Heterologous two-dose Ebola vaccine regimen in pregnant women in Rwanda: a randomized controlled phase 3 trial

Categories: Disease & Virus

September 9, 2025

Study design

This is a parallel, randomized, controlled, open-label, single-center clinical trial of the safety, reactogenicity and immunogenicity of the Janssen two-dose Ebola vaccine regimen in healthy adult pregnant women in Rwanda. The study arms compared pregnant women who received the two-dose Ebola vaccine regimen during pregnancy (group A) or after pregnancy completion (group B). Unvaccinated pregnant group B women served as control. The study was conducted between 6 October 2020 (the date the first participant signed an informed consent form) and 2 March 2023 (the date of the last participant visit) at Gisenyi District Hospital in Rubavu and Gihundwe District Hospital in Rusizi, both located in the Western Province of Rwanda bordering the DRC. The study complies with all relevant ethical regulations. The protocol was approved by the Rwanda National Ethics Committee (821/RNEC/2020), the Rwanda FDA (003/CT/RWANDA FDA/2020) and the Emory University Institutional Review Board (STUDY00000367). The study was prospectively registered with ClinicalTrials.gov under NCT04556526 on 21 September 2020. The study protocol has been published¹⁵.

Participants

Pregnant women residing in the catchment areas of the District Hospitals were recruited from antenatal care clinics and referred to the two district hospitals. Written informed consent to participate was obtained. Inclusion criteria were as follows:

1.

Female (according to their reproductive organs and functions assigned by chromosomal complement).
2.

Eighteen years of age or older at the time of written informed consent.
3.

Healthy based on physical examination, medical history, obstetric history and vital signs performed at screening.
4.

Healthy based on clinical laboratory tests performed at screening. If the results of the clinical laboratory tests are outside of the normal local reference ranges, the participant may be included only if the investigator judges the abnormalities or deviations from normal to be not clinically significant or to be appropriate and reasonable for the population under study. This determination must be recorded in the participant’s source documents and initiated by the investigator. Trace protein in the urine is acceptable if the blood pressure is also normal. Participants must have clinical laboratory test results within the following parameters:
1. a.
  
  Hemoglobin ≥7.0 g dl⁻¹.
2. b.
  
  White blood cells ≥3.4 × 10³ cells per µl.
3. c.
  
  Neutrophils ≥1.1 × 10⁹ cells per l.
4. d.
  
  Lymphocytes ≥1.21 × 10⁹ cells per l.
5. e.
  
  Platelets ≥156 × 10³ cells per µl.
6. f.
  
  Serum creatinine ≤129 µmol l⁻¹
7. g.
  
  Aspartate aminotransferase within 1.5× the upper limit of the normal range for the laboratory conducting the test.
8. h.
  
  Alanine aminotransferase within 1.5× the upper limit of the normal range for the laboratory conducting the test.
9. i.
  
  Total bilirubin ≤25 µmol l⁻¹.
10. j.
  
  Urine protein <1+ by dipstick.
11. k.
  
  Urine blood ≤1+ by dipstick, without evidence of bacteriuria on microscopy.

5.

Confirmed singleton pregnancy by positive urine human chorionic gonadotropin and ultrasound at the time of screening and informed consent, and reconfirmed pregnancy via ultrasound at randomization/day 1. Ultrasound not required on randomization/day 1 if ≤10 days have elapsed since the screening ultrasound and an ultrasound is not indicated for other reasons.
6.

Capable and willing to give informed consent (signed or thumbprint), which includes compliance with the requirements and restrictions listed in the informed consent form and in this protocol, and willing to give informed consent for the infant to participate in the study.
7.

Each potential participant must pass the assessment of understanding by indicating that she understands the purpose, procedures and potential risks and benefits of the study after reading the informed consent and after the investigator or designee has provided detailed information on the study and has answered the potential participant’s questions. Each participant must subsequently sign the informed consent form, indicating that she is willing to participate in the study.
8.

Resides within the catchment area of the study site.
9.

Able and willing to participate for the duration of the study visits and follow-up.
10.

Willing and able to comply with the protocol requirements.
11.

Willing to receive standard prenatal care and planning to deliver at a study district hospital.
12.

Willing to provide verifiable identification and have a photo taken and an iris scan at study entry and follow-up visits.
13.

