Phase I Evaluation of the Safety and Immunogenicity of the Live Attenuated Zika Vaccine rZIKV/D4Δ30-713 in Flavivirus-naïve Adults
Phase: PHASE1
Conditions: Zika Virus
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

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Phase I Evaluation of the Safety and Immunogenicity of the Live Attenuated Zika Vaccine 
rZIKV/D4∆30-713 in Flavivirus-naïve Adults  
 
 
CIR Protocol Number: 
 
318 
 
WCG IRB Protocol Number:  
20181303 
 
UVM IRB Number:  
 
CHRMS 18-0566 
 
 
 
 
FDA IND Number: 
 
 
18187 
 
Ionizing Radiation  
 
 
None 
 
Multi-institution: 
 
 
Yes 
 
Project Assurance: 
FWA# 00000287 (Johns Hopkins Bloomberg School of 
Public Health) 
FWA# 00000727 (University of Vermont Medical Center) 
 
 
 
 
 
FWA# 00000723 (University of Vermont) 
 
 
 
Protocol Version: 
 
 
v7.0  
 
Date:  
 
 
 
February 02, 2022 
 
 
 
 
 
 
Sponsored by: 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
Division of Clinical Research (DCR) 
National Institute of Allergy and Infectious Diseases (NIAID) 
National Institutes of Health (NIH) 
 
 
 
 
 
 
The information contained herein is the property of the National Institute of Allergy 
and Infectious Diseases, National Institutes of Health and may not be reproduced, 
published, or disclosed to others without the written authorization of the National 
Institute of Allergy and Infectious Diseases. 

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Table of Contents 
TEAM ROSTER ......................................................................................................................................... 6 
LIST OF ABBREVIATIONS .................................................................................................................... 9 
1 
PRECIS ............................................................................................................................................. 10 
2 
INTRODUCTION ............................................................................................................................ 12 
2.1 
BACKGROUND – ZIKA ................................................................................................................ 12 
2.2 
BACKGROUND – ZIKV STRAINS ................................................................................................ 14 
2.2.1 
rZIKV/D4∆30-713 Vaccine, Lot ZIKV#118A ........................................................................ 14 
2.2.1.1 
Final Container of rZIKV/D4∆30-713, Lot ZIKV#118A .......................................................................... 14 
2.2.1.2 
Composition of rZIKV/D4∆30-713, Lot ZIKV#118A............................................................................... 14 
2.2.1.3 
Investigational Product Label for rZIKV/D4∆30-713, Lot ZIKV#118A ................................................... 14 
2.3 
RATIONALE ................................................................................................................................ 15 
2.3.1 
Animal Experience ................................................................................................................ 15 
2.3.1.1 
Non-human Primate Study ......................................................................................................................... 15 
2.3.2 
Clinical Experience ............................................................................................................... 17 
2.3.3 
Conclusions ........................................................................................................................... 18 
3 
OBJECTIVES .................................................................................................................................. 19 
3.1 
PRIMARY OBJECTIVES ............................................................................................................... 19 
3.2 
SECONDARY OBJECTIVES .......................................................................................................... 19 
3.3 
EXPLORATORY OBJECTIVES ...................................................................................................... 19 
4 
STUDY DESIGN .............................................................................................................................. 20 
4.1 
OVERALL DESIGN ...................................................................................................................... 20 
4.1.1 
Study Design .......................................................................................................................... 21 
4.1.2 
Sample Size and Placebo Ratio ............................................................................................. 21 
4.1.3 
Duration of Subject Participation ......................................................................................... 21 
4.1.4 
Estimated Duration of the Study ........................................................................................... 21 
4.1.5 
Treatment Assignment ........................................................................................................... 21 
4.1.6 
Blinding ................................................................................................................................. 22 
5 
SELECTION AND ENROLLMENT OF SUBJECTS ................................................................. 22 
5.1 
INCLUSION CRITERIA ................................................................................................................. 22 
5.2 
EXCLUSION CRITERIA ................................................................................................................ 23 
5.3 
SUBJECT WITHDRAWAL AND TERMINATION CRITERIA ............................................................ 25 
5.4 
SPECIAL SITUATIONS: ................................................................................................................ 25 
5.4.1 
Pregnancy: ............................................................................................................................ 25 
5.4.2 
Lost to Follow-up: ................................................................................................................. 26 
5.4.3 
Incarceration: ....................................................................................................................... 26 
5.5 
ACCESS TO MEDICAL RECORDS ................................................................................................ 26 
6 
TEST ARTICLE PREPARATION ................................................................................................ 26 
6.1 
PRE-INOCULATION PREPARATION ............................................................................................. 26 
6.2 
ZIKV VACCINE AND DILUENT STORAGE .................................................................................. 27 
6.3 
ZIKV VACCINE AND DILUENT ACCOUNTABILITY .................................................................... 27 
6.4 
STORAGE DISPOSITION OF USED/UNUSED SUPPLIES ................................................................. 27 
7 
STUDY PROCEDURES .................................................................................................................. 28 
7.1 
RECRUITMENT AND GENERAL SCREENING ............................................................................... 28 
7.2 
SCREENING PROCEDURES .......................................................................................................... 28 
7.3 
INOCULATION PROCEDURE ........................................................................................................ 30 
7.4 
DETAILED STUDY PROCEDURES ................................................................................................ 30 

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7.4.1 
Inoculation and Follow-up .................................................................................................... 30 
7.4.1.1 
Persistence of Zika Vaccine in Bodily Fluids ............................................................................................ 36 
7.5 
SUBJECT TEMPERATURE MEMORY CARD ................................................................................. 37 
7.5.1 
Notification of Staff of Zika-like Symptoms ........................................................................... 37 
7.5.2 
Notification of Staff of COVID-19-like symptoms ................................................................. 37 
7.6 
CLINICAL LABORATORY TESTING ............................................................................................. 38 
7.7 
MEDICAL HISTORY AND CONCOMITANT MEDICATIONS ........................................................... 38 
7.8 
IMMUNOLOGY TESTING ............................................................................................................. 39 
7.8.1 
Antibody Testing .................................................................................................................... 39 
7.8.2 
Other Immunological Assays ................................................................................................ 39 
7.9 
RETENTION OF STUDY SUBJECTS .............................................................................................. 39 
8 
ADVERSE EVENT MONITORING ............................................................................................. 39 
8.1 
DEFINITIONS .............................................................................................................................. 39 
8.1.1 
Adverse Event ........................................................................................................................ 39 
8.1.2 
Medically-attended Adverse Event ........................................................................................ 40 
8.1.3 
Adverse Reaction ................................................................................................................... 40 
8.1.4 
Adverse Event of Special Interest .......................................................................................... 40 
8.1.5 
Pregnancy ............................................................................................................................. 40 
8.1.6 
Serious Adverse Event ........................................................................................................... 41 
8.1.7 
Unexpected Adverse Events .................................................................................................. 42 
8.1.8 
Serious and Unexpected Suspected Adverse Reaction (SUSAR) ........................................... 42 
8.1.9 
Unanticipated Problem ......................................................................................................... 42 
8.1.10 
Unanticipated Problem that is not an Adverse Event (UPnonAE) ................................... 42 
8.1.11 
Protocol Deviation ............................................................................................................ 42 
8.1.12 
Serious Protocol Deviation ............................................................................................... 43 
8.1.13 
Pre-existing Conditions, Worsening of Pre-existing Condition ........................................ 43 
8.2 
ASSESSMENT OF ADVERSE EVENTS ........................................................................................... 43 
8.2.1 
Identification of Adverse Events ............................................................................................ 43 
8.2.2 
Protocol Specific Adverse Event Definitions......................................................................... 44 
8.2.3 
Determination of Severity...................................................................................................... 44 
8.2.4 
Relationship with Receipt of Test Article .............................................................................. 46 
8.2.5 
Adverse Event Action Taken .................................................................................................. 47 
8.2.6 
Adverse Event Outcome ........................................................................................................ 47 
8.2.7 
Adverse Event Seriousness .................................................................................................... 47 
8.3 
ADVERSE EVENT REPORTING .................................................................................................... 47 
8.3.1 
Non-Serious Adverse Events ................................................................................................. 47 
8.3.2 
Serious Adverse Events ......................................................................................................... 47 
8.3.3 
Unanticipated Problems ........................................................................................................ 49 
8.4 
SPONSOR’S REPORTING RESPONSIBILITIES ............................................................................... 49 
8.5 
HALTING RULES ........................................................................................................................ 49 
8.5.1 
Halting Rules for the Study ................................................................................................... 49 
8.5.1.1 
Halting for Unacceptable Reactogenicity................................................................................................... 50 
8.5.1.2 
Halting for Shedding of Zika vaccine in Semen or Vaginal Secretions ..................................................... 50 
8.5.2 
Reporting a Study Halt .......................................................................................................... 50 
If halting criteria are met, a description of the AE(s) or safety issue(s) must be reported by the PI 
within 1 business day to the CSO and the local IRB according to their requirements. ..................... 50 
8.5.3 
Resumption of a Halted Study ............................................................................................... 50 
8.6 
SAFETY OVERSIGHT................................................................................................................... 51 
8.6.1 
Safety Review and Communications Plan (SRCP) ................................................................ 51 
8.6.2 
Sponsor Medical Monitor...................................................................................................... 51 
8.6.3 
NIAID Intramural Data and Safety Monitoring Board ......................................................... 51 

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9 
DATA COLLECTION AND MONITORING .............................................................................. 51 
9.1 
SOURCE DOCUMENTATION AND DATA COLLECTION ................................................................ 51 
9.2 
STUDY DOCUMENTATION .......................................................................................................... 52 
9.3 
RETENTION OF SPECIMENS ........................................................................................................ 52 
9.4 
RETENTION OF RECORDS ........................................................................................................... 52 
9.5 
PROTOCOL COMPLIANCE ........................................................................................................... 53 
9.6 
CLINICAL INVESTIGATOR’S BROCHURE .................................................................................... 53 
9.7 
STUDY MONITORING ................................................................................................................. 53 
10 
STATISTICAL CONSIDERATIONS ............................................................................................ 54 
10.1 
GENERAL DESIGN ...................................................................................................................... 54 
10.2 
STATISTICAL METHODS ............................................................................................................. 54 
10.3 
SAFETY ENDPOINT ..................................................................................................................... 56 
10.4 
IMMUNOGENICITY ..................................................................................................................... 56 
10.5 
PER PROTOCOL ANALYSIS ......................................................................................................... 56 
10.5.1 
Safety Data ........................................................................................................................ 56 
10.5.2 
Virology ............................................................................................................................ 56 
10.5.3 
Immunogenicity Data ........................................................................................................ 56 
10.6 
INTENT-TO-TREAT ANALYSIS ..................................................................................................... 56 
10.6.1 
Safety Data ........................................................................................................................ 56 
10.6.2 
Virology ............................................................................................................................ 56 
10.6.3 
Immunogenicity Data ........................................................................................................ 57 
11 
PROTECTION OF HUMAN SUBJECTS ..................................................................................... 57 
11.1 
INSTITUTIONAL REVIEW BOARDS ............................................................................................. 57 
11.2 
DATA SAFETY MONITORING BOARD ......................................................................................... 57 
11.3 
INFORMED CONSENT ................................................................................................................. 57 
11.4 
RISKS ......................................................................................................................................... 57 
11.4.1 
Venipuncture ..................................................................................................................... 58 
11.4.2 
Risk of Use of Vaginal Cup ............................................................................................... 58 
11.4.3 
Topical Anesthetic Cream ................................................................................................. 58 
11.4.4 
Inoculation ........................................................................................................................ 58 
11.4.5 
Other Risks ........................................................................................................................ 58 
11.5 
BENEFITS ................................................................................................................................... 58 
11.6 
COMPENSATION ......................................................................................................................... 58 
11.6.1 
Compensation for Injury ................................................................................................... 59 
11.7 
CONFIDENTIALITY ..................................................................................................................... 59 
11.8 
BIOHAZARD CONTAINMENT ...................................................................................................... 59 
12 
PUBLICATION POLICY ............................................................................................................... 59 
13 
REFERENCES ................................................................................................................................. 60 
14 
APPENDICES .................................................................................................................................. 64 
14.1 
APPENDIX 1: ADVERSE EVENT GRADING TABLE1 ..................................................................... 64 
14.2 
APPENDIX 2: BRIGHTON CRITERIA FOR GUILLAIN-BARRÉ SYNDROME .................................... 73 
14.3 
APPENDIX 3: GUILLAIN-BARRÉ SYNDROME DISABILITY SCALE ............................................... 74 
14.4 
APPENDIX 4: SCHEDULE OF PROCEDURES POST-INOCULATION WITH ZIKV VACCINE ............ 75 
 
 
LIST OF TABLES 
 
Table 1: Immunization Schedule .................................................................................................. 10 

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Table 2: Replication (Direct Culture in Vero Cells) of rZIKV/D4∆30-713 and Wild-type ZIKV 
in Rhesus Monkeys ............................................................................................................... 15 
Table 3: Replication (RT-PCR) of rZIKV/D4∆30-713 and Wild-type ZIKV in Rhesus Monkeys
............................................................................................................................................... 16 
Table 4: Immunogenicity of rZIKV/D4∆30-713 and Wild-type ZIKV in Rhesus Monkeys ....... 16 
Table 5: Vaccine Candidate rZIKV/D4∆30-713 Protects Monkeys Against ZIKV Challenge ... 16 
Table 6: Comparison of Chimeric Viruses on a DEN4∆30 Background with rDEN4∆30 in 
Humans ................................................................................................................................. 17 
Table 7: Adverse events following one dose of ZIKV/DEN4∆30 or placebo (CIR 318) ............ 17 
Table 8: Serological response to a single dose of ZIKV/DEN4∆30 (n=20) ................................. 18 
Table 9: Solicited Adverse Events/Adverse Reactions ................................................................. 40 
Table 10: Severity Definitions ...................................................................................................... 44 
Table 11: Assessment of Solicited Adverse Events ...................................................................... 45 
 
LIST OF FIGURES 
 
Figure 1: Label for Final Vial of rZIKV/D4∆30-713, Lot (Enlarged Sample) ............................ 14 
 
 
 

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Team Roster 
Principal Investigator: 
 
Anna Durbin, MD 
 
 
 
 
 
Center for Immunization Research 
 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
 
624 N. Broadway, Baltimore, MD 21205 
 
 
 
 
 
410-614-4736  
410-340-6852  
 
Co –investigator 
 
 
Kristen Pierce, MD 
University of Vermont, College of Medicine 
University of Vermont 
 
 
 
 
 
95 Carrigan Drive 
Stafford 220 
 
 
 
 
            Burlington, VT 05405 
 
 
 
 
 
802 847-3819 
 
Clinical Investigators: 
 
Kawsar Talaat, MD 
Noreen Hynes MD, MPH, DTM&H 
Brittany Feijoo, MSN, FNP-BC 
Kam Dhanjani, MPAS, PA-C 
Urvi Patel, PA-C 
Madeleine Blunt, RN 
Beulah Sabundayo, PharmD, MPH 
Center for Immunization Research 
Johns Hopkins School of Public Health 
 
 
 
 
 
624 N. Broadway, Baltimore, MD 21205 
 
 
 
 
 
 
 
410-502-9627 
 
 
 
 
 
443-287-8043 
410-502-7451 
 
 
 
 
 
 
Beth Kirkpatrick, MD  
 
 
 
 
 
Patricia Lutton, CCRC 
University of Vermont, College of Medicine 
University of Vermont 
 
 
 
 
 
95 Carrigan Drive 
Stafford 220 
 
 
 
 
            Burlington, VT 05405 
 
 
 
 
 
802-656-5822 
 
Scientific Investigator: 
 
Stephen S. Whitehead, PhD  
 
Laboratory of Viral Diseases 
 
 
 
 
 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
301-496-7692  
240-281-3767  
 

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OCRPRO Regulatory Affairs: 
Mark S. Miller, PharmD, BCPS, RAC 
 
 
 
 
 
 
CAPT, USPHS 
 
 
 
 
 
 
Office of Clinical Research Policy and Regulatory  
 
 
 
 
 
 
Operations 
 
 
 
 
 
 
National Institute of Allergy and Infectious Diseases 
 
 
 
 
 
 
National Institutes of Health 
 
 
 
 
 
 
5601 Fishers Lane, Room 4B31 
 
 
 
 
 
 
MSC 9820 Bethesda, MD 20892 
 
 
 
 
 
Cell: 240-550-7375 
 
 
 
 
 
Email: mark.miller6@nih.gov  
 
Sponsor Medical Monitor:  
Shirley Jankelevich, MD 
Clinical Monitoring Research Program  
Regulatory Compliance and Human Subjects Protection 
Program 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health 
 
 
 
 
 
Leidos Biomedical Research, Inc 
 
 
 
 
 
5705 Industry Lane 
 
 
 
 
 
Frederick, MD 21702-1201 
 
 
 
 
 
301-846-7322  
 
 
 
 
 
RCHSPSafety@mail.nih.gov 
  
Clinical Study Sites:  
 
Center for Immunization Research 
 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
 
624 N. Broadway  
Baltimore, MD 21205 
 
 
 
 
 
410-955-1622 (phone) 
 
Center for Immunization Research 
Johns Hopkins School of Public Health 
301 Mason Lord Drive Suite 4300 
Baltimore, MD 21202 
 
University of Vermont Vaccine Testing Center 
University of Vermont 
 
 
 
 
 
89 Beaumont Avenue 
Given C205 
 
 
 
 
            Burlington, VT 05405 
 
 
 
 
 
 
University of Vermont Medical Center 
 
 
 
 
 
Clinical Research Center 
 
 
 
 
 
Shepardson 2 
 
 
 
 
 
111 Colchester Avenue 
 
 
 
 
 
Burlington, VT 05401 
 

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Research Laboratories: 
 
Center for Immunization Research 
 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
 
615 N. Wolfe St  
Baltimore, MD 21205 
 
 
 
 
 
410-955-7230 (phone) 
 
University of Vermont Vaccine Testing Center 
University of Vermont 
 
 
 
 
 
95 Carrigan Drive 
Stafford 220 
Burlington, VT 05405 
 
Clinical Laboratories: 
 
Quest Diagnostics 
 
 
 
 
 
1901 Sulphur Spring Road 
 
 
 
 
 
Baltimore, MD 21227 
 
University of Vermont Medical Center 
 
 
 
 
 
111 Colchester Avenue 
 
 
 
 
 
Burlington, VT 05401 
 
 
 
 

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List of Abbreviations 
 
AE 
adverse event 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
AST 
aspartate aminotransferase 
CBC 
complete blood count 
CHIM 
controlled human infection model 
CIR 
Center for Immunization Research 
CLIA 
Clinical Laboratory Improvement Amendments 
CRIMSON 
Clinical Research Information Management System of NIAID 
CRL 
Charles River Laboratories 
CSO 
Clinical Safety Office 
DENV 
dengue virus (serotypes DEN1, DEN2, DEN3, and DEN4) 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
FSH 
Follicle Stimulating Hormone 
GBS 
Guillain-Barré syndrome 
GCP 
good clinical practice 
HBsAg 
hepatitis B surface antigen 
HBV 
hepatitis B virus 
HCG 
human choriogonadotropin 
HCV 
hepatitis C virus 
HID50 
50% human infectious dose  
HIV 
human immunodeficiency virus 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IND 
investigational new drug 
IRB 
institutional review board 
JHSPH 
Johns Hopkins Bloomberg School of Public Health 
LLN 
lower limit of normal 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
NP 
Nasopharyngeal 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
PBMC 
peripheral blood mononuclear cell 
PFU  
plaque-forming units  
PI 
principal investigator 
PRNT50 
plaque reduction neutralization titer 
PT/PTT 
prothrombin time/ partial thromboplastin time 
ROP 
retro-orbital pain 
RT-PCR 
reverse transcriptase polymerase chain reaction 
SAE 
serious adverse event 
SE 
standard error 
SERF 
safety expedited report form 
SOP 
standard operating procedure 
SRCP 
Safety Review and Communications Plan 
SUSAR 
suspected unexpected serious adverse reaction 
ULN 
upper limit of normal 
WBC 
white blood cell 
WHO 
World Health Organization 
WCG IRB 
WIRB- Copernicus Group, Inc.  
wt 
wild-type 
ZIKV 
Zika virus 
 
 
 

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1 
PRECIS  
 
Protocol Title:  
Phase I Evaluation of the Safety and Immunogenicity of the Live 
Attenuated Zika Vaccine rZIKV/D4∆30-713 in Flavivirus-naïve 
Adults 
 
Version: 
 
 
v7.0  
 
Version Date: 
 
February 2, 2022 
 
Phase:  
 
 
Phase 1 
 
Revision History: 
 
v1 25 April 2018 
 
 
 
 
v2 11 July 20218 
 
 
 
 
v3 07August 2018 
 
 
 
 
v4 24 February 2021 
v5 14 June 2021 
v6 06 August 2021 
 
Subjects: 
Healthy male and non-pregnant female subjects 18 to 50 years of 
age, inclusive, with no history of previous flavivirus infection. 
 
Number of Subjects: 
56 flavivirus-naïve subjects will be recruited from metropolitan 
Baltimore/Washington DC and Burlington, Vermont areas. 
 
Study Design: 
Placebo-controlled, double-blind study evaluating safety and 
immunogenicity of a single subcutaneous dose of rZIKV/D4∆30-
713. Subjects will be randomly assigned to receive rZIKV/D4∆30-
713 or placebo in a 5:2 ratio. 
 
