the Study .........................................................................................23
7.9
Efficacy Analysis.........................................................................................................23
7.10
Pharmacokinetic/Pharmacodynamic Analysis.............................................................23
7.10.1 Pharmacokinetic Analysis......................................................................................23
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 4 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.10.2 Pharmacodynamic Analysis...................................................................................24
7.11
Other Outcomes ...........................................................................................................24
7.11.1 Immunogenicity Analysis......................................................................................24
7.11.2 Descriptive Summaries ..........................................................................................30
7.12
Dose Selection Methodology.......................................................................................30
7.12.1 Geometric Mean Titers Analyses...........................................................................31
7.12.2 Seroconversion Rate Analyses...............................................................................31
7.13
Other Immunogenicity Analyses .................................................................................31
7.14
Graphical Presentations ...............................................................................................31
7.14.1 Immunogenicity Figures ........................................................................................31
7.14.2 Primary and Secondary Safety and Tolerability Endpoints...................................32
7.15
Safety Analysis ............................................................................................................33
7.15.1 Solicited Adverse Events Related to Reactogenicity.............................................33
7.15.2 Unsolicited Adverse Event ....................................................................................34
7.15.3 Clinical Laboratory Evaluations ............................................................................35
7.15.4 Vital Signs..............................................................................................................36
7.15.5 12-Lead ECGs........................................................................................................36
7.15.6 Other Observations Related to Safety....................................................................36
7.16
Interim Analysis...........................................................................................................36
7.17
Analyses for the Final Clinical Study Report ..............................................................36
7.18
Changes in the Statistical Analysis Plan......................................................................37
7.18.1 Amendment History...............................................................................................37
7.18.2 Summary of Changes.............................................................................................38
8.0
REFERENCES ..................................................................................................................39
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 5 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
LIST OF IN-TEXT TABLES
Table 7.a
Protocol Defined Visit Windows for Immunogenicity Assessment......................17
Table 7.b
Criterion for Exclusion from the PPS Analyses ....................................................20
Table 7.c
Planned Analyses and Populations for Immunogenicity Endpoints......................29
Table 7.d
Timing and the Types of Analyses with the Respective Output............................37
LIST OF IN-TEXT FIGURES 
Figure 4.a
Schematic of Trial Design .......................................................................................9
Figure 4.b
Staggered Enrollments...........................................................................................11
LIST OF APPENDICES 
Appendix A
Schedule of Trial Procedures.................................................................................40
Appendix B
Serology Plan.........................................................................................................44
Appendix C
Solicited Local and Systemic Adverse Events and Severity .................................49
Appendix D
FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials .....................................................51
Appendix E
CDC Websites........................................................................................................54
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 6 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
3.0
LIST OF ABBREVIATIONS
AE
Adverse Event
ANCOVA
Analysis of Covariance 
ATC
Anatomical Therapeutic Chemical 
CI
Confidence Interval
CRO
Contract Research Organization
CZS
Congenital Zika Syndrome
CSR
Clinical Study Report
DMC
Data Monitoring Committee
eCRF
electronic Case Report Form 
ELISA
Enzyme-linked Immunosorbent Assay
FAS
Full Analysis Set
FAS
Full Analysis Set
FASI
Full Analysis Set for Immunogenicity 
GMT
Geometric Mean Titer
GSD
Geometric Standard Deviation
ICF
Informed Consent Form 
ICH
International Council for Harmonization
Ig
Immunoglobulin
IM
Intramuscular
LoD
Limit of Detection
LLoQ
Lower Limit of Quantification
MedDRA
Medical Dictionary for Regulatory Activities
PIZV
Purified Inactivated Zika Virus Vaccine candidate
PPS
Per-Protocol Analysis Set
PPSI
Per-Protocol Analysis Set for Immunogenicity
PRNT
Plaque Reduction Neutralizing Tests
PT
Preferred Term
RS
Randomized Set
SAE
Serious Adverse Event
SAP
Statistical Analysis Plan
SAS
Statistical Analysis System 
SCR
Seroconversion Rate
SD
Standard Deviation
SOC
System Organ Class
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 7 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
SPR
Seropositivity Rate
SS
Safety Set
ULoQ
Upper Limit of Quantification
WHO
World Health Organization
WHO-DD
World Health Organization Drug Dictionary 
ZIKV
Zika Virus
0008
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 8 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
4.0
OBJECTIVES
4.1
Primary Objectives
To describe the safety of two doses of Purified Inactivated Zika Virus Vaccine candidate (PIZV)
given 28 days apart from three different antigen concentrations (2, 5 or 10 µg) in flavivirus naïve 
and primed healthy adults through 28 days post dose 2.
To select a single vaccine dose level of PIZV for further clinical development.
4.2
Secondary Objectives
To describe the safety of two doses of PIZV given 28 days apart in flavivirus naïve and primed 
healthy adults through the end of the study.
To describe the immune response to PIZV in flavivirus naïve and primed healthy adults at the 
following immunogenicity time points 28 days post dose 1, 28 days post dose 2, and 6 months 
post dose 2.
To describe the persistence of immunity to PIZV at 12 and 24 months post dose 2 in flavivirus 
naïve and primed healthy adults from the placebo group and the PIZV dosing group that will be 
selected for further development.
4.3
Study Design
This is a phase 1, randomized, observer-blind, placebo-controlled, safety, immunogenicity, and 
dose ranging study of PIZV candidate in flavivirus naïve and primed healthy adults aged 18 to 
49 years, inclusive, in ZIKV (Zika Virus) endemic and non-endemic regions.
The trial will last approximately 7 or 25 months for each subject, following a screening period 
prior to Visit 2. The screening period will last for up to 2 weeks (14 days) prior to randomization, 
with the possibility that some subjects (Flavivirus primed subjects only) will need to repeat some 
screening procedures if they were not randomized right after the screening period (further 
clarification is provided in the schedule of trial procedures).
Flavivirus naïve subjects are subjects without detectable serum antibodies against a panel of 
flaviviruses as measured by a reactive antibody-based assay (Luminex) (refer to Appendix B). 
Flavivirus primed subjects are subjects with serum antibodies against a panel of flaviviruses as
measured by a reactive antibody-based assay (Luminex) (refer to Appendix B). 
Approximately 240 subjects aged 18 to 49 years, inclusive, will be enrolled and equally 
randomized into four groups (arms) (1:1:1:1) of approximately 60 subjects, each comprised of 
approximately 30 flavivirus naïve subjects and approximately 30 flavivirus primed subjects. 
Randomization within each cohort (flavivirus naïve versus flavivirus primed subjects) will be 
stratified by flavivirus serostatus and age group: 18 to 29 years, inclusive, versus 30 to 49 years, 
inclusive.
A schematic representation of the trial design is included as Figure 4.a. A schedule of trial 
procedures is provided in Appendix A.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 9 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Figure 4.a
Schematic of Trial Design
There will be two study phases as exhibited in Figure 4.b. 
The first phase of the study will be aimed at enrolling flavivirus naïve subjects only. All subjects 
who sign the Informed Consent Form (ICF) and have confirmed eligibility at Screening Visit 1A 
will be tested for flavivirus serostatus.
Eligible flavivirus naïve subjects enrolled at Screening Visit 1A will be randomized at Visit 2, 
within two weeks after Screening Visit 1A.
Flavivirus primed subjects will not be enrolled during the first phase of the study (i.e., when 
identified at Screening Visit 1A) and will be invited to come back for the second phase of the 
study. They will be contacted after positive recommendations from the external Data Monitoring 
Committee (DMC) are issued following the review of safety and tolerability data in flavivirus 
naïve subjects, when the last flavivirus naïve subject has completed study Visit 4 (28 days post 
dose 1). These flavivirus primed subjects will consequently undergo 2 screening visits: Visit 1A 
during the first phase of the study and Visit 1B at the outset of the second phase of the study. 
Flavivirus primed subjects identified at Screening Visit 1A who do not participate in the second 
phase of the study will be declared screened failure (see Figure 4.b).
Enrollment will be put on hold after all naïve subjects have been enrolled and will resume when 
positive recommendations from the external DMC are issued, as described above.
The second phase of the study will be aimed at enrolling flavivirus primed subjects only.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 10 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Newly identified eligible flavivirus primed subjects at Screening Visit 1B will be enrolled 
(randomized) at (second phase) Visit 2, within two weeks after Screening Visit 1B.
Previously identified flavivirus primed subjects (see Figure 4.b) (who were tested at Visit 1A 
during the first phase and accepted to enter the second phase of the study) will retain their initial 
subject identification (ID) number and undergo an additional screening visit (Visit 1B) to be 
tested for eligibility criteria again, (except for the determination of flavivirus serostatus). 
Flavivirus naïve subjects identified at Screening Visit 1B (during the second phase of the study) 
will be declared screened failure (see Figure 4.b).
To avoid the natural exposure of flavivirus naïve subjects to flaviviruses during the study 
vaccination period, the enrollment into naïve cohort is planned in the sites located in flavivirus 
non-endemic areas only, and the prohibition of traveling into flavivirus endemic areas until
Visit 6. The protocol provides references to flavivirus-endemic areas (refer to Appendix E). 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 11 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Figure 4.b
Staggered Enrollments
* All subjects who were randomized to the placebo group or the PIZV dosing group that was selected for further 
development will attend Visit 9 at Day 393 (12 months post dose 2 and Visit 11 at Day 757 (24 months post dose 2), 
and will also be contacted by phone on Day 575 (Visit 10) for safety follow-up.
Each subject will receive two 0.5 mL doses of either PIZV (2, 5 or 10 µg) or placebo 
intramuscularly (IM) – one dose at Visit 2 (on Day 1) and one dose at Visit 4 (on Day 29) IM 
into the middle third of the deltoid muscle, preferably in the non-dominant arm.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 12 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Each subject will be required to attend 7, 8, 9 or 10 clinical visits and will receive 1 or 2 phone 
calls depending on their flavivirus serostatus at screening and the vaccine dose level they 
received:

Flavivirus naïve subjects identified during the Screening Visit 1A and flavivirus primed 
subjects identified during the Screening Visit 1B will attend 7 visits and will receive 1 phone 
call, and possibly 2 additional visits and 1 phone call (thus a maximum of 9 visits) and 
2 phone calls if they were randomized to the placebo group or the PIZV dosing group that 
was selected for further development; and

