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Published in final edited form as: Lancet Infect Dis. 2023 Aug 3;23(12):1408–1417. doi: 10.1016/S1473-3099(23)00344-4

Safety, tolerability, and immunogenicity of the Ebola Sudan chimpanzee adenovirus vector vaccine (cAd3-EBO S) in healthy Ugandan adults: a phase 1, open-label, dose-escalation clinical trial

Betty Mwesigwa 1, Katherine V Houser 2, Amelia R Hofstetter 2, Ana M Ortega-Villa 3, Prossy Naluyima 1, Francis Kiweewa 1, Immaculate Nakabuye 1, Galina V Yamshchikov 2, Charla Andrews 2, Mark O’Callahan 2, Larisa Strom 2, Steven Schech 4,5, Leigh Anne Eller 4,5, Erica L Sondergaard 4,5, Paul T Scott 4, Mihret F Amare 4,5, Kayvon Modjarrad 4, Amir Wamala 1, Allan Tindikahwa 1, Ezra Musingye 1, Jauhara Nanyondo 1, Martin R Gaudinski 2, Ingelise J Gordon 2, LaSonji A Holman 2, Jamie G Saunders 2, Pamela J M Costner 2, Floreliz H Mendoza 2, Myra Happe 2, Patricia Morgan 2, Sarah H Plummer 2, Somia P Hickman 2, Sandra Vazquez 2, Tamar Murray 2, Jamilet Cordon 2, Caitlyn N M Dulan 2, Ruth Hunegnaw 2, Manjula Basappa 2, Marcelino Padilla 2, Suprabhath R Gajjala 2, Phillip A Swanson II 2, Bob C Lin 2, Emily E Coates 2, Jason G Gall 2, Adrian B McDermott 2, Richard A Koup 2, John R Mascola 2, Aurélie Ploquin 2, Nancy J Sullivan 2, Hannah Kibuuka 1,*, Julie A Ake 4,*, Julie E Ledgerwood 2,*; RV 508 Study Team
PMCID: PMC10837320  NIHMSID: NIHMS1924129  PMID: 37544326

Summary

Background

Sudan ebolavirus (SUDV) can cause severe viral disease, with an average case fatality rate of 54%. A recent outbreak of SUDV in Uganda caused 55 deaths among 164 confirmed cases in the second half of 2022. Although vaccines and therapeutics specific for Zaire ebolavirus have been approved for use during outbreak situations, SUDV is an antigenically distinct virus with no approved vaccines available.

Methods

We conducted an open-label, dose-escalation, phase 1 trial to evaluate the safety, tolerability, and immunogenicity of a monovalent chimpanzee adenovirus 3 vaccine against SUDV (cAd3-EBO S) at Makerere University Walter Reed Project (MUWRP) in Kampala, Uganda. Forty healthy adults without previous receipt of Ebola, Marburg, or cAd3 vectored-vaccines were enrolled between July 22nd and October 1st of 2019 to receive cAd3-EBO S at either 1×1010 or 1×1011 particle units in a single intramuscular vaccination and were followed for 48 weeks. Primary safety and tolerability endpoints were assessed in all vaccine recipients by reactogenicity for the first seven days, adverse events for the first 28 days, and serious adverse events (SAEs) throughout the study. Secondary immunogenicity endpoints included evaluation of binding antibody and T cell responses against the Sudan glycoprotein (GP), and neutralizing antibody responses against the cAd3 vector at four weeks after vaccination. This study is registered with ClinicalTrials.gov, NCT04041570.

Findings

All participants received cAd3-EBO S (n=40/40, 100%), with 38/40 (95%) completing all follow-up visits. The cAd3-EBO S vaccine was well-tolerated with no SAEs. The most common reactogenicity symptoms were pain/tenderness at the injection site (n=34/40, 85%), fatigue (n=29/40, 73%), and headache (n=26/40, 65%), and were mild to moderate in severity. Positive responses for GP-specific binding antibodies were induced by two weeks in 78% (n=31/40) of participants, increased to 85% (n=34/40) of participants by four weeks, and persisted out to 48 weeks in 82% (n=31/38) of participants. Most participants developed GP-specific T cell responses (n=20/34; 59%, 95% CI: 41–75%) by four weeks after vaccination, and neutralizing titers against the cAd3 vector were also increased from baseline (47 IC90, 95% CI: 30–73) to four weeks after vaccination (196 IC90, 95% CI: 125–308).

Interpretation

The cAd3-EBO S vaccine was safe at both doses, rapidly inducing immune responses in most participants after a single injection. The rapid onset and durability of the vaccine-induced antibodies make this vaccine a strong candidate for emergency deployment in SUDV outbreaks.

