Lot-to-lot consistency, immunogenicity, and safety of the Ad26.ZEBOV, MVA-BN-Filo Ebola virus vaccine regimen: A phase 3, randomized, double-blind, placebo-controlled trial

ABSTRACT

This phase-3, double-blind, placebo-controlled study (NCT04228783) evaluated lot-to-lot consistency of the Ad26.ZEBOV, MVA-BN-Filo Ebola vaccine regimen. Participants were randomized (6:6:6:1) to receive the two-dose regimen from three consecutively manufactured lots of Ad26.ZEBOV on Day 1 paired with three consecutively manufactured lots of MVA-BN-Filo on Day 57 (Groups 1–3) or two doses of placebo (Group 4). An additional cohort also received an Ad26.ZEBOV booster or placebo 4 months post-dose 2. Equivalence of the immunogenicity at 21 days post-dose 2 between any two groups was demonstrated if the 95% confidence interval (CI) of the Ebola virus glycoprotein (EBOV GP)–binding antibody geometric mean concentration (GMC) ratio was entirely within the prespecified margin of 0.5–2.0. Lot-to-lot consistency (i.e., consecutive lots can be consistently manufactured) was accomplished if equivalence was shown for all three pairwise comparisons. Results showed that the primary objective in the per-protocol immunogenicity subset (n = 549) was established for each pairwise comparison (Group 1 vs 2: GMC ratio = 0.9 [95% CI: 0.8, 1.1], Group 1 vs 3: 0.9 [0.8, 1.1], Group 2 vs 3: 1.0 [0.9, 1.2]). Equivalence of the three groups for the Ad26.ZEBOV component only was also demonstrated at 56 days post-dose 1. EBOV GP–binding antibody responses (post-vaccination concentrations >2.5-fold from baseline) were observed in 419/421 (99.5%) vaccine recipients at 21 days post-dose 2 and 445/460 (96.7%) at 56 days post-dose 1. In the booster cohort (n = 39), GMCs increased 9.0- and 11.8-fold at 7 and 21 days post-booster, respectively, versus pre-booster. Ad26.ZEBOV, MVA-BN-Filo was well tolerated, and no safety issues were identified.

KEYWORDS:

• Ebola
• Ad26.ZEBOV
• MVA-BN-Filo
• vaccine
• immunogenicity
• clinical trial

Introduction

Since the discovery of Zaire ebolavirus (EBOV) in 1976, Ebola disease has become a global health concern, with >30 outbreaks caused by EBOV reported, mostly in West and Central Africa.^1–3 The largest outbreak to date occurred in 2014 through 2016 across multiple West African countries, causing >11,000 deaths in >28,000 people who contracted EBOV. Most recently, since 2021, there have been five outbreaks of Ebola disease caused by EBOV across the Democratic Republic of the Congo and Guinea responsible for 52 cases and 33 deaths.³ The increasing incidence of Ebola disease outbreaks, most of which have high mortality rates, and their burden on the health-care systems of affected countries has driven the development of novel vaccines.^1,2,4

The heterologous, two-dose EBOV vaccine regimen of Ad26.ZEBOV and MVA-BN-Filo (Zabdeno®/Mvabea®, Janssen-Cilag International NV, Beerse, Belgium) was granted approval under exceptional circumstances from the European Commission in 2020 for prophylactic use in those aged ≥1 year.^5–7 The regimen is prequalified by the World Health Organization (WHO) and is recommended by the WHO Strategic Advisory Group of Experts (SAGE) for prophylactic use in an outbreak setting in lower-risk populations and for prophylactic use in the absence of outbreaks in certain risk groups.^8–10 The regimen has also received regulatory marketing authorizations in Côte d’Ivoire, Gabon, Ghana, Nigeria, Rwanda, Sierra Leone, and Uganda, as well as temporary use authorization in the Democratic Republic of the Congo.

The Ad26.ZEBOV, MVA-BN-Filo vaccine regimen comprises one dose of the monovalent, replication-incompetent, adenovirus type 26 (Ad26) vector–based vaccine encoding the EBOV glycoprotein (GP; Mayinga variant, Ad26.ZEBOV), followed 56 days later by a dose of the recombinant, nonreplicating, modified vaccinia Ankara (MVA)–vectored vaccine encoding GPs from the EBOV (Mayinga variant), Sudan virus (Gulu variant), and Marburg virus (Musoke variant), plus the Taï Forest virus nucleoprotein (MVA-BN-Filo).^6,7 Phase 1, 2, and 3 clinical trials have demonstrated good tolerability and immunogenicity of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen in adults and children.^11–18 In addition to the two-dose regimen, the tolerability and immunogenicity of a booster dose of Ad26.ZEBOV has been evaluated. In a study in adults, in which the booster dose was administered 2 years after the initial dose of the two-dose regimen, the safety profile of the Ad26.ZEBOV booster dose was similar to that of the first Ad26.ZEBOV dose.¹⁶ The booster dose induced a strong anamnestic response; EBOV GP–binding antibody concentrations were approximately 40 times higher at 7 days and 110 times higher at 21 days post-boost compared with immediately before the booster vaccination.¹⁶ Similarly, in both a pediatric study and an adult study, a booster dose of Ad26.ZEBOV in children who had received the two-dose regimen >3 years earlier and in HIV-positive adults who had received the two-dose regimen ≥4.5 years earlier was well tolerated and induced a rapid and robust immune response.^19,20 Thus far, there are no published clinical data on the immunogenicity and safety of an Ad26.ZEBOV booster dose when administered <1 year after the primary vaccine regimen.

Ensuring consistency of a regimen composed of two different vaccines (i.e., Ad26.ZEBOV and MVA-BN-Filo), each produced with a different manufacturing process, can present potential challenges. A common requirement for licensing a vaccine is demonstration that vaccine lots are produced consistently. This study aimed to show lot-to-lot consistency of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen by demonstrating that two-dose vaccine regimens from three consecutively manufactured lots of Ad26.ZEBOV paired with three consecutively manufactured lots of MVA-BN-Filo can elicit equivalent humoral immune responses. In addition, we evaluated the immunogenicity and safety of a booster dose of Ad26.ZEBOV administered 4 months after the second dose of the two-dose regimen.

Methods

Study design and participants

This randomized, double-blind, placebo-controlled, parallel-group, phase-3 study (ClinicalTrials.gov Identifier: NCT04228783) was conducted at seven sites in the United States. Eligible participants were medically stable men or women aged 18–50 years, without known prior exposure to Ebolavirus (including travel to an area with an Ebola disease outbreak <1 month prior to screening, if applicable) or diagnosis of Ebola disease. Participants who had received a candidate Ebola vaccine or a candidate Ad26- or MVA-based vaccine in the past or with known allergy or history of anaphylaxis or other serious adverse reactions to vaccines or vaccine products, including known allergy to chicken or egg protein and gentamicin, were excluded.

