Phase I Evaluation of Two Zika Viruses for Use in Controlled Human Infection Models (CHIM)
Phase: PHASE1
Conditions: Zika Virus
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

 
 
 
Phase I Evaluation of Two Zika Viruses for Use in Controlled 
Human Infection Models (CHIM) 
 
 
CIR Protocol Number: 
 
316 
 
JHSPH IRB Protocol Number: 
IRB00009756 
UVM IRB Number:  
 
 
 
FDA IND Number: 
 
 
19218 
 
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: 
 
 
v10.0 
Date:  
 
 
 
June 7, 2024 
 
 
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. 

CIR 316 Zika CHIM 
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Team Roster 
Principal Investigator 
 
Anna Durbin, MD 
 
 
 
 
 
Center for Immunization Research (CIR) 
 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
 
855 N Wolfe St, Baltimore, MD 21205 
667-306-9772 (phone) 
 
Co –investigator 
 
 
Kristen Pierce, MD 
University of Vermont, College of Medicine 
University of Vermont 
 
 
 
 
 
89 Beaumont Avenue 
Given C205 
 
 
 
 
  
Burlington, VT 05405 
 
 
 
 
 
802-847-3819 (phone) 
 
Clinical Investigators: 
 
Kawsar Talaat, MD, MD 
Noreen Hynes MD, MPH, DTM&H 
Ruby Pritchett, BS 
Megan McKnight, MS, PA-C 
Lina Moscarella, MSN, FNP, AANP-CB 
Sarah Sampson, MSN, FNP 
Beulah Sabundayo, PharmD, MPH 
Center for Immunization Research 
Johns Hopkins School of Public Health 
 
 
 
 
 
624 N. Broadway, Baltimore, MD 21205 
 
 
 
 
 
 
 
410-502-9627 (phone) 
 
 
 
 
 
443-287-8043 (phone) 
410-502-7451 (phone) 
 
 
 
 
 
 
Beth Kirkpatrick, MD  
 
 
 
 
 
Kristen Pierce, MD 
Patricia Lutton, CCRP 
University of Vermont, College of Medicine 
University of Vermont 
 
 
 
 
 
89 Beaumont Avenue 
Given C205 
 
 
 
 
  
Burlington, VT 05405 
 
 
 
 
 
802-656-5822 (phone) 
 
Scientific Investigator: 
 
Stephen S. Whitehead, PhD  
Laboratory of Viral Diseases 
 
 
 
 
 
National Institute of Allergy and Infectious Diseases  
National Institutes of Health 
301-496-7692 (phone) 
240-281-3767 (phone) 

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OCRPRO Regulatory Affairs: 
Mark S. Miller, PharmD, BCPS, RAC 
CAPT, USPHS 
Clinical Regulatory and Operations Specialist/IND 
Manager 
OCRPRO/DCR/NIAID/NIH 
5601 Fishers Lane 
Room 4B31, MSC-9820 
Bethesda, MD 20892 
Office: 240-627-3300 
Cell: 240-550-7375 
Email: mark.miller6@nih.gov  
 
Sponsor Medical Monitor:  
Shirley Jankelevich, MD 
Clinical Monitoring Research Program Directorate 
Frederick National Laboratory for Cancer Research  
Leidos Biomedical Research, Inc 
Support to: Regulatory Compliance and Human Subjects 
Protection Program 
National Institute of Allergy and Infectious Diseases 
National Institutes of Health  
5705 Industry Lane 
Frederick, MD 21704 
301-846-7322 (phone) 
301-846-6224 (fax) 
Shirley.Jankelevich@fnlcr.nih.gov 
  
Clinical Study Sites  
 
Center for Immunization Research 
 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
 
855 N. Wolfe Street, Suites 600 & 601  
Baltimore, MD 21205 
 
 
 
 
 
 
 
ICTR Bayview Clinical Research Unit (Bayview CRU) 
301 Building 
301 Mason Lord Drive Suite 4200 
Baltimore, MD 21224 
 
CIR Isolation Unit  
301 Building 
301 Mason Lord Drive Suite 4300 
Baltimore, MD 21224 
410-550-2725 (phone) 
 
 
 

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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 (CRC) 
 
 
 
 
 
Shepardson 2 
 
 
 
 
 
111 Colchester Avenue 
 
 
 
 
 
Burlington, VT 05401 
 
 
Research Sites 
 
 
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 
 
 
 
 
 
89 Beaumont Avenue 
Given C205 
 
 
 
 
  
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 
 
IRB Sites 
 
 
 
Johns Hopkins School of Public Health IRB 
 
 
 
 
 
615 N. Wolfe Street 
 
 
 
 
 
Suite E1100 
 
 
 
 
 
Baltimore, MD 21205 
 
 
 
 
 
 
UVM Research Protections Office 
213 Waterman Building 
University of Vermont 
Burlington, VT 05401 
 
 
 

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Table of Contents 
Team Roster .................................................................................................................................... 2 
Table of Contents ............................................................................................................................ 5 
List of Tables .................................................................................................................................. 9 
List of Figures ................................................................................................................................. 9 
List of Appendices .......................................................................................................................... 9 
List of Abbreviations .................................................................................................................... 10 
Protocol Precis .............................................................................................................................. 12 
1 
Introduction ........................................................................................................................... 18 
1.1 
Background – Zika ........................................................................................................ 18 
1.2 
Background: ZIKV Strains ........................................................................................... 24 
1.2.1 
ZIKV-SJRP/2016 .................................................................................................. 24 
1.2.1.1 
Final Container of ZIKV-SJRP/2016................................................................ 24 
1.2.1.2 
Composition of ZIKV-SJRP/2016 .................................................................... 24 
1.2.1.3 
Investigational Product Label of ZIKV-SJRP/2016 ......................................... 24 
1.2.2 
ZIKV-Nicaragua/2016 .......................................................................................... 24 
1.2.2.1 
Final Container of ZIKV-Nicaragua/2016 ........................................................ 25 
1.2.2.2 
Composition ZIKV-Nicaragua/2016 ................................................................ 25 
1.2.2.3 
Investigational Product Label of ZIKV-Nicaragua/2016 ................................. 25 
1.3 
Rationale ....................................................................................................................... 25 
1.3.1 
Animal Experience................................................................................................ 25 
1.3.1.1 
Non-human Primate Study ................................................................................ 25 
1.3.2 
Previous Human Experience ................................................................................. 26 
1.3.2.1 
DENV-2 Controlled Human Infection Model (CHIM) .................................... 26 
1.3.2.2 
DENV-3 CHIM ................................................................................................. 26 
1.3.2.3 
ZIKV-SJRP (Cohort 1) ..................................................................................... 27 
1.3.2.4 
ZIKV-Nicaragua (Cohort 2) ............................................................................. 29 
2 
Objectives ............................................................................................................................. 31 
2.1 
Primary Objectives........................................................................................................ 31 
2.2 
Secondary Objectives.................................................................................................... 32 
2.3 
Exploratory Objectives ................................................................................................. 32 
3 
Study Design ......................................................................................................................... 33 
3.1 
Receipt of Test Article .................................................................................................. 33 
3.1.1 
Overall Design ...................................................................................................... 33 
3.1.2 
Study Design ......................................................................................................... 34 
3.1.2.1 
Initial Enrollment of Each Cohort..................................................................... 34 
3.1.2.2 
Enrollment of Remaining 7 Subjects in a Dose Cohort .................................... 34 
3.1.3 
Sample Size and Placebo Ratio Primary Study .................................................... 34 
3.1.4 
Duration of Subject Participation.......................................................................... 35 
3.1.5 
Estimated Duration of the Study ........................................................................... 35 
3.1.6 
Treatment Assignment .......................................................................................... 35 
3.1.7 
Blinding................................................................................................................. 35 

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4 
Selection and Enrollment of Subjects ................................................................................... 36 
4.1 
Inclusion Criteria .......................................................................................................... 37 
4.2 
Exclusion Criteria ......................................................................................................... 37 
4.3 
Subject Withdrawal and Termination Criteria .............................................................. 39 
4.4 
Access to Medical Records ........................................................................................... 40 
5 
Test Article Preparation ........................................................................................................ 40 
5.1 
Pre-inoculation Preparation .......................................................................................... 40 
5.2 
ZIKV and Diluent Storage ............................................................................................ 41 
5.3 
ZIKV Strain and Diluent Accountability ...................................................................... 41 
5.4 
Storage Disposition of Used/Unused Supplies ............................................................. 41 
6 
Study Procedures .................................................................................................................. 41 
6.1 
Recruitment and General Screening ............................................................................. 41 
6.2 
Consenting Process ....................................................................................................... 42 
6.3 
Screening Procedures .................................................................................................... 42 
6.4 
Detailed Study Procedures ............................................................................................ 44 
6.4.1 
Admission to the Inpatient Unit, Inoculation, and Follow-up .............................. 44 
6.4.1.1 
Inpatient Stay Procedures ................................................................................. 44 
6.4.1.2 
Detailed Study Procedures ................................................................................ 45 
6.4.2 
Discharge Criteria ................................................................................................. 54 
6.4.3 
Mitigation Plan Should a Subject Leave the Unit Early ....................................... 54 
6.4.4 
Subject Temperature Memory Card...................................................................... 55 
6.4.5 
Persistence of ZIKV in Bodily Fluids ................................................................... 55 
6.5 
Clinical Laboratory Testing .......................................................................................... 55 
6.6 
Medical History and Concomitant Medications ........................................................... 56 
6.7 
Mosquito Feeding ......................................................................................................... 56 
6.8 
Immunology Testing ..................................................................................................... 57 
6.8.1 
Antibody Testing .................................................................................................. 57 
6.8.2 
Other Immunological Assays ................................................................................ 57 
6.9 
Retention of Study Subjects .......................................................................................... 57 
7 
Adverse Event Monitoring .................................................................................................... 57 
7.1 
Definitions..................................................................................................................... 57 
7.1.1 
Adverse Event ....................................................................................................... 57 
7.1.2 
Medically-attended Adverse Event ....................................................................... 59 
7.1.3 
Adverse Event of Special Interest ......................................................................... 59 
7.1.4 
Suspected Adverse Reaction (SAR) ..................................................................... 59 
7.1.5 
Pregnancy .............................................................................................................. 59 
7.1.6 
Serious Adverse Event (SAE) ............................................................................... 60 
7.1.7 
Unexpected Adverse Events ................................................................................. 61 
7.1.8 
Serious and Unexpected Suspected Adverse Reaction (SUSAR) ........................ 61 
7.1.9 
Unanticipated Problem.......................................................................................... 61 
7.1.10 
Unanticipated Problem That Is Not an Adverse Event (UPnonAE) ..................... 61 
7.1.11 
Protocol Deviation ................................................................................................ 61 
7.1.12 
Serious Protocol Deviation ................................................................................... 62 
7.1.13 
Pre-existing Conditions, Worsening of Pre-existing Condition ........................... 62 
7.2 
Assessment of Adverse Events ..................................................................................... 62 

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7.2.1 
Identification of Adverse Events .......................................................................... 62 
7.2.2 
Protocol Specific Adverse Event Definitions ....................................................... 63 
7.2.3 
Determination of Severity ..................................................................................... 64 
7.2.4 
Relationship with Receipt of Test Article ............................................................. 66 
7.2.5 
Adverse Event Action Taken ................................................................................ 66 
7.2.6 
Adverse Event Outcome ....................................................................................... 67 
7.2.7 
Adverse Event Seriousness ................................................................................... 67 
7.3 
Adverse Event Reporting .............................................................................................. 67 
7.3.1 
Non-Serious Adverse Events ................................................................................ 67 
7.3.2 
Serious Adverse Events ........................................................................................ 67 
7.3.3 
Unanticipated Problems ........................................................................................ 69 
7.4 
Sponsor’s Reporting Responsibilities ........................................................................... 69 
7.5 
Halting Criteria ............................................................................................................. 69 
7.5.1 
Reporting a Study Halt.......................................................................................... 70 
7.5.2 
Resumption of a Halted Study .............................................................................. 70 
7.5.3 
Discontinuation of Study Agent/Intervention ....................................................... 70 
7.6 
Safety Oversight............................................................................................................ 70 
7.6.1 
Safety Review and Communications Plan (SRCP) .............................................. 70 
7.6.2 
Sponsor Medical Monitor ..................................................................................... 70 
7.6.3 
NIAID Intramural Data and Safety Monitoring Board ......................................... 71 
8 
Data Collection and Monitoring ........................................................................................... 71 
8.1 
Source Documentation and Data Collection ................................................................. 71 
8.2 
Study Documentation.................................................................................................... 72 
8.3 
Retention of Specimens ................................................................................................ 72 
8.4 
Retention of Records..................................................................................................... 73 
8.5 
Protocol Compliance ..................................................................................................... 73 
8.6 
Clinical Investigator’s Brochure ................................................................................... 73 
8.7 
Study Monitoring .......................................................................................................... 74 
9 
Statistical Considerations ...................................................................................................... 74 
9.1 
General Design.............................................................................................................. 74 
9.2 
Statistical Methods ........................................................................................................ 74 
9.3 
Safety Endpoint ............................................................................................................. 75 
9.4 
Immunogenicity ............................................................................................................ 75 
9.5 
Per Protocol Analysis .................................................................................................... 75 
9.5.1 
Safety Data ............................................................................................................ 75 
9.5.2 
Virology ................................................................................................................ 76 
9.5.3 
Immunogenicity Data............................................................................................ 76 
9.6 
Intent-to-Treat Analysis ................................................................................................ 76 
9.6.1 
Virology ................................................................................................................ 76 
9.6.2 
Immunogenicity Data............................................................................................ 76 
10 
Protection of Human Subjects .......................................................................................... 76 
10.1 
Institutional Review Boards .......................................................................................... 76 
10.2 
Informed Consent.......................................................................................................... 76 
10.3 
Risks .............................................................................................................................. 76 
10.3.1 
Venipuncture ......................................................................................................... 77 

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10.3.2 
Local Risks Associated with Test Article Administration .................................... 77 
10.3.3 
Risk of Use of Cervico-vaginal Cup ..................................................................... 77 
10.3.4 
Topical Anesthetic Cream..................................................................................... 77 
10.3.5 
Risk from Mosquito Feeding ................................................................................ 77 
10.3.6 
Risk Due to Infection with ZIKV ......................................................................... 77 
10.3.6.1 
Zika Illness .................................................................................................... 77 
10.3.6.2 
Zika Congenital Syndrome ........................................................................... 77 
10.3.6.3 
Guillain-Barré Syndrome (GBS) .................................................................. 78 
10.3.6.4 
Risk of Transmission .................................................................................... 78 
10.3.6.5 
Risk of More Severe Dengue Disease .......................................................... 79 
10.3.6.6 
Other Risks.................................................................................................... 79 
10.3.7 
Risk Mitigation ..................................................................................................... 79 
10.3.7.1 
Prevention of Congenital Zika Syndrome ..................................................... 79 
10.3.7.2 
Mitigation of Risk of GBS ............................................................................ 79 
10.3.7.3 
Mitigation of Risk of Transmission of ZIKV ............................................... 80 
10.3.7.4 
Other Risks.................................................................................................... 80 
10.3.7.5 
Escalation of Care Plan ................................................................................. 80 
10.4 
Benefits ......................................................................................................................... 81 
10.5 
Compensation ............................................................................................................... 81 
10.5.1 
Compensation for Injury ....................................................................................... 81 
10.6 
Confidentiality .............................................................................................................. 81 
10.7 
Biohazard Containment ................................................................................................ 82 
11 
Publication Policy ............................................................................................................. 82 
12 
References ......................................................................................................................... 83 
Appendix A: Adverse Event Grading Table1,2.............................................................................. 88 
Appendix B: Brighton Criteria for Guillain-Barré Syndrome ...................................................... 94 
Appendix C: Guillain-Barré Syndrome Disability Scale .............................................................. 95 
 
 
 

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List of Tables 
Table 1: Schedule of Procedures for Cohorts 1, 2, 3 and 4 .......................................................... 16 
Table 2: ZIKV Viremia in Rhesus macaques ............................................................................... 25 
Table 3: Viremia Induced by the DENV-2 and DENV-3 CHIM Viruses in Normal Healthy 
Volunteers ............................................................................................................................. 27 
Table 4: All Adverse Events Following One Dose of Cohort 1 (ZIKV-SJRP/placebo) .............. 27 
Table 5: Recovery of ZIKV-SJRP from Serum, Whole Blood, CVS, Saliva, and Urine ............. 29 
Table 6: All Adverse Events Following One Dose of Cohort 2a (ZIKV-Nicaragua/Placebo) ..... 30 
Table 7: Recovery of ZIKV-Nicaragua from Serum, Whole Blood, CVS, Saliva, and Urine ..... 31 
Table 8: Solicited Adverse Events ................................................................................................ 59 
Table 9: Severity Definitions ........................................................................................................ 65 
Table 10: Assessment of Solicited Adverse Events ...................................................................... 65 
 
List of Figures 
Figure 1: Estimated Viral RNA Load and Culture Results in Semen from 184 Men (Mead et, al 
2018) ..................................................................................................................................... 20 
Figure 2: Monthly ZIKV Illness Case Counts Reported to PAHO (Vannice et al, 2019) ............ 22 
Figure 3: Label for Final Vial of ZIKV-SJRP/2016 (Enlarged Sample) ...................................... 24 
Figure 4: Label for Final Vial of ZIKV-Nicaragua/2016 (Enlarged Sample) .............................. 25 
Figure 5: Kinetics of ZIKV-SJRP in Serum by Culture and Quantitative PCR ........................... 28 
Figure 6: Kinetics of ZIKV-Nicaragua in Serum by Culture and Quantitative PCR ................... 31 
 
 
List of Appendices 
Appendix A: Adverse Event Grading Table1,2.............................................................................. 88 
Appendix B: Brighton Criteria for Guillain-Barré Syndrome ...................................................... 94 
Appendix C: Guillain-Barré Syndrome Disability Scale .............................................................. 95 
 
 
 

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List of Abbreviations 
b-hCG 
beta-human choriogonadotropin 
AE 
adverse event 
ALT 
alanine aminotransferase 
ANC 
absolute neutrophil count 
AST 
aspartate aminotransferase 
CBC 
complete blood count 
CDC 
Center for Disease Control and Prevention 
CHIM 
controlled human infection model 
CI 
confidence interval 
CIR 
Center for Immunization Research 
CLIA 
Clinical Laboratory Improvement Amendments 
CPK 
creatine phosphokinase 
CRIMSON 
Clinical Research Information Management System of the NIAID 
CRL 
Charles River Laboratories 
CSO 
Clinical Safety Office 
CT 
threshold cycle 
CVS 
Cervico-vaginal secretion 
CZS 
congenital Zika syndrome 
DENV 
dengue virus (serotypes DEN1, DEN2, DEN3, and DEN4) 
DSMB 
Data and Safety Monitoring Board 
FDA 
Food and Drug Administration 
GA1 
ganglioside asialo GM1 
GBS 
Guillain-Barré syndrome 
GCE 
Genome Copy Equivalents 
GCP 
good clinical practice 
HBsAg 
hepatitis B surface antigen 
HBV/HCV 
hepatitis B virus/hepatitis C virus 
HIV 
human immunodeficiency virus 
HLA 
human leukocyte antigen 
ICH 
International Council on Harmonisation of Technical Requirements 
for Pharmaceuticals for Human Use 
ICMJE 
International Committee of Medical Journal Editors 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
IUD 
intrauterine device 
JHSPH 
Johns Hopkins Bloomberg School of Public Health 
LID 
Laboratory of Infectious Diseases 
LLN 
lower limit of normal 
N 
number of subjects 
NIAID 
National Institute of Allergy and Infectious Diseases 
NIH 
National Institutes of Health 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
OR 
odds-ratio 
PAHO 
Pan American Health Organization 
PBMC 
peripheral blood mononuclear cell 
PCR 
polymerase chain reaction 
PFU  
plaque-forming units  
PHEIC 
Public Health Emergency of International Concern 
PI 
principal investigator 
PRNT50 
50% plaque reduction neutralization titer 
PT/PTT 
prothrombin time/ partial thromboplastin time 
RNA 
ribonucleic acid 
ROP 
retro-orbital pain 
RT-PCR 
reverse transcriptase polymerase chain reaction 
SAE 
serious adverse event 
SD 
standard deviation 
SE 
standard error 
SJRP 
São José do Rio Preto, Brazil 

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SOP 
standard operating procedure 
SRCP 
Safety Review and Communications Plan 
SUSAR 
serious and unexpected suspected adverse reaction 
Tmax 
maximum temperature  
TORO 
transfer of regulatory obligations 
ULN 
upper limit of normal 
WBC 
white blood cell count 
WHO 
World Health Organization 
Wt 
wild-type 
ZIKV 
Zika virus 
ZIKV-SJRP 
Zika virus- São José do Rio Preto, Brazil 

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PROTOCOL PRECIS 
Protocol Title:  
Phase I evaluation of two Zika viruses for use in controlled human 
infection models (CHIM) 
Version: 
 
10.0  
Version Date: 
07 June 2024 
Version History:  
v9.0 01 December 2023 
v8.0 01 August 2023 
v7.0 20 February 2023 
v6.0 24 January 2023 
v5.0 14 June 2022 
v4.0 17 September 2021 
v3.0 13 August 2021 
v2.0 23 June 2021 
v1.0 31 July 2019 
Phase:  
 
Phase 1 
Principal Investigator 
Anna Durbin, MD 
 
 
 
 
Center for Immunization Research (CIR) 
 
 
 
 
Johns Hopkins School of Public Health 
 
 
 
 
855 N. Wolfe Street, Suite 600 
 
 
 
 
Baltimore, MD 21205 
 
 
 
 
667-306-9772 (phone) 
 
Co –investigator 
 
Kristen Pierce, MD 
 
 
 
 
University of Vermont, College of Medicine 
 
 
 
 
89 Beaumont Avenue 
 
 
 
 
Given C205 
 
 
 
 
Burlington, VT 05405 
 
 
 
 
802-847-3819 (phone) 
Subjects: 
Healthy male and non-pregnant female subjects between 18 - 40 years of 
age, inclusive, with no history of previous ZIKV or DENV infection. 
No. of Subjects: 
Up to 56 
Study Type:  
Randomized, placebo-controlled, double-blind 
Duration: 
Subjects will be followed for approximately 26 weeks (approximately 180 
days) from the time of inoculation. Subjects will be screened for eligibility 
up to 60 days prior to inoculation on Study Day 0. 
 