Funding

The National Institutes of Health funded the study via an interagency agreement with Walter Reed Army Institute of Research.

Introduction

Over 40 known human outbreaks of Filoviridae viruses have occurred, with increasing regularity since their discovery in 1976.1 Two genera of viruses within the Filoviridae family are known to infect humans: Ebolavirus and Marburgvirus.2 Sudan ebolavirus and Zaire ebolavirus are the two species of the Ebolavirus genus responsible for the majority of outbreaks of Ebola virus disease (EVD).1 A recent 2022 outbreak of Sudan ebolavirus (SUDV) in Uganda caused 55 deaths among 142 confirmed cases,3 a case fatality rate (CFR) of 34%. Among seven prior SUDV outbreaks in Sudan and Uganda between 1976 and 2012, there were a total of 793 confirmed cases and 426 deaths,4 resulting in a CFR of 54%.5 The increased size of outbreaks with consequent spread of filoviruses into additional countries since 2013 emphasizes the urgent need to develop vaccines to limit and prevent future filovirus outbreaks.5 Although two vaccines have attained WHO Prequalification status for use against Zaire ebolavirus (EBOV),6,7 EBOV-specific vaccines are unlikely to confer cross-protective immune responses against the antigenically distinct SUDV8. Therefore, there remains an unmet need for vaccines and therapeutics in SUDV outbreaks.

Currently, the most clinically advanced SUDV-specific vaccine is the chimpanzee adenovirus 3 (cAd3) vaccine reported here. Preclinically, the cAd3-EBO S vaccine was immunogenic, demonstrating protective durability out to 12 months in non-human primates.9 Multiple phase 1/1b trials involving the cAd3 platform in a bivalent Zaire-Sudan cAd3-EBO vaccine have been previously conducted (NCT02354404, NCT02231866, NCT02408913, NCT02368119).10 This bivalent vaccine was shown to have an excellent safety profile and was immunogenic, resulting in both Zaire- and Sudan-specific antibodies and T cell responses by four weeks after vaccination.10 Additional multivalent vaccines are currently undergoing phase 1 clinical testing. The first, a ChAdOx1 biEBOV vaccine expressing both Zaire and Sudan glycoproteins (GPs) is being studied in two ongoing phase 1 trials in the UK and Tanzania (NCT05079750, NCT05301504).11 The second, a multivalent Ad26.Filo/MVA-BN-Filo vaccine regimen, recently reported results demonstrating safety and immune responses against Zaire, Sudan, and Marburg antigens after completion of the prime-boost regimen (NCT02860650).12 Additionally, although the extensive clinical experience with the Zaire-specific rVSV-ZEBOV vaccine may be extrapolated for the Sudan-specific rVSV-SEBOV-GP vaccine, that SUDV-specific product has yet to be evaluated in humans.13,14 To date, there are no Sudan-specific single-dose vaccines for use in endemic areas or during SUDV outbreaks.

The bivalent cAd3-EBO vaccine has displayed promising safety and immunogenicity results in previous trials, however the cAd3-EBO S vaccine has never been evaluated in a monovalent form. The Vaccine Research Center (VRC) and Walter Reed Army Institute of Research (WRAIR), in partnership with the Makerere University Walter Reed Project (MUWRP), evaluated the monovalent cAd3-EBO S vaccine in Kampala, Uganda. In this trial (NCT04041570) we evaluated the safety, tolerability, and immunogenicity of cAd3-EBO S in healthy adults following a single dose of either 1×1010 particle units (PU) or 1×1011 PU of cAd3-EBO S. These doses were found to be safe and immunogenic in prior preclinical and clinical trials evaluating cAd3-vectored vaccines.10,15,16 Importantly, this trial took place in Kampala, Uganda, and therefore evaluated the vaccine in a population regularly threatened by SUDV outbreaks.

Methods

The description of some parts of the Methods is similar to those reported for a trial we recently conducted for a cAd3-vectored Marburg virus vaccine candidate16 because the techniques used were identical/similar.