Table 1: Immunization Schedule 
 
Cohort 
# Subjects 
Month 
Treatment 
Dose 
1 
20 
0 
rZIKV/D4∆30-713 
103 PFU 
8 
0 
Placebo 
0 PFU (diluent) 
2 
20 
0 
rZIKV/D4∆30-713 
104 PFU 
8 
0 
Placebo 
0 PFU (diluent) 
 
Product Description: 
The vaccine rZIKV/D4∆30-713 is a live attenuated chimeric 
vaccine for the prevention of Zika. rZIKV/D4∆30-713 is 
administered in 0.5 mL containing either 103.0 plaque-forming 
units (PFU) or 104.0 PFU. 
Primary Objectives:  

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1) Determine the safety and reactogenicity of a single dose of the LA (Live Attenuated) 
Zika vaccine rZIKV/D4∆30-713, as assessed by the frequency of adverse reactions (AR) 
as defined as vaccine-related adverse events (AEs), graded by severity. 
• The frequency of immediate, systemic, and local AEs and ARs following 
vaccination will be summarized. AEs will be displayed in tabular format, with 
line listings of individual clinical and laboratory AEs classified as immediate, 
systemic, and local events. AEs will be summarized by severity and relationship 
to vaccine by individuals and each group (rZIKV/D4∆30-713 and Placebo). 
2) Determine the immunogenicity of a single dose of rZIKV/D4∆30-713 vaccine in 
flavivirus-naïve subjects as assessed by neutralizing antibody titers to ZIKV at 28, 56, 
and 90 days following vaccination. 
3) Determine if 104 PFU of vaccine is more infectious and induces a higher proportion of 
seroconversion to Zika virus than the 103 PFU dose while maintaining an acceptable 
safety profile. 
 
Secondary Objectives: 
1) Assess the frequency, quantity, and duration of viremia after a single dose of 
rZIKV/D4∆30-713 given at either 103 PFU or 104 PFU. 
2) Evaluate the number of vaccine recipients infected with either 103 PFU or 104 PFU 
rZIKV/D4∆30-713. Infection is defined as recovery of infectious vaccine virus from the 
blood, serum, or urine of a subject and/or by seroconversion to ZIKV (plaque-reduction 
neutralization titer [PRNT50] ≥ 1:10) within the 90-day period following vaccination. 
3) Determine the durability of antibody response 26 weeks after vaccination.  
4) Determine the quantity and duration of ZIKV presence as determined by: 
• The peak virus titer in the blood and the duration of viremia induced by the LA 
Zika vaccine as determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in urine determined 
by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in vaginal secretions 
determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in semen determined 
by RT-PCR and virus culture 
Exploratory objectives: 
1) To evaluate the phenotype of peripheral blood mononuclear cells (PBMCs) at primary 
infection with the rZIKV/D4∆30-713 vaccine.  
2) To evaluate the cellular immune response to primary infection with the rZIKV/D4∆30-
713 vaccine. 
3) To evaluate the innate immune response to primary infection with the rZIKV/D4∆30-713 
vaccine. 
4) To evaluate B and T cell memory responses following primary infection with the 
rZIKV/D4∆30-713 vaccine. 
5) To evaluate the antibody response in saliva to infection with rZIKV/D4∆30-713 as a 
possible diagnostic tool. This will be done in a subset of subjects (those enrolled at the 
Center for Immunization Research [CIR]). 

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Primary Endpoints 
Safety 
Occurrence, intensity and relationship to vaccination of solicited local and general adverse 
events (AEs) within the 28-day follow-up period after vaccination.  
Occurrence, intensity and relationship to vaccination of unsolicited AEs within the 28-day (Days 
0-28) follow-up period after vaccination. 
 
Occurrence of medically-attended adverse events and serious adverse events (SAEs) throughout 
the entire study period. 
Immunogenicity 
Determination of the peak neutralizing antibody titer to ZIKV through 90 days following 
vaccination with either 103 PFU or 104 PFU of rZIKV/D4∆30. 
Secondary Endpoint 
Viremia: Determine the frequency, magnitude, and duration of recovery of vaccine virus from 
the blood, urine, semen (men), and vaginal secretions (women) of subjects who received the 
rZIKV/D4∆30-713 vaccine. Virus titer will be measured by tissue culture (infective virus) and 
PCR. 
Immunogenicity: Determination of the neutralizing antibody titer to ZIKV 6 months following 
vaccination. 
2 
INTRODUCTION 
2.1 
Background – Zika 
 
Zika virus (ZIKV) is a mosquito-borne flavivirus that was first isolated from the blood of a 
sentinel rhesus macaque in the Zika forest of Uganda in 1947 [1]. Sporadic reports of ZIKV 
infection and/or serologic evidence of ZIKV infection were reported from 1951 – 1981 from 
Africa and Asia [2]. The first major outbreak of ZIKV was reported from the Island of Yap in 
Micronesia in 2007 where it was estimated that 72.6% of the population ≥ 3 years of age was 
infected, demonstrating the rapid transmission of ZIKV in a naïve population [3]. ZIKV 
generally causes only a mild infection characterized by rash, low-grade fever, non-purulent 
conjunctivitis and myalgia with most of those infected not reporting symptoms [3, 4]. Nearly all 
symptomatic patients from the Yap island outbreak presented with rash (90%), arthritis/arthralgia 
(65%), and fever (65%). Unlike dengue virus, ZIKV does not cause hemorrhagic manifestations, 
vascular leak syndrome, or liver function abnormalities. Only approximately 19% of subjects 
found to be seropositive to ZIKV in a serosurvey recounted being symptomatic with a Zika-like 
illness. In October 2013, the largest outbreak of ZIKV recorded up to that time began in French 
Polynesia [5, 6]. It was estimated that 28,000 ZIKV infections occurred (~11% of the 
population) with most infections presenting with low-grade fever, rash, arthralgia and 
conjunctivitis. Since its introduction into Brazil, ZIKV has spread to more than 69 countries in 
the Americas, Caribbean, Asia and Africa [7]. 
 

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Brazil first identified an outbreak of ZIKV in its northeast region in early 2015. By September 
increasing numbers of cases of microcephaly were being reported, particularly from regions 
involved in the ZIKV outbreak [8]. Because of the devastating birth defects caused by congenital 
ZIKV infection, the World Health Organization (WHO) declared ZIKV a Public Health 
Emergency of International Concern (PHEIC) on February 1, 2016. Evidence of a link between 
ZIKV infection in pregnancy, particularly during the first trimester, and microcephaly in the 
fetus was collected [4, 9, 10] and a causal association between ZIKV infection and microcephaly 
and/or other birth defects was formally established [11]. Most of the women in whom 
microcephaly of the fetus was diagnosed reported symptoms of ZIKV infection at some point 
during their pregnancy, usually in the first or second trimester; rash was the most common 
symptom reported. However, recent evidence suggests that congenital Zika syndrome (CZS) can 
occur regardless of symptoms in the mother [12, 13]. Although CZS is most likely to occur with 
maternal infection in the first or second trimester, there is increasing evidence that congenital 
anomalies can occur regardless of when during pregnancy the mother was infected with ZIKV. 
In addition, babies who appeared normal at birth are being diagnosed with developmental delay, 
hearing problems, and eye abnormalities after birth. Although the PHEIC ended November 11, 
2016, concern regarding continued ZIKV transmission and congenital infection remains high and 
a safe and effective ZIKV vaccine is a public health priority. 
 
 
Transmission via mosquito vector remains the principal driving force of ZIKV infection and 
disease. Aedes mosquitoes, particularly Aedes aegypti, are thought to be the primary vector for 
ZIKV [14, 15]. Complicating ZIKV control programs, there have been several reports of sexual 
transmission of ZIKV [16-21]. All but two of these cases involve male-to-female transmission; 
there has been one report of female-to-male transmission and one report of male-to-male 
transmission [22, 23]. The cases of sexual transmission have occurred during or just after 
symptomatic illness in all but one case. In that case, an asymptomatic subject transmitted ZIKV 
to his partner [24]. ZIKV RNA has been detected in semen by reverse transcriptase – polymerase 
chain reaction for 2 - 6 months after symptoms have developed [25-27]. However, to date, the 
longest interval between onset of symptoms and recovery of infectious ZIKV from semen is 69 
days [28]. More recent data has found that infectious ZIKV is shed only rarely from ZIKV-
infected men; only 4% of semen samples that were positive for ZIKV by PCR contained 
infectious virus and infectious virus was found only in samples collected within 30 days of 
symptom onset [29]. It is also not known for how long ZIKV can be sexually transmitted, but all 
cases of sexual transmission reported in the literature thus far have occurred within 3 weeks of 
return from an endemic area with all but one of those cases occurring within 2 weeks [21-24, 
30]. Although cases of sexual transmission have occurred, it appears to play a minor role in 
transmission as there have been only 52 documented cases of sexually acquired ZIKV resulting 
from a total of 5,388 travel-associated ZIKV cases in the United States (< 1% of ZIKV cases) as 
of March 21, 2018, https://www.cdc.gov/zika/geo/united-states.html, accessed March 26, 2018) 
and it is not thought to contribute significantly to transmission [31]. 
 
The goal of this project is to evaluate the safety and immunogenicity of the first live attenuated 
ZIKV to be evaluated in humans. The LA ZIKV candidate vaccine rZIKV/D4∆30-713 is highly 
attenuated compared to its parent virus (ZIKV Paraiba/2015). The infectious vaccine virus could 
not be recovered from the blood, saliva, or urine of immunized rhesus macaques following 
administration of a single dose of 104.0 PFU. Should this vaccine have an acceptable safety 

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profile and prove to be immunogenic in flavivirus-naïve healthy adults, the vaccine will need to 
be further evaluated for protective efficacy. The ZIKV epidemic has waned considerably with 
very few cases of ZIKV being reported from Asia and Latin America. For this reason, it is 
unlikely that efficacy of the vaccine will be able to be assessed in a traditional Phase 3 study. It is 
likely that, at this time, efficacy of a promising vaccine candidate will only be able to be assessed 
using a ZIKV controlled human infection model (CHIM) which is currently in development. 
Should the rZIKV/D4∆30-713 vaccine prove to be immunogenic and have an acceptable safety 
profile, we are hoping to include assessment of its efficacy using a ZIKV CHIM as part of our 
clinical development plan.  
 
2.2 
Background – ZIKV strains 
2.2.1 rZIKV/D4∆30-713 Vaccine, Lot ZIKV#118A 
The vaccine candidate rZIKV/D4∆30-713 is a live attenuated virus derived from the Zika virus 
wild-type (wt) strain ZIKV Paraiba/2015 and assembled on a DEN4∆30 genetic background 
using recombinant DNA technology.  
 
A full-length chimeric cDNA clone ZV-Den4∆30 was constructed to generate a viral transcript 
expressing the prM and E structural genes of a contemporary ZIKV strain within the genetic 
background of the DEN4∆30 vaccine strain. The cDNA plasmid, ZV-Den4∆30-713 V1 was 
transfected into qualified Vero cells. Recovered virus was terminally diluted and then amplified 
by serial passaging in Vero cells. The titer was determined by plaque titration in Vero cells. 
From initial transfection through final amplification, only serum-free medium was used for Vero 
cell culture and virus propagation. The rZIKV/D4∆30-713 seed virus was generated in the 
Laboratory of Viral Diseases (LVD), National Institute of Allergy and Infectious Diseases 
(NIAID), National Institutes of Health (NIH), and the vaccine was manufactured at the Charles 
River Laboratories (CRL) Biopharmaceutical Services facility in Malvern, PA.  
2.2.1.1 Final Container of rZIKV/D4∆30-713, Lot ZIKV#118A  
The Final Drug Product was dispensed as 0.6-mL aliquots of approximately 107.3 PFU/mL live 
recombinant Zika virus rZIKV/D4∆30-713 Vero-grown virus vaccine into 2.0-mL sterile 
cryovials and stored at -75°C ± 15°C. 
2.2.1.2 Composition of rZIKV/D4∆30-713, Lot ZIKV#118A 
The Final Drug Product composition is a concentration of PFU/mL live attenuated 
rZIKV/D4∆30-713 (lot ZIKV#118A) in Leibovitz L-15 medium containing 1X SPG. The 
potency of ZIKV/D4∆30-713 (lot ZIKV#118A) is 107.3 PFU/mL. 
2.2.1.3 Investigational Product Label for rZIKV/D4∆30-713, Lot ZIKV#118A 
 
Figure 1: Label for Final Vial of rZIKV/D4∆30-713, Lot (Enlarged Sample) 
 

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LIVE RECOMBINANT ZIKA VIRUS VACCINE 
rZIKV/D4∆30-713 
VERO GROWN VIRUS 
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Date: 13JUN2017 
Vial#: 0000 
Lot:   ZIKV#118A 
Store at -75 ± 15oC 
Charles River Laboratories, Malvern, PA 
2.3 
Rationale 
2.3.1 Animal Experience 
2.3.1.1 Non-human Primate Study 
 
The replication (viremia), immunogenicity, and protective efficacy of the rZIKV/D4∆30-713 (lot 
ZIKV#118A) vaccine candidate was studied in rhesus monkeys. The primary purpose of the 
study was to establish the attenuation phenotype and protective efficacy of the vaccine candidate. 
Monkeys inoculated with the wt parent virus ZIKV-Paraiba/2015 were viremic (as determined 
by direct culture in Vero cells) for 4.25 days with a mean peak titer of 2.1 log10 PFU/mL. This 
was similar to results obtained for an additional wt virus, ZIKV-Nicaragua/2016, in which 
monkeys were viremic for 4.25 days with a mean peak titer of 2.3 log10 PFU/mL. Replication of 
the vaccine candidate rZIKV/D4∆30-713 was below the level of detection (< 0.7 log10PFU/mL) 
in all animals inoculated, thus demonstrating the attenuation phenotype compared to wt ZIKV 
(Table 2). Detection of virus genome by quantitative reverse transcriptase – polymerase chain 
reaction (RT-PCR) in serum also confirmed the attenuation phenotype of rZIKV/D4∆30-713 
(Table 3). rZIKV/D4∆30-713 was not recovered from the saliva or urine of inoculated monkeys 
by RT-PCR further confirming its attenuated phenotype. Regardless of the method for 
measurement of peak virus load following inoculation, the level of replication of rZIKV/D4∆30-
713 was at least 20 - 25 times lower than wt parent ZIKV-Paraiba/2015. Attenuation is likely due 
to chimerization and the presence of the ∆30 mutation. 
 
Table 2: Replication (Direct Culture in Vero Cells) of rZIKV/D4∆30-713 and 
Wild-type ZIKV in Rhesus Monkeys 
Virus 
N 
Dose 
(log10 
PFU) 
% with 
viremia 
Mean no. of 
viremic days 
per monkey 
Mean peak 
virus titer* 
(log10 PFU/mL 
± standard error 
[SE]) 
ZIKV-Paraiba/2015 
4 
4 
100 
4.25 
2.1 ± 0.2 
ZIKV-Nicaragua/2016 
4 
4 
100 
4.25 
2.3 ± 0.1 
rZIKV/D4∆30-713 
4 
4 
0 
0 
  < 0.7 ± 0.0 
* Lower limit of detection = 0.7 log10 PFU/mL 
 
 

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Table 3: Replication (RT-PCR) of rZIKV/D4∆30-713 and Wild-type ZIKV in Rhesus 
Monkeys 
Virus 
N 
Dose 
(log10 
PFU) 
% with 
genome 
detected 
Mean no. of 
genome days 
per monkey 
Mean peak 
titer 
(log10 GE/mL 
 ± SE) 
ZIKV-Paraiba/2015 
4 
4 
100 
7.75 
5.5 ± 0.2 
ZIKV-Nicaragua/2016 
4 
4 
100 
5.25 
5.7 ± 0.1 
rZIKV/D4∆30-713 
4 
4 
100 
2.50 
4.2 ± 0.2 
* Lower limit of detection = 3.2 log10 GE/mL 
 
Due to the attenuated nature of rZIKV/D4∆30-713, the virus was less immunogenic than its wt 
parent virus (Table 4). However, all monkeys inoculated with rZIKV/D4∆30-713 were 
protected against challenge with wt ZIKV and none of the monkeys demonstrated any 
detectable challenge viremia regardless of the detection method (tissue culture or PCR, Table 
5). 
 
Table 4: Immunogenicity of rZIKV/D4∆30-713 and Wild-type ZIKV in Rhesus 
Monkeys 
Virus 
N 
PRNT50 Assay 
(mean reciprocal titer)1 
EC50 RVP Flow Assay 
(mean reciprocal titer)2 
Day 0 
Day 28 Day 42 
Day 0 
Day 28 Day 42 
ZIKV-Paraiba/2015 
4 
< 10 
391 
217 
< 60 
14491 
9809 
ZIKV-Nicaragua/2016 
4 
< 10 
289 
147 
< 60 
15656 
9149 
rZIKV/D4∆30-713 
4 
< 10 
12 
15 
< 60 
871 
451 
 1 
50% Plaque reduction neutralization mean titer (reciprocal) assayed against ZIKV-Paraiba/2015 
 2 
50% Effective serum concentration (mean reciprocal) to block infection of reporter virus particles (RVPs) 
constructed from ZIKV-Paraiba/2015 structural proteins. Assayed by flow cytometry. 
 
Table 5: Vaccine Candidate rZIKV/D4∆30-713 Protects Monkeys Against ZIKV 
Challenge 
Primary inoculation 
N 
Challenge 
dose1 
(log10PFU) 
% with 
challenge 
 viremia 
Mean no. 
of viremic 
days per 
monkey 
Mean peak 
virus titer 
(log10 PFU/mL 
± SE) 
ZIKV-Paraiba/2015 
4 
4 
0 
0 
< 0.7 ± 0.0 
ZIKV-Nicaragua/2016 
4 
4 
0 
0 
< 0.7 ± 0.0 
rZIKV/D4∆30-713 
4 
4 
0 
0 
< 0.7 ± 0.0 
Placebo 
4 
4 
100 
4.75 
2.6 ± 0.2 
1. Challenge virus (ZIKV-Paraiba/2015) was administered subcutaneously to all monkeys on day 56 following 
primary inoculation 
 

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2.3.2 Clinical Experience 
Prior to initiation of this protocol, rZIKV/D4∆30-713 had not previously been studied in 
humans. However, we have studied other recombinant flavivirus vaccines, including chimeric 
flavivirus vaccines on the rDEN4∆30 genetic background [32-35]. rDEN4∆30 has been 
extensively evaluated in humans as both a monovalent dengue virus (DENV)-4 candidate 
vaccine and in tetravalent vaccine formulations [36-41]. The vaccine is well tolerated, induces a 
low-level viremia, and is immunogenic. Chimerization is highly attenuating for flaviviruses and 
has been used as a strategy for flavivirus vaccine development. The recently licensed live 
attenuated tetravalent dengue vaccine consists of four chimeric viruses in which the prM and E 
coding sequence of yellow fever 17D has been replaced with those of DENV-1, DENV-2, 
DENV-3 or DENV-4. We have used chimerization to attenuate the DENV-2 candidate vaccine 
rDEN2/4∆30, the DENV-3 candidate vaccine rDEN3/4∆30, the candidate West Nile vaccine 
rWN/DEN4∆30 and the Langat candidate vaccine rLGT/DEN4 [32-37, 42]. When the prM and 
E coding sequences of rDEN4∆30 were replaced with those of either DENV-2 New Guinea C 
(NGC), DENV-3 Sleman/78, or WNV, the resultant candidate vaccines were less infectious as 
evidenced by a lower incidence of viremia and/or an increase in the 50% human infectious dose 
(HID50) of ~10-fold to more than 10,000-fold (Table 6). 
 
The recombinant ZIKV vaccine rZIKV/D4∆30-713 has been administered at a dose of 103 PFU 
to 20 healthy adult volunteers.  The vaccine was well tolerated at this dose (Table 7). The 
vaccine appears to be over-attenuated at a dose of 103 PFU as fewer than 50% of the vaccine 
recipients were infected with the vaccine, based on virologic and serologic assessments. The 
vaccine virus was not recovered from any vaccinated subject (serum, urine, semen, or vaginal 
secretions), as assessed by culture and PCR. Only 9/20 vaccinated seroconverted to ZIKV 
following vaccination (Table 8), indicating that the vaccine is not sufficiently immunogenic at a 
dose of 103 PFU. 
 