The protocol, protocol amendments, informed consent form, investigator’s brochure, and other relevant documents were submitted to an institutional review board (IRB) by the investigator and were reviewed and approved by the IRB before the study was initiated (Supplemental Table S1). Any protocol amendments required IRB approval before implementation. The study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and that are consistent with Good Clinical Practices and applicable regulatory requirements. Participants or their legally acceptable representatives provided their written consent to participate in the study after having been informed about the nature and purpose of the study, participation/termination conditions, and the risks and benefits of treatment.

Procedures

Following a screening phase of up to 28 days, participants were randomized on Day 1 to receive the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen or placebo. Participants in the main part of the study were randomized 6:6:6:1 to one of four groups. Participants assigned to Groups 1–3 received one of three consecutively manufactured lots of Ad26.ZEBOV (Lots A, B, and C for Groups 1, 2, and 3, respectively) at a nominal dose of 5 × 10¹⁰ viral particles as dose 1 on Day 1, followed by one of three consecutively manufactured lots of MVA-BN-Filo (Lots 1, 2, and 3 for Groups 1, 2, and 3, respectively) at a nominal dose of 1 × 10⁸ infectious units as dose 2 on Day 57 (such that Group 1 received Lot A and Lot 1, Group 2 received Lot B and Lot 2, and Group 3 received Lot C and Lot 3). Participants in Group 4 received two placebo vaccinations (0.9% saline) administered at a 56-day interval. Once randomization in this part of the study was finished, an additional booster cohort of participants was randomized 5:1 to receive either the two-dose heterologous vaccine regimen Ad26.ZEBOV followed by MVA-BN-Filo 56 days later, and then a booster dose of Ad26.ZEBOV 4 months after dose 2 (Group 5), or two placebo vaccinations administered 56 days apart followed by a placebo booster 4 months after dose 2 (Group 6).

Randomization was performed centrally using an interactive web response system and randomly permuted blocks. A computer-generated randomization schedule was prepared before the study by or under the supervision of the sponsor. The study vaccines were prepared by unblinded qualified study site personnel who were not involved in any other study-related procedures and administered in a masked syringe by a blinded study vaccine administrator. Investigators, study site personnel (except those with primary responsibility for study vaccine preparation and dispensing), participants, and sponsor personnel were blinded to the study vaccine allocation.

All participants received the study vaccine or placebo administered by intramuscular injection (0.5 mL) in the deltoid muscle. After each vaccination, participants remained at the study site for at least 30 minutes to evaluate the presence of any acute reactions and solicited local or system adverse events (AEs), or longer if deemed necessary by the investigator (e.g., in the case of grade 3 AEs). At each vaccination, participants received a diary card to document symptoms of solicited local and systemic AEs daily for 7 days post-vaccination; the diary cards were then reviewed by study personnel at the next clinic visit. All participants received a telephone call for safety follow-up at 28 days (±1 week) post-dose 1, 28 days (±1 week) post-dose 2, and 6 months (±15 days) post-dose 2. Blood sampling for immunogenicity assays (8.5 mL per blood draw) was done at clinic visits before vaccination on the days of dose 1 (Day 1) and dose 2 (56 days post-dose 1) administration, and at 21 days (±3 days) post-dose 2. In addition, participants in the booster cohort had blood samples taken for immunogenicity assays on the day of their booster dose and at 7 days (±2 days), 21 days (±3 days), 6 months (±2 weeks), and 12 months (±4 weeks) post-booster dose and a telephone safety follow-up at 28 days (±1 week) post-booster dose.

Mitigations implemented for the COVID-19 pandemic

Shortly after participants received dose 1, the COVID-19 pandemic necessitated imposition of “stay-at-home” orders in the areas where the sites were located, leading to delays in administering dose 2 vaccinations. The protocol was, therefore, amended to widen the window for the second vaccination from ±3 days to −3/+28 days for Groups 1–4 and to ±1 week for Groups 5 and 6, and to allow additional participants to be recruited. In addition, participants were reminded more frequently to come in for their second vaccination (e.g., by text message, phone calls, or e-mails). Sensitivity analyses were then planned using the original time window around the second vaccination.

Assessments

The primary objective was to demonstrate that the two-dose vaccine regimen from three consecutively manufactured lots of Ad26.ZEBOV as dose 1 paired with three consecutively manufactured lots of MVA-BN-Filo as dose 2, administered at a 56-day interval, induce an equivalent humoral immune response, assessed with binding antibody levels against the EBOV GP using enzyme-linked immunosorbent assay (ELISA) at 21 days post-dose 2 vaccination. EBOV GP–binding antibody concentrations were measured using the validated, US Food and Drug Administration–endorsed Filovirus Animal Nonclinical Group (FANG) ELISA. Full details of the assay methods have been published previously.²¹

The secondary objective was to demonstrate that three consecutively manufactured lots of Ad26.ZEBOV as dose 1 induce an equivalent humoral immune response, assessed with binding antibody levels against the EBOV GP, measured using FANG ELISA, at 56 days post-dose 1 vaccination. Determination of the safety and reactogenicity of the two-dose regimen was also a secondary objective, assessed by the frequency of solicited local and systemic AEs until 7 days after each vaccination, unsolicited AEs until 28 days after each vaccination, and serious AEs (SAEs) through the end of the study.

Exploratory endpoints were assessed in the booster cohort and included binding antibody levels against the EBOV GP, measured using FANG ELISA, assessed pre-booster at 4 months post-dose 2 and at 7 days, 21 days, 6 months, and 12 months post-booster dose, as well as solicited local and systemic AEs until 7 days post-booster dose, unsolicited AEs until 28 days post-booster dose, and SAEs until the end of the study. In addition, virus neutralizing antibody levels (90% inhibitory concentration [IC₉₀] titers) against the Ad26 vector were assessed at baseline, pre-booster dose at 4 months post-dose 2, and at 21 days post-booster dose. Full details of the assay for virus neutralizing antibodies have been published previously.^11,22

Statistical analysis

Based on the assumptions for sample size determination for the main part of the study, a total of 144 participants per group receiving Ad26.ZEBOV, MVA-BN-Filo would yield an overall power of at least 90% to show immunogenic equivalence of Groups 1–3 at 21 days post-dose 2 (i.e., with 96.55% power for each of the three pairwise comparisons). To enlarge the safety database, the total number per group was increased to 234 (i.e., an additional 90 participants per group for safety only). A randomization ratio of 6:6:6:1 (Group 1:Group 2:Group 3:Group 4) yielded an overall sample size of 741 participants, with 234 participants each in Groups 1–3 receiving Ad26.ZEBOV, MVA-BN-Filo and 39 participants receiving placebo (Group 4). In the booster cohort, 60 participants were randomized in a 5:1 ratio to receive active vaccination or placebo (Group 5:Group 6), which allowed a reasonable estimation of the humoral immune response to the booster dose.

The primary analysis was performed when all participants in the main part of the study (Groups 1–4) had completed the last study-related visit or discontinued earlier, and all participants in the booster cohort (Groups 5–6) had completed the 6-month post-booster visit or discontinued. The final analysis was conducted when all participants in the study completed the last study-related visit or discontinued.

