Supplementary Online Content

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1 Supplementary Online Content Milligan ID, Gibani MM, Sewell R, et al. Safety and immunogenicity of novel adenovirus type 26 and modified vaccinia Ankara vectored Ebola vaccines: a randomized clinical trial. JAMA. doi: /jama eappendix 1. Supplementary methods eappendix 2. Supplementary results etable 1. Study time and events schedule etable 2. Ebola glycoprotein specific CD8+ responses as assessed by intracellular cytokine staining etable 3. Ebola glycoprotein specific CD4+ responses as assessed by intracellular cytokine staining efigure 1. Ebola glycoprotein specific antibody responses in ELISA units/ml efigure 2. Ebola glycoprotein specific antibody response expressed as endpoint titers efigure 3. Ebola glycoprotein specific T cell responses as assessed by interferon γ ELISpot efigure 4. Intracellular cytokine staining (CD8) efigure 5. Intracellular cytokine staining (CD4) efigure 6. CD8+ Intracellular cytokine staining efigure 7. CD4+ Intracellular cytokine staining (ICS) This supplementary material has been provided by the authors to give readers additional information about their work.

2 eappendix 1: Supplementary methods Inclusion criteria Each potential participant satisfied all of the following criteria at screening to be considered eligible for enrolment in the study: 1. Participant must be able to read and provide consent after completing the informed consent process. 2. Participant must be a man or woman aged 18 to 50 years. 3. Participant must be healthy on the basis of physical examination, medical history, and the investigator s clinical judgment. 4. Participant must meet the following laboratory criteria within 28 days before Day 1 in the main study and within 56 days before Day 1 in the substudy*: Hemoglobin: women: 11.5 g/dl; men 12.5 g/dl Note: based on the local laboratory s normal reference ranges. White blood cell count: 3,501 to 10,999 cells/mm3, inclusive Platelets: 130,001 to 550,000 per mm3, inclusive Urinalysis (clean urine sample): protein and blood <1+, glucose negative Note: for women: in case of menstruation, urinalysis must be postponed but a result should be available before Day 1. Alanine aminotransferase/aspartate aminotransferase (ALT/AST) 1 x institutional upper limit of normal Serum creatinine 1 x institutional upper limit of normal Troponin I 1 x institutional upper limit of normal Fibrinogen 1 x institutional upper limit of normal Prothrombin time 1 x institutional upper limit of normal Activated partial thromboplastin time 1 x institutional upper limit of normal. * If laboratory screening tests are out of range, repeat of screening tests is permitted once, provided there is an alternative explanation for the out of range value. 5. All women of childbearing potential must:

3 Have a negative serum β human chorionic gonadotropin (β hcg) pregnancy test at screening Have a negative urine β hcg pregnancy test immediately prior to each study vaccine administration Practice adequate birth control measures from 28 days before the prime vaccination until at least 3 months after the boost vaccination. The following birth control measures will be considered adequate: Combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation (oral, intravaginal, and transdermal); progestogen only hormonal contraception associated with inhibition of ovulation (oral, injectable, and implantable); intrauterine devices (IUD) and intrauterine hormone releasing systems (IUS); vasectomized partner Abstinence (defined as refraining from heterosexual intercourse during participation in the study [from 28 days before the prime vaccination until at least 3 months after the boost vaccination]) If not heterosexually active at screening, must agree to practice adequate birth control measures if they become heterosexually active during participation in the study (from the start of screening onwards until at least 3 months after the boost vaccination). Agree not to donate eggs (ova, oocytes) for the purposes of assisted reproduction from the start of screening onwards until at least 3 months after the boost vaccination. 6. Women of non childbearing potential, defined as postmenopausal (>45 years of age with amenorrhea for 2 years or any age with amenorrhea for 6 months and serum folliclestimulating hormone [FSH] >40 miu/ml) or surgically sterile (hysterectomy, bilateral tubal ligation, or bilateral oophorectomy), are not required to use the birth control methods as described in Inclusion Criterion #5. 7. A man who has not had a vasectomy and is sexually active with a woman of childbearing potential must use a double barrier method of birth control, such as either condom with spermicidal foam/gel/film/cream/suppository or partner with occlusive cap (diaphragm, cervical/vault caps) with spermicidal foam/gel/film/cream/suppository. In case the female