Study Design and Participants

This study (NCT04041570) was a phase 1, open-label, dose-escalation clinical trial examining the safety, tolerability, and immunogenicity of two different doses (1×1010 PU and 1×1011 PU) of a SUDV chimpanzee adenovirus vector vaccine, cAd3-EBO S, in healthy adults. Scientists at the VRC at the National Institute of Allergy and Infectious Disease (NIAID) of the U.S. National Institutes of Health developed the vaccine and sponsored the trial conducted by MUWRP investigators at the MUWRP Clinical Trial Site in Kampala, Uganda. The study was reviewed and approved by the Makerere University School of Public Health (MUSPH) Institutional Review Board (IRB). Study participants were recruited from the Kampala metropolitan area using IRB-approved written and electronic media. Women who were pregnant, breastfeeding, or planning to become pregnant during the first 24 weeks of the trial were excluded from enrollment. Pregnancy was evaluated during screening and at enrollment with a beta-human chorionic gonadotropin pregnancy test. Eligible participants were 18- to 50-year-old adults in good general health by physical exam and laboratory assessments, without previous receipt of an investigational Ebola, Marburg, or cAd3-vectored vaccine. Full inclusion and exclusion criteria are detailed in the trial protocol (Appendix, page 34–37). All participants provided written informed consent prior to study enrollment. An assessment of understanding was completed during consent, where participants had to score at least 90% within three attempts in order to enroll in the trial.

Vaccine

The recombinant chimpanzee adenovirus 3 SUDV (cAd3-EBO S) vaccine is comprised of a replication-deficient cAd3 vector modified by an E1 region deletion and insertion of an expression cassette for the wild-type GP sequence from the SUDV Gulu strain. The drug substance was manufactured at Advent (Pomezia, Italy), a subsidiary of Okairos (now GlaxoSmithKline). The drug product (VRC-EBOADC086–00-VP), and diluent (VRC-DILADC065–00-VP) were manufactured according to cGMP regulations at the VRC Pilot Plant (VPP), operated by the Vaccine Clinical Materials Program, Leidos Biomedical Research, Inc., Frederick, MD. The investigational product was a sterile, aqueous, buffered solution composed of cAd3-EBO S drug substance filled into single dose vials at 1×1011 PU/mL. This monovalent vaccine was evaluated at two doses in this trial: 1×1010 (henceforth 1010) PU, and 1×1011 (henceforth 1011) PU. Diluent was added to prepare the 1010 PU dose on the day of vaccine administration. The diluent consisted of 10 mM Tris, 10 mM histidine, 5% sucrose (w/v), 75 mM sodium chloride, 1 mM magnesium chloride, 0·02% polysorbate 80 (w/v), 0·1 mM EDTA, and 0·5% (v/v) ethanol.

Study Procedures

Two groups of 20 participants sequentially enrolled according to a dose-escalation plan, each participant receiving a single vaccination on Day 0. Study enrollments for the 1010 PU vaccine dose were limited to one participant per day for the first three participants. Enrollment for the remaining participants in the 1010 PU dose group occurred following a minimum of seven days follow-up and safety review by the protocol safety review team (PSRT). Enrollment of participants into the 1011 PU dose group began in the same manner following a minimum of seven days follow-up on the last 1010 PU dose participant and a dose escalation safety review approval by the PSRT.

All vaccinations were given intramuscularly into a deltoid muscle in a 1 mL volume by needle and syringe. Safety monitoring included a 30-minute post-vaccination observation period, telephone follow-up the next day, and clinical and laboratory evaluations conducted at eight follow-up visits over the 48 weeks of the study. Participants self-reported local and systemic symptoms and the use of concomitant medications for seven days following vaccination. AEs were recorded for 28 days following vaccination and were graded according to the United States Food and Drug Administration (FDA) Guidance for Industry: “Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventative Vaccine Clinical Trials” as well as additional grading parameters for absolute neutrophil counts and arthralgias (Appendix, page 127). The relatedness of AEs to the vaccine product was determined by the investigator, at times informed by discussions with the PSRT.

Outcomes

The primary study endpoints were safety and tolerability of the cAd3-EBO S vaccine. Safety and tolerability of the cAd3-EBO S vaccine were analyzed descriptively in all participants who received the vaccine (modified intent-to-treat), and were defined by the occurrence of solicited local and systemic reactogenicity for seven days following vaccination, change from baseline for safety laboratory measures (complete list in trial protocol), occurrence of adverse events (AEs) for 28 days after vaccination, and the occurrence of serious adverse events (SAEs) and new chronic medical conditions through the last study visit. The secondary endpoints included evaluation of antibody responses to the Sudan GP insert by ELISA, T cell responses by ICS, and neutralizing antibody titers against the cAd3 vector at four weeks after vaccination. In addition, an exploratory endpoint assessed vaccine-induced antibody durability through 48 weeks after vaccination. PBMCs and sera were collected for secondary endpoints at baseline and week four. Sera were evaluated for exploratory endpoints also at days 3 and 7, and weeks 2, 8, 16, 24, and 48. The Anti-Sudan GP IgG ELISA, T cell intracellular cytokine staining, and cAd3 serologic assessment assays, along with the the statistical analyses (performed in R version 4.0.4, with DescTools and Barnard packages), are described in the Appendix, page 7–9.

