Protocol. This trial protocol has been provided by the authors to give readers additional information about their work.

Transcription

1 Protocol This trial protocol has been provided by the authors to give readers additional information about their work. Protocol for: Kennedy SB, Bolay F, Kieh M, et al. Phase 2 placebo-controlled trial of two vaccines to prevent Ebola in Liberia. N Engl J Med 2017;377: DOI: /NEJMoa

2 This supplement contains the following items: 1. Original protocol, final protocol, summary of changes 2. Statistical analysis plan, Immunogenicity analysis plan

3 Partnership for Research on Ebola Vaccines in Liberia (PREVAIL) Version: 1.0 Date: January 30, 2015 Protocol Number: 001 U.S. Project Assurance: FWA Liberian Ministry of Health and Social Welfare Project Assurance: FWA Study Vaccines Provided by: GlaxoSmithKline plc Brentford, Middlesex, United Kingdom Merck & Co., Inc and NewLink Genetics, Inc. Whitehouse Station, New Jersey and Ames, Iowa, United States Liberian Principal Investigator: Stephen Kennedy, MD U.S. Principal Investigator: H. Clifford Lane, MD Sponsored by: Office of Clinical Research Operations and Regulatory Compliance (OCRPRO) National Institute of Allergy and Infectious Diseases (NIAID) National Institutes of Health (NIH)

4 PREVAIL Version 1.0 January 30, 2015 Table of Contents PROTOCOL SYNOPSIS INTRODUCTION Background Ebolavirus Zaire outbreak Investigational Products Rationale for the study and study design Rationale for the study Rationale for the study design METHODOLOGY Study design Primary objectives Primary Hypotheses Secondary objectives Endpoints Sample size and statistical considerations Study cohort Inclusion criteria Exclusion criteria Randomization and blinding STUDY PLAN Baseline data collection Vaccine administration Follow-up data collection Laboratory Tests EVENT DEFINITION, REPORTING AND REVIEW EVD Definite EVD Probable EVD Deaths Event review SAFETY Adverse events Serious adverse events (SAEs) Assessment of causality Follow-up Assessment of SAEs Unanticipated Problems Reporting of Serious Adverse Events and Unanticipated Problems CLINICAL MANAGEMENT Adverse Events and Symptoms and Signs of EVD HIV and Syphilis Study Withdrawal Pregnancy PHARMACY PROCEDURES

5 PREVAIL Version 1.0 January 30, Study Agents ChAd3-EBO Z vaccine rvsv G-ZEBOV Sterile Normal Saline (sodium chloride 0.9 percent injection), USP (preservative free) STUDY AGENT PRESENTATION AND STORAGE Study Agent Labels Study Agent Storage PREPARATION OF STUDY AGENT FOR INJECTION STUDY AGENT ACCOUNTABILITY Documentation Disposition EVALUATION Data analysis Data monitoring PROTECTION OF HUMAN SUBJECTS AND OTHER ETHICAL CONSIDERATIONS Local Review of Protocol and Informed Consent Ethical Conduct of the Study Informed Consent of Study Participants Confidentiality of Study Participants Compensation POST-TRIAL ACCESS TO VACCINES

6 PREVAIL Version 1.0 January 30, 2015 LIST OF ABBREVIATIONS Ad5 Adenovirus type 5 AE Adverse event BDBV Bundibugyo ebolavirus CDC Centers for Disease Control and Prevention ChAd3-EBO Z Replication deficient investigational EBOV vaccine encoded by CRF Case report form CTM Clinical trial material DSMB Data Safety Monitoring Board DTRA Defense Threat Reduction Agency EBOV Ebola virus EC Ethics Committee ELISA Enzyme-linked immunosorbent assay ERC Endpoint Review Committee EVD Ebola virus disease GP Glycoprotein GSK GlaxoSmithKline plc (Brentford, UK) HR Hazard ratio IM Intramuscular IND Investigational New Drug IRB Institutional Review Board LD50 Median lethal dose NIAID National Institute of Allergy and Infectious Diseases NIH National Institutes of Health NK Natural killer NP Nucleoprotein OCRPRO Office of Clinical Research Operations and Regulatory Compliance PCR Polymerase chain reaction PFU Plaque-forming units PHAC Public Health Agency of Canada PID Participant Identification Code RESTV Reston ebolavirus rvsv Recombinant vesicular stomatitis virus SAE Serious adverse event SID Syringe Identification Number SUDV Sudan ebolavirus TAFV Taï Forest ebolavirus Vp Viral particle(s) UP Unanticipated Problem US United States VE Vaccine efficacy WHO World Health Organization VP Viral particles VP40 Viral matrix protein VRC Vaccine Research Center VSV G Vesicular Stomatitis-based ebolavirus vaccine 4