4 partner is using an adequate method of birth control (see Inclusion Criterion #5), a single barrier method of birth control for the male participant is acceptable. Men must also agree not to donate sperm from the start of screening onwards until at least 3 months after the boost vaccination. 8. Participant must be available and willing to participate for the duration of the study visits and follow up. 9. Participant must be willing to provide verifiable identification. 10. Participant must be willing to provide his/her National Insurance/Passport number for the purpose of The Over volunteering Prevention System (TOPS) registration. 11. Participant must have a means to be contacted. Exclusion criteria Any potential participant who met any of the following criteria was excluded from participating in the study: 1. Has been vaccinated with a candidate Ebola vaccine. 2. Has been diagnosed with Ebola disease or exposed to Ebola including travel to West Africa in the last 12 months. West Africa includes but is not limited to the countries of Guinea, Liberia, Mali, and Sierra Leone. 3. Has received any Ad26 or MVA based candidate vaccine in the past. 4. Known allergy or history of anaphylaxis or other serious adverse reactions to vaccines or vaccine products (including any of the constituents of the study vaccines [eg, polysorbate 80, ethylenediaminetetraacetic acid (EDTA) or L histidine for Ad26.ZEBOV vaccine; and tris (hydroxymethyl) amino methane (THAM) for MVA BN Filo vaccine]), including known allergy to egg, egg products and aminoglycosides. 5. Participants with acute illness (this does not include minor illnesses such as diarrhea or mild upper respiratory tract infection) or temperature 38.0ºC on Day 1 will be excluded from enrollment into the study. 6. Chronic active hepatitis B or hepatitis C infection, documented by hepatitis B surface antigen (HBsAg) and hepatitis C antibody, respectively. 7. HIV type 1 or type 2 infection.

5 8. A woman who is pregnant or breast feeding, or planning to become pregnant while enrolled in the study or within 3 months after the boost vaccination. 9. Bleeding or clotting disorders. 10. Any clinically significant acute or chronic medical condition that, in the opinion of the investigator, would preclude participation (eg, history of seizure disorders, autoimmune disease, any spleen disease, active malignancy, active tuberculosis, asthma, other systemic infections, abnormal laboratory safety parameters*). * Grade 1 abnormalities for laboratory tests other than those covered in Inclusion Criterion #4 are not an exclusion criterion. If laboratory tests other than those covered in Inclusion Criterion #4 are out of range (grade 2 or grade 3), repeat of screening tests is permitted once, provided there is an alternative explanation for the out of range value. 11. History of malignancy other than squamous cell or basal cell skin cancer, unless there has been surgical excision that is considered to have achieved cure. Participants with a history of skin cancer must not be vaccinated at the previous tumor site. 12. Post organ and/or stem cell transplant whether or not with chronic immunosuppressive therapy. 13. Major surgery (per the investigator s judgment) within the 4 weeks prior to study entry or planned major surgery through the course of the study. 14. History of myocarditis, pericarditis, cardiomyopathy, transient ischemic attack or stroke, myocardial infarction, angina, coronary artery disease, congestive heart failure, clinically significant arrhythmia (including any arrhythmia requiring medication, treatment, or clinical follow up). 15. Electrocardiogram (ECG) with clinically significant findings, or features that would interfere with the assessment of myocarditis/pericarditis, including any of the following: Conduction disturbance (complete left or complete right bundle branch block or nonspecific intraventricular conduction disturbance with QRS 120 ms, PR interval 210 ms, any second or third degree atrioventricular block, or prolongation of the QT interval corrected according to Fridericia s formula [QTcF] [>450 ms]) Significant repolarization (ST segment or T wave) abnormality. Significant atrial or ventricular arrhythmia; frequent atrial or ventricular ectopy (eg,