Role of Funding Source

NIAID funded the study via an interagency agreement with WRAIR and approved the study design. VRC sponsored and designed the study and conducted the research assays, data analysis, and data interpretation. VRC, WRAIR and MUWRP investigators contributed to writing this report. The trial principal investigator and corresponding author had full access to all the data in the study and had final responsibility to submit for publication.

Results

Between July 22nd and October 1st of 2019, at the Makerere University Walter Reed Project in Kampala, Uganda, 40 healthy adults were enrolled in the study (Figure 1). Participants’ mean age was 29 years (range: 18–44) and 75% were male (n=30/40). Additional participant demographics are included in Table 1. Each participant received a single vaccination of cAd3-EBO S at either 1010 or 1011 PU. All participants completed their secondary endpoint visits, however two (n=2/40, 5%) participants in the 1011 PU dose group moved from the area before completing all follow-up visits. The remaining participants (n=38/40, 95%) completed all protocol-specified visits.

Figure 1: Study Consort Diagram.

Figure 1:

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Forty participants were enrolled into one of two dose groups; safety and immune responses were followed for 48 weeks following vaccination. Two participants in the 1011 PU dose group moved from the area (one after week four and one after week 16). “Follow-up completed” indicates the participant was followed through the duration of the protocol-specified visits.

Table 1:

Demographics

Category Sub-category 1010 PU (n=20) 1011 PU (n=20) Overall (n=40)
SEX a Male 19 (95%) 11 (55%) 30 (75%)
Female 1 (5%) 9 (45%) 10 (25%)
AGE Mean (S.D.) 31.5 (6.9) 26.4 (7.3) 29.0 (7.4)
Range [22, 44] [18, 43] [18, 44]
RACE African Ugandan 20 (100%) 20 (100%) 40 (100%)
BMI b Mean (S.D.) 23.4 (3.5) 23.7 (4.4) 23.6 (3.9)
Range [17.6, 31.1] [19.0, 34.9] [17.6, 34.9]
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a

Sex and race were self-reported by the participants. Sex was reported based on the sex assigned at birth

b

BMI: Body mass index at enrollment, reported as weight in kilograms divided by the square of height in meters.

Data are n (%) or mean (S.D.) unless otherwise noted.

The vaccine was safe and generally well-tolerated. The most common local symptom was pain/tenderness at the injection site (n=34/40, 85%). Solicited systemic reactogenicity (Figure 2) was predominantly mild (n=16/40, 40%) or moderate (n=18/40, 45%). The only exception was one instance (n=1/40, 3%) of severe transient fever in a participant the day after receiving cAd3-EBO S at 1011 PU. This fever was measured at 103.1°F (39.5°C) and lasted less than one day. The most commonly reported systemic symptoms were fatigue (n=29/40, 73%) and headache (n=26/40, 65%). No serious adverse events (SAEs), onset of new chronic medical conditions, or safety pauses occurred during the trial. Five (n=5/40, 13%) participants experienced an AE evaluated as related to vaccination: a single instance of mild dizziness (n=1/40, 3%) beginning on the day of injection and lasting two days, two instances of leukopenia (n=2/40, 5%, one mild and one moderate) beginning three days after vaccination (the moderate leukopenia was resolved 26 days later), and two (n=2/40, 5%) instances of moderate neutropenia beginning on day 3 or day 28 (Table 2). All AEs resolved without sequelae, and leukocyte perturbations resolved by the next complete blood count with the single above-noted exception.

Figure 2: Maximum local and systemic solicited symptoms.

Figure 2:

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Percent of participants (x axis) reporting local or systemic symptoms (y axis) in the seven days following cAd3-EBO S vaccination. For symptoms persisting more than one day, a single count per person at the maximum severity of the symptom was used for the figure. Number of participants reporting each symptom noted in overlaid text.

Table 2.

Adverse events related to cAd3-EBO S vaccination.