Immunogenicity in the main part of the study was analyzed in the immunogenicity subset, which comprised all participants in Groups 1–4 from whom blood samples for immunogenicity assessments were collected. The core immunogenicity analyses were performed on both the per-protocol analysis set for the immunogenicity subset (PP-immunogenicity subset; all randomized and vaccinated participants who received both dose 1 and dose 2 vaccinations within the protocol-defined windows, had at least one post-vaccination evaluable immunogenicity sample, and had no major protocol deviations that could have influenced the immune response) and the per-protocol analysis set for the booster cohort (PP-booster cohort; all vaccinated participants who received dose 1, dose 2, and the booster vaccination [both dose 2 and booster dose within the protocol-defined window], had at least one post-vaccination evaluable immunogenicity sample, and had no major protocol deviations that could have influenced the immune response).

Descriptive statistics (including geometric means with their corresponding 95% confidence intervals [CIs]) were calculated for continuous immunologic parameters at each time point. Frequency tabulations were calculated for discrete (qualitative) immunologic parameters at each time point. For the generation of summary statistics, values of the EBOV GP binding antibody concentrations (ELISA units [EU]/mL) and Ad26 virus neutralizing antibodies (IC₉₀ titers) that were below the lower limit of quantification (LLOQ) were imputed with half of the corresponding LLOQ and values above the upper limit of quantification (ULOQ) were imputed with the corresponding ULOQ. For the calculation of fold changes, the values below LLOQ were imputed with the corresponding LLOQ and values above the ULOQ were imputed with the ULOQ.

To assess the primary objective, each pairwise comparison (i.e., Group 1 vs Group 2, Group 1 vs Group 3, and Group 2 vs Group 3) of the anti-EBOV GP–binding antibody concentrations at 21 days post-dose 2 was based on ratios of the geometric mean concentration (GMC) with corresponding 95% CI. Equivalence of any two groups was shown if the 95% CI of the estimated GMC ratio lay entirely within 0.5 and 2.0. Lot-to-lot consistency was accomplished if equivalence was shown for all three pairwise comparisons. For the secondary objective, each pairwise comparison of the anti-EBOV GP–binding antibody concentrations at 56 days post-dose 1 was based on ratios of the GMCs with corresponding 95% CI. Equivalence and lot-to-lot consistency for Ad26.ZEBOV was demonstrated using the same methods as for the primary objective. Responders were defined as those with an increase in anti-EBOV GP–binding antibody concentrations of >2.5-fold from baseline.

In sensitivity analyses, both the primary and secondary hypotheses of equivalence were also tested based on data from participants who received the second vaccination within the original window (±3 days) around the Day 57 visit.

Safety analyses were performed on the full analysis set, which included all enrolled participants with at least one study vaccine administration documented. AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA), version 24.1. No formal statistical testing of safety data was planned.

Results

Participants

Of 1047 participants screened for the study, 970 were randomized and received dose 1 (Ad26.ZEBOV or placebo) and formed the full analysis set (Figure 1). Of these, 856 (88.2%) participants received dose 2 (MVA-BN-Filo or placebo). Of the 60 participants randomized in the booster cohort, 50 (83.3%) participants received the third dose (Ad26.ZEBOV or placebo). Overall, a total of 849/970 (87.5%) participants completed study vaccination (i.e., received all doses according to the protocol, including the third dose for the booster cohort) and 790 (81.4%) completed the study.

Figure 1. Participant disposition.

Group 1: Lot A of Ad26.ZEBOV as dose 1 and Lot 1 of MVA-BN-Filo as dose 2.

Group 2: Lot B of Ad26.ZEBOV as dose 1 and Lot 2 of MVA-BN-Filo as dose 2.

Group 3: Lot C of Ad26.ZEBOV as dose 1 and Lot 3 of MVA-BN-Filo as dose 2.

Group 4: Placebo as dose 1 and placebo as dose 2.

Group 5: Ad26.ZEBOV as dose 1, MVA-BN-Filo as dose 2, and Ad26.ZEBOV as dose 3 (booster).

Group 6: Placebo as dose 1, placebo as dose 2, and placebo as dose 3 (booster).

*Period from the end of the post-dose 1 period (i.e., from dose 1 administration to 28 days after the first vaccination or the date of last contact [for early study discontinuation], whichever occurred first) until 1 minute prior to dose 2 or the date of last contact (for early study discontinuation), whichever occurred first.

^†Period from the end of the post-dose 2 period (i.e., from dose 2 administration to 28 days after the second vaccination or the date of last contact [for early study discontinuation], whichever occurred first) until 1 minute prior to dose 3, the date of last study visit, or the date of last contact (for early study discontinuation), whichever occurred first.

^‡Period from the end of the post-dose 3 period (i.e., from dose 3 administration to 28 days after the third vaccination or the date of last contact [for early study discontinuation], whichever occurred first) until the date of the last study visit or the date of last contact (for early study discontinuation), whichever occurred first.

^§“Other” reasons included withdrawal of the participant per sponsor request prior to receiving a certified letter confirmation (five participants; certified letters were sent to participants after a certain number of unsuccessful contact attempts as part of the process for a site to consider the participant as lost to follow-up), lost to follow-up due to unclaimed certified letter (one participant), discontinuation due to noncompliance of study visit (one participant), withdrawal of the participant due to being lost to follow-up (one participant) or due to being in a rehab facility (one participant), and refusal of the participant to be contacted by the site (one participant).

AE, adverse event; PP-booster cohort, per-protocol analysis set for the booster cohort; PP-immunogenicity subset, per-protocol analysis set for the immunogenicity subset.

The immunogenicity subset (participants in Groups 1–4 for whom blood samples were collected for analysis) comprised 626 participants; of these, 560 (89.5%) completed study vaccination (Figure 1). Most participants received dose 2 within the extended dose window of −3/+28 days post-dose 1 (526/595 [88.4%] in vaccine Groups 1–3 and 29/31 [93.5%] in placebo Group 4), while 456/595 (76.6%) in Groups 1–3 and 24/31 (77.4%) in Group 4 received dose 2 also within the ±3-day window.

Participant characteristics at baseline were generally similar across study groups in the PP-immunogenicity subset (Groups 1–4; Table 1), PP-booster cohort (Groups 5–6; Table 1), and the full analysis set (Supplemental Table S2). Among all participants (i.e., the full analysis set), the mean age was 35.0 years, 55.1% were female, 64.8% were White, and 29.6% were Black/African American.

Table 1. Baseline characteristics (PP analysis sets).