6 frequent premature atrial contractions, 2 premature ventricular contractions in a row). ST elevation consistent with ischemia or evidence of past or evolving myocardial infarction. 16. History of diabetes mellitus type 1 or type 2, including cases controlled with diet alone. Note: history of isolated gestational diabetes is not an exclusion criterion. 17. Thyroidectomy, or thyroid disease requiring medication during the last 12 months. 18. Uncontrolled hypertension, defined as systolic blood pressure 140 mmhg at enrollment or diastolic blood pressure 90 mmhg at enrollment. Note: In case of diastolic values greater than 90 mmhg or systolic values greater than 140 mmhg, the measurement can be repeated after 30 minutes rest and the participant can be enrolled in case of 2 consecutive measurements fulfilling the inclusion criterion. 19. Major psychiatric illness and/or substance abuse problems during the past 12 months that in the opinion of the investigator would preclude participation. 20. Receipt of live attenuated vaccines from 30 days before Day 1 or receipt of any other vaccine in the period from 15 days before Day Use of experimental therapeutic agents within 3 months from the start of screening. 22. Current or planned participation in another clinical study during the study period. Note: participation in an observational clinical study is allowed. 23. Receipt of blood products or immunoglobulin in the past 3 months. 24. Donation of a unit of blood within 8 weeks before Day 1 or plans to donate blood during participation in the study (from the start of screening onwards). 25. Current or past abuse of recreational or narcotic drugs, which in the investigator s opinion would compromize the participant s safety and/or compliance with the study procedures. Note: urine will be tested to check for current use of amphetamines, benzodiazepines, cocaine, cannabinoids, and opioids. 26. Current alcohol use judged by the investigator to potentially interfere with participant study adherence. 27. History of chronic urticaria (recurrent hives). 28. Chronic or recurrent use of medications which modify host immune response, eg, cancer chemotherapeutic agents, parenteral corticosteroids. 29. Participant cannot communicate reliably with the investigator.

7 30. Participant who, in the opinion of the investigator, is unlikely to adhere to the requirements of the study. 31. Study site employees and family members of the investigator. Pausing criteria The following pausing rules applied to the Sentinel Cohort. If triggered, these pausing rules would prevent the start of enrollment of Cohort 1 and trigger a Data Review Committee (DRC) meeting: 1. One participant experiences anaphylaxis or generalized urticaria clearly attributable to study vaccination within 24 hours of the prime vaccination; OR 2. One participant experiences a serious adverse event that is considered to be related to any of the study vaccines; OR 3. One participant experiences a severe (grade 3) unsolicited adverse event that is considered to be related to any of the study vaccines; OR 4. One participant experiences a severe (grade 3) solicited injection site reaction. (The size [measured in mm] of erythema will not be used as a pausing criterion); OR 5. One participant experiences a severe (grade 3) solicited systemic adverse event considered to be related to any of the study vaccines. (Subjective systemic adverse event corroborated by study personnel.); OR 6. Death of a participant. The following pausing rules applied to Cohort 1 of any group in the main study. These pausing rules would prevent the start of enrollment of Cohort 2 and would trigger a DRC meeting: 1. One participant experiences anaphylaxis or generalized urticaria clearly attributable to study vaccination within 24 hours of prime vaccination; OR

8 2. One participant experiences a serious adverse event that is considered to be related to any of the study vaccines; OR 3. One participant experiences a severe (grade 3) unsolicited adverse event that is considered to be related to any of the study vaccines; OR 4. One participant experiences a severe (grade 3) solicited injection site reaction. (The size [measured in mm] of erythema will not be used as a pausing criterion); OR 5. One participant experiences a severe (grade 3) solicited systemic adverse event considered to be related to any of the study vaccines. (Subjective systemic adverse event corroborated by study personnel.); OR 6. Death of a participant. Occurrence of any of the following events after initiation of Cohort 2 in any group in the main study would lead to suspension of further study vaccination in both the main and the substudy, and would trigger a meeting of the DRC to discuss study suspension or discontinuation of further vaccination: 1. One or more participants experience a serious adverse event that is considered to be related to any of the study vaccines; OR 2. One or more participants experience anaphylaxis or generalized urticaria clearly attributable to vaccination with study vaccine; OR 3. Two or more participants experience a severe (grade 3) (non serious) unsolicited adverse event that is considered to be related to any of the study vaccines; OR 4. Two or more participants experience a severe (grade 3) (non serious) laboratory abnormality (including unexplained hematuria) considered to be related to any of the study vaccines; OR 5. Two or more participants who received at least one dose of study vaccine to date experience the same severe (grade 3) (non serious) solicited injection site reaction. (The size [measured in mm] of erythema will not be used as a pausing criterion); OR 6. Two or more participants who received at least one dose of study vaccine to date experience the same severe (grade 3) (non serious) solicited systemic adverse event considered to be related to any of the study vaccines. (Subjective systemic adverse event corroborated by study personnel.); OR 7. Death of a participant.