 

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Dose Escalation Schedule: 
Cohort 
No. of Subjects1 
  Sex 
Dose 
               Virus 
1 
14 
Female 
102 PFU 
ZIKV-SJRP/2016-184  
2 
14 
Female 
102 PFU 
ZIKV-Nicaragua/2016  
32 
14 
Male 
102 PFU 
ZIKV-SJRP/2016-184 
42 
14 
Male 
102 PFU 
ZIKV-Nicaragua/2016  
 
PFU = plaque-forming units, ZIKV-SJRP = Zika virus-São José do Rio Preto, Brazil 
1. Virus:Placebo ratio, 5:2 
2. Seven subjects from cohort 3 and 7 subjects from cohort 4 will be selected for mosquito-feeding. They 
will also be randomized 5:2 (ZIKV:placebo) to ensure the clinical team remains blinded to the treatment 
assignment. 
 
Product Description: ZIKV-SJRP/2016-184 and ZIKV-Nicaragua/2016 were isolated directly 
from the serum of subjects who were diagnosed with uncomplicated Zika illness. Each virus will 
first be evaluated at a dose of 102 plaque-forming units (PFU). At each subcutaneous dose, the 
virus will first be evaluated in 7 subjects (5 will have received ZIKV, 2 will have received 
placebo). The remaining subjects in each dose cohort will be inoculated with the current dose or 
dose escalation will occur according to predetermined criteria. 
 
Primary Study Objective: Identify a suitable strain of ZIKV for use in safety-assured 
CHIM studies by: 
1. Determining the proportion of infected subjects who demonstrate clinical 
signs/symptoms of Zika infection (fever, rash, conjunctivitis, pruritis, myalgia, arthralgia, 
headache) by ZIKV strain and dose 
2. Determining the quantity and duration of ZIKV in blood, urine, cervico-vaginal 
secretions, semen, and saliva by culture (infectious virus) and by reverse transcriptase – 
polymerase chain reaction (RT-PCR) 
3. Determining if dose correlates with incidence/duration of ZIKV in blood, urine, cervico-
vaginal secretions, semen, and saliva 
A suitable ZIKV strain for CHIM studies is defined as one in which: 
1. ≥ 80% of ZIKV or DENV-naïve inoculated subjects develop detectable viremia, as 
determined by direct virus culture and 
2.   Infectious virus is detected on more than 1 day for ≥ 80% of subjects with detectable 
viremia and 
3. The geometric mean peak titer of virus recovered by culture is 2.0 – 3.0 log10 
PFU/mL 
Ideally, the ZIKV strain would also induce one or more of the following clinical signs in ≥ 50% 
of infected subjects, however this will not be included in dose escalation criteria. 
a) Fever 
b) Rash 
c) Non-purulent conjunctivitis 
d) Myalgia 
e) Arthralgia 
Secondary Study Objectives:  

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1. Characterize the clinical presentation of acute ZIKV infection by assessing the frequency 
of adverse events (AEs), graded by severity, occurring within 28 days of ZIKV 
administration 
2. Longitudinal analysis of virus present in blood, urine, saliva, cervico-vaginal secretions, 
and semen 
3. Time course of the neutralizing antibody response to ZIKV infection 
 
Exploratory Objectives: 
1. Evaluate the cellular immune response to primary infection with ZIKV 
2. Evaluate the innate immune response to primary infection with ZIKV 
3. Evaluate B and T cell memory responses following primary infection with ZIKV 
4. Evaluate the antibody repertoire induced by ZIKV infection 
5. Evaluate the antibody response in saliva to primary infection with ZIKV as a possible 
diagnostic tool 
6. Determine if ZIKV-SJRP and ZIKV-Nicaragua can be transmitted to mosquitoes from 
infected volunteers when administered at a dose of 100 PFU  
 
Safety Considerations / Risk Mitigation: 
1. The majority of ZIKV infections are asymptomatic. With the noted exception of the 
fetus, reactogenicity to ZIKV infection is mild or moderate and self-limiting.  
2. To reduce the risk of mosquito transmission, study subjects will be housed in an inpatient 
facility with an active mosquito-control program during the expected period of viremia, 
Study Days 0 through 9 (discharged on Study Day 9). Data from cohorts 1 & 2 
determined that infectious ZIKV was cleared from blood before Study Day 9.  
3. To reduce the risk of sexual transmission, volunteers must agree to use barrier 
contraception through Study Day 56 (women) or Study Day 90 (men) after inoculation 
with ZIKV. 
4. Data from cohorts 1 & 2 demonstrated that infectious ZIKV was rarely detected in saliva 
or cervico-vaginal secretions following a dose of 102 PFU. 
5. The inoculation is a low dose of 102 PFU. 
6. Virus inoculum will be delivered subcutaneously by needle and syringe and is considered 
less infectious than natural mosquito-borne virus which is augmented by saliva factors. 
7. Sexually-active women of child-bearing potential will be required to use hormonal birth 
control or an intrauterine device to prevent pregnancy while at risk for ZIKV infection 
(we will follow the Center for Disease Control and Prevention (CDC) guidelines of 8 
weeks post-infection (Study day 56); time of infection is defined as inoculation with 
challenge virus). In addition, they will be required to use barrier contraception through 
Study Day 56 in accordance with CDC guidance. 
8. Men will be required to use barrier contraception for 3 months post-infection (Study Day 
90). 
9. At the time of subject screening, serum pregnancy testing will be performed and repeated 
again within 14 days of inoculation, and on the day of inoculation. This will ensure 
women are not pregnant at the time of ZIKV inoculation. 
10. Due to the observed correlation of age and the rare incidence of Guillain-Barré syndrome 
(GBS) following ZIKV infection, only volunteers age 40 years and younger will be 
enrolled.  

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Study Design:  
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, urine 
toxicology screening for opiates and serology screening for previous DENV and ZIKV infection. 
Serum pregnancy testing will be performed on applicable female subjects.  
For subjects who are eligible, receipt of inoculation will be scheduled for Study Day 0. Until 
transmissibility of the two ZIKV strains to mosquitoes is defined by mosquito-feeding on 
volunteers, volunteers will be admitted to the inpatient unit on Study Day 0 and will be 
discharged from the unit on Study Day 9 if discharge criteria are met. This represents the period 
of time when infectious viremia may be present and at a level that could be transmissible to 
mosquitoes.  
 
Volunteers will be evaluated according to the attached Schedule of Procedures. During the 
outpatient and inpatient visits through Study Day 28, the subjects will be evaluated by a clinician 
and will have blood drawn for clinical laboratory studies, virologic assays, and immunologic 
assays. In addition, urine, vaginal secretions, semen, and saliva will be collected at specific time 
points for additional virologic assays. Subjects will have their temperature measured and 
recorded at least twice a day during their inpatient stay, with the exception of the day of 
discharge (Study Day 9). Subjects will return to the clinic on Study Days 9, 10, 12, 14, 16, 21, 
28, 35, 49, 56, 70, 90, 120, and 180 for blood and specimen collection. Study Day 180 will be 
their final visit.  
 
Cohorts 3 & 4 will evaluate the replication kinetics and shedding in semen of both ZIKV strains 
in men. It is expected that the replication kinetics of both ZIKV strains will not be significantly 
different in men compared to women. The transmissibility of both ZIKV strains to mosquitoes 
will be evaluated in cohorts 3 and 4. Based on the peak titers of ZIKV-SJRP recovered from 
volunteers in cohort 1, it is expected that this strain will not be transmissible to mosquitoes and 
future studies utilizing ZIKV-SJRP will be able to be conducted in the outpatient setting. Based 
on the peak titers of ZIKV-Nicaragua recovered from volunteers in cohort 2, it is expected this 
virus will be transmissible to mosquitoes..

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Table 1: Schedule of Procedures for Cohorts 1, 2, 3 and 4 
 
Screen 
Study Day (inpatient interval marked in gray) 
Procedure 
-60 -14 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
12 
14 
16 
21 
28 
35 
56 
70 
90 120 1504 180 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Pregnancy Prevention 
Review 
X 
 
X 
 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Alk Phos, bilirubin 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AST, ALT, serum 
creatinine 
X 
 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
X 
 
 
 
 
 
 
 
 
 
PT/PTT 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urinalysis 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV test 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HCV test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HBV test 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Physical exam 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] [X] [X] 
 
X 
Admit to Unit 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
NP swab for COVID-19 
testing2 
 
 
[X] 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inoculation 
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CBC with differential 
X 
 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
[X] 
 
 
 
 
 
 
 
 
Pregnancy Test (serum)5 
X 
X 
X 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
Blood for serology  
X 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
 
X 
 
X 
 
 
X 
Blood for PBMC  
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
 
X 
 
X 
 
 
X 
Blood for PAXGene  
 
 
X 
X 
X 
X 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
 
 
 
 
 
 
Urine for virus isolation 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
[X] [X] [X] [X] [X] [X] 
 
[X] 
Blood for virus isolation 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
[X] [X] [X] [X] [X] [X] [X] 
 
[X] 
Saliva for virus isolation 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
[X] [X] [X] [X] [X] [X] 
 
[X] 
Cervico-vaginal secretion 
(CVS) for virus isolation5 
 
 
X 
 
 
 
X 
 
 
 
X 
 
X 
X 
X 
X 
X 
[X] [X] [X] [X] [X] [X] 
 
[X] 
Semen for virus isolation6 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
X 
 
X 
X 
[X] [X] [X] [X] [X] [X] 
 
[X] 
Mosquito feeding7 
 
 
 
 
 
 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Oral fluid for serology3 
 
 
X 
 
 
 
X 
 
X 
 
X 
 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
 
X 

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ALT = alanine aminotransferase; AST = aspartate aminotransferase; CBC = complete blood count; HBV = hepatitis B virus; HCV = hepatitis C virus; HIV = human 
immunodeficiency virus; PBMC = peripheral blood mononuclear cell; PT/PTT = prothrombin time/partial thromboplastin time 
1. 
[ ] indicates that these procedures/assays will be applied only if indicated. Sample collection for urine, CVS, semen and saliva for virus isolation on Study Days 120 and 
180 will only be done if indicated. Please see section 6, Study Procedures. 
2. 
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. 
3. 
This will only be done at the Center for Immunization Research (CIR) 
4. 
Phone Call Visit 
5. 
Female subjects only 
6. 
Male subjects only 
7. 
Only 7 subjects from cohort 3 and 7 subjects from cohort 4 will undergo mosquito-feeding. The 7 subjects will be randomized 5:2 (ZIKV:placebo) to ensure the clinical 
team remains blinded to the treatment assignment. 
 
 

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1 
INTRODUCTION 
1.1 
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]. Only 
approximately 19% of subjects found to be seropositive to ZIKV in a serosurvey on Yap island 
recounted being symptomatic with a Zika-like illness. Of the 21% of infected subjects who were 
symptomatic, nearly all presented with rash (90%), arthritis/arthralgia (65%), and fever (65%). 
Unlike dengue virus (DENV), ZIKV does not cause hemorrhagic manifestations, vascular leak 
syndrome, or liver function abnormalities. 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. In retrospective analyses of the French Polynesian 
outbreak, an association between ZIKV infection during the first and/or second trimester and 
microcephaly was reported [7]. Since its introduction into Brazil, ZIKV has spread to more than 
69 countries in the Americas, Caribbean, Asia and Africa [8]. 
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 [9]. 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, 10, 11] and a causal association between ZIKV infection and 
microcephaly and/or other birth defects was formally established [12]. 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 [13, 14]. Although congenital ZIKV syndrome 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. 
ZIKV infection has also been associated with an increased risk of GBS, although a causal 
relationship has not yet been established. In retrospective analyses of the 2013 French Polynesian 
outbreak, an association between GBS and ZIKV infection was reported [15]. Forty-two cases of 
GBS were identified with 88% of those reporting a history of recent viral syndrome a median of 
6 days before the onset of neurological symptoms. Serum from the GBS patients was tested by 

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reverse transcriptase – polymerase chain reaction (RT-PCR) for ZIKV during the hospital 
admission but was negative in all cases. Although serum from these patients was eventually 
found to be positive for ZIKV antibody by either immunoglobulin M (IgM), immunoglobulin G 
(IgG), or neutralization test, the samples were taken up to 3 months post-hospitalization, and it is 
unclear at what time the sample became positive for ZIKV. In contrast, serum from 98 matched 
control patients hospitalized with a non-febrile illness was collected at a single time-point within 
7 days of the admission date of the matched-GBS case. Forty-one of 42 GBS patients were found 
to have neutralizing antibody against ZIKV compared with 54/98 (56%) of control patients for 
an odds-ratio (OR) of 59.7 (non-estimable 95% confidence interval [CI]). However, others 
suggested that the high OR was an artifact of a small sample size and have calculated an OR of 
1.33 (95% CI 0.62 – 2.78) for ZIKV and GBS from this same population when IgG for ZIKV 
from the first blood sample was used as the only relevant evidence of exposure [16]. The single 
time-point was believed to be more relevant and comparable to the single time-point used in the 
control group. Although sera from 6 patients demonstrated high reactivity toward ganglioside 
asialo GM1 (GA1), further testing did not demonstrate any competition between GA1 and ZIKV 
proteins suggesting that there was no molecular mimicry between ZIKV antigens and GA1 [15]. 
The reported incidence of GBS in the French Polynesian outbreak was 0.24/1,000 ZIKV 
infections which is lower than that observed with Campylobacter jejuni infections (0.25 – 
0.65/1,000 infections). Section 10.3.6.3 further discusses the risk of GBS following ZIKV 
infection.  
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 [17, 18]. Complicating ZIKV control programs, there have been several reports of 
sexual transmission of ZIKV [19-24]. 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 5,460 travel-associated ZIKV cases in the 
United States (<1% of ZIKV cases) as of January 2, 2019, (Zika cases in US, accessed May 28, 
2019) and it is not thought to contribute significantly to transmission [25]. All but 2 cases of 
sexual transmission have been from male to female. There has been only one report of female-
to-male ZIKV transmission and one report of male-to-male ZIKV transmission [26, 27]. In all 
but one of the cases of sexual transmission, ZIKV has been transmitted from a symptomatic 
person to his partner. Transmission has occurred during or just after symptomatic illness. In 
only one documented case has ZIKV been transmitted from an asymptomatic person to his 
partner [28]. There have been few longitudinal studies evaluating the sites and duration of ZIKV 
shed in bodily fluids [29, 30]. ZIKV ribonucleic acid (RNA) has been detected in semen by RT-
PCR for 2 - 6 months after symptoms have developed [31-33]. However, to date, the longest 
interval between onset of symptoms and recovery of infectious ZIKV from semen is 69 days 
[34]. Studies have demonstrated that a minority of ZIKV-infected men shed ZIKV in semen. 
Mead et al. found ZIKV by RT-PCR in 60/184 men (33%) and found ZIKV by RT-PCR in only 

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174 of 1,230 (14%) semen samples tested from 
those men [30]. Of the 174 semen samples that 
were positive for ZIKV by RT-PCR, infectious 
virus (as determined by recovery in tissue 
culture) was found in only 4% of the samples. In 
addition, infectious virus was found only in 
samples collected within 30 days of symptom 
onset [30]. Only 6.5% of the men tested shed 
infectious ZIKV in semen. In the Mead study, 
infectious virus was detected in only 3 of 19 
semen samples that were obtained within 30 days 
of illness; infectious virus was not detected in 
any of the 59 samples that were detected after 
day 30 of illness onset (Figure 1). The 3 samples 
that were positive for infectious virus had titers ≥ 
7 log10 RNA copies/mL of semen and were the 
only samples that had titers ≥ 7 log10 RNA 
copies/mL of semen [30]. A second study 
collected 700 semen samples from 97 confirmed 
ZIKV-infected men [35]. Thirty-two of the 97 
men (33%) had at least one semen specimen 
positive for ZIKV by RT-PCR. Infectious virus was isolated from RT-PCR-positive semen 
samples from 8/32 men (25%) or 8/97 total (8.2%). Few subjects had ZIKV shedding detected in 
saliva (15/147; 10.2%) and only 1 woman had ZIKV shedding detected in cervico-vaginal 
secretions (1/50; 2%). Isolation of infectious virus was not performed for saliva or cervico-
vaginal secretion specimens. Aside from the Paz-Bailey study above, few studies have evaluated 
shedding of ZIKV in cervico-vaginal secretions in a longitudinal manner [36-38] and of those 
studies even fewer have evaluated for infectious virus [39]. Viral RNA was generally detected in 
vaginal secretions or brush samples by RT-PCR for ~ 2 weeks post-symptom onset [29, 36-38]. 
There are 2 reports of longer vaginal shedding of ZIKV. The first reported ZIKV vaginal 
shedding in 5 women, 4 of whom were pregnant women [40]. One pregnant woman shed ZIKV 
vaginally for 180 days. Testing for infectious virus was not done. A second case reported vaginal 
shedding at 31 days post-symptom onset [41]. The titer was very low (200 genomic copies/mL) 
and testing for infectious virus was not done. 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 [24, 26-28, 42]. The primary method of viral detection in these studies was by 
RT-PCR. When samples were evaluated for infectious virus by tissue culture, infectious virus 
was found in the minority of samples as noted above. In the Medina study, all but one semen 
sample containing infectious ZIKV were obtained within 15 days of symptom onset. The mean 
titer by polymerase chain reaction (PCR) ± standard deviation (SD) in those 8 semen samples 
that contained infectious virus was 1.8 x108 ± 2.5 x 108 genome copy equivalents (GCE) per mL. 
The mean titer ± SD for serum samples that contained infectious virus was 4.8 x 107 ± 1.1 x 107 
GCE/mL. Medina et al. concluded that specimens with a titer >1.4 x 106 GCE/mL be considered 
infectious [35]. These data suggest that the shedding of infectious ZIKV in semen may occur 
much less frequently and for a shorter duration than presumed by RT-PCR testing and are 
Figure 1: Estimated Viral RNA Load and 
Culture Results in Semen from 184 Men 
(Mead et, al 2018) 

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consistent with epidemiologic data reporting that all but one case of sexual transmission thus far 
have occurred within 3 weeks of return from an endemic area with all but one of those cases 
occurring within 2 weeks. Data from these studies suggest that detection of ZIKV RNA by RT-
PCR may overstate the duration and magnitude of the risk of sexual transmission. In 
summary, these studies determined that: 
 
1. Infectious ZIKV will likely be cleared from the blood well before Study Day 14 post-
infection. 
2. Although ZIKV can be detected by RT-PCR in semen as late as 6 months following 
infection, the shedding of infectious virus is rare, is associated with high titer by RT-
PCR (≥ 7.0 log10 GCE copies/mL semen or threshold cycle [CT] ≤ 27) and occurs within 
30 days of illness. 
3. Samples with a titer > 1.4 x 106 GCE/mL should be considered infectious. 
4. ZIKV was rarely detected in saliva or cervico-vaginal secretions (minimizing the risk of 
transmission from women)  
 
The purpose of this protocol is to develop a ZIKV controlled human infection model 
(CHIM) for use in ZIKV vaccine development and possibly licensure. However, there are 
several issues to consider in the development of a safe and useful ZIKV CHIM. To this end, in 
December of 2016, an ethics consultation was convened by the U.S. National Institutes of Health 
(NIH) in collaboration with the Walter Reed Army Institute of Research (WRAIR) with the 
purpose of identifying if and under what circumstances a ZIKV CHIM could be ethically 
conducted. The report of the consultation was released in February 2017 and can be found at: 
https://www.niaid.nih.gov/sites/default/files/EthicsZikaHumanChallengeStudiesReport2017.pdf. 
The committee identified 2 major risks of a ZIKV CHIM: 
 
1.) the risk of a ZIKV CHIM to the fetus should a pregnant woman become infected and  
2.) the risk to third parties, through mosquito and sexual transmission. 
 
Because of ongoing transmission of ZIKV in endemic areas at that time, the committee 
concluded that these risks did not outweigh the benefits of a ZIKV CHIM as the ongoing ZIKV 
circulation would allow for natural history studies of ZIKV infection to fill knowledge gaps 
related to the kinetics of ZIKV infection, transmissibility, shedding, and clinical illness, and 
would also allow for the conduct of traditional Phase 3 efficacy studies of vaccines and 
therapeutics. The committee did state that if circumstances changed or if the protocol was 
designed to mitigate the risks raised by the committee a specific proposal for a Zika virus human 
challenge study is ethically sound.  

