Conditions: Ebola
Keywords: Liberia Guinea
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 38
Healthy volunteers: no

Brief summary:
Background:

Some people have Ebola virus in their body for months after they recover from Ebola virus disease. Some may have health problems from the virus while others are fine. These people may be able to pass the virus to others. There are currently no drugs for people who have survived Ebola virus disease but still have the virus in their body. A new drug, GS-5734, might help get rid of Ebola virus in semen.

Objective:

To test if GS-5734 helps get rid of Ebola virus in semen and is safe for humans.

Eligibility:

Men who participated in the Ebola survivor study (PREVAIL III) and have evidence of the Ebola virus in their semen

Design:

Participants will be screened with:

Questions

Physical exam

Eye exam

Blood tests

2 semen samples if they have not had it tested recently

Participants must live near the study site in Liberia for 6 months.

Participants will be put into 1 of 2 study groups. They will have an infusion of either GS-5734 or a placebo every day for 5 days. A plastic tube is put into an arm vein. The infusion lasts 1 hour.

Participants will be observed for 1 hour after. They will provide a semen sample on infusion day 4.

After the infusions, participants will have 5 visits in the first month, then 1 per month for 5 more months. These include giving a blood and semen sample.

Blood tests are performed before and after each infusion and the last visit (5 month visit) will also include an eye exam.

When the study is over, if the study drug works and is safe, participants who got the placebo can get the study drug.

Detailed description:
With the unprecedented size of the recent 2014-2016 West African Ebola outbreak, the scientific community is learning a great deal about the psychological and physical consequences of Ebola, Ebola viral persistence in survivors, risk of Ebola disease relapse in survivors, and the potential for survivors to transmit the virus to others. Data from PREVAIL III has demonstrated that persistence of Ebola virus in the semen of male survivors is common. In addition to Ebola virus persistence, Ebola relapse causing clinical disease has been well documented.

There are no licensed therapies for the treatment of Ebola virus disease nor for the clearance of persistent Ebola virus in survivors. A safe, effective therapy that can reduce and/or eliminate persistent Ebola virus from semen would reduce the risk of transmission and enable male survivors to resume normal sexual relations without fear of harming loved ones. The mechanism underlying Post-Ebola Syndrome is as yet unknown, but improvement in Post-Ebola signs and symptoms resulting from GS-5734 treatment would be an added benefit.

This study is a double-blind, randomized, two-phase (treatment and longer-term follow-up), two- arm trial of GS-5734 versus placebo among male Ebola survivors with persistent Ebola virus RNA in their semen. Participants are randomized 1:1 to receive either 100 mg of GS-5734 or placebo once daily by intravenous catheter for 5 days. Informed by transaminase elevations in prior Phase I studies in normal healthy subjects, a risk-mitigation strategy includes a built-in dose de-escalation. Participants will be stratified by country and on the basis of one versus two positive semen samples for Ebola virus RNA using the Cepheid GeneXpert platform assessed within 42 days prior to study enrollment. The early Data Safety and Monitoring Board (DSMB) review in August 2016 concluded there was no need for a cohort dose reduction. The protocol expanded to Guinea in October 2017, where the outbreak ended later. Currently there is an outbreak in the Democratic Republic of the Congo where the study team may evaluate conducting this study following the completion of the outbreak.

Antiviral activity, as well as safety and tolerability, will be assessed during the treatment phase. Longer-term clearance of Ebola virus will be assessed during the 5-month follow-up phase. Primary analyses for the assessment of antiviral activity in the treatment phase will focus on the assay negativity rate (ANR; percentage of genital samples that are negative for Ebola) over the first 28 days of the study, as well as clinical and laboratory adverse events. A sample is considered negative by PCR if the test result is undetectable. Primary analysis for the follow-up phase will focus on the ANR collected monthly from months 2 to 6.

Arms:
arm 1: Male Ebola survivors with persistent Ebola virus in their semen were given GS-5734 100mg intravenously daily for 5 days arm 2: Male Ebola survivors with persistent Ebola virus in their semen were given normal saline intravenously daily for 5 days

Interventions: intervention 1: GS-5734 intervention 2: Placebo Comparator
Intervention descriptions: intervention 1: Daily GS-5734 delivered intravenously (IV) for 5 days intervention 2: Placebo delivered intravenously (IV) for 5 days.

Protocol document:
Prot_SAP_000.pdf: 
PREVAIL IV: Double-blind, Randomized, Two-phase, Placebo-controlled, 
Phase II Trial of GS-5734 to Assess the Antiviral Activity, Longer-term 
Clearance of Ebola Virus, and Safety in Male Ebola Survivors with Evidence 
of Ebola Virus Persistence in Semen 
 
NIAID Protocol Number: 
16-I-N137 
 
Version Number: 
10.0 
 
Date: 
May 13, 2019 
 
Investigational New  
Drug (IND) Number: 
130621 
 
IND Sponsor: 
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) 
 
Local Liberian Medical  
Officer: 
Ian Wachekwa, MD 
 
Safety Medical Officer 
John F. Kennedy Hospital  
 
21 Street, Sinkor 
Monrovia, Liberia 
 
wachekwa@gmail.com  
 
 
 
 
(t) +231880890907 
 
Sponsor Medical Monitor: Alan Lifson, MD, MPH 
 
Professor, Division of Epidemiology and Community Health 
School of Public Health 
University of Minnesota 
1300 S. Second St., Ste. 300 
Minneapolis, MN 55454 
(t) 612-626-9697 
(f) 612-624-0315 
Lifso001@umn.edu 
 
Pharmaceutical Support  
Provided by: 
Gilead Sciences, Inc. 
 


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Conducted by: 
Liberia-US Joint Clinical Research Partnership also known as the 
Partnership for Research on Ebola Virus in Liberia (PREVAIL) 


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Key Roles 
NIH Principal Investigator: Elizabeth S. Higgs, MD, MIA, DTMH 
 
Division of Clinical Research, NIAID 
 
 
 
 
ehiggs@niaid.nih.gov 
 
 
 
 
(c) +301-768-3947 
 
 
 
 
skype: libby.higgs2 
 
 
 
 
 
Liberian Principal 
Investigator:  
Dehkontee Gayedyu-Dennis, MD 
 
 
 
 
PREVAIL  
 
 
 
 
dehkoden@gmail.com 
 
 
 
 
(t) +231-886-538-810 
 
 
Guinean Principal 
Investigator:   
 
Abdoul Habib Beavogui, MD  
Centre National de Formation et de Recherche en Santé Rurale de 
Mafèrinyah 
beavoguia@gmail.com 
(t) +224682045352 
 
 
 
 
UNC Lead Principal  
Investigator:   
 
William Fischer II, MD 
Assistant Professor of Medicine 
Division of Pulmonary and Critical Care Medicine 
The University of North Carolina School of Medicine 
104 Mason Farm Road 
Chapel Hill, NC 27599-7310 
(o) 919-843-9108 
(f) 919-966-9863 
(pager) 919-216-2930 
WFischer@med.unc.edu 
skype: wfischer8 
 
Statistician:   
 
Martha Nason, PhD 
 
 
 
 
Biostatistician 
Biostatistics Research Branch, NIAID 
 
 
 
 
MNason@niaid.nih.gov 
 
Virologists:   
 
Lisa Hensley, PhD 


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Associate Director, Science 
NIH/NIAID/Integrated Research Facility 
(t) 301-631-7200 
Lisa.Hensley@nih.gov  
 
Dr N’Faly MAGASSOUBA, Ph.D 
Université Gamal Abdel Nasser de Conakry.  
Laboratoire des  fièvres hémorragiques en Guinée. 
Commune de Ratoma. Nongo/Conteyah 
BP 5680. Tel: (00224) 622 39 67 60 
E-mail: cmagassouba01@gmail.com 
 
 
 
 
 
 
Clinical Laboratory   
 
Specialist:  
 
 
Marie Hoover, PhD 
CEO 
Advanced BioMedical Laboratories, L.L.C. 
(t) 856-829-9466, ext. 14 
(f) 856-829-9471 
Marie.Hoover@abiolab.com 
 
Protocol Coordinator: 
Sara Albert 
 
 
 
 
Clinical Project Manager 
Leidos Biomedical Research, Inc. 
(t) 301-846-6703 
Sara.Albert@fnlcr.nih.gov 
 
 
Associate Investigators:  
Mosoka Fallah, PhD 
James Soka Moses (Liberia PREVAIL) 
Cliff Lane, MD 
Fatorma Bolay, PhD 
David Wohl, MD 
Stephen Kennedy, MD 
Michael Sneller, MD 
Jamila Aboulhab, MD  
Nene Djiba Barry, MD 
Facely Tolno, MD  
Mandy Kader Konde, MD 
 
Study Sites:   
 
Liberia 
John F. Kennedy Hospital PREVAIL Site 


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Duport Road PREVAIL Site 
 
 
 
 
C.H. Rennie PREVAIL Site 
 
 
 
 
Redemption Hospital PREVAIL Site (recruitment site only) 
 
 
 
 
 
Guinea 
National Center for Training and Research in Rural Health 
“Jean SENECAL” of Maferinyah 
Landreah 
Landreah Annex 
Center of aptitude of the Diploma of Higher Studies Specialized in 
Ophthalmology “CADESSO” 
 
Collaborators: 
Huyen Cao 
Gilead Sciences, Inc. 
333 Lakeside Drive 
Foster City, CA 94404 
(t) 650-372-4407 
Huyen.Cao@gilead.com 
Role: Provide oversight as the clinical research representative from 
Gilead on the PREVAIL IV team. 
 
 
 
 
 
Tomas Cihlar, PhD 
Vice President, Biology 
Gilead Sciences, Inc. 
(t) 650-522-5637 
Tomas.Cihlar@gilead.com 
Role: Provide oversight and expertise on the biological aspects of 
the study agent and its antiviral properties in relation to the study 
objectives. 
 
 
Institutional Review Boards (IRBs): 
NIAID IRB 
5601 Fishers Lane 
Bethesda, MD 20892 
 
National Research Ethics Board of Liberia (NREB)  
Ministry of Health 
JFK Compound 
21 Street, Sinkor 
Monrovia, Liberia 


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National Ethics Committee for Health Research of the Republic of Guinea 
Comité National d'Ethique pour la Recherche en Santé (CNERS) 
BP 634 
Conakry, Guinea 
 
 


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Table of Contents 
Key Roles ........................................................................................................................................ 3 
Table of Contents ............................................................................................................................ 7 
List of Tables ................................................................................................................................ 10 
List of Figures ............................................................................................................................... 10 
List of Abbreviations .................................................................................................................... 11 
Protocol Summary ........................................................................................................................ 13 
Précis ............................................................................................................................................. 17 
1 
Background Information and Scientific Rationale................................................................ 18 
1.1 
Background Information ................................................................................................ 18 
1.2 
Description of the Study Agent (GS-5734) .................................................................... 22 
1.2.1 
General Information .............................................................................................. 22 
1.2.2 
Summary of Previous Preclinical Studies ............................................................. 23 
1.2.3 
Summary of Relevant Clinical Studies ................................................................. 27 
1.2.4 
Additional GS-5734 Human Experience .............................................................. 36 
1.3 
Rationale for Study......................................................................................................... 37 
1.4 
Rationale for Dose Selection .......................................................................................... 38 
1.4.1 
Individual Dose De-escalation Plan ...................................................................... 39 
1.4.2 
Cohort Dose De-escalation Evaluation ................................................................. 39 
1.5 
Rationale for Length of Treatment-Phase ...................................................................... 39 
1.6 
Rationale for Use of the GeneXpert for Ebola Virus RNA Detection ........................... 40 
2 
Study Objectives ................................................................................................................... 40 
2.1 
Primary Objectives ......................................................................................................... 40 
2.1.1 
Treatment Phase .................................................................................................... 40 
2.1.2 
Follow-up Phase.................................................................................................... 40 
2.2 
Secondary Objectives ..................................................................................................... 41 
2.2.1 
Treatment Phase .................................................................................................... 41 
2.2.2 
Follow-up Phase.................................................................................................... 41 
2.3 
Exploratory Objectives ................................................................................................... 41 
3 
Study Design ......................................................................................................................... 41 
3.1 
Description of the Study Design .................................................................................... 41 
3.2 
Study Endpoints ............................................................................................................. 43 
3.2.1 
Primary Endpoints ................................................................................................ 43 
3.2.2 
Secondary Endpoints ............................................................................................ 43 
4 
Study Population ................................................................................................................... 44 
4.1 
Rationale for Subject Selection ...................................................................................... 44 
4.2 
Recruitment Plan & Retention Plans .............................................................................. 44 
4.3 
Subject Inclusion Criteria ............................................................................................... 44 
4.4 
Subject Exclusion Criteria .............................................................................................. 45 
4.5 
Justification for Exclusion of Special Populations ......................................................... 45 
5 
Study Agent/Interventions .................................................................................................... 45 
5.1 
Disposition and Dispensation ......................................................................................... 45 
5.1.1 
Formulation, Packaging, and Labeling ................................................................. 45 
5.2 
Study Agent Storage and Stability ................................................................................. 46 
5.3 
Randomization Procedures ............................................................................................. 46 
5.4 
Blinding and Unblinding Procedures ............................................................................. 46 


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5.5 
Preparation, Administration, and Dosage of Study Agents ........................................... 46 
5.5.1 
Preparation ............................................................................................................ 46 
5.5.2 
Dosing and Administration ................................................................................... 46 
5.5.3 
Dose Adjustments ................................................................................................. 46 
5.6 
Study Product Accountability Procedures...................................................................... 47 
5.7 
Concomitant Medications .............................................................................................. 47 
5.8 
Discouraged Medications ............................................................................................... 47 
6 
Study Schedule ...................................................................................................................... 47 
6.1 
Baseline Ophthalmologic Exam (4 months prior to enrollment) ................................... 47 
6.2 
Pre-Screen (prior to day 0) ............................................................................................. 48 
6.3 
Screening/Week 0 (within 21 days prior to day 1)......................................................... 48 
6.4 
Treatment Phase (day 1 to day 28) ................................................................................. 49 
6.4.1 
Day 1 ..................................................................................................................... 49 
6.4.2 
Days 2, 3, 4, and 5................................................................................................. 49 
6.4.3 
Days 8, 11, 16, 24, and 28 (see Appendix B for windows) .................................. 51 
6.5 
Follow-up Phase ............................................................................................................. 51 
6.5.1 
Weeks 8, 12, 16, 20, and 24 (see Appendix B for windows)................................ 51 
6.5.2 
Week 24 (within ± 6 week window) ..................................................................... 51 
6.6 
Closeout Treatment for Subjects on Placebo Arm ......................................................... 51 
7 
Study Procedures/Evaluations............................................................................................... 51 
7.1 
Clinical Procedures/Evaluations .................................................................................... 51 
7.2 
Laboratory Evaluations .................................................................................................. 52 
7.2.1 
Specimen Preparation, Handling, and Shipping ................................................... 52 
8 
Potential Risks and Benefits ................................................................................................. 52 
8.1 
Potential Risks ................................................................................................................ 52 
8.1.1 
GS-5734 ................................................................................................................ 52 
8.1.2 
Placebo .................................................................................................................. 54 
8.1.3 
Blood Draw/Phlebotomy ...................................................................................... 54 
8.1.4 
Semen Collection .................................................................................................. 54 
8.2 
Potential Benefits ........................................................................................................... 54 
8.3 
Risk-Benefit Assessment................................................................................................ 55 
8.3.1 
Risk Mitigation ..................................................................................................... 55 
9 
Research Use of Stored Human Samples, Specimens, or Data ............................................ 56 
10 Compensation for Inconvenience: Plan for Subjects ............................................................ 57 
11 Assessment of Safety ............................................................................................................ 57 
11.1 Definitions ...................................................................................................................... 57 
11.2 Investigators’ Documentation and Assessment of Adverse Events ............................... 59 
11.3 Reporting Responsibilities and Timelines ..................................................................... 59 
11.4 Investigator Reporting Procedures to the IRBs .............................................................. 60 
11.5 Sponsor’s Reporting Responsibilities ............................................................................ 61 
11.5.1 
Resumption of the Study in the Event that the DSMB Recommends a Pause ..... 61 
11.6 Withdrawal Criteria for an Individual Subject ............................................................... 61 
11.6.1 
Replacement of Withdrawn Subjects or Subjects Who Discontinue Study Agent61 
11.7 Safety Oversight ............................................................................................................. 62 
12 Site Monitoring Plan ............................................................................................................. 64 
13 Statistical Considerations ...................................................................................................... 65 


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13.1 Study Hypotheses ........................................................................................................... 65 
13.1.1 
Treatment Phase .................................................................................................... 65 
13.1.2 
Follow-up Phase.................................................................................................... 65 
13.2 Study Design .................................................................................................................. 65 
13.2.1 
Stratification and Randomization ......................................................................... 65 
13.3 Sample Size Justification ............................................................................................... 65 
13.3.1 
Power Calculations ............................................................................................... 66 
13.4 Statistical Analyses ........................................................................................................ 67 
13.4.1 
Secondary Endpoints ............................................................................................ 67 
13.5 Operational Futility ........................................................................................................ 68 
14 Ethics/Protection of Human Subjects ................................................................................... 68 
14.1 Informed Consent Process .............................................................................................. 68 
14.2 Subject Confidentiality ................................................................................................... 68 
15 Data Handling and Record Keeping ..................................................................................... 69 
15.1 Data Capture and Management ...................................................................................... 69 
15.2 Record Retention ............................................................................................................ 69 
Appendix A: Scientific References ............................................................................................... 70 
Appendix B: Schedule of Procedures/Evaluations ....................................................................... 72 
Appendix C. eGFR Calculation .................................................................................................... 75 
Appendix D: Delineation of Principal Investigator roles for the PREVAIL IV protocol ............ 76 
Appendix E: Compensation for Inconvenience ............................................................................ 77 
 
 
 


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List of Tables 
Table 1. Dosing cohorts in GS-US-399-1812. .............................................................................. 27 
Table 2. GS-US-399-1812: GS-5734 Mean (%CV) Plasma PK Parameters Following a Single 2-
hour IV Infusion of GS-5734 in Healthy Subjects (Cohorts 1-6; Analysis Set: PK) ................... 29 
Table 3. GS-US-399-1812: GS-441524 Mean (%CV) PK Plasma Parameters Following a Single 
2-hour IV Infusion of GS-5734 in Healthy Subjects (Cohorts 1-6; Analysis Set: PK) ................ 29 
Table 4. GS-US-399-1954: GS-5734 Mean (%CV) Plasma PK Parameters Following 
Multiple 1-hour IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; 
Analysis Set: PK)......................................................................................................................... 32 
Table 5. GS-US-399-1954: GS-441524 Mean (%CV) PK Parameters Following Multiple 1-hour 
IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; Analysis Set: PK) ....... 33 
Table 6. GS-US-399-1954: GS-704277 Mean (%CV) PK Parameters Following Multiple 1-hour 
IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; Analysis Set: PK) ....... 34 
Table 7. GS-US-399-1954: Intracellular GS-441524 Phosphorylated Metabolites Mean (%CV) 
PK Parameters in PBMC Following Multiple 1-hour IV Infusions of GS-5734 150 mg in Healthy 
Subjects (Cohorts 1 and 2; Analysis Set: PK) .............................................................................. 34 
Table 8. Median (with the first and third quartile in parentheses) ALT and AST levels in 2,723 
Liberian participants in PREVAIL III: 1083 Ebola survivors and 1640 household and sexual 
contacts. ........................................................................................................................................ 38 
Table 9. Summary of blood samples to be collected for PK analysis.a ........................................ 50 
Table 10. Suggested guidelines for dose de-escalation by the DSMB. ........................................ 64 
Table 11. Power for endpoints under varying assumptions. ......................................................... 66 
 
List of Figures 
Figure 1. Minimum CT(NP,GP) results among those with at least 1 detectable sample from 
PREVAIL III (n=83) ..................................................................................................................... 21 
Figure 2. GS-5734 Metabolism..................................................................................................... 23 
Figure 3. Metabolites: Semen Predose (Ctrough) Concentrations. .................................................. 35 
Figure 4. Study design. ................................................................................................................. 42 
 
 
 


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List of Abbreviations 
AE 
Adverse event/adverse experience 
ALT 
Alanine transaminase  
ANR 
Assay negativity rate (percentage of samples for which Ebola virus is not detected 
by polymerase chain reaction) 
aPTT 
Activated partial thromboplastin time 
AST 
Aspartate transaminase  
BMI 
Body mass index 
CC 
Clinical Center 
CFR 
Code of Federal Regulations 
CNERS 
Comité National d'Ethique pour la Recherche en Santé (National Ethics 
Committee for Health Research of the Republic of Guinea) 
CRF 
 
Case report form 
CSF 
Cerebrospinal fluid 
CSO 
Clinical Safety Office 
CT 
Cycle threshold 
DAIDS 
Division of AIDS 
DCR 
Division of Clinical Research 
DNA 
Deoxyribonucleic acid 
DRC 
Democratic Republic of the Congo 
DSMB 
Data and safety monitoring board 
ECG 
Electrocardiogram 
eGFR 
Estimated glomerular filtration rate 
EVD 
Ebola virus disease 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
GP 
Glycoprotein  
HRPP 
Human Research Protections Program 
IB 
Investigator’s brochure 
ICH 
International Conference on Harmonization 
IM 
Intramuscular 
IND 
Investigational new drug 
INR 
International normalized ratio  
IRB 
Institutional review board 
ISM 
Independent safety monitor 
IV 
Intravenous 
LDL 
Low-density lipoprotein 
LFT 
Liver function test 
LIBR 
Liberian Institute of Biomedical Research  
LOD 
Limit of detection 


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MDRD 
Modification of Diet in Renal Disease  
NIAID 
National Institute of Allergy and Infectious Diseases  
NIH 
National Institutes of Health 
NOAEL 
No observed adverse effect level 
NOEL 
No observed effect level 
NP 
Nucleoprotein 
NREB 
National Research Ethics Board of Liberia 
OAT 
Organic anion transporter 
OCRPRO 
Office of Clinical Research Policy and Regulatory Operations 
OHRP 
Office for Human Research Protections 
PBMC 
Peripheral blood mononuclear cell 
PCR 
Polymerase chain reaction 
PREVAIL 
Partnership for Research on Ebola Virus in Liberia  
PT 
Prothrombin time  
PTM 
Placebo to match 
PTT 
Partial thromboplastin time  
RNA 
Ribonucleic acid 
ROS 
Review of symptoms 
SAE 
Serious adverse event/serious adverse experience 
SERF 
Safety expedited report form 
SBECD 
Betadex sulfobutyl ether sodium  
SMC 
Safety monitoring committee 
SMM 
Study medical monitor 
SOE 
Schedule of events 
SOP 
Standard operating procedure 
SRCP 
Safety review and communication plan 
SUSAR 
Serious and unexpected suspected adverse reaction 
ULN 
Upper limit of normal 
UP 
Unanticipated problem 
UPnonAE 
Unanticipated problem that is not an adverse event 
WHO 
World Health Organization 
 
 
 


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Protocol Summary 
Short Title: 
PREVAIL IV 
 
Clinical Phase: 
II 
 
Sample Size: 
N=60 to 120 
 
Accrual Ceiling: 
N=120 
 
Study Population: 
Men ≥18 years of age with at least one of two semen samples 
demonstrating detection of Ebola virus RNA within 42 days prior 
to enrollment  
 
Accrual Period: 
June 2016-January 2021 
 
Study Duration: 
Start Date: 1 June 2016 
End Date: 1 January 2021 
Total length of individual subject participation: 6 to 7 months 
 
Study Design: 
Blinded, randomized, two-phase (treatment and longer-term 
follow-up), two-arm trial of GS-5734 versus placebo to evaluate 
the safety, tolerability, antiviral activity, and longer-term clearance 
of Ebola virus from male Ebola survivors with persistent Ebola 
virus in semen at screening. 
 