Table 6: Comparison of Chimeric Viruses on a DEN4∆30 Background with rDEN4∆30 in 
Humans 
Vaccine 
Candidate 
Dose (log10 
PFU) 
N 
% with 
viremia 
HID50 
Mean peak 
titer (log10 
PFU/mL) 
Mean day 
of onset ± 
SE 
Mean # 
days of 
viremia ± 
SE 
rDEN4∆30 
[34] 
3 
70 
28 
  < 10 PFU 
0.6 ± 0.1 
10.2 ± 0.6 
1.8 ± 0.3 
rDEN2/4∆30
[34] 
3 
40 
60 
  10 PFU 
0.5 ± 0.03 
9.2 ± 0.6 
3.3 ± 0.6 
rDEN3/4∆30
[34] 
3 
20 
15 
> 105 PFU 
1.0 ± 0.3 
12 ± 0 
4.3 ± 0.7 
WN/DEN4∆
30 [35] 
3 
19 
16 
> 104 PFU 
0.5 ± 0 
15.3 ± 1.9 
2.3 ± 0.7 
 
Table 7: Adverse events following one dose of ZIKV/DEN4∆30 or placebo (CIR 318)  
 
ZIKV/DEN4∆30 
(n=20) 
Placebo 
(n=8) 
P value (1-sided)1 
Local 
 
 
 
 
Erythema 
5% 
0.0% 
0.7143 

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ZIKV/DEN4∆30 
(n=20) 
Placebo 
(n=8) 
P value (1-sided)1 
 
Tenderness 
0% 
0.0% 
n/a 
 
Pain 
0% 
0.0% 
n/a 
 
Pruritus 
0% 
0.0% 
n/a 
 
Induration 
0% 
0.0% 
n/a 
Systemic 
 
 
 
 
Fever 
0% 
0% 
n/a 
 
Headache 
50.0% 
62.5% 
0.8454 
 
Rash 
0.0% 
0.0% 
n/a 
 
Neutropenia2 
0.0% 
0.0% 
n/a 
 
Elevated ALT 
0.0% 
0.0% 
n/a 
 
Myalgia 
20.0% 
25.0% 
0.7922 
 
Arthralgia 
10.0% 
12.5% 
0.8120 
 
Retro-orbital Pain 
5.0% 
12.5% 
0.9259 
 
Fatigue 
25.0% 
25.0% 
0.6940 
 
Muscle weakness 
5.0% 
0.0% 
0.7143 
 
Prolonged PT 
0.0% 
0.0% 
n/a 
 
Prolonged PTT 
0.0% 
0.0% 
n/a 
 
Thrombocytopenia 
0.0% 
0.0% 
n/a 
1. P value is the probability is greater for rZIKV/DEN4∆30 than placebo 
2. Neutropenia was defined as an ANC ≤ 1,000/mm3. 
Table 8: Serological response to a single dose of ZIKV/DEN4∆30 (n=20) 
Geometric mean neutralizing antibody titer (% seroconverted) 
 
Day 28 
Day 56 
Day 90 
Peak titer through 
day 90 
# seroconverted 
(% total) 
85.6 (41.2, 20%) 
66.8 (40.9, 40%) 
71.0 (36.6, 45%) 
56.1 (40.9, 45%) 
(45%) 
1. Seroconversion was defined as a titer of ≥ 10, reciprocal 
2. Geometric mean titer calculated at each time point only for those subjects with a titer of ≥ 10, reciprocal 
3. One subject had a titer of >5120, reciprocal (the ULN). A value of 10,240 was assigned for calculation purposes 
2.3.3 Conclusions 
Based on our experience with other chimeric viruses in the rDEN4∆30 background and the 
replication data of ZIKV/D4∆30-713 in NHP (non-human primates), we expected that this 
candidate vaccine will be attenuated in humans and will induce immune response. For additional 
safety, we initially administered the vaccine at a dose of 103 PFU, a 10-fold lower dose than was 
administered to NHP. The vaccine was not sufficiently infectious or immunogenic at this dose 
and for this reason, the protocol is being amended to evaluate a dose of 104 PFU. Should a dose 
of 104 be insufficiently immunogenic, the protocol may be further amended to evaluate a dose of 
105 PFU. Recent data demonstrating a very low incidence of recovery of infectious wild type 
ZIKV from infected symptomatic men with ZIKV detected by PCR in semen, suggest that the 
risk of sexual transmission may be much lower than was first thought and is not likely to occur 
with this highly attenuated vaccine virus [29]. Indeed, vaccine virus was not recovered from the 
semen of any male volunteer or the cervicovaginal secretions of any female volunteer when 
administered at a dose of 103 PFU. 

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3 
OBJECTIVES 
3.1 
Primary Objectives 
1) Determine the safety and reactogenicity of a single dose of the LA Zika vaccine 
rZIKV/D4∆30-713 given at either 103 PFU or 104 PFU, as assessed by the frequency of 
adverse reactions (AR) as defined as vaccine-related adverse events (AE)s, graded by 
severity.  
• The frequency of immediate, systemic, and local AEs and ARs following 
vaccination will be summarized. AEs will be displayed in tabular format, with 
line listings of individual clinical and laboratory AEs classified as immediate, 
systemic, and local events and AEs will be summarized by severity and 
relationship to vaccine by individuals and each group (rZIKV/D4∆30-713 and 
placebo). 
2) Determine the immunogenicity of a single dose of rZIKV/D4∆30-713 vaccine given at 
either 103 PFU or 104 PFU as assessed by neutralizing antibody titers to ZIKV at 28, 56, 
and 90 days following vaccination. 
3.2 
Secondary Objectives 
1) Assess the frequency, quantity, and duration of viremia after a single dose of 
rZIKV/D4∆30-713 given at either 103 PFU or 104 PFU. 
2) Evaluate the number of vaccine recipients infected with rZIKV/D4∆30-713 given at 
either 103 PFU or 104 PFU. Infection is defined as recovery of infectious vaccine virus 
from the blood, serum, or urine of a subject and/or by seroconversion to ZIKV (plaque-
reduction neutralization titer [PRNT50] ≥ 1:10) within the 90-day period following 
vaccination. 
3) Evaluate the immunogenicity of rZIKV/D4∆30-713 in flavivirus-naïve subjects as 
assessed by the PRNT50 to ZIKV, for each subject at Study Day 28, 56, and 90 post-
administration of LA Zika vaccine.  
4) Determine the durability of antibody response 26 weeks after vaccination.  
5) Determine the quantity and duration of ZIKV presence as determined by: 
• The peak virus titer in the blood and the duration of viremia induced by the LA 
Zika vaccine as determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in urine determined 
by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in vaginal secretions 
determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in semen determined 
by RT-PCR and virus culture 
3.3 
Exploratory Objectives 
The exploratory objectives of this study are: 
 
1) To evaluate the phenotype of peripheral blood mononuclear cells (PBMCs) at primary 
infection with the rZIKV/D4∆30-713 vaccine.  
2) To evaluate the cellular immune response to primary infection with the rZIKV/D4∆30-
713 vaccine. 

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3) To evaluate the innate immune response to primary infection with the rZIKV/D4∆30-713 
vaccine. 
4) To evaluate B and T cell memory responses following primary infection with the 
rZIKV/D4∆30-713 vaccine. 
5) To evaluate the antibody response in saliva to infection with rZIKV/D4∆30-713 as a 
possible diagnostic tool. This will be done in a subset of subjects (those enrolled at the 
Center for Immunization Research [CIR]). 
4 
STUDY DESIGN 
4.1 
Overall Design 
This study is a placebo-controlled, double-blind study in normal healthy, flavivirus-naïve adult 
male and female subjects 18 - 50 years of age, inclusive, recruited from the metropolitan 
Baltimore/Washington, DC and Burlington, VT areas. The purpose of this study is to evaluate 
the safety, reactogenicity, and immunogenicity of a single dose of the live attenuated 
rZIKV/D4∆30-713 vaccine. Placebo recipients are included in the study as a control to better 
assess vaccine-associated versus non-vaccine-associated AEs. Cohort 1 (103 PFU) will be 
enrolled and evaluated first. If the the vaccine is not found to induce seroconversion to ZIKV in 
> 80% of subjects inoculated with 103 PFU of the vaccine, a second cohort of volunteers will be 
enrolled and will be inoculated with 104 PFU of vaccine (or placebo). 
 
After providing written informed consent, subjects will undergo eligibility screening, including 
medical history, physical examination, hematology testing, liver and renal function testing, 
human immunodeficiency virus (HIV) screening, hepatitis B and C screening, urinalysis, 
COVID-19 testing (if not fully vaccinated against COVID-19), and serology screening for 
previous flavivirus infection. Pregnancy testing will be performed on female subjects of 
reproductive potential. Follicle-stimulating hormone (FSH) levels will be collected for women 
who report amenorrhea more than 12 months and less than 24 months. All screening tests must 
be performed within 60 days of inoculation. HIV screening must be performed within 2 weeks of 
inoculation. 
 
All clinically significant abnormalities will be reviewed with subjects and referral for follow-up 
care will be provided. Subjects will be determined to be eligible based on the inclusion and 
exclusion criteria found in Section 5 of this protocol. For subjects who are eligible, the Day 0 
visit will be scheduled for receipt of inoculation. Pregnancy testing will be repeated on 
applicable female subjects on the day of inoculation and during follow-up. Subjects will be 
considered enrolled in the study only after they receive test article (rZIKV/D4∆30-713 or 
placebo). After inoculation, subjects will be evaluated in the clinic for at least 30 minutes. They 
will return to the clinic on study days 4, 6, 8, 10, 12, 14, and 16 for evaluation by a clinician and 
to have blood drawn for clinical laboratory studies, virologic assays, and immunologic assays. In 
addition, urine, vaginal secretions, and semen will be collected at specified time points for 
additional virologic assays. The subjects will also have a clinical evaluation performed at each 
specified visit (see Section 7.4 for a detailed description of study procedures). All subjects will 
be provided with a memory enhancement card on which they will be asked to record their 
temperature 2 times a day for the first 16 days post-inoculation. They will also return to the clinic 
on study days 21, 28, 56, 90, 150, and 180. 

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4.1.1 Study Design 
This study will include two cohorts of 28 flavivirus-naïve male and female subjects, 18 - 50 
years of age. The live attenuated ZIKV vaccine rZIKV/D4∆30-713 will be evaluated. Within 
each cohort, twenty subjects will receive a single dose of either 103 PFU or 104 PFU of 
rZIKV/D4∆30-713 and 8 subjects will receive a single dose of placebo. Subjects will be 
followed for approximately 26 weeks after receipt of LA Zika vaccine.  
 
4.1.2 Sample Size and Placebo Ratio 
Up to fifty-six subjects (40 vaccine recipients and 16 placebo recipients) will be enrolled in the 
study. Twenty subjects per cohort were chosen to receive vaccine for the following reasons:  
• 
This study is designed to build upon the previous Phase 1 studies of other live attenuated 
flavivirus vaccines, which used similar cohorts and the same vaccine to placebo ratio. 
• 
Twenty subjects receiving vaccine will provide adequate power to determine 
seroconversion frequency. We hope to induce a seroconversion frequency of ≥ 80% in 
those subjects who receive the vaccine. Cohort 1 will be enrolled first. If 103 PFU of 
vaccine does not induce seroconversion to ZIKV in ≥ 80% of vaccinated volunteers, 
Cohort 2 will be enrolled. 
• 
We have included placebo-recipients so that we can better assess if common AEs are 
vaccine-related.  
  
4.1.3 Duration of Subject Participation 
Subjects will be followed for approximately 26 weeks (approximately 180 days) from the time of 
the initial inoculation. Subjects will be screened for eligibility up to 60 days prior to inoculation 
on Study Day 0. 
 
4.1.4 Estimated Duration of the Study 
Screening procedures should be completed within 60 days prior to receipt of the test article 
(except HIV testing, which will be completed within 14 days of inoculation). Enrollment will 
occur over a period of 4 - 8 weeks. The study will last for a total of approximately 26 weeks 
from the time the last subject is enrolled (inoculated). 
 
4.1.5 Treatment Assignment 
For each cohort, subjects will be randomly assigned to receive rZIKV/D4∆30-713 or placebo in 
a 5:2 ratio. Treatment assignment will be done using a random number generator to prepare the 
sequence in which subjects are assigned to receive vaccine or placebo. Subjects will be centrally 
randomized by the vaccine preparation study staff at Johns Hopkins Bloomberg School of Public 
Health (JHSPH) in blocks of 7. There will be 4 blocks of 7 subjects. Each block of 7 will include 
5 vaccine recipients and 2 placebo recipients. Subjects will not be enrolled sequentially across 
sites. Once all 7 subjects in a block have reached Study Day 90, the block may be unblinded to 
all staff. Enrollment will occur at both sites with the intent of enrolling 2 blocks at CIR and 2 
blocks at UVM. Should one site have difficulty enrolling subjects, enrollment may be reassigned 
to the other site, however, the initial treatment assignment will remain unchanged.  

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On Study Day 0, once subjects have met all eligibility criteria, study staff will assign the subjects 
sequential ID study numbers from a prepared list. Subjects will be considered enrolled when they 
receive the first dose of test article. Clinical staff will remain blinded to treatment assignment 
until all subjects within a block have reached Study Day 90.  
 
A master log of treatment assignments will be maintained in a record separate from other study 
records. This log will be maintained by the Investigational Drug Service (IDS) pharmacy. It will 
be kept in a locked room with limited access. A sealed envelope containing a copy of the 
treatment assignment will also be kept by the Data and Safety Monitoring Board (DSMB) 
Executive Secretary. The subjects will be informed of their treatment assignment after their 
block has completed Study Day 90. 
4.1.6 Blinding 
This study will be conducted as a double-blind study to avoid biased assessment of AEs. 
rZIKV/D4∆30-713 or placebo will be prepared and drawn into syringes by pharmacy personnel 
who are not involved with the clinical assessment of subjects or performing research assays. 
Syringes are labeled according to standard operating procedures/recommendations. Because 
vaccine diluent is used as the placebo, there will be no difference in the appearance of the 
syringes.  
 
The subject, investigator, and clinical staff will not know which treatment group the subject has 
been assigned to. In addition, other personnel assigned to monitor the study will not know the 
treatment assignment of the subject. Clinical staff will remain blinded to treatment assignment 
until all subjects within a block of 7 subjects have reached study day 90. Once the time point for 
unblinding has occurred (study day 90 for subjects within a block), the principal investigator (PI) 
can request treatment assignments in writing from the unblinded study staff.  
 
If it becomes necessary to unblind a specific subject’s assignment for emergency medical 
management prior to all subjects within a block completing study day 90 post-vaccination, the PI 
will contact the unblinded study staff and obtain the treatment assignment of the subject in 
question. Only that specific subject’s assignment will be unblinded. The sponsor, Office of 
Clinical Research Policy and Regulatory Operations (OCRPRO) and the DSMB executive 
secretary will be notified of the event within 2 business days. This will also be documented in the 
subject’s study chart. 
5 
SELECTION AND ENROLLMENT OF SUBJECTS 
Subjects must meet all eligibility criteria to be enrolled in the study. 
5.1 
Inclusion Criteria 
All of the following criteria must be fulfilled for a subject to qualify for inclusion in this study: 
 
1. Adult male or female between 18 and 50 years of age, inclusive.  
2. Good general health as determined by physical examination, laboratory screening, and 
review of medical history.  

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3. Available for the duration of the study, which is approximately 26 weeks. 
4. Willingness to participate in the study as evidenced by signing the informed consent 
document. 
5. Females only: Female subjects of childbearing potential, with the exception noted below, 
should be willing to use effective contraception and have no plans to undergo IVF (in 
vitro fertilization) during participation in the trial. Reliable methods of contraception 
include hormonal birth control, condoms with spermicide, diaphragm with spermicide, 
surgical sterilization, and intrauterine device. Women must have been on an effective 
method of birth control for at least 30 days prior to enrollment. All female subjects will 
be considered as having childbearing potential, except for women who exclusively have 
sex with women, those who have had a hysterectomy, tubal ligation, or tubal coil (at least 
3 months prior to vaccination), or are considered to be post-menopausal, as documented 
by at least 1 year since last menstrual period with an FSH level in the menopausal range 
or at least 24 consecutive months of amenorrhea. Transgender men who have internal 
female organs and have sex with men will be considered of childbearing potential and 
should be willing to use effective contraception during the trial. Exception: Females who 
have sex with females (exclusively) and have no intention of conceiving a child during 
the study and women whose partners have had a vasectomy will not be required to use 
contraception, however they will be required to use female condoms and/or dental dams 
for at least 1 month following vaccination. For women whose sexual partner has had a 
vasectomy, the vasectomy must have been performed 30 days or more prior to 
enrollment.  
6. Males only: Males should be willing to use barrier contraception for the first 3 months 
following vaccination* and agree to not donate sperm for the duration of the study. 
*Based on CDC guidance for men returning from ZIKV-endemic areas 
5.2 
Exclusion Criteria 
A subject will be excluded from the study if any of the following criteria are met:  
1. Females only: Currently pregnant, as determined by positive β-human 
choriogonadotropin (HCG) test, or breast-feeding.  
2. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, rheumatologic, 
autoimmune, or renal disease based on history, physical examination, and/or laboratory 
studies. 
3. History of any neuroinflammatory disorder i.e. Bell’s Palsy, transverse myelitis 
4. Behavioral, cognitive, or psychiatric disease that, in the opinion of the investigator, 
affects the subject’s ability to understand and cooperate with the requirements of the 
study protocol. 
5. Screening laboratory values of Grade 1 or above for absolute neutrophil count (ANC), 
alanine aminotransferase (ALT), and serum creatinine, as defined in this protocol.  

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6. Any other condition that, in the opinion of the investigator, would jeopardize the safety or 
rights of a subject participating in the trial, or would render the subject unable to comply 
with the protocol. 
7. Any significant alcohol or drug abuse in the past 12 months that has caused medical, 
occupational, or family problems, as indicated by subject history. 
8. History of a severe allergic reaction or anaphylaxis. 
9. Severe asthma (emergency room visit or hospitalization within the last 6 months). 
10. HIV infection, as indicated by screening and confirmatory assays. 
11. Hepatitis C virus (HCV) infection, as indicated by screening and confirmatory assays.  
12. Hepatitis B virus (HBV) infection, as indicated by hepatitis B surface antigen (HBsAg) 
screening. 
13. Any known immunodeficiency syndrome. 
14. History of Guillain-Barrè syndrome. 
15. Current use of anticoagulant medications (this does not include anti-platelet medication 
such as aspirin or non-steroidal anti-inflammatory medications). 
16. Use of immunosuppressive corticosteroids (excluding topical or nasal) or 
immunosuppressive drugs within 28 days prior to or following inoculation. An 
immunosuppressive dose of corticosteroids is defined as ≥ 10 mg of a prednisone 
equivalent per day for ≥ 14 days. 
17. Receipt of a live vaccine within 28 days or a killed vaccine within 14 days prior to 
inoculation, or anticipated receipt of any vaccine during the 28 days following 
inoculation with the exception of COVID-19 vaccines either licensed or under EUA 
which can be given at any time, however all effort will be made to avoid giving COVID-
19 vaccines within the above windows.  
18. Asplenia. 
19. Receipt of blood products within the past 6 months, including transfusions or 
immunoglobulin, or anticipated receipt of any blood products or immunoglobulin during 
the 28 days following inoculation. 
20. History or serologic evidence of previous ZIKV or other flavivirus infection (e.g., 
dengue, yellow fever virus, St. Louis Encephalitis virus, or West Nile virus).  
21. Previous receipt of a flavivirus vaccine (licensed or experimental). 
22. Receipt or anticipated receipt of any investigational agent in the 28 days before or after 
inoculation with the exception of COVID-19 vaccines, either licensed or authorized 
under EUA.  

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23. Refusal to allow specimen storage for future research. 
24. Is in isolation or quarantine for SARS-CoV-2 infection or exposure and cannot complete 
screening or enrollment for this reason. 
5.3 
Subject Withdrawal and Termination Criteria 
A subject will not be considered to have completed the trial if any of the following reasons 
apply. However, any subject who has received vaccine or placebo will be encouraged to remain 
in the study for periodic safety evaluations for the duration of the study at the discretion of the 
investigator. Subjects who have been terminated from the study or withdrawn from the study 
prior to study day 28 may be replaced to ensure sufficient safety data for rZIKV/D4∆30-713 are 
collected. 
 
1. Research terminated by sponsor or investigator – applies if the entire study is terminated 
by the sponsor or investigator for any reason. 
2. Withdrawal of consent – applies to a subject who withdraws consent to participate in the 
study for any reason.  
3. Noncompliant with protocol – applies to a subject who does not comply or is not able to 
comply with protocol-specific visits or evaluations on a consistent basis such that 
adequate follow-up is not possible, and the subject’s safety and the study data’s integrity 
would be compromised by continuing in the trial.  
4. Withdrawn by PI – may occur if the investigator believes that it is in the best interest of 
the subject to be withdrawn from the study.  
5. Other – a category used when previous categories do not apply and requires an 
explanation. 
5.4 
Special Situations: 
5.4.1 Pregnancy:  
Because this is a live replicating virus, although it is attenuated, there is a perceived risk to the 
fetus should a woman be pregnant at the time of vaccination or become pregnant during the 
expected time of vaccine virus replication (expected to be through study day 16 post-
vaccination). For this reason, pregnancy is an exclusion criterion for the study and pregnancy 
prevention counseling will be performed regularly throughout the study. If a subject becomes 
pregnant prior to study day 28 following rZIKV/D4∆30-713 or placebo administration, as 
determined by a positive pregnancy test at the study day 28 visit, the subject will not be included 
in the per-protocol immunogenicity evaluations from her estimated date of conception. However, 
she will be encouraged to remain in the study for periodic safety evaluations and will be 
followed until completion of her pregnancy. The subject will be asked to sign a release of 
medical information form so that records can be obtained from her obstetrician regarding the 
outcome of the pregnancy. Should she become pregnant on or after 28 days following 
administration of rZIKV/D4∆30-713 /placebo, as determined by positive pregnancy test after 
study day 28, the study day 28 sample obtained from the subject will be included in the per-
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until completion of her pregnancy. Current evidence does not suggest any risk to the fetus should 
a woman become pregnant after clearing infectious ZIKV from the blood. For this reason, the 
vaccine should not pose any risk to the fetus should a subject become pregnant after clearing 
infectious vaccine virus from the blood (presumed to be approximately day 16 after post-
vaccination). 
5.4.2 Lost to Follow-up: 
A subject who is not reachable by telephone or other means of communication, and therefore not 
able to be located, is considered lost to follow-up. A subject may be considered lost to follow-up 
and withdrawn from the study once three attempts have been made to contact the subject, 
followed by no response to a certified letter sent to the subject’s last known address requesting 
that the subject contact the clinical site.  
5.4.3 Incarceration: 
If a subject becomes incarcerated during the course of the study, he/she may be terminated from 
the study if his/her period of incarceration will make him/her unable to make scheduled visits. 
5.5 
Access to Medical Records 
The medical history of a subject will be obtained from the subject and recorded. Medical records 
from an outside institution will not be requested unless there is a need to clarify the subject’s 
medical history. Medical records from an outside institution will not be requested without the 
medical release form signed by the subject. 
6 
TEST ARTICLE PREPARATION 
6.1 
Pre-inoculation Preparation 
rZIKV/D4∆30-713 vaccine virus for this protocol will be stored at a NIAID-contracted 
repository until requested by the clinical site. Vials of frozen test agent for administration will be 
formally requested for transfer to the clinical site by the PI/designee after institutional review 
board (IRB) approval for the study has been granted and the Food and Drug Administration 
(FDA) has been in receipt of the protocol for at least 30 days without issuing a clinical hold. The 
test agent may be transferred to the study site prior to IRB approval and FDA review only for the 
purpose of determining the titer of the test agent. 
 