7 PREVAIL Version 1.0 January 30, 2015 WRAIR ZEBOV Walter Reed Army Institute of Research Zaire ebolavirus 5

8 PREVAIL Version 1.0 January 30, 2015 PROTOCOL SYNOPSIS Full Title: Partnership for Research on Ebola Vaccines in Liberia (PREVAIL) Short Title: PREVAIL Clinical Phase: 2/3 IND Sponsor: NIAID/OCRPRO Sample Size: N=28,170 (n=600 in a phase 2 substudy). This is an event-driven trial and sample size may be increased to achieve the target number of primary endpoints. Study Population: Volunteers aged 18 years and older in Liberia who are at risk for Ebola infection Accrual Period: Estimated 4-6 months (longer if necessary) Study Design: Randomized, placebo-controlled trial. Study Duration: Approximately 12 months with a start date of January, 2015 (as long as necessary to obtain the target number of primary event). Rationale: Ebola virus disease (EVD) is spreading in West Africa and there is a critical need for a vaccine to prevent EVD. A large randomized clinical trial is needed to rapidly evaluate candidate Ebola virus vaccines that have completed phase 1 testing. Study Agent Descriptions: The two vaccines to be studied are the ChAd3-EBO Z vaccine and the VSV G- ZEBOV vaccine. The ChAd3-EBO Z vaccine requires a 2 ml administration from a 3 ml syringe; The VSV G-ZEBOV vaccine requires a 1 ml administration from a 3 ml syringe. Since different volumes are required for each vaccine, two placebo preparations will be prepared for administration, syringes with 1 ml of placebo and syringes with 2 ml of placebo. With this approach, the person administering the vaccination may be aware whether the study participant is assigned to the ChAd3-EBO Z arm (vaccine or matching placebo) or the VSV G-ZEBOV arm (vaccine or matching placebo). However, they will not be aware whether the assignment is to active vaccine or placebo. Study participants and clinical staff collecting follow-up data following randomization will be fully blinded. Each study agent is briefly described below. The ChAd3-EBO Z vaccine is comprised of a ChAd3 vector with a DNA fragment insert that encodes the Ebola virus glycoprotein (2 ml IM administration). The VSV G-ZEBOV vaccine is comprised of a single recombinant (vesicular stomatitis virus) VSV isolate (11481 nt) modified to replace the gene encoding the G envelope glycoprotein with the gene encoding the envelope 6

9 PREVAIL Version 1.0 January 30, 2015 glycoprotein from the Ebola virus Zaire strain (ZEBOV) (1 ml IM administration). The 1mL and 2mL placebo control syringes contain normal saline (sodium chloride). Eligible volunteers will be randomized in a 2:1:2:1 allocation to ChAd3-EBO Z (2 ml), 2 ml of placebo, VSV G-ZEBOV (1 ml), or 1mL of placebo. Primary Objective: To determine the efficacy and safety of the ChAd3-EBO Z vaccine and the VSV G-ZEBOV vaccine as compared to placebo (pooled placebo groups). Primary Efficacy Endpoint: Ebola virus disease (EVD) occurring 21 days or more following randomization and confirmed by a positive blood sample (while alive) or a positive blood or buccal swab sample if deceased. Primary Safety Endpoint: Serious adverse experiences within 30 days of vaccination. Secondary Endpoints: Definite EVD occurring at any time after randomization Definite or probable EVD occurring 21 days or more following randomization Definite or probable EVD at any time after randomization Deaths attributed to definite or probable EVD All-cause mortality Serious adverse events over the entire period of follow-up Duration of EVD for surviving participants Grade 3 or 4 events (600 participant substudy) Ebola-specific antibodies over one week and one month of follow-up (600 participant substudy) Vaccine injection site reactions of any grade severity (600 participant substudy) Targeted signs and symptoms during the first month following vaccination of any grade severity (600 participant substudy) Volunteered adverse events of any grade severity (600 participant substudy) Inclusion Criteria: Informed consent Age 18 years Likely to be in the surrounding area of the vaccination center for at least one year Exclusion Criteria: Fever > 38º Celsius History of EVD (self-report) 7

10 PREVAIL Version 1.0 January 30, 2015 Current pregnancy (a negative urine pregnancy test is required for women of child-bearing potential) Breast-feeding an infant Any condition which would limit the ability of the participant to meet the requirements of the study protocol (for example, any serious illness) Procedures: Initially, approximately 10 vaccination centers in existing health clinics will be established in and around Monrovia to consent, vaccinate and follow study volunteers. Depending on the course of the epidemic, additional sites may be added in other parts of Liberia. These centers will be staffed to vaccinate up to 500 volunteers per center each week. A case report form (CRF) with a unique participant identification code (PID) will be completed for each participant consented and screened. At the time of vaccination, a tear-off label on the syringe that includes a unique syringe identification code (SID) will be attached to the baseline CRF. The SID is a unique number from a centrally prepared randomization schedule. This will be the primary link between the blinded vaccine assignment and the PID. Study volunteers and clinical staff following the participants for safety and efficacy outcomes will be fully blinded. Staff administering the vaccine may become aware of which arm the participant is randomized to (ChAd3-EBO Z or VSV G- ZEBOV), but they will not be aware whether active vaccine or placebo was administered. At the time of vaccination, study volunteers will be given an identification card that identifies them as a study participant. The identification card will include contact information in the event they experience vaccine side effects or signs and symptoms of Ebola. Following randomization, participants will be contacted at 1 week and 1 month and monthly thereafter until the close of the study. At each contact, volunteers will be asked questions to assess their health status and any unreported events. Volunteers will be encouraged to contact the vaccination center if they have signs or symptoms of EVD or adverse events. Volunteers will be given instructions by the vaccination center on which Ebola treatment unit (ETU) to go to in the event they have signs and symptoms of EVD. Likewise, if the participant experiences an adverse event requiring hospitalization, they will be given instructions on which hospital to go to for evaluation and treatment. One or two of the vaccination centers will be chosen to conduct a phase 2 substudy on at least 600 volunteers. This substudy, which will entail the collection of additional safety and immunogenicity data, will precede the initiation of enrolment at other vaccination centers. For substudy participants, blood will be drawn prior to vaccination and at 1 week and 1 month follow-up visits for assessing D-dimer, PTT, chemistries, and a complete blood count. HIV and syphilis status will be determined at the baseline (vaccination) visit. Serum and plasma will be stored from each visit for future immunogenicity testing. Adverse 8