Flavivirus primed subjects identified during the Screening Visit 1A will attend 8 visits 
(including Visit 1A and Visit 1B) and will receive 1 phone call, and possibly 2 additional 
visits and 1 phone call (thus a maximum of 10 visits and 2 phone calls) if they were 
randomized to the placebo group or the PIZV dosing group that was selected for further 
development.
All randomized subjects will thus attend Screening Visit(s) (Visit 1A and/or Visit 1B), Visit 2 at 
Day 1, Visit 3 at Day 8 (7 days post dose 1), Visit 4 at Day 29 (28 days post dose 1), Visit 5 at 
Day 36 (7 days post dose 2), Visit 6 at Day 57 (28 days post dose 2), and Visit 8 at Day 211 
(6 months post dose 2). 
All randomized subjects will also be contacted by phone on Day 133 (Visit 7) for safety follow-
up. In addition, all subjects who were randomized to the placebo group or the PIZV dosing group 
that was selected for further development will attend Visit 9 at Day 393 (12 months post dose 2) 
and Visit 11 at Day 757 (24 months post dose 2), and will also be contacted by phone on Day 
575 (Visit 10) for safety follow-up.
Blood samples will be collected at each site visit: i.e., at Screening Visit(s) (Visit 1A and/or 
Visit 1B) for flavivirus serostatus determination and eligibility screening tests (including 
pregnancy testing); at Visits 3 and 5 for routine safety laboratory testing; and at Visits 2, 4, 6, 8, 
and for subjects who will be randomized to the placebo group or the PIZV dosing group that will 
be selected for further development) 9 and 11 for immunogenicity, as well as for further 
development and characterization of assays. 
Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) 
for eligibility screening, and at Visits 3 and 5 for routine safety laboratory testing and for women 
of childbearing potential. Women of childbearing potential will undergo a pregnancy test at 
Visits 2 (blood/serum pregnancy test) and visit 4 (urine pregnancy test) before each 
investigational vaccine/placebo administration.
Each subject will receive diary cards to collect solicited AEs for 7 days after each dose 
(including the days of vaccine/placebo administration), and unsolicited AEs for 28 days after 
each dose (including the days of vaccine/placebo administration). Additional safety assessments 
will include new medical conditions (neurological and neuroinflammatory disorders) with onset 
after the first vaccination, and SAEs collection for the duration of the entire trial. 
Immunogenicity will be assessed for all subjects 28 days after each vaccine/placebo 
administration, and 6 months post dose 2, and for subjects who will be randomized to the 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 13 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
placebo group or the PIZV dosing group that will be selected for further development also 
12 and 24 months post dose 2.
The schedule of trial procedures is exhibited in Appendix A.
4.4 Investigational Study Vaccine 
Takeda’s PIZV is a formalin-inactivated ZIKV vaccine candidate with aluminum hydroxide as 
an adjuvant provided as a liquid in the single-use vials. The investigational vaccine is 
administered IM as a 2-dose regimen containing 0.5 mL of 2, 5, or 10 μg antigen per dose, 
28 days apart. 
4.5
Placebo 
Sodium chloride 0.9% solution is being used as placebo. It is a sterile, clear, colorless liquid 
solution of sodium chloride without preservative designed for parenteral use only. The placebo is 
presented in single-use vials stored at 2°C to 8°C. The placebo is administered IM as a 2-dose 
regimen of 0.5 mL per dose, 28 days apart.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 14 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
5.0
ANALYSIS ENDPOINTS
5.1
Primary Endpoints
Safety and Tolerability of PIZV as determined by:

Percentage of subjects with solicited local reactions (injection site: pain, erythema, swelling, 
and induration), in each severity category, during the 7-day period after administration of 
each dose of PIZV or placebo;

Percentage of subjects experiencing solicited systemic AEs (fever, headache, fatigue, 
malaise, arthralgia, and myalgia), in each severity category, during the 7-day period after 
administration of each dose of PIZV or placebo;

Percentage of subjects experiencing non-serious unsolicited AEs during the 28-day period 
after administration of each dose of PIZV or placebo; and

Percentage of subjects experiencing serious AEs (SAEs) during the 28-day period after 
administration of each dose of PIZV or placebo.
Immunogenicity of PIZV, as determined by:

Geometric mean titers (GMT) of neutralizing anti-ZIKV antibody levels at 28 days post 
dose 2.
5.2
Secondary Endpoints
Safety of PIZV, as determined by:

Percentage of subjects experiencing SAEs throughout the trial.
Immunogenicity of PIZV, as determined by:

GMT of neutralizing anti-ZIKV antibody levels at 28 days post dose 1, 6, 12 and 24 months 
post dose 2 in applicable groups;

Seropositivity rates (SPR) at each immunogenicity time point (28 days post dose 1, 28 days
post dose 2, 6, 12 and 24 months post dose 2) in applicable groups; and

Seroconversion rates (SCR) at 28 days post dose 1 and 28 days post dose 2.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 15 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
6.0
DETERMINATION OF SAMPLE SIZE
The sample size was not determined based on formal statistical power calculations. Stochastic 
simulations suggest that the proposed sample size is deemed adequate under a variety of 
decision-making scenarios.
0016
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 16 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.0
METHODS OF ANALYSIS AND PRESENTATION
7.1
General Principles 
This Statistical Analysis Plan (SAP) was developed upon the International Conference on 
Harmonization (ICH) E3 [1] and E9 [2] Guidelines and information provided in the Study 
Protocol ZIK-101, Version 7.0 dated 12 September 2018 [3]. The Table Shells, Data Listings and 
Figures will be provided in a separate document.
The specific algorithms and computer code developed for all derivation used to generate the 
statistical summaries will be provided in the Derived Database and Technical Programming 
Specifications document.
A blinded data review will be conducted prior to unblinding of subject’s vaccination assignment. 
This review will assess the accuracy and completeness of the trial database, subject evaluability, 
and appropriateness of the planned statistical methods.
All statistical analyses will be conducted using SAS® Version 9.2, or higher.
Immunogenicity and safety endpoints will be summarized descriptively (frequency and percent
for categorical data; and number of subjects with non-missing observations, mean [or geometric
mean], standard deviation [SD] [or geometric standard deviation {GSD}], median, minimum,
and maximum for continuous data, unless specified otherwise) at all relevant study visits, as 
appropriate. 
All confidence intervals about a parameter estimate will be presented using the same number of 
decimal places as the parameter estimate.
Arithmetic means, geometric means and medians will be presented to 1 more decimal place than 
the recorded data. 
The standard deviations (SDs) will be presented to 2 more decimal places than the recorded data. 
Where appropriate, variables will be summarized descriptively by study visit and formulation 
arm. 
For the categorical variables, the count and proportions of each possible value will be tabulated 
by formulation arm. The denominator for the proportion will be based on the number of subjects 
who provided non-missing responses to the categorical variable. For continuous variables, the 
number of subjects with non-missing values, mean, median, SD, minimum, and maximum values 
will be tabulated. 
Unless otherwise specified, all data collected during the trial will be presented in the subject data 
listings. 
7.1.1
Definition of Study Days
Study Day 1 is defined as the date of the first vaccination, as recorded on the eCRF vaccination 
page. Other study days are defined relative to Study Day 1, with Day 1 being the day prior to 
Day 1. 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 17 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Baseline is defined as the last non-missing measurement taken before the first dose of PIZV or 
placebo. Where time is available, the time of the sample collection must be prior to the first dose 
of PIZV or placebo. Day 1 observations taken after the vaccination are considered post-baseline 
values. 
7.1.2
Definition of Study Visit Windows
A window convention will be used to determine the analysis value for a given study visit for 
observed data analysis. 
As defined in the schedule of trial procedures (Appendix A) the day of visit is relative to either 
Day 1 or actual Day of the previous vacation. The analysis windows for immunogenicity 
assessments are constructed around the target days following the window conventions presented 
below.
In addition, for visits where PIZV/Placebo is given analysis windows for immunogenicity
assessment will exclude days after the actual day of the vaccination, as immunogenicity 
assessment of post dose 1 must always be prior to administration of dose 2 of PIZV/Placebo
scheduled for the same visit. 
Based on these rules, Table 7.a exhibits the immunogenicity assessment window for each study 
visit. 
Table 7.a
Protocol Defined Visit Windows for Immunogenicity Assessment
Visit
Target Day
Scheduled 
Vaccination
Visit Window (Study Day / Study Day Relative to 
Dose) (a)
2
Day 1
Dose 1
Study Day 1 0/+7 (b)
4
Day 29
Dose 2
Study Day 29 -4/+7
6
Day 57
Day 29 relative to Dose 2 -4/+7days
8
Day 211
Day 183 relative to Dose 2 -7/+14 days
9
Day 393
Day 365 relative to Dose 2 -7/+97 days
11
Day 757
Day 729 relative to Dose 2 -14/+14 days
(a) Study days are defined relative to study Day 1. Study days relative to dose are defined relative to the actual date 
of the specified dose. 
(b) Immunogenicity assessments of post dose 1 must be prior to the dose 2 vaccination scheduled for the same visit 
and where time is available; the time of the sample collection must be prior to the vaccination time. Day 1 
observations taken after the first dose are considered post-baseline values.
7.1.3
Conventions for Missing Adverse Event Data
If AE information is missing dates, intensity and/or relationship then the following approaches 
will be used so that this information can be used in the statistical analysis. 
7.1.3.1
Unsolicited Adverse Events Missing or Partial Dates
Missing and partial AE start dates will be imputed only to determine the relationship between the 
start dates of the event and the date of the most appropriate vaccination of PIZV/Placebo that the 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 18 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
AE is associated with. An AE will be temporally associated with the correct PIZV/Placebo
vaccination dose using the following rules:

If the available start date of AE indicates that the start is between two consecutive 
PIZV/Placebo vaccination dates, then the AE start date will be imputed such that the AE will 
be assigned to the earlier dose. 

If the available start date of AE is insufficient to distinguish between one (1) or more PIZV 
or placebo administration dates, the event end date will be assessed. If possible, the AE will 
be assigned to the dose in the proximity to the event end date, assuming transient nature of 
the events.

If the available date, both start and end dates, information indicates possible association of 
the AE with multiple doses then the AE start date will be imputed such that the AE is 
assigned to the first possible dose. 

If both start and end dates are completely missing, then the AE will be assigned to the first 
PIZV or placebo administration.
7.1.3.2
Adverse Events Missing Intensity (Severity) and Relationship
Missing information regarding “relationship to PIZV or placebo administration”, related/not 
related, for solicited and unsolicited AEs and “severity”, mild/moderate/severe, for unsolicited 
AEs, will be handled using the worst-case approach. That is, unsolicited AEs with missing 
severity/intensity will be considered as “severe” and solicited and unsolicited AEs with missing 
relationship will be considered as “related”.
Solicited events missing intensity will not be included in the ‘any’ (count) of the respective 
solicited event (by grades). 
7.1.4
Conventions for Missing Concomitant Medication Dates
Missing and partial medication dates will be assessed only to determine the relationship between 
the end date of the medication and the dose of PIZV/Placebo. Medication will be considered 
prior only if partial end date indicates that it was stopped before dose of PIZV/Placebo. 
7.2
Analysis Sets

Safety Set (SS): The safety set will consist of all randomized subjects who received at least 
one (1) dose of PIZV or placebo.

Full Analysis Set (FAS): The FAS will include all randomized subjects who have received 
at least one dose of the investigational vaccine/placebo and provided valid baseline and at 
least one post-vaccination serology result. Subjects will be included in the FAS analysis ‘as 
randomized’. 