After transfer to the clinical site, the vaccine virus will be stored in a locked freezer at -75°C ± 
15°C until time of use. The vaccine virus is supplied as a concentrate that must be diluted with 
PlasmaLyte A, pH 7.4. PlasmaLyte is a sterile, nonpyrogenic isotonic intravenous solution for 
injection Type 1, USP to the proper dose prior to administration. rZIKV/D4∆30-713 will be 
diluted to a final concentration 103.3 PFU/mL (2000 PFU/mL) or 104.3 PFU/mL (20,000 
PFU/mL). A single 0.5 mL dose will be administered subcutaneously such that each subject will 
receive a dose 103 PFU (1,000 PFU) or 104 PFU (10,000 PFU). 
 
After the test agent has been prepared at the clinical site, an aliquot of thawed, undiluted (if 
available), and diluted test agent will be titrated. Additionally, the test agent may be titrated 
periodically to ensure potency. Clinical site pharmacy/lab personnel who are not blinded to 
treatment assignment will be responsible for preparing the vaccine and placebo. The vaccine and 
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will supply a prescription request form to the study staff responsible for test agent preparation 
that will include the protocol number, date of inoculation, dose, route of administration, test 
agent name, test agent lot number, test agent titer (concentration), investigational new drug 
(IND) number, and number of doses to be administered.  
 
The vaccine and placebo will be prepared according to the site’s standard operating procedures. 
rZIKV/D4∆30-713 will be administered at a dose of 103 PFU or 104 PFU subcutaneously. Study 
staff will prepare the correct dose of vaccine (or placebo) for each subject in a biosafety hood 
using aseptic technique. Test agents will be diluted with PlasmaLyte A, pH 7.4 intravenous 
solution, USP. The diluted vaccine and placebo will be drawn up to a volume of 0.5 mL in a 1-
mL syringe and labeled according to standard operating procedures. The labeled syringes will be 
transported at room temperature or on wet ice to the clinic for administration. Test agent must be 
used within 6 hours of being removed from the freezer or refrigerator. Placebo for this study will 
be the vaccine diluent, PlasmaLyte A, pH 7.4. 
6.2 
ZIKV Vaccine and Diluent Storage 
The rZIKV/D4∆30-713 vaccine virus should remain frozen at -75°C ± 15°C until just prior to 
use. Test agent should never be refrozen for reuse in test article preparation. PlasmaLyte will be 
stored per the manufacturer’s recommendation. Vaccine virus and diluent component should be 
opened from new containers for each use. No component should be reused for future vaccine or 
placebo preparation. 
 
Vaccine cannot be used until it is verified by the sponsor as acceptable for use. If the sponsor 
deems any study agent unacceptable for use, it will be quarantined until further directions from 
the sponsor are received. If a study agent is deemed unacceptable for use, it will be disposed of 
per the sponsor’s instruction and a new shipment will be requested. 
6.3 
ZIKV Vaccine and Diluent Accountability 
The unblinded dispenser personnel will maintain an accurate inventory and accountability record 
of each study agent for this study. Partially used vials of study agent or placebo components will 
not be refrozen or reused for future inoculations. 
6.4 
Storage Disposition of Used/Unused Supplies 
After the unblinded dispenser personnel have diluted the study agent and drawn up the syringes 
for administration, they will remove the label from the test agent vial(s) and place the label on 
the test agent preparation form. In this manner, monitoring personnel will be able to verify the 
accountability of all test agent vials used for the study. In addition, the number of test agent vials 
used will be accounted for in the study-specific drug accountability log.  
 
An aliquot of undiluted (if available) and diluted test agents will be titrated by CIR laboratory 
personnel after the test agent has been prepared and delivered to the clinical staff. This is done to 
confirm the potency of the test agent administered to the subjects.  
 
At least 1 aliquot (if available) of diluted test agent will also be frozen and stored at -75°C ± 
15°C at the clinical site for future re-titration if needed. After the syringes have been dispensed 
and aliquots removed for titration, any remaining test agent will be destroyed by the 

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pharmacy/laboratory personnel per standard operating procedures. Any unused diluent/placebo 
from opened vials/bottles will also be destroyed. 
7 
STUDY PROCEDURES 
The following sections provide a detailed listing of the procedures and studies to be performed in 
this protocol at designated time points. 
 
7.1 
Recruitment and General Screening 
Subjects may be recruited from a variety of sources including, but not limited to, subjects 
previously enrolled in vaccine trials at the clinical sites, the use of a center-wide IRB-approved 
screening protocol, and/or by the use of study-specific IRB-approved print, electronic and/or 
other media advertising.  
 
A screening visit may be scheduled after an initial phone screen by clinic staff using an IRB-
approved Pre-Screening Assessment form. The initial phone screen focuses on providing 
background information of the trial and a review of basic inclusion and exclusion criteria.  
 
During the screening process, which may require more than one visit, the subject will read the 
consent form, be encouraged to ask questions, and then complete a comprehension assessment. 
Study staff will review and discuss the answers from the assessment to identify those areas of the 
informed consent form that need further review with the subject. This procedure will help ensure 
that the subject has sufficient understanding of the study before the consent form is signed. The 
subject may either sign the consent form during the screening visit or return after further 
consideration. The subject will have a medical history review, pregnancy prevention counseling 
(if female), physical exam and lab specimens drawn during the general screening process.  
 
7.2 
Screening Procedures 
Subjects will undergo the following screening procedures within 60 days of inoculation: 
 
1. Screening questions for COVID-19. 
 
2. Explain the study and Informed Consent to the subject.  
 
3. Ensure that the subject has successfully completed the Informed Consent 
comprehension assessment, has signed the Informed Consent, and receives a copy of 
the signed Informed Consent.  
 
4. Within 14 days of inoculation, ensure that HIV pre-test counseling has been 
performed and ensure that the subject has agreed to HIV testing (required by MD 
state law only). 
 
5. Elicit a complete medical history, including menstrual, sexual, and contraceptive 
history and history of surgical sterilization for all subjects. 
 

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6. Perform a complete physical examination, including vital signs (height, weight, blood 
pressure, respiratory rate, heart rate, and temperature).  
 
7. Obtain approximately 40 mL of blood for the following laboratory screening tests: 
 
• Prothrombin time/partial thromboplastin time (PT/PTT)  
• Complete blood count (CBC) with differential  
• Alanine aminotransferase (ALT)  
• Aspartate aminotransferase (AST)  
• Total bilirubin  
• Previous flavivirus infection  
• Creatinine  
• Alkaline phosphatase  
• HBV  
• HCV  
 
8. Obtain approximately 5 mL of blood for HIV testing (will be performed within 14 
days of inoculation). Alternatively, a rapid HIV test may be used if a Clinical 
Laboratory Improvement Amendments (CLIA) waiver has been granted to the site. 
 
9. Nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus that 
causes COVID-19 – will be collected from all volunteers regardless of vaccination 
status based on guidance from federal, state, local, and/or institutional health 
officials* or at the discretion of the clinician (may be collected up to 72 hours in 
advance of stated collection time). 
 
*This guidance may be informed by the local COVID infection rate, severity of infections, 
vaccination rates, and other factors as the COVID-19 pandemic continues to evolve and 
change. 
 
 
10. Obtain urine for urinalysis by dipstick testing (glucose, bilirubin, ketones, specific 
gravity, blood, pH, protein, urobilinogen, nitrite, leukocyte esterase).  
 
11. Obtain serum for β-HCG testing in females. A positive β-HCG will exclude the 
subject from the trial. Obtain FSH for females who have not menstruated for greater 
than 12 months but less than 24 months. 
 
12. If necessary, obtain medical release form to confirm method of birth control. 
 
13.  Counsel all female subjects to avoid becoming pregnant during the study and on 
pregnancy prevention during the entire study period. 
 
14. Counsel all male subjects to use barrier contraception for the three months following 
vaccination. 

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7.3 
Inoculation Procedure 
Subjects will receive the LA Zika vaccine rZIKV/D4∆30-713 or placebo on Study Day 0. The 
vaccine will be kept frozen at -75°C ± 15°C until just before use, whereupon it will be thawed, 
diluted to the appropriate dose, and drawn up for administration (see Section 6 for test article 
preparation). The rZIKV/D4∆30-713 vaccine will be kept on wet ice from the time it is thawed 
until it is diluted with PlasmaLyte into its final formulation. The vaccine and placebo 
(PlasmaLyte) will then be delivered at room temperature or on wet ice to clinical staff for 
administration. A volume of 0.5 mL of test article will be delivered by subcutaneous injection in 
the deltoid region of the upper arm with a needle of appropriate gauge and length after wiping 
the injection site with alcohol. 
7.4 
Detailed Study Procedures 
The study procedures to be performed at each visit are listed below. Additional tests may be 
done at the discretion of the PI/provider to evaluate concomitant illness or further evaluate an 
adverse event (AE) experienced by a subject. Photographs may be taken of the injection site. In 
addition, photographs may be taken of other areas of the skin before and/or after inoculation to 
record the characteristics of any rash that may develop. Possible identifiers, such as tattoos, 
jewelry, and clothing will not be included in the photographs, to maintain confidentiality. The 
total volume of blood to be drawn over the 26-week post-vaccination period is approximately 
610 mL. This amount is within NIH guidelines (Medical Administrative Policy 95-9) for adult 
blood donation and should not compromise the health of study subjects. A topical anesthetic 
cream may be used during blood draw at the phlebotomist’s discretion to ease the discomfort of 
the procedure. 
7.4.1 Inoculation and Follow-up 
On the day of inoculation with test article, more subjects may be invited to the clinic than will be 
enrolled. These subjects will be alternates and will be inoculated only if other subjects are not 
available for inoculation or are found to be ineligible on the day of inoculation. Those subjects 
who are alternates will be informed that they are alternates when they are invited to the clinic. 
For study visits with overlapping windows for the visit, subjects will be scheduled so that 2 study 
visits for an individual do NOT occur on the same day. All subjects invited to the clinic will have 
to abide by the facility’s masking policies and COVID-19 related procedures. At each visit, 
volunteers will be asked whether or not they have experienced any COVID-19 symptoms (per 
facility screening guidelines) or if they have had an exposure to someone with COVID-19 prior 
to coming into the clinic. If the volunteer reports symptoms consistent with COVID-19 and they 
are already enrolled in the study, an NP or mid-turbinate swab will be collected and tested for 
COVID-19 on site or at the respective local laboratory. If this is unavailable, the volunteer will 
be referred for local testing. The volunteer will not come into the clinic until the result is 
confirmed to be negative (~45 minutes) or per current facility guidelines for COVID-19 
screening procedures. Should a volunteer test positive for SARS-CoV-2 at any point during the 
study, the volunteer will be asked to quarantine according to current CDC guidelines. We will 
review current CDC guidelines with the volunteer. We will defer all in-person visits until after 
the volunteer has completed their quarantine or if a COVID-19 positive research space is 
available they will be brought to that location. If they test positive for SARS-CoV-2 during the 
first 21-days post-vaccination, scheduled follow-up visits will be done as telehealth visits or at a 
research space designated for COVID-19 positive patients. Based on COVID-19 testing and 

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results availability, NP or mid-turbinate swab may be collected up 72 hours prior to scheduled 
testing visit. 
Study Day 0 (Day of Inoculation with Test Article) 
 
1. Screening questions for COVID-19. 
2. Verify that Informed Consent was obtained and that the consent form was signed by both 
the subject and by the study staff. 
3. Verify that all applicable eligibility criteria have been met. 
4. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. Focused physical exam will include BP, HR, 
temperature, respiratory rate, skin examination, eyes, throat, muscle strength, and biceps 
and patellar deep tendon reflexes. Physical exam may extend beyond these if indicated by 
complaint or physical finding. 
5. Review and document concomitant medications. 
6.  Nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus that 
causes COVID-19 – will be collected based on guidance from federal, state, local, and/or 
institutional health officials* or at the discretion of the clinician (may be obtained up to 
72 hours in advance of study day). 
*This guidance may be informed by the local COVID infection rate, severity of infections, 
vaccination rates, and other factors as the COVID-19 pandemic continues to evolve and 
change. 
 
7. .Obtain approximately 70 mL of blood for CBC with differential, ALT, creatinine, virus 
isolation and culture (PCR/culture), and immunology (PBMCs, plasma, and PAXGene). 
These laboratory studies are drawn as baseline values and will not determine eligibility.  
8. Obtain 5 mL of clean-catch urine for virus isolation & culture. 
9. Obtain vaginal secretion sample (women) for virus isolation and culture (may be 
collected at home by the volunteer). 
10. Obtain semen sample (men) for virus isolation and culture (may be collected at home by 
the volunteer). 
11. Obtain oral fluid for serology (CIR site only).  
12. Perform serum or urine β-HCG testing for female subjects. Ensure the test is negative 
before proceeding; a positive test will exclude the subject from the study as per protocol, 
Section 5.2. 
13. Review pregnancy prevention. 
14. Record vital signs (blood pressure, temperature, heart rate, and respiratory rate). 
15. Administer the test article. 
16. Observe for at least 30 minutes after inoculation and evaluate for immediate 
hypersensitivity.  

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17. Provide education by study staff describing proper use of thermometer, the signs and 
symptoms of potential AEs, and how and when to contact study staff (see Section 7.5.1). 
Study Day 4 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 40 mL of blood for CBC with differential, virology, and 
immunology (PBMCs, plasma, and PAXGene). 
5. Obtain 5 mL of clean-catch urine for virus isolation & culture. 
6. Review pregnancy prevention. 
 
Study Day 6 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 20 mL of blood for CBC with differential, ALT, virology, and 
PAXGene. 
5. Obtain 5 mL of clean-catch urine for virus isolation & culture. 
6. Review pregnancy prevention. 
 
Study Day 8 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus that 
causes COVID-19 – will be collected based on guidance from federal, state, local, and/or 
institutional health officials* or at the discretion of the clinician (may be obtained up to 
72 hours in advance of study day). 
5. Obtain approximately 50 mL of blood for CBC with differential, virology, and 
immunology (PBMCs, plasma, and PAXGene). 
6. Obtain urine sample for virus isolation and culture. 
7. Obtain vaginal secretion sample (women) for virus isolation and culture. Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic. 
8. Obtain semen sample (men) for virus isolation and culture, may be collected at home by 
the volunteer prior to presentation to the clinic. 
9. Obtain oral fluid for serology (CIR subjects only). 
10. Review pregnancy prevention. 
*This guidance may be informed by the local COVID infection rate, severity of infections, 
vaccination rates, and other factors as the COVID-19 pandemic continues to evolve and 
change. 

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Study Day 10 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 20 mL of blood for CBC with differential, virology, and 
PAXGene. 
5. Obtain 5 mL of clean-catch urine for virus isolation & culture. 
6. Review pregnancy prevention. 
 
Study Day 12 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 20 mL of blood for CBC with differential, virology, and 
PAXGene. 
5. Obtain urine sample for virus isolation and culture. 
6. Obtain oral fluid for serology (CIR subjects only). 
7. Review pregnancy prevention. 
 
Study Day 14 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Review pregnancy prevention with the subject. 
5. Nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus that 
causes COVID-19 – will be collected based on guidance from federal, state, local, and/or 
institutional health officials* or at the discretion of the clinician (may be obtained up to 
72 hours in advance of study day). 
*This guidance may be informed by the local COVID infection rate, severity of infections, 
vaccination rates, and other factors as the COVID-19 pandemic continues to evolve and 
change. 
 
6. Obtain approximately 50 mL of blood for CBC with differential, ALT, creatinine, 
virology, and immunology (PBMCs, plasma, and PAXGene). 
7. Obtain 5 mL of clean-catch urine for virus isolation & culture. 
 
Study Day 16 (± 1 day) 

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1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 20 mL of blood for CBC with differential, virology, and 
PAXGene. 
5. Obtain urine sample for virus isolation and culture. 
6. Obtain vaginal secretion sample (women) for virus isolation and culture. Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic. 
7. Obtain semen sample (men) for virus isolation and culture, which may be collected at 
home by the volunteer prior to presentation to the clinic. 
8. Obtain oral fluid for serology (CIR subjects only). 
9. Review pregnancy prevention. 
 
Study Day 21 (± 1 day) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Review pregnancy prevention with the subject. 
5. Obtain approximately 40 mL of blood for immunology (PBMCs, serum, plasma, 
PAXGene), and virology*. CBC and/or ALT or creatinine will be done if prior white 
blood cell (WBC), ANC, platelet count, hemoglobin, or ALT or creatinine results are 
Grade I or above and unresolved, as defined by protocol.  
6. For females, perform β-HCG testing. A positive test will exclude the subject from the 
per-protocol analysis, as per Section 5. 
7. Obtain urine sample for virus isolation and culture.* 
*Virology will be done on Study Day 21 only if virus was recovered from the sample 
drawn at the previous time-point (Day 16) 
 
Study Day 28 (± 2 days) 
1. Screening questions for COVID-19. 
2. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
3. Record vital signs. 
4. Obtain approximately 50 mL of blood for immunology (PBMCs, serum, plasma, 
PAXGene and virology*.  
5. For females, perform β-HCG testing. A positive test will exclude the subject from the 
per-protocol analysis, as per Section 5. 
6. Review pregnancy prevention with the subject.  
7. Obtain urine sample for virus isolation and culture.* 
8. Obtain vaginal secretion sample (women) for virus isolation and culture.* Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic. 

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9. Obtain semen sample (men) for virus isolation and culture, which may be collected at 
home by the volunteer prior to presentation to the clinic.* 
10. Obtain oral fluid for serology (CIR site only). 
* Virology will be done on Study Day 28 only if virus was recovered from the sample 
drawn at the previous time-point (Day 21) 
 
Study Day 56 (+ 14 days) 
1. Screening questions for COVID-19. 
2. Perform interim history for interim complaints (physical exam is optional – will be done 
should subject have any medically-attended AEs since last visit). 
3. Record vital signs. 
4. Obtain approximately 50 mL of blood for virology* and immunology (PBMCs, serum, 
and plasma).  
5. For females, perform β-HCG testing.  
6. Review pregnancy prevention with the subject.  
7. Obtain urine sample for virus isolation and culture.* 
8. Obtain vaginal secretion sample (women) for virus isolation and culture.* Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic. 
9. Obtain semen sample (men) for virus isolation and culture, which may be collected at 
home by the volunteer prior to presentation to the clinic.* 
10. Obtain oral fluid for serology (CIR site only). 
 
*Virology will be done on Study Day 56 only if virus was recovered from the sample 
collected at the previous time-point (Day 28). 
 
Study Day 90 (+ 10 days) 
1. Screening questions for COVID-19. 
2. Perform interim history for interim complaints (physical exam is optional - will be done 
should subject have any medically-attended AEs since last visit). 
3. Record vital signs. 
4. Obtain approximately 40 mL of blood for virology* and immunology (PBMCs, serum, 
and plasma).  
5. For females, perform β-HCG testing.  
6. Review pregnancy prevention with the subject.  
7. Obtain urine sample for virus isolation and culture.* 
8. Obtain vaginal secretion sample (women) for virus isolation and culture. Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic.* 
9. Obtain semen sample (men) for virus isolation and culture, which may be collected at 
home by the volunteer prior to presentation to the clinic.* 
10. Obtain oral fluid for serology (CIR site only). 
 
*Virology will be done on Study Day 90 only if virus was recovered from the sample 
collected at the previous time-point (Day 56). 

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Study Day 150 (+ 14 days) 
1. Screening questions for COVID-19. 
2. Perform interim history for interim complaints (physical exam is optional- will be done 
should subject have any medically-attended AEs since last visit or at the discretion of the 
investigator). 
3. Record vital signs. 
4. Obtain approximately 50 mL of blood for virology* and immunology (PBMCs, serum, 
and plasma).  
5. For females, perform β-HCG testing.  
6. Review pregnancy prevention with the subject.  
7. Obtain urine sample for virus isolation and culture.* 
8. Obtain vaginal secretion sample (women) for virus isolation and culture. Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic.* 
9. Obtain semen sample (men) for virus isolation and culture, may be collected at home by 
the volunteer prior to presentation to the clinic.* 
10. Obtain oral fluid for serology (CIR site only). 
 
*Collection of urine, vaginal secretion, and semen samples and virology (urine, vaginal 
secretion, semen, blood) will be done on Study Day 150 only if virus was recovered from 
the sample collected at the previous time-point (Day 90) or if virology results are not 
available. 
 