11 PREVAIL Version 1.0 January 30, 2015 events will be recorded. After the 1 month follow-up visit, monthly contacts will be identical to that for other randomized volunteers. Enrolment at other vaccination centers at which safety data collection will be limited to serious adverse events will begin after an independent Data and Safety Monitoring Board (DSMB) determines that it is safe to do so. Data Monitoring: Phase 1 studies for the two vaccines have only recently been completed. These phase 1 studies resulted in sufficient information to establish the dose of vaccine to be administered. However, safety data at the doses to be used for the study are limited and there are not safety data in the target population of interest. Thus a substudy of 600 volunteers will be embedded in the clinical trial. These participants will be enrolled at a relatively slow rate so that accumulating safety data from this substudy can be very closely monitored by the independent DSMB. The DSMB will review the safety data from the substudy every week. Based on their review, the DSMB will be asked to make a recommendation on whether and when enrolment should be expanded to include other sites. Participants will continue to be enrolled in the substudy at one or two sites until the DSMB make a recommendation on expansion to other sites. This plan could result in more than 600 participants being enrolled in the substudy. Once full enrolment begins by several vaccination centers, the DSMB will continue to meet every 2-4 weeks. Based on pre-specified monitoring guidelines for the primary endpoint and their review of accumulating safety data, after each of their meetings they will recommend continuing the study as planned, modifying the study, or terminating the study. Recommendations will be made to the study sponsor and the protocol study team. 9

12 PREVAIL Version 1.0 January 30, INTRODUCTION 1.1. Background Ebolavirus Zaire outbreak 2014 The Zaire Ebola virus disease (EVD) outbreak In West Africa was first recognised on 22 March 2014 in Guinea. By December 1, 2014, the epidemic had spread to Liberia, Sierra Leone, Nigeria, Mali, Spain, Senegal and the US, with an estimated 15,935 cases of EVD with 5689 deaths. Without effective interventions, the Centers for Disease Control and Prevention initially estimated there could be 550,000 to 1.4 million cases of EVD by January 20, While this worse case scenario does not appear to be playing out, and there are indications that the rate of new infections has decreased in Liberia, the epidemic continues. The genus Ebolavirus is one of three genera in the family Filoviridae, which along with the genera Marburgvirus and Cuevavirus, are known to induce viral haemorrhagic fever. The 5 distinct species included in the genus Ebolavirus are Bundibugyo (BDBV), Reston (RESTV), Sudan (SUDV), Taï Forest (TAFV), and Zaire (ZEBOV). Ebola virus is a large, negative-strand RNA virus composed of 7 genes encoding viral proteins, including a single glycoprotein (GP). The virus is responsible for causing EVD in humans. In particular, BDBV, ZEBOV, and SUDV have been associated with large outbreaks of EVD in Africa and reported case fatality rates of up to 90%. 2 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 have been summarized by Feldmann and Geisbert. 3 Infection is followed by an abrupt onset of non-specific symptoms such as fever, chills, malaise, and myalgia. The subsequent signs and symptoms indicate multisystem involvement and include systemic (prostration), gastrointestinal (anorexia, nausea, vomiting, abdominal pain, diarrhoea), respiratory (chest pain, shortness of breath, cough, nasal discharge), vascular (conjunctival injection, postural hypotension, oedema), and neurological (headache, confusion, coma) manifestations. Haemorrhagic manifestations consistent with disseminated intravascular coagulation may arise during the peak of the illness and include petechiae, ecchymoses, uncontrolled oozing from venepuncture sites, mucosal haemorrhages, and post-mortem evidence of visceral haemorrhagic effusions. A maculopapular rash associated with varying severity of erythema and desquamation can often be noted by day 5 to 7 of the illness; this symptom is a valuable differential diagnostic feature and is usually followed by desquamation in survivors. Abdominal pain is sometimes associated with hyperamylasaemia 10

13 PREVAIL Version 1.0 January 30, 2015 and true pancreatitis. In later stages, shock, convulsions, severe metabolic disturbances, and, in more than half the cases, diffuse coagulopathy supervene. Laboratory findings include low white blood cell and platelet counts and elevated liver enzymes. In general, the symptoms last for about 7-14 days, after which recovery may occur. Death usually occurs 6 to 16 days after the onset of symptoms. The clinical manifestations of patients with EVD who were treated at an Ebola treatment unit in Monrovia, Liberia between August and October, 2014 have recently been described. 4 Similar to the review by Feldmann and Geisbert, 3 these authors reported that early symptoms of EVD included high fever (up to 40ºC), malaise, fatigue and body aches. After 3 to 5 days, gastrointestinal symptoms began with epigastric pain, nausea, vomiting and diarrhea. Other signs and symptoms included headache, conjunctival infection, chest pain, abdominal pain, arthralgias, myalgias, and hiccups. Clinically significant hemorrhage from the upper or lower gastrointestinal tract or both occurred in fewer than 5% of patients. 4 The virus was isolated from semen 61 days after onset of illness in a man who was infected in a laboratory. 5 Prolonged persistence of virus has been noted in body fluids by PCR in a recent study from Germany. Despite these reports, there have been no documented cases of transmission beyond the 3-week isolation period currently enforced Investigational Products GSK Biologicals investigational ChAd3-EBOV Z vaccine The ChAd3-EBOV Z vaccine is a replication incompetent chimpanzeeadenovirus that expresses the full-length EBOV GP. The envelope sequence comes from the 1976 Mayinga strain. It was developed by the Vaccine Research Center of the National Institute of Allergy and Infectious Diseases (VRC/NIAID) in the United States (US) using the Okairos adenovirus vaccine platform technology, which has subsequently been acquired by GlaxoSmithKline (GSK) Biologicals in May The DNA fragment inserted in the ChAd3 vector encodes the Ebola virus GP, which is expressed on the virion surface and is critical for attachment to host cells and catalysis of membrane fusion. A single dose of viral particles (vp) of ChAd3-EBO Z provided 100% protection against infection with Ebola virus 5 weeks after vaccination in cynomolgous macaques. When macaques were challenged 10 months after vaccination, 2 of 4 given the 1x10 11 dose were protected and 0 of 4 of the 1x10 10 dose were protected. 6 A phase 1 study of 2 doses of a two-component chimpanzee adenovirus type 3 vaccine encoding GP antigens from the Zaire and Sudan species of Ebolavirus found that the vaccine was well tolerated with no serious adverse effects. The higher dose, 2x10 11 particle units (PU) was associated with transient fever in 2 of 10 patients. Both doses studied induced GP-Z specific antibodies; geometric mean titers were of greater magnitude at the 2x10 11 PU dose (2037) compared to 11