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At the time this report was released, ZIKV transmission was already waning.  
Currently, only sporadic cases of ZIKV have been reported to Pan American Health 
Organization (PAHO) (Figure 2) [43]. For this reason, in March of 2018 an NIH/WHO meeting 
was convened to report on pathways to licensure for ZIKV vaccines given the low transmission 
of ZIKV. A ZIKV CHIM was considered as part of a pathway to licensure of a ZIKV vaccine 
and the report concluded that the circumstances and calculus have changed substantially and that 
a ZIKV CHIM could move forward [43]. In designing this protocol, we have addressed the risks 
raised during the 2016 ethics consultation and have provided a detailed risk mitigation strategy. 
We have adopted many of the recommendations of the ethical committee, as outlined in the 2017 
publication.  
The primary objective of this study is to identify and characterize a suitable ZIKV strain 
for use in future ZIKV controlled human infection models (CHIM). The use of a ZIKV CHIM 
will be critical for evaluation and down-selection of candidate ZIKV vaccines. Because ZIKV 
congenital syndrome can occur with asymptomatic infection and infection during any trimester, 
it is likely that a ZIKV vaccine will need to induce protection against ZIKV viremia to prevent 
congenital infection. Prevention of symptoms in the mother may not be sufficient to prevent 
congenital Zika syndrome. The ability of a vaccine to prevent ZIKV viremia (sterilizing 
immunity) can best be evaluated in a CHIM, prior to Phase 2 or Phase 3 studies. In addition, the 
ZIKV epidemic has waned over the past 2 years and modelling exercises predict that, in the 
future, outbreaks may occur only once every 10 years [44]. For this reason, it is not likely that 
there is sufficient predictable ZIKV transmission for a Phase 3 efficacy study to be performed 
and the efficacy of candidate ZIKV vaccines may only be able to be determined by CHIM 
studies. As stated above, the value of a ZIKV CHIM was affirmed at a recent WHO/NIH 
consultation on immune correlates of protection for ZIKV [43]. Because of the current lack of 
ZIKV transmission and the important role that a ZIKV CHIM can play in the pathway to 
licensure of a ZIKV vaccine, the consultation concluded that a ZIKV CHIM was ethically 
justified at this time should the studies sufficiently mitigate the risk of CZS and the risk of third-
party transmission [43]. A ZIKV CHIM could also be critical to the development and licensure 
of diagnostics and therapeutics for ZIKV. 
A secondary objective of the protocol is to further define the clinical presentation, viral 
replication, and sites and duration of ZIKV shedding following infection in healthy adult women 
and men. This study will contribute to the body of knowledge related to ZIKV infection and 
transmission. Although the data from natural ZIKV cases suggest that the risk of sexual 
transmission from men to women does not appear to be high, there are gaps in that knowledge. 
Figure 2: Monthly ZIKV Illness Case Counts Reported to PAHO (Vannice et al, 
2019) 

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We will enroll men to evaluate the shedding of infectious ZIKV in semen to advance knowledge 
of the risk of sexual transmission from men to women. 
The 2 greatest risks of a ZIKV CHIM, as articulated by the ethics consultation, are the 
risk to the fetus should a pregnant woman become infected, and the risk of third-party 
transmission, either by mosquito or by sexual transmission. As recommended by the ethics 
review, cohorts 1 and 2 only enrolled non-pregnant and non-lactating woman. Enrolled women 
were required to use hormonal birth control, an intrauterine device (IUD), or be incapable of 
conceiving (either surgically or biologically) to meet enrollment criteria (Section 4.1). In 
addition, women were tested using a serum pregnancy test at initial screening, within 14 days 
prior to inoculation, and the day of inoculation (prior to receipt of test article) (Section 6). Serum 
pregnancy tests can detect a pregnancy as early as 7 – 10 days post-conception. The embryo 
attaches to the uterine wall during the first 2 weeks of development. Problems with uterine 
attachment and severe cell damage can result in miscarriage. Because the cells of the embryo 
have a greater ability to recover at this stage, exposures during the 3 – 5 weeks post-conception 
are not likely to cause a birth defect [45]. Requiring the use of highly effective birth control and 
serially performing serum pregnancy tests will ensure to the greatest degree possible that only 
non-pregnant women are enrolled in the study. Should a woman have conceived within 7 days of 
ZIKV inoculation, the inoculation would have occurred extremely early post-conception, at a 
time when the expected outcome would either be a spontaneous abortion or a normal pregnancy 
without any resultant birth defect. This strategy minimizes the risk of CZS to the greatest 
possible extent. None of the women enrolled in cohorts 1 or 2 became pregnant during the study. 
As there has been only one documented case of sexual transmission from a woman to a 
man, and data from the Paz-Bailey paper indicated that ZIKV was recovered only infrequently 
from cervico-vaginal secretions. Infectious ZIKV was recovered very infrequently from saliva, 
CVS, and urine following inoculation with ZIKV-SJRP or ZIKV-Nicaragua (Table 5 and Table 
7). Shedding of ZIKV in semen will be evaluated in cohorts 3 & 4, which will enroll men. 
Transmission of both ZIKV strains to mosquitoes will be evaluated in a subset of volunteers in 
cohorts 3 and 4 to assess the possibility of conducting future ZIKV CHIM studies in the 
outpatient setting. The peak infectious titers of ZIKV-SJRP recovered from volunteers in cohort 
1 were well below 3 log10 PFU/mL. Given that mosquitoes ingest only ~2 µL per blood meal, it 
is unlikely that virus at this low titer will be transmitted to mosquitoes. Peak titers of infectious 
ZIKV-Nicaragua were significantly higher than those of ZIKV-SJRP and it is likely that this 
virus will be transmitted to mosquitoes from volunteers enrolled in cohort 4. Volunteers in 
cohorts 3 and 4 will be housed in an inpatient unit from inoculation day through Study Day 8 to 
reduce the risk of mosquito transmission, as was recommended by the ethics consultation. 
Volunteers will not be discharged from the unit until ZIKV titer in serum, urine, saliva, semen, 
and cervico-vaginal secretions is < 1.4 x 106 genomic copies/mL. Additional components of the 
risk mitigation strategy can be found in Section 10.3.7.  
In characterizing the 2 ZIKV strains that will be studied in this protocol, we will conduct 
sampling of blood, urine, cervico-vaginal secretions, semen, and saliva at multiple time-points 
throughout the 6-month duration of subject participation to determine the quantity and duration 
of shedding of ZIKV in these samples. Detection of ZIKV in these samples will be performed by 
RT-PCR as well as by culture of the virus to determine the shedding of infectious virus. The 
incidence of symptomatic illness will be determined, and the clinical characteristics of illness 

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will be described. The data obtained from this study will inform the scientific community 
regarding the sites, magnitude, and duration of infectious ZIKV in women and men. Should a 
safe and effective ZIKV CHIM be developed, it would be extremely valuable in the down-
selection of candidate vaccines and therapeutics and could be used as a critical component of the 
pathway to licensure. 
 
1.2 
Background: ZIKV Strains 
1.2.1 ZIKV-SJRP/2016 
ZIKV-SJRP/2016 was isolated from the serum of a previously healthy non-pregnant adult 
female in São Jose do Rio Preto, (São Paulo State), Brazil who presented with 
uncomplicated ZIKV infection (fever, rash, and conjunctivitis). The virus was isolated 
directly from serum, expanded, and biologically cloned at the Laboratory of Infectious 
Diseases (LID), National Institute of Allergy and Infectious Diseases (NIAID), NIH and 
the clinical lot was manufactured at Charles River Laboratories (CRL) Biopharmaceutical 
Services facility in Malvern, PA. The final drug product, ZIKV-SJRP/2016 (Lot 
ZIKV#117A) was manufactured in qualified Vero cells at CRL using a method 
developed by LID/NIAID/NIH and CRL.  
1.2.1.1 Final Container of ZIKV-SJRP/2016 
The Final Drug Product was dispensed as 1.0 mL aliquots of live, Vero Grown Zika 
virus into 1.8 mL sterile cryovials and stored at -75C 15C. The titer of ZIKV-
SJRP/2016 Lot ZIKV#117A Final Drug Product determined on December 5, 2016 was 
approximately 105.7 PFU/mL. 
1.2.1.2 Composition of ZIKV-SJRP/2016 
The Final Drug Product composition is a concentration of live, Vero Grown Zika virus, 
Lot ZIKV#117A in L-15 medium containing 1X SPG (sucrose 0.128M; KH2PO4, 
0.0038M; K2HPO4, 0.0072M; mono-sodium glutamate, 0.0054M). The potency of 
ZIKV-SJRP/2016 (Lot ZIKV#117A) is 105.7 PFU/mL. 
1.2.1.3 Investigational Product Label of ZIKV-SJRP/2016 
 
 
LIVE ZIKA VIRUS 
ZIKV-SJRP/2016 
VERO GROWN VIRUS 
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Date: 08FEB2017 
Vial#: XXXX 
Lot: ZIKV#117A 
Store at -75 ± 15oC 
Charles River Laboratories, Malvern, PA 
 
 
Figure 3: Label for Final Vial of ZIKV-SJRP/2016 (Enlarged Sample) 
 
1.2.2 ZIKV-Nicaragua/2016 
ZIKV-Nicaragua/2016 was isolated from the serum of a previously healthy 9-year-old 
female patient from Nicaragua who presented with uncomplicated ZIKV infection (fever 

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only). The virus was isolated from serum, expanded, and biologically cloned at the LID, 
NIAID, NIH and the clinical lot was manufactured at CRL Biopharmaceutical Services 
facility in Malvern, PA. The final drug product, ZIKV-Nicaragua/2016 (Lot 
ZIKV#116A) was manufactured in qualified Vero cells at CRL using a method 
developed by LID/NIAID/NIH and CRL. 
1.2.2.1 Final Container of ZIKV-Nicaragua/2016 
The Final Drug Product was dispensed as 1.0 mL aliquots of live, Vero Grown Zika 
virus into 1.8 mL sterile cryovials and stored at -75C 15C. The titer of ZIKV-
Nicaragua/2016 Lot ZIKV#116A Final Drug Product determined on December 5, 2016 
was approximately 106.6 PFU/mL. 
1.2.2.2 Composition ZIKV-Nicaragua/2016 
The Final Drug Product composition is a concentration of live, Vero Grown Zika virus, 
Lot ZIKV#116A in L-15 medium containing 1X SPG (sucrose 0.128M; KH2PO4, 
0.0038M; K2HPO4, 0.0072M; mono-sodium glutamate, 0.0054M). The potency of 
ZIKV-Nicaragua/2016 (Lot ZIKV#116A) is 106.6 PFU/mL. 
1.2.2.3 Investigational Product Label of ZIKV-Nicaragua/2016 
 
 
LIVE ZIKA VIRUS 
ZIKV-NICARAGUA/2016 
VERO GROWN VIRUS 
CAUTION: NEW DRUG LIMITED 
BY FEDERAL (USA) LAW 
TO INVESTIGATIONAL USE 
Date: 15NOV2016 
Vial#: XXXX 
Lot: ZIKV#116A 
Store at -75 ± 15oC 
Charles River Laboratories, Malvern, PA 
 
Figure 4: Label for Final Vial of ZIKV-Nicaragua/2016 (Enlarged Sample) 
 
1.3 
Rationale 
1.3.1 Animal Experience 
1.3.1.1 Non-human Primate Study 
Four rhesus macaques were infected with 2x104 PFU of the Current Good Manufacturing 
Practice (cGMP) material of either ZIKV-SJRP/2016 or ZIKV-Nicaragua/2016 by subcutaneous 
injection. The macaques were examined daily and bled daily from Day 2 post-infection through 
Day 8 post-infection for detection of virus. All 4 animals from each group had ZIKV detectable 
by viral culture on multiple days from Day 2 through Day 5 post-infection. Peak virus was 
recovered on Day 3 or Day 4 post-infection (Table 2). It has been our experience from our 
dengue models that NHP develop viremia earlier than do humans. Of note, the ZIKV that will be 
administered in this protocol will be first given at a dose 200-fold lower than that which was 
administered to rhesus monkeys. 
 
Table 2: ZIKV Viremia in Rhesus macaques 
ZIKV strain 
Dose 
Mean Peak 
Titer (log10 
PFU/mL) 
Peak Titer 
(log10 PFU/mL) 
Mean 
Day of 
Onset 
Mean 
Duration of 

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viremia, days 
(range) 
SJRP/2016 
20,000 PFU 
1.8 ± 0.1 
1.9 
2.3 
3.5 (3 – 4) 
Nicaragua/2016 
20,000 PFU 
2.3 ± 0.11 
2.5 
2.0 (all) 
3.3 (3 – 4) 
PFU = plaque-forming units, ZIKV = Zika virus  
1The mean peak titer of ZIKV SJRP/2016 was significantly lower than that of ZIKV Nicaragua/2016, p < 0.05. 
1.3.2 Previous Human Experience 
1.3.2.1 DENV-2 Controlled Human Infection Model (CHIM) 
We have utilized a DENV-2 and a DENV-3 CHIM to evaluate the protective efficacy of the live 
attenuated tetravalent dengue vaccine candidates TV003 and TV005. The DENV-2 CHIM virus 
was derived from DENV-2 Tonga/74, which caused only mild disease during an outbreak in 
Tonga [46]. Although originally developed as a DENV-2 candidate vaccine, the recombinant 
virus (rDEN230) was not attenuated when compared to its wild-type (Wt) parent in rhesus 
macaques and was not considered for evaluation as a vaccine candidate. However, it was 
repurposed for use as a DENV-2 challenge virus in the dengue CHIM. rDEN230 has been 
administered to more than 50 subjects in 3 different challenge studies. A dose of 1,000 PFU 
induced viremia in 100% of subjects at a mean peak titer of 2.3 log10 PFU/mL, approximately 
100-fold higher than that observed with the attenuated DENV included in TV003 and TV005 
[47, 48]. The mean day of onset of viremia was nearly day 5 post-infection and the mean day of 
peak viremia was nearly 7 days post-infection (Table 3). The level of viremia observed in 
humans was similar to that observed in rhesus macaques (2.3 log10/mL PFU vs 1.7 log10 
PFU/mL) but the duration of viremia was longer in humans (5.2 days vs 2.8 days) [49]. It also 
induced a mild to moderate rash in 88% of subjects to whom it was administered. Fever was not 
observed in any subject. Both TV003 and TV005 were demonstrated to protect 100% of 
vaccinated volunteers against challenge with DEN230 administered 6 months after vaccination 
[48]. 
 
1.3.2.2 DENV-3 CHIM 
The DENV-3 CHIM virus rDEN330 was derived from a Wt DENV-3 (Sleman/78) that was 
noted to cause mild illness during its outbreak in Indonesia [50]. It was originally developed as a 
candidate DENV-3 vaccine virus but was not attenuated in rhesus macaques compared with its 
Wt parent. It was first evaluated in 10 healthy, flavivirus-naïve subjects at a dose of 1,000 PFU 
and induced detectable infectious virus in all 10 subjects. The mean peak titer was 0.5 log10 
lower than that observed with the DEN230 and the absolute peak titer was more than a log 
lower (Table 3). The level of viremia observed in humans was similar to that observed in rhesus 
macaques (1.77 log10/mL PFU vs 1.5 log10 PFU/mL) but the duration of viremia was slightly 
longer in humans (3.4 days vs 2.0 days) [51]. A mild to moderate rash was observed in 80% of 
DEN330 recipients; fever was not observed in any recipient. The DENV-3 CHIM was utilized 
at a dose of 10,000 PFU to evaluate the protective efficacy of TV005 against DENV-3. TV005 
elicited 100% protection against challenge with DENV-3 at 6 months. 
 

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Table 3: Viremia Induced by the DENV-2 and DENV-3 CHIM Viruses in Normal Healthy 
Volunteers 
Virus 
N 
Dose 
Mean peak 
titer ± SE 
(log10 PFU/mL) 
Peak titer 
(log10 PFU/mL) 
Mean 
day of 
peak titer 
± SE 
Mean 
Day of 
onset ± 
SE 
Mean 
duration of 
viremia, days 
± SE 
rDEN230 
57 
1,000 PFU 
2.31 ± 0.07 
3.3 
6.9 ± 0.2 
4.9 ± 0.3 
5.2 ± 0.3 
rDEN330 
10 
1,000 PFU 
1.77 ± 0.15 
2.1 
5.3 ± 0.3 
4.6 ± 0.4 
3.4 ± 0.6 
N = number of subjects, PFU = plaque-forming units, SE = standard error 
1.3.2.3 ZIKV-SJRP (Cohort 1) 
ZIKV-SJRP or placebo was administered to 14 women in cohort 1 randomized 5:2 in 2 blocks of 
volunteers. Ten women received ZIKV-SJRP and 4 women received placebo. Adverse events 
following administration of ZIKV-SJRP or placebo are shown in Table 4. ZIKV-like rash was 
observed in 9/10 volunteers who received ZIKV-SJRP and was the most common adverse event 
related to ZIKV-administration. Non-purulent conjunctivitis occurred in 50% of ZIKV-SJRP 
recipients, arthralgia in 70% and myalgia in 50%. Fever was not observed nor were any signs or 
symptoms consistent with GBS. 
 
ZIKV-SJRP was recovered from 100% of those who received the virus. Both infectious virus 
and virus detected by PCR were recovered from the serum of all 10 infected volunteers (Figure 
5). The virus detected by PCR was below the cut-off for discharge from the inpatient unit at all 
times post-administration. Infectious virus titer peaked at day 5 in 6/10 volunteers. The mean 
peak titer was 46.8 PFU/mL (1.67 log10 PFU/mL). Peak virus titer was below 100 PFU/mL 
(2 log10 PFU/mL) in 8/10 volunteers and below 1,000 PFU/mL (3 log10 PFU/mL in the 
remaining 2 volunteers. Infectious ZIKV-SJRP was found infrequently in CVS, saliva, and urine 
(Table 5). ZIKV-SJRP was found by qPCR in urine, saliva, and CVS in 80%, 70%, and 50% of 
women, although the titers were low and the duration of shedding short (Table 5). ZIKV-SJRP 
was not recovered from any specimen taken from placebo recipients. The low peak titer 
following inoculation with 100 PFU of ZIKV-SJRP suggests that challenge studies may be 
safely performed in an outpatient setting. 
 
Table 4: All Adverse Events Following One Dose of Cohort 1 (ZIKV-SJRP/placebo)  
(CIR 316)  
 
ZIKV-SJRP (n=10) 
N (%) 
Placebo (n=4) 
N (%) 
P value (1-
sided)1 
Local 
 
 
 
 
Erythema 
1 (10.0%) 
0 (0.0%) 
0.7143 
 
Tenderness 
1 (10.0%) 
0 (0.0%) 
0.7143 
 
Pain 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Pruritus 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Induration 
0 (0.0%) 
0 (0.0%) 
n/a 
Systemic 
 
 
 
 
Fever 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Headache 
9 (90.0%) 
3 (75.0%) 
0.9341 
 
Zika-like Rash 
9 (90.0%) 
0 (0.0%) 
0.0050 
 
Neutropenia2 
1 (10.0%) 
0 (0.0%) 
0.7143 

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ZIKV-SJRP (n=10) 
N (%) 
Placebo (n=4) 
N (%) 
P value (1-
sided)1 
 
Nonpurulent 
 
conjunctivitis 
5 (50.0%) 
0 (0.0%) 
0.1259 
 
Elevated ALT 
0 (0.0%) 
1 (25.0%) 
1.0000 
 
Myalgia 
5 (50.0%) 
0 (0.0%) 
0.1259 
 
Arthralgia 
7 (70.0%) 
0 (0.0%) 
0.0350 
 
Retro-orbital Pain 
4 (40.0%) 
0 (0.0%) 
0.7203 
 
Fatigue 
5 (50.0%) 
2 (50.0%) 
0.4286 
 
Muscle weakness 
13 (10.0%) 
0 (0.0%) 
0.7143 
 
Prolonged PT 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Hyporeflexia 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Prolonged PTT 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Thrombocytopenia 
0 (0.0%) 
0 (0.0%) 
n/a 
1. Greater for ZIKV-SJRP 
2. Neutropenia was defined as an ANC ≤ 1,000/mm3.  
3. Muscle weakness was reported by the volunteer but was not detected on physical examination. It lasted 1 
day. 
 
Figure 5: Kinetics of ZIKV-SJRP in Serum by Culture and Quantitative PCR 
 
 
 
 
 
 
 
 
 
 
 
 
 
0
2
4
6
8
10
12
14
16
0
1
2
3
4
5
6
Day post-challenge
Log10 PFU/mL 
Infectious titer - serum
001
002
003
004
007
LLD
009
010
011
013
014
0
2
4
6
8
10
12
14
16
0
1
2
3
4
5
6
Day post-challenge
Log10 GC/mL 
Titer by PCR - serum
001
002
003
004
007
LLQ
Cut-off for discharge
009
010
011
013
014

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Table 5: Recovery of ZIKV-SJRP from Serum, Whole Blood, CVS, Saliva, and Urine 
ZIKV-SJRP recovered by virus culture (infectious virus) (Day 0 – 28) 
Sample 
Frequency 
(N=10) 
Mean peak titer 
(log10PFU/mL) 
Mean day of 
onset 
Mean 
duration 
(days) 
Duration 
range 
(days) 
Serum 
100% 
1.67 
4.0 
4.7 
4 - 6 
Whole blood 
40% 
1.7 
5.3 
2.3 
1 - 3 
CVS 
10% 
2.30 
10.0 
1.0 
1 - 1 
Saliva 
30% 
0.50 
8.7 
1.0 
1 - 1 
Urine 
10% 
0.7 
10.0 
3.0 
3 - 3 
ZIKV-SJRP recovered by quantitative PCR (Day 0 – 28) 
Serum 
100% 
5.11 
2.5 
7.5 
7 - 10 
Whole blood 
100% 
4.19 
4.2 
7.8 
4 - 15 
CVS 
50% 
2.72 
10.7 
3.8 
1 - 7 
Saliva 
70% 
2.69 
10.3 
1.9 
1 – 5 
Urine 
80% 
3.00 
11.5 
3.4 
1 - 10 
 
1.3.2.4 ZIKV-Nicaragua (Cohort 2) 
ZIKV-Nicaragua or placebo has been administered to 14 women in cohort 2 randomized 5:2 in 
2 blocks of volunteers. Ten women received ZIKV-Nicaragua and 4 women received placebo, 
however the treatment assignment has been unblinded for only 7 of the 14 volunteers at this 
time. Adverse events following administration of ZIKV-Nicaragua or placebo are shown in 
Table 6. ZIKV-like rash was observed in 5/5 volunteers who received ZIKV-Nicaragua and was 
the most common adverse event related to ZIKV-administration. Myalgia in 80% of ZIKV-
Nicaragua recipients and arthralgia was observed in 60%. Fever was not observed nor were any 
signs or symptoms consistent with GBS. Non-purulent conjunctivitis was not observed in 
recipients of ZIKV-Nicaragua.  
 