Study Day 180 (+ 28 / - 14 days) 
1. Screening questions for COVID-19. 
2. Perform interim history for interim complaints and focused physical exam, concentrating 
on any acute complaints.  
3. Record vital signs. 
4. Obtain approximately 50 mL of blood for immunology (PBMCs, serum, and plasma).  
5. For females, perform β-HCG testing.  
6. Obtain urine sample for virus isolation and culture.* 
7. Obtain vaginal secretion sample (women) for virus isolation and culture. Vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic.* 
8. Obtain semen sample (men) for virus isolation and culture, may be collected at home by 
the volunteer prior to presentation to the clinic. * 
9. Obtain oral fluid for serology (CIR site only). 
*Collection of urine, vaginal secretion, and semen samples and virology (urine, vaginal 
secretion, semen) will be done on Study Day 180 only if virus was recovered from the 
sample collected at the previous time-point (Day 150) or if virology results are not 
available. 
7.4.1.1 Persistence of Zika Vaccine in Bodily Fluids 
If Zika vaccine is detected in any bodily fluid at Study Day 180, the subject will continue to be 
followed until Zika vaccine is no longer detected in the bodily fluid. To achieve this, the subject 

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will be brought back for unscheduled visits for collection of the specimen of interest (the bodily 
fluid that was positive for Zika vaccine at Study Day 180) and for review of clinical symptoms. 
If clinical symptoms are reported, the subject will undergo clinical evaluation. Persistent 
shedding of Zika vaccine in bodily fluids at Study Day 180 will be reported to the DSMB and the 
FDA as soon as it is detected. The DSMB and the FDA will be asked to provide additional 
guidance as to follow-up procedures, the interval of specimen collection, and the duration of 
specimen collection.  
7.5 
Subject Temperature Memory Card 
Subjects will be provided with a thermometer and a Temperature Memory Card to use as a 
memory aid and will be asked to record temperatures 2 times a day from study day 0 through 
study day 16 post-inoculation.  Study staff will review the subjects’ temperature cards on study 
day visits 4 through 21 to assess for any fevers and compare subject recordings to the study visit 
temperatures. Staff will record all temperatures recorded by the subject and will also separately 
record the maximum temperature for each date (Tmax) on the source document. Subject memory 
cards will not be collected by the study staff as subject’s recordings will not be considered 
required data.  
 
Staff will instruct subjects on how to use the thermometers, to take their temperatures at 
approximately the same time each day, and to take additional temperatures if they feel they have 
elevated temperatures. Subjects will be asked to wait at least 15 minutes after eating, drinking, 
and smoking before taking their temperatures. They will be asked to confirm an elevated 
temperature (≥ 100.4°F) by retaking the temperature after a 20-minute interval and at 1 hour. 
Temperatures not documented to last at least 1 hour will not be considered an AE or included in 
the analysis.  
7.5.1 Notification of Staff of Zika-like Symptoms 
Subjects will be asked to call or otherwise contact the study staff should they experience any 
symptoms of Zika illness. The Zika-like illness symptoms that study staff will review with the 
subjects are the systemic reactogenicity signs included in Table 9. Once notified, study staff will 
arrange to evaluate the subject at the earliest convenience; this may result in an unscheduled visit 
for the subject. 
7.5.2 Notification of Staff of COVID-19-like symptoms 
Subjects will be asked to call or otherwise contact study staff should they experience any 
COVID-19 symptoms. In addition, at each visit, volunteers will be asked whether or not they 
have experienced any COVID-19 symptoms (per facility guidelines) or if they have had an 
exposure to someone with COVID-19 prior to coming into the clinic. If the volunteer reports 
symptoms consistent with COVID-19 and they are already enrolled in the study, an NP or mid-
turbinate swab will be collected and tested for COVID-19 on site or at a local laboratory at any 
time. The volunteer will not come into the clinic until the result is confirmed to be negative. 
Should a volunteer test positive for SARS-CoV-2 at any point during the study, the volunteer 
will be asked to quarantine according to current CDC guidelines. We will review current CDC 
guidelines with the volunteer. We will defer all in-person visits until after the volunteer has 
completed their quarantine or see them in a COVID-19 positive research space if it is available. 
If they test positive for SARS-CoV-2 during the first 21-days post-vaccination, scheduled 
follow-up visits will be done as telehealth visits or in a COVID-19 positive research space. 

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7.6 
Clinical Laboratory Testing 
Using standard techniques, Quest Diagnostics or other CLIA-certified laboratories will perform 
the following tests: 
 
1. CBC plus WBC differential  
2. PT/PTT 
3. ALT, AST, alkaline phosphatase, total bilirubin, creatinine 
4. HIV assay (screening antibody assay with confirmation for positive antibody assays). 
5. Hepatitis B screening by testing for HBsAg  
6. Hepatitis C screening by testing for hepatitis C antibody. If positive, the test is confirmed 
by testing for hepatitis C RNA. If hepatitis C antibody is positive but RNA is negative, 
the subject is assessed as having a past infection that has cleared. In this case, the subject 
is eligible for the study. 
7. Urinalysis (in the event of an abnormal urine dipstick test) 
8. SARS-CoV-2 PCR testing 
9. Serum β-HCG, if required 
Urine and serum β-HCG testing will be performed at the clinical trial site using an FDA-
approved pregnancy test kit. Urine dipstick testing will be performed at the clinical trial site 
using an FDA-approved product. Rapid HIV testing may be performed at the clinical trial site 
using an FDA-approved test kit.  Determination of vaccine virus titer, plaque reduction 
neutralization antibody assays, and cellular immune studies will be done at the clinical trial site 
laboratory. 
 
7.7 
Medical History and Concomitant Medications 
A complete medical history will be collected during screening. Any changes reported in medical 
history during the 28-day post-inoculation period will be assessed as possible AEs. After study 
day 28, the medical history will be updated for any new or changed significant or chronic 
conditions, and to identify if any medically attended AEs have occurred.  
 
Study staff will collect current medications as part of the medical history, including over-the-
counter medications and herbal supplements, at the time of enrollment. All changes or updates to 
medications will be collected through study day 28 following inoculation. After study day 28, 
concomitant medications will be collected to identify new or changed significant or chronic 
conditions. Medications taken for AEs continuing after study day 28 will be recorded throughout 
the trial.  
 

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7.8 
Immunology Testing 
7.8.1 Antibody Testing 
Serum antibody levels to ZIKV will be measured by plaque reduction neutralizing antibody 
assay using standard laboratory protocols. The PRNT50 is defined as the highest dilution of 
antibody that reduces the number of foci or plaques by 50%, compared to the plaque titer of the 
virus alone. 
7.8.2 Other Immunological Assays 
Cytokine, T-cell and B-cell stimulation assays, and PBMC phenotyping may be performed on 
PBMCs. RNA may be isolated from cells and run on microarrays to identify immune pathways 
of interest, such as defining the innate immune response to the vaccine virus. In addition, human 
leukocyte antigen (HLA) typing of samples may be performed as part of assays to map the Zika 
virus epitopes that induce T- or B-cell responses. In addition, an exploratory neutralization 
capacity assay may be performed using target cells that express CD32a (FcγRIIa). This assay 
will evaluate the effect of neutralizing and enhancing antibody in the same assay. ZIKV specific 
antibodies will be measured in oral fluids as an exploratory assay for detection of Zika. 
 
7.9 
Retention of Study Subjects 
We will employ several strategies aimed at retaining subjects through study completion. During 
screening, we will obtain detailed primary locator information, as well as secondary contact 
information. Subjects will also provide information for people who may be contacted if primary 
and secondary means of contact fail. Locator information will be reviewed with subjects at each 
visit (i.e., addresses, phone numbers, email addresses). In addition, birthday cards/holiday cards 
may be mailed to check addresses, and reminders may be sent using various methods (including, 
but not limited to, phone, email, text messaging, electronic media, and postal mail). All data will 
be maintained and updated in a password-protected locator database. 
 
8 
ADVERSE EVENT MONITORING 
8.1 
Definitions 
8.1.1 Adverse Event 
An adverse event (AE) is any untoward or unfavorable medical occurrence in a human subject, 
including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether or not it is 
considered related to the research. 
 
All AEs will be evaluated for severity, action taken, seriousness, outcome, and relationship to the 
investigational vaccine as described in Section 8.2 in this protocol. If a diagnosis is clinically 
evident (or subsequently determined), the diagnosis rather than the individual signs and 
symptoms or lab abnormalities will be recorded as the AE. AEs will be collected through the 28-
day period following inoculation, and any test agent–related AEs (adverse reactions, see below) 
identified in the 28-day post-inoculation period will be followed until resolution.  

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8.1.2 Medically-attended Adverse Event 
A medically attended AE is defined as any visit (other than hospitalization, visit for routine 
health maintenance, or visit for a pre-existing condition that has not worsened) to an emergency 
room (ER), urgent care facility, doctor, nurse, or any other health care provider. Medically 
attended AEs from days 29 through 180 will be collected. Medically attended AEs that are found 
to be unrelated to the study product will be followed until resolution or the end of the study, 
whichever occurs first. 
8.1.3 Adverse Reaction 
An adverse reaction (AR) is an AE that is caused by an investigational agent (drug or biologic). 
AEs that are deemed possibly, probably, or definitely related to study article will be classified as 
adverse reactions. ARs are categorized as solicited ARs and other ARs. Solicited ARs include 
local reactogenicity, systemic reactogenicity, and laboratory events. Solicited ARs are those 
events that the clinician is specifically evaluating during the 28-day post-inoculation period, 
listed in Table 9. All AEs are evaluated using the Adverse Event Grading Table in Section 14.1, 
Appendix 1. 
 
Table 9: Solicited Adverse Events/Adverse Reactions 
Systemic Reactogenicity 
Laboratory Events  
Local Reactogenicity 
Fever 
Decreased Hemoglobin 
Injection Site Pain 
Headache 
Neutropenia 
Injection Site Erythema 
Retro-orbital Pain (ROP) 
Elevated ALT 
Injection Site Tenderness 
Photophobia 
Thrombocytopenia 
Injection Site Induration 
Nausea 
Leukocytosis 
Injection Site Pruritus 
Fatigue 
 
  
Myalgia 
  
 
Arthralgia 
Non-purulent Conjunctivitis 
Muscle Weakness 
 
 
Zika Virus-Like Rash 
 
 
 
8.1.4 Adverse Event of Special Interest 
Guillain-Barré syndrome (GBS) is an AE of special interest. Subjects will be examined for signs 
of muscular weakness at each visit that includes a physical exam and muscle strength will be 
reported as in Section 8.2.2. Should a subject present to an outside physician for evaluation of 
GBS, this will be captured and reported. The Brighton Criteria for diagnosis of GBS will be used 
(Section 14.2, Appendix 2) and the WHO Guillain-Barré Syndrome Disability Scale will be used 
(Section 14.3, Appendix 3). 
 
8.1.5 Pregnancy 
Pregnancy itself is not an AE. However, complications of pregnancies are AEs and may be 
serious adverse events (SAEs). Events that meet SAE criteria during pregnancy, delivery, or in 
the neonate (e.g., congenital anomaly/birth defect) are reportable to the Clinical Safety Office 
(CSO) per the sponsor’s reporting guidelines. Pertinent obstetrical information for all 

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pregnancies will be reported to the CSO via the REDCap reporting system within 1 business day 
from site awareness of the pregnancy on the Pregnancy Notification and Outcome Form.  
Pregnancy outcome data (e.g., delivery outcome, spontaneous or elective termination of the 
pregnancy) will be reported to the CSO within 3 business days of the site’s awareness on a 
protocol-specified form. If a subject becomes pregnant within 28 days of test article 
administration, the subject will be unblinded to determine if she received ZIKV. If she did 
receive ZIKV, she will be referred to an OB/GYN experienced in caring for ZIKV-infected 
pregnant women (Dr. Jeanne Sheffield at JHU). 
 
If the pregnancy occurs prior to study day 28 after rZIKV/D4∆30-713 administration, as 
determined by a positive pregnancy test at the study day 28 visit or before, the subject will not be 
included in the per-protocol analysis. The subject will remain on the study for safety follow-up. 
• If unblinding the treatment assignment would help the subject determine whether she 
wishes to continue her pregnancy, her treatment assignment will be unblinded. 
• The research subject will be advised to notify her obstetrician of study participation and 
study agent exposure. 
• The pregnancy will be reported to the DSMB (if applicable) and/or IRB (if applicable). 
If the pregnancy occurs after study day 28 following rZIKV/D4∆30-713 administration, as 
determined by a positive pregnancy test after the study day 28 visit, the subject will be included 
in the per-protocol immunogenicity analysis.  
8.1.6 Serious Adverse Event 
A serious adverse event (SAE) is an AE that is determined to be “serious,” whether considered 
related to the investigational vaccine or not. SAEs will be collected for the duration of the trial. 
An SAE results in one or more of the following outcomes: 
 
• Death during the period of protocol-defined surveillance 
• A life-threatening event, defined as an event that places a subject at immediate risk of 
death at the time of the event and does not refer to an event that hypothetically might 
have caused death were it more severe 
• Inpatient hospitalization or prolongation of existing hospitalization, defined as at least an 
overnight stay in the hospital or emergency room for treatment that would have been 
inappropriate if administered in the outpatient setting 
• Congenital anomaly or birth defect 
• Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
• Other medically important events* 
*Medical and scientific judgment should be exercised in deciding whether expedited reporting is 
appropriate in other situations, such as important medical events that may not be immediately 

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life-threatening or result in death or hospitalization but may jeopardize the subject or may require 
intervention to prevent one of the outcomes listed above. These will also usually be considered 
serious. 
 
Each AE will be classified by the investigator/designee as serious or non-serious. An SAE needs 
to meet only one or more of the above criteria to be considered serious. 
8.1.7 Unexpected Adverse Events 
An AE is considered unexpected if it is not listed in the Investigator’s Brochure or Package 
Insert (for marketed products) or is not listed at the specificity or severity that has been observed. 
“Expected” does not mean that the event is expected with pharmacologically similar drugs, the 
underlying disease(s), or concomitant medications. It is the responsibility of the IND sponsor to 
make this determination. 
8.1.8 Serious and Unexpected Suspected Adverse Reaction (SUSAR) 
A serious and unexpected suspected adverse reaction (SUSAR) is a suspected adverse reaction 
(SAR) that is serious and unexpected. 
8.1.9 Unanticipated Problem 
An unanticipated problem is any event, incident, experience, or outcome that is: 
 
1.  Unexpected in terms of nature, severity, or frequency in relation to 
a. The research including but not limited to risks that are described in the IRB-
approved research protocol and informed consent document; Investigator’s 
Brochure or other study documents; and 
b. The characteristics of the subject population being studied; 
2. Possibly, probably, or definitely related to participation in the research; and 
3. Places subjects or others at a greater risk of harm (including physical, psychological, 
economic, or social) than was previously known or recognized. (An AE with a 
serious outcome will be considered increased risk). 
8.1.10 Unanticipated Problem that is not an Adverse Event (UPnonAE) 
This is an unanticipated problem that does not fit the definition of an AE, but which may, in the 
opinion of the investigator, involve risk to the subject, affect others in the research study, or 
significantly impact the integrity of research data. Such events would be considered non-serious 
unanticipated problems. For example, we will report occurrences of breaches of confidentiality, 
accidental destruction of study records, or unaccounted-for study drug. 
8.1.11 Protocol Deviation 
A protocol deviation is any change, divergence, or departure from the study design or procedures 
in an IRB-approved research protocol that has an impact on the subject’s rights, safety, or well-
being, and/or the completeness, accuracy, or reliability of the study data. Protocol deviations are 
designated as serious or non-serious and further characterized as: 
1. Those that occur because a member of the research team deviates from the protocol 

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2. Those that are identified before they occur, but cannot be prevented 
3. Those that are discovered after they occur 
8.1.12 Serious Protocol Deviation 
A serious protocol deviation is any change, divergence, or departure from the study design or 
procedures in an IRB-approved research protocol that meets the definition of a Serious Adverse 
Event or compromises the subject’s rights, safety, or well-being, and/or the completeness, 
accuracy, or reliability of the study data. 
 
Non-compliance is the failure to comply with applicable NIH Human Research Protection 
Program (HRPP) policies, IRB requirements, or regulatory requirements for the protection of 
human subjects. Non-compliance is further characterized as 
1. Serious: Non-compliance that: 
a. Increases risks or causes harm to subjects 
b. Decreases potential benefits to subjects 
c. Compromises the integrity of the NIH HRPP 
d. Invalidates the study data 
2. Continuing: Non-compliance that is recurring 
3. Minor: Non-compliance that is neither serious nor continuing 
8.1.13 Pre-existing Conditions, Worsening of Pre-existing Condition 
Stable chronic conditions that are present prior to enrollment and do not worsen are not 
considered AEs and will be accounted for in the subject’s medical history. Exacerbation or 
worsening of pre-existing conditions are defined as AEs and are evaluated using the same criteria 
described in Section 8.2  of this protocol. 
8.2 
Assessment of Adverse Events 
8.2.1 Identification of Adverse Events 
All AEs occurring from the time the subject is enrolled (at administration of investigational agent 
on day 0) through the Study Day 28 will be documented, recorded, and reported. The 
Investigator will assess all AEs with respect to Seriousness, Severity, and Causality 
(relationship to study agent and relationship to research) according to the following guidelines. 
An assessment of safety will include clinical observations and monitoring of hematological, 
blood chemistry, and immunologic parameters. Safety will be evaluated by monitoring the 
subjects for local and systemic adverse events during the course of the trial. Subjects will be 
closely monitored for 30 minutes following inoculation. Additionally, subjects will return to the 
clinic on study days 4, 6, 8, 10, 12, 14, 16, 21, and 28 post-inoculation and may be asked to 
return more often if warranted. It is during this time period that we anticipate AEs related to 
infection with the vaccine virus to manifest themselves; therefore, the subjects will be seen 
frequently. Study staff will review subjects’ reported temperatures and clinical temperatures for 
the first 16 days following inoculation to assess for AEs. At each visit through study day 28 
following inoculation, subjects will be queried about adverse events, including possible vaccine-
related AEs (solicited AEs) and will have a focused physical exam performed. A study clinician 
will be available to subjects by telephone or pager 24 hours a day during the study evaluation 
period. 

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All AEs will be recorded during the period after the subject receives the study vaccine through 
and including post-inoculation study day 28. 
 
All SAEs will be assessed appropriately as AEs and reported following SAE reporting guidelines 
outlined in Section 8.3.2  of this protocol. 
8.2.2 Protocol Specific Adverse Event Definitions 
Fever: Temperature ≥ 100.4°F lasting at least 1 hour 
ZIKV-like Rash: Areas of confluent macular or maculo-papular rash over trunk and 
extremities, blanches when compressed. 
Non-purulent conjunctivitis: Bilateral reddening of the sclera of the eyes without evidence of 
exudate or crusting of the eyelids 
Headache: A pain located in the head, over the eyes, at the temples, or at the base of the skull. 
ROP: Bilateral pain situated behind the orbits of the eye 
Photophobia: An abnormal sensitivity or intolerance to light 
Nausea: Discomfort in the stomach with an urge to vomit 
Fatigue: Excessive tiredness following minimal exertion  
Myalgia: Pain in the muscles that is found in > 2 muscle groups 
Arthralgia: Pain in a joint that is found in > 2 joints. 
Muscle Weakness: Grade 0 - 3 out of 5 on the Oxford Scale. The Oxford Scale grading is as 
follows: 
• 0/5: No contraction 
• 1/5: Visible/palpable muscle contraction but no movement 
• 2/5: Movement with gravity eliminated 
• 3/5: Movement against gravity only 
• 4/5: Movement against gravity with some resistance 
• 5/5: Movement against gravity with full resistance 
8.2.3 Determination of Severity 
The investigator/designee will assess all AE severity using the following classifications. 
Table 10: Severity Definitions  
Severity 
Defined 
Grade 1 (Mild) 
An event that is easily tolerated and may require 1 dose of 
medication/treatment 
Grade 2 (Moderate) 
An event that interferes with daily activity or requires > 1 dose of 
medication/treatment 
Grade 3 (Severe) 
An event that prevents daily activity and requires medical 
intervention 
Grade 4 (Life-
threatening) 
An adverse event that is deemed by the study clinician, the medical 
monitor, or an outside clinician caring for the subject to be life-
threatening 
Grade 5 (Death) 
Any adverse event that results in the death of the subject 
Solicited AE severity grading classifications are listed in Table 10 and  

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Table 11. All other AEs will be graded on severity using the Adverse Event Grading Table in 
Section 14.1, Appendix I. 
 
Table 11: Assessment of Solicited Adverse Events 
Local Reactogenicity 
Grade 
Severity 
Injection Site Tenderness, 
Pruritis, and Pain 
 
1 
 
2 
 
3 
An event that is easily tolerated and may require 1 
dose of medication 
An event that interferes with daily activity or 
requires > 1 dose of medication 
An event that prevents daily activity and requires 
medical intervention 
4 
A life-threatening event 
Injection Site Induration and 
Erythema 
1 
2 
3 
> 0 – 20 mm 
> 20 – 50 mm 
> 50 mm 
4 
Life-threatening 
Systemic Reactogenicity 
Grade 
Severity 
Fever (Oral) 
1 
2 
3 
100.4°F – 101.4°F  
101.5°F – 102.4°F  
≥ 102.5°F  
4 
Life-threatening 
ZIKV-Like Rash 
1 
2 
 
3 
A rash that is present but asymptomatic 
A rash that is symptomatic (pruritus/pain) but 
does not interfere with function 
A rash that is symptomatic and interferes with 
function 
4 
A rash that is life-threatening 
Headache 
ROP 
Photophobia 
Nausea 
Fatigue 
Myalgia 
Arthralgia 
Conjunctivitis 
Muscle weakness 
1 
 
2 
 
3 
 
An event that is easily tolerated and may require 1 
dose of medication/treatment 
An event that interferes with daily activity or 
requires > 1 dose of medication/treatment 
An event that prevents daily activity and requires 
medical intervention 
4 
A life-threatening event 
Solicited Laboratory AEs 
Grade 
Severity 
Hemoglobin (Women) 
1 
2 
3 
9.5 – 10.7 gm/dL 
8.0 – 9.4 gm/dL 
< 7.9 gm/dL 
4 
Life-threatening 
Hemoglobin (Men) 
1 
2 
3 
11 – 12.5 gm/dL 
9.0 – 10.9 gm/dL 
< 8.9 gm/dL 

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4 
Life-threatening 
Neutropenia 
(Reduced ANC) 
1 
2 
3 
750 – 999/mm3 
500 – 749/mm3 
< 500 mm3 
4 
Life-threatening 
Leukocytosis  
(Increased WBCs) 
1 
2 
3 
11,500 – 13,000/mm3 
13,001 – 15,000/mm3 
15,000 or < 1,000/mm3 
4 
Life-threatening 
Thrombocytopenia 
 (Decreased Platelets) 
1 
2 
3 
≥ 100,000 – 120,000/mm3 
≥ 75,000 – 99,999/mm3 
< 74,999/mm3 
4 
Life-threatening 
ALT 
1 
2 
3 
> 1.25 – 2.5 × ULN 
> 2.5 – 5.0 × ULN 
> 5.0 × ULN 
4 
Life-threatening 
1. Any adverse event resulting in death will be given a severity grade of 5. 
 