9 Randomizaton Participants were centrally randomized, using an interactive web response system, to one of four groups (28 day or 56 day interval). Within groups participants were randomly assigned to active vaccine or placebo using randomly permuted blocks. In groups 1 and 3, the block size in the sentinel cohort was 2, with randomization ratio 1:1 (active:placebo), 5 in cohort 1 with randomization ratio 4:1, and 11 in cohort 2 with randomization ratio 10:1. Overall, the randomization ratio was 5:1. In groups 2 and 4, the block size was 6 in both cohort 1 and 2 with randomization ratio 5:1. ELISA Optical densities were read at an absorbance wavelength of 450 nm and optical density responses of samples, positive and negative controls were translated into ELISA units/ml using a reference curve consisting of pooled human vaccine responders representing a 1000 ELISA Units/mL. The reference curve was created by an initial dilution of 1:50 followed by a serial dilution of 10 steps, followed by 4PL fitting. The lower limit of quantification was determined by the analysis of 200 human naïve serum samples at a 1:50 dilution. The optical density results after outlier removal were used to calculate a 95% prediction interval resulting in an optical density value of Back calculation of this value on 20 reference curves resulted in a lower limit of quantification of 36.6 ELISA units/ml. Endpoint titers were determined by expressing or extrapolating the highest serum dilution that produced an optical density reading above the cut off optical density of 0.2. Ad26 neutralization assay Ad26 specific neutralizing antibody titers were assessed by luciferase based virus neutralization assays as described previously and recently optimized and validated for human serum. 1 Serum

10 was heat inactivated and serially diluted by 2 fold dilutions (starting dilution 1:16). Ad26.Luc (108 vp/ml) was added to each well at 500 virus particles per cell. A549 cells were added at 104 cells/well and plates were incubated at 37 C/10% CO 2 for 24 hours. After incubation, plates were frozen and subsequently allowed to thaw at room temperature. Luciferase neolite substrate was added and the lysate was transferred into black/white isoplates. Luminescence counts were recorded on a MicroBeta Trilux. Titers were determined by validated software ANAM (adenovirus neutralization assay macro). The limit of detection for the Ad26 neutralizing antibody assay was 16 IC90, the titer at which 90% neutralization was achieved. T cell assays Samples were stimulated with peptides covering the glycoprotein from Ebola virus (EBOV), Mayinga variant (Think Peptides, UK). A total of 158 peptides (15 amino acid peptides overlapping by 11 amino acids) were pooled into two pools, EBOV GP1 consisting of 77 and EBOV GP2 of 81 peptides. An assay control was used in each of the assays stimulated with a CMV pp65 peptide pool with mer peptides and overlapping by 11 amino acids. Thawing of PBMC for ELISpot and intracellular cytokine staining Frozen PBMC for both assays were thawed and rested overnight at 37 C in RPMI 1640 with 25mM HEPES and L glutamine (Gibco BRL Life Technologies), 10% FBS (Gemini Benchmark), L glutamine (Gibco BRL Life Technologies) and Penicillin Streptomycin (Gibco BRL Life Technologies).