	PP-immunogenicity subset						PP-booster cohort
	Ad26.ZEBOV, MVA-BN-Filo				Placebo	All participants n = 549	Ad26.ZEBOV, MVA-BN-Filo	Placebo	All participants n = 45
	Group 1 n = 176	Group 2 n = 177	Group 3 n = 167	All vaccine groups n = 520	Group 4 n = 29		Group 5 n = 39	Group 6 n = 6
Age, mean (SD), years	35.8 (9.7)	35.6 (9.4)	35.7 (8.9)	35.7 (9.3)	36.1 (8.4)	35.7 (9.3)	32.3 (7.4)	36.3 (6.1)	32.8 (7.3)
Female, n (%)	100 (56.8)	103 (58.2)	99 (59.3)	302 (58.1)	12 (41.4)	314 (57.2)	23 (59.0)	1 (16.7)	24 (53.3)
Race, n (%)
American Indian or Alaska Native	1 (0.6)	0	3 (1.8)	4 (0.8)	0	4 (0.7)	0	0	0
Asian	2 (1.1)	5 (2.8)	6 (3.6)	13 (2.5)	0	13 (2.4)	0	0	0
Black or African American	48 (27.3)	55 (31.1)	41 (24.6)	144 (27.7)	14 (48.3)	158 (28.8)	13 (33.3)	3 (50.0)	16 (35.6)
Native Hawaiian or Other Pacific Islander	1 (0.6)	1 (0.6)	2 (1.2)	4 (0.8)	0	4 (0.7)	0	0	0
White	124 (70.5)	109 (61.6)	110 (65.9)	343 (66.0)	14 (48.3)	357 (65.0)	26 (66.7)	3 (50.0)	29 (64.4)
Unknown	0	2 (1.1)	0	2 (0.4)	0	2 (0.4)	0	0	0
Multiple	0	5 (2.8)	5 (3.0)	10 (1.9)	0	10 (1.8)	0	0	0
Not reported	0	0	0	0	1 (3.4)	1 (0.2)	0	0	0
Ethnicity, n (%)
Hispanic or Latino	21 (11.9)	14 (7.9)	10 (6.0)	45 (8.7)	0	45 (8.2)	0	0	0
Not Hispanic or Latino	155 (88.1)	160 (90.4)	157 (94.0)	472 (90.8)	29 (100.0)	501 (91.3)	39 (100.0)	6 (100.0)	45 (100.0)
Not reported	0	3 (1.7)	0	3 (0.6)	0	3 (0.5)	0	0	0
Body mass index, mean (SD), kg/m^2	30.8 (8.9)	30.5 (7.9)	32.0 (9.5)	31.1 (8.8)	35.8 (8.7)	31.3 (8.8)	30.4 (7.6)	34.4 (8.8)	31.0 (7.8)

Group 1: Lot A of Ad26.ZEBOV as dose 1 and Lot 1 of MVA-BN-Filo as dose 2.

Group 2: Lot B of Ad26.ZEBOV as dose 1 and Lot 2 of MVA-BN-Filo as dose 2.

Group 3: Lot C of Ad26.ZEBOV as dose 1 and Lot 3 of MVA-BN-Filo as dose 2.

Group 4: Placebo as dose 1 and placebo as dose 2.

PP, per-protocol; PP-booster cohort, per-protocol analysis set for the booster cohort; PP-immunogenicity subset, per-protocol analysis set for the immunogenicity subset; SD, standard deviation.

Immunogenicity and equivalence testing

Immunogenicity subset

At baseline, 8.6% of the participants in the PP-immunogenicity subset had positive EBOV GP–binding antibody status, and the overall GMC at baseline in all the Ebola vaccine groups and the placebo group was below the LLOQ (Table 2).

Table 2. Anti-EBOV GP–binding antibody response (PP-immunogenicity subset).

	Ad26.ZEBOV, MVA-BN-Filo				Placebo
	Group 1 n = 176	Group 2 n = 177	Group 3 n = 167	All vaccine groups n = 520	Group 4 n = 29
Day 1 (baseline), n*	155	157	150	462	26
GMC, EU/mL (95% CI)	<LLOQ (<LLOQ, <LLOQ)	<LLOQ (<LLOQ, <LLOQ)	<LLOQ (<LLOQ, <LLOQ)	<LLOQ (<LLOQ, <LLOQ)	<LLOQ (<LLOQ, <LLOQ)
Positive sample, n (%) (95% CI)	14 (9.0) (5.0, 14.7)	13 (8.3) (4.5, 13.7)	14 (9.3) (5.2, 15.2)	41 (8.9) (6.4, 11.8)	1 (3.8) (0.1, 19.6)
56 days post-dose 1 (Day 57, pre-dose 2), n*	175	177	166	518	29
GMC, EU/mL (95% CI)	988 (842, 1159)	994 (865, 1142)	960 (819, 1126)	981 (899, 1071)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	22.4 (18.7, 26.8)	23.4 (20.0, 27.2)	22.8 (19.2, 27.1)	22.9 (20.7, 25.2)	1.0 (0.9, 1.2)
Responders, n/N^† (%) (95% CI)	147/154 (95.5) (90.9, 98.2)	154/157 (98.1) (94.5, 99.6)	144/149 (96.6) (92.3, 98.9)	445/460 (96.7) (94.7, 98.2)	1/26 (3.8) (0.1, 19.6)
21 days post-dose 2 (Day 78), n*	163	163	149	475	27
GMC, EU/mL (95% CI)	11,077 (9806, 12,514)	12,223 (10,665, 14,010)	11,818 (10,320, 13,534)	11,693 (10,844, 12,608)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	254.2 (218.7, 295.5)	290.3 (242.8, 347.1)	286.8 (243.4, 337.9)	276.4 (251.5, 303.9)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	11.1 (9.6, 12.8)	12.6 (10.9, 14.5)	11.8 (10.2, 13.7)	11.8 (10.9, 12.8)	1.0 (1.0, 1.0)
Responders, n/N^† (%) (95% CI)	143/143 (100.0) (97.5, 100.0)	144/145 (99.3) (96.2, 100.0)	132/133 (99.2) (95.9, 100.0)	419/421 (99.5) (98.3, 99.9)	0/24 (0) (0, 14.2)

*Number of participants with data at that time point.

^†Number of responders/number of participants with data at baseline and at that time point.

Group 1: Lot A of Ad26.ZEBOV as dose 1 and Lot 1 of MVA-BN-Filo as dose 2.

Group 2: Lot B of Ad26.ZEBOV as dose 1 and Lot 2 of MVA-BN-Filo as dose 2.

Group 3: Lot C of Ad26.ZEBOV as dose 1 and Lot 3 of MVA-BN-Filo as dose 2.

Group 4: Placebo as dose 1 and placebo as dose 2.

CI, confidence interval; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; EU, ELISA unit; GMC, geometric mean concentration; GMI, geometric mean increase; GP, glycoprotein; LLOQ, lower limit of quantification; PP-immunogenicity subset, per-protocol analysis set of the immunogenicity subset.

At 56 days post-dose 1, 96.7% (95% CI: 94.7%, 98.2%) of the participants in the Ebola vaccine groups (Groups 1–3) had an anti-EBOV GP–binding antibody response, with a GMC of 981 EU/mL (95% CI: 899, 1071). At 21 days post-dose 2, 99.5% (95% CI: 98.3%, 99.9%) of the participants in the Ebola vaccine groups had an anti-EBOV GP–binding antibody response, with a GMC of 11,693 EU/mL (95% CI: 10,844, 12,608). Both the responder rates and the GMCs were similar across all three Ebola vaccine groups at a given time point (Figure 2, Table 2). In the placebo group (Group 4), the anti-EBOV GP–binding antibody responder rate was 3.8% (95% CI: 0.1%, 19.6%) at 56 days post-dose 1 and 0% (95% CI: 0%, 14.2%) at 21 days post-dose 2; the GMC was below the LLOQ at both time points.