Per-Protocol Set (PPS): The PPS will consist of all subjects in the FAS who have no major 
protocol violations (relevant for the analysis, primary immunogenicity assessment or 
persistence assessment) as presented in Table 7.b. These criteria for exclusion of subjects 
0019
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 19 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
from the PPS will be reviewed, approved and documented (in a separate document) before or 
at the same time as SAP, prior to database lock and unblinding as part of the blinded data 
review. Any changes to these criteria after approval of the SAP will be documented and 
approved in a separate document. No further changes will be allowed after database lock and 
unblinding.

PPS for the dose selection: For the dose selection GMT ratio analyses, only subjects with
post-vaccination titers at or above Lower Limit of Quantification (LLoQ) (for this study the 
LLoQ value is set at 26.0) will be included.
All summaries and analyses of safety data will be based on subjects in the SS.
The primary immunogenicity analyses will be based on the PPS, and additional immunogenicity 
analyses will be based on the FAS.
All analyses for the safety population (SS) are to be based on the actual treatment received. 
7.2.1
Major Protocol Violations and Evaluability Criteria 
All reported protocol deviations, including the ones identified as protocol violations and major 
protocol violations, will be listed and presented in the final Clinical Study Report (CSR). 
All protocol violations will be classified as major or not. Subjects with major protocol violations
will be excluded from the PPS to avoid affecting the immunogenicity analysis. The PPS will be 
reviewed and approved prior to database lock. 
Some criterion, but not all, used for the exclusion of subjects from the PPS are exhibited in 
Table 7.b below.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 20 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Table 7.b
Criterion for Exclusion from the PPS Analyses
Protocol Deviation 
Categories 
Criteria for Exclusion
Probable Method of Identification
Study vaccination not per 
protocol
Subject who did not receive both 
doses of PIZV or placebo
Identified programmatically using dosing data
Subject who did not receive both 
doses in the correct interval
Decision will be made on a case by case basis
Subject who did not receive the 
assigned IP in accordance with the 
randomization
Identified by independent statistician to 
maintain the subject level blinding within 
study team
Partial dose administered due to 
spillage during administration 
(administration error)
Identified through source documents and 
provided in blinded fashion to the statistician
Subject who received expired or IP 
that was deemed to be affected by 
the inappropriate storage 
conditions
Identified through source documents and 
provided in blinded fashion to the statistician
Procedure not performed 
per protocol 
Blood sampling for 
immunogenicity analysis outside of 
the window
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
Enrollment criteria not 
met 
Subject who meets any of the 
exclusion criteria as presented in 
the study protocol
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
Subject who did not meet any of 
the inclusion criteria 
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
Prohibitive concomitant 
medications/treatments
Subject who used a prohibited 
medication or was/were 
administered vaccine(s) not 
allowed per protocol
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
Withdrawal from the 
study 
Subjects who discontinue prior to 
the end of study. However, 
subjects who complete Day 57 visit 
and have post-vaccination 
serological results will be included 
in analysis
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
Other
Subjects in flavivirus naïve cohort 
that are seropositive at baseline, as 
determined by the Plaque 
Reduction Neutralizing Tests 
(PRNT) assay (a)
Identified through source documents for 
clinical science review to determine 
evaluability status for each identified subject
0021
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 21 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Table 7.b
Criterion for Exclusion from the PPS Analyses (continued)
Protocol Deviation 
Categories 
Criteria for Exclusion
Probable Method of Identification
Other
Traveling to flavivirus endemic 
countries or flavivirus endemic 
regions of the US/US territories, 
within 4 weeks prior to screening or 
planned travel through to Visit 6 
(applicable only to subjects to be 
enrolled into the flavivirus naïve 
cohort)
Subjects will be identified through source 
documents or clinical science to determine 
whether the subject should be evaluated
(a) Subjects will only be excluded from the PPS for immunogenicity analyses for flavivirus naïve cohort (i.e. will 
not be excluded from the PPS of overall subjects and for flavivirus primed cohort). 
7.3
Disposition of Subjects
The reason for screen failure will be summarized based on all screened subjects.
Disposition of all randomized subjects will be summarized by study site for each cohort
(Flavivirus priming serostatus) and study arm.
The categories will include:

Number of subjects screened;

Number of screen failures;

Number of subjects lab confirmed;

Number of subjects randomized (enrolled) by site, dose group and flavivirus serostatus
(flavivirus naïve, flavivirus primed and overall);

Primary reason for ineligibility for randomization;

Number of subjects who received at least one (1) dose of the PIZV/Placebo;

Number of subjects who received both doses of PIZV/Placebo;

Number of subjects who received only one (1) dose of PIZV/Placebo;

Number of subjects who completed each study visit (by visit);

Number of subjects that completed all study visits;

Number of subjects who prematurely discontinued from vaccine regimen (incomplete dosing 
schedule) with primary reason for discontinuation;

Number of subjects who prematurely discontinued the study with primary reason for 
discontinuation.
0022
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 22 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Major protocol violations leading to exclusion from the PPS will be summarized. All protocol 
deviations will be presented in a subject data listing. 
A subject is assumed ongoing unless he/she completed the end of study eCRF, indicating either 
completion or early termination. The primary reason for study discontinuation will be 
summarized. 
7.4
Demographic and Other Baseline Characteristics
Demographics and baseline characteristics which included age, gender, race (American Indian or 
Alaska Native, Asian, Black or African American, Native Hawaiian/Other Pacific Islander, 
White), body weight, body height and BMI will be summarized descriptively by cohort
(flavivirus naïve and primed) and by study arm based on the SS, FAS and PPS. 
Summary statistics (number of subjects, mean, and median, SD, minimum and maximum) will 
be exhibited for continuous variables and number and percentage of subjects within each 
category will be exhibited for categorical variables. 
Inferential analyses of demographic and baseline characteristics will not be performed. 
Individual demographic and baseline characteristics will be presented in subject data listings. 
For subjects who are screened, but not randomized in the study, these individual subject data as 
well as the date of informed consent, and reason for screen failure will also be presented in 
subject data listings. 
7.5
Medical History and Concurrent Medical Conditions
Medical occurrences before administration of the first dose of investigational vaccine/placebo 
are considered medical history. Medical history and concurrent medical conditions will be coded 
using the Medical Dictionary for Drug Regulatory Activities (MedDRA) coding system. The 
current version of the dictionary will be used. Concurrent medical conditions are conditions that 
are recorded as ongoing at screening. 
A summary table will be provided by system organ class (SOC), preferred term (PT) for each
study arm and cohort, based on subjects randomized.
In addition, all medical history and concurrent medical condition data will be listed by study site, 
dose group and cohort. The subject data listing will contain subject identifier, dose group, SOC, 
PT and start and stop dates of the medical history and concurrent medications. 
7.6
Prior and Concomitant Medications/Vaccinations
Prior and concomitant medications/Vaccinations will be coded using the World Health 
Organization Drug Dictionary (WHO-DD). The current version of the dictionary will be used. 
A prior medication and vaccinations are any medication or vaccination taken before 
administration of PIZV or placebo. A concomitant medication/vaccination is any 
medication/vaccination taken on or after administration of PIZV or placebo.
0023
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 23 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Separate Summary tables for Prior medications and for Concomitant medications, will be 
provided by Anatomical Therapeutic Chemical (ATC) code and dose group based on subjects 
randomized. Separate subject data listings for medication history and concomitant 
medications/vaccinations will be produced by study site and subject number. 
These subject data listing will contain subject identifier, dose group and cohort, ATC code, and 
preferred medication name, dose, frequency, and unit, route of administration, stop date and 
reason for use. 
7.7
Study Vaccine Exposure and Compliance
The investigator records all injections of PIZV or placebo given to the subject in the eCRF. The 
administration date of PIZV or placebo and time information will be listed for each subject. The 
compliance rate will be summarized for the SS analysis by dose group presenting the number 
and percentage of subjects who received both doses and the number and percentage of subjects 
who received the first dose only. 
The duration of follow-up will be summarized based on the SS as a continuous variable and in 
categories of 6, 12 and 24 months intervals from the second (last) vaccine administration. The 
categories will be: <6 months follow-up, 6 months post-dose 2 follow-up (study completers if 
not in groups that go into extension and all who are entering extension), 12 months post-dose 2 
follow-up (only applicable to groups in extension) and 24 months post-dose 2 follow-up
(completers of the extension). 
7.8
Traveling During the Study
For the FV naïve cohort subjects travel is not permitted to any endemic country or region (area) 
until visit 6 (endemic countries and/or regions are described in Appendix E). If the subject 
travels to an endemic area within the period defined per protocol, then the subject may be 
excluded from the PPS, due to the risk of infection with a FV (which would mean the subject 
would no longer be FV naïve). The chance of the subject being infected, during the subjects’ stay 
in an endemic area, will be evaluated (i.e. epidemiological situation during the time of travel will 
be evaluated). All travelling collected for visit 1A and the subsequent visits will be summarized. 
7.9
Efficacy Analysis
Efficacy was not assessed in this study.
7.10
Pharmacokinetic/Pharmacodynamic Analysis
Not applicable.
7.10.1 Pharmacokinetic Analysis
Not applicable.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 24 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.10.2 Pharmacodynamic Analysis
Not applicable.
7.11
Other Outcomes
The primary and secondary endpoints for safety and immunogenicity as presented in Sections 5.1
and 5.2 will be evaluated using the methods detailed below.
7.11.1 Immunogenicity Analysis 
The primary and secondary endpoints for the immunogenicity analyses are presented below and 
will be evaluated using the methods detailed below. The PRNT test is used for determination of 
immunogenicity of the investigational vaccine, by assessing the quantity of neutralizing 
antibodies that bind ZIKA virus in the assay. The assay results are reported as titers (reciprocal 
value of the dilution of the serum from the vaccinated individual that inhibits for 50% the plaque 
formation). 
The GMT will be calculated as the anti-logarithm of Σ (log transformed titer/n), i.e. as the 
antilogarithm transformation of the mean of the log-transformed titer, where n is the number of
subjects with titer information. The geometric standard deviation (GSD) for GMT will be
calculated as the anti-logarithm transformation of the standard deviation of the log-transformed 
titer. The 95% CI will be calculated as the anti-logarithm transformation of the upper and lower 
limits for a two-sided CI for the mean of the log-transformed titers. 
The fold increase from the baseline antibody titers is calculated as the ratio of the post-
vaccination titer level to the pre-vaccination titer level. Considering Q2 PRNT assay’s design the 
minimum of 4-fold increase in titers is considered meaningful. 
The primary population for all immunogenicity analyses will be the PPS. 
Additional immunogenicity analysis will be performed using Zika RVP microneutralization 
assay, with details documented in a separate SAP Addendum. 
7.11.1.1 Definitions for the Immunogenicity Endpoints
The definitions are presented below:

Seronegative subjects: Subjects with no detectable serum antibodies (test results are below 
LOD) as measured by the neutralization assay.