8.2.4 Relationship with Receipt of Test Article 
The clinical investigator will assess all AEs for their relationship to the study agent using the 
following classifications:  
Definitely related: 
Clear-cut temporal association, with no other possible cause 
Probably related: 
Reasonable temporal association, and a potential alternative 
etiology is not apparent 
Possibly related: 
Less clear temporal association; other etiologies also possible 
Unlikely related: 
Temporal association between the AE and the study agent or the 
nature of the event is such that the study agent is not likely to have 
had any reasonable association with the observed illness/event 
(cause and effect relationship improbable but not impossible) 
Unrelated: 
The AE is completely independent of study agent administration, 
and/or evidence exists that the event is definitely related to another 
etiology 
The degree of certainty with which an AE can be attributed to study agent administration will be 
determined by how well the event can be understood in terms of one or more of the following: 
• 
A reaction of a similar nature having previously been observed with this type of vaccine 
and/or formulation or naturally occurring Zika illness 
• 
The event having often been reported in the literature for similar types of vaccine 
products 
All local injection-site reactions will be considered causally related to inoculation. Causality 
assessment is based on information available at the time of the AE assessment. The investigator 
may revise the causality assessment as additional information becomes available. 

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8.2.5 Adverse Event Action Taken 
The investigator/designee will assess the action taken by the subject or the study staff in relation 
to the AE, using the following classifications:  
Action 
1 = None 
2 = Remedial therapy (more than 1 dose of medication required) 
3 = Discontinued study  
4 = Hospitalization 
5 = Other 
8.2.6 Adverse Event Outcome 
The investigator/designee will assess the outcome of the AE, either at resolution or the end of the 
study period, using the following classifications: 
Outcome  
1 = Resolved 
2 = Continuing 
3 = Continuing Chronic Condition 
4 = Unknown, Off-Study before could confirm resolution of AE 
5 = Death 
6 = Unknown 
8.2.7 Adverse Event Seriousness 
The investigator/designee will categorize all AEs as either serious or non-serious using the 
criteria defined in Section 8.1.6 of this protocol.  
Any events defined as serious will also be reported following SAE reporting guidelines outlined 
in Section 8.3 of this protocol. 
 
8.3 
Adverse Event Reporting 
8.3.1 Non-Serious Adverse Events 
Non-serious AEs will be followed to resolution or until the study ends, and will be reported to 
the sponsor as requested, to the IRB according to IRB policies, to the DSMB as required, and to 
the FDA at least annually in the annual report. 
 
AEs meeting the pausing criteria outlined in Section 8.5 of this protocol will be reported to the 
sponsor following the SAE reporting guidelines. AE data will be submitted to the IND Sponsor 
when requested for periodic safety assessments, review of IND annual reports, review of IND 
safety reports, and preparation of final study reports. 
8.3.2 Serious Adverse Events 
Office of Clinical Research Policy and Regulatory Operations Clinical Safety Office 
All SAEs (regardless of relationship and whether or not they are also unanticipated problems) 
must be reported according to the sponsor’s reporting plan by using the REDCap system. Deaths 
and immediately life-threatening SAEs must be reported to the CSO within 1 business day after 
the site becomes aware of the event. All other SAEs must be reported within 3 working days of 
notification of the SAE occurrence to: 

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• REDCap website: https://crimsonredcap.cc.nih.gov/redcap/index.php 
• OCRPRO CSO Phone: 301-846-5301, Fax: 301-846-6224, E-mail: rchspsafety@mail.nih.gov 
 
SAEs that have not resolved by the end of the follow-up period are followed until final outcome 
is known. If it is not possible to obtain a final outcome for an SAE (e.g., the subject is lost to 
follow-up), the reason a final outcome could not be obtained will be recorded by the investigator 
on the AE case report form and according to the sponsor’s reporting guidelines. 
 
SAEs that occur after the study follow-up period (study day 180) that are reported to and are 
assessed by the investigator to be possibly, probably, or definitely related to study drug must be 
reported to the CSO. 
Data and Safety Monitoring Board 
All SAEs must be reported by telephone (followed by written report), email, or fax within 1 
working day of notification of the SAE occurrence to: 
• The DSMB executive secretary – Phone: 301-846-5301, Fax: 301-846-6224, Email: 
niaiddsmbia@mail.nih.gov 
Johns Hopkins University Biosafety Committee 
All SAEs must be reported by telephone (followed by written report), email, or fax within 1 
working day of notification of the SAE occurrence to: 
• The JHU Institutional Biosafety Committee: Phone: 410-955-5918, Fax 410-955-5929 
Institutional Review Board Reporting 
All SAEs will be reported to WIRB-Copernicus Group, Inc (WCG IRB) and UVM IRB as per 
guidelines, respectively, for JHU and UVM as below: 
• WCG IRB Guidelines:  
WCG IRB Phone: 800-562-4789, Fax: 360-252-2498 
www.wcgirb.com for updated reporting guidelines  
• UVM IRB Guidelines: 
o Investigators are required to report AEs that fit the following criteria: 
 Report timelines are based on of the time the investigator or site personnel 
becomes aware of them:  
• A local death report within 48 hours  
• All other local AEs should be reported promptly, not to exceed 7 days 
o Local AEs (whether or not they are serious) that are unexpected, and possibly, 
probably, or definitely related to study participation: 
 Local AE: A negative side effect resulting from the study intervention that 
occurred to a subject enrolled at UVM or another research site under the 
jurisdiction of the UVM IRB.  
 Unexpected: An event does not meet the criteria of unexpected if it is (1) 
included in the current protocol, drug/device brochure, or the informed 
consent; or (2) due to the subject’s underlying disease or predisposing risk 
factors 
 Related: An AE is considered to be related if there is a reasonable 
possibility that the event was caused by the protocol or study 

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interventions. A related event has a strong temporal relationship to the 
drug, device, or intervention, and an alternative cause is unlikely. If it 
cannot be determined whether an event is related, it should be reported as 
“possibly related.” 
o At UVM, all SAEs must be reported to the UVM IRB according to UVM IRB 
guidelines. 
o UVM IRB Phone: 802-656-5040, Fax: 802-656-5041 
8.3.3 Unanticipated Problems 
Unanticipated problems that are also AEs must be reported to the CSO and sent  
sent by using the REDCap system no later than 7 calendar days of site awareness of the event. 
UPs that are not AEs are not reported to the Sponsor CSO. Unanticipated problems that are not 
AEs are not reported to the sponsor CSO. Report unanticipated problems that are also AEs to the 
CSO according to the sponsor’s reporting guidelines. 
 
8.4 
Sponsor’s Reporting Responsibilities 
SUSARs, as defined in 21 Code of Federal Regulations (CFR) 312.32 and determined by the 
IND sponsor, must be reported to the FDA and all participating investigators as IND Safety 
Reports. IND Safety Reports will also be sent to other investigators conducting research under 
the OCRPRO IND and will be shared with other stakeholders per applicable agreements (e.g. 
CRADAs and CTAs). 
 
The sponsor will also submit a brief report of the progress of the investigation to the FDA on an 
annual basis, as defined in 21 CFR 312.33.  
 
AEs that are also unanticipated problems will be summarized by the IND sponsor and distributed 
to investigators. 
 
8.5 
Halting Rules 
“Halting” is discontinuation of study agent for all subjects in a study and suspension of 
enrollment until a decision is made to either resume or permanently discontinue such 
activity.  Subjects are still followed for safety.  
 
8.5.1 Halting Rules for the Study 
If the LA Zika vaccine is considered unacceptably reactogenic, as defined below, additional 
inoculations will be suspended for all subjects and enrollment will be suspended (halted) until 
the DSMB and study sponsor have reviewed the data and recommend that enrollment and study 
agent administration can continue. The PI will notify the DSMB at the time that halting criteria 
are met and obtain a recommendation concerning continuation, modification, or termination of 
the study. The PI will submit the written DSMB summary reports with recommendations to the 
IRB(s). 
 

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The study PI/protocol chair and/or CSO will determine if the study should be halted. In addition, 
the FDA may halt the study at any time following review of any safety concerns. A local IRB 
may halt the study at its site.  
 
8.5.1.1 Halting for Unacceptable Reactogenicity 
Following administration of rZIKV/D4∆30-713, the following criteria will be used to define 
unacceptable reactogenicity of the rZIKV/D4∆30-713 vaccine: 
1. 
One or more subjects experience an SAE (as defined in Section 8.1.6 of this protocol) 
that is determined to be possibly, probably, or definitely related to the administered 
vaccine (as defined in Section 8.2.4. of this protocol); 
2. 
One or more subjects experience anaphylaxis that is possibly, probably or definitely 
related to the administered vaccine; 
3. 
One or more subjects experience a Grade 4 adverse event that is definitely, probably, or 
possibly related to the administered vaccine; 
4. 
Two or more subjects experience the same objective physical finding of severity Grade 
3 that is definitely, probably, or possibly related to the administered vaccine, with the 
exception of Grade 3 erythema at the injection site, as defined in Section 8.2.3. of this 
protocol;  
5. 
Two or more subjects experience the same Grade 3 laboratory abnormality that is 
possibly, probably, or definitely related to the administered vaccine; 
6. 
Two or more subjects experience an ANC ≥ 500/mm3 but < 750/mm3 for > 5 days 
duration1;  
7. 
Two or more subjects experience an ANC < 500/mm3 for any duration1; or 
8. 
Two or more subjects experience a vaccine-associated Zika-like syndrome, defined as 
an infection2 associated with fever and 2 or more of the following symptoms: 
a. 
Grade 2 or greater headache lasting ≥ 12 hours 
b. 
Grade 2 or greater photophobia lasting ≥ 12 hours 
c. 
Grade 2 or greater generalized myalgia lasting ≥ 12 hours 
8.5.1.2 Halting for Shedding of Zika vaccine in Semen or Vaginal Secretions 
1. Should the Zika vaccine be detected in either semen or vaginal secretions following 
vaccination, the study enrollment will be halted pending discussion with the DSMB and 
the FDA. Subjects already enrolled will continue to be followed and additional specimens 
collected per the protocol. 
8.5.2 Reporting a Study Halt 
If halting criteria are met, a description of the AE(s) or safety issue(s) must be reported by the PI 
within 1 business day to the CSO and the local IRB according to their requirements. 
• The PI will notify the DSMB 
• The CSO or designee will notify all other site Investigators by email or through the 
specified pathway 
8.5.3 Resumption of a Halted Study 
The IND sponsor, in collaboration with the PI and DSMB, and in the case of a study halt due to 
shedding of Zika vaccine in semen or vaginal secretions, with the FDA, will determine if it is 
safe to resume the study. The CSO or designee will notify the site investigators of the decision to 
resume the study. The site investigators will notify their local IRB(s) of the decision. 

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8.6 
Safety Oversight 
8.6.1 Safety Review and Communications Plan (SRCP) 
A Safety Review and Communication Plan (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the PI and the IND sponsor CSO that 
delineates the safety oversight responsibilities of the PI, the CSO, and other stakeholders. The 
SRCP also includes the overall plan for conducting periodic safety surveillance assessments. 
8.6.2 Sponsor Medical Monitor 
A medical monitor, Shirley Jankelevich, MD, representing the IND sponsor (OCRPRO), has 
been appointed for safety oversight in this clinical study. The sponsor medical monitor will be 
responsible for performing safety assessments as outlined in an SRCP, as defined in Section 
8.6.1. 
8.6.3 NIAID Intramural Data and Safety Monitoring Board 
The NIAID intramural DSMB includes independent experts that do not have direct involvement 
in the conduct of the study and have no significant conflicts of interests as defined by NIAID 
policy. The NIAID intramural DSMB is constituted to review the safety data of NIAID 
intramural clinical studies that require DSMB oversight, and consists of experts in infectious 
diseases, biostatistics, and clinical trials. The PI’s designee will provide the DSMB executive 
secretary with blinding codes in a sealed envelope in case the DSMB requires this information to 
make its recommendations. The DSMB will review the study prior to initiation and twice a year 
thereafter. The board may convene additional reviews as necessary. Prior to each review, the PI 
will submit a summary of cumulative safety data in a format acceptable (by unblinded cohort if 
requested) to the board. The board will review the study data to evaluate the safety, efficacy, 
study progress, and conduct of the study. Reports of SAEs will be submitted by the PI to the 
board at the same time they are reported to the sponsor and IRB. All unanticipated problems will 
be submitted to the DSMB at the same time they are submitted to the IRB or IND sponsor. IND 
Safety Reports will be submitted to the DSMB by the investigator after their receipt. The PI will 
notify the DSMB of any cases of intentional or unintentional unblinding as soon as possible. The 
PI will notify the board at the time halting criteria are met, and obtain a recommendation 
concerning continuation, modification, or termination of the study. The PI will submit the written 
DSMB summary reports with recommendations to the IRB(s). 
9 
DATA COLLECTION AND MONITORING 
9.1 
Source Documentation and Data Collection 
Complete source documentation (laboratory test reports, hospital or medical records, progress notes, 
observations, etc.) is required for every study subject for the duration of the study. The subject’s 
study record must record his/her participation in the clinical trial and, after unblinding, the 
randomization treatment received (with doses and frequency) or other concomitant medications or 
interventions administered, as well as any adverse reactions experienced during the trial.  
Data from source documentation for subjects enrolled in the study will be entered into the Clinical 
Research Information Management System of NIAID (CRIMSON) Data System. The data entry is 
to be completed on an ongoing basis during the study. Data entry into CRIMSON will be performed 
by authorized individuals, and each individual entering data into CRIMSON will have a unique user 

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ID and password. Corrections to the data system will be tracked electronically (password protected) 
with time, date, individual making the correction, and what was changed.  
 
Corrections to the source document must be made by striking through the incorrect entry with a 
single line (taking care not to obliterate or render the original entry illegible) and entering the correct 
information adjacent to the incorrect entry. Corrections must be initialed and dated by the person 
making the correction whenever possible. Source documentation should support the data collected in 
CRIMSON and must be signed and dated by the person recording and/or reviewing the data.  
 
The investigator is responsible for the accuracy, completeness, and timeliness of the data 
reported to the sponsor in CRIMSON. All data entered into CRIMSON should be reviewed by 
the investigator/designee, and signed as required with written or electronic signature, as 
appropriate. Data reported in CRIMSON should be consistent with source documents or the 
discrepancies should be explained. Source documentation will be made available for review or 
audit by the sponsor or designee and any applicable federal authorities. 
9.2 
Study Documentation 
Study-related documentation will be completed as required by the IRBs, the sponsor, and 
regulatory authorities. Continuing review documentation will be submitted by the investigator to 
the IRBs by the anniversary date of initial review as specified by each IRB. An annual report will 
be submitted by the sponsor to the FDA according to regulations. These reports will provide a 
brief description of the progress of the investigation as outlined in 21 CFR 312.33, and will 
include any protocol revisions if not previously submitted. 
 
The PI will maintain adequate records to account for the disposition of the investigational 
products, including dates of receipt and quantity, current inventory, and dispensation to subjects. 
If the study is terminated, suspended, or completed, the PI will return all unused supplies of the 
investigational product to a NIAID-approved repository or destroy according to the sponsor’s 
recommendation. 
 
9.3 
Retention of Specimens 
All specimens collected as part of this trial will be stored for future research as part of our 
approved biosample repository for vaccine research. These samples may be used to learn more 
about flavivirus infection and other diseases. These samples will not be sold or used to make 
commercial products. All samples stored in the repository will be labeled with the study subject 
ID numbers, which, by themselves, cannot identify study subjects, but are linkable to other 
research databases (e.g., from questionnaires, clinical assessments, logbooks, etc.) generated by 
the main study. The repository database will contain only the study subject ID numbers. A 
master log linking the study subject ID numbers to the names of the subjects will be maintained 
in a password-protected database system with limited access to authorized research team 
members. 
 
9.4 
Retention of Records 
The PI is responsible for retaining all essential documents listed in the International Conference 
on Harmonisation (ICH) Good Clinical Practice (GCP) guidelines. Trial-related documents will 

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be maintained by the investigator in a secure storage facility for a period of 2 years after final 
marketing approval of the vaccine or if 2 years have elapsed since the formal discontinuation of 
the product’s clinical development. These records are also to be maintained in compliance with 
IRB, state, and federal medical records’ retention requirements, whichever is longest. The 
sponsor is required to inform the investigator as to when such documents need no longer be 
retained. Storage of all trial-related documents will be such that confidentiality will be strictly 
maintained to the extent provided by federal, state, and local laws.  
 
It is the PI’s responsibility to retain copies of source documents until receipt of written 
notification to the contrary from NIAID OCRPRO. Study documents should not be destroyed 
without prior written agreement between NIAID OCRPRO and the PI. Should the PI wish to 
assign the study records to another party and/or move to another location, the PI must provide 
written notification of such intent to NIAID OCRPRO, with the name of the person who will 
accept responsibility for the transferred records and/or the new location. NIAID must be notified 
in writing, and written permission must be received by the site prior to destruction or relocation 
of research records. 
 
9.5 
Protocol Compliance 
The PI will conduct the trial in compliance with the protocol agreed to by the sponsor. The 
investigator will not implement any deviation from, or changes to, the protocol without 
agreement, prior review, and documented approval by the sponsor and the IRB that granted 
original approval for the study. The DSMB will be made aware of all protocol revisions (other 
than administrative) and will review any changes to the protocol that involve DSMB oversight or 
involve changes to the study’s data and safety monitoring plan. However, the investigator may 
implement a deviation from, or change in, the protocol to eliminate an immediate hazard(s) to 
subjects without prior IRB or sponsor approval, or when the change(s) involves only logistical or 
administrative aspects of the trial (i.e., change of telephone number[s]). In the event of a medical 
emergency, the PI will perform any medical procedures that are deemed medically appropriate.  
 
As soon as possible, the implemented deviation or change, the reasons for it, and, if appropriate, 
the proposed protocol amendment(s) should be submitted to the Sponsor, IRB, DSMB, and the 
regulatory authorities. 
 
9.6 
Clinical Investigator’s Brochure 
Investigators will receive the current versions of the Clinical Investigator’s Brochure for the live 
attenuated Zika vaccine rZIKV/D4∆30-713, which comprehensively describes all the available 
preclinical experience with the experimental vaccine. If relevant new information becomes 
available during the course of the trial, the investigators will receive a revised brochure or an 
amendment to the current version.  
 
9.7 
Study Monitoring 
As per ICH-GCP 5.18 and FDA 21 CFR 312.50, clinical protocols are required to be adequately 
monitored by the sponsor. This study monitoring will be conducted according to the “NIAID 
Intramural Clinical Monitoring Guidelines.” The sponsor will monitor all aspects of the study, 

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with respect to current GCP, for compliance with applicable government regulations. Prior to the 
start of the study, the investigator will be informed of the frequency of monitoring visits and will 
be given reasonable notification prior to each visit. The objectives of a monitoring visit will be to 
verify the prompt and accurate recording of all monitored data points, and prompt reporting of 
SAEs; to check the availability of signed informed consent forms and documentation of the 
informed consent process for each monitored subject; to compare CRIMSON reports and line 
listings with source data for completeness and accuracy; and to help ensure investigators are in 
compliance with the protocol. The monitors will also inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections, FDA) and 
applicable guidelines (ICH GCP) are being followed. During the monitoring visit, the 
investigator (and/or designee) and other study personnel should be available to discuss the study. 
Study documents must be available for review throughout the course of the study. The sponsor 
will retain original copies of Form FDA 1572 and copies of other study documents as deemed 
necessary. 
 
The investigator (and/or designee) will make study documents (e.g., consent forms, CRIMSON, 
and pertinent hospital or clinical records) readily available for inspection by the local IRB, the 
FDA, the site monitors, and NIAID staff for confirmation of the study data.  
 
A specific protocol monitoring plan will be discussed with the PI and study staff prior to 
enrollment. The plan will outline the frequency of monitoring visits based on such factors as 
study enrollment, data collection status, and regulatory obligations. 
 
10 STATISTICAL CONSIDERATIONS 
10.1 General Design 
The goal of the Phase 1 vaccine trial is to determine the safety and immunogenicity of 
rZIKV/D4∆30-713, a live attenuated Zika vaccine candidate in healthy human subjects. The 
purpose of the Phase 1 trial is to evaluate the reactogenicity, immune response to Zika vaccine 
and the persistence of antibody following subcutaneous administration of a single dose of 
rZIKV/D4∆30-713. 
10.2 Statistical Methods 
This study, like other Phase 1 studies, is exploratory rather than confirmatory; its purpose is to 
estimate event frequencies and patterns of immune responses rather than to test formal statistical 
hypotheses. Descriptive approaches will be used to meet the protocol objectives as stated in 
Section 3 of this protocol, as outlined below. Results will be presented in tabular format, as well 
as graphically where appropriate. 
 