ZIKV-Nicaragua was recovered from the serum and whole blood of 5/5 volunteers who received 
the virus (100%). Both infectious virus and virus detected by quantitative PCR were recovered 
from the serum of all 5 infected volunteers (Figure 6). The virus detected by PCR was above the 
cut-off for discharge from the inpatient unit (> 6.4 log10 GEQ/mL) for only 1 time-point for only 
2 volunteers (both day 6). The titer was below the cut-off for discharge for all other time-points 
and all other volunteers. The mean peak titer of infectious ZIKV-Nicaragua in the serum was 
4,266 PFU/mL (3.63 log10 PFU/mL) which was significantly higher than that of ZIKV-SJRP 
(46.8 PFU/mL; 1.67 log10 PFU/mL),  = 0.01. The higher infectious peak titer following 
inoculation with 100 PFU of ZIKV-Nicaragua suggests that this virus is likely to be transmitted 
to mosquitoes. The mean peak titer in serum by quantitative PCR was also significantly higher 
for ZIKV-Nicaragua compared with ZIKV-SJRP (6.29 log10 GEQ/mL vs 5.11 log10 GEQ/mL, 
respectively, =0.01). 
 
 

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Table 6: All Adverse Events Following One Dose of Cohort 2a (ZIKV-Nicaragua/Placebo)  
(CIR 316)  
 
ZIKV-Nicaragua (n=5) 
N (%) 
Placebo (n=2) 
N (%) 
P value (1-
sided)1 
Local 
 
 
 
 
Erythema 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Tenderness 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Pain 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Pruritus 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Induration 
0 (0.0%) 
0 (0.0%) 
n/a 
Systemic 
 
 
 
 
Fever 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Headache 
4 (80.0%) 
2 (100.0%) 
1.0000 
 
Zika-like Rash 
5 (100.0%) 
0 (0.0%) 
0.0476 
 
Neutropenia2 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Nonpurulent 
 
conjunctivitis 
0 (0.0%) 
1 (50.0%) 
1.0000 
 
Elevated ALT 
2 (40.0%) 
0 (0.0%) 
0.4762 
 
Myalgia 
5 (100.0%) 
1 (50.0%) 
0.2857 
 
Arthralgia 
3 (60.0%) 
0 (0.0%) 
0.2857 
 
Retro-orbital Pain 
4 (80.0%) 
1 (50.0%) 
0.9524 
 
Fatigue 
5 (100.0%) 
2 (100.0%) 
n/a 
 
Muscle weakness 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Prolonged PT 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Hyporeflexia 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Prolonged PTT 
0 (0.0%) 
0 (0.0%) 
n/a 
 
Thrombocytopenia 
0 (0.0%) 
0 (0.0%) 
n/a 
1. Greater for ZIKV-Nicaragua 
2. Neutropenia was defined as an ANC ≤ 1,000/mm3.  
 

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Figure 6: Kinetics of ZIKV-Nicaragua in Serum by Culture and Quantitative PCR 
 
 
Table 7: Recovery of ZIKV-Nicaragua from Serum, Whole Blood, CVS, Saliva, and Urine 
ZIKV-Nicaragua recovered by virus culture (infectious virus) (Day 0 – 28) 
Sample 
Frequency 
(N=5) 
Mean peak titer 
(log10PFU/mL) 
Mean day of 
onset 
Mean 
duration 
(days) 
Duration 
range 
(days) 
Serum 
100% 
3.63 
4.04 
3.0 
5.4 
Whole blood 
100% 
3.52 
3.90 
4.4 
4.2 
CVS 
0% 
n/a 
n/a 
n/a 
n/a 
Saliva 
100% 
0.58 
0.70 
8.0 
3.4 
Urine 
20% 
0.50 
0.50 
6.0 
1.0 
ZIKV-Nicaragua recovered by quantitative PCR (Day 0 – 28) 
Serum 
100% 
6.29 
6.57 
2.4 
11.4 
Whole blood 
100% 
5.62 
6.21 
3.2 
8.8 
CVS 
80% 
2.43 
2.89 
8.5 
4.0 
Saliva 
100% 
2.71 
3.80 
10.8 
2.2 
Urine 
80% 
2.86 
4.23 
8.0 
5.5 
2 
OBJECTIVES 
2.1 
Primary Objectives 
• Determine the suitability of both ZIKV strains for use in a safety-assured Zika CHIM. 
Suitability will be assessed by:  
▪ The frequency and severity of clinical signs and symptoms of infection such as 
fever, rash, conjunctivitis, myalgia, arthralgia, and headache following ZIKV 
administration compared with placebo. 
▪ The infection frequency of ZIKV by strain and by dose. Infection is defined as 
recovery of ZIKV (determined by RT-PCR or culture) from blood, urine, cervico-
vaginal secretions, semen, or saliva and/or by seroconversion to ZIKV (50% plaque 
reduction neutralization titer [PRNT50]≥ 1:10) by 90 days post-administration of 
the ZIKV strain. 
0
2
4
6
8
10
12
14
16
0
1
2
3
4
5
6
7
Day post-challenge
Log10 PFU/mL 
Infectious titer - serum
019
021
020
015
018
LLD
0
2
4
6
8
10 12 14 16 18 20
0
1
2
3
4
5
6
7
Day post-challenge
Log10 GC/mL 
Titer by PCR - serum
019
021
020
015
018
LLQ
Cut-off for discharge
 

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▪ The frequency, magnitude, and duration of ZIKV presence in the blood, urine, 
cervico-vaginal secretions, semen, and saliva by both RT-PCR and virus titration 
in tissue culture by ZIKV strain and dose. 
• A suitable ZIKV CHIM strain is defined as one that: 
a. ≥ 80% of ZIKV and DENV-naïve inoculated subjects develop detectable viremia 
by virus culture (infectious virus) and 
b. Infectious virus is detected on more than 1 day for ≥ 80% of subjects with detectable 
viremia and 
c. The mean peak titer of infectious virus recovered from subjects is 2 - 3.0 log10 
PFU/mL 
Ideally ≥ 50% of ZIKV and DENV-naïve inoculated subjects will develop ≥ 1 of the 
following clinical signs thought to be definitely, probably, or possibly due to the ZIKV 
strain, however this will not be required for dose expansion or dose escalation: 
 
i. Fever 
ii. Rash 
iii. Non-purulent conjunctivitis 
iv. Myalgia 
v. Arthralgia 
A mean peak titer of 2.0 – 3.0 log10 PFU/mL should ensure that virus is readily detected but at a 
level that reduces the risk of symptomatic illness and virus transmissibility. 
2.2 
Secondary Objectives  
• Characterize the clinical presentation of acute ZIKV infection by assessing the frequency 
of adverse events (AEs), graded by severity, occurring within 28 days of ZIKV 
administration.  
• 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 ZIKV 
strain at each tested dose as determined by RT-PCR and virus culture 
▪ The quantity and duration of ZIKV shedding in saliva determined by RT-PCR and 
virus culture 
▪ The quantity and duration of ZIKV shedding in urine determined by RT-PCR and 
virus culture 
▪ The quantity and duration of ZIKV shedding in cervico-vaginal secretions 
determined by RT-PCR and virus culture 
▪ The quantity and duration of ZIKV shedding in semen determined by RT-PCR and 
virus culture 
• Evaluate the kinetics of the serum neutralizing antibody response following primary ZIKV 
infection and determine the peak neutralizing antibody response to ZIKV through Study 
Day 90. 
2.3 
Exploratory Objectives 
The exploratory objectives of this study are the following: 
• Evaluate the cellular immune response to primary infection with ZIKV. 
• Evaluate the innate immune response to primary infection with ZIKV. 

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• Evaluate B and T cell memory responses following primary infection with ZIKV. 
• Evaluate the antibody repertoire induced by ZIKV infection. 
• Evaluate the antibody response in saliva to primary infection with ZIKV as a possible 
diagnostic tool. This will be done in a subset of subjects (those enrolled at the Center for 
Immunization Research [CIR]). 
• Evaluate the transmissibility of ZIKV-SJRP and ZIKV-Nicaragua from infected volunteers 
to mosquitoes. 
3 
STUDY DESIGN  
3.1 
Receipt of Test Article 
3.1.1 Overall Design  
This study is a placebo-controlled, double-blind study in normal healthy adult male and non-
pregnant female subjects 18 - 40 years of age, inclusive, recruited from the metropolitan 
Baltimore/Washington, DC and Burlington, VT areas. The purpose of this study is to evaluate 
the clinical and virologic response to escalating doses of 2 different ZIKV strains administered 
subcutaneously in healthy, ZIKV and DENV-naïve, male and non-pregnant, female adult 
volunteers to identify the most suitable ZIKV strain and dose for use in a ZIKV CHIM. The 
ZIKV CHIM will then be used to evaluate the protective efficacy of candidate ZIKV vaccines 
prior to evaluation of these candidates in Phase 2 clinical trials. Placebo recipients are included 
in the study as a control to better assess ZIKV-associated versus non-ZIKV-associated AEs.  
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, urine 
toxicology screening for opiates and serology screening for previous ZIKV or DENV infection. 
Serum pregnancy testing will be performed on applicable female subjects. All screening tests 
must be performed within 60 days of inoculation. HIV screening must be performed within 2 
weeks of inoculation. Pregnancy screening will be repeated within 14 days prior to inoculation 
and on the day 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 4 of this protocol. For subjects who are eligible, the Day 0 
visit will be scheduled for receipt of inoculation. Serum pregnancy testing will be repeated on 
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 (ZIKV or placebo). Subjects will present 
to the unit on Study Day 0. After eligibility criteria have been reviewed and confirmed, they will 
be inoculated with test article on Study Day 0. Until mosquito-transmissibility data are available, 
subjects will be admitted to the inpatient unit and will be kept in the inpatient unit through Study 
Day 9 (discharged on Study Day 9, for a total of 9 nights), the period of time when viremia is 
expected to be present and at a level that would be transmissible to mosquitoes. If mosquito-
transmissibility data from cohorts 3 and 4 demonstrate that ZIKV-SJRP or ZIKV-Nicaragua 
cannot escape the mid-gut barrier of the mosquito, additional studies of these ZIKV may be 
considered for outpatient evaluation for the duration the subjects’ enrollment. The protocol will 
be revised to include an outpatient enrollment if the transmissibility studies support this. 
For the inpatient portion of the study, subjects will remain in the inpatient unit from Study Day 0 
through Study Day 9 (9 nights, discharged on Study Day 9). If they meet discharge criteria (See 

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Section 6.4.2), they will be discharged from the inpatient unit on Study Day 9. They will be 
evaluated every day during the inpatient stay and will be seen again on Study Day 9, Study Day 
10, Study Day 12, Study Day 14, and Study Day 16. During the outpatient and inpatient visits 
through Study Day 28, the subjects will be evaluated by a clinician and will have blood drawn 
for clinical laboratory studies, virologic assays, and immunologic assays. In addition, urine, 
cervico-vaginal secretions, semen, and saliva will be collected at specific time points for 
additional virologic assays (see Section 6 for a detailed description of study procedures). 
Subjects will return to the clinic on Study Days 21, 28, 35, 56, 70, 90, 120, and 180 for 
evaluation, blood and specimen collection. Study Day 180 will be their final visit. Subjects will 
have their temperature measured and recorded at least twice a day during their inpatient stay, 
with the exception of day of discharge. Temperature will be recorded once by study staff prior to 
discharge. 
3.1.2 Study Design  
This study will include 4 cohorts of 14 ZIKV and DENV-naïve female and male subjects, 18 - 40 
years of age (total: up to 56 subjects). Within each cohort, 10 subjects will receive ZIKV and 4 
subjects will receive a placebo on Study Day 0. Cohorts 1 and 2 (Dose = 102 PFU) will be 
enrolled first and will enroll only women. Cohorts 3 and 4 (Dose = 102 PFU) will enroll men.   
3.1.2.1 Initial Enrollment of Each Cohort 
For each cohort, only the first 7 subjects (5 ZIKV recipients; 2 placebo recipients) will be 
enrolled until all 7 subjects have completed Study Day 28 and the criteria listed in Section 
3.1.2.2 have been met. If the criteria to enroll the remaining 7 subjects from a dose cohort are 
met, enrollment will proceed. For cohorts 3 and 4, mosquito-feeding will be performed on a 
subset of enrolled volunteers. A total of 7 volunteers (randomized 5:2 ZIKV:placebo) from 
cohort 3 and 7 volunteers (randomized 5:2 ZIKV:placebo) from cohort 4 will undergo mosquito-
feeding. 
3.1.2.2 Enrollment of Remaining 7 Subjects in a Dose Cohort 
The remaining 7 subjects in a dose cohort may be enrolled if the following criteria are met: 
1. Halting criteria have not been met and 
2. ≥ 4 subjects in a cohort have detectable infectious virus with a mean peak titer ≥ 1.0 log10 
PFU/mL and 
3. ≥ 4 subjects in a cohort have detectable infectious virus for at least 2 days 
3.1.3 Sample Size and Placebo Ratio Primary Study 
Fourteen subjects (10 ZIKV and 4 placebo recipients) will be enrolled into each dose cohort. 
This sample size was chosen based on our experience in developing the DENV-2 CHIM 
(CIR286, clinical trials.gov NCT01931176, [47]) and the DENV-3 CHIM (CIR304, clinical 
trials.gov NCT02684383).  
Ten subjects are chosen to receive each ZIKV strain at each dose for the following reasons:  
• A sample size of 10 ZIKV recipients and 4 placebo recipients in each cohort will be able to 
detect a statistically significant difference ( = 0.05) in recovery of infectious ZIKV from the 
blood between the 10 ZIKV recipients and 4 placebo recipients at a power of 0.99 with 
≥80% of ZIKV-inoculated subjects having detectable ZIKV viremia and none of the placebo 
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• A sample size of 10 ZIKV recipients and 4 placebo recipients in each cohort will be able to 
detect a statistically significant difference ( = 0.05) in any clinical sign between the 2 
groups at a power of 0.71 if the clinical sign occurs in ≥80% of ZIKV recipients and occurs 
in ≤ 1 placebo recipients. We have included placebo-recipients so that we can determine if 
common AEs are ZIKV-related.  
3.1.4 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. 
3.1.5 Estimated Duration of the Study 
The entire duration of the study is approximately 52 weeks. 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). 
3.1.6 Treatment Assignment 
Subjects will be randomly assigned to receive ZIKV or placebo in a 5:2 ratio within each cohort. 
Inoculation assignment will be done using a random number generator to prepare the sequence in 
which subjects are assigned to receive ZIKV or placebo. Subjects will be centrally randomized 
by the test article preparation study staff at Johns Hopkins Bloomberg School of Public Health 
(JHSPH) in blocks of 7. There will be 2 blocks of 7 subjects for each cohort and there will be a 
total of 4 cohorts enrolled. Each block of 7 will include 5 ZIKV recipients and 2 placebo 
recipients. As described above, each cohort will initially enroll one block of 7 subjects. The 
second block of 7 subjects will be enrolled if criteria in Section 3.1.2.2 are met. Subjects will be 
enrolled sequentially. Cohorts of both ZIKV strains may be enrolled simultaneously. Once all 7 
subjects in a block have reached Study Day 90, the inoculation assignment for that block may be 
unblinded to all staff.  
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 
are inoculated. Clinical staff will remain blinded to inoculation assignment until all subjects 
within a block have reached Study Day 90.  
A master log of inoculation assignments will be maintained in a record separate from other study 
records. This log will be maintained in a locked room with limited access. A sealed envelope 
containing a copy of the inoculation assignment will also be kept by the Data and Safety 
Monitoring Board (DSMB) Executive Secretary.  
The subjects will be informed of their inoculation assignment after their block has completed 
Study Day 90. 
3.1.7 Blinding 
This study will be conducted as a double-blind study to avoid biased assessment of AEs. ZIKV 
or placebo will be prepared and drawn up into syringes by pharmacy/laboratory personnel who 
are not involved with the clinical assessment of subjects or performing research assays. Syringes 
are labeled according to standard operating procedures (SOPs)/recommendations. Because the 

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ZIKV diluent, PlasmaLyte, is used as placebo, there will be no difference in the appearance of 
the syringes.  
The subject, investigator, and clinical staff will not know to which inoculation group the subject 
has been assigned. In addition, other personnel assigned to monitor the study will not know the 
inoculation assignment of the subject. If all volunteers within a block of 7 are able to be 
discharged from the inpatient unit on Study Day 9, the clinical staff will remain blinded to 
inoculation 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 inoculation assignments in writing from the unblinded 
study staff.  
If one or more subjects within a block of 7 does not meet discharge criteria by Study Day 9, 
those subjects must remain in the unit. To avoid undue burden on the subjects who have met 
discharge criteria, only those subjects who did not meet discharge criteria will remain in the unit 
even though this will be unblinding. If the need arises to unblind a specific subject’s assignment 
for emergency medical management prior to all subjects within a block completing Study Day 90 
post-inoculation, the PI will contact the CIR unblinded staff to obtain the inoculation assignment 
of the subject in question. Only that specific subject’s assignment will be unblinded. The 
Sponsor 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. 
4 
SELECTION AND ENROLLMENT OF SUBJECTS 
Subjects must meet all eligibility criteria to be enrolled in the study. Because male-to-female 
sexual transmission of ZIKV is by far the most common pattern of sexual transmission, all male 
volunteers enrolled in the study must agree to use barrier contraception through Study Day 90 
(per CDC guidance) post-enrollment. Female volunteers must use barrier contraception through 
Study Day 56 (per CDC guidance). Men will be enrolled to quantify the shedding of infectious 
ZIKV in semen. These data will better assess the risk of male-to-female transmission. Because 
ZIKV infection during pregnancy can be catastrophic to the fetus, female volunteers of 
childbearing potential will be required to use reliable methods of contraception, which include 
hormonal birth control (implantable, hormonal patch, NuvaRing®, oral contraception, Depo-
Provera injection, etc.), IUD, or surgical sterilization (documented hysterectomy, tubal ligation 
or tubal coil; surgical sterilization procedures must be at least 3 months prior to inoculation) 
following CDC recommendations.  Barrier contraception alone will not be considered a reliable 
method of birth control.  Females who are postmenopausal (as defined as at least 1 year since last 
menstrual period); and females who exclusively have sex with females (and have no intention of 
conceiving a child during the study) are not considered to be of childbearing potential. Female 
volunteers on hormonal birth control must not be on medications or other agents that decrease 
the effectiveness of hormonal birth control. 
 
To reduce the potential risk of transmission, ALL male subjects will be required to use barrier 
contraception through Study Day 90 and all female subjects will be required to use barrier 
contraception through Study Day 56 in accordance with CDC guidance. This includes females 
who exclusively have sex with females, females who are post-menopausal, and subjects who 
have undergone surgical sterilization.  
 

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Should a potential volunteer wish to use hormonal contraception or an IUD, study staff will refer 
her to a provider who can discuss the available contraception options with the subject. The study 
will pay for any costs of hormonal contraception not covered by insurance for the duration of the 
study and for the cost of placement of an IUD not covered by insurance if the subject decides she 
wants to enroll in the study and would like to use one of these methods of contraception. 
4.1 
Inclusion Criteria  
All of the following criteria must be fulfilled for a subject to qualify for inclusion in this study: 
1. Adult ZIKV or DENV-naïve male and non-pregnant females 18 – 40 years of age, 
inclusive.  
2. Good general health as determined by physical examination, laboratory screening, and 
review of medical history.  
3. Available for the duration of the study, approximately 26 weeks post-inoculation. 
4. Must be able to complete the informed consent process and comprehension assessment 
independently and without assistance. 
5. Willingness to participate in the study as evidenced by signing the informed consent 
document. 
6. Willingness to reside in the inpatient unit for 9 days (or longer for safety if necessary) 
following receipt of ZIKV or placebo. 
7. Male subjects: Willingness to use barrier contraception during cervico-vaginal, anal, and 
oral intercourse through Study Day 90 (in accordance with CDC guidance). 
8. Female subjects: Willingness to use barrier contraception during cervico-vaginal, anal, 
and oral intercourse through Study Day 56 (in accordance with CDC guidance).  
9. Female subjects of childbearing potential must be willing to use effective contraception 
while at risk of Zika infection. We will follow CDC guidelines for the use of effective 
contraception of 8 weeks post-infection; time of infection is defined as inoculation with 
challenge virus. Reliable methods of contraception include: hormonal birth control* 
(implantable, hormonal patch, hormonal vaginal ring, oral contraception, Depo-Provera 
injection, etc.), surgical sterilization (hysterectomy, tubal ligation, or tubal coil at least 3 
months prior to inoculation), and intrauterine device. All female subjects will be considered 
having child-bearing potential except for those with post-menopausal status documented 
as at least 1 year since last menstrual period and females who have sex with females 
(exclusively) and have no intention of conceiving a child during the study. Females who 
are not considered to be of childbearing potential will not be required to use contraception 
other than barrier contraception for the purpose of reducing potential transmission.  
*Volunteers on hormonal birth control must not be on medications or other agents that 
decrease the effectiveness of hormonal birth control.  
4.2 
Exclusion Criteria  
A subject will be excluded from enrollment in the study if any of the following criteria are met:  

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1. Currently pregnant, as determined by positive beta-human choriogonadotropin (-hCG) 
test, breast-feeding or planning to become pregnant during the 6-month duration of the 
study.  
2. Evidence of clinically significant neurologic, cardiac, pulmonary, hepatic, rheumatologic, 
autoimmune, or renal disease by history, physical examination, and/or laboratory studies. 
3. Behavioral, cognitive, or psychiatric disease that in the opinion of the investigator affects 
the ability of the subject to understand and cooperate with the requirements of the study 
protocol. 
4. Evidence of recent opiate use based on urine toxicology screen. 
5. Screening laboratory values of Grade 1 or above for absolute neutrophil count (ANC), 
ALT, and serum creatinine, as defined in this protocol.  
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 which 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, by screening and confirmatory assays. 
11. Hepatitis C virus (HCV) infection, by screening and confirmatory assays.  
12. Hepatitis B virus (HBV) infection, by hepatitis B surface antigen (HBsAg) screening. 
13. History of Guillain-Barré syndrome (GBS). 
14. History of seizure disease or peripheral neuropathy 
15. History of any neuroinflammatory disorder i.e. Bell’s Palsy, transverse myelitis 
16. Any known immunodeficiency syndrome, including that caused by malignancy. 
17. Use of anticoagulant medications (use of antiplatelet medication such as aspirin or non-
steroidal anti-inflammatory medication is permitted and will not exclude a subject from 
enrollment). 
18. Use of corticosteroids (excluding topical or nasal) or immunosuppressive drugs within 28 
days prior to or following inoculation. Immunosuppressive dose of corticosteroids is 
defined as ≥10 mg prednisone equivalent per day for ≥14 days. 
19. Receipt of a live vaccine within 21 days or a killed vaccine within the 14 days prior to 
inoculation or anticipated receipt of any vaccine during the 21 days following inoculation 
with the exception of the killed influenza vaccine and COVID-19 vaccines either licensed 
or under EUA which can be given at any time, however all effort will be made to avoid 
giving influenza and COVID-19 vaccines within the above windows.  
20. Asplenia. 
21. 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. 
22. History or serologic evidence of previous ZIKV infection or DENV infection 
23. Previous receipt of a ZIKV or DENV vaccine (licensed or investigational). 
24. Anticipated receipt of any investigational agent in the 28 days before or after inoculation.  
25. Subject has definite plans to travel to a ZIKV-endemic or dengue-endemic area during 
the study. 
26. Previous hypersensitivity to any study product component. 