14 PREVAIL Version 1.0 January 30, 2015 the 2x10 10 PU dose (331) (p=0.001). 7 At the higher dose, these antibody responses were in the range reported to associate with vaccine-induced protective immunity in non-human primate challenge studies. 6,8,9 Safety and immunogenicity data have also been generated from phase 1 studies of the monovalent dose. These studies are described below. Study EBL01 (United Kingdom) In this study, three doses were studied, 1x10 10, 2.5x10 10 and 5x10 10 vp. Twenty participants were to be given each dose. At week 4, geometric mean titer levels were 235 (n=19), 402 (n=20), and 575 (n=20), respectively for the 1x10 10, 2.5x10 10 and 5x10 10 vp doses, For each dose, these geometric means were significantly higher than means at week 2. Most adverse events for participants in this study were of mild severity. The most commonly reported local adverse event was pain at the injection site (35/54 participants; 64.8%). The most commonly reported general adverse events were fatigue (34/54 participants; 63.0%); headache (28/54; 51.9%); myalgia (23/54; 42.6%); malaise (21/54; 38.9%); and feverishness (16/54; 29.7%). At the two higher doses fever was reported by one participant on each dose. In each case, fever occurred within 24 hours and did not persist for more than 24 hours. Three severe adverse events were reported (feverishness, nausea, and malaise), all at the 2.5x10 10 vp dose. Transient mild lymphopenia was observed the day after vaccination for 3 participants in the 2.5x10 10 dose group and 6 participants in the 5x10 10 group. Moderate lymphopenia was observed for 1 participant in the 2.5x10 10 dose group and 2 participants in the 5x10 10 group. Prolonged activated partial thromboplastin was observed in 4 participants within the 2 weeks following vaccination (3 on the 2.5x10 10 dose and 1 on the 5x10 10 vp dose). The elevation fully resolved in 2 participants by 28 days and repeat testing is awaited for the other two participants. Study CVD01 (Mali) In this ongoing study, two doses were studied, 5x10 10 and 1x10 11 vp. Twenty participants were to be given each dose. At week 4, geometric mean titer levels were 523 (n=20) and 1471 (n=11), respectively for the 5x10 10 and 1x10 11 vp doses, These geometric means differed significantly from one another. Safety data are not yet available. Swiss Study 12

15 PREVAIL Version 1.0 January 30, 2015 In this ongoing study, two doses were studied, 2.5x10 10 and 5x10 10 vp. Ten participants were to be given each dose. Data are not yet available. Based upon these data the current plan is to use a dose of 1 x for the ChAd3-EBOV Z vaccine in this trial. Serological studies in 193 healthy individuals belonging to different geographical areas in the United States and Europe showed a low seroprevalence for antibodies to ChAd3, and when present, antibody titres were low. 10 ChAd3 is a Type C adenovirus similar to adenovirus type 5 (Ad5), a common adenovirus infection of humans. ChAd3 vectors display a potency and induce an innate immune profile similar to recombinant Ad5 vectors (rad5). The rad5 vector has been extensively studied in humans. In addition, extensive preclinical studies using rad5 EBOV have demonstrated protection against EBOV challenge. 8,11 Adenovirus type 5 (Ad5; a common adenovirus infection of humans) pre-existing immunity did not appear to cross-react with ChAd3 in mice. 12 ChAd3-based vaccines were capable of inducing an immune response and protection comparable to human Ad5 vectored vaccine even in those with pre-existing immunity against Ad NewLink/Merck Vesicular Stomatitis virus-based vaccine (VSV) The Vesicular Stomatitis Virus-based vaccine (VSV G-ZEBOV) was licensed from the Public Health Agency of Canada (PHAC) by NewLink/Merck and is being developed with the help of the Defense Threat Reduction Agency (DTRA), the Walter Reed Army Institute of Research (WRAIR) and the US Department of Health and Human Services. The VSV G-ZEBOV vaccine is comprised of a single recombinant VSV isolate (11481 nt) modified to replace the gene encoding the G envelope glycoprotein with the gene encoding the envelope glycoprotein from ZEBOV (Kitwit, 1995 strain). The initial studies of the VSV G-ZEBOV vaccine demonstrated 100% protection against lethal ZEBOV challenge in 4 cynomolgus macaques following a single intramuscular immunization of 1 x 10 7 plaque forming units (pfu). 13 At the same dose, this vaccine was also able to protect cynomolgus macaques from homologous aerosol challenge. 14 Delivering 2 x 10 7 pfu of laboratory-grade vaccine by the oral (n=4), intranasal (n=40), or intramuscular (n=2) route 28 days prior to challenge protected cynomolgus macaques from subsequent ZEBOV challenge. 15 Interestingly, the level of neutralizing antibodies was low by all routes of immunization, highlighting the uncertainty regarding immunologic correlates of protection to Ebola virus infection. VSV G-ZEBOV vaccine also protected 4 of 8 (50%) of rhesus macaques when administered 30 minutes following challenge. 16 While all macaques developed fever and lymphopenia, 50% of the animals did not progress to more severe disease, including alterations in clinical chemistry and macular rash. Viremia with rvsv was detected on day 3 post-immunization in most animals. ZEBOV viremia in survivors was 2 to 4 logs lower than in non-survivors. Of note, animals treated 13