Primary Objective 1: Determine the safety and reactogenicity of a single dose of the LA Zika 
vaccine rZIKV/D4∆30-713, as assessed by the frequency of adverse events (AEs) and adverse 
reactions (AR) as defined as vaccine-related adverse events (AE)s, graded by severity.  
The frequency of immediate, systemic, and local AEs and ARs following vaccination will 
be summarized. AEs and ARs will be displayed in tabular format, with line listings of 
individual clinical and laboratory AEs and ARs classified as immediate, systemic, and 

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local events and AEs and ARS will be summarized by severity and relationship to 
vaccine by individuals and each group (rZIKV/D4∆30-713 and placebo). 
 
Primary Objective 2: To determine the immunogenicity of a single dose of rZIKV/D4∆30-713, 
as assessed by: 
• Determination of the serum plaque reduction neutralization titer 50% (PRNT50) to ZIKV 
for each subject at Study Day 28, 56, and 90 post-inoculation. Seroconversion will be 
defined as achieving a PRNT50 ≥ 1:10 at any time-point through Study Day 90 (Day 28, 
56, or 90). The peak PRNT50 to ZIKV through Study Day 90 will be calculated for each 
subject included in the per-protocol an intent-to-treat analysis and the geometric mean 
peak titer for vaccinated subjects will be calculated. 
Secondary Objective 1: Assess the frequency, quantity, and duration of viremia (virus in the 
blood) induced by a single dose of the rZIKV/D4∆30-713 vaccine. The mean peak viremia, 
mean day of onset of viremia, and mean duration of viremia will be calculated. Viremia will be 
detected by culture (infectious virus) and by RT-PCR. 
 
Secondary Objective 2: Determine the number of vaccinees infected with rZIKV/D4∆30-713. 
Infection is defined as recovery of infectious vaccine virus from the blood, serum or urine of a 
subject and/or by seroconversion to ZIKV. Seroconversion will be defined as achieving a 
PRNT50 ≥ 1:10 by Study Day 90 post-vaccination.  
 
Secondary Objective 3: Evaluate the immunogenicity of rZIKV/D4∆30-713 in flavivirus-naïve 
subjects as assessed by the PRNT50 to ZIKV, for each subject at Study Day 28, 56, and 90 post-
administration of LA Zika vaccine. 
 
Secondary Objective 4: Determine the durability of antibody response 26 weeks after 
vaccination. 
 
Secondary Objective 5:  Determine the quantity and duration of ZIKV presence as determined 
by: 
• The peak virus titer in the blood and the duration of viremia induced by the LA Zika 
vaccine as determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in urine determined by RT-
PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in vaginal secretions 
determined by RT-PCR and virus culture 
• The quantity and duration of possible Zika vaccine shedding in semen determined by RT-
PCR and virus culture 
Exploratory Objective 1: To evaluate the phenotype of peripheral blood mononuclear cells 
(PBMCs) at primary infection with the rZIKV/D4∆30-713 vaccine. 
 
Exploratory Objective 2: To evaluate the cellular immune response to primary infection with the 
rZIKV/D4∆30-713vaccine. 
 

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Exploratory Objective 3: To evaluate the innate immune response to primary infection with the 
rZIKV/D4∆30-713 vaccine. 
 
Exploratory Objective 4: To evaluate B and T cell memory responses following primary 
infection with rZIKV/D4∆30-713 vaccine.  
 
Exploratory Objective 5:  To evaluate the antibody response in saliva to infection with 
rZIKV/D4∆30-713 as a possible diagnostic tool. This will be done in a subset of subjects (those 
enrolled at the Center for Immunization Research [CIR]). 
 
10.3 Safety Endpoint 
The primary safety endpoint is the frequency of rZIKV/D4∆30-713-related AEs, as classified by 
both severity and seriousness, through active and passive surveillance. Separate assessments of 
systemic and local reactions will be performed. 
10.4 Immunogenicity 
Zika virus neutralizing antibody titers will be measured on study day 0 (prior to inoculation) and 
Study Days 28, 56, 90, and 180 following vaccination with rZIKV/D4∆30-713. Seropositivity to 
ZIKV will be defined as a PRNT50 of ≥ 1:10. Seroconversion will be defined as a PRNT50 of ≥ 
1:10 to ZIKV by study day 90. Seroconversion will be defined as a PRNT50 of ≥ 1:10 to ZIKV or 
following administration of rZIKV/D4∆30-713. 
10.5 Per Protocol Analysis 
10.5.1 Safety Data 
Subjects who have completed ≥ 5 visits through Study Day 28 will be included in the per-
protocol analysis of the AE / adverse reaction data. All SAEs and AEs of special interest will be 
included in the per-protocol analysis. 
10.5.2 Virology 
Subjects who have completed ≥ 5 visits through Study Day 28 will be included in the per-
protocol analysis of the virology data. 
10.5.3 Immunogenicity Data 
Subjects who have completed at least 2 of the Study Day 28, Study Day 56, and Study Day 90 
visits will be included in the per-protocol analysis of the immunogenicity data. 
10.6 Intent-to-treat analysis 
10.6.1 Safety Data 
All subjects, regardless of the number visits that they missed, will be included in the safety 
analysis. This will include subjects who were replaced. 
10.6.2 Virology 
All subjects, with the exception of those who were replaced, regardless of the number visits that 
they missed, will be included in the virology analysis. 

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10.6.3 Immunogenicity Data 
All subjects, with the exception of those subjects who were replaced, who have had at least one 
specimen collected at an immunogenicity time-point, regardless of the number visits that they 
missed, will be included in the immunogenicity analysis. 
11 PROTECTION OF HUMAN SUBJECTS 
11.1 Institutional Review Boards 
The PI will be responsible for obtaining IRB approval for the study. Before the start of the study, 
the appropriate documents (including, but not limited to, the protocol, Investigator’s Brochure, 
Informed Consent Form, information sheets, and advertisements) will be submitted to the IRB 
for approval. A copy of the study approval (including approval of the informed consent form) is 
to be maintained in the investigator study document binder and a copy will be supplied to the 
sponsor. During the study, the investigator is responsible for providing the IRB with all 
documents subject to review (e.g., protocol amendments, informed consent form updates, 
advertisements, and any written information that may be provided to the subject). Annual reports 
on the progress of the study will be made to the IRB by the investigator in accordance with IRB 
guidelines and government regulations. 
11.2 Data Safety Monitoring Board 
The Sponsor has convened a DSMB to review this study at regular intervals. The DSMB is 
comprised of individuals with expertise in clinical flavivirus vaccine studies, statistics, clinical 
trials, and infectious diseases. The  study team will provide the DSMB with all requested 
data/materials they have requested for review at regular intervals determined by the DSMB. If a 
Halting Rule is triggered, the DSMB will review the event and decide, in collaboration with the 
PI and Sponsor, whether or not the study should continue (Section 8.5). 
11.3 Informed Consent 
In obtaining and documenting informed consent, the investigator and study staff must comply 
with the applicable regulatory requirements, GCP guidelines, and ethical principles. The written 
informed consent form must be approved by the IRB prior to its use. The subject may withdraw 
consent at any time during the course of the trial. A copy of the informed consent document will 
be given to the subject for his or her records. The rights and welfare of the subjects will be 
protected by emphasizing to them that the quality of their medical care will not be adversely 
affected if they decline to participate in this study. 
11.4 Risks 
Risks to the subjects are associated with venipuncture, topical anesthetic cream, and study 
agent(s). These risks are outlined below. In addition, questions asked regarding a subject’s use of 
contraception may make the subject uncomfortable. These questions will be asked in a private 
area and the subject will be made aware that he or she does not have to answer any question that 
they do not feel comfortable answering. Because this is a live vaccine, there is an assumed risk to 
the fetus should the mother receive the vaccine when pregnant.  The vaccine virus is highly 
attenuated and is expected to have only minimal replication in the host. Nevertheless, female 
subjects will be cautioned of the unknown risk of study agents to the fetus and will be advised to 
use effective birth control methods for the duration of the study.  Current evidence does not 
suggest any risk to the fetus should a woman become pregnant after clearing infectious ZIKV 

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from the blood. For this reason, the vaccine should not pose any risk to the fetus should a subject 
become pregnant after clearing infectious vaccine virus from the blood (presumed to be 
approximately day 16 and after post-vaccination). 
11.4.1 Venipuncture 
The total amount of blood to be drawn throughout the 6-month duration of the primary study is 
approximately 500 mL. Risks occasionally associated with venipuncture include excessive 
bleeding, pain, bruising, or hematoma at the site of venipuncture, lightheadedness, and syncope 
(rarely). Infection may occur rarely. 
 
11.4.2 Risk of Use of Vaginal Cup 
The vaginal cup is a commercially-available product used during menstruation. It is contra-
indicated in women with an intrauterine device (IUD) in place. For women who have an IUD in 
place, vaginal secretion specimens will be collected by use of vaginal swab. 
11.4.3 Topical Anesthetic Cream 
Risks occasionally associated with the use of topical anesthetic cream include temporary skin 
discoloration, skin irritation, rash, hives, and rarely, dizziness or drowsiness. 
11.4.4 Inoculation 
Possible local vaccine reactions include pain, swelling, or erythema for 2 to 3 days, 
lymphadenopathy, or pruritus at the injection site. Potential systemic reactions that may occur 
include symptoms of ZIKV such as rash, fever, headache, non-purulent conjunctivitis, 
generalized myalgias, and arthralgias. Immediate hypersensitivity reactions including urticaria, 
anaphylaxis, or other IgE-mediated responses are possible, as with any vaccine. Guillain-Barre 
syndrome has been reported in persons infected with wt ZIKV. 
11.4.5 Other Risks 
Subjects may be asked to defer routine immunization (such as influenza) with live vaccines 28 
days prior or killed vaccines 14 days prior to inoculation or until after 28 days following 
inoculation, with the exception of COVID-19 vaccines. This may increase the risk that the 
subject will be infected with an influenza virus during this period. As with any investigational 
vaccine, there is a theoretical possibility of risks about which we have no present knowledge. 
Subjects will be informed of any such risks should further information become available. 
11.5 Benefits 
Subjects will not receive any direct benefit from participation in this study. They will receive a 
physical examination and laboratory screening for HIV infection, hepatitis B infection, and 
hepatitis C infection. They may potentially develop antibodies against ZIKV virus. It is hoped 
that information gained in this study will contribute to the development of a safe and effective 
vaccine for the prevention of ZIKV infection and to a better understanding of the protective 
immune response to ZIKV infection. 
11.6 Compensation 
Subjects will be compensated up to $80 for screening, up to $150 for inoculation day, and up to 
$80 for each completed scheduled follow-up visit. They will also receive up to a $250 bonus if 
all study visits are completed on time. Subjects will be compensated for the screening only if 

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they are enrolled in the study. Subjects will only be compensated for the visits that they 
complete. In addition, should subjects be asked to return to the clinic after Study Day 180 for 
collection of vaginal secretions (women) or semen (men), they will be compensated up to $80.00 
for each additional visit. Alternates who come to the clinic on vaccination day but who are not 
vaccinated due to fulfillment of enrollment for that day will be compensated up to $150. Subjects 
enrolled in the study will receive a maximum total compensation of up to $1,520 if they 
complete all scheduled visits. Subjects will receive additional compensation, described above, if 
they complete additional visits as required by the protocol. 
11.6.1 Compensation for Injury 
The services at the Johns Hopkins Hospital, the Johns Hopkins Bayview Medical Center, or the 
University of Vermont Medical Center will be available to subjects who require inpatient care 
for any injury resulting from participation in the trial. This short-term medical care will be paid 
for through our contract with NIH. Short-term medical care will be given at a facility determined 
by JHU/UVM and NIH. No long-term medical care or financial compensation for research-
related injuries will be offered by the Johns Hopkins University, Johns Hopkins Hospital, the 
NIH, or the federal government. 
11.7 Confidentiality 
All study-related information will be stored securely at the study site. All subject information 
will be stored in locked file cabinets in areas with access limited to study staff. All laboratory 
specimens, reports, study data collection, and process and administrative forms will be identified 
by coded number only to maintain subject confidentiality. Computer entry will be done using a 
study ID number for each subject and all local databases will be secured with password-protected 
access systems. Forms, lists, logbooks, appointments books, and any other listings that link 
subject study ID numbers to other identifying information will be stored in a separate, locked file 
in an area with limited access. A subject’s study information will not be released without the 
written permission of the subject, except as necessary for monitoring by the Sponsor and/or its 
contractors and the FDA. 
11.8 Biohazard Containment 
As the transmission of HIV and other blood-borne pathogens can occur through contact with 
contaminated needles, blood, and blood products, appropriate blood and secretion precautions 
will be employed by all personnel during the drawing of blood and shipping and handling of all 
specimens for this study, as currently recommended by the Centers for Disease Control and 
Prevention and the NIH.  
 
All infectious specimens will be transported using packaging mandated in the Code of Federal 
Regulations, 42 CFR Part 72. Please also refer to individual carrier guidelines (e.g., Federal 
Express, Airborne Express) for specific instructions. 
12 PUBLICATION POLICY 
The International Committee of Medical Journal Editors (ICMJE) member journals have adopted 
a trials’ registration policy as a condition for publication. This policy requires that all clinical 
trials be registered prior to enrollment of any subject in a public trials registry, such as 
ClinicalTrials.gov, which is sponsored by the National Library of Medicine. The ICMJE defines 
a clinical trial as any research project that prospectively assigns human subjects to intervention 

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or comparison groups to study the cause-and-effect relationship between a medical intervention 
and a health outcome. Studies designed for other purposes, such as to study pharmacokinetics or 
major toxicity, would be exempt from this policy. 
13 REFERENCES 
 
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Trans R Soc Trop Med Hyg 1952;46:509-20. 
2. Hayes EB. Zika virus outside Africa. Emerg Infect Dis 2009;15:1347-50. 
3. Duffy MR, Chen TH, Hancock WT, et al. Zika virus outbreak on Yap Island, Federated States 
of Micronesia. N Engl J Med 2009;360:2536-43. 
4. Brasil P, Pereira JP, Jr., Raja Gabaglia C, et al. Zika Virus Infection in Pregnant Women in 
Rio de Janeiro - Preliminary Report. N Engl J Med 2016 
5. Cao-Lormeau VM, Roche C, Teissier A, et al. Zika virus, French polynesia, South pacific, 
2013. Emerg Infect Dis 2014;20:1085-6. 
6. Musso D, Nilles EJ and Cao-Lormeau VM. Rapid spread of emerging Zika virus in the Pacific 
area. Clin Microbiol Infect 2014;20:O595-6. 
7. Organization WH. WHO Situation Report Zika Virus, Microcephaly, Guillain-Barre 
Syndrome. Vol. 2017, 2017 
8. Schuler-Faccini L, Ribeiro EM, Feitosa IM, et al. Possible Association Between Zika Virus 
Infection and Microcephaly - Brazil, 2015. MMWR Morb Mortal Wkly Rep 2016;65:59-62. 
9. Mlakar J, Korva M, Tul N, et al. Zika Virus Associated with Microcephaly. N Engl J Med 
2016;374:951-8. 
10. Kleber de Oliveira W, Cortez-Escalante J, De Oliveira WT, et al. Increase in Reported 
Prevalence of Microcephaly in Infants Born to Women Living in Areas with Confirmed Zika 
Virus Transmission During the First Trimester of Pregnancy - Brazil, 2015. MMWR Morb 
Mortal Wkly Rep 2016;65:242-247. 
11. Rasmussen SA, Jamieson DJ, Honein MA and Petersen LR. Zika Virus and Birth Defects — 
Reviewing the Evidence for Causality. New England Journal of Medicine 2016;374:1981-7. 
12. Pacheco O, Beltran M, Nelson CA, et al. Zika Virus Disease in Colombia - Preliminary 
Report. N Engl J Med 2016;0:null. 
13. Reynolds MR, Jones AM, Petersen EE, et al. Vital Signs: Update on Zika Virus-Associated 
Birth Defects and Evaluation of All U.S. Infants with Congenital Zika Virus Exposure - U.S. 
Zika Pregnancy Registry, 2016. MMWR Morb Mortal Wkly Rep 2017;66:366-373. 
14. Musso D, Gubler DJ. Zika Virus. Clin Microbiol Rev 2016;29:487-524. 

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15. Ferreira-de-Brito A, Ribeiro IP, Miranda RM, et al. First detection of natural infection of 
Aedes aegypti with Zika virus in Brazil and throughout South America. Mem Inst Oswaldo Cruz 
2016;111:655-658. 
16. Oster AM, Brooks JT, Stryker JE, et al. Interim Guidelines for the Prevention of Sexual 
Transmission ofZika Virus Infection - United States, 2016. MMWR Morb Mortal Wkly Rep 
2016;65 
17. Hills SL, Russell K, Hennessey M, et al. Transmission of Zika Virus Through Sexual Contact 
with Travelers to Areas of Ongoing Transmission - Continental United States, 2016. MMWR 
Morb Mortal Wkly Rep 2016;65:215-6. 
18. Oster AM, Russell K, Stryker JE, et al. Update: Interim Guidance for Prevention of Sexual 
Transmission of Zika Virus - United States, 2016. MMWR Morb Mortal Wkly Rep 
2016;65:323-5. 
19. Foy BD, Kobylinski KC, Chilson Foy JL, et al. Probable non-vector-borne transmission of 
Zika virus, Colorado, USA. Emerg Infect Dis 2011;17:880-2. 
20. Reusken C, Pas S, GeurtsvanKessel C, et al. Longitudinal follow-up of Zika virus RNA in 
semen of a traveller returning from Barbados to the Netherlands with Zika virus disease, March 
2016. Euro Surveill 2016;21 
21. D'Ortenzio E, Matheron S, Yazdanpanah Y, et al. Evidence of Sexual Transmission of Zika 
Virus. N Engl J Med 2016;374:2195-8. 
22. Davidson A, Slavinski S, Komoto K, Rakeman J and Weiss D. Suspected Female-to-Male 
Sexual Transmission of Zika Virus - New York City. MMWR Morb Mortal Wkly Rep 2016 
23. Deckard DT, Chung WM, Brooks JT, et al. Male-to-Male Sexual Transmission of Zika Virus 
- Texas, January 2016. MMWR Morb Mortal Wkly Rep 2016;65:372-4. 
24. Brooks RB, Carlos MP, Myers RA, et al. Likely Sexual Transmission of Zika Virus from a 
Man with No Symptoms of Infection - Maryland, 2016. MMWR Morb Mortal Wkly Rep 
2016;65:915-6. 
25. Atkinson B, Hearn P, Afrough B, et al. Detection of Zika Virus in Semen. Emerg Infect Dis 
2016;22:940. 
26. Matheron S, D'Ortenzio E, Leparc-Goffart I, Hubert B, de Lamballerie X and Yazdanpanah 
Y. Long Lasting Persistence of Zika Virus in Semen. Clin Infect Dis 2016 
27. Barzon L, Pacenti M, Franchin E, et al. Infection dynamics in a traveller with persistent 
shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 
2016. Euro Surveill 2016;21 
28. Arsuaga M, Bujalance SG, Diaz-Menendez M, Vazquez A and Arribas JR. Probable sexual 
transmission of Zika virus from a vasectomised man. Lancet Infect Dis 2016;16:1107. 

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29. Mead PS, Duggal NK, Hook SA, et al. Zika Virus Shedding in Semen of Symptomatic 
Infected Men. N Engl J Med 2018;378:1377-1385. 
30. Russell K, Hills SL, Oster AM, et al. Male-to-Female Sexual Transmission of Zika Virus-
United States, January-April 2016. Clin Infect Dis 2017;64:211-213. 
31. Yakob L, Kucharski A, Hue S and Edmunds WJ. Low risk of a sexually-transmitted Zika 
virus outbreak. Lancet Infect Dis 2016;16:1100-2. 
32. Durbin AP, McArthur JH, Marron JA, et al. rDEN2/4Delta30(ME), A Live Attenuated 
Chimeric Dengue Serotype 2 Vaccine Is Safe and Highly Immunogenic in Healthy Dengue-
Naive Adults. Hum Vaccin 2006;2:255-60. 
33. Wright PF, Ankrah S, Henderson SE, et al. Evaluation of the Langat/dengue 4 chimeric virus 
as a live attenuated tick-borne encephalitis vaccine for safety and immunogenicity in healthy 
adult volunteers. Vaccine 2008;26:882-90. 
34. Durbin AP, Kirkpatrick BD, Pierce KK, Schmidt AC and Whitehead SS. Development and 
clinical evaluation of multiple investigational monovalent DENV vaccines to identify 
components for inclusion in a live attenuated tetravalent DENV vaccine. Vaccine 2011;29:7242-
50. 
35. Durbin AP, Wright PF, Cox A, et al. The live attenuated chimeric vaccine 
rWN/DEN4Delta30 is well-tolerated and immunogenic in healthy flavivirus-naive adult 
volunteers. Vaccine 2013 
36. Durbin AP, Karron RA, Sun W, et al. Attenuation and immunogenicity in humans of a live 
dengue virus type-4 vaccine candidate with a 30 nucleotide deletion in its 3'-untranslated region. 
Am J Trop Med Hyg 2001;65:405-13. 
37. Durbin AP, Whitehead SS, McArthur J, et al. rDEN4 Delta 30, a Live Attenuated Dengue 
Virus Type 4 Vaccine Candidate, Is Safe, Immunogenic, and Highly Infectious in Healthy Adult 
Volunteers. J Infect Dis 2005;191:710-8. 
38. Durbin AP, Kirkpatrick BD, Pierce KK, et al. A Single Dose of Any of Four Different Live 
Attenuated Tetravalent Dengue Vaccines Is Safe and Immunogenic in Flavivirus-naive Adults: A 
Randomized, Double-blind Clinical Trial. J Infect Dis 2013;207:957-65. 
39. Kirkpatrick BD, Durbin AP, Pierce KK, et al. Robust and Balanced Immune Responses to 
All 4 Dengue Virus Serotypes Following Administration of a Single Dose of a Live Attenuated 
Tetravalent Dengue Vaccine to Healthy, Flavivirus-Naive Adults. J Infect Dis 2015;212:702-10. 
40. Durbin AP, Kirkpatrick BD, Pierce KK, et al. A 12-Month-Interval Dosing Study in Adults 
Indicates That a Single Dose of the National Institute of Allergy and Infectious Diseases 
Tetravalent Dengue Vaccine Induces a Robust Neutralizing Antibody Response. J Infect Dis 
2016;214:832-5. 