Seropositive subjects: Subjects with detectable serum antibodies (tested positive at or above 
limit of detection, LOD) as measured by the neutralization assay. 

Seroconverted subjects: Seronegative subjects at baseline with detectable post-vaccination 
serum antibodies (test results are at or above LOD) and seropositive subjects at baseline (FV 
primed) with different fold increases in post-vaccination antibodies from baseline, as 
measured by the neutralization assay.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 25 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Considering Q2 PRNT assay’s design the minimum of 4-fold increase in titers is considered 
meaningful. Seronegative subjects (i.e. with titer < LOD) at baseline with detectable post-
vaccination serum antibodies (test results are at or above LOD) and seropositive subjects at 
baseline (i.e. with titer ≥ LOD) with 4-fold increases in post-vaccination antibody titers from 
baseline are considered as seroconverted subjects.
Subjects tested negative will be assigned the value half of LOD i.e. 5 (10.0/2).
7.11.1.2 Missing and Imputed Immunogenicity Data 
Missing data will be addressed in the immunogenicity analyses following the rules specified 
below:

Missing Immunogenicity Data
Subjects with missing immunological data will be excluded from the immunological analyses. 

Titers Measured Below the Lower Limit of Quantitation (LLoQ)
A titer value measured equal or above Limit of Detection (LOD) (the LOD value is set to 
10.0) and below LLoQ (the LLoQ value is set to 26.0) (≥ LOD and <LLoQ) will be imputed 
to a value that is half of the LLoQ value i.e., (26.0/2 = 13.0) in summaries and analyses, but 
both the raw serology data and imputed data will be included in the listing. 

Titers Measured Below the LLoQ (for the dose selection analysis)
For the GMT ratio analyses subjects with imputed post-vaccination titers will not be 
considered in the analysis to be conservative.
Subjects negative in the test will be assigned ½*LOD (10.0/2 = 5).
7.11.1.3. Analyses of Primary Immunogenicity Endpoints
The primary immunogenicity endpoint is the GMT, and for dose comparison the ratios of 
geometric mean titers (GMT) of neutralizing anti-Zika Virus antibody levels at 28 days post 
dose 2 (Day 57 Visit 6) will be calculated pairwise for the three different PIZV dose levels (2 µg, 
5 µg or 10 µg) in flavivirus naïve adults.
The differences in seroconversion rates at each immunogenicity time point (28 days post dose 1, 
28 days post dose 2, calculated pairwise for the three different PIZV dose levels (2 µg, 5 µg or 
10 µg) in flavivirus naïve adults will also be assessed for the dose comparison.
A sensitivity analysis will be provided using the FAS. 
All pairwise comparisons will be estimated using the PPS, with a sensitivity analysis using the 
FAS without multiplicity adjustment.
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 26 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.11.1.4. Analyses of Secondary Immunogenicity Endpoints
The secondary immunogenicity endpoints are:

The GMT of neutralizing anti-Zika antibody levels at 28 days post dose 1 (Day 29, Visit 4), 
and Visit 6 (Day 57, Visit 6), 6 months (Day 211, Visit 8), 12 months (Day 393, Visit 9) and 
24 months (Day 757, Visit 10) post dose 2 in applicable dose levels (2 µg, 5 µg or 10 µg) or 
Placebo in flavivirus naïve and primed adults. 

Seropositivity rates (SPR) at each immunogenicity time point (28 days post dose 1, 28 days 
post dose 2 and 6, 12 and 24 months post dose 2) in applicable dose levels (2 µg, 5 µg or 
10 µg) or Placebo in flavivirus naïve and primed adults. 

Seroconversion rates (SCR) at 28 days post dose 1 (Day 29 Visit 4) and 28 days post dose 2 
(Day 57, Visit 6) in applicable dose levels (2 µg, 5 µg or 10 µg) or Placebo in flavivirus 
naïve and primed adults. 
Further details related to the dose selection are presented in a separate document, Dose Selection 
Plan for the Purified Inactivated Zika Virus Vaccine (PIZV); version 1 (07 June 2018).
The details of the immunogenicity analyses are presented as below.
GMT analyses
(1) GMT summary analyses (FV naïve, FV primed, overall)
o
Primary analysis: PPS 
Subjects with post vaccine results ≥LOD and < LLOQ will be assigned the titer 
value of 13.0
o
Analysis based on the FAS 
Subjects with post vaccine results ≥LOD and < LLOQ will be assigned the titer 
value of 13.0
o
Sensitivity analysis for PPS and FAS
Subjects with post vaccine results ≥LOD and < LLOQ will be excluded from this 
analysis (i.e. data will be considered missing)
(2) Pairwise comparison of GMTs (GMT ratios) (FV naïve only for dose selection)
o
Primary analysis: PPS
Subjects with Post-vaccination results ≥LLOQ will be included in the analysis, 
while the results ≥LOD and < LLOQ will be considered missing 
o
Analysis based on the FAS 
Subjects with Post-vaccination results ≥LLOQ will be included in the analysis, 
while the results ≥LOD and < LLOQ will be considered missing 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 27 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
o
Sensitivity analysis for PPS and FAS
Subjects with post vaccine results ≥ LOD and < LLOQ will be assigned the titer 
value of 13.0
SCR analyses
(1) SCR rate summary (FV naïve, FV primed, overall) 
FV naïve 
o
Primary analysis: PPS 
Initially seronegative Subjects with post vaccination ≥ LOD defined as 
seroconversion 
o
Analysis based on FAS
Initially seronegative Subjects with post vaccination ≥ LOD defined as 
seroconversion
o
Sensitivity analysis for PPS and FAS
Initially seronegative Subjects with post vaccination ≥ LLoQ defined as 
seroconversion, while the subjects with results ≥ LOD and < LLoQ are excluded 
from analysis (results in this range are considered ‘missing’)
Initially seronegative subjects and initially seropositive subjects in FV naïve cohort 
will be included in the sensitivity analysis
FV primed 
o
Primary analysis: PPS 
Subjects with baseline results ≥ LOD and <LLOQ will be assigned the value of 
LLoQ conservatively at baseline, ≥ 4 fold increased (≥ 104) defined as 
seroconversion 
o
Analysis based on FAS
Subjects with baseline results ≥ LOD and <LLOQ will be assigned the value of 
LLoQ conservatively at baseline, ≥ 4 fold increased (≥ 104) defined as 
seroconversion 
o
Sensitivity analysis for PPS and FAS
Subjects with baseline results ≥LOD and <LLOQ will be assigned the value of 
13 conservatively at baseline, ≥ 4 fold increased (≥ 52) defined as seroconversion
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 28 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
(2) Pairwise comparison SCR (dose selection, FV naïve) 
FV naïve 
o
Primary analysis: PPS 
Initially seronegative Subjects with post vaccination ≥ LOD defined as 
seroconversion
o
Analysis based on FAS
Initially seronegative Subjects with post vaccination ≥ LOD defined as 
seroconversion
o
Sensitivity analysis for PPS and FAS
Initially seronegative Subjects with post vaccination >=LLoQ defined as 
seroconversion, while the subjects with results >LOD and < LLOQ are excluded 
from analysis (results in this range are considered ‘missing’
Initially seronegative subjects and initially seropositive subjects in FV naïve cohort 
will be included in the sensitivity analysis
SPR analyses 
(1) SPR rate summary (FV naïve, FV primed, overall)
FV naïve
o
Primary analysis: PPS 
Subjects with post vaccination ≥ LOD defined as seropositive
o
Analysis based on the FAS
Subjects with post vaccination ≥ LOD defined as seropositive
o
Sensitivity analysis for PPS and FAS 
Subjects with post vaccination ≥ LLoQ defined as seropositive while the subjects 
with results ≥ LOD and < LLoQ are excluded from analysis (results in this range are 
considered ‘missing’)
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 29 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
FV primed 
o
Primary analysis: PPS 
Subjects with post vaccination ≥ LOD defined as seropositive 
o
Analysis based on FAS
Subjects with post vaccination ≥ LOD defined as seropositive
o
Sensitivity analysis for PPS and FAS
Subjects with post vaccination ≥ LLoQ defined as seropositive while the subjects 
with results ≥ LOD and <LLoQ are excluded from analysis (results in this range are 
considered ‘missing’)
(2) Pairwise comparison SPR (FV naïve only)
o
Primary analysis: PPS
Subjects with post vaccination >=LOD defined as seropositive
7.11.1.5 Supportive Immunogenicity Summaries
Supportive immunogenicity summaries will be provided based on the FAS. The planned 
immunogenicity analyses will be based on the PPS and are exhibited in Table 7.c below.
Table 7.c
Planned Analyses and Populations for Immunogenicity Endpoints
Cohorts
Parameter
Time Point
Descriptive
Summaries
Graphical
Presentation
Flavivirus Naïve Healthy Adults
GMT
Day 1 (Visit 2)
PPS, FAS
PPS
Day 29 (Visit 4)
PPS, FAS
PPS
Day 57 (Visit 6)
PPS, FAS
PPS
Day 211 (Visit 8)
PPS, FAS
PPS
Day 393 (Visit 9)
PPS, FAS
PPS
Day 757 (Visit 11)
PPS, FAS
PPS
SPR
Day 1 (Visit 2)
PPS, FAS
PPS
Day 29 (Visit 4)
PPS, FAS
PPS
Day 57 (Visit 6)
PPS, FAS
PPS
Day 211 (Visit 8)
PPS, FAS
PPS
Day 393 (Visit 9)
PPS, FAS
PPS
Day 757 (Visit 11)
PPS, FAS
PPS
SCR
Day 29 (Visit 4)
PPS, FAS
PPS
Day 57 (Visit 6)
PPS, FAS
PPS
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 30 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.11.2 Descriptive Summaries
The geometric mean, standard deviation, median, minimum, maximum and 95% confidence 
intervals will be calculated as the antilogarithm transformation of the mean, standard deviation, 
median and 95% confidence intervals of the log-transformed titers. 
Descriptive statistics for the primary and secondary immunogenicity endpoints, including 
estimates and 95% confidence intervals (95% CI), calculated using the exact Clopper-Pearson 
method [4], for GMT, SPR and SCR, will be provided by time point as per Table 7.c (28 days 
post dose 1 (Visit 2, Day 1), 28 days post dose 2, 6, 12, and 24 months post dose 2 in applicable 
groups) and by dose level.
For seropositive subjects at baseline, the different fold increases in post-vaccination antibodies 
from baseline will also be exhibited as percentage of subjects with a ≥ 4-fold rise at each time 
point by dose level.
Point estimates and 95% CI, calculated using the exact Clopper-Pearson method [4] for ratios in 
GMT and differences in SCR will be provided for each pair of active study arms (i.e., low, 
medium and high doses) of FV naïve cohort, to aid a single PIZV dose level selection.
Details related to the pre-specified pairwise comparisons within study arms (2 µg, 5 µg or 10 µg
dose level) are presented in Section 7.12.
The analysis for seropositivity rates (SPR) will be based on two immunogenicity time points: 
28 days post dose 1 (Day 29 Visit 4) and 28 days post dose 2 (Day 57), for the three PIZV dose 
levels.
The differences in SPR will be analyzed using Newcombe score method [5] with corresponding 
95% CIs, and p-values computed using Fisher’s exact test will be exhibited.
Immunogenicity summaries and analyses will be provided by study arm, overall and each cohort
(flavivirus naïve and primed cohorts).
Descriptive summaries per dose group will not use baseline titer values (in FV primed subjects) 
as the covariate in the analyses, thus ANOVA analyses will be conducted.
7.12
Dose Selection Methodology
The dose selection methods detailed in this section are to be used in conjunction with the 
analyses presented in the dose selection plan. The dose selection analyses are based on the data 
from FV naïve cohort only. The immunogenicity assessment will therefore be based on the 
magnitude of the response, as measured by the ratios of geometric mean titer (GMT) of 
neutralizing anti-ZIKV antibodies, and differences in seroconversion rate between the dosing 
groups. The distribution of neutralizing titers across the population of each dose group will 
provide supplemental information for the dose selection.
The primary immunogenicity analysis will be done using PPS. 
Safety analyses supporting the dose selection will be performed in the flavivirus naïve, flavivirus 
primed cohorts and combined. 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 31 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.12.1 Geometric Mean Titers Analyses
GMT ratios with corresponding 95% CIs and p-values will be estimated from an analysis of 
covariance (ANOVA) model including the log-transformed value of the titer as the dependent 
variable and vaccine dose level (low, medium or high) as independent variables. 
The GMT, GMT ratio and 95% CI will be presented on an anti-log scale of the least-square 
means with associated standard deviations estimated from this ANOVA model. 
This analysis will be performed without multiplicity adjustments.
GMTs with or without imputed values for results ≥LOD and < LLoQ will also be presented as 
depicted in Table 7.c. 
7.12.2 Seroconversion Rate Analyses
Seroconversion in FV naïve subjects is defined as initially seronegative subjects reaching 
seropositivity post vaccination.
7.13
Other Immunogenicity Analyses
The analyses of GMTs, SCRs and SPRs used in the dose selection on the FV naïve cohort will be 
repeated for the FV primed cohort and combined cohorts (FV naïve and FV primed cohorts 
combined). These analyses will also be performed over all relevant time points. 
7.14
Graphical Presentations
Graphical presentations for selected immunogenicity parameters will be provided to explore and 
understand the distributional characteristics for the three dose levels (2, 5 or 10 µg) in the 
flavivirus naïve cohort and FV primed cohort.
Further details are presented below.
7.14.1 Immunogenicity Figures
The primary immunogenicity parameters presented below will be exhibited graphically using the 
PPS.
7.14.1.1 Geometric Mean Figures
The graphical presentations for the GMT analyses will include:

Reverse cumulative distribution curves of antibody titer values for baseline, 28 days post 
vaccination Day 29 (Visit 4), Day 57 (Visit 6), Day 211 (Visit 8), Day 393 (Visit 9) and 
Day 757 (Visit 11) by dose level (2, 5 or 10 µg) and Placebo overlaid.

Line plots of the GMTs plotted for baseline (Day 1, Visit 2), 28 days post vaccination Day 29 
(Visit 4), Day 57 (Visit 6), Day 211 (Visit 8), Day 393 (Visit 9) and Day 757 (Visit 11) by
dose level (2, 5 or 10 µg) and Placebo overlaid. 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 32 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.14.1.2 Seroconversion Rate Figures
The graphical presentations for the seroconversion rates will include:

Bar graphs exhibiting the percentage of subjects with seroconversion including error bars for 
the 95% CIs plotted for 28 days post vaccination 1 Day 29 (Visit 4) and 28 days post 
vaccination 2 Day 57 (Visit 6) by dose level (2, 5 or 10 µg). 
7.14.1.3 Seropositivity Rate Figures
The graphical presentations for the seropositivity rates will include:

Bar graphs exhibiting the percentage of seropositive subjects including error bars for the 95% 
CIs plotted for baseline, 28 days post vaccination Day 29 (Visit 4), Day 57 (Visit 6), Day 211 
(Visit 8), Day 393 (Visit 9) and Day 757 (Visit 11) by dose level (2, 5 or 10 µg).
7.14.1.4 Fold-Rise Figures
The graphical presentations for the fold increases will include:

Bar graphs exhibiting the percentage of subjects with fold increase of 4 and greater including 
error bars for the 95% CIs plotted for baseline, 28 days post vaccination Day 29 (Visit 4), 
Day 57 (Visit 6), by dose level (2, 5 or 10 µg).
7.14.2 Primary and Secondary Safety and Tolerability Endpoints
Safety and tolerability of PIZV will be established by the following endpoints.
7.14.2.1 Primary Safety and Tolerability Endpoints

Percentage of subjects with solicited local reactions (injection site: pain, erythema, swelling, 
and induration), in each severity category, during the 7-day period after administration of 
each dose of PIZV or placebo.

Percentage of subjects experiencing solicited systemic adverse events (AEs) (fever, 
headache, fatigue, malaise, arthralgia, and myalgia), in each severity category, during the 
7-day period after administration of each dose of PIZV or placebo.

Percentage of subjects experiencing non-serious unsolicited AEs during the 28-day period 
after administration of each dose of PIZV or placebo.

Percentage of subjects experiencing serious AEs (SAEs) during the 28-day period after 
administration of each dose of PIZV or placebo.

Percentage of events experiencing serious AEs/AEs causing the withdrawal from the study 
vaccination/study throughout the study. 
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 33 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.14.2.2 Secondary Safety and Tolerability Endpoints

Percentage of subjects experiencing SAEs throughout the trial.
All summaries and analyses will be presented by study arm, overall and each cohort (flavivirus 
naïve and primed cohorts) using SS.
Further details related to the safety analysis are presented below.
Number and percentage of subjects and events with solicited local reactions (injection site: pain, 
erythema, swelling, and induration), in each severity category, during the 7-day period after 
administration of each dose of PIZV or placebo;
Number and percentage of subjects and events with solicited systemic AEs (fever, headache, 
fatigue, malaise, arthralgia, and myalgia), in each severity category, during the 7-day period after 
administration of each dose of PIZV or placebo, or after any PIZV dose;
Number and percentage of subjects and events with non-serious unsolicited AEs during the 
28-day period after administration of each dose of PIZV or placebo; 
Number and percentage of subjects and events with SAEs during the 28-day period after 
administration of each dose of PIZV or placebo, and throughout the study;
Number and percentage of subjects and events with serious AEs/AEs causing the withdrawal 
from the study vaccination/study throughout the study.
7.15
Safety Analysis
All summaries and analyses of safety data will be based on subjects in the SS data. Unless 
otherwise specified, safety data will be summarized by dose for each study arm, overall and each 
cohort (flavivirus naïve and primed cohorts).
Summaries after any PIZV or placebo administration will also be included.
Biologically implausible measurements of body temperature and solicited local symptoms such 
as body temperature < 35ºC or > 42ºC and erythema and swelling > 50.0 cm will be excluded 
from the summaries and analyses but included in the data listings. 
No inferential analyses of safety data will be performed. Unless otherwise specified, data 
imputation will not be performed for any missing adverse event. 
All new medical conditions (including neurological and neuroinflammatory disorders) with onset 
after the first vaccination, deaths, related SAEs and SAEs deemed relevant in the context of 
PIZV safety by the investigator will be summarized. 
7.15.1 Solicited Adverse Events Related to Reactogenicity
Reactogenicity will be assessed for 7 days for solicited local injection site following each dose 
(including day of PIZV or placebo administration) via daily collection of solicited AEs, 
including local reactions at the injection site (pain, erythema, swelling, and induration) and 
systemic AEs (headache, fatigue, malaise, arthralgia and myalgia). In addition, body temperature 
0034
akeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 34 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
(preferably measured as oral route) will be collected (with fever defined as body temperature 
≥38°C/100.4°F.) for 7 days as solicited systemic AEs after each vaccination including the day of 
vaccination. These solicited AEs and their intensity grades are defined in the study protocol, 
Table 10.b [3].
For local solicited AEs erythema, induration and swelling, subjects will record the length of the 
greatest surface diameter. For the systemic solicited AE fever, subjects will record the body 
temperature in either degrees Fahrenheit (ºF) or degrees Celsius (ºC). Fever data will be 
displayed in ºC in summaries and data listings. Intensity grades for erythema, induration and 
swelling will be derived from the recorded diameters and fever will be derived from the recorded 
body temperature measurements, according to Table 10.b provided in the protocol [3]. 
For each solicited AE and fever, the number and percentage of subjects reporting AEs and 
number and percentage of doses followed by an event will be summarized by event severity and 
by relationship to PIZV/Placebo (only for solicited systemic AEs and local solicited AEs are 
considered as related vaccination) for each study arm, overall and each cohort (flavivirus naïve 
and primed cohorts) following each vaccination:

30 minutes post vaccination (solicited local and systemic AEs);

Days 1 through 7 (solicited local and systemic AEs);

Days 1 through 3 and Days 4 through 7 (solicited local and systemic AEs).
For subjects with more than 1 episode of the same event, the maximum severity will be used for 
tabulations.
A summary of Time-to-onset and duration of each event will be presented following each 
administration of PIZV or Placebo. The duration of an event is calculated as the total number of 
all days the subject reported the event during the solicited period of 7 days post vaccination, 
regardless of whether the symptom was reported on consecutive days. 
Prolonged solicited AEs that continued beyond day 7 will be captured on the Adverse Event 
eCRF page, indicated by the checkbox “Is this event a continuation of a Solicited event”. These
prolonged solicited AEs will be analyzed separately from the unsolicited AEs and presented in a 
separate subject data listing. 
7.15.2 Unsolicited Adverse Event
Any AE captured in the Adverse Event eCRF that is a continuation of a solicited AE will not be 
included in any unsolicited AE summaries or subject data listings.
Unsolicited AEs will be assessed for 28 days following administration of each dose of PIZV or 
Placebo (day of administration + 27 days). SAEs and AEs leading to subject withdrawal from the 
trial will be collected throughout the trial for all subjects.
Unsolicited AEs will be summarized up to 28 days after each vaccination. These summaries will 
generally be presented in: overall duration of follow-up, up to 28 days after each vaccination, 
Additionally, AEs from first vaccine dose to 28 days post dose 2 will be summarized. 
0035
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 35 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
SAEs and AEs leading to early termination will be assessed throughout the trial duration and 
presented in 3 ways described for AEs with additional category of events reported throughout the 
trial duration (from first vaccine dose to: 28 days post dose 2, 6 months post dose 2, 12 months 
post dose 2 and 24 months post dose 2) and those with the onset after 28 days post each 
vaccination. 
The number and percentages of subjects with an unsolicited AE will be tabulated at each of the 
following levels:

Overall (Unsolicited AEs, SAEs and Deaths);

By System Organ Class (SOC) and Preferred Term (PT);

Non-serious unsolicited AEs including events with frequency greater than 5% in any study 
arm up to 28 days after vaccination by SOC and PT (in decreasing frequency);

Relationship (not related, related) to vaccine by SOC, PT and intensity; 

By SOC, PT and intensity (mild, moderate and severe).
Subjects reporting more than one occurrence for the term (level) being summarized will be 
counted only once. When relationship or intensity (severity) is concerned the AE with the most 
closely related occurrence or the highest known intensity will be counted. If the investigator did 
not provide the relationship then the relationship will be assumed to be related. If the investigator 
did not provide severity then the worst severity will be assumed. 
Unsolicited AEs, SAEs and new medical conditions (neurological and neuroinflammatory 
disorders) with onset after the first vaccination will be coded using MedDRA and summarized by 
SOC and PT for each study arm.
Safety summaries will be provided by study arm, overall and each cohort (flavivirus naïve and 
primed cohorts).
7.15.3 Clinical Laboratory Evaluations
Routine safety laboratory tests that will be performed on blood and urine samples at screening 
visit for baseline, visit 3 (Day 8, 7 days post dose 1) and Visit 5 (Day 36, 7 days post dose 2) are 
outlined in Table 9.a in the protocol [3].
Subjects with baseline safety laboratory test results within normal limits or not above Grade 1 as 
defined in the FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers (refer 
to Appendix D) are eligible for participation in the study. Toxicity grading scales for laboratory 
abnormalities will be assigned based on the reference values defined in FDA toxicity grading 
scales for healthy adult and adolescent volunteers enrolled in preventive vaccine clinical trials. 
For each laboratory parameter with abnormal results at each time point, the number and 
percentage of subjects and doses will be summarized by grading scale for each study arm, overall 
and each cohort (flavivirus naïve and primed cohorts) following each vaccination. The change in 
the grade of the laboratory values as the shift tables.
0036
of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 36 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.15.4 Vital Signs
The vital signs collected in this study include systolic and diastolic blood pressure, heart rate, 
and body temperature. 
Vital signs will be summarized descriptively by study arm at each applicable visit for overall and 
each cohort (flavivirus naïve and primed cohorts). Individual subject data will be presented in the 
subject data listings by dose group, study site, subject number, collection date and time and 
parameter measured. 
Descriptive statistics (number of subjects, mean, median, SD, minimum and maximum), of vital 
sign parameter (observed and change from previous vaccination) except height, will be 
summarized by study arm at each applicable visit for overall and each cohort (flavivirus naïve 
and primed cohorts). The change in the grade of the vital sign values will be summarized in the 
shift table. 
Only vital signs measurements at the scheduled visits will be included in this summary.
7.15.5 12-Lead ECGs
Not applicable.
7.15.6 Other Observations Related to Safety
For dose selection the following safety summaries will be produced:

Overall rate of fever occurrence after the first and second vaccination by dose;

Severity of systemic reaction (excluding fever) after the first and second vaccination by dose; 
and

Severity of local site reactions after the first and second vaccination by dose.
These summaries will include the occurrence of the event (n/N), probability of the occurrence 
(%), and 95% Confidence interval of the probability of the occurrence. Newcombe score method 
[5] with corresponding 95% CIs and p-values computed using Fisher’s exact test will be used for 
this analysis. 
7.16
Interim Analysis
A first interim analysis will be performed to include immunogenicity and safety data from all 
flavivirus naïve subjects up to Visit 6 (on Day 57, 28 days post dose 2); a second interim analysis 
will be performed for dose selection, and including safety data from all flavivirus primed 
subjects up to Visit 4 (on Day 29, 28 days post dose 1). 
7.17
Analyses for the Final Clinical Study Report
The primary safety and immunogenicity analysis will be performed when all (flavivirus naïve 
and primed) subjects have completed Visit 6 (Day 57) to provide data to support the planning 
and execution of other trials in the development plan of PIZV. The analyses will be performed 
0037
akeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 37 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
by a separate set of unblinded statisticians and programmers at a Clinical Research Organization 
(CRO), who will have access to individual treatment assignments and will not be involved in 
subsequent trial conduct. The study team at Takeda will have access to the group level unblinded 
results and will remain blinded to the individual treatment assignment. The remaining personnel 
(not mentioned before) involved in the conduct of the trial, including those at Takeda, the CRO, 
and the trial sites, will remain blinded to the individual subject treatment assignment until 
unblinding after trial completion (database lock for data through- Visit 11).
The final study report will be written for the data up to Visit 6 (Day 57), and two additional
addenda will be written: one to report the data from Visit 6 (Day 57) to Visit 9 (Day 393, 12
months post dose 2), and one to report the data from Visit 9 (Day 393) until the end of the study, 
i.e., Visit 11 (Day 757, 24 months post dose 2).
The timing and the types of analyses with the respective output is provided in Table 7.d. 
Table 7.d
Timing and the Types of Analyses with the Respective Output
Analysis
Study Time Point
Clinical Study Report/ Data
Interim 
analysis 1
Day 57 (Visit 6) completed 
for FV naïve cohort 
Analysis of immunogenicity and safety. No study report will be 
written.
Interim 
analysis 2
Up to Visit 4 (on Day 29, 28 
days post dose 1) for FV 
primed cohort 
Safety assessment and analysis of dose selection based on the 
immunogenicity data. No study report will be written.
CSR
Day 57 (Visit 6) completed 
for both FV naïve and primed 
cohorts
Primary safety and immunogenicity analysis. CSR will be written.
CSR 
addendum 
1
Visit 6 (Day 57) to Visit 9 
(Day 393, 12 months post 
dose 2)
Additional safety and immunogenicity analysis up to visit 9. CSR 
addendum 1 will be written. 
CSR 
addendum 
2
Visit 9 (Day 393) until the 
end of the study, ie, Visit 11 
(Day 757, 24 months post 
dose 2)
Additional safety and immunogenicity analysis up to visit 11. CSR 
addendum 2 will be written. 
7.18
Changes in the Statistical Analysis Plan 
This SAP contains no changes to the planned analyses described in the protocol, except that 
some additional details are added to some sections for further clarification.
7.18.1 Amendment History
Date
Amendment Number
10 Aug 2018
Initial Analysis Plan
26 Apr 2019
1
0038
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7.18.1
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 38 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
7.18.2 Summary of Changes
This section describes changes to the Statistical Analysis Plan Version 1.0, dated 10 Aug 2018.
The main rationale for this amended SAP is the Amendment to Protocol Version 3.0, dated 12 
Sept 2018. Minor grammatical and editorial changes are included for clarification purposes only.
Section
Description of Change
Cover page
Update on author, SAP and protocol version and date
1.1
Update of approval team
7.1.2
Update of study window per protocol amendment 
7.2.1 and Table 7.b
Clarification added regarding protocol violation and deviation 
summary and listing
Update of method of PPS identification 
7.3
Additional summaries for subject disposition
7.8
Clarification on visits to be summarized
7.15.4
Adding shift table to vital sign, changing “Change from baseline” to 
“Change from previous vaccination” for descriptive summary
7.16 and Table 7.d
Update on planned analysis for interim and CSR addendum per 
protocol amendment
Appendix A
Update on Day 133 and Day 393 window per protocol amendment
0039
ial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 39 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
8.0
REFERENCES
1. ICH. Harmonized Tripartite Guideline E3 Guideline for the Structure and Content of Clinical 
Study Reports. Step 4, 30 November 1995; Available from: 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E3/E3_G
uideline.pdf 
2. ICH. Harmonized Tripartite Guideline E9 Guideline for the Statistical Principles for Clinical 
Trials. Step 4, 5 February 1998; Available from: 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E9/Step4/
E9_Guideline.pdf 
3. A Phase 1, Randomized, Observer-Blind, Placebo-Controlled, Safety, Immunogenicity and 
Dose Ranging Study of Purified Inactivated Zika Virus Vaccine (PIZV) Candidate in 
flavivirus naïve and Primed Healthy Adults Aged 18 to 49 Years. Takeda Vaccines, Inc. 
Protocol No. ZIK-101 Version 7.0 dated 12 September 2018. 
4. The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial. C. J. 
Clopper; E. S. Pearson; Biometrika, Vol. 26, No. 4. (Dec. 1934), pp. 404-413.
5. Newcombe, R., 1998. Interval estimation for the difference between independent 
proportions: comparison of eleven methods. Statist. Med. 17, 873–890.
6. 6. FDA. Guidance for Industry. Toxicity Grading Scale for Healthy Adult and Adolescent 
Volunteers Enrolled in Preventive Vaccine Clinical Trials. 2007 [22 December 2016]; 
Available from: http://www.fda.gov/downloads/BiologicsBloodVaccines/ucm091977.
0040
and Subject to the Applicable Terms of Use
E3_G
ms
E3_G
for Clin
ble T
for Clin
Efficacy
plica
Efficacy
y, Immu
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(PIZV) 
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(PIZV) 
ars. Tak
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ars. Tak
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8.
e
.
the Cas
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the Cas
(Dec. 19
d Su
(Dec. 19
e differe
and
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Property of Takeda: For Non-Commercial Use Only
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 40 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Appendix A
Schedule of Trial Procedures
Visit Number
1A
1B (a)
2
3
4
5
6
7
8
9 (u)
10 (u)
11 (u)
In case of 
early 
termination
Visit Day
Day -15 
to 
Day 1
Day -15 
to 
Day 1
Day 1 Day 8
Day 29 Day 36 Day 57 Day 133 Day 211 Day 393 Day 575 Day 757
Clinical Visit
X
X
X
X
X
X
X
X
X
X
X
Phone Contact
X
X
First and 
second phase 
of the study (b)
Flavivirus naïve (1)
X
X
X
X
X
X
X
X
X
X
X
X
Flavivirus primed (2)
X
X
X
X
X
X
X
X
X
X
X
X
X
Second phase 
of the study (b)
Flavivirus primed (3)
X
X
X
X
X
X
X
X
X
X
X
X
Anchored to visit day
NA
Day 1
+
7 days
Day 1
+
28 days
Day 29
+
7 days
Day 29
+
28 days
Day 29
+
3 ½ mo
Day 29
+
6 mo
Day 29
+
12 mo
Day 29
+
18 mo
Day 29
+
24 mo
Acceptable visit window (days before/ 
days after) (c)
NA
NA
0/+7
-1/+7
-4/+7
-1/+7
-4/+7
-7/+14
-7/+14
-7/+97
-7/+14
-14/+14
Signed informed consent
X
X (t)
Remind study procedure to flavivirus 
primed subjects identified at Visit 1A (2)
X
Assessment of eligibility criteria (d)
X
X
X
Demographics (e)
X
X (t)
Medical history (f)
X
X
X
Travel history
X
X
X
X
X
X
X
X
X
X
X
X
Concomitant medications/vaccinations (g)
X
X
X
X
X
X
X
X
X
X
Physical 
exam (h)
Complete
X
X
Symptom directed
(X)
(X)
(X)
(X)
(X)
(X)
(X)
(X)
Vital signs (i)
X
X
X
X
X
X
X
X
X
X
0041
eda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
Te
8
A
8
9
ica
133
th
133 Day
th
Day
e A
t to
X
c
X
ect
X
ub
X
bjejeje
X
Su
X
Sub
X
a
X
d
1
+
O
+
28
e
28 days
e O
days
Day
nly
Day
y
+7
U
+7
-
s
-4/+
s
4/+
e
al
rc
rc
rci
X
m
X
mm
X
C
X (t)
Co
(t)
Com
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X
n-
X
-C
X
N
X
No
No
No
X
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X
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X
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 41 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Visit Number
1A
1B (a)
2
3
4
5
6
7
8
9 (u)
10 (u)
11 (u)
In case of 
early 
termination
Visit Day
Day -15 
to 
Day 1
Day -15 
to 
Day 1
Day 1 Day 8
Day 29 Day 36 Day 57 Day 133 Day 211 Day 393 Day 575 Day 757
Blood 
collection (j)
Flavivirus serostatus
X
X (t)
Eligibility screening and 
safety laboratory testing
X
X
X
X
Serum pregnancy testing 
(l)
X
X
Immunogenicity and 
assay development
X
X
X
X
X
X
Urine 
collection (k)
Eligibility screening and 
safety laboratory testing 
(urinalysis)
X
X
X
X
Urine pregnancy testing 
(l)
X
X
Randomization (m)
X
Check contraindications to vaccination 
and criteria for delay of vaccination
X
X
Investigational vaccine or placebo 
administration (n)
X
X
Post-vaccination and injection site 
evaluation (o)
X
X
Diary card (p)
Distribution
Days
1-7
Days
8-28
Days
29-35
Days
36-56
Review/collection
Days
1-7
Days
8-28
Days
29-35
Days
36-56
X (q)
Solicited AEs (r)
X
X
Unsolicited AEs (r)
X
X
X
X
X
SAEs and AEs leading to withdrawal or 
discontinuation (s)
X
X
X
X
X
X
X
X
X
X
X
(X)=procedures that are not mandatory but may be performed if deemed necessary; NA=Not applicable.
0042
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Te
9
ca
9 (u)
ab
(u)
le
Day
A
Day 211
Ap
211
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 42 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
(a) Applicable only for flavivirus primed subjects
(b) Staggered enrollment: There will be two study phases