11 IFN γ ELISPOT PBMC ( cells/well) were stimulated with the same peptide pools as used in the ICS (EBOV GP1, EBOV GP2; Think peptide) with a final concentration of 1 μg/ml per peptide. Cells were stimulated in triplicate wells for each EBOV GP peptide pool. Negative control responses were subtracted from peptide stimulated responses on a participant level. Samples were excluded if an average of more than 20 spots/well was detected in the negative controls and if the average value of the PHA response per participant was below 400 spots/well. T cell interferon γ (IFN γ) responses to EBOV GP are expressed as background subtracted spot forming units per 1x10 6 cells. The threshold for detection of a positive response by ELISPOT was ~ IFN γ SFCs/10 6 PBMC (0.005%), based on Russell et al. 2 Intracellular cytokine staining (ICS) from frozen PBMC After resting, 1 x 10 6 PBMC were added to each well. Samples and assay controls were stimulated with peptides (EBOV GP or CMV) for 6 hours with Brefeldin A (Sigma) and anti CD28/anti CD49d (BD Biosciences). Peptide pools (EBOV GP1, EBOV GP2) had a final peptide concentration of 1 μg/ml for each peptide. Staphylococcal Enterotoxin B (SEB) served as a positive control and peptide diluent was used as a negative control. Cells were stained with viable dye, Aqua Dead cell stain (Molecular Probes/Invitrogen) before surface staining was performed at room temperature. After adding FACS Perm II solution (BD Biosciences) cells were stained intracellular at room temperature. A 16 colour staining panel was used with antibodies against; anti CD3 (BioLegend), anti CD4 (BD Biosciences), anti CD8 (BD Biosciences), anti CXCR5 (ebioscience), anti PD 1 (ebioscience), anti CD45RA (BD Biosciences), anti CCR7 (BioLegend), anti CD56 (BioLegend), anti IFNγ, anti TNFα (ebioscience), anti IL 2 (Miltenyi), anti IL 4 (BioLegend), anti IL 21,anti Granzyme B (BD Biosciences), anti CD154 (BioLegend) and anti CD14 (BioLegend). Cells were fixed with 1% of paraformaldehyde (Electron Microscopy Sciences) before acquiring cells with the BD LSR II flow cytometer (BD Biosciences). Analysis was performed using Flow Jo (version 9). Negative control

12 responses were subtracted from peptide stimulated responses. The lower limit of quantification was set at 0.04% based on Horton et al. 3 Fisher s exact test was used to define sample positivity based on total count of tumor necrosis factor α (TNF α), interleukin 2 (IL 2), and/or IFN γ producing T cells for CD4+ and CD8+ cells separately. 3

13 eappendix 2: Supplementary results Serious adverse events The following serious adverse events were experienced; fractured distal radius (MVA/Ad26 D29 recipient); elective tonsillectomy (MVA/Ad26 D29 recipient); bowel perforation following elective colonoscopy (Ad26/MVA D57 recipient); severe gastritis (placebo recipient). None were considered related to the study vaccines.

14 etable1: Study time and events schedule Randomized Groups Open Label Day Group 1 Group 2 Group 3 Group 4 Group 5 1 MVA or placebo (5:1) MVA or placebo (5:1) Ad26 or placebo (5:1) 8 Blood sample Ad26 or placebo (5:1) Ad26 Prime 15 MVA 22 Blood 29 Ad26 or placebo (5:1) Blood sample MVA or placebo (5:1) 36 Blood Blood Blood 50 Blood Blood 57 Ad26 or placebo (5:1) MVA or placebo (5:1) 64 Blood Blood Boost 78 Blood Blood 180 Blood sample* 240 Blood sample* 360 Blood sample* MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein Primary outcome: safety as assessed by: solicited adverse events recorded by participant diary for 7 days following each vaccination all adverse events up to day 21 post boost immunization serious adverse events were monitored throughout the study. Secondary and exploratory outcomes: Blood samples for humoral assays (secondary outcome), peripheral blood mononuclear cell assays (secondary outcome), and intracellular cytokine staining (exploratory outcome). *follow up of active vaccine recipients only Persistence