Study Agent/ 
Intervention Description: 
Daily GS-5734 or placebo delivered intravenously (IV) for 5 days. 
 
Primary Objectives: 
Treatment phase: To compare the antiviral activity over 28 days 
following the administration of 5 days of IV GS-5734 versus 
placebo in male Ebola Virus Disease survivors with evidence of 
Ebola virus RNA in their semen. 
 
 
Follow-up phase: To compare the effect of 5 days of IV GS-5734 
versus placebo on the detection of Ebola virus RNA in semen of 
male survivors of Ebola Virus Disease over 24 weeks.  
 


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Secondary Objectives: 
Treatment phase: 
 To evaluate the safety and tolerability of GS-5734 in male 
Ebola survivors with evidence of Ebola virus RNA in their 
semen. 
 To evaluate the pharmacokinetics of GS-5734 (and metabolites 
as appropriate) in blood and PBMCs. 
 To compare the antiviral activity over 28 days following the 
administration of 5 days of IV GS-5734 versus placebo in 
subjects with: a CT value of either the GP or NP targets of ≤ 39 
in at least one semen sample during the 42-day pre-screening 
period and subjects with one versus two positive semen 
samples during the 42-day screening period.  
 
Follow-up phase:  
 To evaluate 24-week safety outcomes following IV GS-5734 
treatment in male Ebola survivors.  
 To compare individual changes from baseline to 24-week signs 
and symptoms related to “Post Ebola Syndrome” as identified 
in PREVAIL III. 
 To compare the effect of GS-5734 and placebo on change from 
baseline to 24-week signs and symptoms related to “Post Ebola 
Syndrome” identified in PREVAIL III. 
 To compare the effect of IV GS-5734 versus placebo on the 
sustained negativity rate for the detection of Ebola virus RNA 
in semen of male survivors of Ebola Virus Disease over 24 
weeks. 
 
Exploratory Objectives: 
 
 To compare the sensitivity of Ebola viral RNA detection using 
the GeneXpert standard versus “pellet” semen preparation 
methods. 
 To compare changes from baseline in ophthalmologic findings 
between the GS-5734 arm and the placebo arm.  
 To compare Ebola virus RNA sequence changes from 
screening semen samples to day 28 and week 24 semen 
samples when sample volume allows.  


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 To assess participant satisfaction during screening and study 
participation. 
 
 
Primary Endpoints: 
Treatment phase: Assay Negativity Rate (ANR) over days 4, 8, 11, 
16, 24, and 28. 
 
 
Follow-up phase: Assay Negativity Rate (ANR) over weeks 8, 12, 
16, 20, and 24. 
 
 
Secondary Endpoints: 
Treatment phase: 
 Proportion of Grade 1, Grade 2, Grade 3, or Grade 4 aspartate 
transaminase (AST) and alanine transaminase (ALT) levels at 
days 1, 2, 3, 4, 5, 8, 11, 16, 24, and 28. 
 New Grade 3 and Grade 4 adverse events (AEs). 
 Change from baseline in safety labs, including N(%) with an 
increase in grade. 
 Change in targeted signs and symptoms (based on GS-5734 
Phase I study AEs reported as associated with GS-5734 
administration) including: constipation, nausea, vomiting, 
decreased appetite, headache, tremor, pain at infusion site, 
dyspepsia, and others new signs and symptoms reported by 
subjects. 
 Incidence of SAEs. 
 Frequency of discontinuation of study medication due to AEs 
or intolerance.  
 Post-dose concentrations of GS-5734 and metabolites in serum 
on days 1 and 5. 
 Pre-dose concentrations of GS-5734 and metabolites in serum 
on day 5. 
 Steady-state PBMC-associated concentrations of GS-441524 
and its phosphorylated metabolites on Day 5. 
 
Follow-up phase: 
 Sustained Negativity Rate at weeks 8, 12, 16, 20, and 24. 
 Proportion of ALT or AST above baseline, at Grade 1 and 
above at weeks 8, 12, 16, 20, and 24. 


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 Incidence of serious adverse events (SAEs). 
 Change from baseline and 24-week signs and symptoms 
identified as part of the “Post Ebola Syndrome” from 
PREVAIL III data on completed review of symptoms and 
physical exam, depression, and post-traumatic stress disorder 
screening. 
 
Exploratory Endpoints: 
 
 Proportion of Ebola virus RNA detection, either the GP or the 
NP gene targets, at all CT levels in semen. 
 Average minimum CT levels at which Ebola virus RNA is 
detected for GP and NP gene targets in semen. 
 Proportion of Ebola virus RNA detection or either GP or NP 
gene targets of CT ≥ 40. 
 Proportion of abnormal ophthalmologic exams. 
 Proportion of abnormal ophthalmologic exams with an 
inflammatory etiology at baseline which have improved or 
become worse between baseline and week 24. 
 Proportion of Ebola virus RNA sequence changes in the 
polymerase and elsewhere between screening sample day 28  
and 24-week semen samples. 
 
 
 
 
 
 
 
 
 
 
 
 


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Précis 
With the unprecedented size of the 2014-2016 West African Ebola outbreak, the scientific 
community is learning a great deal about the psychological and physical consequences of Ebola, 
Ebola viral persistence in survivors, risk of Ebola disease relapse in survivors, and the potential 
for survivors to transmit the virus to others.  
There are no licensed therapies for the treatment of Ebola virus disease nor for the clearance of 
persistent Ebola virus in survivors. A safe, effective therapy that can reduce and/or eliminate 
persistent Ebola virus from semen would reduce the risk of transmission and enable male 
survivors to resume normal sexual relations without fear of harming loved ones. The mechanism 
underlying Post-Ebola Syndrome is as yet unknown, but improvement in Post-Ebola signs and 
symptoms resulting from GS-5734 treatment would be an added benefit.  
 
This study is a double-blind, randomized, two-phase (treatment and longer-term follow-up), two-
arm trial of GS-5734 versus placebo among male Ebola survivors with persistent Ebola virus 
RNA in their semen. Participants are randomized 1:1 to receive either 100 mg of GS-5734 or 
placebo once daily by intravenous catheter for 5 days. Informed by transaminase elevations in 
prior Phase I studies in normal healthy subjects, a risk-mitigation strategy includes a built-in 
dose de-escalation. Participants will be stratified by country and on the basis of one versus two 
positive semen samples for Ebola virus RNA using the Cepheid GeneXpert platform assessed 
within 42 days prior to study enrollment. The early DSMB review in August 2016 concluded 
there was no need for a cohort dose reduction. The protocol expanded to Guinea in October 
2017, where the outbreak ended later. Currently there is an outbreak in the Democratic Republic 
of the Congo where the study team may evaluate conducting this study following the completion 
of the outbreak.  
 
Antiviral activity, as well as safety and tolerability, will be assessed during the treatment phase. 
Longer-term clearance of Ebola virus will be assessed during the week 8 to week 24 follow-up 
phase. Primary analyses for the assessment of antiviral activity in the treatment phase will focus 
on the assay negativity rate (ANR; percentage of genital samples that are negative for Ebola) 
over the first 28 days of the study, as well as clinical and laboratory adverse events. A sample is 
considered negative by PCR if the result of GeneXpert analysis reads undetectable. Primary 
analysis for the follow-up phase will focus on the ANR collected monthly from months 2 to 6. 
 
 
 


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1 
Background Information and Scientific Rationale 
1.1 
Background Information 
The Zaire Ebola virus disease (EVD) outbreak in West Africa was first recognized in December 
2013 in Guinea. Over the course of the outbreak, EVD spread to Liberia, Sierra Leone, Nigeria, 
Mali, Spain, Senegal, and the US, with over 28,000 cases of EVD in West Africa.1 Although all 
three West African countries have been declared Ebola-free at times, regional flare-ups of EVD, 
twelve at the time of this writing, continue in West Africa. Molecular sequencing is able to link 
these Ebola flares or clusters to transmission from survivors.  
 
Ebola virus is a negative-strand RNA virus composed of 7 genes encoding viral proteins, 
including a single glycoprotein (GP). Five distinct species of Ebola virus have been identified: 
Bundibugyo (BDBV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV), and Zaire 
(ZEBOV). BDBV, ZEBOV, and SUDV have been associated with large outbreaks of EVD in 
Africa with reported case fatality rates of up to 90%. Transmission of Ebola virus to humans is 
not yet fully understood, but is likely due to incidental exposure to infected animals. EVD then 
spreads through human-to-human transmission, with infection resulting from direct contact with 
blood, secretions, organs or other bodily fluids of infected people, and indirect contact with 
environments contaminated by such fluids.  
 
EVD has an incubation period of 2 to 21 days (mean 4 to 10 days). The clinical manifestations of 
acute EVD are generally heralded by an abrupt onset of non-specific symptoms such as fever, 
chills, malaise, and myalgia.2,3 The subsequent signs and symptoms indicate multisystem 
involvement and include systemic (prostration), musculoskeletal (arthralgia, myalgia) and 
gastrointestinal (anorexia, nausea, vomiting, abdominal pain, diarrhea). This may be followed by 
respiratory failure (chest pain, shortness of breath, cough), vascular complications (conjunctival 
injection, postural hypotension, edema), acute renal failure and neurological disease suggestive 
of a meningoencephalitis. Hemorrhagic manifestations consistent with disseminated 
intravascular coagulation may arise during the peak of the illness. In later stages of EVD, shock, 
convulsions, encephalopathy, severe metabolic disturbances, and diffuse coagulopathy may 
develop. Laboratory findings include leukopenia, thrombocytopenia, increased blood urea 
nitrogen, creatinine, creatinine kinase, and elevated liver enzymes. In general, symptoms last for 
about 7 to 14 days, after which either death or recovery occurs. The clinical manifestations of 
patients with EVD in the current epidemic in West Africa include hemorrhage in < 5% of 
patients.4,5 
 
Due to the sporadic nature of prior outbreaks, the remote locations in which they occurred, high 
case-fatality rates, and few survivors, little was known about any long-term sequelae of EVD in 
survivors. In a study of 29 EVD survivors from the 1995 outbreak in Kikwit, DRC, the most 
common symptoms persisting during convalescence were arthralgia, myalgia, abdominal pain, 
and fatigue.6 In a separate report from the Kikwit outbreak, 4 EVD survivors developed uveitis 
during convalescence.7 Through PREVAIL III and other studies of survivors from the 2013-16 
West African Ebola outbreak, the “Post Ebola Syndrome” is being further characterized.  
 
 
 


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Prior to the 2014-2016 Ebola outbreak, data on Ebola virus isolation and/or Ebola viral RNA 
detection from seminal fluid were limited. Ebola virus was isolated from semen at 61 days after 
onset of Ebola illness in a laboratory-acquired infection,8 and Ebola RNA persistence was 
documented out to 91 days in one of five convalescent survivors in the Kikwit DRC outbreak.6 
Recent data from prospective studies from the West African 2014-2016 outbreak demonstrate 
that persistence of seminal Ebola viral RNA is common and can occur at high viral loads.9,10 For 
example, a study in Sierra Leone recruited 93 male survivors between two and nine months after 
initial Ebola symptoms; Ebola virus RNA was detected in 65% of specimens 4 to 6 months after 
onset and in 26% 7 to 9 months after onset11 but longer term longitudinal studies were needed to  
characterize the length and patterns of persistence. 
 
The Partnership for Research on Ebola Virus in Liberia (PREVAIL) is undertaking a 5-year 
study entitled Ebola Natural History Study in Liberia (also known as PREVAIL III), which 
began in Liberia in June 2015. As of  September 2017, there were a total of 4036 subjects 
enrolled in PREVAIL III: 1145 survivors, 2785 close contacts, and 106 birth cohort participants. 
PREVAIL III plans and has enrolled  of their household and sexual contacts; subjects are seen at 
baseline and every six months thereafter. The overall PREVAIL III goals are to characterize in 
Ebola survivors of all ages: the clinical sequelae, the immunology, viral persistence, and the 
potential for transmission of Ebola virus from EVD survivors to close contacts. Within 
PREVAIL III, sub-studies focus on the assessment of persistent virus in body fluids such as 
semen, vaginal fluid, and breast milk, a birth cohort (which involves testing of placenta, cord 
blood, maternal blood at birth, vaginal swabs, and breast milk), and sub-specialty areas such as 
neurology and ophthalmology.  
 
To assess and characterize the persistence of Ebola viral RNA in semen, men enrolled in 
PREVAIL III are invited at their 6-month visit and beyond to undergo counseling and donate 
semen for testing of the presence of Ebola virus RNA. Semen testing is conducted at the Liberian 
Institute of Biomedical Research (LIBR) and the Guinean Viral Hemorrhagic Fever labs using 
the glycoprotein (GP) and nucleoprotein (NP) target genes on the Cepheid GeneXpert platform. 
The GeneXpert platform has shown superior sensitivity for detection of Ebola viral RNA, 
particularly in semen, at low levels of Ebola viral RNA, over other Taqman based assays in both 
published and unpublished studies.12-14   
 
In the Liberian PREVAIL III study men with initial Ebola virus RNA detection are asked to 
continue to donate every two weeks, whereas men with an initial lack of detection, or a negative 
test, return to donate semen for testing every 4-6 weeks. Each time a subject donates semen, he 
returns for the test result and counseling on condom use within two weeks. At that time, he is 
advised on the frequency of follow-up. Upon repeat testing, approximately 30.7% of the 279 
seropositive  male survivors in PREVAIL III have had at least one semen sample with detection 
of Ebola virus RNA. Figure 1 summarizes the CT values for the 83 men who have had at least 
one semen sample with detection of Ebola virus, sorted by date of most recent specimen 
collection. Each individual participant is a row, with samples from the same individual 
connected by horizontal lines. White dots indicate samples where Ebola virus RNA was not 
detected; black dots indicate samples for which Ebola virus RNA was detected with a CT 
between 40 and 45; red dots indicate an observed CT value below 40. The displayed CT value is 
based on the minimum CT of NP or GP from the same sample. Of note, once a male has enrolled 


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in PREVAIL IV his data is no longer included in this plot, so it is slightly biased towards less 
positivity. As of August 2017, 257 male survivors enrolled in PREVAIL III had donated at least 
two semen samples for Ebola virus RNA testing. Seminal Ebola virus RNA shedding appears to 
be intermittent over time and quantitatively variable. The longest reported duration for detection 
of Ebola viral RNA in semen was 40 months following EVD.15 
 
 


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Figure 1. Minimum CT(NP,GP) results among those with at least 1 detectable sample from 
PREVAIL III (n=83) 
 


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The possibility of sexual transmission of filoviruses was first suggested by two cases in Marburg, 
Germany in 1968.16 Based on data from the 2014-2016 outbreak,17 it is now known that Ebola 
virus RNA in seminal fluid can be infectious, supporting previous molecular epidemiologic 
analysis of Ebola flares and cluster outbreaks suggesting that sexual transmission is taking place, 
though the frequency appears low. The first documented case of probable sexual transmission 
occurred in March 2015 in Liberia, from a male survivor to a female via unprotected vaginal 
intercourse, as evidenced by molecular sequencing.18,19 Though the World Health Organization 
(WHO) declared Guinea Ebola-free on December 29th, 2015, after almost two challenging years 
of attempting to control the outbreak, a new EVD cluster began on March 17th, 2016, in 
NZerekore Prefectures, the Koropara sub-prefecture. This outbreak originated from a male 
survivor with low CT value in his semen who sexually transmitted the Ebola virus 470 days after 
his illness.17 The outbreak included 10 persons in Guinea with a 90% mortality in confirmed 
cases and one death and two cases in Liberia.  
 
Safely eradicating Ebola virus RNA from the semen of male survivors might reduce the risk of 
Ebola flares and another large-scale Ebola outbreak in Central and West Africa. The mechanism 
of persistence and location of ongoing replication of Ebola virus leading to detection of Ebola 
virus RNA in seminal fluid is not known. Finally, in addition to Ebola virus persistence, Ebola 
relapse causing meningoencephalitic clinical disease has been well documented.20  
 
Since initiating the study on July 4, 2016, there have been 34 subjects enrolled in Liberia and no 
serious adverse events nor recommended changes to the protocol by the DSMB. The expansion 
to Guinea, where the outbreak ended later, is intended to complete study enrollment. 
 
A focal group discussion about the PREVAIL IV study was held with male survivors from 
PREVAIL III with Ebola virus RNA detected in their semen. These men shared fears and 
concerns around transmitting Ebola virus to their sexual partners, frustrations of needing to use 
condoms when wanting to conceive children, and a desire to “be free” from Ebola virus.  
1.2 
Description of the Study Agent (GS-5734) 
1.2.1 
General Information 
GS-5734 is a diastereomerically pure monophosphoroamidate prodrug of a modified adenine 
nucleoside analog GS-441524. GS-5734 exhibits a potential for in vivo clinical efficacy against 
Ebola virus infection in the treatment of EVD based on the following available data: 
1) Potent in vitro activity against Ebola virus in multiple relevant cell types, including the Ebola 
virus variants isolated during the ongoing outbreak in West Africa. 
2) Preclinical in vitro and in vivo pharmacokinetic profile in non-human primates and other 
relevant animal species indicating high and persistent levels of pharmacologically active 
nucleoside triphosphate metabolite in peripheral blood mononuclear cells (PBMCs); this 
measurement is used as a surrogate for levels in cells relevant for Ebola virus infection, 
supporting once daily IV or intramuscular (IM) administration. 
3) Preliminary safety profile supporting safe administration to non-human primates at doses 
potentially active against Ebola virus infection. 


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4) Potent therapeutic efficacy in Ebola virus-infected rhesus monkeys, the most relevant in vivo 
preclinical model of EVD, at doses well tolerated in non-human primates. The in vivo 
therapeutic efficacy in non-human primates has been demonstrated against multiple Ebola 
virus variants including Makona/2014 isolated during the West African outbreak. 
5) Tissue distribution studies in non-human primates indicate effective penetration and 
distribution of GS-5734 into immune privileged sites (genital tract, eye, and to some extent 
brain) that may represent a persistent reservoir of Ebola virus. 
 
For further information on GS-5734 and the vehicle, refer to the current Investigator’s Brochure 
(IB) for GS-5734. 
1.2.2 
Summary of Previous Preclinical Studies 
1.2.2.1 Preclinical Pharmacology 
GS-5734 has been designed as a prodrug for intracellular delivery of the monophosphate of GS-
441524. Inside cells, the GS-441524 monophosphate undergoes fast conversion to the 
pharmacologically active triphosphate (GS-443902) see Figure 2.  
 
 
Figure 2. GS-5734 Metabolism. 
 
 
 
Figure1.GS 5734Metabolism
Intracellular
Plasma
X


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Efficient metabolism of GS-5734 to triphosphate GS-443902 has been demonstrated in multiple 
cell types relevant for Ebola virus replication. GS-443902 shows good intracellular persistence in 
primary human macrophage (T1/2 = 11 hours) supporting once daily administration. While there 
is no direct biochemical evidence for the inhibition of Ebola virus RNA-dependent RNA 
polymerase (RNA pol) by GS-443902, the compound selectively inhibits respiratory syncytial 
virus (RSV) RNA pol. The primary mechanism of inhibition is the incorporation of GS-443902 
into nascent RNA chain by RSV RNA-dependent RNA-polymerases, causing premature viral 
RNA chain termination. RSV and Ebola virus share a similar mechanism of RNA 
transcription/replication and exhibit a relatively high degree of homology within the active sites 
of their respective RNA pols, suggesting that GS-443902 is likely to directly inhibit Ebola virus 
RNA pol. In contrast, GS-443902 inhibits neither human mitochondrial RNA polymerase nor 
human DNA polymerases α and γ. 
 
GS-5734 is not suitable for oral delivery due to an extensive first-pass hepatic clearance. As a 
result, GS-5734 is being developed for parenteral administration. Multiple studies have been 
conducted to thoroughly characterize the pharmacokinetic profile of GS-5734 itself or the 
diastereomeric mixture GS-466547 administered IM or IV to rats, hamsters, and non-human 
primates. Across all species, the administered prodrug is rapidly cleared from plasma followed 
by the appearance of parent nucleoside GS-441524, which represents the major circulating 
metabolite (Figure 2). Dose-dependent rates of intact prodrug released into plasma were 
observed after IM administration in multiple species. In rhesus macaques, the preferred in vivo 
model for Ebola virus infection, once-daily IM administration of 5 mg/kg GS-466457 for 7 days 
resulted in trough levels of the nucleoside triphosphate GS-443902 in PBMCs above those 
required for 90% inhibition of Ebola virus replication in vitro. Relevant inhibitory concentrations 
of GS-443902 in PBMCs have also been observed in other species used as alternative models of 
Ebola virus infection (cynomolgus macaques and hamsters) following IM or IV administration of 
GS-466547. 
 