Participant Vaccination Dose Related Adverse Events Severity Days After Vaccination Days to Resolution
1 10 10 Leukopenia Moderate 3 26
2 Neutropenia Moderate 28 29
3 10 11 Dizziness Mild 0 2
4 Leukopenia Mild 3 12
5 Neutropenia Moderate 3 12
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The cAd3-EBO S vaccine was immunogenic. Eighty-five percent of participants (n=34/40) developed a positive binding antibody response at four weeks after vaccination, the secondary endpoint for the trial (Figure 3). The geometric mean titer (GMT) at four weeks (Figure 3A; Appendix, page 5) was significantly (p=0.0012 by Welch’s two-sample t test, p=0.0027 by Wilcoxon rank sum test) higher after the 1011 PU dose (449 EC90, 95% Confidence Interval (CI): 249–809) than after the 1010 PU dose (125 EC90, 95% CI: 77–201). In exploratory investigations, additional times after vaccination were assayed (Figure 3B; Appendix, page 5). Importantly, GMT reached near peak levels (277 EC90 for 1011 PU and 102 EC90 for 1010 PU) as early as two weeks after vaccination (Figure 3B), with positive responses in 78% (n=31/40) of participants. Additionally, GMTs were found to be significantly greater after 1011 PU than 1010 PU at most timepoints after vaccination (Figure 3B: Appendix, page 5). The binding antibody responses were durable, with no significant decrease in the GMTs between the week four response and the week 48 response. Indeed, 82% of participants (n=31/38) maintained their positive responder status out to week 48, the final timepoint evaluated (Figure 3C).

Figure 3. cAd3-EBO S vaccine elicits increases in antibody titers that are sustained through 48 weeks post-vaccination.

Figure 3.

Figure 3.

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Baseline-subtracted serum Sudan GP ELISA EC90 titers (y axis) are plotted by (A) vaccine dose (x axis) at the peak (week four) of the response and (B) weeks post-vaccination (x axis) through 48 weeks following vaccination. In (A), each square indicates a participant’s individual titer, and black lines indicate the group GMTs, which are also provided numerically above the population. In (B) squares indicate group GMTs and whiskers are 95% CIs. In (A) and (B), p values result from a Wilcoxon rank-sum test comparing dose groups. (C) Positive response rates at weeks 4 and 48 as defined by a significant increase over baseline titers.

Vaccination with cAd3-EBO S also elicited T cell responses by four weeks after vaccination (Figure 4, Appendix, page 6). Most participants developed a T cell response specific for Sudan GP (n=20/34; 59%, 95% CI: 41–75%). Six participants receiving the 1011 dose had their T cell data omitted from the ICS analysis due to failing quality control parameters. Around half of participants were determined to have a Sudan GP-specific CD4 T cell response (n=19/34; 56%, 95% CI: 38–73%) and a quarter were determined to have a Sudan GP-specific CD8 T cell response (n=9/34; 26%, 95% CI: 13–44%). The responses of all participants with a positive CD4 T cell response after 1011 PU included TNF-α-producing cells (CD4+ TNF+), significantly (p=0.0078) more than the frequency of CD4+ TNF+ responders after 1010 PU (Appendix, page 6). Similarly, a greater proportion of 1011 participants developed IL-2-producing CD4 T cells after vaccination than 1010 recipients (p=0.049). The responses of all participants with a positive CD8 T cell response included IFN-γ-producing cells (CD8+ IFN+). Among non-naïve T cells (Figure 4), defined by exclusion of the naïve CD45RA+ CCR7+ population, both CD4 and CD8 T cell populations responsive to Sudan GP significantly increased after either vaccine dose by the four-week secondary endpoint. The increase from baseline was significantly greater after the 1011 PU dose compared to the 1010 PU dose for CD4 T cells (p=0.0052 by Welch’s two-sample t test, p=0.0030 by Wilcoxon rank sum test), with a similar trend observed for CD8 T cells (p=0.041 by Welch’s two-sample t test, p=0.063 by Wilcoxon rank sum test). The cAd3-EBO S vaccine elicited T cell responses in most participants, with patterns of dose-dependency and CD4 T cell predominance as seen for previously published SUDV GP-containing vaccines.17–19

Figure 4. The frequency of Sudan GP-specific non-naive CD4 and CD8 T cells increases by four weeks after vaccination.

Figure 4.

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Percent of background-subtracted non-naïve (A) CD4 T cells or (B) CD8 T cells (y axis) producing any one of the three tested cytokines (IFN-γ, IL-2 or TNF-α) in response to Sudan GP peptide stimulation at baseline and four weeks post-vaccination by dose group (x axis). Within each violin plot, the black line indicates the median and the colored lines indicate the quartiles. Above each violin plot, the group mean (and standard deviation) is indicated. n=20 for 1010 and 14 for 1011 at each time point. p values result from paired t-tests comparing background-subtracted non-naïve T cell frequencies from baseline to week 4.