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27. Anaphylactic reaction to mosquito bites. 
28. Refusal to allow storage of specimens for future research. 
4.3 
Subject Withdrawal and Termination Criteria 
A subject will not be considered to have completed the trial if any of the following apply. 
However, any subject who has received ZIKV 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 withdrawn will not be replaced. 
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 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 integrity of the study data 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 – is a category used when previous categories do not apply and requires an 
explanation. 
 
 
Special Situations 
Pregnancy:  
To avoid the possibility of becoming infected with ZIKV while pregnant, all female subjects will 
be screened for pregnancy during screening and again within 14 days prior to enrollment and on 
Day 0 prior to the administration of the test article. All female subjects, with the exception of 
females who exclusively have sex with females (and have no intention of conceiving a child 
during the study) and women who are postmenopausal, must agree to use reliable birth control 
while at risk of Zika infection. We will follow CDC guidelines for the use of effective 
contraception of 8 weeks post-infection; time of infection is defined as inoculation with 
challenge virus. Female subjects may be asked to sign a release of medical information form so 
that records can be obtained to confirm the method of birth control. All subjects will receive 
pregnancy prevention counseling from study staff prior to enrollment, during their inpatient stay, 
and at outpatient in-person visits at Days 9, 10, 12, 14, 16, 21 and at each in-person visit after 
Study Day 21. If outpatient in-person visits are greater than 30 days apart, we will perform 
pregnancy prevention counseling by phone. If a subject becomes pregnant at any time during the 
study, she will be followed for the duration of her pregnancy and will be seen in the clinic for 
periodic safety evaluations. She 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. In 
addition, after she has completed her pregnancy, she may be asked to return to the clinic for an 
in-person visit to review the outcome of her pregnancy and the health of the baby. 
Lost to follow-up: 

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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 3 attempts have been made to contact the subject, followed 
by nonresponse to a certified letter to the last known address requesting that the subject contact 
the clinical site.  
Incarceration: 
In the event a subject becomes incarcerated during the study, they may be terminated from the 
study if the period of incarceration will prevent the subject from making the scheduled visits. 
4.4 
Access to Medical Records 
The medical history of a subject will be obtained from the subject and will be documented. 
Medical records from an outside institution may be requested to verify method of contraception 
and may also be requested to clarify the subject’s medical history or clarification of Adverse 
Events as needed. Medical records from an outside institution will not be requested without the 
medical release form signed by the subject. 
5 
TEST ARTICLE PREPARATION 
5.1 
Pre-inoculation Preparation 
ZIKV strains for this protocol will be stored at a NIAID-contracted repository until requested by 
the clinical site. Vials of frozen ZIKV for administration will be formally requested to be 
transferred 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. ZIKV may be transferred to 
the study site prior to IRB approval and FDA review for the purpose of determining the titer of 
the test article only. 
After transfer to the clinical site, each ZIKV strain will be stored in a locked freezer at -75°C ± 
15°C until time of use. Each ZIKV strain is supplied as a concentrate that must be diluted with 
PlasmaLyte to the proper dose prior to administration.  
After the ZIKV strain(s) has been prepared at the clinical site, an aliquot of thawed, undiluted (if 
available), and diluted ZIKV will be titrated. Additionally, the ZIKV strain(s) may be titrated 
periodically to ensure potency. Clinical site pharmacy/lab personnel who are not blinded to 
inoculation assignment will be responsible for preparing the ZIKV strain(s) and placebo. The 
ZIKV strain(s) will be prepared as site-of-injection formulations. Prior to inoculation, the 
PI/designee will supply a prescription request form to the study staff responsible for ZIKV 
strain(s) preparation that will include the protocol number, date of inoculation, dose, route of 
administration, the ZIKV virus name, ZIKV lot number, the titer (concentration), the 
investigational new drug (IND) number, and the number of doses to be administered.  
The ZIKV strain(s) will be prepared according to the site’s SOPs. Study staff will prepare the 
correct dose of ZIKV (or placebo) for each subject in a biosafety cabinet using aseptic technique. 
The ZIKV strains will be diluted with PlasmaLyte, which is a commercially available crystalloid 
solution of intravenous administration. The diluted ZIKV strain(s) (or placebo) will be drawn up 
to a volume of 0.5 mL in a 1 mL syringe and labeled according to SOP. The labeled syringes will 
be transported in a cold container to the clinic for administration. The test article must be used 
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Placebo for this study will be the ZIKV diluent, Plasmalyte, which is commercially available. 
The physical appearance of the placebo and diluted ZIKV strains is colorless and identical. 
5.2 
ZIKV and Diluent Storage 
Each ZIKV strain should remain frozen at -75°C ± 15°C until just prior to use. ZIKV strains 
should never be refrozen for reuse in test article preparation. Diluent components will be stored 
per the manufacturer’s recommendation. ZIKV and diluent components should be opened from 
new containers for each use. No component should be reused for future ZIKV strain or placebo 
preparation. 
Neither ZIKV strain can be used until it is verified by the Sponsor as acceptable for use. If the 
Sponsor deems the ZIKV unacceptable for use, it will be quarantined until further directions 
from the Sponsor are received. If ZIKV is deemed unacceptable for use, it will be disposed of 
per the Sponsor’s instruction and a new shipment will be requested.  
5.3 
ZIKV Strain and Diluent Accountability 
Laboratory/pharmacy test article personnel will maintain an accurate inventory and 
accountability record of each ZIKV strain. Partially used vials of ZIKV or placebo components 
will not be refrozen or reused for future inoculations. 
5.4 
Storage Disposition of Used/Unused Supplies 
After laboratory/pharmacy personnel have diluted the test article and drawn up the syringes for 
administration, they will remove the label from the ZIKV vials and place it on the test article 
preparation form. In this manner, monitoring personnel will be able to verify the accountability 
of all ZIKV vials used for the study. In addition, the number of ZIKV vials used will be 
accounted for in the study specific drug accountability log.  
An aliquot of undiluted (if available) and diluted ZIKV will be titrated according to SOP by 
laboratory personnel after ZIKV has been prepared and delivered to the clinical staff. This is 
done to confirm the potency of ZIKV administered to the subjects.  
At least 1 aliquot (if available) of diluted ZIKV 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 ZIKV will be destroyed by the laboratory personnel 
per SOP. Any unused diluent/placebo from opened vials/bottles will also be destroyed. 
After study completion or termination, all unused study product will be disposed of per sponsor’s 
instructions.  
6 
STUDY PROCEDURES 
The following sections provide a detailed listing of the procedures and studies to be performed in 
this protocol at designated time points. 
6.1 
Recruitment and General Screening 
Subjects may be recruited from a variety of sources including, but not limited to, subjects 
previously enrolled in trials at the clinical sites, the use of a center-wide IRB-approved screening 

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protocol, and/or using study-specific IRB-approved print, electronic and/or other media 
advertising.  
After an initial phone screen (using an IRB-approved Phone Screen/Initial Contact form) by 
clinic staff focused on providing background information of the trial and a review of basic 
inclusion and exclusion criteria, a screening visit may be scheduled.  
6.2 
Consenting Process 
During the screening process, which may require more than 1 visit, the subject will undergo a 
robust informed consent process. At the first screening visit, the subjects may be provided with a 
read only copy of the informed consent form to take home with them. Additional materials 
including a fact sheet (with answers to frequently asked questions and URL for ethics 
documentation) will also be provided. We encourage our subjects to read the consent form at 
home prior to coming to their final screening visit. At this visit, the subject will read the consent 
form, be encouraged to ask questions, and then complete a comprehension assessment. The 
comprehension assessment is designed to test the subject’s knowledge of the most pertinent and 
important details about the study. The subject must be able to complete the informed consent 
process and comprehension assessment independently and without assistance. Site specific 
consenting documents will be used. 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 robust 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 be encouraged to ask 
questions to family, friends or anyone else at home if desired. The subject will also be provided 
with a signed copy of the informed consent. All study-related procedures will occur only after 
the informed consent is signed. 
6.3 
Screening Procedures 
Subjects will undergo the following screening procedures within 60 days of inoculation: 
1. Explain the study and Informed Consent process to the subject. 
2. Ensure that the subject has successfully completed the informed consent comprehension 
assessment, has signed the informed consent, and received a copy of the signed informed 
consent. The comprehension assessment must be completed independently and without 
assistance. 
3. Ensure that HIV pre-test counseling has been performed and documented (within 14 days 
of inoculation).  
4. Elicit a complete medical history, including menstrual and contraceptive history, history 
of surgical sterilization, sexual history, and current sexual practices and method of 
contraception for all subjects. 
5. Obtain a nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus 
that causes COVID-19 – 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). 

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*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. Perform a complete physical examination, including vital signs (height, weight, blood 
pressure, respiratory rate, temperature, and pulse).  
7. Obtain approximately 30 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 ZIKV or DENV infection 
• creatinine 
• alkaline phosphatase 
• HBV 
• HCV 
 
8. Obtain approximately 5mL 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. Obtain urine for urine dipstick testing (glucose, bilirubin, ketones, specific gravity, blood, 
pH, protein, urobilinogen, nitrite, leukocyte esterase) and urine toxicology screen for 
opiates. 
10. Obtain serum for -HCG testing in females. Serum pregnancy will be performed at the 
initial screening visit, within 14 days of inoculation, and on Study Day 0 prior to 
inoculation. A positive -HCG will exclude the subject from the trial. 
11. Obtain medical release form to confirm method of birth control (for applicable female 
subjects). 
12. Counsel all subjects on pregnancy prevention during the entire study period.  
13. Counsel all female subjects to use barrier contraception through Study Day 56. 
14. Counsel all male subjects to use barrier contraception through Study Day 90. 
15. Provide education about residing in the inpatient unit until discharge on Study Day 9 (or 
longer as needed).  
16. Provide education on the use of Cervico-vaginal cup for collection of cervico-vaginal 
secretions.  
17. Provide education on the collection of semen.  
18. Provide education on mosquito feeding (to the subset of volunteers enrolled in the 
mosquito-feeding study). 

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6.4 
Detailed Study Procedures 
The study procedures to be performed at each visit are listed below and in Table 1: Schedule of 
Procedures. Additional tests may be done at the discretion of the PI/Provider to evaluate 
concomitant illness or to further evaluate an adverse event 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. To 
maintain confidentiality, possible identifiers, such as tattoos, jewelry, and clothing will not be 
included in the photographs. The total volume of blood to be drawn over the 26-week post-
inoculation period is approximately 700 mL, which is like donating 1 1/2 units of blood. This 
amount is within NIH guidelines (Medical Administrative Policy 95-9) of 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. 
Additional serum or urine pregnancy tests may be performed, as determined necessary by the 
clinician, to establish the absence of pregnancy at any time during the participation in the study. 
6.4.1 Admission to the Inpatient Unit, Inoculation, and Follow-up 
Subjects will be admitted to the inpatient unit on Study Day 0 and inoculated with ZIKV or 
placebo on Study Day 0. On the day of admission to the inpatient unit, 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. Subjects who may be alternates will be informed that they may be alternates when 
they are invited to the clinic. 
The ZIKV will be kept frozen at -75°C ± 15°C until just before use, whereupon they will be 
thawed, diluted to the appropriate dose, and drawn up for administration (see Section 5 for test 
article preparation). The ZIKV strains and placebo will be kept in a cold container from the time 
they are thawed or removed from the refrigerator until they are delivered 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. For outpatient study visits following discharge, 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. 
6.4.1.1 Inpatient Stay Procedures 
During the inpatient stay, the subject will have vital signs (blood pressure, heart rate, respiratory 
rate, and temperature) taken twice a day, except for day of discharge (vital signs will be collected 
prior to discharge). Should a subject develop a temperature ≥ 100.4°F (38°C), the frequency of 
vital signs monitoring will increase to at least 3 times a day and the subject will be evaluated by 
a clinician at least twice a day until the subject is afebrile for 48 hours. A focused physical 
examination will be performed each day while inpatient and at designated outpatient visits. A 
focused physical exam may include, but is not limited to, blood pressure, heart rate, temperature, 
respiratory rate, skin examination, eyes, pertinent lymph nodes, throat, mental status, pertinent 
cranial nerves, gait coordination (observed gait), muscle strength, and biceps and patellar deep 
tendon reflexes. Additional physical exam components may be done if clinically indicated at the 
provider’s discretion. 
 

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Laboratory studies will be done according to the schedule below. -HCG testing will be 
performed on specimens collected from female volunteers only. Should a subject develop a fever 
≥ 100.4°F (38°C), laboratory studies may be done more frequently at the discretion of the 
clinician. The unit will be staffed 24 hours a day for the duration of the inpatient stay. A clinician 
will evaluate all subjects at least once per day for the duration of the inpatient stay and will be 
available 24 hours a day for the duration of the inpatient stay should he/she be needed. During 
the inpatient stay, additional clinical laboratory studies may be performed to follow-up on any 
clinical laboratory abnormalities that may have developed during the inpatient stay. Additional 
diagnostic studies may be performed should the subject’s clinical condition warrant (e.g., CXR, 
ultrasound, and ECG). Should a subject leave the inpatient unit before the 9-day stay has been 
completed, every effort will be made to follow-up with the subject daily to monitor for 
signs/symptoms of illness. 
6.4.1.2 Detailed Study Procedures  
 
Study Day 0 (Day of Inoculation with Test Article) 
1. Verify that Informed Consent was obtained and that the consent form was signed by both 
the subject and by the study staff. 
2. Verify that all applicable eligibility criteria have been met. 
3. Collect 3.5mL to perform serum -HCG testing (females only). Ensure the test is 
negative before proceeding; a positive test will exclude the subject from the study as per 
protocol, Section 4.2. 
4. Obtain a nasopharyngeal (NP) or mid-turbinate swab to test for SARS-CoV 2 – the virus 
that causes COVID-19 – 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. 
5. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. Physical exam may extend beyond focused if 
indicated by complaint or physical finding. 
6. Admit the volunteer to the inpatient unit. 
7. Record vital signs (blood pressure, temperature, heart rate, and respiratory rate) twice a 
day. 
8. Review and document concomitant medications. 
9. Obtain approximately 57 mL of blood for CBC with differential, virus isolation and 
culture from serum and whole blood (PCR/culture), and immunology (serum, plasma, 
peripheral blood mononuclear cells [PBMCs]), and PAXGene. These laboratory studies 
are drawn as baseline values and will not determine eligibility. 
10. Obtain 5 mL of clean-catch urine for virus isolation & culture. 

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11. Obtain cervico-vaginal secretion or semen sample for virus isolation & culture.  
12. Obtain saliva sample for virus isolation and culture. 
13. Review pregnancy prevention with the subject. 
14. Obtain oral fluid for serology (CIR site only). 
15. Administer the test article. 
16. Observe for at least 30 minutes after inoculation and evaluate for immediate 
hypersensitivity.  
17. Provide education by study staff describing the signs and symptoms of potential AEs, and 
how and when to contact study staff. 
Study Day 1 
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day. 
3. Obtain approximately 15 mL of blood for virus isolation from serum and whole blood 
(PCR and culture) and PAXGene. 
 
Study Day 2 
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day. 
3. Obtain approximately 15 mL of blood for virus isolation from serum and whole blood 
(PCR and culture). 
4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain saliva sample for virus isolation (PCR/culture). 
6. Obtain 2 mL of blood for PAXGene. 
Study Day 3 
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day. 
3. Obtain approximately 15 mL of blood for virus isolation from serum and whole blood 
(PCR and culture) and PAXGene. 
Study Day 4 
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day. 

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3. Obtain approximately 47 mL of blood for CBC with differential, virus isolation from 
serum and whole blood (PCR/culture), immunology (serum, plasma, PBMCs), and 
PAXGene. 
4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
6. Obtain saliva sample for virus isolation (culture/PCR). 
7. Obtain oral fluid for serology (CIR site only). 
Study Day 5 
1. Perform interim history and focused physical exam, concentrating on any acute complaints. 
2. Record vital signs twice a day. 
3. Obtain 13 mL of blood for virus isolation from serum and whole blood (PCR/culture). 
4. Cohorts 3 and 4: Perform mosquito feeding on subset of volunteers. 
Study Day 6  
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day.  
3. Obtain approximately 15 mL of blood for virus isolation from serum and whole blood 
(culture/PCR) and PAXGene. 
4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain saliva sample for virus isolation (PCR/culture). 
6. Obtain oral fluid for serology (CIR site only). 
7. Cohorts 3 and 4: Perform mosquito feeding on subset of volunteers. 
Study Day 7 
1. Perform interim history and focused physical exam, concentrating on any acute complaints. 
2. Record vital signs twice a day. 
3. Obtain 13 mL of blood for virus isolation from serum and whole blood (PCR/culture). 
4. Cohorts 3 and 4: Perform mosquito feeding on subset of volunteers. 
Study Day 8  
1. Perform interim history and focused physical exam, concentrating on any acute 
complaints. 
2. Record vital signs twice a day. 
3. Obtain approximately 52 mL of blood for CBC with differential, AST, ALT, serum 
creatinine, virus isolation from serum and whole blood (culture/PCR), immunology 
(serum, plasma, PBMCs), and PAXGene. 
4. Perform serum -HCG testing (females only). 

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5. Obtain clean-catch urine for virus isolation (PCR/culture). 
6. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
7. Obtain saliva sample for virus isolation (PCR/culture). 
8. Obtain oral fluid for serology (CIR site only).  
Study Day 9 
1. Perform interim history and focused physical exam, concentrating on any acute complaints. 
2. Record vital signs. 
3. Obtain 13 mL of blood for virus isolation from serum and whole blood (PCR/culture). 
4. Review pregnancy prevention and continued use of barrier contraceptives. 
5. Provide volunteer with diary card to record temperature twice a day. 
6. Provide education by study staff describing proper use of thermometer and memory card. 
7. Provide education by study staff describing the signs and symptoms of potential AEs, and 
how and when to contact study staff. 
8. Provide subjects with insect repellent and education on how to prevent mosquito bites 
(May – October).  
9. May discharge from inpatient unit if subject meets discharge criteria (Section 6.4.2). 
Study Day 10 (+ 1 day from 10 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 
2. Record vital signs from subject diary card and perform vital signs. 
3. Obtain approximately 15 mL of blood for PAXGene and virus isolation from serum and 
whole blood (PCR/culture). 
4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain cervico-vaginal secretion sample for virus isolation (PCR/culture). 
6. Obtain saliva sample for virus isolation (PCR/culture). 
7. Obtain oral fluid for serology (CIR site only). 
8. Perform serum -HCG testing (females only). 
9. Review pregnancy prevention and continued use of barrier contraceptives. 
Study Day 12 (± 1 day from 12 days following test article administration)  
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 
2. Record vital signs from subject diary card and perform vital signs. 
3. Obtain approximately 22 mL of blood for CBC with differential, AST, ALT, serum 
creatinine, virus isolation from serum and whole blood (PCR/culture), and PAXGene. 