Results obtained from a balanced excretion and tissue distribution studies in cynomolgus 
monkeys administered a single 10 mg/kg dose of [14C] GS-5734 via the IV route indicate that 
GS-5734 penetrates into sanctuary sites including the brain, eye, and testes. Levels of radioactive 
material recovered from the testes, epididymis, and seminal vesicles were higher than plasma at 
4 hours post administration, suggesting effective uptake of the drug. In addition, both GS-441524 
and its phosphorylated metabolites were detected in semen of male monkeys following the single 
IV dose administration of 10 mg/kg GS-5734. Levels in the eye were approximately half of those 
in plasma at 4 hours. The amount of radioactive material recovered from plasma was 
approximately ten-fold higher than that in the brain at 4 hours. Levels in the brain were persistent 
with similar concentrations maintained through 168 hours, suggesting the potential for marked 
accumulation. Together, these results indicate effective penetration and distribution of GS-5734 
into immune privileged sites that may represent a persistent reservoir of Ebola virus. 
 
While GS-5734 is a substrate for CYP2C8, 2D6, 3A4 in vitro, co-administration with inhibitors 
of these CYP isoforms is unlikely to markedly increase GS-5734 levels as its metabolism is 
likely to be predominantly mediated by hydrolase activity. GS-5734 is a substrate for the organic 
anion transporter 1B1 (OATP1B1) and P-gp. However, the impact of these transporters on 
GS-5734 disposition is likely minimized by the parenteral route of administration. GS-5734 is an 


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inhibitor of CYP3A4, OATP1B1, and OATP1B3 in vitro but its potential to be the perpetrator of 
clinically significant drug-drug interactions is limited by its rapid clearance. No potential for 
induction of enzymes or transporters via the PXR or AhR was detected in reporter cell lines. If 
unanticipated drug-drug interactions are observed, the short-term course of GS-5734 treatment 
may allow for temporary discontinuation of other interacting drugs. 
 
In summary, GS-5734 administered IV exhibits a favorable and consistent pharmacokinetic 
profile as well as efficient delivery of high levels of the pharmacologically active nucleoside 
triphosphate metabolite into cells relevant for Ebola virus replication, supporting clinical 
exploration of the drug as a novel agent for the treatment of EVD. 
 
Further information is available in the IB. 
1.2.2.2 Preclinical Efficacy 
The in vivo efficacy of GS-5734 has been assessed in 2 studies in rhesus monkeys acutely 
infected with Ebola virus (variant Kikwit/1995), a high-mortality model that mimics the course 
of EVD in humans. GS-5734 administered IM at 3 to 10 mg/kg once daily following the onset of 
plasma viremia resulted in significantly prolonged survival time of Ebola virus-infected animals 
without a major impact on the symptoms of EVD. In an independent study, infected animals 
treated with 3 to 10 mg/kg of GS-5734 administered once daily by IV injection for 12 days 
beginning 3 days post Ebola virus inoculation exhibited 100% survival combined with a 
significant suppression of EVD clinical symptoms including amelioration of behavioral 
depression, changes in hematology and coagulation parameters, as well as serum chemistry 
markers of kidney and liver toxicity, and no signs of drug-related toxicities. In comparison, all 
vehicle-treated animals succumbed to Ebola virus infection by day 9. 
 
The profound impact on survival and clinical symptoms of EVD in Ebola virus-infected drug-
treated animals was due to the potent suppression of virus replication. Animals treated with 10 
mg/kg GS-5734 beginning Day 3 post infection exhibited pronounced suppression of plasma 
viral RNA levels with a mean reduction of −3.9 log10 on Day 5 compared to the vehicle-treated 
control group. Plasma viral RNA in three out of six animals treated with 10 mg/kg GS-5734 
decreased below the lower limit of quantification (LLOQ; 8 × 104 RNA copies/mL) by Day 5. 
By Day 12, all animals treated with 10 mg/kg GS-5734 had plasma viral RNA below the lower 
limit of detection for the RT-qPCR assay. Consistent with the profound suppression of Ebola 
virus replication, animals treated with 10 mg/kg GS-5734 showed minimal to no physical signs 
of EVD as indicated by low cumulative physical symptom scores. Fifty percent of animals 
treated with 10 mg/kg GS-5734 remained free of any physical signs of EVD throughout the 
28-day duration of the study. No genotypic resistance was detected in vivo using deep 
sequencing of the entire EBOV RNA pol (L) gene from infected rhesus monkeys treated with 
various GS-5734 dosing regimens. 
 
Similar efficacy of GS-5734 at 10 mg/kg associated with 100% survival rate and profound 
antiviral effect was also demonstrated in rhesus monkeys infected with Ebola virus isolated from 
the most recent West African outbreak (variant Makona/2014). 
 
Further information is available in the IB. 


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1.2.2.3 Preclinical Toxicology 
Parent nucleoside GS-441524 exhibits low in vitro cytotoxicity in several types of human cell 
lines and primary cells (CC50 = 74 to > 200 μM) and low potential for mitochondrial toxicity 
based on results from biochemical and cell-based assays. 
 
The diastereomeric prodrug mixture, GS-466547 (GS-5734 and its diastereomer at phosphorous 
in a ~1:1 ratio), and the parent nucleoside, GS-441524, have a low potential for off-target 
activity. Neither compound significantly inhibited binding to a panel of 87 biological targets 
(receptors, ion channels, enzymes) at a concentration of 10 μM. No notable inhibition of the 
potassium (hERG) channel was observed with GS-466547 or GS-441524 at 30 μM. In Ames 
screening tests, GS-466547 and GS-441524 were not mutagenic. Based on these results, GS-
5734 is considered non genotoxic. 
 
Safety pharmacology studies were conducted to examine the potential effects of GS-5734 on the 
respiratory system, central nervous system, and cardiovascular system after IV administration. In 
a respiratory safety study in rats, GS-5734 had no effect on tidal volume or minute volume; 
however, respiration rates were increased from 0.75 to 6 hours post-dose in animals administered 
≥ 20-mg/kg GS-5734. Respiration rates returned to control levels by 24 hours post-dose, 
resulting in a no observed effect level (NOEL) for respiratory function in male rats of 5 mg/kg, at 
exposures ~2.1-fold above the estimated GS-441524 Cmax at the 150-mg clinical dose. Single IV 
(slow bolus) injection administration of GS-5734 had no effect on the central nervous system of 
rats at dose levels up to 50 mg/kg, the highest dose tested, and was estimated to be at least 17-
fold above the GS-441524 Cmax at the proposed 150-mg dose. In a cardiovascular safety study in 
monkeys, single IV (slow bolus) injection of GS-5734 had no effect on cardiovascular 
parameters up to 10 mg/kg, the highest dose tested. At the 10-mg/kg NOEL, GS-441524 
exposures were ~2.5-fold above the Cmax at the proposed 150-mg dose. The lack of in vivo 
cardiovascular effect is consistent with the weak in vitro inhibition of hERG channel by GS-
5734. The concentration that results in 20% inhibition (IC20; 7.5 μM) and IC50 (28.9 μM) are at 
least 16-fold and 63-fold, respectively, above the estimated free drug concentration (0.458 μM) 
at Cmax of the dose of 150 mg. 
 
The nonclinical toxicology profile of GS-5734 has been characterized through the conduct of 
repeat-dose studies in rats and cynomolgus monkeys with once-daily dosing up to 2 weeks in 
duration, studies to evaluate the genotoxic potential of the compound, and a hemolysis/blood 
compatibility study. Following repeated dosing in rats and monkeys, the kidney was identified as 
the target organ. In both species, clinical chemistry, urinalysis, and/or urinary biomarkers were 
early predictors of the observed kidney changes.  
 
In rats administered GS-5734 via daily IV (slow bolus) injection for 2 weeks, decreases in body 
weight gain and food consumption as well as clinical pathology and microscopic findings 
indicative of kidney injury and/or dysfunction in males administered ≥ 5 mg/kg/day and in 
females administered ≥ 20 mg/kg/day were observed. Effects on body weight gain and food 
consumption as well as clinical pathology and microscopic findings were generally reversible 
after a 4-week recovery period. Female rats were generally less sensitive to GS-5734, and the 
low dose of 5 mg/kg/day was considered the NOAEL in females. 
 


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In monkeys administered GS-5734 via daily IV (slow bolus) injection for 2 weeks, there were no 
changes suggestive of an effect in the kidney and the NOAEL was the high dose of 
10 mg/kg/day. Cynomolgus monkeys administered the diastereomeric prodrug mixture 
GS-466547 once daily via IM injection for 7 days had clinical and anatomic pathology changes 
compatible with renal injury at 15 mg/kg/day; the NOAEL for systemic toxicity was 
7.5 mg/kg/day. 
 
The vehicle used in the IV repeat-dose toxicity studies and the hemolytic potential and plasma 
compatibility study contained 12% [w/v] betadex sulfobutyl ether sodium (SBECD) in water, pH 
3.5 ± 0.1, similar to the vehicle for the proposed Phase 1 clinical study. The toxicity of SBECD 
has been well characterized in the GS-5734 toxicology studies, and in several peer-reviewed 
publications.21-23 SBECD-related microscopic findings of diffuse tubule cell vacuolation and 
focal/multifocal tubule cell hypertrophy in the kidney of rats and monkeys were not considered 
adverse nor associated with any clinical pathology effects indicative of changes in kidney 
function, and have been previously described.22,23 There was no notable exacerbation of the 
SBECD-related effects when administered with GS-5734.  
 
Further information is available in the IB. 
1.2.3 
Summary of Relevant Clinical Studies 
1.2.3.1 
Phase 1 Single Ascending Dose Study GS-US-399-1812 
In the placebo-controlled, double-blind, first-in-human study GS-US-399-1812, healthy 
volunteers were administered single doses of GS-5734 or vehicle in the following dose cohorts: 
Table 1. Dosing cohorts in GS-US-399-1812. 
Cohort 
Subjects 
Dose Level 
1 (n = 10) 
8 Active 
2 PTM 
3 mg GS-5734 or PTM 
2 (n = 10) 
8 Active 
2 PTM 
10 mg GS-5734 or PTM 
3 (n = 10) 
8 Active 
2 PTM 
30 mg GS-5734 or PTM 
4 (n = 10) 
8 Active 
2 PTM 
75 mg GS-5734 or PTM 
5 (n = 10) 
8 Active 
2 PTM 
150 mg GS-5734 or PTM 
6 (n = 10) 
8 Active 
2 PTM 
225 mg GS-5734 or PTM 
 
Administration of single GS-5734 doses of 225 mg or less are complete. A summary of 
preliminary, unaudited, unblinded data from the study follows. 


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Sixty healthy adult males and nonpregnant, nonlactating females of nonchildbearing potential 
were enrolled in the study. Forty-eight subjects received GS-5734 and 12 subjects received 
placebo; all 60 subjects completed the study. Subjects had a median age of 49 years (range: 24-
55 years). Fifty-five percent of subjects were male. Most subjects (90.0%) were white, and most 
were Hispanic or Latino (95%). The median (first, third quartile [Q1, Q3]) body mass index 
(BMI) was 27.4 (25.3, 29.0) kg/m2, and the median (Q1, Q3) creatinine clearance was 112.98 
(103.92, 126.33) mL/min.  
A total of 13 of 48 subjects who received GS-5734 and 1 of 12 subjects who received placebo 
had ≥ 1 treatment-emergent adverse event (AE). No AE was reported for > 1 subject within an 
individual treatment group. Constipation was the only AE reported for > 1 subject in the study, 
occurring in 3 of 48 subjects who received GS-5734 (1 subject each in the 3, 30, and 225 mg 
groups) and 0 subjects who received placebo. Most AEs were Grade 1 (mild) in severity; 
2 subjects, 1 who received GS-5734 75 mg and 1 who received placebo, had AEs (pre-syncope 
and pain in extremity, respectively) that were Grade 2 (moderate) in severity. Two subjects had 
AEs that were considered related to study drug by the investigator: One subject who received 
GS-5734 10 mg had dizziness and 1 subject who received GS-5734 225 mg had ear discomfort 
and pruritus generalized. All of these AEs were Grade 1 in severity, began on Day 1, and 
resolved on the same day.  
No subject had a treatment-emergent laboratory abnormality of maximum severity Grade 4 
during the study. One subject, who received GS-5734 225 mg, had a treatment-emergent Grade 3 
laboratory abnormality, comprising a Grade 3 lipase value of 255 U/L on Day 2. This subject 
also had a Grade 1 amylase value (133 U/L) on Day 2. All other lipase and amylase values for 
this subject were within the normal range, and she had no other signs or symptoms of 
pancreatitis. The only treatment-emergent Grade 2 laboratory abnormalities observed in 
> 1 subject within a treatment group were elevated cholesterol (2 subjects GS-5734 3 mg, 
1 subject GS-5734 30 mg, 1 subject placebo) and elevated low-density lipoprotein (LDL) 
cholesterol (3 GS-5734 3 mg, 1 GS-5734 75 mg, 1 GS-5734 225 mg, 1 placebo). All subjects 
with treatment-emergent Grade 2 elevated total and LDL cholesterol had Grade 1 elevations at 
≥ 1 pre-dose time point. Elevated cholesterol also was the most common treatment-emergent 
Grade 1 laboratory abnormality observed during the study (2 subjects each in the GS-5734 3, 10, 
and 75 mg groups; 1 subject each in the GS-5734 30 and 150 mg groups; 0 subjects in the 
placebo group). 
Three subjects who received GS-5734 had ALT elevations above the upper limit of normal 
(ULN) on Day 5, including 1 subject who received GS-5734 3 mg, 1 subject who received 
GS-5734 150 mg, and 1 subject who received GS-5734 225 mg. The subject who received 
GS-5734 150 mg also had an AST elevation above the upper limit of normal on Day 5. The 
elevated transaminase values were < 1.25 x ULN and, therefore, did not meet Grade 1 toxicity 
grading criteria. These transaminase elevations resolved to within the normal range by the time 
repeat testing was performed 2 days later (on Day 7). Follow-up visit (Day 14) results were 
consistent with pretreatment results for all 3 subjects. No notable changes from baseline were 
observed in total bilirubin, albumin, alkaline phosphatase, or international normalized ratio 
(INR) values for these subjects. No AEs were reported for the 3 subjects.  


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Overall, no consistent patterns of laboratory abnormalities or changes from baseline in laboratory 
parameters were noted during the study. Also, no patterns of clinically relevant changes in vital 
signs or shifts in 12-lead ECGs were observed during the study. 
The plasma PK parameters of GS-5734 and the nucleoside analog metabolite GS-441524 after a 
single 3- to 225-mg dose of GS-5734 administered in the fasted state as a 2-hour IV infusion are 
presented in Table 2 and Table 3. These preliminary data indicate that GS-5734 and GS-441524 
exposures were approximately dose-proportional following single-dose administration of 
GS-5734 3 to 225 mg. The peak plasma concentrations of GS-5734 and GS-441524 were 
achieved approximately 2 and 3.5 to 5 hours, respectively, after the start of infusion. GS-5734 
had a short median t1/2 of ≤ 1 hour, and GS-441524 had a longer median t1/2 of approximately 13 
to 31 hours across the studied dose range.  
Table 2. GS-US-399-1812: GS-5734 Mean (%CV) Plasma PK Parameters Following a 
Single 2-hour IV Infusion of GS-5734 in Healthy Subjects (Cohorts 1-6; Analysis Set: PK) 
PK 
Parameter 
GS-5734  
3 mg  
(n = 8) 
GS-5734  
10 mg 
(n = 8) 
GS-5734  
30 mg 
(n = 8) 
GS-5734  
75 mg 
(n = 8) 
GS-5734  
150 mg 
(n = 8) 
GS-5734  
225 mg 
(n = 8) 
Cmax 
(ng/mL) 
57.5 (31.1) 
220.8 (31.2) 
693.9 (18.6) 
1626.0 (38.6) 
2280.0 (30.1) 
4421.3 (16.0) 
Tmax (hr)a 
2.03  
(2.01, 2.04) 
2.01  
(2.00, 2.03) 
2.02  
(2.00, 2.03) 
2.03  
(2.03, 2.05) 
2.00  
(1.98, 2.04) 
1.97  
(1.95, 1.98) 
t1/2 (hr)a 
- 
0.66  
(0.54, 0.79) 
0.81  
(0.61, 0.91) 
0.90  
(0.82, 1.07) 
0.99  
(0.92, 1.06) 
1.05  
(0.96, 1.21) 
AUCinf 
(hr•ng/mL) 
- 
230.0 (28.4) 
773.9 (22.9) 
1999.6 (27.1) 
2976.1 (19.0) 
5274.7 (11.6) 
a 
Median (Q1, Q3) 
 
Table 3. GS-US-399-1812: GS-441524 Mean (%CV) PK Plasma Parameters Following a 
Single 2-hour IV Infusion of GS-5734 in Healthy Subjects (Cohorts 1-6; Analysis Set: PK) 
PK 
Parameter 
GS-5734  
3 mg  
(n = 8) 
GS-5734  
10 mg 
(n = 8) 
GS-5734  
30 mg 
(n = 8) 
GS-5734  
75 mg 
(n = 8) 
GS-5734  
150 mg 
(n = 8) 
GS-5734  
225 mg 
(n = 8) 
Cmax 
(ng/mL) 
3.2 (10.9) 
9.4 (28.4) 
34.3 (30.9) 
85.8 (23.9) 
152.0 (23.6) 
256.6 (30.2) 
Tmax (hr)a 
5.00  
(4.00, 5.00) 
3.57  
(3.00, 5.00) 
4.00  
(3.25, 4.00) 
4.50 
(3.50, 5.00) 
4.00  
(3.50, 4.00) 
3.50  
(3.00, 4.00) 
t1/2 (hr)a 
12.89  
(7.35, 14.23)b 
22.01  
(19.83, 
25.30) 
27.33  
(22.68 29.62) 
26.88  
(25.44, 
28.90) 
27.38  
(25.94, 
28.77) 
30.59  
(29.46, 
31.13) 
AUCinf 
(hr•ng/mL) 
55.2 (27.6) b 
264.0 (26.7) 
1013.8 (31.1) 
2471.8 (22.7) 
4642.5 (16.2) 
7348.6 (20.7) 
a 
Median (Q1, Q3) 
b 
n = 5 


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The urinary excretion of GS-5734 as unchanged drug and as the GS-441524 metabolite was also 
evaluated in this study for 48 hours from the start of study drug infusion. Following 
administration of single 3- to 225-mg doses of GS-5734, < 10% of the dose was recovered in the 
urine as unchanged drug and approximately 34% to 38% of the dose was recovered as 
GS-441524 over 48 hours. 
Exposure in PBMCs following GS-5734 administration was assessed in this study by measuring 
total GS-441524 levels, which serve as a surrogate for total intracellular phosphorylated species 
of GS-441524. Detectable GS-441524 levels were present in PBMCs after GS-5734 
administration at all doses tested (3 to 225 mg). The median Tmax of GS-441524 in PBMCs was 
variable, ranging from 2 to 9 hours. GS-441524 exposure in PBMCs was persistent for 
≥ 144 hours (6 days; median t1/2 ~38 hours), and a concentration of ≥ 5 μM was maintained for 
the first 24 hours following a single 150-mg dose. 
 