Evidence of normal progress of gestation before randomization (day 1) based on obstetric evaluation (including obstetric history, obstetric examination and fetal ultrasound).
14.

If randomized to group B, must have a negative urine pregnancy test immediately before each study vaccine administration.

Exclusion criteria were as follows:

1.

History of EBOV disease (self-declared or laboratory confirmed).
2.

Has received any candidate Ebola vaccine (except for women found to be pregnant in the UMURINZI study after they received their first dose of vaccine, who are eligible for enrollment into a nonrandomized group under this protocol).
3.

Has received any experimental candidate Ad26- or MVA-based vaccine in the past. Receipt of any approved vaccinia/smallpox vaccine or Ad-based candidate vaccine other than Ad26 at any time before study entry is allowed.
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 (for example, polysorbate 80, ethylenediaminetetraacetic acid or l-histidine for the Ad26.ZEBOV vaccine; tris (hydroxymethyl)-amino methane for the MVA-BN-Filo vaccine)), including known allergies to egg, egg products, chicken proteins and aminoglycosides.
5.

Individuals with acute illness (this does not include minor illnesses such as diarrhea or mild upper respiratory tract infection) or body temperature ≥38.0 °C on day 1 will be excluded from enrollment at that time but may be rescheduled for enrollment at a later date.
6.

Presence of significant conditions (for example, history of seizure disorders, (auto)immune disease or deficiency, any spleen disease, active malignancy, ongoing tuberculosis treatment and other systemic infections) or clinically significant findings during screening of medical history, obstetric history, physical examination, vital signs or laboratory testing for which, in the opinion of the investigator, it would not be in the best interest of the individual (for example, compromise the safety or well-being) or that could prevent, limit or confound the protocol-specified assessments. Individuals who have recently received treatment for acute uncomplicated malaria are eligible for participation if at least 3 days have elapsed from the conclusion of a standard recommended course of therapy for malaria; participants who are acutely ill with malaria at the time of screening should complete therapy and wait an additional 3 days after completion before screening for the study. Individuals with sickle cell traits can be included.
7.

Any of the following during the 6 weeks before screening: (1) confirmed SARS-CoV-2 (COVID-19) infection (positive test), OR (2) suspected SARS-CoV-2 infection (clinical features without documented test results), OR (3) close contact with a person with known or suspected SARS-CoV-2 infection.
8.

History of or underlying liver or renal insufficiency or significant cardiac, vascular, pulmonary (for example, persistent asthma), gastrointestinal, endocrine, neurological, hematological, rheumatological, psychiatric or metabolic disturbances.
9.

History of positive tests for hepatitis B surface antigen (HBsAg) or hepatitis C virus (HCV) antibodies (anti-HCV), or other clinically active liver disease, or testing positive for HBsAg or anti-HCV at screening.
10.

Human immunodeficiency virus (HIV)-infected but not on stable antiretroviral therapy (ART), defined as adherent to the ART regimen for ≥6 weeks before enrollment. Individuals with HIV infection on stable ART may be enrolled.
11.

Obstetric history, including the following:
1. a.
  
  ≥2 consecutive spontaneous abortions.
2. b.
  
  History of preeclampsia or eclampsia.
3. c.
  
  Rhesus-negative multigravida.
4. d.
  
  Grand multigravida (>5 previous pregnancies).
5. e.
  
  Previous late stillbirth (defined as loss of pregnancy at any time after 28 weeks of gestation).
6. f.
  
  Previous low-birth-weight baby or premature delivery (defined as a delivery before 37 weeks of gestation).
7. g.
  
  Previous neonatal death (defined as death of an infant within the first 28 days of life).
8. h.
  
  Previous delivery of an infant with a known or suspected genetic or chromosomal abnormality.
9. i.
  
  History of other significant pregnancy-related or neonatal complications that are judged as likely to affect the safety of the mother or infant or to significantly compromise the collected endpoint data. Previous cesarean section is not an exclusion criterion.

12.

Major surgery within the 4 weeks before screening or planned major surgery through the course of the study (from screening until completion of the study).
13.

Chronic or recurrent use of immunomodulators/suppressors (for example, cancer chemotherapeutic agents or systemic corticosteroids within 6 months before the planned administration of the first dose of study vaccine). Ocular, topical or inhaled steroids are allowed.
14.