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4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
6. Obtain saliva sample for virus isolation (PCR/culture). 
7. Obtain oral fluid for serology (CIR site only). 
8. Perform serum -HCG testing (females only). 
9. Review pregnancy prevention and continued use of barrier contraceptives. 
Study Day 14 (± 1 day from 14 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 
2. Record vital signs from subject diary card and perform vital signs. 
3. Obtain approximately 45 mL of blood for immunology (serum, plasma, PBMCs), 
PAXGene, and virus isolation from serum and whole blood (PCR/culture). 
4. Obtain clean-catch urine for virus isolation (PCR/culture). 
5. Obtain cervico-vaginal secretion sample for virus isolation (PCR/culture). 
6. Obtain saliva sample for virus isolation (PCR/culture). 
7. Obtain oral fluid for serology (CIR site only). 
8. Perform serum -HCG testing (females only). 
9. Review pregnancy prevention and continued use of barrier contraceptives. 
Study Day 16 (± 1 day from 16 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 
2. Record vital signs from subject diary card and perform vital signs. 
3. Obtain approximately 22 mL of blood for CBC with differential, AST, ALT, serum 
creatinine, virus isolation from serum and whole blood (PCR/culture), and PAXGene. 
4. Perform serum -HCG testing (females only).  
5. Obtain clean-catch urine for virus isolation (PCR/culture). 
6. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
(Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to 
the clinic). 
7. Obtain saliva sample for virus isolation (PCR/culture). 
8. Obtain oral fluid for serology (CIR site only). 
9. Review pregnancy prevention and continued use of barrier contraceptives.  
Study Day 21 (± 1 day from 21 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 

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2. Record vital signs. 
3. Perform serum -HCG testing (females only).  
4. Review pregnancy prevention and continued use of barrier contraceptives with all 
subjects. 
5. Obtain approximately 40 mL of blood for immunology (serum, plasma, PBMCs), 
PAXGene and virology* from serum and whole blood (PCR/culture). CBC will be done 
if prior white blood cell count (WBC), ANC, platelet count, or hemoglobin results are a 
Grade I or above and unresolved, as defined by protocol, on Study Day 16. 
6. Obtain clean-catch urine for virus isolation (PCR/culture). 
7. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
(Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to 
the clinic). 
8. Obtain saliva sample for virus isolation (PCR/culture). 
9. Obtain oral fluid for serology (CIR site only). 
10. Review pregnancy prevention and continued use of barrier contraceptives.  
*Whole blood/serum virology will be done only for those subjects who had detectable 
ZIKV in the blood at Study Day 16. 
Study Day 28 (± 2 days from 28 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 
2. Record vital signs. 
3. Obtain approximately 50 mL of blood for immunology (serum, plasma, PBMCs), 
PAXGene and virology* from serum and whole blood (PCR/culture).  
4. Obtain clean-catch urine for virus isolation* (PCR/culture). 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation* (PCR/culture). 
(Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to 
the clinic). 
6. Obtain saliva sample for virus isolation* (PCR/culture). 
7. Obtain oral fluid for serology (CIR site only). 
8. Perform serum -HCG testing (females only).  
9. Review pregnancy prevention and continued use of barrier contraceptives.  
*Whole blood/serum/CVS/semen/saliva/urine virology will be done only for those subjects 
who had detectable ZIKV in the blood at Study Day 21. 
Study Day 35 (± 2 days from 35 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints. 

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2. Record vital signs. 
3. Collect 8 mL blood for whole blood/serum virus isolation* (PCR/culture). Assays will be 
performed only for those subjects who had detectable ZIKV in the blood at Study Day 
28.  
4. Obtain clean-catch urine for virus isolation* (PCR/culture). 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation* (PCR/culture). 
Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to the 
clinic). 
6. Obtain saliva sample for virus isolation* (PCR/culture). 
7. Perform serum -HCG testing (females only).  
8. Review pregnancy prevention and continued use of barrier contraceptives with all 
subjects. 
*Whole blood/serum/CVS/semen/saliva/urine virology will be done only for those subjects 
who had detectable ZIKV in the blood at Study Day 28. 
 
Study Day 56 (± 3 days from 56 days following test article administration) 
1. Perform interim history, including sexual history, and focused physical exam, 
concentrating on any acute complaints.  
2. Record vital signs. 
3. Obtain approximately 40 mL of blood for immunology (serum, plasma, PBMCs)/serum 
virus isolation* (PCR/culture). 
4. Collect 3 mL blood for whole blood virus isolation* (PCR/culture). Assays will be 
performed only for those subjects who had detectable ZIKV in the blood at Study Day 35 
or 49.  
5. Obtain clean-catch urine for virus isolation* (PCR/culture). 
6. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture)*. 
Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to the 
clinic). 
7. Obtain saliva sample for virus isolation* (PCR/culture). 
8. Obtain oral fluid for serology (CIR site only). 
9. Perform serum -HCG testing (females only).  
10. Male volunteers: Review pregnancy prevention and continued use of barrier 
contraceptives. 
*Whole blood/serum/CVS/semen/saliva/urine virology will be done only for those subjects 
who had detectable ZIKV in the blood at Study Day 49. 
Study Day 70 (± 3 days from 70 days following test article administration) 
1. Perform interim history for interim complaints. (Physical exam is optional). 

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2. Record vital signs. 
3. Collect 8 mL blood for whole blood/serum virus isolation* (PCR/culture). Assays will be 
performed only for those subjects who had detectable ZIKV in the blood at Study Day 49 
or 56.  
4. Obtain clean-catch urine for virus isolation* (PCR/culture). 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation* (PCR/culture). 
(Cervico-vaginal cup may be inserted at home by the volunteer prior to presentation to 
the clinic). 
6. Obtain saliva sample for virus isolation* (PCR/culture). 
7. Obtain oral fluid for serology (CIR site only). 
8. Perform serum -HCG testing (females only).  
9. Male volunteers: Review pregnancy prevention and continued use of barrier 
contraceptives with all subjects. 
*Whole blood/serum/CVS/semen/saliva/urine virology will be done only for those subjects 
who had detectable ZIKV in the blood at Study Day 56. 
Study Day 90 (± 7 days from 90 days following test article administration) 
1. Perform interim history for interim complaints. (Physical exam is optional).  
2. Record vital signs. 
3. Obtain approximately 40 mL of blood for immunology (serum, plasma, PBMCs)/serum 
virus isolation* (PCR/culture). 
4. Collect 3 mL blood for whole blood virus isolation* (PCR/culture). Assays will be 
performed only for those subjects who had detectable ZIKV in the blood at Study Day 56 
or 70.  
5. Obtain clean-catch urine for virus isolation* (PCR/culture). Assays will be performed 
only if Day 56 or 70 sample was positive. 
6. Obtain cervico-vaginal secretion or semen sample for virus isolation*. Assays will be 
performed only if Day 56 or 70 sample was positive. (Cervico-vaginal cup may be 
inserted at home by the volunteer prior to presentation to the clinic).  
7. Obtain saliva sample for virus isolation* (PCR/culture). Assays will be performed only if 
Day 56 or 70 was positive. 
8. Obtain oral fluid for serology (CIR site only). 
9. Perform serum -HCG testing (females only).  
10. Review pregnancy prevention. 
*Whole blood/serum/CVS/semen/saliva/urine virology will be done only for those subjects who 
had detectable ZIKV in the blood at Study Day 70. 
Study Day 120 (± 10 days from 120 days following test article administration) 
1. Perform interim history for interim complaints. (Physical exam is optional) 

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2. Record vital signs. 
3. Collect 8 mL blood for whole blood/serum virus isolation* (PCR/culture). Assays will be 
performed only for those subjects who had detectable ZIKV in the blood at Study Day 70 
or 90.  
4. Obtain clean-catch urine for virus isolation (PCR/culture)*. Assays will be performed 
only if Day 70 or 90 sample was positive*. 
5. Obtain cervico-vaginal secretion or semen sample for virus isolation* (PCR/culture). 
Assays will be performed only if Day 70 or 90 sample was positive. (Cervico-vaginal cup 
may be inserted at home by the volunteer prior to presentation to the clinic)*. 
6. Obtain saliva sample for virus isolation* (PCR/culture). Assays will be performed only if 
Day 70 or 90 sample was positive.* 
7. Perform serum -HCG testing (females only).  
8. Review pregnancy prevention. 
*Collection of blood, urine, cervico-vaginal secretion, semen, and saliva samples for 
virology will be done on Study Day 120 only if virus was recovered from the sample 
collected at the previous time-points (Day 70 or 90) or if virology results are not available. 
 
Study Day 150 ± 7 days: The subjects will be contacted, and pregnancy prevention will be 
reviewed. 
Study Day 180 (± 14 days from 180 days following test article administration) 
1. Perform interim history for interim complaints. 
2. Perform focused physical exam. 
3. Record vital signs. 
4. Obtain approximately 40 mL of blood for immunology (serum, plasma, PBMCs)/serum 
virus isolation (PCR/culture). Virus isolation will only be performed for those subjects 
who had detectable ZIKV in the serum at Study Day 90 or 120. 
5. Collect 3 mL blood for whole blood virus isolation (PCR/culture), if necessary. Assays 
will be performed only for those subjects who had detectable ZIKV in the blood at Study 
Day 90 or 120.  
6. Obtain clean-catch urine for virus isolation (PCR/culture). Assays will be performed only 
if the Day 90 or 120 sample was positive*. 
7. Obtain cervico-vaginal secretion or semen sample for virus isolation (PCR/culture). 
Assays will be performed only if Day 90 or 120 sample was positive. (Cervico-vaginal 
cup may be inserted at home by the volunteer prior to presentation to the clinic)*.  
8. Obtain saliva sample for virus isolation (PCR/culture). Assays will be performed only if 
Day 90 or 120 sample was positive*. 
9. Obtain oral fluid for serology (CIR site only). 
10. Perform serum -HCG testing (females only). 

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11. Review pregnancy prevention. 
*Collection of urine, cervico-vaginal secretion, semen, and saliva samples and virology 
(urine, vaginal secretion, saliva) will be done on Study Day 180 only if virus was recovered 
from the sample collected at the previous time-points (Day 90 or 120) or if virology results 
are not available. 
 
Note: Blood, urine, saliva, semen, and cervico-vaginal secretion samples will be collected at the 
indicated visits. PCR and/or viral culture will be performed on the samples collected at the 
indicated time points from Study Day 21 through Day 180 if the sample was positive on samples 
collected on at least one of the previous time-points. If a sample has been negative for 2 
consecutive time-points from Study Day 21 onward, then PCR/culture for ZIKV will not be 
performed on future samples unless there is an indication that the sample may be intermittently 
positive and additional samples need to be tested. Samples will be collected from all subjects 
through Study Day 90 because identifying which subjects may not require a sample would 
unblind the treatment assignment. After Study Day 90, if the samples at the 2 previous time 
points were negative then saliva, cervico-vaginal secretions, blood, semen, and urine will not be 
collected for virus isolation.  
6.4.2 Discharge Criteria 
Subjects will be discharged from the inpatient unit on Study Day 9 if they meet the following 
discharge criteria: 
1. Did not develop fever during the inpatient stay. 
2. If fever did develop, the subject has remained afebrile for 48 hours prior to discharge. 
3. Any continuing solicited AEs are Grade 2 or lower. 
4. The titer of ZIKV in serum, cervico-vaginal secretions, semen, urine, or saliva, is ≤ 1.4 x 
106 genomic copies/mL by Study Day 8 or has been previously demonstrated to be non-
infectious at the detected titer. 
Because of the low inoculum dose to be given, the low titers recovered from NHP, our 
experience with the dengue CHIM, and reported ZIKV literature, subjects are expected to be free 
of infectious transmissible virus in the serum by Day 9. 
6.4.3 Mitigation Plan Should a Subject Leave the Unit Early 
Potential volunteers are carefully screened prior to enrollment to ensure they understand their 
responsibilities and are willing and able to carry them out, including remaining in the inpatient 
unit until discharge criteria are met. Subjects must be willing to stay in the inpatient unit until 
they meet discharge criteria as an inclusion criterion. Subjects are provided with several 
amenities during their inpatient stay including internet access, entertainment (movies, cable 
television, craft projects, games, and classes. In addition, catered meals are provided to subjects 
while on the unit. Should a subject express a desire to leave the unit early, the PI will counsel the 
subject about why he or she is being asked to remain on the unit and counsel him or her to stay. 
However, should a subject insist upon leaving the unit prior to discharge criteria being met, the 
study staff will: 
• Reinforce the risk of mosquito-borne & sexual transmission of ZIKV to third parties 
• Review ZIKV transmission preventive measures with the volunteer 

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• Perform pregnancy prevention counseling 
• Provide mosquito repellent containing DEET and review with volunteer how to use it. This 
will be done during mosquito season (May through October). 
• If possible, arrange daily outpatient follow-up visits for the subject for clinical examination 
and to obtain scheduled blood/urine/saliva/cervico-vaginal secretion/semen samples until 
Study Day 9, after which point they will resume usual outpatient schedule. 
• If subject cannot physically return for follow-up visits, will contact the subject by phone to 
inquire about adverse events and perform pregnancy prevention counseling  
6.4.4 Subject Temperature Memory Card 
Subjects will be provided a thermometer and a “Temperature Memory Card”, to use as a memory 
aid and will be asked to record temperatures 2 times a day on Study Day 9 - Study Day 16. Staff 
will review the temperature card on Study Days 10, 12, 14, 16, and 21 post-inoculation 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 (Tmax) for each date 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 how to use the thermometers, to take their temperatures at 
approximately the same times 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.  
6.4.5 Persistence of ZIKV in Bodily Fluids 
If Zika virus is detected in any bodily fluid at Study Day 180, the subject will continue to be 
followed until Zika virus is no longer detected in the bodily fluid. To achieve this, the subject 
will be brought back for unscheduled visits for collection of the specimen of interest (the bodily 
fluid that was positive for Zika virus 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 virus 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.  
6.5 
Clinical Laboratory Testing 
Using standard techniques, Quest Diagnostics, or other CLIA certified laboratories will perform 
the following tests: 
1. CBC plus white blood cell 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 

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6. Hepatitis C screening by testing for Hepatitis C antibody. If positive, test is confirmed by 
testing for Hepatitis C RNA. If Hepatitis C antibody is positive but RNA is negative, the 
volunteer is assessed as having a past infection that has been cleared. In this case, the 
subject is eligible for the study. 
7. Urinalysis (in the event of an abnormal urine dipstick test) 
8. Serum -HCG, if required 
9. Urine toxicology screen for opiates. 
Serum -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. SARS-CoV-2 testing will be performed at the site using a CLIA-waived test 
(GeneExpert Express) or will be sent to a CLIA-certified clinical laboratory for testing. HIV 
testing may be performed at the clinical trial site using an FDA-approved rapid test kit or sent to 
the clinical laboratory. In the event of a reactive rapid HIV test, a serum sample will be sent to 
the appropriate lab for confirmation. Determination of ZIKV virus titer by RT-PCR and virus 
culture, plaque reduction neutralization antibody assays, and cellular immune studies will be 
done at the clinical trial site laboratory. 
6.6 
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 following test article administration, the medical history will be updated for any new or 
changed significant or chronic conditions.  
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 test article administration. 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 following test article administration 
will be recorded throughout the trial. 
6.7 
Mosquito Feeding 
Feeding of mosquitoes on a subset of subjects enrolled in cohorts 3 and 4 will be conducted as 
described for rDEN430 except the days of feeding will be different [52]. Seven subjects from 
cohort 3 and seven subjects from cohort 4 (each randomized 5:2 ZIKV:placebo) will be selected 
for mosquito feeding. Inclusion of placebo recipients in the mosquito-feeding will be done to 
ensure the clinical team remains blinded to treatment assignment. Five to 7 day-old female Aedes 
albopictus mosquitoes will be brought to the inpatient unit from the insectory of the Laboratory 
of Viral Diseases, National Institutes of Health on study days 5, 6, and 7. Each subject will have 
a pint container holding ten to fifteen Aedes albopictus mosquitoes placed on the ventral surface 
of their forearm. The mosquitoes will be allowed to feed for 10 minutes. This will be performed 
on study days 5, 6, and 7. After feeding, the container will be placed at -20°C for a few minutes 
to “anesthetize” the mosquitoes. After removing the container from the -20° freezer, unfed and 
partially engorged mosquitoes will be removed from the containers and destroyed. Fully 
engorged mosquitoes will remain in the containers and will be immediately returned to the LVD 

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where they will be housed and maintained in the LVD insectory per LVD standard protocol. 
Twenty-one days later the head and midgut tissues of the mosquitoes will be examined for the 
presence of ZIKV antigens. Methods for detection of ZIKV may include polymerase chain 
reaction, immunofluorescence staining, and culture. 
6.8 
Immunology Testing 
6.8.1 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 count of 
virus alone. Other neutralization assays such as microneutralization and flow-based 
neutralization assays may also be utilized as experimental endpoints. 
6.8.2 Other Immunological Assays 
Cytokine, and T-cell and B-cell stimulation assays, and PBMC phenotyping may be performed 
on peripheral blood mononuclear cells. RNA may be isolated from cells and run on microarrays 
to identify immune pathways of interest such as defining the innate immune response to ZIKV. 
In addition, human leukocyte antigen (HLA) typing of samples may be performed as part of 
assays to map the ZIKV epitopes that induce T or B cell responses. 
6.9 
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. 
7 
ADVERSE EVENT MONITORING 
7.1 
Definitions 
7.1.1 Adverse Event 
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 considered related to 
the research. 
All AEs will be evaluated for severity, action taken, seriousness, outcome and relationship to the 
test article as described in Section 7.2 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 each inoculation and any test article-related AEs 
identified in the 28-day post-inoculation period will be followed until resolution.  

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AEs are categorized as solicited AEs and other AEs. Solicited AEs include local reactogenicity, 
systemic reactogenicity, and laboratory events. Solicited AEs are those events that the clinician 
is specifically evaluating during each 28-day post-inoculation period, listed in Table 8. All AEs 
are evaluated using the Adverse Event Grading Table in Appendix A, using severity definitions 
detailed in Section 7,  
 

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Table 9.  
 
All abnormal laboratory findings will be reviewed on a routine basis by the PI to identify 
potential safety signals. An abnormal lab not included on the toxicity table should be assessed 
in a similar fashion to the criteria above. 
7.1.2 Medically-attended Adverse Event 
A medically-attended adverse event is defined as any visit (other than hospitalization or routine 
health maintenance visits) to an emergency room (ER), urgent care facility, doctor, nurse, or any 
other health care provider. Medically-attended adverse events from Day 29 through Day 180 will 
be collected. 
7.1.3 Adverse Event of Special Interest 
Guillain-Barré syndrome (GBS) is an adverse event of special interest for this protocol. Subjects 
will be examined for muscle weakness and hypo- or areflexia at each physical examination. 
Should subjects present with neurological complaints at visits that do not include a physical 
examination, an exam will be performed. The diagnostic certainty of a diagnosis of GBS will be 
determined using the Brighton Criteria (Appendix B) and the disability severity determined using 
the disability scale in Appendix C. Referral to neurology specialist will be issued if indicated. 
7.1.4 Suspected Adverse Reaction (SAR) 
An adverse event for which there is a reasonable possibility that the investigational agent caused 
the adverse event. “Reasonable possibility” means that there is evidence to suggest a causal 
relationship between the test article and the adverse event. A suspected adverse reaction (SAR) 
implies a lesser degree of certainty about causality than adverse reaction that implied a high 
degree of certainty. 
Table 8: Solicited Adverse Events 
Systemic Reactogenicity 
Laboratory Events  
Local Reactogenicity 
 -Fever 
 -Leukopenia 
 -Injection site pain 
 -ZIKV-like rash 
 -Neutropenia 
 -Injection site erythema 
 -Headache 
 -Thrombocytopenia  
 -Injection site tenderness 
 -Retro-orbital pain (ROP) 
 
 -Injection site induration 
 -Photophobia 
 
-Injection site pruritis 
 -Non-purulent conjunctivitis 
 
  
 -Pruritus 
 
 
 -Fatigue 
 
 
 -Myalgia 
 
 
 -Arthralgia 
 
 
 -Muscle Weakness 
 
 
 -Hypo- or areflexia 
 
 
 
7.1.5 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 

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(CSO) per the sponsor’s reporting guidelines. Pertinent obstetrical information for all 
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.  
The pregnancy will be reported to the DSMB and/or IRB (if applicable). 
Per Protocol Analysis in the Event of Pregnancy:  
• If the pregnancy occurs prior to and including Study Day 28 after test article administration, 
the subject will not be included in the per-protocol analysis. The subject will remain in the 
study for safety follow-up. If the pregnancy occurs after Study Day 28 following 
administration of test article, the subject will be included in the per-protocol analysis.  
 
Unblinding Due to Pregnancy:  
• If a subject becomes pregnant within 56 days of test article administration, the subject will 
be unblinded to determine if she received ZIKV and aid in management. 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 occurred after Study Day 
28, the subject will be included in the per-protocol analysis. 
• Post Study Day 56, the subject will be offered the opportunity to have her treatment 
assignment unblinded in the case that this information would help her determine whether 
or not she wishes to continue her pregnancy. She will be included in the protocol per 
analysis. 
• The research subject will be advised to notify her obstetrician of study participation and 
study agent exposure. 
7.1.6 Serious Adverse Event (SAE) 
An SAE is an AE that is determined to be “serious” whether considered related to the 
investigational agent or not. SAEs will be collected for the duration of the trial. An SAE results 
in 1 or more of the following outcomes: 
• Death during the period of protocol-defined surveillance. 
• 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 ward 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 event* 

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*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 
life threatening or result in death or result in 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.” A SAE 
needs to meet only 1 or more of the above criteria to be considered serious. 
7.1.7 Unexpected Adverse Events 
An adverse event 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. 
7.1.8 Serious and Unexpected Suspected Adverse Reaction (SUSAR)  
A SUSAR is a Suspected Adverse Reaction that is both Serious and Unexpected.  
7.1.9 Unanticipated Problem 
An Unanticipated Problem (UP) is any event, incident, experience, or outcome that is: 
1.  unexpected in terms of nature, severity, or frequency in relation to 
a. the research 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; and 
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 harm) than was previously known or recognized. (An AE with a 
serious outcome will be considered increased risk). 
7.1.10 Unanticipated Problem That Is Not an Adverse Event (UPnonAE) 
An unanticipated problem that does not fit the definition of an adverse event, 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 a non-
serious UP. For example, we will report occurrences of breaches of confidentiality, accidental 
destruction of study records, or unaccounted-for study drug. 
7.1.11 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 a major 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 
2. Those that are identified before they occur, but cannot be prevented 

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3. Those that are discovered after they occur 
7.1.12 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 has a major impact on the subject’s rights, 
safety, or well-being and/or the completeness, accuracy or reliability of the study data. 
Non-compliance: 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 
7.1.13 Pre-existing Conditions, Worsening of Pre-existing Condition 
Stable chronic conditions which 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 7.2 in this protocol. 
7.2 
Assessment of Adverse Events 
7.2.1 Identification of Adverse Events 
Assessment of safety will include clinical observations and monitoring of hematological, blood 
chemistry, and immunologic parameters. Safety will be evaluated by monitoring of the subjects 
for local and systemic adverse reactions during the course of the trial. Subjects will be closely 
monitored for 30 minutes following inoculation. Additionally, subjects will be evaluated while in 
the inpatient unit every day during Study Days 0 – 9 and again on Study Days 10, 12, 14, 16, 21, 
and 28. It is during this time period that we anticipate AEs related to infection with the ZIKV 
will manifest themselves, and hence, the subjects will be seen frequently. Study staff will record 
the subjects’ temperature and assess for AEs. At each visit through Study Day 28 following 
inoculation, they will be queried about possible ZIKV-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. They will return to the clinic or 
inpatient unit on Study Day 35, 49, 56, 70, 90, 120, and 180 for sample collection and, at 
designated time points, physical examination. 
All AEs will be recorded during the period after the subject receives the study agent, through and 
including post-inoculation Study Day 28. Signs/symptoms of GBS will be assessed through 
Study Day 180. 
All SAEs will be assessed appropriately as AEs and reported following SAE reporting guidelines 
outlined in Section 7.3.2 of this protocol.  