1.2.3.2 
Phase 1 Multiple Dose Study GS-US-399-1954 
Study GS-US-339-1954 is an ongoing (study conduct complete, data analysis ongoing) 
randomized, blinded, placebo-controlled, Phase 1 study designed to evaluate the safety and 
tolerability of multiple IV doses of GS-5734 compared with placebo and to evaluate the PK of 
GS-5734 and its metabolites following multiple IV doses of GS-5734 in healthy adult subjects. 
The study was conducted at a single center in the United States. 
Two dose cohorts were evaluated in the study. Following screening and Day −1 procedures, 
eligible subjects were confined to the study center beginning on Day −1 and were randomized 
2:1 within each cohort to receive GS-5734 150 mg or matching placebo, respectively. Each 
cohort comprised 2 groups (Groups 1a and 1b and Groups 2a and 2b) of 6 subjects each (4 GS-
5734, 2 placebo). Each subject received GS-5734 or placebo administered IV over a 1-hour 
period once daily for 7 days in Cohort 1 and 14 days in Cohort 2. Subjects were discharged on 
Day 9 for Cohort 1 and Day 16 for Cohort 2 and then returned 7 days after discharge for an in-
clinic follow-up visit.   
Twenty-four healthy subjects were enrolled in the study. Sixteen subjects received > 1 dose of 
GS-5734 and 8 subjects received > 1 dose of placebo. Twenty-two subjects completed the study. 
One subject in Cohort 1 who received GS-5734 and 1 subject in Cohort 2 who received placebo 
discontinued study drug prematurely, both after the fourth dose. Subjects had a median age of 47 
years (range: 19-55 years). Fifty-eight percent of subjects were male. Most subjects (83.3%) 
were white, and all were Hispanic or Latino. The median (Q1, Q3) BMI was 27.5 (25.8, 28.2) 
kg/m2, and the median (Q1, Q3) estimated glomerular filtration rate (by the Cockcroft-Gault 
equation) was 107.96 (101.38, 129.75) mL/min. 
No Grade 3 or 4 AEs, SAEs, AEs leading to study drug or study discontinuation, or deaths were 
reported during the study. However, 1 subject who received GS-5734 discontinued study drug 
after having multiple AEs considered to be related to study drug by the investigator, as described 
further below. The only AEs reported for > 1 subject in a treatment group were constipation 
(3 subjects [37.5%] GS-5734 7-day, 0 subjects GS-5734 14-day, 0 subjects placebo) and 


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dyspepsia (1 [12.5%] GS-5734 7-day, 2 [25.0%] GS-5734 14-day, 0 placebo). All of the AEs 
reported during the study were Grade 1 in severity.  
Nine subjects, 6 of whom received GS-5734 and 3 of whom received placebo, had ≥ 1 AE that 
was considered related to study procedures by the investigator. The AEs included pain in 
extremity (1 subject GS-5734 7-day, 2 subjects GS-5734 14-day); contact dermatitis (1 GS-5734 
7-day, 1 placebo); pruritus (1 GS-5734 7-day, 1 placebo); infusion site extravasation, infusion 
site pain, and infusion site hemorrhage (each 1 GS-5734 7-day); ecchymosis (1 GS-5734 
14-day); and dermatitis and iron deficiency anemia (each 1 placebo). The start dates of the 
procedure-related AEs ranged from Day 1 to Day 15. Time to resolution ranged from < 1 day to 
52 days.  
Four subjects, 3 of whom received GS-5734 and 1 of whom received placebo, had AEs that were 
considered related to study drug by the investigator: One subject in the GS-5734 7-day group had 
constipation, nausea, vomiting, decreased appetite, headache, and tremor. This subject 
discontinued study drug after dosing on Day 4 (reported reason for discontinuation was subject 
decision). The nausea, vomiting, headache, and tremor (left leg and left hand) all started on 
Day 3; the tremor resolved on the same day, and the other 3 AEs resolved between Days 4 and 7. 
The decreased appetite started on Day 5 and resolved on Day 7, and the constipation started on 
Day 8 and resolved on Day 9. Two subjects in the GS-5734 14-day group had dyspepsia that was 
considered related to study drug by the investigator. The dyspepsia started on Day 4 for both 
subjects and resolved on Day 19 for 1 subject and Day 20 for the other subject. One subject in 
the placebo group had diarrhea that was considered related to study drug by the investigator. The 
diarrhea started on Day 7 and resolved on Day 12.  
No subjects had a Grade 3 or 4 treatment-emergent laboratory abnormality during the study. 
Four subjects had treatment-emergent ALT elevations of maximum toxicity Grade 2, and an 
additional 5 subjects had ALT elevations of maximum toxicity Grade 1. These 9 subjects with 
graded ALT elevations included 2 subjects in the GS-5734 7-day group, 6 subjects in the 
GS-5734 14-day group, and 1 subject in the placebo group. Overall, 14 of 16 subjects who 
received GS-5734 (6 in the 7-day group and all 8 in the 14-day group) and 2 of 8 subjects who 
received placebo had a post-dose increase in ALT to a value ≥ 1.5 × their pre-dose ALT value. 
The median time to onset for the ALT elevations for these 16 subjects was 6 days, with a range 
of 5 to 23 days. The median time to resolution of the ALT elevation to < 1.5 the pre-dose value 
was 14 days, with a range of 3 to 64 days. 
Among the 9 subjects with Grade 1 or 2 ALT elevations, 7 (1 in the GS-5734 7-day group and 
6 in the GS-5734 14-day group) also had concomitant elevated AST levels. The start dates of the 
graded ALT and AST elevations varied, ranging from 4 to 24 days after the day of first dose 
administration. The ALT and AST values returned to within the normal range during the study 
for all 9 subjects. None of the 9 subjects with graded ALT or AST elevations had abnormalities 
in total bilirubin, alkaline phosphatase, or albumin during the study. Seven of the subjects also 
had Grade 1 increased prothombin time (PT), which resolved to within the normal range during 
the study for all of the subjects. No clinically significant change in INR occurred in the 
7 subjects. None of the laboratory results for the 9 subjects with transaminase elevations 
indicated systemic sign of drug reaction.  


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Four of the subjects with treatment-emergent graded transaminase elevations had AEs on study: 
One subject was the subject in the GS-5734 7-day group described above with the treatment-
related AEs (constipation, nausea, vomiting, decreased appetite, headache, and tremor) who 
discontinued study drug after the fourth dose. She also had non-treatment-related AEs of pain in 
extremity (a study procedure-related AE), tremor (bilateral hand and left leg), back pain, 
hematochezia, hemorrhage, viral infection, and oral herpes. This subject had a peak ALT value 
of 123 U/L and a peak AST value of 112 U/L, both occurring on Day 7. Another subject, also in 
the GS-5734 7-day group, had a non-treatment-related AE of constipation. The third subject, 
who was in the GS-5734 14-day group, had non-treatment-related AEs of ECG T wave inversion 
(considered not clinically significant by the investigator) and pain in extremity (left and right 
antecubital pain). The fourth subject, also in the GS-5734 14-day group, had a treatment-related 
AE of dyspepsia and a non-treatment-related AE of dizziness. These latter 3 subjects did not 
discontinue study drug or the study.  
The only other treatment-emergent Grade 2 laboratory abnormality observed in > 1 subject in a 
treatment group was elevated total cholesterol, which was observed in 2 subjects in the placebo 
group, both of whom had pre-dose Grade 1 elevations. Of note, Grade 1 laboratory abnormalities 
of increased PT occurred in 10 of 16 subjects who received GS-5734 (4 in the 7-day group, 6 in 
the 14-day group); no subjects who received placebo had Grade 1 increased PT. For all but 2 of 
these subjects, the increases were observed at multiple post-dose time points. The increases 
resolved to within the normal range during the study for all of the subjects. As described above, 
7 of the 10 subjects also had graded transaminase elevations during the study. No clinically 
significant change from baseline in INR was observed in any of the treatment groups; the INR 
ranged from 0.9 to 1.3 across all subjects and time points.  
Overall, no other consistent patterns of laboratory abnormalities or changes from baseline in 
laboratory parameters were noted during the study. Also, no patterns of clinically relevant 
changes from baseline in vital signs or shifts in 12-lead ECGs were observed during the study. 
The plasma PK parameters of GS-5734, the nucleoside analog metabolite GS-441524, and the 
intermediate metabolite GS-704277 after multiple 150-mg doses of GS-5734 administered in the 
fasted state as 1-hour IV infusions over 7 (Cohort 1) or 14 days (Cohort 2) are presented in Table 
4, Table 5, and Table 6. Consistent with a short t1/2 (~1 hour) relative to the once-daily dosing, 
accumulation of GS-5734 did not occur. The nucleoside analog metabolite GS-441524 had a 
longer t1/2 of approximately 21 hours, with an accumulation ratio for AUC after multiple daily 
dosing of approximately 1.9, reaching steady-state by Day 7; no further accumulation was 
observed from Days 7 to 14. The intermediate metabolite GS-704277 had a t1/2 of approximately 
1.6 hours. Slight accumulation of this metabolite was observed after 7 days of dosing 
(accumulation ratio for AUC of 1.4), and further accumulation was observed after 14 days of 
dosing (accumulation ratio of ~1.9). The mechanism of this accumulation is currently unknown. 
Table 4. GS-US-399-1954: GS-5734 Mean (%CV) Plasma PK Parameters Following 
Multiple 1-hour IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; 
Analysis Set: PK) 


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PK Parameter 
GS-5734  
Day 1 
(n = 8) 
GS-5734  
Day 7 
(n = 8) 
GS-5734  
Day 14 
(n = 8) 
Cohort 1 (7 days dosing) 
 
 
 
Cmax (ng/mL) 
2922.5 (21.4) 
3048.6 (21.2) 
NA 
Tmax (hr)a 
1.01 (0.50, 1.05) 
1.05 (1.02, 1.05) 
NA 
t1/2 (hr)a 
0.93 (0.79, 0.99) 
0.97 (0.94, 1.10) 
NA 
AUCtau (hr•ng/mL) 
2347.1 (19.2) 
2476.0 (19.9) 
NA 
Cohort 2 (14 days dosing) 
 
 
 
Cmax (ng/mL) 
3413.8 (26.2) 
3197.5 (15.5) 
3380.0 (23.5) 
Tmax (hr)a 
1.04 (1.03, 1.05) 
1.05 (1.03, 1.07) 
1.05 (1.05, 1.05) 
t1/2 (hr)a 
0.83 (0.78, 0.91) 
1.00 (0.91, 1.03) 
1.03 (0.93, 1.14) 
AUCtau (hr•ng/mL) 
2803.6 (17.8) 
2731.3 (16.9) 
2858.7 (20.2) 
NA = not applicable 
a 
Median (Q1, Q3) 
 
Table 5. GS-US-399-1954: GS-441524 Mean (%CV) PK Parameters Following Multiple 1-
hour IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; Analysis 
Set: PK) 
PK Parameter 
GS-441524 
Day 1  
(n = 8) 
GS-441524 
Day 7 
(n = 8) 
GS-441524 
Day 14 
(n = 8) 
Cohort 1 (7 days dosing) 
 
 
 
Cmax (ng/mL) 
146.4 (25.9) 
231.7 (17.7)  
NA 
Tmax (hr)a 
3.00 (2.00, 4.00)  
3.00 (2.00, 4.00)  
NA 
t1/2 (hr)a 
23.10 (17.14, 24.40)  
23.69 (21.56, 23.84)  
NA 
AUCtau (hr•ng/mL) 
1996.3 (15.4) 
3642.1 (16.3)  
NA 
Cohort 2 (14 days dosing) 
 
 
 
Cmax (ng/mL) 
130.8 (22.0) 
230.0 (19.4) 
230.5 (21.1) 
Tmax (hr)a 
4.00 (2.50, 4.00) 
3.50 (2.50, 4.00) 
2.75 (2.00, 3.50) 
t1/2 (hr)a 
16.39 (15.08, 21.80) 
24.06 (20.25, 33.32) 
21.44 (20.23, 22.00) 
AUCtau (hr•ng/mL) 
1903.1 (16.7) 
3599.5 (15.4) 
3620.7 (15.9) 
NA = not applicable 
a 
Median (Q1, Q3) 
 


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Table 6. GS-US-399-1954: GS-704277 Mean (%CV) PK Parameters Following Multiple 1-
hour IV Infusions of GS-5734 150 mg in Healthy Subjects (Cohorts 1 and 2; Analysis 
Set: PK) 
PK Parameter 
GS-441524 
Day 1  
(n = 8) 
GS-441524 
Day 7 
(n = 8) 
GS-441524 
Day 14 
(n = 8) 
Cohort 1 (7 days dosing) 
 
 
 
Cmax (ng/mL) 
290.6 (21.7)  
386.1 (31.6)  
NA 
Tmax (hr)a 
1.05 (1.03, 1.06)  
1.07 (1.02, 1.17)  
NA 
t1/2 (hr)a 
1.41 (1.19, 1.73)  
1.75 (1.37, 1.97)  
NA 
AUCtau (hr•ng/mL) 
563.1 (21.1)  
753.7 (29.0)  
NA 
Cohort 2 (14 days dosing) 
 
 
 
Cmax (ng/mL) 
274.3 (20.7) 
387.6 (26.3) 
525.4 (23.0) 
Tmax (hr)a 
1.05 (1.03, 1.11) 
1.17 (1.07, 1.17) 
1.17 (1.05, 1.17) 
t1/2 (hr)a 
1.68 (1.53, 1.75) 
1.68 (1.43, 1.83) 
1.61 (1.47, 1.80) 
AUCtau (hr•ng/mL) 
549.9 (22.8) 
789.3 (29.4) 
1077.8 (26.4) 
NA = not applicable 
a 
Median (Q1, Q3) 
 
Following administration of multiple 150-mg doses of GS-5734 for 7 or 14 days, approximately 
8% and 12%, respectively, of the dose was recovered in the urine as unchanged drug and 
approximately 41% and 48%, respectively, of the dose was recovered as GS-441524 over the 
24-hour collection period. The levels of GS-441524 persisted throughout the 24 hour-collection 
period in all subjects after 7 and 14 days of dosing. 
The t1/2 of total GS-441524 phosphorylated metabolites in PBMCs was approximately 40 hours 
(Table 7). Upon multiple once-daily dosing, the accumulation of GS-441524 in PBMCs was 
approximately 2.6- to 3.5-fold. 
 
Table 7. GS-US-399-1954: Intracellular GS-441524 Phosphorylated Metabolites Mean 
(%CV) PK Parameters in PBMC Following Multiple 1-hour IV Infusions of GS-5734 150 
mg in Healthy Subjects (Cohorts 1 and 2; Analysis Set: PK) 
PK Parameter 
GS-5734  
Day 1  
(n = 8) 
GS-5734  
Day 7 
(n = 8) 
GS-5734  
Day 14 
(n = 8) 
Cohort 1 
 
 
 
Cmax (ng/mL) 
2.8 (20.5)  
12.5 (20.7)  
NA 
Tmax (hr)a 
6.00 (1.03, 9.00)  
6.00 (1.00, 12.00)  
NA 
t1/2 (hr)a 
35.26 (33.82, 36.70)  
37.11 (32.22, 40.07)  
NA 
AUCtau (hr•ng/mL) 
90.9 (20.1)  
245.1 (18.9)  
NA 


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Cohort 2 
 
 
 
Cmax (ng/mL) 
4.2 (31.4) 
22.7 (38.2) 
21.6 (32.4) 
Tmax (hr)a 
1.04 (1.03, 3.53) 
1.06 (1.05, 1.07) 
1.06 (1.05, 6.00) 
t1/2 (hr)a 
36.36 (35.97, 41.81) 
34.38 (34.38, 34.38) 
40.16 (28.35, 56.14) 
AUCtau (hr•ng/mL) 
107.4 (17.9) 
392.2 (20.5) 
388.0 (33.8) 
NA = not applicable 
a 
Median (Q1, Q3) 
 
1.2.3.3 
Pharmacokinetic Semen Data from the Phase 1 Multiple Dose Study GS-US-
399-1954 
Parent drug GS-5734 is undetectable in semen (BLQ) due to its short half-life. Levels of pre-
dose (trough) GS-441524 in semen reaches 200250 ng/mL after multiple dosing. The Ctrough of 
GS-441524 in plasma ~110 ng/mL at steady-state. The semen Ctrough levels were approximately 
200 ng/mL at steady state which is about 2x higher than plasma Ctrough (~111 ng/mL) at steady-
state. Both GS-441524 and GS-704277 metabolites were present in semen for up to 7 days after 
the last dose. 
 
Figure 3. Metabolites: Semen Predose (Ctrough) Concentrations. 
 
mean±SD
Cohort1(7-dayQDdosing)
Cohort2(14-dayQDdosing)
MetabolitesSemenPredoseConcentra ons


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1.2.4 
Additional GS-5734 Human Experience 
To date, GS-5734 has been administered on an expanded access basis to 2 patients with Ebola 
infection, a 39-year-old female diagnosed with recrudescent Ebola meningitis and a neonatal 
patient with acute Ebola infection. These patients are described below. 
Recrudescent Ebola Meningitis Case 
This 39-year-old female was originally treated in the United Kingdom for acute EVD in 
December 2014 and January 2015. She subsequently recovered from her acute EVD and 
remained in the United Kingdom. In early October 2015, the patient presented with malaise, 
headache, photophobia, nausea, and vomiting. She underwent lumbar puncture, which revealed a 
pleocytosis, and PCR tests were positive for EBOV in her blood and cerebrospinal fluid (CSF) 
(cycle thresholds [Cts] of 29 and 23.7, respectively). She was diagnosed with Ebola meningitis 
and admitted for inpatient treatment on 08 October 2015. GS-5734 was requested for treatment 
of her case on 09 October 2015. 
Therapy with GS-5734 150 mg IV once daily (every 24 hours) was initiated on 11 October 2015. 
EBOV PCR of blood became negative on 12 October 2015, after the first dose of GS-5734 
(Ct > 40). On the basis of a lack of evident toxicity, both in the patient’s case and in the ongoing 
single ascending dose Study GS-US-399-1812, the dose was increased to 225 mg IV once daily 
on 13 October 2015. The patient received daily GS-5734 225 mg IV for an additional 12 days 
without incident. She completed a 14-day course of GS-5734 on 24 October 2015 (2 days of 
150 mg IV once daily plus 12 days of 225 mg IV once daily). Examination of CSF on 
21 October 2015 (Day 11 of GS-5734 therapy) demonstrated a decrease in EBOV PCR from a 
pretreatment Ct of 23.7 to a Ct of 35.5 on 21 October 2015. EBOV PCR of CSF obtained on 
28 October 2015, 4 days after GS 5734 was discontinued, was negative (Ct > 40). Per 
communication with the treating physician, the patient tolerated GS-5734 well. She experienced 
no AEs related to GS-5734 during her treatment, and there were no apparent laboratory, vital 
sign, or ECG abnormalities attributed to GS-5734. Her neurologic status and the symptoms and 
signs of Ebola meningitis were resolving as of 20 November 2015. 
Acute Ebola Virus Case 
This neonatal black female was born on 27 October 2015 to an Ebola-infected mother. The 
patient’s mother arrived on 23 October 2015 for treatment at the Nongo Ebola treatment center 
(ETC) in Conakry Guinea, operated by Médecins Sans Frontières. The patient’s 2 siblings also 
presented with symptoms of acute EVD on the same day and were transferred to a different 
facility for treatment. The family had recently been exposed to Ebola as a result of contact with a 
family member who died due to EVD as part of the Forecariah branch of the Ratoma Ebola 
transmission chain in Guinea 24. The patient’s mother self-reported a pregnancy of 7 months 
gestation at the time of admission to the Nongo ETC. The mother was diagnosed with EVD on 
24 October 2015 and had a high viral load (Ct = 14.9, PCR method not specified). She delivered 
an infant girl in the ETC on 27 October 2015 and subsequently died a few hours later. 
Upon delivery on 27 October 2015, the neonate appeared mature and weighed 3 kg, consistent 
with a term or near-term gestation. The patient’s capillary and umbilical blood was positive for 
EBOV by PCR on her first day of life, and she was diagnosed with EBOV infection as a result of 


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transplacental transmission. Because of the poor prognosis for neonatal EVD (100% case fatality 
reported), ZMapp™ was obtained for the patient on a compassionate use basis. The patient was 
treated with 4 ZMapp 150-mg infusions between 28 October and 06 November 2015 (Day 2 to 
Day 11 of life). Because she had persistent Ebola viremia and there was a concern for Ebola 
CNS disease, the patient was then treated with a 12-day course of GS 5734 10 mg IV daily from 
14 to 25 November 2015 (Day 19 to 30 of life). Blood, skin, urine, and salivary specimens were 
consistently negative by PCR for EBOV following initiation of GS-5734 therapy. She had no 
apparent AEs or evidence of laboratory abnormalities related to GS-5734 administration. The 
patient recovered and was discharged from the treatment center on 28 November 2015. 
1.3 
Rationale for Study 
Ebola virus persists in at least a proportion of survivors of EVD. Viable Ebola virus has been 
recovered from the ocular fluid of an expatriate survivor months after clearance of viremia.25 
Moreover, Ebola virus was detected by PCR in the semen of a substantial proportion of 100 male 
survivors in Sierra Leone, 65% at 4-6 months and 26% at 7-9 months,11 and 33-38% of 
PREVAIL III male participants with repeat semen sample test results after 6 months of 
participation in PREVAIL III.26 In October 2015, a female expatriate survivor who had 
recovered from EVD over 9 months earlier with documented clearance of viremia, experienced 
an acute febrile illness and meningoencephalitis with high levels of Ebola virus detected in her 
cerebrospinal fluid and blood plasma. This case suggests that persistence of Ebola virus in non-
circulatory compartments may be driving lingering symptoms associated with EVD 
convalescence, be a source for secondary transmission, and serve as a reservoir which can lead to 
recrudescence of severe EVD. 
 
Ebola cluster outbreaks continue to occur in Liberia, Sierra Leone, and Guinea, with molecular 
evidence of transmission documented from the Ebola survivor community. Epidemiologic and 
molecular sequencing data support male survivor sexual transmission to susceptible females. 
Though sexual transmission appears to be uncommon, it has been responsible for at least a 
portion of the 12 Ebola cluster outbreaks that WHO attributes to transmission from survivors to 
others in their communities. During a period when few cases of primary EVD are being reported, 
a study of GS-5734 in EVD male survivors with evidence of Ebola virus persistence in semen 
provides an important opportunity to study the antiviral activity, longer-term genital clearance, 
safety, and pharmacokinetics of GS-5734. Furthermore, the effect of this agent on the dynamics 
of residual virus persisting in compartments such as the genital tract can be explored as can 
changes in clinical symptoms associated with the Post-Ebola Syndrome.  
 
Thorough characterization of the safety and tolerability of GS-5734 will also inform future study 
and use of this agent in Sub-Saharan Africa. Demonstration of an antiviral effect of GS-5734 in 
EVD survivors with detectable Ebola virus in genital fluids would support the use of this agent in 
this population and may be an important tool in preventing recurrent Ebola virus outbreaks in 
West Africa.   
 
While supportive care and treatment of specific symptoms improves survival, there currently are 
no effective therapeutic agents available for the treatment of EVD. The availability of an 
effective antiviral agent with a favorable risk/benefit profile would address a serious unmet 
medical need for the treatment of adults and children infected with Ebola virus. 


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1.4 
Rationale for Dose Selection 
The dose selection of 100 mg IV/day for 5 days, with a built-in dose de-escalation, is based upon 
safety data from the GS-5734 phase I studies in normal human subjects, the clinical 
pharmacokinetic profile in plasma and semen, and written guidance from the Unites States Food 
and Drug Administration (FDA). In late March 2016, the FDA reviewed the data from the Gilead 
Sciences Phase I studies GS-US-399-1812 and GS-US-399-1954. In GS-US-399-1812, the single 
ascending dosing study, three subjects (one subject each receiving 3 mg, 150 mg, and 225 mg 
doses) had transient elevations of transaminases not exceeding Grade 0. In GS-US-399-1954, 
Grade 1 and Grade 2 transaminase elevations occurred in 8 of 16 subjects receiving 7- or 14-day 
multi-dosing of GS-5734 150 mg/day (2 of 8 healthy subjects in the 7-day cohort and 6 of 8 
subjects in the 14-day cohort). The ALT elevations were up to 10 times the subjects’ baseline 
values and returned to normal limits. The onset of transaminase elevations out of the normal 
range occurred as early as day 5 and elevations in transaminases above baseline occurred 
between days 2 and 5. Based on the transaminase elevations, the FDA has placed a partial 
clinical hold on IND 125,566, prohibiting the performance of studies without a favorable 
risk-benefit ratio for subjects. The FDA specified that clinical studies may be performed in EVD 
survivors who have evidence of persistent viral shedding (e.g. documented in semen or vaginal 
fluid) and/or post-Ebola syndrome. The FDA specified that the duration of treatment with GS-
5734 may not exceed 5 days and the dose may not exceed 150 mg/day IV. Multiple-dose clinical 
studies may not be performed in survivors of EVD who have no evidence of persistent viral 
shedding or post-Ebola syndrome as the risk-benefit balance is not favorable for these subjects. 
Data from 2,723 Liberian PREVAIL III participants (including men, women and children) from 
1083 survivors and 1640 close household and sexual contacts, demonstrate no difference 
between the two groups with respect to serum transaminase levels (Table 8). Importantly, these 
levels of transaminases are within the same range as the baseline transaminase levels in healthy 
subjects enrolled in Phase 1 studies with GS-5734. 
 