To better visualize the vaccine-induced cell-mediated and humoral immune responses, the participants were stratified based on positive responses by ELISA or ICS in a post-hoc analysis (Appendix, page 2). Only three participants (9%, n=3/34) were non-responders by either assay. The majority of participants (59%, n=20/34) had positive responses by more than one parameter, with 71% after the 1011 dose (n=10/14) and 50% after the 1010 dose (n=10/20). Half of the participants (n=17/34) had a CD4 T cell response accompanying their antibody response. Further investigation revealed that week four CD4 T cell responses and ELISA titers were positively correlated (r=0.664, p:<0.0001).

The cAd3-EBO S vaccine elicited cAd3 vector-specific neutralizing antibody responses in recipients (Appendix, page 3). There was a significant (p<0.0001) increase in cAd3 neutralization titers for all participants from baseline to week four. However, we found in an post-hoc analysis that the presence of cAd3 vector-specific baseline titers had no impact on week four Sudan GP-specific T cells (CD4 T cells: r=−0.269, p=0.12; CD8 T cells: r=−0.25, p=0.16), and were weakly correlated with week four Sudan GP-specific ELISA titers (r=−0.45, p=0.0041) (Appendix, page 4).

Discussion

In this first report evaluating the monovalent cAd3-EBO S vaccine, we demonstrate the safety, tolerability, and immunogenicity of a vaccine that specifically targets SUDV. The results demonstrate that cAd3-EBO S was well tolerated and immunogenic at both doses evaluated. Importantly, no serious adverse events were experienced by the participants in the study. The binding antibody and T cell responses indicate that the 1011 PU dose may offer enhanced immunogenicity, particularly within the first few weeks after vaccination, without a concerning decrease in tolerability. A safe and efficacious SUDV vaccine is urgently needed; especially in light of the recent outbreak in Uganda, the fifth such outbreak recorded for the country. These findings support advancing this vaccine to phase 2 and 3 clinical testing.

The results reported here represent the first monovalent Sudan vaccine clinical trial published to date. The Sudan GP was previously used as a component of a bivalent cAd3 vaccine (cAd3-EBO) with the GPs from both SUDV and EBOV.10 The safety and immunogenicity findings of this monovalent vaccine trial are consistent with the findings of the prior bivalent vaccine trial.10 While the assays used between the trials were not identical and cannot be directly compared, similar dose-dependent trends in antibody responses were observed in both trials at week four, including a significant difference between dose groups (p=0.01 in the bivalent trial). Furthermore, a similar frequency of participants had a positive response to the SUDV GP after the monovalent (85%) and bivalent vaccines (70–80%).10

In additional clinical trials, Ebola Zaire and Ebola Sudan GPs were tested in DNA or Ad5 vectored platforms.17–19 These bivalent vaccines had similar safety and tolerability as cAd3-EBO S but displayed suboptimal immunogenicity. This resulted in multiple injections being required for the DNA vaccine platforms.18,19 In the Ad5-vectored vaccine trial, individuals with preexisting Ad5 antibody titers had significantly lower vaccine-induced responses.17 Consequently, the cAd3 vector was chosen due to low cAd3 seropositivity worldwide.20,21 Nevertheless, the impact of pre-existing cAd3 titers on week four immunogenicity after cAd3-EBO S was assessed. A weak negative correlation between baseline cAd3 neutralizing antibody titers and week four Sudan GP binding antibody titers was the only significant impact on vaccine-induced responses observed in this trial. Similar vaccine-induced cAd3 neutralization antibody profiles and weak correlations have been reported previously for cAd3-vectored vaccines.10,16 Together the data suggest that receipt of the cAd3-EBO S vaccine will not preclude immunogenicity of subsequent cAd3-vectored vaccination.

Ebola components have also been evaluated in trials utilizing MVA-BN-Filo (which contains Zaire, Sudan, and Marburg antigens) as a boost after cAd3 or Ad26 vector-based vaccines.22,23 Strong humoral responses were noted in those trials, indicating that recombinant adenovirus-based vaccines can be utilized successfully in heterologous prime-boost regimens.12 However, the 56-day interval in the prime-boost regimen may not be ideal for outbreak settings. While strong SUDV-specific responses have been observed following MVA-BN-Filo boost,12 differences in immune assays and readouts confound direct comparisons between the trials’ results. The strong immunogenicity and durability of the single dose cAd3-EBO S vaccine in this trial indicate that it could be an optimal monovalent vaccine to use in outbreak responses, amenable to potential boosting during non-outbreak periods.