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7.2.2 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.  
Headache: a pain located in the head, over the eyes, at the temples, or at the base of the skull. 
Retro-orbital pain: Bilateral pain situated behind the orbits of the eyes 
Photophobia: Abnormal sensitivity or intolerance to light as manifested as discomfort to light in 
a normal lighted room for longer than a few seconds (normal acclimation time). 
Non-purulent Conjunctivitis: Redding of bilateral sclera of the eyes without evidence of 
exudate or crusting of the eyelids 
Pruritis: Generalized itching 
Fatigue: excessive tiredness following minimal exertion.  
Myalgia: pain in the muscles, found in > 2 muscle groups. 
Arthralgia: pain in a joint, 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 
Hypo- or areflexia: Tendon reflexes that are weaker than normal (hyporeflexia) or absent 
(areflexia). Reflexes that are graded as 1* or 0 will be classified as hypo- or areflexia. Grading of 
reflexes are as follows: 
• 0: absent reflex 
• 1+: trace, or seen only with reinforcement 
• 2+: normal 
• 3+: brisk 
• 4+: non-sustained clonus (i.e. repetitive vibratory movements) 
• 5+: sustained clonus 
*Grade 1 tendon reflexes will only be considered hypo- or areflexia if this represents a 
change from the subject’s baseline examination.  
Guillain-Barré Syndrome (GBS): acute or subacute onset of weakness in limbs or cranial 
nerve-innervated muscles, associated with hypo- or areflexia, and a characteristic profile in the 
cerebrospinal fluid (CSF) as defined by the Brighton criteria (Appendix B). A clinical case of 
GBS, evaluated by a health care professional with expertise in neurological examination, should 
meet the following criteria: 

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• Bilateral and symmetric weakness of the limbs (Grade 0 – 3 on the Oxford scale) 
• Decreased or absent deep tendon reflexes in the weak limbs (Grade 0 or 1* on the above 
scale) 
• Monophasic illness pattern; interval between the onset and nadir of weakness ranging from 
12 hours to 28 days with a subsequent clinical plateau;  
• Absence of an identified alternative cause for the weakness.  
*Grade 1 deep tendon reflexes will only be considered acute or subacute muscle weakness if 
this represents a change from the subject’s baseline examination.  
7.2.3 Determination of Severity 
The investigator/designee will assess all AE severity using the following classifications. 
 
 
 

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Table 9: Severity Definitions  
Severity 
Defined 
Grade 1 (Mild) 
Event that is easily tolerated, may require 1 dose of 
medication/treatment 
Grade 2 (Moderate) 
Event that interferes with daily activity or requires more than 1 dose 
of medication/treatment 
Grade 3 (Severe) 
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 a life-
threatening event 
Grade 5 (Death) 
Any adverse event that results in the death of the subject 
Solicited AE severity grading classifications are listed in  
Table 10. All other AEs will be graded in severity using the Adverse Event Grading Table in 
Appendix A: Adverse Event Grading Table1. 
 
Table 10: Assessment of Solicited Adverse Events 
Local Reactogenicity 
Grade 
Severity 
Injection Site Tenderness  
Injection Site Pruritis  
Injection Site Pain 
 
1 
2 
 
3 
Event that is easily tolerated, may require 1 dose of medication 
Event that interferes with daily activity or requires > 1 dose of 
medication 
Event that prevents daily activity  
4 
Life-threatening 
Injection Site Induration 
Injection Site 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 
Rash is present but asymptomatic 
2 
Rash is symptomatic (pruritus/pain) but does not interfere with 
function 
3 
Rash is symptomatic and interferes with function 
4 
Life-threatening 
Headache 
Retro-orbital pain 
Photophobia 
Non-purulent conjunctivitis 
Pruritis 
Fatigue 
Myalgia 
Arthralgia 
Muscle weakness 
Hypo-, areflexia 
1 
 
 
2 
 
3 
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  
4 
Life-threatening 
 
 

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Solicited Laboratory AEs 
Grade 
Severity 
Leukopenia 
1 
2 
3 
2,500 – 3,500/mm3 
1,500 – 2,499/mm3 
1,000 – 1,499/mm3 
4 
<999/mm3 
Neutropenia 
 (reduced ANC) 
1 
2 
3 
750 - 999/mm3 
500 – 749/mm3 
<500 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 
 
7.2.4 Relationship with Receipt of Test Article 
The clinical investigator will assess all AEs for their relationship to the test article using the 
following classifications:  
 
Definitely related: 
Clear-cut temporal association, and 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 test article or the nature 
of the event is such that the test article 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 test article 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 administration of the study test 
article will be determined by how well the event can be understood in terms of 1 or more of the 
following: 
• 
A reaction of similar nature having previously been observed with this type of test article 
and/or formulation or naturally occurring ZIKV-like illness 
• 
All local injection-site reactions will be considered causally related to test article. 
Causality assessment is based on available information at the time of the assessment of 
the AE. The investigator may revise the causality assessment as additional information 
becomes available. 
7.2.5 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  

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4 = Hospitalization 
5 = Other 
7.2.6 Adverse Event Outcome 
The investigator/designee will assess the outcome of the AE, either at resolution or at 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 
 
7.2.7 Adverse Event Seriousness 
The investigator/designee will categorize all AEs either as serious or non-serious, using the 
criteria defined in Section 7.1.6 of this protocol.  
Any events defined as serious will also be reported following SAE reporting guidelines outlined 
in Section 7.3 of this protocol. 
7.3 
Adverse Event Reporting 
7.3.1 Non-Serious Adverse Events  
Non-serious AEs will be followed to resolution, or until the study ends, and 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. At the investigator/designee’s discretion, subjects 
may be contacted up to 30 days after the study follow up period (Study Day 180) regarding non-
serious AEs that are unresolved at Study Day 180.  
AEs meeting the stopping criteria outlined in Section 7.5 of this protocol will be reported to the 
Sponsor following the SAE reporting guidelines.  
7.3.2 Serious Adverse Events 
Office of Clinical Research Policy and Regulatory Operations (OCRPRO) 
All SAEs (regardless of relationship and whether or not they are also UPs) 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 will be reported within 3 working days of notification of the 
SAE occurrence to: 
• 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 CRF and according to the sponsor’s reporting guidelines. 

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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. 
 
DSMB 
All SAEs will 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, E-mail: 
niaiddsmbia@mail.nih.gov 
JHU Biosafety 
All SAEs will 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 
IRB Reporting 
All SAEs will be reported to Johns Hopkins School of Public Health (JHSPH) IRB and 
University of Vermont (UVM) IRB as per guidelines, respectively for JHU and UVM as below: 
 
• JHSPH IRB Guidelines:  
o JHSPH IRB Phone: 1-888-262-3242, Fax: 410-502-0584 
o https://www.jhsph.edu/offices-and-services/institutional-review-board/index.html 
for updated reporting guidelines  
 
• UVM IRB Guidelines: 
o Investigators are required to report AEs that fit the following criteria: 
▪ Report timelines are based upon 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 and 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 ADVERSE EVENT is a negative side effect resulting from the 
study intervention that occurred to a subject enrolled at UVM, FAHC, or 
other 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 may have been caused by the protocol or study 
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.” 
• At UVM, all SAEs will be reported to the University of Vermont IRB (UVM IRB) 
according to UVM IRB guidelines. 

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• UVM IRB Phone: 802-656-5040, Fax: 802-656-5041 
7.3.3 Unanticipated Problems 
Unanticipated Problems that are also adverse events must be reported to the CSO and 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. 
Report UPs that are also adverse events to the CSO according to the sponsor’s reporting 
guidelines or a local IRB UP form. 
7.4 
Sponsor’s Reporting Responsibilities 
Serious, unexpected, suspected adverse reactions (SUSARs) as defined in 21 CFR 312.32 and 
determined by the IND Sponsor will be reported to FDA and all participating Investigators as 
IND Safety Reports.  
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 UPs will be summarized by the IND Sponsor and distributed to Investigators. 
7.5 
Halting Criteria 
If a dose of ZIKV is considered unacceptably reactogenic, as defined below, additional 
inoculations will be suspended for all subjects and suspension of enrollment until the DSMB and 
study Sponsor (OCRPRO) have reviewed the data and recommend that enrollment and study 
agent administration be continued. 
 
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 their site.  
 
Following administration of ZIKV, the following criteria will be used to define unacceptable 
reactogenicity of the ZIKV strain or dose: 
1. 
One or more subjects experience an SAE (as defined in Section 7.1.6. in this protocol) 
that is determined to be possibly, probably, or definitely related to ZIKV (as defined in 
Section 7.2.4. in this protocol), OR 
2. 
One or more subjects experience anaphylaxis that is possibly, probably or definitely 
related to ZIKV, OR 
3. 
One or more subjects experience GBS that is possibly, probably or definitely related to 
ZIKV, OR 
4. 
Two or more subjects experience the same objective physical finding of severity Grade 
3 that is definitely, probably, or possibly related to ZIKV, with the exception of Grade 
3 erythema at the injection site, as defined in Section 7.2.3. in this protocol, OR  
5. 
Two or more subjects experience the same Grade 3 laboratory abnormality that is 
possibly, probably, or definitely related to ZIKV OR 
6. 
Two or more subjects experience an ANC <500/mm3 for any duration1 OR 

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7. 
Two or more subjects experience a ZIKV-like syndrome following administration of 
ZIKV, defined as infection2 associated with fever and 2 or more of the following 
symptoms: 
a. 
Grade 2 or greater headache lasting ≥48 hours 
b. 
Grade 2 or greater photophobia lasting ≥48hours 
c. 
Grade 2 or greater generalized myalgia lasting ≥48 hours 
1 These halting rules resulted from previous discussions between NIAID and the FDA on Phase 1 clinical trials of 
investigational live attenuated DENV vaccines (reference INDs 8463, 13730, and 13886). 
2 Infection is defined as recovery of ZIKVs from the blood, urine, cervico-vaginal secretions, or saliva of a subject 
and/or seropositivity or seroconversion to ZIKV. 
 
7.5.1 Reporting a Study Halt 
If a halting rule is identified by the Study PI/Protocol Chair and/or the CSO, a description of the 
adverse event(s) or safety issue will be reported by the Study PI within 1 business day of sponsor 
awareness to the other Site Investigator. The Study PI will notify the DSMB. The Site 
Investigators will notify their local IRBs. Safety data reports and changes in study status will be 
submitted to the applicable IRB promptly, in accordance with institutional policy. This 
constitutes a minimum criterion, and the decision to halt the trial may be made on the basis of 
any other criteria that, in the judgment of the investigators, FDA, IRB, DSMB, or Sponsor, 
indicate a potentially serious safety concern. 
7.5.2 Resumption of a Halted Study 
The IND Sponsor, in collaboration with the PI and DSMB will determine if it is safe to resume 
the study. The CSO or designee will notify the Site Investigators of the decision on resumption 
of the study. The Site Investigators will notify their local IRB(s) of the decision.  
7.5.3 Discontinuation of Study Agent/Intervention 
Subjects who do not resume study agent/study intervention will continue to be followed for 
safety per protocol or as clinically indicated, whichever is more conservative. 
7.6 
Safety Oversight 
7.6.1 Safety Review and Communications Plan (SRCP)  
A Safety Review and Communication Plans (SRCP) has been developed for the protocol. The 
SRCP is an internal communications document between the PI and the IND Sponsor CSO, which 
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. 
The IND Sponsor and the PI will sign a formal Transfer of Regulatory Obligations 
(TORO)/SRCP which documents that the PI has accepted the responsibility for periodic safety 
surveillance assessments as outlined in 21 CFR 312.32(b). 
 
7.6.2 Sponsor Medical Monitor 
A Medical Monitor representing the IND Sponsor (OCRPRO), will be appointed for oversight of 
safety in this clinical study. The Sponsor Medical Monitor will be responsible for performing 
safety assessments as outlined in a Safety Review and Communications Plan (SRCP) as defined 
in Section 7.6.1. 
 

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7.6.3 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 Intramural 
NIAID clinical studies that require DSMB oversight, and consists of experts in infectious 
diseases, biostatistics, and clinical trials. The PI/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 and deaths 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 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).  
The DSMB will receive summary data in an unblinded manner to determine whether or not dose 
expansion or dose escalation will occur. The DSMB will be responsible for: 
1. Determining if the criteria for a suitable CHIM strain has been met. These criteria are: 
a) ≥ 80% of inoculated subjects develop detectable viremia by virus culture 
and 
b) The mean peak titer of ZIKV recovered is 2 – 3 log10 PFU/mL. 
c) The committee will review the clinical signs and symptoms induced by 
the ZIKV strain, but signs and symptoms will not be required for dose 
escalation. 
2. Recommending whether or not dose-escalation should occur. Dose escalation may occur 
even if the above criteria for a suitable CHIM strain have been met if it is believed that 
more consistent outcomes would be achieved with a higher dose, or if evaluating the 
shedding of virus given at a higher dose would improve the scientific understanding of 
ZIKV infectivity, shedding, and possibly transmission. 
8 
DATA COLLECTION AND MONITORING 
8.1 
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 inoculation 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 the NIAID (CRIMSON) Data System. 

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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 ID and password. Corrections to the data system shall 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 the CRIMSON Data System. 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. 
8.2 
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 the Code of Federal 
Regulations (CFR), Title 21, Part 312.33 (21 CFR 312.33), and will include any revisions of the 
protocol if not previously submitted. 
 
The PI will maintain adequate records to account for the disposition of the investigational 
product, including dates of receipt and quantity, current inventory, and dispensation to subjects. 
If the study is terminated, suspended, or completed, all unused study product will be disposed of 
per sponsor’s instructions. 
8.3 
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 ID 
numbers of the subjects that, 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. 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. 

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8.4 
Retention of Records 
The PI is responsible for retaining all essential documents listed in the International Conference 
on Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good 
Clinical Practice (GCP) Guideline. Trial-related documents will be maintained by the 
investigator in a secure storage facility for a period of at least 2 years since the formal 
discontinuation of clinical development of the product. 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 law.  
 
It is the PI’s responsibility to retain copies of source documents until receipt of written 
notification to the contrary from the OCRPRO of the NIAID. Study documents should not be 
destroyed without prior written agreement between OCRPRO/NIAID 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 OCRPRO/NIAID with the name of the person who 
will accept responsibility for the transferred records and/or their 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. 
8.5 
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 of, 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 data and safety monitoring plan of the study.  
 
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 shall 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 to the 
regulatory authorities.  
8.6 
Clinical Investigator’s Brochure 
Investigators will receive the current versions of the Clinical Investigator’s Brochures for the 
ZIKV strains used in this protocol, which comprehensively describes all the available preclinical 
experience with the ZIKVs. If relevant new information becomes available during the trial, the 
investigators will receive a revised brochure, or an amendment to the current version. 

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8.7 
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, 
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 notifications 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 also will inspect the clinical site regulatory files to 
ensure that regulatory requirements (Office for Human Research Protections-OHRP, 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 the 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, FDA, 
the site monitors, and the 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. 
9 
STATISTICAL CONSIDERATIONS 
9.1 
General Design 
The major objectives of this Phase 1 ZIKV trial are to identify a suitable ZIKV CHIM strain to 
aid in the selection of vaccine candidates and to characterize the clinical and virologic 
characteristics of ZIKV infection. 
9.2 
Statistical Methods 
This study, like other Phase 1 studies, is exploratory rather than confirmatory. As such, the 
primary analyses will assess frequencies of adverse events and describe patterns of challenge 
virus replication and immune response within individuals at multiple timepoints (e.g. pre vs post 
challenge), and within and between groups (challenge virus vs. placebo). 
  
Continuous outcomes (e.g. peak titer of virus shed, antibody titers over time) will be presented 
with the use of summary statistics such as means, medians, quartiles, ranges, etc. as appropriate, 
with data displayed in both tables and figures. Student’s T-tests and linear regression will be 
used to assess differences in continuous outcomes between subjects in each study arm. 
Correlations between 2 continuous outcomes will be evaluated with the use of Pearson or 
Spearman correlation coefficients and graphed as scatterplots.  

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Proportions of subjects with dichotomous outcomes of interest (e.g. presence or absence of: 
specific AEs, viral shedding, seropositivity, four-fold rise in antibody response, etc.) observed in 
each group will be summarized in tables and figures. Differences in proportions between groups 
will be compared with Χ2 tests, Fisher’s exact tests, and logistic regression as appropriate. 
Outcomes will be summarized by severity and/or category where applicable (e.g. immediate, 
systemic, local AE). 
 
The study is powered to detect a difference in the occurrence of virus in blood, urine, cervico-
vaginal secretions, or saliva following administration of one of 2 ZIKV at different doses, 
compared with placebo recipients. A sample size of 10 ZIKV recipients and 4 placebo recipients 
will be able to detect a statistically significant difference ( = 0.05) in recovery of infectious 
ZIKV from the blood between the ZIKV and placebo groups at a power of 0.99 with ≥80% of 
ZIKV-inoculated subjects having detectable ZIKV viremia and 0 placebo recipients having 
detectable ZIKV in blood and body secretions. The detection of ZIKV by RT-PCR and by virus 
culture from blood, urine, cervico-vaginal fluids, semen and saliva in recipients of each ZIKV 
strain will be compared to placebo recipients. In addition, the magnitude and duration of ZIKV 
presence in blood, urine, cervico-vaginal secretions, semen and saliva will be compared to one 
another at each dosing level. The effect of dose escalation on the presence of virus in blood, 
urine, saliva, cervico-vaginal secretions and semen will also be evaluated. Multiple comparisons 
analysis will be used to compare the magnitude and duration of virus presence at each dose level 
of each candidate ZIKV and placebo recipients. 
 
Measuring the transmissibility of ZIKV-SJRP and ZIKV-Nicaragua from infected volunteers to 
mosquitoes is an exploratory endpoint. Following feeding on volunteers, mosquitoes will be 
returned to the NIH for analysis (Section 6.7). The heads and thoraces of the mosquitoes will be 
examined for ZIKV antigen. If antigen is detected in the head of the mosquitoes, it will be 
assumed that the virus escaped the mid-gut barrier and is present in the salivary glands.  
9.3 
Safety Endpoint 
The primary clinical endpoint is the frequency of ZIKV-related AEs, as classified by both 
severity and seriousness, through active and passive surveillance. Separate assessments of 
systemic and local reactions will be performed.  
9.4 
Immunogenicity 
ZIKV neutralizing antibody titers will be measured on Study Days 0, 28, 56, 90, and 180. 
Seroconversion to ZIKV will be defined as a PRNT50 of ≥ 1:10 based on a pre-inoculation 
PRNT50 of <1:5 (seronegative to ZIKV). 
9.5 
Per Protocol Analysis 
9.5.1 Safety Data 
The adverse event data will be included in the per-protocol analysis of all subjects who received 
an inoculation, even if one or more study visits during safety follow-up period were missed. This 
will assure that the largest body of safety data are included in the analysis. 