Table 8. Median (with the first and third quartile in parentheses) ALT and AST levels in 
2,723 Liberian participants in PREVAIL III: 1083 Ebola survivors and 1640 household 
and sexual contacts. 
         
Contacts  
Survivors 
ALT (U/L) 
7 (4-11)  
7 (3-13) 
AST (U/L) 
13 (11-20) 
15 (10-19) 
Note: Self-identification was used to determine survivor status. 
 
Based on the FDA guidance in the partial clinical hold letter, and on the Liberian PREVAIL III 
Ebola survivor participant transaminase data, the dose selected for this study is 100 mg/day for 5 
days with dose de-escalation criteria for individuals and early planned DSMB assessment of 
data. The 100-mg dose represents a 33% reduction from the 150-mg dose allowed by FDA as the 
highest dose that can be investigated under the partial clinical hold. Based on the comparison of 
systemic drug exposure levels, a 100-mg human dose is estimated to be equivalent to 
approximately a 5-mg/kg dose in NHPs. While the efficacy of a 5-mg/kg dose of GS-5734 has 
not been tested in NHPs acutely infected with Ebola, this dose is expected to be efficacious given 
that 10 and 3 mg/kg are associated with 100% and 33%-67% survival rates, respectively, and 
both dose levels demonstrated statistically significant reduction in systemic Ebola viremia 


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compared to control placebo-treated animals. The pharmacokinetic data from the Phase I multi-
dose cohort study demonstrated that the concentration of GS-5734 metabolites in seminal fluid 
are approximately two times that of plasma levels (~220 ng/mL vs. ~100 ng/mL). Thus, a 
100-mg dose of GS-5734 for 5 days may have less hepatotoxicity than the 150-mg dose for 7 
days, while maintaining antiviral activity given the levels of the GS-5734 metabolites in seminal 
fluid are two times those of plasma.  
 
In summary, the dose of 100-mg GS-5734 to be administered by IV infusion for 5 days was 
selected due to the anticipated more favorable toxicity profile compared to the 150-mg dose of 
GS-5734 for 7 days combined with a good potential for antiviral activity manifested in genital 
tract. 
1.4.1 
Individual Dose De-escalation Plan 
In both Phase 1 clinical studies, when hepatic enzyme (ALT/AST) elevations occurred, levels 
generally began to rise by Day 4 or 5, (though transaminase data between day 2 and 5 are not 
available in the multi-dose cohort study) and generally did not exceed the normal range until day 
4 and beyond. A dose of GS-5734 of 100 mg daily over 5 days has been selected for this study, 
with an individual built-in dose de-escalation strategy for infusion days 2-5 for Grade 1 elevation 
in transaminases (1.25 to < 2.5 x ULN DAIDS Table for Grading the Severity of Adverse Events 
November 2014). If the criteria for dose reduction are triggered, the Safety Medical Officer will 
notify the unblinded pharmacist. For Grade 1 elevations in AST and ALT subjects on the GS-
5734 arm will have their dose of study medication reduced to 75 mg IV once daily for the 
remainder of infusion days and subjects on the placebo are will have the vehicle reduced by 25% 
(see Section 5.5.3 for details). If a participant experiences a Grade 2 increase in AST or ALT 
during the infusion days they will be switched to normal saline. 
1.4.2 
Cohort Dose De-escalation Evaluation 
The Data and Safety Monitoring Board (DSMB) will reviewed early safety data to make 
recommendations on the need to reduce the starting dose for all future subjects, and to reduce the 
current dose for those currently being infused (see Section 11.7). The early DSMB review in 
August 2016 concluded there was no need for a cohort dose reduction. There had been 34 
subjects enrolled at the time of DSMB review and no serious adverse events (SAEs) nor 
recommended changes to the protocol by the DSMB. 
1.5 
Rationale for Length of Treatment-Phase 
The objective of the treatment phase is to assess the anti-viral activity of a five day course of GS-
5734 assessed by comparing the ANR between the two study arms. The mechanism and source 
of seminal viral persistence is unknown.  If viral Ebola virus RNA is present it is assumed that 
viral replication is taking place  somewhere in the genital track, perhaps the prostate.  It is also 
not known if the viral replication  is continuous or intermittent, but the intermittent shedding 
suggests that replication may be intermittent as well.  If the mechanism of anti-viral activity is 
through polymerase inhibition and  GS-5734 is effective in stopping viral replication, clearance 
of seminal Ebola virus RNA may take time.  It is not known how long clearance of seminal 
Ebola RNA might take after viral replication ceases. The concentration of GS-5734 in seminal 
fluid metabolites and longer time to clearance from the seminal fluid suggest anti-viral activity 


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may occur over a longer period of time. The long intracellular half-life of active GS-5734 moiety 
of  approximately 42 hours suggest that anti-viral activity may be exerted up to 5 times this half-
life after the last dose and thus anti-viral activity may be present in the seminal compartment for 
up to 15 days. Measuring anti-viral activity out to 28 days will enable an additional 13 days for 
clearance of Ebola virus RNA. Considering all these issues assessing a 28 day period has been 
selected for assessing anti-viral activity. 
1.6 
Rationale for Use of the GeneXpert for Ebola Virus RNA Detection 
The GeneXpert was selected for detection of Ebola virus RNA following extensive testing using 
clinically contrived samples and live Ebola virus in combination with field testing using clinical 
samples. To test the assay performance, semen samples were spiked with live virus and assay 
procedures were optimized and a head to head comparison was performed using identical 
contrived semen samples on the GeneXpert as well as the EZ1 and MGB assays currently in use 
in Liberia. Subsequently, a limit of detection (LOD) was determined for the NP gene target (10 
copies/mL and GP gene target (100 copies/mL) in the GeneXpert assay. In addition, unspiked 
semen from over 30 unique healthy donors was tested to look for the potential of false positives 
(publication pending). Among all these control samples not a single false positive signal was 
detected for either NP or GP target specific PCR. GeneXpert devices were deployed to the 
Liberian Institute of Biomedical Research (LIBR). Incoming semen samples provided by male 
survivors were tested on both the GeneXpert as well as using the EZ-1 and MGB assays. A total 
of 417 samples were tested. Of these, 92 samples had bola virus RNA detected; 88 of the 92 had 
NP detected. Of the 92 samples positive 75 were detected on the GeneXpert only.  
 
The GeneXpert Ebola assay has also been extensively tested for cross reactivity with a number 
of viral, bacterial and parasitic agents. In addition, the sample buffer has been demonstrated to 
inactivate filoviruses by multiple laboratories, minimizing the amount of manipulations required 
with potentially infectious substances and the use of the closed cartridge system reduces the 
potential for cross-contamination. In summary the GeneXpert is being selected as the preferred 
assay platform because: (1) the increased sensitivity compared to existing assays in use, (2) 
reduction in risk to staff deployed in the field handling potentially infectious substances; (3) 
reduced potential for introduction of contaminants and (4) the ease of use. 
2 
Study Objectives 
2.1 
Primary Objectives 
2.1.1 Treatment Phase  
To compare the antiviral activity over 28 days following the administration of 5 days of IV 
GS-5734 versus placebo in male Ebola Virus Disease survivors with evidence of Ebola virus 
RNA in their semen. 
2.1.2 Follow-up Phase 
To compare the effect of 5 days of IV GS-5734 versus placebo on the detection of Ebola virus 
RNA in semen of male survivors of Ebola Virus Disease over 24 weeks.. 


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2.2 
Secondary Objectives 
2.2.1 Treatment Phase 
 To evaluate the safety and tolerability of GS-5734 in male Ebola survivors with evidence of 
Ebola virus RNA in their semen. 
 To evaluate the pharmacokinetics of GS-5734 (and metabolites as appropriate) in blood and 
PBMCs. 
 To compare the antiviral activity over 28 days following the administration of 5 days of IV 
GS-5734 versus placebo in subjects with: a CT value of either the GP or NP targets of ≤ 39 
in at least one semen sample during the 42-day pre-screening period and subjects with one 
versus two positive semen samples during the 42-day screening period. 
2.2.2 Follow-up Phase 
 To evaluate 24-week safety outcomes following IV GS-5734 treatment in male Ebola 
survivors.  
 To compare individual changes from baseline to 24-week signs and symptoms related to 
“Post Ebola Syndrome” as identified in PREVAIL III. 
 To compare the effect of GS-5734 and placebo on change from baseline to 24-week signs 
and symptoms related to “Post Ebola Syndrome” identified in PREVAIL III. 
 To compare the effect of IV GS-5734 versus placebo on the sustained negativity rate for the 
detection of Ebola virus RNA in semen of male survivors of Ebola Virus Disease over 24 
weeks. 
2.3 
Exploratory Objectives 
 To compare the sensitivity of Ebola virus RNA detection using the GeneXpert standard 
versus “pellet” semen preparation methods.  
 To compare changes from baseline in ophthalmologic findings between the GS-5734 arm and 
the placebo arm.  
 To compare Ebola virus RNA sequence changes from screening semen samples to Day  28 
and Week 24 semen samples when sample volume allows.  
 To assess participant satisfaction during screening and study participation. 
 To assess participant satisfaction during screening and study participation. 
3 
Study Design 
3.1 
Description of the Study Design 
This study will be a double-blind, randomized, two-phase (treatment and longer-term follow-up), 
two-arm trial of GS-5734 versus placebo among Ebola survivors in Liberia and Guinea who 
show persistent Ebola virus RNA in their semen at screening (see Figure 4). Participants will be 


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randomized 1:1 to receive either 100 mg GS-5734 or placebo once daily by IV for 5 days. 
Participants will be stratified by country and on the basis of one versus two positive genital 
samples for Ebola PCR taken within 42 days prior to study enrollment.  
 
Antiviral activity as well as safety and tolerability will be assessed during the treatment phase. 
Longer-term clearance of Ebola virus will be assessed during the follow-up phase. Primary 
analyses for the assessment of antiviral activity in the treatment phase will focus on the ANR 
over the first 28 days of the study, as well as clinical and laboratory AEs. ANR is defined to be 
the percentage of genital samples for which Ebola virus is not detected by PCR during a given 
interval (days 3-28 or weeks 8-24). A sample is considered negative by PCR if the NP and GP 
are undetectable by the GeneXpert assay. Primary analysis for the follow-up phase will focus on 
the ANR collected monthly from weeks 8 to 24.  
 
Because data from the Phase 1 studies demonstrate an elevation in liver transaminases, 
individual subject dose de-escalation criteria have been incorporated into the study (see Section 
5.5.3). For cohort dose reductions the DSMB will review early safety data and make a 
recommendation for dose adjustment as described below (see Section 11.7.1.1.1). If the dose-
reduction criteria, Grade 1 elevations during infusion days 1-5, are reached, the dose of GS-5734 
will be reduced to 75 mg/day IV for the rest of the infusion period. For Grade 2 elevations in 
AST or ALT during the study period, participants will be switched to normal saline. 
 
Figure 4. Study design. 
 
2 
PREVAIL IV Viral Persistence Study Design in 
Ebola survivors—proposed design 
Randomized, placebo-controlled, 2-part study: 
Week 24 
Day 1 
Placebo  IV 
QD 
Part 1--Treatment Phase:   
Assess antiviral effect 
GS-5734 
100mgb IV 
QD 
EVD survivors a 
• Males age >18 
• +EBOV PCR 
in genital 
secretions in 
one of two 
samples 42 
days prior to 
randomization 
1:1 
Day 28 
5 day RX 
Part 2--Follow-Up Phase: 
Assess viral eradication 
Frequent genital fluid 
collection 
Monthly genital fluid 
collection 
A Inclustion/Exclusion Criteria detailed in section 4.3 & 4.4 
B Dose reduction to 75mg if criterion are met 


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3.2 
Study Endpoints  
3.2.1 Primary Endpoints 
3.2.1.1 Treatment Phase 
ANR over days 4, 8, 11, 16, 24, and 28. 
3.2.1.2 Follow-up Phase 
ANR over weeks 8, 12, 16, 20, and 24. 
3.2.2 Secondary Endpoints 
3.2.2.1 Treatment Phase 
 Proportion of Grade 1, Grade 2, Grade 3, or Grade 4 aspartate transaminase (AST) and 
alanine transaminase (ALT) levels at days 1, 2, 3, 4, 5, 8, 11, 16, 24, and 28. 
 New Grade 3 and Grade 4 adverse events (AEs). 
 Change from baseline in safety labs, including N(%) with an increase in grade. 
 Change in targeted signs and symptoms (based on GS-5734 Phase I study AEs reported as 
associated with GS-5734 administration) including: constipation, nausea, vomiting, 
decreased appetite, headache, tremor, pain at infusion site, dyspepsia, and others new signs 
and symptoms reported by subjects. 
 Incidence of SAEs. 
 Frequency of discontinuation of study medication due to AEs or intolerance.  
 Post-dose concentrations of GS-5734 and metabolites in serum on days 1 and 5. 
 Pre-dose concentrations of GS-5734 and metabolites in serum on day 5. 
 Steady-state PBMC-associated concentrations of GS-441524 and its phosphorylated 
metabolites on Day 5. 
3.2.2.2 Follow-up Phase 
 Sustained Negativity Rate at weeks 8, 12, 16, 20, and 24. 
 Proportion of ALT or AST above baseline, at Grade 1 and above at weeks 8, 12, 16, 20, 
and 24. 
 Incidence of SAEs. 
 Change from baseline and 24-week signs and symptoms identified as part of the “Post Ebola 
Syndrome” from PREVAIL III data on completed review of symptoms and physical exam, 
depression, and post-traumatic stress disorder screening. 


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3.2.2.3   Exploratory Endpoints 
 
 Proportion of Ebola viral RNA detection, either the GP or the NP gene targets, at all CT 
levels in semen. 
 Average minimum CT levels at which Ebola virus RNA is detected for GP and NP gene 
targets in semen. 
 Proportion of Ebola viral RNA detection of either GP or NP gene targets of CT ≥ 40. 
 Proportion of abnormal ophthalmologic exams. 
 Proportion of abnormal ophthalmologic exams with an inflammatory etiology at baseline 
which have improved or become worse between baseline and week 24. 
 Proportion of Ebola virus RNA sequence changes in the polymerase and elsewhere between 
screening sample day 28  and 24 week semen samples. 
4 
Study Population 
4.1 
Rationale for Subject Selection 
Adult male EVD survivors aged ≥18 years with persistent Ebola virus RNA in their semen based 
on PCR detection of either the NP or the GP Ebola virus gene targets using GeneXpert assay are 
eligible for this study (N=60). Based on preclinical and Phase I safety data, subjects with 
abnormal eGFR, liver transaminases, or prothrombin time will not be eligible for enrollment in 
PREVAIL IV. Women are excluded as the primary endpoint in this study involved detection of 
Ebola virus RNA in semen.  
4.2 
Recruitment Plan & Retention Plans 
Participants may be recruited from the ongoing PREVAIL III (Ebola survivors) study using the 
PREVAIL Social Mobilization and Communications team and Participant Trackers who are 
assigned to follow individual subjects. Ebola survivors may also be recruited from other survivor 
care clinics, research programs, public health programs, or by self-referral. 
4.3 
Subject Inclusion Criteria 
Individuals must meet all of the following criteria to be eligible for study participation: 
 Men ≥18 years of age. 
 One of two semen samples with Ebola virus RNA detection (defined as a positive PCR for 
NP or GP using the GeneXpert assay within 42 days prior to randomization). 
 Willingness to be available for study evaluations for 6 months.  
 Willingness to allow storage of biological samples. 
 Willingness to be followed by a Participant Tracker. 
 Willingness to refrain from alcohol consumption for study days -7 to 14. 
 Willingness to comply with MOH & CDC guidance on using a condom for sexual activity 
and at least through week 24 of the study. 


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4.4 
Subject Exclusion Criteria 
Individuals meeting any of the following criteria will be excluded from study participation: 
 Estimated glomerular filtration rate (see Appendix C) less than 60 mL/min/1.73m2. 
 History of significant renal disease. 
 History of significant liver disease. 
 Evidence of liver disease on physical exam such as ascites. 
 
AST or ALT,  greater than the upper limit of normal, a prothrombin time 1.1 times greater 
than the upper limits of normal, or a total bilirubin > 1.5 times the upper limits of normal (per 
DAIDS toxicity tables version 2.0 Nov. 2014). 
 
Presence of Grade 2 or higher abnormalities for: low hemoglobin, low white blood count 
(WBC), low platelets, or low or high potassium (per DAIDS toxicity tables version 2.0 Nov. 
2014).   
 
Presence of greater than Grade 2 abnormalities for low or high sodium (per DAIDS toxicity 
tables version 2.0 Nov. 2014).   
 Any condition that, in the opinion of the investigator, would compromise the safety of the 
study subject or staff, or would prevent proper conduct of the study. 
4.5 
Justification for Exclusion of Special Populations 
Exclusion of children: Because there are insufficient data regarding GS-5734 dosing or AEs 
available in adults to judge the potential risk in children, children are excluded from this study.   
5 
Study Agent/Interventions 
5.1 
Disposition and Dispensation 
Sufficient quantities of GS-5734 and placebo to match vials will be shipped to the study site 
from Gilead Sciences Clinical Supplies Management (or its designee). Any remaining GS-5734 
injection concentrated solution and/or diluted GS-5734 solution for infusion will be disposed per 
local applicable hazardous waste disposal policies and procedures, or will be maintained in the 
PREVAIL pharmacy. 
5.1.1 Formulation, Packaging, and Labeling 
GS-5734 will be supplied as a sterile, single-use, preservative-free, clear, colorless to slightly 
yellow, aqueous-based concentrated solution containing 5 mg/mL GS-5734 that is to be diluted 
into IV infusion fluids (250-mL normal saline) prior to IV infusion. In addition to the active 
ingredient, GS-5734 injection, concentration 5 mg/mL, contains the following inactive 
ingredients: water for injection, betadex sulfobutyl ether sodium (SBECD), and hydrochloric 
acid and/or sodium hydroxide. Hydrochloric acid and/or sodium hydroxide are used to adjust the 
formulation to a pH of 3.0 to 4.0. 
 
The supplied matching placebo concentrate for solution for infusion is identical in physical 
appearance to the active formulation and contains the same inactive ingredients. 
 


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GS-5734 injection and placebo to match is filled in 50-mL glass vials enclosed with a rubber 
stopper and aluminum seal with a propylene flip-off cap. Each single-use vial contains sufficient 
volume to allow withdrawal of 30 mL of 5 mg/mL of GS-5734 concentrate or placebo. 
 
All labels for GS-5734 and placebo to match vials shall be labeled to meet all applicable 
requirements of the US Food and Drug Administration (FDA) and EU Guidelines to Good 
Manufacturing Practice, Annex 13 Investigational Medicinal Products and/or other local 
regulations as applicable. 
5.2 
Study Agent Storage and Stability 
GS-5734 and placebo to match vials should be stored frozen prior to use. Frozen storage is 
defined as storage in a place in which the temperature is maintained between −25 °C and −10 °C. 
Vials should be thawed prior to use at room temperature. See the pharmacy manual for thawing 
and storage information.  
5.3 
Randomization Procedures 
Once eligibility has been confirmed, subjects will be eligible for randomization. Prior to or 
during the day 1 visit, the investigator or designee will randomize the subject. Subjects will be 
assigned a unique subject number and randomized in a 1:1 ratio to GS-5734 or placebo. For 
subjects participating in PREVAIL III, the PREVAIL III subject number will also be used for 
this study.  
5.4 
Blinding and Unblinding Procedures 
Study team members and subjects will be blinded to treatment arm assignment throughout the 
duration of the study. The Sponsor Medical Monitor can request unblinding in the event of a 
suspected unexpected serious adverse reaction (SUSAR).   
5.5 
Preparation, Administration, and Dosage of Study Agents 
5.5.1 Preparation 
GS-5734 injection and corresponding placebo to match are hypertonic and must be diluted prior 
to administration. Refer to the study pharmacy manual for preparation instructions. 
5.5.2 Dosing and Administration 
The study treatment groups are as follows: 
 Treatment Arm A: GS-5734 100 mg IV over 1 hour, once daily for 5 days. 
 Treatment Arm B: Placebo IV over 1 hour, once daily for 5 days. 
5.5.3 Dose Adjustments 
Individual dose de-escalation will take place as follows with the volume of intravenous study 
agents remaining the same throughout the 5 day dosing period to retain the blind:  
 Due to the potential for hepatotoxicity related to GS-5734 treatment, if a subject experiences 
a Grade 1 elevation of ALT or AST during the 5-day dosing period, the GS-5734 dose will 
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 If a subject in either treatment group experiences a Grade 2 elevation of ALT or AST, he will 
be switched to normal saline.  
 Placebo recipients who experience Grade 1 elevations in  AST or AST will have the amount 
of drug vehicle reduced by 25%.  
 
Note: PREVAIL IV will use the Division of AIDS Table for Grading the Severity of Adult and 
Pediatric Adverse Events” Version 2.0, November 2014, which can be found at: http://rsc.tech-
res.com/document/safetyandpharmacovigilance/daids_ae_grading_table_v2_nov2014.pdf . 
 