Figure 2. Anti-EBOV GP–binding antibody responses (PP-immunogenicity subset).

The GMCs with 95% CIs are shown: the vertical lines represent the width of the CI; the lower and upper horizontal lines depict the lower and upper limits of the CI, respectively; and the middle horizontal line depicts the GMC. Values of the EBOV GP–binding antibody concentrations (EU/mL) that were below the LLOQ were imputed with half of the LLOQ.

Group 1: Lot A of Ad26.ZEBOV as dose 1 and Lot 1 of MVA-BN-Filo as dose 2.

Group 2: Lot B of Ad26.ZEBOV as dose 1 and Lot 2 of MVA-BN-Filo as dose 2.

Group 3: Lot C of Ad26.ZEBOV as dose 1 and Lot 3 of MVA-BN-Filo as dose 2.

Group 4: Placebo as dose 1 and placebo as dose 2.

Day 1: baseline, Day 57: pre-dose 2 (56 days post-dose 1), Day 78: 21 days post-dose 2.

CI, confidence interval; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; EU, ELISA unit; GMC, geometric mean concentration; GP, glycoprotein; LLOQ, lower limit of quantification; PP-immunogenicity subset, per-protocol analysis set for the immunogenicity subset.

Equivalence was established for each pairwise comparison (Group 1 vs Group 2, Group 1 vs Group 3, and Group 2 vs Group 3) of the anti-EBOV GP–binding antibody concentration at 56 days post-dose 1 based on the ratios of the GMCs with corresponding 95% CIs, which were entirely within the prespecified equivalence margin of 0.5 to 2.0 (Group 1 vs Group 2: 1.0 [95% CI: 0.8, 1.2], Group 1 vs Group 3: 1.0 [95% CI: 0.8, 1.3], and Group 2 vs Group 3: 1.0 [95% CI: 0.8, 1.3]; Table 3). Lot-to-lot consistency was demonstrated as equivalence was achieved for all three pairwise comparisons. Furthermore, equivalence was established for the primary objective at 21 days post-dose 2, based on the ratios of the GMCs with corresponding 95% CIs, which were entirely within the prespecified equivalence margin (Group 1 vs Group 2: 0.9 [95% CI: 0.8, 1.1], Group 1 vs Group 3: 0.9 [95% CI: 0.8, 1.1], and Group 2 vs Group 3: 1.0 [95% CI: 0.9, 1.2]), demonstrating lot-to-lot consistency for these comparisons (Table 3).

Table 3. Equivalence assessment of anti-EBOV GP–binding antibody GMCs between paired Ad26.ZEBOV and MVA-BN-Filo lots (PP-immunogenicity subset).

	Comparison Group X		Comparison Group Y		GMC ratio
Comparison: Group X vs Group Y	n	GMC, EU/mL	n	GMC, EU/mL	Group X/Group Y (95% CI)	Equivalent (yes/no)
56 days post-dose 1 (Day 57)
Group 1 vs Group 2	175	988	177	994	1.0 (0.8, 1.2)	Yes
Group 1 vs Group 3	175	988	166	960	1.0 (0.8, 1.3)	Yes
Group 2 vs Group 3	177	994	166	960	1.0 (0.8, 1.3)	Yes
21 days post-dose 2 (Day 78)
Group 1 vs Group 2	163	11,077	163	12,223	0.9 (0.8, 1.1)	Yes
Group 1 vs Group 3	163	11,077	149	11,818	0.9 (0.8, 1.1)	Yes
Group 2 vs Group 3	163	12,223	149	11,818	1.0 (0.9, 1.2)	Yes

Equivalence margin: 0.5 < 95% CI ratio < 2.0.

Group 1: Lot A of Ad26.ZEBOV as dose 1 and Lot 1 of MVA-BN-Filo as dose 2.

Group 2: Lot B of Ad26.ZEBOV as dose 1 and Lot 2 of MVA-BN-Filo as dose 2.

Group 3: Lot C of Ad26.ZEBOV as dose 1 and Lot 3 of MVA-BN-Filo as dose 2.

CI, confidence interval; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; EU, ELISA unit; GMC, geometric mean concentration; GP, glycoprotein; PP-immunogenicity subset, per-protocol analysis set of the immunogenicity subset.

Sensitivity analyses for the equivalence tests at 56 days post-dose 1 and 21 days post-dose 2 were performed to determine the robustness of extending the visit window for the dose 2 vaccination from ±3 days to −3/+28 days. Results of these sensitivity analyses based on data of participants who received dose 2 within the original window versus the widened window were consistent with the results of the primary analysis (Supplemental Table S3).

Booster cohort

At baseline, before primary vaccination, in the PP-booster cohort, one participant in the Ebola vaccine group (Group 5; 1/39 [2.6%]) and no participants in the placebo group (Group 6) had a positive sample for anti-EBOV GP – binding antibodies (Table 4). The GMC at pre-vaccination baseline was below the LLOQ in both the Ebola vaccine group and the placebo group in the booster cohort.

Table 4. Anti-EBOV GP–binding antibody response (PP-booster cohort).