15 etable2: Ebola glycoprotein specific CD8+ responses as assessed by Intracellular cytokine staining Prime on Day 1, Boost on Day 29 Prime on Day 1, Boost on Day 57 Prime on Day 1, Boost on Day 15 MVA-BN-Filo/ Ad26.ZEBOV/ Placebo MVA-BN-Filo/ Ad26.ZEBOV/ Placebo Ad26.ZEBOV/ Ad26.ZEBOV MVA-BN-Filo Ad26.ZEBOV MVA-BN-Filo MVA-BN-Filo Baseline N Median IQ range (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) Day 8 N Median IQ range (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) N Responder % CI (0%; 21.8%) (0%; 21.8%) (0%; 45.93%) (0%; 21.8%) (0%; 21.8%) (0%; 45.93%) (0%; 21.8%) Day 15 N 14 Median 0.02 IQ range (0.02; ) N 14 Responder 4 (28.6%) 95% CI (8.39%; 58.1%) Day 22 N 11 Median IQ range (0.02; ) N 11 Responder 7 (63.6%) 95% CI (30.79%; 89.07%) Day 29 N Median

16 IQ range (0.02; 0.02) (0.0415; ) (0.02; 0.02) (0.02; 0.02) (0.02; ) (0.02; 0.02) N Responder 0 8 (57.1%) (57.1%) 0 95% CI (0%; 21.8%) (28.86%; 82.34%) (0%; 45.93%) (0%; 21.8%) (28.86%; 82.34%) (0%; 45.93%) Day 36 N Median IQ range (0.02; ) ( ; ) (0.02; 0.02) (0.02; ) N Responder 8 (53.3%) 10 (66.7%) 0 6 (50%) 95% CI (26.59%; 78.73%) (38.38%; 88.18%) (0%; 45.93%) (21.09%; 78.91%) Day 50 N Median IQ range (0.02; ) (0.1088; ) (0.02; 0.02) N Responder 8 (53.3%) 11 (73.3%) 0 95% CI (26.59%; 78.73%) (44.9%; 92.21%) (0%; 52.18%) Day 57 N Median IQ range (0.02; 0.02) ( ; ) (0.02; 0.02) N Responder 0 12 (85.7%) 0 95% CI (0%; 21.8%) (57.19%; 98.22%) (0%; 45.93%) Day 64 N Median IQ range (0.02; ) ( ; ) (0.02; 0.02) N Responder 8 (53.3%) 12 (85.7%) 0 95% CI (26.59%; 78.73%) (57.19%; 98.22%) (0%; 45.93%)

17 Day 78 N Median IQ range (0.02; ) ( ; ) (0.02; 0.02) N Responder 7 (46.7%) 11 (78.6%) 0 95% CI (21.27%; 73.41%) (49.2%; 95.34%) (0%; 45.93%) Day 180 N Median IQ range ( ; ) ( ; ) ( ; ) ( ; ) ( ; ) N Responder 10 (71.4%) 10 (66.7%) 13 (92.9%) 9 (75%) 6 (50%) 95% CI (41.9%; 91.61%) (38.38%; 88.18%) (66.13%; 99.82%) (42.81%; 94.51%) (21.09%; 78.91%) Day 240 N Median IQ range (0.02; ) (0.02; ) ( ; ) ( ; ) ( ; ) N Responder 10 (71.4%) 9 (60%) 13 (92.9%) 11 (84.6%) 6 (60%) 95% CI (41.9%; 91.61%) (32.29%; 83.66%) (66.13%; 99.82%) (54.55%; 98.08%) (26.24%; 87.84%) N: number of participants with data and for responders, number of participants with data at baseline and at post baseline time point; IQ: interquartile range CI: exact Clopper Pearson confidence interval; MVA BN Filo: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein; Ad26.ZEBOV: adenovirus type 26 vector vaccine encoding Ebola glycoprotein. Responders are those negative at baseline and positive post baseline, or positive at baseline with at least 3 fold increase from baseline. Follow up time points differed across groups therefore empty cells represent time points where no follow up visit was scheduled;