Received or planned to receive a licensed nonlive attenuated vaccine (for example, tetanus) within 7 days of a study vaccination (that is, before or after). Received or planned to receive a licensed live attenuated vaccine within 4 weeks of a study vaccination (that is, before or after).
15.

Received an investigational drug or investigational vaccine or used an invasive investigational medical device within 3 months before screening, or current or planned participation in another clinical study during the study. Participation in an observational clinical study is allowed.
16.

Receipt of blood products or immunoglobulin within 3 months before screening and/or during participation in the study (except for RhoGAM).
17.

Current or past abuse of alcohol or recreational or narcotic drugs that, in the investigator’s opinion, would compromise the individual’s safety and/or compliance with the study procedures.
18.

History of chronic urticaria (recurrent hives).
19.

Individual cannot communicate reliably with the investigator.
20.

Employee of the investigator or study site, with direct involvement in the proposed study or other studies under the direction of that investigator or study site, including family members of the employees or the investigator.
21.

History of thrombotic thrombocytopenia syndrome (TTS) or heparin-induced thrombocytopenia and thrombosis.

Eligibility was initially limited to women in their second or third trimester of pregnancy until IDMC and the study medical officer reviewed safety data from at least 100 vaccinated pregnant women (group A) and at least 100 nonvaccinated pregnant women (group B), followed for at least 85 days postenrollment. When these data indicated no safety issues, enrollment was extended to women in their first trimester of pregnancy. Trimester at enrollment was categorized as first trimester (0–12 weeks), second trimester (>12–24 weeks) or third trimester (>24 weeks). The estimated gestational age was based on ultrasound for women enrolled in the first trimester. For women enrolled in the second and third trimesters, estimated gestational age was based on ultrasound and the last menstrual period as recommended by the American College of Obstetricians and Gynecologists (ACOG)¹⁶.

For women, sex was based on clinical observation—all women participating in the study were pregnant at some point. For infants, sex observed at birth was reported by investigators. The results are described for infants of both sexes together. No difference by sex of the infant was expected.

Randomization and masking

Eligible pregnant women were randomly assigned 1:1 to receive the two-dose Ebola vaccine regimen during pregnancy (group A) or after pregnancy completion (group B). An allocation template was created by coauthor S.A. at Emory University using simple randomization based on the Microsoft Excel random number generator function. Security-type, opaque envelopes containing the randomization assignment cards were prepared by one study team member and inspected by two other team members to ensure correspondence to the allocation table before sealing the envelopes and deploying them to the sites. The envelopes at each site were placed in a single bin, and participants were invited to reach into the bin to select a single sealed envelope containing a randomization assignment at the time of enrollment. Study staff responsible for enrolling participants and overseeing randomization did not have access to the allocation table; therefore, arm assignment within each envelope was unknown to participants and staff until opened. The randomization assignment was complete and irreversible once the envelope was opened. Birth attendants were not formally notified about the vaccine allocation. There was no masking in this study. An open-label design was selected so that group B women could receive the benefits of the vaccine regimen.

Procedures

All study procedures were performed in the district hospitals, including pre-enrollment screening. A schematic overview of study activities and timeline is shown in Supplementary Fig. 1; detailed schedules of activities are published¹⁵. A target of 2,000 pregnant women ≥18 years of age was to be randomized on day 1. Women randomized to group A received the two-dose Ebola vaccine regimen on days 1 and 57 during pregnancy. Women randomized to group B received the vaccine regimen from 6 weeks postpartum up to 10 weeks postpartum.

Study vaccines were manufactured and provided under the responsibility of Janssen Vaccines and Prevention B.V. Ad26.ZEBOV is a nonreplicating monovalent vaccine that encodes the full-length GP from EBOV Mayinga. The vaccine is produced in the human cell line PER.C6. A 0.5-ml intramuscular injection of 5 × 10¹⁰ viral particles was administered on day 1 (group A) or between 6 and 10 weeks after completion of pregnancy (group B). This vaccine was administered into the deltoid muscle in the upper arm (or thigh if needed). MVA-BN-Filo does not replicate in human cells and is a multivalent vaccine encoding the EBOV Mayinga GP, the Sudan virus Gulu GP, the Marburg virus Musoke GP and the Taï Forest virus nucleoprotein. The EBOV GP expressed by MVA-BN-Filo has 100% homology with the one expressed by Ad26.ZEBOV. A 0.5-ml intramuscular injection of 1 × 10⁸ infectious units of MVA-BN-Filo was administered 56 days (−14 days to +28 days) after the Ad26.ZEBOV dose. This vaccine was administered into the deltoid muscle in the upper arm (or thigh if needed), ideally in the opposite arm/thigh as the first dose (unless the opposite arm/thigh had a condition preventing evaluation of the arm/thigh after injection).