16 PREVAIL Version 1.0 January 30, 2015 with rvsv did not show a decrease in natural killer (NK) cells following challenge but instead showed a substantial increase. The clinical trial material (CTM) for this study has undergone challenge testing in murine and non-human primate models of pre- and post-exposure prophylaxis. In the murine model, the pre-exposure prophylaxis study include mice that were immunized with 20,000, 200, 2 and 0.02 pfu (n=10 per group). The mice were challenged 28 days after immunization with 1000 LD 50 of mouse-adapted ZEBOV. All mice immunized with 200 or 20,000 pfu survived. In the postexposure prophylaxis study, mice were challenged with 1000 LD 50 of mouseadapted ZEBOV and then treated 30 minutes and 2 hours later with graded doses of 2x10 5, 2x10 4, 2x10 3 and 2x10 2 pfu (10 per group). When treated 2 hours after the challenge, doses of 2x10 5, 2x10 4, and 2x10 3 were % effective in preventing death, Treatment within 30 minutes was not effective, with the majority of mice dying at each dose. In rhesus macaques, the pre-exposure prophylaxis study include 4 cynomologous macaques who were inoculated with 1x10 8 pfu and 2 controls. The animals were challenged 28 days later. The two controls died 6 days after challenge and all 4 treated animal survived. In the post-exposure prophylaxis study 12 rhesus macaques were challenged with 3000 TCID 50 of ZEBOV Kitwit. At 30 minutes or 24 hours after challenge, groups of 6 macaques were vaccinated with 2x10 8 pfu of clinical grade rvsv G-ZEBOV GP. Two of 6 (33%) treated 30 minutes after challenge survived but none of the animals treated at 24 hours survived. Two controls died within 10 days. 17 The kinetics of VSV G-ZEBOV vaccine replication in vitro suggest it replicates substantially faster than wild-type ZEBOV and interference with ZEBOV replication via competition for receptor and/or for intracellular resources is a potential mechanism for its effects as a strategy for post-exposure prophylaxis. 18 In contrast to control animals, which undergo a reduction in lymphocytes during infection, vaccinated animals either showed a stable or small increase in CD3+, CD4+, and CD8+ T cells. Several phase 1 studies of the VSV G-ZEBOV vaccine are ongoing. Preliminary results from these studies which support the dose to be used in the trial of 2x10 7 pfu/ml are summarized below. Studies NLG0307 (WRAIR) and NLG0207 (NIH Clinical Center) These two phase 1 studies were designed similarly. Participants were enrolled in 3 cohorts of 13 participants each (10 received the vaccine and 3 received placebo). Data for the first two cohorts, which received the 3x10 6 and 2x10 7 pfu/ml doses, respectively, are available. These data remain blinded so the results presented below for both studies combined include participants who received placebo. Sera from both studies are being tested in 2 laboratories (VRC/NIH and USAMRIID), using ELISAs employing Ebola GP antigens from the Mayinga and 14

17 PREVAIL Version 1.0 January 30, 2015 Kikwit strains, respectively. In the VRC assay, geometric mean titer levels using ELISA assay were similar for each dose group prior to vaccination, 108 and 106 respectively for the 3x10 6 and 2x10 7 pfu/ml dose groups (n=26 per dose). At week 4, geometric means were 457 and 921, respectively, for the 3x10 6 and 2x10 7 pfu/ml dose groups. If one subtracts the 3 lowest values from the NIH groups and the 2 lowest from the WRAIR group each group (assuming these represent the placebo controls) the geometric means (backgrounds subtracted) become 323 and The USAMRIID ELISA was performed on 10 subjects from the WRAIR trial who had received 3x10 6 pfu and had virus in plasma detected by PCR (thus received vaccine, not placebo). Ten of 10 subjects seroconverted and had geometric mean titers of 400 on Day 14 and 1300 on Day 28. Neutralizing antibodies were measured also. Eight of 10 and 10 of 10 of the participants developed neutralizing antibodies by days 14 and 28 respectively, with geometric mean titers of 40 and 300. Safety data from both trials were combined for analysis, and are still blinded, The most common adverse events over 28 days of follow-up (3x10 6, 2x10 7 dose) were headache (15/26 and 15/26); fatigue (15/26, 14/26); myalgia (11/26, 14/26); pyrexia (10/26, 12/26); chills (6/26, 10/26); arthralgia (6/26, 5/26); lymphopenia (7/26, 0/26); nausea (2/26, 5/26); and hyperhidrosis (1/26, 5/26) Most events occurred within 3 days after vaccination. All were grade 1 or 2. There were no cases of arthritis. The main laboratory abnormality were a transient lymphopenia occurring in the first day after inoculation. Three of 52 participants had small oral ulcerations and one had a monocular conjunctivitis. Three of 52 participants had cervical or axillary lymphadenopathy. Viral RNA was measured in plasma or serum by RT-PCR. Viremia was detected in all participants during the first 3 days after inoculation, and was rarely detected on Day 7 and not on day 14. There was no difference in viremia or shedding across the doses tested. Shedding in urine and saliva was detected only in one person. Study CI1401 This study randomized 10 participants to each of 4 groups: 1) 3x10 5 ; 2) 5x10 5 3) 3x10 8 ; and 4) placebo. The data remain blinded. Data on antibody titers are not available. Common systemic adverse events included subjective fever (pyrexia), fatigue, headache, myalgia, chills, and arthralgia. These symptoms were generally mild (grade 1-2), with occasional grade 3 but no grade 4 events. There have been no cases of arthritis. Study NLG0507 This a large phase 1 study being conducted by 8 sites in the United States. Patients will be randomized to one of 5 groups: 1) 3x10 6 (n=64); 2) 2x10 5 (n=64) 3) 1x10 4 (n=64); 4) 1x10 3 (n=64); and 5) placebo (n=74). To date 60 participants have been enrolled. Data are not yet available. Geneva Trials 15