18 etable3: Ebola glycoprotein-specific CD4+ responses as assessed by Intracellular cytokine staining Prime on Day 1, Boost on Day 29 Prime on Day 1, Boost on Day 57 Prime on Day 1, Boost on Day 15 MVA-BN-Filo/ Ad26.ZEBOV/ Placebo MVA-BN-Filo/ Ad26.ZEBOV/ Placebo Ad26.ZEBOV/ Ad26.ZEBOV MVA-BN-Filo Ad26.ZEBOV MVA-BN-Filo MVA-BN-Filo Baseline N Median IQ range (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) Day 8 N Median IQ range (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) (0.02; 0.02) N Responder % CI (0%; 21.8%) (0%; 21.8%) (0%; 45.93%) (0%; 23.16%) (0%; 21.8%) (0%; 45.93%) (0%; 21.8%) Day 15 N 14 Median IQ range (0.02; ) N 14 Responder 5 (35.7%) 95% CI (12.76%; 64.86%) Day 22 N 11 Median IQ range (0.02; ) N 11 Responder 5 (45.5%) 95% CI (16.75%; 76.62%) Day 29 N Median IQ range (0.02; ) (0.02; ) (0.02; 0.02) (0.02; ) (0.02; ) (0.02; 0.02) N

19 Responder 2 (13.3%) 6 (42.9%) 0 1 (7.1%) 4 (28.6%) 0 95% CI (1.66%; 40.46%) (17.66%; 71.14%) (0%; 45.93%) (0.18%; 33.87%) (8.39%; 58.1%) (0%; 45.93%) Day 36 N Median IQ range (0.2852; ( ; ( ; (0.02; 0.02) ) ) ) N Responder 14 (93.3%) 10 (66.7%) 0 8 (66.7%) 95% CI (68.05%; (38.38%; (34.89%; (0%; 45.93%) 99.83%) 88.18%) 90.08%) Day 50 N Median IQ range ( ; ) ( ; 0.238) (0.02; 0.02) N Responder 10 (66.7%) 9 (60%) 0 95% CI (38.38%; (32.29%; 88.18%) 83.66%) (0%; 52.18%) Day 57 N Median IQ range (0.02; 0.02) (0.02; ) (0.02; 0.02) N Responder 0 5 (35.7%) 0 95% CI (0%; 23.16%) (12.76%; 64.86%) (0%; 45.93%) Day 64 N Median IQ range ( ; ( ; ) ) (0.02; 0.02) N Responder 10 (71.4%) 10 (71.4%) 0 95% CI (41.9%; 91.61%) (41.9%; 91.61%) (0%; 45.93%)

20 Day 78 N Median IQ range ( ; ) (0.02; ) (0.02; 0.02) N Responder 8 (57.1%) 8 (57.1%) 0 95% CI (28.86%; (28.86%; 82.34%) 82.34%) (0%; 45.93%) Day 180 N Median IQ range (0.02; ) ( ; ) (0.02; ) (0.02; ) (0.02; ) N Responder 8 (57.1%) 6 (40%) 5 (35.7%) 4 (33.3%) 3 (25%) 95% CI (28.86%; (16.34%; (12.76%; 82.34%) 67.71%) 64.86%) (9.92%; 65.11%) (5.49%; 57.19%) Day 240 N Median IQ range (0.02; ) (0.02; ) (0.02; ) (0.02; ) (0.02; ) N Responder 7 (50%) 5 (33.3%) 4 (30.8%) 2 (15.4%) 1 (10%) 95% CI (23.04%; 76.96%) (11.82%; 61.62%) (9.09%; 61.43%) (1.92%; 45.45%) (0.25%; 44.5%) N: number of participants with data and for responders, number of participants with data at baseline and at post-baseline time point; IQ: interquartile range CI: exact Clopper-Pearson confidence interval; MVA-BN-Filo: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein; Ad26.ZEBOV: adenovirus-type 26 vector vaccine encoding Ebola glycoprotein. Responders are those negative at baseline and positive post-baseline, or positive at baseline with at least 3-fold increase from baseline. Follow up time points differed across groups therefore empty cells represent time points where no follow up visit was scheduled;

21 efigure 1: Ebola glycoprotein specific antibody responses in ELISA units/ml X axis: Study day. Vaccination and randomization occurredd on Day 1. N: number of participants analyzed. For displayed geometric means, confidence intervals and numbers analyzed at every visit see Table 3. MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein. Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein The lines are connecting mean values. Error bars indicate 95% confidence intervals. Arrows represent vaccination time points: blue: MVA vaccination, yellow: Ad26 vaccination 2016 American Medical Association. All rights reserved.