	Ad26.ZEBOV, MVA-BN-Filo	Placebo
	Group 5 n = 39	Group 6 n = 6
Day 1 (baseline), n	39	6
GMC, EU/mL (95% CI)	<LLOQ (<LLOQ, <LLOQ)	<LLOQ (<LLOQ, <LLOQ)
Positive sample, n (%) (95% CI)	1 (2.6) (0.1, 13.5)	0 (0) (0, 45.9)
56 days post-dose 1 (Day 57, pre-dose 2), n	39	6
GMC, EU/mL (95% CI)	1023 (773, 1354)	<LLOQ (<LLOQ, 37)
GMI from pre-dose 1, EU/mL (95% CI)	28.2 (21.3, 37.3)	1.1 (0.9, 1.4)
Responders, n/N* (%) (95% CI)	39/39 (100.0) (91.0, 100.0)	0/6 (0) (0, 45.9)
21 days post-dose 2 (Day 78), n	34	4
GMC, EU/mL (95% CI)	18,877 (14,173, 25,142)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	519.8 (389.3, 694.0)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	17.7 (12.7, 24.7)	1.0 (1.0, 1.0)
Responders, n/N* (%) (95% CI)	34/34 (100.0) (89.7, 100.0)	0/4 (0) (0, 60.2)
4 months post-dose 2 (Day 177, pre-booster), n	39	6
GMC, EU/mL (95% CI)	1919 (1481, 2487)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	52.9 (40.8, 68.5)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	1.9 (1.5, 2.4)	0.9 (0.7, 1.1)
Responders, n/N* (%) (95% CI)	39/39 (100.0) (91.0, 100.0)	0/6 (0) (0, 45.9)
7 days post-booster (Day 184), n	33	5
GMC, EU/mL (95% CI)	17,046 (11,648, 24,946)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	469.3 (320.7, 686.8)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	17.4 (12.7, 23.7)	0.9 (0.7, 1.2)
GMI from pre-booster, EU/mL (95% CI)	9.0 (6.3, 13.0)	1.0 (1.0, 1.0)
Responders, n/N* (%) (95% CI)	33/33 (100.0) (89.4, 100.0)	0/5 (0) (0, 52.2)
21 days post-booster (Day 198), n	30	4
GMC, EU/mL (95% CI)	22,471 (15,420, 32,745)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	618.3 (423.9, 901.8)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	23.4 (15.7, 35.1)	0.9 (0.6, 1.3)
GMI from pre-booster, EU/mL (95% CI)	11.8 (7.9, 17.8)	1.0 (1.0, 1.0)
Responders, n/N* (%) (95% CI)	30/30 (100.0) (88.4, 100.0)	0/4 (0) (0, 60.2)
180 days post-booster (Day 357), n	33	6
GMC, EU/mL (95% CI)	4146 (3049, 5637)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	114.1 (83.9, 155.3)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	4.3 (2.9, 6.3)	0.9 (0.7, 1.1)
GMI from pre-booster, EU/mL (95% CI)	2.2 (1.7, 3.0)	1.0 (1.0, 1.0)
Responders, n/N* (%) (95% CI)	33/33 (100.0) (89.4, 100.0)	0/6 (0) (0, 45.9)
360 days post-booster (Day 537), n	30	5
GMC, EU/mL (95% CI)	2636 (1931, 3598)	<LLOQ (<LLOQ, <LLOQ)
GMI from pre-dose 1, EU/mL (95% CI)	72.5 (53.1, 99.0)	1.0 (1.0, 1.0)
GMI from pre-dose 2, EU/mL (95% CI)	2.6 (1.8, 3.6)	0.9 (0.7, 1.2)
GMI from pre-booster, EU/mL (95% CI)	1.5 (1.1, 1.9)	1.0 (1.0, 1.0)
Responders, n/N* (%) (95% CI)	30/30 (100.0) (88.4, 100.0)	0/5 (0) (0, 52.2)

*Number of responders/number of participants with data at baseline and at that time point.

CI, confidence interval; EBOV, Ebola virus; ELISA, enzyme-linked immunosorbent assay; EU, ELISA unit; GMC, geometric mean concentration; GMI, geometric mean increase; GP, glycoprotein; LLOQ, lower limit of quantification; PP-booster cohort, per-protocol analysis set of the booster cohort.

For participants in the booster cohort who received Ebola vaccine, binding antibody responses were observed in all participants (responder rate of 100%) at all time points after the first vaccination (Table 4). The GMC increased until the assessment at 21 days post-dose 2, decreased by 4 months post-dose 2 (pre-booster dose), and increased again up to 21 days post-booster dose. Compared with pre-booster, the GMCs increased 9.0-fold and 11.8-fold at 7 days and 21 days post-booster, respectively, while compared with 21 days post-dose 2, the GMC was 1.2-fold higher at 21 days post-booster. Compared with 21 days post-booster, the GMC had decreased at 6 months post-booster and decreased further at 12 months post-booster, but still remained 2.2-fold and 1.5-fold higher, respectively, than at 4 months post-dose 2 (Table 4). There were no responders in the placebo group of the booster cohort, and the GMC remained below the LLOQ at all time points.

At baseline before vaccination, positive samples for anti–Ad26-specific neutralizing antibodies (Ad26 nAbs) were observed in two participants in the booster cohort who received Ebola vaccine (2/39 [5.1%]) and none for participants in the booster cohort who received placebo. The overall geometric mean titer (GMT) of Ad26 nAbs at baseline for all participants in the booster cohort was below the LLOQ. At 4 months post-dose 2 (pre-booster), 97.4% (95% CI: 86.5%, 99.9%) of the participants in the Ebola vaccine group had detectable Ad26 nAbs with a GMT of 179 IC₉₀ titer (95% CI: 112, 288) and, compared with pre-dose 1, the GMT increased 9.2-fold pre-booster. At 21 days post-booster dose, 100% (95% CI: 88.4%, 100.0%) of the participants in the Ebola vaccine group had detectable Ad26 nAbs, with a GMT of 1963 IC₉₀ titer (95% CI: 1250, 3085), and the GMT increased 94.7-fold compared with pre-dose 1 and 11.1-fold compared with pre-booster. Negligible correlation was observed between Ad26 nAbs pre-booster and the vaccine-induced EBOV GP–binding antibody concentrations at 21 days post-booster (Spearman correlation coefficient: 0.0243). Weak negative correlations were observed between Ad26 nAbs pre-booster and EBOV GP–binding antibody concentrations at 180 days and 360 days post-booster (Spearman correlation coefficients: −0.1746 and −0.2681, respectively). There were no participants with detectable Ad26 nAbs at any post-baseline time point in the placebo group.

Safety

Solicited AEs

Among the participants in the full analysis set, the percentage of participants with at least one solicited AE tended to be higher after vaccination with Ad26.ZEBOV (dose 1) than after vaccination with MVA-BN-Filo (dose 2; 71.2% vs 51.2%), while it was similar between the dose 1 and dose 2 placebo vaccinations (43.1% vs 39.2%). The percentage of participants with at least one solicited AE after vaccination with Ad26.ZEBOV was similar between dose 1 and the booster dose (71.2% vs 65.1%). Overall, solicited AEs were reported in a higher percentage of participants (post-dose 1 and post-dose 2 combined) who received Ebola vaccines compared with participants who received placebo (74.8% vs 53.4%). Most solicited AEs (local or systemic) were grade 1 or 2 in severity.

Solicited local AEs reported after each dose are shown in Table 5. Solicited local AEs (post-dose 1 and post-dose 2 combined) were reported in 604/912 (66.2%) participants who received Ebola vaccines compared with 19/58 (32.8%) participants who received placebo. Of those who received the booster dose, 25/43 (58.1%) Ebola vaccine recipients and 1/7 (14.3%) placebo recipients reported at least one solicited local AE during the post-booster period. The most frequently reported solicited local AE in the Ebola vaccine groups was vaccination site pain (overall, 58.9% during the post-dose 1 period, 39.1% during the post-dose 2 period, and 58.1% during the post-booster period).

Table 5. Summary of solicited local AEs by derived term and worst severity grade (full analysis set).