18 PREVAIL Version 1.0 January 30, 2015 Participants were randomized either to 1x10 7 or 5x10 7 pfu if they would later work in an Ebola epidemic area; if they were not going to be deployed, participants were randomized to one of these two doses or placebo (3 groups). Among the first 39 participants, 4 cases of mild or moderate arthritis were observed and the study was temporarily halted. As of December 29, 2014, there have been 10 cases of confirmed or probably arthritis among 59 participants (51 who received either the 1x10 7 or 5x10 7 dose). Among the 10 subjects with joint symptoms, the clinical pattern was that of an oligoarthritis, involving 2-4 joints, sometimes with a migratory pattern, which predominantly affected the small joints of the hands and feet, but also the ankles, the knees, the wrists and possibly the sacroiliac joints. The symptoms are mild (grade 1-2). Eight of 10 cases have been diagnosed either by objective clinical signs or by ultrasound; in 2 of the cases no objective clinical signs were present and diagnosis was by ultrasound alone. Severity is mild to moderate (grade 1-2); no serious adverse event has occurred. The arthritis events are self-limited, with median duration of 11 days. The etiology appears to be a viral induced arthritis. Viral RNA was found at low levels in joint fluid from 1 participant. Three of the participants with arthritis also had a sparse maculopapular rash or vesicular lesions on the legs. Viral RNA and virus isolation was confirmed from vesicle fluid from 1 participant. Viral RNA was detected in the plasma of most participants on Day 1 (15 of 19; 79%) and Day 3 (15 of 19 participants, 79%). No viral RNA was detected in any participant on Day 7. Viral RNA loads were low on Days 1 and 3. The median rvsv vaccine copy number/ml of plasma was 328 (range <30-625); the ratio of genome equivalents to infectious units is ~100:1 in the PCR assay, so the levels in plasma represent minimal infectious virus. Immune response data are not yet available. The trial was resumed with the 1x10 5 dose on January 5, Hamburg Trial In this trial, 3 cohorts of participants (n=10 each) will be given the 3x10 6, 2x10 7 or 5x10 7 pfu dose. As of January 6, 2015, 20 participants have been given the first two doses. The most common adverse events in both dose groups were swelling or pain at injection site (8/10 for each dose group) and myalgia (5/10 for each dose group). Five participants had grade 3 (severe) events (2 menstrual pain, 1 neck tension, 1 arthralgia, and 1 weakness). All 20 participants experienced a decrease in lymphocytes within 24 hours which returned to normal between 3 and 7 days. A decrease in platelets was observed in 7 participants; for 4 of these participants levels had returned to baseline between days 3 and 7. An increase of the orally measured body temperature compared to the baseline was observed in 3/10 participants in the 3x10 6 dose group and in 4/10 in the 2x10 7 dose group. The absolute values with a maximum reached about 36 hours after vaccination were between 37.0 C and 38.6 C. One of 20 participants developed an adverse event compatible with arthritis, but had a past history of similar symptoms. Immune response data are not yet available. 16

19 PREVAIL Version 1.0 January 30, 2015 Gabon Trial In this open-label trial, 2 cohorts of healthy adults were to receive 3x10 5 (n=20) or 3x10 6 (n=20) or 1x10 7 (n=20) pfu. As of January 6, 2015, 20 participants have been given the 3x10 5 dose and 8 have received the next highest dose. Data are not yet available Placebo The placebo vials will contain sterile normal saline (sodium chloride). Since the two vaccines require different volumes for injection, two placebos will be used, a 1 ml and a 2 ml preparation Rationale for the study and study design Rationale for the study EVD continues to spread in some areas of West Africa and there is a critical need for a vaccine to prevent EVD. The current outbreak has placed unprecedented burdens upon health care systems and workers. There are two candidate Ebola virus vaccines that have reached an advanced stage of evaluation, the chimpanzee adenovirus 3 (ChAd3-EBO Z) - based vaccine and the Vesicular Stomatitis virus (VSV G-ZEBOV) - based vaccine. This study will evaluate both of these vaccines in a randomized, double-blind, placebocontrolled study in Liberia. This clinical trial to evaluate vaccine efficacy (VE) will provide an accurate assessment of the benefits and risks associated with each candidate vaccine and inform policy on wider scale vaccination in countries at risk Rationale for the study design Experts at the WHO Consultation on Ebola vaccines recommended that these two vaccine candidates be rapidly evaluated for their efficacy and safety without compromising international standards. The experts stated that, if feasible, randomized controlled trials were the design of choice because they would provide the most robust data in the shortest amount of time. This study responds to the WHO recommendations in the following ways: Two vaccines are studied and compared against a single placebo group which is more efficient than two separate trials. With this approach, information on the efficacy and safety of both vaccines will be obtained more rapidly than in two separate trials. A placebo group is used because there is no known licensed treatment to prevent EVD. Blinding afforded by the use of a placebo also reduces bias associated with the ascertainment and documentation of the primary EVD endpoint and safety outcomes. This is the quickest and most reliable way to determine that a vaccine is safe and effective. A prior study of a recombinant adenovirus investigational vaccine for HIV 17