Consideration for cohort dose reduction from 100 mg/day to 75 mg/day via IV:  
 The DSMB reviewed early safety data and did not make a recommendation for a  cohort dose 
adjustment as described (see 11.7.1.1.1).  
5.6 
Study Product Accountability Procedures 
The study pharmacist will be responsible for maintaining an accurate record of the unused study 
agent and an accountability record of study agent supplies for this study. Electronic 
documentation as well as paper copies will be used. Accountability instructions will be included 
in the pharmacy manual. 
5.7 
Concomitant Medications 
Prescription medications taken by subjects as maintenance medications for chronic medical 
conditions, such as hypertension, can be continued during the study and will be recorded on the 
concomitant medication form throughout the study. For this protocol, a prescription medication 
is defined as a medication that can be prescribed only by a properly authorized/licensed clinician. 
5.8 
Discouraged Medications  
Unless advised by a study physician otherwise, study participants will be counseled to refrain 
from taking: non-maintenance, non-prescription medications, over the counter medications, 
herbal supplements. Paracetamol, also known as Tylenol or acetaminophen, is particularly 
discouraged. 
 
Subjects will be advised to refrain from drinking alcohol for 7 days prior to initiation of IV study 
agents through day 14 of the study.   
6 
Study Schedule 
All study visits will take place at the study sites in Liberia and Guinea per study manual of 
operations. A tabular presentation of the schedule of evaluations is presented in Appendix B. 
6.1 
Baseline Ophthalmologic Exam (4 months prior to enrollment) 
The baseline ophthalmologic exam will be done within four months of study enrollment. The 
ophthalmologic exam will include the same set of tests and evaluations included in the 
PREVAIL III Ebola Natural History and data may be taken from PREVAIL III if the 
ophthalmologic exam was conducted within four months prior to enrollment in PREVAIL IV. If 
the ophthalmologic exam was done longer than four months prior to PREVAIL IV study 


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enrollment or participants were not previously enrolled in PREVAIL III the ophthalmologic 
exam will be repeated.  
6.2 
Optional Pre-Screening (prior to screening) 
The following can be performed during the pre-screening period:  
Note: It is anticipated that Pre-Screening will be used in Guinea.  
 Sign pre-screening informed consent for PREVAIL IV.  
 Semen collection for Ebola viral RNA detection using the GeneXpert Ebolavirus Assay. 
 
Note: Semen specimens collected in the context of PREVAIL III are acceptable for meeting 
eligibility criteria for PREVAIL IV if they occur within the -42-day screening window prior 
to randomization. An individual with one semen sample with Ebola virus RNA detected on 
GeneXpert testing within the -42-day window from anticipated randomization during Pre-
Screening or on PREVAIL III can undergo screening and have the second semen sample 
collected during Week 0 (within -21 days prior to day 1). Men coming from other studies or 
programs with or without a history of detectable seminal Ebola viral RNA will have their 
semen retested in a PREVAIL research lab using the GeneXpert. Due to intermittent 
detection of Ebola viral RNA in PREVAIL III, repeat testing is allowable at two-week 
intervals for determination of eligibility in PREVAIL IV.  
 Men within PREVAIL III not currently enrolled in the semen study are eligible for 
enrollment in PREVAIL IV. In Liberia, men can participate in the semen screening at 
PREVAIL sites or if preferable via at-home semen donation. In Guinea, men can participate 
in semen screening either at fixed sites or through mobile screening units.  
6.3 
Screening/Week 0 (within 21 days prior to day 1) 
At screening, the study will be described to the subjects. Subjects will sign the full study 
PREVAIL IV informed consent document before any study procedures (beyond semen screening 
conducted under the Pre-screening informed consent) are performed. The following will be 
performed at the screening visit: 
 Physical exam/signs, medical history (including EVD history), demographics and contact 
information, and symptoms (unless completed within the -42 day screening window under 
PREVAIL III) 
 Vital signs 
 Concomitant medications  
 Depression questionnaire 
 Post-Traumatic Stress Disorder Questionnaire (for those not co-enrolled in PIII) 
 Stigma and Discrimination Questionnaire (for those not co-enrolled in PIII) 
 Blood draw for: 
 ALT/AST 
 CBC with differential  
 Chemistries (Na, K, Cl, CO2, creatinine, glucose, eGFR), total and direct bilirubin  


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 Lipids: triglycerides, cholesterol (HDL, LDL)  
 Prothrombin time (PT), activated Partial thromboplastin time (aPTT), INR, D-dimer 
 Ebola antibody levels 
 HIV and syphilis testing (for Liberian participants not co-enrolled in PREVAIL III 
and all participants in Guinea) 
 Participants with only one semen sample positive for detection of Ebola viral RNA will 
provide another sample within 21 days prior to randomization. Participants who have had 
two semen samples with at least one positive for detection of Ebola virus RNA within the -42 
day window prior to randomization, but the last sample was taken >21 days prior to 
enrollment, will provide an additional semen sample if they are able. Note that the results of 
this sample will not affect eligibility for the study. 
 Randomization will be performed after eligibility is confirmed 
6.4 
Treatment Phase (day 1 to day 28) 
6.4.1 Day 1 
Pre-dose: The following will be performed prior to study agent infusion: 
 Targeted physical exam for signs and symptoms since screening 
 Review of concomitant medications 
 Review of baseline labs 
 Labs per Schedule of Events (SOE) which includes storage of serum sample for future 
serologic hepatitis B and hepatitis C testing 
 Vital signs (within 30 minutes prior to infusion) 
 Insertion of IV catheter  
 
Infusion: After completion of the pre-dose procedures and review of laboratory test results study 
agent will be infused over 1 hour.  
 
Post-dose: The following will be performed after completion of the study agent infusion: 
 Subjects will be observed during and for 60 minutes following the infusion. 
 Vital signs (approximately 30 and 60 minutes after completion of the infusion) 
 AE assessment 
 Physical exam (as needed to assess new signs and symptoms) 
 When site facility conditions allow processing of PK samples, one blood draw for PK 
analysis of GS-5734 and metabolites concentration in plasma (within 5 min after the end of 
infusion). For PK analysis sample schedule see Table 9. 
6.4.2 
Days 2, 3, 4, and 5 
Pre-infusion: 
 Targeted physical exam to assess new signs and symptoms since previous day’s infusion 
 Review of concomitant medications 


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 Review of prior day AST and ALT (by designee) 
 Vital signs (within 30 minutes prior to infusion) 
 AE assessment 
 Insertion of IV catheter if outpatient  
 Insure patency of IV catheter if an inpatient 
 Blood draw for: 
 Per SOE 
 When site facility conditions allow processing of PK samples, one blood sample for 
GS-5734 metabolites concentrations in plasma pre-dose (Day 4 only). For PK 
analysis sample schedule see Table 9. 
 
Infusion: Study agent infusion over 1 hour, with close observation of subject during infusion 
and for 60 minutes after completion of infusion. 
 
Post-infusion: 
 Vital signs (approximately 30 and 60 minutes after completion of infusion) 
 Physical exam (as needed to assess new signs and symptoms) 
 When site facility conditions allow processing of PK samples, blood draw (Day 5 only) for: 
 One blood sample for GS-5734 and metabolites concentrations in plasma (within 5 
minutes after the end of infusion). For plasma PK analysis sample schedule see Table 
9. 
 Blood sample for PBMC GS-441524 and phosphorylated metabolites concentration 
within 1 hour after the infusion. (Note this may be drawn with the blood sample to be 
taken 5 minutes after the infusion.) For PBMC PK analysis sample schedule see 
Table 9. 
 Semen collection (Day 4 only) for:  
 Ebola virus PCR 
 
Table 9. Summary of blood samples to be collected for PK analysis.a 
Sample 
Day 1 
Day 4 
Day 5 
Pre-
infusion 
Post-
infusion 
Pre-
infusion 
Post-
infusion 
Pre-
infusion 
Post-
infusion 
Blood for plasma 
PK 
 
Xb 
Xc 
 
 
Xb 
Blood for PBMC 
PK 
 
 
 
 
 
Xd 
a When site facility conditions do not allow processing of PK samples, these samples will not be drawn and PK 
analysis will be omitted. 
b Collect immediately after the end of infusion (within 5 min if possible) 
c Collect anytime within 1 hour before initiation of infusion 
d Collect within 1 hour after the end of infusion 


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6.4.3 
Days 8, 11, 16, 24, and 28 (see Appendix B for windows) 
 Targeted Physical exam (as needed to assess new signs and symptoms) 
 Review of concomitant medications 
 Vital signs 
 AE assessment 
 Blood draw for other labs per SOE  
 Semen collection for: Ebola virus PCR 
 Participant satisfaction survey (day 28 only) 
6.5 
Follow-up Phase 
6.5.1 Weeks 8, 12, 16, 20, and 24 (see Appendix B for windows) 
 Targeted Physical exam (as needed to assess new signs and symptoms) except for week 24 
which will be a complete review of systems, physical exam to include a depression screen, 
and a post-traumatic stress screen.  
 Review of concomitant medications 
 Vital signs 
 AE assessment 
 Blood draw for labs per SOE 
 Semen collection for Ebola virus PCR 
 Participant satisfaction survey (week 24 only) 
6.5.2 Week 24 (within ± 6 week window)  
 Ophthalmologic exam  
 
Subjects will be completed with the study after the week 24 visit and ophthalmologic visit, 
unless offered closeout treatment as described below. 
6.6 
Closeout Treatment for Subjects on Placebo Arm 
If the treatment is shown to be efficacious and to have acceptable safety and tolerability, the 
subjects who received placebo who continue to have detectable EBOV virus RNA in their semen 
at or after 6 months on study will be offered treatment with GS-5734. 
7 
Study Procedures/Evaluations 
7.1 
Clinical Procedures/Evaluations 
Blood draw: Blood will be drawn according to standard procedures for laboratory evaluations 
and storage. The amount of blood drawn for research purposes will not exceed 10.5 mL/kg or 
550 mL, whichever is smaller, over any 8-week period. 
 


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Semen collection: Collection of semen will occur at PREVAIL clinical study sites, as described 
in the study manual of procedures. 
 
Ophthalmologic Evaluations: Ophthalmologic evaluations will take place at the JFK PREVAIL 
site in Liberia and the Ambroise Paré Clinic site in Guinea, and will only involve non-invasive 
assessments (e.g., visual acuity, intraocular pressure).  
7.2 
Laboratory Evaluations  
Per the SOE, laboratory evaluations include routine chemistry, total and direct bilirubin, liver 
transaminases (ALT/AST), lipids, prothrombin (PT), activated partial thromboplastin time 
(aPTT), and D-dimer, CBC with differential and platelet counts, semen for assessment of EBOV 
RNA, blood and PBMCs for GS-5734 and metabolites, and Ebola antibody levels.  
7.2.1 Specimen Preparation, Handling, and Shipping  
Semen samples collected as part of this study are likely to contain Ebola virus RNA. It is not 
known if the presence of EBOV RNA represents viable Ebola virus. Thus, appropriate protective 
precautions will be taken by all personnel involved in obtaining, handling, and shipping of these 
semen specimens, as currently recommended by the Centers for Disease Control and Prevention, 
the Liberian Ministry of Health, and the Guinean Ministry of Health. 
 
Blood samples collected for GS-5734 and metabolite concentrations in plasma and PBMCs will 
be processed and stored according to Gilead Sciences Laboratory Manual when site facility 
conditions allow proper processing; when conditions do not allow processing for PK analysis, 
samples will not be collected. Processed samples will be shipped to a contract laboratory 
recommended by Gilead Sciences that is qualified to perform quantitative analysis of GS-5734 
and metabolites concentrations in human clinical samples.  
8 
Potential Risks and Benefits 
8.1 
Potential Risks 
8.1.1 GS-5734 
Early signs of kidney injury in humans can readily be detected and monitored using routine 
laboratory tests. In this study, the risk of clinically significant kidney injury in humans will be 
minimized by close monitoring of kidney function. Study subjects will be required to have liver 
function tests at or below the upper limit of normal at the time of screening. Subjects with a 
history of liver disease, renal disease or any other serious or active medical illness will be 
excluded from participation. Serum chemistry assessments, including liver function testing, will 
be closely monitored during the study period. 
 
Based on a review of these Phase I data, a dose selection of 100 mg IV for 5 days has been 
selected with individual dose de-escalation built in for Grade 1 transaminase elevations during 
the 5-day dosing period. A DSMB review is planned after the first 8 randomized subjects (4:4 
GS-5743:Placebo) complete study day 16, for safety and assessment of cohort dose reduction. 
The summarized risk mitigation strategy appears in Section 8.3.1. 
 


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To date in human studies, no serious AEs have occurred in healthy individuals who have 
received at least one dose of GS-5734. GS-5734 has been tested in humans as a single ascending 
dose over a dose range of 3 to 225 mg and in a multi-dose study of 150 mg/day for 7 or 14 days. 
In nonclinical animal studies, toxicity findings were consistent with dose dependent and 
reversible kidney injury and dysfunction.  
While the clinical significance of the nephrotoxicity in animal species is unknown, no evidence 
of nephrotoxicity has been observed through single GS-5734 dose levels up to 225 mg in humans 
or in multi-dose studies of 150 mg/day for 7 and 14 days. The etiology of reversible kidney 
injury observed in rats is consistent with the ability of rat renal organic anion transporters 
(OATs), but not human OATs, to efficiently interact with blood metabolites of GS-5734, 
particularly GS-704277. This effect may lead to proportionally higher intracellular accumulation 
of drug metabolites in renal rat tubules, leading to kidney injury. 
 
An Ebola meningitis patient who received 2 days of GS-5734 150 mg every 24 hours followed 
by 12 days of GS-5734 225 mg every 24 hours without apparent AEs or laboratory changes 
attributed to open-label GS-5734 therapy. Abnormal liver function tests have been observed in 
subjects receiving GS-5734 (as detailed in Section 1.2.3). In summary, in the single ascending 
dose group 3 subjects, 1 subject each in the 3-mg, 150-mg and 225-mg dose groups, had 
elevation of high ALT < 1.25x ULN on day 5 and returned to normal limits by day 7. Overall, 14 
of 16 subjects who received multiple doses GS-5734 150 mg IV in the study had at least 1.5x 
increases in ALT from pretreatment levels, 6 subjects who received 7 days and all 8 subjects 
who received 14 days of study medication. Two of 8 placebo recipients had at least 1.5x 
increases in ALT from pretreatment levels. Grade 1 increased PT occurred in 10 subjects who 
received GS-5743 and no subjects who received placebo; 7 of the subjects also had graded 
transaminase elevations. No clinically significant changes from baseline were observed in total 
bilirubin, alkaline phosphatase, albumin, or INR in subjects who received GS-5734. There were 
no Grade 3 or Grade 4 transaminase elevations and ALT returned to normal range within 15 days 
for the 7-day dosing and by end of the study in the 14-day cohort.  
 
Does levels were well-tolerated in the healthy volunteers enrolled in a single-ascending-dose 
study (GS-US-399-1812) and in healthy volunteers in the GS-5734 multi-dose study of 150 mg 
IV/day with cohorts of 7 or 14 days. One subject in the multi-dose 150 mg/day developed 
anorexia, constipation, emesis, headache, nausea, and shaking of the left leg and left hand 
considered related to study medication and discontinued study medication after the 4th dose. 
This subject had accompanying Grade 2 AST and ALT elevations with normal bilirubin, alkaline 
phosphatase and PT/PTT/INR which returned to normal range. Additional symptoms related to 
GS-5734 treatment occurred in subjects who received GS-5734 included: abnormal sensation in 
the ear, dizziness, constipation, headache, loose stool, and indigestion. These symptoms resolved 
after GS-5734 was stopped 
 
The 100 mg GS-5734 dose in this study will be administered with 6 g of SBECD, which is 
within the range of daily SBECD administration considered safe in humans. The 100-mg dose 
prepared in 0.9% saline will be hypertonic relative to human serum osmolality but approaches 
the normal physiological osmolar range for humans. GS-5734 100 mg administered as a 250-mL 
infusion once daily for 5 days is not anticipated to pose a safety risk to subjects enrolled in this 
study. 


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During animal studies, injection site reactions and associated inflammatory responses were seen 
with repeated daily intramuscular injections of GS-5734. These findings were not observed in 
animals receiving IV injection with a different formulation of the study medication. For this 
clinical study, GS-5734 will only be administered by the IV route. Historically, most injection 
site reactions from parenteral treatments in humans are self-limited in nature and can be treated 
with supportive care alone. No dose-limiting injection site reactions or immuno-allergic reactions 
were observed in the Phase I studies or in the Ebola meningitis patient during her 14-day course 
of GS-5734. 
8.1.2 Placebo 
There are no risks associated with placebo administration. 
8.1.3 Blood Draw/Phlebotomy 
The risks of drawing blood and IV catheter placement include pain, bruising, bleeding, fainting, 
and, rarely, infection. In addition, IV catheter placement may cause phlebitis. 
8.1.4 Semen Collection 
There are no risks associated with the collection of semen. 
8.2 
Potential Benefits 
Participants may receive no benefit from participating in this study. 
 
GS-5734 has shown antiviral activity against Ebola virus in vitro and in NHP models as 
described above. GS-5734 has been administered to a critically ill patient with recrudescent 
Ebola meningitis inclusive of Ebola virus viremia and to a neonate with perinatally acquired 
EVD who had persistent Ebola viremia following 4 doses of experimental ZMapp therapy. The 
adult patient received GS-5734 150 mg IV every 24 hours for 2 days and 225 mg every 24 hours 
for 12 days, and the neonate received GS-5734 10 mg IV every 24 hours for 12 days. Both 
patients recovered with no apparent adverse effects or evidence of laboratory abnormalities 
related to GS-5734 administration. Neither liver nor kidney toxicity was observed in either 
patient with active EVD previously treated with GS-5734 on a compassionate use basis. 
 
Male Ebola survivors have demonstrated shedding of Ebola virus RNA in their semen beyond 18 
months of the onset of their Ebola virus disease. It is not known if symptoms and signs 
associated with what is being called, “Post Ebola Syndrome” are associated with Ebola virus 
persistence. Individuals in the Ebola virus community are aware of the Ebola cluster outbreaks 
thought to be attributable to transmission from the survivor community. In focal group 
discussions, men with Ebola virus RNA found in their semen express worry that they may 
transmit the virus to their loved ones, concern that they want to father children but fears around 
intercourse without a condom. One survivor expressed, that he “wants his freedom back”. If GS-
5734 is effective in reducing or eliminating Ebola virus from the semen of participants, the 
participants may experience the benefit of potentially less transmission risk to their families and 
communities. It is possible that those with post Ebola signs and symptoms may experience an 
improvement in these symptoms. However, there may be no benefit at all.  


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8.3 
Risk-Benefit Assessment 
The selected dose is thought to be a good balance between safety and potential for antiviral 
efficacy. A 10-mg/kg dose of GS-5734 administered intravenously is efficacious in Ebola virus-
infected rhesus monkeys, providing 100% protection against the lethal effects of EVD. The 
pharmacokinetics of a single 10-mg/kg IV dose to rhesus monkeys and a single dose of 150 mg 
to adult human volunteers demonstrated similar systemic concentration-time exposure profiles 
with a short, biphasic half-life of the prodrug GS-5734 and a long terminal elimination half-life 
of the nucleoside metabolite GS-441524. PBMC exposures of total nucleoside were persistent in 
both monkey and humans, with suggestion of greater persistence in humans. In Phase I multi-
dose studies overall, GS-5734 has a favorable pharmacokinetic profile, with concentrations of 
GS-5734 intermediate metabolites in semen approximately two-times that of plasma, thus 
evaluation of a 100-mg dose has potential to have antiviral activity in semen equivalent or 
slightly higher than the 150-mg dose in plasma, which correlates to 100% survival in the NHP 
EVD animal model.  
 
Elevations in transaminases generally did not occur prior to day four or five with 150 mg IV of 
GS-5734. The 33% reduction in dosing to 100 mg IV/day for a reduced duration of 5 days is 
likely to mitigate risk of hepatotoxicity. In addition, the built-in dose de-escalation with 
concomitant monitoring of participants transaminase, PT/aPTT, and creatinine will enable close 
monitoring of patients by the study physicians and aggregate safety data by the DSMB. A risk 
mitigation strategy had been developed for the study (Section 8.3.1).  
8.3.1 Risk Mitigation 
Per Section 8.1.1, despite the absence of serious adverse effects in the GS 7-day and 14-day 
cohorts in the multi-dose Phase I studies of intravenous GS-5734 at a dose of 150 mg/day, the 
prevalence in both cohorts of transient Grade 1 and 2 elevations in hepatic transaminases, and 
temporally related though shorter duration of elevations in prothrombin times in subjects with 
transaminase elevations despite evidence of other hepatic effects is cause for a thoughtful risk-
mitigation strategy. Liver transaminases and prothrombin time returned to normal limits during 
the study. The mechanism for the elevation in transaminases is not known. A risk-mitigation 
strategy for the target population and the target indication, e.g., persistence of Ebola virus in the 
semen of male survivors, warrant a risk mitigation strategy for PREVAIL IV. The risk-
mitigation strategy for PREVAIL IV includes the following:  
i) Restriction of study population to those without a history of significant renal or 
significant hepatic disease, or evidence of hepatic disease on physical exam.  
ii) Restriction of the study population to those with normal results of the following 
within 21 days of initial dosing of study agent: ALT, AST,  
iii) Exclusion of participants with: a total bilirubin > 1.5  times the upper limits of 
normal, or an estimated, GFR < 60 mL/min/1.73m2, or  a PT > 1.1 times the upper 
limits of normal. 
iv) Reduction of the dose of intravenous GS-5724 from 150 mg per day used in the 
GS-5734 multi-dose Phase I Study, to 100 mg intravenous GS-5734 per day.  
v) Built-in adjustments in dosing contingent on ALT or AST elevation: 


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(a) For a Grade I AST or ALT elevations: a 25% dose reduction of GS-5734 from 
100 mg/day to 75 mg/day or 25% by volume reduction of placebo vehicle for 
the remainder of the dosing period with continued blinding of the study team.  
(b) For a Grade 2 elevation in ALT or AST: cessation of administration of study 
agent and switch to normal saline with continued blinding of the study team.  
vi) Reduction in dose duration from the Phase I GS-5734 multi-dose study of 7 days or 
14 days of IV GS-5734 to 5 days of IV GS-5734. 
vii) Enrollment is limited to 4 subjects per week during the first 4  weeks with active 
DSMB involvement looking at a weekly listing of AEs, protocol-determined 
frequency of dose de-escalation, and a formal assessment of the first 8 subjects on 
study inclusive of day 16 laboratory data for all subjects to assess the need for a 
cohort dose reduction (see Section 11.7.1.1.1 DSMB Early Safety Review Plan).  
9 
Research Use of Stored Human Samples, Specimens, or Data 
Intended Use: Samples and data collected under this protocol may be used to better understand 
Ebola infection and Ebola viral persistence in apparent Ebola carriers. Scientists both inside and 
outside of Liberia and Guinea may be involved in studying samples and data from this study.  
 