22 efigure 2: Ebola glycoprotein specific antibody response expressed as endpoint titers. The anti EBOV GP IgG individual responses, as assessed by ELISA (Battelle Biomedical Research Center), are displayed as endpoint titers (the reciprocal of the most dilute titer at which specific IgG remained detectable) with geometric mean and 95% confidence intervals. Placebo groups boosted at Day 29 (n=6) and Day 57 (n=6) are combined inn one panel. X axis: Study day. Vaccination and randomization occurredd on Day 1. N: number of participants analyzed. Data displayed are geometric means with 95% confidence intervals. MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein. Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein 2016 American Medical Association. All rights reserved.

23 efigure 3: Ebola glycoprotein specific T cell responses as assessed by interferon γ ELISpot X axis: Study day. Vaccination and randomization occurredd on Day 1. N: number of participants analyzed. For displayed medians, interquartile ranges and numbers analyzed at every visit seee Table 4. MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein. Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein Arrows represent vaccination time points: blue: MVA vaccination, yellow: Ad26 vaccination 2016 American Medical Association. All rights reserved.

24 efigure 4: Intracellular cytokine staining (CD8) X axis: Study day. Vaccination and randomization occurredd on Day 1. N: number of participants analyzed. Data displayed are medians with interquartile ranges. MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein. Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein Arrows represent vaccination time points: blue: MVA vaccination, yellow: Ad26 vaccination 2016 American Medical Association. All rights reserved.

25 efigure 5: Intracellular cytokine staining (CD4) X axis: Study day. Vaccination and randomization occurredd on Day 1. N: number of participants analyzed. Data displayed are medians with interquartile ranges. MVA: modified vaccinia Ankara vector vaccine, encoding glycoproteins from Ebola virus, Sudan virus, Marburg virus and Tai Forest virus nucleoprotein. Ad26: adenovirus type 26 vector vaccine encoding Ebola glycoprotein Arrows represent vaccination time points: blue: MVA vaccination, yellow: Ad26 vaccination 2016 American Medical Association. All rights reserved.

26 efigure 6: CD8+ Intracellular cytokine staining

27 The proportion of CD8+ T cells from responders secreting any combination of IFN ƴ, IL 2, or TNF α, at Day 15 or 29 postprime, at Day 36, 50, or 78 (21 days post boost) and at Day 180 and Day 240. ICS responses were not observed in CD8+ T cells after MVA BN Filo prime at day 29. The proportion of CD8+ T cells from responders secreting 1, 2 or 3 cytokines is labeled by the blue, green and yellow circumference. N= the number of participants providing data for each pie chart.

28 efigure 7: CD4+ Intracellular cytokine staining (ICS)

29 The proportion of CD4+ T cells from responders secreting any combination of IFN ƴ, IL 2, or TNF α, at Day 15 or 29 postprime, and at Day 36, 50, or 78 (21 days post boost) and at Day 180 and Day 240. The proportion of CD4+ T cells from responders secreting 1, 2 or 3 cytokines is labeled by the blue, green and yellow circumference. N= the number of participants providing data for each pie chart.

30 References 1 Sprangers MC, Lakhai W, Koudstaal W et al. Quantifying adenovirus neutralizing antibodies by luciferase transgene detection: addressing pre existing immunity to vaccine and gene therapy vectors. J Clin Microbiol. 2003; 41(11): Russell ND, Hudgens MG, Ha R et al, Moving to Human Immunodeficiency Virus Type 1 Vaccine Efficacy Trials: defining T Cell Responses As Potential Correlates of Immunity, J Infect Dis. 2003; 187: Horton H, Thomas E, Stucky JA et al. Optimization and validation of an 8 color Intracellular Cytokine Staining (ICS) assay to quantify antigen specific T Cells induced by vaccination. J Immunol Methods. 2007; 323(1):39 54.