Correlates of protection against SUDV in humans remain to be established.24 Studies investigating the correlates of protection for the cAd3-EBO S vaccine in non-human primates (NHPs) are currently ongoing. However, NHP studies involving cAd3-vectored vaccines for EBOV and Marburg have indicated that greater binding antibody titers are associated with increased survival.15,25 The cAd3-EBO S vaccine in this trial elicited rapid and robust binding antibody responses in 85% of participants at week four, which remained durable in 82% of participants out to 48 weeks. These antibody responses were near peak levels just two weeks after vaccination, indicating a rapid onset of immunity, which is critical in outbreak response scenarios. Notably, binding antibody responses were also durable to 48 weeks. This is concordant with previous reports that viral-vectored vaccines induce robust, long-lived immunity.26 Persisting immune responses may facilitate use of a vaccine not only for protection of contacts of infected persons during outbreaks but also for vaccination in endemic areas, particularly for those with potential episodic occupational risk such as health care workers. Future trials of this vaccine should aim to explore this important finding.

Although the role of T cell responses in animal models of SUDV vaccination and challenge studies have yet to be reported, studies of SUDV-infected human samples suggest importance of CD8 T cells in survival after SUDV infection and CD4 T cells in neutralizing antibody responses.27–29 An early T cell recall response to an Ebola infection in vaccinated individuals would be expected to decrease peak viral load, accelerate development of antibody titers and lessen the pathological impact of infection.30 In this trial, half of participants responded to the vaccine with both humoral and CD4 T cell responses. Furthermore, the week four GP-specific ELISA titers and frequency of non-naïve CD4 T cells were correlated. These data support a role for CD4 T cell help in the development of humoral immune responses to the cAd3-EBO S vaccine. However, controlled studies in animal models are needed to further define the potential role of T cells in SUDV infection.

There are several limitations to this trial. This phase 1 study was designed and powered to evaluate the safety and tolerability of the vaccine candidate and not to evaluate the efficacy of cAd3-EBO S. The small trial size and lack of placebo control limit the power of our statistical statements and conclusions on vaccine-induced immune response and optimal dose. In addition, this trial occurred at a single trial site, potentially limiting the generalizability of the results. The trial enrollment was biased towards male participants which confounded our efforts to perform a sex-based analysis of the immunogenicity. Biases in safety assessments also cannot be ruled out, especially due to the open-label study design. The open-label design allowed trial participants and site staff to be aware of vaccine and dose designation, which could have impacted self-reported reactogenicity. In addition, these are the first SUDV-specific trial results to be published, meaning that only limited reference can be made with existing literature and conclusions are difficult to draw based on any such comparisons.

Ebola disease outbreaks are sporadic, unpredictable, and accelerating in frequency with consequent significant threat to global health, yielding a growing imperative to develop effective tools to prevent and treat disease caused by multiple Filoviridae viruses. A potent, tolerable, single dose vaccine like cAd3-EBO S that offers both acute and durable immune responses may be capable of both saving individual lives and reducing viral transmission rates, underscoring the urgent need to expedite advanced testing of this vaccine. The cAd3-EBO S monovalent vaccine has been evaluated in this phase 1 trial and a completed Sabin Vaccine Institute-sponsored phase 1b trial (NCT04723602). A Sabin Vaccine Institute-sponsored phase 2 trial in Uganda and Kenya is being developed, with enrollment planned for later in 2023. The data we present in this manuscript support the utility of the cAd3 vaccine platform in SUDV outbreak responses.

Supplementary Material

Supplemental file

Research in Context.

Evidence before this study

Sudan ebolavirus, along with Zaire ebolavirus, are the two species of the Ebolavirus genus of the Filoviridae family responsible for the majority of human outbreaks of Ebola Virus Disease (EVD). There have been eight recorded outbreaks of Sudan ebolavirus (SUDV), including a recent outbreak in Uganda from September 2022 to January 2023. Despite the availability of approved vaccines and therapeutics for Zaire ebolavirus (EBOV), there are none approved for use against SUDV. Therefore, there is a pressing need to evaluate vaccine candidates specific to SUDV. We searched PubMed from January 1st, 1990, through November 4th, 2022, using search terms “Sudan”, “Ebola”, “SUDV”, “vaccine”, and “clinical trials”. The search yielded reports from phase 1 and 2 clinical trials of several vaccine platforms including DNA, human Ad5- or Ad26-vectored vaccines, and chimpanzee (c)Ad3-vectored vaccines. While trials for the DNA vaccines and Ad5 vector vaccines demonstrated favorable safety results, these platforms were suboptimal due to low immunogenicity of the DNA platform (necessitating multiple doses) and high population immunity against the Ad5 vector (resulting in significantly decreased GP-specific responses in individuals with high pre-existing antibody titers to Ad5). An MVA-BN-Filo boost (containing Zaire, Sudan, and Marburg antigens) following a Zaire-specific Ad26.ZEBOV prime has been shown to induce responses against SUDV following regimen completion, although this regimen is not approved for use against SUDV. A trivalent pan-filo Ad26.Filo prime and MVA-BN-Filo boost recently completed phase 1 evaluation, demonstrating safety and immune responses against Zaire, Sudan, and Marburg antigens following regimen completion. Unfortunately, a 56-day prime-boost interval makes these regimens less desirable during an outbreak response. Another vaccine with published results is a bivalent cAd3-EBO vaccine expressing both Zaire and Sudan glycoproteins (GPs), the Sudan component of which is evaluated as a monovalent vaccine in this report. The cAd3 vaccine platform offers advantages of a favorable safety profile and vaccine-induced immune response, while avoiding the interference from pre-existing immunity that occurs with human adenovirus vaccines and requiring only a single administration for induction of immune responses.