AE, n (%)	Ad26.ZEBOV, MVA-BN-Filo	Placebo
	All groups n = 912	All groups n = 58
Post-dose 1, n	912	58
Any local AE	561 (61.5)	13 (22.4)
Grade 1	477 (52.3)	10 (17.2)
Grade 2	68 (7.5)	1 (1.7)
Grade 3	16 (1.8)	2 (3.4)
Erythema	33 (3.6)	2 (3.4)
Pain	537 (58.9)	11 (19.0)
Pruritus	132 (14.5)	5 (8.6)
Swelling	28 (3.1)	2 (3.4)
Post-dose 2, n	805	51
Any local AE	329 (40.9)	13 (25.5)
Grade 1	291 (36.1)	9 (17.6)
Grade 2	36 (4.5)	1 (2.0)
Grade 3	2 (0.2)	3 (5.9)
Erythema	11 (1.4)	1 (2.0)
Pain	315 (39.1)	12 (23.5)
Pruritus	77 (9.6)	6 (11.8)
Swelling	8 (1.0)	1 (2.0)
Post-booster dose, n	43	7
Any local AE	25 (58.1)	1 (14.3)
Grade 1	22 (51.2)	1 (14.3)
Grade 2	3 (7.0)	0
Erythema	3 (7.0)	0
Pain	25 (58.1)	1 (14.3)
Pruritus	7 (16.3)	0
Swelling	0	0

Participants were counted only once within a period for any given event, regardless of the number of times they actually experienced the event in that period. The event experienced by the participant with the worst toxicity grade was used. If a participant had a missing toxicity grade for a specific AE, the participant was counted in the “Any local AE” row for that AE.

AE, adverse event.

Solicited systemic AEs reported after each dose are shown in Table 6. Solicited systemic AEs (post-dose 1 and post-dose 2 combined) were reported in 560/912 (61.4%) participants who received Ebola vaccines compared with 27/58 (46.6%) participants who received placebo. In those who received the booster dose, 21/43 (48.8%) Ebola vaccine recipients and 3/7 (42.9%) placebo recipients reported at least one solicited systemic AE during the post-booster period. The most frequently reported solicited systemic AE in the Ebola vaccine groups was fatigue (overall, 40.2% during the post-dose 1 period, 22.6% during the post-dose 2 period, and 39.5% during the post-booster period).

Table 6. Summary of solicited systemic AEs by derived term and worst severity grade (full analysis set).

AE, n (%)	Ad26.ZEBOV, MVA-BN-Filo	Placebo
	All groups n = 912	All groups n = 58
Post-dose 1, n	912	58
Any systemic AE	499 (54.7)	20 (34.5)
Grade 1	277 (30.4)	14 (24.1)
Grade 2	141 (15.5)	4 (6.9)
Grade 3	81 (8.9)	2 (3.4)
Arthralgia	169 (18.5)	4 (6.9)
Chills	211 (23.1)	4 (6.9)
Fatigue	367 (40.2)	14 (24.1)
Headache	339 (37.2)	10 (17.2)
Myalgia	288 (31.6)	4 (6.9)
Nausea	150 (16.4)	7 (12.1)
Pyrexia	67 (7.3)	2 (3.4)
Post-dose 2, n	805	51
Any systemic AE	278 (34.5)	17 (33.3)
Grade 1	210 (26.1)	11 (21.6)
Grade 2	57 (7.1)	3 (5.9)
Grade 3	11 (1.4)	3 (5.9)
Arthralgia	51 (6.3)	3 (5.9)
Chills	43 (5.3)	4 (7.8)
Fatigue	182 (22.6)	10 (19.6)
Headache	144 (17.9)	12 (23.5)
Myalgia	107 (13.3)	5 (9.8)
Nausea	49 (6.1)	4 (7.8)
Pyrexia	6 (0.7)	1 (2.0)
Post-booster dose, n	43	7
Any systemic AE	21 (48.8)	3 (42.9)
Grade 1	14 (32.6)	0
Grade 2	5 (11.6)	2 (28.6)
Grade 3	2 (4.7)	1 (14.3)
Arthralgia	6 (14.0)	0
Chills	8 (18.6)	3 (42.9)
Fatigue	17 (39.5)	3 (42.9)
Headache	15 (34.9)	1 (14.3)
Myalgia	11 (25.6)	1 (14.3)
Nausea	6 (14.0)	0
Pyrexia	2 (4.7)	1 (14.3)

Participants were counted only once within a period for any given event, regardless of the number of times they actually experienced the event in that period. The event experienced by the participant with the worst toxicity grade was used. If a participant had a missing toxicity grade for a specific AE, the participant is counted in the “Any systemic AE” row for that AE.

AE, adverse event.

Unsolicited AEs

Unsolicited AEs reported after each dose (which includes both solicited AEs that started within 7 days after vaccination, but were ongoing after this 7-day window after each vaccination and unsolicited AEs reported up to 28 days after each vaccination) are shown in Table 7. Overall, unsolicited AEs (post-dose 1 and post-dose 2 combined) were reported in 112/912 (12.3%) participants who received Ebola vaccines compared with 4/58 (6.9%) participants who received placebo. Among those who received the booster dose, 6/43 (14.0%) Ebola vaccine recipients and 1/7 (14.3%) placebo recipients reported at least one unsolicited AE during the post-booster period. The majority of unsolicited AEs were grade 1 or 2 in severity. Unsolicited AEs that were considered related to the study vaccine (post-dose 1 and post-dose 2 combined) were reported in 61/912 (6.7%) participants who received Ebola vaccines compared with 2/58 (3.4%) participants who received placebo. Among those who received the booster dose, unsolicited related AEs were reported in 2/43 (4.7%) participants in the Ebola vaccine groups and in none of the participants in the placebo groups during the post-booster period.

Table 7. Overall summary of unsolicited AEs and SAEs (full analysis set).

AE, n (%)	Ad26.ZEBOV, MVA-BN-Filo	Placebo
	All groups n = 912	All groups n = 58
Post-dose 1, n	912	58
Any unsolicited AE	80 (8.8)	1 (1.7)
Grade 3	5 (0.5)	0
Related	44 (4.8)	0
Grade 3 and related	3 (0.3)	0
Post-dose 2, n	805	51
Any unsolicited AE	39 (4.8)	3 (5.9)
Grade 3	3 (0.4)	0
Related	23 (2.9)	2 (3.9)
Grade 3 and related	2 (0.2)	0
Post-booster dose, n	43	7
Any unsolicited AE	6 (14.0)	1 (14.3)
Grade 3	0	0
Related	2 (4.7)	0
Grade 3 and related	0	0
Entire study (after first vaccination), n	912	58
Any SAE	14 (1.5)	2 (3.4)
Related	0	0

Participants were counted only once within a period for any given event, regardless of the number of times they actually experienced the event in that period.

AE, adverse event; SAE, serious adverse event.

A summary of SAEs after each dose is shown in Table 7, with individual SAEs listed in Supplemental Table S4. Throughout the entire study, the percentage of participants with SAEs was comparable between the Ebola vaccine groups (14/912 [1.5%] participants) and the placebo groups (2/58 [3.4%] participants). Most SAEs reported in the Ebola vaccine groups and all SAEs reported in the placebo groups were reported outside of the 28-day post-vaccination window for analysis of unsolicited AEs. None of the reported SAEs were considered by the investigator to be related to the study vaccine, and all SAEs were reported in, at most, one participant per group in every period. One death was reported; a participant in the Ebola vaccine booster cohort experienced a fatal gunshot wound at 38 days post-dose 2.