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9.5.2 Virology 
Subjects who were enrolled through Study Day 180, even if they missed one or more visits 
during this time period will be included in the per-protocol analysis of viral shedding. Because 
there are frequent visits and an inpatient stay, missing these time-points is not expected to affect 
the analysis of the duration of virus shedding. 
9.5.3 Immunogenicity Data 
The frequency of seroconversion and peak neutralizing antibody titers will be included in the 
per-protocol analysis for all subjects who were enrolled through Study Day 90 and have samples 
from at least 2 of the following 3 time-points (Day 28, 56, 90). 
9.6 
Intent-to-Treat Analysis 
9.6.1 Virology 
Subjects who were not enrolled through Study Day 180 but who had samples collected for ZIKV 
isolation will be included in the intent-to-treat analysis of viral shedding. 
9.6.2 Immunogenicity Data 
Subjects who did not complete Study Day 90 but did complete Study Day 28 or 56 will be 
included in the intent-to-treat analysis of seroconversion and peak neutralizing antibody titer. 
10 PROTECTION OF HUMAN SUBJECTS 
10.1 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. 
10.2 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. A copy of the informed 
consent document will be given to the subject for his or her records. The subject may withdraw 
consent at any time during the course of the trial. 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 or if they withdraw from the study this study.  
10.3 Risks 
The risks to the subjects are minimal and are generally associated with venipuncture and ZIKV 
infection. These risks are outlined below. There is a potential risk of ZIKV transmission to the 
community by mosquito as well as by sexual transmission. In addition, questions asked 
regarding a subject’s sexual history may make the subject uncomfortable. These questions will 
be asked in a private area and the subject will be made aware that they do not have to answer any 

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question that they do not feel comfortable answering. Subjects will be informed of the known 
risk of ZIKV and birth defects in the fetus. Subjects will be advised to use effective birth control 
methods while at risk for ZIKV infection (we will follow CDC guidelines of 8 weeks for people 
of childbearing potential and 12 weeks for men post-infection time of infection is defined as 
inoculation with challenge virus).  Those of child bearing potential will also be required to use 
barrier contraception through study day 56 to prevent Zika transmission, in accordance with 
CDC guidance. Males will be advised to use barrier contraception through study day 90 to 
prevent Zika transmission in accordance with CDC guidance. 
10.3.1 Venipuncture 
The total amount of blood to be drawn throughout the 6-month duration of the primary study is 
approximately 700 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. 
10.3.2 Local Risks Associated with Test Article Administration 
Local risks associated with subcutaneous administration of test article (either PlasmaLyte or 
ZIKV) include injection site pain, injection site pruritus  injection site erythema or injection site 
induration.  
10.3.3 Risk of Use of Cervico-vaginal Cup 
The cervico-vaginal cup is a commercially-available product used during menstruation. It is 
contra-indicated in women with an IUD in place. For women who have an IUD in place, cervico-
vaginal secretion specimens will be collected by use of cervico-vaginal swab.  
10.3.4 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. 
10.3.5 Risk from Mosquito Feeding 
The mosquito bites that will result from mosquito feeding may cause itching, erythema, and 
welt-formation (normal responses to mosquito bites). Some volunteers may be more sensitive to 
mosquito bites and may have more intense symptoms. Severe allergy to mosquito bites is 
extremely rare. 
10.3.6 Risk Due to Infection with ZIKV 
10.3.6.1 Zika Illness 
ZIKV may cause a mild, transient illness in those who are infected, although the majority of 
infections are asymptomatic. Possible local inoculation reactions include pain, swelling, or 
erythema for 2 to 3 days, lymphadenopathy, or pruritus at the injection site. Systemic reactions 
such as fever, maculopapular, itchy rash, red eyes, headache, and muscle and joint pains have 
been described in patients diagnosed with ZIKV infection.  
10.3.6.2 Zika Congenital Syndrome 
Zika virus has been causally linked to congenital microcephaly [12]. Microcephaly and other 
severe birth defects in the fetus have been linked to ZIKV infection during pregnancy. The risk 
of birth defect in the fetus is highest when infection occurs during the first month of pregnancy 
but can occur with infection in any trimester. There is growing evidence that developmental 
delay, hearing abnormalities, and vision abnormalities may be associated with intrauterine ZIKV 
infection and may become apparent after birth, even when the baby appears to be normal at birth. 

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10.3.6.3 Guillain-Barré Syndrome (GBS) 
GBS is an autoimmune disorder characterized by varying degrees of weakness, autonomic 
dysfunction, and sensory abnormalities. Typically, GBS is manifested by bilateral lower limb 
numbness, tingling, and weakness that can then spread to include the upper extremities. In severe 
cases, the muscles of the diaphragm can be affected which man affect the ability of the patient to 
breath and may require mechanical ventilation. Gradually these symptoms resolve over weeks, 
but it can sometimes take months for resolution to occur. Some patients never fully recover. 
These are due to peripheral nerve or nerve root damage thought to be caused by an autoimmune 
reaction. The annual incidence of GBS worldwide is estimated to be ~ 0.6 – 4 cases/100,000 
population and varies by geography and age group [53-55]. GBS has been associated with 
various infectious agents including influenza, cytomegalovirus, Campylobacter jejuni, 
chikungunya and ZIKV (see Section 1.1 for more detail). Several studies have observed that the 
risk of GBS appears to increase with older age, particularly older than 50 [56-58]. The risk of 
GBS following ZIKV infection is not fully known but is estimated to be around 1 – 2 cases per 
10,000 ZIKV infections in the general population and is irrespective of health status at time of 
infection [15, 59]. This is slightly lower than the estimated risk of GBS following Campylobacter 
infection [15, 60]. 
10.3.6.4 Risk of Transmission 
ZIKV can be sexually-transmitted from men-to-women, men-to-men, and women-to-men (see 
Section 1.1 for more detail). The majority of cases of sexual transmission reported to date have 
occurred just before or during the period of time when the person was symptomatic with ZIKV; 
however, one reported case of sexual transmission has occurred from an individual who did not 
recall any previous symptoms associated with ZIKV infection [28]. The risk of sexual 
transmission appears to be much higher from men-to-women than for women-to-men as ZIKV is 
rarely shed from the female genital tract and when it is shed, it is of short duration [37]. The 
reported cases of sexual transmission have occurred within 3 weeks of return from a ZIKV-
endemic area with most of those occurring within 2 weeks of return [42] (see Section 1.1 for 
more detail). Study subjects will be housed in an inpatient unit for Study Days 0 – 9 (discharged 
on Study Day 9). All subjects will be counseled on the risks of congenital Zika syndrome and 
will be required to use barrier contraception through Study Day 56 per CDC guidelines. 
Condoms will be provided at no cost to subjects in the study. 
The major route of ZIKV transmission is by mosquito. It is believed that Aedes aegypti is the 
primary vector for ZIKV but that Aedes albopictus may also transmit the virus. Aedes aegypti 
mosquitoes are not known to inhabit the study areas, although Aedes albopictus species are 
present. Aedes albopictus mosquito-pools have not been identified as positive for ZIKV in the 
current outbreak. The virus must achieve a sufficient level of viremia in infected volunteers to 
support transmission by mosquitoes (generally >3 log10 PFU/mL). It is not anticipated that 
subjects will achieve this level of viremia for any extended length of time during this study. As 
an added protection, subjects will be housed in an environmentally-controlled inpatient unit 
until Study Day 9 to prevent any possible transmission by mosquito until mosquito-
transmissibility studies have been performed and the data from these studies support 
outpatient studies. 
In accordance with American Red Cross blood donation guidelines, we will inform volunteers 
that they cannot donate blood for at least 6 months following ZIKV infection and that they 

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should inform blood or plasma donation centers that they were enrolled in the trial and received 
ZIKV challenge (for those who were not placebo recipients). 
 
10.3.6.5 Risk of More Severe Dengue Disease 
Previous infection with one DENV serotype can predispose to more severe dengue following 
subsequent infection with a second, different DENV serotype ≥ 18 months after the first 
infection [61, 62]. Modeling studies from a cohort study from Nicaragua suggested that a 
previous Zika infection may increase the probability of symptomatic and more severe DENV-2 
infection occurring 3 years after Zika infection [63]. It is unknown how long the enhanced risk of 
severe dengue following Zika infection persists. However, it is thought that the risk of severe 
dengue following subsequent heterotypic DENV infection can last years to decades. 
10.3.6.6 Other Risks 
Subjects may be asked to defer routine immunization (such as influenza) until after 21 days 
following inoculation. Subjects will be offered influenza vaccination at no charge before they are 
enrolled in the study (≥ 14 days prior to enrollment [killed vaccine] or ≥ 21 days prior to 
enrollment [live vaccine]), or after Study Day 21 of the study. This may increase the risk that the 
subject will be infected with an influenza virus during this period. As with any investigational 
product, 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. 
10.3.7 Risk Mitigation 
10.3.7.1 Prevention of Congenital Zika Syndrome 
Female volunteers who are pregnant or breast-feeding are not eligible for participation in the 
study. Female volunteers will be screened for pregnancy on 2 occasions prior to enrollment, on 
the day of ZIKV administration, and at regular intervals thereafter. All volunteers will be 
counseled on pregnancy prevention at regular intervals throughout the study and they will be 
provided with barrier contraception through study day 56 (women) or study Day 90 (men). All 
volunteers must agree to the use of barrier contraception for the specified duration. Female 
volunteers of child-bearing potential must also agree to the use of effective birth control (defined 
in Section 4.1) while at risk for ZIKV infection (we will follow CDC guidelines of 8 weeks post-
infection (Study Day 56); time of infection is defined as inoculation with challenge virus). 
Female volunteers who exclusively have sex with females and females who are postmenopausal 
will be granted an exception to this requirement as they are not at risk for pregnancy from sexual 
intercourse.  
10.3.7.2 Mitigation of Risk of GBS 
It is estimated that the overall risk of GBS associated with ZIKV is 1 – 2 cases per 10,000 ZIKV 
infections. GBS is known to occur more commonly in subjects older than 50 and in subjects with 
history of GBS or autoimmune disease [54]. To reduce the risk of GBS, enrollment will be 
limited to subjects 18 – 40 years of age without history of autoimmune disease or history of 
GBS. Subjects will be examined daily while in the inpatient unit, approximately weekly from 
Day 14  – Day 70, then again at Study Day 90, 120, and 180. This examination will include a 
neurological assessment. Should a volunteer exhibit neurologic signs or symptoms suggestive of 
GBS or other neurologic disorder, the subject will be evaluated by a neurologist and treatment 

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recommended. If hospitalization is needed, the volunteer will be hospitalized at the sponsor’s 
expense. 
10.3.7.3 Mitigation of Risk of Transmission of ZIKV 
To reduce the risk of ZIKV transmission, subjects will be housed in the inpatient unit from Study 
Day 0 through Study Day 9 (discharged on Study Day 9 if discharge criteria are met). ZIKV is 
generally able to be recovered from the blood for only a few days before and after ZIKV 
symptoms occur. A recent longitudinal study detected ZIKV by PCR in 88% of ZIKV-infected 
subjects [29]. ZIKV was detected in the serum at a level thought to be infectious (CT ≤ 27) only 
until approximately day 8 post-symptom onset. It is unlikely that infectious ZIKV will be 
detected in blood at a level that could be transmitted beyond Day 8 post-infection. Volunteers 
will not be discharged from unit unless the titer of ZIKV in serum, urine, saliva, cervico-vaginal 
secretions and semen is < 1.4 x 106 genomic copies/mL by Study Day 8. At discharge from the 
inpatient unit, subjects will be given commercially available mosquito repellent containing 
DEET during mosquito season (May – October) and will be instructed to use it when outdoors 
through Study Day 28. In addition, this trial will be conducted as a dose-escalation study to 
ensure that only the lowest dose necessary to induce the desired virologic and clinical outcomes 
will be used. A very low dose of ZIKV (100 PFU) will first be evaluated. Lower doses of ZIKV 
are likely to induce lower titers in the blood and therefore reduce the risk of transmission. It is 
anticipated that the level of viremia induced in this study is below the level required for mosquito 
transmission (~ 3.0 log10 PFU/mL). To reduce the risk of sexual transmission of ZIKV, subjects 
will be counseled on the use of barrier contraception through Study Day 56 (women) or Study 
Day 90 (men) and must agree to the use of barrier contraception when they engage in intercourse 
during this time period. Condoms will be provided to all subjects at no cost for the duration of 
the study. In addition, virus inoculum will be delivered by needle and syringe and is considered 
less infectious than natural mosquito-borne virus which is augmented by saliva factors. 
10.3.7.4 Other Risks 
Subjects will be counseled regarding the timing of routine immunizations such as the flu vaccine. 
When available, subjects will be offered the flu vaccine at no cost to them during screening or 
within the acceptable time periods during the study. Topical anti-itch medication 
(diphenhydramine and hydrocortisone cream/ointment) will be available to the volunteers upon 
request if the mosquito bite symptoms are bothersome to the volunteer. Systemic antihistamines 
will be available to treat the itch from the Zika-like rash or, if needed, the itch from mosquito 
bites. 
10.3.7.5 Escalation of Care Plan 
Should a volunteer develop new onset muscle weakness, change in deep tendon reflexes, or 
evidence of dysautonomia, or any other change from baseline exam that the clinician feels 
warrants further evaluation, the volunteer will be assessed by a formal consultation with a 
neurologist, or will be transferred by ambulance to the Emergency Room associated with the 
University of Vermont Medical Center or the Bayview Medical Center. Following evaluation, 
should a volunteer require hospitalization, she will be admitted to the University of Vermont 
Medical Center or the Bayview Medical Center for further management.  

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10.4 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 to ZIKV. It is hoped that 
information gained in this study will contribute to the development of safe and effective ZIKV 
vaccines and to a better understanding of ZIKV infection, ZIKV disease, and the risk of ZIKV 
sexual transmission. Should a potential volunteer wish to begin using hormonal contraception or 
an IUD, she will be referred to a practitioner to discuss her contraception options. Should she 
decide to use either hormonal contraception or an IUD and be otherwise eligible for enrollment 
in the study, the Sponsor will pay for her contraception for the duration of the study (oral 
contraception, NuvaRing®, hormonal patch, Depo-Provera injection, etc.), or for placement of 
longer-acting contraception (hormonal implant or an IUD). The Sponsor will also pay for 
removal of the long-term contraception should the subject desire. 
10.5 Compensation 
Subjects will be compensated up to $200 for screening, up to $300 for each inpatient day & night 
(Study Days 0 through 8, discharged on Day 9), up to $125 for each completed scheduled 
follow-up visit (Study Days 9, 10, 12, 14, 16, 21, 28, 35, 56, 70, 90, 120 and 180), and up to $50 
for the scheduled phone call on Study Day 150. They will receive $100.00 for each mosquito-
feeding session. They will also receive a $300 bonus if all study visits and procedures are 
completed on time and if they adhere to all inpatient policies and the code of conduct. Subjects 
may only receive a portion of the study visit payment and/or bonus if not all study visits or 
procedures are completed on time or for failure to follow the code of conduct agreement. 
Subjects will be compensated for the screening only if they are enrolled in the study. Subjects 
will only be compensated for the visits that they complete. We may invite more potential 
volunteers to come to the unit on Study Day 0 than we will enroll at that time. This is to ensure 
that we are able to enroll the planned number of volunteers in the case that someone is not 
eligible on Study Day 0 or does not come to the unit. Alternates who come to Day 0 but who are 
not inoculated due to fulfillment of enrollment for that day will be compensated for screening 
and for one outpatient day ($325.00). Subjects enrolled in the study will receive a maximum total 
compensation of $5,175.00. Payment will be distributed by check or other mechanism such as 
Clincard. 
 
10.5.1 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.  
10.6 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 

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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. 
10.7 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. 
11 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 
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. 
 
 

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Appendix A: Adverse Event Grading Table1,2 
Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
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 
Local Reactogenicity 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Injection Site Tenderness 
Tenderness that is easily 
tolerated 
Tenderness that interferes with 
daily activity 
Tenderness that prevents daily 
activity 
Injection Site Pain 
Pain that is easily tolerated 
Pain that interferes with daily 
activity 
Pain that prevents daily 
activity 
Injection Site Pruritus 
Pruritus that is easily tolerated 
Pruritus that interferes with daily 
activity 
Pruritus that prevents daily 
activity 
Injection Site Induration 
>0 - 20 mm 
>20 - 50 mm 
>50 mm 
Injection Site Erythema 
>0 - 20 mm 
>20 - 50 mm 
>50 mm 
Systemic Reactogenicity 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Fever (Oral) 
100.4° F – 101.4°F 
101.5°F – 102.4°F  
≥102.5°F  
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 
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 
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 
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 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
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 
Pruritus  
Pruritus that is easily tolerated, 
may require 1 dose of 
medication/treatment 
Pruritus that interferes with daily 
activity or requires >1 dose of 
medication 
Pruritus that prevents daily 
activity 
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 
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 
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 
Muscle Weakness 
Muscle weakness that is easily 
tolerated, may require 1 dose of 
medication/treatment 
Muscle weakness that interferes 
with daily activity of requires >1 
dose of medication 
Muscle weakness that prevents 
daily activity 
Hypo/areflexia 
Hypo/areflexia that is easily 
tolerated, may require 1 dose of 
medication/treatment 
Hypo/areflexia that interferes 
with daily activity of requires >1 
dose of medication 
Hypo/areflexia that prevents 
daily activity 
Solicited Lab AEs 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Leukopenia 
2,500-3,500/mm3 
1,500-2,499/mm3 
1,000-1,400/mm3 
Neutropenia (Reduced 
ANC)2 
≥750-999/mm3 
≥500-749/mm3 
<500/mm3 
Thrombocytopenia 
(Reduced Platelets) 
100,000 – 120,000/mm3 
75,000 - 99,999/ mm3 
<74,999/mm3 
Other Laboratory Values 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Leukocytosis (Increased 
WBCs) 
11,500 - 13,000/mm3 
13,001 - 15,000/mm3 
>15,000 or <1,000/mm3 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Hemoglobin (female) 
9.5 - 10.7 gm/dL 
8.0 - 9.4 gm/dL 
<7.9 gm/dL 
Hemoglobin (male) 
11.0 - 12.5 gm/dL 
9.0 – 10.9 gm/dL 
<8.9 gm/dL 
PT 
>1.0 - 1.25 x upper limit of 
normal (ULN) 
>1.25 - 1.5 x ULN 
>1.5 x ULN 
PTT 
>1.0 - 1.66 x ULN 
>1.66 - 2.33 x ULN 
>2.33 x ULN 
ALT 
>1.25 - 2.5 x ULN 
>2.5 - 5.0 x ULN 
>5.0 x ULN 
Creatinine 
1.5 - 1.7 mg/dL 
>1.7 – 2.0 mg/dL 
>2.0 mg/dL 
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 
Fibrinogen, Increased 
>ULN to 600 mg/dL 
>600 mg/dL 
N/A 
Creatine phosphokinase 
(CPK) 
≥4 x ULN- 6 x ULN 
>6 x ULN- 10 x ULN 
>10 x ULN 
Sodium: Hyponatremia 
130 – 134 mEq/L 
123 – 129 mEq/L 
<122 mEq/L 
Sodium: Hypernatremia 
145 – 150 mEq/L 
151 – 157 mEq/L 
>158 mEq/L 
Potassium: Hypokalemia 
3.1 – 3.2 mEq/L 
2.9 – 3.0 mEq/L 
<2.8 mEq/L 
Potassium: Hyperkalemia 
5.2 – 5.5 mEq/L 
5.6 – 6.0 mEq/L 
>6.1 mEq/L 
Phosphate: 
Hypophosphatemia 
2.0 – 2.2 mg/dL 
1.5 – 1.9 mg/dL 
<1.4 mg/dL 
Calcium (Corrected for 
Albumin): Hypocalcemia 
1.95 – 2.04 mmol/L 
1.75 – 1.94 mmol/L 
<1.74 mmol/L 
Calcium (Corrected for 
Albumin): Hypercalcemia 
2.51 – 2.88 mmol/L 
2.89 – 3.13 mmol/L 
>3.14 mmol/L 
Magnesium: 
Hypomagnesemia 
0.60 – 0.74 mmol/L 
0.45 – 0.59 mmol/L 
<0.44 mmol/L 
Bilirubin 
(hyperbilirubinemia) 
>1.0 – 1.5 x ULN 
>1.5 – 2.5 x ULN 
>2.5 ULN 
Glucose: Hypoglycemia 
(Nonfasting, No Prior 
Diabetes) 
55 – 69 mg/dL 
40 – 54 mg/dL 
<39 mg/dL 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Glucose: Hyperglycemia 
(Nonfasting, No Prior 
Diabetes) 
116 – 160 mg/dL 
161 – 250 mg/dL 
>251mg/dL 
Triglycerides 
— 
400 – 750 mg/dL 
>751 mg/dL 
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 
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 
Other Cardiovascular 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Cardiac Arrhythmia 
Asymptomatic; transient 
dysrhythmia, no therapy required 
Recurrent/persistent 
dysrhythmia; symptomatic 
therapy required 
unstable dysrhythmia, 
hospitalization and therapy 
required 
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 
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 
Pericarditis 
Mild/moderate asymptomatic 
effusion, no therapy 
Symptomatic effusion, pain, 
EKG changes 
Tamponade or 
pericardiocentesis or surgery 
required 
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 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
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 
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 
Oral 
Discomfort/Dysphagia 
Difficulty swallowing but able to 
eat and drink 
Unable to swallow solids 
Unable to drink fluids; IV 
fluids required 
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 
Other Respiratory 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
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 
Dyspnea 
Dyspnea on exertion 
Dyspnea with normal activity 
Dyspnea at rest 
Other Neurologic 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Nuchal Rigidity 
--  
-- 
Presence of Nuchal rigidity 
Neuropsychological 
 
Mild confusion or cognitive 
impairment 
Moderate confusion or cognitive 
impairment 
Severe confusion or cognitive 
impairment 
Neurocerebellar 
Slight incoordination or 
dysdiadochokinesia 
Intention tremor, dysmetria, 
slurred speech, or nystagmus 
Ataxia requiring assistance to 
walk or arm incoordination 
interfering with ADLs 

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Event 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
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 
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 
Paresthesia  
(Burning, Tingling, etc.) 
Mild discomfort; no therapy 
required 
Moderate discomfort; non-
narcotic analgesia required 
Severe discomfort; or narcotic 
analgesia required with 
symptomatic improvement 
Other Dermatologic 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Dermatitis 
Rash is present but asymptomatic 
Rash is symptomatic 
(pruritus/pain) but does not 
interfere with function 
Rash is symptomatic and 
interferes with function 
Other Urinalysis 
Grade I – Mild 
Grade II – Moderate 
Grade III – Severe 
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 Cervico-vaginal 
Bleeding) 
Microscopic only,  
6-10 rbc/hpf 
>10 rbc/hpf 
Gross, with or without clots;  
or RBC casts 
Other Miscellaneous  
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Malaise 
Malaise that is easily tolerated 
Malaise that interferes with daily 
activity 
Malaise that prevents daily 
activity 
 
1. Grade 4 will be assigned to any adverse event that is determined to be potentially life-threatening. Grade 5 will be assigned to any adverse event that 
results in death 

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2. These values for neutropenia are values that have been used in dengue controlled human infection model studies and dengue live attenuated vaccine 
studies 
Appendix B: 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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Appendix C: 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