Added value of this study

This trial describes the outcomes from the first SUDV-specific vaccine trial evaluating a single dose cAd3-EBO S vaccine, demonstrating that the vaccine is safe, well tolerated, and immunogenic. We observed a SUDV GP-specific binding antibody response in 85% (n=34/40) of participants four weeks after vaccination, which was durable in 82% (n=31/38) of participants out to 48 weeks.

Implications of all the available evidence

These trial results support the utility of this SUDV-specific vaccine, cAd3-EBO S. The rapid development of a durable antibody response following a single vaccination highlights the suitability of this vaccine in SUDV outbreak responses. Importantly, the cAd3-EBO S vaccine product has been evaluated in more individuals than any other single-dose SUDV-specific vaccine, having completed phase 1 studies in both monovalent and bivalent cAd3-EBO formulations. The results described here, in addition to the previously published cAd3-vectored bivalent EBO vaccine clinical trial results, demonstrate that the cAd3-EBO S vaccine is safe and produces durable binding antibody and T cell responses against the SUDV GP by four weeks after vaccination.

Acknowledgements

We thank the clinical trial participants for their contribution to furthering vaccine research. We acknowledge the Vaccine Production Plant and Vaccine Clinical Materials Program for provision of the vaccine for the clinical trial. We acknowledge the Emmes corporation for assistance with data management and protocol development. We acknowledge our colleagues Lesia K. Dropulic for her review of the manuscript, and Merlin L. Robb, Melanie D. McCauley, Casey Storme, Jacqueline Sievers, and Jayda Jones for technical and project management support. The RV508 Study Team at MUWRP included Jacqueline Namugabo, Claire Beingana, Maureen G. Mukyala, Joseph Wandege, Christine Nanteza, Brenda Atwijuka, Juliet Kizanye, Gertrude Nassanga, Joanita Namuli, Herbert Kityo, Mathias Ssekitoleko, Roy Nassaka, Hilda Mutebe, and Mable Kabahubya. Study team members at the VRC included Alicia Widge, Sarah E. O’Connell, Obrimpong Amoa-Awua, Sandeep R. Narpala, Britta Flach, Josephine Cox, Allison Beck, Mercy Guech, Ellie Seo and Judy A. Stein. The National Institutes of Health funded the study via an interagency agreement with Walter Reed Army Institute of Research.

Footnotes

Declaration of Interests

NJS is listed on patents involving cAd3-vectored vaccines. All other authors declare no competing interests.

Disclaimer: The views expressed are those of the authors and should not be construed to represent the positions of the U.S. Army, the Department of Defense, the U.S. National Institutes of Health, or the Henry M. Jackson Foundation for the Advancement of Military Medicine. The investigators have adhered to the policies for protection of human subjects as prescribed in Army Regulation 70–25.

Data Sharing Statement

Data generated in this study is available as de-identified participant data on ClinicalTrials.gov (NCT04041570, https://www.clinicaltrials.gov/ct2/show/NCT04041570) from 24 November 2021 with no end date. The study protocol, statistical analysis plan, and informed consent form are available on ClinicalTrials.gov (https://www.clinicaltrials.gov/ProvidedDocs/70/NCT04041570/Prot_SAP_ICF_000.pdf). Additional data may be made available upon reasonable request to the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file
NIHMS1924129-supplement-Supplemental_file.pdf (5MB, pdf)

Data Availability Statement

Data generated in this study is available as de-identified participant data on ClinicalTrials.gov (NCT04041570, https://www.clinicaltrials.gov/ct2/show/NCT04041570) from 24 November 2021 with no end date. The study protocol, statistical analysis plan, and informed consent form are available on ClinicalTrials.gov (https://www.clinicaltrials.gov/ProvidedDocs/70/NCT04041570/Prot_SAP_ICF_000.pdf). Additional data may be made available upon reasonable request to the corresponding author.

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