20 PREVAIL Version 1.0 January 30, 2015 yielded the unexpected result that those receiving the vaccine had an increased incidence of infection. This was despite evidence of protection in the non-human primate model, thus underscoring the uncertainty in moving from animal studies to human studies and the potential for harm with an investigational vaccine. The trial will be initiated in Monrovia, Liberia in order to rapidly establish the necessary clinical trial infrastructure to do the study in a population that has experienced a high rate of EVD. Depending on the course of the epidemic, additional sites may be added in other parts of Liberia. Several phase 1 studies for the two vaccines to be evaluated have recently been completed. These phase 1 studies have resulted in sufficient information to establish the dose of each vaccine to be administered. However the safety database in a West African population at the doses to be used is limited. Thus a substudy of 600 volunteers at one or two sites will be embedded in this clinical trial. The safety data from this substudy will be closely monitored by an independent Data and Safety Monitoring Board (DSMB). This substudy is referred to as a phase 2 substudy because it will provide important information on safety and immunogenicity typically included in a phase 2 clinical trial. This substudy will precede the initiation of enrolment at other vaccination centers. The DSMB will review the data from this substudy and be asked to make a recommendation on when enrolment should be expanded to other sites. In addition, the same independent DSMB will closely monitor the trial to rapidly identify any safety problems during the course of the full study and to determine if there is early evidence that one or both of the vaccines are clearly and substantially beneficial or early evidence that one or both of the vaccines are clearly of no benefit. 2. METHODOLOGY 2.1. Study design This is a randomized, placebo-controlled trial of two EVD vaccines. Since different volumes are required for each vaccine, two placebo preparations will be prepared for administration, syringes with a 1 ml and a 2 ml saline fill volume. Study volunteers will be randomized in a 2:1:2:1 to ChAd3-EBO Z (2 ml), 2 ml of placebo, VSV G-ZEBOV (1 ml), or 1 ml of placebo (see Figure 1). The trial is event-driven with a target of 112 primary events for each vaccine versus the pooled placebo comparison. Thus, follow-up will continue until the targeted number of primary events occurs or until the independent DSMB recommends otherwise. To complete the trial in one year, it is estimated that over 27,000 volunteers will need to be enrolled over 4 months and followed for at least 8 months, to achieve the target number of events required to reliably establish the efficacy of each vaccine. For example, as illustrated in Figure 1 below, if the one 18

21 PREVAIL Version 1.0 January 30, 2015 year incidence of EVD is 1.0% in the pooled placebo group, 9,390 volunteers per arm are required (28,170 volunteers total) (see section 2.6 for details) to obtain the desired number of events. Initially, the study will be conducted at multiple sites in and around the city of Monrovia, Liberia. Depending on the course of the epidemic, additional sites may be established in other parts of Liberia. Figure 1 Study design overview Liberia residents aged 18 years N=28,170 Randomized 2:1:2:1 VSV G-ZEBOV Vaccine (1 ml) (N = 9,390) VSV G-ZEBOV Placebo (1 ml) (N = 4,695) ChAd3-EBO Z Vaccine (2 ml) (N = 9,390) ChAd3-EBO Z Placebo (2 ml) (N = 4,695) First 600 volunteers at one or two designated vaccination centers Subsequent 27,570 volunteers Visits at week 1 and month 1 Contact at week 1 and month 1 Monthly contact through a common closing date 19

22 PREVAIL Version 1.0 January 30, Primary objectives To determine the efficacy of a single intramuscular dose of the ChAd3 EBO-Z vaccine compared with a pooled placebo group in the prevention of definite EVD occurring 21 days or more following randomization. To determine the efficacy of a single intramuscular dose of the VSV G- ZEBOV vaccine compared with a pooled placebo group in the prevention of definite EVD occurring 21 days or more following randomization. All randomized participants will be provided information on how to minimize risk of infection with Ebola Primary Hypotheses Vaccination with the ChAd3 EBO-Z will reduce the incidence of definite EVD occurring 21 days or more following randomization compared with pooled placebo. Vaccination with the VSV G-ZEBOV will reduce the incidence of definite EVD occurring 21 days or more following randomization compared with pooled placebo. The success criterion for each vaccine versus pooled placebo comparison requires the lower bound of the 97.5% confidence interval for vaccine efficacy be greater than 0% Secondary objectives To compare the ChAd3 EBO-Z vaccine and the VSV G-ZEBOV vaccine with pooled placebo for the following other outcomes: Serious adverse events within 30 days of vaccination Definite EVD occurring at any time after randomization Definite or probable EVD occurring 21 days or more following randomization Definite or probable EVD at any time after randomization Deaths attributed to definite or probable EVD All-cause mortality Serious adverse events at any time after randomization Duration of EVD for surviving participants Grade 3 or 4 events (600 participant substudy) Ebola-specific antibodies over one week and one month of follow-up (600 participant substudy) Vaccine injection site reactions of any grade severity (600 participant substudy) Targeted signs and symptoms during the first month following vaccination of any grade severity (600 participant substudy) 20

23 PREVAIL Version 1.0 January 30, 2015 Volunteered adverse events of any grade severity (600 participant substudy) 2.5. Endpoints The primary efficacy endpoint is definite EVD occurring 21 days or more following randomization through a common calendar closing date for the trial. Definitions are given in section 4.1. Because there may be uncertainty about the EVD diagnoses, all suspected EVD events will be collected and reviewed by an Endpoint Review Committee (ERC). The ERC will be asked to assign a certainty level to their diagnosis of EVD. Deaths will also be reviewed by the ERC and classified as due to EVD or other causes. In addition to the inclusion of deaths due to EVD as part of the primary endpoint, this information will be used to assess whether the vaccines influence the EVD case-fatality rates. The primary safety endpoint is serious adverse events occurring within 30 days of vaccination. Serious adverse events will be reported for the entire duration of follow-up, which will end on a common closing date for all volunteers. Serious adverse events that occur any time after randomization are a secondary endpoint Sample size and statistical considerations This is an event-driven trial with 112 primary events required for each of the two vaccine versus placebo comparisons. The following assumptions were made in computing the event targets and sample size. The primary analysis will be modified intention to treat using EVD outcomes that occur 21 days or more following randomization; a Cox model for time to EVD with a single indicator for the active vaccine will be used to estimate the hazard ratio (HR) for each pair-wise comparison: ChAd3 EBO-Z vaccine versus pooled placebo and VSV G-ZEBOV versus pooled placebo. For each primary comparison there is 1:1 allocation for vaccine and placebo (1 ml and 2 ml placebo groups will be pooled). Type 1 error = (2-sided), adjusted for the two primary comparisons (0.05/2). Power = 0.90 to detect vaccine efficacy of 50% (1-HR)x100. Based on these assumptions, the target number of primary events is 112 for each vaccine versus pooled placebo comparison. 21