Throughout the entire study, unsolicited AEs leading to study vaccination discontinuation were reported in 15/912 (1.6%) participants in the Ebola vaccine group and in 1/58 (1.7%) participant in the placebo groups. All of these AEs were considered by the investigator to not be related to the study vaccine, except for four related AEs in one participant (fatigue, arthralgia, myalgia, and chills) and one related AE in another participant (headache), all of which were reported during the post-dose 1 period.

Discussion

This study demonstrated consistency of the two-dose Ad26.ZEBOV, MVA-BN-Filo vaccine regimen using three consecutively manufactured lots of Ad26.ZEBOV paired with three consecutively manufactured lots of MVA-BN-Filo at 21 days post-dose 2. Consistency is a particular challenge with this Ebola vaccine regimen as it is composed of two different vaccines produced using different manufacturing processes; despite these unique challenges, the equivalence criterion was met. It is also notable that lot-to-lot consistency was assessed and met by Ad26.ZEBOV alone as well, at the pre-dose 2 time point. Overall, the results indicate that there is a consistency in manufacturing as judged by the immunogenicity of the vaccine.

In the main study, almost all participants (99.5%) in the Ebola vaccine groups were responders, as demonstrated by post-vaccination EBOV GP–binding antibody concentrations >2.5-fold the baseline value at 21 days post-dose 2, with a similarly high response rate across the vaccine groups. At 21 days post-dose 2, EBOV GP–binding antibody GMCs were shown to be robust and consistent with those reported in other studies.^13,16,23 High responder rates (95.5%–98.1%) were also observed across the three vaccine groups at 56 days post-dose 1. Both Ad26.ZEBOV and MVA-BN-Filo are nonreplicating in human cells. An advantage of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen over live replicating vaccines is its suitability for use in pregnant women and infants. Additionally, since the Ad26.ZEBOV, MVA-BN-Filo vaccine does not replicate in human cells, a steep drop in anti-GP antibodies is observed from 21 days post-dose 2 to about 6 months post-dose 2. However, a stable plateau follows, which is maintained for at least 4.5 years post-dose 1, after which a strong anamnestic response is observed upon vaccination with an Ad26.ZEBOV booster dose.^20,24 At 1 year, the responder rate observed with the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen is also high, ranging across studies between 49% and 100% in adults and 70% and 100% in children.²⁴

The study also evaluated a booster dose of Ad26.ZEBOV given 4 months after dose 2 of the two-dose regimen, an earlier booster administration than that given in previous studies. In the current study, binding antibody responses were observed in all participants who received the booster dose (responder rate of 100%) at all post-baseline time points. Although an anamnestic response was clearly activated, as evidenced by a 9.0-fold increase in GMCs within 7 days of booster administration, the magnitude of the anamnestic response in the current study was lower than in other studies conducted across different countries and populations where an Ad26.ZEBOV booster dose was administered at a variety of later time points (between 1 and 4.5 years post-dose 1).^13,16,19,20 The vaccine label allows for a booster dose for those at high risk of being in contact with the Ebola virus after 4 months of completing the two-dose course of vaccination.⁶ The results of this study confirm the immunogenicity benefit of the booster given at 4 months post-dose 2 and adds to the overall evidence of the effectiveness of the booster given as early as 4 months or up to at least 4.5 years post-dose 1 that could guide future vaccination strategies, which is particularly useful given the unpredictable nature of Ebola outbreaks.

In general, the safety and tolerability profile of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen in the current study was similar to safety profiles observed in prior studies.^11–18 The vaccines were well tolerated, and no safety signals were detected. Most solicited and unsolicited AEs were mild to moderate (grade 1 or 2) in severity and, throughout the entire study, rates of unsolicited AEs leading to discontinuation of study vaccination were low across groups. No SAEs were considered related to the study vaccine.

As with many clinical trials conducted in recent years, the impact of the COVID-19 pandemic on this study was substantial, as the study was at the stage of recruitment and vaccination when the pandemic started. Due to imposed “stay-at-home” orders, approximately 22% of participants of the immunogenicity subset who were enrolled at the time did not return to the site within the protocol-defined windows for dose 2 administration. Additional participants were therefore enrolled in the immunogenicity subset to maintain approximately 90% power for the primary and secondary hypotheses testing. In addition, the protocol was amended to widen the window for the second vaccination from ±3 days to − 3/+28 days, and participants were reminded more frequently to come in for their second vaccination. While extension of the dose interval may be seen as a limitation of the study results, it is notable that more than three-quarters of participants (77%) in the immunogenicity subset received their second dose within the original 56 days ±3 days window. Furthermore, sensitivity analyses showed that widening of the second vaccination window did not affect vaccine-induced immune responses and that the primary analysis on the immunogenicity subset was robust. Similar findings were observed from a sensitivity analysis in a previously published study of the Ad26.ZEBOV, MVA-BN-Filo vaccine regimen, in which 18% of participants received the second dose outside of the protocol-defined window of 56 days, with actual intervals for these participants ranging from 96 to 147 days.¹⁶ The analysis showed that participants who received a delayed second dose had similar antibody responses to those who received their second dose within the protocol-defined window.

In conclusion, consistency of three consecutively manufactured lots of Ad26.ZEBOV paired sequentially with three consecutively manufactured lots of MVA-BN-Filo was successfully demonstrated, with robust EBOV GP–binding antibody responses observed across all vaccine groups. An Ad26.ZEBOV booster dose administered 4 months post-dose 2 elicited a strong and rapid anamnestic response. Ad26.ZEBOV, MVA-BN-Filo was well tolerated and, overall, no safety issues were identified.

Author contributions statement

NG, AG, JD, JH, KL, CR, and MD were involved in the conception and design of the study. NG, CM, AG, JD, BK, LB-J, JH, KL, CR, and MD contributed to the analysis and interpretation of the data. NG, CM, AG, and JD contributed to drafting of the manuscript. All authors revised the manuscript critically for intellectual content, approved the final version to be published, and agree to be accountable for all aspects of the work.

Prior presentation

The design and findings of this study were previously presented as a poster at the 16th Vaccine Congress, on 12–14 September 2022, at Lake Garda, Italy.

Acknowledgments

The authors wish to acknowledge Chan Tang, of Janssen Vaccines & Prevention B.V., for contributions to this study. Medical writing support was provided by Michelle Hughes, of Lumanity Communications Inc., and was funded by Janssen Vaccines & Prevention B.V.

Disclosure statement

NG, JD, and MD were employees of Janssen at the time the study was conducted. CM, AG, BK, LB-J, JH, KL, and CR are current employees of Janssen. All authors are stockholders of Johnson & Johnson.

Data availability statement

The data sharing policy of Janssen Pharmaceutical Companies of Johnson & Johnson is available at https://www.janssen.com/clinical-trials/transparency. As noted on this site, requests for access to the study data can be submitted through the Yale Open Data Access [YODA] Project site at http://yoda.yale.edu.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website at https://doi.org/10.1080/21645515.2024.2327747.

Additional information

Funding

This study was sponsored by Janssen Vaccines & Prevention B.V.