The first phase of the study will be aimed at enrolling flavivirus naïve subjects only (1).
All subjects who sign the ICF and have confirmed eligibility at Screening Visit 1A will be tested for flavivirus serostatus.
Eligible flavivirus naïve subjects enrolled at Screening Visit 1A (1) will be randomized at Visit 2, within two weeks after Screening Visit 1A.
Flavivirus primed subjects will not be enrolled during the first phase of the study (ie, when identified at Screening Visit 1A (2)) and will be invited to come 
back for the second phase of the study. They will be contacted after positive recommendations from the external DMC are issued following the review of 
safety and tolerability data in flavivirus naïve subjects, when the last flavivirus naïve subject has completed study Visit 4 (28 days post dose 1). These 
flavivirus primed subjects will consequently undergo 2 screening visits: Visit 1A during the first phase of the study and Visit 1B at the outset of the second 
phase of the study. Flavivirus primed subjects identified at Screening Visit 1A who do not participate in the second phase of the study will be declared 
screened failure.
Enrollment will be put on hold after all naïve subjects (1) have been enrolled and will resume when positive recommendations from the external DMC are 
issued, as described above.

The second phase of the study will be aimed at enrolling flavivirus primed subjects only (2 and 3).
Newly identified eligible flavivirus primed subjects at Screening Visit 1B (3) will be enrolled (randomized) at (second phase) Visit 2, within two weeks 
after Screening Visit 1B.
Previously identified flavivirus primed subjects (2) (who were tested at Screening Visit 1A during the first phase and accepted to enter the second phase of 
the study) will retain their initial subject identification (ID) number and undergo an additional screening visit (Visit 1B) to be tested for eligibility criteria 
again (except for the determination of flavivirus serostatus). 
Flavivirus naïve subjects identified at Screening Visit 1B (during the second phase of the study) will be declared screened failure.
(c) If the subject has a temporary clinically significant active infection (as assessed by the investigator) or temperature ≥38.0°C/100.4°F, within 3 days of 
intended investigational vaccine/placebo administration, the investigational vaccine/placebo administration may be delayed as per the assessment of the 
investigator within the allowed visit window. The visit windows of Visit 3 and Visit 4 should be calculated from the day of the first vaccination. Following 
the same rule, the visit windows of Visit 5 and Visit 6 should be calculated from the day of the second vaccination.
(d) One single subject identification (ID) number will be assigned to each subject at Screening Visit (1A or 1B). Eligibility by review of relevant 
inclusion/exclusion criteria will be documented before enrollment.
(e) Demographic information, to be obtained at Screening Visit (1A or 1B), will include age (date of birth), sex, race, and ethnicity as provided by the subject.
(f)
Medical history will be collected at Screening Visit(s) (Visit 1A and/or Visit 1B) and at Visit 2 (Day 1) and will include any medical history that may be 
relevant to subject eligibility for trial participation such as prior vaccinations, concomitant medications, and previous and ongoing illnesses or injuries. 
Relevant medical history can also include any medical history that contributes to the understanding of an AE that occurs during trial participation, if it 
represents an exacerbation of an underlying disease/preexisting problem. Adverse occurrences before administration of the first dose of investigational 
vaccine/placebo are considered medical history.
(g) All medications, vaccines and blood products taken or received by the subjects within 3 months prior to the start of the trial are to be recorded on the source 
document (patient record) and entered on the Prior and Concomitant Medications eCRF. The use of antipyretics and/or analgesic medications within 24 
hours prior to vaccination must be identified and the reason for their use (prophylaxis versus treatment) must be described in the source documents and the 
eCRF. 
0043
Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 43 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
(h) Complete physical examination will be performed at Screening Visit(s) (Visit 1A and/or Visit 1B). All subsequent symptom-directed physical examinations, 
performed if deemed necessary or indicated by review of the subject’s medical history, should assess clinically significant changes from the baseline 
examination. The findings should be documented in the subject’s source document; findings consistent with the definition of AEs or SAEs must be 
transcribed into the electronic Case Report Form (eCRF). For any procedures at the site, the investigator shall follow his/her standard practice.
(i)
Vital signs include (however, not limited to) systolic/diastolic blood pressure, pulse rate, respiratory rate, and temperature, and should be documented in the 
subject’s source document and transcribed into the eCRF. Follow standard of care for trial population and operational feasibility. 
(j)
Blood samples will be collected at each site visit: ie, at Screening Visit(s) (Visit 1A and/or Visit 1B) for flavivirus serostatus determination (approximately 
10 mL of blood) and eligibility screening tests (including pregnancy testing) (approximately 10 mL of blood), at Visits 3 and 5 for routine safety laboratory 
testing (approximately 10 mL of blood), and at Visits 2, 4, 6, 8, and (for subjects who will be randomized to the placebo group or the PIZV dosing group that 
will be selected for further development) 9 and 11 for immunogenicity, as well as for further development and characterization of assays (approximately 
60 mL of blood). Should a blood sampling be performed during a vaccination visit, this sampling must occur before the administration of the investigational 
vaccine or placebo. The maximum volume of blood taken at any single visit will be between approximately 10 mL and 60 mL, and the approximate 
maximum total volume of blood for the whole trial will be 280 mL to 410 mL depending on the subject’s flavivirus serostatus at screening and the vaccine 
dose level the subject received. All samples will be collected in accordance with acceptable laboratory procedures. Blood samples will be processed and 
stored at the trial site as described in the provided Laboratory Manual. 
(k) Urine samples will be collected for all subjects at Screening Visit(s) (Visit 1A and/or Visit 1B) and at Visits 3 and 5; and for women of childbearing 
potential at Visits 2 and 4 before each investigational vaccine/placebo administration for pregnancy testing. Urine samples for eligibility screening and 
routine safety laboratory testing will be sent to the central laboratory. Urine pregnancy tests will be performed at the study site using kits provided by the 
sponsor.
(l)
Serum pregnancy tests will be conducted in the central laboratory and urine pregnancy tests will be done at the study site using kits provided by the sponsor. 
Subjects must have a negative urine -hCG pregnancy test prior to receiving any dose of investigational vaccine/placebo. 
(m) If eligible, the subject will be randomized at Visit 2. 
(n) The investigational vaccine/placebo will be administered in the deltoid muscle of the non-dominant arm (approximately at the top middle third).
(o)
After vaccine/placebo administration at Visit 2 (on Day 1) and at Visit 4 (Day 29), the subject will be observed for at least 30 minutes for observation of 
solicited AEs, unsolicited AEs, and measurement of body temperature.
(p) Diary cards will be handed out to the subjects at Visits 2, 3, 4 and 5, and reviewed with the subject and collected at the following visit. 
(q) In case of early termination during the vaccination period only.
(r)
After each dose, solicited AEs (for 7 days including the day of vaccine/placebo administration) and unsolicited AEs (for 28 days including the day of 
vaccine/placebo administration) will be collected on diary cards by each subject. The investigator will transcribe these data into eCRF. 
(s) SAEs and AEs leading to withdrawal from the trial or discontinuation of vaccine/placebo administration will be collected for the duration of the entire trial. 
SAEs will be reported to the sponsor within 24 hours of the investigator becoming aware of the event. AEs leading to early termination will be recorded by 
the investigator.
(t)
Only for newly screened subjects at Screening Visit 1B (i.e., not for flavivirus primed subjects who were tested at Screening Visit 1A and accepted to enter 
the second phase of the study).
(u) All subjects who were randomized to the placebo group or the PIZV dosing group that was selected for further development will attend Visit 9 at Day 393 
(12 months post dose 2) and Visit 11 at Day 757 (24 months post dose 2), and will also be contacted by phone on Day 575 (Visit 10) for safety follow-up.
0044
mercial Use Only and Subject to the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 44 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Appendix B
Serology Plan
0045
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 45 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
CONFIDENTIAL
0046
e Terms of Use
Te
Property of 
of Takeda: For Non-Commercial Use Only and Subject to the Applicable 


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 46 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
CONFIDENTIAL
0047
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
Te
rty of Takeda: For Non-Commercial Use Only and Subject to the Applicable 


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 47 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
CONFIDENTIAL
0048
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
Te
rty of Takeda: For Non-Commercial Use Only and Subject to the Applicable 