24 PREVAIL Version 1.0 January 30, % of volunteers in the pooled placebo group will develop EVD after 12 months. Volunteers will be enrolled over a 4 month period and followed for a minimum of 8 months (average follow-up is 10 months and range is 8 to 12 months) Deaths unrelated to EVD and losses to follow-up will occur at the rate of 1% per month. With these additional assumptions, 28,170 volunteers, 9,390 per active vaccine arm and 4,695 per each of the two placebo groups, are needed to obtain the required number of primary events. There is uncertainty about these assumptions. As Table 1 below indicates, sample size to achieve the target number of events varies substantially by the control event rate. Thus, sample size assumptions will be re-evaluated using pooled (both vaccine groups and the pooled placebo groups combined) EVD outcome data during the trial by the protocol team. Based on these evaluations, key design parameters may be changed. For example, enrolment may continue until the target number of events is nearly achieved. Table 1: Combined sample size (and number per group) for comparing each vaccine versus placebo for EVD (90% power to detect 50% vaccine efficacy; (2-sided) level of significance). Percent with Ebola after 12 Months in the Pooled Placebo Group (%) Combined Sample Size (number per arm) ,560 (12,520) ,170 (9,390) ,660 (7,220) ,580 (5,860) ,790 (4,930) ,780 (4,260) ,250 (3,750) For the phase 2 substudy of 600 volunteers (200 on each arm) at one of the vaccination centers, Table 2 gives the power to detect a statistically significant difference in the proportion with grade 3 or 4 adverse events between those in the vaccine group and those in the pooled placebo group for a range of true event rates. For this outcome and the immunogenicity outcome, there is no 22

25 PREVAIL Version 1.0 January 30, 2015 adjustment to type 1 error for the two pair-wise comparisons planned. Power is good (80% or greater) to detect an adverse event that occurs in at least 6% of vaccines and no more than 1% of volunteers in the pooled placebo group, or one that occurs in at least 13% of vaccine recipients and no more than 5% of those in the pooled placebo group. Table 2. Power to detect a difference in Grade 3 or 4 adverse events at one month between the vaccine group and the pooled placebo group. Calculations are based on a two-sided unconditional score test at the.05 level (2-sided) level of significance, with no adjustment for multiple comparisons. Sample Size Proportion with Event Pooled placebo arm Vaccine arm Pooled placebo arm Vaccine arm Power n=200 n= Table 2 is also relevant for potential observed rates of immune response. For example, if 5% of the 200 pooled placebo group participants have an immune response to Ebola at month one, we will have good power to find an immunological effect of the vaccine as long as the response rate is at least 13%. Assuming low rates of antibody levels among the placebo recipients, rates in the vaccine groups by themselves are of interest. Confidence intervals for different observed immune responses in the vaccine groups are given in Table 3. The widths of the 95% confidence intervals for the response rates in one of the vaccine groups for four possible observed immune responses are shown. If 70% of the 200 participants assigned to one of the vaccine groups has a positive immune response, the 95% confidence interval for the true response rate will be from 63% to 76%. Table 3 Exact Binomial Confidence Intervals for a range of possible observed percents with an immune response Observed Rate of Immune Response N=200 30% (24%, 37%) 50% (43%, 57%) 70% (63%, 76%) 90% (85%, 94%) 23

26 PREVAIL Version 1.0 January 30, 2015 Like the sample size for the main study, there is uncertainty about the sample size assumptions for the phase 2 substudy. Thus, sample size assumptions will be re-evaluated by the protocol team using pooled (both vaccine groups and the pooled placebo groups combined) immunogenicity and safety data prior to completing enrolment Study cohort The study will be initiated in approximately 10 vaccination centers in existing health facilities in and around Monrovia, Liberia. If necessary depending on the course of the epidemic, additional vaccination centers may be established in other parts of Liberia. As part of the public health campaign to prevent EVD, there will be widespread communication about the trial to encourage volunteers to go to a vaccination center near where they live. There will be significant outreach efforts that will target health care workers and other persons likely to have contact with patients with EVD, e.g. ambulance drivers, burial crews. In general, efforts will be made to include high risk individuals based on the epidemiology of the disease Inclusion criteria The inclusion criteria for the study are broad reflecting the target population that would eventually receive an efficacious vaccine. Informed consent Age 18 years Likely to be in the surrounding area of the vaccination center for at least one year Exclusion criteria Fever > 38.0º Celsius History of EVD (self-report) Current pregnancy (a negative urine pregnancy test is required for women of child-bearing potential) Breast-feeding an infant Any condition which would limit the ability of the participant to meet the requirements of the study protocol (for example, any serious illness) 2.8. Randomization and blinding Randomization occurs when the vaccine/placebo is administered. Eligible volunteers will be randomized in a 2:1:2:1 allocation to ChAd3-EBO Z (2 ml), 2 ml of placebo, VSV G-ZEBOV (1 ml), or 1 ml of placebo. The randomization schedule will be prepared using permuted block randomization to ensure the 24