As shown in Supplementary Fig. 1, a target subset of 300 women was to be enrolled and provide blood samples for immunogenicity assessment of the two-dose regimen administered during pregnancy (group A) and after pregnancy (group B).

Group A women were followed until at least 6 weeks after pregnancy completion to capture adverse maternal/fetal outcomes of interest. Group B women were followed until at least 4 weeks after dose 2. Participants in the immunogenicity subset were followed for 12 months after dose 1. Therefore, the duration of individual participation could range from approximately 6 to 23 months. All infants were followed for 14 weeks postdelivery.

Women who tested positive for pregnancy during the UMURINZI Ebola Vaccination Campaign after they received dose 1 were offered screening and enrollment into the present study to receive dose 2 in a controlled setting (group C). These women were not randomized but were followed for safety outcomes for 6 weeks after pregnancy completion (women) or 14 weeks postdelivery (infants).

There were three amendments to the original protocol. Protocol amendment 1 was issued on 8 July 2020. The overall reason for the amendment was to incorporate comments from the ethics committee on the initial protocol, which was never approved by the Health Authority. The study was only initiated under protocol amendment 1. Protocol amendment 2 was issued on 22 July 2021. The overall reason for the amendment was to include information about TTS observed with Janssen’s COVID-19 vaccine and clarification of the safety notification process. Main changes included the exclusion of participants with a known history of TTS or heparin-induced thrombocytopenia with thrombosis, as well as those who planned to or received a COVID-19 vaccine within a disallowed timeframe before study vaccination. Additionally, a 24-h reporting timeframe for TTS events was added, and potential conditions that should be reported within 24 h were listed. Protocol Amendment 3 was issued on 24 November 2021. The overall reason for the amendment was to include additional language around TTS, as recommended by the United States FDA on 17 September 2021. Main changes included an update of the description of TTS observed with Janssen’s COVID-19 vaccine to align with the language recommended by the FDA and inclusion of the recommendation to evaluate any potential risk factors for TTS in participants based on medical history.

Outcomes

Primary and secondary objectives and corresponding outcomes are shown in Table 4. Brighton Collaboration case definitions for primary adverse outcomes of interest are shown in Extended Data Table 3. Outcomes were abstracted from medical records linked across participants using a unique participant identifier and biometric data¹⁷. All AEs were recorded using medical terminology in the source documents and case report forms. AEs were coded in accordance with the ‘Medical Dictionary for Regulatory Activities v25.1’ using the lower-level term as the description most closely related to the investigator’s terminology, a preferred term describing a group of closely related lower-level terms, and the system organ class, which is the broad category including related preferred terms. The reporting of safety data included the incidence, severity and relatedness.

Table 4 Trial objectives and outcomes

Clinical outcomes were collected via hardcopy paper records and captured and validated electronically in RedCap Cloud, with additional validation performed using SAS v9.4. Immunogenicity samples were analyzed using the Filovirus Animal Nonclinical Group (FANG) enzyme-linked immunosorbent assay (ELISA) to assess concentrations of anti-EBOV GP-specific binding antibodies.

Immunogenicity assessments

Venous blood samples were collected for immunogenicity assessments at protocol-defined visits. IgG binding antibodies against EBOV GP were measured using the validated and FDA-endorsed FANG ELISA, performed by Q2 Solutions Vaccine Testing Laboratory.

Statistical analysis

The study is not powered for any formal statistical hypothesis testing. A target sample size of 1,000 participants in group A and 1,000 participants in group B was determined based on feasibility and the estimated background incidence of key pregnancy outcomes. With a sample size of 1,000, the probability of observing at least one AE occurring at a rate of 1 in 1,000 was 63%. If no SAEs or AEs were observed in group A, this would provide 95% confidence that the true incidence was ≤0.3%. The width of the 95% CI for observing an AE at a rate of 1 in 1,000 is 0.006. An overenrollment of ≤10% was permitted. For a 10% overenrollment, the width of the 95% CI for observing an AE at a rate of 1 in 1,000 is 0.005.