Storage: Access to stored samples will be limited using either a locked room or a locked freezer. 
Samples and data will be stored using codes assigned by the investigators. Data will be kept in 
password-protected computers. Only investigators will have access to the samples and data.  
 
Tracking: Samples and data acquired under this protocol will be tracked per the PREVAIL data 
management system. 
 
Disposition at the Completion of the Protocol: 
 In the future, other investigators (both at NIH, in Liberia, Guinea and outside) may wish to 
study these samples and/or data. In that case, Institutional Review Board (IRB) approval 
from the NIAID IRB, the National Ethics Research Board in Liberia, and the National Ethics 
Committee for Health Research in Guinea must be sought prior to any sharing of samples 
and/or data. Any clinical information shared about the sample would similarly require prior 
IRB approvals.  
 Samples will be maintained in the PREVAIL Repository in Liberia and at the Maferinyah 
and Conakry study sites in Guinea.  
 
Reporting the Loss or Destruction of Samples/Specimens/Data to the IRB: 
 Any unanticipated loss of samples or data will be reported to the IRBs.  
 Subjects may decide at any point not to have their samples stored. In this case, the principal 
investigator will advise study personnel to destroy all known remaining samples and report 
what was done to both the subject and to the IRB. 


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10 Compensation for Inconvenience: Plan for Subjects 
Participants in the study will be compensated for their time and inconvenience. Compensation 
will be provided at each regularly scheduled study visit/contact. The governments of each 
country will recommend the amount of compensation to be provided.  
11 Assessment of Safety 
A manual of operations will describe procedures for safety reporting. Below a brief summary is 
provided. 
11.1 Definitions 
Adverse Event: An AE is any untoward or unfavorable medical occurrence in a human subject, 
including any abnormal sign (e.g., abnormal physical exam or laboratory finding), symptom, or 
disease, temporally associated with the subject’s participation in the research, whether or not 
considered related to the research. 
 
Adverse Reaction (AR): An AE that is caused by an investigational agent (drug or biologic). 
 
Suspected Adverse Reaction (SAR): An AE for which there is a reasonable possibility that the 
investigational agent caused the AE. ‘Reasonable possibility’ means that there is evidence to 
suggest a causal relationship between the drug and the AE. An SAR implies a lesser degree of 
certainty about causality than AR, which implies a high degree of certainty. 
 
Causality (relatedness): Likelihood that the event is caused by the study agents. Causality will 
be assessed considering the factors listed under the following categories: 
 
Reasonable possibility of relatedness 
 reasonable temporal relationship 
AND 
 little evidence for a more likely alternative etiology 
 
No reasonable possibility of relatedness  
 does not have a reasonable temporal relationship 
OR 
 reasonable evidence for a more likely alternative etiology 
 
Note: Other factors should also be considered for each causality category when appropriate. 
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. 
 
Serious Adverse Event (SAE): An SAE is an AE that results in one or more of the following 
outcomes:  
a. Death 
b. A life-threatening event (i.e., an immediate threat to life) 


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c. Hospitalization 
d. Prolongation of existing hospitalization 
e. Persistent or significant incapacity or substantial disruption of the ability to conduct 
normal life functions 
f. Congenital anomaly or birth defects in the offspring of a study participant 
g. Other important medical events, that, based upon appropriate medical judgment, may 
jeopardize the participant and may require medical or surgical intervention to prevent one 
of the outcomes listed in this definition. (Examples of such medical events include 
allergic bronchospasm requiring intensive treatment in an emergency room or at home, 
blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse.) 
 
Note: For this protocol laboratory-confirmed malaria events that do not require hospitalization 
will not be considered as SAEs. 
 
Unexpected Adverse Event: An AE is unexpected if it is not listed in the IB or is not listed at 
the specificity or severity that has been observed. It is the responsibility of the IND sponsor to 
make this determination. 
 
Serious and Unexpected Suspected Adverse Reaction (SUSAR): A SUSAR is an AE that is 
serious, related to the study agent, and unexpected. 
 
Severity: The Investigator will grade the severity of each AE according to the “Division of 
AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events” Version 2.0, 
November 2014, which can be found at:  
http://rsc.tech-
res.com/document/safetyandpharmacovigilance/daids_ae_grading_table_v2_nov2014.pdf 
 
Unanticipated Problem (UP): A UP is any event, incident, experience, or outcome that 
1. is 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, IB, or other study documents; and 
b. the characteristics of the subject population being studied; and 
2. has a reasonable possibility of relatedness to participation in the research (as defined 
above); 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. (Per the IND 
sponsor, an AE with a serious outcome will be considered increased risk.) 
 
Unanticipated Problem that is not an Adverse Event (UPnonAE): A UP that does not fit the 
definition of an AE, but which may, in the opinion of the investigator, involve risk to the subject, 
affect others in the research study, or significantly impact the integrity of research data. Such 
events would be considered non-serious UPs. For example, we will report occurrences of 
breaches of confidentiality, accidental destruction of study records, or unaccounted-for study 
drug. 


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11.2 Investigators’ Documentation and Assessment of Adverse Events 
All AEs occurring from the first dose of the study drug through the end of the treatment phase on 
day 28 will be documented, assessed, and recorded in the CRF. During the follow-up phase all 
Grade 3 and Grade 4 AEs, and all grade transaminase elevations, will be documented, assessed, 
and recorded on CRFs.  
 
At each contact with the subject, information regarding AEs will be elicited by appropriate 
questioning and examinations and will be: 
 documented in the subject’s medical record/source document, and 
 assessed with respect to seriousness, severity (intensity or grade), and causality (see criteria 
listed above). 
 
If a diagnosis is clinically evident (or subsequently determined), the diagnosis rather than the 
individual signs and symptoms or lab abnormalities will be documented as the AE. 
 
All abnormal laboratory findings will be reviewed on a routine basis by the DSMB to identify 
potential safety signals.  
11.3 Reporting Responsibilities and Timelines 
Except for events limited to a laboratory abnormality, all UPs, SAEs, and deaths are recorded by 
the investigator on CRFs and submitted to the data center.  
 
SAEs must be submitted within 24 hours after the investigator becomes aware the event has 
occurred. All deaths are considered SAEs and must be reported as such, as well as being reported 
on a Death CRF. 
 
A pregnancy in a sexual partner during the treatment phase of the study will be reported to site 
investigators by participants and the outcome of the pregnancy will be recorded on a CRF. 
 
Safety data will be submitted to the DSMB for safety monitoring as described below (Section 
11.7) and to the IND sponsor when requested for periodic safety assessments, review of IND 
annual reports, review of IND safety reports, and preparation of final study reports. 
 
Suspected Unexpected Serious Adverse Reactions (SUSARs) will be reported to the FDA by the 
IND Sponsor within the legal timeframe: 
 7 calendar days in case of death or life-threatening events after the sponsor's initial 
receipt of the information; relevant complementary information should be collected and 
reported within 8 extra-days. 
 15 calendar days for all other events after the sponsor's initial receipt of the information; 
relevant complementary information should also be collected and reported within 8 extra-
days. 


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11.4 Investigator Reporting Procedures to the IRBs 
Investigators are responsible for reporting to the NIAID IRB, National Research Ethics Board 
(NREB) of Liberia, and National Ethics Committee for Health Research (CNERS) in Guinea as 
outlined below. 
11.4.1.1 Definitions 
Protocol Deviation: Any change, divergence, or departure from the IRB-approved study 
procedures in a research protocol. 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. 
3. Those that are discovered after they occur. 
 
Serious Protocol Deviation: A deviation that meets the definition of an SAE or compromises 
the safety, welfare, or rights of subjects or others. 
 
Non-compliance: The failure to comply with applicable NIH Human Research Protections 
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 participants; 
b. decreases potential benefits to participants; 
c. compromises the integrity of the NIH HRPP; or 
d. invalidates the study data. 
2. Continuing: non-compliance that is recurring. 
3. Minor: non-compliance that is neither serious nor continuing. 
11.4.1.2 Expedited Reporting to the NIAID IRB, NREB, and CNERS 
Serious and non-serious UPs, deaths, serious deviations, and serious or continuing non- 
compliance will be reported within 7 calendar days of investigator awareness. SAEs that are 
related to the research (i.e., meet causality definition of reasonable possibility of relatedness 
above) will be reported to the NIAID IRB, NREB , and CNERS within 7 calendar days of 
investigator’s awareness, regardless of expectedness.  
11.4.1.3 Waiver of Reporting Anticipated Protocol Deviations, Expected UPnonAEs, and 
Deaths to the NIAID IRB, NREB, and CNERS 
Anticipated deviations in the conduct of the protocol will not be reported to the NIAID IRB, 
NREB, or CNERS unless they occur at a rate greater than anticipated by the study team. 
Expected AEs will not be reported to the NIAID IRB, NREB, or CNERS unless they occur at a 
rate greater than that known to occur in Ebola survivors. If the rate of these events exceeds the 
rate expected by the study team, the events will be classified and reported as though they are 
UPs.  


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11.4.1.4 Annual Reporting to the NIAID IRB, NREB, and CNERS 
The following items will be reported to the NIAID IRB, NREB, and CNERS in summary at the 
time of continuing review: 
 Serious and non-serious UPs.  
 Expected SAEs that are related to the research. 
 SAEs that are not related to the research. 
 All AEs, except expected AEs granted a waiver of reporting.  
 Serious and non-serious protocol deviations.  
 Serious, continuing, and minor non-compliance. 
 Any trends or events which in the opinion of the investigator should be reported. 
11.5 Sponsor’s Reporting Responsibilities 
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 IND sponsor will also submit an IND Annual Report of the progress of the investigation to 
the FDA as defined in 21 CFR 312.33. 
11.5.1 Resumption of the Study in the Event that the DSMB Recommends a Pause 
If the study is paused (see Section 11.7.1.1), the IND sponsor, in collaboration with the principal 
investigator and the DSMB, will determine if it is safe to resume the study. The principal 
investigator will notify the IRBs of the decision on resumption of the study. 
11.5.1.1 Discontinuation of Study Agent 
Subjects who meet the pre-determined criteria for discontinuation of the study agent will 
continued to be followed for safety.  
11.6 Withdrawal Criteria for an Individual Subject 
An individual subject will be withdrawn for any of the following reasons: 
 An individual subject’s decision. (The investigator should attempt to determine the reason for 
the subject’s decision.) 
 Non-compliance with study procedures to the extent that it is potentially harmful to the 
subject or to the integrity of the study data. 
 The investigator determines that continued participation in the study would not be in the best 
interest of the subject. 
11.6.1 Replacement of Withdrawn Subjects or Subjects Who Discontinue Study Agent 
The following subjects may be replaced:  
 Subject who withdraw or are withdrawn from the study prior to administration of study agent 
on day 1 may be replaced.  


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11.7 Safety Oversight 
11.7.1.1 Data and Safety Monitoring Board 
The NIAID Intramural independent Data and Safety Monitoring Board (DSMB) with ad hoc 
representation from Liberia and Guinea will monitor the study. The DSMB will be composed of 
experts in clinical trials, ethics, and a representative from West Africa. None of the members of 
the study team will be part of the DSMB or have access to the closed data unless the DSMB 
made a recommendation for early stoppage based upon safety concerns. Prior to initiation and 
throughout the study the board will review the study data to evaluate the safety, efficacy, study 
progress, and conduct of the study 
 
The trial will be conducted under the direction of an the protocol team. Members of the protocol 
team will be blinded to interim results by treatment group. The protocol team will monitor the 
enrollment and follow-up of participants, including the collection of event documentation and 
the completeness of study data. The study statistician will prepare open reports for the DSMB 
that contain this information along with pooled ANR summaries, pooled SAE information, 
pooled transaminase, prothrombin time, other lab data, and other pooled data (such as 
demographics). The study statistician will also prepare closed reports that break these data out by 
treatment group. The protocol team will meet with the DSMB during an open session prior to the 
DSMB’s closed review of treatment differences. The study statistician will attend these closed 
sessions to help clarify the closed report. The protocol team may request that the DSMB convene 
if SAEs occur that are considered to be related to the study drug. 
 
The DSMB will review the study prior to initiation and will review the safety data (laboratory 
changes, injection site reactions, AEs, dose de-escalations, dosing cessations, and SAEs) from 
the study every week via emailed weekly safety reports. In addition, the DSMB will meet to 
review safety data after a total of 8 participants have enrolled and completed 16 days on the 
study to enable assessment of safety data during anticipated drug exposure. After the initial 
meeting, the DSMB will continue to meet every 2-4 weeks depending on patient accrual. The 
DSMB will monitor safety closely throughout the trial and may pause enrollment in the event of 
deaths or SAEs that are considered drug-related. Based on their reviews, they will be asked to 
make a recommendation on whether enrollment should continue, pause, discontinue, or if there 
should be a cohort wide dose reduction. 
 
The DSMB may also pause enrollment or request that participants be notified of an increased 
frequency of unanticipated or anticipated adverse effects (e.g., elevated transaminases). The 
DSMB will also review the completeness of follow-up and other aspects of study conduct. After 
each meeting they will recommend continuing the study as planned, modifying the study, or 
terminating the study. 
 
The DSMB will be provided with the protocol and the pre-specified dose de-escalation and dose 
cessation criteria. All SAEs, all UPs, and all IND Safety Reports will be reported by the principal 
investigator to the DSMB at the same time they are submitted to the IRBs or IND sponsor. The 
principal investigator will notify the DSMB of any cases of intentional or unintentional 
unblinding as soon as possible. The principal investigator will notify the board at the time dose-
reduction or cessation criteria are met. If the study is paused the DSMB, IND sponsor, and 


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Principal investigator will collaborate to determine if it is safe to resume the study. The PI will 
submit the written DSMB summary reports with recommendations to the IRB(s). 
 
While the DSMB will be provided these guidelines for monitoring the study, they will be asked 
to use their expert and independent judgment concerning modification or early termination of the 
study. It is understood that this complex decision depends on multiple factors.  
11.7.1.1.1 Early Safety Review Plan 
The DSMB will review safety data after the first 8 participants (n=4/group) have completed the 
five day dosing period and lab safety data through day 16. It is expected that 8 will be enrolled 
on each arm at this time point, and 6 will have received 5 days of study product. Upon 
completion of the review, the board will make a recommendation to either continue the study at 
the 100-mg GS-5734 dose or reduce the dose to 75 mg for the remainder of the study. If the 
DSMB finds that a large proportion of participants on the active arm are meeting the criteria for 
de-escalation of dose, the DSMB should consider recommending the dose be reduced for all 
participants. As a guideline, if 4 or more of this first set of participants (4 through day 16; 6 
through dosing; 8 enrolled) on the active arm have met the criteria for dose de-escalation or 
experienced grade 1 or higher elevations in AST or ALT, a de-escalation for the remainder of the 
study would be appropriate. Table 10 shows recommended thresholds for the DSMB decision to 
recommend study dose de-escalation, based on an assumed enrollment of 2/group (94 total) per 
week for the first 4 weeks, followed by 4 per group (8 total) on subsequent weeks. The table 
gives guidelines for DSMB meetings starting at approximately day 23, when 16-day follow-up 
data on the first 4 people per group should be available, and meeting every two weeks up to the 
point where there is at least 16-day follow-up available on approximately 50% of the study 
participants. At the last study point listed below, approximately study week 8, we estimate 48 of 
60 participants will be enrolled. Under the assumptions used here, we would expect all 
participants to be enrolled within two weeks of this time-point, so it is unlikely that study-wide 
dose de-escalation at time points after this would be suggested. 


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Table 10. Suggested guidelines for dose de-escalation by the DSMB. 
Approx 
study 
day 
(week) 
Number 
of 
enrolled, 
per arm 
Number 
of with 
5 days 
dosing, 
per arm 
Number 
of with 
16 days 
follow-
up, per 
arm 
Number of Subjects that 
meet dose de-escalation 
criteria OR grade 1 or 
higher AST or ALT 
Number of Subjects 
with Grade 2 or higher 
AST or ALT 
Treatment 
Arm 
Placebo 
Arm 
Treatment 
Arm 
Placebo 
Arm 
23 (4) 
8 
6 
4 
≥4 
0 
≥1 
0 
37 (6) 
16 
12 
8 
≥5 
0 
≥1 
0 
51 (8) 
24 
20 
16 
≥6 
0 
≥2 
0 
65 (10) 
30 
28 
24 
N/A 
N/A 
N/A 
N/A 
Assumes enrollment of 2 subjects on active drug/week enrolled weeks 1-4 in each group, then 
4/week 
12 Site Monitoring Plan 
According to the International Conference on Harmonization Good Clinical Practice (ICH GCP) 
guidelines, Section 5.18, and FDA 21 CFR 312.50, clinical protocols are required to be 
adequately monitored by the study sponsor. This study monitoring will be conducted according 
to the “NIAID Intramural Clinical Monitoring Guidelines.” Monitors under contract to the 
NIAID/OCRPRO will visit the clinical research site to monitor aspects of the study in 
accordance with the appropriate regulations and the approved protocol. The objectives of a 
monitoring visit will be: 1) to verify the existence of signed informed consent documents and 
documentation of the consent process for each monitored subject; 2) to verify the prompt and 
accurate recording of all monitored data points, and prompt reporting of all SAEs; 3) to compare 
abstracted information with individual subjects’ records and source documents (subjects’ charts, 
laboratory analyses and test results, physicians’ progress notes, nurses’ notes, and any other 
relevant original subject information); and 4) 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 visits, the investigator (and/or 
designee) and other study personnel will be available to discuss the study progress and 
monitoring visit. 
 
The investigator (and/or designee) will make study documents (e.g, consent forms) and pertinent 
hospital or clinical records readily available for inspection by the IRBs, the FDA, the site 
monitors, and the NIAID staff, or other designees, for confirmation of the study data.  
 
A specific protocol monitoring plan will be discussed with the principal investigator 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. 


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13 Statistical Considerations 
13.1 Study Hypotheses 
13.1.1 Treatment Phase  
Male survivors of EVD who receive 5 days of IV GS-5734 will have Ebola virus RNA detected 
in a lower proportion of samples over the first 28 days, as compared to those who receive 
placebo. 
13.1.2 Follow-up Phase 
Male survivors of EVD who receive 5 days of IV GS-5734 will have Ebola virus RNA detected 
in a lower proportion of samples over the first 24 weeks, as compared to those who receive 
placebo. 
13.2 Study Design 
This study will use a parallel groups design.  
13.2.1 Stratification and Randomization 
All participants must have at least two semen samples before enrollment, at least one of which 
has detectable Ebola virus RNA, in order to be eligible for this study. Subjects will be stratified 
based on whether screening samples both had detectable Ebola virus RNA, or only one of the 
two had detectable Ebola virus RNA. Recruitment will focus on PREVAIL III participants with 
evidence of repeated samples with detectable Ebola virus RNA or CT values <39 on a single 
sample to try to enroll people likely to have detectable RNA on two screening samples, but 
participants may be enrolled from outside of PREVAIL III as well. Subjects will also be 
stratified based on the country in which they reside. There are substantial differences between 
countries in how the epidemic unfolded and how those with EVD were treated. 
 
Subjects will be randomized 1:1 within the strata defined above.  
13.3 Sample Size Justification 
Preliminary data from PREVAIL III was used to estimate the rates of detectable virus in semen 
of survivors. Among 134 men who contributed at least two semen samples, we analyzed the last 
two semen samples per person. One hundred men had undetectable CT at both time points, 13 
(10%) were detectable at both time points, and 21 (16%) had mixed detectability 
(detectable/undetectable or undetectable/detectable). Based on these estimates, 26% of male 
survivors may be eligible for the study.  
 
We use this data to estimate the proportion of men who might be eligible for the study as well as 
the “success” rate among placebos. The enrollment is limited by the number of available subjects 
with detectable Ebola virus in semen at screening. Based on semen testing in PREVAIL III, 
approximately 26% of male survivors will have detectable seminal Ebola viral RNA when tested 
longitudinally during the first 1-2 year window. This percentage may decrease with time.  
 


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Based on the power calculations below, complete data on 50 men (25/arm) should be adequate to 
find a difference under many assumptions. However, as the power calculations do not take into 
account missing data, nor is it clear what type of heterogeneity or treatment effect might be more 
realistic, the study will attempt to enroll at least 60 people (30/group), with a target of at least 20 
participants with two screening samples with a detectable CT or at least 30 participants have an 
Ebola NP or GP CT value using the Cepheid of <39. If the total sample size of 60 is enrolled 
without 20 people with two detectable samples, or at least 30 participants with an Ebola NP or 
GP CT value using the Cepheid of <39 we will continue to enroll until one of these criteria is 
met up to a total of 120 people if possible. 
13.3.1 Power Calculations 
The table below lists the simulated power to find a significant difference in treatment-phase 
ANR and follow-up ANR for various true rates of ANR in the placebo and GS-5734 arms. With 
50 people (25/arm), we will have reasonable power (>80%) to find a difference in both the 
treatment-phase ANR and the follow-up ANR if the effect of treatment is to cut the probability 
of a detectable sample by 50% for each person at each time, starting at day 3. If the effect of 
treatment is instead that 40% of people experience sterilization starting on day 3, such that their 
probability of a detectable sample drops to 0 (except for false positives), and the rest of the 
people are unaffected, then the power varies depending on the amount of heterogeneity in the 
probabilities.   
 
ANR p-value based on t-test on proportion negative at 6 (day 3 to day 28) or 5 (week 8 to 
week 24) visits. All power estimates based on 1,000 simulations, 1% false positive rate, and 5% 
false negative rate, alpha=.05, no adjustments for multiple comparisons. 
 