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41. Kirkpatrick BD, Whitehead SS, Pierce KK, et al. The live attenuated dengue vaccine TV003 
elicits complete protection against dengue in a human challenge model. Science Translational 
Medicine 2016;8:330ra36-330ra36. 
42. Pierce KK, Whitehead SS, Kirkpatrick BD, et al. A Live Attenuated Chimeric West Nile 
Virus Vaccine, rWN/DEN4Delta30, Is Well Tolerated and Immunogenic in Flavivirus-Naive 
Older Adult Volunteers. J Infect Dis 2017;215:52-55. 

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14 APPENDICES 
14.1 Appendix 1: Adverse Event Grading Table1 
Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
General Severity 
Grading 
Event that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Event that interferes 
with daily activity or 
requires more than 1 
dose of 
medication/treatment 
Event that prevents 
daily activity and 
requires medical 
intervention 
Event that has life 
threatening 
consequences and/or 
urgent intervention is 
indicated 
Local Reactogenicity 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Injection Site 
Tenderness 
Tenderness that is easily 
tolerated 
Tenderness that interferes 
with daily activity 
Tenderness that 
prevents daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Injection Site Pain 
Pain that is easily 
tolerated 
Pain that interferes with 
daily activity 
Pain that prevents daily 
activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Injection Site Pruritus 
Pruritus that is easily 
tolerated 
Pruritus that interferes 
with daily activity 
Pruritus that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Injection Site 
Induration 
> 0 - 20 mm 
> 20 - 50 mm 
> 50 mm 
Life-threatening 
consequences; urgent 
intervention indicated 
Injection Site 
Erythema 
> 0 - 20 mm 
> 20 - 50 mm 
> 50 mm 
Life-threatening 
consequences; urgent 
intervention indicated 
Systemic 
Reactogenicity 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Fever (Oral) 
100.4° F – 101.4°F 
101.5°F – 102.4°F  
≥ 102.5°F  
- 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Headache 
Headache that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Headache that interferes 
with daily activity or 
requires > 1 dose of 
medication 
Headache that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Retro-orbital Pain 
(ROP) 
ROP that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
ROP that interferes with 
daily activity or requires 
> 1 dose of medication 
ROP that prevents daily 
activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Non-purulent 
Conjunctivitis 
Easily tolerated, may 
require 1 dose of 
medication/treatment 
Interferes with daily 
activity or requires > 1 
dose of 
medication/treatment 
Prevents daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Photophobia 
Photophobia that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Photophobia that 
interferes with daily 
activity or requires > 1 
dose of medication 
Photophobia that 
prevents daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Nausea 
Nausea that is easily 
tolerated 
Nausea that interferes 
with daily activity or 
requires > 1 dose of 
medication 
Nausea that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Fatigue 
Fatigue that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Fatigue that interferes 
with daily activity or 
requires > 1 dose of 
medication 
Fatigue that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
Myalgia 
Myalgia that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Myalgia that interferes 
with daily activity or 
requires > 1 dose of 
medication 
Myalgia that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Arthralgia 
Arthralgia that is easily 
tolerated, may require 1 
dose of 
medication/treatment 
Arthralgia that interferes 
with daily activity or 
requires > 1 dose of 
medication 
Arthralgia that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
ZIKV-like Rash 
Rash is present but 
asymptomatic 
Rash is symptomatic 
(pruritus/pain) but does 
not interfere with 
function 
Rash is symptomatic 
and interferes with 
function 
Life-threatening 
consequences; urgent 
intervention indicated 
Solicited Lab AEs 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Leukopenia 
2,500 - 3,500/mm3 
1,500 - 2,499/mm3 
1,000 - 1,400/mm3 
Life-threatening 
consequences; urgent 
intervention indicated 
Neutropenia (Reduced 
ANC)2 
≥ 750 - 999/mm3 
≥ 500 - 749/mm3 
< 500/mm3 
Life-threatening 
consequences; urgent 
intervention indicated 
Thrombocytopenia 
(Reduced Platelets) 
100,000 – 120,000/mm3 
75,000 - 99,999/ mm3 
< 74,999/mm3 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Laboratory 
Values 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Leukocytosis 
(Increased WBCs) 
11,500 - 13,000/mm3 
13,001 - 15,000/mm3 
≥ 15,000 or < 
1,000/mm3 
Life-threatening 
consequences; urgent 
intervention indicated 
Hemoglobin (female) 
9.5 - 10.7 gm/dL 
8.0 - 9.4 gm/dL 
< 7.9 gm/dL 
Life-threatening 
consequences; urgent 
intervention indicated 
Hemoglobin (male) 
11.0 - 12.5 gm/dL 
9.0 – 10.9 gm/dL 
< 8.9 gm/dL 
Life-threatening 
consequences; urgent 
intervention indicated - 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
PT 
> 1.0 - 1.25 x ULN 
> 1.25 - 1.5 x ULN 
> 1.5 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated 
PTT 
> 1.0 - 1.66 x ULN 
> 1.66 - 2.33 x ULN 
> 2.33 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated - 
ALT 
> 1.25 - 2.5 x ULN 
> 2.5 - 5.0 x ULN 
> 5.0 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated - 
Creatinine 
1.5 - 1.7 mg/dL 
> 1.7 – 2.0 mg/dL 
> 2.0 mg/dL 
Life-threatening 
consequences; urgent 
intervention indicated - 
Fibrinogen, Decreased 
100 mg/dL to < lower 
limit of     
                          normal 
(LLN) 
50 - 99 mg/dL 
<50 mg/dL, or 
associated with gross 
bleeding, or associated 
with disseminated 
coagulation 
Life-threatening 
consequences; urgent 
intervention indicated - 
Fibrinogen, Increased 
> ULN to 600 mg/dL 
> 600 mg/dL 
N/A 
Life-threatening 
consequences; urgent 
intervention indicated 
Creatine 
phosphokinase (CPK) 
≥ 4 x ULN- 6 x ULN 
> 6 x ULN- 10 x ULN 
> 10 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated 
Sodium: 
Hyponatremia 
130 – 134 mEq/L 
123 – 129 mEq/L 
< 122 mEq/L 
Life-threatening 
consequences; urgent 
intervention indicated - 
Sodium: 
Hypernatremia 
145 – 150 mEq/L 
151 – 157 mEq/L 
> 158 mEq/L 
Life-threatening 
consequences; urgent 
intervention indicated - 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Potassium: 
Hypokalemia 
3.1 – 3.2 mEq/L 
2.9 – 3.0 mEq/L 
< 2.8 mEq/L 
Life-threatening 
consequences; urgent 
intervention indicated - 
Potassium: 
Hyperkalemia 
5.2 – 5.5 mEq/L 
5.6 – 6.0 mEq/L 
> 6.1 mEq/L 
Life-threatening 
consequences; urgent 
intervention indicated 
Phosphate: 
Hypophosphatemia 
2.0 – 2.2 mg/dL 
1.5 – 1.9 mg/dL 
< 1.4 mg/dL 
Life-threatening 
consequences; urgent 
intervention indicated - 
Calcium (Corrected 
for Albumin): 
Hypocalcemia 
1.95 – 2.04 mmol/L 
1.75 – 1.94 mmol/L 
< 1.74 mmol/L 
Life-threatening 
consequences; urgent 
intervention indicated 
Calcium (Corrected 
for Albumin): 
Hypercalcemia 
2.51 – 2.88 mmol/L 
2.89 – 3.13 mmol/L 
> 3.14 mmol/L 
Life-threatening 
consequences; urgent 
intervention indicated 
Magnesium: 
Hypomagnesemia 
0.60 – 0.74 mmol/L 
0.45 – 0.59 mmol/L 
< 0.44 mmol/L 
Life-threatening 
consequences; urgent 
intervention indicated 
Bilirubin 
(hyperbilirubinemia) 
> 1.0 – 1.5 x ULN 
> 1.5 – 2.5 x ULN 
> 2.5 ULN 
Life-threatening 
consequences; urgent 
intervention indicated - 
Glucose: 
Hypoglycemia 
(Nonfasting, No Prior 
Diabetes) 
55 – 69 mg/dL 
40 – 54 mg/dL 
<39 mg/dL 
Life-threatening 
consequences; urgent 
intervention indicated 
Glucose: 
Hyperglycemia 
(Nonfasting, No Prior 
Diabetes) 
116 – 160 mg/dL 
161 – 250 mg/dL 
> 251mg/dL 
Life-threatening 
consequences; urgent 
intervention indicated 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Triglycerides 
— 
400 – 750 mg/dL 
> 751 mg/dL 
Life-threatening 
consequences; urgent 
intervention indicated - 
Liver Transaminase 
(LFTs) 
GGT 
AST 
Alk Phos 
 
> 1.25 – 2.5 x ULN 
> 1.25 – 2.5 x ULN 
> 1.25 – 2.5 x ULN 
 
> 2.5 – 5.0 x ULN 
> 2.5 – 5.0 x ULN 
> 2.5 – 5.0 x ULN 
 
> 5.0 x ULN 
> 5.0 x ULN 
> 5.0 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated 
Pancreatic Amylase 
Lipase 
> 1.0 – 1.5 x ULN 
> 1.0 – 1.5 x ULN 
> 1.5 – 2.0 x ULN 
> 1.5 – 2.0 x ULN 
> 2.0 x ULN 
> 2.0 x ULN 
Life-threatening 
consequences; urgent 
intervention indicated - 
Other Cardiovascular 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Cardiac Arrhythmia 
Asymptomatic; transient 
dysrhythmia, no therapy 
required 
Recurrent/persistent 
dysrhythmia; 
symptomatic therapy 
required 
unstable dysrhythmia, 
hospitalization and 
therapy required 
Life-threatening 
consequences; urgent 
intervention indicated 
Hypertension 
Transient, increase > 20 
mm Hg diastolic BP; no 
therapy required 
Recurrent; chronic 
increase > 20 mm Hg 
diastolic BP; therapy req. 
acute therapy required 
outpatient or 
hospitalization possible 
Life-threatening 
consequences; urgent 
intervention indicated 
Hypotension 
Transient orthostatic 
hypotension with heart 
rate increased by 20 
beats/min or decreased by 
< 10 mm Hg systolic BP, 
no therapy required 
Symptoms or BP 
decreased by < 20 mm 
Hg systolic, correctable 
with oral fluid therapy 
Mean arterial pressure 
< 60 mm Hg, IV fluids 
required, or 
hospitalization 
Life-threatening 
consequences; urgent 
intervention indicated 
Pericarditis 
Mild/moderate 
asymptomatic effusion, 
no therapy 
Symptomatic effusion, 
pain, EKG changes 
Tamponade or 
pericardiocentesis or 
surgery required 
Life-threatening 
consequences; urgent 
intervention indicated 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Hemorrhage, Blood 
Loss 
Minimal blood loss, 
asymptomatic, no therapy 
required 
Symptomatic blood loss 
and no transfusion 
required 
Massive blood loss or > 
2 units transfused 
Symptomatic AND 
transfusion of 1-2 units 
of blood or packed red 
cells 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Gastrointestinal 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
 
Vomiting 
Mild or transient; 2 – 3 
episodes per day or mild 
vomiting lasting < 1 week 
Moderate or persistent; 4 
– 5 episodes per day; or 
vomiting lasting ≥ 1 
week, therapy required 
Severe vomiting of all 
food/fluids in 24 hours 
or orthostatic 
hypotension or IV 
therapy required 
Life-threatening 
consequences; urgent 
intervention indicated 
Diarrhea 
Mild or transient; 3 - 4 
loose stools per day or 
mild diarrhea lasting less 
than 1 week 
Moderate or persistent; 5 
- 10 loose stools per day 
or diarrhea lasting ≥ 1 
week, therapy required 
> 10 loose stools/day 
bloody diarrhea; or 
orthostatic hypotension 
or electrolyte 
imbalance, > 2 L IV 
fluid required 
Life-threatening 
consequences; urgent 
intervention indicated 
Oral 
Discomfort/Dysphagia 
Difficulty swallowing but 
able to eat and drink 
Unable to swallow solids 
Unable to drink fluids; 
IV fluids required 
Life-threatening 
consequences; urgent 
intervention indicated 
Constipation 
Constipation less than 78 
hours and requires 
medication for relief 
Moderate abdominal pain 
78 hours with impaction, 
requiring therapy 
Requiring disimpaction 
or hospital treatment 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Respiratory 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Bronchospasm Acute 
Transient; no therapy; 
FEV1 or peak flow 
reduced to 70- < 80% 
Therapy required; 
normalizes with 
bronchodilator; FEV1 or 
peak flow 50 - 69% 
No normalization with 
bronchodilator; FEV1 
or peak flow 25 – 49%, 
retractions 
Life-threatening 
consequences; urgent 
intervention indicated 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Dyspnea 
Dyspnea on exertion 
Dyspnea with normal 
activity 
Dyspnea at rest 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Neurologic 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Nuchal Rigidity 
--  
-- 
Presence of Nuchal 
rigidity 
Life-threatening 
consequences; urgent 
intervention indicated 
Neuropsychological 
 
Mild confusion or 
cognitive impairment 
Moderate confusion or 
cognitive impairment 
Severe confusion or 
cognitive impairment 
Life-threatening 
consequences; urgent 
intervention indicated 
Neurocerebellar 
Slight incoordination or 
dysdiadochokinesia 
Intention tremor, 
dysmetria, slurred speech, 
or nystagmus 
Ataxia requiring 
assistance to walk or 
arm incoordination 
interfering with ADLs 
Life-threatening 
consequences; urgent 
intervention indicated 
Neuromotor 
Mild weakness in muscle 
of feet, but able to walk; 
and/or mild increase in 
reflexes 
Moderate weakness in 
feet or legs, e.g. unable to 
perform deep knee bend, 
mild weakness in hands, 
loss of previously present 
reflex or development of 
hyperreflexia. 
Marked distal weakness 
Life-threatening 
consequences; urgent 
intervention indicated 
Neurosensory 
Mild impairment 
(decreased sensation in 
focal area or symmetrical 
distribution) 
 
Moderate symmetrical 
impairment, mild 
impairment that is not 
symmetrical 
 
Severe impairment 
(decreased or loss of 
sensation to knees or 
wrists) or loss of 
sensation of at least 
moderate degree in 
multiple areas or 
functions 
Life-threatening 
consequences; urgent 
intervention indicated 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Paresthesia  
(Burning, Tingling, 
etc.) 
Mild discomfort; no 
therapy required 
Moderate discomfort; 
non-narcotic analgesia 
required 
Severe discomfort; or 
narcotic analgesia 
required with 
symptomatic 
improvement 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Dermatologic 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Dermatitis 
Rash is present but 
asymptomatic 
Rash is symptomatic 
(pruritus/pain) but does 
not interfere with 
function 
Rash is symptomatic 
and interferes with 
function 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Urinalysis 
Grade I – Mild 
Grade II – Moderate 
Grade III – Severe 
Grade IV – Life 
Threatening 
Proteinuria: Random 
Urine 
1+ 
2+ - 3+ 
4+ 
- 
Proteinuria: 24 Hour 
Urine 
200 mg – 1 g loss/day or 
< 0.3% or < 3 g/L 
> 1 – 2 g loss/day or 
0.3% - 1.0% or 3 – 10 g/L 
> 2 g loss/day or > 
1.0% or > 10 g/L 
- 
Hematuria (In the 
Absence of Vaginal 
Bleeding) 
Microscopic only,  
6-10 rbc/hpf 
> 10 rbc/hpf 
Gross, with or without 
clots;  
or RBC casts 
Life-threatening 
consequences; urgent 
intervention indicated 
Other Miscellaneous  
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Grade IV – Life 
Threatening 
Malaise 
Malaise that is easily 
tolerated 
Malaise that interferes 
with daily activity 
Malaise that prevents 
daily activity 
Life-threatening 
consequences; urgent 
intervention indicated 
1Grade 4 will be assigned to any AE that is determined to be potentially life-threatening. Grade 5 will be assigned to any AE that results in 
death 
 
 

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14.2 Appendix 2: Brighton Criteria for Guillain-Barré Syndrome 
Level 1 of diagnostic certainty 
Level 2 of diagnostic certainty 
Level 3 of diagnostic certainty 
 
Bilateral and flaccid weakness of  
            the limbs 
 
Bilateral and flaccid weakness of  
            the limbs 
 
Bilateral and flaccid weakness of 
 
the limbs 
       Decreased or absent deep tendon      
 
reflexes in weak limbs 
 
Decreased or absent deep tendon 
 
reflexes in weak limbs 
 
Decreased or absent deep tendon 
 
reflexes in weak limbs 
 
Monophasic illness pattern; and 
 
interval between onset and nadir of 
 
weakness between 12 hours and 28 
 
days; and subsequent clinical  
            plateau 
 
Monophasic illness pattern; and 
 
interval between onset and nadir of 
 
weakness between 12 hours and 28 
 
days; and subsequent clinical  
 
plateau 
 
Monophasic illness pattern; and 
 
interval between onset and nadir of 
 
weakness between 12 hours and 28 
 
days; and subsequent clinical 
 
plateau 
 
Cytoalbuminologic 
 dissociation  
 
(i.e.  
laboratory normal value  
 
and CSF total  laboratory normal 
 
value and CSF total white cell 
 
count < 50 cells/µL 
 
Absence of identified alternative 
 
diagnosis for weakness 
 
Absence of identified alternative 
 
diagnosis for weakness 
 
Electrophysiological findings 
 
consistent with GBS 
 
CSF total white cell count < 50 
 
cells/µL (with or without CSF 
 
protein elevation above laboratory 
 
normal value); OR 
 
electrophysiological studies 
 
consistent with GBS if CSF not 
 
collected or results not available 
 
 
 
 

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14.3 Appendix 3: Guillain-Barré Syndrome disability scale 
 
0. Healthy 
1. Minor symptoms or signs of neuropathy but capable of manual work / capable of running 
2. Able to walk without support of as stick (5 meters across an open space) but incapable of manual work / running 
3. Able to walk with a stick, appliance of support (5 meters across an open space) 
4. Confined to bed or chair bound 
5. Requiring assisted ventilation (for any part of the day or night) 
6. Death 
 
From: Assessment and management of Guillain-Barré syndrome in the context of Zika virus infection 
Interim guidance update; 18 August 2016, WHO/ZIKV/MOC/16.4 Rev.1 
 
 
 

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14.4 Appendix 4: Schedule of Procedures Post-inoculation with ZIKV Vaccine 
 
 
Study Day  
 
Procedure  
Screen 
0 
4 
6 
8 
10 
12 
14 
16 
21 
28 
56 
90 
150 
180 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Alk.Phos, bilirubin, AST 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HCV test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBV test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Flavivirus antibody 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV-2 PCR (NP 
or mid-turbinate swab) 
X7 
X7 
 
 
X7 
 
 
X7 
 
 
 
 
 
 
 
Physical examination  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X]1 
[X]1 
[X]1 
X 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Inoculation 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X]4 
 
 
 
 
 
ALT / Creatinine 
X 
X 
 
X6 
 
 
 
X 
 
[X]4 
 
 
 
 
 
Pregnancy Test 
X 
X 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
Serology (blood) 
 
X 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
PBMC collection 
 
X 
X 
 
X 
 
 
X 
 
X 
X 
X 
X 
X 
X 
PAXGene collection 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
 
Urine for virus isolation 
 
X 
X 
X 
X 
X 
X 
X 
X 
[X]2 
[X]2 
[X]2 
[X]2 
[X]3 
[X]3 
Blood for virus isolation 
 
X 
X 
X 
X 
X 
X 
X 
X 
[X]2 
[X]2 
[X]2 
[X]2 
[X]2 
 
Vaginal secretion for 
virus isolation 
 
X 
 
 
X 
 
 
 
X 
 
[X]2 
[X]2 
[X]2 
[X]3 
[X]3 
Semen for virus isolation 
 
X 
 
 
X 
 
 
 
X 
 
[X]2 
[X]2 
[X]2 
[X]3 
[X]3 
Oral fluid for serology5 
 
X 
 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
1. 
[X]1 indicates that the physical exam is optional and will be performed if the subject has had a medically attended adverse event since the last visit or at the discretion of the investigator 
2. 
[X]2 indicates that samples will be collected. Viral culture and PCR is optional and will be done only if virus was recovered from the sample drawn at the previous time-point 
3. 
[X]3 indicates that samples for virology will be collected on study day if virus was recovered from the sample drawn at the previous time-point or virology results are not available 
4. 
[X]4 indicates that the lab is optional and will be done if the previous sample was a grade 1 or higher 
5. 
This will only be done at JHU CIR 
6. 
ALT testing only 
7. 
Nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus that causes COVID-19 – will be collected from all volunteers regardless of vaccination status based on 
guidance from federal, state, local, and/or institutional health officials* or at the discretion of the clinician (may be collected up to 72 hours in advance of stated collection time). 
8. 
*This guidance may be informed by the local COVID infection rate, severity of infections, vaccination rates, and other factors as the COVID-19 pandemic continues to evolve and change.. NP 
or mid-turbinate swabs may be collected at other time-points if volunteer has COVID-like symptoms.