Data were analyzed using SAS v9.4. No formal statistical hypothesis testing was planned. To aid understanding of the uncertainty around the estimates, CIs for primary endpoint estimates and the risk differences between groups A and B were computed post hoc. The CI of the risk differences should be interpreted with caution, given the highly inflated type I error due to the numerous primary outcomes. Data from group C (n = 5) were analyzed separately and are not included here. Group C is not expected to influence the conclusions of the study. The unvaccinated pregnant women in group B served as the control group for the vaccinated pregnant women in group A. For group A, the primary analysis dataset for baseline demographic characteristics and safety data was the FAS, which included all randomized participants who received at least one dose of the study vaccine regimen, regardless of protocol deviations, and all infants born to these women. For group B, the primary analysis dataset for baseline demographic characteristics and safety data was the EAS, which included all women enrolled and all infants born to these women.

Immunogenicity assessments were performed on the PPI analysis set, which included all women who provided informed consent for immunogenicity sample collection, received both dose 1 and dose 2 within the protocol-defined window and did not have major protocol deviations that could impact immunogenicity outcomes (for example, another vaccination and immunomodulating medications). Infants in the PPI analysis set were those born to group A women in the PPI analysis set who also provided immunogenicity samples.

The number and percentage of participants screened, enrolled/randomized, vaccinated and completed follow-up were summarized following the Consolidated Standards of Reporting Trials guidance. Major (defined as those that would jeopardize the ability to evaluate study endpoints) and minor protocol deviations were described by group. Baseline demographic characteristics were described by group. The frequency and percentage of primary adverse outcomes of interest were tabulated by group and trimester. Adverse maternal/fetal outcomes of interest were described from randomization until 6 weeks postpartum. Women who discontinued the study without reporting any adverse maternal/fetal outcomes were excluded from the analysis. Additionally, the frequency and percentage of maternal and infant SAEs were tabulated by group; participants were counted only once for any given event, regardless of the number of times they experienced the event.

Missing data were not imputed. Participants who discontinued the study without reporting any maternal/fetal outcomes were excluded from the analyses because of completely missing information on the pregnancy outcome. Efforts were made to query the sites for missing severity of the AE or missing relationship of the AE with the study vaccine. An AE with a missing severity or relationship was considered as an AE reported, but was considered as not reported for the severity or relationship. For example, an AE with missing severity was considered as an AE reported for the analysis of any grade, but was not considered for the analysis of grade 3.

GMCs with corresponding 95% CIs were calculated for the continuous immunological parameter of anti-EBOV GP-specific binding antibody concentration at each time point. Sample positivity (defined as the proportion of samples with a quantifiable response) and the proportion of participants who were responders (defined as the proportion of participants with a greater than 2.5-fold increase in anti-EBOV GP-binding antibody concentration compared to baseline) were calculated at each time point, along with their corresponding Clopper–Pearson exact 95% CIs. All values below the lower limit of quantification (LLOQ) were imputed with half the LLOQ for calculation of GMC, and values greater than the upper limit of quantification (ULOQ) were imputed with the 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 evaluate potential associations between anti-EBOV GP-specific binding antibody concentrations in women at different time points (predose 1, 21 days postdose 2, postpartum day 1 and 365 days postdose 1), cord blood at postpartum day 1 and infants at 14 weeks of age, a post hoc correlation analysis was performed. Partial Spearman correlation coefficients controlling for the trimester of the mother at randomization were calculated, and correlation plots were generated.

An IDMC consisting of independent experts in obstetrics, pediatrics and statistics evaluated safety data twice at the outset of the study and then three times monthly. The study was prospectively registered with ClinicalTrials.gov under NCT04556526 on 21 September 2020.

Ethics and inclusion statement

Local researchers based at Center for Family Health Research (CFHR) were included throughout the research process, including study design, study implementation, data ownership, intellectual property and authorship of publications. Publications are inclusive of local and regional research relevant to our study. The research is locally relevant and conducted with local leadership and in collaboration with the Rwandan Ministry of Health. Roles and responsibilities were agreed upon among collaborators ahead of the research. The study did not include formal plans for local capacity building. The study was approved by local ethics review committees. The study did not pose any health, safety, security or other risk to researchers. The study could cause risk to participants, and participant safety procedures are described in the manuscript.