Table 11. Power for endpoints under varying assumptions. 
N 
Pi=P(detectable|person i) 
Control group 
Treatment 
Effect  
Power d3-
d28 ANR 
Power w8-
w24 ANR 
50 
pi=.5 
.5  
(multiplicative 
on probability) 
starting d5 
.96 
.98 
pi ~Beta(10,10) 
.93 
.97 
pi ~U(0,1) 
.95 
.98 
pi ={. 1 𝑤𝑖𝑡ℎ 50% 𝑐ℎ𝑎𝑛𝑐𝑒
. 9 𝑤𝑖𝑡ℎ 50% 𝑐ℎ𝑎𝑛𝑐𝑒 
.97 
.99 
50 
pi=.5 
.4               
(Probability 
sterilize) 
starting d5 
.77 
.84 
pi ~Beta(10,10) 
.85 
.89 
pi ~U(0,1) 
.16 
.23 


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pi ={. 1 𝑤𝑖𝑡ℎ 50% 𝑐ℎ𝑎𝑛𝑐𝑒
. 9 𝑤𝑖𝑡ℎ 50% 𝑐ℎ𝑎𝑛𝑐𝑒 
.52 
.67 
 
13.4 Statistical Analyses 
The primary analysis of treatment-period ANR will be conducted once subjects have completed 
their 28-day follow up and reviewed by the DSMB. The follow-up ANR can be conducted once 
all subjects have completed the week 24 visit. ANR will be computed as the proportion of assays 
in a specified window that are negative. For the primary analysis, any returned CT value will be 
considered a positive PCR. 
 
The primary analysis will be based on an Intention-to-Treat framework, in which participants are 
analyzed in the group to which they are randomized, including participants who reduce or 
discontinue their individual dosing. In the event that the DSMB recommends a study-wide dose 
reduction, the primary analysis will remain all people randomized to treatment, regardless of 
dose, compared to all people randomized to control, but the DSMB can recommend increasing 
the sample size at that point so there are sufficient people at the lower dose for secondary 
analyses.  
 
The primary analyses of ANR during the treatment phase and during the follow-up phase will be 
based on a two-sided permutation test comparing the groups (i.e. using a 2 sample t-test to define 
the test but use its permutation distribution to compute significance). This will be stratified by 
number of detectable samples during screening and country, as was done for randomization. No 
multiplicity adjustment will be imposed on the primary analyses of the ANR from the two 
different time periods.  
 
As the expectation is that the study will be fully enrolled before we have sufficient data collected 
on the follow-up phase, there are no formal stopping rules for efficacy or futility.  
 
This study could generate considerable missing data as a consequence of the dose de-escalation 
plan and unsuccessful donations at individual visits. If there is substantial missingness (more 
than just a few) then the previous analysis will need to adjust for differences in the number of 
observations among participants as the proportions from different participants will have different 
variances under the null hypothesis. This will be accomplished by replacing the 2 sample t-test 
with a t-test of the regression coefficient associated with an indicator variable for group 
membership (i.e. active or placebo) from a weighted linear regression analysis with weights 
given by the number of study visits for which a participant successfully made a donation (these 
weights are a consequence of inverse relationship between the variance of a binomial proportion 
and the number of trials). 
13.4.1 Secondary Endpoints 
Secondary analyses or safety and tolerability endpoints will include tabulation of grade 2 and 
higher laboratory values, including but not limited to ALT and AST, and adverse events by 
treatment arm. 
 


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Secondary analyses will examine the effect of defining a positive CT value to be any reading of 
40 or lower, as well as the main analysis based on a per-protocol definition of treatment arm. 
Other secondary analyses will include per protocol analyses, Generalized Estimating Equations 
models for the serial binary endpoints of detectability, exact tests of proportions for binary 
endpoints such as Sustained Negativity Rate (no detectable Ebola RNA at any included time 
point), and censored regression models to compare CT trajectories will also be performed.  
 
In the event that the study-wide dose is reduced to 75 mg, a secondary analysis will be restricted 
to people on both arms enrolled after the dose reduction.   
13.5 Operational Futility 
Since not all male survivors are interested in enrolling in PREVAIL III, any confirmed male 
survivor interested in participating in this study will be considered for screening/enrollment. 
14 Ethics/Protection of Human Subjects 
14.1 Informed Consent Process 
Informed consent is a process where information is presented to enable persons to voluntarily 
decide whether or not to participate as a research subject. It is an ongoing conversation between 
the human research subject and the researchers which begins before consent is given and 
continues until the end of the subject's involvement in the research. Discussions about the 
research will provide essential information about the study and include: purpose, duration, 
experimental procedures, alternatives, risks, and benefits. Subjects will be given the opportunity 
to ask questions and have them answered. 
 
The subjects will sign the informed consent document prior to undergoing any research 
procedures. The subjects may withdraw consent at any time throughout the course of the trial. A 
copy of the informed consent document will be given to the subjects for their records. The 
researcher will document the signing of the consent form in the subject’s record. The rights and 
welfare of the subjects will be protected by emphasizing to them that the quality of their medical 
care will not be adversely affected if they decline to participate in this study. 
14.2 Subject Confidentiality 
All records will be kept confidential to the extent provided by federal, state, and local law. The 
study monitors and other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records. Records will be kept locked and all computer entry and networking programs will be 
done with coded numbers only. Clinical information will not be released without written 
permission of the subject, except as necessary for monitoring by the IRBs, the FDA, NIAID, 
OHRP, the pharmaceutical supporter, or the sponsor’s designee. 


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15 Data Handling and Record Keeping 
15.1 Data Capture and Management  
Study data will be maintained on CRFs and collected directly from subjects during study visits 
and telephone calls, or will be abstracted from subjects’ CRFs from PREVAIL III when 
available. Source documents include all recordings of observations or notations of clinical 
activities and all reports and records necessary to confirm the data abstracted for this study. Data 
entry onto CRFs will be performed by authorized individuals. The investigator is responsible for 
assuring that the data collected are complete, accurate, and recorded in a timely manner. 
15.2 Record Retention 
The investigator is responsible for retaining all essential documents listed in the ICH GCP 
guidelines. Study records will be retained for a minimum of 5 to 7 years and in compliance with 
institutional, IRB, state, and USG federal, Liberian and Guinean medical records retention 
requirements, whichever is longest. All stored records will be kept confidential to the extent 
required by USG, Liberian, and Guinean federal, state, and local law. 
 
Should the investigator wish to assign the study records to another party and/or move them to 
another location, the investigator will 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. Destruction or relocation of research records will not proceed 
without written permission from OCRPRO/NIAID. 
 
 
 


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Appendix A: Scientific References 
1. 
de Vries DH, Rwemisisi JT, Musinguzi LK, et al. The first mile: community experience 
of outbreak control during an Ebola outbreak in Luwero District, Uganda. BMC Public 
Health. 2016;16:161. 
2. 
Bwaka MA, Bonnet MJ, Calain P, et al. Ebola hemorrhagic fever in Kikwit, Democratic 
Republic of the Congo: clinical observations in 103 patients. The Journal of infectious 
diseases. 1999;179 Suppl 1:S1-7. 
3. 
Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet. 2011;377(9768):849-862. 
4. 
Bah EI, Lamah MC, Fletcher T, et al. Clinical presentation of patients with Ebola virus 
disease in Conakry, Guinea. N Engl J Med. 2015;372(1):40-47. 
5. 
Schieffelin JS, Shaffer JG, Goba A, et al. Clinical illness and outcomes in patients with 
Ebola in Sierra Leone. N Engl J Med. 2014;371(22):2092-2100. 
6. 
Rowe AK, Bertolli J, Khan AS, et al. Clinical, virologic, and immunologic follow-up of 
convalescent Ebola hemorrhagic fever patients and their household contacts, Kikwit, 
Democratic Republic of the Congo. Commission de Lutte contre les Epidemies a Kikwit. 
The Journal of infectious diseases. 1999;179 Suppl 1:S28-35. 
7. 
Kibadi K, Mupapa K, Kuvula K, et al. Late ophthalmologic manifestations in survivors 
of the 1995 Ebola virus epidemic in Kikwit, Democratic Republic of the Congo. The 
Journal of infectious diseases. 1999;179 Suppl 1:S13-14. 
8. 
Emond RT, Evans B, Bowen ET, Lloyd G. A case of Ebola virus infection. British 
medical journal. 1977;2(6086):541-544. 
9. 
Sissoko D, Duraffour S, Kerber R, et al. Persistence and clearance of Ebola virus RNA 
from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling 
study. Lancet Glob Health. 2017;5(1):e80-e88. 
10. 
Etard JF, Sow MS, Leroy S, et al. Multidisciplinary assessment of post-Ebola sequelae in 
Guinea (Postebogui): an observational cohort study. Lancet Infect Dis. 2017. 
11. 
Deen GF, Knust B, Broutet N, et al. Ebola RNA Persistence in Semen of Ebola Virus 
Disease Survivors - Preliminary Report. N Engl J Med. 2015. 
12. 
Pettitt J, Higgs E, Fallah M, et al. Assessment and Optimization of the GeneXpert 
Diagnostic Platform for Detection of Ebola Virus RNA in Seminal Fluid. The Journal of 
infectious diseases. 2017;215(4):547-553. 
13. 
Pinsky BA, Sahoo MK, Sandlund J, et al. Analytical Performance Characteristics of the 
Cepheid GeneXpert Ebola Assay for the Detection of Ebola Virus. PLoS One. 
2015;10(11):e0142216. 
14. 
Semper AE, Broadhurst MJ, Richards J, et al. Performance of the GeneXpert Ebola 
Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study. 
PLoS Med. 2016;13(3):e1001980. 
15. 
Group PIS, Sneller MC, Reilly C, et al. A Longitudinal Study of Ebola Sequelae in 
Liberia. N Engl J Med. 2019;380(10):924-934. 
16. 
Martini GA, Schmidt HA. [Spermatogenic transmission of the "Marburg virus". (Causes 
of "Marburg simian disease")]. Klinische Wochenschrift. 1968;46(7):398-400. 
17. 
Diallo B, Sissoko D, Loman NJ, et al. Resurgence of Ebola Virus Disease in Guinea 
Linked to a Survivor With Virus Persistence in Seminal Fluid for More Than 500 Days. 
Clinical infectious diseases : an official publication of the Infectious Diseases Society of 
America. 2016;63(10):1353-1356. 


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18. 
Mate SE, Kugelman JR, Nyenswah TG, et al. Molecular Evidence of Sexual 
Transmission of Ebola Virus. N Engl J Med. 2015;373(25):2448-2454. 
19. 
Christie A, Davies-Wayne GJ, Cordier-Lassalle T, et al. Possible sexual transmission of 
Ebola virus - Liberia, 2015. MMWR Morbidity and mortality weekly report. 
2015;64(17):479-481. 
20. 
Jacobs M, Rodger A, Bell DJ, et al. Late Ebola virus relapse causing 
meningoencephalitis: a case report. Lancet. 2016;388(10043):498-503. 
21. 
Hafner V, Czock D, Burhenne J, et al. Pharmacokinetics of sulfobutylether-beta-
cyclodextrin and voriconazole in patients with end-stage renal failure during treatment 
with two hemodialysis systems and hemodiafiltration. Antimicrobial agents and 
chemotherapy. 2010;54(6):2596-2602. 
22. 
Luke DR, Tomaszewski K, Damle B, Schlamm HT. Review of the basic and clinical 
pharmacology of sulfobutylether-beta-cyclodextrin (SBECD). Journal of pharmaceutical 
sciences. 2010;99(8):3291-3301. 
23. 
Stella VJ, He Q. Cyclodextrins. Toxicologic pathology. 2008;36(1):30-42. 
24. 
World Health Organization (WHO). Emergencies preparedness, response: New Ebola 
cases confirmed in Guinea as WHO warns of more possible flare ups. Available at: 
http://www.who.int/csr/disease/ebola/new-ebola-cases-confirmed-guinea/en/. Accessed 
18 March. World Health Organization. 2016. 
25. 
Varkey JB, Shantha JG, Crozier I, et al. Persistence of Ebola Virus in Ocular Fluid during 
Convalescence. N Engl J Med. 2015;372(25):2423-2427. 
26. 
Fallah M. A cohort study of survivors of Ebola virus infection in Liberia (PREVAIL III). 
Paper presented at: Conference on Retroviruses and Opportunistic Infections 
(CROI)2016; Boston. 


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Appendix B: Schedule of Procedures/Evaluations 
Evaluations/ 
Procedures 
Pre-
Screena 
Screening/ 
Week 0 
(within 21 
days prior 
to Day 1) 
Day 1f 
(Infusion) 
Day 
2 
Day 
3 
Day  
4 
Day 
5 
Day 
8 
(+1) 
Day 
11  
(+1)  
Day 
16 
(±2) 
Day 
24 
(±2) 
Day 
28f 
(+3) 
Wk. 
8g 
(±14 
days) 
Wk. 
12 
(±14  
days) 
Wk. 
16 
(±14 
days) 
Wk. 
20 
(±14 
days) 
Wk. 24g 
(+5 
days) 
End of 
Study 
Pre-
dose 
Post-
dose 
Pre-Screening Informed 
Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
 
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Demographics 
(participant and 
contacts) 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Targeted Physical 
exam/signs & 
symptoms 
  
  
X 
Xd 
Xd 
Xd 
Xd 
Xd 
Xd 
Xd  
Xd 
 Xd  
Xd 
Xd 
Xd 
Xd 
Xd 
  
Vital signs 
  
X 
Xc 
Xc 
Xc 
Xc 
Xc 
Xc 
Xd  
Xd   
X 
 Xd  
Xd  
X 
X 
X 
X 
X 
Completed Medical 
Exam & ROS/ 
medication history 
  
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X 
Medical and EVD 
history a 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Depression 
Questionnaire 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
PTSD Questionnaire a 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Stigma and 
Discrimination 
Questionnaire a 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Randomize subject 
  
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Infusion 
  
  
  
X 
X 
X 
X 
X 
  
  
  
  
  
  
  
  
  
  


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Assessment for Adverse 
events 
  
  
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Review of concomitant 
meds 
  
X 
X 
  
X 
X 
X 
X 
X 
 X 
X 
 X 
X 
X 
X 
X 
X 
X 
Participant satisfaction 
survey 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
 
X 
Blood Samples 
 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Chemistries, total and 
direct bilirubin,  
AST/ALT, and lipids 
  
X 
X 
  
Xj 
Xj 
Xj 
Xj 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
CBC with diff  
  
X 
X 
  
Xj 
Xj 
Xj 
Xj 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Prothrombin time (PT), 
aPTT, INR, and  
D-dimer 
  
X 
X 
  
Xj 
Xj 
Xj 
Xj 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Leftover serum and 
plasma storage 
  
X 
Xn 
  
Xj 
Xj 
Xj 
Xj 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
GS-5734 PK and 
metabolites - plasmao 
  
  
  
Xh 
  
  
 Xj 
Xh 
  
  
  
  
  
  
  
  
  
  
PBMCs for intracellular 
concentrations - PKo 
  
  
  
  
  
  
  
Xi 
  
  
  
  
  
  
  
  
  
  
eGFR 
  
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Ebola antibody levels 
  
X 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
X 
Semen Samples  
Ebola PCR with CT for 
NP & GP 
Xb 
 Xm 
  
  
  
 
Xk   
  
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Ophthalmologic Exam 
Xe 
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
  
Xl 
HIV and Syphilis Test  
 
Xp 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
a Participants in Guinea can sign a pre-screening informed consent form for semen collection and testing. Procedures conducted under PREVAIL III will be accepted for 
PREVAIL IV pre-screening. 
b Positive PCR for NP or GP using the GeneXpert assay at pre-screening in at least one of two or more semen samples within 42 days of randomization.  
c On infusion days, vital signs will be obtained within 30 minutes prior to start of infusion and 30 minutes and 60 minutes after infusion completes.  
d Performed only if needed to evaluate new symptoms/complaints. 
e Baseline ophthalmologic exam to be conducted within 4 months of enrollment in PREVAIL IV. The baseline ophthalmologic exam can be extracted from PREVAIL III 
data if done within 4 months prior to enrollment in PREVAIL IV.  Ophthalmologic exams must be repeated if conducted > 4 months prior to enrollment in PREVAIL IV 
or if they were not previously done.  
f Beginning & end of Treatment phase with DSMB evaluation planned for end of treatment phase. 
g Beginning & end of Follow up phase. 
h Draw immediately after infusion if possible and not later than 5 minutes after infusion. 


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i Draw within 1 hour after infusion, but may be drawn with the PK plasma sample drawn within 5 minutes after infusion. 
j Pre-infusion sample. 
k Day 4 semen samples can +1 e.g. on days 4 or 5 but only one semen sample is needed during infusion.  
l End of study ophthalmologic exam to be done at 24 weeks  +/- 6 weeks 
m An additional semen sample will be collected during the screening period if the participant has only had one sample with Ebola viral RNA detected or already had two 
previous semen tests with at least one positive detection of Ebola virus RNA and the last semen sample was collected greater than 21 days prior to enrollment. Note that 
the results of this sample do not affect subject eligibility. A participant with one previous semen sample with positive Ebola virus RNA detected within the -42-day 
window can provide the second required sample for randomization during the -21 day screening period, if they are able. The results of this sample will be used to stratify 
the participant and should be done on days -21 to -3.  
nA portion of excess serum will be used for future serologic testing for hepatitis B and hepatitis C. 
o When site facility conditions do not allow processing of PK samples, sample should not be collected. 
p In Guinea and in Liberia for participants not co-enrolled in PREVAIL III. 
 


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Appendix C. eGFR Calculation 
A normal serum creatinine does not necessarily reflect a normal Glomerular Filtration Rate 
(GFR). Serum creatinine alone is not a sensitive indicator for mild to moderate kidney injury. 
Estimate glomerular filtration rate (GFR) can be estimated e(GFR) from measured serum 
creatinine. Normal estimated GFR (eGFR) has been calculated from population level studies 
(NHANES III) in the US and have been extensively validated for Caucasians and African 
Americans and will be used in this study because similar data is not available for Liberia. The 
eGFR will be estimated using the Chronic Kidney Disease Epidemiology Collaboration formula 
(CKD-EPI) for PREVAIL IV. The West African male population is muscular and lean with low 
BMI, in a tropical climate without climate control, and a cultural tendency not to drink water. 
The CKD-EPI has not been validated in the African non-American population. eGFR varies by 
age; the table below will be used to determine normal age eGFR to determine eligibility.  
 
eGFR Method: The CKD-EPI equation for African American males is: 
 
Black 
Male 
≤ 0.9 
GFR = 163 × (Scr/0.9)-0.411 × (0.993)Age 
Black 
Male 
> 0.9 
GFR = 163 × (Scr/0.9)-1.209 × (0.993)Age 
 
Ref: Levey AS, Stevens LA, et al. A New Equation to Estimate Glomerular Filtration Rate. Ann Intern Med. 2009; 
150:604-612. 
 
The equation does not require weight or height variables because the results are reported 
normalized to 1.73 m2 body surface area, which is an accepted average adult surface area. 
 
The table below shows population estimates for mean (average) estimated glomerular filtration 
rate (eGFR) by age. These means, derived from the NHANES III survey of over 10,000 
individuals in the US, demonstrate that eGFR varies across age groups and that kidney function 
tends to decline with age.  
 
Reference Table for Population Mean eGFRs From NHANES III 
Age (Years) 
Mean eGFR* 
18a-29 
116 mL/min/1.73 m2 
30-39 
107 mL/min/1.73 m2 
40-49 
99 mL/min/1.73 m2 
50-59 
93 mL/min/1.73 m2 
60-69 
85 mL/min/1.73 m2 
70+ 
75 mL/min/1.73 m2 
a Lower age range adjusted from 20years to 18 years of age 
 


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Appendix D: Delineation of Principal Investigator roles for the PREVAIL IV protocol 
 
Overview: All  investigators interact with subjects, participate in screening, oversee infusions, 
oversee study staff, review safety data, participate in design, and recruitment efforts. All PIs 
work with the study staff, the operations staff, the data management center, the pharmacists, and 
other members of the PREVAIL IV study team. The investigator team shares responsibility for 
subject safety and communication with the safety office.  
Dr. Dennis resides in Liberia and is present at the site on a daily basis. She serves as the primary 
oversight to the medical monitors and the interface with the John F. Kennedy Hospital (JFK). Dr. 
Dennis provides daily leadership to the PREVAIL IV study team at JFK and Duport Road.  
Dr. Bea resides in Guinea and provides oversight of the study on a daily basis. He serves as the 
oversight to the medical monitors and the interface with the sites and the Guinea MOH Trial 
Steering Committee.  
Dr. Higgs has primary responsibility for the writing of the protocol and interface with the 
DSMB, Gilead, the FDA, and the IRBs. Dr. Higgs has overall responsibility for ensuring that the 
implementation of the protocol complies with applicable regulations and policies. Dr. Higgs 
travels back and forth to Liberia and Guinea. 
In addition to the responsibilities outlined in the "overview" above, Dr. Fischer travels back and 
forth to Liberia and Guinea as needed and takes a lead role in recruiting subjects not affiliated 
with the PREVAIL studies previously. Dr. Fischer speaks French and English and is helpful in 
bridging the team into one functional unit. Dr. Fischer has primary responsibility over the 
emergency and safety SOPs.   
 
 
 


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Appendix E: Compensation for Inconvenience  
Subjects will be compensated for their time and inconvenience as follows per current PREVAIL 
standards: 
 
 Pre-screening information session and consent: $20 
 Semen collection ( $20/visit) 
 Follow up visit for results without any additional tests/evaluations ($10) 
 Screening (up to 4 separate visits): $20/each = up to $80 
 Study Infusion Days 1-5: $30 (x5) = $150 
 Follow-up visits during the treatment phase: $20 (x5) = $100 
 Follow-up visits for the longer term follow-up phase: $20 per month x 5 = $100 
 Study completion bonus after 15 visits over 6 months: $50 
 
If the subjects presents for information only, they will be reimbursed $20. In addition, subjects 
may receive per diem compensation according to site-specific guidelines 
 
Compensation for participants in Guinea will be in the equivalent GNF conversion.