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 48 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
CONFIDENTIAL
0049
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
Te
rty of Takeda: For Non-Commercial Use Only and Subject to the Applicable 


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 49 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Appendix C
Solicited Local and Systemic Adverse Events and Severity 
Table C-1
Solicited Local and Systemic AEs
Local AEs (injection site):
Pain
Erythema
Swelling
Induration
Systemic AEs:
Fever (a)
Headache
Fatigue
Malaise
Arthralgia
Myalgia
(a) Based on recorded body temperature, fever is defined as body temperature ≥38.0°C/100.4°F regardless of 
method taken (Brighton Case Definition).
0050
bject to the Applicable Terms of Use
ms 
ca
jec
Property of Takeda: For Non-Commercial Use Only and Subj
erature ≥
ubje
erature ≥


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 50 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Table C-2
Severity of Solicited Safety Parameters
1.
Adverse Event
2.
Intensity Grade
3.
Severity/Intensity
Pain at injection site
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity with or without treatment
3
Severe: Prevents daily activity with or without treatment
Erythema at injection 
site (a)
0
<25 mm
1
Mild: >25 – ≤50 mm
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Induration at injection 
site (a)
0
<25 mm
1
Mild: >25 – ≤50 mm
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Swelling at injection site
(a)
0
<25 mm
1
Mild: >25 – ≤50 mm 
2
Moderate: >50 – ≤100 mm
3
Severe: >100 mm
Fever (b)
Record body temperature in °C/°F
Headache
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity with or without treatment
3
Severe: Prevents normal activity with or without treatment
Fatigue
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Malaise 
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Arthralgia
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
Myalgia
0
None
1
Mild: No interference with daily activity
2
Moderate: Interference with daily activity
3
Severe: Prevents daily activity
(a)
Subjects are to record greatest surface diameter in mm in the Diary.
(b)
Based on recorded body temperature, fever is defined as body temperature ≥38.0°C/100.4°F regardless of method taken.
0051
f Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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Myalgia
of


Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 51 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Appendix D
FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials
Toxicity grading scales for laboratory abnormalities should consider the local laboratory 
reference values when the parameter limit values are defined. The characterization of laboratory 
parameters among some populations of healthy adults and adolescents may require the exercise 
of clinical judgment. Source: U.S. Department of Health and Human Services, Food and Drug 
Administration, and Center for Biologics Evaluation and Research, September 2007 [6]. 
Table D-1
Table for Laboratory Abnormalities (Serum)
Serum (a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life 
Threatening
(Grade 4) (b)
Sodium – Hyponatremia mEq/L
132 – 134
130 – 131
125 – 129
< 125
Sodium – Hypernatremia mEq/L
144 – 145
146 – 147
148 – 150
> 150
Potassium – Hyperkalemia mEq/L
5.1 – 5.2
5.3 – 5.4
5.5 – 5.6
> 5.6
Potassium – Hypokalemia mEq/L
3.5 – 3.6
3.3 – 3.4
3.1 – 3.2
< 3.1
Glucose – Hypoglycemia mg/dL
65 – 69
55 – 64
45 – 54
< 45
Glucose – Hyperglycemia
Fasting – mg/dL
Random – mg/dL
100 – 110
110 – 125
111 – 125
126 – 200
>125
>200
Insulin requirements or 
hyperosmolar coma
Blood Urea Nitrogen
BUN mg/dL
23-26
27 – 31
> 31
Requires dialysis
Creatinine – mg/dL
1.5 – 1.7
1.8 – 2.0
2.1 – 2.5
> 2.5 or requires dialysis
Calcium – hypocalcemia mg/dL
8.0 – 8.4
7.5 – 7.9
7.0 – 7.4
< 7.0
Calcium – hypercalcemia mg/dL
10.5 – 11.0
11.1 – 11.5
11.6 – 12.0
> 12.0
Magnesium – hypomagnesemia mg/dL
1.3 – 1.5
1.1 – 1.2
0.9 – 1.0
< 0.9
Phosphorous – hypophosphatemia mg/dL
2.3 – 2.5
2.0 – 2.2
1.6 – 1.9
< 1.6
CPK – mg/dL
1.25 – 1.5 x 
ULN (c)
1.6 – 3.0 x 
ULN
3.1 –10 x 
ULN
> 10 x ULN
Albumin – Hypoalbuminemia g/dL
2.8 – 3.1
2.5 – 2.7
< 2.5
--
Total Protein – Hypoproteinemia g/dL
5.5 – 6.0
5.0 – 5.4
< 5.0
--
Alkaline phosphate – increase by factor
1.1 – 2.0 x 
ULN
2.1 – 3.0 x 
ULN
3.1 – 10 x 
ULN
> 10 x ULN
Liver Function Tests –ALT, AST increase by factor
1.1 – 2.5 x 
ULN
2.6 – 5.0 x 
ULN
5.1 – 10 x 
ULN
> 10 x ULN
Bilirubin – when accompanied by any increase in Liver 
Function Test increase by factor
1.1 – 1.25 x 
ULN
1.26 – 1.5 x 
ULN
1.51 – 1.75 x 
ULN
> 1.75 x ULN
Bilirubin – when Liver Function Test is normal; 
increase by factor
1.1 – 1.5 x 
ULN
1.6 – 2.0 x 
ULN
2.0 – 3.0 x 
ULN
> 3.0 x ULN
Cholesterol
201 – 210
211 – 225
> 226
---
Pancreatic enzymes – amylase, lipase
1.1 – 1.5 x 
ULN
1.6 – 2.0 x 
ULN
2.1 – 5.0 x 
ULN
> 5.0 x ULN
0052
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 52 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
(a)
The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. 
Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
(b)
The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory 
abnormalities as Potentially Life Threatening (Grade 4). For example. a low sodium value that falls within a grade 3 
parameter (125-129 mEq/L) should be recorded as a grade 4 hyponatremia event if the subject had a new seizure associated 
with the low sodium value.
(c)
“ULN” is the upper limit of the normal range.
Table D-2
Table for Laboratory Abnormalities (Hematology)
Hematology (a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Hemoglobin (Female) – gm/dL
11.0 – 12.0
9.5 – 10.9
8.0 – 9.4
< 8.0
Hemoglobin (Female) change 
from baseline value – gm/dL
Any decrease – 1.5 1.6 – 2.0
2.1 – 5.0
> 5.0
Hemoglobin (Male) – gm/dL
12.5 – 13.5
10.5 – 12.4
8.5 – 10.4
< 8.5
Hemoglobin (Male) change from 
baseline value – gm/dL
Any decrease – 1.5 1.6 – 2.0
2.1 – 5.0
> 5.0
WBC Increase – cell/mm3
10,800 – 15,000
15,001 – 20,000
20,001 – 25, 000
> 25,000
WBC Decrease – cell/mm3
2,500 – 3,500
1,500 – 2,499
1,000 – 1,499
< 1,000
Lymphocytes Decrease –
cell/mm3
750 – 1,000
500 – 749
250 – 499
< 250
Neutrophils Decrease – cell/mm3
1,500 – 2,000
1,000 – 1,499
500 – 999
< 500
Eosinophils – cell/mm3
650 – 1500
1501 - 5000
> 5000
Hypereosinophilic
Platelets Decreased – cell/mm3
125,000 – 140,000 100,000 – 124,000
25,000 – 99,000
< 25,000
PT – increase by factor 
(prothrombin time)
1.0 – 1.10 x ULN
(b)
1.11 – 1.20 x ULN
1.21 – 1.25 x ULN
> 1.25 ULN
PTT – increase by factor (partial 
thromboplastin time)
1.0 – 1.2 x ULN
1.21 – 1.4 x ULN
1.41 – 1.5 x ULN
> 1.5 x ULN
Fibrinogen increase – mg/dL
400 – 500
501 – 600
> 600
--
Fibrinogen decrease – mg/dL
150 – 200
125 – 149
100 – 124
< 100 or associated with gross 
bleeding or disseminated 
intravascular coagulation 
(DIC)
(a)
The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. 
Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
(b)
“ULN” is the upper limit of the normal range.
0053
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 53 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Table D-3
Table for Laboratory Abnormalities (Urine)
Urine (a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Protein
Trace
1+
2+
Hospitalization or dialysis
Glucose
Trace
1+
2+
Hospitalization for hyperglycemia
Blood (microscopic) – red blood 
cells per high power field
1 - 10
11 - 50
> 50 and/or 
gross blood
Hospitalization or packed red 
blood cells (PRBC) transfusion
(a) The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate.
Table D-4
Table for Clinical Abnormalities (Vital Signs)
Vital Signs (a)
Mild
(Grade 1)
Moderate
(Grade 2)
Severe
(Grade 3)
Potentially Life Threatening
(Grade 4)
Fever (°C) (b)
(°F) (b)
38.0 – 38.4
100.4 – 101.1
38.5 – 38.9
101.2 – 102.0
39.0 – 40
102.1 – 104
> 40
> 104
Tachycardia – beats per minute
101 – 115
116 – 130
> 130
ER visit or hospitalization for 
arrhythmia
Bradycardia – beats per minute (c)
50 – 54
45 – 49
< 45
ER visit or hospitalization for 
arrhythmia
Hypertension (systolic) – mm Hg
141 – 150
151 – 155
> 155
ER visit or hospitalization for 
malignant hypertension
Hypertension (diastolic) – mm Hg
91 – 95
96 – 100
> 100
ER visit or hospitalization for 
malignant hypertension
Hypotension (systolic) – mm Hg
85 – 89
80 – 84
< 80
ER visit or hospitalization for 
hypotensive shock
Respiratory Rate – breaths per 
minute
17 – 20
21 – 25
> 25
Intubation
(a) Subject should be at rest for all vital sign measurements.
(b) Oral temperature; no recent hot or cold beverages or smoking.
(c) When resting heart rate is between 60 – 100 beats per minute. Use clinical judgement when characterizing 
bradycardia among some healthy subject populations, for example, conditioned athletes.
Source: Appendix E of ZIK-101 Protocol Amendment 2 Version 6.0, dated 11 December 2017.
0054
the Applicable Terms of Use
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Purified Inactivated Zika Virus Vaccine (PIZV)
Trial No. ZIK-101
Page 54 of 54
Statistical Analysis Plan Version 2.0
26 April 2019
Appendix E
CDC Websites

World Map of Areas with Risk of Zika: https://wwwnc.cdc.gov/travel/page/world-map-areas-
with-zika

Travel & Dengue Outbreaks: https://www.cdc.gov/dengue/traveloutbreaks/index.html

Yellow Fever Maps: https://www.cdc.gov/yellowfever/maps/index.html

Geographic Distribution of Japanese Encephalitis Virus: 
https://www.cdc.gov/japaneseencephalitis/maps/index.html
Source: Appendix D of ZIK-101 Protocol Amendment 2 Version 6.0, dated 11 December 2017.
0055
Property of Takeda: For Non-Commercial Use Only and Subject to the Applicable Terms of Use
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